Genomics, Proteomics and Secondary Metabolites ...

- 1 -

Genomics, Proteomics and Secondary

Metabolites Biosynthesis Research on

Streptomyces asterosporus DSM 41452

Dissertation zur Erlangung des Doktorgrades

der Fakultät für Chemie und Pharmazie

der Albert-Ludwigs-Universität Freiburg im Breisgau

Vorgelegt von

Songya Zhang

Aus Zhengzhou, China

2018

- 2 -

Dekan: Prof. Dr. Manfred Jung

Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber

Referent: Prof. Dr. Andreas Bechthold

Korreferent: Prof. Dr. Irmgard Merfort

Drittprüfer: Prof. Dr. Oliver Einsle

Datum der Promotion: 20.04.2018

- 3 -

Erklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbstständig und nur unter

Verwendung

der angegebenen Literatur und Hilfsmittel angefertigt sowie Zitate kenntlich gemacht

habe.

- 4 -

Acknowledgements

I would like to take this opportunity to express my appreciation to my supervisor, Prof. Dr.

Andreas Bechthold, for his support, encouragement and guidance throughout my study in this

outstanding research environment at the Fakultät für Chemie und Pharmazie in Freiburg

University. His enthusiasm and attitude towards science and research will definitely affect my

life.

I am particularly thankful to my co-advisor Prof. Dr. Irmgard Merfort for kindly reviewing this

thesis, her generous help during my study, for being the supervisor of my doctoral committee.

I desire to convey my earnest appreciation to Prof. Dr. Oliver Einsle for being the member of

my doctoral committee. In addition, I feel grateful to Dr. Lin Zhang for his help to determine

protein structure and professional advice on my research project.

I would also wish to thank Prof. Dr. Stefan Günther for reviewing the manuscript. I also want

to express my greatest thanks to Dennis Klementz for his reliable help in terms of

bioinformatics analysis and for helping revise my manuscript.

My deep gratitude as well goes to Prof. Dr. David Zechel for his discussion about my research

project, review my paper in earnest. His attitude toward academic research really motivates

me a lot.

I desire to thank Dr. Claudia Jessen-Trefzer for her kind help about my project and her helpful

discussions, and for her amendment to the manuscript. Also, I am grateful to Dr. Thomas

Pauluat for the NMR data analysis and his professional suggestion on the manuscript.

I am really grateful to Dr. Max Cryle from Monash University for his kindly providing substrate

and earnest guidance on the P450 project. In addition, I want to thank Dr. Greule Anja for her

suggestion and effort to solve this interesting scientific question.

I also want to express my greatest gratitude to Dr. Verónica I. Dumit and Dr. Mingjian Wang

from Center for Biological Systems Analysis in Freiburg University for their assistance,

professional suggestion and guidance on proteomics analysis.

I want to thank Prof. Dr. Jun Yin from Georgia State University, Dr. Stephen G. Bell from

University of Adelaide, Mr. Gunter Stier from Heidelberg University for their kindness of

providing plasmids.

- 5 -

I am appreciated Prof. Dr. Xin-zhuan Su and Dr. Richard Eastman from National Center for

Advancing Translational Sciences/NIH for helping compound activity test and manuscript

review.

I would like to thank all my colleagues who helped me throughout my PhD study. I would like

to thank Dr. Gabriele Weitnauer for her kind help and unselfish assistance in many ways. I

would like to express my warmest gratitude to Marcus Essing, Sandra Groß, Elizabeth Welle

and Frau Weber for their contribution to the lab. Especially, I like to thank Marcus Essing for

strong technician support for maintaining equipment running in the lab. My gratitude also

goes to Sandra Groß for tireless help in the lab, and for reviewing my thesis. I also want to

thank Elizabeth Welle for strong technician assistance on the LCMS, HPLC system in the lab. I

want to thank Sandra Cabrera, Dr. Susanne Elfert, Tanja Herbstritt and Judy Wang for their

kind help. I would like to thank Dr. Roman Makitrynskyy for his kind help during my study. In

the meanwhile, I would like to thank all my current and previous colleagues working in the lab

of AG Bechthold for their selfless help and for sharing me various kinds of perspectives and

outlook to the life, world. I wish all of them have a wealthier and more successful future.

