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Poster Session - kormb.or.kr

May 02, 2022

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Page 1: Poster Session - kormb.or.kr

Poster Session

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A_Antibiotics, Antifungals, and Antiviral Compounds

A-1

Synthesis and Characterization of Carboxymethyl Cellulose/Silver Nanobiocomposites Using a Solution Plasma Process with Silver Electrodes

Jung Wan Kim1,2, Hyo-Yeon Kwon1, Yu-Been Ko2, Ji-Min Kim2, and Sang-Yul Lee3 1Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea 2Department of Bioengineering and NanoBio Engineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 3Department of Material Engineering, Korea Aerospace University,Korea, Goyang 10540, Republic of Korea

Solution plasma process (SPP) was adapted to synthesize biocomposites of carboxymethyl cellulose (CMC)/silver nanoparticles (AgNPs). Unlike previous studies using AgNO3 as the precursor and tungsten electrodes, silver electrodes were used in this study for the synthesis ofpure CMC/AgNP biocomposites. CMC/AgNPs were synthesized by discharging plasma at 800 V, 30 kHz for 7 min in 0.3, 0.5, 0.75, or 1.0%CMC solutions. UV-Vis spectroscopy showed a peak at 406, 408, 407,and 406 nm with absorbance of 44.7, 37.3, 35.3, and 34.9, respectively.The absorbance was much higher than those obtained in the previous studies (3-3.5 at 440 nm). Blue shift transition of the peaks and high absorbances could be due to the formation of smaller particles and increased surface plasmon resonance (SPR), respectively. FTIR analysisshowed no change in the molecular structure of CMC during SPP. Antimicrobial activity was investigated against 6 pathogens (Escherichiacoli, Vibrio vulnificus, Pseudomonas aeruginosa, Staphylococcus aureus,Bacillus cereus, Candida albicans). CMC (1.0%)/AgNP biocompositeshowed minimal inhibition concentrations (MIC) in a range of 58.5-78μg AgNPs/ml for the bacteria and 415.6 μg /ml for the yeast. V. vulnificusbecame more resistant to the biocomposites when culture in low salt medium, having MIC of 195 μg/ml. The results indicated that synthesisof antimicrobial biocomposites were possible using Ag electrodes instead of AgNO3. Keywords : Solution plasma process (SPP), CMC/AgNP biocomposites,antimicrobial activity

A-2

Antimicrobial Effect of MSF, a Novel Material Created by Fusion of Probiotics and Filipendula glaberrima Nakai Extract

Gyeong hun Choi1, Boram Jeon1, Ki Young Kim1, Sanghyun Lee2, Yun Ji Lee3, Dae Kyun Chung1,4,5*, and Hangeun Kim5* 1Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea 2Department of Plant Science and Technology, Chung-Ang University, Anseong 17546, Republic of Korea 3Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Eumseong 27709, Republic of Korea 4Skin Biotechnology Center, Kyunghee University, Suwon 16229, Republic of Korea 5Research and Development Center, Skin Biotechnology Center Inc., Yongin 17104, Republic of Korea

Filipendula glaberrima Nakai has been used as traditional painkiller orto cure frostbite and burn. The conventional therapy suggests that this plant may have antimicrobial effects, but the efficacy of Filipendula glaberrima Nakai extracts on antimicrobial activity in HaCat cells waspoor. hBD3 is known as a peptide with antimicrobial activity, and especially shows excellent efficacy against Staphylococcus aureus. Toincrease the antimicrobial efficacy, we prepared MSF, which is L. plantarum K8 lysates that manufactured after fermentation in a mediumcontaining F. glaberrima Nakai extract. MSF significantly increased hBD3 transcripts and inhibited the internalization of S. aureus to HaCatcells as compared to Filipendula glaberrima Nakai extracts only or L.plantarum K8 lysates only. In the current study, we developed a new material with enhanced antimicrobial efficacy through the fusion of probiotics and the extracts of natural products. This novel material suchas MSF could be used to develop cosmetic materials with antibacterialeffects. Keywords : Filipendula glaberrima Nakai, hBD3, MSF

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A-3

Characterization of Alginate/AgNP Biocomposites Synthesizedby Solution Plasma Process Using Silver Electrodes as an Antimicrobial Agent

Suyeon Kim2, Su-Jin Kang1, Sang-Yul Lee3, and Jung-Wan Kim1,2*

1Department of Bioengineering and Nano Bio Engineering, Graduate School of Incheon National University, Incheon 22008, Republic of Korea 2Division of Bioengineering, Incheon National University, Incheon 22008, Republic of Korea 3Department of Material Engineering, Korea Aerospace University, Goyang 10540, Republic of Korea

Previously, alginate-AgNP biocomposites with antimicrobial activitieswere synthesized by a facile one-step solution plasma process (SPP) using AgNO3 as a precursor, alginate from brown algae as the matrix, and tungsten electrodes. In this study, more efficient synthesis of cleanerbiocomposites was attempted using Ag electrodes without the precursor.Plasma was discharged in 0.75% alginate solution using a pulsed field unipolar power supply at 800 voltages with 30 kHz of frequency for 2,3, or 7 min with Ag electrodes with a gap of 1 mm. UV-Vis spectroscopyshowed a peak at 407 ~ 413 nm, having blue-shift compared to the resultsobtained using AgNO3 (415-440 nm). Also, the peaks had absorbance of 21.55~ 45.08, which was much higher than those of the previous results(1~2.5). It implied that the sizes of AgNPs produced using Ag electrodesmight be smaller and had higher surface plasmon resonance. The resultsof FTIR showed that chemical bonds of alginate were not changed. Theminimum inhibitory concentration of against E. coli, V. vulnificus, P. aeruginosa, S. aureus, B. cereus, were 9.12~ 18.24 ㎍/ml; C. albicans, 60.8μg/ml of AgNPs that were synthesized using 0.75% alginate and discharging plasma for 2 min. Therefore, the SPP using Ag electrodes seemed to be a much more efficient way to produce antimicrobial agentsthan the previous SPP. Keywords : Solution plasma, alginate-silver nanoparticle biocomposite,antimicrobial agent

A-4

Multi-Omics Based Characterization of Inhibition of Methicillin-Resistant Staphylococcus aureus by lactobacillus species

Sunghyun Jo, Won-Suk Song, Jae-Seung Lee, Hyo-Jin Jeon, Ji-Eun Kwon,Ji-Hyeon Park, Ye-Rim Kim, and Yun-Gon Kim* Department of Chemical Engineering, Soongsil University, Seoul 06978, Republic of Korea

As demands for new strategies to control methicillin-resistant Staphylococcusaureus (MRSA) increase, studies that gain insight from gut bacteria regarding growth inhibition and anti-virulence strategies have been reported. Although it was reported in several studies that Lactobacillusspecies, lactic acid-producing bacteria, have antibacterial activity against MRSA, the molecular mechanism under these phenomena is unclear. In here, we evaluated the inhibition effect against MRSA Lactobacillus species isolated from stools of infants and performed a multi-omics study about the highest growth inhibitory strains. Firstly, we observed that MRSA growth inhibition was not correlated with extracellular pH level, affected by lactobacillus. In the multi-omics study, it was observed that MRSA significantly upregulated arginine deiminase system, proteins related protein synthesis, and downregulatedquorum-sensing system, capsular polysaccharide synthesis proteins inthe lactobacillus co-culture model. In this study, the underlying mechanism involved in growth inhibition and anti-virulence of MRSAmediated by lactobacillus was discovered. It is expected that these resultscan provide insight into probiotics- pathogen interaction mechanisms and new opportunities for antibiotics development. Keywords : MRSA, Lactobacillus, multi-omics

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A-5

Manipulation of Bacterial Mechanisms for Engineering Applications

Han-Shin Kim1, So-Young Ham2, Hee-Deung Park2,3, Kyung-Chul Kim1,Minsu Son1, Kyung-Taek Koh1, and Jeong-Hoon Park4 1Korean Peninsula Infrastructure Special Committee, Korea Institute of Civil Engineering and Building Technology (KICT), Goyang-si, Gyeonggi-do 10223, Republic of Korea 2School of Civil, Environmental and Architectural Engineering, Korea University, Seoul 02841, Republic of Korea 3KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea 4Clean Innovation Technology Group, Korea Institute of Industrial Technology (KITECH), Jeju-Si, Republic of Korea

Biofilms are composed of a single or multiple species of bacteria embedded in extracellular polymeric substance (EPS), which affects increasing antibiotic resistance through restriction of the transport of antibiotics to the bacteria cells. An alternative approach to treatment withantimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found mandarin peel extract (MPE)'s ability to inhibit P. aeruginosaPA14 biofilm formation. The result showed that 62-71% and 59-65% reduction in biofilm volume and thickness by 0.002-0.2% MPE treatment, respectively. Furthermore, MPE decreased production of EPS, whereas increased bacterial swarming motility. These results leadsto the hypothesis that MPE reduced level of a second messenger c-di-GMP, which involved in bacterial biofilm development. To thes thishypothesis, a reporter-based measurement assay was used to evaluate the c-di-GMP inhibitory effect of MPE. The result showed that MPE slightly but significantrly reduced the c-di-GMP level through increasedphosphodiesterase activity. Taken together, these finding suggest that MPE as a biofilm inhibitor has new potential for pharmacological and industrial applications. Keywords : Mandarin peel extract, biofilm inhibition, c-di-GMP

A-6

Synthesis and Antimicrobial Activity of Cyclic mBjAMP1 from Branchiostoma japonicum

Solmin Kim, Hysuk Yun, and Chul Won Lee*

Department of Chemistry, Chonnam National University, Gwangju 61186, Republic of Korea

Multidrug resistant (MDR) bacteria are rapidly increasing due to excessive use of antibiotics. Antimicrobial peptides (AMP) are in the spotlight as an important materials to overcome this problem. However,the disadvantage of AMP is that it has low bioavailability due to its unstable form. To overcome this, mBjAMP, one of the antimicrobial peptides, was newly designed and synthesized in a cyclic form (cyclotideform). Cyclotide has the advantage of being structurally stable againstchemical, enzymatic and thermal conditions due to its multiple disulfidebonds and head to tail cyclization. mBjAMP1 consists of 21 amino acidresidues and was isolated from Branchiostoma japonicum. And it contains two cysteines in the primary sequence. First, a wild-type sampleof mBjAMP1 was synthesized through solid-phase peptide synthesis (SPPS). After purification by HPLC, air oxidation was used to oxidizethe thiol group on the two cysteine residues to form a disulfide bond. Cyclic mBjAMP1 (C-mBjAMP1) was generated through head-to-tail cyclization. The CD spectrum of C-mBjAMP1 was used to confirm theconformational changes by cyclization of mBjAMP1. In addition, a minimum inhibitory concentration (MIC) was determined to assess theimprovement of antimicrobial activity of the peptides. Keywords : Antimicrobial peptides, cyclotide, bioavailability

A-7

Antifungal Metabolites Produced by Bacillus siamensis Isolated from Rhizosphere Soil of Weeds

Jeongeun Kim, Jueun Kim, and Chul Won Lee*

Department of Chemistry, Chonnam National University, Gwangju 61186, Republic of Korea

The establishment of strategy for dealing with plant disease is the oneof the most important tasks in agricultural industry. The Ralstonia solanacearum, one of the severe plant fungal pathogens that occur in thewet tropics, subtropics and some temperature regions of the world, arewell known for cause serious damage to tomato and other solanaceousplants. We isolated Bacillus strain from rhizosphere soil of weeds whichshowed antifungal activity against R. solanacearum. The strain was identified as Bacillus siamensis by the 16S rRNA sequence analysis. TheLC-MS analysis of the culture supernatant of B. siamensis suggested thatit produces various cyclic lipopeptides including iturins (m/z 1043.6 and1057.8), fengycins (m/z 1464.5 and 1492.0) and surfactins (m/z 994.8, 1009.0, 1023.1 and 1037.3). To obtain the pure lipopeptides, crude metabolites were precipitated by adjusting culture media to pH 2 and the precipitate was dissolved in acetonitrile. Then the crude lipopeptides were further purified using preparative reverse phase-high performanceliquid chromatography (RP-HPLC). The determination of minimal inhibitory concentration (MIC) and hemolysis assay were carried out toconfirm the activities and toxicities of the purified lipopeptides. The molecular structure of the purified peptides was confirmed by 2D NMRspectroscopy. Keywords : Ralstonia solanacearum, cyclic lipopeptides, antifungal compounds

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A-8

Verification of Antibacterial Activity of Ag/Cu Nanoparticles against Several Pathogenic Bacteria

Ji Young Lee1, Chan Hun Yoon3, Young Ju Jeong3, and Dae Ook Kang2*

1Division of Life Science and Research Institute of Life Science, Gyeongsang National University, Jinju, Gyeongnam 52828, Republic of Korea 2Department of Bio Health Science, Changwon National University, Changwon, Gyeongnam51140, Republic of Korea 3Young Dong Tech. Co., Ltd., Changwon, Gyeongnam 51399, Republic of Korea

The antibacterial activities of nanoparticles of silver and copper werestudied by using food-borne pathogenic bacteria as test microorganismsincluding such as Escherichia coli, Salmonella enteritidis, Listeria monocytogenes,Shigella boydii, Bacillus cereus and Staphylococcus aureus. Agar welldiffusion method showed Ag/Cu nanoparticles (Ag/CU NP) had antibacterial activity against all tested microbes. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) ofAg/CU NP were determined and compared. The antibacterial activity of Ag/CU NP were not affected by temperature and pH changes. Cell wall and membrane were severely damaged by treatment of Ag/CU NPto bacterial cells, which was observed via SEM. The antibacterial activitywas retained for up to three months at both 4℃ and -20℃. The Ag/CUNP inhibited growth of L. monocytogenes in beef, boiled rice and milkstored at 4℃ for 30 days. Taken these together, the Ag/CU NP might beapplied to food storage containers. Keywords : Ag/Cu nanoparticles, antibacterial activity, pathogenic bacteria

A-9

Antimicrobial Use of Bacteriochlorophyll a to Inhibit the Growth of Cyanobacteria Which Cause Harmful Agal Boom

Hyeonjun Kim1, Eui-Jin Kim2, and Jeong K. Lee1*

1Department of Life Science, Sogang University, Seoul 04107, Republic of Korea 2Microbial Research Department, Nakdonggang National Institute of Biological Resources, 137, Donam 2-gil, Sangju-si, Gyeongsangbuk-do 37242, Republic of Korea

Chlorophyll synthase (ChlG) esterifies chlorophyllide a with a C20-moiety of either geranylgeranyl or phytyl group to form chlorophylla (Chl a), a main pigment of the O2-evolving photosynthetic organisms.Likewise, bacteriochlorophyll synthase (BchG) esterifies baceriochlorophyllidea with the same C20-moiety to form bacteriochlorophyll a (Bchl a), a mainpigment of anoxygenic photosynthetic bacteria. Interestingly, ChlG ofSynechocystis sp. PCC6803 is competitively inhibited not only by bacteriochlorophyllide a but also by BChl a with an inhibition constant(Kic) of 39.5 ± 2.2 μM, whereas the activity of ChlG was not affected by Chl a. Consistently, the exogenous addition of BChl a to the culture ofSynechocystis sp. PCC6803 results in the retardation of its photosyntheticgrowth in a dose-dependent manner, while Chl a does not show any effect. The growth-inhibitory effect of BChl a was further illustrated with othercyanobacterial species; Anabaena variabilis, Anabaena circinalis, andMicrocystis aeruginosa, which are the representative cyanobacterial species for algal blooms in river. Taken together, BChl a arrests the cyanobacterial growth by inhibiting ChlG and thus Bchl a may be practically used to control the cyanobacterial blooms. Keywords : Cyanobacteria, chlorophyll synthase, bacteriochlorophyll a

A-10

Screening for Inhibitors of the Cyclic(Phe-Pro) Signal in Pathogenic Vibrio Species

JaeJun Lim1, Keun-Woo Lee1, In-Hwang Kim1, and Kun-Soo Kim1,2* 1Department of Life Sciences, Sogang University, Seoul 04107, Republic of Korea 2Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 04107, Republic of Korea

To obtain chemicals interrupting a signal closely associated with the virulence expression in the human pathogen Vibrio vulnificus, we screened total 6,566 chemicals of the Chembank collection cordially provided by the Korean Chemical Bank for candidates that repress the expression of the gene leuO, which encode a master regulatory proteinfor the virulence pathway associated with the signal molecule cyclic-Phe-Pro (cFP). For this, we constructed a screening system in which luxAB-reporter genes were transcriptionally fused to the promoterregion of leuO. The resulting strain was added with each of the collectionchemicals dissolved in DMSO, and the Lux activity was quantitativelymeasured in 96-well plates employing DMSO and purified cFP as negative and positive controls, respectively. Through three rounds of screening, we could narrow down to two compounds which represses the reporter expression to a 30% level but do not affect growth of cells.These final candidates SG0010 and SGR0020 significantly inhibit the expression of leuO of V. vulnificus in concentration-dependent manner,and, furthermore, inhibit transcription of ctxAB of V. cholerae and vvhBAof V. vulnificus, which encode exotoxins known to be a target of the cFP-ToxR-LeuO signaling pathway. These compounds not only reducedresistance to ROS, but also significantly increased survivability of miceinfected by V. vulnificus. These compounds exerted no significant harmful effects either on growth of V. vulnificus cells or viability of human epithelial cell line HaCaT as measured by the MTT assay. Theseresults suggest that these two compounds can serve as agents controlling the virulence of pathogenic Vibrio species. Keywords : Vibrio vulnificus, chemical compound, pathogenicity

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A-11

Sargassum fusiforme and Its Components Inhibit Respiratory Syncytial Virus Infection In Vitro and In Vivo

Kiramage Chathuranga, Asela Weerawardhana, and Jong-Soo Lee* College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy.Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effectsagainst respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration ofSFE significantly reducedRSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation.Moreover, oral inoculation of SFE significantly improved RSV clearancefrom the lungs of BALB/c mice. Interestingly, the phenolic compoundseicosane, docosane, and tetracosane were identified as active componentsof SFE. Treatment with a non-cytotoxicconcentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection. Keywords : Sargassum fusiforme, therapeutic effects, RSV

[The National Research Foundation of Korea (2018M3A9H4078703) and KRIBB Research Initiative Program (KGM9942011)]

A-12

Inhibition of Respiratory Syncytial Virus Replication In Vitro and In Vivo by Stichopus japonicus (selenka) Extract

Yehjin Cho, Kiramage Chathuranga, and Jong-Soo Lee*

College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

The sea cucumber Stichopus japonicus (Selenka) is an invertebrate animal inhabiting the coastal sea around Korea, Japan, China, and Russia. It is one of the highest commercially valuable species as seafoodand it has been commonly used for centuries in indigenous and folk medicine. Although it has many therapeutic effects, antiviral activity against RSV has not been reported in detail. In this study, we show thatextracts from S. japonicus has antiviral effects against Respiratory Syncytial Virus (RSV) in vitro cell cultures and an in vivo mouse model.Treatment of human respiratory tract cell line (HEp2) with a non-cytotoxicconcentration of S. japonicus extract significantly reduced RSV replication,RSV-induced cell death, RSV gene transcription and RSV protein synthesis. Moreover, the treatments significantly diminish syncytial formation after RSV infection in HEp2 cells. Time dependent treatmentof S. japonicus after RSV infection in HEp2 cells showed that treatmentwith two-hour post infection of virus infection can provide better resultby demolishing further replication of RSV virus in Hep2 cell line. Interestingly, oral inoculation with S. japonicus extract significantly improved viral clearance in the lungs of BALB/c mice. Collectively, these results suggest that extracts of S. japonicus could use as a potent natural anti-RSV candidate. Keywords : Stichopus japonicus (selenka), RSV, therapeutic effects

[The National Research Foundation of Korea (2018M3A9H4078703) and KRIBB Research Initiative Program (KGM9942011)]

A-13

Studies on a Bacteriophage Specific for Antibiotic-Resistant Escherichia coli Isolated from Agricultural Environment

So-Hui Park, In Young Choi, Amani Sliti, Ye-Rim Park, and Mi-Kyung Park* School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

Antibiotic-resistant (AR) bacteria are broadly present in the agriculturalenvironment and their negative effects have significantly risen in the pastfew decades. The purpose of this study was to isolate and characterizeAR Escherichia coli and its phage. Thirty-seven strains of E. coli wereisolated from agricultural environment and IMViC test was performed prior to 16S rRNA sequencing. The presence of virulence genes and antibiotic resistance were investigated using PCR and E test, respectively.The E. coli-specific phage was isolated and purified from slaughter wastewater. Morphological characteristics, host range, and stability ofthe purified phage were performed using TEM, dot assay, and plaque assay. This phage had an icosahedral head (65.44 ± 8.90 nm in length,10.08 ± 2.59 nm in width) and a tail (14.76 ± 3.58 nm in length, 2.03 ±0.59 nm in width). In addition, it showed clear plaques against 11 strainsof E. coli including E. coli CMD05061 (an AR E. coli) and was stable under various ranges of pHs (3 - 11) and temperatures (-20 - 60℃). Thisstudy demonstrated that E. coli-specific phage could be potentially useful as a biocontrol agent against AR E. coli. Keywords : Escherichia coli, antibiotic resistance, bacteriophage

A-14

Heterologous Epression of the Nystatin-Like Pseudonocardia Polyene B1 Biosynthetic Gene Cluster

JiHee Park, Hee-Ju Nah, Si-Sun Choi, and Eung-Soo Kim*

Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea

Many natural products produced by actinomycetes have been used as antibiotics for decades in various fields. Especially, Polyene antibiotics such as nystatin A1 and amphotericin B belong to a large family of veryvaluable antifungal polyketide compounds. Nystatin-like Pseudonocardiapolyene (NPP) A1 which contains disaccharide moiety in tetraene macrolide backbone has been reported to be produced by a rare actinomycetes called Pseudonocardia autotrophica. Previously, a pharmacokinetically-improved NPP derivative named NPP B1 was developed by substituting two amino acids in the enoyl reductase domainin module 5 of the nppC. Although the NPP B1 productivity was increased by various strategies including elimination of the native plasmid in P. autotrophica and overexpression of pathway-specific regulatory genes, its titer is still far from satisfactory. To overcome thislow-titer issue, the NPP B1 biosynthetic gene cluster (BGC) from P. autotrophica was isolated via bacterial artificial chromosome (BAC) system, followed by homologous and heterologous expressions in several Streptomyces hosts. More details related to NPP B1 BGC expression and its biological activities will be discussed. Keywords : NPP B1, heterologous expression, Bacterial Artificial Chromosome (BAC)system

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A-15

Isolation of Novel Streptomyces via Antifungal Bioassay-Based Screening

Yeon-Jin Yoo, Heung-Soon Park, Si-Sun Choi, and Eung-Soo Kim*

Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea

Because of the side effects of chemical pesticides, research is proceed to develop microbial-based biological pesticides as the need for eco-friendly pesticide development has been raised. To develop an actinomycetes-based biological pesticide that produces a variety of useful secondary metabolites, we selected antifungal-active actinomycetesthrough antifungal activity tests on F. oxysporum and C. albicans using2400 actinomycetes cultures medium. The selected strains conducted an additional antifungal activity test for 10 plant pathogenic fungi and selected one candidate strain for high antifungal activity. It was also pot-tested with selected strains, and the control efficacy was 61.6% forstrawberries, 72.7% for tomatoes, and 60.0% for peppers. It showed highcontrol efficacy Whole genome sequencing of this strain was performedto identify the putative biosynthetic gene clusters responsible for the antifungal compounds. the data was analyzed by antiSMASH. In silicoanalysis by antiSMASH, Candidate BGCs were identified at those showed high homology responsible for the biosynthesis of rifamycin with antifungal activity in the NRPS family. Additionally, we identifiedBGCs that showed high homology with elaiophyin and mediomycin inType 1 PKS. These BGCs are predicted to show antifungal activity. Asa result, the newly-screened actinomycetes species could be the promising candidates for development of novel antifungal agents or eco-friendly microbial pesticides. Keywords : Antifungal, actinomyces, microbial pesticides

A-16

Antimicrobial Spectrum and Characterization of Purified Recombinant Micro Halocin HB384, Derived from Halophiles

Sang-Jae Lee1*, Ga Eul Jeong1, Dariimaa Ganbat1, YuJeong Yeom1, Jio Kim1, Dong-Woo Lee2, Seong-Bo Kim3, Yong-Jik Lee4, and Han-SeungLee1

1Major in Food Biotechnology and Research Center for Extremophiles & Marine Microbiology, Silla University, Busan 46958, Republic of Korea 2Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 3Bio-Living Engineering Major, Global Leaders College, Yonsei University, Seoul 03722, Republic of Korea 4Department of Bio-Cosmetics, Seowon University, Chung-Ju 28674, Republic of Korea

The problem of resistance due to excessive abuse of antibiotics is the cause of the birth of multidrug-resistant bacteria, and the problem hasnot been solved until now. Therefore, as a solution to the problem of existing antibiotics, attention was paid to the search for antimicrobial peptides (AMPs) whose resistance problem was not revealed. In this study, AMPs derived from halophiles were obtained based on the geneinformation of halocin peptides. It was named HB384, and the gene encoding HB384 was cloned into pGST vector and the recombinant HB384 was expressed in E. coli BL21. The recombinant protein was purified by GST affinity chromatography, and the molecular weight of HB384 was 3.14 kDa. Disk diffusion assays were performed to evaluateantimicrobial activity. HB384 showed antimicrobial activity against Gram-positive bacteria S. aureus, B. subtilis and Gram-negative bacteriaS. typhimurium, E. coli. Moreover, HB384 was stable at 99℃ for 8 hours,as the concentration increased, antimicrobial activity against pathogen.In less than 6 µg, HB384 almost reached the maximum antimicrobial activity against pathogen. When comparing the number of moles of antimicrobial activity substances per area of the clear zone, the antimicrobial activity of HB384 against B. subtilis was 3.2 times betterthan that of ampicillin. As a result, purified HB384 is expected to be applicable not only as a functional peptide material, but also as a substitute for existing antibiotics. Keywords : Micro halocin, antimicrobial kinetic activity, GST affinitychromatography

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A-17

Screening of Antagonistic Bacteria Having Antifungal Activity against Phytopathogenic Fungi of Chilli Pepper

MyeongSeon Ryu, Se Won Park, Hee-Jong Yang, Ji Won Seo, Jinwon Kim,and Do-Youn Jeong*

Microbial Institute for Fermentation Industry (MIFI), Sunchang 56048, Republic of Korea

Chilli anthracnose, Sclerotinia stem rot (SSR), Phytophthora blight of chilli pepper are caused by fungal pathogens such as Colletotrichum acutatum and Sclerotinia sclerotiorum, and Phytophthora species threatening the production of chilli pepper. To search biological controlagents (BCAs) of phytopathogenic fungi, five kinds of useful Bacillus-likeisolates were selected from field soil of Sunchang in Korea. The selectedisolates were characterized by production of enzyme, siderophore and indole-3-acetic acid (IAA). Also, antifungal activity against three of thephytopathogenic fungi that frequently occur in chilli pepper was tested. Among them, PBS-68 strain had the most excellent antifungal activity.Physiological characteristics of PBS-68 strain also confirmed by analysis of carbohydrate fermentation patterns and enzyme productionability. Based on the experimental results, PBS-68 strain was finally selected as a candidate for BCA. BLASTn search of the 16S rRNA genesequence via National Center for Biotechnology Information (NCBI) database indicated that the isolate, PBS-68 strain matched Bacillus subtilis (GenBank accession no. NR027552) with similarity values of 99.59% (1442/1448 bp). Based on the above results, B. subtilis PBS-68strain is expected to be used as a BCA for the chilli pepper pathogenic fungi. Keywords : Antagonistic bacteria, Bacillus subtilis, biological control agents

[This work was supported by a grant from the Establishment of IntegratedBiobank for Agriculture, Food and Livestock Microbiome Project funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA)]

A-18

Strategic Approach for Reducing Aflatoxin Levels in Korean Traditional Nuruk

Jeonghyun Yun1 and Jang-Eun Lee1,2*

1Department of Food Biotechnology, University of Science and Technology, Daejeon 34113, Republic of Korea 2Research Group of Traditional Food, Research Division of Strategic Food Technology, Korea Food Research Institute, Jeollabuk-do 55365, Republic of Korea

Nuruk is a fermentation starter used for brewing alcoholic beverages. Since Nuruk is made from uncooked grains with naturally inoculated microorganisms, it can be infected with Aspergillus flavus, which produces carcinogenic aflatoxin (AF). To determine the safety of alcoholic beverages and reduce the AF levels in Nuruk, this study evaluated total AF levels in Nuruk and the transfer of AF from Nurukto alcoholic beverages and isolated AF-producing fungi and antifungallactic acid bacteria (LAB). Out of 61 Nuruk, 14 exceeded the Korean permissible total AF level of 15 ppb. Only AF G1 is transferred to alcoholic beverages at a rate of about 1.2% to 1.3%. The LC-MS-based non-targeted metabolomics approach allowed for the selection of two LAB species, Weissella paramesenteroides and Pediococcus pentosaceus,exhibiting high antifungal activity. They reduced AF via inhibition of mycelial growth, AF production, and AF surface binding. This presentstudy was firstly considered the safety of Korean traditional alcoholic beverages in terms of the overall fermentation process starting from Nuruk.

Keywords : Aflatoxin, lactic acid bacteria, Nuruk

A-19

Antibiofilm and Antifungal Activities of Mmedium-Chain Fatty Acids against Candida albicans

Sagar Kiran Khadke, Jin-Hyung Lee, Yong-Guy Kim, and Jintae Lee*

School of Chemical Engineering, Yeungnam University, Gyeongsan 38541, Republic of Korea

Candida albicans is an opportunistic pathogen responsible for candidiasisand colonizes host tissues and implant devices via biofilm formation. These biofilms are tolerant to conventional antifungal therapeutics andthe host immune system. The transition of yeast cells to hyphae is a crucialstep in C. albicans biofilm development, and the quorum-sensing molecule farnesol inhibits this transition. We hypothesized that fatty acids mimicking farnesol might influence hyphal and biofilm formationby C. albicans. Amid 31 saturated and unsaturated fatty acids, six medium-chain saturated fatty acids, such as heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and lauric acid, efficiently inhibited C. albicans biofilm formation by more than 75%at 2 µg/ml with MICs in the range 100–200 µg/ml. These six fatty acidsat 2 µg/ml and farnesol at 100 µg/ml inhibited hyphal growth and cell aggregation. The addition of fatty acids to C. albicans cultures decreasedthe productions of farnesol and sterols. Furthermore, the downregulationof several hyphal and biofilm-related genes triggered by heptanoic or nonanoic acid closely resembled the changes instigated by farnesol. In addition, nonanoic acid, the most effective compound, diminished C. albicans virulence in a Caenorhabditis elegans model. Our results suggest that medium-chain fatty acids effectively inhibit hyphal growthand biofilm formation than farnesol. Keywords : Candida albicans, fatty acids, quorum sensing

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A-20

Iodoindoles as an Inhibitor of Biofilm Formation and Rapid Killing of Multidrug-Resistant Acinetobacter baumannii Strains and Other Microbes

Chaitany Jayprakash Raorane, Jin-Hyung Lee, and Jintae Lee*

School of Chemical Engineering, Yeungnam University, Gyeongsan 38541, Republic of Korea

Multi-drug resistant Acinetobacter baumannii is well-known for its rapid acclimatization in hospital environments. The ability of the bacterium to endure desiccation and starvation on dry surfaces for up to a month results in outbreaks of health care-associated infections. Previously, indole and its derivatives were shown to inhibit other persistent bacteria. We found that among 16 halogenated indoles, 5-iodoindole swiftly inhibited A. baumannii growth, constrained biofilmformation and motility, and killed the bacterium as effectively as commercial antibiotics such as ciprofloxacin, colistin, and gentamicin.5-Iodoindole treatment was found to induce reactive oxygen species, resulting in loss of plasma membrane integrity and cell shrinkage. In addition, 5-iodoindole rapidly killed three Escherichia coli strains, Staphylococcus aureus, and the fungus Candida albicans, but did not inhibit the growth of Pseudomonas aeruginosa. This study indicates the mechanism responsible for the activities of 5-iodoindole warrants additional study to further characterize its bactericidal effects on antibiotic-resistant A. baumannii and other microbes. Keywords : Acinetobacter baumannii, antibiotics, biofilm

A-21

Antifungal Activity of Bacillus subtilis NK 2 against Phytopathogen

MyeongSeon Ryu, Ji Won Seo, Hee-Jong Yang, Su-Ji Jeong, and Do-Youn Jeong*

Microbial Institute for Fermentation Industry(MIFI), Sunchang 56048, Republic of Korea

This study was conducted to isolate bacteria having antifungal activityagainst various phytopahogens by producing multifunctional biocontrolagents. The use of biological fungicides can effectively reduce the consumption of chemical fungicides or toxic substances to control plantdiseases. For the screen of biological fungicides, a total of 157 differentbacterial isolates were obtained from undamaged soil by repeated cultivation in Sunchang, Korea, and screened for antibiotics agents, siderophores, and extracellular enzymes (protease, cellulase, and amylase) production. Among the isolates, NK2 strain with superior enzymatic and antifungal activity was selected for further experiments. The NK2 strain was identified as Bacillus subtilis by 16S rRNA sequenceanalysis. Finally, physiological and biochemical characteristics of B. subtilis NK2 were examined. The characteristic research results suggestthat B. subtilis NK2 has useful multifunctional biocontrol ability againstvarious phytopathogens. Keywords : Antifungal, Bacillus subtilis

[This work was supported by a grant from the Establishment of IntegratedBiobank for Agriculture, Food and Livestock Microbiome Project funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA)]

A-22

Screening and Isolation of a Novel Polyene-Producing Streptomyces Strain Inhibiting Phytopathogenic Fungi in the Soil Environment

Heung-soon Park, Si-Sun Choi, and Eung-Soo Kim*

Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea

Microbial-based eco-friendly biological substances are needed to protect crops from phytopathogenic fungi and replace toxic chemical fungicides that cause serious environmental issues. To develop the substances, Bioassay-based antifungal screening of approximately 2,400 Streptomyces strains led to the isolation of 149 strains as tentativeantifungal producers. Among them, one Streptomyces strain was selectedas a potential novel fungicide producer to protect various crops in the soil environment through an in vitro antifungal assay against various phytopathogenic fungi. Whole-genome sequencing of the Streptomycesstrain and an anti-SMASH genome mining approach revealed an approximately 150-kb polyene biosynthetic gene cluster in the chromosome.The target compound responsible for the anti-phytopathogenic activitywas a giant linear polyene compound highly homologous to the previouslyreported neotrafibiricin A (NTF A). These results suggest that a bioassay-based screening of a novel antifungal Streptomyces strain followed by its genome mining for target compound BGC characterizationwould be an efficient approach to isolating a novel candidate phytopathogenicfungicide that can protect crops in the soil environment. Keywords : Streptomyces, phytopathogenic fungi, antifungal activity

A-23

Black Phosphorus-Ddecorated Nanoparticle as a Revertant of Polymyxin B Usage against Mcr-1-Mediated Resistant Escherichia coli

Hyejin Cho and Kwang-sun Kim*

RNA-Nano Biochemistry Laboratory(RNBL), Department of Chemistry Pusan National University, Busan 46241, Republic of Korea

Treatment of multidrug-resistant Gram-negative bacterial infections bypolymyxins has been impeded by the occurrence of resistance by Mcr-1,mobilized colistin resistance. Here, we report the synthesis of a nickel (Ni) doped Zinc oxide (NZO) combined with black phosphorus (BP) (NZB) nanocomposite and its effect on the action of polymyxin B (PolB) against polymyxin-resistant Escherichia coli carrying mcr-1 gene. Checkerboard assays showed that the combination of NZB and PolB showed a positive synergy against Mcr-1-expressing E. coli cells, resulting in a more feasible integration of PolB into E. coli. Further mechanistic studies found that the synergy is attributed by the charge neutralization of the E. coli cell surface by NZB. Finally, we determinedthe lowest synergistic concentration of NZB with PolB and the concentration was biocompatible to mammalian cell in vitro. Therefore,NZB is the first biocompatible nano-adjuvant to polymyxins against polymyxin-resistant E. coli cells, recognizing the physical status of bacteria instead of known adjuvants targeting cellular gene products. Therefore, NZB has the potential to repurposing polymyxins as leadinglast-resort antibiotics to combat polymyxin-resistant Gram-negative bacterial infections. Keywords : Nano-adjuvant, mobilized colistin resistance (mcr-1), charge neutralization

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A-24

Toxicological Effects of Polyvinyl Chloride and Low Density Polyethylene Microplastics on Earthworm

Songhee Lee1, Eunhea Jho2*, and Sooim Shin1*

1Department of Biotechnology and Bioengineering, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea 2Department of Agricultural and Biological Chemistry, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea

Despite of benefits derived from plastic use, accumulation of plastic inecosystems, is becoming an increasing environmental concern. Researchon microplastic toxicity has mainly focused on aquatic environments, while studies of that on terrestrial ecosystems are limited. The aim of this study was to examine the potential toxic effects of widely used Polyvinyl chloride(PVC) and Low density polyethylene(LDPE) on earthworm in oxidative stress. To investigate the potential effects of microplastic, level of ROS/RNS, amount of GSH, activity of SOD, andATP synthesis were measured with different concentrations of microplasticexposure. Although activity of SOD was not changed, the amounts of ROS and amount of GSH were increased in both PVC and LDPE groups.However, ATP synthesis was decreased in the PVC treated group and increased in the LDPE treated group. For PVC, an increase in antioxidantdefense led to the elimination of ROS, but failed for LDPE treated group.These results indicate that each microplastic have different oxidative stress mechanisms in the earthworm. These findings will provide implications for risk of microplastic in terrestrial ecosystems. Keywords : Polyvinyl chloride, low density polyethylene, oxidative stress

A-25

Changes of Antibiotic Resistance from Influent and Effluent in Wastewater Treatment Plants of Han River, Seoul, South Korea

Hanseob Shin, Yongjin Kim, and Hor-Gil Hur* School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea

Antibiotic resistance has emerged as critical health problem by overuseand misuse of antibiotics. Wastewater treatment plants (WWTPs) are considered as a sink and a source of antibiotic resistance. In this study,we applied culture dependent and independent method for investigationof antibiotic resistance in two WWTPs. The number of multidrug resistant (MDR) bacteria carrying antibiotic resistance genes (ARGs) decreased after treatment process of WWTPs, even though they wereresistant to corresponding antibiotics. Of the MDR bacteria, Gammaproteobacteriaclass seemed dominant in both influent and effluent, and mainly carriedARGs, possibly suggesting they are main carrier of ARGs in WWTPs.In addition, SmartChip analysis showed that the number and the abundance of ARGs were lower in the effluent than influent. However,some ARGs persisted the treatment processes. ARGs in WWTPs seemedto be correlated with mobile genetic elements (MGEs), especially integrons and insertional sequences. Both culture dependent and independent methods indicated that ARGs were reduced after treatmentprocess but significant concentration of ARGs still remained in effluentsof WWTPs. In addition, even in the mitigation of ARGs of MDR bacteria, they were already evolved and equipped with intrinsic resistance throughselective pressure by contaminants in WWTPs. This study suggests thatWWTPs work for mitigation of antibiotic resistance, at the same time,act as a hotspot of evolution of antibiotic resistance. Keywords : Wastewater treatment plants, antibiotic resistance

A-26

Characterization of a New Antimicrobial Agent against Bovine Mastitis-Causing Staphylococcus aureus RF122

Minhye Shin1, Daye Mun1,2, Hye Jin Choi1, Shelley M. Payne2, and Younghoon Kim1*

1Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea 2Department of Molecular Biosciences, College of Natural Science, The University of Texas at Austin, Austin TX 78712, USA

Bovine mastitis is one of the most prevalent and costly diseases in the dairy industry, and Staphylococcus aureus RF122 is the most prevalentpathogens causing intra-mammary infections in dairy cows. Iron is absolutely required for the bacterial growth, virulence associated with colonization and survival from the host immune system. The bacterialferrous iron transporter protein FeoB functions as a major iron transporter in prokaryotes that has been shown to play a crucial role in virulence of some pathogenic bacteria. In this study, we present a novelunconventional antibacterial agent that inhibits FeoB in vitro enzyme activity, bacterial growth, and virulence factor expression related to milkquality. The small molecule synergistically enhanced bacterial antibioticsusceptibility and was also effective against a broad range of Gram-positive pathogens, suggesting therapeutic potential to overcomethe emergence of antibiotic-resistant bacteria against conventional antibiotics. This novel inhibitor will may represent a promising biotechnological application for preventing S. aureus-induced bovine mastitis in the milk and dairy industry. Keywords : Bovine mastitis, ferrous iron transporter, Staphylococcus aureus

A-27

Study on High Antibacterial Activity Condition of Sodium Hypochlorite Solution at Low Concentration

Kwang-Hwan Jhee*, Hyeon-Bin Son, Won-Bin Bae, and Yeong-Jun JeonDepartment of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea

Sodium hypochlorite (NaClO) is a disinfectant widely used in hospitalsand food industries, and has antibacterial activity against not only bacteria but also fungi and viruses. The antibacterial activity of NaClOis thought to depend on the concentration of hypochlorous acid (HClO)in the solution rather than hypochlorite ion (ClO-). The pH of the solutiondetermines how much HClO is formed. Therefore, when we used NaClO,acid is used to lower the pH and increase the antibacterial activity. Tomaintain HClO solution in a stable form, obtaining maximize its antimicrobial activities, and minimize undesirable side products, the pHmust be maintained between 3.5 and 5. HClO easily penetrates bacterialcell due to its neutrality and attacks. The results of the time kill test wereobtained by exposure to 4 ppm for 1 minute showed 99.9% antibacterialactivity. In addition, the correlation between the change in chlorine concentration and antibacterial activity according to the factor of temperature, humidity and the mixing of surfactant or chlorhexidine (CHX) was confirmed. Our results provide conditions of high antimicrobial activity while minimizing toxicity with a low NaClO concentration. Keywords : Hypochlorous acid, sodium hypochlorite, time kill test

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A-28

Properties of Antimicrobial Peptide Lactoferricin and Lactoferrampin:Expression and Purification from E. coli

Hanseul Seo, Ju-Un Park, Jung-Min Park, So-Min Park, Na-Rin Kim, andHyune-Hwan Lee*

Lab of Molecular Biotechnology, Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yong-in 17035, Gyeonggi-do, Republicof Korea

Short peptide lactoferricin (LFcin) and lactoferrampin (LFampin) which are derived from human lactoferrin are known as antibacterial, antifungal, and even anti-viral peptide. LFcin is found at N-terminal domain and LFampin is at C-terminal, respectively. In this study, we modified the original amino acid sequences of both peptides to more strong one by comparison of several sequences. The strong LFcin peptide, HLSA and mLFampin were chosen and synthesized and the antibacterial and antifungal activity were analyzed. Both peptides showed strong and broad specificities. On the other hand, we tried to express both peptides in E. coli. For the expression, Mxe intein was usedas fusion partner to obtain the short peptide more easily by cutting withDTT. For the purification by affinity column, CBD(chitin-binding domain) or (His)6-tag was used. After cutting the expressed fused proteinwith DTT, both peptides were purified with the chitin-affinity column or Ni-NTA column. The purified peptides were identified by SDS-PAGE.The isolated peptides showed strong antibacterial activity against Staphylococcus aureus as indicator. Here we report the results. Keywords : Lactoferricin, antimicrobial peptide, lactoferrampin

A-29

Biocontrol of Carbapenem-Resistant Klebsiella pneumonia (a CRE) Using Bbacteriocin-Mediated Antagonism in the Gut Microbiota

Seohee Jeong and Heenam Stanley Kim*

Division of Biosystems and Biomedical Sciences, College of Health Sciences, Korea University, Seoul 02841, Republic of Korea

Klebsiella pneumonia is a commensal bacterium residing in the humangastrointestinal (GI) tract and a prevalent nosocomial pathogen, causingpulmonary diseases, pyogenic liver abscess, and bacteremia. Carbapenem-resistant K. pneumonia (CRKP), particularly, has been a serious problemto human health due to its multidrug resistance. In this study, we characterized 68 K. pneumoniae isolates that produce various bacteriocinswith specific killing activity against diverse K. pneumoniae strains. Among these, three strains (Kpn101, Kpn102, Kpn103) were selected as potential biocontrol agents for CRKP infections based on their potentkilling effect against CRKP strain ATCC-BAA 1705. We found that at least in strain Kpn102, a plasmid confers the bacteriocin-mediated killing activity. Our data suggest that bacteriocin-producing commensalK. pneumoniae can be developed as an effective and safe tool to eliminateCRKP in the gut, without causing collateral damage on the gut microbiotaas in current antimicrobial therapies Keywords : Gut microbiota, Klebsiella pneumoniae, multidrug-resistance

A-30

Comparison of 5 Types of Oral Cleansers

Seon Kyeong Kim2, Ji-Min Gwak2, Eun-Jin Kim1, Gyeong-Woo Lee2, Young-Jung Lee3, Hae-In Jeon4, and Bae-Il Kwon1*

1Department of Dental Hygiene, Silla University, Busan 46958, Republic of Korea 2Department of Energy and Chemistry, Silla University, Busan 46958, Republic of Korea 3Department of Food Engineering, Silla University, Busan 46958, Republic of Korea 4Department of Chemical Engineering, Busan 46958, Republic of Korea

An oral cleanser is a medicine that rinses the inside of the mouth and isused to remove bacteria left in the mouth after brushing teeth. As the Corona 19 situation worsens recently, the risk of tooth decay due to oralbreathing while wearing a mask, and demands for oral cleanliness products are increasing to solve problems such as bad breath caused bywearing a mask. As demand for oral cleansers has increased, there has also been an increase in interest in whether or not they are effective in reducing oral bacteria. Therefore, we will conduct a study to select fivetypes of oral cleansers sold throughout the city based on any criteria andsee how effective they are and how different they are. Among the criteriafor specifying oral cleansers, the highest priority was "products to be usedby many people", and we selected oral cleansers that had different effectsto emphasize in order to smooth out the difference in effectiveness between products. In addition, he established criteria for variables in smooth experiment progress. Before entering this experiment, the best visibility LB Agar was selected and used through an experiment comparing BAP, MacConkey, and LB Agar in the candidate group. In this experiment, we tried to reach a conclusion by selecting only the values of the results of the same experiment, such as wounds in the mouthand medication for oral treatment, to adjust the oral environment as muchas possible. Keywords : Oral cleanser, LB Agar, bacteria in the oral cavity

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A-31

Cross-Resistance to Different Classes of Antibiotics in Salmonella Typhimurium

Jirapat Dawan and Juhee Ahn*

Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea

The sequential antibiotic treatment has recently gained attention in clinical medicine to control multidrug-resistant (MDR) bacteria. However, relatively few studies have been conducted on the effect of sequential antibiotic therapy on the evolution of cross-resistance. Therefore, in this study, we aimed to investigate the antimicrobial activity of sequential antibiotic treatments against MDR SalmonellaTyphimurium in association with the development of antibiotic cross-resistance. One-half of the MIC of ceftriaxone (CEF [1/2]), ciprofloxacin (CIP [1/2]), polymyxin B (POL [1/2]), or tetracycline (TET [1/2]), followed by 1×MIC of CEF [1], CIP [1], POL [1], and TET[1] were used to evaluate antimicrobial activities against S. Typhimurium (STWT), ciprofloxacin-induced S. Typhimurium (STCIP),and clinically isolated S. Typhimurium (STCLI). The development of cross-resistance were assessed based on the viability changes and relative fitness. The expression of efflux pump-related genes were evaluated by using a quantitative RT-PCR assay. The serial exposuresof STWT, STCIP, and STCLI to CIP [1/2]+CIP [1] and STWT to TET [1/2]+TET [1] showed the highest viability. The CIP [1/2]+POL [1] treatment showed an increase in fitness cost and a decrease in viabilityfor all strains. The efflux pump-related genes were overexpressed in allstrains exposed to serial antibiotics. Consequently, the sequence of serialantibiotic treatments affected the development of cross-resistance to antibiotics. This study provides useful information for designing effective antibiotic treatments against antibiotic-resistance bacteria. Keywords : Salmonella, serial antibiotic treatments, multidrug resistant

A-32

Characteristics of Collaterally Susceptible and Resistant Acinetobacter baumannii Exposed to Antibiotics

Jirapat Dawan, Nana Nguefang Laure*, and Juhee Ahn*

Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea

The emergence and spread of antibiotic-resistant Acinetobacter baumannii is a major public health concern. Bacteria can evolve collateral susceptibility and resistance to additional antibiotics. In this study, we aimed to evaluate the relative fitness, collateral susceptibility, and collateral resistance of wild-type A. baumannii KACC 12454 (ABKACC), wild-type A. baumannii CCARM 12088 (ABCCARM), polymyxin B-(PMB-) adapted ABKACC, PMB-adapted ABCCARM, stabilized ABKACC, and stabilized ABCCARM to ciprofloxacin (CIP), meropenem (MER), PMB, tetracycline (TET), and tobramycin (TOB).Compares to wild-type ABKACC, the susceptibility of PMB-adapted ABKACC was decreased to PMB (from 2 to 128 μg/ml) but increased toCIP (from 2 to 1 μg/ml), MER (from 16 to 1 μg ml-1), TET (from 16 to2 μg/ml), and TOB (from 64 to 16 μg/ml). The resistance of PMB-adaptedABCCARM was increased to CIP and PMB when compared to ABCCARM,showing MIC 32 and 64 μg/ml respectively. However, the stabilized ABKACC, and stabilized ABCCARM were lost their resistance activity to allantibiotics except CIP and TET treatments. The presence of β-lactamaseand efflux pump inhibitors increased the susceptibilities of CIP, MER, PMB, TET, and TOB in all strains. Stabilized ABKACC, stabilized ABCCARM, and PMB-adapted ABCCARM, showed higher levels of relativefitness than PMB-adapted ABKACC. Therefore, the collateral susceptibilityand collateral resistance of A. baumannii were vary depending on the antibiotic exposure. Keywords : Acinetobacter baumannii, collateral sensitivity, collateral resistance

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A-33

Interaction between Atibiotic-Sensitive and Antibiotic-Resistant Salmonella Typhimurium in Associated with Antibiotic Resistance during Antibiotic Exposure

Jun Hwan Kim, Jirapat Dawan, and Juhee Ahn*

Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea

Bacteria communities are heterogeneous, including antibiotic-sensitiveand antibiotic-resistant bacteria. The heterogeneous populations can involve in bacteria competitive and cooperative interactions, resulting in the development of antibiotic resistance. In this study, we aimed to evaluate the mutual interaction between antibiotic-sensitive and antibiotic-resistant Salmonella Typhimurium. The single and mixed culture of S. Typhimurium ATCC 19585 (STS) and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 (STR) with 1×MICceftriaxone (CEF) were used to determine the viability, β-lactamase activity, and gene expression. The susceptibility of STR to CEF was decreased with increasing inoculum densities from 102 to 107 CFU/ml,showing more than 5-fold increase of MIC50. Compared to single culture,the number of STS in mixed culture was increased up to 108 CFU/ml inthe presence of CEF after 20 h of incubation at 37℃. Furthermore, the mixed culture showed the highest β-lactamase activity as 18 μmol/ min/ml, consistent to the highest relative expression of β-lactamase- related genes (blaTEM). Therefore, the β-lactamase produced from STR

can protected STS from CEF, thus the β-lactamase production play an important role in inducing cooperative interaction between STS and STR

in mixed culture. This study provides valuable information for understanding the mutual interaction within bacteria heterogeneous populations.

Keywords : Salmonella, β-lactamase, mutual interaction, heterogeneouspopulations.

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A3B0100830416)]

A-34

Synergism of Enrofloxacin and Sulfamethoxazole/Trimethoprim and Its Clinical Application in Pig Infections

Eon-Bee Lee, Seung-Chun Park*, and Biruk Tesfaye BirhanuLaboratory of Veterinary Pharmacokinetics and Pharmacodynamics, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of Korea

Antibiotic resistance to certain antimicrobials has been constantly increasing worldwide due to the indiscriminate use of drugs, which hasbeen recognized as a global livestock problem as well as pig production.Given this background, new approach to develop the antimicriobials isnecessary for clinical use. Several previous studies have shown that combinations of antibiotics with different modes of action can reducethe amount of antimicrobial agents to minimize antimicrobial resistanceand side effects. Combination therapy is proposed as a major breakthroughin suppressing the development of resistant bacteria and exploring newmixtures in veterinary medicine. Enrofloxacin (ENFX) is currently effective in the bactericidal activity of various gram-negative and gram-positive bacteria in pigs in some countries, including Korea. Sulfamethoxazole (SFX) / Trimethoprim (TRM) mixture is widely usedas an antimicrobial agent that inhibits microorganisms. Unfortunately,no previous studies have implemented the combination (ENFX-SFX/ TRM) to achieve the effect of antimicrobial activity even if the optimalratio of SFX/TRM has been proven to be 5:1 in pigs. In this study thecombinatory effects of the ENFX-SFX/TRM combination on six bacterial species (E. coli, Salmonella spp., P.multocida, A.pleuropneumonia,M.hyopneumoniae, B.bronchiseptica) was designed whether the combinationexhibited a synergistic effect, or not. Antimicrobial pharmacodynamicswere performed for minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), fraction inhibitory concentration (FIC) and time kill assays. To confirm the safety of the mixture, a singleoral toxicity was conducted. Also, it is important to establish a residual washout period. Therefore, we set the withdrawal period after clinical application of this formulation to healthy pigs. From the results of the antimicrobial pharmacokinetics, we were able to obtain the optimal combinational ENFX 25 + SFX/TRM 75 ratio. On the other hand, it wasdetermined as a safe formulation with an LD50 of 5000 mg/kg or morein a single oral toxicity test. Taking the above results together, it was confirmed in this study that the ENFX+SFX/TRM combination productis safe and has excellent antibacterial effect. Therefore, the optimal dosage regimen and efficacy using an integrated pharmacokinetic- pharmacodynamic model in diseased pigs should be additionally demonstrated. Keywords : Combination therapy, antibiotic resistance, enrofloxacin- Sulfamethoxazole/Trimethoprim

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Bacteriocinogenic and Safety Properties of Bacillus tequilensis ST816CD Isolated from Kimchi

Gee-hyeun Choi1, Clarizza May Dioso2, Joanna Ivy Irorita Fugaban1, Wilhelm Heinrich Holzapfel1, and Svetoslav Dimitrov Todorov1 1ProBacLab, Department of Advanced Convergence, Handong Global University, Pohang 37554, Gyeongbuk 37554, Republic of Korea 2HEM Inc., Pohang, Gyungbuk 37554, Republic of Korea

Traditional fermented food products are considered as a trend in the search for healthier lifestyle. In the last decade, different bacterial speciesinvolved in the fermentation processes of food products were evaluatedas probiotic candidates for human and other animal application. However, safety evaluations on strain basis need to be considered as anessential milestone on the recommendation for application of any new strain. This project aims to identify and characterize bacteriocin- producing strains for biocontrol of Staphylococcus spp., clinical and food-associated pathogens, and to evaluate their safety. Bacillus tequilensis ST816CD, identified via 16S rRNA sequencing, was isolated from artisanal kimchi from the Pohang region. Treatment of thecell-free supernatant of B. tequilensis ST816CD with proteolytic enzymes resulted in deactivation of the produced inhibitory metabolite/sagainst Staphylococcus species. However, B. tequilensis ST816CD most probably expresses more than one type of antimicrobial metabolite/s since this hypothesis was confirmed by PCR analysis indicating the presence of surfactin (srfa), subtilosin (sbo), and ituricin(ituc) genes in the DNA of B. tequilensis ST816CD. The stability of the expressed antimicrobials was found to be not affected after exposed todifferent temperatures (4-100℃), pH (2-10), and chemicals (NaCl, Tween 80, SDS, and skim milk). Bacteriocin activity of B. tequilensis ST816CDagainst S. simulans KACC 13241 and S. auricularis KACC 13252 wasat most 1600 AU/mL, which was tested during 24 hours after incubationat 37℃. ST816CD was found to produce gelatinase, γ-hemolysin, andbiogenic amines. PCR-based analysis showed that ST816CD does not harbor potentially beneficial adhesion genes (map, mub, eftu, ef1249, ef2380, ef2662, and prg) and genes related to the production of folate (folPE, folKQ, pabB, and pabC) and antimicrobials (bli, thu, coa, nis, and ped) but has gad genes encoding glutamate decarboxylase (GAD)for GABA production. Molecular-based screening for the presence of virulence genes in ST816CD showed an incomplete operon for hemolysin (hblABC) and no evidence for enterotoxin gene (nheABC) and vancomycin-resistant genes (vanABCDEG). The presence of the strains with virulence activity and biogenic amines production in the traditional fermented food products needs to be regarded as undesiredand potential health hazard to the consumers. Moreover, due to lack ofstandardization, preparation of the traditional fermented food productscan be hidden and unexpected problems related to their safety and quality.Good manufacturing practices are an assurance for the food safety. Keywords : Bacillus tequilensis, bacteriocin, food safety

A-36

Antibacterial Properties of Bacillus subtilis ST829CD against Emerging Pathogens Staphylococcus simulans and Staphylococcus auricularis

Joanna Ivy Fugaban1, Clarizza May Dioso2, Wilhelm Heinrich Holzapfel1,and Svetoslav Dimitrov Todorov1

1ProBacLab, Department of Advanced Convergence, Handong Global University, Pohang, Gyeongbuk 37554, Republic of Korea 2HEM Inc., Pohang, Gyeongbuk 37554, Republic of Korea

Bacteriocin production is considered an advantageous property for various beneficial cultures. In addition to their potential as biopreservatives.Bacillus spp., widely distributed with varied roles in nature, has been associated with different fermented food products. Their ability to produce a wide variety of antimicrobial by-products and enzymes has paved their way to the spotlight as a promising biotechnological tool forvarious industrial applications. This study aimed to screen and characterizea bacteriocin-producing strain from traditional fermented food productsand evaluate their safety and functional properties. A potential producerof antimicrobial metabolites isolated from Korean fermented food products identified as B. subtilis ST829CD through 16S rRNA sequencing.The produced inhibitory substance(s) was determined to be partially inactivated upon treatment of its CFS with proteolytic enzymes, indicating an array of possible active inhibitory metabolite/s. To furtherconfirm the production of other antimicrobial compounds, molecular- based screening for genes coding for lichenicidin (bli), surfactin (srfa),iturin (ituc), and subtilosin (sbo) was carried out. The bacteriocin activities were determined against S. simulans and S. auricularis at 800AU/mL and 400 AU/mL, respectively. The mechanism of action was demonstrated to be cell lytic. Phenotypic safety evaluation showed B. subtilis ST829CD to be gelatinase positive and a producer of biogenicamines. PCR detection of potential virulence factors confirmed the absence of genes coding for enterotoxin (nheABC) and hemolysin (hblABC) enzyme production. Furthermore, beneficial properties werescreened genotypically showing that adhesion genes (map, mub, eftu, ef1249, ef2380, ef2662, prg), GABA-associated genes (gad), folate-codinggenes (folPE, folKQ, pabB, pabC), and additional bacteriocin-coding genes (bli, thu, coa, nis, ped) were all absent. Bio-molecular approachesprovide information on the possible production of diverse antimicrobialmetabolites by the same bacterial strain and their possible role(s) in thestability, safety, and beneficial properties of traditional fermented foodproducts. Additionally, traditional skills and technologies employed forthe preparation of fermented foods are considered as an essential factorin their preservation and microbial biodiversity. Thus, such traditional fermented food products can be valuable sources of new strains that are producers of various bioactive metabolites and with potential biotechnologicalapplications. Keywords : Bacillus subtilis, Staphylococcus spp., antimicrobials

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A-37

Structure Elucidation and Bioactivities of Bacteria-Derived Compounds Separated from Unique Habitats

Young Eun Du1, Woong Sub Byun1, Eun Seo Bae1, Seok Beom Lee2, Sunghoon Hwang1, Yern-Hyerk Shin1, Bora Shin1, Yong-Joon Jang3, Yeonjung Lim4, Suckchang Hong2, Jongheon Shin1, Sang Kook Lee1, Jang-Cheon Cho4, Sang-Jip Nam5, Seung-Il Nam6, and Dong-Chan Oh1*

1Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea 2Research Institute of Pharmaceutical Science and College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea 3Natura center of Life and Environment, Seoul National University, Seoul 08826, Republic of Korea 4Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea 5Department of Chemistry and Nanoscience Ewha Womans University, Seoul 03760, Republic of Korea 6Korea Polar Research Institute, Incheon 21990, Republic of Korea

Investigating the secondary metabolites produced by bacteria associatedwith unique habitats, such as insect or extreme environments has becomea promising strategy for discovering novel bioactive compounds. Formicins A-C (1-3) were discovered from Streptomyces sp. SFA33, associated with wood ant (Formica yessensis). The structures of formicin A and formicin B were elucidated to be structurally unique indenone thioesters bearing an N-acetylcysteamine moiety based on 1D/2D NMR and UV spectroscopy with MS/MS analysis. The absolute configuration of formicin C was determined by applying the phenylglycinemethyl ester (PGME) method followed by 1H chemical-shift analysis. Formicin A inhibited the growth of human triple negative breast cancer(TNBC) cells. Two new secondary metabolites, svalbamides A (4) andB (5), were isolated from a culture extract of Paenibacillus sp. SVB7 that was isolated from deep sea sediment from Svalbard archipelago inthe Arctic Ocean. The combinational analysis of HR-MS and NMR spectroscopic data revealed the structures of 4 and 5 as being lipopeptidesbearing 3-amino-2-pyrrolidinone, D-valine, and 3-hydroxy-8-methyldecanoicacid. The absolute configurations of the amino acid residues in 4 and 5were determined using the advanced Marfey’s method with deducing the stereochemistry of 3-hydroxy-8-methyldecanoic acid based on quantum mechanics-based calculations. Svalbamides A and B inducedquinone reductase activity in Hepa1c1c7 murine hepatoma cells. Keywords : Unique habitat, wood ants, arctic ocean

A-38

Airborne Virus Reduction Effect of Violeds® in Indoor Space

Yeongmin Yoon, Heeho Bae, Chunghoon Lee, and Kunsub ChungBio Research Team, Seoul Viosys, Gyeonggi-do 15429, Republic of Korea

After the COVID-19 pandemic, social concerns are rising about the increase in transmission infection caused by airborne viruses in indoorspaces. Ultraviolet rays are emerging as a factor that reduces viruses. However, there are no studies on how to reduce airborne viruses using ultraviolet ray in indoor space. In this study, we provided the virus reduction effect of Violeds®. The Violeds®, developed by Seoul Viosys,is ultraviolet LED technology that guaranteed safety by optimizing thewavelength, irradiation dose, time, angle, and distance of ultraviolet. Wechecked virus reduction rate of air circulation device that is applied fanand Violeds® by space area of indoor space, air flow of fan and irradiationdose of Violeds®. As a result, Violeds® reduced airborne virus by morethan 90% in a space of 120m3. As a result of confirming the reduction effect according to the air flow condition, we confirmed the reductioneffect of 90% at the air flow of 36m3/min or more when driving for 30 minutes. Under the condition of 60m3/min air flow in a 120m3 space, we confirmed that it was possible to reduce airborne viruses even in a shorttime by showing a reduction effect of more than 90% in 20 minutes ofapplying Violeds®. These results were similar to the experimental resultsin KTL (Korea Testing Laboratory). In KTL, the air circulation device applied Violeds® reduced airborne virus by 99% in 30 minutes in a 60m3

space. These results show that Violeds® effectively reduces airborne viruses in indoor spaces, and furthermore, it is considered to be a sufficient guide for the use of Violeds® for airborne virus reduction purposes.

Keywords : Violeds®, Indoor space, airborne virus reduction

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B_Bioactive Compounds and Functional Foods

B-1

Functional Evaluation of Gardenia jasminoides Ellis through Fermentation with Lactic Acid Bacteria

So Hee Park1,2, Chun-Zhi Jin1, Jong Min Lee3, Min-Kyoung Kang1, Dong-Jin Park1, and Chang-Jin Kim1,2*

1Industrial Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 2Department of Bio-Molecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea 3Department of Biotechnology, Pukyong National University, Busan 48513, Republic of Korea

Gardenia jasminoides Ellis (Rubiaceae), one of the well known traditional herbal medicines, which is used as functional food in our modern life owe to its multi-functions: anti-obesity, diabetic, oxidant, inflammatory and insomnia. Genipin and geniposide are both bioactivecomponents of G. jasminoides. Genipin is aglycon part of geniposidestructurally, and has much better activity comparing with geniposide. Though, the only problem is that G. jasminoides contains genipin in a micro-scale. In this study, our purpose is to evaluate G. jasminoides bybio-conversion of geniposide to genipin through fermentation with GRAS level lactic acid bacteria (LAB). More than 180 LABs were isolated from Korean traditional food (Kimchi) and screened with G. jasminoides through fermentation. Lactobacillus sp. #1 and Lactobacillussp. #13, two LABs strains had the strongest conversion ability. Activitytests of fermented and non-fermented G. jasminoides were carried out by Stitch program and glucose uptake assay. Bio-conversion have G. jasminoides showed much higher glucose uptake activity in L6 rat myoblast cell. This study may provide a new insight into bio-conversionof compounds from traditional herbal medicines for potentially improving functions to treat diseases.

Keywords : Gardenia jasminoides, lactic acid bacteria, fermentation

[This research was supported by a grant (NRF-2013M3A9A5076601)from a study on the strategies of improving the value of microbial resources funded by Ministry of Science and ICT of the Korea Government and The KRIBB Research Initiative Program (KGM 5492113)]

B-2

Bio-Conversion of Korean Traditional Medicines through Fermentation with Lactic Acid Bacteria

So Hee Park1,2, Chun-Zhi Jin1, Jong Min Lee3, Min-Kyoung Kang1, Dong-Jin Park1, and Chang-Jin Kim1,2*

1Industrial Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 2Department of Bio-Molecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea 3Department of Biotechnology, Pukyong National University, Busan 48513, Republic of Korea

Adult (lifestyle) diseases, usually they are caused by abnormal life pattern (especially modern life) and very complicated to treat due to itsmany different reasons of causes. Targeting based (cell based) treatmentworks well in a short time, but in a long-term plan it is not enough. Koreantraditional medicines and lactic acid bacteria (LAB), they are one of solutions to solve these complex problems. The mechanisms and functions are being clearly explained recently, and they are being generally accepted to use as functional food and medicinal practices. The aim of this study, is to improve effects of Korean traditional medicine through bio-conversion of its active components by fermentation with LABs. 200 strains of LABs were singly isolated from Korean traditionalfood (Kimchi), and among them, three were new strains, 14 strains hadbio-conversion ability on Illicium verum, Gardenia jasminoides and some others also. This study may provide new insights into bio-conversion of compounds from traditional medicines for potentially treatment of adult disease.

Keywords : Bio-conversion, lactic acid bacteria, Korean Traditional Medicine

[This research was supported by a grant (NRF-2013M3A9A5076601) from a study on the strategies of improving the value of microbial resources funded by Ministry of Science and ICT of the Korea Government and The KRIBB Research Initiative Program (KGM 5492113)]

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B-3

Development of Soy Functional Food and Biomedicinal Materials Using Enzymes Secreted by Bacillus sp.

Jong-Hoon Kim1, Doo Seob Choi1, MinJi Jang1, Ye-Jin Jung2, Kyung-Hyun Oh2, Kyung-Ho Han2, Mi-Sun Kwak1, and Moon-Hee Sung1,2* 1Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea 2Kookminbio Corporation, Seoul 02826, Republic of Korea

Soybeans are an effective and common source of vegetable protein in the human diet. The origin of soybean originated in Northeast Asia, andis said to have been cultivated in China for the first time, and cultivationmarks have remained in Korea since the Bronze Age. Soymilk is a representative processed food of soybean and has been spotlighted as a complete food that can replace milk. In particular, whole soymilk contains pureed soybean and shells, so it is rich in various biomedicinalmaterials and dietary fiber. We have considered how to use the useful substances contained in whole soybean milk more efficiently and createadded value. The soybean extract was biocatalyzed using GRAS fermented food microbial resources, and its components were comparedand analyzed. It was confirmed that the amount of useful materials increased through the action of various enzymes. This result is expectedto develop postbiotics, as a functional food and biomedicinal materialsor as a candidate for a new drug. This research was supported by KoreaEnvironmental Industry and Technology Institute(KEITI) grant funded by the Ministry of Environment of Korea. Keywords : Soybean, enzyme, postbiotics

B-4

Evaluation of Enzymatic Hydrolysate of Porcine Whole Blood for Production of Valuable Amino Acids

Seonghun Kim1,2 1Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup 56212, Republic of Korea 2Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Daejeon 34113, Republic of Korea

Porcine blood is an agricultural byproduct in pork meat processing industry. Although some portion of blood could be used as food, it wasconsidered as disposal of wastes. Nevertheless, it could be a potential bioresource for bioconversion into high valuable products. In this study, we evaluated and analyzed the enzymatic hydrolysate of porcine wholeblood for the production of amino acids. Porcine whole blood hydrolyzedby protease cocktails, then formulated to dried powder form to analyze the content of soluble compounds. Peptide contents, microbiome profiles, amino acid composition, residual antibiotics, and other metabolites were analyzed in the blood hydrolysate. All together these analyses showed that the different peptide contents and microbiome profiles were identified in the hydrolysates prepared by two protocols.In addition, 287 compounds were identified by metabolite profile analysis through liquid chromatography‐mass spectrometry/mass spectrometry (LC-MS). Interestingly, these metabolite profile analysisshowed overall, 82 metabolites in glycolysis/ glyconeogenesis, TCA cycle, amino acid biosynthesis, and glutamate metabolism. Moreover, twenty essential amino acids were quantitatively analyzed and the certain amino acids are highly contented in the lysate. Based on these analysis, the blood lysate could be utilizable to produce valuable aminoacids. Therefore, this evaluated analytical data could contribute the potential utilization of mixture amino acids generated from a blood byproduct as the supplementary compounds for further application. Keywords : Porcine whole blood, metabolite profile, valuable amino acids

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B-5

The Potential of Microbial-Producing Violacein for a Variety of Purposes in Biotechnology

Songhee Jeong, Serin Kim, Gaon Park, Yujin So, Yu Jeong Lee, Hyun JuKim, and Sang Jun Lee*

Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea

Violacein is a bacterial-producing and indole-containing purple pigmentthat is synthesized by condensation of two tryptophan molecules throughsuccessive catalysis of the five enzymes VioA, VioB, VioE, VioD, andVioC. The natural pigment can serve as a bacterial defense mechanismagainst specific predators, helping violacein- producing bacteria surviveeffectively in various environments. It has diverse biological functionsincluding antiviral, antioxidant, antifungal, and antibacterial activities.In particular, previous studies have shown that violacein extracted from Chromobacterium violaceum can induce apoptosis in a variety of cancercells including leukemia cell lines. In this study, we tested anticancer activity of violacein extracted from microbial Massilia sp. cell. As a result, we observed the cytotoxic effect of violacein on colon cancer cells through WST-8 assay. Keywords : Violacein, anticancer activity, colon cancer cells

B-6

A Glycosylated-Metabolite β-Gentiobiosylpaeoniflorin of Paeonia Lactiflora bioconverted by Leuconostoc sp. Alleviates Metabolic Syndrome in High Fat Diet-Fed Mice

So Hee Park1,2, Jong Min Lee1,3, Chun-Zhi Jin1, Min-Kyong Kang1, Dong-Jin Park1, and Chang-Jin Kim1,2* 1Industrial Bio-Materials Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea 2Department of Bio-Molecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 3Department of Biotechnology, Pukyong National University, Busan 48513, Republic of Korea

This study investigated the anti-metabolic syndrome effects of bioconverted substance through fermenting Paeonia lactiflora with Leuconostoc sp. HPLC/MS and NMR spectral data analysis proved thatthe dramatically increased metabolite by fermentation product was β-gentiobiosylpaeoniflorin(βGPF), which is a glycosylated form of paeoniflorin(PF). In animal experiments, 70 male C57BL/6 mice werefed normal, high fat, high fat diet supplemented with 1000 mg/kg/day non-fermented extract, fermented extract, and 100 mg/kg/day single compound of metformin, PF or βGPF. After 6 weeks of oral gavage, thefermented extract not only reduced body weight gain by 44.83% compared to the high fat diet group, but also decreased by 19.9% compared to non-fermented extract. In the case of a single substance, the βGPF group showed 19.6% lower weight gain than the PF group. Similar results were revealed in indicators of diabetes and cardiovasculardisease. Therefore, these results demonstrate that P. lactiflora fermented extract containing βGPF, which is the main effective components shouldbe considered as a potential supplement for alleviating metabolic syndrome.

Keywords : β-gentiobiosylpaeoniflorin, anti-metabolic syndrome, bioconversion

[This research was supported by a grant (NRF-2013M3A9A5076601) funded by Ministry of Science and ICT of the Korea Government]

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B-7

Comparison of Antioxidant Effects of Chrysanthemum indicum L. Extracts

Ji-Hye Choi, Se-Ram Cho, Chan-Hwi Park, Jin-Woo Hwang, Ji-Eun Lee, Sang-Oh Kwon, and Sung-Gyu Lee*

Department of Medical Laboratory Science, College of Health Science Dankook University, Cheonan-si, Chungnam 31116, Republic of Korea 2R&D Center, S&D Co., Ltd., Republic of Korea

Purpose : This study was conducted to investigate the antioxidant activityof extracts with various extraction conditions such as extraction time andtemperature of Chrysanthemum indicum L. Method: Extracts of C. indicum L. 1,2,3 and the content of total polyphenols and flavonoids, andthe ability to scavenging free groups of ABTS and DPPH were measuredto study the antioxidant function. The treatment of C. indicum L. to RAW264.7 macrophages induced cellular damage by the production of nitricoxide and the toxicity of samples was evaluated by measuring cell viability. RESULTS: The polyphenol content of C. indicum L. 3 extractwas 61 μg/mg, which had the highest content. Radical scavenging abilitywas also excellent in C. indicum L. 3.

Keywords : Chrysanthemum indicum L., antioxidant, anti-inflammation

B-8

Oral Administration of Weissella confusa WIKIM51 (Wilac D001) Reduces Body Fat Mass by Modulating Lipid Biosynthesis and Energy Expenditure in Diet-Induced Obese Mice

Sung-soo Park1, Jieun Lee2, Ji Ye Mok3, Misun Yun1, Hee Eun Jo1, YoungJoon Oh1, Sang Min Park3, and Hak-Jong Choi1*

1Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea 2SME Service Department, World Institute of Kimchi, Gwangju 61755, Republic of Korea 3Pharmsville Co., LTD., Seoul 07793, Republic of Korea

Obesity is closely associated with profound dyslipidemia, insulin resistance,and fatty liver diseases. Recent reports have suggested that the alterationof gut ecosystem can significantly impact obesity and diabetes. In this study, we found that Weissella confusa WIKIM51 (Wilac D001) isolatedfrom kimchi significantly reduced lipid accumulation in 3T3-L1 adipocytes and that oral administration of Wilac D001 to the mice markedly reduced a high fat diet (HFD)-induced increase of body weightgain, fat mass, and fatty liver. In addition, plasma levels of triglyceride,leptin, and insulin were significantly lowered in mice fed a HFD withWilac D001 than those in HFD-fed mice. Furthermore, Wilac D001 obviously suppressed the expression of the genes involved in adipogenesisand fatty acid synthesis such as PPARγ, C/EBPα, FAS, and SREBP-1c,whereas the expression of PPARα and PGC-1α, the genes related in energy expenditure, was elevated. Together, these results indicate that intake of Wilac D001 efficiently suppresses the development of obesityand fatty liver caused by HFD feeding. Keywords : Obesity, Weissella confusa WIKIM51 (Wilac D001), probiotics

B-9

Improvement of Anti-Obesity Efficacy by the Combination of Compounds from Coptis chinensis and Silybum marianum in High Fat Diet-Fed Obese Mice

Young Geol Yoon, Jin Hyung Lee, Young Hoon Choi, and Hyon Ju Im Department of Biomedical Science, Jungwon University, Goesan 28024, Republic of Korea

In this study, we investigated whether the combined administration ofthe main components derived from Coptis chinensis and Silybum marianum was effective in improving hyperlipidemia and anti-obesity efficacy using a high fat diet (HFD)-fed obese mouse model. HFD- induced obese mice were supplemented with the berberine (BBR) and silibinin (SBN) combination (BBR-SBN) along with the HFD for 8 weeks. Body weight and food intake were measured every week and thelevels of total cholesterol, triglyceride and HDL-cholesterol were analyzed at the end of the experiment. Consumption of HFD in the micecaused rapid increases in body weight and the levels of total cholesteroland triglycerides compared to those of the normal control (NC) group. However, supplementation of BBR-SBN in these obese mice significantlyreduced body weight gain and suppressed the levels of total cholesteroland triglyceride with the increment of HDL-cholesterol level. Abdominalfat weight was significantly increased in the HFD-fed group, and the adipocytes within the epididymal adipose tissue were found to have expanded sizes compared to the NC group. However, in the BBR-SBNadministered group, abdominal fat weight was significantly reduced andthe sizes of the adipocytes were comparable to those in the NC group.Moreover, the deposition of white giant vesicular fat cells in liver tissuesseen in the HFD-fed group were considerably reduced in the BBR-SBN group. These results suggest that the BBR and SBN combinations havetremendous potential as an anti-obesity agent by significantly reducingbody weight gain as well as lowering serum lipid levels and thus improving anti-obesity efficacy in HFD-induced obese mice. Keywords : Aanti-obesity, hyperlipidemia, high-fat diet

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B-10

Enhanced Antioxidant Activities of Fermented Milk Casein Based on Synbiotic Interaction between Lactobacillus gasseri KML39 and Cudrania tricuspidata Leaf Extract

Hyosu Choi and Nam-Su Oh*

Department of Food and Biotechnology, Korea University, Sejong 30019, Republic of Korea

The object of this study was to examined the antioxidant activities of fermented milk casein on synbiotics interaction between Lactobacillusstrains and Cudrania triscuspidata(CT) leaf extract. In addition, the growth kinetics of the selected strain Lactobacillus gasseri KML39 (KML 39) were measured during fermentation. The antioxidant activitiesof fermented casein milk were determined based on the ferric reducingantioxidant power assay (FRAP), 2,2-diphenyl-1-picrylhydrazyl radicalscavenging activity (DPPH) and 2,2’-azino-bis (3-ethylenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). Further, the total polyphenolcontents (TPC) and total flavonoid contents (TFC) were measured usinga colorimetric method. As a result, The addition of CT into casein resultedin enhanced antioxidant capacity relative to that in the casein. Moreover,fermentation with all Lactobacillus strains in the presence of CT remarkablyincreased both DPPH radical scavenging activity and reducing power. In particular, CT-supplemented casein fermented by KML39 showed thehighest radical scavenging activity and the antioxidant capacities during fermentation gradually increased due to the release of bioactive metabolitesuntil the exponential growth phase. Therefore, this study indicated that KML 39 more suceesfully utilized the CT-related nutrients during fermentation with improved health- promoting effects. Keywords : Fermented milk casein, Lactobacillus gasseri, Cudrania triscuspidata leaf extract

B-11

Sargahydroquinoic Acid Isolated from Sargassum serratifolium Attenuates PMACI-Induced Human Basophilic KU812F Cells Activation

Yongseok Kim1, Taisun Shin2, Kap Seong Choi1, Jiyeon Chun1, GinnaeAhn3, and Sun-Yup Shim1* 1Department of Food Science and Biotechnology, Sunchon National University, Suncheon 57922, Republic of Korea 2Division of Food and Nutrition, Chonnam National University, Gwangju 61186, Republic of Korea 3Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu 59626, Republic of Korea

Basophils and mast cells are characteristic effector cells in allergic reaction. Sargahydorquinoic acid (SHQA), potent antioxidant compound,isolated from marine algae, Sargassum serratifolium with various biochemical properties. In the present study, we investigated the inhibitory effects of sargahydroquinoic acid (SHQA) in phorbol myristate acetateand calcium ionophore, A23187 (PMACI)-induced human basophilic KU812F cells activation. SHQA reduced PMACI- induced intracellularreactive oxygen species (ROS) and calcium levels. Western blot analysisrevealed that SHQA down-regulated activation of ERK, p38 and NF-kBin a dose-dependent manner. Moreover, SHQA suppressed the productionof the cytokines, interleukin (IL)-1b, IL-4, IL-6 and IL-8. It inhibited PMACI-induced KU812F cells degranulation by reducing b-hexosaminidaserelease. Furthermore, SHQA increased mRNA levels and protein expression of extracellular antioxidant enzyme, superoxide dismutase (SOD)3. These results. These results suggest that S. seratifoliumcontaining SHQA is a marine-derived potential therapeutic functional materials for the inhibition of effector cell activation in allergic disorders. Keywords : Sargahydorquinoic acid, Sargassum serratifolium, human basophilic KU812F cells

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B-12

Metabolomic Characteristics of Stimulating Psychotropic Drug Users’ Urine Samples – in a View of Untargeted Analysis

Jin Woo Park1,2 and Junghyun Son1,3* 1Doping Control Center, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea 2Department of Life Science, College of Natural Science, Hanyang University, Seoul 04763, Republic of Korea 3Department of Biological Chemistry, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea

Stimulating psychotropic drugs (phenmetrazine, phentermine, and methylphenidate) are phenyl ethyl amine compounds that excite sympatheticnerves and affect the central nervous system. We investigated metabolomicfeatures of Stimulating psychotropic drug users’ urine samples comparedwith negative control groups in an untargeted analysis. Samples were scanned with an orbitrap high-resolution mass spectrometer (ShimadzuUFLC-XR - Q Exactive PlusTM (Thermo Scientific)), Each one was scanned in Full scan mode in triplicate, and ddMS2 scan once for compound identification. Among the compounds significantly differentin concentration between drug users and controls, xanthine metabolites,which are adenosine receptor antagonists and phosphodiesterase inhibitors, were detected higher in drug users’ groups than negative control ones. We believe it is associated with wakefulness, tracheal dilation, and increased heart rate. Also, isovanillic acid, metabolite of dopamine, was detected higher in the drug user group. We expect the untargeted analysis method may contribute to diagnose the stimulant drug abuse. Keywords : Stimulating psychotropic drug, untargeted analysis, metabolomics

B-13

Screening of Single-Stranded DNA Aptamers for Specific Parathyroid Hormone

In-Hwan Oh1, Woo-Ri Shin1, Simranjeet Singh Sekhon1, Sung Min Woo2, Ji-Young Ahn1, and Yang-Hoon Kim1* 1Major in Microbiology School of Biological Sciences College of Natural Sciences Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, Chungbuk 28644, Republic of Korea 2Department of Food Science and Biotechnology, Shin Ansan University, 135 Sinansandaehak-Ro,Danwon-Gu, Ansan 15435, Republic of Korea

PTH (Parathyroid hormone) secreted by the parathyroid gland plays a key role in regulating calcium in the blood. Abnormal PTH secretion canlead to a variety of disorders, such as hypocalcemia, hyperphosphatemia,osteosclerosis, and exostosis. Therefore, when there is a change in the calcium concentration in the blood, PTH in the blood is detected as a testto find the exact cause. However, it is difficult to detect PTH due to the various forms of PTH and its short half-life. Aptamer, a short single- stranded DNA or RNA, has the characteristic of specifically binding toa target substance, and is known to exist stably in various environments.In this study, aptamer specific for PTH was selected through SELEX process and SPR analysis. Western blot analysis using aptamers showsthat selected aptamers specifically bind to PTH. In addition, the dockingsimulation shows that selected aptamers bind to different sites of the PTH.

Keywords : Aptamer, Parathyroid Hormone (PTH), SELEX

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MSIT) (No. 2020R 1A2C1009463). This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A0604 6235)]

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B-14

Anti-Inflammatory Effect of Quercus aliena Blume Insect Gall Extract

Da Som Kim1, Won-Jae Chi1*, Eun Hee Bae1, Hyehyun Hong2, TaeJin Park2, and Seung-Young Kim2 1Microorganism Resources Division, National Institute of Biological Resources, Republic of Korea 2Department of Pharmaceutical Engineering and Biotechnology, Sun Moon University, Republic of Korea

Quercus aliena Blume contain various kinds of phytochemical substancesand are known to have a wide range of physiological activities such asanti-inflammatory, anti-allergic and anti-cancer effects. It also has the characteristic of forming an insect gall against the stimulus of insects. Insect gall is known to have a variety of pharmacological actions, but the basic mechanism of inhibitory effects on inflammation in RAW 264.7macrophages of Q. aliena Blume insect gall (QBG) has yet to be investigated. And it has the characteristic of forming an insect gall againstthe stimulus of insects. And it is characterized by forming a gall insectagainst the magnetic poles of the insect. Therefore, in this study, we investigated the anti-inflammatory of QBG on LPS-treated RAW 264.7cell. We found that pretreatment with QBG inhibited nitric oxide (NO),prostaglandin E2 (PGE2) and pro-inflammatory cytokines. In addition,QBG significantly inhibited inducible nitric oxide synthase (iNOS) andcyclooxygenase-2 (COX-2) on LPS-treated RAW 264.7 cell. Based onthese results, we concluded that QBG could be a new source for the ingredients of cosmetics and pharmaceuticals. Keywords : Quercus aliena, insect gall, anti-infllammation

B-15

The Potential Therapeutic Applications of Linusorbs

Youn Young Shim1,2,3,4, Ji Hye Kim 3, Jae Youl Cho 3, and Martin J.T. Reaney 1,2,3,*

1Department of Plant Sciences, University of Saskatchewan, Saskatchewan, Canada 2Prairie Tide Diversified Inc., Saskatchewan, Canada 3Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea 4Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan University, Guangdong, P.R.China

Flaxseed (Linum usitatissimum L.) has been associated with numerous health benefits. The flax plant synthesizes an array of biologically activecyclic peptides or linusorbs (LOs, a.k.a. cyclolinopeptide) from three or more ribosome-derived precursors. [1–9-NαC]-linusorb B3 and [1–9-NαC]-linusorb B2, suppress immunity, induce apoptosis in human epithelial cancer cell lines, and inhibit T-Cell proliferation. The mechanism of LOs action is unknown. Using gene expression analysisin nematode cultures and human cancer cell lines we have observed thatLOs exert their activity, in part, through induction of apoptosis. Specific LO properties include: 1) reversibly binding to human serum albumin;2) induce heat shock protein (HSP) 70A production in Caenorhabditiselegans (exposure of nematode cultures to [1–9-NαC]-linusorb B3 induced a 30% increase in production of the HSP 70A protein); and 3) modulate regulatory genes in apoptosis in human lung epithelial cancerlines. These diverse activities indicate that LOs might induce apoptosisin cancer cells or act as versatile platforms to deliver a variety of biologically active molecules for cancer therapy.

B-16

Integrated Metabolomics and Volatolomics for Comparative Evaluation between Soybean and Its Fermented Products

Sanghee Lee1, Sunmin Lee1, Seung Hwa Lee2, Hae Jin Kim2, and ChoongHwan Lee1* 1Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea 2Experiment Research Institute, National Agricultural Products Quality Management Service, Gyeongsangbuk-do 39660, Republic of Korea

Soybeans are fermented in a several products, including doenjang and cheonggukjang, which have different nutritional and flavoring benefits,and microbial colonies. To compare doenjang(D) and cheonggukjang(C),4 samples including raw material soybean(S) and intermediate meju(M)were comprehensively examined using metabolomics and volatolomicsthrough GC-TOF-MS, UHPLC-LTQ-Orbitrap-MS, and HS-SPME-GC-MSbased methods. Multivariate analysis based on UHPLC-LTQ-Orbitrap-MS and GC-TOF-MS datasets displayed that products C, D were separated from raw material S. M, an intermediate of D, is in the path from S to D, indicating that metabolite has changed through M. The HS-SPME-GC-MS datasets showed clustering for D segregated apart from S, C, M datasets. Most volatile compounds including sulfur compounds, 2(3)-methylbutanal, esters, aldehydes and furans were moreabundant in D. Pyrazines, 3-methyl-1-butanol, maltol, methoxyphenols werehigher in C. These compounds are referred to as off-flavor, suggesting characteristic musty flavor of the cheonggukjang. The relative contentsof the amino acids, fatty acids, isoflavone aglycones and non-DDMP soyasaponins content were higher in D with long fermentation period. According to the correlation analysis, these compounds showed positivecorrelation with antioxidant phenotypes. The study can present to producers and consumers nutritional, functional, and organoleptic characteristics of fermented soy products and the selection criteria. Keywords : Fermented soy product, metabolomics & volatolomics, metabolic pathway

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B-17

Analysis of Flavonoids and Phenolics in Syzygium formosum Leaves by a Liquid Chromatography-Tandem Mass Spectrometry

Khanh Hong Thi Hoang1, My Tuyen Thi Nguyen1, Nan Young Lee3, JongTae Park2,3, and Jaehan Kim1* 1Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Republic of Korea 2Department of Food Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea 3705, Building W1, Cooperation Center for Industry-University and Research Institute, Chungnam National University, 99, Daehark-ro, Yuseong-gu, Daejeon 34134, Republic of Korea

Flavonoids and phenolic components are secondary metabolites of theplant that have beneficial functionalities of anti-bacterial, anti-oxidative,anti-allergic, or anti-inflammatory. Syzygium formosum (SF) has long been used in traditional medicine by the Vietnamese for the treatment of skin-related diseases such as rashes, atopic dermatitis, and psoriasis.To understand the underpinning mechanism of skin relief functionality,the quantitative profile of phytochemicals in leaf extract has been performed. By using UPLC-triple quadrupole mass spectrometry, eightflavonoids including catechin, EGCG, quercetin, and five glycoconjugatesof myricetin and quercetin as well as four unidentified flavonoid glycoconjugates were found. Myricetin-3-O-rhamnose exhibited the highest content with 53.04 mg/g dry leaf among flavonoids which occupied 50% of total flavonoids, followed by catechin with 21.62 mg/gdry leaf, and quercetin-3-O-arabinose with 17.94 mg/g dry leaf. The SFleaves with high flavonoid content could be further applied in the foodand pharmaceutical industries. Keywords : Syzygium formosum, flavonoids

B-18

Identification of Pyridomycin and Its Derivatives from Streptomyces sp. W3009 Using Tandem LCMS Analysis

Byeongsan Lee1,2, Kyung Taek Heo1,3, Dong-Jin Park4, Chang-Jin Kim4,Jae-Hyuk Jang1,3, BangYeon Hwang2, and Young-Soo Hong1,3* 1Anticancer Agent Research Center, Korea Reaserch Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea 2College of Pharmacy, Chungbuk National University, Cheongju 28160, Republic of Korea 3KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34141, Republic of Korea 4Industrial Bio-materials Research Center, Korea Reaserch Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea

Pyridomycin, a natural product with potent antituberculosis activity, inhibits a major drug target, the InhA enoyl reductase. Biochemical and three dimensional structural studies show that pyridomycin functions as a competitive inhibitor at both the NADH cofactor binding site and the lipid substrate binding pocket of InhA. Hence multiple academic andpharmaceutical efforts have led to the discovery of direct InhA inhibitors.Streptomyces are one of the most important sources for the discovery ofnew antibiotics successfully introduced to the market or still used in clinical trials. However, the frequency of rediscovery of old known compounds is quite high, making it an important hurdle to overcome innew drug discovery programs for molecules derived from active natural products. Thus, in an effort to isolate novel natural products, the use ofmethods based on dereplication with LC-MS has steadily increased to identify and characterize new derivatives of known compounds. In thepresent work, we have applied LC-MS-guided screening to select candidate pyridomycin and its unreported derivatives from an in-houselibrary of Actinomycetes culture broths. We identified a Streptomyces sp.W3009 strain that produces pyridomycin by mining of a LC-MS library. In addition, we found several related substances in this Streptomyces sp. W3009 strain using a comparison analysis of the MSfragmentation pattern analysisand the GNPS molecular networking. Among them is an unknown derivative with unusual MS fragments (m/z411 and m/z 373), which are presumed to have been substituted the 2-butan-2-ylidene moiety on C2 position of pyridomycin. Keywords : Pyridomycin derivatives, dereplication, tandem mass

[Supported by grants the NRF fund(NRF2020R1I1A206871311 and NRF2013M3A9A5076601]

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B-19

Optimized Astaxanthin Extraction from Haematococcus pluvialis with Novel α-Quartz Nanoplates

Nakyeong Lee1, Gyuseop Moon2, Sungwook Chung2, and You-Kwan Oh2* 1Department of Bioresource Collection, Honam National Institute of Biological Resources, 99, Gohadoan-gil, Mokpo-si, Jeollanam-do 58762, Republic of Korea 2School of Chemical and Biomolecular Engineering, Pusan National University, Busan 46241, Republic of Korea

Astaxanthin, a reddish color ketocarotenoid in Haematococcus pluvialis,has attracted attention for its strong antioxidant activity, which is responsible for its protective properties against inflammation, cancer, diabetes, cardiovascular diseases. But the robustness cell walls on the red cyst stage are composed of a trilaminar layer which is containing a non-hydrolyzable biopolymer called sporopollenin. These multiple cellwalls are considered major technical bottlenecks because they protect themselves from chemical solvent penetration. The astaxanthin extractionfrom the 90-day-cultured Haematococcus cyst cells was performed with two-dimensional α-quartz nanoplates using an ultrasonication bathfor 5 min. The mixture of dichloromethane/ methanol-water (1:1 v/v) was selected as the optimal extract solvent. The maximum astaxanthinextraction efficiency was 99% under the 800 mg-nanoplates/L condition.On the other hand, it was 59% under control condition. This study wasshowed energy-efficient, sustainable approaches for astaxanthin extractionfrom cyst cells with applied nanotechnology. Keywords : Haematococcus pluvialis, astaxanthin, extraction

B-20

Comparison of Human Milk Oligosaccharides Concentration in Four Asian Countries

My Tuyen Thi Nguyen1, Khanh Hong T. Hoang1, Yong-Ki Kim2, Ji A Jung2,Dan Li3, Xuan Hong M. To4, Beenish Israr5, Hyun Joo An6, and Jaehan Kim1* 1Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Republic of Korea 2Maeil Asia Human milk Research Center, Maeil Dairies Co. Ltd., 63 Jinwiseo-ro, Jinwi-myeon, Pyeongtaek, Gyeonggi-do 17706, Republic of Korea 3College of Food Science and Engineering, Changchun University, Changchun 130022, P.R.China 4Faculty of Nursing and Medical Technology, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City 700000, Vietnam 5Faculty of Food, Nutrition and Home Science, University of Agriculture, Faisalabad 38000, Pakistan 6Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea

Human milk oligosaccharides (HMOs) are recognized as prebiotics, andtheir abundance in human milk is a critical element of evolution’s strategy to establish and guide the development of baby food. We havecollected 208 human milk samples from four Asian countries, includingKorea (n=102), China (n=54), Vietnam (n=26), and Pakistan (n=26). Theratio of secretor/non-secretor was kept at 77/23 for all countries. The concentrations of 15 major HMOs were analyzed by liquid chromatographycoupled with mass spectrometry (LC/QQQ). The result showed that theconcentrations of total HMOs in four Asian countries were in the rangeof 5.0 – 11.6 g/L. Pakistani mothers’ milk had the highest concentrationof HMOs, showing 11.6 ± 5.1 g/L, which is 2 – 2.3 times higher than thosein Korean and Chinese (5.0 – 5.8 g/L). The concentration of HMOs inVietnam was 7.4 ± 1.8 g/L. In Asian milk, 2’-FL and 3-FL are the two most abundant oligosaccharides, accounted for 17.2 – 28.6% and 16 –34.2 % of total HMOs, respectively. Vietnam had the highest ratio of sialylated HMOs, which occupied 26.1% (1.9 g/L). Meanwhile, Koreaand China had the lowest concentrations of sialylated HMOs, showing0.5-0.6 g/L (10.9 –11.1% of total HMOs). The sialylated HMOs in Pakistani mothers’ milk was 1.7g/L, slightly less than in Vietnam, and accounted for 14.5% of total HMOs. The obtained data could provide the dynamic range of major HMOs in Asian countries, guiding the recommended nutrient intake (RNI) in infant formula and food. Keywords : Human milk oligosaccharide, mass spectrometry, concentration

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B-21

Comprehensive Analysis of Triterpenoids and Their Carboxylic Acid in Syzygium formosum (Wall.) Masam Leaves by Mass Spectrometry

Jae Yoon Lim1, Hyun jun Lee1, A hyun Lee1, Nguyen Thi My Tuyen1, Jaehan Kim1*, Nan young Lee2, and Jong tae Park2 1Department of Food and Nutrition, Chungnam National University, Republic of Korea 2705, Building W1, Cooperation Center for Industry- University and Research Institute,Chungnam National University, 99, Daehark-ro, Yuseong-gu, Daejeon 34134, Republic of Korea

Triterpenoids are the carbon skeleton components composed of six isoprene units. Lupenol and amyrins with a carboxyl group are determined to be triterpenoic acids that have various beneficial functionsof anti-inflammatory, anti-diabetes, or anti-cancer. Syzygium formosum(Wall.) Masam (SF) leaves and their extract has been used for traditionalherbal medicine in Vietnam. Despite their health benefits functionalities,comprehensive analysis is difficult due to the structural similarity andthe molecular weight closeness. The type of triterpenoic acid is determined by the number and position of hydroxyl groups in the pentacyclic structure with carboxyl group. For the identification and, further, quantitation of actual bioactive components in leaf extract, the positional isomers have to be separated appropriately. In this study, wedevelop a method by the comparison of product ion ratio for the comprehensive analysis of triterpenoids and their carboxylic acids having the same molecular weight and formula. The method, then,-wasapplied to the SF leaves extract to verify the profile and quantitation of triterpenoids in the extract. For the industrial use of the leaf extract, thescale up and the optimization of the extraction process was monitored. Keywords : Syzygium formosum (Wall.) Masam leaves, triterpenoids, mass spectrometry

B-22

Physiological Activities of Methanol Extracts from Geranium eriostemon Fisher ex DC

Yu Jin Oh, Min-Sung Lee, Jae Woo Kim, Yeong-Su Kim*, and Dae WookKim*

Wild Plants Industrialization Research Division, Baekdudaegan National Arboretum, Bonghwa 36209, Republic of Korea

Geranium eriostemon Fisher ex DC. (GE) is an alpine plant, which is widely distributed throughout northern parts of Korea. This study investigated the physiological properties, antioxidative, anti-inflammatory,α-glucosidase and tyrosinase inhibitory acitivities of the methanolic extract of GE. The antioxidant activity of GE extracts were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity assay and an improved 2,2’-azino-bis-[3-ethylbenzothiazolinesulphonate] (ABTS) radical cation decolorization assay in vitro. The results of the ABTS and DPPH antioxidants activity showed that the GEextracts exhibited the IC50 values of 66.55 μg/ml and 21.73 μg/ml, respectively. The anti-inflammatory activities of GE extracts on lipopolysaccharide (LPS)-induced RAW 264.7 cells were investigated.GE extracts potently inhibited the production of NO in LPS-induced RAW 264.7 cells, with IC50 value of 29.70 μg/ml. The results of the α-glucosidase and tyrosinase inhibitory activities of GE extracts exhibitedthe IC50 values of 2.67 μg/ml and 201.36 μg/ml, respectively. Therefore,these results suggest that the GE extracts has antioxidative, anti-inflammatory, α-glucosidase and tyrosinase inhibitory activities thus appearing to be a potential as a functional material. Keywords : Geranium eriostemon Fisher ex DC., anti-oxidant, anti-inflammation

B-23

Anti-Inflammatory Effect of Elaeagnus umbellata Leaf Extract on RBL-2H3 Mast Cells and HaCaT Keratinocytes

Jae-Yeul Lee1,2, Se-Ho Park1,2, Kwang-Hwan Jhee1, and Seun-Ah Yang3*

1Department of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea 2Institute of Natural Science, Keimyung University, Daegu 42601, Republic of Korea 3Department of Food Science and Technology, Keimyung University, Daegu 42601, Republic of Korea

Atopic dermatitis is a prevalent chronic inflammatory skin disease. The keratinocytes play a role in maintaining skin homeostasis through recruiting immune cells, such as mast cells, macrophage, and T cells, via the production of chemokines and cytokines. In this study, we investigated the effects on anti-inflammatory of ethanol extract of Elaeagnus umbellata leaves (EUL). In order to investigate the anti-allergic effect of the EUL, we performed the inhibition of β-hexosaminidase release in RBL-2H3 mast cells. The results suggestedthat the release of β-hexosaminidase was suppressed by the extract (24% inhibition at 100 μg/ml). In addition, the secretion of macrophage- derived chemokine (MDC), regulated on activation, normal T cell expressed and secreted (RANTES), thymus and activation-regulated chemokine (TARC) were effectively inhibited by the treatment of EULin tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-stimulatedHaCaT keratinocytes. Furthermore, the phosphorylation of STAT1 andIκBα were suppressed by the EUL in TNF-α/IFN-γ-stimulated HaCaTkeratinocytes. These results suggest that EUL is potential anti- inflammatory mediators for the prevention of inflammatory skin diseases such as atopic dermatitis. Keywords : Anti-inflammatory, keratinocyte, Elaeagnus umbellata

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B-24

Alteration of Bacterial Community in Dog Feces fed a Heme-Rich Single Cell Protein Diet

Seungki Lee1,2 and Pil Kim1,2* 1Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi 14102, Republic of Korea 2Hemolab Co., Bucheon, Gyeonggi 14102, Republic of Korea

Many anaerobic bacteria inhabiting in animal gut are absent the heme biosynthesis, thereby characterized as no respiratory and sensitive to reactive oxygen species. To observe the alteration of bacterial community in animal gut by supplying a heme-rich single cell protein (SCP), four-weeks domesticated dogs were fed with A-diet (control nutrient), B-diet (control nutrient + heme-rich SCP), C-diet (control nutrient + probiotics), and D-diet (control nutrient + probiotics + heme-rich SCP) for 6 days and their fecal microbiomes were analyzed. Total bacterial communities were 172 and 202 species from the A- andB-diet-fed-dog feces. Total bacterial communities from the C- and D-diet-fed-dog feces were 136 and 159, respectively. Proportion of Firmicutes phylum, where many lactic acid bacteria are belonged to, from the A- and B-diet-fed-dog feces were 92 % and 99% whereas proportion of Proteobacteria phylum, where many Gram-negative bacteria are belonged to, were 8 % and 0.5 %. Those from the C- and D-diet-fed-dog feces were 79% and 70 % for Firmicutes and 0.8% and0.5 % for Proteobacteria, respectively. Therefore, supplementation of a heme-rich SCP nutrient increased the number of bacterial species withreduction of the Proteobacteria phylum proportion in dogs. The possiblereasons in heme-rich-SCP-fed animal gut are further discussed. Keywords : Heme-rich-single cell protein, microbiome, bacterial community

B-25

Aquafaba Derived from Korean Soybeans: A Versatile Vegan Egg Replacer

Yue He1, Youn Young Shim 1,2,3,4, Ji Hye Kim4, Jae Youl Cho4, and MartinJ.T. Reaney1,2,3,*

1Department of Plant Sciences, University of Saskatchewan, Saskatchewan, Canada 2Prairie Tide Diversified Inc., Saskatchewan, Canada 3Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan University, Guangdong, P.R.China 4Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea

The substitution of animal-based foods (meat, eggs, and milk) with plant-based products can increase global food supply. Recently, pulse cooking water (a.k.a. Aquafaba) was described as a cost-effective alternative to egg in gluten-free, vegan cooking and baking applications.Aquafaba (AQ) forms stable edible foams and emulsions with functionalproperties that are similar to those achieved with eggs. However, the functional ingredients of AQ are usually discarded after food preparation. This study developed highly functional food ingredients using Korean-made soybean AQ. In this study, a zero-waste and cost-effective hybrid process that uses a small number of efficient stepswere developed for processing Korean soybean cultivars (ver. Backtae,Seoritae, and Jwinunikong) to recover an effective oil emulsifier, AQ. The AQ product from Backtae (yellow soybean) achieved superior emulsion properties (92%) to AQ from other cultivars and produced more stable food oil emulsions. Therefore, this study will potentially leadto gluten-free, vegan products for vegetarians and consumers with animal protein allergies. This is the first report on the production of AQ,an egg white substitute derived from cooked soybeans.

B-26

Regulation of Amyloid-β Accumulation in Alzheimer’s Diseases Condition by Aquaporin-4 Expression

Jae-Yeul Lee1,2, Se-Ho Park1,2, Kwang-Hwan Jhee1, and Seun-Ah Yang3*

1Department of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea 2Institute of Natural Science, Keimyung University, Daegu 42601, Republic of Korea 3Department of Food Science and Technology, Keimyung University, Daegu 42601, Republic of Korea

Aquaporin-4 (AQP4) is known as a water membrane transporter protein.Recent studies indicate that AQP4 deficiency causes the pathophysiologicalprocesses such as spatial memory, and language-associated learning. Inthis study, we investigated the roles of AQP4 on Aβ clearance in insulinand amyloid-β (Aβ) peptide-treated astrocytic C6 cells under physiologicaland pathological conditions. Insulin is known to upregulate AQP4 protein expression in C6 cells, and we found that the high level of insulin(100 g/ml) inhibited AQP4 expression. In addition, the high concentrationof insulin treatment was suppressed the brain-derived neurotrophic factor (BDNF), which is known as a cell aging marker, and glial fibrillaryacidic protein (GFAP) compared to untreated cells. High level insulin treatment makes the inhibition of low density lipoprotein receptor- related protein 1 (LRP1) in C6 cells, and reduced the expression levelof matrix metalloproteinase (MMP)-2 and MMP-9 by suppressing the AQP4. Our data indicate that AQP4 plays key roles for Aβ clearanceon C6 cells. Taken together, our data suggested that controlling of AQP4possibly provide a basic idea for developing a novel in vitro system forthe research of Aβ-associated neurodegenerative diseases. Keywords : Amyloid-β, astrocyte, insulin

B-27

The Protection Effect of Zizania latifolia Extract on Scopolamine-Induced Memory Impairment in Balb/cJ Mice

Jae-Yeul Lee1,2, Se-Ho Park1,2, Kwang-Hwan Jhee1, and Seun-Ah Yang3*

1Department of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea 2Institute of Natural Science, Keimyung University, Daegu 42601, Republic of Korea 3Department of Food Science and Technology, Keimyung University, Daegu 42601, Republic of Korea

Zizania latifolia, known as Manchurian wild rice, is the only member of the wild rice genus Zizania native to Asia including China, Korea, andJapan. In this study, we investigated the protective effect of Z. latifoliaextract (ZLE) and tricin, which is major compound of Z. latifolia, in insulin and amyloid-β (Aβ)-treated C6 cells and scopolamine-inducedmemory impairment in Balb/cJ mice. ZLE and tricin significantly increased the cell viability compared with insulin and Aβ-treated cells.In addition, the expression of aquaporin-4 (AQP4), glial fibrillary acidicprotein (GFAP), brain-derived neurotrophic factor (BDNF), low-densitylipoprotein receptor-related protein 1 (LRP1), matrix metalloproteinase(MMP)-2, and MMP-9 were decreased by insulin and Aβ treatment. However, ZLE or tricin treatment were significantly increased comparedto insulin and Aβ treated cells. Also, treatment with ZLE or tricin to themice exhibits the necrosis suppression and Aβ accumulation in hippocampaltissue. The protein expression associated with Aβ accumulation (apolipoprotein E4; ApoE4, insulin-degrading enzyme; IDE, LRP1, MMP-2, and -9) was also regulated by ZLE or tricin treatment. Theseresults confirm that ZLE or tricin exhibits a potential to be a functionalfood with a memory improvement. Keywords : Amyloid-β, tricin, Zizania latifolia

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B-28

Metabolomic-Based Comparison of Traditional and Industrial Doenjang Samples with Antioxidative Activities

SongHui Soung1, Sunmin Lee1, Seung Hwa Lee2, Hae Jin Kim2, Na-RaeLee1, and Choong Hwan Lee1* 1Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea 2Experiment Research Institute, National Agricultural Products Quality Management Service, Gyeongsangbuk-do 39660, Republic of Korea

A variety of doenjang are manufactured by many food companies usingdifferent ingredients and fermentation processes, and thus, the qualitiessuch as taste and flavor are very different. Therefore, in this study, we compared many products with 19 traditional doenjang (TD) and 17 industrial doenjang (ID). For that, we performed non-targeted metaboliteprofiling and multivariate statistical analysis using GC-TOF-MS and UHPLC-LTQ-Orbitrap-ESI-MS/MS to discover distinct metabolites intwo types of Doenjang. Amino acids, organic acids, isoflavone aglycones,non-DDMP soyasaponins and hydroxyisoflavones were relatively higher in TD. Dipeptides, lysophospholipids, isoflavone glucosides andDDMP-conjugated soyasaponin were abundant in ID, which are precursors of above-mentioned metabolites in TD. The higher contentsof amino acids, isoflavone aglycone and hydroxyisoflavone in TD werepositively correlated with antioxidant activities. Interestingly, in TD, itshowed the relatively higher contents of biogenic amines such as tyramine, 2-phenylethylamine, tryptamine, histamine, and agmatine, which are either beneficial or toxic depending on concentration. We alsoobserved relatively higher antioxidant (ABTS, FRAP, and TPC) and enzyme activities (protease and β-glucosidase) in TD. Based on our results, it may provide valuable information of Doenjang to consumersand manufacturers which can be used while selecting and developing new products. Keywords : Non-targeted metabolite profiling, biochemical phenotypes,correlation analysis

B-29

Screening and Characteristics of Lactic Acid Bacteria Strains as Cosmetic Ingredient

MyeongSeon Ryu, Hee-Jong Yang, Su-Jin Shin, Jin Won Kim, Gwang SuHa, and Do-Yeon Jeong*

Microbial Institute for Fermentation Industry (MIFI). 61-27, Minsokmaeul-gil,Sunchang-eup, Sunchang-gun, Jeonbuk 56048, Republic of Korea

In this study, we tried to screen Lactic acid bacteria as multi-function material having antibacterial, antioxidant and whitening activity, from250 kinds of traditional fermented korea kimchi and cheonggukjang. wespread lactic acid bacteria on selection medium, and incubate at 30℃ for 2 days. We screened total 337 lactic acid bacteria, and their antibacterial activity was tested against total 6 antibacterial strain usingan agar diffusion assay. We were isolated 16s rRNA sequence analysis.As a result, Lactobacillus plantarum, Lactobacillus paraplantarum, Leuconostoc mesenteroides, Lactobacillus pentosus and Lactobacillusparacasei were identified. The selected strains investigated lactic acid bacteria characteristic API 20 ZYM kit, API 50 CHL kit, antioxidant andwhitening activity (antioxidant-DPPH/SOD, L-DOPA and elastase inhibitor activity). According to the study, we were selected 10 lactic acidbacteria with highly anti-bacterial and cosmetics activity. The most antioxidant (DPPH 19.44%, SOD 67.14%) and whitening (L-DOPA 17.56%, elastase inhibition 31.66%) active strain is SRCM210299. Weexpect this microorganism to be used in the inner-beauty industry.