Last but not the least I would like to show my gratitude to my family for their backing,

especially my loving wife: Jing Zhu, for her unconditional support and passion. I owe all the

success of my PhD study to my family.

- 6 -

Wissenschaftliche Publikationen und Akademische Aktivitäten

Wissenschaftliche Publikationen

I. Songya Zhang, Andreas Bechthold. Iteratively Acting Glycosyltransferases, 2nd edition of

Handbook of Carbohydrate-Modifying Biocatalysts, 2016, Pages 321-348, Pan Stanford

Publishing.

II. Songya Zhang, Jing Zhu, Tao Liu, Suzan Samra, Huaqi Pan, Jiao Bai, Huiming Hua, Andreas

Bechthold, a Novel Glycosylated Polyketide from the Terrestrial Fungus Myrothecium sp.

GS-17. Helvetica Chimica Acta, 2016, 99 (3), 215-219.

III. Songya Zhang, Jing Zhu, David Zechel, Claudia Jessen-Trefzer, Richard T. Eastman, Thomas

Paululat, Andreas Bechthold. Novel WS9326A derivatives and one novel Annimycin

derivative with antimalarial activity are produced by S. asterosporus DSM 41452 and its

mutant, ChemBioChem, 2017, 19(3), 272-279.

IV. Arne Gessner, Tanja Heitzler, Songya Zhang (cofirst), Christine Klaus, Renato Murillo,

Hanna Zhao, Stephanie Vanner, David L. Zechel, Andreas Bechthold. Changing

Biosynthetic Profiles by Expressing bldA in Streptomyces Strains. ChemBioChem, 2015,

16(15):2244-2252.

V. Greule Anja, Songya Zhang, Thomas Paululat, Andreas Bechthold. From a Natural Product

to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from

Streptomyces diastatochromogenes Tü6028. Journal of visualized experiments: JoVE,

2017, (119): 54952.

VI. Anja Greule, Marija Marolt, Denise Deubel, Iris Peintner, Songya Zhang, Claudia Jessen-

Trefzer, Christian De Ford, Sabrina Burschel, Shu-Ming Li, Thorsten Friedrich, Irmgard

Merfort, Steffen Lüdeke, Philippe Bisel, Michael Müller, Thomas Paululat, Andreas

Bechthold. Wide distribution of foxicin biosynthetic gene clusters in Streptomyces

strains-an unusual secondary metabolite with various properties. Frontiers in

microbiology, 2017, 8:221.

VII. Songya Zhang, Dennis Klementz, Jing Zhu, Stefan Günther, Andreas Bechthold. The

complete genome sequence of S. asterosporus DSM 41452, a high producer of the

- 7 -

neurokinin A antagonist WS9326As. Journal of Biotechnology (under review).

VIII. Arslan Sarwar, Zakia Latif, Songya Zhang, Andreas Bechthold, Biological control of potato

common scab with rare Isatropolone C compound produced by Streptomyces sp. A1RT,

Frontiers in microbiology (under review).

IX. Songya Zhang, Mingjian Wang, Dennis Klementz, Jing Zhu, Verónica I. Dumit, Andreas

Bechthold. Comparative Proteomic Analysis of S. asterosporus DSM 41452 reveals the

AdpA regulon in a native non-sporulating Streptomyces species by SILAC. Applied

microbiology and Biotechnology (in preparation).

X. Songya Zhang, Lin Zhang, Anja Greule, Jing Zhu, Oliver Einsle, Max Cryle, Andreas

Bechthold, Structural Characterization of Cytochrome P450WS9326A, mediates the

formation of the olefinic bond to generate the dehydrotyrosine formation in WS9326A

Biosynthesis, ACS chemical biology (in preparation).

Poster Präsentation

I. Songya Zhang, Lin Zhang, Anja Greule, Jing Zhu, Max Cryle, Oliver Einsle, Andreas

Bechthold. Structural Characterization of Cytochrome P450 Sas16, mediates the

formation of the olefinic bond to generate the dehydrotyrosine formation in WS9326As

Biosynthesis. RTG 1976 Symposium 2017: Unique Cofactor-dependent Enzymes in

Microbes, 10/2017, Freiburg, Germany

II. Songya Zhang, Dennis Klementz, Mingjian Wang, Jing Zhu, Stefan Günther, Verónica I.

Dumit, Andreas Bechthold. Complete genome sequencing and comparative Proteomic

Analysis of S. asterosporus DSM 41452 reveals the AdpA regulon in a native non-

sporulating Streptomyces species. International VAAM-Workshop 2017: Biology of

Bacteria Producing Natural Products, 09/2017, Tübingen, Germany.

III. Songya Zhang, Jing Zhu, Andreas Bechthold. WS9326A Derivatives from S. asterosporus

DSM 41152: Chemical Structure and Biosynthesis. International VAAM-Workshop 2016:

Biology of Bacteria Producing Natural Products, 09/2016, Freiburg, Germany

IV. Songya Zhang, Jing Zhu, Roman Makitrynskyy, Olga Tsypik, Andreas Bechthold.

Connecting Chemotype, Phenotype and Genotype, revealing the Gene Regulatory

Mechanism of Morphological Development and Secondray Metabolism in S. asterosporus

- 8 -

DSM 41452.Tag der Forschung der Universität Freiburg 2016, 07/2016, Freiburg,

Germany

V. Songya Zhang, Jing Zhu, Tao Liu, Suzan Samra, Huiming Hua, Andreas Bechthold.

Exploiting and Elucidation of a new Glycosylated Polyketide from Fungus Myrothecium

sp., 2016 VAAM Annual Conference, 03/2016, Jena, Germany

VI. Jing Zhu, Songya Zhang, Andreas Bechthold. Revealing the Hidden “domain skipping”

Biosynthetic Mechanism in the Annimycin Polyketide Synthase from S. asterosporus

DSMZ 41452. VAAM workshop, 09/2016, Freiburg, Germany

VII. Jing Zhu, Xiaohui Yan, Anja Greule, Songya Zhang, Andreas Bechthold. Exploring the

Biosynthetic Capability of Ganefromycin by Direct Cloning and Heterologous Expression,

Annual Conference 2016 of the Association for General and Applied Microbiology (VAAM),

03/2016, Jena, Germany

VIII. Anja Greule, Songya Zhang, Andreas Bechthold. Foxicins: Ortho-Quinone Derivates

produced by Polyketomycin Producer Streptomyces diastatochromogenes Tü6028,

International Symposium on the Biology of Actinomycetes, 10/2014, Kuşadasi, Turkey

- 9 -

Abstract

Many important industrial strains for antibiotic production belong to the genus of

Streptomyces. They are characterized as special bacteria with a complex fungus-like life cycle

(Ohnishi et al. 2005). The sporulation of Streptomyces has been clearly demonstrated to have

a significant association with the production of antibiotics (Chandra and Chater 2014). Non-

sporulation mutants fail to generate the aerial mycelium due to different reason. To date at

least 20 reported genes are involved in the aerial mycelium formation (Takano et al. 2003). We

previously reported that the defective bldA gene prevents the generation of aerial hyphae and

the formation of secondary metabolites in Streptomyces calvus by inhibiting the expression of

the TTA-containing adpA gene (Gessner et al. 2015; Hackl and Bechthold 2015). However, our

following research found that the constitutive expression of bldA in some “bald” Streptomyces

strains didn’t efficiently restore the sporulation, which attracts our interests. One of the strain

is Streptomyces asterosporus DSM 41452. Experimental data indicate that there is a potential

unknown mechanism causing the “poorly sporulating” phenotype in S. asterosporus DSM

41452. In this dissertation, the complete genome of S. asterosporus DSM 41452 was

sequenced and annotated. By detailed comparative genome sequence analysis, a transposon

gene was found upstream of adpA gene of S. asterosporus DSM 41452 which hinder the

transcription of adpA. By complementation of adpA gene with a functional promoter in this

strain, the sporulation was restored.