Keywords : Lactic acid bacteria, whitening activity, antioxidant activity

[This research was supported by Traditional Culture Convergence Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (NRF- 2016M3C1B5907049)]

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B-30

Production of γ-Aminobutyric Acid(GABA) by Enterococcus casseliflavus PBio05 from Oenanthe javanica

Semi Choi, Jeong A Kim, Geun Su Kim, Do Young Kwon, Sang gu Kim,Sang yun Lee, and Kang Wook Lee*

Pulmuone Institute of Technology, Cheongju 28164, Republic of Korea

In this study, a new lactic acid bacterium (LAB) that could produce gamma-aminobutyric acid (GABA)was isolated from Oenanthe javanica (water celery) and identified as an Enteroccoccus casseliflavusstrain. Effects of GABA-producing conditions including the type of medium, growth temperature, initial pH, growth time, MSG concentration,and carbon source on the production of GABA by this strain were thendetermined to optimize GABA production. First, the optimal medium for the growth of E.casseliflavus was BHI medium at 35℃. After 24 h of growth using BHI medium, the relative absorbance was 0.5117, higherthan that with MRS (OD600 = 0.16, 24h) or TSB (OD600 = 0.4364, 24h). At initial pH 7.0 (56.59 ± 0.07 mM) with MSG concentration at 5%to 7% (5%, 55.75 ± 0.06 mM; 7.0%, 58.33 ± 0.09 mM) using maltose carbon source (1%, 54.55 ± 0.56 mM), optimal level of GABA was produced when BHI medium was used for culturing the E. casseliflavusstrain at 35℃ Properties of these strains are being investigated and willbe presented at the meeting. Keywords : γ-Aminobutyric acid, Enteroccoccus casseliflavus, Oenanthejavanica

B-31

Lucidin 3-methyl ether Isolated from Rubia philippinensis Inhibits the Proliferation of Multiple Myeloma Cells via Wnt/β-catenin Pathway

SuBeen Shin, Taekyung Nam, Younglim Son, Yewoon Oh, Yeamin Han,and Sangtaek Oh*

Department of Bio & Fermentation Convergence Technoloty, Kookmin University, Seoul 02707, Republic of Korea

The genus Rubia has long utilized as a traditional herbal medication andstudied that exerts a variety of biological activities including anti-cancer,anti-inflammatory, and anti-osteoporotic activities. However, much research has not been done on the mechanism of action and active metabolites of Rubia philipinensis. Multiple myeloma (MM) has been demonstrated that non-phosphorylated β-catenin highly expresses in most primary MM cells. We found that R. philippinensis extracts inhibitedWnt3a-condition media (Wnt3a-CM)-induced β-catenin response transcription (CRT) using a cell-based reporter system. Among naturalcompounds isolated from the dichloromethane extract of R. philippinensis,lucidin 3-methyl ether was an active compound which induced β-cateninphosphorylation at Ser33/Ser37/Thr41 via GSK3β-independent mechanismthus causing proteasome degradation and then suppressed CRT. Also, lucidin 3-methyl ether inhibited the expression levels of target genes ofβ-catenin including cyclin D1, c-myc and axin-2 in multiple myelomacells. Furthermore, lucidin 3- methyl ether was demonstrated that inhibited proliferation and promoted apoptosis of MM cells through Annexin V-FITC/PI staining and caspase-3/7 activity assay system. Theresults suggested that lucidin 3-methyl ether exerts anti-proliferative activity by suppressing the Wnt/β-catenin signaling and has potential as a therapeutic agent for the MM treatment. Keywords : Lucidin 3-methyl ether, Wnt/β-catenin signaling, multiplemyeloma

[This research was supported by Korea Environmental Industry and Technology Institute(KEITI) grant funded by the Ministry of Environmentof Korea]

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B-32

Anti-Aging and Antioxidants Effects of New Coumarins from Fraxinus rhynchophylla

Beom Zoo Lee1,2, Chau Ha Pham3,4, Ik Soo Lee5, Soon-Kyu Jeong1, SulhaeLee1, KwangWon Hong2, and Hee Min Yoo3,6*

1Chemland Co., Ltd., Gunpo IT Valley, Gunpo 15850, Republic of Korea 2Department of Food Science and Biotechnology, College of Life Science and Biotechnology, Dongguk University, Goyang 10326, Republic of Korea 3Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea 4Department of Microbiology and Molecular Biology, Chungnam National University (CNU), Daejeon 34134, Republic of Korea 5Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea 6Department of Bio-Analytical Science, University of Science and Technology (UST), Daejeon 34113, Republic of Korea

Fraxinus rhynchophylla Hance is a deciduous tree belonging to the Oleaceae family and F. rhynchophylla that grows in areas such as Korea,China, Europe, and North America. The bark of F. rhynchophylla Hanceis referred to as Jinpi and has been used as a herbal ingredient for folkremedies since ancient times. In this study, a total of 12 coumarin compounds – three novel compounds and nine known compounds – were isolated from an extract of F. rhynchophylla Hance. Furthermore, afterdetermining the physicochemical properties and chemical structures ofeach compound, a series of experiments were conducted to investigate the anti-aging and antioxidants effects of the compounds. Additionally,the intracellular and mitochondrial ROS scavenging activities of fraxicoumarin A were examined. As a result, treatment with fraxicoumarin A scavenged ROS generated by treatment with H2O2. The treatment offraxicoumarin A also prevented cell death by effectively inhibiting thedepolarization of the mitochondrial membrane (Δψm). FraxicoumarinA exhibited potent antioxidant activity as it resulted in a tenfold or higherincrease in catalase mRNA levels in addition to a sevenfold or higher increase in glutathione S-transferase (GST) mRNA levels. Moreover, fraxicoumarin A was determined as an effective anti-aging componentas it increased the activity of nuclear factor erythroid-2-related factor2 (NRF2) and heme oxygenase-1 (HO-1) to provide effective protectionagainst oxidative stress. Thus, this study concludes that fraxicoumarinA could potentially serve as a antioxidants for oxidative damage. Keywords : Fraxinus rhynchophylla Hance, anti-aging, antioxidants

B-33

Antioxidants Activity of Inularins from Inula britannica

Dae Kil Jang1,2, Zijun Li3,4, Ik Soo Lee5, Han-Seung Shin2, and Hee Min Yoo3,6*

1StarlingForce Co., Ltd., Seoul 08511, Republic of Korea 2Department of Food Science and Biotechnology, College of Life Science and Biotechnology, Dongguk University, Goyang 10326, Republic of Korea 3Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea 4College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 61186, Republic of Korea 5Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea 6Department of Bio-Analytical Science, University of Science and Technology (UST), Daejeon 34113, Republic of Korea

Sunbokhwa is the herbal medicine that refers to the flower of Geumbulcho (Inula britannica Linne. Var. Japonica Thunb) that belongsto the Asteraceae family. It is known to have the medicinal effect of treating water and can improve the color of blood and its flow in vessels.In this research, a series of studies were conducted on the extraction andpurification of useful ingredients from sunbokhwa extract with severalbenefits such as antioxidant effects. In addition, studies were conductedon the physical and chemical properties of the ingredients, the antioxidant and the mechanisms of action. Six eudesmane sesquiterpene lactone compounds were extracted from the Inula britannica extract, purified, and named Inularins A-F. As a result of investigating the freeradical scavenging activity of Inularins using HaCaT skin keratinocytescells, the amount of free radicals decreased upon treatment with Inularins. This effect was more pronounced in the case of Inulain D, withthe amount free radicals decreasing upon treatment with Inularin D. Boththe Inularin A and D compounds demonstrated potent antioxidant effects. The mitochondrial membrane potential (MMP) was found to increase upon treatment with both Inularin A and D. In addition, westernblot analysis showed that the expressions of Nrf2 and HO-1 were significantly increased upon treatment with Inularin A and D compounds, highlighting how the compounds effectively controlled oxidative stress. Keywords : Inula britannica, Inularins, antioxidant

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Assessment of 2’FL and 6’SL Mixture in an LPS-Induced Gut Inflammation In Vivo Model

Gyuwan Kim and Sae hun Kim*

Department of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul 02841, Republic of Korea

Human milk oligosaccharides(HMOs) are indigestible carbohydrates in human milk, which contribute to infant microbiome and immune systemdevelopment. 2’-fucosyllactose(2’-FL) and 6’-sialyllactose(6’-SL) aretwo major components of HMOs. Our previous study demonstrated theanti-inflammatory effects and gut barrier protein expression enhancing effects of a 2’-FL and 6’-SL mixture in an HT-29 cell (human colorectal adenocarcinoma cell) in vitro model. Therefore, in order to optimize thedosage of 2’-FL and 6’-SL mixture with anti-inflammatory effects against inflammation induced by LPS, a study using an C57BL/6 micein vivo model was conducted. 3 weeks old C57BL/6 mice were pre-treated with different dosage of HMOs mixture (low, medium, highdosage) by oral gavage for 2 weeks. LPS was injected (1mg/kg) from day 10 to day 14 by intraperitoneal injection. In the intestine, high dosage of HMOs exhibited higher anti-inflammatory effects than the low or medium dosage. In contrast, expressions of gut barrier proteins such asClaudin 4 and ZO-1 were enhanced more in low dosage than medium or high dosage. Interestingly, while LPS injection decreased diversity of the gut microbiota, gut microbiota diversity was maintained by HMO treatment to levels similar to the control. In summary, anti-inflammatoryeffects in the gut is a key property which affects gut barrier function, gutmetabolism, and gut microbiome. In this respect, high dosage of HMOsis suggested as the most optimal treatment for gut inflammation. Keywords : HMOs, anti-inflammatory effects, gut inflammation

B-35

Piceatannol, a Resveratrol Analog, Attenuates Dermatophagoides farinae-Induced Atopic Dermatitis Like Symptoms in NC/Nga Mice

Chang Hyung Lee2, and Jong-eun Kim1

1Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea 2Department of Food Science and Technology, Korea National University of Transportation, Jeungpyeong 27909, Republic of Korea

Piceatannol is a resveratrol metabolite commonly found in red wine, grapes, and passion fruit seeds. Several studies investigated piceatannol’simmune-modulating effects on processes relevant to allergic reactions.However, the relevance between piceatannol and atopic dermatitis (AD) has not been reported yet. Therefore, this study sought to investigate piceatannol’s effects in animal and cell line models. AD-like symptomsand skin lesions were induced by repeated topical application of Dermatophagoides farinae extract (DFE) on NC/Nga mice’s skin. Piceatannol was topically applied five times for four weeks. Piceatannol’smolecular mechanism was studied on the TNFα/IFNγ induced HaCaT cell line.Topical application of piceatannol attenuated DFE-induced AD-like symptoms as investigated by skin thickness, dermatitis score,scratching time, and skin water loss. Histopathological analysis showedthat piceatannol suppressed DFE-induced eosinophil and mast cell infiltration into the skin. These observations occurred concomitantly with the down regulation of inflammatory markers, including serum TARC, MDC, and IgE. Also, piceatannol alleviated Th2 cytokines suchas IL-4 and IL-13 in skin tissue. Piceatannol decreased phosphorylationof JAK-STAT protein in TNFα/IFNγ induced HaCaT cell line. A molecular docking study showed that piceatannol strongly interacts withJAK1, suggesting a possible piceatannol mode of action. Piceatannol, a metabolite of resveratrol, has potential therapeutic efficacy for treating AD by targeting JAK1. Keywords : Piceatannol, atopic dermatitis, skin

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B-36

Heavy Metal-Induced Oxidative Stress Tolerance of Microalgae through Symbiotic Effect of Phycospheric Bacterium

Kichul Cho, Grace Choi, Seung Seob Bae, Yong-Min Kwon, Dawoon Chung, Eun Jong Yoo, and Dae-sung Lee*

Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea, Chungchungnam-do 33662, Republic of Korea

Due to the high potential for industrial applicability, microalgae considered as a fulture green bioresource. Despite of their potential, lowbiomass productivity in outdoor mass cultivation system still limit industrial application of microalgae. To overcome the problem, we testedalgal stress resistance property with help of symbiotic algal phycosphericbacterium. Diverse brakish microalgae were isolated from Geum riverestuary (Seocheon, South Korea), and the highest biomass productivitywas observed in chlorophyta Scenedesmus obliquus. The optimal culture condition of S. obliquus was determined by lab-scale high- throughput screening test. After 10 times subculture of xenic algal culture, we isolated phycospheric bacterium Paracoccus marcusii. Interstingly, the isolated phycospheric bacterium plays significant rolefor heavy metal stress tolerance. The inhibitory concentration (IC50) against cobalt chloride was analyzed, and lipid peroxidation along withthe increased cellular reactive oxygen species (ROS) was observed in a concentration-dependent manner. After co-cultivation with phycosphericbacteria with S. obliquus at IC50 of cobalt chloride, significantly enhanced algal growth and reduced lipid peroxidation was observed compared to mono-culture condition. Furthermore, the productivity oftotal carotenoid was significantly enhanced via co-cultivation with phycospheric bacteria. Our results speculated that phycospheric bacteriaP. marcussi play a significant role as a helper of stress tolerance, and thisecological engineering potentially applicable for the value-added microalgal biomaterial production. Keywords : Microalgae, bacteria, symbiosis

B-37

In Vitro Functional Potential of Fermented Milk Supplemented with Fruit Pulps

Tais Fernanda Borgonovi1, Mateus Kawata Salgaço2, Katia Sivieri2, Svetoslav Dimitrov Todorov3, Sabrina Neves Casarotti4, and Ana Lúcia Barretto Penna1 1Sao Paulo State University – UNESP, Department of Food Engineering and Technology, 15054-000, São José Do Rio Preto, Sao Paulo, Brazil, 2Departament of Food and Nutrition, Faculty of Pharmaceutical Sciences, Sao Paulo State University,14800-903, Araraquara, Sao Paulo, Brazil, 3ProBacLab, Department of Advanced Convergence, Handong Global University, Pohang, Gyeongbuk 37554, Republic of Korea 4Federal University of Rondonópolis - UFR, Institute of Natural and Exact Sciences, 78736-900, Rondonópolis, MT, Brazil

The addition of fruit pulp (FP) to fermented milk (FM) isa strategy to formulate new value-added fermented products with functional properties. This study aimed to evaluate chemical and microbiologicalcharacteristics of fermented milk products supplemented with FP and fermented by Lacticaseibacillus casei SJRP38, Lactiplantibacillus plantarum ST8Sh and Streptococcus thermophilus TA080. Moreover, effect of FM products towards the human intestinal microbiota was evaluated, using the SHIME® model. FM without pulp (FMC) and supplemented with 1% passion fruit pulp (FMP) or buriti fruit pulp (FMB) were prepared. Chemical composition, fatty acid profile and bacterial viability were evaluated on first day of storage. The pH values,acidity, total phenolic compounds (TPC) and antioxidant activity (AA)were assessed after 1 and 14 days of storage. In the SHIME® in vitro model, inoculated with fecal sample from healthy donors, FM preparations were administered individually for 5 days, interspersed with washout (5 days). In each period, samples were withdrawal from the SHIME® in vitro model simulating the ascending colon to assess bacterial viability (lactobacilli, streptococci, bifidobacteria, clostridia and total anaerobic bacteria) and short-chain fatty acids (SCFA) levels.The chemical analysis of all preparations met the standards required for FM (moisture, 88-89%; protein, 3.5-3.7%; ash, 1.2-1.4%). FMB showed higher oleic acid levels (66.38%) compared to FMC (31.02%). The addition of FP reduced the viability of bacterial cultures in all preparations; however, populations remained higher than 108 CFU/mL.There was no difference on pH values among preparations. FMP and FMB showed the highest acidity on 1st day compared to FMC, however,on 14th day FMP and FMC had similar acidity, while FMB was lowerthan FMC. Presence of FP in the FM increased TPC (FMC 0.08; FMP 0.10; FMB 0.09 mg GAE/100 g) and AA (FMC 0.09; FMP 0.20; FMB 0.18 µmol Trolox/g) on 14th day of storage. Addition of FPs to all FMsincreased the production of acetate and butyric acid, however only FMBincreased the production of propionic acid. The viability of the bacteria remained stable in monitored periods, except for FMB, where streptococci levels decreased. The addition FP enhanced the functionalproperties of FM, indicating its potential as part of the functional food. Keywords : Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), functional fermented dairy product, fruit pulp fruit

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Anti-Obesity Effects of Ecklonia cava Ethanol Extract against High-Fat Diet Induced Obesity in Rats

Muhammad Aleem Abbas, Naila Boby, and Seung-Chun Park*

Laboratory of Veterinary Pharmacokinetics and Pharmacodynamics, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of Korea

Ecklonia cava is an eatable brown alga that has comprehensive health benefits. This brown alga has been known to have prolific therapeutic effects including anti-diabetic, anti-cancer, anti-inflammatory, and antioxidant due to phlorotannin’s enriched chemical profile. The E.cava(70% ethanol) extract efficacy against diet-induced obesity has not beenelucidated yet. To evaluate the antiobesity effects of EC at gross and biomolecular levels we have employed a rat high-fat diet (HFD) inducedobesity model. The EC was analyzed by GC-MS and LC-MS to elucidateits chemical profile. Then, a normal diet and a high-fat diet with or withoutpretreatment of EC were given to the male Sprague Dawley rats. Liverenzymes, plasma lipids, and tissue sections of liver and adipose tissue were examined and messenger RNA expression of adipogenic genes wasstudied to elucidate the obesity related mechanism of EC. EC significantlyreduced body weight, adipose tissue mass, and hyperglycemia as compared with rats fed only with HFD. The plasma lipids and liver enzymes decreased dose-dependently in EC-supplemented HFD rats. The mRNA expression of adipogenic genes (PPARγ, SREBP-1C, and LPL) was downregulated in EC supplemented HFD rats. In our study, we aim to evaluate the antiobesity effects and mechanism of the extractand our findings suggested that EC antiobesity effects are mainly relatedto its potential to modulate the plasma lipids through adipogenic generegulation. Based on the findings from the treatment of E.cava (70% ethanol) extract under the animal model we propose the EC can be a potential therapeutic remedy to improve obesity-associated pathologieswith the chemical enriched profile. Keywords : Anti-obesity, adipogenic genes, liver enzymes

B-39

Protective Effect of Pyrus Ussuriensis Maxim. Extract against Ethanol-Induced Gastritis in Rats

Naila Boby and Seung-Chun Park *

Laboratory of Veterinary Pharmacokinetics and Pharmacodynamics, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of Korea

Pyrus ussuriensis Maxim (Korean pear) is well known for hundreds ofyears as traditional herbal medicine for asthma, cough, and atopic dermatitis in Korea and China. Although it was originally shown to possess anti-inflammatory, antioxidant, and anti-atopic properties, its gastroprotective effects have not been explored. The aim of the presentstudy was to evaluate the protective effects of the P. ussuriensis Maximextract (PUE) against ethanol-induced gastritis in rats. In the present study, the profile of the bioactive compounds of PUE was elucidated by gas chromatography and mass spectroscopy (GC-MS) and High- performance liquid chromatography (HPLC) analysis. Gastroprotectionof PUE at different doses (250 and 500 mg/kg body weight) prior to ethanol ingestion was evaluated by an in-vivo gastritis model of rats. Thehealing properties of PUE were evaluated immunohistochemical localization of leucocytes common antigen, ulcer score, and histologicalexamination. To explicate the mechanisms of gastroprotection by PUE, its antisecretory action and plasma prostaglandin E2 (PGE2), gastric mucosal cyclic adenosine monophosphate (cAMP), and histamine levels were evaluated. PUE has strong antioxidant effects with IC50 value of 56.18 µg/mL and 22.49 µg/mL for 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’- Azino-di- (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) inhibition (%), respectively. Additionally, GC/MS and HPLC analysis elucidated several bioactive compounds profile of PUE. Pre-treatment of PUE can significantly (p < 0.05) decrease the ulcer indexby preventing gastric mucosal lesions, erosion, and cellular degeneration.In addition, immunohistochemical analysis revealed that PUE markedlyattenuated the leucocytes infiltration in a dose-dependent manner. Theenhancement of the PGE2 level and attenuation of the cAMP level alongwith inhibition of histamine release by the PUE pretreatment appearedto be directly linked to the cytoprotective and healing effects of PUE.On the other hand, the downregulation of the H+/K+ ATPase pathway along with muscarinic receptor (M3R) and histamine receptor (H2R) inhibition was also involved in the gastroprotection by PUE; however,it did not affect the expression of cholecystokinin-2 receptors (CCK2R).Considering the analyzed parameters, no sign of toxicity was observedby PUE. It was concluded that PUE represents an auspicious therapeuticoption to reduce the risk of gastritis on the basis of the findings of thisstudy. Keywords : GC-MS, HPLC analysis, leucocytes common antigen, Prostaglandin E2 and H+/K+ ATPase

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B-40

The Specific Complexation Assay of Homocysteine and Cysteine with Cucurbit[7]uril at Different pH Using a Fluorescent Labeling Reagent

Kwang-Hwan Jhee1, Won-Bin Bae1, Hyeon-Bin Son1, Yeong-Jun Jeon1,and Daesuk Bang2 1Department of Applied Chemistry, Kumoh National Institute of Technology Gumi 39177, Republic of Korea 2Department of Chemical Engineering, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea

The total homocysteine (Hcy) level in plasma or serum is a sensitive indicator of vitamin B12 and folate deficiencies. Hcy and cysteine (Cys)both have similar chemical structures, their detection is quite difficultfor the early diagnosis in physiological conditions. We introduce a sensitive fluorescence method for the detection of Hcy. After pre-incubating cucurbit[7]uril (CB[7]) and Hcy and Cys in advance, a fluorescent labeling reagent (NBD-Cl) was used to assay of Hcy and Cys.Fluorescence was recorded by exciting 465 nm and measuring the emission 540 nm. At different pH (7.0, 7.5, 8.0, 8.5 and 9.0), when Hcy and Cys form complexes with CB[7], the fluorescence intensity decreases. The proportion of complexes formation was calculated compared to the control. Our data showed that the fluorescence intensityof Hcy at 540 nm was not decreased at pH 7 and that of Cys decreased.However, at pH 9, the fluorescence intensity of Hcy was decreased 40%and Cys was not decreased. These results suggest that CB[7] can act asa host by binding selectively with Hcy, but not Cys at different pH. Thisspecific host-guest reaction at various pH can apply for a simple and useful method for determining the Hcy concentration in plasma or serum. Keywords : NBD-Cl, Homocysteine, Cucurbit[7]uril

B-41

Selective Binding Properties of Homocysteine and Cysteine with Cucurbit[7]uril by pH Change

Kwang-Hwan Jhee1*, Won-Bin Bae1, Hyeon-Bin Son1, Yeong-Jun Jeon1,and Daesuk Bang2 1Department of Applied Chemistry, Kumoh National Institute of Technology Gumi 39177, Republic of Korea 2Department of Chemical Engineering, Kumoh National Institute of Technology Gumi 39177, Republic of Korea

An abnormal level of homocysteine (Hcy) in plasma is a risk factor forcardiovascular diseases and Alzheimer’s diseases etc. Due to a high degree of similarity in both structure and chemical properties between Hcy and cysteine (Cys), their detection is quite difficult. We investigatedthe Hcy assay method using Ellman’s reagent (DTNB) at different pH.After pre-incubating Hcy and Cys with cucurbit[7]uril (CB[7]), DTNBwas added to detect the sulfhydryl group (-SH) of Hcy and Cys. If free form of –SH were exist, DTNB can react with –SH and will give absorbance at 412 nm. We performed the DTNB assay at different pHvalues (6.0, 7.0, 7.5, 8.0, 8.5 and 8.8) and calculated the proportion of complexation formation by measuring the absorbance decreases at 412 nm when Hcy and Cys form complexation with CB[7]. Our results exhibited that the absorbance of Hcy and Cys at 412 nm decreased by2.4% and 12.5% at pH 6.0, respectively, and decreased by 36.6% and3.2% at pH 8.8. It was found that Cys hardly formed complexation withCB [7], and Hcy formed more than 36% complexation at pH 8.8. Theselective binding information of Hcy and Cys at different pH to the CB[7]can provide a simple and useful method to determine Hcy concentrationin plasma or serum. Keywords : Homocysteine, pH, Cucurbit[7]uril

B-42

Fluorescence Assay of Homocysteine and Cucurbit[7]uril Complex by NBD-Cl

Kwang-Hwan Jhee*, Hyeon-Bin Son, Won-Bin Bae, and Yeong-Jun JeonDepartment of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea

We performed the complexation assay homocysteine and cucurbit[7] uril (CB[7]) using UV-visible spectrophotometer and fluorescence spectrophotometer. After preincubating cysteine (Cys) and homocysteine(Hcy) with cucurbit[7]uril (CB[7]), 4-Chloro-7-nitrobenzofurazan (NBD-Cl)was used to detect Cys and Hcy. NBD-Cl reacts with sulfhydryl groups to produce highly sensitive chromogenic and fluorogenic reagent. In ourassay system, fluorescence intensity was measured even at a concentrationof 1 nmol. We can detect the fluorescence decrease only preincubatingHcy and CB[7] sample. As the concentration of CB[7] increased, the fluorescence intensity of Hcy decreased up to 50%. This suggests that the -SH group of Hcy is buried inside CB[7]. The different complexationability of Cys and Hcy to CB[7] can provide a sensitive and useful methodfor determining Hcy concentration. Previously, we reported three waysto determine the Hcy-CB[7] complexation by the assays for chemically,enzymatically and immunologically. Our results show that ethanethiolgroup (-CH2CH2SH) of Hcy was blocked by the cavity of CB[7]. Weanticipate that this probe will be of great benefit for the studying Cys andHcy in biological systems. Keywords : 4-Chloro-7-nitrobenzofurazan, Cysteine, Homocysteine

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5-Hydroxy-2-Methyl-Chroman-4-One Isolated from Endogenous Lichen Fungus Daldinia fissa as a Selective Monoamine Oxidase B Inhibitor

Geum seok jeong1, Myung-Gyun Kang2, Sang-Ah Han3, Ji-In Noh1, Jong-Eun Park1, Sang-Jip Nam4, Daeui Park2, Sung-Tae Yee1, and HoonKim1*

1Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea 2Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon 34114, Republic of Korea 3College of Pharmacy, Ewha Womans University, Seoul 03760, Republic of Korea 4Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea

Total 195 extracts of endogenous lichen fungus (ELF) derived from Ukraine were evaluated for inhibitory activities against monoamine oxidase(MAO)-A, MAO-B, acetylcholinesterase (AChE), butyrylcholinesterase(BChE), and as well as antioxidant activity. Among them, ELF13 extractisolated from Daldinia fissa showed the highest inhibition of MAO-B. From the ELF13 extract, 5-hydroxy-2-methyl-chroman-4-one (HMC) was isolated and it was found that HMC selectively inhibited MAO-B (IC50 = 3.23 µM), compared to MAO-A (IC50 = 13.97 µM). HMC is a reversible competitive inhibitor and has a Ki value of 0.896 µM. HMCwas non-toxic to normal and cancer cells up to 50 µM, and was predictedto have blood-brain barrier permeability and high gastrointestinal absorption in in silico pharmacokinetics. In docking simulation, HMCshowed higher binding affinity to MAO-B (-7.3 kcal/mol) than to MAO-A (-6.1 kcal/mol), and it formed a hydrogen bond with Cys172 of MAO-B, while it did not form hydrogen bond in MAO-A. From theseresults, it is suggested that HMC can be regarded as a candidate substancefor the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Keywords : Daldinia fissa, 5-hydroxy-2-methyl-chroman-4-one, selectivemonoamine oxidase B inhibitor

B-44

Purification and Characterization of Neuropeptides from the Starfish, Asterias amurensis

Mi Jeong Jo1, Hye-Jin Go2, Nam Gyu Park2, and Gun-Do Kim1* 1Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea 2Department of Biotechnology, Pukyong National University, Busan 48513, Republic of Korea

SMPs from pyloric caeca tissue were purified from Asterias amurensisand their characteristics were investigated. These peptides were purifiedin three to seven steps of reverse-phase, gel-filtration and ion-exchange HPLC. The primary structure of the purified SMPs was determined byMALDI-TOF mass spectrometer, automated amino acid sequencer andLC-MS/MS. SMP1 : FGGKGAFDPLSAGFTD (1586.72 Da)SMP2 : FGGSRGAFDPLSAGFTD (1701.81 Da)SMPs were identified as peptides similar to starfish myorelxant peptide (SMP) derived from starfish Patiria pectinifera. In order to investigate the pharmacologicalactivities of purified peptide, each peptide was introduced to various muscle systems of echinoderms. First, SMPs were measured for relaxingactivity in various muscle systems of starfish. In apical muscles and tubefeet of starfish, the well-known relaxing peptides S1, S2 (derived fromAsterias rubens) and SMP (derive from P. pectinifera) were used as comparative peptides. In apical muscle and tube feet of P. pectinifera,SMP1 and SMP2 showed relaxing activity. Also, in A. amurensis, SMP1and SMP2 showed relaxing activity in apical muscle, but much lowereffects in tube feet. In compressor and retractor muscle of sea urchin, SMPs did not show any response. These results suggest that SMP function as broad relaxants in the starfish muscle, which mean that thecontributions are different for each muscle system.Cloning and sequencing of a cDNA encoding the SMP results show that it comprised1959 bp including open reading frame (ORF) of 1080 bp. The ORF ofSMP was translated into 359 amino acids. The SMP precursor protein had homology with myorelaxant peptide of P. pectinifera and myorelaxant-type peptide of A. rubens. And SMP was relatively highexpressed in radial nerve cord but it was expressed in other tissue (tubefeet, apical muscle, stomach and pyloric caeca). The gene expression wasvery low only in gonad. Collectively, SMPs were the muscle relaxing peptides first identified in A. amurensis. SMPs were identified as potential neuropeptides exhibiting various physiological activities in vivo in echinoderms. Keywords : Neuropeptide, purification, starfish

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B-45

Improvement Effect of Soybeans Fermented with Bacillus subtilis SCGB 574 on High Fat Diet-Deteriorated Large Intestinal Health in Rat Model

Jaeho Choi1 and Tatsuya Unno1,2*

1Subtropical/Tropical Organism Gene Bank, Jeju National University, Jeju 63243, Republic of Korea 2Faculty of Biotechnology, College of Applied Life Sciences, SARI, Jeju 63243, Republic of Korea

Obesity is associated with impaired intestinal epithelial barrier function,which contribute to host systemic inflammation and metabolic dysfunction.Korean traditional fermented foods, fiber-rich bean products, have beenvarious biological activities in anti-inflammatory responses, but has notreported the large intestinal health. In this study was investigated the intestinal health promoting effects of soybeans fermented with Bacillussubtilis SCGB 574 on high fat diet (HFD)-induced obesity model. SDrat were fed either a HFD (60% of kcal from fat) or HFD supplemented with 10% soybeans fermented with Bacillus subtilis SCGB 574 powder(B574) for 10 weeks. B574 treatment significantly decreased the HFD-induced body and fat weights. B574 treatment significantly attenuated the HFD-induced serum alanine aminotransferase (ALT)/aspartateaminotransferase (AST) activities and blood glucose level. Also, B574treatment promoted HFD-reduced mucin (MUC2) and tight junction components (ZO-1, Claudin-1, and Occludin-1) mRNA expression in large intestine tissue. Additionally, histopathological evaluation showedthat 574 treatment attenuated the HFD-increased inflammatory cells infiltration and epithelial damages in large intestine tissue. This indicatesthat B574 supplement improves HFD-deteriorated large intestinal health by the activation of mucin and tight junction components. Keywords : Bacillus subtilis SCGB 574, HFD, large intestinal health

B-46

Evaluation of Beneficial Properties of Lactobacillus gasseri ST16HK as a Potential Probiotic Candidate for the Oral Cavity

Hamin Kim, Joanna Ivy Irorita Fugaban, Wilhelm Heinrich Hozlapfel, andSvetoslav Dimitrov Todorov ProBacLab, Department of Advanced Convergence, Handong Global University, Pohang, Gyeongbuk 37554, Republic of Korea

Conditions in the human oral cavity provide optimal conditions for different microorganisms’ growth and colonization. Evolutionary processes allowed establishment of hurdles and mechanisms inherent to the human oral cavity for an effective reduction and control of differentpathogens. However, the environmental conditions in the human mouth can be optimal for the colonization of different beneficial microorganisms.The presence of several LAB can be considered beneficial since this characteristic can help in maintaining a well-balanced and healthy microbial community. Therefore, this study was aimed to isolate beneficial strains from the oral cavity of healthy volunteers and evaluatedthese strains as potential oral probiotic candidates. The screening processwas based on the isolation of LAB, differentiation, identification and safety assessment, and series of experiments directing the selection of appropriate candidates with beneficial properties. Isolate ST16HK, originated from the oral cavity of a Caucasian volunteer, was identifiedas Lactobacillus gasseri ST16HK according to 16S rRNA sequencing.Physiological and phenotypic tests showed no hemolytic, proteinase, orgelatinase activities, neither production of biogenic amines. Strain ST16HK can be considered as safe based on negative results for the detection of efaA, cyt, IS16, esp, asa1 and hyl virulence genes, and the absence of vancomycin resistance (vanABCDEG) genes. ST16HK was resistant to tobramycin, but not to other antibiotics applied in this study.Cell-to-cell antagonism indicated that ST16HK was able to inhibit the growth of B. cereus, B. pumilus, S. mitis, Lb. paracasei, L. monocytogenes,S. delphinii, and S. gordonii. ST16HK does not produce β-galactosidase;positive results were found for eftu and gad genes (adhesion and GABAproduction), but not for genes related to folate production. ST16HK presented a high level of cell surface hydrophobicityat 85.10%. The studied strain was able to survive in a wide range of pH values (4.0 to 8.0) and ox-bile concentrations (up to 0.1%). ST16HK showed good survival in two artificial saliva models for 30 minutes and in a simulatedGIT passage model for 4h. Interaction of ST16HKwith commonly used drugs from different generic groups and oral hygienic products were evaluated and the MIC determined to predict possible negative consequences from a combined application of some drug/hygienic products and the studied probiotic candidate. Keywords : Oral cavity probiotics, Lactobacillus gasseri, antagonisticactivity

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An Integrative Pharmacology-Based Analysis of Trifolium pratense L. in the Ovariectomized Rats

Yixian Quah1, Seung-Jin Lee2*, and Seung-Chun Park1* 1College of Veterinary Medicine, Kyungpook National University, 80 Daehak-ro, Daegu 41566, Republic of Korea 2Reproductive and Development Toxicology Research Group, Korea Institute of Toxicology, Daejeon 34114, Republic of Korea

Trifolium pratense (red clover) ethanolic extract (TPEE) has been usedas a popular over-the-counter remedy for their immense potential in thetreatment of menopause complications including osteoporosis and cardiovascular disease. This study was designed to elucidate the anti- osteoporosis and cardiovascular disease parameters as well as gut microbiota composition in ovariectomized (OVX) rats for TPEE. The present study also aimed to elucidate the interrelationship between the levels of serum lipid and cholesterol related to menopausal symptomsand gut microbiota composition after treatment of TPEE in the OVX rats. Briefly, female Sprague-Dawley OVX rats were treated with different dosages of TPEE for 12 weeks. Bone metabolism, osteoblastic and osteoclastic gene expression, bone microarchitecture, uterus and bone histology, serum and bone biomarkers as well as gut microbiota composition were evaluated. Compared to the OVX rats, TPEE treatmentsdown-regulated nuclear factor kappa-B ligand (RANKL), alkaline phosphatase (ALP), and up-regulated estrogen receptor (ER) β gene expression. Reduced serum C-terminal telopeptides of type 1 collagen(CTX-1) level and improvement in the estrogen dependent characteristicsof the uterus on the lining of the lumen were observed in the TPEE intervention group. TPEE-treated OVX rats showed significant reductionin serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDLlevels but showed increase in HDL level. TPEE intervention was seento reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlationanalysis at the phylum level revealed that Bacteriodetes and Proteobacteriawere strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlated with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels. In short, our analyses indicate that TPEE showed moderate anti-osteoporosis effect but promising estrogenic effect. TPEE possesses hypocholesterolemiceffects and shows potential for reducing risk factors for coronary heartdiseases Keywords : Trifolium pratense, menopause, gut microbiota

B-48

Therapeutic Potential of EGCG, a Green Tea Polyphenol, for Treatment of Coronavirus Diseases

Junsoo Park*, Rackhyun Park, Minsu Jang, Yea-In Park, and Yeon-Jung Park Division of Biological Science and Technology, Yonsei University, Wonju 26493, Republic of Korea

Epigallocatechin gallate (EGCG) is a major catechin found in green tea,and there is mounting evidence that EGCG is potentially useful for thetreatment of coronavirus diseases, including coronavirus disease 2019(COVID-19). Coronaviruses encode polyproteins that are cleaved by 3CL protease (the main protease) for maturation. Therefore, 3CL protease is regarded as the main target of antivirals against coronaviruses.EGCG is a major constituent of brewed green tea, and several studies have reported that EGCG inhibits the enzymatic activity of the coronavirus3CL protease. Moreover, EGCG has been reported to regulate other potential targets, such as RNA-dependent RNA polymerase and the viralspike protein. Finally, recent studies have demonstrated that EGCG treatment interferes with the replication of coronavirus. In addition, the bioavailability of EGCG and future research prospects are discussed. Keywords : Coronavirus, SARS-CoV-2, EGCG

B-49

Screening for Probiotics by Fermentation to Increase Bioactive Ingredients of Antler Velvet

Jiseon Yoo, Juyeon Lee, Minkyoung Kang, Bohyun Yun, and Sangnam Oh*

Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea

Antler velvet is widely used as a medicinal material in traditional medicine it has various effects such as anti-aging, antioxidant, or immunity enhancement. In this study, to increase the functionality of domestic antler velvet, probiotic bacteria were selected to enhance bioactive ingredients including gamma-aminobutyric acid, uronic acid,and sialic acid of antler velvet by fermentation. Seventeen Lactobacillusspp. (L. sakei, L. rhamnosus, L. brevis, and L. plantarum) strains isolatedfrom kimchi and infant feces, which showed beneficial effects on the lifespan of C. elegans, were selected as suitable strains for antler velvet fermentation. To ferment antlers velvet with strains, 2% antler extract and 17 suitable strains were independently cultured in the minimal prebiotic medium for two days. Next, to investigate whether Lactobacillusspp. strains increase the content of the specific component, we measured uronic acid, sialic acid, GAG, GABA, total nitrogen, amino acid, and protein content of fermented antler velvet extract. Especially, L. rhanmnosus LFR20-004 and L. sakei LFR20-007 out of 17 strains significantly changed the bioactive components as well as total nitrogenand amino acids on the second day of fermentation. Taken together, wesuggest that domestic antler velvet fermented with LFR20-004 and LFR20-007, increasing bioactive molecules and expecting to provide more functionality of antler. The value of domestic antlers as fermentedfoods might be expected to increase through performing in vitro and invivo to reveal the biological mechanisms to promote health impacts.

Keywords : Antler velvet, bioactive ingredients, probiotics

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C_Biodegradation and Bioremediation

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C-1

Semi-Rational Engineering of a Cold-Active Acetyl Xylan Esterase for Substrate Selectivity

Nandanwar, Shweta Bharat Borkar, and Hak Jun Kim*

Department of Chemistry, Pukyong National University, Busan 48513, Republic of Korea

Cold-active acetyl xylan esterase catalyzes the hydrolysis of glucose penta-acetate and xylan acetate, reversibly producing acetyl xylan from xylan. It showed relatively broad substrate specificity toward several acetylated compounds and cephalosporin antibiotics. The acetylation activity of this enzyme may be effective for the semi-synthesis of newantibiotics. To make this enzyme to accommodate broader substrates forthe modification of cephalosporin antibiotics, we used covalent dockingusing Autodock vina and HotSpot3D webserver. We found the 7 amino acid residues critical for substrate binding. Theses residues were substituted by site saturation mutagenesis and the substitutes showing better activity against linalyl acetate and terpinyl acetate were screened and identified by sequencing. The affinity of more active mutants wereevaluated using Autodock.

Keywords : Semi-rational approach, cold active enzyme, promiscuity

C-2

Isolation and Characterization of Pseudomonas sp. JPS-CO2 Using Carbon Monoxide as a Sole Carbon Source

Il lae Jung1,2 1Radiation Biology Team, Korea Atomic Energy Research Institute, Daejeon 34057, Republic of Korea 2Department of Radiation Science and Technology, University of Science and Technology, Daejeon 34113, Republic of Korea

Carbon monoxide is a representative harmful gas among air pollutants,and various attempts have been made to remove carbon monoxide fromthe atmosphere. The research team is developing a new bio-based air purification system using microorganisms to remove carbon monoxidefrom the atmosphere. To this end, I first tried to isolate a new bacterial strain that can grow by using carbon monoxide in the atmosphere as thesole carbon source. First, carbon monoxide was supplied to a specificbioelectrochemical reactor (3V, 10-s pulse) to promote microbial metabolism, and mixed strains obtained from the environment were inoculated into the reactor and cultured further for 4 weeks. As a resultof microbial enrichment cultivation, a new strain that can grow dominantly was finally obtained, and as a result of 16-rRNA analysis of this strain, it was named as Pseudomonas sp. JPS-CO2. As a result ofinvestigating the availability of carbon monoxide on the isolated microbial strain, it was found that 0.5% of carbon monoxide supplied to the reactor was effectively removed after 10 days of cultivation. This result is expected to be developed as a new concept of bio-air purificationsystem to remove carbon monoxide from the atmosphere in the future.

Keywords : Air pollutants, carbon monoxide, Pseudomonas

C-3

Biodegradation of Plastics in Anaerobic Condition by Enterobacter spp. Isolated from Gut of Tenebrio molitor

Mingeun Kang1, Minhye Shin1, Sangdon Ryu1, Hye Jin Choi1, Daniel Lee1,Woong Ji Lee1, Hayoung Kim1, An Na Kang1, Daye Mun1, Sangnam Oh2*,and Younghoon Kim1*

1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea 2Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea

Polystyrene, also widely known as StyrofoamTM, is a polymer of styrene and one of a major plastic that pollutes the aquatic environments. Largequantities of polystyrene have been introduced into the environment, resulting in the hazardous accumulation of the material in ecosystems.To solve this problem, it is utmost importance on how to effectively treatthe polystyrene wastes. In this study, 105 strains were isolated from thegut of mealworm (Tenebrio molitor) larvae, which is known to chew anddigest polystyrene. Ten bacterial strains successfully grew on selective-media containing polystyrene as a sole carbon source. Subsequent incubation of the strains on polystyrene-media produced clear zones around the bacterial colonies, indicating that they would be functionallyinvolved in polystyrene degradation. 16S amplicon sequencing resultssuggested that the isolated strains are closely related to Enterobacter spp.Especially, Strain of SLAM LG3 formed a clade which was distinct from all other Enterobacter species, suggesting that SLAM LG3 is a promisingcandidate as a novel polystyrene-degrading bacterium. In combinationwith the functional studies, our study provides potentials of novel polystyrene-degrading microorganisms to be applied for petroleum- based plastic degradation.

Keywords : Tenebrio molitor, Polystyrene (PS), degradation

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C-4

Metagenomic Analysis of Eco-Friendly Treatment Process of Textile Dye Wastewater Bioaugmented with the Novel Microbial Consortium CES-1 at a Full-Scale System

Aalfin Emmanuel Santhanarajan1, Woo Jun Sul2, Keunje Yoo3, Hoon JeSeong4, Hong-Gi Kim5, and Sung-Cheol Koh6*

1Korea Maritime and Ocean University, Department of Envirenmental Engineering, Busan 49112, Republic of Korea 2Chung-Ang University, Department of Systems Biotechnology, Anseong 17546, Republic of Korea 3Korea Maritime and Ocean University, Department of Envirenmental Engineering, Busan, Republic of Korea 4Chung-Ang University, Department of Systems Biotechnology, Anseong 17546, Republic of Korea 5Bayo, Inc. R&D, Jinju, Republic of Korea 6Korea Maritime and Ocean University, Department of Envirenmental Engineering, Busan 49112, Republic of Korea

The presence of very small amounts of dyes in water (less than 1 ppm for some dyes) is highly visible and affects the aesthetic merit, water transparency and gas solubility in waterbodies. The hypothesis is that the azo-dye could be degraded via pathways including azo-dye reduction,aromatic compound degradation, amino acid metabolism, nitrogen andphosphorus cycle and TCA cycle. The activities of dye wastewater treatment plant were evaluated before and after the addition of CES-1. Total genomic DNA samples were extracted from different treatments with different time variables. The effect of CES-1 bioaugmentation onthe microbial community structures and functional genes of each treatmentstep was explored by high-throughput sequencing and metagenomic analysis. Through the addition of CES-1, the sludge reduction rate over20 months after the bioaugmentation was 26% (reduction from 6.18 to5.00 in sludge per ton of influent COD). The removal rate of COD increased up to 97.8% in 50 days and the removal rate of other parametersincreased as well. Massilia timonae and Verminephrobacter sp. were dominant in the bioaugmented samples. The metagenomic analysis revealed the key enzymes involved in azo dye and aromatic compoundwere well annotated including azo-reductase (acpD), catechol 2,3-dioxygenase(dmpB), protocatechuate 4,5-dioxygenase, alpha chain and beta chain (ligA and ligB). These enzymes were dominant in CES-1 and three separate groups of sample such as primary aeration (PA), secondary aeration (SA) and sludge digestion of control (PA_0303_17 and SA_ 0303_17), 50 days (PA_0629_17, SA_0629_17 and SD_0303_17) and531 days’ (PA_1026_18, SA_1026_18 and SD_1026_18). TCA cycle provides abundant NADH and FADH2 to the electron transfer chain, NADH and FADH2 eventually transfer electrons to the azo dyes throughcytochrome. Among them, NdoR and nahA is combined with the complex structure to transmit electrons to azo dyes. This proves the bioaugmentation of CES-1 in the treatment made an impact on enhancing the metabolic pathways in dye degradation. fadE, ENO and Glucanobacter_unclassified were clustered with PA_1026_ 18 and commonly present in all the bioaugmented samples after 531 days. The high consistency of the analyses mentioned above indicated that the network analysis is a reasonable and powerful tool to provide us new insights and eligible biomarker in complex environmental examples. The intrinsic relationshipsbetween the microbial community structures and functions of enzymesfor the dye metabolism were analyzed using sophisticated algorithms. This understanding of metabolic pathways in the dye wastewater treatment system will undoubtedly contribute to an optimized and efficient operation of the treatment system and any other wastewater treatment systems.

Keywords : Bioaugmentation, biodegradation, metagenomics

C-5

Biosorption of As(III) Using Saccharomyces cerevisiae SRCM 501804 Isolated from Korean Turbid Rice Wine: Isotherm and Kinetic Studies

MyeongSeon Ryu, Jinwon Kim, Hee-Jong Yang, Gwangsu Ha, Su-Ji Jeong, Sua Im, Su-Jin Shin, Ji-Won Seo, Se Won Park, and Do-Youn Jeong*

Microbial Institute for Fermentation Industry (MIFI), Sunchang 56048, Republic of Korea

In this study, Saccharomyces cerevisiae SRCM 501804 (Genbank accession No. MZ068240) was isolated from Korean turbid rice wine (Makgeolli). Dried biomass of S. cerevisiae SRCM 501804 showed superior biosorption capacity of trivalent arsenic (As(III)) in aqueous solution. The characterizations of the S. cerevisiae SRCM 501804 wereperformed by phylogenetic analysis, Fourier transform infrared spectroscopy (FT-IR), MINTEQ, and point of zero charge (pHpzc). Additionally, the influence of pH (2.0-10.2), biomass dosages (0.01-0.09g), contact times (0-120 min) and initial concentration (51.2-281.5 mg/L) on the biosorption of As(III) were evaluated. Dried biomass of S. cerevisiae SRCM 501804 removed 90.1% of the As(III) from a 40.8mg/L within 1 h. The biosorption isotherm and kinetic results showed the Langmuir isotherm and Pseudo-second order models well fitted. Thebiosorption experiments showed that S. cerevisiae SRCM 501804 couldbe used as effective biosorbent for the As(III) biosorption in aqueous solution.

Keywords : Saccharomyces cerevisiae, trivalent arsenic, biosorption

[This work was supported by a grant from the Establishment of IntegratedBiobank for Agriculture, Food and Livestock Microbiome Project funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA)]

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C-6

Characterization of DDT-Degrading Soil Bacteria Isolated from a DDT-Contaminated Soil

Jae-Hyung Ahn1, Geun-Hyung Choi2, Joon-hui Chung1, Ja-Yeon Lee1, Myoung-Hwa Jung1, In-Cheol Park1, Da-Yeon Kim1, Si-Hyun An1, and Younggun Yoon1

1Bioremedation Team, National Institute of Agricultural Sciences, 166 Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do 55365, Republic of Korea 2Agromaterial Assessment Division, National Institute of Agricultural Sciences, 166 Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do 55365, Republic of Korea

Dichlorodiphenyltrichloroethane (DDT) is one of organochlorine insecticides, which was prohibited for its toxicity in most of the worldin the 1970s and 1980s. However, it has been frequently detected in various environments for its non-biodegradability and can have adverseeffects on organisms over a long time. We isolated six DDT-degradingbacterial strains from a DDT-contaminated soil in Korea. They were affiliated with the genera Flexivirga, Rhodopseudomonas, Paralcaligenes,Nitrobacter, Mesorhizobium, and Oleiharenicola, respectively. They degraded 14~77% of 1.5 mg/L of 4,4’-DDT within 34 days in minimummedia, among which, Paralcaligenes sp. KSB-10 showed the highest degradation rate (77%). Interestingly, DDE and DDD, the known metabolites of DDT were not detected in the HPLC chromatograms atday 34 for all the isolates. When strain KSB-10 was inoculated into minimum media containing various concentrations (2.3~9.3 mg/L) of technical grade of DDT (4,4’-DDT:2,4’-DDT=1:0.4) and incubated for 19 days the degradation rate decreased from 54% to 19% as the DDT concentration increased. In addition, the degradation rates of 2,4’-DDTwere generally lower than those of 4,4’-DDT, indicating that the formeris more difficult to degrade than the latter or the degradation rate decreases at lower concentrations.

Keywords : DDT, biodegradation, bacteria

[This work was supported by the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea (project no. PJ01420002)]

C-7

Biological Upgrading of Ethanol-Assisting Depolymerized Lignin: from Waste to Value-Added Product

Linh Thanh Nguyen, and Eun Yeol Lee* Department of Chemical Engineering (Integrated Engineering). Kyung Hee University, Yongin 17104, Republic of Korea

Waste lignin is dramatic increasing from pulp, paper, and biofuel production. More than 100 million tons of waste lignin is being generatedannually. Although many attempts have been made to establish sustainable lignin valorization, but it remains challenging. In this work, we present a new approach for lignin valorization by biological co-upgrading lignin and its organic solvent - ethanol from depolymerizationprocess into protocatechuic acid and polyhydroxyalkanoic acid. Pseudomonas putida KT2440 with high efficient ethanol utilization wasused for biological upgrading of ethanol-assisting depolymerized lignincontaining various lignin monomers at a concentration of 77 mg/mL. Toproduce value-added products, P. putida KT2440 was engineered by knocking out the protocatechuate 3,4-dioxygenase, construction of formaldehyde utilization pathway, and reconstruction acetaldehyde direct conversion to acetyl-coenzyme A using aldehyde dehydrogenasefrom Dickeya zeae. Additionally, we demonstrate the promoting of the utilization of formaldehyde on the growth and production of value-addedproducts. Finally, the engineered strains are able to produce 6.73 ± 0.26mg/L of PCA with a 17.5% (w/w) yield of total lignin monomers, and 303.66 ± 26.75 mg/L of PHA with 21.26% (w/w) of dry cell weight from0.5 mL of ethanol-assisting depolymerized lignin. This study representsa sustainable approach for lignin valorization, which employs ethanol-solvent of depolymerization of lignin as the co-substrate withoutseparation process.

Keywords : Lignin valorization, polyhydroxyalkanoic acid, co-upgrading

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C-8

Are There Any Fungi that Can Decompose of Plastic in Soil Fungal Communities?

Myoung-Ho Joo1, Guan Yong1, HanNa Choe1, Chi Nam Seong2, and Mi-Kyung Lee1*

1Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup 56212, Republic of Korea 2Department of Biology, College of Life Science and Natural Resources, Sunchon National University, Suncheon 5792, Republic of Korea

In agriculture, plastics play an important role in increasing production,such as controlling weeds, maintaining soil temperature, and preventingwater loss. But, Plastic (agricultural waste) was known to accumulate in the soil as one of the main factors of environmental pollution and affect the soil ecosystem. To solve the soil pollution caused by plastics, we conducted a study to find fungi capable of degrading plastic in the fungalcommunity of agricultural plastic waste. To find fungi capable of degrading plastic, collected agricultural waste plastic from agriculturalland, fallow land, and dumpsite. And then, each sample's DNA was extracted and through Next-Generation sequencing analysis. As a result,identified the fungi of 522 strain and each sample's dominant species.These results suggest that there may be fungi that can degrade agricultural plastic in the fungi community of agricultural plastic waste.Further study, we will analyze the characteristics of bio-degradable fungiof plastic by using fungi isolated from agricultural plastic waste. This data will provide a new experimental field of perspective for bio-remediation.

Keywords : Biodegradation, fungi, plastic

C-9

Cesium Removal Using R. erythropolis

June Lee, Chang-Hyun Jeon, and Woong Kim*

Department of Environmental Engineering, Kyungpook National University, Daegu 41566, Republic of Korea

Nuclear power generation is eco-friendly and economical because it canobtain a lot of energy with a small amount of fuel compared to fossil fuels.It is being developed as a sustainable energy source to secure a stable power supply and carbon dioxide reduction. However, due to the wastewater from nuclear power plants and radioactive materials releasedfrom Fukushima, Chernobyl nuclear power plant accidents, the need forenvironmental restoration research is increasing. Cesium is a representativeradioactive material. Adsorption, coagulation, precipitation, reverse osmosis, and ion exchange are used to remove cesium, but there are limitsto practicality due to the complexity of the treatment method with highcost. To overcome the problems of the existing processes, an economical,easy-to-treat, and eco-friendly bioremediation method is newly attractingattention in radioactive waste treatment. In this study, the aerobic bacteriaR. erythroplis was used to remove cesium dissolved in aqueous solution.As a result of conducting the experiment under various conditions, its cesium removal efficiency is up to 86% after 24 hours of incubation. During bacterial growth, the maximum removal efficiency of cesium was showed when the stationary phase is start. As a result of analyzingthe bacteria using FE-TEM, cesium was intensively accumulated in some specific points inside of the bacteria. In addition, we can check thatthe bacteria which removed cesium grew 3-4 times longer than controlbacteria. Removal of heavy metals and radioactive materials using bacteria is eco-friendly and has high economical, which shows the possibility of solving environmental pollution caused by nuclear power plant wastewater or accidents.

Keywords : Bacteria, bioremediation, Cesium

C-10

Analysis of Microbial Communities in Waste Plastic Films and Soils Collected from a Landfill Site in Gochang-gun, Korea and Their Potential to Degrade Polyethylene Films

Joon-hui Chung, Si-Hyun An, Da-Yeon Kim, Younggun Yoon, In-CheolPark, and Jae-Hyung Ahn*

Bioremedation Team, National Institute of Agricultural Sciences, 166 Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do 55365, Republic of Korea

Non-degradability of plasticware has been recognized as serious environmental problem. Biodegradation of plastics by microorganismshas a potential to alleviate the rapid accumulation of plastic wastes. Toidentify and isolate the microbes to degrade plastics, we carried out themetagenomic study for waste plastic films and nearby soils collected from a landfill site located in Gochang-gun, Korea, in which plastic wastes have been buried for more than 20 years. The analysis of bacterialand fungal communities has been in progress using DNA and RNA extracted from the waste films and soils. Physical and chemical parameters of landfill soils were measured and observed as high pH values. Fourier-transform infrared (FT-IR) spectroscopy to the films showed peaks related to –OH functional group implied as erosion of thefilms. When pure low-density polyethylene (LDPE) films have been cultured with landfill soil and waste film samples for 2 months, functionalgroups containing oxygen such as –OH group appeared in the FT-IR spectroscopy analysis, suggesting partial oxidation of LDPE films. Bacteria and fungi have been isolated from the oxidized LDPE films forfurther study. These results suggest that the microbiome enriched in theplastic films has potential to degrade LDPE films.

Keywords : Polyethylene, biodegradation, metagenome

[This work was supported by the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea (project no. PJ01497401)]

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C-11

A Study on the Selection of Water Purification Species Using Microalgae Biofilm

Dae Geun Kim1, Junho Lee2, Sehoon Oh2, and Yujin Lim2 1LED Agri-Bio Fusion Technology Research Center, Jeonbuk University, Jeollabuk-do, Iksan 54596, Republic of Korea 2College of Environmental and Bioresource Sciences, Jeonbuk University, Jeollabuk-do, Iksan 54596, Republic of Korea

Contamination of livestock wastewater, fertilizers, pesticides, etc., which appear as nonpoint sources of pollution, cannot be predicted andprepared, so it is necessary to rely on the purification of rivers themselves.Microalgae have been reported to remove nitrogen and phosphorus fromthe water and to remove many of the pollutants in the water as a primary producer that grows through photosynthesis. It has been confirmed thatmany kinds of microalgae grow wild in Korea, and in particular, the efficiency of the treatment of nutrients is known according to the type of microalgae. Therefore, microalgae growing in domestic rivers were selected and compared with their ability to remove toxic heavy metals.In order to effectively carry out these studies, microalgae clusters that form colonies in rivers were collected. In addition, the ability of the colony to remove contaminants was first confirmed. After that, the effects of individual microalgae constituting the colony were compared.Microalgae colonies were mainly found in puddles of water or in calm spots in rivers, and had viscous or filamentous forms. Microalgae suchas Coccomyxa sp., Cladophora sp., Acutodesmus sp., And Gloeocystissp. Showed different treatment efficiencies for nitrogen, phosphorus andheavy metals. Chlococcum sp. and Gloeocystis sp. Showed high removalefficiency of nitrogen and phosphorus. While Acutodesmus sp. showedhigh removal efficiency of heavy metals such as Cu, Pb and Zn. From these results, the removal of toxic heavy metals through microalgae is effective. If the microalgae treatment characteristics of the selected microalgae are appliedto the streams in the event of pollution, it is expected to increase the efficiency of the river's self-cleaning by increasing the biological purification capacity.

Keywords : Microalgae, biofilm, purification

C-12

One-Pot Chemical and Biological Depolymerization of PET Using a Novel Biocompatible Catalyst, Betaine

Doyeon Kim, Dong Hyun Kim, Dong Oh Han, and Kyoung Heon Kim*

Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Republic of Korea

Due to its unique physical characteristics, poly(ethylene terephthalate)(PET) has been broadly usedin numerous industries. However, PET poses significant environmental problems worldwide due to itslow decomposition and recycling rate. Since replacing PET with other substances is almostimpossible, an efficient procedure for PET recyclingis needed in a circular economy. Here, wedesigned an integrated processconsisting of depolymerizing PET and converting PET monomers intohigh value-added products with a one-pot process for the paradigmshift to a method for resourcerecovery of PET components. The centerof our approach is the use of a biocompatible and greencatalyst, betaine,in the glycolysis process that allows the entire PET glycolysis slurry to be used as asubstrate that can directly be applied to additional bioprocesses without any separation steps. Basedon the density functional theory (DFT) analysis, by not only synergistic effects betweenthe anion andcation groups of betaine but also strong hydrogen interactions between PET, EG, and betaine,betaine effectively catalyzedPET depolymerization. Through the PET glycolysis with betaine and theoptimized enzyme hydrolysis for the PET glycolysis slurry, PET wasdecomposed to terephthalate(TPA, 31.0 g/L, 62.8% mol/mol) and ethylene glycol (EG, 11.7 g/L, 63.3% mol/mol) at high yields andhigh titers. This process has been applied to the conversion of the PET hydrolysate (TPA and EG) toprotocatechuic acid (PCA) and glycolic acid (GLA), respectively. We suggest that this one-pot chemo- bioprocess Integrating chemical glycolysis, enzymatic hydrolysis, andbioconversion for PETdepolymerization and recycling, is highly promising for the upcycling of waste PETs.

Keywords : PET recycling, betaine, depolymerization

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C-13

Direct Removal of Harmful Cyanobacterial Species Using an Adsorption Process and the Potential Applications as Lipid Source

Yun Hwan Park1, Ho seon Kim1, Ka Young Kim1, Hyun Soo Kim3, JaewonPark4, Sok Kim1,2*, and Yoon-E Choi1*

1Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Republic of Korea 2OJeong Eco-Resilience Institute, Korea University, Seoul 02841, Republic of Korea 3Department of Electronic Engineering, Kwangwoon University, Seoul 01897, Republic of Korea 4Department of Electrical and Electronic Engineering, Southern University of Science and Technology, Shenzhen, P.R. China

In the present study, we applied an adsorption process to remove and recover the harmful pollutant Microcystis aeruginosa using polyethyleneimine(PEI)-modified chitosan-waste biomass composite fiber sorbent (PEI-CBF). The enhancement of the positive binding sites on the sorbent after PEI modification is important factor to ensure the removal potential of the adsorption processing for M. aeruginosa cell removal from an aqueous solution. The PEI-CBF could remove 90.5% of M. aeruginosacells without requiring additional processes, while CBF removed 22.7%under the same experimental conditions. In the cell removal process using PEI-CBF, the M. aeruginosa cells were bound to the PEI-CBF without cell lysis and damage. From the cell-loaded PEI-CBF, 95.3% of adsorbed cyanobacterial cells were recovered via a desorption processin an alkalic solution and ultrasonication. In addition, the total lipid content of the recovered M. aeruginosa cells was similar as that of non-adsorbed M. aeruginosa cells. Furthermore, the cell removal performance of regenerated sorbents was almost entirely maintained. Our adsorption process can be applied as a breakthrough technology toallow the conversion of the environmental pollutant M. aeruginosa to energy resources by recovering and controlling the cells without substrate loss by cell lysis.

Keywords : Microcystis aeruginosa, adsorption, waste to resource

C-14

Dynamics of Microbial CH4-Oxidation Potential in Rhizosphere during Rhizoremediation of Diesel-Contaminated Soil

JiHo Lee and Kyung-Suk Cho*

Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Methane is a representative non-CO2 greenhouse gas. Diesel-contaminatedsoil is one of the potential methane gas sources. For soil remediation,it is necessary to develop the technology that can satisfy not only pollutant degradation but also CH4 mitigation. In this study, microbial CH4-oxidationpotential in rhizosphere was evaluated during rhizoremediation of diesel-contaminated soil planted with maize and tall fescue. . The CH4oxidation rates in the maize rhizosphere were constantly increased from1.1 μmol·g-dry soil-1·h-1 at 0 d to 1.5 μmol·g-dry soil-1·h-1 at 83 d. TheCH4 oxidation rates in the tall fescue remained almost constant at 1.1 μmol·g-dry soil-1·h-1 for 83 d. The dynamics of CH4 oxidation functional gene, pmoA, in the maize and tall fescue rhizosphere showed the similar pattern to that of CH4 oxidation rate in each rhizosphere. In the maize rhizosphere, the abundance of methanotrophs ranged 0.61~2.67%, andits abundance in the tall fescue rhizosphere was 0.79~2.99%. The dominant methanotrophs were almost the same in the maize and tall fescue rhizosphere. Sphingopyxis, Methylocapsa, Methylosarcina, Methylococcus, Methylocystis were dominant methanotrophs. The useof these methanotrophs in rhizoremediation process is expected to mitigate CH4 emission during the remediation of contaminated soil.

Keywords : Rhizoremediation, methane oxidation rate, methanotrophs

[This study was supported by the National research Foundation of Korea (NRF) grant funded by the Korea government, the Ministry of Scienceand ICT (MSIT) (2019R1A2C2006701)]

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C-15

Evaluation of N2O-Reducing Activity in Rhizosphere during Rhizoremediation of Diesel-Contaminated Soil

Ji-yoon Kim and Kyung-Suk Cho*

Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Diesel-contaminated soil is considered a major anthropogenic N2O source. Various physico-chemical and environmental factors in soil canaffect nitrogen cycle including N2O-reduction. However, there are few researches on N2O-reduction activity in diesel-contaminated soil. In thisstudy, N2O-reduction performance in rhizosphere was characterized during the rhizoremediation of diesel-contaminated soil. Three planting treatments (no planting, maize and tall fescue planting) of out door potexperiment were conducted for 110 days. In all experimental groups, theN2O-reduction activity gradually decreased as the remediation time elapsed. That is, the N2O-reduction activity decreased with decreasingresidual diesel concentration in soil. The N2O-reduction rates in the soilwithout planting were higher than those in the soil planting with maizeor tall fescue. However, the nosZI, functional gene for N2O reductase,abundances were not correlated to the N2O reduction activities in all experimental groups. The nosZI abundances increased after 83 day in the no planting soil and tall fescue planting soil (150.15 - 291.48 gene copy number ∙ g-dry soil-1). The nosZI abundances in the maize plantingsoil increased after 19 day (170.58 - 273.74 gene copy number ∙ g-dry soil-1). Based on bacterial community structures, the abundances of major N2O-reducing bacteria such as Hyphomicrobium, Sphingomonas,Pseudomonas increased during the initial period (0 - 19 d). Their abundances at the day 19 were 14, 15 and 24 % in the no planting soil,tall fescue planting soil, and maize planting soil, respectively. The abundances in all experimental groups gradually decreased to 7 - 8 % until the day 40, and they maintained during 40 - 110 day. These resultscan be used to establish strategy for the mitigation of N2O emission duringrhizoremediation of contaminated soil.

Keywords : Rhizoremediation, nitrous oxide reduction, diesel-contaminatedsoil

[This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) (NRF-2019R1A2C2006701)]

C-16

Effect of Compost Amendment on Bioremediation Performance of Diesel-Contaminated Soil

Hyoju yang and Kyung-Suk Cho*

Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Bioremediation using microbial activity is well-known for the remediation technology of contaminated soil. Compost amendment canincrease microbial activity by supplying available nutrients, resulting in the enhancement of bioremediation efficiency. Compost addition canalso affect the CH4 and N2O emission and the soil microbial communitystructure. In this study, to evaluate the compost amendment effect on bioremediation performance, low nutrient barren soils mixed with different ratios of compost (0, 5, 10 and 20%, w/w) were contaminatedwith diesel of 10,000 mg-TPH·kg-dry soil-1. The diesel removal efficiency was improved by increasing the compost addition amount. After 103d, the diesel removal efficiency was 54% in the soil withoutcompost, and it was 85% in the soil with 20% compost. The CH4 oxidation potential in all treatments increased during diesel bioremediation. The CH4 oxidation potential rate in the soil with 20% compost was 1.79 timeshigher than that in the soil without compost. However, the N2O reductionpotential decreased over time, and there was no significant effect of compost amendment. The compost amendment affected bacterial communitystructures, and the abundances of Immundisolibacter, Acidibacter and Terrimonas were increased by compost amendment. The information obtained in this study can be utilized as a meaningful reference for the innovation of bioremediation technology.

Keywords : Bioremediation, compost amendment, diesel contaminatedsoil

[This study was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government, the Ministry of Scienceand ICT (MSIT) (2019R1A2C2006701)]

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C-17

Degradation of Bisphenol A by Sphingobium sp. A3 Isolated from Contaminated Soil

Hye Kyeong Kang, Hyun Mi Jin, Ji Young Jung, and Mi Hwa Lee* Microbial Research Department, Nakdonggang National Institute of Biological Resources, 137, Donam 2-gil, Sangju-si, Gyeongsangbuk-do 37242, Republic of Korea

Bisphenol-A (BPA) is an endocrine-disrupting compound (EDC) that mimics the function of estrogen causing damage to reproductive organs.Bacterial BPA degradation has been consider as a cost effective and eco-friendly method compared with physical and chemical methods. In this study, we found BPA-degrading bacteria from soil and characterizedits BPA-degradation efficiency. The soil sample was collected at a wastedump site and a BPA-degrading enrichment culture was conducted aerobically using mineral salts medium with BPA as sole carbon source.We isolated the strain A3 from the BPA-degrading enrichment culture,and the evaluation of its BPA degradation ratio showed that the strain A3 degraded more than 70% of 200 ppm of BPA in the medium for 24 hours. The bacterial identification and phylogenetic analysis based on 16S rRNA gene sequence showed that the strain A3 was closely relatedwith Sphingobium yanoikuyae ATCC 51230T, and when tested on R2A medium, the growth of strain A3 was observed at a temperature rangeof 15 to 35℃ and pH range of 5.0-9.5.

Keywords : Biodegradation, bisphenol A, Sphingobium sp. A3

C-18

Effect of Rouxiella sp. S1S-2 on In Vitro Assay for Odor Reduction in Livestock Eenvironment

Young Ho Nam, Ji Young Jung, and Mi Hwa Lee*

Microbial Research Department, Nakdonggang National Institute of Biological Resources, 137, Donam 2-gil, Sangju-si, Gyeongsangbuk-do 03760, Republic of Korea

Odor generation is one of the most common concern in livestock farm. In this study, among various odorous compounds, we focused on microbial odor control against ammonia (NH3) and amine. For this, weperformed in vitro assay to screen bacterial strains harboring odor reduction potential. The evaluation of NH3 and amines removal efficiencies using gas detector tubes, from control to bacterial-treated pig manure slurry in odor emission chamber, showed that strain S1S-2showed a high removal efficiency against NH3 and amine odor. Especially, the treatment of 5% (w/v) freeze-dried powder showed removal efficiency of > 85% for NH3 and amines. The bacterial identification using 16S rRNA gene sequencing showed that the strain S1S-2 belong to a strain of the genus Rouxiella. In conclusion, we screened the strain Rouxiella sp. S1S-2 harboring high potential of NH3and amines removal and this strain could be a candidate in the future fieldexperiment studies for odor management in livestock.

Keywords : Rouxiella sp. S1S-2, odor reduction, NH3 and amine

C-19

Isolation of Plant Growth-Promoting Rhizobacteria Having Heavy Metal Tolerance and Diesel Degradation Capacity

Soo yeon Lee, Yun-Yeong Lee, and Kyung-Suk Cho*

Department Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Soil contamination with hazardous pollutants such as heavy metals andoil causes the disturbance of the soil biota due to decreasing the microbialactivity and diversity. For the remediation of contaminated soils, rhizoremediation is attracting attention as a promising technology. Theintroduction of promising microbial agents into contaminated soil is oneof the strategies to enhance rhizoremediation performance. In this study,heavy metals (Cu, Cd, and Pb) tolerant-rhizobacteria were isolated fromthe rhizosphere of Zea mays and Festuca arundinacea, which were cultivated in heavy metals/diesel-contaminated soil. Additionally, plant growth promoting (PGP) traits and diesel removability of the isolateswere evaluated. Among the 44 isolates, three strains with outstanding PGP traits (IAA, siderophore, and ACC deaminase productivity), heavymetal tolerance, and diesel removability were selected and identified. Novosphingobium sp. CuT1 showed a high resistance of heavy metals at 10 mM of Cu and Pb, this bacterium had a diesel removability of 40.5%. Sphingomonas sp. PbM2 and Bacillus sp. PbT3 could grow at 1 mM ofheavy metals, and the strain PbT3 exhibited a diesel removability of 77.7%. These results suggest that the isolated rhizobacteria have potential as the candidates for enhancing the rhizoremediation performancesoil contaminated with heavy metals and diesel.

Keywords : Heavy metal tolerance, plant growth-promoting rhizobacteria,rhizoremediation of contaminated soil

[This study was supported by the National research Foundation of Korea (NRF) grant funded by the Korea government, the Ministry of Scienceand ICT (MSIT) (2019R1A2C2006701)]

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C-20

Effect of Petroleum Hydrocarbons Concentration on Rhizoremediation and CH4 Emission

Yun-Yeong Lee, Yoonjoo Seo, Minyoung Ha, Jiho Lee, Hyoju Yang, and Kyung-Suk Cho* Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Rhizoremediation is a sustainable approach for petroleum hydrocarbons(PHs)-contaminated soils using the synergism between plants and microorganisms in rhizosphere. This study investigated the effects of initial PHs concentrations on rhizoremediation performance, CH4emission, and bacterial community structures in soil planted with tall fescue (Festuca arundinacea). The soils were artificially contaminatedwith diesel at initial concentrations of 5,000 (T5), 10,000 (T10), 30,000(T30), and 50,000 (T50) mg-TPHs·kg-soil-1. Five kg of soil was put in a pot, and 10-12 tall fescues seedlings per a pot were planted. The potexperiments were conducted for 85 days. PHs concentrations did not decrease up to day 22, but significantly declined on day 37. On day 85,the PHs removability was 49.8% (T5), 67.2% (T10), 43.6% (T30), and36.5% (T50). CH4 emission was affected by initial PHs concentration, in which CH4 emission was approximately 4 times higher in the highestinitial PHs concentration (T50) than that of the lowest one (T5). PHs removability exhibited a positive relationship with Rhizobium, Halothiobacillus, and Geobacter, while CH4 emission was positively associated with Stenotrophomonas, Acidicapsa, Gemmatirosa. The results in this study provided a comprehensive information for both rhizoremediation and CH4 emission in PHs-contaminated soils.

Keywords : Rhizoremediation, CH4 emission, petroleum hydrocarbon

[This study was supported by the National research Foundation of Korea (NRF) grant funded by the Korea government, the Ministry of Scienceand ICT (MSIT) (2019R1A2C2006701)]

C-21

Degradation of Polyethylene Terephthalate (PET) through Extracellular PETase in Corynebacterium glutamicum and Surface - Displayed PETase in Pichia pastoris

Chae-Eun Kim, Hyuk-Jin Kwon, and Hyune-Hwan Lee*

Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyeonggi-do 17035, Republic of Korea

Poly (ethylene terephthalate) (PET) is the rigidly structured polyester that is used in fabrics and storage materials. PET causes several naturalproblems because chemical degradation of polyester plastic requires high-temperature, high-pressure. This process makes large amounts oftoxic substances that could destroy the environment. Therefore, it is necessary to find ways to decompose PET in an eco-friendly way. Meanwhile, PET hydrolase (PETase) expressed from Ideonella sakaiensishas been found to have a very high effect on PET decomposition, and this is thought to be the starting point for solving environmental problemscaused by plastic. In this study, we designed recombinant PETase from Corynebacterium glutamicum and Pichia pastoris, respectively. We used secretion signal to make PETase from C. glutamicum to move outof the cell wall in C. glutamicum, and the enzymatic activity of PETasewas verified. Also, we designed a whole-cell biocatalyst by displayingPETase on the surface of Pichia pastoris SMD1168. PETase was fusedto a TIP1 DNA(TIP630), encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae. The localizationof the surface-expressed PETase was confirmed by flow cytometric analysis and immunofluorescence confocal microscopy using FITC-labeledsecondary antibody. The degradation of PET to MHET, BHET, and TPAusing both recombinant strains was analyzed by HPLC. We report the results here.

Keywords : PETase, secretion signal, glycosylphosphatidylinositol (GPI)-anchored protein

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C-22

Optimization of Cultural Conditions for Rhodococcus sp. 3-2 for Biodegradation of Fungicide Benomyl and Carbendazim

Jun-Kyung Park1, Ho-Dong Lim1, Kong-Min Kim1, Jung-Hwan Ji1, In-Cheol Park2, Jae-Hyung Ahn2, and Gui Hwan Han1*

1Center for Industrialization of Agricultural and Livestock Microorganisms (CIALM), 241, Cheomdangwahag-ro, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea 2Bioremediation Team, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea

The development and increased utilization of chemical fertilizers for agricultural purposes are recognized all over the world. Despite their usefulness, to protect crops and livestock, they have potential risks to food safety, ecosystem and all the living things. Likewise, benomyl andcarbendazim that are considered, as a carcinogen is a major pollutant detectable in the environment. In this regard, Rhodococcus sp. was reported to reduce the harmful effects of benomyl and carbendazim. Therefore, in this work, the growth conditions of microorganisms suchas Rhodococcus species that have an efficiency of biodegrading of benzimidazole fungicides was evaluated. Initially, optimization of growth parameters such as carbon source, nitrogen source, mineral source, temperature, pH and agitation speed for Rhodococcus sp. weredetermined in lab scale. In addition, for the mass production and fieldapplication of Rhodococcus species for agriculture purposes, cost efficient production with higher efficacy using the economical carbon and nitrogen sources are warranted. Therefore, for enhanced growth ofRhodococcus sp., we have developed a defined media and optimizationwere performed for their growth types. Furthermore, the storage stabilityof Rhodococcus sp. in, liquid (L) and new types of formulation in powderform(P) was developed and optimized for their effect on fungicide at different temperature for at least 6 months. Based on the results, L andP were found to maintain their viable cell count at > 1.0 x 108 CFU/g atlow-temperature conditions. Firstly, growth optimization of Rhodococcussp. in 5L jar-fermenter with the optimized defined medium were carriedout and OD at 600nm, pH, viable cell count was determined. Secondly,industrial scale culturing with the optimized defined media (1.5 ton) were carried out, and viable cell counting the liquid samples and powder types.Further, the degradation of benomyl and carbendazim by liquid or powder form of Rhodococcus sp. in the field will be studied to increasethe crop yield without causing harm to ecosystem and approaching thesustainable agricultural system.

Keywords : Residual pesticide, biodegradation, microorganisms

C-23

Biological Upgrading of 3,6-anhydro-L-galactose from Red Seaweeds to a New Platform Chemical, 3,6-anhydro-L-galactitol for Producing Bioplastics

Jahyun Lee, Dong Hyun Kim, and Kyoung Heon Kim* Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Republic of Korea

Producing petrochemicals from renewable biomass instead of fossil fuels has received much attention in the manner of sustainable development. Recently, marine algae are gaining importance owing to their advantages over lignocellulosic feedstock. Particularly, red microalgae have higher carbohydrate contents and simpler composition among marine algae. In red macroalgal carbohydrates, 3,6-anhydro- L-galactose (AHG) is the main monomeric sugar composing agarose along with D-galactose. However, AHG is not a common sugar and ischemically unstable. Thus, not only monomeric AHG but also red macroalgal biomass itself cannot be efficiently converted or utilized. Tobreak through this problem, we biologically upgraded AHG to its sugar alcohol, 3,6-anhydro-L-galactitol (AHGol), an anhydrohexitol, a new platform chemical that much stable than AHG. For the upgrade of AHGto AHGol, agarose passes a chemical hydrolysis process, producing agarobiose (AB) and a biological process, subsequently, converting ABto AHGol using metabolically engineered Saccharomyces cerevisiae foreffective production of AHGol with high titers and yields. The producedAHGol was further converted to another platform chemical for plastics,isosorbide. To our knowledge, this is the first that demonstration of upgrading a red macroalgal biomass component to a platform chemicalvia a new biological route, by using an engineered microorganism.

Keywords : Red-microalgae, chemo-bio process, 3,6-anhydro-L- galactitol(AHGol)

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D_Cell Culture and Biomedical Engineering

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D-1

Evaluation of Biological Activity of Lactic Acid Bacteria and Bacillus Isolated from Fermented Food

MinJi Jang1, Ye-rin Kim1,2, Gun Jay2, Young-joong Kim2, Mi-sun Kwak1,and Moon-Hee Sung1,2* 1The Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea 2Kookminbio Corporation, Seoul 02826, Republic of Korea

Kimchi is a traditional fermented food in Korea and is known to containvarious physiological activities such as carotene, dietary fiber, indicatingvarious functions such as antioxidant, anticancer. We separated Lactobacillusand Bacillus from Kimchi. As a result, 89 Lactobacillus species and 101Bacillus species were isolated, and we conducted experiments with eightof these strains, six Lactobacillus plantarum and two Bacillus velezensis.Cytokine is a protein immunomodulator secreted from immune cells. Tumor Necrosis Factor alpha (TNF-α) is a cytokine produced in macrophagesthat induces infection, inhibits tumor production and viral replication, and induces apoptosis of tumor cells. Interferon beta (IFN-β) is producedin virus-infected cells and acts on the cells themselves and surroundingthem to increase resistance to viral infection. We measured the TNF-α,IFN-β secretion of selected Lactobacillus and Bacillus with RAW264.7,a mouse-derived macrophage cell line. So, we found Lactobacillus andBacillus, which have the best secretion function. We hope that they canbe applied as functional biomaterials in the future.

Keywords : Lactobacillus, Bacillus, cytokine

[This research was supported by Korea Environmental Industry and Technology Institute (KEITI) grant funded by the Ministry of Environmentof Korea]

D-2

Enterotoxin Express to Attenuated Bacteria Cancer Targeting and Therapy in Mouse Tumor Model

Jam-Eon Park, Seung-Hyeon Choi, Ji Yung Choi, and Seung-Hwan Park*

Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Jeonbuk-do, Republic of Korea

Bacteria-mediated cancer therapy was capable of actively targeting andefficiently removing solid tumor. Also, tumor targeting bacteria could be used as mediators that can specifically deliver anticancer agents to tumors. To utilize these functions into bacteria, we engineered attenuatedSalmonella typhimurium, which express to Clostridium perfringensenterotoxin (CPE). CPE is known as cytolysin, which specifically bindsto claudin (CLDN)-4. CPE gene expression was regulated by pBAD promotor. We confirmed the expression and secretion of CPE induced by L-arabinose at engineered bacteria. In vitro, CLDN-4 positive breastcancer cell lines (BCCs) were treated with CPE to confirm binding toCDLN-4 and cytolysis of CPE. Consequently, CPE was co-localized with CLDN-4 and bring cell death of BCCs. In vivo, engineered bacteriaadministered to murine/human breast tumor-bearing mice intravenously,was successfully localized to tumor tissue and gene expression was induced by L-arabinose. The engineered bacteria significantly suppressedtumors. CLDN-4 expression level in the CPE-expressed bacteria group was down-regulated compared bacteria groups without L-arabinose. Therefore, engineered bacteria that carry a CPE gene for targeted CLDN-4 positive cancer therapy can be designed as a theranostic agent.

Keywords : Clostridium perfringens enterotoxin, Claudin-4 positive breast cancer, Salmonella typhimurium

D-3

Nanoperforator: A New Way to Defeat Enveloped Viruses

Younghun Jung1, Hyunseok Oh2, and Dae-Hyuk Kweon1,2* 1Institute of Biomolecular Control, Sungkyunkwan University, Suwon 16419, Republic of Korea 2Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea

Lipid-bilayer nanodiscs encircled by membrane scaffold proteins (MSPs) have been used to study membrane proteins of interest in a physiological environment. Here, we suggest novel nanodisc-based approach to deal with influenza virus infection. Mechanistically, nanodiscs carrying gangliosides bind to influenza virions and are co-endocytosed into host cells. At low pH in the endosome, the nanodiscsrupture the viral envelope, trapping viral RNAs inside the endolysosomefor enzymatic decomposition. To improve stability and antiviral effect,we further established dimeric or large nanodisc with ganglioside receptors whose belt proteins were circularized. Through increased cooperativity of dimeric nanodisc or enlarged area of large nanodisc, theyexhibited enhanced antiviral potency in vitro. We showed that ease ofperforation involved in viral inhibition by analyzing membrane fusionand perforation between nanodiscs and viral envelope. Circularized dimeric nanodiscs were more thermally stable than conventional nanodiscs. In addition, PEGylation on cysteine residue of large nanodiscenhanced thermal stability and proteolytic resistance. With improvementsin antiviral efficacy and stability, we expect in vivo potential of antiviralnanodiscs. Our results suggest a new class of antiviral that induces irreversible physical damage of enveloped viruses and its structural variations to improve in vivo efficacy. In conclusion, the lipid nanostructure provides new dimension for antiviral activity of decoy molecules.

Keywords : Nanodisc, enveloped virus, perforation

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D-4

Development of Dual-Functional Aptamer That Inhibit the Pancreatic Cancer Cell Growth

Woo-Ri Shin1, Hee-Young Cho1, Sang Yong Kim2, Ji-Hyang Wee2, Ji-Young Ahn1, and Yang Hoon Kim1*

1Major in Microbiology School of Biological Sciences College of Natural Sciences Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, Chungbuk 28644, Republic of Korea 2Department of Food Science and Biotechnology, Shin Ansan University, 135 Sinansandaehak-Ro, Danwon-Gu, Ansan 15435, Republic of Korea

Pancreatic cancer is the 4th leading cause of cancer death in the world.It is hard to diagnose early and is not easily treated once it spread to otherorgans. Thus, novel therapeutic strategies are urgently needed. Here wedeveloped an RNA aptamer against CTHRC1 and ALPPL2 protein forthe prevention of pancreatic cancer. The RNA aptamer that has been treated with pancreatic cancer, will be targeted ALPPL2 expressed onthe surface of pancreatic cancer cell and downregulated the CTHRC1 function by blocking the Smad signal for cell growth. We selected the RNA aptamer against CTHRC1 and ALPPL2 protein using in vitro transcription SELEX. Then, we analyzed the binding structure of the protein-RNA aptamer complex, which is confirmed the RNA aptamer bind to the active site of the protein using the MOE program. RNA aptamers of each binding to CTHRC1 and ALPPL2 were conjugated toproduce an aptamer complex with two functions that specifically bindto the surface and inhibit the growth of pancreatic cancer cells. This dual-functional aptamer complex had a high affinity to each target protein active site.

Keywords : Pancreatic cancer, aptamer, SELEX

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2019R1I1A1A01062365)][This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2020R1A6A1A06046235)]

D-5

Fabrication of pH-Responsive Nanoparticles Comprising Alginate and Chitosan with Bursting Release

Li Jiaoyang1, Hui Jin2*, and Shin Sik Choi 1,3*

1Department of Energy Science and Technology, Myongji University, Yongin 17058, Republic of Korea 2Korea Innovative Medicines Consorsium Seoul 06666, Republic of Korea 3Department of Food and Nutrition, Myongji University, Yongin 17058, Republic of Korea, Department of Energy Science and Technology, Myongji University, Yongin 17058, Republic of Korea

Drug delivery system using pH-responsive nanoparticles has been extensively explored due to the controlled release in the gastrointestinaltract with minimizing side effects. Herein, we fabricated nanosized vehicles comprising alginate and chitosan through the electrostatic complexation and investigated their pH-responsive bursting release ofcargo protein. The size and absolute value of potential of nanoparticlesat pH 2.0 were significantly higher than those at pH 7.4 indicating thatthe particles were aggregated at pH 2.0 while collapsed at pH 7.4. BSA,a model cargo protein was encapsulated with high efficiency ratio (about100 μg/mL) and dramatically released at pH 7.4 (about 80%). However,the cargo protein was rarely released in the acidic (pH 2.0) condition resulting in only 1% of release ratio for 2 h. Results demonstrate that ourpH-responsive nanoparticle system has a potential for the oral- administered drugs or vaccines.

Keywords : Nanoparticle, bovine serum albumin (BSA), bursting release

D-6

Diagnosis of Lung Cancer Using Odorant Markers and Caenorhabditis elegans

Nari Jang1, Arvie Camille V. de Guzman2*, and Shin Sik Choi1,2* 1Department of Food and Nutrition, Myongji University, Yongin 17058, Republic of Korea 2Department of Energy Science and Technology, Myongji University, Yongin 17058, Republic of Korea

Early diagnosis of cancer is important to reduce the mortality rate of patients and to increase the success rate of treatment. Along with the earlydiagnosis, an easy, economical, rapid and non-invasive diagnostic method is also required for reducing the number of severe cancer patients.Many researches have been recently studied to discover and identify anodorant molecule specifically from tumor tissues or cancer patients’ blood, saliva and exhaled breath. A previous study has suggested that 2-ethyl-1-hexanol might be a biomarker or one of the specific volatile organic compound (VOC) occurring in the lung cancer tissue and patient.In this study, we investigated a novel lung cancer diagnosis system usingC. elegans, a nematode which responded to cancer-specific odorant molecules with chemotaxis behaviors, attraction or avoidance. C. elegans strains showed either positive or negative preference dependingon the strain species and the sample concentrations suggesting that a specific neuronal regulator genes execute the olfactory preference andlocomtional behavior. Taken together, the C. elegans olfactory sensing of odorant molecules from cancer tissue has a potential for an easy, economical, rapid and non-invasive cancer diagnostic tool.

Keywords : C. elegans, diagnosis, biomarker

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D-7

Cultivation of Euglena gracilis Using Nnoodle and Rice Cake Wastes as Nutrient Sources for Paramylon Production

Shin Myung Kim, Jee Young Kim, Cho Rok Jin, and Yoon-E Choi*

Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Republic of Korea

Paramylon, a β-1,3-glucan, is a commercially valuable polysaccharideas a nutirtional and medical supplement derived from Euglena gracilis.In paramylon production, the cost of microalgal cultivation is one of theimportant considerations. In this study, we evaluated cost-effective complex mediums for heterotrophic growth of E. gracilis utilizing various nutrient residues. Noodle and rice cake wastes were selected asmajor carbon sources accounting for about 80% of the medium cost. Forthe composition of the medium, an industrial by-product, corn steep solid(CSS), was additionally supplied as a nitrogen source. In addition, enzymatic hydrolysis was performed using three types of enzymes: α-Amlyase (AE), Amyloglucosidase (AGE) and their mixture (AE+AGE) to utilize glucose as a carbon source for accumulation of paramylon. As a result, it was shown that biomass and paramylon productivities were enhanced by up to 38.7% and 40.1% in complex medium compared to the synthetic (modified Hutner's) medium. This indicates that food residues can be a good supplement to the medium foreconomical production of paramylon.

Keywords : Euglena gracilis, paramylon, enzymatic hydrolysis

D-8

The Combination Effects of Zinc and Adiponection supplementation on Porcine In Vitro Embryo Development

Muhammad Rosyid Ridlo1,3, Eui Hyun Kim1, and Geon A Kim2*

1Department of Theriogenology and Biotechnology, Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea 2Department of Biomedical Laboratory Science, School of Medicine, Eulji University 11759, Uijeongbu, Republic of Korea 3 Department of Bioresource Technology and Veterinary. Vocational College Universitas Gadjah Mada, Yogyakarta, Indonesia

Zinc (Zn) is an important factor during oocytes in vitro maturation andmany other physiological function. In addition, Adiponetine (ADQ) improved porcine somatic cell nuclear transfer embryo development during in vitro culture (IVC) in our previous study. The purpose of thisstudy is to investigate the effect of combination treatment during IVM and IVC on porcine oocytes. Cleavage rate, blastocyst rate, and total cellnumber after parthenogenetically activation (PA) by electrical stimulationwere studied. Zinc concentration added during IVM is 1 μg/ml, ADQconcentration supplemented during IVC after PA is 15 μg/ml, and the combination treatment was supplementation of Zn 1 μg/ml during IVMand ADQ 15 μg/ml during IVC respectively. Cleavage rate showed thatZn-ADQ (85.87 ± 0.65) resulted highest rate compared to control (79.1 ± 0.32) and Zn treatment (86.23 ± 0.34) (p<0.05). Moreover, the cleavagerate revealed Zn treatment significantly higher than control. Blastocystrate showed significant different between all group treatment control (19.56 ± 0.42), Zn (25.32 ± 0.37), and Zn-ADQ (29.94 ± 0.56) (p <0.05).The Zn-ADQ treatment revealed highest blastocyst rate than other groups. Total cell number counting revealed that Zn-ADQ (71.22 ± 1.18)and Zn (68.91 ± 1.22) were higher than control (56 ± 0.97) (p <0.05), while Zn-ADQ and Zn treatment showed no significant different. We concluded that combination treatment during IVC and IVM utilizing Znand ADQ has the best result among Control and Zn treatment groups toembryo development in porcine.

Keywords : Zinc, adiponectin, pig embryo

[This study was supported by the National Research Foundation (#2018R1D1A1B07048765), Cooperative Research Program for Agriculture Science and Technology Development (#PJ014990022021) and Eulji University in 2020]

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D-9

Application of Polyethylenimine-Chitosan Composite Fiber to Induce Astaxanthin Accumulation in Haematococcus pluvialis

JaeBeen Lee1, Yun Hwan Park1, Sok Kim1,2, and Yoon-E Choi1* 1Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Republic of Korea 2OJeong Eco-Resilience Institute, Korea University, Seoul 02841, Republic of Korea

The ability of Haematococcus pluvialis, a unicellular green microalga,to accumulate a high amount of astaxanthin has gained attention. Variousstress-inducing factors were used to increase astaxanthin production inH. pluvialis cultures. Addition of a synthetic cationic polymer, polyethyleneimine (PEI), was recently suggested as a new way to increase astaxanthin accumulation. However, there is a disadvantage that the injection of the solution for stress stimulation cannot be recovered due to one-time use, and the stimulation is reduced by concentration dilution. In this study, we investigated the effects of reusable and recoverable PCF (PEI-Chitosan fiber) treatment on H. pluvialis. According to findings, PCF can cause astaxanthin accumulation.When comparing coating and composite methods for fabricating the PCFs, it was discovered that composite induced astaxanthin accumulation,while coating had little effect. PEI, chitosan, and cross-linker conditionswere investigated in order to maximize PCF effect. Optimal PEI concentration was observed at 0.1M. In the case of chitosan, 2% small molecular weight chitosan was used for its role as a backbone and stability, and 0.1mL/L of Epichlorohydrin was used as a crosslinking agent. Content of astaxanthin was 324 ± 12 pg per cell, which was 4.4 times higher than untreated group. PCF should serve as a novel approachto enhance astaxanthin production.

Keywords : Haematococcus pluvialis, polyethylenimine, astaxanthin

D-10

Targeted Delivery of Immunogenic Cell Death Inducer and IDO1 siRNA via Anti-CD44/PD-L1 Aptamer-Conjugated Liposome

Minhee Kim, Jongsam Lee, and Dong-Eun Kim*

Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05902, Republic of Korea

Cancer cells have various immune evasion mechanisms to induce tolerance against immune cells by turning the tumor microenvironment(TME), such as overexpression of programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase-1 (IDO1). To reverse and enhance the immune response in tumor site, we designed targeted co-delivery system of IDO1 siRNA and doxorubicin (DOX, immunogenic cell death inducer) using dual-aptamer conjugated liposome (aptamosome). Aptamosome was conjugated with dual DNA aptamers such as CD44 and antagonistic PD-L1 DNA aptamers to specifically deliver the drugs into targeted cancer cells as well as to inhibitPD-1/PD-L1 interaction between cancer cell and T-cell. We demonstrated that aptamosomes loaded with DOX and IDO1 siRNA were readily delivered into the cancer cells through an aptamer-mediatedendocytic manner. Moreover, the aptamosome was shown to increase immunogenic cell death and suppress the expression of IDO1 in target cancer cells with a higher efficiency as compared to the plain liposome.We suggest that combinatory treatment of DOX and IDO1 siRNA with dual-aptamer conjugated liposome can exhibit synergistic anticancer effects by reversing immune evasive cancer cells into cancer cells withimmune-favorable TME.

Keywords : Targeted delivery, liposome, aptamer

D-11

The Effect of Korean Dendropanax morbifera Extracts on the Alzheimer's Condition Cells

Kwang-Hwan Jhee1, Yeong-Jun Jeon1, Se-Ho Park1,2, Hyeon-Bin Son1, Won-Bin Bae1, and Seun-Ah Yang3 1Department of Applied Chemistry, Kumoh National Institute of Technology Gumi 39177, Republic of Korea 2Institute of Natural Science, Keimyung University, Daegu 42601, Republic of Korea 3Department of Food Science and Technology, Keimyung University, Daegu 42601, Republic of Korea

Recent research suggests that the "cerebral lymphatic system", called the "glymphatic system", is responsible for the removal of extracellular waste proteins from nerve cells through perivascular pathways. And Aquaporin-4 channel protein (AQP4) is an integral part of this systemand has been implicated in neuropathology such as Alzheimer's disease.As a result, it has been reported that the elimination of the Alzheimer- related proteins, amyloid β and tau are reduced according to the glymphatic system disturbances due to AQP4 deficiency. Dendropanaxmorbiferus is known to improve brain cognition by inhibiting AChE. However, there is no study between the relationship between the AQP4activity and Dendropanax morbiferus. We tried to determine whether Dendropanax morbiferus leaf and branches extract increases AQP4 andbrain-derived neurotrophic factor (BDNF), which is known as a cell aging marker, glial fibrillary acidic protein (GFAP) and low density lipoprotein receptor-related protein 1 (LRP1) or not. When 500 ug/ml of Dendropanax morbiferus extract was added, the protein expression levels of BDNF and GFAB were increased 20% and 17%, respectivelyand LRP1 and AQP4 were slightly increased. Our data suggest that Dendropanax morbiferus extract is helpful in suppressing Alzheimer's.

Keywords : Alzheimer, Dendropanax morbifera, AQP4

D-12

The Effect of the Red Grape Extracts on the Alzheimer's Condition Cells

Kwang-Hwan Jhee1, Yeong-Jun Jeon1, Se-Ho Park1,2, Hyeon-Bin Son1, Won-Bin Bae1, and Seun-Ah Yang3 1Department of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea 2Institute of Natural Science, Keimyung University, Daegu 42601, Republic of Korea 3Department of Food Science and Technology, Keimyung University, Daegu 42601, Republic of Korea

Glymphatic system is responsible for the removal of extracellular wasteproteins from nerve cells through perivascular pathways. And Aquaporin-4 channel protein (AQP4) is an integral part of this system.It has been reported that the elimination of the Alzheimer-associated protein amyloid β is reduced by glymphatic system disturbances due toAQP4 deficiency. When the natural extract activates AQP4, it may beexpected to suppress Alzheimer's. We tried to find the effect of red grapeextracts, which are known to be good for brain cognitive function, on the Alzheimer's condition cells. We investigated the protein expressionlevels of aging marker protein BDNF (brain-derived neurotrophic factor), GFAP (glial fibrillary acidic protein) and the beta amyloid protein scavenging protein LRP1 (low-density lipoprotein related protein 1) by western blot. When 100~500 ug/ml red grape extracts wereadded, BDNF and GFAP increased 10% and 23-38%, respectively. AndLRP1 and AQP4 were slightly increased compared to control. Our datasuggest that red grapes extracts can be a natural food to help suppress the Alzheimer's disease.

Keywords : Aquaporin-4, Alzheimer's condition cells, glymphatic system

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D-13

Expression of IGF1 and IGF1R in the Female Reproductive Organs in Dogs

Eun Pyo Kim1, Geon A Kim3*, and Jae ho Shin2 1Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea 2Department of Clinical Pathology, University of Health Science, Eulji University, Seonganam 13135, Republic of Korea 3Department of Clinical Pathology, University of Health Science, Eulji University, Uijeongbu 11759, Republic of Korea

Insulin-like growth factor 1(IGF1) and IGF1R are implicated in the regulation of female reproductive function of granulosa and cell proliferation, but in dogs, our knowledge is limited to the possible roleof the IGF1 system in ovary. Furthermore, the expression of IGF1 andits receptor (IGF1R) has not yet been described in dogs. In this study, we aimed to describe the protein localization of IGF1 and IGF1R in thefemale reproductive organs including ovary, oviduct and uterus of dogs.Female reproductive organs from three female dogs were collected byroutine ovariohysterectomy. Cortex of ovary and uterine horn of bifurcation were cut into 0.5 cm3 and fixed into 10% formalin. Oviductswere isolated from fat tissue with forceps and could be put into 10% neutral formalin. Detection of IGF1 and IGF1R proteins in ovarian follicle, oviduct and uterus was carried out with an indirect immunoperoxidasemethod using rabbit polyclonal antibodies. The IGF1 expression of follicular walls of ovaries were identified in all tested dogs. In oviduct,luminal epithelial cells and cilia showed positive signals and uterus alsoshowed similar expressions for IGF1 and IGF1R. In conclusion, IGF1and IGF1R may have a role in ovary as well as oviduct and uterus of dog.

Keywords : IGF1 and receptors, dog, female reproductive organ

[This research was funded by the Center for Companion Animal Research (CCAR), Rural Development Administration, Republic of Korea, grant number PJ014990022021]

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E_Cellular Nutrition in Health and Disease

E-1

Autophagy Down-Regulates NLRP3-Dependent Inflammatory Response of Intestinal Epithelial Cells

Yewon Yun and Dong-Eun Kim*

Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05902, Republic of Korea

Dysregulation of inflammation induced by noninfectious stress conditions,such as nutrient deprivation, causes tissue damage and intestinal permeability, resulting in the development of inflammatory bowel diseases. We investigated the effect of autophagy on cytokine secretionrelated to intestinal permeability under nutrient deprivation. Activationof NLRP3 inflammasomes, which were excessively activated in the short-term exposure to starvation, was diminished during the inductionof autophagic process in the long-term exposure to starvation. When autophagy was inhibited, starvation-induced NLRP3 inflammasomes and IL-1β production were significantly enhanced. A prolonged nutrientdeprivation resulted in an increased epithelial mesenchymal transition (EMT), leading to intestinal permeability. Under the nutrient deprivationcondition, IL-17E/25, which is secreted by IL-1β release, increased epithelial permeability through enhanced EMT. Thus, our results suggest that upregulation of autophagy maintains the intestinal barrier by suppressing the activation of NLRP3 inflammasomes and the releaseof their products, including proinflammatory cytokines IL-1β and IL-17E/25 under nutrient deprivation.

Keywords : Autophagy, NLRP3 inflammsome, nutrient deprivation

E-2

Regulation of Inflammation Using Autophagy in Intestinal Epithelium Cells under Inflammatory Condition

Boran Yoon and Dong-Eun Kim*

Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05902, Republic of Korea

NLRP3 inflammasome is a critical intracellular component of the innateimmune system composed of the NLRP3 protein, procaspase-1, and ASC. It engages in activation of caspase-1 and secretion of IL-1β andIL-18 in response to broad range of stimuli from pathogen-associatedmolecular patterns (PAMPs) and danger-associated molecular patterns(DAMPs). We hypothesized that macroautophagy, a homeostatic mechanism that removes unnecessary or dysfunctional organelles through a lysosomal degradation pathway, downregulates NLRP3 activation in intestinal epithelial cells under inflammatory stimulating condition with PAMPs and DAMPs. We observed that induction of macroautophagy with Torin1 causes NLRP3 inflammasome components,NLRP3 and ASC to be degraded in intestinal epithelial cells under inflammatory condition. During the progression of autophagy, degradation of macroautophagy marker, p62 was coincidental with decrease in NLRP3 and ASC expression in the cells under inflammatorycondition exerted by LPS. Based on these observations, we currently investigate whether TRAF6 and MARCH7 are involved in ubiquitinationand subsequent recruitment of inflammasome components such as NLRP3 and ASC to autophagy receptor, p62 in intestinal epithelial cellsunder inflammatory condition.

Keywords : Autophagy, inflammation, NLRP3 inflammasome

E-3

Activation of AMPK-Induced Autophagy Attenuates Apoptotic Degeneration in Retinal Pigment Epithelial Cells

Yujin Park, and Dong-Eun Kim*

Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05902, Republic of Korea

Diabetic condition often causes nutrient deprivation and subsequent accumulation of oxidative stresses in retinal pigment epithelium (RPE),resulting in diabetic eye disease and age-related macular degeneration (AMD). During these stressful conditions, RPE is degenerated with accumulated dysfunctional protein aggregates. However, these proteinand damaged organelle aggregates are readily disposed by macroautophagyto maintain cellular protein homeostasis. Despite of autophagic proteostasis in RPE, cellular apoptosis occurs due to prolonged stress conditions. We investigated whether these apoptosis and autophagy aremediated by AMPK. We observed that AMPK acts as the main regulator of cell survival and death metabolism in RPE cells with its two primingphosphorylation steps. The first step occurring during the short-term (<12h) starvation is that AMPK activates autophagy, resulting in degradation of the apoptotic markers such as Noxa, Bmf, and p53. Thesecond step under prolonged starvation causes AMPK to induce apoptosisthat leads to RPE degeneration. Given that this AMPK-autophagy- apoptosis axis plays a crucial role in determining cell fate, we suggestthat apoptosis was accelerated in RPE cells under nutrient deprivationcondition when autophagy is inhibited or overactivated.

Keywords : Autophagy, AMPK, nutrient deprivation

E-4

Two-Way Choice Feeding Assays in Drosophila

Youngseok Lee1,2, Jiun Sang1, and Bhanu Shrestha1

1Department of Bio & Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea 2Interdisciplinary Program for Bio-Health Convergence, Kookmin University, Seoul 02707, Republic of Korea

Food preference is a fundamental behavior, which allows animals to choose nutritious foods, while rejecting toxic foods. In this protocol, wedescribe two-way choice feeding assays (binary food choice assay) andnew technique for ingestion assay (DrosoX) using Drosophila melanogaster.These are simple, sensitive, and reproducible methods that detect preference or aversive characteristics of the food. We expect that theseassays are sufficient to study mechanism behind molecular and cellular behavioral response towards any tastants.

Keywords : Protocols, fruit fly, feeding assay

[This work was supported by grants to Y. L. from the Basic Science Research Program of the National Research Foundation of Korea (NRF)funded by the Ministry of Education (NRF-2021R1A2C1007628) andKorea Environmental Industry and Technology Institute (KEITI) grantfunded by the Ministry of Environment of Korea]

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F_Fermentation and Food Technology

Poster S

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F-1

Comparison of Antioxidant, Antibacterial Efficacy and Bioactive Components of Chamaecyparis obtusa Leaf Extract and New Material CPE-1 through Bioconversion

Soo Ah Jeong, Chae Won Park, Da hee Kim, Seong Hyun Oh, and BeongOu Lim*

Department of Applied Biochemistry, College of Biomedical & Health Health Science, Konkuk University, Chungju 27478, Republic of Korea

Chamaecyparis obtusa leaves used in this study contain various components and are well known to be rich in phytoncide, phenolic compounds and flavonoids. The purpose of this study is to develop a novel CPE-1 material through biological conversion of C. obtusa leaves,and to compare the differences in antioxidant activity, antibacterial activity and components of C. obtusa leaf extract and CPE-1. C. obtusaleaves were extracted using 99% ETOH and CPE-1 was prepared througha 10-day fermentation process. The biological activation effect of the prepared two samples was confirmed and the antibacterial effect was investigated. As a result, both C. obtusa leaf extract and CPE-1 showedstrong antioxidant efficacy. Compared to the existing C. obtusa leaf extract, CPE-1 extract had higher flavonoid content, and DPPH scavenging activity and reducing power also showed higher antioxidantactivity. The antibacterial effect of C. obtusa leaf extract and CPE-1 wasfound in both Staphylococcus aureus and Bacillus, but the existing C. obtusa leaf extract showed higher antibacterial activity. Finally, the composition changes of the two samples were confirmed through component analysis of C. obtusa leaf extract and CPE-1. Previous studies have confirmed that gallic acid is the largest component in C. obtusa leafextract. Under the same conditions, it was confirmed that the gallic acidcontent of CPE-1 increased by about 40% compared to the existing extract content. In addition, we identified a new peak through bioconversion. In conclusion, in this study, we developed a new materialthat has a higher physiological activity than C. obtusa leaf extract throughbioconversion. In addition, both C. obtusa leaf extract and CPE-1 werecompared with strong antioxidant and antibacterial effects, and excellenteffects were confirmed. This is a result suggesting that CPE-1 can be usedas a functional food and cosmetic material.

Keywords : Chamaecyparis obtusa, bioconversion, biological activity

F-2

Optimization of Time for the Fermentation of Turmeric, Ganghwa mugwort, Glasswort, Annual Wormwood, and Omija Using Indigenous Strains

Jeong-Yeon On and Soo-Ki Kim*

Department of Animal Science and Technology, Konkuk University 120 Neungdong-ro Gwangjin-gu, Seoul 05029, Republic of Korea

This study was conducted to determine the optimum time for the fermentation of some medicinal plants using indigenous strains. A totalof five medicinal plants namely turmeric (Curcuma longa), Ganghwa mugwort (Artemisia princeps), glasswort (Salicornia europaea), annualwormwood (Artemisia annua), and omija (Schisandra chinensis) wereused. Indigenous microorganisms were isolated from these plants. Enterococcus faecium was used as a starter culture for the fermentationof turmeric, glasswort, and Ganghwa mugwort. Lactobacillus plantarum and Wickerhamomyces anomalus were used as starter cultures toward the fermentation of annual wormwood and omija, respectively. For fermentation, each of the plant powder at a final concentration of 5% (w/v) was added to their respective media (annualmugwort - 1/5 of de Man Rogosa Sharpe (MRS) medium; turmeric, Ganghwa mugwort, and glasswort - Bacillus minimal medium (BMM),omija - 1/5 of yeast malt (YM) medium)) and sterilized after adjustingthe pH to 7.0 ± 0.50. Then 1% (v/v) of the respective starter culture wasinoculated and fermentation was carried out at 37 ℃ (turmeric, annualwormwood, Ganghwa mugwort, and glasswort) and 30 ℃ (omija) undershaking conditions (100 rpm) for 48 h. In case of all the medicinal plants,the viable cell counts increased from their initial values (~ log 7 cfu/ml) in the range of log 8 - 9 cfu/ml. Concurrently, the pH values were alsodecreased to ~ 5.2 – 6.2. The results indicated that the optimum time for fermentation of all the medicinal plants was 16 h based on high viable cell count and low pH. The antioxidant compounds and antioxidant activities of the non-fermented and fermented plant extracts were also determined.