Proteomics has always been an efficient method to investigate the cellular physiology and

metabolism of an organism. In this thesis, we first time employ SILAC-based comparative

proteomic approach to profile the AdpA regulon in the native non-sporulating S. asterosporus

DSM 41452. In our study, more than 1200 proteins were identified, including proteins involved

in strain’s metabolism, cellular processing and signaling, information storage and processing,

etc. Most importantly, we managed to demonstrated that SILAC approach can be efficiently

applied for Streptomyces proteomics research.

In terms of its secondary metabolites of S. asterosporus DSM 41452, from the genome of S.

asterosporus DSM 41452, we found the gene clusters for WS9326A (Johnston et al. 2015) and

- 10 -

Annimycin (Kalan et al. 2013), which both have been detected in S. calvus ATCC 13382 were

identified through bioinformatics analysis of genome sequence of S. asterosporus DSM 41452.

Six compounds, WS9326A and its derivatives WS9326B, WS9326D, WS9326E, WS9326F, and

WS9326G were isolated from the scale-up fermentation of S. asterosporus DSM 41452.

Surprisingly, two new WS9326A derivatives SY11 and SY12 were isolated from one Annimycin-

defect mutant strain S. asterosporus DSM 41452::pUC19Δ3100spec, structures of SY11 and

SY12 were partially characterized by mass spectrometry and NMR. The boundary of WS9326A

gene cluster was determined by disrupting gene orf(-1) and sas1 at the terminus of the gene

cluster. In-frame gene knockout of the gene encoding the N-methyltransferase(MTase) in

module 2 of WS9326A NRPSs resulted in the disruption of WS9326A production, suggesting

that the methylation of the tyrosine residue is essential for the substrate recognition by the

downstream condensation domain. Gene inactivation of sas13 by single crossover seem didn’t

influence the production of WS9326A, which exclude the possibility of sas13 participating the

formation of the nonproteinogenic dehydrotyrosine residue in WS9326A production. In

addition, in-frame gene deletion of sas16 by PCR-targeting method led to the loss of WS9326A,

and the production of WS9326A was restored after the complementation of gene sas16.

Therefore, this gene is proposed to participate in the formation of the dehydrotyrosine moiety.

For an in-depth understanding of the biochemical role of Sas16 during the biosynthesis of the

N-methyl-dehydrotyrosine protein, the gene was heterologously expressed for in vitro

enzymatic assay. We successfully elucidated the protein structure of Sas16 to reveal the

underlying molecular basis of substrate selectivity of this Cytochrome P450 enzyme.

Keywords: S. asterosporus DSM 41452; Complete genome sequencing; Sporulation; AdpA;

Mutagenesis; Proteomics; SILAC; LC-MS/MS; Secondary metabolites; WS9326A; Derivative;

NMR spectroscopy; NRPS, Gene cluster; Gene inactivation; Gene deletion; Dehydrotyrosine;

Double bond; Biosynthesis; Protein expression; Crystallization; X-ray Crystallography; Protein

structure; P450 cytochrome; Enzyme catalytic assay;

- 11 -

Abstrakt

Streptomyceten, als eine Art wichtiger industrieller Stamm für die Herstellung von Antibiotika,

wurden über Jahrzehnte hinweg untersucht. Es wurde als spezielles Bakterium mit einem

komplexen pilzartigen Lebenszyklus charakterisiert (Ohnishi et al. 2005). Die Sporulation von

Streptomyceten wurde eindeutig als signifikant mit der Produktion von Antibiotika in

Verbindung gebracht (Chandra und Chater 2014). Nicht-Sporulations-Mutanten erzeugen das

Luftmyzel aus unterschiedlichen Gründen nicht. Bis heute sind mindestens 20 beschriebene

Gene an der Luftmyzelbildung beteiligt (Takano et al. 2003). Wir berichteten bereits, dass das

defekte bldA-Gen die Bildung von Lufthyphen und die Bildung von Sekundärmetaboliten in

Streptomyces calvus verhindert, indem es die Expression des TTA-haltigen adpA-Gens hemmt

(Gessner et al. 2015; Hackl und Bechthold 2015). Unsere folgenden Untersuchungen haben

jedoch ergeben, dass die konstitutive Expression von bldA in einigen "kahlen"

Streptomyceten-Stämmen, die von der DSMZ gekauft wurden, die Sporulation nicht effizient

wiederherstellt, was unser Interesse geweckt hat. Einer der Stämme ist Streptomyces

asterosporus DSM 41452. Das Phänomen zeigt an, dass ein potentieller unbekannter

Mechanismus den "schwach sporulierenden" Phänotyp in S. asterosporus DSM 41452

verursacht. In dieser Dissertation wurde das vollständige Genom von S. asterosporus DSM

41452 sequenziert und kommentiert. Durch detaillierte vergleichende Genomsequenzanalyse

wurde ein Transposon-Gen stromaufwärts des adpA-Gens von S. asterosporus DSM 41452

gefunden, das die Transkription von adpA behindert. Durch Komplementierung des adpA-

Gens mit einem funktionellen Promotor in diesem Stamm wurde die Sporulation

wiederhergestellt.

Die Proteomik war schon immer eine effiziente Methode, um die zelluläre Physiologie und

den Stoffwechsel eines Organismus zu untersuchen. In dieser Arbeit verwenden wir zum

ersten Mal den SILAC-basierten komparativen Proteomik-Ansatz, um das AdpA-Regulon im

nativen nicht-sporulierenden S. asterosporus DSM 41452 zu profilieren. In unserer Studie

wurden mehr als 1200 Proteine identifiziert, einschließlich Proteine, die am Stamm-

Stoffwechsel, der zellulären Verarbeitung und Signalisierung, Informationsspeicherung und -

- 12 -

verarbeitung usw. beteiligt sind. Am wichtigsten ist, dass wir gezeigt haben, dass der SILAC-

Ansatz in der Streptomyceten-Proteomik effizient angewendet werden kann.

In Bezug auf seine Sekundärmetabolite von S. asterosporus DSM 41452 wurden Gencluster

von WS9326A (Johnston et al. 2015) und Annimycin (Kalan et al. 2013), die zuvor in S. calvus

ATCC 13382 gefunden wurden, durch bioinformatische Analyse der Genomsequenz von S.

asterosporus DSM 41452 identifiziert. Sechs Verbindungen, WS9326A und seine Derivate

WS9326B, WS9326D, WS9326E, WS9326F und WS9326G wurden aus der Scale-Up-

Fermentation von S. asterosporus DSM 41452 isoliert. Überraschenderweise wurden zwei

neue Analoga SY11 und SY12 isoliert aus einem Annimycin-Defekt-Mutantenstamm S.

asterosporus DSM 41452::pUC19Δ3100spec und ihre Strukturen teilweise gemäß den

Massenspektrometrie- und NMR-Daten charakterisiert. Die Titer von WS9326A in der

entsprechenden S. asterosporus-Mutante waren leicht verbessert. Darüber hinaus wurde die

Grenze des WS9326A-Genclusters durch Aufbrechen des Gens orf (-1) und sas1 am Terminus

des Genclusters bestimmt. In-frame-Gen-Knockout des Gens, das die N-Methyltransferase

(MTase) in Modul 2 der WS9326A-NRPSs kodiert, führt zur Unterbrechung der WS9326A-

Produktion, was auf die Bedeutung des Methyl-Tyrosins für die Substraterkennung durch die

Kondensationsdomäne hinweist. Die Geninaktivierung von sas13 durch single crossover

scheint die Produktion von WS9326A nicht zu beeinflussen, was die Möglichkeit ausschließt,

dass sas13 an der Bildung des nichtproteinogenen Dehydrotyrosinrests in WS9326A beteiligt

ist. Zusätzlich führte die In-frame-Gen-Deletion von sas16 durch doppelten Crossover zum

Verlust von WS9326A, und die Produktion von WS9326A wurde nach der Komplementation

des Gens sas16 wiederhergestellt. Daher wird vorgeschlagen, dass dieses Gen an der Bildung

des Dehydrotyrosins in WS9326As beteiligt ist.