Keywords : Fermentation, medicinal plants, indigenous microorganisms

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F-3

Antioxidant Activity of Nypa Fruticans Wurmb Fermentation Extracts by Bacillus subtilis

Ji-Hye Choi1, Chan-Hwi Park1, Se-Ram Cho1, Sung-Gyu Lee1, Jin-Woo Hwang1, Ji-Eun Lee1, Sang-Moon Kang2, Yong-Seung Joun2, and Hyun Kang1* 1Department of Medical Laboratory Science, College of Health Science Dankook University, Cheonan-si, Chungnam 31116, Republic of Korea 2R&D center, ANPEP Inc., Republic of Korea

Nypa fruticans wurmb (NFW) means “bamboo shoot of sea” and is a plantthat lives tropical wetland of Southeast Asia. In this study, ethanol extracts of NFW fermented under various conditions by Bacillus subtiliswere examined antioxidant activity. All NFW fermentation ethanol extracts showed higher polyphenol contents than NFW ethanol extracts.Especially, polyphenol content of 3F was 81.64 mg/g and it is higher thanother fermentation NFW extracts. Also, NFW fermentation ethanol extracts tends to decrease RC50 value in radical scavenging activity andshowed higher activity than NFW ethanol extracts in FRAP assay. Then,We used HaCaT cell for examining cytotoxicity of fermentation NFW extracts. It was confirmed that the fermented NFW extract tends to be less toxic than the NFW extract at a concentration of 50 ㎍ / mL or less.All results suggest that fermentation NFW extracts seems to be a functional materials. Among the 6 extracts, 3F has strongest antioxidantactivity and is expected to be a new functional material.

Keywords : Nypa fruticans wurmb, antioxidant, fermentation

F-5

Characterization of Fermented Food-Derived GRAS Lactobacillus for Industrial Production

Doo Seob Choi1, Young Cheol Jung2, Sojeong Heo3, Do-Won Jeong3, Mi-Sun Kwak1, and Moon-Hee Sung1,2* 1Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea 2Kookminbio Corporation, Seoul 02826, Republic of Korea 3Department of Food and Nutrition, Dongduk Women’s University, Seoul 02748, Republic of Korea

Lactobacillus can be easily found in popular and common fermented foods such as kimchi, fermented milk, and dairy products. Lactobacillus,which has been ingested by humans for a long time along with fermentedfoods, is also proven to be safe. In addition to fermented foods, lactic acid bacteria are gaining popularity as a raw material for healthy functional foods that help human intestinal health. To separate lactic acidbacteria with different characteristics from general lactic acid bacteria,lactic acid bacteria that grow at low temperatures and decompose proteins were isolated using meat aged at low temperatures. Since it wasisolated from aged meat, it was necessary to confirm its safety, and bychecking the genome of the isolated lactic acid bacteria, it was confirmedthat it is suitable for GRAS (Generally Recognized as Safe). To check whether the separated lactic acid bacteria can be applied industrially, theculture scale was increased and finally cultivated in a 500L fermentor. The lactic acid bacteria powder obtained by recovering, freeze-drying,and pulverizing the lactic acid bacteria in a GMP facility is used as a rawmaterial for health functional foods. It can be used in probiotics and postbiotics. We have presented the possibility of industrial-scale as a result of these studies.

Keywords : Lactobacillus, fermentation, industrialization

[This research was supported by Korea Environmental Industry and Technology Institute(KEITI) grant funded by the Ministry of Environmentof Korea]

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F-6

Evaluation of Bacillus subtilis D2-7 for the Use of Functional Starter during Soybean Fermentation

Ye-Ji Jang, Myong-Hui Han, Woosoo Jeong, Hee-Min Gwon, Soo-HwanYeo, and So-Young Kim*

Department of Agrofood Resources, National Institute of Agricultural Science, RDA, Wanju 55365, Republic of Korea

In this study, we evaluated 17 out of the genera Bacillus isolated fromthe fermented soybean sauces in terms of antibiotic resistance assay, antibacterial, fibrinolytic activities, and taste sensing analysis. Of them,the Bacillus subtilis D2-7 isolate was selected to the antibiotic- sensitivestrain against 8 kinds of antibiotics (chloramphenicol, clindamycin, kanamycin, erythromycin, gentamicin, streptomycin, tetracycline, andvancomycin) according to the criteria of EFSA. The antibacterial activityagainst E. coli, S. typhimurium, and B. cereus was measured as inhibitionzone, which of sizes were 3.4, 2.8, and 0.8 mm, respectively. Moreover,the D2-7 isolate had the highest fibrinolytic activity as 230% comparedto the control (plasmin). By using the E-tongue analysis, the isolate D2-7belonged to the high level within 20% of the umami ranking. Consequently, the B. subtilis D2-7 could be widely expected to spread as functional starter during soybean fermentation.

Keywords : Bacillus subtilis D2-7, functional starter, soybean fermentation

F-7

Comparison of Enzyme Activities of Isolated Fungi from Commercial Nuruk

Sujeong Lee, Hee-Min Kwon, So-Young Kim, Soo-Hwan Yeo1, and Woosoo Jeong* Fermented and Processed Food Science Division, Department of Agrofood Resource, NIAS, RDA, Wanju 55365, Republic of Korea

Nuruk produced from wheat was collected in the various area, its characteristics were analyzed, and the enzyme activity of the fungi isolated from each Nuruk was analyzed and compared. The pH and amino acid were analyzed with general components of 5 kinds of Nuruk (HN,KJ, SU, JJ, SH). In addition, as a result of measuring the enzyme activityof each Nuruk, the HN Nuruk was measured to have the highest α-amylase activity 6,489.8 U/mg, glucose-forming activity 1,258.6 U/mg, and acidic carboxypeptidase activity 1,679.8 U/mg. Based on these results, the difference in the characteristics of each Nuruk was confirmed. Subsequently, the identification results of fungi isolated from Nurukwere presented, and the enzyme activity of respective fungi was measured. The fungi isolated from Nuruk were identification toAspergillus sp., Rhizopus sp., Lichtheimia sp., and Mucor sp., and variousmold could be excavated. The enzyme activity of the isolated fungi wasparticularly high in the α-amylase activity. Among them, all the fungi isolated from HN Nuruk were measured relatively high, and the highestwas 162.5 U/mg. From these results, it is expected that fungi with excellent enzyme activity will be selected and utilized to contribute to quality improvement during Nuruk production.

Keywords : Nuruk, fungi, enzyme activity

F-8

Latilactobacillus curvatus BYB3 Isolated from Kimchi Alleviates DSS-Induced Colitis in Mice through Inhibition IL-6 Production

Xing WangDivision of Animal Science, Chonnam National University, Gwangju 61186, Republic of Korea

In recent studies, it has been researched that probiotics have health-promoting effects such as intestine immune modulation. In this paper, the authors focus on the immunomodulatory properties of Latilactobacillus curvatus strain, former named L. curvatus, which wasisolated from kimchi. In a 14-day dextran sulfate sodium (DSS)-inducedcolitis mouse model, treatment with L. curvatus BYB3 significantly decreased the disease activity index score, colon length, and lost of weight in the mice. In addition, histological analyses showed that treatment with L. curvatus BYB3 protected the structural integrity of theintestinal epithelial layer and mucin-secreting goblet cells from DSS-induced damage, with only slight infiltration of immune cells. To evaluate the molecular mechanisms underlying L. curvatus BYB3- mediated IL-6 production, the in vivo anti-inflammatory effects of L. curvatus BYB3 were examined in a dextran sodium sulfate (DSS)- induced colitis mouse model. L. curvatus BYB3 induced significantlylower levels of IL-6 in with that induced by DSS. These results indicate that L. curvatus BYB3 may have health-promoting effects via immunemodulation, and may thus be applicable for therapy of various inflammatory diseases.

Keywords : Latilactobacillus curvatus, IL-6, probiotics

F-9

Kimchi-Derived Limosilactobacillus fermentum JNU532 Inhibits Melanogenesis in B16F10 Melanoma Cells

Ziyao Meng and Sejong Oh*

Division of Animal Science, Chonnam National University, Gwangju 61186, Republic of Korea

Melanin is a natural skin pigment produced by specialized cells called melanocytes, it is a major determinant of skin color and provides defenseagainst the harmful effects of ultraviolet radiation-induced skin damageto a certain extent. However, the abnormal accumulation of melanin maylead to the development of certain skin diseases. In this study, the antioxidant and antimelanogenic activities of the cell-free supernatant (CFS) of twenty probiotic candidates were evaluated. Based on the results of 60% 2,2-diphenyl-1-picrylhydrazyl scavenging activity, 21%2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) scavenging capacity, and a 50% ascorbic acid equivalent ferric reducing antioxidantpower value, six selected probiotic candidates were selected as the strain with the higher antioxidant potential. The antimelanogenic properties and noncytotoxic activity of selected probiotic candidates were elucidated using B16F10 cells. Following which, Limosilactobacillus fermentum JNU532 was selected. Tyrosinase activity was reduced by 16.7% and with >23.2% reduction in melanin content upon in CFS-treated B16F10 cells, In addition, the mechanism underlying the antimelanogenic activity of L. fermentum JNU532 was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR)and western blotting. Therefore, L. fermentum JNU532 may be considered a potentially useful, natural depigmentation agent.

Keywords : Melanin, Limosilactobacillus, antimelanogenic

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F-10

Effect of Fermentative Starter Cultures on the Dynamics of Bacterial and Viral Communities and Metabolites during Kimchi Fermentation

Mi-Ja Jung, Se Hee Lee, Tae Woong Whon, and Seong Woon Roh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

Lactic acid bacteria in kimchi are the major fermentative microorganismsderived from raw ingredients, and affect quality, safety, and nutritional and organoleptic properties of the final product. In our previous study, the key strains, Weissella koreensis CBA3615, Latilactobacillus sakeiCBA3614, and Leuconostoc gelidum CBA3613, were established as the raw ingredient-originated fermentative microbes, and their metabolic properties were also confirmed using gnotobiotic kimchi. In this study,in order to find out the role of these strains in the natural fermentationrather than in axenic environment, starter cultures were inoculated intokimchi with wild indigenous microbial community and the dynamics ofbacterial communities and metabolites were analyzed during the fermentation. In addition, since bacteriophages within the viral community in kimchi can directly affect fermentation by influencing bacterial function and composition, the diversity and composition of DNA viral communities were determined based on metagenomic approach and compared with those of the corresponding bacterial communities. Our results provide insights into the ecological role of LAB starters in kimchi fermentation and the potential impact of bacteriophages as modulators of bacterial communities associated withthe fermentation properties of kimchi.

Keywords : Kimchi, lactic acid bactera, multi-omics

F-11

Optimization of Production Time for Mass Cultivation Based on Kefir Grains

Young-Seon Kim, Hye-Young Youn, and Kun-Ho Seo*

Center for One Health, College of Veterinary Medicine, Konkuk University, 120, Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea

Kefir is a dairy product produced by kefir grains(KG) which contain lactic acid bacteria and yeast. The KG increase yield of fermentation products that affect beneficial ability of kefir. In the present study, weoptimized the production time of mass cultivation of KG using 1L of pasteurized milk. The KG were cultured under 24 (A group) and 48-hour(B group) interval conditions at room temperature, respectively. The pHof kefir and whey was evaluated by pH meter after the last experiment day. To evaluate pure kefir grains, A and B groups of kefir grains were lyophilized by using a freeze-dryer. Interestingly, kefir grains of B groupincreased from 44.3g to 165.05g (about 272.57 %), compared to A groupfrom 44.81g to 151.83g (238.83 %). The pH of kefir and whey is 3.84± 0.05 and 3.34 ± 0.02 in A group and 3.18 ± 0.03 and 3.30 ± 0.06 in Bgroup, respectively (p > 0.05). Weight of kefir grains decreased after freeze drying treatment from 50g to 6.53g ± 0.02g (about 13.06 %) in A group and to 6.67g ± 0.05g (13.33 %) in B group. It is noteworthy thatproduction rate of kefir grains was increased at 48-hour of cultivation time and no significant difference with pH between A and B group. Ourresults suggest that cultivation time is the critical factor of the optimization of kefir grain biomass.

Keywords : Kefir, Kefir grains, mass cultivation

F-12

Investigation of Growth, Functional and Safety Characteristics of Nuruk-Derived Fungi

Woosoo Jeong, Sujeong Lee, Hee-Min Kwon, So-Young Kim, and Soo-Hwan Yeo*

Fermented and Processed Food Science Division, Department of Agrofood Resource, NIAS, RDA, Wanju 55365, Republic of Korea

Fungi were isolated from Nuruk in each region and identified using ITSand 18s rRNA sequencing. The fermentation characteristics, functionality,and safety of 8 types of fungi were evaluated. Growth characteristics generally had a wide range, and optimal conditions were 28~30℃ and pH 5~7. Among 8 types of strains, the strains showing the highest enzymeactivity were Aspergillus oryzae SS1 (alpha-amylase 1.75 U/g, glucoamylase1392.2 U/g), and A. luchensis 74-5 (acidic protease 11.96 U/g) respectively. In the functional investigation, antihypertensive and antidiabetic activity were confirmed. The strain with the best antidiabeticactivity was Lichthemia ramosa 74-3, and the activity was 806% compared to the standard material. Antihypertensive activity was expressed as fibrinolysis activity and ACE inhibitory activity. Lich. ramosa 81-1 had the best antihypertensive activity. The safety of the fungus was confirmed by the presence of the toxin gene and antibiotic resistance. Aflatoxin genes aflR, ver1, and omtA were identified, and two aflatoxin genes (aflR, omtA) were detected in A. oryzae 59-5 and SS1. Antibiotic resistance, based on the Ministry of Food and Drug Safety, was generally resistant to antibiotics, and only four types of fungiwere not resistant to gentamicin antibiotics.

Keywords : Fungi, Nuruk, characteristics

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F-13

Characterization of Latilactobacillus curvatus MS2 Isolated from Korean Traditional Fermented Seafood and Cholesterol Reduction Effect as Synbiotics with Isomalto-oligosaccharide in BALB/c Mice

Su-Jeong Lee1, Chae Eun Kim2, SeYoung Oh3, Won Je Jang1,4, Mi-HyeonJeon1, Da-In Noh1, Young-Sun Lee1, Hee-Kyung Son1, Chan-Hee Kim5,Jong Min Lee4, and Eun-Woo Lee1*

1Biopharmaceutical Engineering Major, Division of Applied Bioengineering, Dong-Eui University, Basan 47340, Republic of Korea 2Cellnlife Inc., Seoul 08377, Republic of Korea 3Biospectrum Inc., Seoul 16827, Republic of Korea 4Department of Biotechnology, Pukyong National University, Busan 48513, Republic of Korea 5Department of Marine Bio-Materials and Aquaculture, Pukyong National University, Busan 48513, Republic of Korea

This study investigated the properties of Latilactobacillus curvatus MS2isolated from Korean traditional fermented seafood as probiotics and theeffect of reducing cholesterol as a synbiotic with isomalto-oligosaccharide(IMO) in BALB/c mice. The isolated strain showed high resistance to acids and bile acids and exhibited a high DPPH scavenging capacity of 72.27 ± 0.38%. In the intestinal adhesion test using HT-29 cells, the adhesion rate of MS2 was 17.10 ± 1.78%, which was higher than the adhesion rate of the other investigated probiotics. MS2 showed good antimicrobial activity against food-borne pathogens, especially Staphylococcusaureus, S. epidermidis, Escherichia coli, and Vibrio vulnificus. MS2 hasbeen identified as a safe microorganism because it does not produce β-glucuronidase and β-glucosidase, which are harmful to humans. This strain had high availability for IMO among the prebiotics of fructo-oligosaccharide, inulin and IMO. Oral administration of MS2 andIMO to BALB/c mice for 5 weeks resulted in a significant reduction in blood cholesterol levels by regulating liver lipid metabolism. These results suggest that the combination of MS2 and IMO has potential forapplication in functional foods.

Keywords : Synbiotics, Latilactobacillus curvatus, isomalto- oligosaccharide

F-14

Phylogenetic Diversity and Possibility as Probiotics in Bacteria from Shindari of Jeju Traditional Fermented Food

Youngsoo Ryu, and Moonsoo Heo*

Marine Applied Microbes and Aquatic Organism Disease Control Lab, Department of Marine Life Science, Jeju National University, Jeju 63243, Republic of Korea

Since olden times, barley was the typical crop in the soils of Jeju Islanddue to its topographical features. People in Jeju has eaten Shindari. Shindari is a fermented drink of Jeju which is made from leftovers of cooked barely and nuruk for short fermentation periods. Although Makgeolli and Shindari have a lot in common in the terms of its fermentation period and materials, research on Shindari is still in its early stage compared to the the study of Makgeolli. In this study, we examinedmajor bacterial species of Shindari. and we confirmed the antibacterialactivities of isolated strain against fish and human harmful bacteria. Finally, we studied whether Shindari is suitable as a probiotics. Amongthe isolates, the Pediococcus genus and Bacillus genus were the most predominant with 25%, followed by Cronobacter genus 25%, Enterococcusgenus 16%, Aneurinibacillus genus 5%, Klebsiella genus 4%, and Paenibacillus genus 2%. In the antibacterial activity results, growth inhibition was observed for all bacteria except fish disease bacteriumPhotobacterium damselae subsp. piscicida and human harmful bacterium Streptococcus mutans. Finally the potential result of as probiotics, It is expected that the Shindari can be used as probiotics by surviving a toxic and strong acid environment.

Keywords : Shindari, phylogenetic analysis, probiotics

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F-15

Probiotic Potential Comparison of Lactobacillus plantarum Isolated from Kimchi, Pickle, and Human with Standard Probiotic Strains

Ho Jae Lee1, Hyelyeon Hwang1, Chang Hee Jeong1, Hyejin Sohn2, Tae Woon Kim1, Sung Gu Han2, and Sung Wook Hong1* 1World Institute of Kimchi, Gwangju 61755, Republic of Korea 2Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 05029, Republic of Korea

Fermented foods are rich in beneficial probiotics and have been associated with a range of health benefits from strong digestive health to support immune system. In this study Lactobacillus plantarum WiKim0112, isolated from Korean radish water kimchi, was investigated for probiotic properties in vitro and compared with standardprobiotic strains of the species; L. plantarum KACC 11451 (origin pickle) and L. plantarum ATCC BAA-793 (origin human oral). Most ofstrains showed a high survival rate under gastrointestinal tract conditionssuch as artificial gastric juice (pH 2.5) and 0.3% bile salts, and heat treatment. Neutralized cell free culture supernatants of L. plantarumstrains were capable of inhibiting food-borne pathogenic bacteria including E. coli O157:H7, Listeria monocytogenes, and Staphylococcusaureus. Moreover, L. plantarum WiKim0112 showed approximately 40% of DPPH and 10% of ABTS radical scavenging activity. L. plantarum WiKim0112 significantly decreased pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and Most of L. plantarum strains increased anti-inflammatory cytokines (IL-4, IL-10, IFN-γ) gene expression in LPS-stimulated Caco-2 cells. Favorable probiotic properties of L. plantarum WiKim0112 along with antioxidant and pro-inflammatory activity imply its potential for clinical or technologicalapplications.

Keywords : Lactobacillus plantarum, probiotic properties, Kimchi

F-16

Optimization of Culture Conditions for Production of Natural Nitrite from Kimchi Cabbage and Radish Powder Using Staphylococcus hominis subsp. hominis WiKim0113

Hyelyeon Hwang, Ho Jae Lee, Tae Woon Kim, Chang Hee Jeong, and SungWook Hong* World institute of Kimchi, Gwangju 16755, Republic of Korea

Synthetic nitrite is considered an undesirable preservative for meat products; thus, controlling synthetic nitrite concentrations is importantfrom the standpoint of food safety. The aim of this study was to determinethe optimal medium and culture conditions for growth of Staphylococcushominis subsp. hominis WiKim0113 to produce natural nitrite. Kimchicabbage or radish powders could be used as potential substitutes of nitritedue to their high nitrate content. We used 16S rRNA gene sequence analysis to select a culture starter with excellent nitrite productivity, which we subsequently identified as S. hominis subsp. hominis WiKim0113. It exhibited a 45.5% reduction rate of nitrate to nitrite, withnitrate levels reduced to 25% in the medium containing 200 ppm sodiumnitrate. We found that contents of nitrite in the medium containing 5% kimchi cabbage powder and 5% radish powder were 51mg/L(3.0 fold increases, relative to control) and 15 mg/L(2.5 fold increases, relative to control), respectively, using S. hominis subsp. hominis WiKim0113.Inoculated fermentation can shorten the fermentation cycle and increasethe content of nitrite. These results demonstrated that fermented kimchicabbage and radish powders can be used as high potential nitrite replacerin meat industry.

Keywords : Kimchi cabbage, Staphylococcus hominis, nitrite

F-17

Real-Time PCR Assay for Identification of Lactobacillus plantarum Group Species by Comparative Genomic Analysis

Dayoung Kim, Eiseul Kim, and Hae-Yeong Kim*

Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea

The Lactobacillus plantarum group consists of four closely species or subspecies including L. pentosus, L. paraplantarum, L. plantarumsubsp. argentoratensis, and L. plantarum subsp. plantarum. These strains have a very high similarity between the 16S rRNA gene, resultingin a problem that can be misidentified. In this study, we searched for genesspecific to L. plantarum group through comparative genomic analysis and developed real-time PCR targeting specific genes. Genome analysisrevealed specific genes: GHKL domain-containing protein, (MFS)-typetransporter YcnB, and hypothetical protein. The developed real-time PCR was able to accurately detect genes specific to species or subspeciesfrom 41 different lactic acid bacterial strains. The standard curves for quantification were established using 105 to 109 CFU/mL of L. plantarumgroup species. This method was successfully applied to 32 probiotic products and could detect mislabeled products. Also, this method was able to qualitatively and quantitatively detect the strains of L. plantarumgroup in fermented food up to the subspecies level. Our method can beapplied to accurately determine the species or subspecies nomenclatureand to detect L. plantarum group species in various food and environmentsamples.

Keywords : Lactobacillus plantarum group, comparative genomics, real-time PCR

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F-18

Potential Characteristics of the Starter Lactic Acid Bacteria Isolated from Cheese Aging Cave in Imsil

Da Hye Song, Seok Geun Jeong, and Yu Jin Choi*

Imsil Cheese and Food Research Institute, 50, Doin 2-gil, Seongsu-myeon, Imsil, Jeollabuk-do 55918, Republic of Korea

The purpose of this study is to evaluate the LAB isolated from the firstcheese aging cave in Imsil, as a starter for dairy products with active ingredients for immunity and antioxidant activity. We isolated many LABs and identified to lactobacillus sp., lactococcus sp., and pediococcussp. by 16S rRNA analysis. And these LABs have been evaluated not only as a cheese making starter, but also for its antioxidant, cytotoxic and anti-inflammatory potential. The LABs were showed resistance to acidsand bile bovine. Milk protein degradation showed excellent activity inall strains. Compared to LGG, Lactococcus lactis subsp. lactis CCL20004,Pediococcus pentosaceus CCL20007 and Lactobacillus graminis CCL20017led to a significant increase in DPPH radical scavenging activity. Cytotoxicity were performed at raw 264.7 cell, and it was confirmed thatthere was no cytotoxicity. In the production of nitric oxide, Lactobacillusbrevis CCL20007 and Lactobacillus fuchuensis CCL20021 showed higher values than LGG. Taken together, Many LABs displayed antioxidant and antiinflammatory activity. Especially, Lactococcus lactis subsp. CCL20004 could be used as starter, and exhibited the mostbeneficial probiotic activities with antioxidant and antiinflammation properties.

Keywords : Lactic acid bacteria, probiotic, starter

F-19

Immune Activity of Plant Origin Lactic Acid Bacteria in RAW 264.7 Macrophages

Da Hye Song1, Neul I Ha2, Hee Gyeong Jeong2 and Yu Jin Choi1*

1Imsil Cheese & Food Research Institute, 50, Doin 2-gil, Seongsu-myeon, Imsil, Jeollabuk-do 55918, Republic of Korea 2Jangheung Research Institute for Mushroom Industry, Jangheung 59338, Republic of Korea

Probiotic bacteria can interact with the gut microbiome to strengthen theimmune system, enhance immune responses, and induce appropriate immune signaling pathways. Thus, the present study aimed to investigate the Immune effects of Lactobacillus acidophilus JMIL-001, Pediococcuspentosaceus JMIL-002, Lactobacillus fermentum JMIL-003 and Lactobacillus plantarum ICFPL-001 isolated from Kimchi using RAW264.7 cells treated with lipopolysaccharide (LPS). Our results showed that treatment with these lactic acid bacteria decreased nitric oxide andprostaglandin E2 production via downregulation of the inducible nitricoxide synthase and cyclooxygenase-2. In addition, treatment with theselactic acid bacteria suppressed the expression of pro-inflammatory cytokines, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Therefore, these data suggest that probiotic bacteria possesses immuneresponse potential and provide a molecular basis regarding the development of functional probiotic products.

Keywords : Lactic acid bacteria, immune, probiotic

[This study was carried out with the support of ́ R&D Program for Forest Science Technology (Project No. 2020198A00-2122-BA01)´ provided by Korea Forest Service(Korea Forestry Promotion Institute)]

F-20

Optimal Conditions for γ-Aminobutyric Acid (GABA) Production in the Seaweed-Based Medium by L. plantarum Y7

Min-Sun Kim, Jaegon Kim, Myong-Hyun Lee, Gyeong-Hwuii Kim, Geun-Ah Kim, and Sung-Sik Yoon*

Department of Biological Science and Technology, Yonsei University, Wonju 26493, Republic of Korea

Gamma-aminobutyric acid (GABA) is a non-protein amino acid that hasbeen reported to be involved in hypertension, irritability, insomnia, andstress. This multi-functional GABA is synthesized by glutamate decarboxylase(GAD) enzyme that converts the irreversible decarboxylationof glutamate to GABA. Earlier study claimed that Lactiplantibacillus plantarum Y7(L. plantarum Y7) was a higher GABA producer in the defined medium containing monosodium glutamate(MSG), which is controvercial food additive. It is well known that seaweed species givesavory taste due to glutamic acid, a precursor of GABA via fermentation.In search for natural glutamic acid substitute, its content was comparedamong the natural sources including seaweeds, resulting that sea tangle(Saccharina japonica) was chosen to extract the soluble glutamicacid. Using L. plantarum Y7 as a GABA producing strain, GABA production was examined in the medium containing monosodium glutamate (MSG) in this study. Effect of carbon and nitrogen sources were studied on GABA production by L. plantarum Y7 in the seaweed-based medium. Analysis of GABA produced was determinedusing both HPLC and GABase enzyme assay. Among the seven nitrogensources tested, highest amount of GABA was produced in the mediumabove with yeast extract 2% combined with tryptone 4% (74.16 ± 0.72μg/ml) and yeast extract 4% combined with tryptone 3% (88.29 ± 0.65μg/ml). It was noted that carbon sources had no significant effect on theGABA production. The results of present study suggest that sea tangle water extract is a promising natural glutamic acid substitute in GABAfermentation, not efficient as is MSG fermented in the seasoning industry.

Keywords : GABA fermentation, monosodium glutamate, Lactiplantibacillusplantarum

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F-21

Characterization and Optimization of GABA-Producing Fructophilic L. planatrum FBT215 from Kimchi

Jaegon Kim, Gyeong-Hwuii Kim, Min-Sun Kim, Myong-Hyun Lee, Geun-Ah Kim, and Sung-Sik Yoon*

Department of Biological Science and Technology, Yonsei University, Wonju 26493, Republic of Korea

Gamma-aminobutyric acid (GABA), the inhibitory neurotransmitter, intervenes in numerous physiological functions. The aim of this study is to screen GABA-producing lactic acid bacteria (LAB) and optimize GABA production in various conditions. Prescreening of potential GABA-producing LAB was performed on an acidified-MRS agar plate.The GABA amount was quantified by GABase enzymatic assay and high-performance liquid chromatography (HPLC). The effect of cultureconditions according to temperature, initial pH, time, carbon and nitrogen sources, L-monosodium glutamate (MSG), and vitamin B group was determined in a modified-MRS broth via one-factor-at-a-time(OFAT) strategy. The antibiotic resistance was confirmed by disk diffusion method. The complete genome sequence was analyzed. A totalof 100 LAB isolates were isolated from Korean fermented foods. Amongthese, Lactiplantibacillus plantarum (L. plantarum) FBT215 presented the highest GABA production, 104.02 μg/ml, and could tolerate acidic pH and bile salts. The LAB produced 4960.6 μg/ml GABA in modified-MRS broth (adjusted pH 7.5) containing 1% fructose, 3% tryptone, and 200 mM MSG, at 37℃ as a result of OFAT strategy. L. plantarum FBT215 was shown to have colistin resistance. The genomesize of the LAB was 3,150,001 bp, and 2,898 of 3,003 open reading frames were annotated. This study provides newly isolated L. plantarumFBT215 could be potentially a promising strain for the production of GABA itself in food industry.

Keywords : GABA, Lactiplantibacillus plantarum, optimization

F-22

Anti-Oxidant and Anti-Inflammatory Effects of Abelmoschus manihot L. Extracts Fermented with Bacillus licheniformis CP6 from Korea South Sea

Joo Young Yang1, Yu Jeong Yeom1, Hae Rang Lee1, Ga Eul Jeong1, Seong-Bo Kim2, Yong-Jik Lee3, Mi-Hwa Park4*, and Sang-Jae Lee1*

1Major in Food Biotechnology and Research Center for Extremophiles & Marine Microbiology, Silla University, Busan 46958, Republic of Korea 2Bio-Living Engineering Major, Global Leaders College, Yonsei University, Seoul 03722, Republic of Korea 3Department of Bio-Cosmetics, Seowon University, Chung-Ju 28674, Republic of Korea 4Depatrment of Food and Nutrition, Silla University, Busan 46958 Republic of Korea

In this study, we investigated the anti-oxidant and anti-inflammatory effects of fermented Abelmoschus manihot L. extracts in lipopolysaccharide(LPS)-stimulated RAW 264.7 mouse macrophages. The fermentation was performed using Bacillus sp. in mixture of Abelmoschus manihotL. at 37℃ for 1days (Named CP6-1D). DPPH radical scavenging activity of CP6-1D was increased in a 73% to 76%. The CP6-1D suppressed reactive oxygen species (ROS), nitric oxide (NO) production and the expression of iNOS and COX-2. Also, CP6-1D showed inhibitory effecton the production of pro-inflammatory cytokines such as interleukin-6(IL-6), interleukin-1β (IL-1β and tumor necrosis factor-α (TNF-α). Moreover, nuclear translocation of nuclear factor-κB (NF-κB) and phosphorylation of mitogen-activated protein kinases (MAP kinase) were strongly suppressed by CP6-1D in LPS-stimulated RAW 264.7 cells. The present results indicate that CP6-1D has an antioxidant and anti-inflammatory properties. Base on this results, it is expected to be able to provide basic information that can be used as a new natural functional materials in various fields such as functional foods, cosmetics,and pharmaceutical materials.

Keywords : Abelmoschus manihot L., Bacillus sp., fermentation

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F-23

Modulation of Fish-Gut Microbiota by LAB-Fermented H. illucensas Fish Feed's Substitutes

Hyunsol Jo, Sujeong Park, and Sunmee Hong*

Marine Industry Research Institute for East sea rim 22 Haeyanggwahak-gil, Jukbyeon-myeon,Uljin-gun, Gyeongsangbuk-do 36315, Repubilc of Korea

Probiotics, Lactic Acid Bacteria (LAB) are well-known as valuable functional food and/or feed to promote specific health benefits. And Hermetia illucens (Hi; black solder fly) is attracting attention as an alternative protein that supplies nutrients for fish, animal and pets etc. Additionally, Hi is rich in lipids and minerals, has higher protein contentthan other insects. This study was conducted bioconversion in Hi extract(HiE) using the lactic acid bacteria (LAB; L. lactis and L. plantarum)to produce fish meal substitutes. It was extracted in hot water at 60℃during 4 h as not to destroy the nutrient components of the Hi and then fermented by LAB during 24 h (HiLAB). We confirmed that HiLAB hadantibacterial activity against fish pathogen, antioxidant effect and production of γ-Aminobutyric acid (GABA). For feeding experiment on 600 juvenile Marbled Flounder (Pleuronectes yokohamae; Py) during10 weeks, two type produced diet containing similar quantities of protein,lipid, carbohydrate and other minerals with 4.5 % LAB (experiment sample) and 4.5% soybean meal (control). In gut-microbiota of Py after10 weeks by NGS analysis, 11 LAB strain (99.9%) with L. lactis etc (96.88%) was identified in Py’s gut feeding HiLAB. We suggested thatfish feed additives of 4.5% HiLAB could be utilized to promote the growth and to improve of gut environment of other fish strain or pets aswell as Py.

Keywords : Fish feed, Hermetia illucens, lactic acid bacteria

[This work was suppoted by IPET through Agri-Bio industry Technology Development Project, funded by MAFRA (121047-2 and 119027-3)]

F-24

Anti-Cancer Effect of Kalopanaxsaponin B, Metabolite of Kalopanax pictus Extract by Intestinal Microflora in Mice

Eunha ChoRadioisotope Research Division, Quantum and Convergence Sciences, Korea Atomic Energy Research Institute, Daejeon 34057, Republic of Korea

The stem bark of Kalopanax pictus (family Araliaceae) has been usedin China as a medicament for the treatment of inflammation-related diseases since ancient times. In preliminary study, the extract of Kalopanaxpictus has been studied to have anti-carcinogenic, anti-inflammatory andanti-diabetic effects. In this research, we investigated the metabolism of kalopanax pictus in mice. When K. pictus extract was orally administered to mice, kalopanaxsaponin B was detected in the feces ofmice. Orally administered K. pictus extract was metabolited to kalopanaxsaponin B by intestinal microflora in mice. As a result of measuring cell-death ability of kalopanaxsaponin B in cancer cell A549,it was confirmed that the ability increased when used in combination withthe radioisotope Lu-177 that emits radiation. From these results, it seemsthat if the extract of K. pictus is taken together during radiation therapyfor anti-cancer, it is converted into active metabolites by intestinal microflora, thereby enhancing the efficacy.

Keywords : Kalopanax pictus, Kalopanaxsaponin B, metabolite

F-25

Anti-Inflammatory Effect of Dstylium racemosum Leaf Gall Extract Applying Biorenovation Techique

Da Som Kim1, Won-Jae Chi1*, SoonOk Kim1, Hyehyun Hong2, TaeJin Park2, and Seung-Young Kim2 1Microorganism Resources Division, National Institute of Biological Resources, Republic of Korea 2Department of Pharmaceutical Engineering and Biotechnology, Sun Moon University, Asan-si, Chungcheongnam-do 31460, Republic of Korea

Biorenovation is a method of modifying the structure of a broad range of substrates such as chemical and plant extract by microbial enzymes to improve biological efficacy compared with its reaction substrates. Bacillus sp. JD3-7 was isolated from traditional soy sauce of Jeju Islandand used for biorenovation in this study. in vitro anti-inflammatory activity of extract from Distylium racemosum leaves with galls applyingbiorenovation technique (DRB) was investigated in lipopolysaccharide(LPS)-stimulated RAW264.7 macrophages. During the entire experimentperiod, DRB treated with 100, 200, 400 μg/ml showed improved cell viability in RAW264.7 cells. and.in these concentrations, DRB is inhibited the production of NO and prostaglandin E2 (PGE2), interleukin-1β (IL-1β), interleukin-6 (IL-6) expression of inducible NOsynthase (iNOS), cyclooxygenase-2 (COX-2). Our study shows that DRB can control inflammation by effectively inhibiting the productionof various inflammatory mediators. Our study shows that DRB can control inflammation by effectively inhibiting the production of variousinflammatory mediators. It also suggests that the application of biorenovation could be useful in developing anti-inflammatory materials.

Keywords : Dstylium racemosum, all, anti-infllammation

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F-26

Zero Waste Concept in Biorefinery; Production of Bioethanol and Lignocellulose Nanofibers

Hye Jee Kang, Bo Som Lee, Mi Dan Kang, Ga Young Kim, Seung EunLee, and Young Hoon Jung*

School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

Utilization of all the produced by-products from each process becomemore important to make biorefinery industry succeed. However, most studies are focused on the production of biosugars and biofuels throughthe multi-complex unit processes including pretreatment, saccharification,and fermentation. In this study, we are suggesting the zero waste conceptsin biorefinery by production of both bioethanol and lignocellulose nanofibers (LCNF). After acidic pretreatment of rice husk at solids loadings of 15% w/v, 1% sulfuric acid, and 190℃ with the varying durations of 5, 20, and 60 min, the pretreated rice husk was enzymaticallyhydrolyzed with 20 FPU/g substrate at 50℃ for 48 h, showing the maximum saccharification yield of 32.23%. The liquor fraction from thehydrolyzed biomass slurry was fermented by using Saccharomyces cerevisiae and the solids fraction was mechanically treated for the production of LCNF. This leads to high amount of ethanol production (5.09 g/L). Also, It was confirmed that solid by-products could be utilized for LCNF with the sedimentation ratio of up to 0.54 for 24 h. As a resulft,this zero waste concept in biorefinery may contribute more to industrialization.

Keywords : Biomass, biorefinery, Lignocellulose nanofiber

F-27

Production and Identification the Bifidogenic Growth Stimulator, DHNA in Protaetia brevitarsis Extract

Su Jeong Park, HyunSol Jo*, and SunMee Hong*

Department Research and Development, MIRE, Uljin 36315, Republic of Korea

To increase the beneficial effects of Protaetia brevitarsis juice for humanhealth, DHNA and bifidogenic growth-stimulator(BGS) in Protaetia brevitarsis extract (PbE) was fermented by Lactobacillus plantarum (Lp) and Weissella paramesenteroides (Wp). DHNA concentration in PbE observed in aerobic culture with 1% starch was higher than the othercarbon source, dextrose, sucrose, maltose, etc. The fermented commercialPb juice (PbTea), and fermented PbE, showed positive peaks for DHNAin TLC and HPLC analysis. Also, BGS and antioxidant activity of the fermented PbTea with 1% starch was higher than fermented PbE and PbTea in which was not fermented. Conversion of DHNA to the membrane-bound demethylmenaquinone is an important reaction in a precursor of menaquinone (MK; VitK2) biosynthesis. And, we cloned Men genes in Lp and Wp which enconding this enzyme and produced these enzyme protein in BmN cells by baculo system. These Men enzymes were expected to promote production of DHNA in fermentedPbE and PbTea. In addition, DHNA-enriched PbE and PbTea could bedeveloped as feed or food with regulating agent its microbiota.

Keywords : DHNA, menaquinones, Weissella sp.,

[This work was supported by IPET through Agri-Bio industry Technology Development Project, funded by MAFRA (121047-2 and 119027-3)]

F-28

Strategies for the Economic Production of Natural Edible Black Pigment Using Aspergillus niger

Yunsun Jeong, and Hakdong Shin*

Department of Food Science and Biotechnology, Sejong University, Seoul 05006, Republic of Korea

Most of the natural edible black pigments are produced from squid ink,which has disadvantages such as high prices and unstable supply. As analternative, we have utilized Aspergillus niger to secure an economicallyproducing method of black pigments usable in the food industry. A. nigerDBD-0406 was selected and optimized culture conditions (pH 7.0, 37℃,and YM medium with 1% of dextrose content) for optimal back spore formation. Also, an economic culture technique based on soybean by-products was established. The melanin synthesis pathway of A. nigerwas determined using inhibitors. It was shown that the agar containingtricyclazole and phthalide, which suppressed reductase of DHN melaninpathway, inhibited the production of black spores. The entire genome information of the A. niger DBD-0406 was obtained with a total genomelength was 36.9 Mb, 14 contigs, and 10,902 genes. The loci of six majorgenes related to the spore pigment were identified, and they were not clustered with each other. In addition, fecal samples from 24 healthy adults were collected and the effect of A. niger on the gut microbiota wasevaluated using in vitro fecal incubation model. There were no significant differences in bacterial species diversity, structure, and composition, suggesting the edible potential of this live strain. This studyprovides strategies for the economic production of alternative natural black pigment using black fungus based on genomic and microbiome analysis.

Keywords : Fungi, Aspergillus niger, pigment

F-29

Genome Analysis and Induction of Leuconostoc Prophage Derived from Kimchi

Songhee Kim and Jong-Hyun Park*

Department of Food Science and Biotechnology, Gachon University, Sungnam 13120, Republic of Korea

Bacteriophage of some dairy starter lactic acid bacteria (LAB) is presentin the state of prophage integrated into the bacterial chromosome and it can affect the fermentation for starter culture. In the kimchi fermentation, Leuconostoc has contributed as a principal starter, but studies on the phage are still unknown. In this study, prophage of kimchi-derived Leuconostoc stains was investigated and the inductionwas performed. Except for one strain, kimchi-derived Leuconostoc hadat least one prophage region with including questionable and incompleteones, which ranged from 0.6 to 6.2 genome %. The capsid proteins for 10 among 17 prophages were identified as belonging to the Siphoviridaefamily. The superinfection exclusion system and methylase suggested by the co-evolution of phage and bacteria were found in several prophage genes. Prophage induction was not confirmed in H2O2 and organic acids, which are environmental factors of kimchi. However, only induction with mitomycin C was able to confirm, but could not reinfection. The induced phage morphology was confirmed as Siphoviridae family. Therefore, it suggested that Leuconostoc had several prophage infections and the presence of some genes might be helpful to the bacteria and phage co-existence in kimchi microecosystem.

Keywords : Kimchi, Leuconostoc, prophage

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F-30

Evaluation of Probiotic Potential of Bacillus amyloliquefaciens NY12-2

Soohwi Yang, Da Hye Kim, Sun Young Kong, and Nam Soo Han*

Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea

Bacillus (Ba.) amyloliquefaciens is currently used for soybean fermentationproviding desirable organoleptic characteristics, improved food safety,and enrichment of nutrients. The aim of this study was to investigate theprobiotic properties of the strain such as tolerance to gastrointestinal juice, adherence capacity to Caco-2 cells, biogenic amine production, and hemolytic activity. As results, the viability of Ba. amyloliquefaciensNY12-2 was higher than Lacticaseibacillus rhamnosus GG (LGG) after180 min incubation in acid condition and similar to LGG in bile condition.Also, the strain was safe to be non-biogenic amine producer and non- hemolytic bacterium. Ba. amyloliquefaciens NY12-2 showed lower adhesion ability compared with LGG and Lactiplantibacillus plantarumWCFS1. In brief, Ba. amyloliquefaciens NY12-2 tested in this study hadtolerance on gastrointestinal juice and were safe for use in food, while its adherence level on Caco-2 cell was relatively low. Further studies suchas immune modulation and anti-oxidative activity are necessary to discover its health promoting activities.

Keywords : Bacillus, probiotics, safety property

F-31

Fermented Chrysanthemum indicum Linne on the Effects of Skin Barrier and Maintaining Moisture

YoungJi ChoiDepartment of Commercialization Support, Honam National Institute of Biological Resources, 99, Gohadoan-gil, Mokpo-si, Jeollanam-do 58762, Republic of Korea

Chrysanthemum indicum Linne(CI) has long been used in traditional medicine for the treatment of inflammatory diseases. In addition, it is usually known as an important medicinal and aromatic plant widely usedas a carminative, digestive, lactogogue, and diuretic, and for treating respiratory and gastrointestinal disorders. The skin barrier protects against the invasion of pathogens, fends off chemical and physical assaults, and protects against extensive water loss. In this study, the effects of the fermented CI (FCI) on strengthening the skin barrier and maintaining moisture, as well as their antifungal activity, were investigated in human keratinocyte (HaCaT) cells. The expression of loricrin, involucrin, filaggrin, hyaluronic acid synthase genes were measured by reverse transcription polymerase chain reaction (RT-PCR)and Real time PCR. These results suggest that the FCI could be very useful in cosmetics for the treatment of dermatological diseases.

Keywords : Chrysanthemum indicum, fermentation, maintaining moisture

F-32

Isolation of Electroactive Pseudomonas Strain Using Specified Media and Tungsten Nanoparticle for Enhanced Bioelectricity Generation in Microbial Fuel Cell

Himanshu Khandelwal, and Jung Rae Kim*

School of Chemical and Biomolecular Engineering, Pusan National University, Busan 46241, Republic of Korea

Pseudomonas sp. has been known to have diverse capability of feedstockconversion and bioremediation for recalcitrant organic contaminants, thus extensively studied for its metabolic pathway and recombination purposes. Recently, Pseudomonas sp. has been highlighted for production of valuable commodity, bioremediation, biosensor and wastewater treatment, therefore many Pseudomonas strains have beenscreened by various isolation strategies. Some Pseudomonas sp. showedan electrochemical activity which transfer their respiratory electron to carbon electrode with simultaneous electricity generation in a microbialfuel cell. Bacterial cells carry out direct electron transfer by forming biofilm and/or indirect transfer via electron shuttle to deliver respiratoryelectron. In this study, we isolated a novel Pseudomonas strain using bluewhite screening method using tungsten nanoparticle. The enrichment stage used a designed growth media to pose selective pressure for growthof Pseudomonas from mixed inoculum. Pseudomonas aeruginosa sp.(PBH03) was isolated and was tested for electrochemical and bioconversion activity (Poster)

Keywords : Electroactive bacteria, Tungsten nanoparticle, Pseudomonas

F-33

Assessment of CO2 Production Gene Expression in Saccharomyces cerevisiae by Real-Time PCR

Kang Uk Kim, Joo Hyun Lee, Seung Chul Yang, Hee Jung Lim, Hye Jin Kim, and Seung Wook Kim*

R&D Center, Ottogi Corporation, Anyang-si 14060, Republic of Korea

The carbon dioxide (CO2) gas is one of the most important factor in bakingand brewing process. It produces as a by-product when yeast performsaerobic respiration and alcoholic fermentation. Saccharomyces cerevisiaecauses CO2 production as the yeast feed on the glucose source. It is important to identify efficient methods for measuring CO2 gas production of S. cerevisiae because the rates of CO2 production in yeastdetermine the quality of fermented foods such as bread and beer. In aerobic respiration condition, CO2 production-related genes of S. cerevisiae are known PDC (Pyruvate decarboxylase), KGD (α-Ketoglutarate dehydrogenase, and IDH (Isocitrate dehydrogenase). Inthis study, 227 of S. cerevisiae strains were isolated and identified fromvarious fermentation foods in Korean traditional markets. After that 5 strains were selected which have high CO2 gas production by fermentation test, we identified real-time PCR experimental conditionsto assess the expression level of CO2 production-related genes (PDC, KFD, and IDH). Consequently, we consider that quantitative expressionlevel analysis of genes by real-time PCR would be helpful for the furtherresearch in S. cerevisiae.

Keywords : Saccharomyces cerevisiae, carbon dioxide, gene expression

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The Optimization of Bio-Capsule Formation to Improve the Fermentation Efficiency

Hyobin Moon1, Hye-Won Park1, Chan-Woo Kim1, Kyu-Taek Choi1, Jun-Su Choi1, and Heui-Dong Park1,2*

1Department of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 2Institute of Fermentation Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

In order to industrialize fermentation technologies, methods must be developed to increase the productivity of fermentation products and reduce production costs. The immobilization technology has the advantage of having the high cell density, improved yield, reduced riskof microbial contamination, ease to control processes, improved reproducibility, and possible reuse. The method of immobilization usingcarrageenan and alginate as a support has the disadvantage of requiringa slightly higher temperature, inhibiting cell enzyme activity and indicating cytotoxicity. On the other hand, the method in which two different microbes (mainly fungi and yeast) is naturally immobilized during incubation without external supports and chemical bonds have the advantage of reducing production costs by being used to shorten processes, such as simultaneous saccharification and fermentation. In this study, we aim to establish optimal conditions for the immobilizationmethod in which yeast is naturally adhered to the mycelium of filamentous fungus, and to find out the improvement of fermentation efficiency and reusability using bio-capsules, spherical mycelium attached with yeast.

Keywords : Optimization, bio-capsule, fermentation

F-35

Development of Immobilized Saccharomyces Yeast Starter on Apple Pomace for Cider Production

Hyewon Park1, Hyo-Bin Moon1, Chan-Woo Kim1, Kyu-Taek Choi1, Jun-Su Choi1, and Heui-Dong Park1,2*

1Department of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 2Institute of Fermentation Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

As the people's dietary standards improve, the intake of fruits has changed, consuming many processed foods. In the case of apple, a largeamount is consumed in the form of squeezed juice, and a significant amount of by-product is produced in the process of making juice. Thevast amount of apple is required to manufacture apple juice, cider, jam,and vinegar, generating large volumes of residue, known as apple pomace. Being perishable and highly biodegradable, the disposal of these wastes represents a serious environmental problem. Often only 20% is retrieved as animal feed and the rest 80% goes to landfill, is incinerated or is sent to composting sites which results in release of greenhouse gases. Therefore, alternative methods of utilization of apple pomace waste are needed. In this study, in order to improve the qualityof cider by using apple pomace waste as a yeast immobilization carrier. Freeze-drying technology was used to make it easier for yeast to be attached to apple pomace. After immobilization yeast on a frozen dry apple pomace, drying technologies (freeze-drying, air-blast drying) were examined to investigate optimal survival rate of yeast cells. This study proposed the potential of the use of apple pomace as a high-qualitycider.

Keywords : Immobilization, apple pomace, cider

F-36

Development of Air-Blast Drying Method for Improving Preservation of Yakju Yeast Starter

Chan-Woo Kim1, Hyo-Bin Moon1, Hye-Won Park1, Jun-Su Choi1, Kyu-Taek Choi1, and Heui-Dong Park1,2* 1Department of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 2Institute of Fermentation Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

In this study, we aimed to establish optimal preservative conditions ofair-blast drying to increase survival rate of Yakju yeast starter. Air-blastdrying is effective to improve the industrial availability of the strains. Among 512 yeasts (isolated from persimmon, apple, grape, aronia and nuruk) isolated from various fruits and nuruk, two Saccharomyces and2 non-Saccharomyces yeasts were selected by comparing low- temperature resistance, volatile ester compounds and sensory evaluation. Experiments were conducted alongside the optimization of protectant and rehydration conditions using different types of sugars and rehydration solutions to enhance the viability and storability of air-blastdried yeast cells. Six types of sugars (fructose, glucose, maltose, raffinose, sucrose, trehalose) and four types of rehydration solutions (distilled water, 1× phosphate buffered saline, 0.85% NaCl, and 1% peptone water) and three types of antioxidants (1, 3, 5 mM of L-ascorbicacid, glutathione, tocopherol) were used as an extra protectant to examinestarter survival rate immediately after air-blast drying and to assess anychanges in starter viability during storage. These studies contribute to the development of the domestic brewing industry technology and to improve the quality of Korean traditional liquor.

Keywords : Yeast starter, air-blast drying, Yakju

F-37

Eco-Friendly Oxalic Acid Production Using A. niger Strain

JiYi Kim1, Nari Jang1, Jiaoyang Li2, and Shin Sik Choi1,2* 1Department of Food and Nutrition, Myongji University, Yongin 17058, Republic of Korea 2Department of Energy Science and Technology, Myongji University, Yongin 17058, Republic of Korea

Oxalic acid is a useful material used in various fields such as medicine, pulp, and food. Oxalic acid is being produced by chemical synthesis; however, it has problems such as environmental pollution and hazardous manufacturing process. Therefore, it is necessary to manufacture oxalicacid using an eco-friendly and cost-effective microbial fermentation system. Aspergillus niger, a filamentous fungus has a metabolism that produces organic acids including oxalic acid and citric acid. To utilizemethanol, a byproduct of C1 gas, methane refinery, the methanol- resistant A. niger strain was used for the production of oxalic acid. In our previous study, the methanol-resistance/conversion A. niger strain cultivated in a semi-solid medium containing 5-15% methanol and corncob powder produced oxalic acid up to 123 g/kg. In this study, we confirmed that more than 2 g/L/ day oxalic acid was produced from methanol and xylose as two carbon sources through the optimized 2-stepculture process. Both alcohol dehydrogenase enzyme activity assay andgenome analysis concreted the biological conversion of methanol and xylose into the value-added organic acids. These results demonstrate thatthe biological process using the modified A. niger strain has a potentialfor the eco-friendly production of oxalic acid. Keywords : Aspergillus niger, methanol, oxalic acid

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Exploration of Probiotic Property of Lactobacillus plantarum FFC 248 Isolated from Korean Traditional Fermented Food

MyeongSeon Ryu, Ho Jin Jeong, Hee Gun Yang, Se Won Park, Hee-JongYang, and Do-Youn Jeong*

Microbial Institute for Fermentation Industry (MIFI), Sunchang 56048, Republic of Korea

Lactic acid bacteria (LAB) are a kind of bacteria that convert saccharidesto lactic acid and have been ingested in the various kinds of fermented foods like a kimchi, makgeolli, and yogurt. Lately, some species of LABare studied as potential probiotic supplements because of their physiological activity such as bile salt hydrolase (BSH) and antibacterialactivity against pathogenic bacteria. In this study, LAB were isolated from various kinds of Korean traditional fermented foods and it's probiotic properties were determined. All LAB isolates were tested in antibacterial activity, BSH activity, and DPPH scavenging activity. Celladhesion test of three LAB with the superior antibacterial and BSH activity was conducted by using HT-29 cells. FFC 248 showed higher BSH activity and cell adhesion ability on HT-29 cells than the others. Based on these results, we selected FFC 248 for further experiments. FFC248 strain is identified as Lactobacillus plantarum by 16S rRNA gene sequencing and API kit analysis which was named as Lactobacillus plantarum FFC 248. These results indicate potential ability of L. plantarum FFC 248 as a probiotics.

Keywords : Lactic acid bacteria, pobiotics

[This research was supported by Traditional Culture Convergence Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (NRF-2016M3C1B5907049)]

F-39

Optimization of Medium to Improve Protease Activity Uing Response Surface Methodology by Bacillus amyloliquefaciens SRCM115785

MyeongSeon Ryu, Hee Gun Yang, Gwang Su Ha, Se Won Park, Ho JinJeong, Hee Jong Yang, and Do Youn Jeong*

Microbial Institute for Fermentation Industry(MIFI), Sunchang 56048, Republic of Korea

The aim of this work was to establish the medium composition for enhancing production of protease using response surface methodology(RSM). Bacillus amyloliquefaciens SRCM115785 as protease producerwas finally selected by protease productivity analysis among the six candidates isolated from Korean traditional food(Makgeolli, Kimchi,Doenjang) and identified by 16S rRNA gene sequencing. Effect of eleven initial media components such as carbon (glucose, starch, sucrose) and nitrogen sources (yeast extract, peptone, malt extract, beefextract), mineral factors (K2HPO4, KH2PO4, CaCl2, (NH4)2SO4) for enhancing the protease production were assessed by Plackett-Burman experimental design. Glucose, yeast extract and beef extract were chosenas promising medium components for further optimization studies. Central composite design (CCD) experiment was used to establish the optimal concentrations of respective variables, which indicated that glucose 6.75 g/l, yeast extract 12.42 g/l and beef extract 17.48 g/l wereoptimal concentrations. Coefficient of determination (R2) of derived equation from the RSM regression for the protease production was mathematically reliable with 0.9997. At optimum parameters, 54.73 unit/ml of maximum protease increased by 206% when compared with LB medium used as unoptimized medium (26.53 unit/ml) and our statistical model was confirmed by subsequent validation experiments.High-performance of protease-producing microorganisms and establishment of effective protease fermentation process can be benefitfor industrial application.

Keywords : Bacillus amyloliquefaciens, protease activity, optimization

[This research was supported by Traditional Culture Convergence Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (NRF-2016M3C1B5907049)]

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F-40

Potential Probiotics Activity of Bacillus velenzensis SRCM 19473 Isolated from Traditional Fermented Soybean Products and Its Application

MyeongSeon Ryu, Ji Won Seo, Su-Ji Jeong, Hee-Jong Yang, and Do-Youn Jeong*

Microbial Institute for Fermentation Industry(MIFI), Sunchang 56048, Republic of Korea

Bacillus velezensis is known to have high enzyme activity such as protease, cellulase, lipase, and amylase, potential probiotic activity, highanti-bacteria activity. The FDA stated that non-toxigenic and non-pathogenic strains of B. velezensis are widely available and have been safely used in a variety of food applications. In this study, we isolatedBacillus-like bacteria from traditional fermented soybean pastes, and confirmed a probiotic properties including safety evaluation such as Bacillus cereus toxin gene and harmful molecules. Selected strains wereable to survive in acidic and bile conditions, and had high extra-cellularenzyme activities, broad-spectrum against pathogenic bacteria, and adherence to HT-29 cells. Final selected strain, SRCM119473 was identified as B. velezensis by 16S rRNA sequence analysis. We manufacturedthe Cheonggukjang with SRCM119473 and compared with type stain 168 about amino nitrogen and free amino acid contents as well as extra-cellular enzyme activities. These results suggest that the SRCM119473has a high potential property as probiotic resource for commercial application such as soybean fermented products and synbiotic materials.

Keywords : Bacillus velenzensis, probiotics, Cheonggukjang

[This research was supported by Traditional Culture Convergence Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (NRF-2016 M3C1B5907049)]

F-41

Characterization of Lactobacillus paracasei JSRL18-60 with Probiotic Properties as a Starter in Manufacturing Fermented Milk

MyeongSeon Ryu, Su-Ji Jeong, Hee-Jong Yang, Ji Won Seo, and Do-Youn Jeong*

Microbial Institute for Fermentation Industry(MIFI), Sunchang 56048, Republic of Korea

In this study, eighty-eight lactic acid bacteria (LAB) were isolated fromKorean traditional fermented foods for use as yogurt. Among the isolates,thirteen LAB strains with curd formation ability were selected. Among these, five LAB strains exhibited notable antibacterial activity against pathogens, such as Bacillus cereus, Staphylococcus aureus, and Listeriaivanovii. JSRL18-60 strain showed superior probiotic properties, suchas high adhesion capacity to Caco-2 colorectal adenocarcinoma cells, and tolerance to low pH, temperature, and condition of the bile. Basedon these results, JSRL18-60 strain was selected for further evaluation and identified by 16S rRNA sequencing as Lactobacillus paracasei. Themaximum growth of L. paracasei JSRL18-60 was determined after 30h of fermentation by measuring the optical density (1.74), viable cell number (9.61 log CFU/mL), and dried cell weight (2.30 g/L). Finally, we manufactured fermented milk using skimmed milk and L. paracaseiJSRL18-60, and the optimal pH condition (pH 4.48) of the fermented milk was reached within 21 h of fermentation. Moreover, the log of the total cell count of L. paracasei JSRL18-60 was retained for at least 10 days at 9.48-9.36 log CFU/mL. The pH and acidity levels of the fermented milk containing L. paracasei JSRL18-60 were changed frompH 4.48 and 0.89% to pH 4.38 and 0.98%, respectively, in 10 days. Thesefindings suggested that L. paracasei JSRL18-60 could be used as a starterfor producing fermented milk with probiotic properties.

Keywords : Lactobacillus paracasei, probiotic, yogurt

[This research was supported by Traditional Culture Convergence Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (NRF-2016 M3C1B5907049)]

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F-42

Metabolic Engineering and High Cell Density Culture of Escherichia coli to Enhance the Productivity and Improve Properties of Glycogen

MInsu Kim, Hyun-ah Yu*, and Jong-Tae Park*

Department of Food Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea

Glycogen has been known as an energy storage compound for living organisms, but its crucial roles in various biological processes were recently unveiled. In addition, glycogen possesses unique properties such as high water-solubility, slow-digestibility, and dendrimeric properties because of its unique branched structure. Thus, it can applyin various industries including food, cosmetics, and pharmaceuticals industries. In our previous studies, when maltose was used as the maincarbon source, the malP knocked-out Escherichia coli mutant accumulates over 20-fold polysaccharides than the parental strain. Intriguingly, long-unbranched maltodextrins were dominantly accumulatedin the ΔmalP mutant. Insufficient expression of glycogen branching enzyme, which encoded by glgB, could be the possible reason. Therefore,in this study, the promoter of glgB was replaced by the malPQ promoterof E. coli that is well-coordinating in E. coli maltose metabolism and stronger than the original. Additionally, Vibrio vulnificus glgB which exhibits greatly short-chain transferring activity replaced the original glgB of E. coli. The defined medium was used for the high cell densityculture, and maltose syrup was fed as the carbon source. Optimizing fermentation condition was failed due to the rapid acetate accumulationand cell lysis at the stationary phase. To reduce the glucose-1-phosphatepressure on the glycolysis pathway glgC and glgA expressions were improved by the promoter replacement. The new recombinant strain showed greatly reduced acetate overflow and cell lysis during the fermentation. Dry cell weight (DCW) reached over 55 g/L. The maximalaccumulation rate of glycogen was 0.5 g/g DCW. When a proper purification process is established, this strain would be highly valuablefor the industrial production of glycogen.

Keywords : Glycogen, metabolic engineering, high cell density culture

F-43

Metabolic Shift of Klebsiella pneumoniae L17 the Reduced Agent Iron in Fermentation of Glycerol to 1,3-Propanediol Using qPCR

Daseul Kong, and Jung Rae Kim*

School of Chemical and Biomolecular Engineering, Pusan National University, Busan 46241, Republic of Korea

1,3-PDO is a value-added product that used as adhesives, laminates. Forefficient production of 1,3-PDO, sustainable and non-toxic regeneration of NADH is of great importance. ZVI (Zero-Valent Iron) can provide reducing equivalent for 1,3-PDO synthesis from glycerol as an electrondonor. Klebsiella pneumoniae has a 1,3-PDO production pathway fromglycerol and has been extensively investigated as exoelectrogens. In thisstudy, we attempt to produce 1,3-PDO from glycerol by using an electrochemically active strain, K. pneumoniae L17, and ZVI as an electron donor. As a result, the production of 1,3-PDO using ZVI hasincreased significantly to 24.23 ±1.33 mol/l. These results implicate thatZVI can regulate the bioconversion of electroactive strain such as L17,therefore improve glycerol conversion into value-added platform chemicals.

Keywords : 1,3-propanediol, zero-valent iorn, qPCR

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Safety Evaluation of Fermented Microorganisms Isolated from Korean Traditional Fermented Foods

Do-Hee You1, Soo-Jeong Lee1, Dong-Geun Park1, You-Tae Kim2, and Ju-Hoon Lee1,2,3* 1Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea 3Department of Food and Animal Biotechnology, Seoul National University, Seoul 08826, Republic of Korea

The safety of four probiotic strains (three Lactiplantibacillus plantarum,and one Bifidobacterium bifidus) from three Korean traditional fermented foods were evaluated through genotypic analysis (DNA analysis for virulence genes and antibiotic resistance genes) and phenotypic (minimum inhibition concentration assay for nine antibiotics,hemolysis test, cell cytotoxicity assay). In genotypic analysis, their whole genome sequences were used to perform antibiotic resistance geneand virulence factor detection by searching Comprehensive AntibioticResistance Database(CARD) and Virulence Factor Database(VFDB).Antibiotic resistance gene and virulence factor should not be founded on their sequence due to possibility of horizontal gene transfer to otherbacteria. As a result, they had not any acquired antibiotics resistance geneor virulence factor, it means that they are safety in silico. Minimum inhibitory concentration assay (MIC) for nine antibiotics with ETEST®strip showed that their MIC values were lower than the European Food Safety Authority (EFSA)’s cut-offs. Hemolysis test was performed by streaking the probiotic strains on tryptic soy agar with 5% sheep blood.The results showed that there are no beta-hemolytic traits on agar, meaning that none of all strain has hemolytic activity. Furthermore, cellcytotoxicity assay conducted via LDH assay kit. As a result, all tested probiotics strains had no cytotoxicity on CaCO-2 cells, whereas pathogen strain Bacillus cereus ATCC 14579 revealed high cytotoxicity.These results suggest that these probiotics strains are safe in accordancewith international standards.

Keywords : Probiotics safety, antibiotics resistance, toxicity

F-45

In vitro Characterization and Evaluation of Fermented Microorganisms Isolated from Korean Traditional Fermented Foods for Industrial Applications

Dong-Geun Park1, You-Tae Kim2, and Ju-Hoon Lee1,2,3* 1Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea 3Department of Food and Animal Biotechnology, Seoul National University, Seoul 08826, Republic of Korea

Total 1,589 bacteria were previously separated from eight Korean traditional fermented foods and four probiotic strains (two Lactobacillusplantarum, two Lactococcus lactis) were selected. These strains wereevaluated in two characteristic categories of food processing (heat tolerance and oxygen tolerance) and probiotic functions (cholesterol lowering, mucin adhesion, and Caco-2 cell adhesion) with Lb. rhamnosus GG as control. Heat tolerance test revealed that survival ratesof Lb. plantarum (< 90.3%) and Lc. lactis (< 59.3%) at 42℃ were lowerthan that of the control strain (90.9%). Oxygen tolerance test showed thatsurvival rates of Lb. plantarum (> 76.2%) and Lc. lactis (> 72.2%) werehigher than that of the control strain (71.2%). Furthermore, mucin and Caco-2 cell adhesion activities showed that they have over 90.4% and 81.2% adhesion rate, respectively, with no significant difference from the control strain. Also, cholesterol lowering activities of four probioticsstrains showed 66.1% to 74.8% higher than that of control (35.5%). Therefore, all test results suggest that these selected probiotics may have required characteristics and functions for food industrial applications.

Keywords : Probiotics, cholesterol lowering, functional evaluation

F-46

Cloning and Characterization of Three D-Lactate Dehydrogenase Genes from Newly Isolated Sporolactobacillus terrae EFEL 7000

Yayun Cheng, and Nam Soo Han* Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Sciences and Biotechnology, Chungbuk National University, Cheongju 28644, Republic of Korea

PLA (polylactic acid) is a highly versatile biodegradable and environmentally friendly polymer which could be the substitute for synthetic plastics derived from petroleum. D-PLA shows better stabilityperformance than L-PLA due to higher melting temperature. We isolatedSporolactobacillus terrae EFEL 7000 from rhizome soil, having excellent D-lactate production with high purity. The aim of this study was to identify the genes responsible for D-lactate formation in Sp. terraeEFEL 7000 and characterization of these enzymes to facilitate the production of pure D-lactate. For this goal, we cloned three genes (ldhd1,ldhd2, and ldhd3) responsible for D-lactate production from Sp. terraeEFEL 7000, and those genes were overexpressed in Escherichia coli StarBL21(DE3) using an inducible pET-21a(+) vector. Three genes encodedD-lactate dehydrogenases having 341, 334, and 335 amino acids, respectively. These enzymes were purified by Ni-NTA column chromatography and molecular weights were determined by SDS-PAGE. The condition for measurement of enzyme activity was optimized and kinetic parameters such as Km and Kcat were determined.This is the first study on the characterization of D-lactate dehydrogenasesfrom Sp. terrae EFEL 7000, and it contributes to understanding the catalytic mechanism of those D-lactate dehydrogenases and leads to developing a biotechnological method to produce D-lactate.

Keywords : D-Lactate dehydrogenase, Sporolactobacillus terrae EFEL7000, D-Lactate

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F-47

Enhancement of Bioethanol Production from Eucheuma denticulatum Using by Various Yeasts Adapted to Galactose

Jieun Kim, Ji Won Yang, Gwi-Taek Jeong, and Sung-Koo Kim*

School of Marine, Fisheries and Life Science, Pukyong National University, Busan 48513, Republic of Korea

The use of fossil fuels has increased rapidly, accounting for about 86%of the current world energy use, and environmental problems such as global warming are emerging worldwide due to the use of such fossil fuels. Therefore, there is a growing interest in renewable biofuels to combat warming. Among them, bioethanol using seaweed is a fuel thatcan replace gasoline and does not compete for food resources comparedto other biomass. Eucheuma denticulatum is a red seaweed that has themost carbohydrate. Therefore, in this study, the thermal acid hydrolysis and enzymatic saccharification were performed using E. denticulatum,and fermentation was performed using yeasts suitable for high galactoseconcentration to increase the yeast absorption to galactose rate. The pretreatment efficiency (EP) of optimal thermal acid hydrolysis conditions,which were determined as 10% (w/v) slurry content and 300 mM HNO3at 121℃ for 90 min, was 44.6% with 31 g/L of monosaccharide. Enzymatic saccharification with 8-24 U/ml Cellic CTec2 were performed at 50℃ for 72 h. The maximum monosaccharide concentrationof 45.9 g/L with efficiency of enzymatic saccharification (ES)= 66.2% was obtained with 20 U/ml Cellic CTec2 at pH 5.0, 50℃, and 200 rpm for 72 h. HMF (5-hydroxymethylfurfural) was removed by absorption using 1 to 2% activated carbon for 2 min. Ethanol fermentation was performed using Saccharomyces cerevisiae, Candida lusitaniae and Kluyveromyces marxianus adapted to non-adapted and high galactose.

Keywords : Eucheuma denticulatum, adaptive evolution, bioethanol

F-48

ODFM, An Omics Data Resource from Microorganisms Associated with Fermented Foods

Tae Woong Whon, Se Hee Lee*, and Seong Woon Roh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

ODFM is a data management system that integrates comprehensive omics information for microorganisms associated with various fermentedfoods, additive ingredients, and seasonings (e.g. kimchi, Korean fermented vegetables, fermented seafood, solar salt, soybean paste, vinegar, beer, cheese, sake, and yogurt). The ODFM archives genome,metagenome, metataxonome, and (meta)transcriptome sequences of fermented food-associated bacteria, archaea, eukaryotic microorganisms,and viruses; 131 bacterial, 38 archaeal, and 28 eukaryotic genomes arenow available to users. The ODFM provides both the Basic Local Alignment Search Tool search-based local alignment function as well as average nucleotide identity-based genetic relatedness measurement,enabling gene diversity and taxonomic analyses of an input query againstthe database. Genome sequences and annotation results of microorganismsare directly downloadable, and the microbial strains registered in the archive library will be available from our culture collection of fermentedfood-associated microorganisms. The ODFM is a comprehensive database that covers the genomes of an entire microbiome within a specific food ecosystem, providing basic information to evaluate microbial isolates as candidate fermentation starters for fermented foodproduction.

Keywords : Fermented foods, microorganisms, omics data

F-49

Isolation and Expression of Lycopene Cyclase Involved in the C40 Carotenoid Astaxanthin Biosynthesis in Paracoccus marcusii MBLB0836

Chae Rin Yun1, Eui-Sang Cho1, and Myung-Ji Seo1,2* 1Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

Astaxanthin as a representative of red cyclic C40 carotenoids is one of the most industrially important carotenoids for the applications into foodcolorant, animal feed, and neutraceutical. Lycopene cyclase (CrtY) converts lycopene to β-carotene by the formation of two rings at each end of the linear lycopene structure in the astaxanthin biosynthesis. Here, we isolated the CrtY gene in Paracoccus marcusii MBLB0836 (CrtYPmMBLB0836) that was previously isolated from Salicornia europaea L. as representative halophyte. The astaxanthin production byP. marcusii MBLB0836 was elucidated by HPLC analysis. The clonedCrtYPmMBLB0836 was functionally expressed in Escherichia coliBL21(DE3) using inducible expression vector pET28a. After expressionand purification by Ni-NTA affinity chromatography, the recombinantCrtYPmMBLB0836 exhibited a major soluble band with approximately42 kDa by SDS-PAGE analysis. The HPLC analysis also demonstratedthat the purified recombinant CrtYPmMBLB0836 successfully catalyzed the formation of β-carotene from lycopene as substrate. Thisstudy will provide a wide base of knowledge on P. marcusii-derived lycopene cyclase in the astaxanthin biosynthesis at the molecular level.

Keywords : Paracoccus marcusii, astaxanthin, lycopene cyclase

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF- 2019R1A2C1006038) and by the Basic Science Research Programs through the NRF funded by the Ministry of Education (NRF- 2016R1D1A1B03931582)]

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Isolation of Lactic Acid Bacteria Producing 4,4'-Diaponeurosporene as C30 Carotenoid from Korean Fermented Vegetable Food, Kimchi

Hanseul Lim1, Deok Jun Yoon2, and Myung-Ji Seo1,2* 1Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea 2Department of Bioengineering and Nano-Bioengineering,Graduate School of Incheon National University, Incheon 22012, Republic of Korea

Carotenoids are abundant pigments in nature with having antioxidant activity as major striking feature. Carotenoids has been thus applied todevelop natural colorant, functional food and animal feeding materials.Lactic acid bacteria (LAB) are known to produce various antioxidants,such as superoxide dismutase and thioredoxin reductase. In particular, the specific species of Lactobacillus, Enterococcus and Pediococcushave been reported to be the producers of 4,4'-diaponeurosporene as antioxidant C30 carotenoid. The current study describes the isolation ofLAB producing 4,4'-diaponeurosporene from Korean fermented vegetable food, Kimchi. We isolated 64 strains showing the yellow-coloredpigments and genetically screened the 4,4'-diaponeurosporene-producingLAB by investigating the presence of its core biosynthetic genes; diapophytoene synthase (CrtM) and diapophytoene desaturase (CrtN) by PCR analysis. The 4,4'-diaponeurosporene production was monitoredby HPLC and UV/VIS absorption spectrum analyses. Finally, the selected strains were identified to be Lactobacillus plantarum and L. pentosus. In conclusion, this research will contribute the scientific information regarding potential probiotics producing natural and rare 4,4'-diaponeurosporene, and the applicability of functional food materials fortified with C30 carotenoid.

Keywords : 4,4'-diaponeurosporene, Lactobacillus,, carotenoid

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF- 2019R1 A2C1006038) and by the OTTOGI HAM TAIHO FOUNDATION]

F-51

Heterologous Expression and Characterization of Glutamate Decarboxylase from Lactococcus garvieae MJF010 Isolated from Human Feces

Hyo Jung Lim1, Eui-Sang Cho1, and Myung-Ji Seo1,2* 1Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

Gamma-aminobutyric acid (GABA), a non-protein amino acid, is an inhibitory neurotransmitter in the brain and spinal cord of mammals withbiological activities including anti-hypertensive, anti-diabetic, anti- inflammatory, and antioxidant effects. GABA is mainly synthesized viaglutamate decarboxylase (GAD), which catalyzes the decarboxylation of L-glutamate. In this study, we heterologously expressed in Escherichia coli and biochemically characterized the recombinant GADof Lactococcus garvieae MJF010 (rGADLgMJF010), which was isolated from human feces in accordance with the IRB regulation at Incheon National University (INUIRB-20-67). The optimal temperatureand pH of the rGADLgMJF010 activity was determined to be 35˚C andpH 5.0, respectively. CaCl2, MgCl2, and ZnCl2 increased the rGADLgMJF010activity, whereas AgNO3 decreased it significantly. The Km, Vmax and kcatvalues of the rGADLgMJF010 for L-glutamate were 2.94 mM, 0.023 mM/min, and 12.3 min-1, respectively. The rGADLgMJF010 activity was independent on the presence of exogenous PLP, implying the expressed form of rGADLgMJF010 in E. coli BL21(DE3) as GAD-PLPcomplex.

Keywords : Lactococcus garvieae, gamma-aminobutyric acid, glutamate decarboxylase

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF-2019 R1A2C1006038) and by the OTTOGI HAM TAIHO FOUNDATION]

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Isolation of Gamma-Aminobutyric Acid-Producing Enterococcus Strains from Human Fecal Samples

Ji-Su Baek1, Hyo Jung Lim2, and Myung-Ji Seo1,2* 1Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea 2Department of Bioengineering and Nano-Bioengineering, Gradiate School of Incheon National University, Incheon 22012, Republic of Korea

Gamma-aminobutyric acid (GABA) as a ubiquitous non-protein amino acid is primarily synthesized by the α-decarboxylation of L-glutamate,which is catalyzed by glutamate decarboxylase (GAD). Main producerof GABA has been reported to be lactic acid bacteria (LAB) which iscommonly considered to be generally recognized as safe (GRAS) bacteria. This study describes the isolation of GABA-producing LAB from human fecal samples (IRB regulation at Incheon National University, INUIRB-20-67). Seventeen strains were selected for GABA-producers from 79 feces-derived isolates after the TLC analysisscreening. The isolates showed the high 16S rRNA gene sequence similarity to the type strains of Enterococcus faecium (14 isolates) andE. avium (3 isolates). The GABA production analysis by GABase assayshowed that E. faecium MJ1-1 showed the highest GABA productionlevel (3.59 mM), followed by E. avium MJ2-13 (3.56 mM), and E. faecium MJ1-4 (3.46 mM). The studies on the enhancements of GABAproduction by these strains within an Enterococcus genus and functionalcharacterizations of Enterococcus-derived GADs are currently conducted.This study could support the expandability of GABA- producing rare LAB, in particular Enterococcus strain as commensal microorganismin the intestines of humans.

Keywords : Gamma-aminobutyric acid, human feces, Enterococcus

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF-2019R1A2C1006038) and by the OTTOGI HAM TAIHO FOUNDATION]

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Optimization of Culture Conditions for the Production of Deinoxanthin as C40 Carotenoid by Deinococcus yunweiensis KCTC 3955T

Chang Jae Lee1, Eui-Sang Cho2, Dong-Ho Seo3, Jong-Hyun Jung4, and Myung-Ji Seo1,2* 1Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea 2Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 3Department of Food Science and Technology, College of Agriculture and Life Science, Jeonbuk National University, Jeonju 54896, Republic of Korea 4Radiation Research division, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea

Deinoxanthin is a unique C40 carotenoid of the Deinococcus genus, inparticular majorly produced from radiation-resistant D. radiodurans. Deinoxanthin was proven to exhibit significantly stronger reactive oxygen species (ROS)-scavenging activity which contributes to cell stability resistant to desiccation, ionizing radiation, and hydrogen peroxide. In this study, we evaluated the deinoxanthin production fromsix strains of the genus Deinococcus using HPLC and UV–VIS spectrumanalyses. Among them, D. yunweiensis KCTC 3955T showed the highestdeinoxanthin production (2.03 μg/ml) in the basic medium (5 g/L of tryptone, 1 g/L of glucose, and 3 g/L of yeast extract). To enhance its deinoxanthin production, the culture medium influencing its productivitywas optimized via one-factor-at-a-time (OFAT) strategy, resulted in the selection of glycerol and soytone as carbon and nitrogen sources, respectively. In addition, the most significant factors including mediumcomponents and metal ions with each value were optimized using centralcomposite design. Finally, the optimized medium enhanced the deinoxanthin production by 1.26-folds compared to basic medium.

Keywords : Deinococcus yunweiensis, deinoxanthin, culture optimization

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF-2019 R1A2C1006038) and by the Basic Science Research Programs throughthe NRF funded by the Ministry of Education (NRF-2016R1 D1A1B03931582)]

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High Performance Enzymatic Synthesis of Maltoheptaose Palmitate and Evaluation of Its Thermal and Emulsifying Properties

Phu Cuong Nguyen1, My Tuyen Thi Nguyen2,3, Jae-Han Kim2, Soon-TaekHong1, Dae-Ryeol Kim1, and Jong-Tae Park1* 1Department of Food Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea 2Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Republic of Korea 3College of Agriculture, Can Tho University, Can Tho City 900000, Vietnam

In this study, enzymatic synthesis of a novel maltoheptaose palmitate (G7-PA) was carried out by esterification of G7 and PA with the catalystsof immobilized Candida antarctica lipase B (CALB). First of all, the response surface methodology based on three variables (DMSO concentration, the molar ratio of G7/PA, and the amount of immobilizedenzyme) at three levels was adopted to optimize the conditions of the G7-PA synthesis. The optimal conditions for achieving 92.75% of conversion yield were 0.2 of the G7/PA molar ratio, 33.5 U of immobilized CALB per 1 g of PA in 10% DMSO during 24 h of incubationat 60℃. The structure of G7-PA was identified by 1D & 2D NMR spectroscopy, MALDI-TOF/MS, and differential scanning calorimetry.Results showed that the ester bond was located at C-6 of the glucose atthe reducing end of the G7. The G7-PA monoester showed the melting temperature at 56.3 ℃ that was 6.5 ℃ lower than that of the free PA, andexhibited a different endothermic pattern from the G7. G7-PA was compared with the commercial sucrose-PA (S-PA) in terms of emulsion-forming ability and stability at extreme conditions. The emulsion prepared with S-PA was flocculated under highly acidic conditions due to S-PA hydrolysis, while the G7-PA emulsion was stableat pH 3 due to the resistance of G7-PA against the acid hydrolysis. Theseresults demonstrated that G7-PA has excellent emulsifier potentials, andits applications of in foods, cosmetics, and drug delivery under extremepH conditions are promising.

Keywords : Maltoheptaose, lipase, sugar esters

F-55

Culture Optimization for the Production of 4,4'-diaponeurosporene as a C30 Carotenoid by Lactobacillus plantarum subsp. plantarum KCCP11226

Deok Jun Yoon1, Eui-Sang Cho1, Chi Young Hwang1, and Myung-Ji Seo2*

1Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

Lactobacillus plantarum is well-known probiotics species with being recently reported the production of C30 carotenoid as organic pigment with antioxidant property. In this study, cell culture condition of Lactobacillus plantarum subsp. plantarum KCCP11226 producing 4,4'-diaponeurosporene was optimized for the enhancement of carotenoid production. The one factor-at-a-time (OFAT) method was employed to determine the optimal carbon and nitrogen source, respectively, resulting in lactose and beef extract for the optimal carbonand nitrogen source, respectively. In addition, the response surface methodology (RSM) based on Box-Behnken experimental design wasused to enhance the production of 4,4'-diaponeurosporene by optimizingthe level of lactose, beef extract, pH, and temperature. Finally, this studycould confer the strategy for the enhancement of 4,4'-diaponeurosporeneas C30 carotenoid by optimizing the culture conditions of Lactobacillus plantarum subsp. plantarum KCCP11226, which could be useful information for the application of 4,4'-diaponeurosporene in diverse bio-industries.

Keywords : Culture optimization, 4,4'-diaponeurosporene, Lactobacillusplantarum subsp. plantarum

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF-2019 R1A2C1006038) and by the OTTOGI HAM TAIHO FOUNDATION]

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Bacterioruberin as C50 Carotenoid from Haloferax marinum MBLA0078: Culture optimization and antioxidant activity

Eui-Sang Cho1, Chi Young Hwang1, Chae Rin Yun1, Dong-Hyun Jung2,and Myung-Ji Seo1,3* 1Department of Bioengineering and Nano-Bioengineering, Graduate Schoolof Incheon National University, Incheon 22012, Republic of Korea 2Bacteria Research Team, Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea 3Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

Halophilic archaea are known to widely inhabit various high-salt environments. Colonies of most haloarchaea are pigmented pink or reddue to the presence of bacterioruberin (BR) as C50 carotenoid, which has been reported to protect extreme halophiles from the negative effects ofradiant energy from sunlight and harmful osmotic stress caused by lowconductivity. In this study, a new potent BR producer, Haloferax marinum MBLA0078 was isolated from seawater nearby YoungheungdoIsland. The classical one-factor-at-a-time (OFAT) method determined glucose and fish peptone were the best carbon and nitrogen source, respectively. Thereafter, a sequential statistical strategy was used to optimize bacterioruberin production from Haloferax marinum strain MBLA0078. Based on Plackett–Burman (PB) experimental design, factors affecting BR productivity were screened and BR production was enhanced by response surface methodology (RSM). We identified the characteristic three-fingered profile and relative composition of BR andits precursor by TLC and HPLC. The radical scavenger activity of BRwas evaluated by ABTS, FRAP, and DNA nicking assay, respectively.These results suggest that the BR extracts from Haloferax marinumMBLA0078 can be a useful antioxidant for food and biotechnologicalapplication.

Keywords : Bacterioruberin, Haloferax marinum, culture optimization

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF- 2019R1A2C1006038) and by the Basic Science Research Programs through the NRF funded by the Ministry of Education (NRF- 2016R1D1A1B03931582)]

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Culture Optimization for C50 Carotenoid Bacterioruberin-ProducingHalophilic Archaeon Halorubrum sodomese MBLA0099 Isolated from Seawater

Chi Young Hwang1, Eui-Sang Cho1, Deok Jun Yoon1, and Myung-Ji seo1,2*

1Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

One strain MBLA0099 was isolated from seawater nearby Yeoungheungisland, Republic of Korea (37°15'22.36"N 126°30'03.35"E) as showingthe production of red-pigment and identified to be Halorubrumsodomense. The red-pigment was thereafter identified to be bacterioruberin as C50 carotenoid after HPLC analysis. In order to enhance the carotenoid production, the culture of Hrr. sodomenseMBLA0099 was optimized with controlling the carbon and nitrogen sources, C/N ratio, and salinity concentration in the culture medium. Thecarotenoid production was maximized with optimizing 0.2% (w/v) sucrose and 0.773% yeast extract as the carbon and nitrogen source, respectively, under the C/N ratio of 1:1. The effects of salinity concentration on the C50 carotenoid production by strain MBLA0099 were also investigated using NaCl,and MgSO4, resulting in the optimalconcentration of 20% and 7%, respectively. Finally, the optimized culture conditions of strain MBLA0099 enabled the increase of C50carotenoid production with 2.06-fold compared to the basal medium ATCC 1176.

Keywords : Halorubrum sodomense, bacterioruberin, culture optimization

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MIST) (NRF- 2019R1A2C1006038) and by the Basic Science Research Programs through the NRF funded by the Ministry of Education (NRF- 2016R1D1A1B03931582)]

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Characteristics of Dominant Lactic Acid Bacteria in Kimchi Fermented at Low Temperature for a Long Time

Soyeong Mun, and Hae Choon Chang*

Department of Food and Nutrition, Kimchi Research Center, Chosun University, Gwangju 61452, Republic of Korea

In this study, we characterized lactic acid bacteria (LAB), which are dominant (over 70%) LAB in kimchi fermented below 0℃ for 2~3 months. Thus ten strains were selected among 30 strains of LAB isolatedfrom kimchi. All strains were identified as Weissella koreensis by 16s rRNA gene sequence analysis. Acid production from carbohydrate wereanalyzed using API 50 CHL kit and three strains (LAB 1, 9, 10) showingdifferent metabolism patterns with type strain were finally selected forfurther study. Growth at different temperature (5~30℃) and pH (pH 4.0~9.0) were examined. The three LAB strains showed optimum growth at 30℃ (A600 : 2.30~2.66). In optimum pH for LAB growth,two strains showed optimum growth at pH 8.0 (A600 : 3.22~3.26) andthe other showed at pH 6.5~7.0 (A600 : 2.31~2.32). The LAB strains were stable at –2~20℃ for 24~72 h (8.54~8.96 log CFU/mL). In acid and alkali tolerance test, they were quite stable at pH 6.0~8.0 for 24 h,however their viable cells were gradually decreased, and then finally detected 2.44~3.99 log CFU/mL cells at pH 8.0 for 72 h.

Keywords : Weissella koreensis, Kimchi, low-temperature

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Optimization of Rice-Bran Fermentation Using Cholesterol-Lowering Effect of Lactiplantibacillus plantarum EM

Songhee Moon, and Hae Choon Chang*

Department of Food and Nutrition, Kimchi Research Center, Chosun University, Gwangju 61452, Republic of Korea

Rice-bran fermentation were optimized using Lactiplantibacillus plantarum EM, which showed strong cholesterol-lowering activity. Inorder to optimize the nutrient source for L. plantarum EM, carbon sources, nitrogen sources, minerals, and vitamins were added to 20% rice-bran. When 3% of corn steep liquor and 1% of glucose were addedinto the rice-bran, the numbers of viable cells were the highest (9.78 logCFU/mL). Optimized initial pH and temperature for the fermentation were pH 6.0 and 30℃. The fermented rice-bran showed antibacterial activities (200-400 AU/mL) against food-born pathogens as well as antifungal activities (100-400 AU/mL) against food spoilage molds. Thefermented rice-bran was further hot air-dried at 55℃ for 16 h and ground(HFRB). As a result of the cholesterol-lowering effect of HFRB, cholesterol-lowering ability of HFRB was 1.7 times higher than that ofL. plantarum EM dead cells. In addition, the phytic acid content of the HFRB decreased 2.1 times compared to that of raw rice-bran. In sensoryevaluation of HFRB, it showed higher overall acceptability than raw rice-bran. The results indicate that the fermented rice-bran (HFRB) canbe used as a functional food candidate.

Keywords : Rice-bran, Lactiplantibacillus plantarum, fermentation

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Characterization of Psychrotrophic Lactic Acid Bacteria Isolated from Kimchi

Minkyeong Jo, and Hae Choon Chang*

Department of Food and Nutrition, Kimchi Research Center, Chosun University, Gwangju 61452, Republic of Korea

We tried to isolate psychrotrophic lactic acid bacteria (LAB) from kimchi. Five strains of LAB, which can grow well at low temperature (25℃↓) were selected among the 24 strains of LAB isolates. The fiveLAB isolates showed maximum growth at 25℃. The five isolates wereidentified as Leuconostoc mesenteroides ssp. dextranicum with 99.9%identities using the API 50CHL kit. However, the isolates were identified as Leuconostoc gelidum ssp. aenigmaticum (two strains of isolates with 100% and 99.93% identities) and Leuconostoc gelidum ssp. gasicomitatum(three strains of isolates with 100% identities) based on 16S rRNA genesequences. In haem-stimulated aerobic growth test, growth of the two strains did not stimulated by haem. While growth of the other three strainswere stimulated by haem addition at aerobic condition. In exopolysaccharide(EPS) production assay, all five isolates produced EPS. In acid resistancetest of LAB isolate, all five strains were stable at pH 8.0 for 72 h. In theheat resistance test, the five isolates were stable at -2~10℃ for 72 h. Inthe salt resistance test, the five isolates were stable at 1~9% (w/v) NaClfor 72 h.

Keywords : Kimchi, lactic acid bacteria, psychrotrophic

F-61

Influence of the Main Ingredients of the Fermented Vegetable Food, Kimchi, on Microbial Succession and Metabolite Profile

Hye Seon Song, Se Hee Lee, Joon Yong Kim, and Seong Woon Roh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

Kimchi is spontaneous fermented food by lactic acid bacteria originatedfrom raw ingredients. To investigate the effect of these ingredients on food fermentation, four types of the food that only differed in their mainraw ingredients (kimchi cabbage, green onion, leaf mustard, and youngradish) were manufactured and investigated. The major microorganisms identified were the Leuconostoc gelidum, Weissella kandleri, and Lactobacillus sakei groups, but distribution of the species was confirmedto differ depending on the sample type. The three species were mainlydistributed in the food made of kimchi cabbage and young radish, andthe Lac. sakei group was hardly found in the food made of green onionand leaf mustard. Additionally, metabolite analysis showed that the freesugar, organic acid, ethanol, and amino acid profiles differed based on sample type. This study indicates that main ingredients can be an important factor in determining the microbial community and metabolitecomposition.

Keywords : Fermented food, lactic acid bacteria, Kimchi

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Development of qPCR-Based Assay Method for Restriction Endonuclease Activity of CAS9-sgRNA Ribonucleoprotein Complexes

Tri Minh Nguyen, Seul-Ah Kim*, and Nam Soo Han*

Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Science, Chungbuk National University, Cheongju 28644, Republic of Korea

Cas9-sgRNA ribonucleoprotein complexes (RNPs) have been extensivelyused for genome editing of various microorganisms. However, the methods to assay Cas9-sgRNA RNPs activity during reaction remain achallenge. The aim of this study was to develop the qPCR-based assaymethod for restriction endonuclease activity of Cas9-sgRNA RNPs. To achieve this goal, dextransucrase gene (dsr) of Leuconostoc citrium wasamplified by PCR and it was used as a target DNA to be cleaved by Cas9-sgRNA RNPs. Cas9 protein was purified from the broth cultureof recombinant E. coli BL21 cells harboring pET28a-Cas9-His by using Ni-NTA column. The sgRNA365 and sgRNA433 to bind dsr DNA wereprepared by in vitro transcription. The amount of Cas9 protein and sgRNA for RNPs was optimized to be 1000ng/µl and 200ng/µl, respectively. The RNPs and dsr amplicon were incubated at 37℃ for 10minutes, stopped by boiling at 95oC for 5 minutes, and the concentrationsof remaining dsr DNA were measured by qPCR. As result, RNP1 (Cas9-sgRNA365) and RNP2 (Cas9-sgRNA433) showed 2.4 x 10-3

pmole/minute/mg and 5.2 x 10-3 pmole/minute/mg of specific endonuclease activity, respectively. Shortly, RNP2 activity was higherthan RNP1 and this was consistent with that of agarose gel electrophoresis. In this study, qPCR-based assay of restriction endonuclease activity was successfully established and it can be effectively used to measure Cas9-sgRNA RNPs activity.

Keywords : CRISPR/Cas9, Real Time PCR, restriction endonuclease activity

F-63

Establishment of Alcohol Fermentation Conditions for Production of Strawberry Wine

Eun jung Yim, Seul Ki Park, Hyeon Jin Kang, Seung Wha Jo, Hyo Bin Oh, and Do Youn Jeong Microbial Institute for Fermentation Industry (MIFI), Sunchang County 56048, Republic of Korea 2Institute of Jinan Red Ginseng, Jinan County 55442, Republic of Korea

The optimal alcohol fermentation conditions and yeast strains were selected for the production of strawberry wine. First, experiments weredesigned with different levels inoculation rate(0, 1, 5%), stirring speed(0, 80, 150 rpm), incubation temperature(27, 35℃), sugar content(no add sugar, 22°brix) and raw material concentration(50, 70, 100%) for the selection of optimal alcoholic fermentation conditions. According to the design of the experiment, the fermentation characteristics(pH, acidity, sugar content, alcohol content, viable cell count) were measured after incubation for 4 days. As a result, the alcoholcontent was the highest in 1% yeast inoculation, 150 rpm, 27℃, 22°brix,and 100% raw material condition. In order to select strain, 13 yeasts werecultured for 4 days under optimal fermentation conditions, and contentof alcohol and ellagic acid were measured. As a result, it was confirmed that Saccharomyces cerevisiae SRCM101802 strain has higher alcoholand ellagic acid contents than other strains.

Keywords : Strawberry, wine, alcohol fermentation

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Quality Characteristics and Bioactivity Analysis of Vinegar Fermented Using Omija

Seulki Park, SeungWha Jo, EunJung Yim, HyeonJin Kang, and DoYounJeongMicrobial Institute for Fermentation Industry(MIFI), Sunchang 56048, Republic of Korea

This study was conducted to selected optimal acetic fermentation conditions and acetic acid bacteria for the production of omija vinegar.First, experiments were designed with different levels inoculation rate(0,10, 20%), incubation temperature(27, 35℃), stirring speed(0, 80, 150rpm), alcohol content(5, 10, 15%) and raw material concentration (50, 70, 100%) for the selection of optimal acetic fermentation conditions. According to the design of the experiment, the fermentationcharacteristics(pH, total acidity, viable cell count) were measured after incubation for 3 days. The total acidity was measured the highest underconditions of 10% inoculation of strain, 150 rpm, 5% alcohol contents, and 100% raw material concentration. To select the strain, 12 kinds of acetic acid bacteria were cultured for 3 days under optimal fermentationconditions, pH, total acidity, and viable cell count were measured. As a result, it was confirmed that Acetobacter pasteurianus SRCM102476strain has total acidity than other strains. As a result of analyzing the organic acid components of omija vinegar, the main organic acids were acetic acid, succinic acid, and citric acid, and their contents were 18884.50, 19868.00, and 14375.00 ppm, respectively. In addition, the main free sugars were maltose, glucose and fructose, and their contentswere 581.00, 101.50, 113.50 ppm, respectively. As a result of bioactivityanalysis, it was confirmed that after fermentation, α-glucosidase inhibitory and angiotensin-converting enzyme inhibitory activity of omija vinegar, it increased 160%, 130% respectively.

Keywords : Omija, vinegar

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Establishment of Acetic Fermentation Conditions for Production of Rhus Verniciflua Vinegar

Hyeon Jin Kang, SeungWha Jo, EunJung Yim, SeulKi Park, and DoYounJeongMicrobial Institute for Fermentation Industry(MIFI), 61-27, Minsokmaeul-gil,Sunchang-eup, Sunchang-gun, Jeonbuk 56048, Republic of Korea

The optimal acetic fermentation conditions and acetic acid bacteria wereselected for the production of Rhus verniciflua vinegar. First, experiments were designed with different levels inoculation rate (0, 1, 5, 10%), stirring speed (0, 80, 150rpm), yeast extract content (0, 0.5, 1%), alcohol content(0, 5, 10%) and raw material concentration (10, 50, 100%) for the selection of optimal acetic fermentation conditions. According to the design of the experiment, the fermentation characteristics (pH, total acidity, viable cell count) were measured afterincubation for 4 days. As a result, the total acidity was measured the highest under conditions of 10% inoculation of strain, 150rpm, 1% yeastextract, 5% alcohol, and 100% raw material. In order to select strain, 10acetic acid bacteria were cultured for 4 days under optimal fermentationconditions, and fermentation characteristics (pH, total acidity, viable cellcount) and functional(antioxidant, antidiabetic, anti-obesity activity) were measured. As a result, it was confirmed that Acetobacter pasteurianus SRCM102408 strain has higher antioxidant, antidiabetic and anti-obesity activities than other strains.

Keywords : Rhus verniciflua, vinegar

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Suitability of Limosilacobacillus reuteri as Health Promoting Starter for Different Types of Kimchi Fermentation

Gayun Kim, and Nam Soo Han*

Brain Korea 21 Center Health Industry, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea

Limosilacobacillus reuteri (LBRE14) is known to have gastrointestinalstability, capacity to restore the gut epithelial barrier and anti- inflammatory activity as a probiotic. The aim of this study was to evaluatethe health-promoting starter potential of LBRE14 in Baechu kimchi (BK) and Nabak kimchi (NK). To achieve this goal, BK and NK wereprepared with and without LBRE14 inoculation and Leuconostoc (Le.)mesenteroides, a commercial kimchi starter, was used as a positive control. BK and NK reached their proper pH (4.2~4.5) and pH (3.9~4.2)on the 7th day and 3rd day of fermentation, respectively. GC/MS analysisshowed that sulfides volatile metabolites such as diallyl disulfide and allyl methyl sulfide were identified dominant compounds in the initial fermentation of both kimchi. Statistical assessment of the volatile datavia heatmap analysis demonstrated that the two types of kimchi inoculated with LBRE14 produced a lot of acetic acid. In BK inoculatedwith LBRE14, free sugar remained, lysine, and threonine increased thanother kimchi inoculated without LBRE14. On the other hand, NK inoculated with LBRE decreased threonine and alanine. The sensory evaluation test revealed that NK with LBRE14 showed the highest preference. In conclusion, LBRE14 is a suitable probiotic starter for Nabak kimchi because it fermented well and promotes the unique tasteof kimchi.

Keywords : Probiotic, Kimchi, fermentation

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Bacteroides xylanisolvens Plays a Key Role to Shape Gut Microbial Ecosystem via Cross-Feeding

Cheon Seong Won, and Nam Soo Han*

Brain Korea 21 Center Health Industry, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea

The gut microbial ecosystem accounts for 70% of all microorganismsin our body and is maintained by using polysaccharides as a carbon source. However, most gut microorganisms cannot metabolize polysaccharides and studies on how gut microbes use polysaccharidesto maintain the gut microbial ecosystem are limited. The aim of this studywas to investigate the role of Bacteroides xylanisolvens, which is knownto hydrolyze various polysaccharides, on the gut microbial ecosystem. For this, single culture and co-culture were conducted in a medium consisting of dextran, resistant maltodextrin, and inulin, and polysaccharide degradation was analyzed by TLC and HPLC. As results,the single culture studies showed that, among 32 species tested, B. xylanisolvens utilized those polysaccharides most efficiently which theothers poorly used. Co-culture of B. xylanisolvens with other bacteria showed that polysaccharides were hydrolyzed into oligomers and otherbacteria made a growth using them. In conclusion, these results suggestthat the ability of B. xylanisolvens to hydrolyze polysaccharides could allow the maintenance of gut microbial ecosystem in the gut through cross-feeding.

Keywords : Bacteroides xylanisolvens, cross-feeding, gut microbial ecosystem

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Inhibition of ACE2, a COVID-19 Binding Receptor, by Extracts of Protein-Based Fermented Foods

SungHoon Hong, Dong Hyeon Lee*, and Nam Soo Han* Brain Korea 21 Center Health Industry, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea

Angiotensin-converting enzyme 2 (ACE2) is part of renin–angiotensin–aldosterone system and expressed in a variety of tissues including respiratory tract, myocardium, kidney, and gastrointestinal mucosa. ACE2 acts as a functional receptor for coronavirus infection and severalpeptide molecules are known as potential ACE2 inhibitors. In this study,we investigated ACE2 inhibiting properties of 7 different protein-basedfermented foods including Cheonggukjang, Doenjang, soy sauce, myeongranjeot (salted pollock roe), ojingeojeot (salted squid), cheese,and yogurt. When FPLC was conducted to separate the peptides in the samples by molecular weight, peptides between 1000 to 1600 Da whichwere consisted of 9 to 14 amino acids showed strong inhibition activitieson ACE2. By using the equivalent amount of samples, Cheonggukjangshowed the highest inhibitory activity which inhibited 50% of ACE2. Doenjang and myeongranjeot showed the second and third highest activities which were 35% and 26% inhibition, respectively. In conclusion, this study shows that the peptides of 1000~1600 Da molecular weight in above three samples have strong ACE2 inhibitoryproperties. Further research on protective effect of fermented food against coronavirus mediated infection is needed.

Keywords : ACE2, fermented food, ACE2 inhibitory peptide

F-69

Growth Properties of Tetragenococcus halophilus Isolates from Various Jeotgals and Their Aminopeptidase Activities

TaeJin Kim1, Se Jin Lee1*, and Jeong Hwan Kim1,2* 1Division of Applied Life Science (BK Four), Graduate School, 2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea

Tetragenococcus halophilus strains were isolated various jeotgals, the traditional Korean fermented seafood. Strains with proteolytic activitieswere selected by spotting on TSB agar containing skim milk (2%, w/v)and NaCl (5%, w/v), and identified through 16S RNA gene sequencing.Selected isolates were cultivated on MRS broth under different conditions: incubation temperature (4,15,25,30,37,45℃), and initial pHof growth medium (4~11), and NaCl concentration (12,15,18,21,24%,w/v). It was confirmed that T. halophilus strains grew best under the conditions of 25℃, 12% NaCl, and at pH 7. Five strains (CY54, CY298,MG86, MS149, and MC254) with relatively higher protease activity were selected and their aminopeptidase activity was measured. Amongthem, T. halophilus CY54 showed 5.42 ± 0.001U/mL, 6.28 ± 0.001U/mL,7.22 ± 0.001U/mL, 6.41 ± 0.001U/mL, and 5.70 ± 0.001U/mL for Ala-pNA, Arg-pNA, Glu-pNA, Met-pNA, and Lys-pNA as the substrate, respectively.

Keywords : Tetragenococcus, growth, aminopeptidase

F-70

Characteristics of Meju Fermented by Co-Culturing of Four Different Starters

Ji Yeon Yoo1, and Jeong Hwan Kim1,2* 1Division of Applied Life Science (BK21 Four), Graduate School, 2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea

Meju, a type of fermented soybean paste, is used as a starter in the preparation of various Korean traditional soybean-based foods. Therefore, it is important to improve the quality characteristics of meju,and various microorganisms are used for this purpose. In this study, Tetragenococcus halophilus, Bacillus subtilis, Aspergillus oryzae and Zygosaccharomyce rouxii were used together as starters to prepare meju.Meju 1 was not inoculated with a starter (control). Meju 2 was prepared by inoculating B. subtilis, A. oryzae and Z. rouxii. Meju 3 was prepared by inoculating 4 organisms (T. halophilus, B. subtilis, A. oryzae and Z. rouxii). The three meju samples were fermented for 2 months at 25℃and analyzed every week. The quality characteristics of meju were determined by measuring the viable cell counting, pH, titratable acidity(TA), reducing sugar, volatile basic nitrogen (VBN), amino-type nitrogen (ANN), ammonia-type nitrogen (AMN), moisture content, biogenic amine, crude protein and crude lipid, and will be presented at the meeting.

Keywords : Meju, fermentation, co-culture

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F-71

Properties of Rice Bran Fermented with Selected Lactic Acid Bacteria Producing γ-Aminobutyric Acid

Hye Sung Jeon1, and Jeong Hwan Kim1,2* 1Division of Applied Life Science (BK21 Four), Graduate school, 2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea

Lb. brevis G144 and Lb. curvatus K285 were isolated from Galchi Jeotgal and Gat-Kimchi. Rice bran (20%, w/v) was inoculated with eachLAB (106 CFU/g), and fermented at 35℃. Rapid increase of Lactobacillus was observed after 24 h during 120 h fermentation. In ricebran fortified with glutamate (0, 0.5, 1%) and fermented with Lb. curvatus K285, pH decreased from 6.3 at 0 h to 5.7 at 24 h, and decreasedafter 24 h until 120 h. In rice bran fortified with glutamate (0.5, 1, 3%)and fermented with Lb. brevis G144, pH increased and TA decreased. In rice bran fortified with glutamate (3%) and fermented with Lb. curvatus K285, pH increased 6.3 at 0 h to 6.8 at 48 h, then decreased to6.0 at 120 h. TA was changed in an inverse manner. GABA content increased from 7.35 mM and 7.44 mM in no glutamate added rice branwith Lb. brevis G144 and Lb. curvatus K285. In rice bran fortified with glutamate 0.5, 1, 3% and fermented with Lb. brevis G144, GABA contentincreased 31.50 mM, 34.18 mM, and 35.27 mM. In rice bran fortifiedwith glutamate 0.5, 1, 3% and fermented with Lb. curvatus K285, GABAcontent increased 35.35 mM, 62.06 mM, and 162.23 mM. Amino acidssuch as valine, ornithine, and phenylalanine etc., were increased in ricebran fermented with Lb. brevis G144 and Lb. curvatus K285.

Keywords : Rice bran, GABA, Lactobacillus

F-72

Transferable Gene in Plasmid of Staphylococcus equorum KS1030 Exhibiting Acquired Lincomycin Resistance

Sojeong Heo1, Tao Kim1, Hongeun Na1, Gawon Lee1, Jong-Hoon Lee2, and Do-Won Jeong1*

1Department of Food and Nutrition, Dongduk Women’s University, Seoul 02748, Republic of Korea 2Department of Food Science and Biotechnology, Kyonggi University, Suwon 16227, Republic of Korea

Staphylococcus equorum KS1030 from the traditional high-salt-fermentedseafood jeotgal exhibited the lincomycin resistance and contained smallplasmid pSELNU1 carrying the lincosamide O-nucleotidyltransferasegene lnuA. The pSELNU1 showed the horizontal transfer from strain KS1030 into other Staphylococcus species. Meanwhile, pSELNU1 didnot possessed the conjugal element, thus we determined the genome sequences of strain KS1030 to understand the transfer mechanism of pSELNU1. The complete genome of S. equorum KS1030 consists of asingle circular 2,862,845-bp chromosome and four circular plasmids. The G+C content of the genome is 33.16%. The genome was predictedto contain 2,903 ORFs, 61 tRNA genes, and 22 rRNA genes. The currentcomplete genome of S. equorum KS1030 revealed that one of the four plasmids, a 13 kb plasmid designated pKS1030-3, possessed transferrableelements including the relaxase gene rlx (GenBank accession no. JL104_RS14645), which nicks an origin of transfer to initiate the processof DNA mobilization and transfer known as bacterial conjugation. Comparative genomic analysis revealed that the presence of rlx is strain-specifically prosperity.

Keywords : Staphylococcus euquorum, relaxase, lincomycin resistance

F-73

Optimizing Media and Engineering Escherichia coli for Overproducing Colanic Acid

Na Ree Han, Hyeong Min Han, and Kyoung Heon Kim*

Departmrnt of Biotechnology, Korea University, Seoul 02841, Republic of Korea

Colanic acid (CA) is a major exopolysaccharides of bacterial cells. Sinceit has several biological activities, CA has a valuable application in industries. However, the culture media and conditions have not been optimized and studied so far. In this study, CA producing Escherichia coli strain (ΔwaaF) was constructed and the culture medium was statistically optimized for mass CA production. Glucose and tryptone were found to be the optimal carbon source and nitrogen source, respectively. Fractional factorial design demonstrated that tryptone andNa2HPO4 were the essential components for CA production. Through more optimization of culture medium, we gained 1910.0 mg/L of CA, which is about 12-times higher than the amount of CA obtained when using the non-optimized medium. Moreover, the evaluated value of CAwas 2052.8 mg/L, comparable with a previous experimental value thatwe obtained. This study shows a strong demonstration of medium optimization for higher production of CA, and leads up to further application for achieving maximum production of CA.

Keywords : Colanic acid, Escherichia coli strain ΔwaaF, medium optimization

F-74

Bioethanol Production from Gloiopeltis furcate Using Saccharomyces cerevisiae with the Deletion of GPD1 and GPD2

Jiwon Yang, Jieun Kim, Gwi-Taek Jeong, and Sung-Koo Kim*

School of Marine, Fisheries and Life Science, Pukyong National University, Busan 48513, Republic of Korea

The seaweed, Gloiopeltis furcate (red seaweed) was fermented to produce bioethanol. A total monosaccharide concentration from thermalacid hydrolysis pretreatment of G. furcate was 23.77 g/L with 10 % (w/v) seaweed slurry treated with 200 mM HCl at 121 ℃ for 90 min. After thethermal acid hydrolysis, enzymatic saccharification was carried out with 16 U/mL Viscozyme L, CTec2 or mixture of Viscozyme L and CTec2 (1:1) to G. furcate hydrolysates. Based on the ES value, CTec2 producedthe highest hydrolysis efficiency of monosaccharides. Therefore, CTec2 was selected as the optimal enzyme for enzymatic saccharification ofG. furcate hydrolysate. A total monosaccharide concentration of 55.3 g/L was obtained after thermal acid hydrolysis and enzymatic saccharification. Ethanol production was carried out using wild type (WT) Saccharomyces cerevisiae CEN.PK2-1, each ∆GPD1, ∆GPD2and ∆GPD1∆GPD2. Gene GPD1 and GPD2 are Glycerol-3-phosphatedehydrogenase which it related to the glycerol production from S. cerevisiae. Glycerol production pathway was blocked by the deletion of GPD1 and GPD2 to increase the ethanol production using CRISPRCas-9 tool.

Keywords : Bioethanol, fermentation, CRISPR Cas9

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F-75

Statistical Optimization of Parameters for KOH Pretreatment to Improve Glucose Recovery from Chestnut Shells

Kang Hyun Lee, Jeongho Lee, Seunghee Kim, Hyerim Son, and Hah Young Yoo*

Department of Biotechnology, Sangmyung University, 20, Hongjimun 2-Gil, Jongno-Gu, Seoul 03016, Republic of Korea

As the global population grows and the demand for food increases, thegeneration of food waste is also increasing. Chestnut shells (CNS), one of the by-products of food processing, were estimated to be generated more than 550 thousand tons worldwide in 2019. In this study, parametersof KOH pretreatment were optimized to improve enzymatic hydrolysisof CNS with the aim of increasing sugar recovery from the CNS. The concentration of the KOH solution for pretreatment was selected as 3%(w/w) by a fundamental experiment, and the experiment was designed to optimize parameters such as temperature, time, and solid/liquid ratioby response surface methodology. The optimal condition was determined as follows: temperature of 75ºC, time of 2.8 h and solid/liquidratio of 77.1 g/L. The glucan content and enzymatic digestibility of theCNS pretreated under optimal conditions were found to be 83.2% and 48.4%, respectively, and were improved by about 1.8 and 3.8-fold, respectively, compared to before the pretreatment. Finally, the enzymatical hydrolysates from the CNS are expected to be beneficiallyutilized for microbial fermentation in near future.

Keywords : Chestnut shells, KOH pretreatment, enzymatic hydrolysis

F-76

Fermented Soybean Extract by Bacillus subtilis Modulates Immune Status in Mice

Md. Sekendar Ali1,2,3, Biruk Tesfaye Birhanu1, Muhammad Aleem Abbas1, and Seung-chun Park1* 1Laboratory of Veterinary Pharmacokinetics and Pharmacodynamics, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of Korea 2Department of Biomedical Science and Department of Pharmacology, School of Medicine, Brain Science and Engineering Institute, Kyungpook National University, Daegu 41944, Republic of Korea 3Department of Pharmacy, International Islamic University Chittagong, Kumira, Chittagong 4318, Bangladesh

The present study was designed to investigate the effects of fermented soybean extract (FSE) on the neutrophil migration and phagocytosis, splenocyte proliferation, T and B lymphocyte proliferation, and cytokines expression on BALB/c mice as well as anti-inflammatory effects on RAW264.7 cells. Soybean was fermented using Bacillus subtilis HA and the investigation was carried out for 21 days by oral administration on five weeks old female mice arranging into six groups. The immune response was examined by splenocytes counting using trypan blue dye exclusion method, and splenocyte was activated by lipopolysaccharides (LPS) and concanavalin A (Con A) and the proliferation was determined by measuring the absorbance. Neutrophilmigration and phagocytosis were assessed by using CytoselectTM 24-well cell migration and Cytose-lectTM 96-well phagocytosis assay,respectively. T and B lymphocytes population were determined by flowcytometric analysis. Subsequently, quantification of cytokines was performed using sandwich ELISA from blood plasma. The results showed that the neutrophil migration and phagocytic activities, splenocyte proliferation, level of CD3+, CD4+, CD8+ T and CD18+ Bcells, and cytokines IL-2, IL-4 and IFN-γ expression were increased bydose-dependent manner. However, the IL-6 and TNF-α levels were decreased after treatment of FSE. Also, the extract considerably inhibitsthe LPS (lipopolysaccharide)-induced nitric oxide (NO) production andmRNA expression of iNOS and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and Cox-2) as well as TLR4 in RAW 264.7 cells (p <0.05). Furthermore, there are some important and predominant compounds thathave been identified in FSE by gas chromatography-mass spectroscopy (GC-MS) and liquid chromatography with tandem mass spectroscopy (LC-MS/MS) analysis. Taken together, we showed that the FSE increased the number of lymphocytes and promotes the neutrophils migration and phagocytosis, and proliferates T and B lymphocytes as well as activates the pro-inflammatory cytokines. These effects may playa role in increasing non-specific immunity, while suppressing of somecytokines indicating the immunomodulatory effects of the extract. Molecular docking study showed that daidzin, daidzein and genistin could potentially bind to human high affinity FcγRI-IgG complex to beinvolved in immunological response.

Keywords : Fermentation, immune response, cytokines

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G_Foodborne Pathogens and Food Safety

G-1

Diversity of the Heavy Metal Resistance Genomic Island in Listeria monocytogenes and Other Listeria spp.

Sangmi Lee1, Cameron Parsons2, Yi Chen3, Zahra Hanafy2, Eric Brown3,and Sophia Kathariou2 1Department of Food and Nutrition, Chungbuk National University, Chengju, Chungbuk 28644, Republic of Korea 2Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695-7624, USA, 3Division of Microbiology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD 20740-3835, USA

Listeria genomic island 2 (LGI2) is a chromosomal island that aboundswith heavy metal detoxification systems (cadmium and arsenic) and isoften found among serotype 4b hypervirulent clones of the foodborne pathogen Listeria monocytogenes, but not among non-pathogenic Listeria spp. Here, we provide the first evidence of a novel LGI2 variant (LGI2-3) chromosomally harbored by two L. welshimeri strains from an urban watershed. LGI2-3 was overall similar to LGI2 but bore a cystathionine beta-lyase gene, which was previously identified only inLGI2-1, as well as a novel cadAC cassette (cadA7C7) that resembled cadA4C4 in that it was associated with a lower level of cadmium resistance than other cadAC cassettes. Analysis of CadA sequences revealed two amino acids that might be indispensable in modulating the tolerance level to cadmium. Transfer of LGI2-3 to L. monocytogenes wasnot observed in the conjugation experiments between the LGI2-3- harboring L. welshimeri strain and L. monocytogenes strains. Further characterization of this island could enhance our knowledge on the physiologically important accessory genes that contribute to the geneticdiversity of Listeria spp.

Keywords : Listeria genomic island 2 (LGI2), heavy metal resistance, Listeria welshimeri

G-2

Investigation of Dual Binding Effect of Salmonella Enteritidis-Specific Phage by Exposure to Various pH Conditions

Si Eun Kang, Damilare Emmanuel Adeyemi, Jaein Choe, Hyeju Jung, andMi-Kyung Park*

School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

Salmonella Enteritidis-specific (SE) phage was previously isolated froman eel farm for using as a dual binding agent in magnetoelastic (ME) biosensor method. The head of SE phage needs to bind with ME sensorwhile the tail binds with target pathogen with charge interaction. The purpose of this study was to investigate pH effect on the SE phage immobilization on ME sensor and finally on the performance of ME biosensor method. Various pHs of SM buffer were exposed to ME sensor for 60 min for the confirmation of the sensor stability and the surfaceof the ME sensor was observed using an microscope at every 5-min interval. SE phage (108 PFU/mL) was suspended with various pHs of SM buffer and immobilized on the ME sensor surface prior to the exposure of S. Enteritidis (108 CFU/mL) for 1h at RT. The resonant frequency (RF) of phage-immobilized sensor was measured before andafter exposure to S. Enteritidis. The phage and bacterial attachment on ME sensor was observed using TEM and SEM, respectively. The opticalmicroscope image revealed the gold coating of ME sensor at the exposureof pH 1 and pH 3 was peeled. RF shift of pH 7 was the significantly greaterthan others (p < 0.05). The best phage immobilization was observed atpH 7 with a density of 21 ±. The number of S. Enteritidis (5.03 ± 1.33 CFU/100 µm2) bound on ME sensor at pH 7 was significantly greater than others (p < 0.05). It was concluded that SM buffer at pH 7 providedthe best condition of SE2 phage with ME sensor and target pathogen as a dual binding agent.

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G-3

Molecular Cloning of RBP Gene and Characterization from Bacteriophage ØCJ19 Active against Enterotoxigenic Escherichia coli

Gyeong-Hwuii Kim1, Jaegon Kim1, Jong Pyo Chae2, Jong Soo Jeon2, JunOk Moon2, Geun-Ah Kim1, Min-Sun Kim1, Myung-Hyun Lee1, and Sung-Sik Yoon1* 1Department of Biological Science and Technology, Yonsei University, Wonju 26493, Republic of Korea 2Institute of Biotechnlogy, CJ CheilJedang, Suwon 16495, Republic of Korea

Enterotoxigenic Escherichia coli (ETEC) is a major pathogenic Escherichia coli that causes diarrhea and swelling in piglets after weaning. Bacteriophage is a useful biological agent for safe control of ETEC as an antibiotic substitute. In this study, the characteristics and genetic analysis of the phage ØCJ19 that infects ETEC was performed.In addition, molecular cloning experiments were performed to understand the interaction between phage and host bacteria. Phage ØCJ19 belongs to the family Myoviridae. One-step growth curve showeda latent phase of 5 min and burst size of approximately 20 phage particles/infected cell. Phage infectivity was stable for 2 h between 4℃and 55℃, and the phage was stable between pH 3 and 11. Genetic analysisrevealed that phage ØCJ19 has a total of 49,567 bases and 79 open readingframes (ORFs). The full genomic sequence of phage ØCJ19 showed themost similarity to an Escherichia phage, vB_EcoS_ESCO41. There were no genes encoding lysogeny, toxins, virulence factors, or antibioticresistance in this phage, suggesting that this phage can be used safely as a biological agent to control ETEC. Cloning experiments were performed on the putative receptor-binding protein (RBP) gene of phageØCJ19, which is responsible for recognition and binding between phageand host bacteria. The putative RBP gene ligated to the pET30 expressionvector was transformed into BL21 competent cells. As a result of geneticanalysis of the pET30a vector ligated with RBP, the regions corresponding to the RBP gene were completely matched. These resultsshowed the potential to improve food safety by applying the phage ØCJ19 to animal foods contaminated with ETEC. In addition, the molecular biological results can be used as a basis for the study of thephage-host binding ability of the RBP gene of phage that recognizes hostbacteria, and will contribute to the study of host range expansion through mutagenesis of RBP.

Keywords : Bacteriophage, enterotoxigenic Escherichia coli, receptor- binding protein

G-4

Evaluation of a Rapid Detection Based on Real-Time qPCR Targeting Male-Specific Coliphages Derived from Shellfish in Korea

Gyungcheon Kim, and Hakdong Shin*

Department of Food Science and Biotechnology, College of Life Science, Sejong University, Seoul 05006, Republic of Korea

Severe norovirus outbreaks due to the consumption of contaminated shellfish have been recently reported. Due to the difficulty of culturing norovirus, male-specific phage has been suggested as an indicator species. The double agar layer method, which is a conventional methodfor Male-specific coliphages (MSCs) detection, has moderate sensitivity and requires incubation hours. The purpose of this study is to detect MSCs-contaminated shellfish samples without incubation hours. The virome sequence profiles were obtained from the oyster samples with the MSC detection in the double agar layer method. Assembled contigs were analyzed to identify biomarker regions within MSCs-contaminated samples. Specific primers were designed to amplify detected biomarker sequences and the specificity was identifiedwith in silico environment. The real-time qPCR method with newly designed primers showed higher detection sensitivity for MSCs. This result suggests that the virome information could be useful to identify a biomarker for indicator virus.

Keywords : Shellfish, male-specific coliphages, real-time qPCR

G-5

Determination of Pathogenic Escherichia coli, Listeria monocytogenes, Campylobacter spp., and Hygiene Indicator Bacteria from Cheese Samples in Farmstead Cheese Manufacturing and Retail Market in Korea

Hyeon-Jin Kim, Hye-Young Youn, and Kun-Ho Seo*

Center for One Health, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea

Cross-contamination through cows, workers, and utensils can affect theincreased microbial load of the cheese. In present study, we monitoredthe contamination levels of aerobic bacteria count, coliform, Escherichia coli, pathogenic E. coli(PE), Listeria monocytogenes(LM),Campylobacter coli(CC), and Campylobacter jejuni(CJ) of either foodprocessing step and final products from farmstead cheese manufacturing(FCM) and retail market located in Gyeong-gi and Seoul, respectively. PE, LM, CC, and CJ and hygiene indicator bacteria were calculated according to the standards of ministry of food and drug safety(MFDS). PE, LM, CC, and CJ were not detected in all cheese products and passedthe standard of MFDS. Interestingly, contamination levels of aerobic bacteria count in teat skin and grilled cheese were not significantly different in FCM except spring (p >0.05). Moreover, enteropathogenic E. coli was isolated from teat skin in spring and summer. Aerobic bacteria were detected in both cheese vat(0.53±0.92 and 0.77±0.68 log CFU/g)and worker’s gloves(0.67±0.58 and 0.83±0.75 log CFU/g) at farmsteadcheese manufacturing in summer and fall. Disinfection of facilities andsanitation of workers will be necessary to reduce cross-contamination of microorganisms from the cheese processing stage.

Keywords : Cheese, foodborne pathogen, hygiene indicator bacteria

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G-6

Growth Curve, Biofilm Formation, and Antimicrobial Susceptibility of Kocuria salsicia Isolated from Cheese Brine from Farmstead Cheese Manufacturing in Korea

Hye-Young Youn, Hyeon-Jin Kim, and Kun-Ho Seo*

Center for One Health, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea

Bacteria of the genus Kocuria can survive in extreme environments andcan cause infections in humans, including catheter-related bacteremia. In the present study, we investigated the growth curve, biofilm formation,and antimicrobial susceptibility of K. salsicia strains (KS1-9) isolated from cheese brine from a farmstead cheese manufacturing in Korea. Allisolates showed growth at 15% salt concentration and at 15, 25, 30, 37,and 42℃. Notably, K. salsicia KS6 and KS8 showed slow growth at 5℃.In the biofilm formation analysis via crystal violet staining, the isolate KS6 exhibited the highest biofilm formation ability at various temperatures. Remarkably, at 25 and 30℃, KS3, KS6, and KS8 showedhigher biofilm formation ability than that of Staphylococcus aureusATCC 25923. The antimicrobial resistance of the isolates was evaluatedusing the VITEK® 2 system, and the highest number of isolates were found to be resistant to marbofloxacin and nitrofurantoin (both 9/9, 100%), followed by enrofloxacin (7/9, 77.8%). Notably, three of the nineisolates (33.3%) showed multi-drug resistance. These findings indicatethat it is critical to ensure the hygienic handling of the brine used in farmstead cheese manufacturing to prevent contamination with potential human pathogens.

Keywords : Kocuria salsicia, cheese brine, farmstead cheese manufacturing

G-7

Microbial Contamination Levels of Fermented Milk Samples in Farmstead Fermented Milk Manufacturing, Fermented Milk Manufacturing Plant, and Retail Market in South Korea

Hye-Young Youn, Hyeon-Jin Kim, and Kun-Ho Seo*

Center for One Health, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea

Fermented milk is widely consumed worldwide because of its beneficial effects. Although fermented milk has low pH-induced lactic acid, aceticacid, and alcohol, foodborne pathogens can grow in inadequate sterilization and processing stages. Pathogenic Escherichia coli(PE), Listeria monocytogenes(LM), Campylobacter coli(CC), Campylobacterjejuni(CJ), and hygiene indicator bacteria(HIB; aerobic bacteria, E. coli,and coliform) were investigated by season in a total of 221 samples (environmental samples, raw materials, processing stages, and final products)in farmstead fermented milk manufacturing(FFM), fermentedmilk manufacturig plant(FMP), and retail market in 2020. PE, LM, CC,and CJ and HIB were evaluated according to the standards of ministryof food and drug safety(MFDS). As a result, PE, LM, CC, CJ, and HIBwere not detected in 125 final products out of 221 samples, which passedthe microorganism standard of MFDS. Interestingly, enteropathogenicE. coli were isolated from bovine teat skin in spring in FFM. In processingstages of FMP, there were significant seasonal difference in aerobic platecount: the average count in fall (2.56 ± 0.03, 2.20 ± 0.13, 2.88 ± 0.06,3.09 ± 0.01, and 2.55 ± 0.12 log CFU/g, respectively) was higher than that of summer (0.33 ± 0.58 log CFU/g, p<0.001). This result meansthat hygiene management in the fermented milk processing stages shouldoccur throughout the four seasons, not just in summer.

Keywords : Fermented milk, foodborne pathogen, hygiene indicator bacteria

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G-8

Isolation and Characterization of Two Bacteriophages That Specifically Infect Escherichia coli O157:H7

Minjin Oh, Jaeeun Kim, and Byoung Sik Kim*

Department of Food Science and Engineering, ELTEC College of Engineering, EwhaWomans University, Seoul 03760, Republic of Korea

Despite of ongoing developments on food industry, food poisonings caused by bacterial pathogens are steadily occurring. Escherichia coli O157:H7 is a major food-borne pathogen causing serious illnesses likehemolytic colitis and hemolytic uremic syndrome. Recent warning on antibiotic resistance broadly spread among pathogenic bacteria is also of concern for Escherichia coli O157:H7. In this study, we isolated two E. coli O157:H7 bacteriophages, ECCF01 and ECCF02, from chicken fecal samples and characterized them as alternative antimicrobialscontrolling E. coli O157:H7. Both phages specifically infect E. coli strains with O157 serotype but not affect non-pathogenic strains such as MG1655 and DH5α. They also do not infect other food-borne bacteriaincluding other enteric pathogens like Shigella flexneri and SalmonellaEnteritidis. Electron microscopic analyses indicated that ECCF01 belongs to the Myoviridae family. Moreover, ECCF01 and ECCF02 werestable in a broad range of pH(pH 3-10) and temperature(4-60℃) conditions. Therefore, these phages could be used as sustainable food preservatives controlling pathogenic E. coli O157:H7 in various foods.

Keywords : Bacteriophage, Escherichia coli O157:H7, foodborne pathogens

G-9

Variations of the MARTX Toxin Gene in Food-Borne Pathogens Vibrio vulnificus and Vibrio cholerae Isolated From Korea

Changyi Cho1, Hanhyeok Im2, Sang Ho Choi2, and Byoung Sik Kim1* 1Department of Food Science and Engineering, ELTEC College of Engineering, Ewha Womans University, Seoul 03760, Republic of Korea 2Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea

Vibrio food poisoning occurs in summer when sea temperature rises. Among Vibrio species causing food-borne illness, V. vulnificus and V. cholerae produce a potent protein toxin, the MARTX (Multifunctional Autoprocessing Repeats-in-ToXin) toxin. This toxin is very large in sizeand consists of several toxic effector domains, cysteine protease domain,and repeats-containing regions. Here, Vibrio strains isolated from Korean seawater and seafood samples were examined and draft genome sequence of some isolates were inspected for the presence of MARTX toxin gene, rtxA. The obtained rtxA genes were further analyzed bioinformatically to classify the type of MARTX toxin and to identifyputative effector cleavage sites. Among the analyzed nine rtxA genes,four different types of MARTX toxins were deduced. Notably, we alsofound that one isolate producing the toxin is actually V. mimicus, not V.cholerae, suggesting the spread of this potent toxin gene among variousmarine bacteria. Considering ongoing climate changes and naturally occurring horizontal gene transfer and homologues recombination among rtxA genes, the surveillance for the MARTX toxin genes in seafood-borne pathogens seems to be urgent.

Keywords : Vibrio spp., MARTX toxin, seafood

G-10

Development of a New Analysis Method for Doping Agents in Food Ingredients

Hana Park1,2, and Junghyun Son1,3* 1Doping Control Center, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea 2Department of Biotechnology, Yonsei University, College of Life Science and Biotechnology, Seoul 03722, Republic of Korea 3Department of Biological Chemistry, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea

Food was found to be a potential source of inadvertent doping in sports.Despite regulations, anabolic hormones are illegally used to promote livestock growth, resulting in the presence of hormone residues in animals. Athletes who have consumed such meat may be tested positivefor anabolic agents in doping control. Consumption of non-castrated pigs, which are naturally rich in steroid hormones, and pigs fed feed contaminated with mycotoxins can also result in a positive doping test. There have been several cases of athletes claiming to be tested positivefor doping by consuming food contaminated with doping agents. However, the procedures to prove that the intake was unintentional arevery difficult and it is a reality that players themselves must pay attentionto all food and be responsible for the results. The strategy of our anti-doping research is focused on protecting athletes from inadvertentdoping. Based on actual cases, we selected food ingredients and developed a method to pre-treat them in a form suitable for the dopinganalysis method being used. In the future, we will use this proposed method to reveal the possibility of the existence of doping agents in mostfood ingredients and to establish a ‘food ingredients doping analysis model’ that can be practically analyzed in athletes' villages.

Keywords : Inadvertent doping, anabolic hormones, food contamination

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G-11

Three-Dimensional Structure Analysis of PAT Protein- Aptamer Complex by X-Ray Diffraction

Jin-Pyo Lee1, Woo-Ri Shin1, Simranjeet Singh Sekhon1, Sung Min Woo2,Ji-Young Ahn1, and Yang-Hoon Kim1*

1Major in Microbiology School of Biological Sciences College of Natural Sciences Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, Chungbuk 28644, Republic of Korea 2Department of Food Science and Biotechnology, Shin Ansan University, 135 Sinansandaehak-Ro, Danwon-Gu, Ansan 15435, Republic of Korea

GM crops give us advantages such as resistance to pests and improvedproductivity, but they cause various side effects such as stability and environmental pollution. A detection system for monitoring GM cropsis important. We applied it to the detection of GM crops using an aptamerthat recognizes and binds to the structure of various targets such as proteins, and compounds. We selected the aptamer that specifically bindsthe herbicide-resistant protein Phosphinothricin N-acetyltransferase (PAT), using the SELEX method. PAT protein was crystallized to confirminteractions through structural analysis. The Bar gene was cloned in pET-21a and expressed by E.coli BL21 (DE3). The protein was purifiedby three-step chromatography: Affinity, Ion-exchange and Gel filtrationchromatography. Sitting drop vapor diffusion was used for crystallizationat 291K temperature. Analyzing the structure of protein-aptamer complex, can improve the specificity of aptamer to the target.

Keywords : PAT protein, aptamer, protein-aptamer complex

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2020R1A6A1A06046235)] [This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2020R1A2C1009463)]

G-12

Characterization and Employment of a Novel Salmonella-Specific Phage Isolated from Slaughter Wastewater

Heejeong Lee, Su-Hyeon Kim, Yu-Bin Jeon, and Mi-Kyung Park*

School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

The purpose of this study was to isolate, characterize, and employ a Salmonella-specific phage as a green approach. Salmonella-specific phage was isolated and purified from a slaughter wastewater. Its morphology was observed by TEM for viral classification. The pH andtemperature stabilities of the phage were investigated by exposure to various pHs (1 to 12) and temperatures (–70 to 70℃). The specificityof the phage was determined against 43 pathogens by a dot assay. Phageefficiency was investigated by in vitro challenge assay at a MOI of 0.01,0.1, 1.0, 10, and 100. Employment of phage to control Salmonellacontamination in fresh produce was investigated using onion as a modelsystem. The phage was highly specific to only Salmonella spp. and belonged to the Myoviridae family with icosahedral head (105.79 ± 11.21nm) and contractile tail (115.38 ± 30.96 nm) lengths. The phage was stable in wide pHs (3-11) and temperatures (–20-60℃). The phage’s efficiency in vitro was maintained up to 8 h even at MOI of 0.1. The number of Salmonella was significantly reduced by 3.8 ± 0.5 log CFU/gin onion when treated with Salmonella-specific phage. This study demonstrated that the Salmonella-specific phage is promising as a biocontrol agent against Salmonella in fresh produce applications.

Keywords : Phage, Salmonella, green approach

G-13

Characterization of the Family Tectiviridae Bacteriophage Infecting Acinetobacter baumannii

Jae Hyung Kim, Soyul Im, and Minsik Kim*

Department of Food and Nutrition, Brain Korea 21 FOUR, College of Human Ecology, Yonsei University, Seoul 03722, Republic of Korea

Acinetobacter baumannii is a coccobacillus Gram-negative bacterium,causes persistent nosocomial infections such as pneumonia and bacteremia. Along with a broad spectrum of antibiotic resistance, problem of A. baumannii has been more complicated due to isolation ofthe pathogen from foods and environment recently. In this study, an A.baumannii-infecting bacteriophage PhiAB03 from sewage was characterized for the purpose of development of novel antimicrobials. Based on morphological and genomic analysis, PhiAB03 classified intothe family Tectiviridae featuring with small dsDNA genome envelopedby inner proteinaceous lipid membrane and outer icosahedral capsid, andflexible spike protein on the capsid. Seven out of 17 predicted ORFs from~14 kb genome encoded structural and assembly/packaging-related proteins, while holin and DNA polymerase were also found. PhiAB03 could specifically infect 13 foodborne Acinetobacter spp. isolates showing intermediate resistance against cefotaxime among 75 strains tested as well as A. baumannii ATCC 17978. The infectivity of PhiAB03was maintained in varied conditions, ranged from 4 to 50℃, and pH 2 to 11. After further analysis on the host lytic properties, PhiAB03 couldbe developed as one of antimicrobial candidates to control A. baumannii.

Keywords : Bacteriophage, Acinetobacter baumannii, Tectiviridae

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G-14

Characterization of Bacteriophage PBCC01 Isolated from Retail Carrot for Bacillus cereus Control

Hanbin Seol, and Minsik Kim*

Department of Food and Nutrition, Brain Korea 21 FOUR, College of Human Ecology, Yonsei University, Seoul 03722, Republic of Korea

Bacillus cereus, one of major foodborne pathogens producing enteric and emetic toxin, are found anywhere including foods. Due to commons’reluctance to chemicals and wide spread of drug-resistance, bacteriophagehas recently got a spotlight again as alternative antimicrobials. Here, weisolated B. cereus-infecting phage PBCC01 from retail carrots and characterized it to examine potential as novel antimicrobials. Phage PBCC01 belonging to the family Myoviridae could infect 5 B. cereus strains including 2 food isolates among 13 strains tested. Phage infectivity was stably maintained from 4 to 60℃ and from pH 5 to 10for an hour. About 55% of phages were attached to the host B. cereusATCC 27348 in 5 min with divalent cations. Eclipse and latent periodsof PBCC01 were determined as 20 and 40 min, respectively, and the burstsize was calculated as ~600 PFU/infected cell. Host bacterial growth was effectively inhibited for 7 (MOI of 0.1-1) to 12 hr (MOI of 10) by PBCC01treatment. Two putative enzymes, which probably hydrolyze the host peptidoglycan layers, were predicted from 94.6-kb genome, indicatingthe potential as useful enzybiotics. Altogether, phage PBCC01 and its lytic enzymes could be developed as promising candidates of alternativebiocontrol agents against B. cereus.

Keywords : Bacteriophage, Bacillus cereus, antimicrobials

G-15

Improvement of Thermal Stability by Adaptive Evolution of Salmonella Phage

Jieon Lee, Doyeon Kim, and Minsik Kim*

Department of Food and Nutrition, Brain Korea 21 FOUR, College of Human Ecology, Yonsei University, Seoul 03722, Republic of Korea

Phages with lytic activity against pathogens are considered as alternativeantimicrobials. Wild-type (WT) phages isolated from nature, however,are often inappropriate to apply in certain practical settings such as thermal processing, due to inadequate characteristics. Here, we adaptively evolved (AE) the newly isolated Salmonella phage JE01 by repeated challenges against sub-lethal heat treatment to exhibit improved thermal stability. The infectivity reduction of WT JE01 wasmore than 4-log PFU/ml at 73 or 74℃ for an hour, but that of AE phagesthrough sequential repeated exposure to the same temperature was onlyabout 1-log. Genomic analysis revealed several missense mutations in AE phages compared to WT. Interestingly, all the mutations were placed at four genes including orf61 for putative tape measure protein, orf65for tail fiber protein, and adjacent orf63 for hypothetical protein, implying the association of phage tail module with the enhanced thermal stability. The infectivity and adsorption rate of AE phages was similarto the WT, though reduction of burst size (~60%) and shorten of latentperiod (~33%) might be interchanged as fitness costs. Taken together, this adaptive evolution strategy could be applied to other WT phages to improve their usability as antimicrobials.

Keywords : Bacteriophage, adaptation, antimicrobials

G-16

Enhanced Staphylococci Lytic Activity of Bacteriophage by Repeated Chemical Challenges

Hyoju Choi, and Minsik Kim*

Department of Food and Nutrition, Brain Korea 21 FOUR, College of Human Ecology, Yonsei University, Seoul 03722, Republic of Korea

As bacteriophage therapy has regained attention for recent decades as alternative control measures for pathogens, there have been numerous approaches of engineering phages to promote therapeutic efficiency. Here, we analyzed a Staphylococcus aureus-specific phage with enhanced bactericidal activity generated by repeated challenges of sodium pyrophosphate. After 30 times of sequential treatment of phageSA3821 with the chemical and subsequent screening, the surviving phage SA3821M was isolated. SA3821M showed increased thermal tolerance and improved lytic activity against its host compared to the parental SA3821. As sodium pyrophosphate, a chelating agent, has beenreported to cause genetic changes in virions, the SA3821M genome hadvarious mutations and deletions compared to the SA3821 genome. Especially, the majority of genetic changes (60.6%) were placed at theregion encoding tail tape measure protein (TMP), suggesting that thisprotein dictating a tail length and facilitating the injection of viral DNAinto the host might be the hot key for the enhanced lytic activity of SA3821M. Based on the results, we propose the applicability of simplepractical challenges of sodium pyrophosphate to obtain more effectivephage with mutations in host-interacting apparatus to fight pathogens.

Keywords : Bacteriophage, tail tape measure protein, Staphylococcus aureus

G-17

Control of Foodborne Acinetobacter spp. Using Bacteriophage Cocktail

Soyul Im, Doyeon Kim, and Minsik Kim*

Department of Food and Nutrition, Brain Korea 21 FOUR, College of Human Ecology, Yonsei University, Seoul 03722, Republic of Korea

Acinetobacter spp. has become a global threat due to its high antibioticresistance rate. They are a major cause of nosocomial infections and werealso have been detected in foods recently, requiring urgent alternative control means. Here, we identified 41 Acinetobacter isolates in 7 different species from distributed raw foods in Korea and applied a phagecocktail as an alternative biocontrol agent. Most isolates (95.1%) showedfull or intermediate resistance to cefotaxime, and 12.2% of isolates werenonsusceptible to even “the last-line antibiotics,” carbapenems, in diskdiffusion susceptibility test. Myoviral phage PhiAB01 and podoviral phage PhiAB02 isolated from sewage could simultaneously infect the food isolates A. calcoaceticus YMFM 405 and A. baumannii YMFM 601. Phage cocktail consisting of PhiAB01 and PhiAB02 effectively inhibited the bacterial growth in vitro for 12 h (MOI=10), and increasedthe survival rate of Galleria mellonella larvae from YMFM 601 infectionin vivo for 6 d (MOI=1, 10). In addition, the pathogen loads from artificially spiked ground pork with RIFR YMFM 601 were significantlyreduced through 6 h by the phage cocktail at both 4℃ and 25℃. Takentogether, the developed phage cocktail could be a candidate agent to biocontrol foodborne drug-insensitive Acinetobacter spp.

Keywords : Bacteriophage, Acinetobacter spp., biocontrol

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G-18

Construction of Gene Deletion Derivative of Cronobacter sakazakii-Specific Lytic Phage

Doyeon Kim, and Minsik Kim*

Department of Food and Nutrition, Brain Korea 21 FOUR, College of Human Ecology, Yonsei University, Seoul 03722, Republic of Korea

Although genetically modified bacteriophages have been received increased attentions to enhance its applicability, engineering of lytic phage is challenging due to its short residence time in host bacteria as well as limited encapsulation capacity of the head. Here, we applied a CRISPR-Cas9 system to construct an infectious gene deletion mutant of virulent phage HJC01 inhibiting Cronobacter sakazakii. Phage HJC01 has 49 kb dsDNA genome and functions of most predicted genes(56%) are unknown. Through orf22-targeting spacer-mediated dsDNAbreak and subsequent homologous recombination, orf21, 23 (hypotheticalproteins) and orf22 (Dam methylase) were erased from the HJC01 genome. The resultant mutant HJC01Δ21_23 was not altered significantlyfrom the HJC01 in particle morphology and host infection manner. However, the burst size was decreased by 40% along with 5 min-delayedburst time, and consequently resulting in smaller plaques. Since the deleted genes were non-essential for phage viability, other foreign genessuch as reporter cassettes can be introduced into the room given by thedeletion to develop useful engineered HJC01 derivatives. The strategyused in this study also could be applied to other virulent phages to engineer their applicability to fight with the pathogens.

Keywords : Bacteriophage, Cronobacter sakazakii, CRISPR

G-19

Isolation and Characterization of Bacteriophages for Control of Pathogenic Escherichia coli

Taehee Won1, Jinshil Kim1,2, Eunbyeol Ahn1, and Sangryeol Ryu1,2*

1Department of Food and Animal Biotechnology, Department of AgriculturalBiotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Pathogenic Escherichia coli is well-known for causing a variety of serious diseases in humans and global emergence of antibiotics-resistantpathogenic E. coli necessitates development of alternatives to antibiotics.Bacteriophages (phages) are one of the promising alternatives to antibiotics but they typically have a narrow host range that phage cocktails are generally used for phage therapy to broaden host ranges of phages. In this study, two phages (ELT1 and ELT3) were isolated fromretail raw chicken meats. Both phages were classified as the Myoviridaefamily based on the transmission electron microscopy. ELT1 has 109 ±5 nm tail and 90 ± 5 nm head and ELT3 has 105 ± 5 nm tails and 91 ± 5 nm head. The whole-genome sequencing results demonstrated that thegenome size of phages ELT1 and ELT3 is 170,727 bp and 163,510 bp, respectively. Each phage can infect 11 strains out of 18 pathogenic E. coli strains including enterohemorrhagic E. coli and enterotoxigenic E.coli and combining these two phages can control 15 out of 18 strains tested, suggesting that these phages can be useful components for phagecocktail against pathogenic E. coli.

Keywords : Pathogenic Escherichia. coli, bacteriophage, phage cocktail

G-20

Survival and Growth Characteristics of Foodborne Pathogen in Romaine Lettuce

Nayeseul Kim, Chaerin Kim, Dawoon Kim, Myung-In Jeong, Kwang KyoOh, Bo-Eun Kim, Jae-Gee Ryu, Iksung Jeon, and Kyoung-Yul Ryu*

Microbial Safety Team, Agro-Food Safety & Crop Protection Department, National Institute of Agricultural Science (NIAS), Rural Development Administration (RDA), Wanju, Jeollabuk-do 55365, Republic of Korea

Foodborne pathogens exist in plant tissue for survival and growth. Preliminary experiments on the localization and growth in lettuce leaf surface revealed an increased (1.0 log CFU/mL) recovery of pathogen.It was no difference in pathogen population when compared to leaves with and without wound. Most of the inoculated pathogen existed insidethe wounded leaf but rarely on rough surface of the unwounded leaf. Since foodborne pathogens utilize nutrients for survival and growth from fresh vegetables, the romaine lettuce extracts was prepared to determinethe minimal concentration for survival and growth of foodborne pathogens. Escherichia coli O157:H7, Listeria monocytogenes and Pectobacterium carotovorum grew vigorously in romaine lettuce extracts at 100%, 10%, 1% and the density of the pathogens reached to 4-5 log CFU/mL. At 0.1% concentration, the density of E. coli O157:H7,L. monocytogenes and P. carotovorum were 2.74, 1.29 and 2.91 log CFU/mL respectively. The result suggested that foodborne pathogens can survive and growth when contacted with romaine leaf extract under0.1% concentration. Therefore it is very important to increase the safetyby minimize the wound during harvesting, storage and distribution of lettuce.

Keywords : Foodborne pathogen, romaine wound, survival

G-21

Comparison of Pathogenicity of Pectobacterium carotovorum Isolated from Potato, Chinese Cabbage and Lettuce

Dawoon Kim, Nayeseul Kim, Chaerin Kim, Myung-In Jeong, Kwang KyoOh, Bo-Eun Kim, Jae-Gee Ryu, Iksung Jeon, and Kyoung-Yul Ryu*

Microbial Safety Team, Agro-Food Safety & Crop Protection Department, National Institute of Agricultural Science (NIAS), Rural Development Administration (RDA), Wanju, Jeollabuk-do 55365, Republic of Korea

Pectobacterium carotovorum deteriorates the quality of most vegetablescausing typical soft rot symptom. The pathogenicity of 36 P. carotovorumisolates from different hosts was investigated by inoculating the pathogen (7 log/ml) into potatoes, carrots, Chinese cabbage and lettuceand the development of soft rot symptom were evaluated after 24h incubation at 28℃ and 10℃. The degree of pathogenicity was classifiedinto strong, intermediate and weak based on the development soft rot symptom. Strong symptom occurred in order of potatoes (95.5%) > carrots (18.2%) > lettuce (4.5%) > Chinese cabbage (4.5%) while the aggressiveness of pathogen was diverse. Most isolates showed strong symptom at 30℃ and the incidence of typical symptom was different from 100% in potato to 55.6% in lettuce. However, the aggressivenessof pathogen was lowered from 58.3% in potato to 16.7% in carrot and Chinese cabbage at 10℃. Also, Chinese cabbage isolates collected fromHaenam area revealed strong pathogenicity to all the vegetables and theincidence was 100% in potato and 82% in lettuce. But the lettuce isolatescollected from Gimje area showed less aggressiveness in lettuce (1.3%)and Chinese cabbage (8.8%). These results are expected to be used as basic data for characterization of Pectobacterium strains and evaluation of antibiotics susceptibility in Korea.

Keywords : Pectobacterium, pathogenicity, safety

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G-22

Construction of Synthetic Reporter Strain for Screening of EHEC Virulence Inhibitors

Sin Young Hong, and Byoung Sik Kim*

Department of Food Science and Engineering, ELTEC College of Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes attaching and effacing lesions on intestinal epithelial cells. Since production of Shiga toxin is enhanced upon antibiotic treatments,alternative agents controlling EHEC strains are of necessary. The aim of this study is to find alternatives that target virulence factors and reducethe toxicity of EHEC. The locus of enterocyte effacement (LEE) is essential for the virulence of EHEC, and most of the LEE genes are positively regulated by a Ler protein. GrlA, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. Indeed,relative gene expression of lee genes was decreased in grlA mutant compared with the wild-type. To construct a synthetic reporter strain forscreening of GrlA inhibitors, E. coli DH5α was co-transformed with pSY2101, carrying the arabinose-inducible grlA gene, and with pSY2104, a reporter plasmid carrying the ler promoter fused with luxoperon. When the reporter strain was treated with varying amounts of L-arabinose, a concentration-dependent bioluminescence was observed.This result suggests that the constructed synthetic reporter strain can beused for future high throughput screening to find out GrlA inhibitors diminishing virulence of EHEC.

Keywords : EHEC, Inhibitor, reporter strain

G-23

Characteristics and Antibiotic Resistance of Bacillus cereus Isolated from Agricultural Environment

Jieun Jung, Hyeon-Suk Jin, Seung-Mi Seo, Kwang Kyo Oh, Myeong-InJeong, Bo-Eun Kim, Jae-Gee Ryu, and Kyoung-Yul Ryu*

Microbial Safety Team, Agro-food Safety & Crop Protection Department, National Institute of Agricultural Science (NIAS), Rural Development Administration (RDA), Wanju, Jeollabuk-do 55365, Republic of Korea

This study aimed to investigate the enterotoxigenic characteristics and antibiotic resistance of 162 B. cereus isolated from garlic chives, soils,compost, and irrigation water. Isolation rate of B. cereus was from 0.63 to 4.39 log CFU/g in garlic chives, soil, composts, and irrigation water.In 103 isolates from garlic chives, soil, compost and irrigation waterhaving β-hemolytic activity, 45.6 and 74.8% of B. cereus possessed hemolytic enterotoxin genes (hblACD) and non-hemolytic enterotoxingenes (nheABC), respectively. However, 100% of B. cereus isolates from irrigation water had six enterotoxin genes. All of B. cereus isolates showed sensitive response against 7 non β-lactam antibiotics. However,32% of B. cereus isolated from garlic chives and soil showed intermediateresistance to rifampin, clindamycin, erythromycin, and tetracycline. 12.6% of B. cereus isolated from compost and irrigation water had intermediate resistance to rifampin, clindamycin, and tetracycline. Irrigation water was considered to be major source of B. cereuscontaining multiple toxin genes and exhibiting intermediate resistanceto non β-lactam antibiotics. Therefore, it suggests that continuous monitoring against contamination pathway of B. cereus in agriculturalenvironment for the safety.

Keywords : Bacillus cereus, enterotoxin, antibiotic resistance

G-24

Construction of Reporter Strain for Screening Inhibitors that Control Curli Fimbriae Expression in EHEC

Jihye Park, and Byoung Sik Kim*

Department of Food Science and Engineering, ELTEC College of Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Novel approaches to inhibit virulence of foodborne pathogens are needed for the emergence of antibiotic-resistant pathogens. An adhesivefactor that is responsible for surface attachment is one of the Enterohemorrhagic Escherichia coli (EHEC) virulence factors. Curli, adhesive fimbriae of EHEC, is essential for biofilm formation and celladhesion. CsgD is a major transcriptional regulator and positively regulates the expression of curli fimbriae by directly binding to the regulatory region of curli genes. In this study, we constructed a reporterstrain to identify inhibitors that prevent EHEC infections. To constructa reporter strain, E. coli DH5αwas co-transformed with an arabinose- inducible CsgD expression plasmid and a CsgD-activated lux reporter plasmid. We demonstrated that relative luminescence was increased dependently on the concentration of adding arabinose. This result suggests that the constructed reporter strain can be used to screen CsgD specific inhibitors reducing the adherence of EHEC on foods.

Keywords : EHEC, adhesion, inhibitor

G-25

Antibacterial Activity of Natural Preservatives against Staphylococcus aureus by Using Statistical Experimental Design

Yeong Jin Park, Do-Un Lee, Cho Eun Kang, Na-Kyoung Lee, and Hyun-Dong Paik*

Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 05029, Republic of Korea 2WithBio, InC., Neungdongro 120, Seoul 05029, Republic of Korea

Staphylococcus aureus is one of the most common food and skin pathogens which causes gastroenteritis and skin infections. Methicillin-resistant S. aureus (MRSA) is resistant to some antibiotics like penicillinand associated with more health problems. Therefore, natural preservatives for effectively inhibiting MRSA and for human safety arenecessary. In this study, hydrothermal extracts and 70 % ethanol extractsof cinnamon, onion peel, and chestnut inner shell and ε - polylysine (PL)were used to screening the antibacterial activity against a cocktail of threestrains of S. aureus (ATCC 25923, ATCC 33594, and ATCC 33591). TheMIC of each preservative against the cocktail of three strains of S. aureuswere 4, 8, 16, 4, 8, 8, and 1 mg/mL. Following Plackett-Burman Design,two natural preservatives and PL were selected for the next statistical experimental design. The combination of these natural preservatives increased the antibacterial effect on S. aureus more than a single kindof natural preservatives. Based on these results, the synergistic effect ofmultiple natural preservatives including PL is confirmed and using of these complex preservatives can be applied in food and cosmetics industry.

Keywords : Staphylococcus aureus, natural preservatives, statistical experimental design

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G-26

Isolation and Characterization of Novel Bacteriophages BS1 and BS3 Infecting Bacillus cereus

Su Min Son1, Jinshil Kim1,2, and Sangryeol Ryu1,2* 1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Bacillus cereus is a spore forming bacterium that produces numerous toxins. Recently, imprudent use of antibiotics has led to prevalence of drug-resistant bacteria, which is a huge threat to human health. Bacteriophage(phage) is a natural antimicrobial agent that can control multidrug resistant bacteria. Our goal is to isolate and characterize novel phages specific for B. cereus. Phages BS1 and BS3 were isolated from sludgeand vegetables, respectively. Both phages were classified as the Myoviridaefamily based on transmission electron microscopy. Whole-genome sequencing revealed that phages BS1 and BS3 have 160,629 bp and 158,241 bp long circular DNA, respectively. When exponentially growingB. cereus ATCC 14579 was infected with phage BS1 [multiplicity of infection (MOI) of 0.1] or phage BS3 (MOI of 1), reduction in opticaldensity at 600 nm was observed within 3 hours and the inhibition activitywas maintained up to 12 hours. This study concludes that phages BS1 and BS3 are promising biocontrol agents against B. cereus.

Keywords : Bacillus cereus, bacteriophage, biocontrol

[This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (ProjectNo. PJ01574702)" Rural Development Administration, Republic of Korea]

G-27

Development of Novel Endolysin Active against Gram-Negative Bacteria

JoonBeom Kim1, Jinwoo Kim1, and Sangryeol Ryu1,2* 1Department of Food and Animal Biotechnology, Department of AgriculturalBiotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

The emergence of antibiotics-resistant bacteria has caused serious globalpublic health problems. Endolysin, a bacteriophage-derived cell wall-degrading enzyme, is drawing attention as a promising solution forcontrolling them. While endolysin has been successfully used to controlGram-positive bacteria, it is difficult to use native endolysin against Gram-negative bacteria because the peptidoglycan layer of Gram-negativebacteria is protected by the outer membrane. In this study, we tried to make recombinant endolysins by fusing endolysin with various bacteriophage-derived transmembrane domains that have possibility of translocating Gram-negative bacterial outer membrane. We selected 8 candidate genes from SPN1S phage genome and constructed 24 recombinant endolysins by fusing them with SPN1S endolysin or B4 endolysin. The antibacterial activities of fusion proteins against Escherichia coli MG1655, Salmonella Typhimurium LT2 were compared and several of them were found to have noticeable antibacterial effect against the Gram-negative bacteria. These results suggest that many unknown genes from phages may expand the scope of endolysin engineering for developing endolysin active against the Gram-negative bacteria.

Keywords : Gram-negative bacteria, recombinant endolysin

G-28

Application of the Broad Spectrum Lytic Phage HJP1 to Control Multidrug-Resistant Salmonella Isolates from Retail Raw Chicken

Haejoon Park1, Jinshil Kim1, Hyeongsoon Kim1, Eunshin Cho1, HyeeunPark1, and Sangryeol Ryu1,2* 1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Multidrug-resistant (MDR) Salmonella has been recognized as a leadingcause of foodborne diseases that threaten public health. Biofilms formedby MDR Salmonella in food processing facilities are a source of cross-contamination of foods. In this context, the use of lytic bacteriophages (phages) has been re-highlighted as a biocontrol agent that can simultaneously control MDR Salmonella and its biofilms. In thisstudy, we isolated a lytic siphophage HJP1 from retail raw chicken andused it to control MDR Salmonella strains that have a strong capabilityof forming biofilms. HJP1 phage exhibited extensive lytic activity against MDR Salmonella and robustness to withstand a wide range ofpH (4-12) and temperature (30-60℃). On stainless steel and glass, the HJP1 phage was able to inhibit the biofilm formation by 53.9-56.8% and reduce preformed biofilms by around 47.4-48.5%. Furthermore, a foodapplication test on chicken meat and milk showed that HJP1 phage effectively reduced cross-contamination by MDR Salmonella Thompsonfrom stainless steel to an undetectable level. Our findings suggest that the HJP1 phage is a promising antimicrobial agent for the control of biofilm formation and cross-contamination by MDR Salmonella in thefood industry.

Keywords : Foodborne pathogen, multi-drug resistant, bacteriophage

G-29

Characterization of a Vibrio vulnificus Mutant Deficient in Putative fimZ Gene which Only Present in Clinical Genotype (biotype 1C)

Joon-GI Kwon1, Jeong-Eun Kwak2, Do-Hee You2, Ho-Jeong Choi2, andJu-Hoon Lee1,2,3* 1Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea 3Department of Food and Animal Biotechnology, Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Vibrio vulnificus is the pathogen isolated from the estuarine waters andvariety of seafood. It has been subdivided into three biotypes. Biotype 1 is responsible for the majority of human infections. In addition, biotype 1 are composed of two genotypes: a clinical genotype (biotype 1C), and an environmental genotype (biotype 1E). To identify their virulence factors and clinical characteristics, complete genome sequences of clinical genotype and environmental type were obtained from the NCBI database and compared genome sequences using pan-genome approach.Subsequent pan-genome analysis revealed that biotype 1C have putative fimZ gene, but biotype 1E doesn’t have. The putative fimZ gene deficientmutant loss their ampicillin and vancomycin resistance and showed a colonial morphology change from opaque form to translucent form. Also, the mutant type displayed significantly reduced expression of CPSassociated genes. A virulent opaque form produces capsular polysaccharide(CPS) and a translucent phenotype produces little or no CPS. Moreover,the mutant type formed significantly more biofilm than wild type. Theseresults suggest that putative fimZ gene affect the CPS production of clinical strains and indicate that this gene may be associated with virulence factor of clinical strains.

Keywords : Vibrio vulnificus, clinical genotype, food-borne pathogen

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G-30

The Role of yeiE Gene in Resistance to Sulfite and Oxidative Stresses in Cronobacter sakazakii ATCC 29544

Eunshin Cho1, Jinshil Kim1,2, Seokho Hong1, Nam-Chul Ha1, and Sangryeol Ryu1,2* 1Department of Food and Animal Biotechnology, Department of AgriculturalBiotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Cronobacter sakazakii is a gram-negative pathogen that causes meningitisand sepsis in neonates. Previously, yeiE, a LysR-type transcriptional regulator, has been demonstrated to play an essential role in invasion intoepithelial cells, biofilm formation, and resistance to oxidative stress inC. sakazakii. Analysis of the crystal structure of YeiE revealed that sulfitewas supposed to act as a ligand. Here, we investigated the effects of yeiEon the defense against sulfite and oxidative stresses in C. sakazakii. Wecompared the growth of the yeiE mutant in M9 media containing MgCl2instead of MgSO4 in the presence of varying amounts of sulfite. The checkerboard assay results using sulfite and H2O2 showed that the resistance of C. sakazakii to H2O2 increased depending on the amount of sulfite. The yeiE mutant exhibited growth defect compared to the wild type in the presence of 0.5 mM or more sulfite. Furthermore, the yeiE mutant had 32-folds higher sensitivity to sulfite and 2-folds higher sensitivity to H2O2. The gas generation by the yeiE mutant increased compared to the wild-type in the presence of 100 mM H2O2, indicatingthat yeiE mutation caused a higher catalase activity in C. sakazakii. These results suggest that yeiE plays a crucial role in the bacterial resistance to sulfite and oxidative stresses.

Keywords : yeiE, sulfite, oxidative stress

G-31

Selective Isolation of Campylobacter Using Bacteriophages

Jinshil Kim1,2, Jeong In Hur1, Haejoon Park1, Sangryeol Ryu1,2*, and Byeonghwa Jeon3* 1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea 3Environmental Health Sciences, School of Public Health, University of Minnesota, St Paul, MN 55108, USA

Due to the fastidious nature, the isolation of Campylobacter is often hampered by fast-growing competing bacteria. Especially, extended- spectrum beta-lactamase (ESBL)-producing Escherichia coli has beenreported to affect the isolation of Campylobacter. To address the issue,we developed a new isolation method for Campylobacter using a bacteriophage (phage) cocktail capable of inhibiting ESBL-producing E. coli. Five phages (JEP1, 4, 6, 7, and 8) were screened and used to construct a phage cocktail. Interestingly, the phages exhibited differentlevels of infection efficiency depending on the phylogenetic group of E. coli. The combination of phages targeting the different phylogeneticgroups of ESBL-producing E. coli increased the infection frequency ashigh as 91%. Moreover, the phage cocktail showed antimicrobial synergy against ESBL-producing E. coli in combination with the antibiotics of Campylobacter-selective supplements. Also, the isolationprocedures were further optimized by adjusting culture conditions, suchas temperatures and selective media combined with the phage cocktail.This is the first demonstration showing that phages can be utilized as antimicrobial supplements to improve selective isolation of target bacteria by eliminating their bacterial competitors.

Keywords : Bacteriophage, Campylobacter, antimicrobial-resistant Escherichia coli

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G-32

Characterization of Novel Staphylococcus aureus Bacteriophage DSP11 and DSP12

Deokwoo Bae1, Chanyoung Lee2, and Sangryeol Ryu1,2* 1Department of Food and Animal Biotechnology, Department of AgriculturalBiotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Staphylococcus aureus is a Gram-positive pathogen that causes food poisoning. With the emergence of antibiotic-resistance bacteria, such as methicillin-resistant S. aureus (MRSA), the bacteriophages and its derived protein, endolysins, are emerging as alternatives to antibiotics.In this study, staphylococcal bacteriophages DSP11 and DSP12 were isolated from the animal feces and pig internal organ, respectively. Transmission electron microscopy (TEM) analysis revealed that both bacteriophages belong to the Siphoviridae family with an isometric headand a non-contractile tail. To determine the ability of the phages to suppress growth of bacteria, the optical densities of the S. aureus RN4220cultures infected with DSP11 or DSP12 were monitored. DSP11 showedthe best growth inhibition ability when MOI of 0.1 was used; the growthinhibition started 3 h post-infection and maintained up to 24 h. DSP12 showed the best growth inhibition ability when MOI of 10 was used; thegrowth inhibition started 1 h post-infection and persisted up to 10 h. Bothbacteriophages showed strong inhibition zones against various strains of S. aureus even though their host ranges are comparatively narrow, suggesting that their endolysins may have a greater potential as a biocontrol agent.

Keywords : Staphylococcus aureus, bacteriophage

G-33

Effects of mcr-1 on Virulence of Enterohemorrhagic Escherichia coli ATCC 43894

Eunbyeol Ahn1, Jinshil Kim1,2, Byeonghwa Jeon3*, and Sangryeol Ryu1,2*

1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea 3Environmental Health Sciences, School of Public Health, University of Minnesota, St Paul, MN 55108, USA

Dissemination of the mobilized colistin resistance (mcr) gene by plasmids in pathogenic Escherichia coli is a concern to global public health. In this study, we investigated the impact of mcr-1 gene on the virulence of enterohemorrhagic E. coli (EHEC) by comparing EHEC harboring plasmid containing mcr-1 gene and its isogenic mcr-1 mutant.The mcr-1-harboring plasmids were easily transferred to EHEC ATCC43894 by conjugation and remained stable in ATCC 43894 even after 10 consecutive subcultures. Although the acquisition of mcr-1 did notaffect the growth of ATCC 43894, it decreased the ability of biofilm formation and swimming motility. We also found that the mcr-1increased bacterial adherence to HEp-2 cells while it decreased invasiveness of ATCC 43894. The acquisition of the mcr-1 in ATCC 43894 made the cell surface more hydrophilic. Interestingly, band patterns of core-lipid A region and O-units analyzed by deoxycholate- polyacrylamide gel electrophoresis (DOC-PAGE) were altered by the mcr-1, suggesting that the mcr-1 modified the polysaccharide as well aslipid A. Collectively, our results propose that the acquisition of mcr-1not only conferred colistin resistance but also can affect virulence in ATCC 43894.

Keywords : mcr-1, enterohemorrhagic Escherichia coli, antibiotic resistance

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G-34

Virulent Phage Engineering Using Type I-E CRISPR-Cas System for Efficient Control of Pectobacterium carotovorum

Hyeongsoon Kim1, and Sangryeol Ryu1,2* 1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Virulent (lytic) phages are promising antimicrobials to control variousantibiotics-resistant pathogenic bacteria including plant pathogens. Lytic phages are normally used for biocontrol because gene transfer is lower compared with the temperate phages. However, no efficient gene-editing tool is available for engineering of virulent phages inPectobacterium carotovorum (Pc). In this study, we tested whether thelysis cassette genes from phage POP72 that has superior lytic activity could enhance the lytic activity of the virulent phage PK10. PK10 genome is edited by homologous recombination with a donor plasmid harboring the desired gene and homologous flanking arms. Efficiency of recombinant phage screening was enhanced by overexpression of endogenous type I-E CRISPR-Cas system of Pc. The POP72_53 genefrom POP72 phage was inserted downstream of the gene encoding majorcapsid protein of PK10 genome and the PK10 harboring the POP72_53gene showed higher lytic activity against Pc. The simple gene-editing method using type I-E CRISPR-Cas system of Pc would be a useful tool to enhance the antibacterial properties of virulent phages for efficient control of Pc.

Keywords : Virulent phage engineering, CRISPR-Cas system, Pectobacteriumcarotovorum

G-35

Characterization and Application of a New Shigella flexneri 2a Infecting Phage SFP20 for Food Safety

Ho-Jeong Choi1, and Ju-Hoon Lee1,2* 1Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea 2Department of Food and Animal Biotechnology, Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Shigella is an important causative agent of Shigellosis causing diarrhea.To control this pathogen, a new novel lytic bacteriophage SFP20 was isolated from a sewage sample. Host range assay showed that SFP20 specifically infect S. flexneri 2a strain 2457T. TEM morphology observation revealed that SFP20 belongs to the family Podoviridae. One-step growth analysis revealed 5 min eclipse time, 10 min latent time,and 20.3 PFU/cell burst size. In addition, Challenge assay showed 3.95log reduction of S. flexneri 2a in 2 h. The stability test showed it is stableat the range of temperature (-20 to 60℃) and pH (5 to 11). The completegenome sequence of SFP20 consists of 39.5-kb containing 47 (ORFs) without gene associated to toxin, and virulence factors. There was no infection of rfaL gene knock-outed mutant of host, suggesting that O-antigen of LPS is the host receptor for its infection. To apply this phagefor food safety, moreover, SFP20 was treated to fresh cabbage and milksamples contaminated with S. flexneri 2a. This phage treatment with 106

CFU/ml of S. flexneri 2a showed that no detection of S. flexneri 2a upto 6 h in the cabbage (MOI = 105) and significant reductions in milk, suggesting that SFP20 can be used as a natural food preservative in various food applications.

Keywords : Shigella flexneri, bacteriophage, food-borne pathogen

G-36

Evaluation of Real-Time PCR Detection Method of Diarrheal and Emetic Bacillus cereus

Won Jeong Park, Ga Yeon Lim, Min Jung Lee, Jinhee Hwang, and Soon Han Kim*

Food Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Cheongju, Republic of Korea

Bacillus cereus cause diarrheal and vomiting type food poisoning. Symptoms are generally mild and short-lived. It is widespread in natureand considered prevalence in products of raw grains. To develop a sensitive and reliable real-time PCR methods for detection of hblD(enterotoxin) and ces (emetic toxin), specific primers and probe sets weredesigned. The inclusivity and exclusivity of real-time PCR methods wasevaluated using 31 strains (14 enterotoxic and 17 emetic strains) and 82non-target strains. To determined the sensitivity and linearity of real-timePCR, standard curves of hblD and ces were generated using gene synthesis products. As the result, real-time PCR method of hblD and ceswere shown to be highly specific and sensitive for target strains. Thesewere 100% inclusive for target B. cereus and 100% exclusive for non-target B. cereus. Both methods had estimated detection limits of 100

copies. The accuracy of methods was high (R2 > 0.99) for all three diluentsbased on linear regression analysis. This study provide more efficient detection methods of diarrheal and emetic B. cereus.

Keywords : Bacillus cereus, enterotoxin, emetic toxin, real-time PCRmethod

G-37

Characterization and Genomic Analysis of a Geobacillus stearothermophilus Bacteriophage GR1

Dahee Choi, and Minsuk Kong*

Department of Food Science and Technology, Seoultech, Seoul 01811, Republic of Korea

Geobacillus stearothermophilus is a thermophilic, spore-forming bacterium, which is mainly responsible for flat sour spoilage of dairyproducts and low-acid canned foods. Due to its thermal resistance characteristics of spores, the rapid detection of this bacterium is essential to the food industry. To identify a novel bioprobe for G. stearothermophilus,we isolated a bacteriophage GR1 from roadside soil. Transmission electron microscopy (TEM) studies showed that GR1 belongs to the Siphoviridae family, which has an 88 nm icosahedral head and 187 nmnon-contractile tail. Among 10 strains of G. stearothermophilus, GR1 can only infect 3 strains (3/10, 30%), and it cannot infect Bacillus cereus,B. amyloliquefaciens, B. subtilis, Alicyclobacillus acidoterrestris, and Clostridium perfringens, indicating its high host specificity. Genomic analysis showed GR1 has 79,837bp long circular double-stranded DNA with an average CG content of 32.34%. Among 108 open reading frames(ORFs) predicted, we identified an amidase-containing endolysin (LysGR1). The C-terminus of LysGR1 contains tandem repeats of LysMmotifs (PF01476), suggesting that they function as a cell wall binding domain of the endolysin. In addition, the LysM motifs showed a verylow degree of homology to previously reported endolysins, implying thatLysGR1 is novel. Considering there is few bacteriophages infecting G.stearothermophilus were isolated, we suspect that GR1 and LysGR1 provide potential bioprobes specifically targeting G. stearothermophilus.

Keywords : Bacteriophage, endolysin, Geobacillus stearothermophilus

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G-38

Safety Assessment of Tetragenococcus halophilus Isolates from Jeotgals

Yunji Kang, and Jeonghwan Kim*

Division of Applied Life Science Life Sci. (BK21 Four), Graduate School, Gyeongsang National University, Jinju 52828, Republic of Korea

We assessed the safety of 10 Tetragenococcus halophilus strains isolatedfrom various Jeotgals, a traditional Korean high-salt-fermented seafood. For determining resistance against common antibiotics, minimum inhibitory concentrations were examined by disks impregnated with different concentrations of each antibiotic. All strains were sensitive to ampicillin, erythromycin,penicillin G, and Rifampicin, but resistant togentamicin, streptomycin, neomycin, nalidixic acid based on theEnterococcus breakpoint values provided by the European Committeeon Antimicrobial Susceptibility testing in 2015. A total of 8 isolates wereresistant to cephalothin, chloramphenicol and ciproflaxacin (80%), 7 isolates were resistant to tetracycline (70%), and 2isolates were resistantto vancomycin (20%). None of the strains exhibited a- and b-hemolyticactivities. Through this work, we could select T. halophilus isolates forpossible use as starters for fermented foods such as soy sauce and jeotgal.

Keywords : Tetragenococcus halophilus, minimum inhibitory concentration(MIC), hemolytic activity

G-39

Contribution of Putative Peptidases to Helical Cell Shape in Campylobacter jejuni

Yewon Jung1, Jinshil Kim1,2, Hyungho Lee3, and Sangryeol Ryu1,2*

1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea 3Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea

Campylobacter jejuni is a leading cause of foodborne illness worldwide.As an epsilon-proteobacterium, C. jejuni has helical cell shape, which is also important for its pathogenesis. Previously, it has been reported that peptidoglycan peptidases (Pgp) 1-3 in C. jejuni contribute to maintaining helical cell shape by cleaving peptidoglycan crosslinking.To characterize Pgp-associated genes in this study, we selected four genes that encode homologs of Helicobacter pylori cell shape-determining (Csd)proteins, such as peptidases and transferases. Single gene knockout mutants of each gene were constructed to examine morphological changes. The transmission electron microscopy (TEM) analysis revealedthat two knockout mutants of cj1087c or cj0131 lost helical cell shape.Complementation of cj1087c knockout mutant fully restored helical cellshape but not in cj0131 knockout mutant. Furthermore, both knockout mutants caused defection in motility, one of important virulence factorsof C. jejuni. The cj1087c knockout mutant was defective for invasion into human intestinal epithelial Caco-2 cells compared to wild type. Collectively, both cj1087c and cj0131 can influence cell morphology andultimately affect pathogenic properties of C. jejuni.

Keywords : Campylobacter jejuni, helical cell shape, peptidoglycan peptidase

G-40

Evaluation of Foodborne Pathogen Contamination Levels in Livestock Feces Using 16S rRNA Genes Analysis

Gi Beom Keum1, Hyeri Kim1, Sheena Kim1, Jae Hyoung Cho1, Eun Sol Kim1, Ju-Hoon Lee2, and Hyeun Bum Kim1* 1Department of Animal Resources Science, Dankook University, Cheonan 31116, Republic of Korea 2Department of Food Animal Biotechnology, Department of Agricultural Biotechonology, Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

The importance of foodborne pathogens increases as the consumption of Livestock products expands worldwide. The foodborne pathogens originated from the farm environments, including feces, contaminatedfeeds and water are important sources of the foodborne illness. In this study, microbial communities and foodborne pathogens from livestockfeces were inspected using high throughput 16S rRNA gene sequencingand polymerase chain reaction targeting various virulence genes. A totalof 100 fecal samples were collected from cattle (n=33), pig (n=34) andchicken (n=33) for monitoring of pathogens, these 100 fecal samples (cattle(n=33), pig (n=34) and chicken (n=33)) were used for metagenome sequencing. Identification of foodborne pathogens using metagenomic analysis showed that Listeria, Salmonella, Staphylococcus,Bacillus and Clostridium were identified in chicken fecal samples. WhileCampylobacter, Salmonella, Staphylococcus, Bacillus and Clostridiumwere detected in pig feces, Campylobacter, Bacillus and Clostridiumwere detected in cattle fecal samples. The cattle fecal microbiota presented higher relative abundance of Oscillospira, Treponema and Ruminococcus. Pig fecal microbiota was mostly composed of Prevotella, Treponema and Oscillospira. Furthermore, the chicken fecalmicrobiota was significantly enriched in Lactobacillus, Clostridium andAcetobacter. The present study may help to control the spread of foodborne pathogens in the farm environments, which is a natural source of these microorganisms.

Keywords : Foodborne pathogens, livestock, microbiome

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G-41

Speculating the Roles of RclA in Copper Resistance in Salmonella

Youngjin Cho1, Jungik Cho2, and Hyunjin Yoon1,2* 1Department of Molecular Science Technology, Ajou University, 206 World cup-ro, Yeongtong-gu, Suwon 16499, Republic of Korea 2Department of Applied Chemistry and Biological Engineering, Ajou University, 206 World cup-ro, Yeongtong-gu, Suwon 16499, Republic of Korea

RclA, an NADH-dependent Cu2+ reductase, is known to be induced under stressful conditions inside macrophages. We observed that rclA could be induced by NaClO and H2O2, which are plausible stressors inside macrophages. Salmonella lacking rclA was significantly attenuated in survival upon NaClO treatments, indicating an importantrole of RclA in bacterial resistance to NaClO. Interestingly, NaClO alsoincreased the expression of cueR, copA, cueO, cueP, and golTSB, whoseproducts comprise the cytoplasmic Cu+ export systems and the absenceof RclR, a transcriptional regulator for rclABC operon, dampened the transcriptional response of these Cu+ resistance-associated genes uponNaClO treatments. This result stimulated us to investigate the possibilityof correlation between NaClO and Cu+ resistance via RclA: NaClO-induced RclA might accelerate cytoplasmic accumulation of Cu+ and in turn stimulate the production of Cu+ export systems to detoxifythe accumulated Cu+. To test the possibility, we compared bacterial resistance to Cu2+ between rclA-induced and -not induced conditions. Salmonella resistance to Cu2+ was not influenced by RclA. These resultsshow that, despite RclR-dependent induction of the Cu+ transport systems, RclA did not influence Cu2+ resistance.

Keywords : RclA, NaClO, copper

G-42

Identification of Resistance Factors Required for Escherichia coli Survival against Acid and Copper Stresses

Yeeun Kim1, Kyung-Ah Park1, Seohyeon Lee1, and Hyunjin Yoon1,2* 1Department of Molecular Science Technology, Ajou University, Suwon 16499, Republic of Korea 2Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon 16499, Republic of Korea

Pathogenic Escherichia coli resistant to acidic conditions poses a risk to food safety, especially in acidic foods such as Kimchi. In order to identify the bacterial factors required for acid resistance, transcriptomicanalysis was conducted on acid-resistant enterotoxigenic E. coli (ETEC) strains and the genes with significant changes in their expression under acidic pH were selected as putative resistance factors against acid stress.These genes included genes associated with the glutamate-dependent acid resistance (GDAR) system and copper resistance. Not only the lackof gadA, gadB, gadAB, or ybaST encoding components of GDAR systembut also the lack of cueR encoding a regulator for copper resistance genessignificantly attenuated bacterial viability after acid treatments. Interestingly, E. coli strains lacking cueR or ybaST were also attenuatedin survival after copper treatments, suggesting the cooperative killing activities between acid and copper. Meanwhile, treatments with 3-mercaptopropionic acid or aminooxyacetic acid, probable inhibitors ofGDAR system, abolished the growth and adaptation of E. coli under acidic stress conditions. These results suggest that GDAR system can be a promising target to prevent acid resistance from pathogenic E. coli.

Keywords : E. coli, glutamate-dependent acid resistance (GDAR) system, copper resistance

G-43

Characterization of a Bacillus cereus-Specific Bacteriophage B13S and Its Endolysin

Booyoung Yu, Minsuk Kong*, and Jena KimDepartment of Food Science and Technology, Seoultech, Seoul 01811, Republic of Korea

Bacillus cereus is a ubiquitous soil bacterium responsible for food poisoning and nongastrointestinal infections. Due to the emergence of antibiotic-resistant B. cereus strains, there is an urgent need for alternatives to antibiotics. To address these problems, we isolated B. cereus-specific bacteriophage B13S from the sewage and analyzed its genome. B13S, belonging to the Myoviridae family, has 155.77-kb double-stranded circular DNA containing 222 open reading frames. B13S forms clear plaques and there are no genes encoding integrases or repressors in its genome, suggesting that B13S is a virulent phage. Although B13S infects only 2 strains of 24 B. cereus strains, its host strains (ATCC 21768 and NCCP 11313) are not killed by other phages isolated in our lab, suggesting that B13S might be a valuable componentof a phage cocktail to control B. cereus. In addition, its endolysin (PlyB13S), which contains a glycoside hydrolase 25 domain (PF01183)as an N-terminal catalytic domain, caused rapid cell lysis and showed much broader lytic spectrum than its parental phage, suggesting that PlyB13S is an alternative biocontrol agent against B. cereus.

Keywords : Bacillus cereus, bacteriophage, endolysin

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G-44

Proteomics Profiles in Cold Stress Tolerant Campylobacter jejuni under Refrigeration Conditions

Jeong In Hur1, Jinshil Kim1,2, Sangryeol Ryu1,2*, and Byeonghwa Jeon3*

1Department of Food and Animal Biotechnology, Department of AgriculturalBiotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea 3Environmental Health Sciences, School of Public Health, University of Minnesota, St Paul, MN 55108, USA

Campylobacter jejuni is a thermotolerant foodborne pathogen that optimally grows at 42℃. Since C. jejuni is most frequently transmittedto humans by contaminated poultry, it is important to understand the mechanisms underlying its survival at refrigeration temperatures. Using85 C. jejuni strains isolated from retail raw chicken, we measured theirviability at 4 ℃ for 21 days under aerobic and microaerobic conditionsand observed different levels of survival. A multilocus sequence typing(MLST) analysis showed that clonal complex (CC)-45 and CC-21 weredominant in cold-sensitive (CS) and cold-tolerant (CT) strains, respectively.Aerotolerance seemed to play a role in the survival of C. jejuni underaerobic conditions. Also, CT strains survived better on chicken skin stored at 4℃ than CS strains. Then, proteomics profiles were comparedbetween CS and CT strains using two-dimensional gel electrophoresis (2DGE), identifying 24 proteins whose expression levels were altered by cold stress. Among those, mutation of periplasmic nitrate reductase(napA) rendered C. jejuni more sensitive to cold stress than the wild type.The results in this study demonstrated some strains of C. jejuni are highly tolerant to cold stress, and possibly napA may contribute to their cold stress tolerance.

Keywords : Campylobacter jejuni, cold tolerance, two-dimensional gelelectrophoresis

G-45

The Duality of Two Dairy Related Strains of Streptococcus infantarius subsp. infantarius

Svetoslav Dimitrov Todorov1,2, Carliane Ribeiro de Matos3, Hévila Oliveira Salles3, Bernadette Dora Gombossy de Melo Franco2, Wilhelm Heinrich Holzapfel1, and Karina Maria Olbrich dos Santos3,4 1ProBacLab, Department of Advanced Convergence, Handong Global University, Pohang, Gyeongbuk 37554, Republic of Korea 2Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Departamento de Alimentos e Nutrição Experimental, Laboratório de Microbiologia de Alimentos, São Paulo, SP, Brazil, 3EMBRAPA Caprinos e Ovinos, Sobral, CE, Brazil, 4EMBRAPA Agroindustria de Alimentos, Rio de Janeiro, RJ, Brazil

The genus Streptococcus includes various species, remarkably different in their behavior, applications, virulence and safety. Taxonomically Streptococcus infantarius subsp. infantarius belonging to the Streptococcus bovis group, which includes several pathogen species; however, has been found as predominant species in some African dairyproducts that are widely consumed and considered to be safe. Streptococcus infantarius subsp. infantarius safety may be questioneddue to the association of this species with clinical cases. In this study, isolates from dairy origin were selected based on their bacteriocinogenic potential and differentiated by their RAPD-PCR profiles. Two strains were identified by 16S rRNA sequencing as St. infantarius subsp. infantarius and investigated regarding their potential beneficial properties and factors related to virulence and safety. A series of in vitrotests included properties related to survival in the gastrointestinal tractand beneficial intestinal activities. Production of bacteriocin/s, detectionof related genes and partial characterization of expressed antimicrobialprotein were evaluated. Genes related to folate biosynthesis were detected in both studied strains. Evaluation of physiological tests relatedto strains virulence, adhesion, resistance to antibiotics and detections ofvirulence and biogenic amines production related genes were also investigated. Taking in consideration all the aspects of the specific natureof St. infantarius subsp. infantarius K1-4 and K5-1 (beneficial propertiesand virulence characteristics), both strains cannot be considered safe forhuman or other animals application, even though they have been isolated from dairy products. This study is highlighting the importance of evalution for presence of potential virulence factors in newly characterized strains in order to be confident in their safety. Moreover,just because a strain was isolated from food and no one reported sick afterconsuming the product, does not automatically confirm strains safety. Analyzing and setting the entire picture, it may be concluded that a potentially pathogenic bacteria has developed strong mechanisms for survival. Based on the effective adaptation of St. infantarius subsp. infantarius and its association with fermented dairy products, better hygienic practices need to be applied by artisanal producers of dairy products to control its presence in the dairy products.

Keywords : Streptococcus infantarius subsp. infantarius, bacteriocins, safety

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G-46

Characteristics of Ciprofloxacin-Resistant Salmonella from Retail Chicken Meat in Korea

Minhyeok Cha1, Junyoung Kim2, Hyo-Sun Kwak3, Eunkyung Shin2, Seokhwan Kim4, Jae-Gee Ryu5, Young-Min Chi6, and Gun-Jo Woo1* 1Laboratory of Food Safety and Evaluation, Department of Biotechnology, Korea University Graduate School, Seoul 02841, Republic of Korea 2Division of Bacterial Diseases, Centre for Laboratory Control of Infectious Diseases, Korea Disease Control and Prevention Agency, Cheongju, Republic of Korea 3Department of Food Science and Biotechnology. Kyung Hee University, Gyeonggi-do 17104, Republic of Korea 4Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Cheongju 28159, Republic of Korea 5Microbial Safety Team, National Academy of Agricultural Science, Rural Development Administration, Wanju, Republic of Korea 6Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea

Fluoroquinolone is widely used in both humans and animals, especially in the poultry industry. Recently, fluoroquinolone resistance has increased in Salmonella, a major food-borne pathogen, from poultry farms. We investigated 183 strains of Salmonella spp. isolated from chicken meat sold in retail and 13 strains from poultry farm and slaughterhouse environments in Korea from 2010 to 2017. Eighteen serotypes were identified including 58 (29.6%) S. Virchow and 44 (22.5%) S. typhimurium strains. Antimicrobial susceptibility was determined via broth microdilution method. Ciprofloxacin resistance was observed in 59 of the 196 Salmonella isolates, including 32 (54.2%)S. Virchow, 14 (23.7%) S. Hadar, and 8 (13.6%) S. typhimurium. Ser83Phe mutations were detected in GyrA (45/59), with the majorityoccurring in S. Virchow ST16 and S. Hadar ST33 (31 and 13 strains, respectively). Overall, 56 amino acid substitutions were found in Ser83and Asp87 of GyrA or Thr57 of ParC, all of which reduced ciprofloxacinsusceptibility. One or both qnrA and qnrS plasmid-mediated quinoloneresistance genes were identified in 17 Salmonella isolates. Twelve isolates exhibited efflux pump gene overexpression affecting sensitivity.The findings suggest that the antimicrobial selection pressure of fluoroquinolone in chicken farms may have affected Salmonellacharacteristics in chicken meat, especially the risk of antimicrobial resistance via dissemination of resistant strains. Therefore, continuousfluoroquinolone resistance monitoring is necessary to develop appropriate strategies for antimicrobial resistance management in poultry production.

Keywords : Fluoroquinolone, Salmonella, antimicrobial resistance

G-47

Characteristics of E. coli O157:H7-Specific Phage and Biofilm Inhibition of Novel Phage Lipopolysaccharide Depolymerase

Dowon Park, and JongHyun Park*

Department of Food Science and Biotechnololgy, College of Bionano Technology, Gachon University, Seongnam 13120, Republic of Korea

Escherichia coli O157:H7 is a food-borne pathogen that causes seriousdiseases such as hemolytic uremic syndrome and bloody diarrhea. Biofilm formed by the pathogen increases the resistance to environmental stresses and makes the pathogen more dangerous. To prevent the outbreak in the food industry, a novel T1-like phage BECP10targeting specifically E. coli O157:H7 were isolated, characterized, andsequenced. From phage genome annotations, only the tailspike protein(TSP) sequence of BECP10 had very low homology with the reportedT1-like phages, implying that TSP was related to the phage uniquenesson host infection. To identify this unique host spectrum, TSP gene designated as Dpo10 was cloned and purified. Dpo10 showed O-polysaccharide degrading activity interestingly only in the E. coliO157 strains and exhibited the opaque haloes as evidence. Dpo10 exhibited strong enzymatic activities across the wide ranges of temperature (25℃ to 65℃) and pH (5.6 to 9). Dpo10 did not inhibit thegrowth of E. coli O157:H7, however, could inhibit biofilm formation by99% on polypropylene, glass, and stainless steel. Additionally, Dpo10 increased significantly the complement-mediated serum lytic activitiesagainst E. coli O157:H7. This is the first report on O157:H7-specific depolymerase derived from siphophage and Dpo10 could be a promisinganti-biofilm agent against E. coli O157:H7.

Keywords : E. coli O157:H7 biofilm, bacteriophage, phage depolymerase

G-48

Microbiological and Molecular Characteristics of Biofilm-Producing Staphylococcus aureus Isolated from Korean Dairy Farm Environments

Sangdon Ryu1, Minhye Shin1, Daye Mun1, Woong Ji Lee1, Hye Jin Choi1,An Na Kang1, Mingeun Kang1, Hayoung Kim1, Daniel Lee1, Sangnam Oh2*, and Younghoon Kim1*

1Deptartment of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea 2Deptartment of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea

In dairy products, raw milk acts as a mediator for major foodborne pathogenic bacteria. However, the sources of pathogens that contaminatemilk are often unclear. This research investigated the presence of hygieneindicator bacteria (total aerobic bacteria, psychrotrophic bacteria, coliform,and yeasts/molds) and pathogenic microorganisms (Salmonella spp., Escherichia. coli O157:H7, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Campylobacter jejuni/coli)in Korean dairy farm environments. The microbiological analysis showed that a few sites, such as vat bottoms, room floors, drain holes, and niches, showed high microbial loads in most dairy farms. Based onquantitative microbial tests, Bacillus cereus was detected in three farms,and Staphylococcus aureus was detected in only one farm. Among them,Staphylococcus aureus JDFM SA01 detected from a milk filter was confirmed to be toxic in Caenorhabditis elegans life span experiments.Subsequently, RNA-seq was performed to analyze the characteristics ofS. aureus JDFM SA01 biofilm formation. In biofilms, the significant upregulation of genes encoding microbial surface components recognizing adhesive matrix molecules promotes adhesion, which mightclarify the improved biofilm viability and biomass. This study providedinsights into the prevalence of pathogenic bacteria and microbial contamination levels across Korean dairy farms. Keywords : Korean dairy farm, pathogenic microorganisms, Staphylococcus aureus

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H_Functional Genomics and Systems Biology

H-1

Flux Balance Analysis with Ensemble Biomass (FBAwEB): An Approach for Mitigating Biomass Composition Uncertainties

Yoon-Mi Choi1,2, Dong-Hyuk Choi2, Yi Qing Lee2, Lokanand Koduru3, Nathan Lewis4,5, Meiyappan Lakshmanan1*, and Dong-Yup Lee1,2* 1Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01, Centros 138668, Singapore, 2School of Chemical Engineering, Sungkyunkwan University, 2066, Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do 16419, Republic of Korea 3Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Singapore 138673, Singapore, 4Departments of Pediatrics and Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA, 5Novo Nordisk Foundation Center for Biosustainability at the University of California San Diego, La Jolla, CA 92093, USA

The biomass equation is a critical component in genome-scale metabolicmodels (GEMs): it is one of most widely used objective functions withinconstraint-based flux analysis formulation, describing cellular driving force under the growth condition. The equation accounts for the quantities of all known biomass precursors that are required for cell growth at certain conditions. Moreover, published GEMs also adopt relevant information from other species to derive the biomass equation when any of the macromolecular composition is unavailable. However,it is often reported that the macromolecular composition of an organismcould change across environmental conditions. Thus, the validity of same biomass equation across multiple conditions, and across species at times, is questionable. Here, we first investigated the qualitative andquantitative variations of macromolecular compositions of three modelorganisms, namely Escherichia coli, Saccharomyces cerevisiae and Cricetulus griseus, across different environmental/generic variations. We observed that while macromolecular building blocks such as DNA,RNA, protein, and lipid composition vary notably, variations in monomeric units such as nucleotides and amino acids is not appreciable.We further confirmed that the monomeric compositions could be estimated using high throughput omics data such as genome, transcriptome and proteome. Based on these observations, we subsequently propose a new framework - “Flux Balance Analysis with Ensemble Biomass (FBAwEB)” to account for the natural variation in macromolecular composition due to differences in growth conditions. During simulations, FBAwEB perform multiple simulations using ensemble biomass equations and then aggregate the results to represent the net metabolic fluxes for a particular given condition. Overall, the currentanalysis clearly highlights the importance of accounting for the naturalvariation in the biomass equation.

Keywords : Biomass equation, flux balance analysis, natural variation of cellular composition

[This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(iPET) funded by the MAFRA (32136-05-1-HD050)]

H-2

Screening of Aptamers against Bst Polymerase for Increasing Reverse Transcriptase Activity

Do-Young Kim1, Woo-Ri Shin1, Simranjeet Singh Sekhon1, Sang Yong Kim2, Ji-Hyang Wee2, Ji-Young Ahn1, and Yang-Hoon Kim1* 1Major in Microbiology School of Biological Sciences College of Natural Sciences Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, Chungbuk 28644, Republic of Korea 2Department of Food Science and Biotechnology, Shin Ansan University, 135 Sinansandaehak-Ro, Danwon-Gu, Ansan 15435, Republic of Korea

Loop-Mediated Isothermal Amplification(LAMP) is an isothermal nucleic acid amplification technique that uses Bst polymerase, instead of Taq polymerase, and unlike conventional PCR methods. All reactionsare carried out at isothermal temperatures. Bst polymerase not only mediates DNA amplification but also has weak reverse transcriptase activity. The concentration of aptamer measured after every SELEX round will aid in selecting the desired aptamer pool. SPR analysis will help determine the binding affinity of bst polymerase with aptamer. Thestructural analysis will be carried out using the MOE software. The potential application of this study includes diagnosing RNA virus-mediated disease quickly and simply effectively in the field.

Keywords : SELEX, aptamer, LAMP

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MEST) (No. NRF-2019R1A2C1010860). This research also was supported by BasicScience Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2020R1A 6A1A06046235)]

H-3

Stimulated Astaxanthin Production in Paracoccus haeundaensis Using Co-Culture Mehtod with Lactobacillus fermentum

Seong Seok Choi1, Mi Young Son1, and Gun-Do Kim2* 1Basic Science Research Institute, Pukyong National University, Busan 48513, Republic of Korea 2Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea

In this study, we confirmed an increase in astaxanthin production in Paracoccus haeundaensis by co-culture with lactic acid bacteria. Among various lactic acid bacteria, Lactobacillus fermentum strain wasselected as a suitable strain for co-culture with P. haeundaensis. Underoptimal conditions, when P. haeundaensis and L. fermentum were co-cultured, the increase was about 2.5 times compared to that of P. haeundaensis single culture. Real-time qPCR was performed to confirmthe transcriptional levels of carotenoid biosynthetic genes such as crtE,crtB, crtI, crtY, crtZ and crtW in P. haeundaensis in co-culture comparedto single culture of P. haeundaensis. Among 6 carotenoid biosynthesisgenes, the relative RNA level of the crtI gene was significantly increasedby 3.2 times, the crtY gene increased by 1.9 times, and the crtZ gene increased by 1.9 times to co-culture compared to single culture of P. haeundaensis. These results are thought to be useful as basic data for increasing carotenoid production in carotenoid biosynthetic microorganisms.

Keywords : Paracoccus haeundaensis, Lactobacillus fermentum, co-culture

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H-4

Purification and Characterization of Type II Restriction Endonuclease from Fervidobacterium islandicum AW-1

Dariimaa Ganbat1, Dong-Woo Lee2, Seong-Bo Kim3, Gae-Won Nam4, Han-Seung Lee1*, and Sang-Jae Lee1* 1Major in Food Biotechnology and Research Center for Extremophiles and Marine Microbiology, Silla University, Busan 46958, Republic of Korea 2Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 3Bio-Living Engineering Major, Global Leaders College, Yonsei University, Seoul 03722, Republic of Korea 4Department of Bio-Cosmetics, Seowon University, Chung-Ju 28674, Republic of Korea

Prime purpose of restriction-modification system is to defend the bacterial cell against macrophages. Type II restriction endonucleases occur in bacterial kingdom and identification of this system is importantfor development of genetic tools. Whole genome sequence of Fervidobacterium islandicum AW-1 revealed the presence of three adjacent ORFs coding for the restriction endonuclease and the corresponding methyltransferase. Gene encoding a type II restriction endonuclease was cloned and the recombinant enzyme was expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity with Ni-NTA affinity chromatography and molecular weight was 36 kDa. DNA containing two GATC sites from pUC19 was used as substrate. The effect of temperature, pH and metal ion were studied to determine optimal reaction condition. Optimal temperature for the restriction endonuclease activity was 65-70℃. Specific DNA cleavage was obtained at pH range 5.0-10.0 and 5-10 mM MgCl2. Manganese and cobalt could replace magnesium as a cofactor for activity. Recombinant enzyme was stable at 70℃ for more than two hoursand at 75℃ for 30 minutes. The recognition sequence of the restrictioncleavage is /GATC. This enzyme named as FisI, is a thermostable isoschizomer of the mesophilic prototype restriction endonuclease DpnII.

Keywords : FisI, Fervidobacterium islandicum AW-1, type II restrictionenzyme

H-5

Genetic Polymorphisms of HLA-DRA Are Associated with Hepatitis in the Korean Population

MiJin Hong, Mingyeong Kim, Eun-Hye Choi, Dahyun Hwang, and Hyun-Soek Jin* Department of Biomedical Laboratory Science, College of Life and Health Sciences, Hoseo University, Asan, Chungnam 31499, Republic of Korea

Hepatitis is an inflammation of the liver and commonly caused by Hepatitis B virus (HBV). There are several factors that cause hepatitis,however, a genetic association has recently begun to be studied. Especially, human leukocyte antigen (HLA) is an essential component of the immune response mechanism and there are many studies provingthe significant association between genetic polymorphism of HLA andhepatitis. To investigate of genetic association with hepatitis, SNPs from393 hepatitis cases and 8936 control cases were compared from KoreanAssociation Resource (KARE) cohort in Korea. As a result, 14 SNPs in HLA-DRA significantly associated with hepatitis. Among SNPs, rs9268644 showed the most statistically significant correlation with hepatitis (p-value= 1.92 ⅹ 10-5, OR= 0.64, 95% CI= 0.53~0.79). We usedregulomeDB to identify potentially functional SNPs and as a result, 8 SNPs are located in the genomic eQTL region and can affect transcriptionfactor binding and gene expression. The genotype-based mRNA expression analysis (GTEx Portal) showed that the group of minor alleleof HLA-DRA showed increased mRNA expression. Increased HLA-DRA expression causes increased HLA expression, and this mightreduce incidence of hepatitis. In conclusion, the SNPs in HLA-DRA genemay contribute to reduce risk of hepatitis in a Korean population.

Keywords : HLA-DRA, Hepatitis, genetic polymorphisms

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H-6

The Transcription Unit Architecture of Streptomyces lividans TK24

Yongjae Lee1, Namil Lee1, Yujin Jeong1, Soonkyu Hwang1, Woori Kim1,Suhyung Cho1, Bernhard O. Palsson2,3,4, and Byung-Kwan Cho1* 1Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 2Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA, 3Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, USA, 4Novo Nordisk Foundation Center for Biosustainability, 2800 Kongens Lyngby, Denmark

Streptomyces lividans is an attractive host for production of heterologousproteins and secondary metabolites of other Streptomyces species. To fully harness the pharmaceutical potential of S. lividans, understandingits metabolism and genetic regulatory elements is essential. This studyaimed to determine its transcription unit (TU) architecture and elucidateits diverse regulatory elements. Total 1,978 transcription start sites and1,640 transcript 3′-end positions were identified, which were integratedto determine 1,300 TUs, consistent with transcriptomic profiles. The conserved promoter was found as 5′-TANNNT and 5′-TGAC, representingthe -10 and -35 elements, respectively. Analysis of transcript 3′-end positions revealed the presence of distinctive terminator sequence and the RNA stem structure responsible for the determination of the 3′-boundary of a transcript. The TU information and regulatory elements identified will serve as invaluable resources for understanding the complex regulatory mechanisms of S. lividans and to elevate its industrial potential.

Keywords : Streptomyces, transcription unit, regulatory element

[This work was also supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031957) and the Bio & MedicalTechnology Development Program (2018M3A9F3079664) through theNational Research Foundation of Korea funded by the Ministry of Science and ICT] [This work was also supported by the Novo Nordisk Foundation (NNF10CC1016517) and the Bio & Medical Technology DevelopmentProgram (2018M3A9F3079664) through the National Research Foundation of Korea funded by the Ministry of Science and ICT]

H-7

Elucidating the Regulatory Elements for Transcription Termination and Posttranscriptional Processing in the Streptomyces clavuligerus Genome

Soonkyu Hwang1,2, Namil Lee1,2, Donghui Choe1,2, Yongjae Lee1,2, WooriKim1,2, Yujin Jeong1,2, Suhyung Cho1,2, Bernhard O. Palsson3,4,5, and Byung-Kwan Cho1,2,6*

1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 2KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 3Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA, 4Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, USA, 5Novo Nordisk Foundation Center for Biosustainability, 2800 Kongens Lyngby, Denmark, 6Innovative Biomaterials Research Center, KAIST Institutes, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea

We identified the transcriptional regulatory elements in β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27064 by determining a total of 1,427 transcript 3′-end positions (TEPs) using theterm-seq method. Termination of transcription was governed by three classes of TEPs, of which each displayed unique sequence features. Thedata integration with transcription start sites and transcriptome data generated 1,648 transcription units (TUs) and 610 transcription unit clusters (TUCs). TU architecture showed that the transcript abundancein TU isoforms of a TUC was potentially affected by the sequence contextof their TEPs, suggesting that the regulatory elements of TEPs could control the transcription level in additional layers. We also identified TUfeatures of a xenobiotic response element (XRE) family regulator andDUF397 domain-containing protein, particularly showing the abundance of bidirectional TEPs. Finally, we found that 189 noncodingTUs contained potential cis- and trans-regulatory elements that playeda major role in regulating the 5′ and 3′ UTR. These findings highlight the role of transcriptional regulatory elements in transcription termination and posttranscriptional processing in Streptomyces sp.

Keywords : Streptomyces, term-seq, transcription unit

[This work was supported by the Novo Nordisk Foundation (NNF10 CC1016517 to BOP), and the Bio & Medical Technology Development Program (2018M3A9F3079664 to B-KC) through the National Research Foundation of Korea (NRF) funded by the Ministry of Scienceand ICT (MSIT)]

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H-8

Fluorometric Detection of Hepatitis C Virus RNA with PCR-Primed Rolling Circle Amplification of Viral RNA

Jamin Ku, and Dong-Eun Kim*

Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05902, Republic of Korea

Direct detection of viral RNA is very challenging due to its large size and unstable nature unlike DNA. RNA also forms secondary structures that hinder annealing with intended DNA primer at ambient temperature.We developed a fluorometric method for detection of Hepatitis C virus(HCV) RNA (9,600 nts) as long-sized RNA by combining reverse transcription (RT)-PCR of viral RNA and subsequent Rolling Circle Amplification (RCA) of amplicon DNA. HCV genomic RNA extractedfrom patient’s blood serum was reverse-transcribed and portion of 5′-untranslated region (UTR) in cDNA was amplified through RT-PCR with reduced number of heat cycles (10 cycles). We found that reducedPCR cycle is more beneficial to provide priming condition for subsequent RCA than ordinary 30 cycles of PCR. Amplicon DNAs werelater heat-denatured and cooled, by which closed-circular template andhairpin-type primer DNAs were annealed to the antisense strand of amplicon DNA. This ternary complex of DNA molecules were subjectto RCA for target DNA amplification at ambient temperature. Amplifiedsingle-stranded DNA harboring tandem repeats of G-quadruplex structures were fluorescently detected with Thioflavin T. Our tandem amplification of viral RNA would be useful to detect scarce amount of viral RNA within 1.5 h.

Keywords : Fluorometric detection of long-sized viral RNA, PCR-primed rolling circle amplification, reduced PCR cycle

H-9

Fluorometric Detection of SARS-CoV-2 Viral RNA Using Tandem Isothermal Gene Amplification

Hyojin Lee, and Dong-Eun Kim*

Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05902, Republic of Korea

Detection of SARS-CoV-2 through fast, accurate, and sensitive testingis the most important and fundamental step to cope with COVID-19 epidemic. We developed a sensitive fluorometric assay for detecting SARS-CoV-2 viral RNA with PCR-free method. This assay system based on tandem isothermal gene amplification is composed of ternary Rolling Circle Amplification (t-RCA) and subsequent strand displacement amplification coupled with RCA (SDA-RCA). In t-RCA, viral RNA forms a ternary initiation complex with the hairpin primer andRCA circular template. The t-RCA product, which contains multiple-repeated dumbbell-like structures, participate in the SDA reaction. The SDA reaction produces large amounts of short single-stranded DNAs (ssDNAs), which act as primers for the second RCA reaction. A long stretch of ssDNA containing repeated copies of the G-quadruplex was produced in the second RCA. Subsequently, fluorometric detection of amplified viral gene was accomplished by monitoring emission of strong fluorescence by Thioflavin T that intercalates into the G-quadruplex. Only in the presence of viral RNA,fluorescence was quantitatively detected due to viral gene amplification.Hence, our tandem gene amplification can detect SARS-CoV-2 viral RNA at constant temperature within 1 h.

Keywords : SARS-CoV-2, isothermal amplification, fluorometric detection

H-10

Characterization of Conidial-Specific Transcription Factor csgA in Aspergillus spp.

He jin Cho1, and Hee-Soo Park1,2*

1School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 2Department of Integrative Biology, Kyungpook National University, Daegu 41566, Republic of Korea

Aspergillus spp. mainly reproduce through asexual reproduction, producingasexual spore called conidia. The process of conidia formation is controlled by a variety of transcription factors. In this study, we characterized one of conidial specific GAL4-like transcription factor CsgA which contains the GAL4-like zinc-finger domain. The role of csgA was investigated in two Aspergillus species, the model organism A. nidulans (Ani) and the aflatoxin producer A. flavus (Afl). The AnicsgAdeletion mutant strains showed increase in conidiation and fungal growth. The AnicsgA deletion mutant showed delayed production of sexual structure called cleistothecia. Overexpression of AnicsgA showedextreme decrease in conidiation but increased cleistothecia production.The production of sterigmatocystin increased in the AnicsgA deletion mutant conidia than wild-type conidia. Also, the AnicsgA deletion mutants showed increase in trehalose amount and thermal stress resistance. These results suggest that csgA plays critical role in conidiation,fungal development and mycotoxin production in A. nidulans. To test whether the function of CsgA is conserved in other Aspergillus species,we produced deletion mutants in A. flavus. The AflcsgA deletion mutantshowed remarkable decrease in fungal growth, but conidiation increasedin dark condition. The AflcsgA deletion mutant exhibit defect in sclerotialproduction, which is sexual structure of A. flavus. The AflcsgA deletion mutant showed increase in trehalose amount but decreased productionof aflatoxin. In conclusion, CsgA plays crucial role in proper conidiationand fungal growth and regulates secondary metabolites production in both A. nidulans and A. flavus.

Keywords : Aspergillus nidulans, Aspergillus flavus, GAL4-like transcription factor

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H-11

Characterization of a Forkhead Gene fkhB in Aspergillus nidulans

Seo yeong Jang1, and Hee-Soo Park1,2* 1Department of Integrative Biology, Kyungpook National University, Daegu 41566, Republic of Korea 2School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

The Forkhead domain genes are generally known to be important for meiosis and cell cycle regulation. Six Forkhead genes have been foundin Aspergillus nidulans. Transcriptomic analysis found a variety of thespore-specific genes in A. nidulans. Among them, fkhB was specificallyexpressed in asexual spores. Deletion of fkhB affected both sexual and asexual development. The fkhB deletion mutant strain exhibited decreased the colony size and produced reddish asexual spores unlike the wild-type. The number of conidia in the fkhB deletion mutant strainwas significantly decreased compared to wild type strains. These resultspropose that the fkhB gene is involved in asexual sporogenesis. The deletion of fkhB increased the sensitivity to heat stress, decreased the amount of conidial trehalose, and decreased conidial viability. In addition, the absence of fkhB resulted in a decrease in sterigmatocystinproduction. Taken together, these results show that fkhB is essential forproper bacterial growth, development, and sterigmatocystin productionin A. nidulans.

Keywords : Aspergillus nidulans, Forkhead

H-12

Genome-Wide Analysis of Spore-Specific Transcription Factors in Aspergillus nidulans

YeEun Son1, and Hee-Soo Park1,2* 1School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 2Department of Integrative Biology, Kyungpook National University, Daegu 41566, Republic of Korea

Aspergillus nidulans, a representative filamentous model organism, mainly propagates by forming the asexual spores, called conidia. Theconidia formation process (conidiation) is regulated by various transcription factors, including BrlA, AbaA, WetA, and Velvet regulators. Previous our transcriptomic analysis identified the nineteenspore specific genes encoding transcription factors. Then phenotypes ofeach deletion mutant strains were analyzed in A. nidulans. In this study,we characterized one of the spore-specific-C2H2 zinc finger A SscA, encoding C2H2 zinc finger transcription factor. The sscA deletion mutants showed defect conidiation, sexual development and decreasedconidial viability. The ΔsscA mutant conidia were more sensitive to thermal, oxidative, UV and cell wall stresses than wild-type (WT) conidia. The amount of trehalose in sscA deletion strains was decreasedcompared to WT. Deletion of sscA causes induced germ tube formation in the presence and absence of glucose. Absence of sscA led to increasein the amount of sterigmatocystin in the conidia. The sscA overexpressingstrain exhibited abnormal asexual and sexual development and showedrapid hyphal growth. Overall, these results demonstrate that SscA areessential for proper asexual and sexual development, conidial maturation and secondary metabolites in A. nidulans.

Keywords : Aspergillus nidulans, conidia, transcription factor

H-13

Transcriptomic Profile of Plant-Beneficial Chryseobacterium sp. NBC122 under Salinity Stress

Jungwook Park1, Hyejung Jung2, Gil Han2, Young-Su Seo2, and Sang Mi Yu1* 1Environmental Microbiology Research Team, Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea 2Department of Integrated Biological Science, Pusan National University, Busan 46241, Republic of Korea

Salinity stress is the devastating abiotic stresses that negatively affectedthe crop productivity by more than 50%. Recent global warming depletesavailable freshwater resources and increases saline contamination of agricultural soils. We previously isolated Chryseobacterium sp. NBC122, which promotes plant growth and salinity tolerance in napa cabbage (Brassica rapa subsp. pekinensis). However, the details in response to salinity stress in Chryseobacterium sp. NBC122 are unclear.Interestingly, the bacterial growth of Chryseobacterium sp. NBC122 was delayed in bacterial culture condition (Luria-Bertani medium) containing 200 mM NaCl, but not in plant growth condition (Murashigeand Skoog medium). We employed a transcriptomic approach to betterunderstand the salinity tolerance mechanism. Our results revealed that260 genes of Chryseobacterium sp. NBC122 are highly induced duringsalinity stress in plant growth condition. Of these, many genes were associated with antioxidant function and the degradation of xenobioticcompounds. On the other hand, down-regulated genes were distributedin carbon and energy metabolisms. Consequently, these findings elucidated the mechanism of salinity tolerance in Chryseobacterium sp.NBC122 and could be considered as an important guide to applicationfor crop protection.

Keywords : Chryseobacterium sp. NBC122, transcriptome, salinity stress

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H-14

The Association Study between ADAM17 and Allergic March in Korean Population

Jaemee Jung, Hyun-Seok Jin and Dahyun Hwang*

Department of Biomedical Laboratory Science, College of Life and Health Sciences, Hoseo University, Asan, Chungnam 31499, Republic of Korea

Asthma is a representative inflammatory disease with airway obstruction that causes breathing difficulties. There are many factors toinduce asthma, however, TNF-α may play an important role to induce inflammation. Therefore, therapeutic potential of TNF alpha-targetingagent is being considered to treat asthma. In this study, we analyzed whether genes and clinical values are related to asthma in a Korean population. First, we analyzed the genetic polymorphisms of ADAM17(also called TNF-α converting enzyme or TACE) between 193 asthmapatients and 3,228 normal controls. As a results, 15 SNPs in ADAM17showed statistically significant association with asthma. Among them, rs643211 SNP showed the greatest statistical correlation with asthma (P-value = 0.00041, OR = 1.95, 95% CI = 1.35~2.82. The genotype-basedmRNA expression analysis showed that the group of minor allele of ADAM17 showed increased mRNA expression, and also this groups showed increased risk of asthma. This means that increased ADAM17expression contributed to the increase in asthma. Secondary, we compared 21 clinical values in serum between asthma and normal group.As a results, CRP (increased), total cholesterol (increased), and total bilirubin (decreased) showed statistically significant association with asthma. CRP is a representative marker of inflammation and positive relationship has been reported between raised CRP level and current asthma. Bilirubin has recently begun to attract attention as therapeutic agents because bilirubin has strong antioxidative activity in body. Thereare many researches that increased bilirubin protected inflammatory diseases. In conclusion, gene and clinical values may contribute to development of asthma in a Korean population.

Keywords : ADAM17, SNP, allergic march

H-15

Glutaric Acid Production by Systems Metabolic Engineering of an L-lysine-Overproducing Corynebacterium glutamicum

Taehee Han1, Gi Bae Kim1, and Sang Yup Lee1,2,3* 1Metabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Cross- Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea 2BioInformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 3BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea

There has been increasing industrial demand for five-carbon platform chemicals, in particular, glutaric acid, a widely used building block chemical for the synthesis of polyesters and polyamides. Here we reportthe development of an efficient glutaric acid microbial producer by systems metabolic engineering of an L-lysine-overproducing Corynebacteriumglutamicum BE strain. Based on our previous study, an optimal syntheticmetabolic pathway comprising Pseudomonas putida L-lysine monooxygenase(davB) and 5-aminovaleramide amidohydrolase (davA) genes and C. glutamicum 4-aminobutyrate aminotransferase (gabT) and succinate- semialdehyde dehydrogenase (gabD) genes was introduced into the C.glutamicum BE strain. Through system-wide analyses including genome-scale metabolic simulation, comparative transcriptome analysis,and flux response analysis, 11 target genes to be manipulated were identified and expressed at desired levels to increase the supply of direct precursor L-lysine and reduce precursor loss. A glutaric acid exporter encoded by ynfM was newly discovered and overexpressed to further enhance glutaric acid production. Fermentation conditions including oxygen transfer rate, batch-phase glucose level, and nutrient feeding strategy were optimized for the efficient production of glutaric acid. Fed-batch culture of the final engineered strain produced 105.3 g/L of glutaric acid in 69 h without any byproduct. The strategies of metabolicengineering and fermentation optimization described here will be usefulfor developing engineered microorganisms for the high-level bio-basedproduction of other chemicals of interest to industry.

Keywords : Metabolic engineering, Corynebacterium glutamicum, multi-omics

[This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (Grant number 2020M3A9I503788311) and funded by the Ministry of Scienceand ICT, Republic of Korea]

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I_Host-Microbe Interactions and Pathogenesis

I-1

Manufacture of Capsules for Attachment of Cosmetic Fabric Using Camellia sinensis Seed Oil Containing Skin Microbiome

JuHyun Lee1, Chaeyun Baek1*, Jaewoo Jeon2, and Woojin Kim2 1COSMAX BTI, Gyeonggi-do 13486, Republic of Korea 2Korea Dyeing & Finishing Technology Institute, Daegu 41706, Republic of Korea

Through our previous studies, it was confirmed that many Staphylococcusstrains exist in healthy skin in their twenties. Based on this result, theskin microbiome Strain CX, which is expected to have a good effect onskin condition, was isolated, and it was confirmed that the culture medium of Strain CX has skin improvement effects such as skin moisturizing and skin barrier improvement. In order to continuously acton the skin for the effects of Strain CX, sustained-release melamine/ formaldehyde microcapsules loaded with Camellia sinensis seed oil containing skin microbiome were prepared by emulsion polymerizationand analyzed for properties. The size, shape and structural characteristicsof the prepared microcapsules were observed through SEM and FT-IR,and the release behavior of the supported C. sinensis seed oil containingskin microbiome was confirmed through UV absorbance. It is thought that it can be applied to textile materials that are continuously used in everyday life, such as clothing and bedding materials, by mixing microcapsules carrying the manufactured C. sinensis seed oil containingskin microbiome into yarn or attaching them to fabric. Through this, it is expected to be able to give continuous skin improvement effect.

Keywords : Skin microbiome, Staphylococcus, cosmetic fabric

I-2

Complement Inactivation Strategy of Staphylococcus aureus Using Decay-Accelerating Factor and the Response of Infected HaCaT Cells

Kyoung Ok Jang1, Jong hyo Hong1*, Dae Kyun Chung1,2,3*, and HangeunKim2* 1Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea 2Research and Development Center, Skin Biotechnology Center Inc., Yongin 17104, Republic of Korea 3Skin Biotechnology Center, Kyung Hee University, Suwon 16229, Republic of Korea

Staphylococcus aureus is a Gram-positive bacteria that can cause sinusitis, respiratory infections, skin infections, and food poisoning. Recently, it was discovered that S. aureus infects epithelial cells, but theinteraction between S. aureus and the host is not well known. In this study,we confirmed S. aureus can be internalized by HaCaT cells using the ESAT-6-like protein EsxB and amplified within the host cells by escaping host immunity. S. aureus inhibits the activity of the complementsystem by increasing CD55 to survive on the surface of host cells. S. aureus sufficiently amplified within the host and released through the initiation of other cell death. On the other hand, the infected host cellsincrease their surface expression of UL16 binding protein 1 to inform immune cells that they are infected and try to be eliminated. These hostdefense systems seem to involve the alteration of tight junctions and theinduction of ligand expression to activate immune cells. Taken together,our study elucidates a novel aspect of the mechanisms of infection andimmune system evasion for S. aureus

Keywords : Staphylococcus aureus, HaCaT cell, host defense system

I-3

Integrative Multi-Omics Analysis for Characterization of Inhibitory Effects on Adipocyte Differentiation and Lipid Accumulation by Akkermansia muciniphila

Jae-Seung Lee, Won-Suk Song, Sung-Hyun Jo, Hyo-Jin Jeon, Ji-Eun Kwon, Ji-Hyeon Park, Ye-Rim Kim, and Yun-Gon Kim*

Department of Chemical Engineering, Soongsil University, Seoul 06978, Republic of Korea

Obesity is a public health problem and induces metabolic diseases suchas type 2 diabetes and cardiovascular diseases. Recent reports suggest that A. muciniphila, which is known as next generation probiotics, reduces body weight and blood glucose level in obese host. However, the beneficial effects of this microbe on adipocytes were only reports in cell phenotypic changes or alterations of markers related to obesity. Therefore, molecular level studies are necessary for comprehensive understanding of the inhibitory effects by A. muciniphila. In this study,the changes of biological mechanism in 3T3-L1 cells under treatment with the cell lysate of A. muciniphila were investigated by multi- omics-based approaches. Firstly, the cell lysate of A. muciniphilasuppressed differentiation and lipid accumulation of 3T3-L1 cells in cellular level. Moreover, inhibitory effects of the bacterial lysate on 3T3-L1 cells were observed in mRNA expression levels, fatty acid production, proteomic and metabolomic analysis results. Taken together, multi-omics study suggested anti-adipogenic and anti- lipogenic effects on 3T3-L1 cells treated with the cell lysate of A. muciniphila. These results may provide a new insight for discovering anti-obesity targets in the cellular components of A. muciniphila.