Um die biochemische Rolle von Sas16 während der Biosynthese des N-Methyl-Dehydro-

Tyrosin-Proteins zu verstehen, wurde Sas16 heterolog für In-vitro-Enzymtests exprimiert. In

der Zwischenzeit haben wir erfolgreich die Architektur von Sas16 aufgeklärt, um die

zugrundeliegende molekulare Basis der Substratselektivität dieses Cytochrom P450 Enzyms

aufzudecken

13

Table of Contents

Contents

Acknowledgements ............................................................................................................... - 4 -

Wissenschaftliche Publikationen und Akademische Aktivitäten .......................................... - 6 -

Abstract ................................................................................................................................. - 9 -

Table of Contents .................................................................................................................... 13

List of Figures ........................................................................................................................... 17

List of Tables ............................................................................................................................ 22

List of abbreviations ................................................................................................................ 24

Chapter 1. Introduction and Background ................................................................................ 26

1.1 Streptomyces and its AdpA regulon System ................................................................. 26

1.2 Streptomyces Genome Features ................................................................................... 30

1.3 Proteomics for Streptomyces ........................................................................................ 32

1.4 Antibiotics discovery and their action mechanism ....................................................... 33

1.5 Natural Product Biosynthesis mechanism ..................................................................... 36

1.5.1 Polyketide ............................................................................................................... 38

1.5.2. Nonribosomal peptides ......................................................................................... 42

1.5.3 Tailoring enzymes ................................................................................................... 48

1.5.4 Cytochrome P450 enzyme ...................................................................................... 50

1.6 Research Aims ............................................................................................................... 54

Chapter 2. General Materials and Methods ............................................................................ 56

2.1 Chemicals and Antibiotics ............................................................................................. 56

2.2 Enzymes and Kits ........................................................................................................... 58

2.3 Media ............................................................................................................................. 58

2.4 Software and Bioinformatics Tools ............................................................................... 60

2.5 Buffers and Solution ...................................................................................................... 61

2.5.1 Buffers for plasmid isolation from E. coli ............................................................... 61

2.5.2 Buffers for isolation of genomic DNA from Streptomyces ..................................... 62

2.5.3 Buffers for DNA gel electrophoresis ....................................................................... 62

2.5.4 Buffers and solutions for protein gel electrophoresis (SDS-PAGE) ........................ 63

2.5.5 Buffer for protein samples preparation of SILAC ................................................... 63

2.5.6 Buffers for protein purification .............................................................................. 64

14

2.5.7 Buffers for Sas16 enzymatic assay ......................................................................... 65

2.5.8 Solutions for blue/white selection of E. coli ........................................................... 65

2.5.9 Buffer and solutions used for the Malachite Green phosphatase assay ................ 65

2.6 General Methods ........................................................................................................... 66

2.6.1 Cultivation of strains Streptomyces and E. coli ...................................................... 66

2.6.2 Plasmid Isolation from E. coli ................................................................................. 66

2.6.3 Genomic DNA Extraction of Streptomyces ............................................................. 67

2.6.4 PCR Amplification ................................................................................................... 67

2.6.5 DNA fragment purification by agarose gel electrophoresis ................................... 68

2.6.6 Plasmid construction .............................................................................................. 69

2.6.7 DNA Transformation into E. coli ............................................................................. 69

2.6.8 Plasmid from E. coli to Streptomyces by intergeneric conjugation ........................ 70

2.6.9 Gene disruption by single crossover ...................................................................... 71

2.6.10 Targeted Gene deletion by double crossover method......................................... 72

2.6.11 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)............ 72

Chapter 3. The complete genome sequence of Streptomyces asterosporus DSM 41452 ...... 73

3.1 Background .................................................................................................................... 73

3.2 Materials and Methods ................................................................................................. 74

3.2.1 Primers fragments used in this study ..................................................................... 74

3.2.2 Plasmid information ............................................................................................... 74