Keywords : Akkermansia muciniphila, adipocytes, multi-omics

I-4

The LC-MS/MS Based Multi-Omics Analysis to Investigate Clostridium difficile Infection via In Vitro Coculture Device, the Mimetic Intestinal Host-Microbe Interaction Coculture System, MIMICS

Jieun Kwon, Sung-Hyun Jo, Won-Suk Song, Jae-Seung Lee, Hyo-Jin Jeon, Ji-Hyeon Park, Ye-Rim Kim, and Yun-Gon Kim*

Department of Chemical Engineering, Soongsil University, Seoul 06978, Republic of Korea

Clostridium difficile is a Gram-positive bacterium and is considered as an enteric pathogen. C. difficile colonizes the host with intestinal dysbiosis and produces virulence factors, Toxin A and B. Although thecytotoxicity of C. difficile has been well elucidated, the exact pathogenic mechanisms and biological pathways of C. difficile infection (CDI) arenot yet clear. Here, we developed a CDI model using MIMICS to investigate C. difficile-host interactions throughout the early (12 hr) andlater (48 hr) stages of infection. We also apply an LC-MS/MS-based multi-omics approach to study the physiological changes in CDI-induced gut epithelial cells at the molecular and cellular levels. Theproteomic analysis confirmed that proteins involved in cellular responses such as stress, immune responses and DNA damage responsesare upregulated at early stages of infection. In addition, a representativeCDI phenotype has been identified in the late stage of the infection. Ourin vitro CDI model and multi-omics approach not only provide a betterunderstanding of host-anaerobic pathogen interactions but may also aidin the treatment of C. difficile and other anaerobic infections.

Keywords : Clostridium difficile infection, host-pathogen interaction, multi-omics analysis

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I-5

Anti-Inflammatory Effect of Fermented Herbal Medicines in HaCaT

Chaeyun Baek, Yonghwan Lim*, Minji Kim, and Misun Kim COSMAX BTI, Gyeonggi-do 13486, Republic of Korea

Herbal medicines have been used for medical treatment since ancient times in Asian countries, including Korea, China, and Japan. Althoughseveral studies have recently been conducted to determine the empiricaleffects of herbal medicines, studies on direct effects on the skin are inadequate. Although different from country to country, fermentation technology is used in various food industries worldwide. This fermentation technology has an advantage in that it increases effectivesubstances and produces new substances through metabolic processes.Glycyrrhiza uralensis, Cirsium japonicum var. maackii and Lonicera japonica are herbal medicines used to treat dermatitis, and we studiedfermentation methods to increase the efficacy of these medicines. As aresult of fermentation using the Bifidobacterium sp. DS008 we found,the inflammatory factors IL-1α, IL-1β, and IL-6 were reduced by 28.3%,43.5%, and 74.0%, respectively. In addition, skin barrier factors such asfilaggrin and HAS3 were increased by 33.4 and 3.5 times, respectively.After 24 hours of fermented product treatment, the cell regeneration rateincreased by 21.5%. The fermented product can help improve skin, and has high utility value in the cosmetic field in the future.

Keywords : Herbal medicines, fermentation, bifidobacterium

I-6

Ameliorative Effects of Kimchi-Originated Lactic Acid Bacteria Treatment on Alcohol-Induced Liver Injury in Mice

Juseok Kim, Seong Woo Ahn, Seul Ki Lim, Hak-Jong Choi, Seong WoonRoh*, and Se Hee Lee*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

Alcoholic liver disease (ALD) is a common health problem globally. ALD includes fatty liver, cirrhosis, steatosis, fibrosis, and ultimately hepatocellular carcinoma. To induce ALD in mice, alcohol was administrated for 10 days, and kimchi-originated lactic acid bacteria (LAB): Lactiplantibacillus plantarum DSR J266 and Lactobacillusbrevis DSR J301 (LpLb), and Lacticaseibacillus rhamnosus GG (LGG)were orally administrated at the same period. In the alcohol-diet (AD) group, serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly increased compared to thosein the non-alcohol-diet (ND) group but relatively decreased in the LpLband LGG-administrated groups. The level of IL-6 was relatively decreased in the LpLb and LGG groups than in the AD group. Many vesicles of fat accumulation were found in liver tissues of the AD group,but the number of vesicles was remarkably reduced in the LpLb and LGGgroups, which might be that the administration of the LAB alleviates thefat accumulation in the liver tissue. The expression of genes such as FAS,MIP-2, TNF-α, and α-SMA was increased in the AD group but decreased in the LpLb and LGG groups. The expression of tight junction-related genes in the intestine was decreased in the AD group but relatively increased in the LpLb and LGG groups. Escherichia-Shigella genus significantly increased in the AD group, whereas the genus was relativelydecreased in the LpLb and LGG groups, confirming the efficacy of administration of the LAB. As a result, it was confirmed that alcohol intake might cause health abnormalities in the liver and intestines, but the LAB intake helped recover these symptoms.

Keywords : Kimchi-originated lactic acid bacteria, alcoholic liver disease

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I-7

Lactococcus sp. NY-1 Ameliorates the Degeneration of Nigral Dopaminergic Neurons and Motor Deficits in a MPTP-Induced Parkinson Mouse Model

Namhee Kim1, Young Seo Jang1,2, Hyo Kyeong Park1, Sulhee Lee1, YoungJoon Oh1, and Hak-Jong Choi1* 1Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea 2Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Republic of Korea

Parkinson's disease (PD) is a common neurodegenerative disease, characterized by loss of dopaminergic neurons and motor deficits suchas tremor, rigidity, and slow movement. Recent studies have shown thatpathogenesis of PD is closely related to the bidirectional communicationin the gut-brain axis, and probiotics may inhibit PD progression via modulation of the gut-brain axis. In this study, we investigated the neuroprotective effects of a lactic acid bacterium, Lactococcus sp. NY-1(NY-1), isolated from kimchi in a PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP). Treatment with MPTP generated significant motor deficit, dopaminergic neuronal(tyrosine hydroxylase positive) death, striatal dopamine reduction, andneuroinflammation. Administration of NY-1 in drinking water for 4 weeks before the MPTP injection significantly alleviated the motor deficit in rotarod test and forelimb grip strength test. We also found thatNY-1protected against impairment of TH-positive neurons and attenuated microglial proliferation and astrocyte reactivity in the substantia nigra pars compacta following MPTP treatment. Furthermore,administration of NY-1 to PD mice recovered non-motor symptom including depression and anxiety in open field test. In conclusion, NY-1could prevent MPTP induced-motor deficits, dopaminergic neuron loss,and neuroinflammation. These results highlight the role of probiotics forbrain health, and their potential usage for prevention of neurodegenerativediseases such as PD.

Keywords : Parkinson's disease, gut-brain axis, lactic acid bacteria

I-8

Immunomodulatory Effects of a Halophilic Bacterium Isolated from Kimchi on Innate and Adaptive Immunity

Seul Ki Lim1, Min-Sung Kwon1, Young Joon Oh1, Sang-Pil Choi1, Ga HeeChoi1,2, Haegyo Jeong1, and Hak-Jong Choi1* 1Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea 2Division of Animal Science, Chonnam National University, Gwangju 61186, Republic of Korea

Halophilic microorganisms are salt loving bacteria that inhabit at variousareas of high salt concentration. Although numerous halophilic bacteriahave been isolated from kimchi, little is known about their physiologicalfunctionality. In this study, we isolated a halophilic bacterium from kimchi, Gracilibacillus kimchii K7, and evaluated its immunomodulatoryproperties by in vitro co-culture assays with different types of immunecells, such as mouse bone marrow-derived dendritic cells (BMDCs) andsplenocytes isolated from OT-II transgenic mice. G. kimchii K7 triggeredthe release of interleukin (IL)-10 and IL-12 p70 from BMDCs and significantly decreased IL-4 production and increased IL-10 and interferon-γ production from ovalbumin-restimulated splenocytes. These results indicate that treatment of G. kimchii K7 induces the suppression of Th2 response. Furthermore, we evaluated the ability of G. kimchii K7 to suppress the Th2 responses in vivo. In a 2,4- dinitrochlorobenzene-induced atopy dermatitis (AD)-like murine model, oral administration of G. kimchii K7 reduced AD-like skin lesionsand serum immunoglobulin E (IgE) levels. These results suggest that ahalophilic bacterium, G. kimchii K7 is a strong immunomodulatory strain and may be an applicable as a novel therapeutic agent for the treatment of AD.

Keywords : Halophilic Bacterium, immunomodulatory effect, atopy dermatitis

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I-9

Antiviral Activity of Lactobacillus reuteri (BSA 218) against Influenza Virus In Vitro and In Vivo

Niranjan Dodantenna1, Buddhika Nuwan Gamage1, Young Hyo Chang2,and Jong-Soo Lee1* 1College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea 2ABS Research Support Center, KRIBB, Daejeon 34141, Republic of Korea

Probiotics are well known substances that stimulate the growth of gut microbiota and enhance host immune responses. Recent studies are focusing on promising approaches for preventing various viral infections through probiotics. In this study, a novel probiotic candidateLactobacillus reuteri (BSA 218) strain was selected and studied about its effect on the production of pro-inflammatory cytokines and against viral infections in vitro and in mouse model. Results revealed that pre-treatment of BSA 218 live culture mixture and BSA 218 supernatantsonly significantly inhibit the replication of Influenza A virus (PR8), Vesicular Stomatitis Virus (VSV), Newcastle Disease Virus (NDV), andHerpes Simplex Virus (HSV) in RAW264.7 cells. Such antiviral properties can be elucidated by the induction of antiviral molecules in type I IFN signalling pathway. Interestingly, we found induced secretion of type I IFNs, pro-inflammatory cytokines and transcriptional activation of antiviral genes in vitro and in vivo following BSA 218 treatment. Furthermore, daily oral administration of BSA 218 revealedprophylactic effect against lethal doses of highly pathogenic InfluenzaA subtypes H1N1, H3N2 and H5N2 led to the recovery of weight lossand inhibit viral titer in mice lungs. Taken together, these results suggested that the novel candidate probiotic strain BSA 218 would be an alternative promising approach for developing an anti-viral/ anti-influenza candidate.

Keywords : Lactobacillus reuteri BSA 218, anti-viral activity, influenza

[The National Research Foundation of Korea (Grant no. 2019R 1A2C2008283) and KRIBB Research Initiative Program (KGM 9942011)]

I-10

Foot-and-Mouth Disease Virus Structural Protein VP4 Antagonizes the Host Type-1 Interferon Signalling

Ashan Subasinghe, N.A. Nadeeka Nethmini, and Jong-Soo Lee*

College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

Foot-and-mouth disease (FMD) is a highly infectious virus and is a causative agent of acute vesicular diseases that affect pigs, cattle, and other domestic and wild hoof animals. Foot-and -mouth disease virus (FMDV) genome encodes several viral proteins that can enforce multiplestrategies to escape host defense mechanisms including the type-I interferon (IFN) pathway. In this study, we demonstrate that the downregulation of type-I IFN pathway by FMDV structural protein VP4in vitro. In the virus replication study, overexpression of FMDV-VP4 downregulates the IFN signalling in porcine (PK15, PAM, LFBK) andhuman (HEK293T) cell lines, thus increase Vesicular stomatitis virus (VSV-GFP) and Influenza virus (PR8-GFP) replication. In addition, FMDV-VP4 transient transfection or stably over-expression exhibited a minimal secretion of pro-inflammatory cytokine and negatively regulates the transcription of IFN stimulatory genes. Correlate with the virus replication phenotypes, FMDV-VP4 significantly reduces the phosphorylation of signaling molecules in the type-I IFN and NF-κB pathways mainly TBK1, IRF3, STAT1, IκBα, and p65 in PK15, PAM and HEK293T cell lines. Collectively, these results suggest that FMDV-VP4 negatively regulates the type-I IFN pathway and lead to severe FMDV infection.

Keywords : FMDV, VP4, Type-I interferon (IFN)

[The National Research Foundation of Korea grant (2018M3A 9H4079660) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM9942011)]

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I-11

The Structural Protein VP3 of Foot-and-Mouth Disease Virus Inhibits Interferon Signaling by Targeting MAVS Aggregation

Pathum Ekanayaka, Byeong-Hoon Lee, and Jong-Soo Lee*

College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

As a structural protein of Foot-and-mouth disease virus (FMDV), VP3plays a vital role in virus assembly and inhibiting the interferon (IFN) signal transduction to promotes the FMDV replication. Previous studiesdemonstrated that FMDV VP3 blocks the type-I IFN response via inhibiting the mRNA expression of the mitochondrial antiviral- signaling protein (MAVS), however, the underline mechanism is poorlyunderstood. Here we identified that the specificity of VP3 interaction with MAVS for the negative regulation of type-I IFN antiviral responsesfor effective replication of FMDV. Further, we describe that the transmembrane (TM) domain is the specific region of MAVS which interacts with the FMDV VP3. The TM domain of MAVS governs the mitochondria localization of MAVS and it is a key factor in type-I IFNsignaling transduction via MAVS aggregation. Thereby, the interactionof FMDV VP3 with the TM domain of MAVS leads to the inhibition ofMAVS mitochondria localization, self-association and aggregation, resulting in the suppression of type-I IFN response as shown in the results.Collectively, these data provide a clear understanding of a key molecularmechanism used by the FMDV VP3 for the suppression of IFN responses via targeting MAVS.

Keywords : FMDV VP3, MAVS, aggregation

[The National Research Foundation of Korea grant (2018M3A9 H4079660) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM9942011)]

I-12

Inhibition of RIG-I and MDA5 Mediated Type-1 Interferon Signaling by Foot-and-Mouth Disease Virus Non-Structural Protein 2B

Asela Weerawardhana, Md Bashir Uddin, and Jong-Soo Lee*

College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

The highly contagious foot-and-mouth disease (FMD) caused by the foot-and-mouth disease virus (FMDV) is an acute disease affecting cloven-hoofed animals. FMDV nonstructural protein 2B plays an important role in rearrangement and disruption of host cell membranes,also known for its function as a viroporin. In addition, FMDV 2B involvein modulating host immune signaling. Here we examined the ability ofFMDV 2B to evade host immune response by targeting retinoic acid-inducible gene-1 (RIG-I) and melanoma differentiation-associated gene-5 (MDA5). FMDV 2B target RIG-I for ubiquitination and proteosomal degradation by recruiting E3 ubiquitin ligase ring finger protein 125 (RNF125), and target MDA5 for apoptosis induced Caspase-3 and Caspase-8 dependent degradation. Furthermore, FMDV2B 125-154 amino-acid region is important for both RIG-I and MDA5 degradation. These results suggest that FMDV 2B negatively regulatesthe type I interferon pathway and advances the pathogenesis and lead for severe FMD infections.

Keywords : Foot-and-Mouth Disease Virus 2B, RIG-I, MDA5

[The National Research Foundation of Korea grant (2018M3A9H 4079660) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM9942011)]

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I-13

Foot-and-Mouth Disease Virus VP1 Interacts with MAVS to Inhibit Type-I Interferon Signaling Pathway and VP1 E83K Substitution Results in Virus Attenuation

Pathum Ekanayaka1, Seo-Yong Lee1,2,3, Thilina U.B. Herath1, Jong-Hyeon Park2*, and Jong-Soo Lee 1* 1College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea 2Animal and Plant Quarantine Agency, Gyeongsangbuk-do 39660, Republic of Korea 3FVC, Gyeongsangbuk-do 39660, Republic of Korea

VP1, a pivotal capsid protein encoded by the foot-and-mouth disease virus (FMDV), plays an important role in receptor-mediated attachmentand humoral immune responses. Previous studies show that amino acidchanges in the VP1 protein of cell culture-adapted strains of FMDV alterthe properties of the virus. In addition, FMDV VP1 modulates host IFNsignal transduction. Here, we examined the ability of cell culture- adapted FMDV VP1(83K) and wild-type FMDV VP1(83E) to evade host immunity by blocking mitochondrial antiviral signaling protein (MAVS)/TNF Receptor Associated Factor 3 (TRAF3) mediated cellularinnate responses. Wild-type FMDV VP1(83E) interacted specifically with C-terminal TRAF3-binding site within MAVS and this interactioninhibited binding of TRAF3 to MAVS, thereby suppressing interferon-mediated responses. This was not observed for cell culture-adapted FMDV VP1(83K). Finally, chimeric FMDV harboring VP1(83K) showed very low pathogenicity in pigs. Collectively, these data highlighta critical role of VP1 with respect to suppression of type-I IFN pathwayand attenuation of FMDV by the E83K mutation in VP1.

Keywords : FMDV VP1, E83K substitution, Type-I IFN pathway

[The National Research Foundation of Korea grant (2018M3A9 H4079660) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM9942011)]

I-14

Foot-and-Mouth Disease Virus 3C Protease Antagonizes Interferon Signaling via RIG-I and MDA5, and C142T Substitution Attenuates the FMD Virus

Pathum Ekanayaka1, Sung Ho Shin2, Jong-Hyeon Park2*, and Jong-SooLee 1* 1College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea 2Animal and Plant Quarantine Agency, Gyeongsangbuk-do 39660, Republic of Korea

3C protease (3Cpro), a chymotrypsin-like cysteine protease encoded bythe foot-and-mouth disease virus (FMDV), plays an essential role in processing the FMDV P1 polyprotein into individual viral capsid proteins in FMDV replication. Previously, it has been shown that 3Cpro

is involved in the blockage of the host type-I interferon (IFN) responsesby FMDV. However, the underlying mechanisms are poorly understood. Here, we demonstrated that the protease activity of 3Cpro contributed tothe degradation of RIG-I and MDA5, key cytosolic sensors of the type-IIFN signaling cascade in proteasome, lysosome and caspase-independentmanner. And also, we examined the degradation ability on RIG-I and MDA5 of wild-type FMDV 3Cpro and FMDV 3Cpro C142T mutant which is known to significantly alter the enzymatic activity of 3Cpro. The results showed that the FMDV 3Cpro C142T mutant dramatically reduce the degradation of RIG-I and MDA5 due to weakened protease activity. Thus, the protease activity of FMDV 3Cpro governs its RIG-I and MDA5 degradation ability and subsequent negative regulation of the type-I IFNsignaling. Importantly, FMD viruses harboring 3Cpro C142T mutant showed the moderate attenuation of FMDV in a pig model. In conclusion,our results indicate that a novel mechanism evolved by FMDV 3Cpro tocounteract host type-I IFN responses and a rational approach to virus attenuation that could be utilized for future vaccine development.

Keywords : FMDV 3Cpro, RIG-I and MDA5, C142T mutation

[The National Research Foundation of Korea grant (2018M3A 9H4079660) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM9942011)]

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I-15

Unwinding the Role of Pseudouridylation in the Pathogenesis of Fungal Pathogen Cryptococcus neoformans

Seung-Heon Lee1, Jin-Young Kim1, Seong-Ryong Yu1, Anna F. Averette2,Joseph Heitman2, and Yong-Sun Bahn1* 1Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 2Department of Molecular Genetics and Microbiology, Medicine, and Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA

Cryptococcus neoformans is a basidiomycetous fungal pathogen that causes systemic cryptococcosis and meningoencephalitis in immunocompromisedindividuals. Due to its clinical importance, revealing the factors that canaffect its life cycle is critical for development of antifungal and anticryptococcal drugs. Among the various factors, pseudouridylationof RNA is the most abundant type of post-transcriptional modification.Pseudouridylases isomerize uridine into pseudouridine, which subsequently affects the stability of RNA structure. In Saccharomyces cerevisiae, eight proteins exist as stand-alone pseudouridylases, and each protein has specific catalytic sites and roles. To unravel the biological functions of the enzymes in C. neoformans, we identified sixputative pseudouridylases in C. neoformans by using BLAST search inthe FungiDB database with protein sequences of the known pseudouridylasegenes. To characterize the function of pseudouridylases, we constructedmore than two independent strains for 5 putative pseudouridylase genes and examined their phenotypic traits under various conditions. CBF1, which is essential gene in S. cerevisiae, is also suspected to be essentialin C. neoformans since the repressive strains showed growth defect. Among the genes, DEG1 and PUS7 seemed to have major roles in stressresponses such as high temperature and antifungal drugs. In vivo virulence study using insect model and mice virulence study based on Signature-Tagged Mutagenesis (STM) showed reduced virulence of pus7Δ strains, indicating that PUS7 might mediate pseudouridylationof virulence-related genes. Identification of the pseudouridine sites requires N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CMC), which binds to pseudouridine specifically andprevents elongation of cDNA. By comparing the termination rate on the uridine sites of RNAs in both treated/non-treated samples, we can identify pseudouridylated RNA transcripts. By comparing and analyzing pseudouridylated RNA transcripts of PUS genes deletion mutants, we will identify virulence related pseudouridine transcript and characterize their roles in pathogenicity of C. neoformans.

Keywords : Cryptococcus neoformans, pseudouridylation, RNA modification

I-16

Unraveling the Mechanism and Function of CEX1

Tae-Hyun Kim, and Yong-Sun Bahn*

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised individuals. Thecell adhesion ability and the power to pass through Blood-Brain-Barrier(BBB) play a critical role in the development of meningitis. Previous study revealed the correlation of TF and kinase with BBB adhesion and crossing. We focused on CEX1 which is a kinase gene involved in bothBBB adhesion and crossing. We tagged the RFP on CEX1 to check localization on basal and ER stress and check the phenotype for cex1Δstrains. HXL1 splicing was conducted for ER stress sensitivity, the mostobvious phenotype of cex1Δ, and it was found that the phenotype by ER stress of cex1Δ strains was not HXL1 dependent. We performed the GFPchromosomal tagging on secretion proteins which are co-work with Cex1p in Saccharomyces cerevisiae as a COP1 complex. We performedGFP chromosome tagging on secretive proteins operating with Cex1p in S. cerevisiae as a COP1 complex in C. neoformans to verify localizationrelationships through fluorescence microscopy and conduct western blot and co-IP experiments. Through these studies, we can find out if CEX1 contributes to tRNA trafficking by doing COP1 complex in C. neoformans as in S. cerevisiae.

Keywords : CEX1, Cryptococcus neoformans, BBB

I-17

Functional Interaction between Microbiome and Transcriptome ofBroiler Jejunum Affected by Chronic Heat Stress Condition

Young-Jun Seo, Chiwoong Lim, Seok-Won Lim, and Jun-Mo Kim*

Department of Animal Science and Technology, Chung-Ang University, Anseong-si, Gyeonggi-do 17546, Republic of Korea

Heat stress has a effect on animal health and production performanceHS causes multiple physiological disturbances, such as endocrine disorders, immune dysregulation, and so on. And also HS has harmfuleffects on the structure of intestinal tissue, absorption and utilization ofnutrients including microbiota. The purpose of this study was to confirmthe functional interaction between microbiome and transcriptome of broiler jejunum affected by chronic heat stress condition. A total of 60 7-week-old broilers were divided into two groups, thermoneutral control(CON) and heat stress condition (HS). The jejunal mucosa and contentsare collected each groups. In microbiome profiling, functional pathway analysis revealed protein metabolic pathways and lipid metabolic pathways. Then in trancriptome profiling, we identified pathways related with protein and lipid metabolism. We apply metabolite information to connect between functional pathways between microbiomeand transcriptome. Through interaction analysis, it shows the microbiomefunctional pathways and host functional pathways are correlated. In conclusion, microbial composition and expression of genes related withprotein and lipid metabolism in heat stress condition. Pathways interaction analysis results suggest that microbiome and transcriptomepathways interact each other in heat stress condition. Further research is needed to metabolite quantification to identify detail metabolic process between microbiota and host.

Keywords : Heat stress, multi-omics integration, broiler

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I-18

Therapeutic Potential of Trimethylamine Degrading Bacteria against Atherosclerosis

JuneBeom Kim1,2, and Sang Sun Yoon1* 1College of Medicine, Yonsei University, Yonsei-ro 50-1, Seoul 03722, Republic of Korea 2Department of Microbiology and Immunology, Brain Korea 21 Project for Medical Sciences, Republic of Korea

There have been many studies indicating the correlation between gut microbiome and cardiovascular health. Some gut metabolites are reported to be effective to alleviate the cardiovascular disease, while some other metabolites are known to either cause or aggravate the disease. According to previous studies, trimethylamine is a potential threat to human’s cardiovascular health as it tends to be converted trimethylamine N-oxide in the blood, potent to cause atherosclerosis. Inthis research, trimethylamine, gut metabolite derived from choline, is the main target to be degraded and inhibited. For that, murine modelswith high trimethylamine and trimethylamine N-oxide concentration would be set with high choline diet. Methylophilus methylotrophus, phomicrobium vulgare, Hyphomicrobium aestuarii, and recombinant E.coli containing trimethylamine dehydrogenase gene of Methylophilus methylotrophus will be cultured in various trimethylamine or trimethylamineN-oxide present in vitro conditions, which test their significance on degradation. Also, the feces of high choline diet fed mice will be culturedon selective medium plates of which colonies are also going to be testedfor the ability to degrade trimethylamine. According to the results fromin vitro, the bacteria will be tested through gavage on murine models withhigh-choline diet. The data will be evaluated to see whether the bacterialstrains can significantly lower the trimethylamine and trimethylamineN-oxide level in vivo.

Keywords : Gut microbiome, cardiovascular disease, trimethylamine

I-19

Sirtuin 3 Promotes Innate Host Defense against Mycobacteroides abscessus Infection

Young Jae Kim1,2,3,4, Sang Min Jeon1,2,3,4, Prashanta Silwal1,4, Chorong Park1,2,3,4, Kyeong Tae Kim1,2,3,4, Wonhyoung Seo1,2,3,4, Taylor Roh1,2,3,4, and Eun-Kyeong Jo1,2,3,4* 1Department of Microbiology, 2Department of Medical Science, 3Brain Korea 21 FOUR Project for Medical Science, 4Infection Control Convergence Research Center, Chungnam National University School of Medicine, Daejeon 35015, Republic of Korea

The global incidence of infection with rapid growing nontuberculous mycobacteria is increasing, however, host defense factors are poorly understood. In this study, we investigated sirtuin 3 (SIRT3), a mitochondrialprotein deacetylase, in host defense against Mycobacteroides abscessus(Mabc) infection. The level of SIRT3 was decreased in macrophages andmouse lung tissues after Mabc infection. Mycobacterial growth and pathologic inflammation were increased in macrophages and lung tissues from SIRT3 knockout (KO) mice compared with wild-type (WT)mice during infection with Mabc. After Mabc infection, lung tissues andmacrophages from SIRT3 KO mice exhibited significantly higher levelof inflammatory cytokines and chemokines except interferon-γ and interleukin-12 p40, than WT mice. Moreover, macrophages from SIRT3KO mice showed more damaged mitochondria and mitochondrial reactive oxygen species (mitoROS) than those from WT mice during Mabc infection. Especially, mRNA expression and protein levels related to mitochondrial oxidative phosphorylation were decreased in the lungsfrom SIRT3 KO mice compared with those from WT mice infected withMabc. We found that treatment of resveratrol (RSV), a widely used activator of SIRT1/3, repressed Mabc-induced mitoROS in macrophagesfrom WT mice, in a SIRT3-dependent manner. We further showed thatadministration of RSV suppressed bacterial growth and tumor necrosisfactor mRNA expression in the lung tissues from mice after infectionwith Mabc. Together, these data suggest that SIRT3 is required for hostdefense through reduction of pathologic inflammation and regulation of mitochondrial homeostasis via suppression of mitoROS against Mabc infection.

Keywords : Sirtuin 3, Mycobacteroides abscessus, mitochondrial reactive oxygen species

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Phylogenetic Analysis in Accordance with the Lectin like Adhesins in Enterohemorrhagic Escherichia coli (EHEC) Strains

Seung-Hak Cho1*, Jin-Sung Seo1, Kwang-Jun Lee1, and Cheorl-Ho Kim2

1Division of Zoonotic and Vector Borne Disease Research, Center for Infectious Diseases Research, National Institute of Health, Cheongju-si 28159, Chungcheongbuk-do, Republic of Korea 2Glycobiology Unit, Department of Biological Science, Sungkyunkwan University, Suwon 16419, Republic of Korea

Background: During the infection of Enterohemorrhagic Escherichia coli(EHEC), lectin-like adhesins mediate an adherence to host intestinaltract. It is important to identify and understand the lectin-like adhesinsfor vaccine development. Moreover, phylogenetic analysis and investigation of their evolution is required for understanding of the characteristics. In this study, we analyzed evolutionary characteristics of the predicted lectin-like adhesins for the construction of lectin-glycaninteraction network (LGI).Materials/Methods: We performed a phylogenetic analysis according to FimH gene that is a famous adhesinof EHEC using Ugene v37 with the results of our former study. For thephylogenetic analysis, MUSCLE algorithm was used for multiple sequence alignment and PHYLIP neighbor joining algorithm was usedfor the estimation of tree. In the tree estimation step, a total of 1,000 bootstrapping replication was performed. In addition, we referenced epidemiological information from the BioSample data of EHEC strains.Results: In the former study, we predicted a total of 163,528 outer-membrane embedded proteins from the genome of 2,285 strains of EHEC. Virulence-related outer membrane protein, Porin, Outer membrane usher protein, PapC, Coiled stalk of trimeric autotransporteradhesin and intimin/invasion bacterial adhesion mediator protein were the major function of the genes according to the GO-terms. We investigated 732 strains of EHEC that have an epidemiological information for sampling date and location. According to the phylogenetic analysis, 732 EHEC strains were classified into five groupsthat most of strains are devided into two groups and rest of them are included in three groups. The big two groups represent Asia/Pacific and Europe, repectively.Conclusions: The phylogenetic analysis of the predicted lectin-like adhesin showed that the EHEC strains are deferentially adapted in between Asia/Pacific and Europe. Moreover, the putative lectin-like adhesin will be confirmed by glycan array experiments. The lectin-glycan interaction (LGI) network of EHEC andtheir evolutionary analysis will provide researchers a good resource for vaccine or drug development.

Keywords : Enterohemorrhagic Escherichia coli(EHEC), lectin-glycaninteraction network (LGI), phylogenetic analysis

I-21

The Promising Effects of Fecal Microbiota Transplantation for Colitis-Associated Cancer in AOM/DSS Mouse Model

HyunWoo Son1, Jae-Ho Shin1*, and Eun Soo Kim2* 1Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea 2Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Republic of Korea

Living organisms have an extensive microbial community called microbiome. Most of the entire can be found in the digestive tract andis the mostly located in the large intestine. This is called the gut microbiome. This gut microbiome has numerous cells and genes, so it performs various functions within the host. Changes in the gut microbiome are attracting people's attention as it has been shown to be a factor influencing several diseases. Therefore, recovery of dysbiosis to normobiosis through fecal microbiota transplantation (FMT) is suggested as a promising treatment. In fact, FMT is actually used as atreatment for Clostridioides difficile infection, and is known to be veryeffective in Crohn's disease and ulcerative colitis. However, there arenot many studies on colon cancer yet. We conducted a study using a mouse model of colitis-associated cancer (CAC) induced by azoxymethane(AOM)/dextran sulfate sodium (DSS) to see the therapeutic effect of FMT on colitis-associated cancer (CAC). A total of four groups [Control,Non-FMT, normobiosis mouse FMT (nFMT), and dysbiosis mouse FMT (dFMT)] were tested for 47 days. On day 47, PET-CT was performed to quantify colon cancer. After that, it was sacrificed, histological analysis of the colon (length, tumor size, tumor number, H&E staining), spleen size, and FITC dextran permeability were checked, and the gut microbiota and functional gene abundance were confirmed using fecal samples. Genomic DNA sequencing was performed using Illumina Novaseq 6000 sequencing platform. The results suggest that FMT may be an effective option for treating CAC.

Keywords : Fecal microbiota transplantation (FMT), Colitis-Associatedcancer (CAC), gut microbiome

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Unraveling Correlation between Virulence and Metabolism Regulation via c-di-GMP and sgrS in Salmonella

Jiwon Baek1, Eunsuk Kim1, and Hyunjin Yoon1,2* 1Department of Molecular Science Technology, Ajou University, 206 World cup-ro, Yeongtong-gu, Suwon 16499, Republic of Korea 2Department of Applied Chemistry and Biological Engineering, Ajou University, 206 World cup-ro, Yeongtong-gu, Suwon 16499, Republic of Korea

Cyclic-di-GMP, a circular RNA dinucleotide synthesized by diguanylatecyclases, functions as a second messenger in a variety of physiologicalprocesses, including motility, biofilm formation, and virulence factor production. In this study, the role of cyclic-di-GMP in virulence and metabolism regulation in Salmonella Typhimurium was investigated. Overexpression of a diguanylate cyclase AdrA significantly decreasedthe expression of Salmonella Pathogenicity Island (SPI)-1 genes. Moreover, the overexpression of cyclic-di-GMP down-regulated the expression of ptsG, ptsH, manX, manY and manZ, which encode components of sugar phosphotransferase systems (PTS). These PTS genes have been known to be down-regulated by small RNA sgrS. Therefore, the possibility of coordinated regulation of SPI-1 and PTS by cyclic-di-GMP and sgrS was examined. The expression of PTS geneswas negatively regulated by Cyclic-di-GMP and sgrS independently each other or in combination. Interestingly, SPI-1 genes were also down-regulated by sgrS overexpression but their mRNA stability was not directly influence by sgrS. This study, for the first time, suggests thepossibility of regulatory correlation between cyclic-di-GMP and sgrSin the regulation of sugar transport systems and SPI-1.

Keywords : Salmonella, SPI-1, c-di-GMP

I-23

A Study of Pathogenesis and Gene Regulation at Zoonotic and Plant Pathogens in Sub-Inhibitory Concentration of Environmental Pollutants

Jeong Woo Park, BuKyeong Yoon, and Ki Hwan Moon* Division of Marine Bioscience, Korea Maritime and Ocean University, Busan 49112, Republic of Korea

Pollutants emitted into the environment rapidly diffuse and dilute, causing direct and indirect damage to living organisms, including bacteria. Our previous in silico study showed that the sub-inhibitory concentration (sub-IC) of pollutants, phenol and formalin, can be modulate the gene expression of pathogenic bacterium, Edwardsiella piscicida. Here, we selected two representative aquatic and terrestrial pathogens Vibrio vulnificus and Pseudomonas syringe pv. tomato. Eachpathogen was exposed different environmental pollutants, sodium dodecyl sulfate (SDS) and the cigarette ash, respectively. SDS is a surfactant included in detergents or toothpaste and is a typical aquatic environmental pollutant. Cigarette ash is emitted from smokers, about 20 percent of the world's population. Minimal Inhibitory Concentration(MIC) assays were performed with V. vulnificus and P. syringe with eachmatching pollutant. Based on MIC data, we cultured each bacterium withsub-IC of pollutant, then extracted RNAs to check virulence genes expression with future differentially expressed gene (DEG) analyses andqRT-PCR. Although this research is in very early stage, we hypothesized that the virulence factor will be altered its expression level with sub-IC of pollutants, which may modulate the pathogenesis mechanisms of pathogens in the hosts. Our study will provide basic knowledge aboutthe overall pathogenesis and understanding the correlation between environmental pollutants and pathogenic bacteria.

Keywords : Cigarette ash, SDS, sub-inhibitory concentration

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I-24

Crosstalk of Major Three MAPKs Governs the Thermosensitivity of Cryptococcus neoformans

Yu-Byeong Jang, and Yong-Sun Bahn* Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

Cryptococcus neoformans causes meningoencephalitis regardless of immune system disruption and is responsible for approximately 600,000deaths annually. Mitogen-activated protein kinases (MAPK), the pivotalkinases of eukaryotes, play major roles in metabolism and stress response. There are three major MAPKs including Cpk1, Mpk1 and Hog1 and two minor MAPKs (Cpk2 and Mpk2) in C. neoformans. Eventhough the study of individual major MAPK pathways have progressed extensively, the research concerning the crosstalk among MAPK pathways have yet to be elucidated. We aim to understand the crosstalkamong MAPKs to explain the complex signaling pathway regulating thevirulence. We constructed the double and triple MAPK deletion mutants(mpk1Δ hog1Δ, cpk1Δ hog1Δ, mpk1Δ cpk1Δ, mpk1Δ cpk1Δ hog1Δ) andverified characterizing how MAPK crosstalk regulate the downstreamfactors. We discovered through phenotypic analysis that all three majorMAPKs independently play a role in thermosensitivity. To identify theregulation mechanisms with the crosstalk of MAPKs, we observed the changes of phosphorylation of MAPKs in all double MAPK mutants under high temperature. In our previous studies, it is known that Hog1 of C. neoformans serotype A H99 was highly phosphorylated at basalcondition. When moved up to host temperature, Hog1 was dramaticallydephosphorylated like the Hog1 from Candida albicans. As a wild type,cpk1Δ and mpk1Δ showed dephosphorylation of Hog1 at 37℃. However, mpk1Δ cpk1Δ showed more severe dephosphorylation of Hog1 in response to 37℃. In addition, through qRT-PCR, we screenedthe downstream transcription factors (TFs) which are essential or thermosensitive from our TF database. We discovered that MAPKs regulate the expression of transcription factors: HXL1, HSF1, CRZ1, SRE1, PZF1, and MBS2. Hog1 mainly regulates the mRNA expressionlevel and Mpk1 and Cpk1 regulate minor under host temperature. Furthermore, we wondered if the heat shock changed the localizationof transcription factors. Until now, we confirmed host temperature did not change the localization of Hsf1 and Crz1. We plan to tagging remaintranscription factors and observe the translocation of transcription factors under thermal upshift. Therefore, we aim to provide insight intothe regulation of thermal response by complex MAPK crosstalk.

Keywords : MAPK, Cryptococcus, crosstalk

I-25

Systematic Study of Host-Derived Cues for the Regulation of Pathogenicity-Related Transcription Factors in Human Fungal Pathogen Cryptococcus neoformans

Seong-Ryong Yu, Minjae Lee, Kyung-Tae Lee, and Yong-Sun Bahn*

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

Cryptococcus neoformans is a causative agent of fungal meningoencephalitis,which results in more than 180,000 deaths annually. Nevertheless, its treatment option is limited mainly due to a lack of complete understanding of how the pathogen interacts with the host during infection and disease progression. Although a number of signaling pathways involved in the pathogenicity of C. neoformans have been characterized in past years, it remains elusive how complex signaling pathways are coordinated and regulated during the whole infection process. Previously we performed NanoString-based in vivo transcriptionprofiling of 183 kinases, 178 transcription factors, and 139 phosphatasesduring the whole infection process of C. neoformans. Here we focusedon 12 transcription factors, including PDR802, BZP4, HOB5, ZNF2, FZC39, FZC30, SRE1, HLH1, STB4, MLN1, MET32, and GAT201 ofwhich in vivo expression were highly induced during host infection butdid not exhibit evident in vitro phenotypes. To elucidate their in vivo functions, the expression level of the 12 genes were measured in hostmimic condition (HMC). We found that all of them were highly inducedby HMC. Next, we dissected the HMC signals that trigger the inductionof the 12 transcription factors. We found that PDR802 was highly induced by body temperature and carbon starvation. Also, MLN1 deleted mutantsshown growth defects when they have maltose. In conclusion, we provided insight into signaling pathways modulating the pathogenicityof C. neoformans.

Keywords : Cryptococcus neoformans, transcription factor, Host-cue

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I-26

Adenylyl Cyclase and Protein Kinase a Play Critical and Distinct Roles in Antifungal Drug Resistance and Pathogenicity of Candida auris

Ji Seok Kim, and Yong-Sun Bahn*

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

Candida auris is a globally emerging multidrug-resistant fungal pathogen causing invasive human infection and associated with high fatality diseases in immunocompromised patients. Accordingly, the importance of research on C. auris is increasing. In this study, we characterized cyclic adenosine monophosphate (AMP) pathway of C. auris which has been considered to be one of the most important signaltransduction pathway and related to the growth and virulence of pathogenic fungal species. Among the various genes associated with thecAMP pathway, we constructed knockout strains for the adenylyl cyclaseCYR1, PKA gene catalytic subunit TPK1, TPK2, and regulatory subunitBCY1 which are expected to play an important role in the signaling process and related to growth and pathogenicity of C. auris. We also created not only single knockout strains of TPK1 and TPK2, but also tpk1Δ tpk2Δ, to figure out the role of these two genes and how they are controlled by BCY1. Then we conducted a phenotypic analysis of eachmutants to find out what stress these genes are involved in. In addition,we confirmed differences in biofilm formation between wild-type and mutants, and we found that BCY1 and TPK1 served as positive regulator.As a results, these findings show that PKA genes are involved in the formation of C. auris biofilm. We also found that the expression level of multi-drug resistance related genes such as CDR1, CDR2, MDR1, MDR2, ERG11, and TAC1 are controlled by cAMP pathway genes. Itindicates that cAMP pathway regulates the drug resistance of Candida auris, either directly or indirectly. Consequently, these results will indicate that targeting cAMP pathway genes in C. auris could serve asan effective alternative to antifungal therapy against emerging multidrug-resistant fungal pathogen C. auris.

Keywords : C. auris, multi-drug resistance, cAMP pathway

I-27

Effect of Microplastic Exposure on the Pathogenicity of Zoonotic Pathogen, Edwardsiella piscicida

Ju Bin Yoon1, Sungmin Hwang2, and Ki Hwan Moon3* 1Department of Convergence Study on the Ocean Science and Technology, Korea Maritime & Ocean University, Busan 49112, Republic of Korea 2Department of Biology, Duke University, Durham, NC 27708, USA, 3Division of Marine Bioscience, Korea Maritime & Ocean University, Busan 49112, Republic of Korea

Due to the increase of industrial activities, new types of anthropogenicpollutants are rapidly generated. Microplastic (MP) is spotlighted as a new aquatic pollutant that can be threaten both large animals and microorganisms. Our previous study with classical aquatic pollutants, phenol and formalin, showed that the subinhibitory concentration of pollutants can be utilized as signaling molecules, and modulate genesexpression. Here, we exposed zoonotic pathogen, Edwardsiella piscicida, with 0.05 μm-sized MP, then performed the TEM imaging toobserve the microbial community and cell morphology. The TEM datashows that MP particles were possibly entering the bacteria, and a noticeable change in the cell membrane. Transcriptome analyses were performed to identify the differentially expressed genes with MP exposure, resulting that genes involved in major virulence factor, type VI secretion system, and carbohydrate transport and metabolism pathways were regulated. The genome-wide gene expression data corresponded to the results of a qRT-PCR. The in vivo zebrafish infectionassay shows that MP exposed E. piscicida became a hypervirulence. Although this result was contrary to our hypothesis, we assumed that MPexposed E. piscicida could be escaped the host’s immune system at thebeginning stage of infection, then restores its virulence factors withoutpollutants in the host. This study imply that pollutants inflow into theaquatic environment is able to influence pathogenesis mechanisms of pathogens.

Keywords : Edwardsiella piscicida, microplastic, pathogenesis

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I-28

Rapid Identification is an Answer for the Efficient Control/Treatment of Vancomycin Resistant Enterococci

Jorge Enrique Vazquez Buchelli1, Joanna Ivy Irorita Fugaban1, Yosep Ji2,Svetoslav Dimitrov Todorov1, and Wilhelm Heinrich Holzapfel1 1ProBacLab., Department of Advanced Convergence, Handong Global University, Pohang, Gyeongbuk 37554, Republic of Korea 2HEM Inc., 404, Ace Gwanggyo Tower 3, 77, Changnyong-daero 256-gil, Yeongtong-gu, Suwon-si, Gyeonggi 11 16229, Republic of Korea

The incidence of Vancomycin_Resistant_Enterococci (VRE), as a majorcause of nosocomical infections, has become an issue of worldwide concern since vancomycin has hitherto been considered as the last lineof defense again this pathogen. The numbers of VRE infections have increased rapidly since their first appearance in the 1980s. In 2017, VREcaused more than 54,500 infections among hospitalized patients and resulted in almost 10% mortality in USA. Possible spread of the antibioticresistance determinants via horizontal gene transfer to beneficial bacterial strains can be a real scenario. On the other hand, timely detection of VRE in patients with bacterial infections can be a critical factor in decision on an antibiotic treatment. However, with current diagnostic methods, the detection may take more than 24 hours. Taking into consideration the importance of early diagnosis of VRE caused infections, an urgent need exists for the rapid, sensitive, specific detection of this kind of microorganism. The Fluorescent_In_ Situ_Hybridization with Flow_Cytometry (FISH-FLOW) approach provides the potential to detect and quantify from 104 bacterial cells andto target a specific gene at the same time in a single sample of which theresults can be obtained in a matter of hours. For this research, 6 differentVRE strains were differentiated by RAPD-PCR, identified by 16S_rRNA partial sequencing and shown to be positive for vanA and vanB genes by PCR and also for virulence genes associated with enterococci. For identification of VRE by FISH-FLOW we designed PNA (peptide nucleic acid) probes specific for the genus Enterococcusand for the vanA resistance gene. The obtained results indicate that thedesigned PNA specific probes can be used to detect and quantify VREstrains in mixed bacterial populations. The obtained FISH-FLOW results were validated by standard microbiological techniques and the behavior of the studied VRE strains evaluated in the presence of growthlimiting factors. The results suggest that not all cells in the populationof a resistant strain contain or express the resistance gene under antibiotic free conditions. The results show that FISH-FLOW can be regarded as a rapid and reliable way for detection of VRE strains and can be developed in the future as an approach for clinical diagnosis.

Keywords : Vancomycin Resistant Enterococci (VRE), Fluorescent In situ Situ Hybridization with Flow cytometry (FISH-FLOW), rapid diagnostic

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J_Infection and Immunity

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J-1

The Inhibitory Effect of Behenic Acid on Biofilm Formation of Staphylococcus aureus

Seo Young Kim, and Tae Jong Kim*

Department of Forest Products and Biotechnology, Kookmin University, Seoul 02707, Republic of Korea

Staphylococcus aureus is opportunistic pathogen which presents on skin, nose, and respiratory of human. S. aureus in our body forms a biofilm composed of proteins and polysaccharide on the biotic or abioticsurface to survive in environmental stress. The cells in the biofilm are 50 to 500 times more resistant to antimicrobial agents than planktonic cells. Therefore, the biofilm formation is a reason for chronic infections.S. aureus is the main pathogenic bacteria of infection in diabetic foot ulcers, which 15-25% of diabetic patients suffer from, and the higher theblood sugar level, the more severe the symptoms of infection. In this study, we evaluated the effect of behenic acid on biofilm formation of S. aureus in range of normal blood sugar level (0.1%) and diabetes level (0.2% ~ 0.6%). The biofilm formation was quantitatively analyzed bycrystal violet assay. Behenic acid inhibited the biofilm formation by a concentration-dependent manner at all tested glucose concentration. Especially, behenic acid showed the higher biofilm inhibition with 0.1%glucose than with 0.2 ~ 0.6% glucose. We propose that behenic acid isan inhibitor against the biofilm formation of S. aureus in the human body.

Keywords : Staphylococcus aureus biofilm, behenic acid, diabetic footulcer

J-2

Optimal Method for Identification of Staphylococcus spp.

Minji Seong, Se-Kyung Oh, Kil-Soo Kim, and Eui-Suk Jeong*

Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea

Staphylococcus is a genus of Gram-positive facultative anaerobic organisms and under the microscope, they appear round, and form in grape-like clusters. The staphylococcus infection can cause a wide variety of diseases in humans and animals through either toxin production or penetration. These bacteria commonly inhabit the skin andnose where they are innocuous, but may enter the body through cuts orabrasions which may be nearly invisible. And, Staphylococcus spp., iszoonosis, which is easily infected from humans to animals. Animal experiments are essential to biological and medical research. High quality laboratory animal is most important in the experiment for get reliability and reproducibility data. However, most of the infections inlaboratory animals are closely related to the hygiene of breeders and/orresearcher. However, it is not easy to identify bacterial of similar colonymorphology and morphology diagnosis is subjective. In this study, we comparing conventional diagnosis methods for objectively identificationof Staphylococcus spp., Our PCR assay will be used to improve qualitycontrol in laboratory animals and laboratory animal facilities.

Keywords : Staphylococci, PCR assay, quality control

J-3

Effects of Pediococcus pentosaceus Strains Isolated from Three Different Types of Kimchi in ICR Mice Infected with Escherichia coli or Salmonella Typhimurium

Hanjin Oh, Jihwan Lee, Yongju Kim, Jaewoo An, Seyeon Chang, Youngbin Go, Dongcheol Song, Hyunah Cho, and Jinho Cho*

Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk 28644 Republic of Korea

One hundred twenty ICR male mouse with initial body weight of 26±2g(5 weeks old) were randomly assigned to six treatments for 2 week feeding trial to determine the effect of Pediococcus pentosaceus strainswhich are isolated from three different types of Kimchi in ICR mice infected with Escherichia coli or Salmonella Typhimurium. Six groupswere Normal control group without E. coli and S. Typhimurium orally administrated (NC-; n = 20), Normal control group with E. coli or S. Typhimurium orally administrated (NC+; n = 20), L. plantarum orally administrated group after E. coli or S. Typhimurium orally administrated(LP; n = 20), P. pentosaceus strain A orally administrated group afterE. coli or S. Typhimurium orally administrated (PSA; n = 20), P. pentosaceus strain B orally administrated group after E. coli or S. Typhimurium orally administrated (PSB; n = 20), P. pentosaceus StrainC orally administrated group after E. coli or S. Typhimurium orally administrated (PSC; n = 20) on each E. coli infected groups and S.Typhimurium infected groups. LP and PSC had significantly(p < 0.05)improved in growth performance compared with other groups except forNC- in E.coli infected mice group. NC+ were significantly lower (p <0.05) growth performance compared with other groups except for NC-in S.Typhimurium infected mice groups. In E.coli and Salmonella count in intestine, LP and PSC groups had significantly lower(p < 0.05) countsthan NC+, and PSB groups. In conclusion, LP and PSC strains are isolatedfrom kimchi can act as probiotics by inhibiting E. coli and S. Typhimurium.

Keywords : Kimchi, probiotic, intestinal microorganisms

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J-4

Efficacy of Novel Oil Adjuvant CAvant®SOE for Foot and Mouth Disease Vaccine

W A Gayan Chathuranga1, Young-Hoon Ahn2, Young-Jung Shim2, Eun-Hee Kim2, Sung Ho Shin3, Hyundong Jo3, Jong-Hyeon Park3, Sung-Sik Yoo2*, and Jong-Soo Lee1* 1College of Veterinary Medicine, Chungnam National University, Daejeon 34314, Republic of Korea 2Choong Ang Vaccine Laboratory Co., Ltd., Daejeon 34055, Republic of Korea 3Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of Korea

Foot and Mouth Disease (FMD) is a notifiable contagious disease of cloven-hoofed mammals. A high potency vaccine that stimulates the hostimmune response is the foremost strategy used to prevent disease persistence in endemic regions. FMD vaccines comprise inactivated virus antigens whose immunogenicity is potentiated by immunogenic adjuvants. Oil-based adjuvants have clear advantages over traditional adjuvant vaccines; however, there is potential to develop novel adjuvantsto increase the potency of FMD vaccines. Thus, we aimed to evaluate the efficacy of a novel water-in-oil emulsion, called CAvant®SOE, as a novel vaccine adjuvant for use with inactivated FMD vaccines. We found that inactivated A22 Iraq virus plus CAvant®SOE induced better antigen-specific humoral and cell-mediated immune responses in micethan a commercial vaccine. Moreover, A22 Iraq-CAvant®SOE inducedslightly higher production of neutralizing antibodies than the commercial vaccine. Intramuscular immunization of pigs using A22 Iraq-CAvant®SOE also led to higher and longer-lasting virus-neutralizingantibody titers than immunization with the reference adjuvant. A singledose of A22 Iraq-CAvant®SOE elicited effective control of virus shedding, with no detectable clinical symptoms; it also protected againstchallenge with heterologous FMDV. Levels of protection correlated strongly with vaccine-induced neutralizing antibody titers. Collectively,these results indicate that CAvant®SOE-adjuvanted vaccine is a promising candidate for control of FMD in pigs.

Keywords : Foot and mouth disease virus, adjuvant, CAvant®SOE

[The Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Grant No. 318039-3) and the National Research Foundation of Korea (2018M3A9H4078703)]

J-5

Toll-like Receptor 7 Agonist GS-9620 as an Effective Adjuvant in Influenza Vaccines

Dhammika Haluwana1, W.A. Gayan Chathuranga1, Meehyein Kim2, andJong-Soo Lee1* 1College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea 2Virus Research Group, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea

GS-9620 is a synthetic dihydropteridinone derivative that was identifiedas a TLR7 specific small-molecule agonist. GS-9620 has previously been shown to suppress hepatitis B virus (HBV) in various animal models. Though published knowledge of GS-9620 fulfils the criteria asa potent immune-stimulant, there is no documented usage of immune-stimulant for the vaccine adjuvant currently. In this study, we evaluate the mode of action and efficacy of GS-9620 as an immune-stimulant for the vaccine adjuvant. In vitro, GS-9620 showedthe induction of TLR7-mediated robust cytokine production and immune response gene expression in human and murine immune cells.In vivo, the effect of GS-9620 as an immune-stimulant was examined with influenza recombinant protein sM2HA2 and inactivated A/PuertoRico/8/34 virus (iPR8). Consequently, intramuscular administration ofsM2HA2 and intranasal administration of iPR8 with GS-9620 inducedsignificantly higher antigen-specific humoral and cell-mediated immune responses than well-known TLR7 agonist Imiquimod. Further,it enhanced the lung virus clearance and protective efficacy of the sM2HA2 subunit antigen and iPR8 vaccine against the lethal challengeof divergent influenza subtypes (H5N2, H9N2, H1N1 and PR8) in a murine model. Our results collectively suggest that GS-9620 can providean effective mucosal and systemic immune-stimulant for adjuvant of both subunit and inactivated whole virus vaccine.

Keywords : Toll-like receptor 7, GS-9620, adjuvant

[The National Research Foundation of Korea (2018M3A9H4078703, 2019M3E5 D5066834)]

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CAvant WO-60 as an Effective Immunological Adjuvant for Avian Influenza and Newcastle Disease Vaccine

W A Gayan Chathuranga1, Eun-Seo Lee1, Young-Jung Shim2, Young-hoon Ahn2, Sung-sik Yoo2, and Jong-Soo Lee1* 1College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea 2Choong Ang Vaccine Laboratories Co., Ltd., Daejeon 34055, Republic of Korea

Despite current vaccines used in poultry being immunogenic, the number of pathogens including avian influenza virus (AIV) and Newcastle disease virus (NDV) causes enormous economic losses to the global poultry industry. To solve this problem, vaccine efficacy can befurther improved with the introduction of effective adjuvants. Here, wedeveloped a new adjuvant, CAvant® WO-60, that safely and effectivelyenhances both the immunogenicity of conserved influenza antigen sM2HA2 and inactivated whole H9N2 antigen (iH9N2): it induced bothhumoral and cell-mediated immunity in mice and provided 100% protection from challenge with 10 LD50 of A/Aquatic bird/Korea/ W81/2005(H5N2) and A/Chicken/Korea/116/2004 (H9N2) AIV. Importantly, immunization of chickens with CAvant® WO-60 emulsifiediH9N2+inactivated NDV LaSota (iNDV) bivalent inactivated vaccine induced seroprotective levels of antigen-specific antibody responses. Thus, the new adjuvant CAvant® WO-60 would be a promising adjuvant for poultry vaccines.

Keywords : CAvant® WO-60, adjuvant, Water-in-Oil

[The Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Grant no. 316043-3, Grant No. 318039-3) and the NationalResearch Foundation of Korea (2018M3A9H4078703)]

J-7

Efficacy of Recombinant Subunit Vaccine Candidate against Foot-and-Mouth Disease Virus Serotype O and A in Pig

W A Gayan Chathuranga1, Jong-Hyeon Park2*, and Jong-Soo Lee1* 1College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea 2Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of Korea

Foot-and-mouth disease virus (FMDV) causes an acute, severe and highly contagious disease of cloven-hoofed animals whose control relieson efficient vaccination. Although currently, available inactivated virusvaccines have proved to be effective in FMD control, concerns regardingtheir safety have been raised during the vaccine formulation process. Therefore, it is necessary to develop safe and effective alternative vaccine to replace the traditional inactivated vaccine. In this study, we developed two multi-epitope recombinant protein OVM and AVM, OVM antigen comprised of tandem repeats of antigenic site of two SouthKorea isolates O/Andong/SKR/2010 and O/Jincheon/SKR/2014 with classical vaccine strain O1/Manisa/Turkey/69, AVM antigen comprisedof tandem repeats of antigenic site of South Korea isolates A/Pocheon/KOR/2010 with two classical vaccine strain A22/Iraq/24/64and A/Malaysia/97 and evaluated their efficacy with immunogenic adjuvant ISA201. In the mouse model, OVM-AVM vaccine candidate induced effective antigen-specific humoral and cell-mediated immuneresponses and effectively protected from mouse-adapted O/Jincheon/ SKR/2014, O/VET/2013 and A/Malaysia/97 virus challenge. Moreover,intramuscular immunization of pigs with OVM-AVM vaccine candidateeffectively protected from O/Jincheon/SKR/2014 and A/SKR/4/2018 challenge. Together, our results suggested that the developed OVM-AVMvaccine candidate provides opportunities for safer and effective vaccineproduction that may replace the traditional inactivated vaccine for theprevention and control of FMD in pigs in the future.

Keywords : FMDV, OVM-AVM, multi-epitope recombinant protein

[The Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Grant no. 318039-3) and the National Research Foundation of Korea (2018M3A9H4078703)]

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J-8

E3 Ubiquitin Ligase RNF 172 Negatively Regulates Innate Immune Signal by Targeting Nuclear Factor-κB Essential Modulator

Kiramage Chathuranga, and Jong-Soo Lee*

College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

Nuclear Factor-κB Essential Modulator (NEMO) is a key regulatory protein that functions during NF-κB and Interferon-mediated signalingin response to extracellular stimuli as well as pathogen infection. Smoothregulation of NEMO signaling is essential for the host innate immune responses and maintenance of homeostasis. In this study, we report thatthe E3 ligase RNF 172 (also known as membrane associated RING-CH2 or MARCH2) is a novel negative regulator of NEMO-mediated signaling upon bacterial or viral infection. RNF 172 directly interactedwith NEMO during the late time of infection and catalyzed the K-48-linked ubiquitination of Lys326 on NEMO, which then resulted in its proteasomal degradation. Ultimately, CRISPR/Cas9 mediated deletion of RNF 172 exhibited a marked resistance along with increased innate immune responses against viral or bacterial infections both in vitroand in vivo. In addition, RNF 172-/- mice were more susceptible to a LPSchallenge due to the massive production of cytokines. Collectively, thesefindings provide new insights into the molecular regulation of NEMO and suggest an important role for RNF 172 in homeostatic control of innate immune responses.

Keywords : RNF 172, NEMO, ubiquitination

[The National Research Foundation of Korea (Grant no. 2019R1A 2C2008283) and KRIBB Research Initiative Program (KGM9942011)]

J-9

Differentially Expressed Genes in a Bacterial Wilt Resistant Tomato Plant Transplanted with Two Different Soil Microbial Fractions

Hyoung Ju Lee1, Kihyuck Choi1, Joo Hwan Kwon1, Junesung Lee2, Seon-In Yeom2, and Seon-Woo Lee1* 1Department of Applied Biology, Dong-A University, Busan 49315, Republic of Korea 2Department of Agricultureal Plant Science, Division of Applied Life Science (BK21 Plus Program), Gyeongsang National University, Jinju 52828, Republic of Korea

Bacterial wilt (BW) caused by Ralstonia solanacearum greatly reducesthe production of Solanaceae crops including tomato plant. In the previous study, BW-resistant tomato cultivar transplanted with uplandsoil microbial fraction (UpMF) showed the strong resistance to BW compared to the control, while the BW-resistance was completely abolished by forest soil microbial fraction (FoMF) transplant. To compare differentially expressed genes (DEGs) between two different microbiota transplant, RNA-Seq was performed using Illumina HiSeq system. Interestingly, most of genes involved in plant-microbe interactions was equally expressed irrespective of microbiota transplant.However, DEG analysis identified a total of 32 DEGs that showed robustdifferential expression under two different MFs and at three different time points. Most of these DEGs were associated with signaling pathwayin tomato plants. Among 32 genes, 15 and 17 genes showed the increasedand decreased expression in UpMF transplant compared to FoMF transplant, respectively. To compare the expression of DEGs, quantitative reverse transcriptional PCR is under investigation using RNAs isolated from the tomato plants transplanted with UpMF and FoMF. This result suggests that microbiota-specific signaling occurs intomato plant to modulate BW resistance in a BW-resistant tomato plant.

Keywords : Bacterial wilt, tomato plant, differentially expressed genes

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J-10

Oral Immunization with Cell Extracts of SARS-CoV-2 Spike Protein Antigen-Expressing Recombinant Lactococcus lactis in Mice

Biao Xuan, and Eun Bae Kim*

Department of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon 24341, Republic of Korea

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beganto spread around the world and became a global pandemic named coronavirus disease (COVID-19). At present, many COVID-19 vaccinesare being developed, and most of these vaccines are administered by intramuscular injection. The aim of the study is to develop an oral vaccinefor COVID-19 by using SARS-CoV-2 spike (S) protein antigen- expressingrecombinant Lactococcus lactis and test it in mice. SARS-CoV-2 S protein receptor binding domain (RBD) S1 subunit as an antigen and itwas transformed into wild type L. lactis IL1403, and its expression wasconfirmed by western blot. Cell extracts of recombinant L. lactis were orally administered into mice. Western blot experiment detected intracellulartarget antigen of the recombinant L. lactis, however, extracellular target antigen was not detected. After immunization with priming, 1st and 2nd

boosting, antigen specific serum IgG and fecal IgA were measured and the immunization group was 1.5-fold (p = 0.002) and 1.4-fold (p = 0.016)higher than control group, respectively. Our results indicate that cell extracts of SARS-CoV-2 S protein antigen-expressing recombinant L. lactis induces mice to produce antigen-specific antibody. This strategymay potentially be used in development of oral vaccines.

Keywords : Coronavirus, L. lactis, oral vaccine

[This study was supported by NRF (NRF-2019R1A2C1009406)]

J-11

Anti-Inflammatory and Anti-Fibrotic Effects of Nocardiopsis sp. 13G027 Extract in Transforming Growth Factor β1-Induced Nasal Polyp-Derived Fibroblasts

Grace Choi1, Geum Jin Kim2,3, Hyukjae Choi2,3, Jeong Min Lee1, Mi-JinYim1, Il-Whan Choi4, and Dae-Sung Lee1* 1Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea, Chungcheongnam-do 33662, Republic of Korea 2College of Pharmacy, Yeungnam University, Gyeongsangbuk-do 38531, Republic of Korea 3Research Institute of Cell Culture, Yeungnam University, Gyeongsangbuk-do38531, Republic of Korea 4Department of Microbiology and Immunology, Inje University College of Medicine, Busan 49267, Republic of Korea

Nocardiopsis species produce bioactive compounds such as anti- microbial agents, anti-cancer substances, and toxins. However, no reporthas described their anti-inflammatory and anti-fibrotic effects during nasal polyp (NP) formation. In the present study, we investigated the anti-inflammatory and anti-fibrotic effects of Nocardiopsis sp. 13G027extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophagesand transforming growth factor (TGF)-β1-stimulated nasal polyp- derived fibroblasts (NPDFs). Nitric oxide (NO) production was analyzed using the Griess reaction. Prostaglandin E2 (PGE2) release was determined using sandwich enzyme-linked immunosorbent assay. Theexpression of mitogen-activated protein kinases (MAPKs) and proteinkinase B (Akt) in LPS-induced RAW 264.7 cells and α-smooth muscleactin (α-SMA), type I collagen (Col-1), fibronectin, and phosphorylatedsmall mothers against decapentaplegic 2/3 (Smad2/3) in NPDFs was measured using Western blotting. Treatment with the 13G027 extract significantly suppressed NO and PGE2 production. The extract suppressed the LPS-induced the phosphorylation of MAPKs and Akt andthe DNA-binding activity of activator protein-1 (AP-1). The expressionof pro-fibrotic components such as α-SMA, Col-1, fibronectin, and Smad2/3 was inhibited in TGF-β1-exposed NPDFs. Therefore, the current findings suggested that Nocardiopsis sp. 13G027 is a valuable candidate for the treatment of inflammatory disorders such as NP formation.

Keywords : Nasal polyp, Nocardiopsis sp. 13G027, anti-inflammatoryand anti-fibrotic

[This work was supported by a research grant 2021M00500 from the National Marine Biodiversity Institute of Korea, Republic of Korea]

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J-12

Comparative Analysis of Chikungunya Virus for Reliable and Sensitive Detection Using qPCR, ddPCR and RPA

Dongju Park1,2,3, Zohaib Ul Hassan1,2,4, Changwoo Park1,2,5, Seungbum Kim3, and Seil Kim1,2,4*

1Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea 2Convergent Research Center for Emerging Virus Infection, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea 3Department of Bioscience and Biotechnology, Chungnam National University, Daejeon 34134, Republic of Korea 4Department of Bio-Analysis Science, University of Science & Technology (UST), Daejeon 34113, Republic of Korea 5Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea

Background: Chikungunya virus (CHIKV) is the pathogen of re-emergingepidemic disease accompanying abrupt febrile illness. Until now, millions of people have been infected worldwide. However, vaccines anddrugs against CHIKV are not yet available. In the absence of vaccines and drugs against CHIKV, the reliable and sensitive assays for CHIKVdiagnostics are crucial for the clinical decision. Methods: For reliableand accurate detection of CHIKV, quantitative PCR (qPCR), droplet digital PCR (ddPCR) and Recombinase Polymerase Amplification (RPA) assays targeting to Capsid protein (C) and Envelop protein 2 (E2)genes were developed. Both qPCR and ddPCR assay used same probesand primer pairs. The primer pairs and probes for RPA was designed and tested according to manufacturer’s instruction. Results: All assays weretested using viral RNA (or cDNA) in low concentration. qPCR and ddPCR show the similar limit of detection. Both qPCR and ddPCR assaycan detect few copies of CHIKV genomes. The RPA assay can detect CHIKV genomes of low copy number within 15 minutes. Conclusions:Both qPCR and ddPCR for CHIKV were very sensitive and reliable for the detection of CHIKV. The RPA assay is more rapid than PCR-basedassay though the sensitivity of RPA assay was lower than that of PCR-based assay.

Keywords : Chikungunya virus, quantitative PCR and droplet digital PCR, Recombinase Polymerase Amplification

J-13

Comparison of Sensitivity and Quantification of SARS-CoV-2 using Two PCR-Based Methods

ChangWoo Park1,2,3, Min kyu Park1,5, and Seil Kim1,2,4*

1Microbiological Analysis Team, Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea 2Convergent Research Center for Emerging Virus Infection, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea 3Department of Agricultural Biotechnology, Seoul National University (SNU), Seoul 08826, Republic of Korea 4Department of Bio-Analysis Science, University of Science & Technology (UST), Daejeon 34113, Republic of Korea 5College of Bioscience and Biotechnology, Chungnam National University (CNU), Daejeon 34134, Republic of Korea

World Health Organization (WHO) announced that Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had become a pandemic on March 11. SARS-CoV-2 is a newly discovered human coronavirus and highly transmittable and has rapidly spreading worldwide, highlighting the essential role of diagnosis in preventing thespread of the epidemic. Currently, the diagnosis of SARS-CoV-2 infection is mainly done by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) methods. Although the RT-qPCRmethod is regarded as a gold standard test on emergency situations, theresult of RT-qPCR is relative quantification and Ct value can be varied by primer pairs, reaction mixture and etc. To assess the methods, comparison of RT-qPCR and ddPCR with various primer pairs were done. The cultured viral genomic RNA and the extracted RNA from clinical samples were used as templates for the assays. The Ct values ofRT-qPCR assay with various primer pairs were varied with the same template, while the results of ddPCR were similar regardless to primerpairs, indicating ddPCR assay can be used for the diagnosis and quantification of SARS-CoV-2 genomic RNA.

Keywords : Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Droplet digital PCR (ddPCR)

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J-14

Pathogenic Bacterial Shiga Toxins Involve Pro-Inflammatory Cytokine Production Via p38 MAPK/MK2/TTP Pathways

Seo-Young Park1,3,4, Yu-Jin Jeong1, Kyung-Soo Lee1,2, Jongsun Park3,4*,and Moo-Seung Lee1,2* 1Environmental Diseases Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-Gu, Daejeon 34141, Republic of Korea 2Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 127 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea 3Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea 4Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea

Multi-functional bacterial exoprotein Shiga toxins (Stxs) produced by Shigella dysenteriae serotype 1 and certain Escherichia coli are responsible for causing hemorrhagic colitis that may progressively leadto hemolytic uremic syndrome (HUS) and central nervous system (CNS). The precise pathophysiological mechanism between Stxs and the toxin-induced inflammation has not been completely understood. Numerous studies have defined the p38 mitogen-activated protein kinase (MAPK) and its downstream target MAPK-activated protein kinase 2 (MK2) signaling pathway in a variety of cell types. We identifiedpreviously unknown role of Tristetraprolin (TTP) as MK2 substrate in Stxs-intoxicated cells. We have observed that Stxs induce phosphorylationof the MK2 at residue Thr334 and TTP in the toxin receptor Gb3-positivecells including macrophage-like differentiated THP-1 (D-THP-1) and human proximal tubule epithelial cell line HK-2 while not in the Gb3-negative human T84 colon carcinoma cells. Thus, Stxs selectivelymediate MK2 and TTP activation in a Gb3-dependent manner. TTP-knockdown by using targeted siRNA in the D-THP-1 cells treatedwith Stx2a upregulate the expression of TNF-α, IL-8, MCP-1 and MIP-1α at transcriptional and translational levels. In conclusion, the MK2-TTPsignaling pathway regulates Stx-mediated inflammatory response.

Keywords : Shiga toxin, MAPK-activated protein kinase 2 (MK2), Tristetraprolin(TTP)

J-15

A Murine CD8+ T Cell Epitope Identified in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein

Jihyun Yang1, Eunjin Kim1,2, Jong-Soo Lee2 and Haryoung Poo1*

1Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea 2College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has posed a devastating threat world-wide. The receptor-binding domain (RBD) of the spike protein is one of the most important antignes for SARS-CoV-2 vaccines, while the analysis of CD8 cytotoxic T lymphocyte activity in preclinical studies using mouse models is critical for evaluating vaccine efficacy. Here, we immunized C57BL/6 wild-type mice and transgenic mice expressing human angiotensin- converting enzyme 2 (ACE2) with the SARS-CoV-2 RBD protein to evaluate the IFN-γ-producing T cells in the splenocytes of the immunized mice using an overlapping peptide pool by an enzyme-linkedimmunospot assay and flow cytometry. We identified SARS-CoV-2 S395-404 as a major histocompatibility complex (MHC) class I-restrictedepitope for the RBD-specific CD8 T cell responses in C57BL/6 mice.

Keywords : SARS-CoV-2, CD8 cytotoxic T lymphocyte, epitope

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K_Microbial Ecology and Diversity

K-1

Unexpected Candidate Phyla Radiation Microbiome in the Human Gut Environment

Joon Yong Kim, Tae Woong Whon, Se Hee Lee, and Seong Woon Roh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

The Candidate Phyla Radiation (CPR) has been recently described as the large evolutionary radiation of bacterial candidate phyla and superphyla. Most CPR members are uncultivable and have a small genome. In the CPR group, only strain TM7x belonging to the class Saccharimonadia was isolated and identified from a human associatedsaliva sample. In this study, I attempted to discover the CPR microbiomein human faecal samples using a filtration method. When the filtrationstep was incorporated in the sample preparation, microbial communityrichness was increased, while evenness was decreased. In addition, therelative abundance of CPR was dramatically increased in filtered samples, with a variety CPR group members detected. Most of these members were assigned to the class Saccharimonadia, which containedthe strain TM7x, but the classes Parcubacteria, Microgenomatia, ABY1, and CPR2 related taxa were also detected. These classes had notbeen described in human related samples before this study. The study revealed that many CPR members thrived in the human faecal samples.Further study based on metagenomics approach will expand the understanding of the role of the CPR in the human gut microbiome.

K-2

The groESL ISR Sequence-Based Species-Specific Identification of GRAS and Non-GRAS Lactiplantibacillus as an Alternative to 16S rRNA Sequencing

Jong Min Lee1, So Hee Park2, Won Je Jang1, So Young Park1, So-JeongKim1, Eun Ji Lee1, Dong Nyoung Oh1, Sun Jay Yoon1, Sung Jin Jeong1,Nan Kyung Kim1, Da Won Jeong1, and Chang-Jin Kim2* 1Department of Biotechnology, Pukyong National University, Busan 48513, Republic of Korea 2Industrial Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea

The untranscribed DNA sequences of the intergenic spacer regions (ISR)in the groESL were analyzed to resolve the ambiguity within phylogenetically close GRAS and non-GRAS species. The sequencingresults of amplified polymerase chain reaction (PCR) products using groESL-ISR-specific primers accurately distinguished L. plantarumand L. pentosus, which were not distinguished by 16S rRNA sequences.Furthermore, the 20 selected major probiotics species were divided intoseveral groups according to the length (22-91 bp) and homology (65-99%) of their ISR sequences, and this discrimination was consistentwith core-genome-based differentiation. Therefore, ISR sequence- based species identification showed more accurate results than 16S rRNA sequence-based identification and represented genome-based speciation.

Keywords : Identification, groESL Intergenic spacer region, Lactiplantibacillus

K-3

Value Evaluation of Actinobacterial Resource Based on Microbial Characteristics

So Hee Park1,2, Chun-Zhi Jin1, Min-Kyoung Kang1, Dong-Jin Park1, andChang-Jin Kim1,2* 1Industrial Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 2Department of Bio-Molecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea

Actinomycetes, as a producer of diverse secondary metabolites which have the primary importance in medicine, cosmetic, agriculture and foodproduction until now. Though the utilization and application of these actinomycetes were still insufficient due to less information. It is necessary to evaluate microbial resources so that the researchers can dofurther experiments quickly and efficiently as possible without screeningwork. The purpose of this study is to support and help researchers through infrastructure construction and quantitative analysis of microbes. We focus on 16S rRNA sequencing for taxonomic analysis, enzyme (protease,amylase, lipase, cellulose) assay, physiological characters of each stain (growth temperature, media pH, salt tolerance) and LC/MS profiles of culture broth. We have more than 15,000 actinomycetes’ information and culture broth could be supplied. It is helpful for high-throughput screening and speed up experiments. Visit us: http://mrscc.net/.

Keywords : Actinomycetes library, functional study, high-throughput screening

[This research was supported by a grant (NRF-2013M3A9A5076601) from a study on the strategies of improving the value of microbial resources funded by Ministry of Science and ICT of the Korea Government]

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K-4

Spirihalobacter sediminis gen. nov., sp. nov., Isolated from Sediment

Hyekyoung Yang1, Ha Jung Moon1, Hong Sik Im1, Yochan Joung2, Byungkwon Kim3, Jungjo Han3, and Sang-Seob Lee1,2* 1Integrative Biotechnology, Sungkyunkwan University, 2066, Seobu-ro, Jangan-gu,Suwon-si, Gyeonggi-do 16419, Republic of Korea 2Korea Environmental Microorganisms Bank (KEMB), 2066, Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do 16419, Republic of Korea 3Gyeonggi Province Maritime and Fisheries Research Institute, Ansan-si, Gyeonggi-do, Republic of Korea

A Gram-stain-negative, motile, aerobic, helical-shaped bacterial strains,designated MAW1-3T, was isolated from sediment collected from the seashore of West Sea, Republic of Korea. The isolate was catalase-positive and oxidase-positive. Strain MAW1-3T grew at 20-45℃ (optimum 42℃), pH 6-8 (optimum pH 7), and in the presence of 0.5-9.0 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain MAW1-3T belonged to the family Rhodospirillaceae. The strain is grouped into a clade with genus genusCaenispirillum, Haematospirillum and Novispirillum, sharing the highest sequence similarities with Caenispirillum salinarum (93.88%).The only respiratory quinone was ubiquinone-10 (Q-10). The polar lipidprofile comprised phosphatidylglycerol, phosphatidylethanolamine, phospholipid and amino lipid. The major fatty acids were C18:1 ω7c, C16:0,summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH). The Whole-genome of strain MAW1-3T revealed a genome size 3.6 Mbp. TheDNA G+C content was 69.9 mol%. On the basis of phenotypic and phylogenetic data, strain MAW1-3T represented a novel genus and species, for which the name Spirihalobacter sediminis gen. nov, sp. nov.is proposed. The type strain of is MAW1-3T (=KCTC 82704T).

K-5

Mariniroseobacter mytili gen. nov., sp. nov., Isolated from Mussel

Hajung Moon1, Hyekyoung Yang1, Hong Sik Im1, Hyeyoung Sung2, Yochan Joung2, and Sang-Seob Lee1,2* 1Integrative Biotechnology, Sungkyunkwan University, 2066, Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do 16419, Republic of Korea 2Korea Environmental Microorganisms Bank (KEMB), 2066, Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do 16419, Republic of Korea

A novel bacterium, designated MH4019T, was isolated from mussel in Wando, Republic of Korea. The isolate was gram staining-negative, aerobic, rod-shaped, oxidase- and catalase- positive. Strain MH4019T

grew at 15℃-30℃ (optimum 30℃) and pH 6-8 (optimum pH 7) and inthe presence of 0.5-5.0% (w/v) NaCl (optimum 3%). Phylogenetic analyses based on the 16s rRNA gene sequence showed that strain MH4019T belonged to the family Rhodobacteraceae in the class Alphaproteobacteria and was closely related to the type strains of Thalassobius maritimus, Pseudooceanicola atlanticus with 95.8% and94.43% 16s rRNA gene sequence similarities, respectively. The major fatty acids were summed feature 8(C18:1ω7c and/or C18:1ω6c), iso-C18:00.The predominant respiratory quinone was Q-10. The major polar lipidswere phosphatidylglycerol, two unknown phospholipids and four aminolipids. The DNA G+C content was 59.68 mol%. Strain MH4019T

represents a novel genus of the family Rhodobacteraceae, for which thename Mariniroseobacter mytili gen. nov., sp. nov. is proposed for this isolate, and the type strain is MH4019T (=KCTC 82703T).

K-6

Importance and Value of Microbial Resources for Nematode Control

Min-Kyoung Kang, Jong-Hoon Kim, Dong-Jin Park, Kwang-Hee Son*, and Chang-Jin Kim*

Industrial Bio-Materials Research Center, Korea Research Institute of Bioscience and Biotechnology 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea

The growing troubles of facility cultivation farmers are increasing dayby day due to the increasing damage of nematodes every year. Nematodessurvive regardless of the environment such as soil, irrigation water, anddirt and cause damage to crops. The annual loss alone is about 250 billionwon, and the cost of nematodes for nematode control is also estimated at 70 billion won. Methods of controlling nematodes are largely dividedinto chemical control, physical control, and biological control. Chemicalcontrol is a method of treating nematode drugs such as pesticides and organic farming materials, physical control is a method of using solarheat, and biological control is a method of using natural enemies. Of these, the chemical control effect is the greatest, but because pesticidescan cause residual problems, organic farming materials are expensive and the efficacy of drugs is inferior, but many farmers are looking for organic farming materials. Therefore, we sought to find a resource for controlling nematodes from natural products. Among natural products,microorganisms produce a variety of beneficial secondary metabolites.In this study, potential nematode control microorganisms were searched from a microbial library, and the possibility of natural organic materialswas suggested. In addition, through this study, we propose the value ofmicroorganisms for the control of plant diseases in the future.

Keywords : Nematode, microbial, natural resource

[This research was supported by a grant (Project No. NRF-2013 M3A9A5076601) from the Ministry of Science, ICT and Future Planning of the Korea Government and was supported by Korea Instituteof Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) through (Project No. 321110-4 “Crop Viruses and PestsResponse Industry Technology Development”) Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)]

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K-7

Understanding New Mechanism of Access and Benefit-Sharing of Genetic Resources under the Nagoya Protocol

Young Hyo Chang, and Minho An*

ABS Research Support Center, Korea Research Institute of Bioscience and Biotechnology(KRIBB), Daejeon 3414, Republic of Korea

As South Korea became the party to the Nagoya Protocol (ABS) and enforced a national law in accordance, these changes have increased public awareness on sovereign rights over genetic resources and the ABS. These also apply to the research fields of microbiology. Microorganisms in everyday life, even those of the same species, can be dispersed everywhere on the Earth, and for this reason, we cannot easily determine their country of origin. However, researchers are nowrequired to specify where it had been isolated. It signifies, if researchersisolates an activated strain of microorganisms and wants to write a research article or apply for a patent on the study results, they have to prove that there was no act of biopiracy throughout the sample collectionprocess. Furthermore, the biological resource banks that manage varioussamples of microorganisms must verify in the position of recipient whether the strains that a donor is willing to deposit have well respectedthe ABS. To cope with these changes in research environment, a researcher must understand the overall process of research activities andeach step of the ABS. In this regard, our study will help researchers deal with the ABS procedures of resource providing country, from access togenetic resources to benefit-sharing of their research results, and be readyto minimize potential damages caused by the ABS.

Keywords : Nagoya protocol, genetic resources, access and benefit- sharing

[This research was supported by a grant (Project No. NRF-2013 M3A9A5076601) from the Ministry of Science, ICT and Future Planning of the Korea Government and was supported by Korea Instituteof Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) through (Project No. 321110-4 “Crop Viruses and PestsResponse Industry Technology Development”) Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)]

K-8

Isolation of New Type and Unreported Endophytic Fungi from Plant’s Roots in Gyeongsangdo Province, Korea

Doo Ho Choi, and Jong-Guk Kim* School of Life Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea

Because of their contribution in nature as de-composer or symbiosis orpathogen, countless surveys regarding fungi have been studied. In surveyregarding diversity analysis of isolated fungal species, total 824 individuals of endophytic fungi were isolated from 19 kinds of plantsliving in Gyeongsang Province. During the identification based on fungal morphology and phylogenetic analysis of the internal transcribedspacer (ITS) rDNA region, the isolated fungi group was classified as 4phyla, 10 classes, 20 orders, 33 families, 57 genera, and 148 species. Among the isolated strains, 5 kinds of unreported fungal specieses anda new fungal species were identified. For more detail of fungal identification, not only ITS but β-tubulin (BenA), calmodulin (CaM) andRNA polymerase II second largest subunit (RPB2) were used. Based onthe morphological characters, analysis of taxonomy and molecular analysis, 8 individuals of endophytic fungi were identified as below; NIBRFG0000505325 as unreported Aspergillus insuetus; NIBRFG 0000505326 as unreported A. nomius; NIBRFG0000505327 as unreportedPenicillium hetheringtonii; NIBRFG0000505328 as unreported P. sublectaticum; NIBRFG0000505331 as unreported P. jacksonii; KACC48990-KACC 48992 as new species named P. ulleungdoense. By reporting the research of fungi in Gyeongsang province environment, the data could be meaningful for understanding the geographical distribution of Ascomycetes on Gyeongsang province and Korea.

Keywords : Endophytic fungi, morphological characters, analysis of taxonomy

K-9

Description of Novel Strain Parashewanella sp. 202IG2-18 from Marine Sponge

Soobin Kim and Jin Sook Park*

Department of Biological Science and Biotechnology, Hannam University, Daejeon 34054, Republic of Korea

A Gram-stain-negative, rod-shaped, aerobic, pale yellow-pigmented bacterium, designated strain 202IG2-18, was isolated from the marin sponge collected from the South Sea coast of the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences indicated thatstrain 202IG2-18 belonged to the family Shewanellaceae and showed the highest 16S rRNA gene sequence similarity with Parashewanella tropica MEBiC05444T (97.05%). Strain 202IG2-18 grew at 18-30℃, with and optimum of 25℃. The pH range for growth was between 5.5-9.5,with optimum of pH 7.0-7.5 The range of NaCl concentration for growthwas between 1.0-5.0 % (w/v), with an optimum of 2.0-3.0 %. The DNAG+C content of strain 202IG2-18 was 39.80 mol%. The major fatty acidswere C15:0 iso (25.68%), C17:1 ω8c (13.50%), C16:1 ω7c / C16:1 ω6c (12.68%). The major respiratory quinone was ubiquinone-8. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, two amino phospholipids and two unidentified phospholipids. On the basis of the polyphasic analyses, strain 202IG2-18 is considered to representa novel species of the genus Parashewanella .

Keywords : Marine spongd, new species, shewanella

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K-10

Isolation and Identification of a Novel Bacterium from Toxin-Producing Dinoflagellate Centrodinium punctatum

Yue Jiang, Lingmin Jiang, Yuxin Peng, Ki Hyun Kim, Jiyoung Lee, and Zhun Li*

Biological Resource Center/Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea

A Gram-stain-negative, aerobic, rod-shaped bacterium (strain R2A-3) was isolated from the toxin-producing dinoflagellate Centrodinium punctatum, optimized for growth at 25℃, pH 8.0, in the presence of 0-7%(w/v) NaCl, and identified as a novel genus and new species based on a polyphasic taxonomic approach. Phylogenetic analyses based on 16SrRNA gene and 92 core genes sets revealed that the strain R2A-3 belongsto the family Nevskiaceae in the class Gammaproteobacteria and represented an independent taxon separated from other genera. 16S rRNA gene of strain R2A-3 showed the highest sequence similarity to Fontimonas thermophila HA-01T (94.1%) and Sinimarinibacterium flocculans NH6-24T (93.2%), and less than 92.8% similarity with othergenera in the Nevskiaceae family. The genome length of strain R2A-3 was 3608892 bp with 65.2% G + C content. Summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c) as the major fatty acids (>10%). Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolaminewere demonstrated as the major polar lipids. According to phylogenetic,phenotypic, chemotaxonomic features and genomic, the R2A-3T strainrepresents a new species in the new genus of the family Nevskiaceae.

Keywords : Novel genus, marine, dinoflagellate

K-11

Impact of Osmotic Pressure on Growth and Intracellular Metabolic Network of Staphylococcus aureus KCTC 3881

Godfrey Mwiti, Hyungseok Choi, and Jaehan Kim*

Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Republic of Korea

Found as a commensal in 30% of the human population, Staphylococcusaureus is not only responsible for Staphylococcal food borne disease butalso an osmotolerant organism with the ability to survive potentially dryand stressful environments. To better understand the response of this highly versatile pathogen under high osmotic stress, we cultivated S. aureus KCTC 3881 strain at varying NaCl concentrations of 5g/L, 90g/L,125 g/L and 150 g/L. Using glucose as the initial carbon source, cell growth associated with the dynamics of substrate and product changesin fermentation media has been monitored. The results showed NaCl hada dose-dependent effect of increasing the lag phase with a subsequent acceleration in growth. The specific growth rate was highest at NaCl concentration of 90g/L and lowered in 125 g/L and 150g/L when compared to the control. Interestingly, S. aureus KCTC 3881 kept theircellular growth metabolizing lactate and acetate the fermentation end products up to 125g/L. The cell yield coefficient on glucose decreased with increasing NaCl concentration. Upon depletion of glucose, S. aureus KCTC 3881 was able to continue to grow on lactate and acetateup to 125g/L of NaCl. Meanwhile acetate use as growth substrate was decreased to near zero in 125g/L.

Keywords : Staphylococcus aureus, environmental stress, metabolic network

K-12

Description of Novel Strain Vibrio sp. 188UL20-2 from Mytilus corusus

Kim Jae Yeon, and Jin sook Park*

Department of Biological Science and Biotechnology, Hannam University, Daejeon 34054, Republic of Korea

A Gram-stain-negative, rod-shaped, aerobic, pale yellow-pigmented bacterium, designated strain 188UL20-2, was isolated from the Mytiluscorusus collected from the Ulleungdo coast of the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences indicated thatstrain 188UL20-2 belonged to the family Vibrionaceae and showed thehighest 16S rRNA gene sequence similarity with Vibrio marisflavi WH134T (96.59 %). Strain 188UL20-2 grew at 18-37℃, with and optimum of 25℃. The pH range for growth was between 5.0-10.0, with optimum of pH 7.5-8.0 The range of NaCl concentration for growth wasbetween 1.0-4.0 % (w/v), with an optimum of 3.0 %. The DNA G+C content of strain 188UL20-2 was 45.40 mol%. The major fatty acids were C16:1 ω7c / C16:1 ω6c (27.98%), C18:1 ω7c / C18:1 ω6c (15.78%), C16:0 (12%).The major respiratory quinone was ubiquinone-8. The polar lipids werephosphatidylethanolamine, phosphatidylglycerol, amino phospholipidsand two unidentified phospholipids. On the basis of the polyphasic analyses, strain 188UL20-2 is considered to represent a novel species of the genus Vibrio.

Keywords : Vibrio, new species, Mytilus corusus

K-13

Pelomonas riviphilus sp. nov., Isolated from River Water of Namgang

Hong Sik Im1, Yochan Joung2, and Sang-Seob Lee1* 1Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, 2066 Seobu-ro Jangan-gu Suwon-si Gyeonggi-do 16419, Republic of Korea 2Institute of Biotechnology and Bioengineering, Sungkyunkwan University, 2066 Seobu-ro Jangan-gu Suwon-si Gyeonggi-do 16419, Republic of Korea

A Gram-staining-negative, aerobic, motile by flagellum, rod-shaped, designated strain YR-4T was isolated from the stream water of Namgangin Jinju, the Republic of Korea. Phylogenetic analysis based on the 16SrRNA gene sequence revealed that strain YR-4T formed a lineage withingenus Pelomonas of the family Comamonadaceae. Phylogenetic analysis also showed that strain YR-4T was most closely related to Pelomonas saccharophila DSM 654 T (98.1 % 16S rRNA gene sequence similarity), Pelomonas aquatica CCUG 52575T (98.0 %), Mitsuaria chitosanitabida NBRC 102408T (97.7 %), and Pelomonas puraquae CCUG 52769T (97.6 %), and Roseateles depolymerans KCTC42856T (97.0%). The growth was observed at 10-37 ℃ (optimum at 37℃), pH 6-8 (optimum at pH 7), 0-2.0 % NaCl (optimum at 0 %). The major fatty acids of the bacterial strain were C16:0 and summed feature3 (iso-C15:0 2-OH and/or C16:1ω7c). The genome size of strain YR-4T was3.6 Mbp and the G+C content was 70.2 mol%. Based on phenotypic, genotypic, and phylogenetic analysis, strain YR-4T represents a novel species of the genus Pelomonas, for which the name Pelomonasriviphilus sp. nov. is proposed. The type strain is YR-4T (= KEMB 1603-002T= KCTC 82705T).

Keywords : Pelomonas, Comamonadaceae, taxonomy

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K-14

Pedobacter endophyticus sp. nov., an Endophytic Bacterium Isolated from Carex pumila

Yuxin Peng1,2, Lingmin Jiang1, and Jiyoung Lee1* 1Korean Collection for Type Cultures (KCTC), Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea 2Department of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea

An aerobic, Gram-stain-negative, weak-motile, short rod-shaped bacterial strain, designated JBR3-12T, was isolated from the Carex pumila plants, and its taxonomic position was investigated by using a polyphasic taxonomic approach. The strain produced a pink pigment onTSA. Grew optimally at 25oC, pH 8, and in the presence of 3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showedthat strain JBR3-12T formed a lineage within genus Pedobacter and mostclosely to Pedobacter sandarakinus DS-27T (98.0%) and Pedobacter agri PB92T (97.6%). The DNA G+C content of the genome was 41.3 mol%, the whole genome length was 5426070 bp. The major fatty acidsof JBR3-12T were iso-C15:0, summed feature 3 (comprising C16:1 ω6cand/or C16:1 ω7c) and iso-C17:0 3-OH. The predominant polar lipid isphosphatidylethanolamine. The predominant quinone is menaquinone-7(MK-7). Based on its phenotypic, phylogenetic, genomic, and chemotaxonomicfeatures, strain JBR3-12T is proposed to represent a novel species of genus Pedobacter, for which the name is Pedobacter endophyticus sp.nov. The type strain is JBR3-12T (= KCTC 82363T= NBRC 114901T)

Keywords : Pedobacter endophyticus sp. nov., novel species, Carex pumila

K-15

Isolation of Mariniflexile maritimus sp. nov., a Novel Bacterium in the Family Flavobacteriaceae

So-Ra Ko1, Ve Van Le1,2, Long Jin3, Sang-Ah Lee1,2, Chi-Yong Ahn1,2*,and Hee-Mock Oh1,2* 1Cell factory Research Centre, Korea Research Institute of Bioscience & Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 2Department of Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea 3College of Biology and the Environment, Co-Innovation Centre for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210-037, P.R.China

A novel Gram-stain-negative, rod-shaped, aerobic, non-motile bacterialstrain, designated M5A1MT, was isolated from seawater collected from the South Sea of Korea. Based on 16S rRNA gene sequence similarity,strain M5A1MT was closely related to Mariniflexile gromovii KMM 6038T (95.3%), Mariniflexile fucanivorans SW5T (95.2%), Mariniflexilesoesokkakense RSSK-9T (95.1%). Genome-based phylogenetic analyses revealed that strain M5A1MT formed a distinct cluster with thetype strains of the genus Mariniflexile. The major cellular fatty acids constituents (> 5% of the total fatty acids) were iso-C15:0, anteiso-C15:0,iso-C15:0 3-OH, iso-C15:1 G, iso-C16:0 3-OH, and iso-C17:0 3-OH. The respiratory quinone was identified as MK-6. The major polar lipids werephosphatidylethanolamine and one unidentified polar lipid. The genomic DNA G+C content of strain M5A1MT was determined to be 37.7 mol%. On the basis of phenotypic, phylogenetic, and chemotaxonomic characteristics, strain M5A1MT is considered to represent a novel species within the genus Mariniflexile, for which the name Mariniflexile maritimus sp. nov. is proposed.

Keywords : Mariniflexile, polyphasic characterization, Flavobacteriaceae

K-16

Neobacillus endophyticus sp. nov., an Endophytic Bacterium Isolated from Selaginella involvens Roots

Lingmin Jiang, and Jiyoung Lee*

Korean Collection for Type Cultures (KCTC) Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea

A Gram-positive, facultatively anaerobic, rod-shaped, endospore- forming,oxidase-positive, and catalase-negative strain designated as BRMEA1T

was isolated from the surface-sterilized Selaginella involvens roots. Growth of strain BRMEA1T was found to occur at pH 6.0-8.0 (optimumpH 7.0), 15-50 ℃ (optimum 25-30℃), in absence of NaCl. Phylogeneticanalysis based on 16S rRNA gene sequences indicated that strain BRMEA1T formed a lineage within the genus Neobacillus (family Bacillaceae) and showed the highest sequence similarity with Neobacillusdrentensis DSM 15600T (98.3%), N. fumarioli KCTC 13885T (98.2%),and less than 98.2% 16S rRNA gene sequence similarity with the othermembers of the genus Neobacillus. Whole-genome analysis of strain BRMEA1T comprised a circular chromosome (5,632,809 bp in size) with38.5% G + C content. Digital DNA-DNA hybridization (DDH) values revealed that strain BRMEA1T showed 20.5% and 22.0% genomic DNArelatedness with the closest species, N. drentensis DSM 15600T and N.fumarioli KCTC 13885T, respectively. The strain BRMEA1T whole- genome showed the presence of 11 specific conserved signature indels(CSIs) for genus Neobacillus. The major cellular fatty acids (>10%) ofstrain BRMEA1T were found to be iso-C15:0 and anteiso-C15:0, while the majorpolar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine,and phosphatidylglycerol. Polyphasic analysis results revealed that BRMEA1T represented a novel species of the genus Neobacillus, with the proposed name Neobacillus endophyticus sp. nov. The type strain is BRMEA1T (= KCTC 43208T = CCTCC AB 2020071T).

Keywords : Polyphasic taxonomy, novel species, endophytic bacterium

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K-17

Metabolic Determinants of Non-Obligate Pairwise Cross-Feeding between Aspergillus and Bacillus species

Digar Singh, and Choong Hwan Lee*

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea

Metabolite cross-feeding (MCF) is an important driving force which influence microbial interactions. Fungal-bacterial interactions plays pivotal role in various food fermentative artisans including koji, meju,soy sauce, kimchi, cheese, and wine. We designed a non-obligatory pairwise MCF between the wild-type strains of A. oryzae and B. amyloliquefaciens. Cross-feeding Aspergillus metabolite to Bacillusthrough media conditioning affected higher growth rates coupled with early onset of biofilm formation. Multivariate analyses (MVA) based onthe untargeted LC-MS datasets displayed clear clustering between the MCF-treated and control sample extracts with higher relative abundanceof iturins, fengycins, and surfactins. Conversely, cross-feeding of Bacillus metabolites into Aspergillus cultivation system significantly inhibited conidiation and growth. MVA showed clustered patterns between the MCF-treated and control Aspergillus extracts. Cross-fed cyclic surfactins (Bacillus origin) were transformed to their linear derivatives supposedly through protease mediated lactone ring hydrolysis in Aspergillus broth. Further, the Aspergillus origin metabolites including polyketide, sesquiterpenoid, alkaloid and oxylipins were significantly increased in cross-fed cultures. Pairwise MCF selectively benefitted Bacillus growth but inhibited Aspergillussuggesting an ammensalic interaction. These findings may help designing a fermentative bioprocess with optimal productivity and robustness.

Keywords : Metabolite cross-feeding, LCMS, metabolomics, Aspergillus,Bacillus

K-18

Probiotic and Reduction of Odorous Compound Characteristics of Lactobacillus reuteri SRCM210547 Isolated from Hanwoo Feces

MyeongSeon Ryu, Su-Jin Shin, Su A Im, Jin Won Kim, Gwang su Ha, Hee-Jong Yang, and Do-youn Jeong*

Microbial Institute for Fermentation Industry(MIFI), Sunchang 56048, Republic of Korea

This study was aimed to isolate lactic acid bacteria (LAB) having probiotics characteristics such as reducing effect of ammonia and hydrogen sulfide gas from fecals (Hanwoo). First, all isolates were screened for suppression ability of common odor compounds (NH3 andH2S) produced in livestock. Additionally, isolates were investigated several probiotics properties such as antioxidant, antimicrobial, and enzyme activities (protease, cellulase, and β-galactosidase). Among theisolates, SRCM210547 showed higher antioxidant activity (26.87%) than other strains. Finally, SRCM210547 was selected to further experiments, and which was named as Lactobacillus reuteri SRCM210547by 16S rRNA sequencing analysis. Additionally, SRCM210547 was analyzed to their acidic, bile resistance, and adhesion ability to intestinalepithelial cell (HT-29). As a result, L. reuteri SRCM210547 Showed 65%and 90% of higher survival rate in acidic condition (pH 2.0) and bile resistance (0.3% oxgall). Also, Adhesion abillity showed 65.58% compared to L. rhamnosus GG. These results suggest that L. reuteriSRCM210547 has potential for application as probitics with effect of improvement to livestock environment.

Keywords : Adhesion ability, ammonia, hydrogen sulfide

[This work was supported by a grant from the Establishment of IntegratedBiobank for Agriculture, Food and Livestock Microbiome Project funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA)]

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K-19

Identification and Genome Sequencing of a Novel Algicidal Bacterium in the Genus Sabulilitoribacter

Ji-Sung Oh, and Dong-Hyun Roh*

Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju 28644, Republic of Korea

A novel algicidal bacterium against Skeletonema costataum, designatedstrain BrNp1-15, was isolated from coastal seawater, Boryeong of Korea. The isolate was Gram-stain-negative, aerobic, rod-shaped and yellow pigmented. Strain BrNp1-15 was able to hydrolyze esculin andgelatin, and possessed alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cysteine arylamidase,trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase,β-galactosidase, α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidaseactivities. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain BrNp1-15 belonged to the family Flavobacteriaceaeand it was most closely related to Sabulilitoribacter multivoransM-M16T (97.6%, sequence similarity). The whole genome sequence ofstrain BrNp1-15 comprised a circular chromosome, 3,580,797 bp in length. The G+C content of the genome was 31.4 mol%, and its genomecontained 3,140 protein-coding genes, 42 tRNA genes and 6 rRNA genes.

Keywords : Sabulilitoribacter, Algicidal bacterium, genome sequencing

[Supported by NRF-2017R1D1A3B04033871]

K-20

Soil Micro-and Mycobiome and Climate Factors are Associated with the Decline of Korean Firs(Abies koreana) in Regions of Mt. halla, Jeju Island, Korea

Minsu Jeong1, Jae-Ho Shin1*, Min-Ji Kim1, Setu Bazie Tagele1, DaRyungJung1, and Young Jae Jo2 1 School of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea 2Department of Applied Plant Sciences, University of Gondar, Gondar 196, Ethiopia

The cause of the decline of the Korean fir tree in various regions of Mt.Halla in Jeju is unknown, despite various ecological and geographic investigations of the Korean fir. In this paper, inter-kingdom interactionsbetween micro- and Myco-biome in Korean fir rhizosphere soil sampleswere examined with network analysis for the cause of decline. Abundantsoil samples of healthy condition Korean firs (HKF) and dead Korean firs(DKF) were collected (19 per condition), and the diversity, physicochemicalassociation, and inter-Kingdom network structure were analyzed with the soil microbiota. Nutrient-related physicochemical factors such as potassium and sodium ions, which had significant differences betweenthe two conditions, had a high correlation with the bacterial and fungalcommunities of HKF. The HKF and DKF were significantly clustered by the fungal community, and Shannon diversity and evenness of fungalcommunities was relatively higher in DKF. Ectomycorrhizal fungal genera such as Russula, Inocybe, Sebacina, and Hydnotrya were clearlyabundant in HKF, while the proportion of plant pathogen or saprotrophwas high in DKF. The inter-kingdom network indicates that HKF networks have high transitivity and density, considerably larger and more complex when compared to those of DKF. Loss of nutrients suchas potassium sodium resulting from differences in sand composition mayaffects the Korean fir tree and soil microbiota communities in Mt. Hallain Jeju.

Keywords : Inter-kingdom, Korean fir, microbiome

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K-21

Lacihabitans sedimenticola sp. nov., and Lacihabitans aurantiacussp. nov., Isolated from Freshwater Sediment

Ji-Hye Han, and Seoni Hwang Microbial Research Department, Nakdonggang National Institute of Biological Resources(NNIBR), Sangju-si 37242, Republic of Korea

Two orange colored, Gram-stain negative, non-motile, aerobic, and rod-shaped bacteria, designated strains CS3-21T and CCS-44T, were isolated from sediment of the Nakdong River and Chodang cave in Republic of Korea, respectively. Their 16S rRNA gene sequences demonstrated that these strains belong to the genus Lacihabitans of thefamily Spirosomaceae. Strains CS3-21T and CCS-44T were closely related to Lacihabitans soyangensis HME6675T (98.35 and 98.65% similarities) and Lacihabitans lacunae HME7103T (96.01 and 96.08%similarities). Both strains shared common chemotaxonomic characteristics comprising MK-7 as the main isoprenoid quinone, iso-C15:0 and Summed feature 3(C16:1 ω7c and/or C16:1 ω6c) as the predominant cellular fatty acids, and phosphatidylethanolamine (PE) asthe major polar lipid. The genomic DNA G+C contents of strains CS3-21T and CCS-44T were 36.51 and 37.1mol%, respectively. The average nucleotide identity and digital DNA-DNA hybridization valuesbetween these strains and their closest relatives were below the cut-offvalues of threshold for species demarcation. On the basis of the polyphasic evidence presented here, both strains represents as novel species of the genus Lacihabitans, for which the name Lacihabitans sedimenticola sp. nov. (CS3-21T = KCTC 52753T) and Lacihabitans aurantiacus sp. nov. (CCS-44T = KCTC 52754T) are proposed.

Keywords : Lacihabitans, freshwater sediment, novel species

K-22

Oxazolidinone-Resistant Enterococci Strains Isolated from Swine Farms

Shabnam Aghamoradi, Jae-Young Oh, Lee Sang Hyeon, and Jong-ChanChae*

Division of Biotechnology, Jeonbuk National University, Iksan 54596, Republic of Korea

Enterococci are gastrointestinal commensals that can colonize a varietyof environments such as human, animals, soil, water and food. The resistance of enterococci is important issue to transmission of antimicrobial resistance and public health. A total of 907 strains (660Enterococcus faecalis and 247 Enterococcus faecium) were isolated from 8 swine farms and 2 slaughterhouses. Antimicrobial susceptibilitiesof two entecococcal species suggested highly resistance to tetracycline,aminoglycoside, chloramphenicol, and ciprofloxacin. In addition, 108 (16.3%) E. faecalis strains were resistantto linezolid. The optrA gene wasdetected in most of the linezolid (oxazolidinone)-resistant E. faecalis(LREF) strains and eight LREF strains contained the poxtA gene knownto be located in the plasmid. Furthermore, cfr variation, cfr(D) gene wasfound by whole genome sequencing analysis. Through the conjugationof the LREF strains carrying poxtA, it was confirmed that the plasmid was transferrable to the other Enterococcus.

Keywords : Swine farm, linezolid-resistant, Enterococus faecalis

K-23

Characterization of Halobellus ruber sp. nov., a Novel Halophilic Archaeon Isolated from Korean Solar Saltern Using Genomic and Production Analyses

Chi Young Hwang1, Eui-Sang Cho1, Deok Jun Yoon1, and Myung-Ji Seo1,2*

1Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

A novel halophilic archaeon, designated strain MBLA0160T was isolated from Korean solar saltern in Sorae. According to predicted functional proteins of strain MBLA0160T, amino acid transport and metabolism were the highest categories. Genome rapid annotation showed that amino acid and derivatives was the most subsystem feature counts. Based on pan-genomic analysis, strain MBLA0160T and other species of the genus Halobellus (Hbs.) had 7,621 POGs: 1,461 core POGs, 2,341 accessory POGs, and 3,819 unique POGs, respectively. Theunique POGs distribution showed that the strain MBLA0160T had 97 annotated unique KEGG, which were mainly included metabolism andenvironmental information processing. Ortholog average nucleotide identities (OrthoANI) and in silico DNA-DNA hybridization (isDDH)values between the strain MBLA0160T and other strains of the genus Hbs. were under 84.4% and 28.1%, respectively. The antiSMASH analysis of strain MBLA0160T also showed the presence of the terpenebiosynthetic gene cluster, in particular C50 carotenoid, of which production was confirmed by HPLC analysis as detecting bacterioruberinand its isomer and precursors. In conclusion, the halophilic archaeon Hbs. ruber sp. nov. MBLA0160T could be considered to be novel potential bacterioruberin-producer.

Keywords : Halobellus, bacterioruberin, solar saltern

[This research was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03931582) and by National Institute of Biological Resources funded by the Ministry of Environment (NIBR202102109)]

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K-24

Comparative Genome Analysis of Three Novel Haloferax sp. Isolated from Seawater and Sediment nearby Youngheungdo Island

Eui-Sang Cho1, In-Tae Cha2, Won-Jae Chi2, Seong Woon Roh3, and Myung-Ji Seo1,4* 1Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Microorganism Resources Division, National Institute of Biological Resources, Incheon 22689, Republic of Korea 3Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea 4Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

Three novel halophilic archaea, designated MBLA0076T, MBLA0077T,and MBLA0078T, were isolated from seawater and sediment nearby Youngheungdo Island. The genome size of MBLA0076T, MBLA0077T,and MBLA0078T was 3.56, 3.48, and 3.48 Mb. The 16S rRNA gene similarity of the three strains share 98.5-99.5 %, whereas their relatedtype species of the genus Haloferax have less than 98.5%. Phylogeneticanalysis based on the 16S rRNA and rpoB′ gene sequence indicated that the three isolates belonged to the genus Haloferax. The values of OrthoANI and isDDH were indicated below the species delineation threshold. Pan-genomic analysis showed that most core POGs of the three novel strains and other related taxa were related to amino acid transport and metabolism in the COG category and KEGG pathway. Thelargest proportion of unique POGs were categorized into amino acid transport and metabolism (MBLA0076T), carbohydrate transport and metabolism (MBLA0077T), and energy production and conversion (MBLA0078T) in the COG database. Based on the comparative genomicanalysis, we propose that MBLA0076T, MBLA0077T, and MBLA0078T

represent type strains of the genus Haloferax and suggest the names Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., respectively.

Keywords : Haloferax, comparative genomics, pan-genomic analysis

[This research was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03931582) and by National Institute of Biological Resources funded by the Ministry of Environment (NIBR202102109)]

K-25

Description of Nocardioides luti sp. nov., Isolated from Clay Mineral and Genome Mining for the Alkylresorcinol Biosynthetic Gene Cluster

Deok Jun Yoon1, Eui-Sang Cho1, Chi Young Hwang1, Young-Do Nam2, So-Lim Park2, Seong-Il Lim2, and Myung-Ji Seo1,3* 1Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Research Group of Healthcare, Korea Food Research Institute, Wanju 55365, Republic of Korea 3Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

Novel species strain KIGAM211T named as Nocardioides luti was isolated from kaolinite as one of clay minerals collected from Sancheongin Korea. After whole genome sequencing analysis of strain KIGAM211T, its complete genome was characterized to have 4.52 Mbof genome length with 72.3 mol% G + C content, comprising a total of 4,294 genes containing 4,210 coding genes. The genome of strain KIGAM211T specifically contained much more genes involved in aminoacid transport and metabolism (E) and lipid transport and metabolism (I), compared to the genomes of related strains. The OrthoANI and isDDH values between KIGAM211T and its neighbor strains were belowthe generally accepted species boundaries for 96% and 70%, respectively. Pan genome contains a total of 17,788 genes containing 1,556 core genes and 6,285 unique genes. Genome mining for secondarymetabolite biosynthetic gene clusters resulted in the presence of potentialalkylresorcinol biosynthetic gene cluster containing type-III PKS, SAM-dependent methyltransferase, and FAD-dependent oxidoreductase,of which similarities was about 78% between them of other Nocardioidesspecies. In conclusion, KIGAM211T could be proposed to be novel producer of alkylresorcinol as amphiphilic lipid with antioxidant and antibacterial activities.

Keywords : Nocardioides, alkylresorcinol, kaolinite

[This research was supported by Collaborate Research Program of theKorea Food Research Institute (KFRI) and the Korean Institute of Geoscience and Mineral Resources (KIGAM)]

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K-26

Bacteroides faecium sp. nov., Isolated from Human Faeces

Juseok Kim, Hye Seon Song, Joon Yong Kim, Yujin Kim, and Seong WoonRoh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

A novel bacterial strain, CBA7301T, was isolated from human faeces andwas characterised using a polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequences revealed thatstrain CBA7301T belonged to the genus Bacteroides, in the family Bacteroidaceae. The similarity between the 16S rRNA gene sequence of strain CBA7301T and that of its most closely related species, namely Bacteroides faecichinchillae JCM 17102T, was 96.2%, and the averagenucleotide identity value between these two strains was 77.9%. The genome size was 6782182 bp, and the DNA G+C content was 42.8 mol%.Strain CBA7301T was Gram-stain-negative, strictly anaerobic, and rod-shaped. The optimal growth of this organism occurred at 30 ℃, pH7.0, and 0.5% (w/v) NaCl. The respiratory quinones were the menaquinones 9 and 10. The predominant polar lipids were phosphatidylethanolamine, unidentified phospholipids, and unidentifiedamino phospholipids. The major cellular fatty acid was anteiso-C15:0. According to the polyphasic taxonomic analysis, strain CBA7301T

represents a novel species in the genus Bacteroides, which we named Bacteroides faecium sp. nov.; the type strain is CBA7301T (=KCCM 43355T).

Keywords : Human faeces, bacterial taxonomy, bacteroides

K-27

Fulvivirga lutea sp. nov., a Marine Bacterium Isolated from Seawater

Seung Seob Bae*, Yoon-Hee Jung, Yong Min Kwon, Dawoon Choung, Grace Choi, Kichul Cho, Woon-Jong Yu, Dea-Sung Lee, and KyunghwaBaek National Marine Biodiversity Institute of Korea, Chungcheongnam-do 33662, Republic of Korea

A strictly aerobic, Gram-stain-negative, gliding, rod-shape, marine bacteria, designated strain S481T was isolated from a surface seawater sample collected at Gunsan marina, in the West Sea of the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain S481T formed monophyletic clade with members ofgenus Fulvivirga, showing 93.7-95.8% sequence similarity to the type strains. The strain S481T has a single circular chromosome of 4.13 Mbpwith a DNA G+C content of 37.3 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between strain S481T and all genome-sequenced species of the genus Fulvivirga werebelow 71.2 and 18.8% respectively, indicating low values than standardcut-off for species delineation. Growth was observed at 30-42 ℃ (optimum 37 ℃), at pH 6-8 (optimum pH 7), and with 0-6% NaCl (optimum 1-2). The major fatty acids (>10%) were iso-C15:0, C16:1ω5cand iso- C15:1 G. The respiratory quinone was MK-7. The major polarlipids were identified as phosphatidylethanolamine, three unidentifiedaminolipids and five unidentified lipids. Based on the results of phenotypic characterization, phylogenetic analysis and genome-basedcomparison, strain S481T represents a novel species in the genus Fulvivirga, for which we propose the name Fulvivirga lutea sp. nov. Typestrain is S481T (=KCTC 82209T =JCM 34505T). [Supported by a grantfrom MABIK in-house program (2021M00500)]

Keywords : Seawater, flammeovirgaceae, Fulvivirga lutea

[Supported by a grant from MABIK in-house program (2021M00500)]

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K-28

Genome of Methicillin-Resistant Staphylococcus epidermidis Isolated from Residential Environment

Jong-Chan Chae, Eobin Park, Haeseong Lee, Jae-Young Oh, and Kui-JaeLee*

Division of Biotechnology, Jeonbuk National University, Iksan 54596, Republic of Korea

Staphylococcus epidermidis is a gram-positive opportunistic pathogenwhich is known as a coagulase-negative bacterium and a major pathogen ofnosocomial infection. Currently, methicillin-resistant Staphylococcus epidermidis (MRSE) is attracting public concern. Strain Z0118SE0272was isolated from the surface of sofa in the residence of veterinarian where human skin contact was frequently occurring. It was identified as S.epidermidis by mutS gene. Based on genome sequences, a chromosome (2.5 Mb) and three plasmids (3kb, 1.7kb, 2.5kb) were determined. The genome contained 2,437 coding genes, 7 5S, 6 16S, 623S rRNAs, and 61 tRNAs. In addition, dfrC, norA, mecA, ANT(4`)-Ib, blaZ genes were found in chromosome corresponding to antibiotic resistance activities against trimethoprim, ciprofloxacin, cefoxitin, kanamycin, and penicillin. The detected genes were consistent with the result of antibiotic susceptibility for the strain. Also, SCCmec structurewas identified by SCCmec finder. In the upstream of mecA, the SCCmecsubtype IV was found which was composed of ccrA2, ccrB2, orfX withIS431. Downstream of mecA, ccrC was located. Consequently, most similar structure in strain Z0118SE0272 was SCCmec type IV (2B&5 ZH47).

Keywords : Staphylococcus epidermidis, methicillin, SCCmec

K-29

Identification of Vancomycin-Resistant Enterococcus faecium in Aquatic Environments

Lee Sang Hyeon, Jae-Young Oh, Hyeonbin Kim, and Jong-Chan Chae*

Division of Biotechnology, Jeonbuk National University, Iksan 54596, Republic of Korea

In trying to detect vancomycin-resistant enterococcus (VRE) in naturalrivers and effluents from livestock wasterwater treatment plants, four VREs were isolated. All VRE strains were identified as Enterococcus faecium and were resistant to penicillin, ampicillin, ciprofloxacin, nitrofurantoin, vancomcyin, and teicoplanin. While one strain belonged to ST192 in the MLST results, the other 3 strains were identified as ST1421 lacking pstS, one of the 7 housekeeping genes. Also, all of themharbored esp and hyl virulence genes indicating that they might be pathogenic. As a result of full genome sequencing of the VRE strains,the vanA gene cassette along with Tn1546 located on a plasmid. Recently, vancomycin-resistant E. faecium ST1421 strains which a pstSgene was deleted in were reported from human feces of patients. In comparison of genetic structures of vanA gene cassettes, the same genetic structure was sharing among the VREs isolated in this study andthe human feces. It is suggesting that clinical isolates are already existingin natural environments which might cause a public health problem.

Keywords : Vancomycin, Enterococcus faecium, genome

K-30

Competitive Interaction of Bacillus spp. and Dominant Fungi in Meju

Seong-Eui Yoo, and In-Hyung Lee*

Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea

Bacteria and fungi affect each other’s growth and metabolic activitiesin their natural habitats by various bacterial and fungal interactions (BFI). Diverse bacteria and fungi are observed in traditional meju. Thecomposition of microbial flora are important factors in the fermentationprocess. In order to get insights on their involvement in mejufermentation, we analyzed interactions between bacteria and fungi. Weselected the dominant species, Bacillus spp. as representative bacteriaspecies, and for representative fungal species, we selected zygomycetesbased on their dominance during the early fermentation period, and Aspergillus spp. on the middle and late fermentation periods, all of whichwere isolated from traditional meju. When bacteria and fungi were mix-cultured, the growth of both bacteria and fungi were significantly decreased compared to the single culture regardless of species. The growth of fungi was more decreased compared to that of bacteria, suggesting competitive interactions between them. Metabolic analysistracing the utilization of carbon sources from soybeans using a Phenotype Microarray plate PM1 revealed that the niche overlapping indexes (NOI) between bacteria and fungi were less than 0.9, suggestingthat they used different carbon sources. Therefore, exploitation competition, which is the competition for nutrients, was not the main cause of growth decrease. When bacteria and fungi were co-cultured using a transwell plate, in which testing strains are separated by a membrane but medium is shared, the growth of fungi was also decreasedbut to a lesser extent than mixed culture. Therefore, the fungal growth was mainly affected by interference competition from direct physical contact in addition to secreted bacterial metabolites. These studies on the interactions between bacteria and fungi shed light on the basis of florachange during the process of meju fermentation. This research was supported by Korea Environmental Industry and Technology Institute(KEITI) grant funded by the Ministry of Environment of Korea.

Keywords : Bacterial and fungal interaction (BFI), competitive interaction, meju

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Aminipila terrae sp. nov., a Strictly Anaerobic Bacterium Isolated from River Sediment

Yeon Bee Kim, Se Hee Lee, Tae Woong Whon, and Seong Woon Roh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

In this study, aimed at investigating and characterising river sedimentbacteria, we isolated a Gram-stain-positive, rod-shaped, obligate anaerobic bacterium, strain CBA3637T, from the sediment of the GeumRiver. This strain grew at 10-40℃ (optimum, 30℃), 0-1% NaCl (optimum,0%), and pH 7-8 (optimum, pH 7). The 16S rRNA gene sequence comparison revealed Aminipila butyrica DSM 103574T to be the closestrelative of strain CBA3637T (96.6-96.7% similarity); and both strains clustered together in phylogenetic analysis. The genome of strain CBA3637T was found to consist of a single chromosome (3.51 Mbp; 36.98% G+C content). Comparative genomic analysis of the strain CBA3637T with A. butyrica DSM 103574T revealed that strain CBA3637T

possessed five unique pathways related to polyamine biosynthesis, lipopolysaccharide metabolism, pyrimidine metabolism, and cofactor and vitamin metabolism. Strain CBA3637T contained C14:0, C16:0, and C18:1 ω9c as the major fatty acids, and diphosphatidylglycerol as the major polar lipid. No respiratory quinone was observed. Biochemical, chemotaxonomic, and genotypic data revealed the strain CBA3637T isa representative of a novel species within the genus Aminipila, for which the name Aminipila terrae is proposed. The type strain is CBA3637T

(=KACC 21651T =DSM 110662T).

Keywords : Aminipila terrae, Geum river, polyphasic taxonomy

K-32

Optimization of DNA Extraction and Amplification Conditions for Airborne Fungal Community Analysis

Soo-Kyung Kang, Yun-Yeong Lee, and Kyung-Suk Cho*

Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03670, Republic of Korea

To investigate the source of air pollution and its harmfulness, it is requiredto analyze fungal community structures in bioaerosol. However, only few studies are conducted on the airborne fungal community structuresand also the methods of DNA extraction and amplification are weekly established. In this study, the optimum conditions for DNA extraction and its amplification from bioaerosol samples were established. The samples were collected in quartz filters using a TSP high volume air sampler on the roof of the Engineering building, Ewha Womans University. For fungal DNA extraction, the filter was cut into small piecesand placed in the lysis buffer. To enhance the yield of DNA extraction,the mixture was additionally shaken for 15 min. Consequently, the DNAextraction yield was about 3 times higher in the shaking method than thatof the non-shaking method. After then, PCR was performed targeting the fungal ITS1 and ITS2 regions. To amplify ITS2 region, PCR was conducted with forward primers (ITS3, ITS3R and 5.8SR) and reverseprimers (ITS4 and ITS4-B). For ITS1 region, the combination of ITS1(f-), ITS5 (f-) and ITS2 (r-) was used. As a result, the fungal DNA was more clearly amplified when primer sets targeting the ITS2 region wereutilized. Additionally, the combination of ITS3 (forward) and ITS4-B (reverse) was recommended to amplify fungal ITS2 region.

Keywords : Airborne fungi, DNA extraction, ITS region primer

[This study was supported by the National research Foundation of Korea (NRF) grant funded by the Korea government, the Ministry of Scienceand ICT (MSIT) (2019R1A2C2006701)]

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K-33

Syntrophic Relationship among Flavobacterium sp. TCH3-2 and Synthetic Community Members Involved in Tomato Plant Growth Promotion

Hyojun Seo, Eunjin Kim, Jong Uk Son, Aryan Rahimi-Midani, Sang-MooLee, Hyoung Ju Lee, Kihyuck Choi, and Seon-Woo Lee*

Department of Applied Bioscience, Dong-A University, Busan 49315, Republic of Korea

The rhizosphere microbial communities affecting the plant growth hasshown to play a greater role throughout the plant life. Therefore, understanding the role of these communities is crucial. Previously wehave isolated a Flavobacterium sp. named as TCH3-2 which its syntrophic interactions with other bacterial species (SynCom) enhancethe growth of tomato plants. However, relationship between the TCH3-2and each bacterial strain of SynCom has not been understood yet. In thisstudy, we examined the syntrophic relationship among TCH3-2 and SynCom members. We conducted microbial-interaction tests using V-shape and top agar methods. The V-shape method was conducted byco-culturing TCH3-2 and SynCom members in a known inoculation siteson TSM media. In addition, the top agar method was conducted by inoculating single strain of SynCom on TCH3-2 lawn prepared in TRMmedia. The V-shape method showed that the growth of TCH3-2 was promoted in minimum distance of 1 cm from each member of SynCom.On the other hand, top agar method results indicated that presence of TCH3-2 can stimulate the growth of Ketobacter sp. and Archangium sp.of SynCom. Taken together, our study suggested a syntrophic interactionamong TCH3-2 and SynCom members to promote the growth of tomatoplants.

Keywords : Flavobacterium sp. TCH3-2, synthetic community, syntrophy

K-34

Companilactobacillus pabuli sp. nov., a Lactic Acid Bacterium Isolated from Animal Feed

Ji Young Jung, Hye Kyeong Kang, Hyun Mi Jin, and Mi Hwa Lee*

Microbial Research Department, Nakdonggang National Institute of Biological Resources, 137, Donam 2-gil, Sangju-si, Gyeongsangbuk-do 37242, Republic of Korea

One gram-positive, facultative anaerobic, catalase-negative, non-motile,non-spore-forming, and rod-shaped lactic acid bacterium strain, denotedas NFFJ11T, isolated from total mixed fermentation (TMF) feed in SouthKorea, was characterized through polyphasic approaches, including sequence analyses of the 16S rRNA gene and housekeeping genes (rpoAand pheS), determination of average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH), fatty acid methyl ester (FAME) analysis, and phenotypic characterization. Phylogenetic analyses based on 16S rRNA, rpoA, and pheS gene sequences revealedthat the strain NFFJ11T belong to the genus Companilactobacillus. The16S rRNA gene sequence of strain NFFJ11T exhibited a high similarityto Companilactobacillus formosensis S215T (99.66%), Companilactobacillusfarciminis Rv4 naT (99.53%), Companilactobacillus crustorum LMG 23699T (99.19%), Companilactobacillus futsaii YM 0097T (99.06%), Companilactobacillus zhachilii HBUAS52074T (98.86%), and Companilactobacillus heilongiiangensis S4-3T (98.66%). However, average nucleotide identity (ANI) and in-silico DNA-DNA hybridization (isDDH) values for these type strains were in the range of79.90-92.93% and 24.50-49.30%, respectively, which offer an evidencethat the strain NFFJ11T belong to a novel species of the genus Companilactobacillus. The cell wall peptidoglycan type was A4α (L-Lys-D-Asp) and the G+C content of the genomic DNA was 35.7 mol%. The main fatty acids of strain NFFJ11T were C18:1 ω9c (43.3%),C16:0 (20.1%), and summed feature 7 (18.3 %; comprising any combination of C19:1 ω7c, C19:1 ω6c, and C19:0 cyclo ω10c). Throughpolyphasic taxonomic analysis, it was observed that strain NFFJ11T isa novel species belonging to the genus Companilactobacillus, for whichthe name Companilactobacillus pabuli sp. nov. is proposed. The type strain is NFFJ11T ( = KACC 21771T  = JCM 34088T).

Keywords : Lactic acid bactera, new species, animal feed

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K-35

Studies of Marine-Derived Fungi in Korea

Dawoon Chung, Yong Min Kwon, Seung Sub Bae, and Grace Choi Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea, Seocheon-gun 33662, Republic of Korea

Marine-derived fungi play an important role in marine ecosystem and have tremendous potential as sources of bioactive compounds. In this work, we investigated peer-reviewed research articles studying marine-derived fungi in Korea published by December 2019. A total of84 articles was reported from 2002 and 2019, describing 818 strains of266 species belonging to 3 phyla, 8 classes, 21 orders, 43 families, and76 genera. At the genus level, Penicillium/Eupenicillium/Talaromycesfungi were most prevalent (40.2% of all species), followed by Aspergillus/Eurotium/Neosartorya (13.9%). In terms of sampling sitesand isolation sources, approximately 67% of fungal strains were originated from the eastern (44.6% of all strains) and western coasts (22.1%). The major isolation sources were coastal plants, marine animals, sediment, and marine algae. Among th 818 fungal strains, 292strains (35.7%) appeared to produce natural products (with antimicrobial,antioxidant, antidiabetic, anticancer, and antivirus activities), or enzymes(β-glucosidase, caseinase, cellulase, and gelatinase). In addition, Penicillium jejuense and Trichoderma rugulosum isolated from marineanimals and plants were reported as marine-derived novel species in Korea. This report was aimed to provide current knowledge of marine-derived fungi in Korea and also to facilitate studies of marine fungi for industrial applications.

Keywords : Marine-derived fungi, Korea, Penicillium/Aspergillus

K-36

Discovery for High Efficient Amylase and Protease Producers Derived from Matured Compost

Yongjoon Yoon1, Gahee Lee1, Trigueros Orozco Sara1,2, Woo Young Bang3, Sung Ho Choi3, Byoung-Hee Lee3, and Ki Hwan Moon1* 1Division of Marine Bioscience, Korea Maritime & Ocean University, Busan 49112, Republic of Korea 2Major in Marine Sciences and Biotechnology, Universitat Catolica de Valencia, Valencia 46001, Spain, 3National Institute of Biological Resources, Environmental Research Complex, Incheon 22689, Republic of Korea

Manure composting is a good approach for the nutrients stabilization,and the reduction of pathogens and odors in compost. For accelerating compost maturation, a well-established microbial ecosystem and highly efficient enzymes derived from the microbes that composed the ecosystem are required. In this year, the compost maturity test is legallymandated and implemented by Korea Ministry of Environment to evaluate the quality of compost released into the environment. Differenttypes of anthropogenic wastes were utilized for ingredients of manurecomposting, such as food wastes and livestock manure. Leachate and harmful gases generated from immature compost adversely affect the terrestrial environment by altering the pH of the soil. In this study, we isolated bacteria that exhibit highly efficient amylase, and protease activities from matured compost using skim milk and starch agar, respectively. To identify each isolates, 16S rDNA sequencing will be performed. The physiological and biochemical characteristics of isolates will be investigated by API kits and various colorimetric enzymeassay kits. Even though this research is in very early stage, the isolates that contain large organic molecules degrading enzymes can be accelerated the fermentation process of compost which may help the farms by reducing production and management times, and increasing production efficiency of matured compost.

Keywords : Amylase, manure composting, protease

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K-37

Characterization of Streptococcus bovis/Streptococcus equinus Complex (SBSEC) Isolated from Korean Domestic Ruminants

Seonyoung Park1,2, Hyemin Kwon1, In Ho Lee1, Bo Yeong Seo1, Ji HyungKim1*, and Seongwon Seo2* 1Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea 2Division of Animal and Dairy Sciences, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Republic of Korea

Streptococcus bovis/Streptococcus equinus complex (SBSEC) includeslactic acid-producing bacteria considered as the causative agent associated with acute rumen lactic acidosis in intensive ruminants. Considering the limited information on the detailed characteristics anddiversity of SBSEC in Korea and the emergence of antimicrobial resistance (AMR), we investigated the diversity of SBSEC from domestic ruminants and verified the presence of antimicrobial resistancegenes (ARGs) against several antimicrobials with their phenotypic resistance. Among 51 SBSEC isolates collected, two SBSEC members (S. equinus and S. lutetiensis) were identified; sodA-based phylogeneticanalyses and comparisons of overall genome relatedness revealed potential plasticity and diversity. The AMR rates of these SBSEC againsterythromycin, clindamycin, and tetracycline were relatively lower thanthose of other SBSEC isolates of a clinical origin. An investigation of the ARGs against those antimicrobials indicated that tetracycline resistance of SBSECs generally correlated with the presence of tet(M)-possessing Tn916-like transposon. However, no correlation between the presence of ARGs and phenotypic resistance to erythromycin and clindamycin was observed. Although a limited number of animals and their SBSEC isolates were examined, this studyprovides insights into the potential intraspecies biodiversity of ruminant-origin SBSEC and the current status on antimicrobial resistance of the bacteria in the Korean livestock industry.

Keywords : Streptococcus bovis, bovine acidosis, genome

K-38

Hannaella euonymus sp. nov., Isolated from Forsythia koreaana

Jeong-Seon Kim, Mi ran Lee, Soon-Wo Kwon, Young-Joon Ko, and Soo-Jin Kim* Agricultural Microbiology Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju-gun, Jeollabuk-do 55365, Republic of Korea

The yeasts were isolated from forsythia to study the diversity of yeast.A total of 17 species of yeast were isolated from the flowers in Suncheon,Korea, and the strain FST2-2 was expected to be novel species. This novel yeast, which belongs to the family Bulleribasidiacease in order Tremellale, was named Hannaella euonymus. The nucleotide sequenceof the D1/D2 region of the large subunit rRNA gene of the strain FST2-2was compared with the type strains of genus Hannaella. As a result of comparing the sequences of strain FST2-2 with H. zeae CBS 10801T andH. kunmingensis CBS JCM 11006T, each of them had 96.7% homology(difference of 20nt among the sequences 589nt). When sequence comparison of internal transcribed spacer regions (primer pair ITS1-ITS4), strain FST2-2 showed 95.88% homology with H. zeae CBS10801T and 95.41% with H. kunmingensis CBS JCM 11006T. The strainFST2-2 assimilated these carbon sources (inulin, sucrose, raffinose, melibiose, galactose, lactose and trehalose etc.) and nitrogen sources (potassium nitrate, ethylamine hydrochloride and creatine etc.) except soluble starch and sodium nitrite. No growth occurs in the absence ofcalcium pantothenate, riboflavin, pyridoxine, biotin, folic acid, myo-inositol, and niacin. Based on these results, strain FST2-2 could beclassified as a novel species of the genus Hannaella, for which the nameHannaella euonymus sp. nov. is proposed.

Keywords : Yeast, novel species, Hannaella

[Supported by the National Institute of Agricultural Sciences, RDA, Korea. (PJ013549062021)]

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L_Microbial Genetics, Physiology, and Metabolism

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L-1

The Relationship between Composition Change of Cellular Fatty acid and Prebiotic Effect on Staphylococcus aureus and Staphylococcus epidermidis by Linoleic Acid

Shin Jisoo, and Tae-Jong Kim*

Department of Forest Products and Biotechnology, Kookmin University, Seoul 02707, Republic of Korea

Staphylococcus aureus is a representative harmful bacteria that causesvarious skin diseases. Meanwhile, Staphylococcus epidermidis is known to be beneficial because of its peptides which inhibit pathogenicbacteria. And a substance that promotes beneficial bacteria selectivelyis called prebiotiocs. In previous study, we found that linoleic acid inhibited the growth of S. aureus but promoted the growth of S. epidermidis. In this study, we measured changes in fatty acid composition of strains by linoleic acid treatment. The cells with Abs600=0.05 were incubated with linoleic acid for 3 hours. The cellular fatty acids were extracted and analyzed by the method of MIDI Sherlock MIS. The treatment with linoleic acid increased significantly the cellularcontents of linoleic acid and its metabolized fatty acids in both strains.It proposes that linoleic acid was incorporated into the cells. We proposethat the cytoplasmic membrane is the target for linoleic acid incorporation and the physiological property change of the cell membrane may a reason for strain specific growth change by linoleic acid.

Keywords : Linoleic acid, skin-prebiotic, bacterial membrane physiology

L-2

Comprehensive Functional Analysis of Putative Lipase-Encoding Genes in the Plant Pathogenic Fungus Fusarium graminearum

Sieun Kim1, Soyoung Choi1, Jiyeun Park1, Jiyoung Shin2, Jung-Eun Kim2, Rina Hong1, Gyung Ja Choi3, Yin-Won Lee1, and Hokyoung Son1,2* 1Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea 2Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 3Eco-Friendly New Materials Research Group, Research Center for Biobased Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea

Fusarium graminearum is the important plant pathogenic fungus that causes Fusarium head blight (FHB) on wheat and barley and ear rot onmaize. Fusarium infections result in reduced grain yield and contamination of mycotoxins, such as trichothecenes and zearalenone, which are harmful to humans and animals. To understand the molecularmechanisms underlying fungal development, virulence, and mycotoxinproduction, large collections of F. graminearum mutants (e.g., for transcriptional regulators, kinases, and phosphatases) and their phenome database have been constructed. Lipases, which catalyze the hydrolysis of long-chain triglycerides, participate in various biological pathways in most living organisms. In pathogenic microbes, phospholipases are expected to be important virulence factors since phospholipids are major components of the cell membrane and are involved in virulence-related signal transduction pathways. Some secreted fungal lipases are known to be involved in plant pathogenicity.However, the roles and molecular mechanisms of most fungal lipasesare largely unknown. In this study, we identified 86 putative lipase-encoding genes (LIPs), based on the similarity of their amino acidsequences and conserved structural domains, in F. graminearum and generated a genome-wide deletion mutant collection. We analyzed phenotypic changes, such as vegetative growth, asexual and sexual reproduction, and pathogenicity, of the mutants. Many mutants showedimpaired phenotypes in various developmental stages suggesting that individual LIPs are involved in particular biological processes. The ongoing works are focusing on the elucidation of the potential roles of LIPs in diverse biological processes, such as virulence, cell homeostasis,and nutrient acquisition, in F. graminearum. This study is the first comprehensive functional analysis of LIPs, and it provides a valuable genetic resource for further basic and applied biological researches in filamentous fungi including plant pathogens.

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L-3

Genome-Wide Functional Analyses of Putative WD40-Repeat Repeat Proteins in Fusarium graminearum

Soyoung Choi1, Jiyeun Park1, Sieun Kim1, Hee ji Moon1, Na hyun Lee1, Ji Young Shin2, Jung-Eun Kim2, Gyung Ja Choi3, Yin-Won Lee1, and Hokyoung Son1,2* 1Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea 2Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 3Therapeutic & Biotechnology Division, Center for Eco-Friendly New Materials, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea

WD40-repeat (WD40) proteins constitute one of the most abundant protein families in eukaryotes. WD40 proteins serve as rigid interactionplatforms for the assembly of large protein complexes which participatein diverse cellular processes, such as protein modifications, transcriptional and translational regulation, and signal transduction pathways. Despite the importance of WD40 proteins in acting as hubs for cellular networks, few studies have examined biological roles and molecular mechanisms of them in fungi. Fusarium graminearum is animportant plant pathogenic fungus causing Fusarium head blight on wheat, barley as well as ear rot on maize worldwide. Infection of thisfungus results in severe yield losses and contamination with mycotoxins, which have a remarkable impact on human and animal health. To understand the complex molecular mechanisms underlying development and pathogenesis of this fungus, mutant collections of F. graminearum have been extended globally. In this study, I identified 157genes containing WD40 domains and generated the mutant library of these genes via targeted gene deletion in F. graminearum. Until now, I constructed deletion mutants for 139 WD40 genes, whereas 25 genes seem to be essential. These mutants are being assayed to build a comprehensive phenotype database for vegetative growth, sexual and asexual reproductions, virulence, mycotoxin production and various stress responses. Meanwhile, dozens of virulence-related WD40 geneswere preliminary selected to characterize novel protein complexes related to virulence. And I have been trying to establish a streamlined experimental platform for a large-scale interactome study using affinity purification mass spectrometry (AP-MS) in this fungus. As a pilot experiment, I successfully identified putative interaction partners of a Fgwd101 protein, a homolog of yeast Sec13 using an AP-MS experiment, and co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays are being performed to confirm protein-protein interactions. A mutant set of WD40 genes andthe workflow for interaction studies constructed in this research wouldenable my research group to characterize novel protein complexes regulating fungal virulence. This is the first comprehensive functional characterization of the WD40 genes and will be a milestone for future interactome studies in plant pathogenic fungi.

Keywords : WD40, Fusarium graminearum, Protein Protein Interaction(PPI)

L-4

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius

Areum Lee, Hyeju Jin, and Jaeho Cha*

Department of Microbiology, Pusan National University, Busan 46241, Republic of Korea

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S.acidocaldarius has not identified clearly. The objective of this study isto identify the gene cluster, which is predicted to be involved in glycogenmetabolism, and to confirm the role of the glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31 was increased in theearly and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, GlgA and AmyA were shown to be responsible for the synthesis of glycogen, whereas GlgX and Gaa appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the geneexpression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among MR31, Δglg and ΔglgX, the death rateof Δglg mutant was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

Keywords : Sulfolobus acidocaldarius, glycogen metabolism, role of glycogen

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L-5

Different Patterns in Co-Culture of Staphylococcus aureus with Other Staphylococcus spp.

Seonmi Lee and Woo Jun Sul*

Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea

Staphylococcus is the most common microorganism on the skin along with Corynebacterium and Cutibacterium. Their interactions provide abaseline for studies that explore the role of bacterial communities on theskin. Among them, Staphylococcus aureus is a representative bacteria causing a broad array of skin infections. As S. aureus act as a pathogenon the skin, controlling them can be an effective way to alleviate skin diseases. 28 types of Staphylococcus spp. were isolated from the humanskin by swab. We observed growth modality both axenic culture growthof Staphylococcus strains and co-culture of the strains with S. aureusUSA 300 trying to find whether they kill S. aureus or not. We used Redfluorescence Protein (RFP) expressing pHC48 electroporated to S. aureus USA300 to sort out them. In our study, we found that one S. hominis (S38) kill S. aureus USA 300 at the wide range of pH, but theother S. hominis (S39) does not kill the USA 300 under pH 7 but tendsto kill the USA 300 as the environment gradually changes to pH 4. Additionally, there are some S. aureus compete with USA 300 under pH7. We suggest that there are some mechanisms in the group of Staphylococcus spp. inhibiting the formation of S. aureus biofilm according to pH environment.

Keywords : Skin microbiome, Staphylococcus aureus, bacterial co-culture

L-6

LC-MS/MS-Based Proteomic and Metabolomic Approaches for Investigation of Clostridioides difficile Inhibition Mechanism by Probiotics

Hyo-Jin Jeon, Sung-Hyun Jo, Won-Suk Song, Jae-Seung Lee, Ji-Eun Kwon, Ji-Hyeon Park, Ye-Rim Kim, and Yun-Gon Kim*

Department of Chemical Engineering, Soongsil University, Seoul 06978, Republic of Korea

Clostridioides difficile infection (CDI) is a life-threatening disease andis known to have a very high recurrence rate. Recently, new approachesusing probiotic therapy have been reported for recurrent CDI treatment.However, the mechanisms behind the inhibitory effect of C. difficile byprobiotics are unclear. To investigate the mechanisms of C. difficile inhibition, we observed the response of C. difficile caused by probioticsthrough combined proteomic and metabolomic studies. Firstly, in orderto select the probiotic strains that exhibit the highest inhibitory effect onC. difficile, we evaluated the inhibitory activity of Lactobacillus and Bifidobacterium species against C. difficile. The proteomic analysis showed that proteins involved in proline-dependent regulation, iron uptake, electron transfer, and riboflavin metabolic pathway were significantly changed. In addition, targeted metabolomics showed an overall decrease in the Stickland reaction, which is major energy-generation metabolism of C. difficile. Finally, we indicated thatthese metabolic changes caused a reduction in the growth rate and toxinproduction of C. difficile. In conclusion, our multi-omics approaches suggested the inhibitory mechanisms of C. difficile by probiotics at the molecular level.

Keywords : Clostridioides difficile, probiotics, multi-omics

L-7

Integrative Multi-Omics Approaches for Prebiotic Effects of the Inulin on Faecalibacterium prausnitzii

JiHyeon Park, Won-Suk Song, Sung-Hyun Jo, Jae-Seung Lee, Hyo-Jin Jeon, Ji-Eun Kwon, Ye-Rim Kim, and Yun-Gon Kim* Department of Chemical Engineering, Soongsil University, Seoul 06978, Republic of Korea

The human gut commensal bacteria Faecalibacterium prausnitzii is wellknown for its anti-inflammatory effects that improve host intestinal health. Although several studies reported that inulin, one of the well-known prebiotics, increases the abundance of F. prausnitzii in theintestine, the mechanism under this effect of inulin remains unclear. Here, we applied LC-MS/MS-based multi-omics approaches to confirmthe effects of inulin on F. prausnitzii. Interestingly, the proteomic analysis revealed that the putative proteins involved in sucrose utilization of F. prausnitzii are upregulated in the presence of inulin. Toinvestigate the function of the proteins, we cloned the target genes, andobserved the ability for sucrose degradation. In addition, we demonstrated that the sucrose degradation activity in F. prausnitziiculture media is enhanced by using inulin as a carbon source comparedto glucose. Taken together, this study suggests that the reduction of sucrose induced by inulin could potentially improve host health and prevent gut dysbiosis like type 2 diabetes which is associated with sucrose.

Keywords : Faecalibacterium prausnitzii, prebiotics, mlti-omics

L-8

Effects of the spoT and toxR Genes on Glycogen Metabolism and Virulence of Vibrio vulnificus

Ji Min Kim1, Su-Jin Kang1*, Sae Bom Yoon2*, and Jung Wan Kim1,2* Department of Bioengineering and Nano-Bio Engineering, Graduate School of Incheon National University, Incheon 22012, Republic of Korea 2Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

Vibrio vulnificus is a marine pathogen that can cause septicemia. The bacterium experiences nutrient-rich state in host and nutrient-poor statein aquatic environment. Glycogen accumulation would be a way to ensure supply of energy under unfavorable conditions. Especially, for pathogens with alternating habitats such as V. vulnificus, modulation of glycogen metabolism would be critical in their successful persistence in nature. MalT is a transcriptional regulator for the maltose regulon inE. coli. The malT mutation in V. vulnificus resulted in loss of both virulence and glycogen accumulation. It also affected expression of various enzymes such as SpoT, a bifunctional (p)ppGpp synthase/ hydrolase, and ToxR, a cholera toxin transcriptional activator. Both wereexpressed much less in the mutant than in wild type. As an effort to understand inter-relationship among these genes, each gene was knocked-out via conjugation. The toxR mutant accumulated glycogen twice as much as wild type when excess maltodextrin was added to the culture medium, while the spoT mutant showed slight difference only. These implied that toxR have direct impact on glycogen metabolism. InC. elegans killing assay, spoT exhibited stronger virulence than wild type probably due to accumulation of (p)ppGpp, while toxR attenuated virulence of V. vulnificus. More biofilm was formed by the malT and thetoxR mutants, but slightly less by the spoT mutant than wild type undercertain conditions.

Keywords : malT, spoT, toxR, glycogen metabolism, virulence

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L-9

Characterization of Cold-Tolerant Saccharomyces cerevisiae Cheongdo Using Phenotype Microarray

Bonbin Gu1, Kyung-Mi Jung2, Jongbeom Park1, Jueun Jang1, Seok-HwaJung1, Sang Han Lee1, and Soo Rin Kim1*

1School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 2Cheongdo Peach Research Institute, Gyeongsangbuk-Do Agricultural Technology Administration, Cheongdo 38315, Republic of Korea

The cold-tolerant yeast Saccharomyces cerevisiae is industrially usefulfor lager fermentation, high-quality wine, and frozen dough production.S. cerevisiae Cheongdo is a recent isolate from frozen peach samples which has a good fermentation performance at low temperatures and desirable flavor profiles. Here, phenotype microarray was used to investigate industrial potentials of S. cerevisiae Cheongdo using 192 carbon sources. Compared to commercial wine yeast S. cerevisiaeEC1118, Cheongdo showed significantly different growth rates on 34 substrates. The principal component analysis of the results highlightedthat the better growth of Cheongdo on galactose than on EC1118 was the most significant difference between the two strains. The intact GAL4gene and the galactose fermentation performance at a low temperatures suggested that S. cerevisiae Cheongdo is a promising host for industrialfermentation rich in galactose, such as lactose and agarose.

Keywords : Phenotype microarray, cold-tolerance yeast, galactose

L-10

Anti-Inflammatory Effect of Lactobacillus plantarum IDCC 3501 and Its Safety Evaluation

Seung A Chae1, Soo-Yeon Yang1, Won Yeong Bang1, O-Hyun Ban1,2, Soo-Jung Kim3, Young Hoon Jung2*, and Jungwoo Yang1* 1Ildong Bioscience, 17 Poseunggongdan-ro, Pyeongtaek-si, Gyeonggi-do 17957, Republic of Korea 2School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 3Department of Integrative Food Bioscience and Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea

This study investigated the anti-inflammatory activity of L. plantarumIDCC 3501 isolated from kimchi (Korean fermented food) and its safety.When lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were treated with cell-free supernatant from L. plantarum IDCC 3501,the mRNA expression level of inflammatory markers (i.e., TNF-α, IL-1β, and IL-6) was significantly reduced. The decreased cell viability byLPS was recovered and NO production in LPS-induced cell was also decreased. The genes responsible for antibiotic resistance and virulencewere not detected from the genome analysis of this strain. Consistent withthis, minimal inhibitory concentrations against various antibiotics, biogenic amines and D-lactate production, as well as enzymatic and hemolysis activities indicated that L. plantarum IDCC 3501 did not produce any harmful compounds during fermentation. Also, no acute toxicity and mortality were observed in a murine mouse model when feeding with L. plantarum IDCC 3501. Based on our findings, L. plantarum IDCC 3501 is safe and beneficial for human consumption.

Keywords : Probiotics, anti-inflammatory effect, safety evaluation

L-11

Enhanced Ceramides Production by Lactobacillus rhamnosus IDCC 3201 and Its Proposed Mechanism

Minjee Lee, Won Yeong Bang, Soo-Yeon Yang, Seung A Chae, Gwang-Seob Kim, O-Hyun Ban, and Jungwoo Yang*

IBS Research Center, Ildong Bioscience, Gyeonggi-do 17957, Republic of Korea

The use of probiotics has been applied for a variety of fields (e.g., immunesystem, mental health, and heart). In this study, the feasibility of lysatesfrom L. rhamnosus IDCC 3201 was evaluated for cosmetic ingredients.More specifically, enhanced ceramides production by the lysates and itsproposed machanism were investigated through in vitro and genome analysis. In results, enhanced spingomyelinase activity and thereby increased ceramides production by the lysates from L. rhamnosus IDCC3201 was observed. Furthermore, it was found that the existence of gbagene in L. rhamonsus IDCC 3201 was attributed to enhanced ceramidesproduction. Finally, it was verified that the lysates of L. rhamonsus IDCC3201 was regarded as safe for its use as cosmetic materials. Thus, thesefindings have significant implications that might lead to the developmentof functional and safe cosmetic products from probiotics.

Keywords : Ceramides, probiotics, skin health

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L-12

CRISPR/Cas9-Mediated Bacteriophage λ Genome Engineering

Ho Joung Lee, Hyun Ju Kim, and Sang Jun Lee*

Department of Systems Biotechnology, and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea

Phage therapy has emerged as an alternative way for the treatment of clinical pathogens when the antibiotics cannot be used, for instance, inthe case of MRSA (Methicillin Resistant Staphylococcus aureus) infection. Phage therapy means the control of bacterial infections usingvarious bacteriophages. In recent, the antibiotic-resistant microbial strains and Shiga-toxin producing Escherichia coli (STEC) have been grown as critical threats in the public health and medical care system.There are steady reports of outbreaks and deaths due to those pathogensworldwide. However, those multiple-antibiotic-resistant bacterial infections have limited specific treatment and were hardly developed yet. Therefore, an effective bacteriophage engineering system is neededfor the development of effective bacteriophages in phage therapy. In thisstudy, we chose the Escherichia coli-specific temperate bacteriophageλ as a model system to establish CRISPR/Cas9-mediated bacteriophagegenome engineering system. The CI repressor protein is the key factor that controls the life cycle of bacteriophage λ, and the life cycle changeis the concentration of CI protein-dependent. In this study, a lysogenic λ E. coli harboring cas9 gene in the chromosome was constructed. However, culture at the higher temperature for the rapid growth of lysogenic E. coli makes the lysogenic cells enter the lytic state, due to cI857 single nucleotide mutation, which makes the CI repressor became heat-labile and interferes with iterative genome engineering of the lysogenic λ cells. The cI857 mutation was corrected using the target-mismatched Cas9 method. As a result, the thermo-stability of lysogenic λ cells was confirmed. And its infectivity remained the same. Throughout this study, an accurate bacteriophage genome engineeringsystem by CRISPR/Cas9 was established.

Keywords : CRISPR/Cas9, bacteriophage genome editing, phage therapy

L-13

Microbial Single Base Genome Editing Aided by Target-Mismatched sgRNAs

Ho Joung Lee, Hyun Ju Kim, and Sang Jun Lee* Department of Systems Biotechnology, and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea

CRISPR/Cas system has revolutionized genome editing technology. CRISPR/Cas9 is consists of a single molecular guide RNA (sgRNA) andCas9 nuclease, which recognizes a specific sequence and its PAM (protospacer adjacent motif), and consequently cleaves the target DNAs.Since their function is modularized, therefore, the CRISPR/Cas9 has been used for negative selection tools that cleave unchanged target DNAsby site-specific mutagenesis, leaves the edited target DNAs, finally thecells with the desired mutation could be obtained. In this study, multiplebase-pair mutations were introduced in the galK gene of Escherichia coliby the CRISPR/Cas9 negative selection. However, the single base editedcells were rarely obtained since the CRISPR/Cas9 also cleaves the singlebase edited target DNAs despite mismatched sequence(s), which is called mismatch tolerance. To solve this problem, 1~2 bp mismatch(es)with the unedited target DNA was introduced into sgRNAs in advance.The unedited target DNAs were cleaved despite mismatch because of mismatch tolerance, however, the single base edited target could be survived due to the increased mismatches. As a result, the single base edited cells were obtained with high editing efficiency by using target-mismatched sgRNA method. Furthermore, general rules for the design of target-mismatched sgRNAs were optimized in the randomly selected 16 genome-wide targets. Consequently, this study shows that the target-mismatched sgRNA method is very effective for single nucleotide editing in microbial genomes.

Keywords : CRISPR/Cas9, microbial genome editing, mismatch tolerance

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L-14

Correlation of Citrate Synthase Homologous Overexpression and Acetate Metabolism Activity under Oxygen Limited Conditions in Pseudomonas putida

Sakkuntala Mutyala, and Jung Rae Kim*

Department of Chemical Engineering, Pusan National University, Busan 46241, Republic of Korea

Citrate synthase encoded by gltA regulates entry of acetyl-CoA into TCAcycle. This,citrate synthase gene expression is NADH dependent. Hence, assumed that thecitrate synthase copies might be lower under oxygen limited conditions. In thisstudy, homologous overexpressed gltAPseudomonas putida mutant was compared with wild type for acetate metabolism under oxygen limited conditions. Further, monitored thecurrent production ability of Pseudomonas putida with increasing gltA expressionin anaerobic microbial fuel cell. Thus, this study may help in understandingthe importance of TCA cycle marker enzyme citrate synthase for acetate utilizationof P. putida.

Keywords : Microbial fuel cell, acetate metabolism, citrate synthase

L-15

Identification at Novel Glutamine Transporters in Escherichia coli

Jiwon Park1, and Ok Bin Kim1,2,3* 1Interdisciplinary Program of EcoCreative, Ewha Womans University, Seoul 03760, Republic of Korea 2Department of Life Science, Ewha Womans University, Seoul 03760, Republic of Korea 3Department of EcoScience, Ewha Womans University, Seoul 03760, Republic of Korea

In Escherichia coli, glutamine is required as a nitrogen source for a rangeof biosynthetic reactions and plays a central role in nitrogen assimilation.All nitrogen sources entered E. coli are metabolized after converted to glutamine and glutamate. Since E. coli only detects intracellular nitrogen sources, it is important to study glutamine transporters to understand E.coli nitrogen metabolism. E. coli actively accumulates glutamine through various transport mechanisms but, there is only one identifiedglutamine transporter (GlnHPQ). After deletion of glnH, the growth ofmutant strain(ΔglnH) did not significantly decrease in minimal mediumcontaining glutamine as sole nitrogen source. Therefore, it is expected that there are other glutamine transporters other than GlnHPQ. E. coligenome processes several candidate genes for glutamine transporter. Forexample, the gene yaaJ encoding an uncharacterized transporter that shows similarity with AlsT of Staphylococcus aureus (42%) and Bacillussubtilis (43%). AlsT (alanine/sodium symporter) has been recently suggested as a main glutamine transporter in S. aureus. To identify analternative glutamine transporter in E. coli, the several candidate geneswere deleted, and measured the effect on growth with glutamine as a nitrogen source. The transcriptional changes of candidates were analyzed depending on the type of nitrogen source. Then, the uptake of[14C]-glutamine by candidate transporter was analyzed.

Keywords : Escherichia coli, glutamine, transporter

L-16

Signaling Networks Involved in the Sexual Reproduction Cycle of Cryptococcus neoformans

Jin-Young Kim, Yujin Lee, Yeonseon Lee, and Yong-Sun Bahn*

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

The fungal pathogen Cryptococcus neoformans causes cryptococcosisby the inhalation of infectious spores generated by unisexual or bisexualreproduction. To understand complex signaling networks modulating the developmental process, a complete understanding of genome-scaletranscription factors (TFs) and kinases is needed. Previously we reportedthat 37 TFs and 42 kinase mutants constructed in C. neoformans MATαH99 strain background exhibited altered mating efficiency. To further elucidate the mating regulatory mechanism, we constructed knockout mutants of the mating-regulating TFs and kinases in YL99 strain-MATαisogenic strain of H99 strain-to monitor unilateral and bilateral mating, and to perform an analysis of their function in the developmental process. We constructed 30 gene-deletion strains representing 15 TFs and 20 gene-deletion strains representing 10 kinases and are currently constructing gene-deletion strains for the remaining mating-regulatingTFs and kinases. For confirmed mutant strains, we are examining matingphenotypes during bilateral mating: mating pheromone production, cellfusion efficiency, filamentous growth, formation of basidia and basidiospores. Furthermore, we are examining transcript profiles of mating-regulating TFs and kinases at different developmental stages ofsexual reproduction. Ultimately, this study will focus on mapping and discovering the functions of the mating-regulating TFs and kinases, andelucidating complex signaling networks in the developmental process of C. neoformans.

Keywords : Cryptococcus, sexual development, mating

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L-17

Identification of DrBrx Interacting Proteins in Deinococcus radiodurans

Soyoung Jeong1,2, Jong-Hyun Jung1, Sangryeol Ryu2,3, and Sangyong Lim1* 1Research Division for Biotechnology, Korea Atomic EnergyResearch Institute, Jeongeup 56212, Republic of Korea 2Department of Food and Animal Biotechnology, Seoul NationalUniversity, Seoul 08826, Republic of Korea 3Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea

Many bacteria have a functional thiol redox system comprising the dithiol-containing redoxin proteins such as thioredoxin(Trx) and glutaredoxin(Grx), as well as peroxiredoxin(Prx). These proteins not only scavenge reactive oxygen species(ROS) but modulate the activitiesof the target protein by using the reactivity of the cysteine residue. Deinococcus radiodurans as major radiation resistant bacterium, has apowerful antioxidant system. Unlike other bacteria, D. radioduranspossesses a putative homolog of BrxC(DR_1832) containing an only onecysteine residue. In previous study, we revealed that it has peroxide-scavenging function. Mutation in DR_1832 also sensitized cells to H2O2. Herein, we investigate the brx-interactome in D.radiodurans. Based on the reaction mechanism, we generated DR_1832 mutant lacking the resolving formation of intermolecular disulfide bonds and identified 25 D.radiodurans proteins potentially interacting with DR_1832. These proteins are involved in cellular metabolism including glycolysis and amino acid biosynthesis, and stressresponses. Especially, among the candidates, OxyR and Nudix proteinsexhibited binding signal. And also in ELISA assay, OxyR showed strongaffinity with DR_1832. Taken together, we suggested that DR_1832 ispossibly involved in the antioxidant system by modulating the target protein such as OxyR.

Keywords : Deinococcus, bacilliredoxin, OxyR

L-18

Elucidating the Role of the Casein Kinase 2 in the Pathogenicity of the Human Fungal Meningitis Pathogen Cryptococcus neoformans

Yeseul Choi, and Yong-Sun Bahn* Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

The opportunistic human fungal pathogen Cryptococcus neoformanscauses fatal meningoencephalitis both in immunocompromised patientsand immunocompetent individuals. However, the therapeutic options for treatment of cryptococcosis are currently highly limited. As a potential antifungal drug target, kinases have been considered to be goodcandidates and play important regulatory roles in cellular mechanismsand virulence of fungal pathogens. In previous studies, we found Cka1, a serine/threonine eukaryotic kinase, is involved in regulation of cell growth, cell cycle, cellular morphology and pathogenicity of C. neoformans. In this study, we aim to figure out the regulatory mechanismof casein kinase 2 (CK2) complex, whose catalytic subunit is Cka1. Wefound one catalytic subunit Cka1 and two regulatory subunits which areCkb1 and Ckb2 as a putative CK2 complex subunit. We identified the physical interactions between each subunit of casein kinase 2 using co-IP.The two regulatory subunits form dimer, and a catalytic subunit attachesto each regulatory subunit to form a tetramer. We also constructed singleand double knockout mutants of regulatory subunits to compare the phenotypes with cka1Δ. The regulatory subunits were involved in antifungal drugs susceptibility, oxidative stress and DNA damaging responses. To find out that CK2 is related with cell cycle, we performed FACS analysis, and the knock out mutants show cell cycle disruption. Also, the knock out mutants shows abnormal and swollen cell morphology in SEM and TEM analysis. These data demonstrate that CK2 acts as a cell cycle regulator and each subunit plays distinct roles.In addition, CK2 is known as an upstream regulator in human, we figuredout the Hog1 phosphorylation under oxidative stress. The regulatory subunits single knock out mutants showed similar phenotype like WT. Interestingly, the Hog1 phosphorylation level decreased at basal in thecatalytic subunit, Cka1 and regulatory subunits double knock out mutants. As a result, Cka1 plays major roles and Ckb1/Ckb2 have minorroles in casein kinase 2 complex. This study will provide a comprehensive Cka1 cellular mechanism to develop an antifungal drug.

Keywords : Cryptococcus neoformans, casein kinase 2, antifungal drug

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L-19

Analysis of Spontaneous Gene Deletion in Bacteriophage MK1

Mooseung Kim1, and Sangryeol Ryu1,2* 1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Center for Food Bioconvergence, Seoul National University, Seoul 08826, Republic of Korea

Deletion or mobilization of various genes during DNA replication is amajor force in evolution. Here, we isolated an Escherichia coli phageMK1 that forms a turbid plaque with a halo. We observed that about 0.01% of the MK1 plaques became clear even after several rounds of single plaque purification step. The phage forming a clear plaque (MK1C) was isolated and its genome was compared with that of MK1.The whole genome sequencing of MK1 and MK1C revealed that MK1C had point mutations at MK1_029 (hypothetical protein) and intergenic regions of unidentified genes, between MK1_064 and MK1_066, between MK1_072 and MK1_073, and after MK1_096. Moreover, MK1C lost 4.5 kb DNA including 3’ part of MK1_020 (putative tail spike), MK1_021 (hypothetical protein), MK1_022 (hypothetical protein), and 5’ part of MK1_023 (putative tail fiber). The 14 bp DNA homology regions were found at both ends of the deleted 4.5 kb DNA, suggesting that MK1C may be derived from MK1 by spontaneous deletion of the 4.5 kb DNA from MK1 through homologous recombination. Deletion of the 4.5 kb DNA from MK1 by utilizing CRISPR Cas9 system made plaques clear while MK1_029 deletion didnot affect plaque morphology, indicating that the genes in the 4.5 kb DNAregion affect plaque morphology.

Keywords : Bacteriophage, spontaneous mutation, DNA recombination

L-20

Optimizing Improvement of Microalgal Growth Using Silver Nanoparticles

Chang-hyun Jeon, June Lee*, and Woong Kim* Department of Environment Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea

The utilization of microalgal biomass for the energy production has garnered worldwide interest due to the reduction of thermal power andde-nuclear power generation that it can achieve. In addition, microalgalbiomass has been utilized in various chemical and pharmaceutical industries for the production of industrially important value added products. In previous studies, the use of Localized Surface Plasmon Resonance (LSPR) on metal nanoparticles significantly improved the growth of the photosynthesis pigment content of algal cultures. LSPRis the effect of the interaction between the conduction band electrons ofthe metal nanoparticles and the electromagnetic field. With this effect,metal nanoparticles backscatter specific wavelengths and deliver them to the microalgae to help pigment synthesis. We derived 0.24 mM Silvernanoparticles and 175 μmol/m2s of light intensity as the optimal conditions for the cultivation of Chlorella vulgaris using silver nanoparticlesbased on Response Surface Methodology. In this study, we incubatedC. vulgaris by synthesizing silver nanoparticles to improve the accumulationof microalgal biomass. We determined the improvement in C. vulgarisgrow when cultivation using optimal conditions. Optical density, dry cellweight (g/L), chlorophyll contents (mg/L), and lipid contents (g/L) of C. vulgaris using a silver nanoparticles solution were 27%, 26%, 11%,and 31% better, respectively, than the control without silver nanoparticlessolution over two weeks of cultivation. The results show promise in termsof gaining access to high value-added pigments for a myriad of applications in fields ranging from medicine to materials science, and in developing sustainable technologies for the future.

Keywords : LSPR, microalgae, nanoparticle

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L-21

Elucidating the Role of C2H2 Zinc-Finger Transcription Factor Zfc2 Involved in the Pathogenicity and Virulence of Cryptococcus neoformans

Soojin Yu1, Seong-Ryong Yu1, Yee-Seul So1, Dong-Gi Lee1, Gena Lee Meyers1, Héctor M. Mora Montes2, Gustavo H. Goldman3, and Yong-SunBahn1* 1Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 2Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato,Noria Alta s/n, Col. Noria AltaGuanajuato, Gto. CP 36050, México, 3Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) and Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Brazil

Cryptococcus neoformans is a fungal pathogen that causes meningoencephalitis, and it is responsible for more than 600,000 deathsglobally every year. Among the 45 pathogenicity-related transcription factors that we identified through large-scale in vivo and in vitro virulence and infectivity assays, a unique C2H2-type zinc finger transcription factor, ZFC2, stood out. ZFC2 was exceptional for three reasons: 1) ZFC2has no ortholog in any other known fungal transcription factors, 2) displays different phenotypic traits compared to the usual pathogenicity-related phenotypes of C. neoformans, 3) exhibits highly attenuated virulence in the insect killing model and reduced lung infectivity in themurine infection model. For these reasons, we aim to elucidate the roleZFC2 plays in the pathogenicity of C. neoformans. In spot assays, zfc2∆showed dramatically increased resistance against tunicamycin (TM), which inhibits the first step of N-glycosylation mediated protein folding.Under basal conditions, Zfc2 was enriched in the nucleus, but after 30minutes of treatment with TM, Zfc2 translocated into the cytoplasm andre-located back into the nucleus after 90 minutes. Because ER stress response is mainly governed by the unfolded protein response (UPR) pathway, we compared the splicing levels of HXL1 in the zfc2∆ mutantwith those in the wild type (WT) strain. Unexpectedly, splicing levelsof HXL1 mRNA in zfc2∆ were similar to that of the WT strain with orwithout TM treatment. To understand the role of Zfc2, RNA-seq basedtranscriptome analysis of zfc2∆ compared to the WT was performed. Under basal conditions, a total of 782 genes were differentially regulated(cut-off: over 2-fold changes) in the zfc2∆ mutant compared to the WT,of which 312 genes were upregulated while 470 were downregulated,suggesting that Zfc2 could serve as both transcriptional activator and repressor. However, under ER stress condition (0.2 μg/ml TM, 90 min),total of 170 genes were differently regulated in zfc2∆ compared to theWT, of which 137 genes were upregulated and 33 were downregulated.Moreover, electron microscope imaging showed that capsule formation,a cryptococcal virulence factor, was notably increased in zfc2∆ compared to the WT. Overall, our research aims to provide an extensiveunderstanding of a unique pathogenic transcription factor of C. neoformans.

Keywords : ZFC2, zinc finger transcription factor, Cryptococcus neoformans

L-22

Escherichia coli as a Model System to Study the Mitochondrial Mutations in Biliary Atresia

Ji-Young Lee1, Hong Koh2, Kyoung Su Kim1, Jung mo Lee1, and Dong-Woo Lee1* 1Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 2Department of Pediatrics, Yonsei University College of Medicine, Severance Children’s Hospital, Severance Pediatric Liver Research Group, Seoul 03722, Republic of Korea

Biliary atresia (BA) is a hepatic disease characterized as blockage of bileflow caused by the absence of extrahepatic ducts. Even after a successfulKasai surgery, a considerable number of patients exhibit hepatocellulardysfunction due to mitochondrial mutations. Previously, we identified the BA-specific SNP-like mitochondrial mutations in the ND2 and ND4subunits of the complex I. To investigate the effect of those mutations on the structure and function of complex I, we generated Escherichia coli mutant strains lacking the ND2 and ND4 subunits as microbial counterparts, respectively. The ΔnuoN and ΔnuoM mutants exhibited severe growth defects under anaerobic conditions, whereas they, including Δndh, a knock-out strain of NuoA-N isozyme, showed a similar pattern to WT under aerobic conditions. To examine the effectof BA-specific mutations on bacterial growths, we generated several E.coli mutants and characterized their growth patterns. Remarkably, somemutant strains showed growth defects due to a lack of peripheral alternative respiratory chains similar to those of ΔnuoN and ΔnuoM under anaerobic conditions, indicating that the mutations hinder the cellular growth through the impaired catalytic function of complex I. Therefore, we suggest that these E. coli mutant strains can be used asa model system to study the effect of mitochondrial mutations in complexI.

Keywords : Biliary Atresia, mitochondrial complex I, Escherichia coli

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L-23

A New Antibiotic Tolerance Mechanism in Burkholderia cenocepacia Reduces Formation of Reactive Oxygen Species

Dongju Lee, and Heenam Kim* College of Health Sciences, Korea University, Seoul 02841, Republic of Korea

Burkholderia cenocepacia is an opportunistic pathogen belonging to the Burkholderia cepacia complex (Bcc) that cause severe pneumonia in cystic fibrosis patients. Because of resistance to multiple antibiotics, a new antibiotic resistance can exacerbate the problem which is difficultto cure Bcc infections. We selected for the resistant isolates against tobramycin, one of the antibiotics currently used for B. cenocepacia infections, and obtained three null mutations in the gene encoding adenosylcobinamide-phosphate synthase, an enzyme involved in coenzyme B12 biosynthesis. The mutants showed the resistance to various aminoglycosides and β-lactams, and no changes in the growthrate. These mutations can decrease the activity of coenzyme B12-dependent methylmalonyl-CoA mutase, which supply succinyl- CoA to the tricarboxylic acid (TCA) cycle. This may, in turn, cause a reduction of hydroxyl radicals, the key killing agents for the bactericidalantibiotic-mediated cell death mechanism. Although the mode of actionof aminoglycosides has been known simply as protein synthesis inhibition, our data demonstrate that oxidative killing has the major role,and explain why aminoglycosides are mostly ineffective against infections against anaerobic pathogens that do not use the aerobic respiration.

Keywords : Antibiotic tolerance, ROS, aminoglycosides

L-24

Corynebacterium glutamicum Gene cg3082 Plays a Regulatory Role in Metal Transport

Won-Woo Choi1, Younhee Kim2, and Heung-Shick Lee1* 1Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-ro, Sejong-si 30019, Republic of Korea 2Department of Korean Medicine, Semyung University, 65 Semyung-ro, Jecheon-si, Chungbuk 27136, Republic of Korea

In this study, we assessed the function of the cg3082 gene of Corynebacteriumglutamicum, which is predicted to encode a metalloregulatory protein.Deletion of the cg3082 gene did not affect the growth of the mutant cells,suggesting that the gene does not function as an essential gene under ordinary growth conditions. However, the Dcg3082 cells showed resistance to nickel, which was added to the growth medium, whereas cg3082-overexpressing cells showed increased sensitivity to the metal.RT-qPCR analysis identified increased transcription of the cg3082 gene,when cells were grown in the presence of nickel, cadmium, or manganese, suggesting that the cg3082 gene may participate in metal transport as a regulatory protein. In addition, the Dcg3082 cells showedincrease resistance to oxidants, such as hydrogen peroxide and cumene hydroperoxide. Collectively, these findings suggest that the cg3082 genemay play a negative regulatory role in the efflux of metals, involving nickel, manganese, and cadmium.

Keywords : Corynebacterium glutamicum, nickel, oxidative stress

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1I1A3A01051388)]

L-25

The NCgl0127 Gene of Corynebacterium glutamicum Plays Role in Cysteine Metabolism

Han-Deul Yang1, Younhee Kim2, and Heung-Shick Lee1* 1Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-ro, Sejong-si 30019, Republic of Korea 2Department of Korean Medicine, Semyung University, 65 Semyung-ro, Jecheon-si, Chungbuk 27136, Republic of Korea

In this study, we analyzed the NCgl0127 gene, which was annotated as an ABC-type transporter in Corynebacterium glutamicum. To elucidatethe function of the gene, we constructed a NCgl0127 deleted mutant strain (ΔNCgl0127) and found the growth of the strain on minimal medium was severely retarded as compared to that of the with wild-type strain. Dramatically, the growth was recovered in the medium containingL-cysteine. The transcriptional levels of the genes metB, cysI, cysK, cysE,which are involved in cysteine biosynthesis and sulfur assimilation pathway, were decreased in the ΔNCgl0127 strain. Surprisingly, except cysI, which is involved in the conversion of sulfite to sulfide, the transcription of the genes metB, cysK, and cysE, were recovered in the medium supplemented with L-cysteine. In addition, supplementation ofthe medium with sulfide supported the growth of the ΔNCgl0127strainon minimal medium. Further, the NCgl0127-overexpressing strain (P180-NCgl0127) showed increased sensitivity to oxidant diamide and SDS. Collectively, these results suggest that the NCgl0127 gene of C. glutamicum affect the assimilatory metabolism of sulfur.

Keywords : Corynebacterium glutamicum, ABC transporter, L-cysteine

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1I1A3A01051388)]

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Regulation of Redox-Metabolism by the Negative Autoregulatory Transcription Factor OsnR in Corynebacterium glutamicum

Haeri Jeong1, Younhee Kim2, and Heung-Shick Lee1* 1Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-ro, Sejong-si 30019, Republic of Korea 2Department of Korean Medicine, Semyung University, 65 Semyung-ro, Jecheon-si, Chungbuk 27136, Republic of Korea

The osnR gene of Corynebacterium glutamicum specifically regulates genes involved in redox-dependent stress responses. In this study, we show its DNA-binding ability as a negative transcriptional factor. Overexpression of the gene (P180-osnR) affected genes linked to the redoxreactions and mycothiol metabolism. Analysis involving ChIP-seq identified not only the osnR gene but also cg0026, which seem to functionin membrane-associated redox metabolism, as the targets. DNA bindingof OsnR to the regulatory region of its own gene was stimulated by oxidant diamide, suggesting that OsnR functions as a negative autoregulatory transcription factor, whose activity is modulated by theredox status of the cell. Moreover, the transcription level of σ factor-encoding gene sigH was decreased by 50% in the P180-osnR cellsas compared to that of the wild type cells. Accordingly, in vivo assays,performed in the presence of reductant DTT, suggested 30% increasedbinding of the OsnR protein to the sigH regulatory region. Collectively, our data suggest that the osnR gene not only functions as a specific regulator for genes involved in redox-dependent stress responses but alsoparticipates in the global regulation by controlling the transcription of other master regulators, such as sigH.

Keywords : Corynebacterium glutamicum, redox metabolism, auto- regulation

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1I1A3A01051388)]

L-27

Amino Acid Availability Regulates the Protein Levels of Peptidoglycan Endopeptidases in Escherichia coli

Byoung jun Choi, Si Hyoung Park, Yung Jae Kim, and Chang-Ro Lee*

Department of Biological Sciences, Myongji University, Yongin, Gyeonggido 17058, Republic of Korea

Peptidoglycan (PG) hydrolases play diverse physiological roles, such as cytokinesis, PG synthesis, quality control of PG, PG recycling, and antibiotic resistance, but the regulatory mechanisms of their expressionare poorly unveiled. In this study, we have revealed novel mechanismsregulating the protein levels of the synthetically lethal PG endopeptidases MepS and MepM. A mepS mepM double mutant was lethal in amino acid-rich medium, whereas it exhibited almost normal growth in minimal medium, suggesting the unnecessity of MepS and MepM in minimal medium. Protein levels of MepS and MepM dramatically decreased in the minimal medium. The decrease in MepS level in the minimal medium was governed by Prc, a periplasmic proteaseinvolved in the proteolysis of MepS, whereas the level of MepM in theminimal medium was not affected by Prc. Phenotypic and biochemicalanalyses demonstrated that protein levels of MepS and MepM increasedin the presence of aromatic amino acids and glutamate in the medium,respectively. Therefore, these results indicate that the protein levels of MepS and MepM are affected by distinct amino acid availability- dependent regulatory mechanisms.

Keywords : Peptidoglycan hydrolase, peptidoglycan endopeptidase, amino acid availability

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L-28

Comparative Genomics Determines Strain-Dependent Secondary Metabolite Production in Streptomyces venezuelae Strains

Woori Kim1, Namil Lee1, Soonkyu Hwang1, Yongjae Lee1, Jihun Kim1, Suhyung Cho1, Bernhard Palsson2,3,4, and Byung-Kwan Cho1,4* 1Department of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 2Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA, 3Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, USA, 4Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Lyngby, Denmark

Streptomyces venezuelae is well known to produce various secondary metabolites (SMs). Although many strains have been classified as S. venezuelae species, genomic differences and diversity in SM productionbetween the strains have never been compared. Here, we report completegenome sequences of three S. venezuelae strains harboring chloramphenicol and jadomycin biosynthetic gene clusters (BGCs). With these high-quality genome sequences, we revealed that the three strains share more than 85% of total genes and most of the SMBGCs.Despite such conservation, the strains produced different amounts of chloramphenicol and jadomycin, indicating differential regulation of SM production at the strain level. By comparing the chloramphenicoland jadomycin BGCs among the three strains, we found sequence variations in many genes, the non-coding RNA coding regions, and binding sites of regulators, which affect the production of the SMs. Weanticipate that these genome sequences of closely related strains wouldserve as useful resources for understanding the complex secondary metabolism.

Keywords : Comparative genomics, Streptomyces venezuelae, biosyntheticgene cluster

[Supported by Bio & Medical Technology Development Program (2018M3A9F3079664 to B.-K.C.) through the NRF, funded by the MSIT, and the Novo Nordisk Foundation (NNF10CC1016517)]

L-29

Discovering the Neuroprotective Effect of Lactobacillus in Oxidative Stress Induced HT22 Cells by Using nLC-ESI-MS/MS

Chau Pham1,2, Hyunesoo Kwon1,3, Youngeun Kim1,3, Soojin Lee2*, DukjinKang1*, and Hee Min Yoo1,4 *1Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea 2Department of Microbiology and Molecular Biology, Chungnam National University (CNU), Daejeon 34134, Republic of Korea 3School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea 4Department of Bio-Analytical Science, University of Science and Technology (UST), Daejeon 34113, Republic of Korea

Neurodegenerative, a consequence of the dysfunction and death of neuron cells and central nervous system (CNS) including the most common disease such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disorder (HD) affect more than 1 million people inthe world and leading the most cause death after cancer. In recent decades,the effects of the gut microbiota on our brain and behavior have been documented. Notably, intestinal microflora takes part in bidirectional communication mechanisms between the gastrointestinal tract and thecentral nervous system. Moreover, several studies in the animal model provide evidence that probiotics, lactobacillus treatment reduce anxiety- and depression-like behavior. In addition, probiotics may act beneficially by improving the intestinal barrier and immune responses.However, the underlying molecular mechanisms of Lactobacillus in theneuron cell have been not elucidated. Therefore, the aim of this study to uncover the effect of Lactobacillus on neuroprotection in the oxidativestress-induced in the hippocampus cell line (HT22 cells). Oxidative stress is well known that has been contributed to the progression of thenumber of neurodegenerative diseases. We used H2O2 as a signal to induce oxidative stress in the HT22 then treatment cell-free supernatantlactobacillus to assess the neuroprotection effect. Fluorescence- activated cell sorting (FACS) analysis was performed to evaluate the ROS level and cell induce apoptosis population. Our data indicated thatLactobacillus significantly reduces the ROS accumulation and protectsthe cell from H2O2 induces cell death in comparison to the control group.The western blotting analysis also revealed that Lactobacillus upregulationthe expression of an anti-apoptotic protein. Further, in order to investigatethe regulatory protein of lactobacillus in the HT22 oxidative stress, proteomics was used with tandem mass tag (TMT) via nLC-MS/MS analysis. Totally, more than 3000 proteins were identified and quantifiedin 6 replication. Among them, 66 proteins are up-regulation and 47 proteins are downstream regulation. We strongly believe that our methodcan be applied to find new biomarkers for neuron disorders.

Keywords : Short-chain-fatt- acid (SCFAs), neurodegenerative disease,Lactobacillus

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L-30

Systematic Analysis of Radiation Resistance and DNA Damage Signaling Network in the Radiation-Resistant Fungus, Cryptococcus neoformans

Kwang-Woo Jung Radiation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea

Cryptococcus neoformans is a basidiomycete pathogen, causing life-threatening cryptococcosis, and is a radiation-resistant fungus, which is often found in highly radioactive environments. Our previousstudies revealed that a novel bZIP transcription factor, Bdr1, governs DNA damage responses by controlling the expression of DNA repair genes, and Rad53 and Chk1 kinases played redundant and discrete rolesin genotoxic stress. However, comprehensive regulatory mechanisms of radiation resistance and DNA damage response at the transcriptionfactor (TF) network level remains unidentified. To elucidate radiation-resistant mechanisms at the TF network level, we attempted three systematic approaches using phenotypic analyses with genome-wide of TF mutant library, transcriptional regulation of TF, andphosphoproteome analysis. We found that strains deleted with HCM1, FZC1, ADA2, or FZC1 exhibited radiation sensitivity. Furthermore, weidentified that MBS1, DDT1, GAT7, YAP1, MIZ1, and GAT1 were also involved in the DNA damage response via the systematic analyses of 155 TF mutants in response to DNA damage stress. We demonstrated that transcriptional levels of 16 TFs [12 TFs were upregulated and 4 TFsare downregulated] were changed after radiation exposure in a Rad53-Bdr1 pathway independent manner. Among them, perturbationof HCM101 or FZC5 in the bdr1Δ mutant renders strains more susceptible to radiation than each mutant. In phosphoproteome analysis,we found the phosphorylation levels of proteins involved in the signaltransduction, transcription, translation, and RNA processing, and modification were altered during the recovery process after radiation exposure. Therefore, this study would improve understanding of the network of the radiation-resistance and DNA damage response in the radiation-resistant fungus, C. neoformans.

Keywords : Cryptococcus neoformans, DNA damage, transcription factor

L-31

Identification and Enzymatic Activities of Marine-Derived Fungi Trichoderma spp.

Woon-Jong Yu, Ji Yeon Lim, and Dawoon Chung* National Marine Biodiversity Institute of Korea (MABIK), Seocheon-gun, Chungcheongnam-do 33662, Republic of Korea

Fungi in marine environments play an important role in nutrient regeneration cycles. The genus Trichoderma, is considered as one of thefungi that are efficient producers of extracellular enzymes. National Marine Biodiversity Institute of Korea (MABIK) collects, preserves, and studies marine-derived fungi in Korea. Among the culture collections, we selected Trichoderma species to elucidate enzyme activities. Based on the morphology and phylogenetic analyses using ribosomal DNA internal transcribed spacer (ITS) and Translation elongation factor 1α (TEF-1α), 6 strains were identified as T. hamatum,T. koningiopsis, T. citrinoviride, T. afroharzianum, T. capillare and T. asperellum. Activities of 4 widely used enzymes including amylase, lipase, protease and chitinase were examined in these Trichoderma spp.on plate assay methods. All tested Trichoderma isolates displayed chitinase and lipase activities. Amylase activities weren't observed in these six isolates. In addition, protease activities were found in T. koningiopsis, T. citrinoviride, T. capillare. Enzyme assay of chitinase indicated strong cell-wall degrading enzyme activities of these Trichoderma isolates. Enzyme assays showed that Trichoderma spp. notonly demonstrate ecological roles but also have great potential in industrial applications.

Keywords : Fungi, Trichoderma spp., enzyme activities

[This work was supported by a grant from National Marine BiodiversityInstitute of Korea (MABIK,2020M00500)]

L-32

Biogenic Silver Nanoparticles Synthesized from Aggregatimonas sangjinii

Yong Min Kwon, Kyung Woo Kim, Dawoon Chung, Jaoon Young HwanKim, and Eunseo ChoNational Marine Biodiversity Institute of Korea, Seocheon 33662, Republic of Korea

Silver nanoparticles (AgNPs) are one of the most crucial and remarkable nanomaterials involved in the applications of medical, bioremediation,drug delivery etc. The synthesis of biogenic AgNPs has been applied asan alternative to physical and chemical synthesis. The extracellular AgNPs synthesis using Aggregatimonas sangjinii F202Z8 was confirmedby the absorbance peak at 450 nm in UV-Vis spectrophotometer. Further,presence of F202Z8-formed AgNPs was confirmed by transmission electron microscopy and energy dispersive X-ray spectroscopy analysis.On transmission electron microscopy, the AgNPs were spherical or amorphous structure in shape and ranged from 10 to 103 nm. The averagesize and concentration of AgNPs was 48 nm and 1.5×109 ml-1 using thenanoparticle tracking analysis, respectively. The synthesized AgNPs exhibited a broad spectrum of antibacterial effect against a variety of pathogenic Gram-positive and Gram-negative bacteria. The minimal inhibitory concentration values of F202Z8-formed AgNPs against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus was found to be 80, 40, 30 µg/ml-1, respectively. The highest antibacterial activity showed against S. aureus, a highly virulent pathogenic bacteriathat causes food poisoning, skin infection, and sepsis. This study suggests that A. sangjinii F202Z8 is a potential candidate for efficientsynthesis of AgNPs and may be suitable for the formulation of new typeas bactericidal materials.

Keywords : Silver nanoparticles, Aggregatimonas sangjinii, antibacterialactivity

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L-33

Genome-Wide Analysis of WD40 Repeated Proteins Involved in Pathogenicity of Cryptococcus neoformans

Jin-Tae Choi, Yu-Byeong Jang, Seong-Ryong Yu, Yujin Lee, and Yong-Sun Bahn*

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

Cryptococcus neoformans is one of the major human fungal pathogenswhich causes death by meningoencephalitis. The cell signaling regulation in various environments and host conditions is known to be important for the survival and virulence of C. neoformans. Among the cell signaling regulation, protein-protein interaction (PPI) is known as an essential phenomenon. Though the WD40 domain is one of the mostcommon and abundant domains related to PPIs, the role of WD40 domain-containing proteins remains elusive. We focused on 130 WD40 domain-containing genes, and constructed knockout mutants and examined their in vitro phenotypic traits under 30 different growth conditions. For the genes which could not delete, we replaced the promoter to conditionally regulate the expression of target genes. As aresult, we discovered Rav1, which known as a subunit of the regulator of ATPase of vacuoles and endosomes (RAVE) complex in the model yeast, was related to cellular growth on various temperature and stress conditions and the production of virulence factors in C. neoformans. Doa1, which is involved in protein degradation, was related to oxidativestress response and antifungal drug susceptibility. Interestingly, doa1Δ mutants were highly susceptible to fluconazole, but resistant to fludioxonil. Swd1, known as a histone methyltransferase, was related to DNA damage response, tunicamycin susceptibility, and capsule production. Through further study, we can dissect the roles of WD40 proteins and PPI partners on various stress responses and host interactionof C. neoformans, and these findings will provide comprehensive insightto develop the PPI inhibitors as novel antifungal agents.

Keywords : WD40, Crytococuccus, pathogenicity

L-34

Mutations in ArgS Arginine-tRNA Synthetase Confer Additional Antibiotic Tolerance Protection to Extended-Spectrum-Beta-Lactamase-Producing Burkholderia thailandensis

Jongwook Park, and Heenam Stanley Kim* College of Health Sciences, Korea University, Seoul 02841, Republic of Korea

Highly conserved PenI-type class A β-lactamase in pathogenic membersof Burkholderia species can evolve to extended-spectrum β-lactamase(ESBL), which exhibits hydrolytic activity toward third-generation cephalosporins, while losing its activity toward the original penicillin substrates. We describe three single-amino-acid-substitution mutationsin the ArgS arginine-tRNA synthetase that confer extra antibiotic tolerance protection to ESBL-producing Burkholderia thailandensis This pathway can be exploited to evade antibiotic tolerance induction in developing therapeutic measures against Burkholderia species, targeting their essential aminoacyl-tRNA synthetases.

Keywords : ArgS, stringent response

L-35

Electron Transfer System via Flavin and Flavocytochrome of Shewanella Oneidensis MR-1

Serah Choi, and In Seop Chang*

School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea

Electroactive microorganisms form a biofilm on the electrodes of a bioelectrochemical system and exchange electrons with the electrode through an electron transfer system. Shewanella oneidensis MR-1 is oneof the representatives of electrochemically active microorganisms. It hasseveral types of electron transport systems. For example, they use flavin,a mediator secreted by the outer cell membrane, to transfer electrons without physical contact, and induce electrons through flavocytochrome,where the flavin binds as a cofactor for redox proteins in the outer membrane. In this study, the electron transport system corresponding tothe oxidation and reduction peaks of the cyclic voltammetry method wasspecified based on the differential pulse voltammetry method that can clearly distinguish the electron transport system. As the biofilm of S. oneidensis MR-1 is developed at the anode of the bioelectrochemical system, the reduction reaction predominates for electron transfer through flavin, and the oxidation reaction predominates for electron transfer through flavocytochrome in the cyclic voltammogram.

Keywords : Microbial electrochemical system, biofilm, electron transfer system

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L-36

Metabolic Properties of Methanol Utilization in Syngas-Fermenting Bacterium, Eubacterium limosum KIST612

Ji-Yeon Kim, Mungyu Lee, Nulee Jang, Byeongchang Kang, and In SeopChang* School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea

Methanol could be used to solve the problem of low solubility of gases substrates by activating the reductive acetyl-CoA pathway. In this study,properties of methanol metabolism of Eubacterium limosum KIST612were investigated to optimize the methanol supply conditions during syngas fermentation. Methanol could be used by mta-encoding methyltransferase (ELI_XXXX-ELI_YYYY) in E. limosum KIST612.Methanol utilization only started in the presence of CO2. In addition, the result of proteomic analysis showed similar regulation patterns of carbonmetabolism (glycolysis, the reductive acetyl-CoA pathway, and methanol metabolism) at methanol condition compared with hydrogencondition. It indicated that methanol could be used to enhance the consumption of hydrogen during syngas fermentation. We proved it through profiles of autotrophic fermentation in E. limosum KIST612.At hydrogen with methanol condition, it was confirmed that hydrogen consumption accelerated, and more gases were converted into liquid fuel(acetate and butyrate) compared to the without methanol conditions.

Keywords : Syngas fermentation, methanol metabolism, acetogen

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M_Microbial Genomes and Metagenomics

M-1

Genomic Properties of a Novel Methane Oxidizing Strain Methylocystis sp. MJC1

Sannizabekov, and Eun Yeol Lee*

Department of Chemical Engineering, College of Engineering, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do 17104, Republic of Korea

Methylocystis sp. MJC1 is a promising biocatalytic platform for methaneconversion into value-added products. To facilitate its development as a platform strain, we present the complete genome sequence of M. sp. MJC1. We assembled,compared and functionally annotated M. sp. MJC1's genome. Comparative genomics reaffirmed M. sp. MJC1 as a separate type II methanotroph species with Methylococcus parvus beingits closest strain.Genome functional annotation has shown that M. sp. MJC1 has all major type II methanotroph biochemical pathways such as methane monooxygenase, methanol dehydrogenase, tetrahydromethanopterinpathway, serine cycle, TCA cycle and EMC pathway.Together, these findings open a possibility for future practical applications such as development of genome-scale model, or applying metabolic engineeringstrategies to M. sp. MJC1.

Keywords : Methylocystis, methanotrophs, MJC1

M-2

Analysis of Genome Sequence of Bacillus subtilis subsp. subtilis MD 32, Isolated from Kimchi

Ji Young Lee1, Gyu Sung Cho3, Charles M. A. P. Franz3, and Dae Ook Kang2* 1Division of Life Science and Research Institute of Life Science, Gyeongsang National University, Jinju, Gyeongnam 52828, Republic of Korea 2Department of Bio Health Science, Changwon National University, Changwon, Gyeongnam51140, Republic of Korea 3Max Rubner-Institut, Federal Research Institute for Nutrition and Food, Department of Microbiology and Biotechnology, Kiel, Germany

Diverse bacterial strains were isolated from fermented foods and testedfor antibacterial activity. One Strain (MD 32) producing bacteriocin wasselected for further study and identified as Bacillus subtilis by 16S rRNAgene analysis. The genome was sequenced using an Illumina MiSeq platform, and the genome size was 4,238,856 bp with a GC content of43.41 mol%. The genome encoded 4,396 proteins, with 45 tRNAs, 6 rRNAs, and 5 noncoding RNAs. Orthologous average nucleotide identity (ANI) was calculated with closely related type strains using theorthologous ANI tool(OAT). The ANI values for the MD 32 strain withrespect to Bacillus type strains B. subtilis subsp. subtilis KCTC 1028T,Bacillus licheniformis ATCC 14580T, and Bacillus atrophaeus NRRL NRS-213T were 98.11%, 72.46%, and 79.32%, respectively. This indicated that strain MD 32 could be identified as a B. subtilis subsp. subtilis strain. No plasmid-related sequences and none of the typical Bacillus toxin genes weredetected. Two antibiotic resistance genes [mph(k) and aadk] were present. A sbo-alb (sboA and albA to albG) operon involved in bacteriocin production could be identified, which contained the subtilosin A gene (sboA) and the subtilosin immunity gene(albB). The bacteriocin gene-positive B. subtilis subsp. subtilis MD 32could potentially be of use in the food and feed industries for applicationas a natural biopreservative.

Keywords : Bacillus subtilis subsp. subtilis MD 32, bacteriocin, genomesequencing

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M-3

Genome Sequence of Weissella koreensis Strain HJ, a Probiotic Bacterium Isolated from Kimchi

Eiseul Kim1, Hae Choon Chang2, and Hae-Yeong Kim1* 1Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea 2Department of Food and Nutrition, Chosun University, Gwangju 61452, Republic of Korea

In this study, we have performed the draft genome sequencing and genome analysis of W. koreensis strain HJ. This strain was isolated fromkimchi and showed 98.8% identity with the 16S ribosomal RNA geneof W. koreensis strain S-5623. Genome assembly of the reads produced15 contigs (9,113 to 396,269 bp, N50 length 347,703 bp). W. koreensisHJ contains a 1,427,571 bp genome size, with a GC content of 35.5%. The draft genome of W. koreensis HJ has 1,418 protein-coding genes, 55 RNAs, and 179 subsystems. The genome of W. koreensis HJ containedprobiotic-related genes, such as the F0F1 ATP syntheses genes related to acid tolerance, choloyglycine hydrolase genes related to bile tolerance, and NADPH-quinone oxidoreductase genes related to oxidative stress resistance. The comparative genomic analysis of five W. koreensis strains yielded a pan-genome of 1,620 genes, core-genome of 1,140 genes, accessory-genome of 276 genes, and unique-genome of 204 genes. Of these genes, there are 79 unique genes that had only W. koreensis HJ, including NAD(P)H-binding protein, low-temperature requirement protein A, LicD family protein, glycosyltransferase, adhesin, and hypothetical proteins. Our results suggest that W. koreensisHJ can be considered a novel probiotic candidate strain.

Keywords : Weissella koreensis, genome sequencing, probiotics

M-4

Genomic Comparison Revealed Region-Enriched Genes of African Swine Fever Virus (ASFV)

Seungyeon Choi, Jongbin Park, and Eun Bae Kim* Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Republic of Korea

Since the introduction of African swine fever virus (ASFV) into Georgiain 2007 from Africa, the disease has spread from European continent toAsia, which is causing severe economic losses in the swine industry dueto the lack of knowledge of any good vaccines. Several studies alreadycompared its genome, but they did not consider regional characteristics.Here, we analyzed 89 ASFV genomes originated from Africa, America,Asia and Europe using comparative genomics to identify the regionalcharacteristics. We downloaded the genomes from NCBI GenBank andclassified them based on country and continent. From the genome comparison, we found 605 orthologs from 89 genomes, which includes54 core and 551 accessory genes. Of the accessory genes, a total of 15 genes were identified to be region- enriched genes (1, 2, and 12 genesfrom America, Europe, and Asia, respectively). However, further studiesare required to find biological and geographical meaning of such region-enriched genes. This work will present an improved genetic basisto develop new strategies to reduce the risk of ASFV transmission to domestic pigs. Keywords : ASFV, genome

M-5

Analysis of Microbial Community on Dotted Gizzard Shad (Konosirus punctatus) in South Korea

Jun Hyeok Kwon, Su Jin Yum, and Hee-Gon Jeong* Department of Food Science and Technology, Chungnam National University, Dajeon 34134, Republic of Korea

Dotted gizzard shad, known as Jeon-eo in South Korea, is one of the popular seafood in summer. To characterize the bacterial communitiesincluding potential pathogens, total DNA on wild-caught dotted gizzardshads were isolated and analyzed based on 16S rRNA genes sequencing.The microbiota were composed mainly by Proteobacteria(84.62%), Firmicutes(8.15%), and Bacteroidetes(7.12%) phyla. At the family level Vibrionaceae(31.50%), Pseudoalteromonadaceae(27.35%) and Shewanellaceae(9.18%) were dominant. Vibrio(28.35%), Pseudoalteromonas(26.88%),Shewanella(9.18%) and Psychrobacter(6.85%) were dominant genus. Among them, the relative abundance of Vibrio genus were high. Therefore, common food-borne pathogens belonging to Vibrio genus such as V. cholerae, V. parahaemolyticus and V. vulnificus detected andquantified by qRT-PCR. Only V. parahaemolyticus was detected in 22 samples out of 34 samples (64.71%, 5.26Ⅹ102 mean CFU/g). Althoughfurther studies are needed, this study can be used to improve the food safety of seafoods.

Keywords : Dotted gizzard shad, microbiota, food-borne illness

M-6

Draft Genome and a Novel Plasmid Analysis of Enterococcus hirae YS00198 from Pig Feces

Jeong Ho Yoo, and Eun Bae Kim* Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea

Most Enterococcus hirae are known as pathogens that cause inflammationin the intestine of animals and humans. However, several strains havethe potential of probiotics. Here, we analyzed the draft genome and a novel plasmid of E. hirae YS00198 isolated from piglet feces. We extracted genomic DNA of E. hirae YS00198 and sequenced by MGIseq-2000. Sequenced reads were assembled by using SPAdes andobtained genome was annotated by RAST for gene prediction. We detected the virulence factors and antibiotic resistance genes by using CARD and BAGEL4. The draft genome is 2,948,865 bp (36.7% G+C contents) in length and novel one circular plasmid with 3,347 bp (32.8%G+C content). Through genome annotation, a total of 2,896 protein- coding sequences (CDS) and 41 tRNA were found. Chromosome contains Enterolysin A and three antibiotic resistance genes (tetM, ErnB,AAC(6’)-Iid). The plasmid has several restriction enzyme sites including XcmI and SpeI. We found that G+C contents of the plasmid and chromosomic CDS were significant different. Our findings suggestthe potential of the plasmid of E. hirae YS00198 for Lactic Acid Bacteriavector system.

Keywords : Enterococcus hirae, genome analysis, plasmid

[This study was supported by National Research Foundation (NRF, NRF-2019R1A2C1009406)]

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M-7

Draft Genome and a Novel Plasmid of Lactiplantibacillus plantarum GD00040 from Korean Cucumber Kimchi

Gi Bae Choi, and Eun Bae Kim*

Department of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon 24341, Republic of Korea

Lactiplantibacillus plantarum is generally recognized as safe that are generally used in the food industry. Lpb. plantarum strains were commonly found in the vegetables, fruits, and fermented foods. In thisstudy, we sequenced and analyzed the genome of Lpb. plantarumGD00040 isolated from Korean cucumber kimchi. Genomic DNA wasextracted and prepared DNA Libraries were sequenced using MGIseq system. Obtained sequencing reads were filtered by cut adaptor and assembled by SPAdes 3.15.1. To predict the presence of functional genes, virulence factors (VF), and antibiotic resistance genes (ARGs), we compared using various annotation database including RAST, CARD, and BAGEL4. The genome was assembled into 122 contigs (contig length ≥ 500 bp) with 3,392,132 bp in length. Through the genome annotation, 54 RNA genes, and 3,399 coding sequences were predicted. Plantaricin J and pediocin resistance genes were found. In addition, wefound and analyzed a circular plasmid in Lpb. plantarum GD00040. It has several restriction enzyme sites (SacI, NcoI, etc) and is 3,963bp in length. These results will be useful when we make a new plasmid vectorsystem for lactic acid bacteria.

Keywords : Lactiplantibacillus plantarum, genome analysis, plasmid

[This study was supported by the National Research Foundation of Korea(NRF-2019R1A2C1009406)]

M-8

Characterization of the Kale (Brassica oleracea L.) Microbiota and Shifts in Microbial Composition during Different Storage Conditions

Su Jin Yum1, Jun Hyeok Kwon1, Seung Min Kim2, and Hee-Gon Jeong1*

1Department of Food Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea 2Department of Human Ecology, Korea National Open University, Seoul 03087, Republic of Korea

Kale (Brassica oleracea L.) is one of the most popular vegetables andcan be consumed immediately without further treatment. The microbiota of kale is a potential source of food-borne pathogens, but the presence of pathogens on kale has not evaluated. We analyzed the microbiota ofkale in 2 sites (gwangju and yeoju) at August using 16S rRNA gene amplicon sequencing. The overall microbiota structure was significantlydifferent between 2 sites. Observed OTUs and simpson’s indices were significantly different between the 2 sites. Firmicutes (59.52±26.21%)and Proteobacteria (39.80±26.51%) were predominant phyla. The predominant orders were Bacillales (57.60±27.61%), Enterobacteriales (31.51±29.77%), and Pseudomonadales (7.08±7.95%). At the genus level, Bacillus (15.05±20.75%), Acinetobacter (5.05±7.48%), Pantoea(4.93±7.69%), Escherichia (4.09±14.36%), and Pseudomonas (1.95±2.26%)were found dominantly. The pathogenic species such as B. cereus, A. lwoffii, P. agglomerans, E. coli (EAEC, EHEC, ETEC, EPEC), and P.aeruginosa were detected and quantified by qRT-PCR. We also analyzedthe changes of endogenous microbiota in kale during storage at 4℃ and30℃. The proportion of K. pneumoniae were the highest at 30℃ afterwashing (8.50 to 59.95%). This study can contribute toward a better understanding of the kale microbiota and provides insights into the roleof washing and storage conditions in the fresh vegetables.

Keywords : Microbiota, kale, food-borne pathogen

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M-9

Metagenomic Mining for Antibiotic Selectable Markers for Vector Systems

Jongbin Park, and Eun Bae Kim*

Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Republic of Korea

The wide use of antibiotics in the animal industry increases the prevalence of antibiotic resistance genes (ARGs). Exploration and application techniques are needed to prevent the spread of ARGs. Recently, several studies reported the profile of ARGs in the animal farmusing the environmental metagenome sequences. In this study, we explored chloramphenicol resistance genes (CRGs) into the chicken farm environments and evaluated them for application in vector engineering. We collected feces and litter samples from various domesticchicken farms and extracted DNA for sequencing. Metagenome sequences were aligned to reference chloramphenicol resistance genes from CARD (Comprehensive Antibiotic Resistance Database) database. CRGs detected from the metagenomes were amplified usinggene-specific primers and sequenced. We constructed vectors, and theywere transformed into E. coli DH5α and tested on the chloramphenicolcontained LB agar for resistance. Novel variant CRGs were aligned withcmx, pp-flo, and catI (> 90% sequence coverage). Obtained our sequences represented 96% of sequence identity when compared with references. We confirmed that our constructs have phenotypic resistanceto chloramphenicol. Our findings provide new insights into the metagenomic approaches for selectable markers in plasmid vector systems.

Keywords : Metagenome, vector, selectable marker

[This study was supported by National Research Foundation (NRF, NRF-2019R1A2C1009406)]

M-10

Comparison of Microbial Community Profiling on Traditional Fermented Soybean Products (Doenjang, Cheonggukjang) Using Next Generation Sequencing

MyeongSeon Ryu, Gwangsu Ha, JinWon Kim, Sua Im, Su-Jin Shin, Hee-Jong Yang, and Do-Youn Jeong* Microbial Institute for Fermentation Industry (MIFI), 61-27, Minsokmaeul-gil,Sunchang-eup, Sunchang-gun, Jeonbuk 56048, Republic of Korea

In order to identify and characterize the microbial communities in twotypes of Korean fermented paste made from whole soybeans, Doenjangand Cheonggukjang, microbial communities were analyzed using nextgeneration sequencing. According to the taxonomic results, the overallmicrobial composition was then compared between the groups. Firmicuteswas the most common phylum in both groups, representing 94.48% and99.69% in Doenjang and Cheonggukjang groups, respectively. Proteobacteriawas the second dominant phylum, accounting for 4.34% and 0.235% inthe both groups, respectively. The most dominant genus of the microbiotaof the both Doenjang and Cheonggukjang was Bacillus. Bacillus and Tetragenococcus were significantly more abundant in Doenjang than inCheonggukjang, where as Lactic acid bacteria such as Enterococcus, Lactobacillus, Leuconostoc and Weissella is significantly more abundant in Cheonggukjang than in Doenjang. Especially, pathogens including Bacillus cereus, Escherichia coli and Staphylococcus aureusthat cause food poisoning were more abundant in Doenjang than in Cheonggukjang.

Keywords : Cheonggukjang, Doenjang, Next Generagtion Sequencing

[This work was supported by “Traditional food safety monitoring program” under the Ministry of Agriculture, Food and Rural Affairs andpartly Korea Agro-Fisheries and Food trade corporation in 2021]

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M-11

Distinct Shifts in Microbial Community Composition by Rainfall during Microcystis Bloom in Nakdong River, Korea

Mingyeong Kang1,2, Ve Van Le1,2, So-Ra Ko1, Sang-Ah Lee1,2, and Chi-Yong Ahn1,2* 1Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea 2Department of Environmental Biotechnology, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea

The various environmental factors could play a role in the formation and collapse of Microcystis bloom. Among them, rainfall has important influences not only on Microcystis but also on the microbial community that interacts with Microcystis. To elucidate the distinct changes in the diversity and the composition of the microbial community by rainfall, we employed 16S rRNA high-throughput sequencing from June to October in 2019 and 2020 at three sample sites in the Nakdong River, Korea. Throughout the investigation period, Microcystis blooms have different phases of Pre-Bloom, Bloom_1, Rainy Season, Bloom_2, andPost-Bloom at all sampling sites. The Microcystis abundance was influenced by rainfall and phase-specific transitions of the microbial communities accompanied at both phylum and class levels. In general,bacteria diversity and the relative abundance of Gammaproteobacteriaincreased in the Post-Bloom period with the collapse of Microcystis bloom. However, such a transition pattern appeared earlier with heavy rainfall during the Rainy Season. Through network analysis, the microbial composition of the rainfall-associated microbial cluster affected other modules associated with Microcystis. In conclusion, rainfall significantly influenced the bacteria communities associated with Microcystis blooms and the understanding of microbial interactionsduring Microcystis blooms and rainfall could be improved by high-throughput sequencing.

Keywords : Rainfall, Microcystis bloom, Nakdong River

M-12

Complete Genome Sequence of Halo-Tolerant Thermophile, Bacillus licheniformis CP6, Showing Degradation of Plant-Derived Protein

Yong-Jik Lee1, Gae-Won Nam1, JiYeon Woo1, YunJi Jo1, JiMin Lim1, JinSeong Lee1, Mi-Hwa Park2, YuJeong Yeom3, Seok-Cheol Cho4, and Sang-Jae Lee3* 1Department of Bio-Cosmetics, Seowon University, Cheong-Ju 28674, Republic of Korea 2Department of Food and Nutrition, College of Medical and Life Science, Silla University, Busan 46958, Republic of Korea 3Major in Food Biotechnology and Research Center for Extremophiles & Marine Microbiology, Silla University, Busan 46958, Republic of Korea 4Departmentof Food Science and Engineering, Seowon University, Cheong-Ju 28674, Republic of Korea

A Bacillus licheniformis CP6 strain was isolated from seawater at Tongyeong in Korea for finding alkaline protease-producing thermophiles. This strain can grow at up to 60oC and can grow a diverse concentration of NaCl (0% to 20%) and pH (7 to 9) together with the production of protease. The proteolytic ability of this strain was confirmed in culture experiments with plant-derived proteins. Therefore, to search for genes involved in proteolysis, we conducted a genomic analysis of this strain. The complete genome comprises 4,313,909 bp with a G+C content of 46.0%, 4,404 coding genes, and 105RNA genes, respectively. Strain CP6 was identified as B. licheniformis using genome sequencing service offered in ChunLab and the RAST analysis revealed it harbored 23 protease-related protein degradation meaning that it can be used as a source of protease production for the eco-friendly industrial application.

Keywords : Bacillus licheniformis, halo-tolerant, thermophile

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M-13

Prevalence, Diversity and UV-light Inducibility Potentials of Bacillus subtilis Prophages, and Their Possible Roles in Host Properties

Haftom Abraha1,5, Kwang-Pyo Kim1,4*, Donghyun Shin4, Youbin Choi1,4,Woobin Hyun2, Jae Won Lee1, Hai Seong Kang3, So Min Seo1, Desta BerheSbhatu5, Min Kyeong Cho1, Ga Yeong Kim1, and Joo Young Cho1 1Department of Food Science and Technology, Jeonbuk National University, Jeonju 54896, Republic of Korea 2Indulgence Food Research Laboratory, Namyang Dairy Products Co. Ltd, Sejong 30055, Republic of Korea 3Food Safety Evaluation Department (Food Microbiology Division), National Institute of Food and Drug Safety Evaluation, Cheongju 28159, Republic of Korea 4Department of Agricultural Convergence Technology, Jeonbuk National University, Jeonju 54896, Republic of Korea 5Department of Biological and Chemical Engineering, Mekelle Institute of Technology, Mekelle University, PO Box 1632, Mekelle, Ethiopia

Bacillus subtilis is an important bacterial species for its various industrial, medicinal and agricultural applications. Prophages are known to play vital roles in host properties. Nevertheless, studies on prophages and temperate phages of B. subtilis are relatively limited. Inthe present study, in-silico analysis of prophages is carried out in sequenced B. subtilis strains to investigate their prevalence, diversity, features, and possible roles. Besides, UV-light prophage inducibility potential is investigated in large collection of B. subtilis. In-silico analysis of 190 genomes of B. subtilis strains revealed that 77.89% of them contained intact prophages (242 in total) that exist as integrated and/or plasmid forms. Phylogenomic analysis revealed the rich diversityof the prophages distributed in 14 clusters and 7 singletons. The analysisof putative prophage proteins indicated prophage involvement in encoding proteins linked to immunity, bacteriocin production, sporulation, and resistance of the B. subtilis host that can enhance its adaptability to diverse environments. Induction study in 91 B. subtilis strains demonstrated that UV-light treatment was instrumental in producing infective phages in 18.68% of them with wide range of hostspecificity. The high prevalence and inducibility potentials of prophagesobserved in this study imply that prophages may play vital roles in the B. subtilis host.

Keywords : Bacillus subtilis, prophages, prophage induction

M-14

Characterization of a Cryptic Plasmid, pTH32, from Tetragenococcus halophillus 32

MinJae Kim1, and Jeong Hwan Kim1,2*

1Division of Applied Life Science (BK21 Four), Graduate school, 2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Republic of Korea

Tetragenococcus species are halophilic lactic acid bacteria, which growwell in the presence of NaCl up to 20%. We isolated tetragenococci fromjeotgals and inoculated into tryptic soy broth (TSB) containing 5% NaCland cultivated for 3 days. Then plasmid DNAs were prepared and agarosegel electrophoresis was done to examine plasmid profiles of tetragenococci. One strain, T. halophilus 32, possessed a small-sized plasmid (pTH32). When pTH32 was digested by restriction enzymes EcoRI and HindIII, two fragments of 1.1 and 2.1 kb in size were produced.Each fragment was extracted from an agarose gel and ligated with E. colicloning vector, pUC19. Currently, DNA sequencing on these 2 insertsare under progress now. DNA sequence analysis will reveal several characteristics of pTH32 such as G+C content, open reading frames (ORFs). Based on the characteristics of the sequence that will be revealed, pTH32 or part of it will be used for the construction of a Tetragenococcus - E. coli shuttle vectors. Such vectors will help for thestudies on Tetragenococcus species and also for the improvements of tetragenococci through genetic engineering.

Keywords : Tetragenococcus, plasmid, sequence

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M-15

Antibiotic Resistomes and Occurrence of Multidrug Resistant Bacteria during Wastewater Treatment Processes

Mingyeong Kang, Jihye Yang, and Woojun Park* Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Republic of Korea

Wastewater treatment plants (WWTP) receiving many contaminant resources might be a potential reservoir of antibiotic resistance genes (ARGs) and occurrence of multidrug resistant (MDR) bacteria through horizontal gene transfer (HGT). Environmental samples from five different WWTP processes during summer and winter seasons were collected to monitor ARGs resistomes and culturable MDR bacteria under eight antibiotics including gentamicin and azithromycin. Nanopore-based ARG abundance and bacterial community analyses suggested the possibility of MDR bacterial generation by accumulationof the ARGs. Activated and mixed sludges exhibited strong tendency to have lower bacterial diversities as well as ARGs due to selective forces favoring special microorganisms during aeration processes. Escherichia coli strains being enriched in WWTP (up to 71%) were dominant in all samples and high abundance of Cloacamonas acidaminovorans was detected only in the anaerobic digested sludge (60-79%). Two ARG types [sulfonamide resistance genes (sul1) and aminoglycoside resistance genes (aadA13, aadA2, and ant(3”)-Ia)] were prevalent throughout all processes. Total counts of culturable MDRbacteria such as Niabella, Enterococcus, and Chryseobacterium speciesincreased gradually during aerobic WWTP processes. Further genomicanalyses of all MDR bacteria isolated from our samples will give us insight into ARG-types determination, presence of MGEs, locations ofgenomic islands, and the origins of ARGs.

Keywords : Metagenome, sludge, Nanopore

[This work was supported by grants from the National Research Foundation of Korea (No. NRF-2020M3A9H5104237)]

M-16

Metagenomic and Genomic Approaches for Antibiotic Resistome in Zoo Animal Manures

Jihyeon Min, Sohyeon Yun, Pureun Kim, and Woojun Park*

Laboratory of Molecular Environmental Microbiology, Department of Environmental Sciences and Ecological Engineering, Korea University, Seoul 02841, Republic of Korea

Animal manures are considered as a reservoir of antibiotic resistance genes (ARGs), which might lead to occurrence of multidrug resistant (MDR) bacteria. Animal fecal samples collected from 11 herbivorous animals including sable antelope (SA), long-tailed goral (LTG), and common eland (CE) at a public zoo in the summer and winter were examined for the presence of ARGs using metagenomics and culturableMDR bacteria along with whole genome sequencing approaches. Eightantibiotics including meropenem and azithromycin were used to isolate culturable MDR strains, showing that manures from three animals (SA,LTG, and CE) have 104-fold higher culturable MDR bacteria such as Chryseobacterium, Sphingobacerium and Chitinophaga species, whereas a few MDR bacteria from water buffalo, rhinoceros, and elephant were cultivated against all tested antibiotics. The high ThreeMDR-bacteria rich-samples along with composite samples having all mixed 11 manures were further analyzed using nanopore-based metagenomic analyses during summer and winter seasons. ARGs including lnu(C), tet(Q) and mef(A) were common in all tested samples.The SA and CE samples having tulathromycin (a macrolide antibiotic)treatment appeared to harbor high copy of mef(A) genes. Beta-lactamsresistance genes (cfxA, cfxA3, cfxA4 and cfxA5) were prevalent in the feces of LTG and CE, but not in SA’s manures. Our further genomic analyses will provide insight into dissemination of ARGs from animal and generation of MDR bacteria in zoo animal manures.

Keywords : Herbivores, antibiotic, metagenome

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M-17

Metabolomics Analysis of Piglets Fed Contaminated with Deoxynivalenol Using Liquid Chromatography Mass Spectrometry

Jin Young Jeong, Minji Kim, Sang Yun Ji, Byeonghyeon Kim, Youl ChangBaek, Hwa Seol Park, and Hyunjung Jung Animal Nutrition & Physiology Team, National Institute of Animal Science, Wanju 55365, Republic of Korea

Deoxynivalenol (DON), a Fusarium species, is an important mycotoxinin foods and feedstuffs. The purpose of this study was to assess the metabolic profiles of piglets (Landrace x Yorkshire) fed with purified DON liver and jejunum tissue using liquid chromatography mass spectrometry. DON was mixed with the diet using commercial toxin at1, 3, 10 mg/kg in feed for 28 d. Results were analyzed by multivariateanalysis to elucidate the potential compounds. The DON-treated groupsobserved the discriminating metabolites in the jejunum tissue, but no differences in the liver. The profiling revealed the potential metabolitessuch as glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, andvaline, leucine and isoleucine biosynthesis. Aspartic acid, glucose, serine, and glycine were identified high sensitivity as candidate biomarkers via variable importance in the projection plot in piglets fed with DON (FDR < 0.05, p < 0.05). Additionally, Masson's trichrome stains in liver and jejunum tissue were used to visualize of collagen fibers. In conclusion, these findings suggest that the potential biomarker compounds can explain the physiological changes for piglets fed contaminated with DON.

Keywords : Piglet, deoxynivalenol, metabolite

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N-1

Metabolic Modeling Approach to Elucidate Role of Opportunistic Pathogen Candida albicans in Dysbiotic Gut Microbiome of Crohn’s Disease

Yi Qing Lee1, Yoon-Mi Choi1, Dong Seok Kim1, Su Kyung Kim1, MinoukLee1, Meiyappan Lakshmanan2, Mi Jin Kim3, Yon Ho Choe3, and Dong-Yup Lee1* 1School of Chemical Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 16419, Republic of Korea 2Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01, 138668, Singapore 3Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06355, Republic of Korea

Candida albicans is a dimorphic, opportunistic fungal pathogen that exists as a normal part of the human gastrointestinal microbiota. In healthy individuals, C. albicans exists as a commensal, but it can alsoundergo yeast-to-hyphae transition which contributes to its colonizationand pathogenicity, especially in immunocompromised individuals. Despite recent findings that suggests colonization of C. albicans may lead to dysbiosis in the gut and cause various inflammatory pathologiesincluding Crohn's disease (CD), the mechanism of C. albicansmorphogenesis and their subsequent interactions with the gut microbiome still remains unclear. Hence, it is important to elucidate suchmechanisms to be targeted as a cure for CD. In this study, we built a genome scale metabolic model (GEM) of C. albicans, iCal986, consisting of 986 genes, 2150 reactions and 1771 metabolites. iCal986 was manual curated and validated with culture, gene essentiality, carbonand nitrogen source data. Next, using the Gene Inactivity Moderated by Metabolism and Expression (GIMME) algorithm, we built two condition specific GEMs for both yeast and hyphal states by integratingrelevant transcriptomic data to iCal986. Subsequently, a reaction essentiality analysis was used to identify anti-virulent targets specific to each morphological state. In addition, we simulated pairwise interactions between C. albicans and other gut microbes present in CDsamples. As a result, we were able to identify anti-virulent targets to inhibit yeast-to-hyphae transition, and elucidate the mechanism of gut dysbiosis by characterizing metabolic interactions between C. albicansand the gut microbiome, while identifying potential gut microbes that can suppress its virulence.

Keywords : Candida albicans, Crohn's disease, genome scale metabolic model

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the MSIT (No. 2020R1A2C2007192)]

N-2

Description of Anaerostipes faecalis sp. nov., a New Segmented Filamentous Bacterium Isolated from Swine Faeces

Ji Young Choi1, Seung-Hyeon Choi1, Jam-Eon Park1, Ji-Sun Kim1, Se WonKang1, Jiyoung Lee1, Mi-Kyung Lee1, Jung-Sook Lee1, Ju Huck Lee1, Yeongjin Hong2* and Seung-Hwan Park1*

1Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea 2Department of Microbiology, Chonnam National University Medical School, Gwangju 61186, Republic of Korea

A novel, strictly anaerobic, gram-negative, segmented filamentous bacterium (SFB), strain AGMB03513T, was isolated from the faeces ofa 5-month-old pig. Comparative analysis of 16S rRNA gene sequencesindicated that strain AGMB03513T forms a lineage within the genus Anaerostipes and is most closely related to A. butyraticus DSM 22094T

(= KCTC 15125T, 95.8%), A. hadrus DSM 3319T (= KCTC 15606T, 95.5%), A. caccae DSM 14662T (= KCTC 15019T, 94.0%), and A. rhamnosivorans DSM 26241T (= KCTC 15316T, 93.4%). Phylogeneticanalysis based on the 16S rRNA gene and whole genome sequencing analysis revealed that its closest relatives are members of the family Lachnospiraceae and that the closest related is A. butyraticus. Strain AGMB03513T grows at temperatures of between 30 and 45°C within a pH range of 7.0 to 9.0, and in medium containing up to 1.5% NaCl. Cells were found to utilize d-glucose, d-mannitol, d-lactose, d-saccharose,d-maltose, d-xylose, l-arabinose, d-mannose, and d-sorbitol, and acetatewas identified as the major end product of metabolism. The DNA G+Ccontent of the strain is 37.0 mol%. The major components of cellular fattyacids were C12:0, C16:0, and C18:0. On the basis of phenotypic, phylogenetic,biochemical, chemotaxonomic, and genomic characteristics, we considerit reasonable to assign novel species status to strain AGMB03513T, forwhich we propose the name Anaerostipes faecalis sp. nov. The type strainAGMB03513T (=KCTC 25020T=NBRC 114896T).