3.2.3 Genomic DNA preparation and whole-genome sequencing .................................. 75

3.2.4 Genome assembly and annotation ........................................................................ 75

3.3 Results and Discussion................................................................................................... 76

3.3.1 General genome features ....................................................................................... 76

3.3.2 Gene clusters related with Secondary metabolism................................................ 79

3.3.3 bldA and adpA gene in S. asterosporus DSM 41452 .............................................. 84

3.3.4 Function verification of adpA gene ........................................................................ 86

3.3.5 Phylogenetic and orthologous analysis .................................................................. 87

3.4 Conclusion ..................................................................................................................... 89

Chapter 4. Comparative Proteomic Analysis of Streptomyces asterosporus DSM 41452 ...... 91

4.1 Introduction ................................................................................................................... 91

4.2 Materials and Methods ................................................................................................. 93

4.2.1 Primers fragments used in this study ..................................................................... 93

4.2.2 Plasmid information ............................................................................................... 94

15

4.2.3 Strain constructed and used in this study .............................................................. 94

4.2.4 Bacterial strain and culture condition .................................................................... 95

4.2.5 Functional AdpA overexpression in S. asterosporus DSM 41452 ........................... 95

4.2.6 Construction of Arginine and Lysine auxotrophic mutant of S. asterosporus DSM

41452 ............................................................................................................................... 95

4.2.7 Bacterial culture for SILAC test ............................................................................... 96

4.2.8 Protein sample preparation for LC-MS/MS analysis .............................................. 97

4.2.9 Mass Spectrometry Measurement ......................................................................... 98

4.2.10 Protein Identification ............................................................................................ 99

4.3 Results and Discussion................................................................................................... 99

4.3.1 Complementation of the functional adpA gene in S. asterosporus DSM 41452 .... 99

4.3.2 In silico analysis of AdpA in S. asterosporus DSM 41452 ...................................... 101

4.3.3 Construction of Arginine and Lysine auxotrophic mutant of S. asterosporus DSM

41452 ............................................................................................................................. 103

4.3.4 Statistical analysis of proteomics data ................................................................. 106

4.3.5 Proteomic analysis of the effects of AdpA in S. asterosporus DSM 41452 .......... 108

4.4 Conclusion and Outlook .............................................................................................. 115

Chapter 5. Research on the secondary metabolites of Streptomyces asterosporus DSM 41452

and the biosynthesis of WS9326As ....................................................................................... 117

5.1 Background .................................................................................................................. 117

5.2 Materials and Methods ............................................................................................... 118

5.2.1 Primers fragments used in this study ................................................................... 118

5.2.2 Plasmid information ............................................................................................. 120

5.2.3 Strain constructed and used in this study ............................................................ 121

5.2.4 Genome sequencing and bioinformatic Analysis ................................................. 121

5.2.5 Generation of gene sas13 disruption mutant in S. asterosporus DSM 41452 ..... 122

5.2.6 Generation of gene sas16 disruption mutant in S. asterosporus DSM 41452 ..... 122

5.2.7 Strain information, Fermentation, Extraction ...................................................... 123

5.2.8 Isolation of Compound WS9326A, B, D, E, F, G .................................................... 123

5.2.9 Sample analysis by HPLC-MS ................................................................................ 123

5.2.10 NMR methods and General instrument for structural characterization ............ 124

5.2.11 Structure information of compound 1-6 ............................................................ 124

5.2.12 Antiparasite assay method and materials .......................................................... 125

5.3 Results and Discussion................................................................................................. 125

16

5.3.1 Chemical structure Elucidation of WS9326A derivatives from S. asterosporus DSM

41452 ............................................................................................................................. 125

5.3.2 Discovery of two new WS9326A analogs by disrupting Annimycin production in S.

asterosporus DSM 41452 .............................................................................................. 129

5.3.3 Antiparasitic activity assay of WS9326As ............................................................. 132

5.3.4 Characterization of the WS9326A gene cluster in S. asterosporus DSM 41452. . 133

5.4 Conclusion ................................................................................................................... 150

5.5 Appendix ...................................................................................................................... 152