Keywords : Anaerostipes faecalis, segmented filamentous bacterium, swine faeces

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N-3

A Behavioral Changes Following Oral Administration of Soy Enzyme Treatment Material to Drosophila, an Animal Model of Parkinson's Disease

Joo-Yeon Jang1, Dae-Ho Kim1, Jong-Hoon Kim1, Jiun Sang1, Kyung-Ho Han2, Mi-Sun Kwak1, Youngseok Lee1, and Moon-Hee Sung1,2* Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea 2Kookminbio Corporation, Seoul 02826, Republic of Korea

Soymilk is a liquid soybean processed product, also known as soybeanmilk, and is a water extract of soybeans from which insoluble components have been removed. Soymilk contains proteins that aid inthe metabolism of nutrients to make it easier to digest. Enzymatic treatment was performed on the basis of this soymilk, and then severalexperimental groups were made to investigate whether they could havea positive behavioral effect. Climbing assay to determine motility and Survival assay to determine viability were conducted, and the Drosophila group used in the experiment was w1118 type known as wild Drosophila and DJ-1βex54 type known as Parkinson-model. Parkinson'sdisease is a disease that shows symptoms of ataxia. That is, the DJ-1βex54

type Drosophila, a Parkinson's model Drosophila, exhibits features suchas muscle structural deformation or dyskinesia as aging occurs, and theclimbing rate and survival rate, which are values representing motility,decrease compared to general Drosophila. When oxidative stress appears, Parkinson's model Drosophila have lower motility and survivalrates compared to wild-type fruit flies, and H2O2 was added to each sample in a 1% ratio to artificially provide such an environment. Climbing rates were observed for 3 days and their survival rates were observed for about 2 weeks. This research was supported by Korea Environmental Industry and Technology Institute(KEITI) grant funded by the Ministry of Environment of Korea.

Keywords : Drosophila, climbing rate, survival rate

N-4

Bacteriophages Modulating Gut Proteobacteria Inducing Inflammatory Bowel Disease

Yoonjung Hwang1,2, and Heejoon Myung1,2,3* 1Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yong-In 17035, Republic of Korea 2Bacteriophage Bank of Korea, Hankuk University of Foreign Studies, Yong-In 170365, Republic of Korea 3LyseNTech, Yong-In, Republic of Korea

Gastrointestinal tract microbiota plays an important role in the regulationand pathogenesis of many gastrointestinal diseases. It is known that Inflammatory Bowel Disease (IBD) is caused by imbalance of the intestinal microbial community, particularly the abundance of Proteobacteria as an indicator. In this study, IBD was induced in mice that had been orally administered with a cocktail of phages targeting Proteobacteria, and the changes in intestinal microbiome and disease activity index (DAI) were observed. The group fed with the phage cocktail showed a similar DAI to the control group. However, analysisof intestinal microbial community through 16S rRNA sequencing revealed that Proteobacteria was decreased in the phage-treated group. Surprisingly, a species level analysis showed that the decreased Proteobacteria were not the hosts of the bacteriophages in the cocktail.Further research is needed to interpret this phenomenon.

Keywords : Bacteriophage, IBD, microbiome

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N-5

Explainable Artificial Intelligence Reveals Shifts in Human Gut Microbiome Composition Linked to Phenotypic Distinction of Healthy Lifestyle Improvement

Pham QuangHuy1, Trang Nguyen2, and Jae-Ho Shin1* 1School of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea 2Minerva Schools at Keck Graduate Institute, San Francisco, California, USA

Alterations in the human gut microbiome have been noticed in a varietyof conditions such as inflammatory bowel disease, obesity, diabetes, autism and much remains to be well-educated about the links between the microbiome and human health. The fusion of artificial intelligencewith rich microbiome datasets can offer an improved understanding ofthe microbiome’s role in human health. To gain actionable insights it isessential to consider both the predictive power and the transparency ofthe models by providing explanations for the predictions. We combinedthe collection dataset in published study of human gut microbiome samples from navy trainees (n = 66) and healthy people (n = 38) withthe application of an explainable artificial intelligence (EAI) approach that provides accurate predictions of phenotypes with explanations of healthy lifestyle improvement between two cohorts. The limitation of previous published dataset study is that it is essential to consider both the predictive power of the models and the transparency of the recommendations by providing an explanation for the predictions instead of investigating one-by-one single genus or species. The explanations are showed in terms of shifts in the relative abundance ofkey microbes that drive the predictions. The changes in microbial composition linked to diet can accelerate the development of personalized indicator for healthy gut microbiome. This suggests that easily accessible microbiome samples could be used to investigate health‑related phenotypes, offering potential for non‑invasive diagnosisand condition monitoring. Our machine learning approach sets the stagefor novel effort focused on understanding the complex relationships between microbial communities and phenotypes. Our explainable model can be applied to predict any status from microbiome samples andhas the potential to accelerate the development of microbiome‑based personalized therapeutics and non‑invasive diagnostics.

Keywords : Artificial intelligence, human gut microbiome, phenotypicdistinction

N-6

Study of Astaxanthin Extraction Method for Vegan Seafood Development : Optimal Culture Conditions and Efficient Extraction Method of Xanthophyllomyces dendrorhous

Youkyeonh Lee, and Jeongeun Hyeon*

Department of Food Science and Biotechnology, Sungshin Women's University, Seoul 01133, Republic of Korea

In the food industry, interest in vegan foods is continually growing. Asof 2018, the global meat substitute market size was $4.4 billion, recordingan annual average growth rate of 6.3% until 23, and stable growth is expected. However, most of research has been developed as a substitute for livestock products. This study designed the optimal culture conditions of Xanthophyllomyces dendrorhous to harvest natural astaxanthin to be used in the development of vegan seafood, and studieda method of efficiently extracting astaxanthin from X. dendrorhous. Inthe research of optimal culture conditions, a design is proposed in whichshaking culture is performed for at least 4 days to maximum 9 days by irradiating white light. For mass culture, the optimal culture period should be adjusted according to the Scale Up level. In the research of efficient extraction method of astaxanthin, the enzyme reaction methodshowing 0.5x10-4mg/L is suggested as a more suitable method than theSoy Oil stirring method showing the concentration of 0.3x10-5mg/L. It is expected that the development of functional raw materials that can beapplied to a wider range of seafood products will be possible throughresearch on the basis of vegan seafood using microorganisms.

Keywords : Astaxanthin, enzyme reaction method, Xanthophyllomyces dendrorhous

N-7

Monitoring of Anthropogenic Impact on Antarctic Environment

JinJu Kim1, Dockyu Kim2, Sanghee kim2, and Woo Jun Sul1* 1Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 2Division of Polar Life Sciences, Korea Polar Research Institute, Yeonsu-gu 21990, Incheon, Republic of Korea

Antarctica is considered the last pristine continent. However, as humanactivity increases in Antarctica, microbes and environmental hazardousfactors may enter the Antarctic environment. Here, we analyzed the dissemination and accumulation of antibiotic resistance genes from thewastewater of the Antarctic Sejong Station to the Antarctic ecosystem by anthropogenic activities through metagenomic analysis of wastewater,seawater, control water, algae, limpet, and penguin intestine samples. The characteristics of bacterial community and antibiotic resistance genes significantly different according to the environment. The most dominant resistance genes were multi-drug resistance, peptide antibiotics,trimethoprim, MLSB, and aminoglycoside resistance genes. 5 plasmidcontigs with the vgaC gene, an MLSB resistance gene, were identifiedin 5 different penguin intestinal samples. 5 plasmid contigs with the vgaCgene are considered identical plasmids because they have more than 95%identity. Hence, this may suggest the possibility that one plasmid will propagate to another penguin intestine. Our results suggest that increasedhuman activity may have contributed to increased antibiotic resistanceand mobility in the Antarctic ecosystem.

Keywords : Antibiotic resistance genes, antarctic ecosystem, metagenome

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N-8

The Effects of Ethyl Alcohol on Fecal Bacterial Structure

Minjeong Kim, and Hakdong Shin*

Department of Food Science and Biotechnology, Sejong University, Seoul 05006, Republic of Korea

The ethyl alcohol has a bactericidal effect and is therefore used for sterilization purposes. The concentration of ethyl alcohol in alcoholic beverages is generally less than 50%, but studies on the effect of alcoholconsumption on the intestinal microbiome are still insufficient. This study aims to investigate whether ethyl alcohol affects the gut microbiome based on in vitro gut microbiome incubation model. Fecalsamples (n=19) were obtained from the healthy adults, then it is transferred to an anaerobic chamber immediately after collection. Feceswere homogenized with basal medium, then cultured with variable contents of ethyl alcohol (5%, 10%) for 24 hours. For the microbiota analysis, bacterial 16S rRNA gene amplicon sequencing was performed(V4 region, MiSeq platform). Microbial structural compositional changes were observed in ethanol-treated groups, in relation to the control group. Beta diversity using the weighted Unifrac distance between the control and both ethanol-treated groups significantly different. Compare to the control, microbial taxa at the phylum level, Firmicutes/Bacteroides ratio gradually increased with ethanol contentsand the significant decreases of genera Bacteroides abundance were observed in both ethanol-treated groups. As a result of this study, we confirmed that ethanol can affect the intestinal environment, and consistent changes follow according to the ethanol content.

Keywords : Ethyl alcohol, microbiome

N-9

The Effect of Cacao Consumption on Gut Microbiome Using in vitro Fecal Microbiome Incubation Model

Sunil Jung, and Hakdong Shin* Department of Food Science and Biotechnology, College of Life Science, Sejong University, Seoul 05006, Republic of Korea

Food consumption can affect the gut microbiome, and cacao is one of the foods that modern people frequently consume in the form of chocolateand cocoa. Cacao has many beneficial effects on healths such as reducingLDL cholesterol, cancer risk, and improving heart function. This studyaimed to identify whether cacao has beneficial effects on the gut microbiome using in vitro system. The fecal samples were collected from14 Korean subjects, inoculated in basal medium, and cultured under anaerobic conditions for 24h. For microbial analysis, the 16S rRNA gene-based sequencing was used with QIIME2 pipeline for bioinformaticanalysis. As a result, the 0.1% and 1% of cacao addition groups showed a higher microbial diversity tendency, in relation to the control group. Compared with the control group, the cacao 1% group showed an increased relative abundance of beneficial bacteria like Agathobacterand Bifidobacterium. Unlike the cacao 1% group, the cacao 0.1% groupshowed no significant difference in bacterial compositions. For the prediction of the functional profile of the microbial community, PICRUSt was used and there were metabolic differences between groups. This study suggests that the consumption of cacao can be beneficial to the gut microbiome but further study is needed to revealhow consumption of cacao affects the gut microbiome.

Keywords : Microbiome, cacao

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The Effects of Garlic and Ginger on the Profile of Gut Microbiota Based on in vitro Fecal Microbiome Incubation Model

Jina Ha, and Hakdong Shin*

Department of Food Science and Biotechnology, College of Life Science, Sejong University, Seoul 05006, Republic of Korea

Garlic is one of the food ingredients most consumed by Koreans. Also, ginger is a food ingredient that Koreans consistently consume as tea andspicery, etc. According to recent studies, both garlic and ginger have beneficial effects on human health. This study aims to identify the effectsof garlic and ginger on the bacterial structure of the gut microbiome. Wecollected the fecal samples from healthy Korean donors (n=20) for in vitro fecal microbiome incubation model. After inoculation of fecal samples, the inoculum added each ingredient of garlic and ginger was incubated for 24 hours in anaerobic conditions. Subsequently, to analyzemicrobial changes in this study, a 16S rRNA gene-based sequencing technique (V4 region, Illumina Miseq) was applied with QIIME2 pipeline. As a result of analyzing fecal cultures for each material, we confirmed that there are differences in the composition of the gut microbial community. The abundance of beneficial bacteria such as Bifidobacterium was increased in samples added garlic. In addition, thechanges in beneficial metabolic functions of both garlic and ginger-added groups were detected with predicted PICRUSt analysis results. In this study, the possibility was confirmed that garlic and gingerhave an improving effect on the gut microbial community.

Keywords : Garlic, ginger, gut microbiome

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Longitudinal Microbial Profiling Reveals the Influence of Personal Stool Frequency on the Gut Microbiome

Gwoncheol Park, and Hakdong Shin*

Department of Food Science and Biotechnology, Sejong University, Seoul 05006, Republic of Korea

Stool frequency is regarded as one of the direct factors that represent bowel function, and this indicator could be measured easily by recording.The negative correlation between stool frequency and colonic transit time (CTT) has been demonstrated, and stool frequency is expected to have a significant correlation with predicted colonic microbial metabolism and the profile of the gut microbiota. We used self-reportedstool frequency combined with 16S rRNA amplicon profiles of fecal samples obtained through six-time points to identify the relationship between stool frequency and gut microbial profiles. We observed that a decrease in stool frequency was associated with an increase in microbialdiversity and abundant predicted functions and that the microbial composition changes with stool frequency. Especially, in the group withfrequent bowel movements, the Ruminococcus genus was identified asa core microbiome and higher relative abundance. This study will provide basic information on stool frequency, a factor that characterizes the gut microbiome. To identify the influence of stool frequency more accurately, a comprehensive study based on long-term longitudinal observation to identify the lasting characteristics according to the stoolfrequency is needed.

Keywords : Stool frequency, gut microbiome

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Probiotic and Functional Characterization of Bifidobacterium pseudocatenulatum SLAM E6 Isolated from Infant Feces Using C. elegans Model

Hayoung Kim1, Daye Mun1, Woong Ji Lee1, Hye Jin Choi1, An Na Kang1,Mingeun Kang1, Daniel Lee1, Minhye Shin1, Sangdon Ryu1, Sangnam Oh2*, and Younghoon Kim1* 1Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea 2Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea

Bifidobacterium is famous for beneficial functions as probiotic agent forprevention and treatment of various gastrointestinal disorder. However,Bifidobacterium pseudocatenulatum hasn’t been studied very much asprobiotics agent thus we isolated and researched its functional characterization using C. elegans. C. elegans model is organism to haveits whole genome sequence. Because of various characteristics includingits genome feature, it is used in various research. In this study, we isolatedB. pseudocatenulatum SLAM E6 from infant feces by using anaerobicculture system and performed C. elegans lifespan assay, killing assay andadhesion assay. In lifespan assay, B. pseudocatenulatum significantly extended C. elegans lifespan compared to E. coli OP50 strain. Moreover,it could protect C. elegans from pathogenic bacteria including Staphylococcus aureus newman, E. coli O157:H7 EDL933. Finally, westudied its adhesion ability to C. elegans intestinal tract. As a result, B.pseudocatenulatum has remarkable adhesion ability similar to Lactobacillus rhamnosus GG. In this study, we confirmed B. pseudocatenulatum has outstanding capability as probiotics.

Keywords : Bifidobacterium, C. elegans, probiotics

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Multi-omics Based Characterization of Gut Microbiome in Weaning Piglets Model Supplemented with Multi-Strain Probiotics

Woongji Lee1, Minhye Shin1, Sangdon Ryu1, An Na Kang1, Daye Mun1,Hayoung Kim1, Hye Jin Choi1, Mingeun Kang1, Daniel Lee1, Sangnam Oh2*, and Younghoon Kim1*

1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea 2Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea

For pig industry, early-life microbial colonization is an important driver for the development and maturation of the gut. So, in recent years, variousstudies have been conducted on the effects of probiotics on the intestinaltract and performance of piglets. In this context, we investigated the effect of multi-strain probiotics on the growth and health of piglets, and the characteristics of gut microbiome through multi-omics analysis. Multi-strain probiotics (MSPs) were composed of 4 Lactobacillus whichwere isolated from healthy piglets fecal and evaluated probiotics properties. In the metagenomic approaches, MSPs supplement altered the piglet gut microbiota composition and especially, Lactobacillus wassignificantly increased. Similar to the metagenomic results, Lactobacilluswas dominant species in piglet gut microbiota composition in culturomicapproaches. Lastly, we analyzed the piglets fecal through LC/MS andGC/MS. As a result, supplement MSPs induced a significant regulation of metabolite in the gut of piglets. In conclusion, supplement MSPs toweaning piglets changes gut microbiome composition and regulated gut metabolite. These results provide us with new insights into the new strategy in pig industry.

Keywords : Multi-strain probiotics, gut microbiome, weaning piglets

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Characterization and Evaluation of the Probiotic Properties of the Spore-Forming Bacillus coagulans

Hye Jin Choi1, Minhye Shin1, Sangdon Ryu1, Daye Mun1, Woongji Lee1,An Na Kang1, Mingeun Kang1, Hayoung Kim1, Daniel Lee1, Sangnam Oh2*, and Younghoon Kim1* 1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea 2Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea

Bacillus coagulans is a gram-positive, spore-forming, and lactic acid-producing bacterium. It is known to form spores to survive in the host's gastrointestinal tract functioning as probiotics that could play a beneficial role in the host health. In this study, we evaluated probiotic characteristics, including acid, bile and heat tolerance as well as antimicrobial activity, of SRCM121381 and SRCM214335 isolated from human breastmilk. Both strains showed high tolerance against acidand bile, while heat tolerance was relatively low than a positive controlLactobacillus rhamnosus GG (LGG). In addition, we used FIMM (Fermentor for Intestine Microbiota Model) system to evaluate effectsof on the diversity change of intestinal microorganisms in animal intestine based on Next Generation Sequencing (NGS). The addition ofSRCM121381 and SRCM214335 resulted in increased population of Romboutsia and Turicibacter at the genus level, while the abundance ofEscherichia decreased significantly. Our results provide the potential use of as probiotics for further application in functional foods and feedadditives development.

Keywords : Probiotics, B. coagulans, spore-forming bacteria

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Enhanced Longevity and Immune Response of Caenorhabditis Elegans Supplemented with Potential Probiotic Strains Isolated from Canine Feces

An Na Kang1, Daye Mun1, Woong Ji Lee1, Hye Jin Choi1, Hayoung Kim1,Mingeun Kang1, Daniel Lee1, Minhye Shin1, Sangdon Ryu1, Sangnam Oh2*, and Younghoon Kim2* 1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea 2Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea

Aging process is an inexorable universal process among all living organisms including the companion animals. Accompanying the rapidgrowth of the pet-conomy industry, the current field of aging research has been noticing the role of probiotics enhancing household animalshealthy lifespan. Despite the continuous growth of the probiotic marketfor companion animals, the kind and coverage of probiotic microorganismis still limited. In this study, we investigated the fecal bacterial compositions of household canines and isolated lactic acid bacteria, thepotential probiotics and examined the anti-aging activities using Caenorhabditis elegans model. Total 305 lactic acid bacteria were isolated from various species of household canines under aerobic culturomic approach and identified using 16s rRNA sequencing. They were fed to aging N2 var. Bristol strain of C. elegans and 2 lactobacillusspecies and 2 enterococcus species were evaluated to be effective in enhancing longevity and immune response of the aged nematode. Moreover, they improved the elderly C. elegans development, stress response and especially the mechanism involved in mitochondrial energy metabolism. Therefore, as the demand for the extended qualitytime with the companion animals is increasing, probiotics market has been in need and the potential probiotic strains isolated from canine fecesfrom this study exhibited promising anti-aging activity through the agingC. elegans model.

Keywords : Culturomics, probiotics, anti-aging

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The Lotion Containing Enterococcus faecalis SL-5 Extract Changes the Acne Skin Microbiome

Bo-yun Choi1, Hye Sung Han2, Sun Hye Shin2, Jun Ki Hong2, Sang HyunLim3, Doo Heon Son3, Ju Bin Kim1, Kui Young Park2, and Woo Jun Sul1*

1Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 2Department of Dermatology, Chung-Ang University Hospital, Seoul 06973, Republic of Korea 3Cellbiotech, R&D Center, Gimpo-si 10003, Republic of Korea

Colonization of pilosebaceous follicles by Cutibacterium acnes is considered one of the key factors inducing acne by affecting the skin'sinflammatory response and skin microbes and innate immunity. Enterococcus faecalis SL-5, a LAB strain isolated from the human gut,is known to have significant antimicrobial activity against gram-positivebacteria, especially C. acnes. Therefore, we identified that the extract of E. faecalis SL-5 would improve the condition of acne skin patients,and we tried to research the changes in the skin microbiome accordingly.20 acne patients were offered vehicle lotion and the lotion containing E. faecalis extract. The skin surface samples and sebum samples wereperformed DNA extraction, and after sequencing using the Illumina MiSeq platform, microbiome analysis was performed through QIIME2.As a result, it was shown that the phylogenetic diversity of the skin microbiome was more clearly decreased by E. faecalis extract, suggesting that some microbes among skin microbes were removed bybacteriocin contained in E. faecalis extract, leaving only species at a closedistance in the phylogenetic tree. In addition, changes in microbial community were observed according to the improvement of acne by applying a lotion containing the E. faecalis extract.

Keywords : Enterococcus faecalis SL-5 extract, lotion, acne skin microbiome

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Expression and Characterization of Recombinant beta-N-Acetylhexosaminidase from Akkermansia muciniphila

Gi-Seob Hong1, Seung Yeon Yoo1, Joon-Young Hur1, Ji Yeong Park1, Ji-UIm1, Dong-Woo Lee3, Sang-Jae Lee1,2, and Han-Seung Lee1,2*

1Major in Food Biotechnology, Silla University, Busan 46958, Republic of Korea 2The Research Center for Extremophiles & Marine Microbiology, Silla University, Busan 46958, Republic of Korea 3Department of Biotechnology,Yonsei University, Seoul 03722, Republic of Korea

Akkermansia muciniphila is a mucin-degrading bacterium in human intestinal tract and is presumed to play an important role in human health.A. muciniphila genome have more than 110 carbohydrate-activated enzyme genes (Carbohydrate-Active enZYmes, CAZy) and fourteen genes were annotated as hexosaminidases or N-acetylhexosaminidases.Six putative hexosaminidases were cloned and expressed in Escherichiacoli BL21(DE3) and purified by Ni-NTA affinity chromatography. Amuc_1669, a member of Glycosyl hydrolase family 20 (GH20), and Amuc_2109, a member of Glycosyl hydrolase family 3 (GH3) hydrolyzedpNP-GlcNAc, which indicates that both enzymes are N-acetyl-glucosaminidases.However, Amuc_1669 hydrolyzed pNP-GalNAc but not chitobiose, while Amuc_2109 hydrolyzed chitobiose but not pNP-GalNA. In addition, both enzymes did not show activity on colloidal chitin, carboxymethylcellulose (CMC), xylan, and cellobiose. The enzyme’s optimal pH and temperature were 6.5 and 40℃.

Key words : Akkermansia muciniphila, beta-N-acetylhexosaminidase,Glycosyl hydrolase

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Screening of Intestinal Microbes Capable of Degrading Starch and Tributyrin from Porcine Feces Using In Vitro Clearing Zone Assay

Jong-Heum Park, Ki-Nam Yoon, Beom-Seok Song, and Jae-Kyung Kim*

Korea Atomic Energy Research Institute, Daejeon 340578, Republic of Korea

Intestinal microflora is an integral part of animal organisms, includinghuman. These microbes improve their host's metabolic ability and provide them with beneficial effects such as enhanced digestion/ absorption of nutrients and maintenance of immune function. We examined 100 bacterial strains isolated from porcine fecal samples fortheir amylolytic and lipolytic activities. Each strain was streaked onto blood agar and incubated in an anaerobic chamber. Cultured colonieswere then harvested for suspending in phosphate buffered saline and homogenized with a bead ruptor for 5 min to obtain strain lysates. A clearing zone assay for amylolytic and lipolytic activity was performedusing the lysates, starch and tributyrin as substrates were used for the evaluation of each activity. Our results showed that a total of 19 lysatesin 100 bacterial strain lysates were able to degrade starch, and 7 lysatesof them had stronger activities. However, a series of further evaluationsare required to clarify what type of amylolytic activity provided by theseven strains. In addition, most of strain lysates used in the test were able to degrade tributyrin, and 31 lysates of them showed strong activities. Further elucidation of their lipolytic acitivity on tributyrin is also needed.

Keywords : Intestinal microflora, strain lysate, amylolytic and lipolyticactivities

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Anti-Inflammatory Effects of Porcine Gut Microbiota GM97 on LPS Stimulated RAW 264.7 Cells

Jae-Kyung Kim1, Ki-Young Song1, Ki-Nam Yoon1,2, Beom-Seok Song1,and Jong-Heum Park1 1Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea 2Department of Food Science and Technology, Graduate School of Chonnam National University, Gwangju 61186, Republic of Korea

Gut microbiota (GM) is well known to plays an important role in healthand disease prevention of human and animals. In previous study, GM97was selected as an anti-inflammatory probiotics candidate through the screening test using 100 different GM species isolated from piglet feces.The present study was revealed that anti-inflammatory mechanisms of GM97 in RAW264.7 macrophages cells stimulated with bacterial lipopolysaccharide (LPS). GM97 did not show any cytotoxicity in RAW264.7 cells up to 50 ug/mL from CCK-8 and trypan blue dye exclusion assay. Moreover, Nitric oxide (NO) production was dramaticallyreduced by GM97 in a concentration-dependent manner. GM97 also decreased TNF-α, IL-1β and IL-6 pro-inflammatory cytokine secretionlevels and increased IL-10, which is an anti- inflammatory cytokine in LPS induced RAW264.7 cells. In addition, GM97 effectively reduced overexpression of TLR4 and NF-κB-mediated signaling pathway gens,as well as pro-inflammatory cytokine genes. Finally, the level of proteinexpression of mitogen activated protein kinases (MAPKs) inhibited bypretreatment of GM97. In conclusion, GM97 would be a good probioticcandidate through the protective effects on inflammation against GM dysbiosis.

Keywords : Porcine gut microbiota, anti-inflammation, RAW 264.7 cells

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Effect of Pediococcus Probiotic Strain for Tuberculosis Drug Development

Youjin Yoon1,2, Hoonhee seo2, Sukyung Kim2, Youngkyung Lee1,2, and HoYeon Song1,2* 1Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea 2Microbiome R&D institute, Probiotics Microbiome Convergence Center, Asan 31538, Republic of Korea

Tuberculosis is a very serious infectious disease that threatens humanityand therefore the development of drugs is necessary. Currently, tuberculosis treatment is effective in controlling the growth of Mycobacterium tuberculosis, but there is a risk of re-infection and sideeffects, taking 6-18 months to be treated. Rifampicin, isoniazid, and pyrazinamide, the most widely used anti-tuberculosis drugs now, can significantly change the composition of intestinal microflora and develop versatility and broad drug resistance. It also causes side effectsof increased bacterial burden and low diversity dysbiosis. Therefore, weintend to develop an anti-tuberculosis drug that minimizes side effects and shortens the duration of administration, unlike other anti- tuberculosis drugs, using the probiotic P. acidilactici PMC202. As a result of the CFU experiment using P. acidilactici PMC202, it was confirmed that the activity decreased at 1.56%, 078%, and based on thisexperiment, additional studies such as toxicity analysis and confirmationof mechanisms are required.

Keywords : Mycobacterium tuberculosis, isoniazid, probiotics

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Behavior and Gut Microbial Effects of Probiotics on Stressed Mice and Assessment of Synbiotic Combination with Commercial Prebiotics

Jiyoung Park1, Jaegwang Song2, Hyung Wook Kim2, and Hakdong Shin*

1Department of Food Science and Biotechnology, College of Life Science, Sejong University, Seoul 05006, Republic of Korea 2Department of Bio-integrated Science and Technology, College of Life Sciences, Sejong University, Seoul 05006, Republic of Korea

As the 'Gut-Brain axis' has been proposed, interest in the link betweenmicrobes and mental health is increasing. Probiotic strains have positiveeffects on both physical and mental health and prebiotics promote the growth of beneficial bacterial composition in the human gut environments. To select effective probiotics that reduce the changes caused by stress-induced depression, C57/BL6J mouse model was used.For each group (n=16), probiotics or PBS were ingested for 6 weeks, andstress was applied for 4 weeks. Fecal samples were collected 3 times andthe behavioral experiment was conducted. To investigate the effects ofprebiotics on the selected probiotics, in vitro fecal incubation model wasconducted using feces from 24 adults. Synbiotic combinations with 4 probiotics and 2 prebiotics were used. Bifidobacterium bifidus 193 andLactobacillus plantarum 182 were selected as probiotics through the prediction of functionality, microbial diversity, composition, and abundance. It showed a positive change in open field test, Elevated plusmaze test, and Y maze spontaneous alternation test. As a result of the change in microbial composition and diversity in in vitro fecal incubationmodel, the possibility that the effect of probiotics can be increased by combination with prebiotics was confirmed.

Keywords : Probiotics, prebiotics, gut-brain axis

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Efficacy of Lactobacillus Fermentum Isolated from the Vagina of a Healthy Woman against Carbapenem-Resistant Klebsiella Iinfections In Vivo

Hanieh tajdozian1,2, Hoonhee seo2, Sukyung Kim2, Md Abdur Rahim1,2, Saebim Lee2, and Ho-Yeon Song1,2* 1Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea 2Microbiome R&D institute, Probiotics Microbiome Convergence Center, Asan 31538, Republic of Korea

Carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiellapneumoniae carbapenemase (KPC) are increasingly reported worldwideand have become more and more resistant to nearly all antibiotics duringthe past decade. The emergence of K. pneumoniae strains with decreasedsusceptibility to carbapenems, which are used as a last resort treatmentoption, is a significant threat to hospitalized patients worldwide as K. pneumoniae infection is responsible for a high mortality rate in the elderly and immunodeficient individuals. This study used Lactobacillusfermentum as a candidate probiotic for treating CRE-related infectionsand investigated its effectiveness. We treated mice with L. fermentumoriginating from the vaginal fluid of a healthy Korean woman and evaluated the Lactobacilli’s efficacy in preventive, treatment, non-establishment,and colonization mouse model experiments. Compared to the control, pre-treatment with L. fermentum significantly reduced body weight lossin the mouse models, and all mice survived until the end of the study. The oral administration of L. fermentum after carbapenem-resistant Klebsiella(CRK) infection decreased mortality and illness severity during a 2-weekobservation period and showed that it affects other strains of CRK bacteria. Also, the number of Klebsiella bacteria was decreased to below5.5 log10 CFU/ml following oral administration of L. fermentum in thecolonization model. These findings demonstrate L. fermentum’s antibacterial activity and its potential to treat CRE infection in the future.

Keywords : CRE, L. fermentum, CRK

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Genomic Analysis of Halophilic bacterium, Lentibacillus sp. CBA3610, Derived from Human Feces

Seung Woo Ahn, Tae Woong Whon, Se Hee Lee, and Seong Woon Roh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

Lentibacillus species are gram variable aerobic bacteria that live primarily in halophilic environments. Previous reports have shown thatbacteria belonging to this species are primarily isolated from salty environments or food. We isolated a bacterial strain CBA3610, identifiedas a novel species of the genus Lentibacillus, from a human fecal sample.In this report, the whole genome sequence of Lentibacillus sp. CBA3610is presented, and genomic analyses are performed. Complete genome sequence of strain CBA3610 was obtained through PacBio RSII and Illumina HiSeq platforms. The size of genome is 4,035,571 bp and genesestimated to be 4,714 coding DNA sequences and 64 tRNA and 17 rRNAwere identified. The phylogenetic analysis confirmed that it belongs tothe genus Lentibacillus. In addition, there were genes related to antibioticresistance and virulence, and genes predicted as CRISPR and prophagewere also identified. Genes related to osmotic stress were found according to the characteristics of halophilic bacterium. Genomic differences from other Lentibacillus species were also confirmed through comparative genomic analysis. Strain CBA3610 is predicted tobe a novel species of Lentibacillus through phylogenetic analysis andcomparative genomic analysis with other species in the same genus. Thisstrain has antibiotic resistance gene and pathogenic genes that can affect humans. In future, the information derived from the results of several genomic analyses of this strain is thought to be helpful in identifying the relationship between halophilic bacteria and human gut microbiota.

Keywords : Lentibacillus sp. CBA3610, complete genome sequence, gutmicrobiota

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Thermal Stress Induces Changes of Cecal and Jejunal Microbiota and Functional Analysis of Broiler

Chiwoong Lim, Young-Jun Seo, and Jun-Mo Kim* Department of Animal Science and Technology, Chung-Ang University, Anseong-si, Gyeonggi-do 17546, Republic of Korea

Chicken gastrointestinal microbiota is important factor for host nutrition, and health. Jejunum is the central organ of absorption and mucosa surfaces offer sites for first line health and immunity promotionof broilers under heat stress condition. Cecum contains the most microorganisms and is known to ferment and digest crude fiber to provideextra energy to the host. However, little information of the relationshipbetween heat stress and the gastrointestinal microbial ecosystem of poultry, especially broilers. The objective of the present study is aimedat elucidating the effects of heat stress in the cecal and jejunal microbiomeusing 16S rRNA sequencing methods. A total of 120 7-wk-old broiler were allotted to 10 replicates in a completely randomized design. Broilers were divided into two groups, six each: thermoneutral control (CON) and heat stress condtion (HS). 16s rRNA isolated from broiler’sjejunum and cecum of each group. Phylogeny analysis revealed differences between cecal and jujunal microbiome. After heat stress erysipelotrichaceae was increased in jejunum, which is known to be associated with inflammation-related disorders of the gastrointestinal tract. In the case of the cecum under heat stress, abundance of Clostridiales was increased and bacteroids was decreased. There was a significant changes in both the jejunum and the cecum, and it couldbe potential microoraganisms related to broiler’s gastrointestinal changes under heat stress.

Keywords : Heat stress, broiler, functional genomics

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Screening of Lactobacillus Strains Exhibiting Anti-Cancer Potential Property

Chang Hoon Hwang, Na-Kyoung Lee*, and Hyun-Dong Paik *

Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 05029, Republic of Korea 2WithBio, InC., Neungdongro 120, Seoul 05029, Republic of Korea

Recent research has focused on anticancer property of Lactobacillusstrains isolated from fermented foods. Their anticancer effect was causedby inducing apoptosis on cancer cells. However, sepsis, which can occurwhen cancer patients take living organism, can cause serious situationsin patients with reduced immunity due to cancer. Therefore, this experiment was conducted with heat-killed Lactobacillus strains. For first screening, the Lactobacillus strains which have anti-inflammatoryeffect were selected by nitric oxide (NO) assay. The cancer cells weretreated with different concentrations of heat-killed Lactobacillus strainand the cytotoxicity were assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. In particular, the heat-killed cells of L. plantarum (KU15120 and KU15149) and L. brevis(KU15159 and KU15176) appeared to be the most effective at inhibitingthe growth of AGS, DLD-1, and LoVo cells in a dose-dependent mannerhaving 35–76% survival compared with negative control at 9 log CFU/ml. In addition, the morphology of cancer cells treated with heat-killed Lactobacillus strain showed their effects by microscopy observation. And then, the related factors to apoptosis, such as bax, bcl-2,caspase-3, and caspase-9, were detected by RT-PCR. However, they didnot show a valid change. In conclusion, these results might suggest thepotentials for prophylactic effects against cancer.

Keywords : Probiotics, apoptosis, necrosis

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Development of Staphylococcus-Targeted Antimicrobial Agent

Md Abdur Rahim1,2, Hoonhee Seo2, Sukyung kim2, Hanieh Tajdozian1,2, YoungKyoung Lee1,2, and Ho-Yeon Song1,2* 1Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea 2Microbiome R&D Institute, Probiotics Microbiome Convergence Center, Asan 31538, Republic of Korea

Staphylococcus aureus (S. aureus) is one of the most significant multidrug-resistant bacteria, causing serious healthcare-associated andcommunity-acquired infections globally. The current antibiotic regimenare becoming ineffective against this pathogen due to resistance, in addition, they disrupt the normal microbiota highlighting the urgent needfor the development of a new S. aureus-specific antibacterial providingboth high efficacy and safety. Alpha-viniferin (α-viniferin), a bioactivephytochemical compound, has been reported to have excellent anti-Staphylococcus efficacy as a topical agent. However, so far, thereare no clinical trials that have been conducted to elucidate its efficacyto the best of our knowledge. The goal of this study was to investigate the antibacterial efficacy of α-viniferin against S. aureus in a ten daysclinical trial. Based on the results, α-viniferin showed 50% minimum inhibitory concentrations (MIC50 values) of 7.8 µg/ml in culture broth medium. α-Viniferin was administered in the nares three times a day for10 days using a sterile cotton swab stick. Nasal swab specimens were collected before (0 days) and after finishing the trial (10th day) and thenanalyzed. In the culture and RT-PCR-based analysis, S. aureus was reduced significantly p < 0.01. In addition, 16S ribosomal RNA-basedmetagenomics analysis showed that S. aureus reduced from 51.03% to23.99% at the genus level. RNA-seq analysis was also performed to gaininsights into molecular mechanisms of α-viniferin against S. aureus, which revealed that some gene groups were reduced in 5-fold FC cutoff,2x MIC conditions. The study results demonstrate α-viniferin as a potential S. aureus-specific drug candidate.

Keywords : α-viniferin, clinical trial, MRSA

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Development of a New Probiotic Candidate for the Treatment Mycobacterium tuberculosis

YoungKyoung Lee1,2, Hoonhee seo2, Sukyung Kim2, Md Abdur Rahim1,2,Yoojin1,2, and Ho-Yeon Song1,2* 1Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea 2Microbiome R&D Institute, Probiotics Microbiome Convergence Center, Asan 31538, Republic of Korea

Mycobacterium tuberculosis is the leading cause of death from an infectious disease worldwide. The outbreak of strains of Tuberculosis resistant to multiple anti-tuberculosis drugs is increasing. It is urgentlyrequired to develop new drug candidates that are safe and effective enough to combat the emergence of resistance. Developing a candidatesubstance with a mechanism and concept different from that of conventional antibiotics is urgent. For a long time, probiotics are also used in functional food supplements and they are known to useful to behealthy. Thus, probiotics are mainly used as a new class of anti- mycobacterial agents in infectious diseases recently. We report on a promising approach of live biotherapeutic products, the strain of Lactobacilli, that inhibits M. tuberculosis growth. It reduces intracellularM. tuberculosis H37Rv in RAW 264.7 cells and didn’t show any dysbiosis in the artificial human gut microbial community simulator andno toxicity was found in the Guinea Pig model during 14 days of continuous oral treatment. Based on these results, it can be a good drugcandidate for the treatment of Tuberculosis.

Keywords : Anti-tuberculosis effect, probiotics, Mycobacterium tuberculosis

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Metabolomics Based Chemotaxonomic Classification of Lactic Acid Bacteria and Bifidobacterium spp.

Kim Su Hyun1, Su Young Son1, Su Min Lee1, Jeon-Kyung Kim2, Dong Hyun Kim2, and Choong Hwan Lee1* 1Department of Bioscience and Biotechnology, Kon Kuk University, Seoul 05029, Republic of Korea 2Neurobiota Research Center, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea

Lactic acid bacterium (LAB) and Bifidobacterium spp. belongs to probiotics that known for promoting human health. As the popularity of these probiotics increases, the global probiotic market is continuouslyexpanding, and the demand for new functional strains is increasing. In the screening of candidate strains and efficacy of probiotics, understanding and utilization of probiotics is required, while related studies are very insufficient. In this study, we investigated the metaboliccharacteristics of 6 groups (BI, LA, LB, LC, OT, EC) based on biochemical and taxonomic classification (BI=Bifidobacterium. animalis, B. bifidum, B. breve, B. longum, B. pseudocatenulatum; LA=Lactobacillus acidophilius, L. amylovorous, L. crispatus, L. delbrueckii. L.gasseri; LB=L. casei, L. paracasei, L. rhamnosus, L. curvatus, L. plantarum; LC=L. mucosae, L. reuteri; OT= Enterococcusfaecalis, Lactococcus lactis, Pediococcus acidilactici, EC= Escherichia coli). We performed metabolomics-based chemotaxonomyof 64 strains by gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) and ultra-high performance liquid chromatography (UHPLC)-Orbitrap-MS/MS combined with multivariate statistical analysis to find the distinguished metabolic marker of each group. TheLC-MS-based dataset showed more clear clusters for each group than GC-MS-based dataset. In LA group, the levels of most fatty acids andlipids were relatively high compared with other groups. In LB group, the most hydroxy fatty acids were relatively high compared with othergroups. The LC group showed a relatively increasing pattern of compounds such as phenyllactic acid and indolelactic acid like LB. BI and OT groups showed relatively high content of most amino acid and peptide compounds. OT group is characterized by higher level of carbohydrates. The metabolomics-based chemotaxonomy could provide a detailed view of the differences and similarities among LABand Bifidobacterium spp. In conclusion, our study suggested that metabolomics-based chemotaxonomy is an effective approach to identify metabolic characterization of probiotic candidate.

Keywords : Probiotics, metobolomics, chemotaxonomy

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Neoagarooligosaccharides Alleviate Body Weight Gain and Modulate Gut Microbiota in Obese Rats

Robie Vasquez1, Ju Kyoung Oh1, In-Chan Hwang1, Jeong Min Yoo1, Je Hyeon Lee2, Soon-Kwang Hong3, and Dae-Kyung Kang1* 1Division of Bio-Resources Science, Dankook University, Cheonan, Republic of Korea 2DYNE BIO Inc., Seongnam 13209, Republic of Korea 3Department of Biological Science and Bioinformatics, Myongji University, Yongin 17058, Republic of Korea

Some oligosaccharides function as prebiotics and improve the dysbiosisof the gut microbiota, which consequently affects the physiological properties of the host. In this study, modulation of the gut microbiota andphysiological changes by neoagarooligosaccharide (NAO) administrationto obesity-induced rats were investigated. NAO supplementation improved obesity and its associated parameters as well. In addition, principal coordinates analysis showed that the intestinal microbial structure in obese rats changed by NAO administration, which enrichedseveral taxa associated with weight loss, specifically Eubacterium fissicatena and Ruminococcaceae UCG-005. On the other hand, the abundances of Lachnospiraceae NK4A136, uncultured Ruminococcaceae,Eubacterium xylanophilum and uncultured Desulfovibrionaceae, whichwere all positively associated with body weight gain and obesity/ diabetes-related metabolic factors, decreased by NAO administration. These results indicate that the effects of NAOs on the improvement ofobesity and glucose/lipid metabolism in obesity- induced rats may beassociated with their modulatory effect on gut microbiota. The functionof gut microbiota should be further investigated to elucidate the mechanisms by which NAOs act on gut microbiota and alleviate obesity.

Keywords : Gut microbiota, obesity, neoagarooligosaccharide

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Enrichment of the Novel Bacterial Strains from Rhizosphere Soil Using Various Carbon Sources and Antibiotics

Minseo Choi, Hyoung Ju Lee, Jee Eun Heo, Kihyuck Choi, and Seon-WooLee*

Department of Applied Bioscience, Dong-A University, Busan 49315, Republic of Korea

The importance of the role of the rhizosphere microbiota in plant growth and health has been recognized. However, it is not well known how thestructure of the root microbiome is reorganized to improve the health of the host plant. Several recent studies have shown that the structure of soil microbiome was modulated according to carbon sources of rootexudates. Our previous study showed that the microbiome fraction (MF)of upland rhizosphere soil fully supported the bacterial wilt (BW) resistance in a BW-resistant tomato cultivar Hawaii 7996 compared to the control. In this study, we hypothesized that supplementation of carbon sources could induce the structural change of rhizosphere microbiome and enrich novel bacterial species which can result in subesquent isolation of keystone species. To test this hypothesis, different carbon sources and antibiotics were supplemented in rhizosphere MF, which was collected from rhizosphere soil of tomato plant treated with upland MF. Microbiome structure was analyzed usingthe soils enriched in two consequent time point and we attemted to isolateunique bacterial species based on the microbiome results. Here, we isolated 154 bacteria species representing 6 phyla and 39 distinct species,including 7 novel species. Our results provide new insights into the potential use of carbon sources and antibiotics as stimulants for the cultivation of novel species in the rhizosphere soil environment of plants.

Keywords : Rhizosphere microbiome, enrichment culture, novel strain

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Rhizophere Microbiome Enhance the Bacterial Wilt Resistant in Tomato Plant

Joo Hwan Kwon, Kihyuck Choi, Hyoung Ju Lee, Pyeong An Lee, Jee EunHeo, Raees Khan, and Seon-Woo Lee* Department of Applied Bioscience, Dong-A University, Busan 49315, Republic of Korea

Plant-associated microbiome plays an important role in plant disease resistance. Previously, we found that the upland microbiome fraction (MF) fully supported the resistance to bacterial wilt (BW) in a BW-resistanttomato cultivar Hawaii 7996, while forest soil MF transplant dramaticallysuppressed the BW resistance in Hawaii 7996. Then, we aimed to identifywhich microbiome compartment of tomato root causes this differentialBW-resistance by microbiota transplant in tomato plant. In present study,we separately transplanted rhizosphere and endosphere microbiome extracted from upland MF-transplanted tomato roots and assessed the BW-resistance by either rhizosphere or endosphere microbiome. BW-resistance in Hawaii 7996 plant was apparent by transplant of rhizosphere microbiome, but not by that of endophytic microbiome. To validate this result, we constructed synthetic community (SynCom) comprising nine bacterial isolates from rhizosphere soil of upland MF-transplanted tomato, based on microbiome analysis. After 14 dayspost inoculation of R. solanacearum SL341, treatment of SynCom reduced the BW occurrence in tomato by 1.6-fold, compared to controlplant. In addition, each SynCom members did not show antagonistic activity against R. solanacearum SL341 on the R2A or 1/2 TSA agar medium. Overall, our results suggest that the rhizosphere microbiome by upland MF transplant can fully modulate the expression of BW- resistance in the tomato plant.

Keywords : Tomato, microbiome, bacterial wilt

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Anaerococcus faecalis sp. nov., Isolated from Swine Faeces

Seung Yeob Yu, Byeong Seob Oh, Seoung Woo Ryu, Won Jung Choi, andJu Huck Lee*

Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea

An obligate anaerobic, Gram-stain-positive, non-spore forming, non-motile, catalase and oxidase-negative, coccoid-shaped bacterium designated AGMB00486T was isolated from swine faeces. The optimal growth of the isolate occurred at pH 8.0 and 37℃. Furthermore, the growth was observed in the presence of up to 4% (w/v) NaCl but not atsalinity levels higher than 5%. The phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain AGMB00486T was a member of the genus Anaerococcus and that the isolate was most closelyrelated to Anaerococcus vaginalis KCTC 15028T (96.7% 16S rRNA gene sequence similarity) followed by Anaerococcus hydrogenalisKCTC 15014T (96.7%) and Anaerococcus senegalensis KCTC 15435T

(96.3%). Whole genome sequence analysis determined that the DNA G+C content of strain AGMB00486T was 30.1 mol%, and the genomesize, numbers of tRNA and rRNA genes were 2,268,866 bp, 47 and 8, respectively. The average nucleotide identity values between strain AGMB00486T and the three related type strains were 77.0, 77.4 and 77.2%, respectively. The major cellular fatty acids (> 10%) of strain AGMB00486T were C14:0, C16:0 and C16:0 DMA. Accordingly, these distinct phenotypic and phylogenetic properties revealed that strain AGMB00486T represents a novel species, for which the name Anaerococcus faecalis sp. nov. is proposed. The type strain is AGMB00486T (=KCTC 15945T=CCTCC AB 202009T).

Keywords : Taxonomy, Anaerococcus, swine faeces

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Difference of Microbiome and Mycobiome according to Various Duration and Amount of Brassica juncea(green mustard) Treatment

RyeongHui Kim1, Setu Bazie Tagele2, Tae Hyung Park2, DoKyung Lee2, Min-Kyu Park2, and Jae-Ho Shin1,2* 1Department of Integrative Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea 2School of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea

Biofumigation comprehensively refers to chopping brassica plants containing Glucosinoltes(GSLs) and incorporating them in the soil. GSLs released from chopped plants are hydrolyzed to ITC by water andmyrosinase. The released ITCs and incorporated plant nutrients are known to affect soil bacteria and fungi.In many studies, experiments related to the increase of plant production with the soil fumigation effectof biofumigation are being conducted. However, there is no established method exactly as to how much biofumigant should be treated, and how long should be treated.In this study, the degree of changes in bacteria and fungi according to the amount of biofumigant and biofugation treatmenttime was studied.The soil, which was heavily damaged by phytophthorablight, was brought from the field. After dividing the same amount of soil in a small zipper bag, biofumigant is treated with 0.5%, 1%, 2%, and5% (biofumigant/soil) respectively. All treated soils are incubated at room temperature for 1, 2, 3, and 4 weeks. Samples that have been incubated are uncovered and sampled, and samples after one month andtwo months are also sampled. The sampled soil was subjected to 16s rRNA gene and ITS rRNA gene sequencing to perform microbiome andmycobiome analysis.

Keywords : Biofumigation, microbiome, mycobiome

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The Effect of Ramadan Fasting on Gut Microbiome

Gyudae Lee, Kyeongmo Lim, and Jae-Ho Shin*

Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea

Muslims fast from sunrise to sunset in the ninth month of each year inthe islamic calendar. Depending on their location, their fasting times range from 10 to 21 hours per day, and they do not eat or drink during the fasting period. This is different from the usual intermittent fasting, which only forbids 16 hours of food intake. As widely known, intermittent fasting is known to be very helpful for health, such as lossingweight and preventing diabetes. The experiment was conducted to findout the effect on the intestinal microbial community through Ramadanfasting. We collected fecal, and dietary recalls from 20 Muslins in Korea,including 15 Pakistani and 5 Nigerian. Fecal sampling was conducted total of 3 times during the Ramadan fasting period, and 2 times after finishing ramadan. The microbiome community was analyzed through16S rRNA amplicon sequencing. As a result of the analysis, it was confirmed that there was a difference in alpha and beta diversity beforeand after fasting, and as a result of confirming the change in specific taxonomy at the genus level, it was confirmed that the relative abundanewas significantly higher during fasting than after fasting in the case of Coprococcus and Lachnospiraceae_NK4A136_group. In the case of Lachnospiraceae, it is a representative butyrate-producing bacterium and is a beneficial bacterium that maintains the intestinal environmentin an anaerobic state. The result demonstrated that intermittent fastingincreasing butyrate-producing bacteria, which can possibly effect on human health.

Keywords : Intermittent fasting, Ramadan, Lachnospiraceae

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Anaerosporobacter faecicola sp. nov., Isolated from Faeces of Korean Cow

Seung-Hyeon Choi, Jam-Eon Park, Ji Young Choi, Ji-Sun Kim, Se Won Kang, Jiyoung Lee, Mi-Kyung Lee, Jung-Sook Lee, and Seung-Hwan Park*

Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea

A novel bacterial isolate designated as strain AGMB01083T was isolatedfrom Korean cow faeces deposited in the National Institute of Animal Science (Wanju, Republic of Korea). The bacterium is an obligate anaerobic, Gram-strain-positive, and motile. Cells are straight or curvedrod-shaped, flagella and spores are observed. Growth occurs between 20-40 °C (temperature optimum of 35 °C), at pH 7-9 (pH optimum of7), and in the presence of 0.5-1.0% (w/v) NaCl. Based on the 16S rRNAgene sequence analysis, the strain belongs to the genus Anaerosporobacterand is most closely related to A. mobilis HY-37-4T (=KCTC5027T, similarity, 95.7%). The DNA G+C content is 36.2 mol%, determined bythe whole-genome sequence. The average nucleotide identity value between strain AGMB01083T and strain A. mobilis HY-37-4T is 75.5%,below the interspecies identity threshold value. The major cellular fatty acids (>10%) of strain AGMB01083T are C16:0, C16:0 dimethyl acetal (DMA), and C16:0 3-OH. Based on the phylogenetic, phenotypic, biochemical, chemotaxonomic, and genomic characterization, strain AGMB01083T is proposed to be a novel species, named Anaerosporobacterfaecicola, in the genus Anaerosporobacter. The type strain is AGMB01083T (=KCTC 15857T=NBRC 114517T).

Keywords : Korean cow, taxonomy, novel bacterium

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A Novel sporolactobacillaceae bacterium, KGMB08714T, Isolated from Healthy Korean Faeces

Min Kuk Suh1, Ji-sun Kim1, Han-sol Kim1, Mi kyung Eom1, Kook-II Han1,Se Won Kang1, Ju Huck Lee1, Seung-Hwan Park1, and Jung-Sook Lee1,2*

1Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea 2University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea

A novel sporolactobacillaceae bacterium, KGMB08714T was isolatedfrom a faecal sample of a healthy Korean. Strain KGMB08714T was facultatively anaerobic, Gram-positive, motile and spore-forming rod-shaped, which formed ivory-white colonies on Reinforced Clostridial Medium agar with 5% horse blood. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain KGMB0714T wasclosely related to Sporolactobacillus vineae SL153T, with a 95.58% 16SrRNA gene sequence similarity. The major end product of short-chainfatty acid (SCFA) was lactate during fermentation. Based on the phylogenetic and phenotypic characteristics, KGMB08714T describedas a novel species of the family Sporolactobacillus.

Keywords : Sporolactobacillus, gut microbiome

[This research was supported by the Bio & Medical Technology Development program of the National Research Foundation of Korea (NRF-2016M3A9F3946674) funded by the Ministry of Science and ICT (MIST) of the Republic of Korea and a grant from the Korea Research Institute of Bioscience & Biotechnology (KRIBB) Research Initiative Program]

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A Novel Bacteroides Species, KGMB07931T and KGMB10229, Isolated from Human Faeces

Han Sol Kim1, Ji-Sun Kim1, Min Kuk Suh1, Mi Kyung Eom1, Kook-Il Han1,Ju Huck Lee1, Seung-Hwan Park1, Se Won Kang1, and Jung-Sook Lee1,2*

1Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea 2University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea

Two anaerobic, Gram-negative strains designated KGMB07931T and KGMB10229, were isolated from two different healthy Korean faecal samples. Pylogenetic analysis based on the 16S rRNA gene sequenceindicates that the KGMB07931T and KGMB10229 sequences are verysimilar to each other, and the two strains are also closely related to the strains Bacteroides uniformis ATCC 8492T (97.5 %), Bacteroides rodentium JCM 16496T (96.6 %), Bacteroides fluxus YIT 12057T (94.5%). Their optimal temperature, NaCl concentration and pH for growthwere 37 °C, 0-0.5 % (w/v) NaCl and pH 7.0, respectively. The major cellular fatty acids (>10 %) of strain KGMB07931T were anteiso-C15:0and C18:1cis9. The major menaquinones are MK-9 and MK-10. The DNAG+C content based on whole genome sequences was 46.3 mol%. On thebasis of phenotypic and phylogenetic data, strains KGMB07931T and KGMB10229 represents a novel species in the genus Bacteroides. Thetype strain is KGMB07931T.

Keywords : Bacteroides sp. nov., human faeces, taxonomy

[This work was supported by the Bio and Medical Technology Development program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) of the Republicof Korea (NRF-2016M3A9F3946674) and a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Researchinitiative program]

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Faecalicatena faecalis sp. nov., Isolated from Swine Faeces

Byeong Seob Oh, Seoung Woo Ryu, Seung Yeob Yu, Won Jung Choi, andJu Huck Lee*

Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea

An obligately anaerobic, Gram-stain-positive, non-motile, non-spore- forming and rod-shaped strain AGMB00832T was isolated from swinefaeces. The isolate was a member of the genus Faecalicatena and mostclosely related to Faecalicatena orotica KCTC 15331T (97.2%, 16S rRNA gene sequence similarity) according to the 16S rRNA gene sequence-based phylogenetic analysis. Based on the whole genome sequence analysis, The DNA G+C content of strain AGMB00832T was38.8 mol%. Besides, the genome size and numbers of rRNA and tRNAgenes were 3,098,210 bp, 12 and 45, respectively. In biochemical analysis, strain AGMB00832Twas shown to be negative for catalase, oxidase and urease. Furthermore, the isolate was positive for β-glucosidase, β-glucuronidase, glutamic acid decarboxylase, proline arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase.The major cellular fatty acids (>10%) of the isolate were C14:0, C16:0 andC18:1ω11t DMA. On the basis of polyphasic evidences, strain AGMB00832T

represents a novel species of a genus Faecalicatena, for which the nameFaecalicatena faecalis sp. nov. is proposed. The type strain is AGMB00832T (= KCTC 15946T =NBRC 114613T).

Keywords : Swine faeces, gut microbiota, taxonomy

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Gut Microbiome-Based Bacterial Signatures for Non-Invasive Dectection of Invasive Cervical Cancer

Daryung Jung1, Gi-Ung Kang2, Min-Sueng Kim2, Yoon Hee Lee3,4,5, SeYoung Jeon4,5, Hyung Soo Han5,6, Gun Oh Chong3,4,5*, and Jae-Ho Shin1,2*

1Department of Biomedical Convergence Science & Technology, Kyungpook National University, Daegu 41566, Republic of Korea 2Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea 3Department of Obstetrics and Gynecology, School of Medicine, Kyungpook National University, Daegu 41404, Republic of Korea 4Department of Obstetrics and Gynecology, Kyungpook National University Chilgok Hospital, Daegu 41404, Republic of Korea 5Clinical Omics Research Center, School of Medicine, Kyungpook National University, Daegu 41940, Republic of Korea 6Department of Physiology, School of Medicine, Kyungpook National University, Daegu 41405, Republic of Korea

The gut microbiome is being increasingly implicated in diagnosis of various diseases, including both digestive diseases and non-digestive diseases. However, the evidence for the differences in invasive cervicalcancer (ICC) in gut microbiome remains unclear. In this study, we investigated whether the gut microbiome differed in ICC patients compared with healthy controls in our cohorts. And, we developed a diagnostic model for predicting ICC, and identified bacterial signaturesfrom gut microbiome that contributed in classifying ICC using ampliconsequencing data. Subjects including 21 healthy women (control group)and 25 women with clinically confirmed ICC (cancer group) were included. We carried out 16S rRNA amplicon sequencing using the Illumina Miseq platform. The alpha diversity analysis revealed that cancer group is lower compared to control group in terms of diversity index, especially statistically significant in Shannon’s index. Beta diversity analysis showed a clear separation between control and cancergroup. Furthermore, there were significant differences in the taxonomicprofiles between control and cancer group. And, we analyzed Linear discriminant analysis effect size (LEfSe) to validate the taxonomic differences at the genus level. Correspondingly, 7 bacteria genera wereidentified through a 10-fold cross-validation on a random forest modelfor distinguishing cancer with control group. This model had high diagnostic utility (area under the curve [AUC] = 0.952) for predicting ICC. Our research provides exploration of ICC using the gut microbiomeand this non-invasive diagnostic model could be widely used to complement the clinical assessment.

Keywords : Microbiome, cervical cancer, machine learning

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Field Test of Selected Entomopathogenic Fungi for the Management of Thrips palmi

Hyeju Jeong, Sungnam Choi, Hwasook Kwak, Jihee Han, and JaekyeongSong*

Agricultural Microbiology Division, National Institutes of Agricultural Sciences, RDA, Wanju-gun 55365, Republic of Korea

Thrips palmi is pest that are problematic in greenhouse cultivation fruitsand vegetables. Nymph and adult insects of Thrips palmi are harmful to the leaves, flowers, stems, and even fruits of plants. So it reduces thecommercial value. The development of insecticide resistance due to repeated treatment of chemical insecticides makes it difficult to controlthrips at farm. Therefore, the selection of useful microorganisms to effectively control thrips is on the rise. In the previous experiment, entomopathogenic fungi with high pathogenicity to thrips were selected. Two strains of them were tested at field. The strains Isaria sp. S3-2 andMetarhizium sp. S5-3 were treated three times to cucumber seedling at7 days intervals. As a result, the number of thrips per seedling was 43.1in the control group, 7.1 in the Isaria sp. S3-2 strain treated group, and12.4 in the Metarhizium sp. S5-3 strain treated group. Since the population of thrips in the selected entomopathogenic fungi treatment group is lower than that of the control group, the selected microorganismsare thought to be effective in controlling thrips.

Keywords : Thrips palmi, entomopathogenic fungi, biological control

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Characterization of Glutamate Decarboxylase (GAD) from Latilactobacillus curvatus K285 Isolated from Gat-kimchi

Se Jin Lee and Jeong Hwan Kim*

Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University, Jinju 52828, Republic of Korea

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from gat-kimchi, a Korean traditional vegetable food. LAB wereisolated by spreading diluted samples on MRS agar plates. A GABA-producing strain was isolated from 2,000 LABs by TLC of culture supernatant containing MSG (mono sodium glutamate). The isolate, K285, was identified as Latilactobacillus curvatus by 16S rRNAgene sequencing. Growth characteristics of Lb. curvatus K285 were examined for 120 h at different conditions: pH (3-10), temperature (-1℃-45℃), and NaCl (0%-10%) respectively. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB, indicating the gadCB operon structure. gadB was overexpressed in Escherichia coli BL21 (DE3) and recombinant GAD was purified through Ni-NTA column (GE Healthcare, Sweden). The size was 54 kDaby SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 50°C and the activity was dependent on pyridoxal 5’-phosphate. The Kmand Vmax of GAD were 5.35 ± 0.27 mM and 0.041 ± 0.0008 mM/min,respectively, when glutamate was used as the substrate.

Keywords : GABA, GAD gene cloning, gat-Kimchi

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Comparison of Fecal and Cecal Microbiota in Mice Fed a High Fat Diet or a Normal Diet

Sunwoo Lee, Vineet Singh, and Unno Tasuya*

Major of Molecular Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju 63243, Republic of Korea

Intestinal microflora plays an important role in maintaining our health,and research in the field is increasing. In general, fecal and cecal microbiota are targeted to investigate gut microbiota shifts during dietary intervention. In this study, we compared fecal and cecal gut microbiotain mice undergoing different dietary interventions. The mice(n = 7) werefed either a high fat diet(HFD) or a normal diet(ND) for 12 weeks. Fecaland cecal gut microbiota were analyzed through Miseq sequencing. We applied Mothur to process sequence data, STAMP and R to perform microbiota comparison, and PICRUSt to predict metabolic activities. The results from the beta-diversity analysis showed differences in microbial diversity between ND and HFD, likewise observed betweenfecal and cecal microbiota. Differential abundance analysis showed significant difference in some genera such as probiotic strains (i.e. Lactobacillus and Bifidobacterium), Bacteroides, and Helicobacter. Therefore, our results suggest that the conclusions obtained from dietaryinterventions could vary depending on which microbiota was analyzedand what type of dietary intervention was taken. This study provides fundamental information about the role of microbiota in dietary intervention studies.

Keywords : Microbial diversity, fecal and cecal, diet intervention

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The Anti-Inflammation Effect of a Strain of Lactobacillus rhamnosus via Gut-Brain Axis in Irritable Bowel Syndrome Mice Model Under Chronic Stress

Jihyun Kim, and Sae Hun Kim*

College of Life Science and Biotechnology, Korea University, Seoul 02841, Republic of Korea

In modern society, people face demanding competition and feel chronicstress. Chronic stress can cause both physical and psychological illnesssuch as irritable bowel syndrome (IBS) and depression. Although it is recently studied that gut microbiota can influence the host’s health via the gut-brain axis, the mechanism of the effect of single bacterial strain via the gut-brain axis is unknown. In this study, C57BL/6 J male mice were administered a strain of L. rhamnosus ad-libitum. Treatment groupswere exposed to unpredictable chronic mild stress (UCMS) and received10 mg/kg of loperamide. Mice that were fed L.rhamnosus-supplementeddiet showed improved UCMS-induced dysregulation, upregulating themRNA expression of tight junction protein, brain-derived neurotrophicfactor and serotonin and downregulating pro-inflammatory markers inthe intestine and the brain. Therefore, the supplement of L. rhamnosusis expected to improve cognitive impairment as well as to attenuate inflammation via gut-brain axis in IBS mice model.

Keywords : Probiotics, gut-brain axis, anti-inflammation

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Fungal Microbiome of Deoxynivalenol : Modulating Intestinal Morphology and Microbiota in Piglets Ileum Tissue

Jin Young Jeong, Minji Kim, Sang Yun Ji, Byeonghyeon Kim, Youl ChangBaek, Hwa Seol Park, and Hyunjung JungAnimal Nutrition and Physiology Team, National Institute of Animal Science, Wanju 55365, Republic of Korea

Deoxynivalenol (DON) causes diseases and physiological responses tohumans and livestock. In particular, it affects feed intake and productivityin pigs. This study was conducted to identify of the intestinal microbiotaand morphology in piglets fed DON contaminated feeds with differentconcentrations. We performed the ileum contents from four groups of piglet (fed either a standard diet, or a diet with 1, 3, 10 mg/kg purifiedDON). Fungi from the ileum contents of 2-way hybrid piglets (Landrace× Yorkshire) were characterized by ITS ribosomal RNA gene sequencing based on MiSeq platform, and then taxonomy and diversity were used q2-feature-classifier and q2-diversity to generate interactive visualizations,respectively. DON exposure reduced feed intake and body weight compared with control groups. 1,385,732 gene sequences were generatedwith total sequences sampled (10,000). Exposure to DON significantlyaffected the ileum fungal microbiota, especially Ascomycota, which wasthe most abundant. We estimate that Ascomycota plays an important rolein regulating DON for detoxification. The gut mycobiota was predominant by yeast, with an abundance of phylum Ascomycota in morethan 90% of the piglets. The diversity communities were estimated withpiglets fed exposure to DON. Additionally, we observed collagen fibersin ileum tissue using Masson's trichrome staining. It stained a collagenfibers in the submucosa of piglet ileum. Acute exposure to DON inducesdisturbances of gut microbial communities. In conclusion, these findingssuggest that the intestinal microbiome can provide better understandingof the gut microbiota association with growth performance in piglet exposure to DON

Keywords : Piglet, deoxynivalenol, microbiome

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Peptoniphilus faecalis sp. nov., Isolated from Swine Faeces

Seoung Woo Ryu, Byeong Seob Oh, Seung Yeob Yu, Won Jung Choi, andJu Huck Lee*

Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea

An obligately anaerobic, Gram-positive, non-motile, coccus-shaped bacterial strain designated AGMB00490T was isolated from swine faeces. 16S rRNA gene sequence-based phylogenetic analysis indicatedthat the isolate belongs to the genus Peptoniphilus and that the most closely related species is Peptoniphilus gorbachii KCTC 5947T (97.22% 16S rRNA gene sequence similarity). Whole genome sequence analysisdetermined that the DNA G+C content of strain AGMB00490T was 31.2 mol% and moreover that the genome size and numbers of tRNA and rRNA genes were 2,129,517 bp, 34, and 10, respectively. Strain AGMB00490T was negative for oxidase and urease; positive for catalase,indole production, arginine arylamidase, leucine arylamidase, tyrosinearylamidase, and histidine arylamidase; and weakly positive for phenylalanine arylamidase and glycine arylamidase. The major cellularfatty acids (>10%) of the isolate were determined as C16:0 and C18:1ω9c.Strain AGMB00490T produced acetic acid as a major end product of metabolism. Accordingly, phylogenetic, physiological, and chemotaxonomicalanalyses revealed that strain AGMB00490T represents a novel species for which the name Peptoniphilus faecalis sp. nov. is proposed. The type strain is AGMB00490T (= KCTC 15944T = NBRC 114159T).

Keywords : Microbiome, swine faeces, Peptoniphilus

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Identification of Vaginal and Fecal Microbiome Characteristics of Hanwoo

Soyoung Choi, JiHye Cha, Minji Song, JuHwan Son, Mi-rim Park, Yeong-jo Lim, and Woncheoul Park*

Animal Genomics and Bioinformatics Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Republic of Korea

Improving reproduction ability is very important in the beef cattle industry. However, the study of Hanwoo (Korea native cattle) is mainlyfocused on meat quality and growth. In addition, the microbiome studyof reproductive system and feces about healthy Hanwoo is very insufficient. Therefore, in this study, the microbiome information of thevagina and feces of Hanwoo confirmed characteristics of the microbialcomposition. A total of 31 clinically healthy Hanwoo in Cheongju wereenrolled in this present study. They have delivered healthy calves morethan once. We collected fecal and vaginal samples during the breedingseason. All samples were performed the paired-end sequencing using the Next-generation sequencing of the V3-V4 region of 16S rRNA genevia the Illumina MiSeq platform. After removing low quality reads andchimeras using QIIME2, samples had averaged 56,926 reads per sampleranging from 9,457 to 124,197. We confirmed the sequencing quality and OTUs of each samples through the alpha rarefaction curve. We founded the vaginal microbial composition more complex than fecal microbiome. In both groups, vaginal and fecal samples, were identifiedcommon and important microbes. And PCoA showed that significantlydifferences between two groups, and LEfSe analysis detected which bacterial taxons differed. The results of our study were able to confirm the microbial composition of the vaginal and fecal samples in healthy Hanwoo. Our study suggests that when conducting microbiome study in the future, samples should be collected and analyzed according to theexperiment purpose. Furthermore, the microbiome of the vagina and feces, which is related to reproductive ability, could be used as the potential biomarker.

Keywords : Hanwoo, vaginal microbiome, fecal microbiome

N-47

Feeding Levels on Ruminal Microbial Community of Growing Korean Native Goat (Capra hircus coreanae)

Jinwook Lee, Bong-Hwan Choi, Sung-Soo Lee, Dong-Kyo Kim, Eun-DoLee, and Kwan-Woo Kim*

National Institute of Animal Science, Rural Development Administration, Hamyang 50000, Republic of Korea

The objective of this study was to investigate the effects of feeding levelson ruminal bacterial community of growing Korean native goats. Fivegrowing bucks (19.5 ± 1.4 kg) with an average age of 5 months were randomly allocated to one of five feeding levels (maintenance [M], 1.1x M, 1.2 x M, 1.4 x M, and 1.6 x M). The experimental design was 5 × 5 Latin square design and all animals were housed individual pens (1.2m × 0.9 m). At the end of feeding trials, rumen samples were collected before morning feeding using stomach tube. Alpha diversity did not differ among dietary treatment groups. Assessment of the relative abundances revealed 9 phyla of which Firmicutes and Bacteroidetes were found to be the most dominant, but the relative abundance of thesephylum did not differ between treatment groups. The relative abundanceof Synergistetes was influenced quadratically (p < 0.05) at the phylum levels. At the genus levels, only the abundance of Muribaculum was increased linearly (p < 0.05). The relative abundance of Anaerohabdus,Intestinimonas, Anaerovorax, Emergencia, Alkalibacter, Gracilibacter,Oribacterium, Roseburia, Breznakia, Thermanaerovibrio and Anaeroplasmawere significantly influenced by quadratic model (p < 0.05). Among the23 predominant genera (relative abundance > 1%), eight genus includingSaccharofermentans, Ruminococcus and Paludibacter showed high correlations between growth performance and ruminal fermentation parameters. These results indicated that dietary feeding levels affects ruminal microbial community, and further affects growth performanceand ruminal fermentation. These results serve as a basis for the establishment of goat feeding programs in farm scale of republic of Korea.

Keywords : Growing goat, Rumen microbiome, feeding level

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Feeding Levels on Rumen Bacterial Community of Fattening Korean Native Goat (Capra hircus coreanae)

Jinwook Lee, Bong-Hwan Choi, Sung-Soo Lee, Dong-Kyo Kim, Eun DoLee, and Kwan-Woo Kim*

National Institute of Animal Science, Rural Development Administration, Hamyang 50000, Republic of Korea

Ruminal microbial community was influenced by various factors including dietary feeding levels, feed type and growing phase. The aim of this study was to explore the effects of feeding levels on ruminal bacterial community and to investigate the correlation between ruminalbacterial abundance and growth performance/fermentation of fatteningKorean native goat using 16S rRNA sequencing. Five Korean native goat(Capra hircus coreanae) bucks (38.0±1.7 kg) with an average age of 2.4year-old were randomly allocated to one of five feeding levels (maintenance [M], 1.1 x M, 1.2 x M, 1.4 x M, and 1.6 x M). The experimental design was 5 × 5 Latin square design and all animals werehoused individual pens (1.2 m × 0.9 m) for an adaption period of 2 weeksand collection period for 1 weeks. Rumen samples were collected beforemorning feeding using oral stomach tube. Assessment of the relative abundances revealed 10 phyla of which Firmicutes and Bacteroidetes were found to be the most dominant, but the relative abundance of thesephylum did not differ between treatment groups. The relative abundanceof Proteobacteria was decreased linearly (p < 0.001) at the phylum levels.At the genus levels, the relative abundance of Butyrivibrio was decreasedlinearly (p < 0.01) and quadratically (p < 0.05). The abundance of Breznakibacter, Ruminococcus and Ballitalea were decreased linearly (p < 0.05), but the abundance of were Duncaiella, Millionella, Parapedobacter and Tyzzerella increased linearly (p < 0.05). The relativeabundance of Oribacterium was increased quadratically (p < 0.01), butthe abundance of Kineothrix was decreased quadratically (p < 0.05). Among the 24 predominant genera (relative abundance > 1%), 14 genusincluding Buryrivibrio, Ruminococcus and Selenomonas were associated with the growth performance and ruminal fermentation characteristics. These results suggest that higher feeding levels of growing goat increases digestibility and nutrient utilization efficiency in the rumen, and further affects ruminal microbial community. These results serve as a basis for the establishment of goat feeding programs in farm scale of republic of Korea.

Keywords : Fattening goat, Rumen microbiome, feeding level

N-49

Effect of a Bioconverted Product of Lotus corniculatus Seed on the Axillary Microbiome and Body Odor

Min-Ji Kim1, Setu Bazie Tagele1, HyungWoo Jo2, Min-Chul Kim1, YeonGyun Jung1, Yeong-Jun Park1, Jai-Hyun So3, Hae Jin Kim4, Ho Jin Kim4, Dong-Geol Lee2, Seunghyun Kang2, and Jae-Ho Shin1* 1Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea 2R&I Center, COSMAX BTI, Seongnam 13486, Republic of Korea 3National Development Institute of Korean Medicine, 94, Hwarang-ro, Gyeongsan, Gyeongsangbuk-do 38540, Republic of Korea 4Experiment Research Institute, National Agricultural Products Quality Management Service, Gimcheon, Gyeongsangbuk-do 39660, Republic of Korea

The skin microbiome, especially the axillary microbiome, consists of odor-causing bacteria that decompose odorless sweat into malodor compounds, which contributes to the formation of body odor. Plant-derived products are a cheap source of bioactive compounds thatare common ingredients in cosmetics. Microbial bioconversion of natural products is an ecofriendly and economical method for productionof new or improved biologically active compounds. Therefore, in this study, we tested the potential of a Lactobacillus acidophilus KNU- 02-mediated bioconverted product (BLC) of Lotus corniculatus seed toreduce axillary malodor and its effect on the associated axillary microbiota. A chemical profile analysis revealed that benzoic acid wasthe most abundant chemical compound in BLC, which increased following bioconversion. Moreover, BLC treatment was found to reduce the intensity of axillary malodor. We tested the axillary microbiome of18 study participants, divided equally into BLC and placebo groups, andrevealed through 16S rRNA gene sequencing that Staphylococcus, Corynebacterium, and Anaerococcus were the dominant taxa, and someof these taxa were significantly associated with axillary malodor. Afterone week of BLC treatment, the abundance of Corynebacterium and Anaerococcus, which are associated with well-known odor-related genes that produce volatile fatty acids, had significantly reduced. Likewise, the identified odor-related genes decreased after the application of BLC. BLC treatment enhanced the richness and networkdensity of the axillary microbial community. The placebo group, on theother hand, showed no difference in the microbial richness, odor associated taxa, and predicted functional genes after a week. The resultsdemonstrated that BLC has the potential to reduce the axillary malodorand the associated odor-causing bacteria, which makes BLC a viable deodorant material in cosmetic products.

Keywords : Bioconversion, skin microbiome, cosmetics

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N-50

The Pathway of Propionate Production and Description of a Novel Propionate Producing Gut Bacterium AP1T

Ji-Sun Kim1, Min Kuk Suh1, Mi Kyung Eom1, Kook-Il Han1, Han-Sol Kim1, Keun Chul Lee1, Ju Huck Lee1, Seung-Hwan Park1, Se Won Kang1,Jam-Eon Park1, Byeong Seob Oh1, Seung Yeob Yu1, Seung-Hyeon Choi1,Seoung Woo Ryu1, Ji Young Choi1, and Jung-Sook Lee1,2* 1Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea 2University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea

A novel Gram-stain-positive, non-motile, non-spore-forming, rod-shapedand strictly anaerobic bacterium, designated AP1T, was isolated from a healthy Korean faeces. Comparative analysis of 16S rRNA gene sequences showed that strain AP1T was most closely related to Merdimonas faecis BR31T (94.3 %) and the average nucleotide identity(ANI) value between strain AP1T and M. faecis BR31T was 75.6 %. Interestingly, strain AP1T produced the highest concentration of propionate compared with the other reference strains. In gut commensalbacteria, propionate can be produced via mainly three metabolic pathways such as propandiole pathway, acrylate pathway, and succinatepathway. To investigate the metabolic pathway(s) utilized by strain AP1T

to produce propionate, it was analyzed the genome sequences and KEGGpathway. It was found that strain AP1T is not able to produce using abovethree pathways that most gut bacteria use. It was thought that strain AP1T

produced a large amount of propionate though the amino acid catabolicpathway, that was also supported by increased propionate after adding amino acids. Based on the phylogenetic, phenotypic and chemotaxonomiccharacteristics, strain AP1T represents a novel propionogenic species ina new genus within the family Lachnospiraceae. It is also expected thatpropionogenic bacterium AP1T can be used as new generation probioticsto target various metabolic syndrome as propionate-producing consortiumdoes.

Keywords : Propionate, novel, gut microbiota

[This research was supported by a Bio & Medical Technology Development program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) of the Republicof Korea (NRF-2016M3A9F3946674), and a grant from the Korea Research Institute of Bioscience & Biotechnology (KRIBB) Research initiative program]

N-51

Chronic Exposure to Antibiotics Modulates Gut Microbiome and Immune/Stress-Related Genes in the Fishes

Jun Hyeok Yang1, Sungmin Hwang2, Seungki Lee3, and Ki Hwan Moon1*

1Division of Marine Bioscience, Korea Maritime & Ocean University, Busan 49112, Republic of Korea 2Department of Biology, Duke University, Durham, NC 27708, USA, 3National Institute of Biological Resources, Environmental Research Complex, Incheon 22689, Republic of Korea

Antibiotic pollutants in the milieu escalate the selective pressure on microbial flora and promote the increase of antibiotic resistance in theaquatic environment. The indiscriminate use of antibiotic agents can affect not only microorganisms but also their hosts in both aquatic and terrestrial environments. Previous studies have shown that chronic antibiotic exposure affected to growth, reproduction and hormones in freshwater medaka. In this study, we hypothesized that chronic antibioticexposure can alter the composition of gut microbiome in the host, and modulate expression levels of immune and stress-related genes. To setup the antibiotic concentration for chronic exposure assays, we selectedsix antibiotics: ampicillin, erythromycin and chloramphenicol for the Korea native ricefish (Oryzias latipes), and oxytetracycline, sulfamethoxazole/trimethoprim and erythromycin for zebrafish (Danio rerio), respectively.The isolated bacteria from intestine of fish were used for minimal inhibitory concentration (MIC) test of each antibiotics. Based on MIC data, we established the antibiotics concentration for chronic exposureassay. Fishes were exposed for a month to each antibiotics. Then, total DNA from fish intestine was purified for future gut microbiome analyses. RNA was also purified to check immune and stress-related gene expression with future differentially expressed genes (DEG) analyses. Our study will be provided a solid reason to halt indiscriminateuse of antibiotic agents.

Keywords : Antibiotics, chronic exposure, fish gut microbiome

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Microbiome of Seoul Metropolitan Rapid Transit Subway (SMRT) Aerosol: Unraveling the Seasonal Variations of Airborne Microbial Assemblages of SMRT, South Korea

Zohaib Ul Hassan1,2,3, and Seil Kim1,2,3* 1Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea 2Convergent Research Center for Emerging Virus Infection, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea 3Department of Bio-Analytical Science, University of Science & Technology (UST), Daejeon 34113, Republic of Korea

Background: Subway systems are requisite for urban societies; however,microbiological characteristics of subway aerosols are relatively unknown. SMRT has grown to one of the world’s largest public transit networks s, with 9 lines lengthening 327 km (including 290 km underground). The average number of passengers travel per day is 7.2 million and per year is 2.6 billion. With an epic increase in commuters in Seoul, the subway network also gaining in reputation and is progressively being used as the primary means for public transportation.Methods: In this study, we determined the seasonal airborne microbialdiversity profiles at SMRT stations by sequencing 16S rRNA and ITS.Particulate matter samples were collected from air purifiers installed inthe platform area of SMRT subway stations. Three stations including themost crowded one were selected for the sampling. A) Gangnam, B) Suyu,C) Indeagwon. The sampling was carried out each season during 2019.After extracting the total DNA from all seasonal samples, PCR was performed with Illumina overhang adapter primers for the V3-V4 regionof the 16S rRNA gene and ITS2 region of the ITS gene. The amplified products were further purified and sequencing libraries were made. Sequencing was carried with the Illumina Miseq sequencing system (Illumina, USA). Results: The SMRT microbiome showed extensive taxonomic diversity, with the most common bacterial genera at the subway stations associated with the skin. Overall, stations included inthis study harbored different phylogenetic communities based on α-diversity and β-diversity comparisons. Microbial assemblages also varied depending upon the season sampled, as well as the station. Conclusions: This study is the first extensive microbiome study at one of the world’s largest mass transit subway systems. The study shows thatthe microbial composition of the SMRT subway stations comes from adiverse combination of environmental, human sources, season, and thelifestyle of commuters.

Keywords : Microbiome, subway, seasonal diversity

N-53

Assessment of Incubating Media Composition for In vitro Gastrointestinal Digestion-Fecal Fermentation : GID-FF

Dabin Jeon1, and Tatsuya Unno1,2* 1Faculty of Biotechnology, School of Life Sciences, SARI, Jeju National University, Jeju-si 63243, Republic of Korea 2Subtropical/Tropical Organism Gene Bank, Jeju National University, Jeju-si 63243, Republic of Korea

Recent gut microbiome studies revealed a number of important roles ofgut microbiome in human health. For the most of gut microbiome studies, animal experiments are considered essential. There are time-consuming,high-cost, and even ethical issue in animal experiment. We applied in vitro gastrointestinal digestion and fecal fermentation (GID-FF) to investigate the effects of various function of foods or medicines on gutmicrobiota. While incubating gut microbiota in this system, three kindsof media (Phosphate-buffered saline media; PBS, Basal culture media;BCM, and New Basal culture media ; NBCM) were tested using threehealthy people’s fecal samples. Our results showed that microbiota shiftsdepended on incubation time, carbon source, and type of bile salts. Briefly, As incubation time increased, the abundance of Bacteroides andBilophila was increased. Also, it showed the association between carbonsources and the abundance of Dorea, Bacteroides, Fusicatenibacter, andParabacteroides. Therefore, we suggest that type of media should be carefully selected depending on type of samples(i,e, food and medicine).

Keywords : GID-FF, fecal microbiome, incubation media

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N-54

The First Successful Fecal Microbiota Transplant for Iranian Recurrent CDI Patients with Uunderlying Inflammatory Bowel Disease and the Alterations of Gut Microbiota

Youngjae Jo1, Minsung Kim1, Jae-Ho Shin1*, Abbas Yadegar2, and MinSoo Jung1 1Department of Applied Biosciences, Kyungpook National Univeristy, Daegu 41566, Republic of Korea 2Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Fecal microbiota transplant (FMT), a novel alternative to existing antibiotic treatment, has emerged for the therapeutic treatment of recurrent Clostridioides difficile infection (rCDI) and concurrent inflammatory bowel disease (IBD). Although FMT has been used for decades worldwide, more research about effects of FMT on gut microbiome is necessary. Particularly, there have been no previous studies focusing on Iranian patients. This study examines how gut microbiota change in Iranian patients with inflammatory bowel disease(seven with ulcerative colitis and one with Crohn’s disease) before andafter FMT. FMT was performed to 8 patients via colonoscope. The patients experienced no adverse effects and IBD flare-up, and all recovered to full health. Regardless of type of IBD, rCDI patients all hada lack of Bacteroidetes compared to donor samples. Following FMT treatment, the proportions of Bacteroidetes were increased until a normalrange was achieved. More specifically, Prevotella which are hierarchically ranked below Bacteroidetes, were found to increase significantly following FMT; Prevotella was also found to correlated negatively with inflammation, suggesting that Prevotella may be a keyfactor for resolving CDI and IBD. Gut microbial diversities were foundto increase while dysbiosis indices decreased; however, the distance ofmicrobial communities that were not narrowed and varying donor stool engraftment indicate that gut bacterial communities of recipients do notchange compared to donors. FMT leads to significant alterations of thebacterial community structure in rCDI patients with IBD. Although themicrobial composition of rCDI patients after FMT treatment did not change compared to donors, most patients recovered their own beneficialbacteria. Moreover, the decreasing in microbial dysbiosis index and increasing in phylogenetic diversity after FMT demonstrate the importance of both FMT treatment and subsequent management. Collectively, our findings show that FMT helped reprograming the gutmicrobiome of rCDI patients.

Keywords : Fecal microbiota transplant, microbiome, prevotella

N-55

Bacterial Community Composition Change in Temperate Octocoral, Scleronephthya gracillimum Responding to Season and Heat Stress

Seonock WooMarine Biotechnology Research Center, KIOST, Busan Metropolitan City 49111, Republic of Korea

Coral diversity is affected by climate change. Environmental impacts can induce the change of relationship between coral and its symbiotic microbial community. Furthermore the microbial community change could lead the coral diseases considered one of the reasons of coral deathand the opportunistic infections in corals exposed to the increased temperature. In this study we collected a temperate octocoral, Scleronephthyagracillimum in Jeju,Korea and exposed to various seawater temperature(26, 28 and 30℃ ) to compare the composition of bacterial community using the Next Generation Sequencing technique. The results showed total 88 species of bacteria were found in S. gracillimum in wild conditionand they were classified to 72 genus, 41 family, 32 order, 16 class and 13 phylum. Soft coral, S. gracillimum were enriched in OTUs from thefamilies Hahellaceae, Mycoplasmataceae, Alteromonadaceae, Anaplasmataceae,and Rhodobacteraceae. The number of bacteria species belong to following 8 families, Flavobacteriaceae, Bacillaceae, Comamonadaceae,Alteromonadaceae, Pseudoalteromonadaceae, Hahellaceae, Pseudomonadaceaeand Vibrionaceae robustly increased in responses to the heat stress in 26,28 and 30ºC groups and the species Mesoflavibacter sabulilitoris, Vibriotubiashii, Pseudomonas azotoformans, Oceanospirillum beijerinckii, Neptuniibacter Caesariensis, and Amphritea spongicola showed proportionally increase by temperature. The number of Endozoicomonaselysicola showed an increase up to 26ºC then decrease in 28 and 30℃ groups.

Keywords : Scleronephthya gracillimum, environmental impacts, OTUs

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Microbiome Analysis of the Deep Dea Stalked Barnacle, Neolepas marisindica from Onnuri Vent Field in Central Indian Ridge

Seonock Woo1, and Nayoung Lee2 Marine Biotechnology Research Center, KIOST, 2Risk Assessment Research Center, KIOST, Republic of Korea

All animals on Earth form associations with microorganisms, includingprotists, bacteria, archaea, fungi, and viruses. In the ocean, marine animal–microbial relationships has not been studied much like as terrestrial animals. The potential for microbiomes to influence the health, physiology, behavior, and ecology of marine animals could alter currentunderstandings of how marine animals adapt to change, and especiallythe growing climate-related and anthropogenic-induced changes already impacting the ocean environment. This new stalked barnacle, Neolepas marisindica, is morphologically distinct from the Pacific dwelling N. zevinae and N. rapanuii and was recently classified as follows: Arthropoda; Crustacea; Hexanauplia; Scalpellomorpha; Eolepadidae; Neolepas. We analyzed the microorganism composition of N. marisindica collected from Solitaire and the Onnuri Vent Field on the Central Indian Ridge in expedition of the research vessel ISABU (RVISABU). The first results were shown in this presentation to understand‘ who inside’ using the targeted small subunit (SSU) ribosomal RNA (rRNA) gene. The further investigation combined with function gene expression study of N. marisindica is undergoing to explain the key ofmetabolism of living, the features of hydrothermal vent animals with genes function analysis and also the information about host and symbiontcoadaptation and evolutionary process.

Keywords : Neolepas marisindica, onnuri vent field, the Central IndianRidge

N-57

Seasonal Microbiome Composition Change of Soft Coral and Seawater in the South Sea, Korea

Seonock Woo1, and Yejin Jo2 1Marine Biotechnology Research Center, KIOST, 2Risk Assessment Reseaerch Center, KIOST, Republic of Korea

The warm current running from tropical Philippines through subtropicalTaiwan to the temperate region Korea, the Kuroshio transfers heat fromlower to higher latitudes. It warmed most rapidly in 1981–1998, when sea surface temperatures rose by 1.5°C (0.9°C/decade), almost 7 timesthe global rate. This affected the physiological responses of corals as wellas the corals habitat range. We chose Eleutherobia rubra, a azooxanthellate soft coral around Korea to investigate the microbiomecomposition and to compare with the habitat seawater microbiome change along the year. This study focused on the identification of the microbiome from E. rubra and seawater from its habitat and its diversityand the microbiome composition change in four seasons. As results, Proteobacteria was mainly found with 53.5% and secondly Bacteroideteswith 9.9% from all coral samples and Proteobacteria with 47.6% and Bacteroidetes with 25.6% from seawater. The family, Endozoicomonadaceaewith 26% and Spirochaetaceae with 8.5% were found from the coral samples along the four seasons and the family, Flavobacteriaceae with16.4% and Pseudomonadaceae with 10.8% from seawater. Endozoicomonaselysicola was the species showing the highest count rate from the coralsamples and Psuedomonas gramnis was the species showing the highestcount rate from the seawater samples and any endozoicomonas specieswere found in the seawater samples.

Keywords : E. rubra, microbiome, South Sea

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O_Plant Microbiology

O-1

Nematicidal Activity of Aureothin Family Isolated from Actinomyces

Min-Kyoung Kang, Jong-Hoon Kim, Dong-Jin Park, Kwang-Hee Son*, and Chang-Jin Kim*

Industrial Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea

Plant parasitic nematodes are parasitic on all parts such as plant growthpoints, leaves, stems, and roots, and ingest cell fluid as nutrients throughbulbous needles. Parasitic nematodes can be classified into external parasitics, which are inflicted by the outside according to their infestationhabits, internal parasitics that enter the tissues and take up nutrients byinserting the head into the tissues. In domestic facility cultivation, the damage of root-knot nematodes is very severe. In the case of melon, thedetection rate is higher than 80%, and the detection rate is high and thedensity is high, so most of the fields cannot be cultivated without nematode control. There is also pine wilt disease (PWD), which is caused by pine wood nematode among plant diseases. Abamectin, a compoundcurrently used as a nematode control, is used in agriculture, but it is timeto take other measures due to problems such as water insolubility and resistance. Therefore, we tried to find compounds with nematicidal activity from actinomyces, which are known to produce many antibiotics. The active compounds were isolated from the selected strainsby screening for bactericidal activity against Bursaphelenchus xylophilus from actinomyces. The nematode active compound was isolated and purified from extracts from two strains. Compounds wereidentified and properties were confirmed through MS/MS and NMR analysis. It was confirmed that a total of three compounds had nematicidal activity, and it was confirmed that the structure is an aureotinderivative. Therefore, the efficacy of the strain extract containing the active compound was evaluated in greenhouse conditions. Since it showed excellent efficacy, it is suggested that it could be developed as a potential pesticide of aureotin-based compounds.

Keywords : Plant disease control, actinomyces, aureothin

[This research was supported by a grant (Project No. NRF-2013M3 A9A5076601) from the Ministry of Science, ICT and Future Planningof the Korea Government and was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) through (Project No. 321110-4 “Crop Viruses and PestsResponse Industry Technology Development”) Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)]

O-2

Identification and Characterization of a New Fungal Pathogen Causing Root Rot Disease of Gastrodia elata in Korea

Kang Minjeong, Eun-Kyung Bae, Chanhoon An, and Eung-Jun Park*

Forest Biotechnology Division, National Institute of Forest Science, Suwon 16631, Republic of Korea

Gastrodia elata (G.elata), a myco-heterotrophic orchid family (Orchidaceae), depends on two fungal partner: Mycena spp. and Armillaria mellea, seed germination and plant growth, respectively. Recently, more than 70% of G.elata has loss in the actual yield in Koreadue to root rot disease. In this study, we aim to identify the new fungalpathogen causing root rot of G.elata. We have observed necrotic spots of G.elata from Anseong (37˚0´N 127˚16´E), one of the largest G.elataproduction areas in Korea. Symptoms showed round and grayish brownspots, which eventually coalesced into larger black lesions on tubers. Thesix pure cultures were recovered from necrotic lesions of symptomatictubers. For fungal identification and characterization, fungal genomic DNA was isolated from tuber of G.elata, and amplified from two fungalloci, the internal transcribed spacer regions 1 and 4 and 5.8S nrDNA (ITS), and the translation elongation factor alpha (TEF-α). Three pathogenic fungi were identified the new fungal pathogen through phylogenetic analysis, morphological observation, and the pathogenicitytest, while other 3 fungi were identified and formally described as Clonostachys rosea. Therefore, these results may assist to make the proper method to control root rot disease in the fields of G.elata in Korea.

Keywords : Gastrodia elata (G.elata), Internal Transcribed Spacer (ITS), Translation Elongation Factor alpha (TEF-α)

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O-3

Isolation, Characterization and Culture Conditions for Multifunctional Biocontrol Agents against Phytopathogenic Botrytis cinerea and Ralstonia solanacaum

Sun-il Seo, Haeseong Park, Ji Hwan Lim, and Pyoung Il Kim*

Center for Industrialization of Agricultural and Livestock Microorganisms, Jeonbuk, Republic of Korea

Botrytis cinerea and Ralstonia solanacearum are critical pathogens which causes bacterial wilt and gray mold, respectively in various crops,including pepper, tomato, and potato. Once infected, it is very importantto block the infection early because the both pathogens rapidly spreadsto healthy crops. There are many microbial agents capable of controllingbacterial wilt and gray fungi, respectively, but simultaneous management strategies of mitigate both strains are very limited. In thisstudy, 500 strains were isolated and characterized from soil and the surface of plants for simultaneous controlling Botrytis cinerea and Ralstonia solanacearum infections. Firstly, evaluation of anti- pathogenic activity of the isolates was performed by using paper diskdiffusion assay. As a result, we found 3 bacterial isolates showing strongantifungal activity against the plant pathogens. 16S rDNA analysis revealed that they are members of Bacillus subtilis, Bacillus velezensisand Bacillus siamensis. Among three strains, B. subtilis JC-6 showedthe best anti-pathogenic activity. Especially, this strain showed simultaneous mitigation of Botrytis cinerea and Ralstonia solanacearum.Prior to agricultural field application, we investigated medium optimization for mass cultivation of B. subtilis JC-6 with low costs. 5carbon sources (glucose, sucrose, glycerol, maltose and soluble starch)and 5 nitrogen sources (soy bean flour, yeast extract, soytone, peptone and tryptone) were tested. The optimized carbon and nitrogen source wasglucose and yeast extract. As a result, B. subtilis JC-6 showed 2.5x109total cells/ml and the endospore cells were 2.0x109 under the optimizedculture conditions using 100L fermenter. It is obvious that B. subtilis JC-6 will be effectively used for multifunctional biological agent of bacterial wilt and gray mold disease.

Keywords : Botrytis cinerea, Ralstonia solanacaum, biocontrol agents

O-4

A Cocktail of Bacteriophage Suppresses Bacterial wilt in Tomato Plant and Alters Rhizosphere Microbiome Structure

Roniya Thapa Magar, Seung Yeup Lee, Hyo Jeong Kim, and Seon-Woo Lee*

Department of Applied Bioscience, Dong-A University, Busan 49315, Republic of Korea

Bacteriophages (phages) have been proposed as an alternative to pesticides to kill bacterial pathogens of plants. However, the effective strategy of phage biocontrol is variable and poorly understood. Here, wecharacterized phages infecting Ralstonia pseudosolanacearum, for theirbiocontrol efficacy and their effect on microbiome structure in tomato plants. Among 33 isolated phages, two distinct phages RpT1 and RpY2having comparable genomic and similar morphological features withinthe Autographiviridae family. Both phages had a broad host range and persisted in a wide range of natural environmental conditions such as various soils, temperatures, and pH over a prolonged period. Two phageswere treated in tomato plants at various times with several concentrations,either individually or in cocktail. Treatment of phages 3 days later of pathogen inoculation with 10-fold higher than pathogen density demonstratedbiocontrol activity against bacterial wilt both in cocktail and separately.Additionally, bacterial community analysis in the rhizosphere revealedthat the composition and abundance of phage-treated plants were alteredwith selective microbiota. Results suggest that the decreased disease incidence was explained by lysis of R. pseudosolanacearum in rhizospheresoil and possible enrichment of plant-beneficial bacterial species. Altogether,our results provide insights into developing the effective phage therapyto manage bacterial wilt and its effect on microbiome composition.

Keywords : Bacteriophage, biocontrol, Ralstonia pseudosolanacearum

O-5

Characterization of Newly Isolated Bacteriophage PhiPC01 to Control Pectobacterium carotovorum subsp. carotovorum

Jae Hyung Kim, Gio Kim, and Minsik Kim*

Department of Food and Nutrition, Brain Korea 21 FOUR, College of Human Ecology, Yonsei University, Seoul 03722, Republic of Korea

Pectobacterium carotovorum subsp. carotovorum is one of the most important phytopathogens causing bacterial soft rot. The disease bringshuge economic loss as it occurs on diverse crops such as carrot, cabbage, and potato during storage and distribution processes as well as cultivation. To control the pathogen, we newly isolated Pectobacterium-targeting bacteriophage PhiPC01 from sewage sampled from TancheonWater Treatment Center. Based on the morphological and genomic analysis, phage PhiPC01 having icosahedral head and short tail is assumed to be classified into the family Autographiviridae. Total 50 open reading frames (ORFs) were predicted from the 39.9-kb dsDNA genome, and most of the functional proteins were similar to other autographiviruses PPWS4, PP81, and PP47 infecting Pectobacterium spp.. PhiPC01 was able to infect not only P. carotovorum but also P. brasiliense, and stably retains its infectivity at a wide range of temperatures (4-50 ℃) and pH levels (5-11). Currently, we are tryingto evaluate other bacteriolytic properties of PhiPC01and its putative endolysin (ORF4) to develop them as useful tools in controlling phytopathogenic P. carotovorum.

Keywords : Bacteriophage, Pectobacterium carotovorum, phytopathogen

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O-6

Species Diversity of Endophytic Fungi Isolated from Root of Quercus spp. in the Limestone Area

Hyeok Park1,2, and Ahn-Heum Eom1* 1Department of Biology Education, Korea National University of Education, Cheongju 28173, Republic of Korea 2Department of Bioresource Collection, Honam National Institute of Biological resources, Mokpo 58716, Republic of Korea

Endophytic fungi live in all tissues of plant and do not cause disease symptoms. They secrete secondary metabolites such as alkaloids whichcan defend plants from herbivore or external pathogens. Recently, endophytic fungi are becoming more important because of the study results that these metabolites showed antimicrobial or anticancer effects.Limestone refers to a rock that contains at least 50% of the carbonate minerals. Soils formed from limestone differ in physical and chemicalproperties from non-limestone soil. In this study, we tried to confirm thedifference of endophytic fungal diversity according to the Ca2+

concentration and the pH level in the limestone area. We collected theroot samples from the rhizosphere of Quercus spp., and isolated the endophytic fungi from the roots. We observed the morphological characteristics of the fungal strains, and analyzed internal transcribed spacer (ITS) rDNA sequences. As a result, 48 fungal species from 29 genera were identified. We confirmed that the species diversity and thedominant species differ according to the Ca2+ concentration and the pHlevel of the soil. This result suggested that the soil environment of limestone areas can affect the endophytic fungal diversity in plant roots.

Keywords : Endophytes, fungi, symbiosis

O-7

Culturable Bacterial Composition of Rhizosphere Soil of Alpine Leek underneath Palmate Maple

Sung-Je Yoo, Eun Hye Na, Yong Hwa Jeong, and Shin Ja Park*

Future Agriculture Division, Sangju Agricultural Technology Center, Sangju 37154, Republic of Korea

Alpine leek (Allium victorialis) is a medical plant with anti-cancer andanti-oxidant functions and used as a food ingredient due to unique taste.It was reported that habitats of alpine leek was closely related with neighboring plants such as hardwood; ecological important value of alpine leek was higher in maple layer. Root-associated microbial composition was also affected by maple layer; out of these, some bacteria could play an useful role in plant growth promotion. In this study, weisolated bacterial strains from rhizosphere of alpine leek surrounded ornon-surrounded by palmate maple, and investigated their composition.As a result, we identified 21 strains from alpine leek grown in the bottom of palmate maple, the strains were belonging to different 11 genera including Streptomyces, Curtobacterium and Lelliottia, whereas, Pseudomonas (25%) and Bacillus (57.1%) were commonly abundant in rhizosphere soils of alpine leek regardless surrounding plants. Thesefindings demonstrated that bacterial compositions in rhizosphere of alpine leek were could be related to neighboring plant including palmatemaple. Further study, we will elucidate the effects of 21 strains on alpineleek growth and productivity.

Keywords : Alpine leek, palmate maple, identification

O-8

The Interaction between PGPB and Tannin on the Growth and Early Development of Plants and Its Implication on Tannin-Mediated Allelopathy in Temperate Forests

Jung-Ah Cho1, and Sang-im Lee2* 1College of Transdisciplinary Studies, School of Undergraduate Studies, DGIST, Daegu 42988, Republic of Korea 2Laboratory of Integrative Animal Ecology, Department of New Biology, DGIST, Daegu 42988, Republic of Korea

Tannins are highly abundant compounds in ecosystems and have long been assumed to be involved in allelopathic interaction among plants. Despite well-known importance of ecological interactions between plants and microbes, the potential role of tannin in mediating these interactions has not been well addressed. In this study, we investigatedthe influence of tannin on the growth of plant growth-promoting bacteria(PGPB) that are found under the oak and pinetrees and their plant-growth-promoting (PGP) effects. We isolated 67 bacteria in the soil under the oak and pine trees in South Korea, and selected eight speciesof PGPB and tested their combinatory effect with tannin on the early development and the growth of Arabidopsis thaliana. We found that thePGP effect in response to tannin depended on the species of PGPB. In particular, two species belonging to Lysinibacillus promoted the growthand early development of plants only when tannin was absent in the soil,whereas two species belonging to Paenibacillus showed visible PGP effect only when tannin was present. Our results suggest that tannin, anallelochemical, has a strong effect on plant-microbial interaction in a temperate ecosystem. The mechanism of species-specific effects of PGPB in response to tannin needs further investigation.

Keywords : PGPB, tannin, allelopathy

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O-9

The Ideal Stretagy for Powerful Antioxidant Producing Microalga Biomass (Haematococcus lacustris) Increment by Co-Cultivation with Key-Species of Bacterium Identified by Metagenomic Analysis

Sang-Ah Lee1,2, and Chi-Yong Ahn1,2* 1Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 2Department of Environmental Biotechnology, KRIBB School of Biotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea

A green unicellular alga (Haematococcus lacustris NIES144) has been known for its diverse beneficial carotenoid pigments, such as β-carotene, canthaxanthin, and astaxanthin. Specifically, carotenoid pigments such as β-carotene (BC), canthaxanthin (CX), and astaxanthin (AX) synthesized by microalgae are attracting international attention asthe world is becoming an aging society. These pigments are used as functional food of antioxidants and anti-aging for humans. They are produced also by plants and fish, but not suitable for foods due to soil contamination and heavy metal accumulation in top predator fish. However, in the case of microalgae, high biomass could be obtained ina small-scale incubator, and it is safer from heavy metals or contaminations. BC, CX, and AX are biosynthesized by different representative microalgae strains. Dunaliella salina was recognized first as producing a high concentration of intracellular BC. Chlorosarcinopsis and Dactylococcus produce CX as a strong antioxidant material. AX, a powerful antioxidant, is also produced by H. lacustris under stress conditions such as nutrients depletion. H. lacustris use the phytoene as an AX precursor produced by the methylerythritol phosphate pathway in the plastids and mevalonate terpenoid pathway cytoplasm. Therefore, H. lacustris can produce various kinds of pigments, thereby being an industrially valuable candidate for diverse antioxidation raw materials without complicatedprocesses. During carotenoid biosynthesis in microalgae, the normal algal plastids are degraded and the chlorophyll-a content is decreased. In this way, diverse carotenoid pigments are produced at different compositions, depending on algal strains or conditions. However, the slow growth of this alga is the major obstacle for producing the industrialscale of astaxanthin. To solve this weak point, algal growth-promotingbacterial strains that co-existed in the algal phycosphere were identifiedaccording to the NGS metagenomics analysis. This study focused on defining algal growth positive bacteria community modules, and majorabundant taxa that had a positive correlation with algal growth were isolated (Sphingomonas hankookenesis, Paenarthrobacter ureafaciens,and three species of Microbacterium sp.) and each of algal growth-promoting effect patterns were evaluated. For the astaxanthin industrialization, this metagenomic data analysis will give solving the ideal strategy for the slow growth of H. lacustris some insight.

Keywords : NGS-metagenome, Haematococcus lacustris, Key-role species

O-10

Comparative Proteomic Analysis of Erwinia amylovora Cultured in Three Different Conditions

Sang-Wook Han, Lynn Heo, and Jongchan Lee Department of Plant Science and Technology, Chung-Ang University, Anseong 17546, Republic of Korea

Erwinia amylovora is a Gram-negative plant pathogenic bacterium causing fire blight disease on apple and pear. Although the disease is a serious threat to Rosaceae plants worldwide, in 2015, the disease was firstly reported in Korea. Therefore, most studies regarding the virulenceof the bacterium have been focused on foreign strains. Here, we report differentially abundant proteins from Korean strain TS3128 isolated from Anseong grown three different conditions: LB (rich medium), HMM (hrp inducing medium), and MBMA (amylovoran inducing medium). Using a label-free shotgun proteomic analysis, a total of 969,998, and 1033 proteins were commonly detected in the three biologicalreplicates cultured in LB, HMM, and MBMA, respectively, and were subjected to comparative analysis and clustering the orthologous groups(COGs) classification. In LB vs. HMM, the abundance of proteins belonging to groups E, F, H, J, M, and P were mostly changed. Specifically, proteins related to the type III secretion system and iron-related proteins were identified. In the second set (LB vs. MBMA),the expression of proteins associated with amylovoran production and carbohydrate metabolism were changed. In addition, many proteins related to cell wall/membrane/capsule, including TonB-dependant receptors and porins, were also found. In HMM vs. MBMA, the numbers of proteins whose abundance was changed were lower than the other twosets (LB vs. HMM and LB vs. MBMA). These results lead to new insightsinto understanding biological and cellular mechanisms that responded to different conditions. It also provides fundamental information to elucidate virulence functions in E. amylovora isolated in Korea.

Keywords : Fire blight, proteomics, Erwinia amylovora

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O-11

Optimal Culture Conditions of Antagonistic Bacillus thuringiensis BC-046 against Gray Mold caused by Botrytis cinerea on Ginseng

Jae Hyoung Yi, Hak Kyu Kim, Young Moon Mo, Ki Uk Lee, and NamYong UmGinseng and Medicinal Plants Research Institute, Gangwon ARES, Cheorwon 25054, Republic of Korea

Gray mold disease is one of the major diseases of ginseng. We selectedBacillus thuringiensis BC-046, a gray mold antagonist, in a previous study. The control of gray mold disease was mainly chemical control using disinfectants. However, research on biological control has been actively conducted in recent years due to the occurrence of resistant strains and environmental pollution problems due to the abuse of chemical pesticides. Therefore, for the development of an antagonist microbial agent, the culture characteristics of the BC-046 strain were tested. The growth curve of BC-046 was measured, and the time of thestationary phase was confirmed based on the growth curve. The culturecharacteristics of BC-046 were measured for growth according to the influence of the optimum medium, temperature, stirring speed, pH, carbon source, carbon source concentration, nitrogen source, nitrogen source concentration, and inorganic salts. As a result of setting the optimal culture conditions of BC-046, the growth induction period wasshorter than that of the existing culture conditions, and the cell mass increased by 42% compared to the previous one after 10 hours of culture.This result is thought to be helpful in establishing a mass production system for the practical use of BC-046. In addition, it is thought that thereis a need for follow-up studies, such as selection of cryoprotectants forlong-term storage and antagonistic activity.

Keywords : Ginseng, gray mold antagonist, optimal culture conditions

O-12

Establishment of Fire Blight (Erwinia amylovora) Inoculation Conditions for Each Part of Apple Seedling

Moran Lee, Hyeju Jeong, Jihee Han, and Jaekyeong Song*

Agricultural Microbiology Division, National Institutes of Agricultural Sciences, RDA, Wanju-gun 55365, Republic of Korea

The injuries caused by the fire blight pathogen, Erwinia amylovora, make not only the current crop year’s products but also the pear and appleindustries extremely destructive and dangerous in a region or country. Management of fire blight is difficult by limitations on use of antibioticsin agriculture, development of antibiotic resistance, and limited efficacyof alternative control agents. Infections commonly occur during blooming or on late blooms during the three weeks following petal fall. Many use of highly susceptible rootstocks to fire blight, such as Malling 9, Malling 26, etc. has increased the danger that infected blocks are ableto suffer serious damage. Microorganisms offer the further prospect forapplication to improve the plant protection because they are generally recognized as “natural” compounds able to influence the safety and quality of fruits. We performed biological assay to find inoculation conditions for each part (flower, branch, leaf, seedling) of M9 and Crabapple (Malus prunifolia) seedlings prior to the useful microorganism experiment. As a result, the optimal inoculation concentration of E. amylovora for bioassay with flowers, branches, leaves and seedlings wasfound to be 106 CFU/ml. The result showed that this bioassay can be used as basic data for biological analysis to control fire blight pathogens usingeffective microorganisms.

Keywords : Fire blight, apple, Erwinia amylovora

O-13

The Cooperation between Flavobacterium sp. and Nnative Rhizosphere Microbiota Enhance the Growth of Tomato Plant

Jong Uk Son, Eunjin Kim, Sang-Moo Lee, Aryan Rahimi, Pyeong An Lee,Hyoung Ju Lee, Kihyuck Choi, and Seon-Woo Lee*

Department of Applied Bioscience, Dong-A University, Busan 49315, Republic of Korea

The rhizosphere microorganisms affect plant fitness such as plant growthand disease resistance. Previously, we showed that a Flavobacterium sp.TCH3-2 strain isolated from tomato rhizosphere soil promote tomato growth with indigenous microbial community, but not by alone. In thisstudy, we investigated the effect of changes in rhizosphere and endospheric microbiome by TCH3-2 on tomato growth. The 16S rRNAgene amplicons were sequenced by MiSeq sequencer and analyzed usingQIIME2 pipeline. The 16S rRNA amplicon sequencing data showed thatthere was significant difference between the TCH3-2 treated and untreated control group in tomato rhizosphere and endosphere microbiome at 3- and 5-weeks post inoculation. To validate microbiomedata, we constructed a synthetic community (SynCom) with several bacterial isolates from tomato rhizosphere soil and assessed the plant growth-promoting activity of TCH3-2 and SynCom in sterilized nurserysoil. The inoculation of TCH3-2 or SynCom did not showed plant growth-promoting activity, but the mixed treatment significantly increased the growth of tomato. Intriguingly, the treatment of heat-killedTCH3-2 with SynCom failed to promote the growth of tomato plants. In addition, we found that the plant growth-promoting compound are secreted by the combination of SynCom and TCH3-2 using a hydroponic system. Taken together, these data showed that Flavobacterium sp. TCH3-2 strain might enhance plant growth by collaborating with beneficial rhizosphere microbiota.

Keywords : Flavobacterium sp., PGP, SynCom

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O-14

Characterization of Potential Plant Growth-Promoting Rhizobacteria as Biological Agents with Antifungal Activity, Plant Growth-Promoting Activity, and Mineral Solubilizing Activity

Song Min Lee1, Ji-Youn Kim1, Hee Sook Kim1, Ka-Yoon Oh1, Sang-HyeonLee2, Jeong Su Jang1*, and Kwang Hui Lee1 1Food Research Center, Angel Co., Ltd., Busan 46988, Republic of Korea 2Department of Pharmaceutical Engineering, Silla University, Busan 46958, Republic of Korea

The purpose of this study is to confirm the antifungal activity, plant growth-promotion activity, and mineral solubilizing activity of 18 typesof bacteria isolated purely from rhizosphere soil. The potential of isolatesof the genus Bacillus and Pseudomonas as biocontrol agents was confirmed through the antifungal activity of these isolates. This activitywas judged to be due to the activities of various hydrolytic enzymes onthe cell wall of plant pathogenic fungi and the activity to produce siderophores. In addition, most of the isolates have been found to haveACC deaminase production activity, IAA production activity, and nitrogen fixation activity. These characteristics are believed to have a positive effect on root development and growth, and productivity of cropsby reducing the concentration of stress ethylene under environmental stressconditions in which plants are commonly exposed. In addition, as a resultof confirmation test for the solubilizing activity of the isolates for phosphoric acid, silicon, calcium carbonate, and zinc, some isolates werefound to have mineral solubilizing activities. Inoculation of these isolates during plant growth is expected to help plant growth by converting nutrients necessary for plant growth into usable forms that can be absorbed by plants. The availability of 18 isolated strains as a biocontrol agent was suggested through the results of antifungal activity, plant growth-promotion activity, and mineral solubilizing activity.

Keywords : Plant growth-promoting rhizobacteria, mineral solubilizingactivity, biocontrol agent

O-15

Improvement of Biomass and Lipid Production under Light Emitting Diodes (LEDs) Wavelength of Tetraselmis gracilis

Sang baek Lee, Sung-Koo Kim*, Gwi-Taek Jeong, So Hee Kim, and UiHun LeeSchool of Marine, Fisheries and Life Science, Pukyong National University, Busan 48513, Republic of Korea

Microalgae are one of the means to reduce the concentration of carbondioxide in the atmosphere by absorbing carbon dioxide, the main greenhouse gas in the atmosphere. Microalgae can be used as a feedstockfor products such as food, fed and cosmetics. It has also been consideredas a promising feedstock for biofuel production. LEDs have advantagesthat are different from other artificial light sources such as small size oflight source, excellent energy efficiency, and being able to irradiate onlyspecific wavelengths. Therefore, it is suitable for culturing microalgae that grows efficiently at a specific wavelength. Experiment was conducted with a two-phase culture system. The first phase experimentoptimized nitrate concentration and photoperiod. Nitrate concentration was set at 80, 160, 240 and 320 mg L-1. As a result of the experiment,T. gracilis produced 0.45, 0.63, 0,72 and 0.84 g dcw L-1 of biomass, respectively, and the productivity (dcw/day) was the highest at 160 mg/L.The cultivation period could be shortened through the photoperiod (12:12, 18:6 and 24:0 h light/dark cycle) experiment. The second phaseimproves lipid content of microalgae by applying the LED wavelength,nitrate depletion, pH and salt.

Keywords : Microalgae, lipid, light emitting diodes

O-16

The Effect of Light-Emitting Diodes (LEDs) Photoperiod and Light Intensity on Growth and Lipid Production of Chlamydomonas hedleyi

Uihun Lee, Sung-Koo Kim*, Gwi-Taek Jeong, So Hee Kim, and Sang Baek Lee School of Marine, Fisheries and Life Science, Pukyong National University, Busan 48513, Republic of Korea

Microalgae are attracting attention as an alternative to grain biofuels dueto their high lipid productivity, fast growth rate, and non-edible resources. Without competing with food and feed crops, and growing awareness of global energy issues, microalgae are now at the forefrontof research as a sustainable and environmentally friendly alternative. It is the next generation biomass with almost no restrictions on arable landwith higher oil yields than currently available agricultural crops. In this study, to find the optimal LED photoperiod and light intensity, the LEDphotoperiod was set to 12:12 h, 18:6 h and 24:0 h light/dark cycle andlight intensity was set to 300, 400, 500 and 600 µmolm-2s-1. The maximumefficiency is achieved by dividing the biomass production phase (the first phase using red LED, 625 nm) and the lipid production phase (the secondphase using green LED, 520 nm). In the first phase, C. hedleyi producedmaximum biomass about 0.95 g dcw/L at 10 days under the 24:0 h light/dark cycle and 500 µmolm-2s-1. In the second phase, the highest lipidproduction about 41.3% was obtained in the culture after exposing to green LEDs under the 12:12 h light/dark cycle and 400 µmolm-2s-1 at second phase day 2.

Keywords : Microalgae, light-emitting diodes, light conditions

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O-17

Enhancement of Growth and Lipid Production of Pavova lutheri Using Phytohormones at LED Wavelength in 60 L Bioreactor

So Hee Kim, Ui Hun Lee, Sang Baek Lee, Gwi-Taek Jeong, and Sung-KooKim*

School of Marine, Fisheries and Life Science, Pukyong National University, Busan 48513, Republic of Korea

Microalgae have been focused as a possible source of new generationbiofuels because of fast growth, high biomass productivity and high lipidaccumulation. Furthermore, long chain fatty acids found in microalgaehave important functions as health supplements. Plant growth regulators(PGRs) can contribute to plant growth and development. PGRs are chemical messengers involved in a broad spectrum of physiological andbiochemical processes of higher plants at very low concentration. In thisstudy, first phase culture was optimized for each concentration using of 2, 4-dichlrophenoxyacetic acid (2, 4-D), indole-3-acetic acid (IAA), and1-naphthalene acetic acid (NAA). And auxin-based PGRs 2, 4-D, IAA, and NAA were each mixed with gibberellic acid (GA) and cultured forsynergistic effects. After that, lipid accumulation by the concentration of abscisic acid (ABA) was optimized in the second phase culture. Afteroptimizing the type and concentration of phytohormones, photoperiods 12:12, 18:6, and 24:0 h were optimized. The optimized condition wasa mixture of 5 mg/L of 2, 4-D and 20 mg/L of GA, and the photoperiodwas 24:0 h light and dark cycle. At this time, the biomass reached 1.16g/L, produced 0.54 g/L higher biomass compared to the control withoutusing the phytohormones, and the cultivation period was also shortenedby 5 days.

Keywords : Microalgae, phytohormones, light-emitting diodes

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P_Protein Engineering and Evolution

Poster S

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P-1

Characterization of Cold-Adapted Protease from Janthinobacterium livid PAMC 25641

Dongheon Lee, and Jong-il Choi*

Department of Biotechnology and Bioengineering, Chonnam National University, 77 Yongbong ro, Buk gu, Gwangju 500 757, Republic of Korea

Because psychrophilic microorganisms have adapted to cold environments,enzymes are also particularly active at moderate and low temperatures.The cold-adapted protease produced by Janthinobacterium lividumPAMC 25641 was extracted using anion-exchange chromatography, with specific activity of 264 U/mg and an overall field of 12.5% and 110kDa. Optimal pH range and temperature was 7.0-7.5 and 40℃. K+ and Na+ metal ions stimulated enzyme activity with 118% and 115% respectively. Enzyme activity was lowered by PMSF, while it has not been affected by DTT, Tween-20, ethanol and EDTA. Protease gene wasinserted into the pET-28a(+) and transformed to E.coli BL21(DE3). Itsheterologous protease from soluble fraction of the cell lysate had 17.22U/mg enzyme activity.

Keywords : Janthinobacterium lividum PAMC 25641, cold-adapted protease

P-2

Enzymatic Depolymerization of Ulvan by Using Ulvan Lyase from Marine Microbes for Effective Low Molecular Weight Ulvan Production

Yewon Jo, and Jeong eun Hyeon*

Department of Food Science and Biotechnology, College of Knowledge-Based Services Engineering, Sungshin Women's University, Seoul 01133, Republic of Korea

Ulvan, a polysaccharide derived from marine algae, is structurally similar to mammalian glycosaminoglycans, so it is used in biomedicalresearch and medical applications. The biological activity of green algalpolysaccharides is affected by its molecular weight. Compared to highmolecular weight ulvans, low molecular weight ulvans exhibit more reactivity, higher solubility and easier absorption. Therefore, a low molecular weight ulvan depolymerization method is required to obtainuseful substances. In this study, we did several designs to enzymaticallydepolymerize ulvan. First, ulvan lyase, which acts on the initial decomposition of ulvan polysaccharides, was constructed as an enzyme complex by dockerin-cohesin interaction. And then, green algae were macerated with 2% cellulase and incubated at 55℃, and lyophilized ulvan polysaccharide was extracted by adding 1% hydrogen peroxide and reacted with the enzyme complex. As a result, the ulvan lyase enzymecomplex constructed by the dockerin-cohesin interaction depolymerizedulvan 1.8 times more than the control group. This ulvan depolymerizationmethod can be used to develop pharmaceutical technology using variousproperties of low molecular weight ulvan such as anti-coagulation, antioxidant, and anti-tumor.

Keywords : Ulvan lyase, ulvan depolymerization, dockerin-cohesin

P-3

Enhanced Production of Multifunctional Oligosaccharide through Porphyran Degradation by Construction of Artificial Enzymes from Marine Microbes Derived

JooHee Han and Jeong Eun Hyeon*

Department of Food Science and Biotechnology, College of Knowledge-Based Services Engineering, Sungshin Women's University, Seoul 01133, Republic of Korea

Porphyran, a polysaccharide composed of red algae, is a source of a multifunctional oligosaccharide material and new biomass raw materials with various physiological activities. The breakdown of porphyrans into oligosaccharide is a process that provides excellent andpromising alternative resources, which is obtained by the glycolysis using various porphyranolytic enzymes in several stages. In this study,porphyran was extracted by grinding, sterilization, ethanol reaction, extraction, heat treatment and drying method from porphyra Yezoensis, and extracted porphyran was used as a substrate for the study. In addition,we have established a method for efficient hydrolysis through an enzymecomplex obtained through cohesin-dockerin interaction that degrades natural algal polysaccharides. The cohesin-dockerin interaction is designed by genetically binding dockerin module to the end of an existing enzyme, and then attaching cohesin module to obtain a protein complex.The designed protein complex has been shown to approximately 2.0 times its activity on the substrate, which can be considered as a usefulway to obtain efficient oligosaccharides or monosaccharides through hydrolysis of red algae.

Keywords : Porphyran, new biomass raw materials, multifunctional oligosaccharide material

P-4

Improved Antibacterial Efficacy of Endolysins Through Protein Engineering

Yongwon Jung, Jonghwi Kim, Taehwan Jeong, and Heejoon Myung*

Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yong-In, Mo-Hyun, Gyung-gi Do 17035, Republic of Korea

Endolysin is a bacteriophage-encoded enzyme that degrades bacterial cell wall when a bacteriophage bursts out in lytic replication cycle. While wild-type endolysins are promising, protein engineering offers many opportunities to increase its enzyme activity. In this study, we engineeredthree types of endolysins. We constructed chimeric endolysins throughfunctional domain shuffling and fusion with two types of antimicrobialpeptide (AMP), KR12 or Cecropin A. An endolysin typically consists of enzymatically active domain (EAD) and cell wall binding domain (CBD). It is known that LysAm24 from phage AM24 infecting Acinetobacter baumannii has a broad bactericidal activity against variousGram-negative bacteria. So, we constructed a chimeric endolysin composed of CBD from LysAm24 and EAD from a Pseudomonas aeruginosa phage PBPA90. An endolysin’s efficacy might be hampereddue to the presence of outer membrane in Gram-negative bacteria. SinceAMPs destroy outer membrane, we fused KR12 with endolysin PA90and Cecropin A with endolysins 10-24(13), PBEC30 and PBEC56. Theengineered endolysins showed improved antibacterial efficacy againstvarious Gram-negative pathogens including P. aeruginosa, Klebsiella pneumoniae, A. baumannii, and Escherichia coli.

Keywords : Engineered endolysin, domain shuffling, antimicrobial peptide

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P-5

Heterologous Expression of Human Glutamine Transporters in Escherichia coli

Eyoung kim1, and Ok Bin kim1,2,3* 1Department of EcoScience, Ewha Womans University, Seoul 03760, Republic of Korea 2Interdisciplinary Program of EcoCreative, Ewha Womans University, Seoul 03760, Republic of Korea 3Department of Life Science, Ewha Womans University, Seoul 03760, Republic of Korea

There have been many reports on the correlation between amino acid transporters and diseases. Especially, glutamine is central role in growthof cancer cells. Human glutamine transporter SLC1A5 (solute carrier family 1 member 5) has 3 variants (var1, var2, var3). Among them, thevar3 was shown to promote cancer growth by expressing it in the mitochondria of cancer cells. There are several difficulties to express SLC1A5 in E. coli. The problem that lies codon bias can be solved by pRARE2 plasmid which supplies tRNAs for 7 rare codones (AGA, AGG,AUA, CUA, GGA, CCC, and CGG). We used four diverse vector systems with different advantages: pET30b, pQE80L, pBAD30, pColdI. The pET system is one of the most powerful system but is only applicablein BL21 series due to T7 promoter. In contrast, the other 3 vectors can be easily used with any E.coli host strain. The pQE system combines apowerful T5 promoter (recognized by E. coli RNA polymerase) with adouble lac operator repression module to provide tightly regulated, high-level expression. In lac promoter system there could be leaky expression. In contrast, pBAD system has araPBAD promoter which need arabinose absolutely for induction. And pBAD30 is low-copy plasmid due to the p15A ori, and it helps with proper expression of membrane proteins. pColdI has cold shock gene cspA promoter which only can be transcripted below 15℃. In pCold system, The synthesis ofhost-derived proteins is suppressed and the POI(protein of interest) can be obtained at a low temperature. pBAD30 vector system is inducible by arabinose and controlled by catabolite repression. In contrast, the other vectors have T7 promoter (pET30b), T5 promoter (pQE80L), andcspA promoter (pColdI), which do not require active CRP to start transcription. So culture media for expression of SLC1A5 cloned in pBAD30 should be prepared without glucose. The final obstacle to using pBAD30 vector was that it has same ori with pRARE2: p15A. Since pRARE2 has to be used for expression for all vector system, the ori p15Aof pRARE2 was replaced to ori pBR322.

Keywords : SLC1A5, vector systems

P-6

Metal-Mediated Protein Assembly Using a Genetically Incorporated Metal-Chelating Amino Acid

Sooin Kim, Hyun Soo Lee*, Sanggil Kim, and Hyunjung YooDepartment of Chemistry, Sogang University, 35 Baekbeomro Mapogu, Seoul 04107, Republic of Korea

Many natural proteins function in oligomeric forms, which are critical for their sophisticated functions. The construction of protein assemblieshas great potential for biosensors, enzyme catalysis, and biomedical applications. In designing protein assemblies, a critical process is to create protein–protein interaction (PPI) networks at defined sites of a target protein. Although a few methods are available for this purpose, most of them are dependent on existing PPIs of natural proteins to someextent. In this report, a metal-chelating amino acid, 2,2′-bipyridylalanine (BPA), was genetically introduced into defined sites of a monomeric protein and used to form protein oligomers. Depending on the number of BPAs introduced into the protein and the species of metal ions (Ni2+and Cu2+), dimers or oligomers with different oligomerization patternswere formed by complexation with a metal ion. Oligomer sizes couldalso be controlled by incorporating two BPAs at different locations withvaried angles to the center of the protein. When three BPAs were introduced, the monomeric protein formed a large complex with Ni2+. In addition, when Cu2+ was used for complex formation with the protein containing two BPAs, a linear complex was formed. The method proposed in this report is technically simple and generally applicable tovarious proteins with interesting functions. Therefore, this method would be useful for the design and construction of functional protein assemblies.

Keywords : Peptides and proteins, metals, oligomers

P-7

Conversion of Racemic Unnatural Amino Acids to Optically Pure Forms by a Coupled Enzymatic Reaction

Sooin Kim, Hyun Soo Lee*, Hannae Lee, and Dongchan Kim Department of Chemistry, Sogang University, 35 Baekbeomro Mapogu, Seoul 04107, Republic of Korea

Genetic code expansion (GCE) technology is a useful tool for the site-specific modification of proteins. An unnatural amino acid (UAA)is one of the essential components of this technique, typically requiredat high concentration (1 mM or higher) in growth medium. The supplyof UAAs is an important limitation to the application of GCE technology,as many UAAs are either expansive or commercially unavailable. In thisstudy, two UAAs in a racemic mixture were converted into optically pure forms using two enzymes, the d-amino acid oxidase (RgDAAO) fromRhodotorula gracilis and the aminotransferase (TtAT) from Thermus thermophilus. In the coupled enzyme system, RgDAAO oxidizes the d-form of UAAs in a stereospecific manner and produces the corresponding α-keto acids, which are then converted into the l-form ofUAAs by TtAT, resulting in the quantitative and stereospecific conversion of racemic UAAs to optically pure forms. The genetic incorporation of the optically pure UAAs into a target protein produced a better protein yield than the same experiments using the racemic mixtures of the UAAs. This method could not only be used for the preparation of optically pure UAAs from racemic mixtures, but also thebroad substrate specificity of both enzymes would allow for its expansionto structurally diverse UAAs.

Keywords : Genetic code expansion, unnatural amino acids, D-amino acid oxidases

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P-8

Synthesis of γ-valerolactone with High Enantiopurity Via a Two-Step Chemoenzymatic Conversion of Levulinic Acid

Yurim Kim1, Hae Ji Jeon1, Jiyeoung Jeong1, Gyulim Oh1, Dohoon Lee2, and Young Joo Yeon1* 1Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea 2Green Chemistry and Materials Group, Korea Institute of Industrial Technology (KITECH), Cheonan 31056, Republic of Korea

Conversion of levulinic acid to enantiomerically pure γ-valerolactone is important for applications in green solvent, fuel and flavor industries.Levulinic acid is obtainable from cheap, abundant and environmentallyfriendly sources such as cellulosic biomass. Currently, there have been difficulties in producing (R)-γ-valerolactone with a high yield and enantiopurity from levulinate. In this study, levulinic acid was transformed into (R)-γ-valerolactone through (R)-4-hydroxyvaleric acid in two chemoenzymatic steps. For the first step, engineered 3-hydroxybutyrate dehydrogenase (e3HBDH) catalyzed levulinic acidreduction to (R)-4-hydroxyvaleric acid. This was followed by the secondstep where 1% sulfuric acid lactonized the (R)-4-hydroxyvaleric acid to(R)-γ-valerolactone. In conclusion, (R)-γ-valerolactone was synthesizedwith approximately 100% yield and >99% enantiomeric excess from thefree form of levulinic acid. References 1. Dohoon Lee, Young Joo Yeon,Enantioselective chemoenzymatic synthesis of (R)-γ-valerolactone from levulinic acid (2020), Process Biochemistry, 90, 113-117

Keywords : Chemoenzymatic conversion, levulinic acid, (R)-γ-valerolactone

P-9

Intervening Variable Sequence Regions of Homologous Sugar Isomerases Define Thermal Adaptation Traits

Jae Yoon Sung1, Keunyoung Yoo1, Yunhye Joo1, Sun-Mi Shin2, Sang-Jae Lee3, Immanuel Dhanasingh4, Ngoc Kim Quyen Hoang4, Thịnh-Phát Cao4, Seong-Bo Kim5, Sung Haeng Lee4, and Dong-Woo Lee1* 1Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 2School of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea 3Major in Food Biotechnology, Silla University, Busan 46958, Republic of Korea 4Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju 61452, Republic of Korea 5Department of Bio-Living Engineering, Yonsei University, Seoul 03722, Republic of Korea

Hyperthermophilic enzymes share similar structural folds with mesophiliccounterparts. Nevertheless, they show extraordinary resilience and robustness to stability. We hypothesized that homologous recombinationof core metabolic enzymes could efficiently generate stable offspringsin extreme environments. To understand the contribution of interveningvariable sequences of a core metabolic enzyme to its intrinsic and extrinsic properties, here we generated 625 chimeras using homologousrecombination from five distantly-related L-arabinose isomerases (AIs) from extant origins. We chose 78 properly folded chimeric AIs with altered temperature and pH optima for stability and determined the three-dimensional crystal structures of hyperthermophilic and chimericAIs. The fitness landscape integrated with intermolecular interaction- energy analysis revealed adaptive genetic traits involving intra-subunitsalt bridges and inter-subunit metal-binding sites. Those residues responsible for the thermostability of AIs are exclusive in variable sequence regions. Interspecific displacement of region-specific variablesequences through homologous recombination may be exploited to provide an unprecedented repertoire of homologous enzymes to environmental fitness.

Keywords : Recombination, self-assembly, environmental adaptation

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P-10

The Influence of Two Short Loop on the Thermostability of Pullulanase Type I from Extremophilic Bacteria Deinococcus radiodurans

Seul-Ki Yang1, Min-Kyu Kim1, Sangyong Lim1, Cheon-Seok Park2, andJong-Hyun Jung1* 1Radiation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea 2Graduate school of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 17104, Republic of Korea

Commercial α-amylases in industrial processing are required thermostability.Deinococcus radiodurans is a extremophile and their proteins showedextraordinary stability although their optimum growth is 30℃. DrPul is pullulanase in D. radiodurans, its optimum temperature was 55-60℃but strongly reduced its stability (half-life, 1.5 min). Multiple alignmentanalysis showed that Drpul had metal binding site. In the presence of metal ions, its Tm value was shifted and exhibited 182 fold higher stability at 45℃ compared to the without calcium ion. The structural analysis indicated that metal ion was coordinated by two short loops. Toreveal the effect of loop on the stability Asp574, Tyr576, Asn917 residueswere replaced to Ala. The mutants commonly showed low binding affinity toward metal ion and stability was not boosted by calcium ion. Interestingly, when both loops were tightly linked by disulfide bond, thehalf-life of the mutant was 3.6 fold higher than wild type in the absenceof calcium ion at 45℃. It supported that the immobilization of the loopscould be a effective method for thermostability improvement.

Keywords : Thermostability, metal binding site, loops

P-11

Vector Optimization for Soluble Expression of Dengue Viral Non-Structural Protein 1 in Escherichia coli

Eun-Jeong Kim, and Soo-Im Shin*

Department of Biotechnology and Bioengineering, College of Engineering, Chonnam National University, Gwangju 61186, Republic of Korea

Non-structural protein 1 (NS1) of dengue virus is one of the factors thatdevelop the diseases of severe dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF) during the viral pathogenesis. However, the mechanism of NS1 in the progression of the disease is barely understood. Besides, efficient processes for the production of recombinant NS1 as a soluble form have not been established. In thisstudy, we constructed soluble NS1 expression systems using two different vectors. We genetically engineered 6xHis-tagged NS1 gene within pUC19 or pET11a and expressed it in Escherichia coli system,which is widely used and easily scaled-up. Notably, to acquire soluble NS1, we fused the periplasm targeting signal sequence of amicyanin protein that is encoded by mauC gene from Paracoccus denitrificans, at the end of the N-terminus of NS1. The two expression systems withdifferent vectors are assessed for their soluble protein production abilityand confirmed expression of NS1 protein in the periplasm in E-coli. Ourwork will further help to find the role of NS1.

Keywords : Recombinant NS1, protein expression optimization

P-12

Biochemical Identification of a New 1,3-α-3,6-anhydro-L-galactosidase Involved in Agar Degradation in Streptomyces coelicolor A3(2

Maral Tsevelkhoroloo, Soon-Kwang Hong*, and Chang-Ro Lee*

Department of Biological science, Myongji University, Yongin-Si, Gyeonggi-do 17058, Republic of Korea

The putative lacZ super family protein, SCO3479, from an agar degrading bacterium Streptomyces coelicolor A3(2) was characterizedbiochemically. The SCO3479 was annotated as beta-galactosidase andexpected to be involved in agar hydrolysis based on in silico analysis. The DNA fragment (2988-bp) encoding the putative β-galactosidase (NP_627681.1) was cloned into pET28A(+) vector and expressed in E.coli BL21(DE3). The expressed SCO3479 (995 aa) was estimated as amonomeric protein with MW of 108 kDa by gel-filtration chromatography.The SCO3479 protein had α-neoagarobiose hydrolase (NABH) activity,cleaving α-(1,3) linkage of neoagarooligosaccharides to produce 3,6-anhydro-L-galactose and D-galactose. The optimal condition for SCO3479 was pH 6 and 30℃. The enzyme activity was enhanced by MgCl2 and NaCl. SCO3479 also hydrolyzed neoagarotetraose into 3,6-anhydro-L-galactose and neoagarotriose, and NA6 into 3,6-anhydro-L-galactose and neoagaropentaose, indicating that SCO3479 is a neoagarooligosaccharide hydrolase enzyme, cleaving α-1,3-glycosidic linkages and thus releasing 3,6-anhydro-L-galactose from the non-reducingend of various NAOSs.

Keywords : Agar, Streptomyces coelicolor,, beta-galactosidase

[Supported by " Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2020R1F1A1060789)]

P-13

Binding of a Nanodisc to Antibody Converts Antiviral Mode-of-Action of Broad Neutralizing Antibody against Influenza Viruses

Jaehyeon Hwang, Younghun Jung, Seokoh Moon, Hyunseok Oh, SoominKim, Kyeong Won Kim, Jeong Hyeon Yoon, and Dae-Hyuk Kweon*

Departmnent of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea

Many studies have been conducted on broadly neutralizing antibodies (bNAbs) against influenza viruses. However, these virustatic antibodieslacking virucidal activity have shown limited antiviral potency. Here, we developed a nanodisc providing the immunoglobulins with virucidalefficacy by rupturing the viral envelope, resulting in augmentation of antiviral effects. Nanodiscs carrying a Fc-binding peptide sequence bound to bNAbs targeting stem region of hemagglutinin (HA) were co-endocytosed into host cells. At low pH in the endosome, the antibody-nanodisc conjugate (ANC) inhibited HA-mediated membranefusion due to the antibody’s HA binding while viral envelope was disrupted by the HA that evaded arrest by antibody through nanodisc-mediated virus perforation. Antiviral activity of bNAb was amplified due to the dual mode-of-action of ANC. Administration of theANC, especially with a large nanodisc, reduced morbidity and mortality compared with bNAbs alone in mouse models. Our results suggest thatthe bNAbs with a virustatic mode-of-action could be converted a virucidethrough conjugation to nanodisc.

Keywords : Broadly neutralizing antibodies, nanodiscs, influenza viruses

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P-14

Production of Recombinant GPCR Human Sweet Taste Receptor and Interaction with Sweeteners by Docking Simulation

Gihyeon Chae, Si-Wook Jang, Na-Hee Jung, Hyun-A Kim, and Kwang-Hoon Kong*

Department of Chemistry, Chung-Ang University, Seoul 06974, Republic of Korea

The perception of sweet taste is initiated by the activation of specific chemo-sensory receptor T1R2-T1R3. In previous studies, we found thatthe sweet-tasting protein brazzein was approximately 1800 times sweeter than sucrose, and the mutated brazzein variant with 3 amino acidswas over 22000 times sweeter than sucrose. However, the mechanismof brazzein’s sweetness is still unknown. In this study, recombinant sweettaste receptor was produced in a bacterial expression system and the interaction with sweet-tasting protein was investigated through computer structure modeling and protein-protein docking simulations. Recombinant hT1R2 and hT1R3 proteins were expressed using Escherichia coli expression system and purified by His-tag affinity chromatography. Because hT1R2 and hT1R3 are transmembrane protein, transmembrane domain(TMD) was eliminated to enhance the efficiency of protein expression. Through computer structure modelingand protein-protein docking simulation, the protein structure models of the receptor and one of its ligands, brazzein, were predicted and dockedwith each other to generate five potentially important amino acids, Lys174, Asp188, His189, Tyr416, and Pro440 on T1R2 subunit locatednear the binding pocket. From these results, we were able to partially elucidate the mechanism of tongue-to-brain sweetness by providing important clues as to how sweetness is triggered.

Keywords : Taste receptor, recombinant protein, GPCR

P-15

Heterologous Expression and Characterization of Molybdenum-Containing Formate Dehydrogenase (FDH) from Eubacterium limosum KIST612 in Escherichia coli

Minji Kim, Stacy Simai Reginald, Hyeryeong Lee, Serah Choi, Basit Sharif, and In Seop Chang* Department of School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea

Carbon dioxide (CO2) has received considerable attention as a sourcefor value-added chemicals and fuels. Although CO2 is abundant and low-cost material, it has major drawbacks in terms of difficulties of thermodynamics and kinetics to be converted as chemical feedstocks dueto the size of energy requirement for activating CO2 reduction. To overcome the challenges and establish efficient CO2 reduction, it is necessary to have an effective catalyst. Among the various catalyst forCO2 reduction, biological catalysts (i.e., enzyme) are promising due totheir intrinsic characteristics such as high specificity to substrate. Herein,we focused on a CO2 reducing enzyme, formate dehydrogenase (EC 1.17.1.9) which is capable of catalyzing CO2 reduction and formate oxidation. FDHs existed in a diverse array of organisms can be dividedinto metal-dependent FDHs and metal-independent FDHs. The FDH from E. limosum KIST612 is selected as it is one of the sufficiently studiedacetogens which have natural metabolic pathway to fix atmospheric CO2as their substrate. One of the gene clusters encoding the FDH, the operonELI_0994~0997 is identified for their CO2 reducing roles in the Wood-Ljungdahl pathway. In this study, all genes in this cluster are successfully transformed in E.coli and overexpressed under anaerobic condition. These recombinant FDHs are purified and analyzed for theirCO2 reduction and formate oxidizing activities.

Keywords : CO2 reduction, Formate dehydrogenase (FDH), Eubacteriumlimosum KIST612

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P-16

Gold Binding Peptide Fused Glucose Dehydrogenase Gamma-Alpha Complex (GDHγα) on Nanopatterned Electrode Surface Fabricated via E-beam Lithography

HyeRyeong Lee1, Taehoon Yi2, and In Seop Chang1* 1School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea 2Division of Liberal Arts and Sciences, GIST College, Gwangju Institute of Science and Technology, 123 Cheomdan- gwagiro, Buk-gu, Gwangju 61005, Republic of Korea

In this study, using the FAD-dependent glucose dehydrogenase (FAD-GDH) as a model enzyme, it is aimed to control the enzymatic orientation of the enzyme via site-specific fusion of gold binding peptidein enzyme. Most importantly, positioning of synthetized fusion proteinwould be regulated at nano-level, using nano-patterned electrode fabricated through e-beam lithography technique. For this, 1) the synthetic glucose dehydrogenase (GDH), consisting of the site-specificexpression of a gold binding peptide (GBP) on the α-subunit of GDH which enables close proximity between enzymatic active and electrodesurface as well as DET of enzyme-electrode interface will be used; 2) the number of GBP repeats fused to FAD-GDH would be optimized in terms of enzymatic catalytic activity and binding affinity; 3) The nano-patterned electrode would be fabricated via e-beam lithography for its unit pattern to have diameter similar to that of target enzyme; 3)the fusion protein will be immobilized on the nano-patterned electrodesurface and the binding morphology of enzymatic nanopatterns wouldbe investigated.

Keywords : Enzyme nano-patterning, enzyme electrode, binding specificity

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Q_Synthetic Biology and Metabolic Engineering

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Q-1

Metabolic Engineering of Methylosinus trichosporium OB3b for Production of Lysine and Cadaverine from Methane

Ok Kyung Lee, Thu Thi Nguye, Sanzhar Naizabekov, and Eun Yeol Lee*

Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 17104, Republic of Korea

Recently, methane is evaluated as an attractive low-cost feedstock thatcan be used in biotechnological production of value-added products suchas food and polymers. In this study, we present the bioconversion of methane to lysine and cadaverine using a metabolically engineered typeII methanotroph, Methylosinus trichosporium OB3b. The metabolicallyengineered strain, OB3b/cad4 was able to produce 0.28 g/L of cadaverinewith a volumetric productivity of 6.52 mg/g DCW/day by a gas bioreactor system. These results demonstrate the feasibility of food andpolymers production from methane as a sole carbon source using an engineered type II methanotrophic strain for the first time.

Keywords : Methylosinus trichosporium OB3b, methane, cadaverine

Q-2

Data-Driven and Model-Guided Systematic Framework for Feed Media Optimization in CHO Cell Culture

Donghyuk Choi1, Jongkwang Hong2, Yaron R. Silberberg3, Uiseon Park3,Beehak Hong3, Fumi Shozui4, and Dong-Yup Lee*

1School of Chemical Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 16419, Republic of Korea 2Division of Biological Science and Technology, Yonsei University, 1 Yonseidae-gil, Wonju, Gangwon-do 26493, Republic of Korea 3Osong Customer Service center, Ajinomoto Genexine Co., Ltd. 194-25, Osongsaengmyeong 1-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea 4Cell Culture Media Group, Material Development Section, Material & Technology Solution Labs, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc. 1-1, Suzuki-cho, Kasawaki-ku, Kasawaki-shi, 210-8681, Japan

Mammalian cell cultures remain the primary means of producing therapeutic biologics which has multi-million-dollar market. Over the last decade, production yield of CHO cell cultures has dramatically increased through significant improvements in media and feed compositions, feeding strategy, and bioprocess conditions. However, feed media development has been mainly driven by empirical approacheswithout fundamental understanding of cellular and metabolic characteristicsin response to the various feed conditions. Recently, more systematic approaches have been exploited for exploring condition-specific CHOcell metabolism based on the CHO genome-scale metabolic model (GEM). Therefore, we utilized statistical- and model-guided approachto understand in-depth intracellular metabolism with different feed compositions. Subsequently, we propose highly influential feed mediacomponents which may affect culture performance. This research can be utilized as a strategy to implement optimization of feed composition and feeding strategies based on mechanistic understanding.

Keywords : Feed media development, Chinese hamster ovary cell, genome-scale metabolic model

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Q-3

Establishment of Soluble Expression of Psychrophilic Enzymes Using Chaperone System

Borkar, Sondavid Nandanwar, and Kim Hak Jun*

Department of Chemistry, Pukyong National University, Busan 48513, Republic of Korea

Recombinant protein production is a vital process in biotechnology, which requires proper protein folding to maintain its function. Psychrophilic enzymes are actively used in the biotechnology industrybecause they maintain high activity at low temperatures, but are unstable that easily aggregates and forms inclusion bodies (improper folding) thataffect the protein function. Proteins that do not emerge in fully soluble,well-folded, and active form in a heterologous expression system are known as DTE (difficult-to-express) proteins. To improve protein yieldand quality, several strategies have been implemented such as co-expression of chaperones to minimize aggregation and enhance proteolytic stability. In this study, we used BL21(DE3) E. coli system that co-express the GroELS chaperone. The GroEL is a large double-ring-shaped oligomeric 14-mer chaperonin, which binds and encapsulates a substrate polypeptide along with its co-chaperonin GroES, which caps the central cavity of GroEL and facilitates the correctfolding of proteins in the presence of ATP. We investigated the role ofchaperone GroELS from E. coli and Antarctic psychrophilic bacteriumPsychrobacter sp. PAMC21119 for the soluble expression of DTE 51 enzymes (7 mesophilic and 44 psychrophilic enzymes). E. coli BL21 (DE3) showed soluble expression of 1 protein at 15℃, E. coli BL21 (DE3)-pGro7 showed 2 proteins at 30℃ and 3 proteins at 15℃, and E. coli BL21 (DE3)-PsyGroELS showed 8 proteins at 10℃. Here, we present the preliminary screening results of soluble expression of DTEenzymes.

Keywords : Difficult-to-express proteins, molecular chaperones, PsyGroELS

Q-4

Improvement of 1-Deoxynojirimycin Production by Bacillus subtilis subsp. Inaquosorum

Khai Ngoc Nguyen1,2, Yunah Kim1,2, Sawarot Maibunkaew1,2, Jisoo Park1,2, Mien Thi Nguyen1,2, Doo-Byoung Oh1,2, and Ohsuk Kwon2,3* 1Environmental Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea 2Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea 3SME Support Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea

In this study, we confirmed that Bacillus subtilis subsp. inaquosorumKCTC 13429 (B. subtilis IWT) is able to produce 1-deoxynojirimycin (DNJ), a potent α-glucosidase inhibitor having antidiabetic and antiviralactivities. By using UV irradiation, a mutant B. subtilis I.247 was thenisolated with approximately 50% increased DNJ titer than wild-type strain. In addition, sorbitol and yeast extract were identified as the bestcarbon and nitrogen sources, respectively for maximizing DNJ production by B. subtilis I.247. Under optimum conditions (3.4% sorbitol and 2.4% yeast extract at 32ºC) predicted using response surfacemethodology (RSM), B. subtilis I.247 was able to produce 359 mg/L afterfive days of cultivation. Moreover, the DNJ production by B. subtilisI.247 in optimum condition was further increased to 773 mg/L upon transformation with a vector expressing a gabT1-yktc1- gutB1 DNJ biosynthetic gene cluster. Our results strongly suggest B. subtilis IWT as a promising candidate for further strain development for industrial scale DNJ production.

Keywords : 1-Deoxynojirimycin, Bacillus subtilis subsp. inaquosorum,culture optimization

Q-5

Accurate Single Base Editing of Bacterial Genome by Target-Mismatched CRISPR/Cas9

Ho Joung Lee, Hyun Ju Kim, and Sang Jun Lee*

Department of Systems Biotechnology, and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea

CRISPR/Cas system has evolved the genome editing field. CRISPR/Cas9 is modularized with a single molecular guide RNA (sgRNA) and Cas9 nuclease, which recognizes a specific sequence andits PAM (protospacer adjacent motif), and consequently cleaves the target DNAs. Therefore, the CRISPR/Cas9 has been used for negative selection tools that cleave unedited target DNAs during site-specific mutagenesis, leaves only the cells with edited target DNAs, finally we could obtain the cells with wanted mutation. In this study, we introducedmultiple base-pair mutations in the galK gene of Escherichia coli cellsusing the CRISPR/Cas9 negative selection method. However, we couldrarely obtain the single base edited cells because the CRISPR/Cas9 alsocleaves the single base mismatched target DNAs, and it is called mismatch tolerance. To solve this issue, we introduced 1~2 bp mismatch(es) with the unedited target DNA into sgRNAs in advance. The unedited target DNAs were cleaved due to mismatch tolerance, however, the single base edited target could survive because of the increased mismatches. As a result, we could obtain the single base editedcells with high editing efficiency by using target-mismatched sgRNA method. Furthermore, general rules for the design of mismatched sgRNAs were optimized in the randomly selected 16 genome-wide targets. Our study shows that the target-mismatched sgRNA method isvery effective for single nucleotide editing in microbial genomes.

Keywords : Microbial genome editing, CRISPR/Cas9, mismatch tolerance

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Q-6

Syntrophic Culture of Methanotroph and E. coli for Efficient Conversion Methane to Mevalonate

Ji In Baek1,2, Ji Yeon Yoo1, Hyewon Lee1, Dae-Hee Lee1,3, Dong-MyungKim2, and Seung-Goo Lee1,3*

1Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea 2Department of Chemical Engineering and Applied Chemistry, Chungnam National University, 99 Daehak-ro, Daejeon 34141, Republic of Korea 3Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea

Methane has been recognized as a high potent greenhouse gas. Also, itis an important renewable resource for the production of biochemicals and fuels. While methane conversion is useful in biotechnological applications, it is technically challenging due to high stability of methane. Methanotrophs have emerged as promising biocatalysts because of their ability to utilize methane at ambient temperature and pressure. Methanotrophs use methane as a sole carbon and energy source,and modulate non-methanotrophs metabolism by secreting organic acids in nature. While methanotrophs have a high methane conversionactivity, its use for biochemical synthesis has been limited becuase the lack of genetic tools for elaborate metabolic engineering. Here, we developed syntrophic system of methanotrophs and partners that are easy to genetically engineered. First we utilized Methylococcus capsulatusBath as an efficient and robust biocatalyst to convert methane into organic acids. Next, we used E. coli SBA01, which is an evolutionary strain with high tolerance to organic acids and high ability to producebiochemicals from them. As a results, from methane, we synthesized mevalonate (MVA), a precursor of various terpenoids such as isoprene,bisabolol, lycopene, and so on. We expect this highly modular syntrophicmicrobial system can be applied to various useful productions.

Keywords : Methanotrophs, shytrophy, synthetic

Q-7

Regulation of Microbial Transcriptional Initiation Using CRISPR Interference

Bumjoon Kim, Hyun Ju Kim, and Sang Jun Lee*

Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea

Protospacer adjacent motif(PAM) sequence is an essential factor to operate CRISPR/Cas9 system. We define PAM flexibility of CRISPR interference using dCas9 and dCas9-NG in Escherichia coli. CRISPR/dCas9 derived from CISPR/Cas9 with two point mutations (D10A, H840A) that deactivate endonuclease activity. dCas9 protein guided by crRNA can specifically bind to target DNA sequence withoutDNA cleavage. CRISPR/Cas9 technology has limitations in designingtarget-specific single-guide RNA (sgRNA) due to the dependence of PAM sequence(5'-NGG) for binding target DNAs. In this study, we constructed a chromosomally integrated L-arabinose inducible dCas9 and dCas9-NG systems(ΔaraBAD:PBAD-dcas9(-NG))-KmR) in E.coli. Plasmids carrying various crRNAs with target sequences specific for the gal promoter (-10 region) were transformed into dCas9(-NG) expressingE. coli. Our study shows dCas9(-NG) has more expanded PAM sequences than Cas9(-NG). dCas9 protein can recognize 5’-NGG, NAG,NTG, NGA, and dCas9-NG protein can recognize 5’-NGG, AGA, TGC,GGT, AAG PAM sequences and fully repress target gal promoter and regulate transcription expression and metabolite of D-galactose.

Keywords : CRISPR interference, dCas9(-NG), PAM dependence

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Q-8

Enhanced Production of D-Ribose in Transketolase-Deficient Bacillus subtilis PLK1

Minji Bu1, Kang-Hyo Lee1, Kyung-Un Kim1, Ho-Sun Yoo1, Dae-Hyuk Kweon2*, Kyoung Heon Kim3*, and Yong-Cheol Park1* 1Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea 2Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Seobu-ro 2066, Suwon, Gyeonggi-do 16419, Republic of Korea 3Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Republic of Korea

D-Ribose, a ribosyl residue of ribonucleic acid and ATP, is able to be widely applied in foods, cosmetics and animal feed additives. D-Ribose can be chemically synthesized from D-glucose, D- gluconic acid and also be biologically produced by transketolase-deficient microbial strains. When the substrate glucose is taken up, a common d-ribose producer ofthe transketolase-deficient Bacillus subtilis ATCC 21951 consumes D-ribose produced from D-glucose. so it is necessary to develop a mutantstrain unable to metabolize D-ribose. Chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was applied to develop a transketolase-deficient mutant of B. subtilis PLK1, which showed a faster glucose uptake rate than B. subtilis ATCC 21951 and no consumption of D-ribose. Medium optimization was made to improve the D-ribose producing-performance of B. subtilis PLK1. The composition of optimal culture medium was determined to be 70 g/L glucose, 15 g/L yeast extract, 5 g/L KH2PO4, 5 g/L K2HPO4, 1 g/L MgSO4· 7H2O, 15 g/L CaCO3, 1 g/L citric acid, and pH 7.0. As a result of fed-batch fermentation in a 2.5 jar fermentor, a D-ribose concentrationof 48.3 g/L, a molar yield of 91 %, and a productivity of 0.34 g/L·hr wereobtained.

Keywords : D-Ribose, chemical mutagenesis, Bacillus subtilis

Q-9

Metabolically-Engineered Production of (–)-α-Bisabolol in Recombinant Saccharomyces cerevisiae

NaYoung Lim, Tae-Yeob Kim, Haeseong Park, Hyeoncheol Lee, Yong-HaSeo, and Yong-Cheol Park* Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea

(–)-α-Bisabolol is one of isoprenoids present in German chamomile anda potent cosmetic ingredient with whitening, skin-smoothing, antibacterial and anti-inflammentary activities. In this study, metabolicengineering strategies were attempted to produce (–)-α-bisabolol in Saccharomyces cerevisiae. The codon-optimized MrBBS gene coding for (–)-α-bisabolol synthase from Matricaria recutita was expressed inS. cerevisiae for (–)-α-bisabolol production. The resulting strain (DM)produced 9.5 mg/L of (–)-α-bisabolol in 24 h of batch culture. Additionally, the mevalonate pathway was intensified by introducing atruncated HMG1 gene coding for HMG-CoA reductase and ERG10encoding acetyl-CoA thiolase. The resulting strain (DtEM) produced a 2.9-fold increased concentration of (–)-α-bisabolol than the DM strain.To increase the acetyl-CoA pool, the ACS1 gene coding for acetyl-CoAsynthetase was also overexpressed in the DtEM strain. Finally, the DtEMA strain produced 124 mg/L of (–)-α-bisabolol with 2.7 mg/L-h of productivity in a fed-batch fermentation, which were 13 and 6.8 timeshigher than the DM strain in the batch culture, respectively. Conclusively,these metabolically-engineered approaches might pave the way for thesustainable production of other sesquiterpenes in engineered S. cerevisiae.

Keywords : (–)-α-Bisabolol, Saccharomyces cerevisiae, MrBBS

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Q-10

The Ethylmalonyl-CoA Pathway for Methane-Based Biorefineries: A Case Study of Using Methylosinus trichosporium OB3b, a Type II Methanotroph, for Producing 2-Hydroxyisobutyric Acid and 1,3-Butanediol from Methane

Dung Hoang Anh Mai, Thu Nguyen Thi, and Eun Yeol Lee*

Department of Chemical Engineering (Integrated Engineering), Kyung Hee University, Yongin 17104, Republic of Korea

With the synthetic methylotrophs are still at early stages and synthetic methanotrophs are nonexistent, genetically engineered methanotrophsare another feasible option for the valorization of methane to sustainablyproduce valuable products. Extensive research efforts have been devotedto transform methanotrophs into biocatalysts that can capture and valorize atmospheric methane to mitigate global warming. However multiple knowledge gaps and the lack of validation for extrapolated knowledge pose significant challenges for metabolic engineering of methanotrophs. Compared to type I methanotrophs, characterized by the ribulose monophosphate (RuMP) pathway, type II methanotrophs withthe ethylmalonyl-CoA (EMC) pathway can achieve better carbon efficiency through multiple carboxylation nodes, in which one CO2 andone HCO3

- are re-assimilated into biomass, making them preferable forbiomass-derived products. In the current study, to lay the groundwork for future studies, transcriptome analysis and genome-scale metabolic models were employed to evaluate the potential regulatory nodes of theEMC pathway of Methylosinus trichosporium OB3b. The interplay of poly(3-hydroxybutyrate) (PHB) pathways and entry genes in the EMCpathway were identified to be involved in flux diversion from EMC pathway. We further demonstrated the production of two EMC pathway-derived products 2-hydroxyisobutyric acid (2HIBA) and 1,3-butanediol (1,3-BDO) from methane. By inducing the PHB overflowmechanism, and deleting phaC that encodes polyhydroxybutyratesynthase, 2HIBA and 1,3-BDO titers were enhanced to 30.0±1.827 mgL-1 and 68.54±1.381 mg L-1, respectively. To the best of our knowledge,this is the first report on the production of EMC-derived compounds frommethane.

Keywords : Methylosinus trichosporium OB3b, 2-HydroxyisobutyricAcid, 1,3-Butanediol

Q-11

Mining of Secretion Tag for Multiple Peptide Delivery into Host Cells through the Engineered Secretion System in Bacteria

Juyeon Hong, Jaewon Ha, and Miryoung Song*

Department of Bioscience and Biotechnology, Hankuk University of Foreign studies, 81, Oedae-ro, Mohyeon-eup, Cheoin-gu, Yongin-si, Gyeonggi-do 17035, Republic of Korea

Biological function of protein is tightly related with cellular localizationwhich is directed by genetically encoded signals as RNA with specific secondary structure or peptide sequences. Especially, Salmonella typhimurium secrete certain set of proteins with such signals throughsecretion apparatus called type 3 secretion system (T3SS) to survive within host cells. Here, we mined tags for the secretion through the synthetic type 3 secretion system, which serves as a delivery apparatusencoded in the engineered bacteria for medical applications. Since the synthetic T3SS (synT3SS) was constructed by genetic manipulation ofSalmonella pathogenicity island 1 (SPI1), the tags were selected from effectors secreted through native SPI1-encoded T3SS. Six genes encoding each effector were placed under the control of constitutive promoter to avoid the biased effect of expression regulation on ColE1plasmid, respectively, with cognate chaperon gene when necessary. Theresulting plasmid was individually introduced into SPI1-defective Salmonella strain carrying synT3SS and the secretion was evaluated bywestern blot analysis. The secreted titer of two proteins, SptP and SopB,were as high as that from wild type Salmonella containing same plasmidfor each protein. SipA and SopD from supernatant were detected slightlylower than that from control strain. However, lowest amount of SopA and SopE2 were identified from the culture supernatant of Salmonellawith synT3SS. We are currently working on identifying signal sequencesof these effectors, in order to use them as secretion tags of therapeuticproteins. Different secretion tags will enable secretion of multiple proteins encoded on the same plasmid. These will aid delivery of multipletherapeutic proteins with different effects, thereby maximizing therapeutic effects on cancer.

Keywords : Drug delivery, synthetic T3SS, therapeutic protein

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Q-12

Enhanced Bactericidal Effect of Engineered Endolysin against Acinetobacter baumannii

Jeonghyun Lim, and Miryoung Song*

Department of Bioscience and Biotechnology, Hankuk University of Foreign studies, Yong-in 17035, Republic of Korea

Multidrug resistant in pathogenic bacteria and rapid spread of drug resistance mechanisms are major concerns in public health. Bacteriophage encoded endolysins can be used as new class of antimicrobial agents against bacterial infection. In previous study, Pseudomonas bacteriophage encoded endolysin PA90 has shown antibacterial effect on various species of bacteria. However, there was only minor bactericidal effect on gram negative bacteria including Acinetobacter baumannii probably due to the defect in penetrating outermembrane. Therefore, we introduced specific outer membrane destabilizing peptide to improve the antibacterial function of PA90 as an artilysin. Here, we fused cell penetrating peptide DS4.3 (DS4.3::PA90) or outer membrane destabilizing peptide thanatin (Thanatin::PA90) at the N-terminal of PA90, respectively. The activityof resulting artilysins were evaluated by colony forming unit (CFU) reduction assay using A. baumannii. At lowest concentration (62.5 nM),reduction of A. baumannii number was 103 times higher by DS4.3::PA90treatment but no bactericidal effect was observed with PA90 treated cells.Interestingly, all bacteria were killed by the addition of DS4.3::PA90 orThanatin::PA90 at highest concentration as 250 nM, respectively, while50% of cells were remained by PA90 treatment. These results suggest that fusion of outer membrane penetrating peptide or destabilizing peptides can enhance antibacterial effect of endolysin against even gramnegative bacteria, which imply the potential of artilysin as an alternativeantibiotics.

Keywords : Bacteriophage, anti-bacterial agents, artilysin

Q-13

Screening of High D-Ribose Production Bacillus subtilis Mutant with FLIPRbs

SeungJoo Kim1, Kyeong-Won Kim1, Won-beom Park1, Jeong-Hyeon Yoon1, Yong-Cheol Park2, and Dae-Hyuk Kweon1* 1Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Gyeonggido 16419, Republic of Korea 2Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 02707, Republic of Korea

D-Ribose is a five-carbon monosaccharide. D-Ribose made from glucose through the pentose phosphate pathway (PPP), It is an expensivesugar that is a synthetic material for riboflavin, inosine monophosphate,ATP, and analogues of Nucleotides used in antiviral drugs. To increaseribose productivity using Bacillus subtilis, random mutation was performed using 2-Aminopurine. The FLIPRbs biosensor can measure the concentration of ribose by Fluorescence Resonance Energy Transfer(FRET) phenomenon, which appears by CFP on the N-terminal of the ribose binding protein and YFP on the C-terminal. 17 strains with a highlevel of ribose production were selected, and strains that produced riboseof up to 18.2 g/L in the flask fermentation for 72 hours were found, and the control strain produced ribose of 12.5 g/L in the same culture time However, there was a colony-by-colony variation in which the productivity of ribose was different for each colony. We plan to stabilizethe selected mutant strains and proceed with RNA sequencing.

Keywords : Ribose, Bacillus subtilis, pentose phosphate pathway

Q-14

Multiplexed Fortimicin Biosynthetic Pathway Integration in Escherichia coli K12

Hwiso Ryu1+, Ji Yoon Lee1+, Yu Rin Seol1, and Je Won Park2* 1Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea 2School of Biosystems and Biomedical Sciences, Korea University, Seoul 02841, Republic of Korea

Fortimicins, one of the aminocyclitol-containing aminoglycoside antibiotics utilized in clinicals, are produced form soil actinomycete Micromonospora olivasterospora DSM43868. Within a couple of years,we have been developed the heterologous expression of the fortimicin biosynthetic gene constructs using plasmid-based co-expression system. However, compared with the extrachromosomal plasmid-aidedexpression, chromosomal integration should be desirable due to enhanced stability, moderated cell-to-cell variability, and needlessness of selection markers (or antibiotics) for plasmid maintenance. Herein, we report multiplexed gene constructs integration for the heterologousproduction of fortimicin biosynthetic intermediates (FOR-FU 10 in the first round and FOR-KK1 in the second round, respectively) in E. coli, clearly demonstrating that the integrated recombinants could produce higher titers compared with plasmid-based expressions. We expect this approach can be readily expandible to diverse biosynthetic pathways andrecombinant chasses to create robust high-titer microbial cell factories.

+ These authors contributed equally to this work

Keywords : Chromosomal integration, aminocyclitol antibiotics, heterologous recombination

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Q-15

Chromosomal Integration of Aminocyclitol-Containing Pseudo-Trisaccharide Hybrid Biosynthetic Pathway

Chang Rae Kim1, Minsuk Seo1, and Je-won Park2* 1Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea 2School of Biosystems and Biomedical Sciences, Korea University, Seoul 02841, Republic of Korea

As growing concerns on multi-drug resistant bacteria, or superbugs, weused to design and construct aminoglycoside biosynthetic gene sets tocreate novel aminoglycoside analogs. Of these analogs, 6-xylosyl-IST-FU-10 was synthetized by introducing a glycosyl transferase encoding gene derived from the gentamicin biosynthetic gene cluster to the gene construct responsible for the biosynthesis of anistamycin biosynthetic intermediate, IST-FU10. Genes arranged in theconstruct was synthetized and assembled into poly-monocistronic bio-parts using BioBricks assembly. This study aims to overcome the problem of low productivity of the biosynthetic pathway expressed viaplasmid in E. coli. We integrated the construct into the chromosome ofthe host E. coli BL21(DE3) using recombination method. The double stranded breakage of the chromosome using intron-encoded endonuclease, I-SceI, raised lambda-red recombination efficiency for the integration of the large (7245 bp) 6-xylosyl-IST-FU-10 biosyntheticconstruct. As a result, the productivity of 6-xylosyl-IST-FU-10 in the integrated strain increased 1.75 folds compared to the plasmid bearingstrain. Chang Rae Kim and Minsuk Seo contributed equally to this work

Keywords : Chromosomal integration, aminoglycoside hybrid, pathwayengineering

Q-16

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

Seulgi Kang1,2, Yoseb Song1,2, Jongoh Shin1,2, Sangrak Jin1,2, Jiyun Bae1,2,Suhyung Cho1,2, and Byung-Kwan Cho1,2* 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 2KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea

Acetogens are naturally capable of metabolizing carbon monoxide (CO),a component of syngas, for autotrophic growth in order to produce biomass and metabolites via the Wood–Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution for growth improvement ofEubacterium limosum ATCC 8486 under CO conditions. As a result, the growth rate of evolved population was significantly increased to 1.44 folds. To identify the causal mutations related to growth improvement, we performed whole genome resequencing of each population. Interestingly, we found a key mutation in CO dehydrogenase acetyl-CoAsynthase (CODH/ACS) complex coding gene. To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth fitness of the single clone was comparable tothose of the evolved populations and wild type strain under CO conditions. Consequently, this study demonstrates that the mutations inthe CODH/ACS complex affect autotrophic growth enhancement in thepresence of CO as well as the CO tolerance of E. limosum ATCC 8486.

Keywords : Acetogens, carbon monoxide, adaptive laboratory evolution

[This work was supported by the C1 Gas Refinery Program (2018M3D3A1A01055733 to B.-K.C.) through the National ResearchFoundation of Korea (NRF) funded by the Ministry of Science and ICT(MSIT)]

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Q-17

Acetogenic Bacteria Utilize Light-Driven Electrons as an Energy Source for Autotrophic Growth

Sangrak Jin1,2, Yale Jeon3, Min Soo Jeon3, Jongoh Shin1,2, Yoseb Song1,2,Seulgi Kang1,2, Jiyun Bae1,2, Suhyung Cho1,2, Jung-Kul Lee4, Dong RipKim3, and Byung-Kwan Cho1,2* 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 2Innovative Biomaterials Research Center, KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 3Department of Mechanical Engineering, Hanyang University, Seoul 04763, Republic of Korea 4Department of Chemical Engineering, Konkuk University, Seoul 05029, Republic of Korea

Acetogenic bacteria utilize cellular redox energy to convert CO2 to acetate using the Wood–Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as inorganic materials such as photoresponsive semiconductors. Here, we developeda nanoparticle-microbe hybrid system, in which chemically synthesizedcadmium sulfide nanoparticles (CdS-NPs) were displayed on the cell surface of the industrial acetogen, Clostridium autoethanogenum. Thehybrid system converts CO2 into acetate without the requirement of additional energy sources such as H2 and only utilizes light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum utilizes electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicated that genes encoding the metal ion or flavin bindingproteins were highly upregulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition ofthese cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

Keywords : Acetogenic bacteria, artificial photosynthesis, cadmium sulfide nanoparticle

[This work was supported by the C1 Gas Refinery Program (2018M3D3A1A01055733 to B.-K.C.) through the National ResearchFoundation of Korea funded by the Ministry of Science and ICT]

Q-18

Engineering Bacteroides thetaiotaomicron to Produce Butyrate Based on a Genome-Scale Metabolic Model-Guided Design

Kang San Kim, Yoseb Song, Donghui Choe, Minjeong Kang, and Byung-Kwan Cho*

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea

Bacteroides thetaiotaomicron represents a major symbiont of the humangut microbiome that is increasingly viewed as a prospective candidate strain for microbial therapeutics. This chiefly owes to several intrinsic features of B. thetaiotaomicron including the prevalence in the humangut, ability to stably colonize and modify the intestinal niches, and prospective role in the alleviation of gut inflammations. To streamline design and manipulation of this strain, we reconstructed a genome-scalemetabolic model (GEM) using the previous GEM iAH991 as the template. Validation of the updated model iKS1119 revealed that the insilico predictions were in close agreement with the experimentally measured parameters, suggesting that the model can be employed for the reliable prediction of B. thetaiotaomicron phenotypes. We used butyrate as a proof-of-concept to test whether B. thetaiotaomicron harbors the capacity for the heterologous production of biomolecules. Genomic integration of butyrate biosynthetic pathway from Clostridiumacetobutylicum into a wild-type B. thetaiotaomicron yielded around 30mg/L of butyrate. The GEM-guided rational strain design improved thefinal titer by up to 3-folds. This study shows that B. thetaiotaomicron may serve as an effective strain for live microbial therapeutics in human.

Keywords : Bacteroides thetaiotaomicron, genome-scale model, butyrate production

[This project was supported by the Korea Bio Grand Challenge (2018M3A9H3024759 to B.-K.C.), the Basic Science Research Program (2018R1A1A3A04079196 to S.C.), the Bio & Medical Technology Development Program (2018M3A9F3079664 to B.-K.C.)from the Ministry of Science and ICT (MSIT) through the National Research Foundation (NRF) of Korea]

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Q-19

Optimizing the Methylerythritol-4-phosphate Pathway in Corynebacterium glutamicum for Improving Squalene Production

Dong Hun Kang, Han Min Woo*, Hyeonbae Lim, and Jaehyun Park Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

Microbial production of squalene has driven the development of microbial cell factories, triggered by the limitation of low-yielding bioprocesses from plants and illegal harvesting shark liver. In addition,RNA-guided CRISPR technologies have been applied to microbial cellfor rapid development of metabolic engineering in microbial cell factories for making it produce desired product like squalene. Those technologies have various potential to specific gene knockdown and knockout depending on what kind of CRISPR Cas systems you choose.In our study, we have metabolically modified Corynebacterium glutamicum using CRISPR Cas systems to produce squalene from glucose. For this, we have used three strategies, one of which is CRISPR-dCas9 based gene interference for precursor rebalancing, redox balancing and blocking the competing pathway by targeting combinatorial genes (gapA, gdh, and idsA). Second one is CRISPR-Cas12a based gene knockout by introducing STOP mutation to 16TH codon (E) in idsA gene. The last one is a plasmid based over-expression of key enzymes (dxs, idi, and ispA) belong to MEP pathway and truncated squalene synthase. As a result, we found the bestsqualene production strain with blocking competing pathway by repressing the idsA gene using high-throughput cultivation. Using thisstrain, 5.4 ± 0.3 mg/g dry cell weight (DCW) and 105.3 ± 3.0 mg/L squalene were analyzed, which was a 5.2-fold increase over the parentalsqualene production strain. After that, we additionally optimized MEPpathway using the last strategy. As a result, we identified additional twokey enzymes (IspDF) other than the known enzymes (Idi and Dxs). Thus,the combination of those two studies is expected to obtain the strain producing squalene more than previously engineered strain.

Keywords : Corynebacterium glutamicum, squalene, CRISPR

Q-20

Microbial Biosynthesis and Secretion of Hydrophobic Squalene Using Engineered Corynebacterium glutamicum and Its Application

Jaehyun Park, Dong Hun Kang, and Han Min Woo*

Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), Suwon 16419, Republic of Korea

Microbial production of hydrophobic chemicals requires host engineering to enhance the production by transporting the compounds across the cell membrane or increasing the capacity of the intracellular storage, in addition to pathway engineering. Herein, we engineered Corynebacterium glutamicum to produce a hydrophobic squalene as a model product. Protein engineering of the key enzymes were resulted in a 4.6-fold improvement of squalene production. We notably identifiedthat squalene was secreted in the defined culture medium without any treatments during the batch fermentation. Further, different cell lengthswere investigated for the secretion by analyzing both intracellular and extracellular squalene productions using the CRISPR-Cas-assisted engineered strains. To increase the total squalene production, a lactate-bypass was designed to increase the pyruvate level that can be reloaded from lactate utilization, by inactivating the pyruvate carboxylase. Fed-batch fermentation resulted in 1406 ± 16 mg/L of tSQ production (32% of tSQ in the medium). In addition, a biocompatible dodecane overlay were used to mitigation of intracellular squalene to the cell/dodecane-mixed layer, which occupied 87.7 % of tSQ in 10% of the culture volume. For its cosmetic applications, bio-squalene/ dodecane nanoemulsions were formulated and analyzed. In this study, an uncovering of the secretion and pathway engineering of lactate-bypass will help for the further engineering of microbial cells forother hydrophobic isoprenoids production and its application.

Keywords : Corynebacterium glutamicum, squalene, biosynthesis

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Q-21

Biocontainment and Inducer Free System for Scalable Cultivation of Engineered Cyanobacteria Using Industrial Flue Gas

Jigyeong Son1, Hyun Jeong Lee1,2, Sun Young Choi3, and Han Min Woo1,2*

1Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea 2BioFoundry Research Center, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea 3SOL, Inc, BK Tower 2F, 28 Beopwon-ro 11-gil, Songpa-gu, Seoul, Republic of Korea

Recent public attention to a shift from fossil fuel-derived chemical production to self-sustaining chemical production has accelerated the development of industrial-scale microalgal production as a promising feedstock for fermentative biochemical production with regard to conversion and valorization of . We created an engineered cyanobacterialstrains for heterologous isoprenoid chemicals from as model platform.For industrial scalable fermentation of these strains, GMO issue and compensating the production cost is limitation. Biocontainment is solution for biosafety and environmental risk and Inducer-free system is cost-effective in scalable fermentation. Here, we developed biocontainment and inducer-free system for large-scale cultivation forengineered cyanobacteria for isoprenoid production using industrial fluegas in a closed photobioreactor .

Keywords : Cyanobacteria, biocontainment, industrial scalable-cultivation

Q-22

Sustainable Production of the Calorie-Free Sweetener Precursor, ent-kaurenoic Acid from CO2 Using Engineered Cyanobacteria

Sungcheon Ko, and Han Min Woo*

Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

Metabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to fatty acid-derived chemicals as bio-solar cell factories. Here we report a complete biosynthesis of ent-kaurene (ent-KN), ent-kaurenoic acid (ent-KA) from CO2 in engineered cyanobacteria toward diterpenoids production of the calorie-free sweetener. To synthesize ent-KN, we designed the diterpene synthetic module (DS module) that a prenyl phosphate converts kaurene into theengineered cyanobacteria (the OverMEP modules) in which the methylerythritol 4-phosphate pathway was optimized to increase the precursors either farnesyl diphosphate (FPP) or geranylgernaly pyrophosphate (GGPP) from CO2. ent-KN were detected in the cell extracts and medium by LC-MS. Next, we set out to produce ent-KA froment-KN by reconstituting the cytochrome P450 module (CP module). Engineered S. elongatus PCC 7942 produced 2.9 ± 0.01 mg L-1 ent-KAdirectly from CO2, respectively. This study is the first report of photosynthetic production of ent-kaurenoic acid from CO2 in cyanobacteria.

Keywords : Cyanobacteria, terpenoid, calorie-free sweetener

Q-23

Application of the CRISPRi-Derived Technologies for Engineering of Cyanobacteria

Mieun Lee1,2, Sun Young Choi1,2, and Han Min Woo1,2* 1Department of Food Science and Biotechnology, Sungkyunkwan university, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea 2BioFoundry Research Center, Institute of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

Cyanobacteria are photosynthetic bacteria considered to be a desirablemicrobial platform for synthetic biology, due to their CO2 utilizing ability, relative fast growth rate, and genetic malleability. In order to make the genetic engineering of cyanobacteria more easily done, CRISPRi technology was introduced into the Synechococcus elongatusPCC 7942 system. First application is interference with dCas12a enzyme, derived from Cas12a, demonstrating the repression strength ranging 53 to 94 %, and successful multiple target repression. After confirming its effectiveness, it was applied to squalene production, repressing regarding genes, acnB and cpcB2, resulting enhanced squalene production, 9.5 mg/L/OD and 7.6 mg/L/OD, respectively. Second application was constructing logic NAND gate using CRISPRiwith dCas9 enzyme, and light-responsive promoter. The S. elongatusPCC 7942 cells were subjected to combination of chemical inducer andlight/dark alternating environmental stimuli. Resulting circuit responsewas observed by detecting fluorescence signal of yellow fluorescence protein, whose signal was reversed by regulation of the dark-inducing promoter purF, and CRISPRi. These results show effective CRISPRi-derived application in cyanobacteria S. elongatus PCC 7942,and potential for further metabolic engineering approaches with developed tools.

Keywords : Cyanobacteria, CRISPRi

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Q-24

Advanced Bacterial Genome Engineering Tool Using High-Fidelity Cytosine Base Editing with Reduced DNA Off-Targets

Yubeen Heo1,2, Gue-Ho Hwang3, Han-Min Woo1,2*, and Sang-Su Bae3*

1Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea 2BioFoundry Research Center, Institute of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea 3Department of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, 222, Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea

Sophisticated genome editing technology with high-resolution has demanded as an essential tool for re-designing microbial cycle dependent on purpose. Rat APOBEC1-derived cytosine base editor (CBE) representative technology for genome editing induced DNA off-targets that result from unintended SNV on genomic DNA, which limited significancy of CBE as programmed specific genome editing tool. We apply high-fidelity CBEs for more advanced bacteria engineering and evaluate capacity of high-fidelity CBE by generating premature termination codons (PTCs) in desired genes (CBE-STOP) in Corynebacterium glutamicum as a GC-rich model bacterium. We constructed several single gene-inactivated strains for three genes (ldh,idsA and pyc) and ultimately developed a strain with five genes (ldh, actA,ackA, pqo and pta) that were inactivated sequentially for enhancing succinate production. Whole-genome sequencing (WGS) data of thesemutants showed that genome-wide random point mutations despite of desired phenotypes. Off-targets were accumulated rationally dependent on duration time exposed to CBE. C. glutamicum mutants with high-fidelity CBEs (YE1-BE3 and BE3-R132E) employed for CBE-STOPrevealed drastically reduced sgRNA-independent off-targets with increased precision. We evaluate CBEs as precise genome engineeringtool considering both efficient on-target editing and minimized genome-wide DNA off-targets for developing microbial cell factory.

Keywords : Cytosine base editor, genome editing, DNA off-target effects

Q-25

A Hybrid Embden-Meyerhof-Parnas Pathway to Decipher a Metabolic Link between Carbon and Phosphorus in Corynebacterium glutamicum

Hyejeong Cho1, Phil Kim1, Yoojin Lee1, and Han Min Woo1,2* 1Department of Food Science and Biotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea 2BioFoundry Research Center, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

The low carbon yield from fundamental Embden–Meyerhoff–Paranas (EMP) pathway for sugar catabolism, anabolism, and energy metabolism produces infavorable economics. Herein, we constituted ahybrid EMP that replaced the first phase of the EMP in Corynebacteriumglutamicum with non-oxidative glycolysis (NOG) and revealed a link between sugar and phosphate metabolism. Although carbon was conserved via NOG, the energy metabolism was significantly limited. In accordance with synthetic glucose kinase activity and phosphoketolase on the hybrid EMP, cell growth was completely recovered in the C. glutamicum pfkA mutant strain. Notably, we have revealed a phosphate-replenishing pathway that involved trehalose biosynthesis for the generation of inorganic phosphate (Pi) sources in the hybrid EMP when external Pi supply was limited. Thus, the re-designed hybrid EMP pathway with balanced carbon and phosphorusstates can be employed in the efficient microbial platform for biochemical production.

Keywords : Non-oxidative glycolysis, phosphorus metabolism, Corynebacterium glutamicum

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Q-26

One-Step Multiplex Genome Editing to Accelerate the Genome-Based Discovery of New Antibiotics in Bacillus

Mansu Kim1,2, Da-Eun Jeong1, Ha-Rim Kim1, and Soo-Keun Choi1,2* 1Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 2Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, Korea University of Science and Technology(UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea

Genome-based identification of new antibiotics is emerging as an alternative to traditional methods. However, uncovering hidden antibiotics under the background of known antibiotics remains a challenge. To over this problem using a quick and effective genetic approach, we developed a multiplex genome editing system using a cytosine base editor (CBE). The CBE system achieved simultaneous double, triple, quadruple, and quintuple gene editing with efficiencies of 100%, 100%, 83%, and 75%, respectively, as well as the 100% editingefficiency of single targets in Bacillus subtilis. Whole-genome sequencing of the edited strains showed that they had an average of 8.5 off-target single-nucleotide variants at gRNA-independent positions. The CBE system was used to simultaneously knock out five known antibiotic biosynthetic gene clusters to leave only an uncharacterized polyketide biosynthetic gene cluster in Paenibacillus polymyxa E681. The polyketide showed antimicrobial activities against gram-positive bacteria, but not gram-negative bacteria and fungi. Therefore, our findings suggested that the CBE system might serve as a powerful toolfor multiplex genome editing and greatly accelerating the unraveling ofhidden antibiotics in Bacillus and Paenibacillus species. [

Keywords : Base editor, multiplex genome editing, Bacillus

This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF-2018M3A9F3079565); and the KRIBB Research Initiative Program]

Q-27

Identification of Genetic Regulatory System Controlled by Carbon Monoxide in Eubacterium limosum KIST612

Byeongchan Kang, Soyoung Oh, Hyunsoo Kang, Ji-Yeon Kim, Young-Joon Ko, and In Seop Chang*

School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea

Transcription in prokaryotes proceeds as the related gene units consistof a single operon, and sometimes there are also operon units that are regulated by several promoters. The operon regulates the expression level of genes on a unit so that bacteria can quickly adapt to environmentaland substrate conditions. Eubacterium limosum KIST612 is a Gram-positive, obligately anaerobic, and acetogenic bacterium. This strain can use carbon monoxide as the sole energy and carbon sources. In E. limosum KIST612, it was confirmed that the transcription level ofgenes is regulated depending on the substrate or environmental conditions. This pattern of transcriptional regulation has also been confirmed in genes for consuming carbon monoxide, a representative substrate that E. limosum KIST612 can use. Transcription regulation bythese transcription factors can be confirmed not only through the phenotype during the growth, but also through the level of transcription.To investigate the regulation of the level of transcription of a gene related to carbon monoxide consumption, we analyzed the operon unit of the related gene by co-transcription analysis and identified the transcriptionregulator(s) in the strain.

Keywords : Eubacterium limosum KIST612, carbon monoxide, operon

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Q-28

Studies of Molecular Profiling and Optimization for Growth and PHB Production Conditions in Rhodobacter sphaeroides

Yu Rim Lee, Soo Youn Lee, Ji Yye Lee, Jin-Suk Lee, and Sangmin Lee*

Gwangju Bio/Energy R&D Center, Korea Institute of Energy Research, Gwangju 61003, Republic of Korea

In the recent climate change regime, the industrial demand for sustainable materials to replace petroleum-derived polymers continuesto increase. Polyhydroxybutyrate (PHB) is especially interested as a substitute for polypropylene. Accumulating evidence shows that PHB is generated as a carbon storage material in various microorganisms. Theeffects of growth conditions on PHB production have been widely studied in chemolithotrophs, particularly in Rhodobacter. However, theresults on PHB production in Rhodobacter have been somewhat inconstant due to different strains and experimental conditions, and it is currently unclear how diverse environmental factors are connected with PHB production. Here, we report optimized growth conditions for PHB production and show that the growth conditions are closely associated with reactive oxygen species (ROS) regulation. PHB accumulates in cells up to approximately 50% at the highest level underdark aerobic conditions as opposed to light aerobic/anaerobic conditions. According to the time course, PHB contents increased at 48hours and then gradually decreased. When observing the effect of temperature and medium composition on PHB production, 30℃ and acarbon/nitrogen ratio of 9:1 or more were found to be most effective. Among PHB biosynthetic genes, PhaA and PhaB are highly correlatedwith PHB production, while PhaC and PhaZ observed little change inoverall expression levels. We found that peroxidase activities and expression levels of antioxidant-related genes in cells under dark conditions were relatively high, whereas the amount of hydrogen peroxide was comparatively low compared to the light conditions. Theseresults suggest optimal culture conditions for growth and PHB production and the importance of ROS-scavenging signaling with regardto PHB production.

Keywords : Polyhydroxybutyrate, growth conditions, reactive oxygen species

Q-29

Engineering of Escherichia coli to Grow Solely on CO2 and Formic Acid

Cindy Pricilia Surya Prabowo1,2, Junho Bang1,2, Chang Hun Hwang1,2, Jung Ho Ahn1,2, Jong An Lee1,2, and Sang Yup Lee1,2,3* 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon 34141, Republic of Korea 3BioInformatics Research Center and BioProcess Engineering Research Center KAIST, Daejeon, Republic of Korea

The development of synthetic autotrophs or formatotrophs has been anappealing issue as a solution to environmental concerns, and the conversion of useless C1 compounds to value-added chemicals was needed. The development of Escherichia coli strains capable of growing exclusively on CO2 and FA has previously been reported. The final celldensities, though, were so low (less than OD600 of 1). In this paper, we present the results for the development of E. coli capable of growing torelative higher cell density solely on CO2 and FA (more than OD600 of 11). This E. coli strain is expected to be useful as a platform strain capableof growing solely on CO2 and FA and generating valuable chemicals solely through C1 compound assimilation.

Keywords : Escherichia coli, C1 compound assimilation, metabolic engineering

[This work was supported by the C1 Gas Refinery Program through theNational Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2016M3D3A1A01913250)]

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Q-30

Production of 4-Amino-1-butanol by Corynebacterium glutamicum

Cindy Pricilia Surya Prabowo1,2, Jae Ho Shin1,2, Jae Sung Cho1,2, Tong UnChae1,2, and Sang Yup Lee1,2,3* 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon 34141, Republic of Korea 3BioInformatics Research Center and BioProcess Engineering Research Center KAIST, Daejeon 34141, Republic of Korea

4-Amino-1-butanol (4AB) serves as an intermediate compound for drugs and a precursor of biodegradable polymers used for gene delivery.4AB is produced by chemical synthesis from petroleum resources. To date, the production of 4AB from renewable resources has not been reported. This study reported for the first time the production of 4AB from glucose by metabolically engineered Corynebacterium glutamicum. The production used a newly designed pathway comprisinga putrescine aminotransferase (encoded by ygjG) and an aldehyde dehydrogenase (encoded by yqhD) from Escherichia coli. The genes were introduced to a putrescine producing C. glutamicum strain developed in this study to produce 4AB. Then, further metabolic engineering strategies such as fine-tuning the ygjG and yqhD expressionlevels, eliminating competing pathways, and optimizing culture conditions were applied. The final engineered strain reached 24.7 g/L of 4AB in fed-batch culture.

Keywords : Metabolic engineering, Corynebacterium glutamicum, amino alcohols

[This work was supported by the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries)from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557)]

Q-31

Microbial Production of Acrylic Acid through Novel β-Alanine Route

Yoo-Sung Ko1,2, Je Woong Kim1,2, Tong Un Chae1,2, Chan Woo Song1,2, and Sang Yup Lee1,2,3* 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), Institute for the BioCentury, Korea Advanced Institute of Scien, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea 3BioProcess Engineering Research Center and BioInformatics Research Center, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea

Acryli acid (AA) is an important industrial chemical used for several applications including superabsorbent polymers and acrylate esters. Here, we report the development of a new biosynthetic pathway for theproduction of AA from glucose in metabolically engineered Escherichia coli through the β-alanine (BA) route. The AA production pathway waspartitioned into two modules: an AA forming downstream pathway anda BA forming upstream pathway. We first validated the operation of thedownstream pathway in vitro and in vivo, and then constructed the downstream pathway by introducing efficient enzymes (Act, Acl2, andYciA) screened out of various microbial sources and optimizing the expression levels. For the direct fermentative production of AA from glucose, the downstream pathway was introduced into the BA producing E. coli strain. The resulting strain could successfully produce AA fromglucose in flask cultivation. AA production was further enhanced by expressing the upstream genes (panD and aspA) under the constitutiveBBa_J23100 promoter. Replacement of the native promoter of the acsgene with the BBa_J23100 promoter in the genome increased AA production to 55.7 mg/L in flask. Fed-batch fermentation of the final engineered strain allowed production of 237 mg/L of AA in 57.5 h, representing the highest AA titer reported to date.

Keywords : Metabolic engineering, Escherichia coli, Acrylic acid

[This work was further supported by Hanwha Solutions through KAIST-Hanwha Solutions Future Technology Institute] [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556 and NRF-2012M1A2A20 26557)]

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Q-32

High-Level Production of 3-Hydroxypropionic Acid by Metabolically Engineered Escherichia coli Using Glycerol as a Sole Carbon Source

Yoo-Sung Ko1,2, Je Woong Kim1,2, Tong Un Chae1,2, and Sang Yup Lee1,2,3*

1Metabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Laboratory, Department of Chemical and Biomolecular Engineering (BK21, 2BioProcessEngineering Research Center, KAIST, Daejeon, Republic of Korea 3BioInformatics Research Center, KAIST, Daejeon, Republic of Korea

3-Hydroxypropionic acid (3-HP), an industrially important C3 chemical,was overproduced in a high level from glycerol/crude glycerol as a sole carbon source by metabolically engineered Escherichia coli with a potential for an industrial scale production. A glycerol-dependent 3-HPbiosynthetic pathway was constructed with the introduction of heterologousgenes and subsequent screening of those genes combinations was performed. Through a fed-batch fermentation, 49.9 g/L of 3-HP was produced. Further improvement in 3-HP production was succeeded bymanipulating fermentation condition leading to produce 76.2 g/L of 3-HP with a yield of 0.457 g 3-HP/g glycerol and with a productivity of1.98 g/L. This strain also produced 61.0 g/L of 3-HP with a yield of 0.594 g 3-HP/g crude glycerol and with a productivity of 2.28 g/L/h when takingcrude glycerol as its sole carbon source.

Keywords : Metabolic engineering, 3-Hydroxypropionic acid, glycerol

[The work was supported by the Technology Development Program toSolve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) and by Hanwha Chemical through KAIST-Hanwha Solutions Future Technology Institute]

Q-33

Metabolic Engineering of Escherichia coli and Corynebacterium glutamicum to Produce Methyl-anthranilate, a Grape-Flavoring Compound

Jae Sung Cho, Zi Wei Luo, and Sang Yup lee*

Department Chemical and Biomolecular Engineering, KAIST, Republic of Korea

Methyl anthranilate (MANT), a chemical that gives off grape scent and flavor, is a widely used compound in flavoring foods and drugs, but it is currently produced by petroleum-based processes. Here, we report thedirect fermentative production of MANT from glucose by metabolicallyengineered Escherichia coli and Corynebacterium glutamicum strains harboring a synthetic plant-derived metabolic pathway. Optimizing thekey enzyme anthranilic acid (ANT) methyltransferase1 (AAMT1) expression, increasing the direct precursor ANT supply, and enhancingthe intracellular availability and salvage of the cofactor S-adenosyl- L-methionine required by AAMT1, results in improved MANT production in both engineered microorganisms. Furthermore, in situ two-phase extractive fermentation using tributyrin as an extractant is developed to overcome MANT toxicity. Fed-batch cultures of the finalengineered E. coli and C. glutamicum strains in two-phase cultivation mode led to the production of 4.47 and 5.74 g/L MANT, respectively, in minimal media containing glucose. The metabolic engineering strategies developed here will be useful for the production of volatile aromatic esters including MANT.

Keywords : Methyl-anthranilate, Corynebacterium glutamicum, Escherichia coli

[This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineriesfrom the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea (Grants NRF-2012M1A2A2026556 and NRF- 2012M1A2A2026557)]

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Q-34

Genome Engineering of Corynebacterium glutamicum Using CRISPR/Cas9-Based Recombineering

Jae Sung Cho, Kyeong Rok Choi, Cindy Pricilia Surya Prabowo, Jae HoShin, Dongsoo Yang, Jaedong Jang, and Sang Yup Lee*

Department of Chemical and Biomolecular Engineering, KAIST, Republic of Korea

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, relies heavily onrandom mutagenesis and rather inefficient double crossover events. Here, we report a rapid genome engineering strategy to scarlessly knockout one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single- stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecTfor efficient curing such that multiple gene targets can be done iterativelyand final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study thecombinatorial deletion effects of three different genes on the productionof γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum.

Keywords : CRISPR/Cas9, Corynebacterium glutamicum, recT

[This work was further supported by Hanwha Chemical through KAIST-Hanwha Chemical Future Technology Institute)][This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineriesfrom the Ministry of Science and ICT through the National Research Foundation of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557)]

Q-35

Corynebacterium glutamicum as a Biofactory for 5-Aminovaleric Acid Production

Taehee Han1, Jae Ho Shin1, Seok Kyun Park1, Young Hoon Oh1, Jae Woong Choi1, and Sang Yup Lee1,2,3* 1Metabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Cross- Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea 2BioProcess Engineering Research Center, KAIST, Daejeon, Korea 3BioInformatics Research Center, KAIST, Daejeon 34141, Republic of Korea

5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical especially for synthesis of polymers and other industrially valuable chemicals. Employment of lysine 2-monooxygenase encodedby the davB gene and 5-aminovaleramidase encoded by the davA genehas been successful for enzymatic conversion of L-lysine to 5AVA. Inaddition, a recombinant Escherichia coli strain expressing the davB anddavA genes was developed for the bioconversion of L-lysine to 5AVA. Corynebacterium glutamicum, an efficient L-lysine producing microorganism, is a highly promising platform to develop of direct fermentative production of 5AVA using L-lysine as a precursor for 5AVA.In this report, metabolic engineering was done in C. glutamicum to enhance the fermentative production of 5AVA from glucose.

Keywords : Metabolic engineering, systems biology, Corynebacterium glutamicum

[This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineriesfrom the Ministry of Science and ICT through the National Research Foundation of Korea [NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557]

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R_Whole Cell Biocatalysis and Bioprocess Engineering

Poster S

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R-1

RRaman Based In-line Monitoring: AKey Technique for Bioprocess Digital Twin towards Advanced Biomanufacturing

Cheol-Hwan Park, Seo-Young Park, and Dong-Yup Lee*

School of Chemical Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 16419, Republic of Korea

Despite increasing demand for high product quality of biotherapeutic products such as monoclonal antibodies, biomanufacturing processes are still operated based on experience due to highly complex and uncertain nature. In order to overcome this limitations, various sensorsand process analytical techniques (PAT) have evolved to characterize the bioprocesses, and especially, in-line monitoring technique based on Raman spectroscopy has been significantly developed to closely monitor the dynamic behaviors of biomanufacturing processes. The aimof this review is to provide an overview of current Raman based in-linemonitoring and to discuss applications in mammalian cell culture processes, mainly by Chinese hamster ovary (CHO) cells which are themost widely utilized host organism for industrial production of biotherapeutics. We observe that the Raman monitoring technique has the ability to measure important quality-related process variables. We observed, however, that these Raman based monitoring requires complex data handling via robust chemometric models to extract validprocess information. The review concludes with perspectives for Ramanbased in-line and real-time techniques to characterize the manufactureof biopharmaceuticals with consistent product quality and improved performance. In short, the in-line Raman monitoring system could be one of the most feasible practical PATs for in-line and real-time processmonitoring technique for a simultaneous analysis of multi-attributes inbioprocess, guiding the process monitoring efforts as a part of digital twinfor advanced biomanufacturing.

Keywords : Raman spectroscopy, in-line monitoring, CHO cells

R-2

Increasing Resistance to Inhibitory Components by Iron Oxide Supplementation in Biohydrogen Production

Jeong-Hoon Park1, Do-Hyung Kim2, Jeong-Jun Yoon2, Youngsun Hong1,Seung Jin Oh1, Young Jin Yang1, and Tae-Guen Lim1,3 1Sustainable Technology and Wellness R&D Group, Korea Institute of Industrial Technology (KITECH), Jeju-si 63243, Republic of Korea 2Green and Sustainable Materials R&D Department, Korea Institute of Industrial Technology (KITECH), Cheonan-si 31056, Republic of Korea 3Department of Civil, Environmental and Architectural Engineering, Korea University, Seoul 02841, Republic of Korea

Hydrogen is mainly supplied as petrochemical-based byproduct and extracted gas from fossil fuels. In order to avoid economic and environmental problems by conventional chemical-based hydrogen production process, many studies for eco-friendly hydrogen productionare conducted actively. In particular, the hydrogen production by biological process has the advantages that the process is relatively simpleand waste resources can be used as substrate. In the case of using wasteresources or biomass are used as a substrate, a series of processes are required to remove contained toxic substances. In this study, the effect of iron oxide (Fe2O3) supplementation on hydrogen production was investigated as a strategy to overcome the decrease in hydrogen productivity by toxic substances. The effects on toxic substances and ironoxide supplement were evaluated in each conditions of 0-2.9 g/L of formic acid, 0-5.7 g/L of levulinic acid and 0-2.0 g/L of 5-HMF. Unlike5-HMF, formic acid and levulinic acid showed a tendency to increasehydrogen productivity at a certain concentration. The concentration offormic acid and levulinic acid calculated by kinetic analysis were 1.1 g/L and 3.0 g/L, respectively, for the maximum hydrogen productivity.Iron oxide supplement was found to be able to overcome the inhibitoryeffects up to about 2.5 g/L of formic acid, 5.7 g/L of levulinic acid and2.1 g/L of 5-HMF.

Keywords : Biohydrogen, iron oxide, inhibition

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R-3

Production of Biodegradable Plastic Polymer, Poly-3-hydroxybutyrate (PHB) by Newly Isolated Methanotroph

Ok Kyung Lee, Seunggi Kang, and Eun Yeol Lee*

Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 17104, Republic of Korea

Plastics are used in many applications because of their durability, stability and low cost, but it is known to release methane during their decomposition, which has a negative impact on global warming. Methanotroph could be a consolidated solution to solving two major environmental problems simultaneously by using methane as a sole carbon source and accumulating the methane as poly-3-hydroxybutyrate(PHB), a biodegradable plastic polymer. Unfortunately, methanotrophspecies isolated and reported to date are quite limited. In this study, wereport the isolation and characterisation of novel methanotroph with theability to produce up to 38% PHB of dry cell weight from cattle shed. The new type II methanotroph, Methylocystis strain was characterizedusing phylogenetic analysis and electron microscopy analysis. Responsesurface methodology (RSM) was applied to improve PHB production in a flask scale. The use of RSM optimized medium yielded up to 1.77 ± 0.5 g/L in biomass, 43.2 ± 0.5 wt% PHB of dry cell weight, which improved the biomass and PHB content to 2.5 and 1.13-fold, respectively.

Keywords : Poly-3-hydroxybutyrate, methane, methanotroph

R-4

Whole Cell Biotransformation of C12 Fatty Derivatives Using E. coli Two-Cell Systems Expressing Carboxylic Acid Reductase and Alcohol Dehydrogenase

Kwon-Young Choi1,2, Tae-Young Cha1, Yong Yuk1, and HyunA Park2 1Department of Environmental and Safety Engineering, Ajou University, Suwon, 16499, Republic of Korea 2Department of Envrionmental Engineering,Ajou University, Suwon 16499, Republic of Korea

Here, we present the whole cell biotransformation of 1-dodecanoic, ω-hydroxydodecanoic acid and α,ω-dodecanedioic acid using whole cellbiotransformation of E. coli BW25113DfadD expressing carboxylic acid reductase (CAR) and alcohol dehydrogenase (ADH) enzymes. Among the screened 13 CAR enzyme, CAR enzyme from Mycobacteriumabscessus showed the highest 1-dodecanoic acid reduction activity of 53.12 % conversion in single cells of heterologous CAR and endogenous ADH. Next, the host cells were engineered to simultaneously expressYarrowia lypolytica ADH with the GroES/EL-DnaK/J/E chaperone in a single host system, while two cell systems used two strains of E. coliexpressing CAR-Sfp and ADH-GroES/EL-DnaK/J/E, respectively. In results, additional ADH expression was not effective in a single host system, whereas two cell system significantly increased α,ω-dodecanedioic acid conversion by total 71.3%; α,ω-dodecanediol (68.2%) and ω-hydroxydodecanoic acid (3.1%), respectively. Interestingly,the CAR4714 enzyme could converted ω-hydroxydodecanoic acid intoα,ω-dodecanediol up to 97.17% conversion in 17 h (12.43 mg/L/h).

Keywords : C12 fatty derivatvies, CAR, ADH

R-5

Media Optimization for Medium Chain Length PHA Production from Pseudomonas sp. Using Alkane Mixture (n-Octane, n-Decane, and n-Dodecane)

Sojin Park1,2, Jeong-Jun Yoon1*, Yung-Hun Yang3, and Jong-Min Jeon1

1Department of Green & Sustainable Materials R&D, Korea Institute of Industrial Technology (KITECH), Chungnam 31056, Republic of Korea 2Department of Production Technology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea 3Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea

Polyhydroxyalkanoate (PHA) is a biodegradable plastic that replaces the petroleum based and has diverse physicochemical properties depends on the monomer composition. In particular, the medium-chain-length polyhydroxyalkanoates (mcl-PHA) have elastomeric properties and aremore suitable for high value-added applications. The aim of this study was media optimization for mcl-PHA production by Pseudomonasspecies using alkane mixture. To optimize the mcl-PHA production, it was determined the mixture ratio of alkanes by mixture analysis method,and the contents of n-octane, n-decane and n-dodecane in media were9.15%, 6.44% and 4.29%, respectively. Moreover, the optimal concentration of nitrogen and phosphorus for PHA production were 0.05% and 1.0%, respectively. When Pseudomonas sp. was cultured in the above medium, the cell growth and PHA yield were obtained 3.41g/Lof cell dry weight and 2.05 g/L PHA with 60wt% PHA content.

Keywords : Medium chain length PHA, alkane mixture, Pseudomonasspecies

[This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Ministry of Science and ICT (NRF- 2020R1A2C2102381) and Korea Institute of Industrial Technology (KITECH-EO210014)]

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R-6

Characterization of Halophilic Microorganisms Isolated from the Fermented Seafood

Bo Gyoung Choi1, Dariimaa Ganbat1, Ga Eul Jeong1, YuJeong Yeom1, HaeRang Lee1, Jae Seung Lim1, Yong-Jik Lee2, Mi-Hwa Park3, Han-SeungLee1, and Sang-Jae Lee1* 1Major in Food Biotechnology and Research Center for Extremophiles & Marine Microbiology, Silla University, Busan 46958, Republic of Korea 2Department of Bio-Cosmetics, Seowon University, Chung-Ju 28674, Republic of Korea 3Depatrment of Food and Nutrition, Silla University, Busan 46958 Republic of Korea

To identify the diversity of halophilic bacteria from fermented seafoods,86 strains were isolated and phylogenetic analysis was carried out basedon the result of 16S rRNA gene sequencing. The isolated strains were divided into 3 phyla, 7 families, 9 genera, 24 species. Bacilli class, themain phyletic group, comprised 84.9% with 4 families, 6 genera and 19species of Bacillaceae, Planococcaceae, Staphylococcaceae and Enterococcaceae. The strains were tested for amylolytic, cellulolytic, lipolytic, proteolytic activity and 55 strains showed at least one enzymeactivity. Furthermore, auxin activity was determined in two strains. These results indicated that isolated strains have the possibility of the application for the food and feed industries and importance of genetic resource in Korea.

Keywords : Fermented seafood, halophiles, food & feed industry

R-7

Cloning and Expression of Acido-Thermophilic α-Amylase coding gene from Alicyclobacillus sacchari for Isomaltodextrin Production

Bo Gyoung Choi1, Dariimaa Ganbat1, Gi-Seob Hong1, Seung Yeon Yoo1, Joon Young Hur1, Ji Yeong Park1, Ji-u Lm1, Dong-Woo Lee2, Seong-BoKim3, Han-Seung Lee1, Sang-Jae Lee1*

1Major in Food Biotechnology and Research Center for Extremophiles & Marine Microbiology, Silla University, Busan 46958, Republic of Korea 2Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 3Bio-Living Engineering Major, Global Leaders College, Yonsei University, Seoul 03722, Republic of Korea

This study investigates a novel acido-thermophilic a-amylase, an enzyme required to produce Isomaltodextrin (IMD). In the manufacturingprocess of Isomaltodextrin, food-grade starch is treated with α-amylaseto cleave the amylose and amylopectin chain into shorter units. It is thenheated until liquefied. α-Amylase (α-1.4 D-glucan glucanohydrolase) (EC 3.2.1.1) hydrolyzes the α-1,4 glucosidic bonds of starch and produced glucose-containing oligosaccharides. In particular, α-amylase,which remains active in the presence of denaturants, exhibits activity over a wide range of temperature and pH, making it stable at high temperatures, lowering cost, enhancing substrate solubility, and loweringcontamination originated from mesophilic microorganisms, which is why it is preferred in industry. The coding gene of acidophilic Alicyclobacillus sacchari DSM 17974 alpha-amylase (ASAA) was cloned and expressed in E. coli BL21. Analysis of the sequence revealedan open reading frame of 2,715 bps that encodes a protein of 905 aminoacid residues with a calculated molecular mass of 98.3 kDa. The isoelectric point (pI) of the enzyme was about 4.46. The recombinant ASAA was purified to homogeneity by heat treatment, Ni-NTA chelateaffinity chromatography, thrombin treatment to remove His-tag from thefusion protein, and gel filtration. This work will contribute to the development of acido-thermostable α-amylase, one of the key enzymesin the production of isomaltodextrin.

Keywords : Isomaltodextrin, Acido-thermophilic α-amylase, Alicyclobacillussacchari DSM 17974

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R-8

Photoautotrophy CO2 Electrosynthesis Using Rhodobacter sphaeroides in Bioelectrochemical System

Shuwei Li and Jung Rae Kim*

School of Chemical and Biomolecular Engineering, Pusan National University Busan 46241, Republic of Korea

To alleviate the greenhouse effect and climate change, it is of great importance to develop sustainable CO2 capture and utilization. The microbial electrosynthesis (MES) has been highlighted to convert CO2to value added metabolites and intermediate chemicals by using electrochemically active bacteria as biocatalyst and electricity as reducing power. However, the MES only produced simple organic acidsor alcohols from CO2. In this study, we presented a novel bioelectrosyntheticpathway for polyhydroxybutyrate (PHB) production using a photosyntheticbacteria, Rhodobacter sphaeroides. The electrode attached R. sphaeroidesdirectly uptakes electrons from the electrode surface, while the suspendedR. sphaeroides utilize the electrochemically evolved hydrogen as electron mediator to convert CO2 to polyhydroxybutyrate (PHB). The results show that MES-driven electrons and protons transfer enabled direct conversion of CO2 into higher-value added organic matter suchas PHB.

Keywords : CO2 electrosynthesis, PHB, Rhodobacter sphaeroides

R-9

Bioconversion of Ricinoleic Acid to (E)-11-(heptanoyloxy) undec-9-enoic Acid by Recombinant Escherichia coli Overexpressingan Alcohol Dehydrogenase and a Baeyer-Villiger Monooxygenase

Sueyoung Lim1, Yong-Han Cho1, Min-Geun Shin1, Kyungmoon Park2*, and Yong-Cheol Park1* 1Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea 2Department of Bio and Chemical Engineering, Hongik University, Sejong 30016, Republic of Korea

For efficient bioconversion of ricinoleic acid to (E)-11-(heptanoyloxy)undec-9-enoic acid (11-HOUA), a major intermediate of brassylic acid,a recombinant Escherichia coli overexpressing an alcohol dehydrogenasefrom Micrococcus luteus and a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440 was constructed and subjected to whole-cell biotransformation process. Feeding of glucose or glycerol facilitated both the supply of NAD+ and NADPH cofactors and the preparation of high-density cell biocatalyst. By the glucose feeding strategy, 30.8 g/L of the engineered E. coli cells produced 29.7 mM of 11-HOUA with 1.9 mM/h of productivity, which were 1.8 and 1.6 times higher than the same biotransformation without the glucose feeding, respectively. Intermittent addition of glycerol increased 11-HOUA productivity by 16% compared to that by the glucose feeding. Finally, 34.5 mM of 11-HOUA concentration, 77% conversion and 2.2 mM/h productivity were obtained using 31.6 g/L of cell biocatalyst along withthe glycerol addition. It was concluded that supplementation of additional carbon sources in biotransformation process would be a potentstrategy to increase the performance of fatty acid bioconversion.

Keywords : Ricinoleic acid, (E)-11-(Heptanoyloxy) undec-9-enoic acid, recombinant Escherichia coli

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Production of Azelaic Acid from Nonanoic Acid and Its Esters by Direct Biotransformation of Candida tropicalis

Inji Yeo, Ji-Young Kim, Min-Woo Jun, Yeong-Eun Cho, and Yong-CheolPark*

Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea

Azelaic acid is an α,ω-dicarboxylic acid able to be used as a polyester monomer and strong antibacterial agent. Through chemical oxidation of oleic acid at a hazardous condition, azelaic acid is produced commercially. In this study, a sustainable bioconversion process using whole cell biocatalyst was developed. To directly convert nonanoic acid,and its esters to azelaic acid and to increase the total yield of azelaic acidduring the ozone decomposition. after ethyl nonanoate addition whole cell of Candida tropicalis ATCC20962 immediately cleaved ethyl nonanoate to nonanoic acid, and then converted nonanoic acid into azelaic acid with the aid of nonane addition and continuous glucose supply. Finally, a fed-batch biotransformation by continuous feeding ofpure nonanoic acid resulted in the production of 30.1 g/L azelaic acid with 0.30 g/L-h productivity and 90% molar yield. By combination of the ozonolysis and our process, a maximum of 95% molar carbon yieldof azelaic acid to oleic acid was estimated. This is the first report that nonanoic acid and its esters were directly and biologically transformedto azelaic acid with over 90% yield, and would be a groundwork for the biotransformation of fatty acids with under nine carbons to the corresponding α,ω-dicarboxylic acids.

Keywords : Azelaic acid, nonanoic acid, biotransformation

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Bioconversion of Acetone to Acetol in Methylomonas sp. DH-1 biocatalyst by Heterologous Expression Novel Protein from Methylacidiphilum sp. IT6

Tin Hoang Trung Chau, and Eun Yeol Lee*

Department of Chemical Engineering (Integrated Engineering), Kyung Hee University, Yongin 17104, Republic of Korea

Acetol (hydroxyacetone) is a well-known intermediate or starting material for various industrial applications. Newly isolated strain Methylacidiphilum sp. IT6 can assimilate acetone for growth with the high expression recorded in the pmoCAB3 operon and the operon- flanking hypothetical protein IT6_09410 (Genbank accession number QSR88567). Besides, Methylomonas sp. DH-1 was claimed to accumulated acetone due to the deficiency of converting enzymes. In this study, we expressed this hypothetical protein IT6_09410 of Methylacidiphilum sp. IT6 by integrating into Methylomonas sp. DH-1and evaluating the acetone assimilation of the mutant as a whole-cell biocatalyst. The maximum accumulation, average productivity, and specific productivity of acetol were 18.291 mM, 1.52 mM/h and 0.158 mmol/g cell/h under the optimized conditions. Methylomonas sp. DH-1 with heterologous expressed protein IT6_09410 demonstrated the potential to be deployed as a whole-cell biocatalyst for the assimilationof acetone into acetol, which could be further converted into value-added materials.

Keywords : Acetone, acetol, Methylomonas sp. DH-1

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Quick Start-Up and High Power Density of Microbial Fuel Cell Enhanced with a Polydopamine/Polypyrrole Graphite Felt

Minsoo Kim, Lishuwei, Eunseo Kim, and Jung Rae Kim*

School of Chemical Engineering, Pusan National University, Busan 46241, Republic of Korea

Microbial fuel cells (MFCs) convert chemical energy directly to electrical energy via electrochemically active microorganisms. The interactions between microbes and the surface of anode play an importantrole in capturing the respiratory electrons from bacteria. This is becauseit influences biofilm growth and extracellular electron transfer (EET). Therefore, anode performance is a key factor for the power density ofMFC. In this study, a novel type of anode was developed by electrodepositionusing polydopamine (PDA), polypyrrole (PPY), and graphite felt (GF).The prepared PDA-PPY-GF reached 600mW/m2, which was 1.5, 1.15,and 1.13 times greater than those of the GF, PDA-GF, PPY-GF, respectively. PDA has superior hydrophilicity and adhesive force for strong interaction with biofilm, and PPY provides electrochemically active sites for electron transfer between biofilm and anode. Results fromRaman Spectroscopy, Brunauer-Emmett-Teller (BET) and Contact angle analysis show an increase in the physicochemical properties. These results show that modification provides a promising anode electrocatalyst for high-performance MFC to enhance waste treatmentfor the purpose of future bioprocesses.

Keywords : Microbial fuel cell, polydopamine, polypyrrole

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Room-Temperature Cell Disruption for Astaxanthin Recovery from Haematococcus pluvialis Cyst Using Ultrathin Nanoplates and Imidazolium-Based Ionic Liquids

Lakshmi Narasimhan Aditya1, Nakyeong Lee1,2, Gyuseop Moon1, Young-Eun Kim1, Myeonghwa Park1,2, Bolam Kim1,2, Sungwook Chung1,and You-Kwan Oh1,2*

1School of Chemical Engineering, Pusan National University (PNU), Busan 46241, Republic of Korea 2Institute for Environment & Energy, Pusan National University (PNU), Busan 46241, Republic of Korea

High-efficiency, energy-saving extraction is a critical step for the recovery of heat-labile astaxanthin pigments from mature Haematococcuspluvialis with a rigid, thick cell-wall structure. Recently, as ‘green’ solvents, ionic liquids (ILs) have been extensively applied for algal biorefinery purposes. However, when applied to H. pluvialis, their efficiencies are low or high-temperature conditions are required. Here,we report the technical feasibility of room-temperature extraction of astaxanthin from H. pluvialis cyst cells by combining ultrathin α-quartznanoplates with ethyl-3-methylimidazolium ([Emim])-based ILs. When pretreated with nanoplates, the cell walls of intact cyst cells wereseverely lacerated, thereby facilitating subsequent extraction of astaxanthinby 4 [Emim]-based ILs with SCN, DEP, HSO4, and Cl anions. At room temperature (28℃) conditions, the astaxanthin extraction efficiencies were significantly improved by 5 times regardless of the IL type, compared to the controls without NPL pretreatment, resulting in high astaxanthin recoveries of ~81%.

Keywords : Haematococcus pluvialis, astaxanthin, ionic liquid

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Modular Process Design of Cinnamaldehyde Production through Engineering of Escherichia coli and Corynebacterium glutamicum

Jaewoo Son, Hyunbae Bang, In Hyeok Choi, Chang Gyu Lim, Jun HongJang, Ji Won Cha, Eun Jung Jeon, Min Gi Sohn, Hyo Jin Yun, and Ki JunJeong*

Department of Chemical and Biomolecular Engineering, KAIST, Daejeon 34141, Republic of Korea

Cinnamaldehyde (CAD) is a major ingredient of cinnamon oils that is produced by cinnamon plants. Currently, CAD is drawing attention sinceit has potential to be applied in foods and pharmaceuticals. Nevertheless,conventional production process for CAD such as extraction from plantsor chemical synthesis is not eco-friendly, thus limits the utilization of CAD for downstream applications. Here, we developed modularly microbial-based bioprocess system for high production of CAD. As thefirst process module, maximum titer of L-phenylalanine (L-Phe) couldbe achieved up to 38.6 g/L at a pilot-scale (30 L) fed-bath fermentationthrough the genome-scale metabolic engineering in E. coli. Next, as a second module, a highly efficient bioconversion process using C. glutamicum as a whole-cell biocatalyst was developed for high production of trans-cinnamic acid (t-CA) from L-Phe as a substrate. Weengineered C. glutamicum in which phenylalanine ammonia lyase fromStreptomyces maritimus could be expressed to levels as high as 39.1 %of the total proteins. In addition, through the optimal conditions (temperature, pH, and base source) and recycling system coupled withmembrane filtration with a bioreactor, 8.1 g/L of t-CA from 12 g/L ofL-Phe could be achieved. For the last module, C. glutamicum was engineered for efficient bioconversion t-CA into CAD for a whole-cellbiocatalyst-based fermentation process. An expression module of carboxylic acid reductase from Mycobacterium phlei was constructed,and the putative dehydrogenase involved in the conversion of CAD to cinnamyl alcohol related genes were deleted for preventing loss of CADin C. glutamicum. To this end, by deletion of the gene involved in thereverse conversion of CAD back to t-CA, we developed engineered C.glutamicum that produced 1.1 g/L of CAD for 1.2 g/L of t-CA.

Keywords : Corynebacterium glutamicum, Escherichia coli, Cinnamaldehyde

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Factors Affecting Product Formation and Performance in Syngas Fermentation by Eubacterium limosum KIST612

Mungyu Lee, Nulee Jang, Ji-Yeon Kim, Soyoung Oh, and In Seop Chang*

School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea

Researchers are reporting various studies to secure eco-friendly energyfrom stable resources due to the unstable oil price volatility, global warming and population growth issues. These studies are focusing to produce various chemicals and energy by environmentally friendly wayfrom various resources existing in the world. The energy production process using microorganisms is more eco-friendly than the other processes, and has the advantage of less secondary toxic by-products than chemical processes. Especially, in the case of gas fermentation, utilization of waste gas from gasification and industries, are very usefuland attractive Synthesis gas (syngas) is a fuel gas mixture that mainly include CO, CO2 and H2. The biofuels and chemicals can be produced from Syngas by various microorganisms. Acetogens can produce biofuels and chemicals through Wood-Ljungdahl pathway by using COas sole carbon and energy source. Mutants are being developed to produce novel products or increase the productivity of existing products.However, the performance of product production from microbes has tobe considered by various parameter not only basic growth curve. Eventhe same microorganisms may exhibit higher productivity or worse results depending on the actual process operation method. In this study,the product (acetate and butyrate) production changes according to operation methods using Eubacterium limosum KIST612, moreover introduce the operation method according to the target product.

Keywords : Bioreactor operation strategy, syngas fermentation, acetate