Chapter 6. Biochemical characterization of Cytochrome P450 Sas16 .................................. 162

6.1 Research Background .................................................................................................. 162

6.2 Materials and Methods ............................................................................................... 165

6.2.1 Primers fragments used in this study ................................................................... 165

6.2.2 Plasmid information ............................................................................................. 166

6.2.3 Strain constructed and used in this study ............................................................ 166

6.2.4 Cloning of sas16 gene into pET28 vector ............................................................. 167

6.2.5 Purification of Sas16 ............................................................................................. 167

6.2.6 CO difference spectrum of Sas16 ......................................................................... 168

6.2.7 Substrate binding study ........................................................................................ 168

6.2.8 Crystallization and Data Collection ....................................................................... 168

6.2.9 Structure Determination and Refinement ........................................................... 169

6.2.10 Malachite Green Phosphatase Assay of Acetyltransferase (A) domain ............. 169

6.3 Results and Discussion................................................................................................. 170

6.3.1 Multiple sequence alignment of Sas16 ................................................................ 170

6.3.2 Vector Construction, Expression and Purification of Sas16 ................................. 176

6.3.3 Crystallization and Structure determination of Sas16 ......................................... 178

6.3.4 CO difference spectrum of Sas16 ......................................................................... 184

6.3.5 Substrate binding studies of Sas16 ....................................................................... 185

6.3.6 Construction of Sas16 enzymatic assay ................................................................ 187

6.3.7 Sas13 protein expression and purification ........................................................... 198

6.3.8 A domain (module 2 of Sas17) protein expression and purification .................... 200

6.4 Conclusion ................................................................................................................... 202

Reference .............................................................................................................................. 205

17

List of Figures

Figure 1. 1. Typical colonies of Streptomyces (A) and Characteristic life cycle of Streptomyces (B).

........................................................................................................................................................ 27

Figure 1. 2. Schematics representing the transcription mechanism of adpA triggered by A-factor.

........................................................................................................................................................ 28

Figure 1. 3. The AdpA regulatory cascade leading to the morphological development and secondary

metabolite production .................................................................................................................... 29

Figure 1. 4. Clinical used antibiotics discovered from Streptomyces .............................................. 34

Figure 1. 5. Examples of natural product assembled by diverse building blocks ............................ 36

Figure 1. 6. A schematic overview of the origin of the precursor building blocks for secondary

metabolism.. ................................................................................................................................... 37

Figure 1. 7. Polyketides with diverse structure and function from natural product. ...................... 39

Figure 1. 8. The type I PKSs modular, deoxyerythromolide-B-synthase (DEBS) for erythromycin

biosynthesis;.................................................................................................................................... 40

Figure 1. 9. Examples of iterative PKSs involved in the biosynthesis of lovastatin, doxorubicin and

chalcone .......................................................................................................................................... 41

Figure 1. 10. Representative NRPS derivatives produced by microorganism ................................. 43

Figure 1. 11. Architecture of NRPS synthetase and the biosynthesis mechanism of NRPS compound

........................................................................................................................................................ 44

Figure 1. 12. Examples of different NRPS assembly method. ......................................................... 46

Figure 1. 13. Examples of nonproteinogenic amino acid biosynthesis in NRPS .............................. 48

Figure 1. 14. Active nautral product with post-tailoring modifications .......................................... 49

Figure 1. 15. Special tailoring enzyme catalyzing Favorskiise rearrangement ................................ 50

Figure 1. 16. (A) The overall structure of P450 protein (exemplified by Orf6* (CYP165D3)); (B) Top

view of heme ................................................................................................................................... 51

Figure 1. 17. The catalytic cycle of cytochrome P450 enzymes ...................................................... 52

Figure 1.18. Examples of P450 enzymes with diverse function involved in the biosynthesis of natural

product ............................................................................................................................................ 54

Figure 2. 1. Schematic representation of gene inactivation via single crossover and gene deletion

via double crossover. ....................................................................................................................... 72

Scheme 3.1. Genomic overview of S. asterosporus DSM 41452. .................................................... 72

Figure 3.1. (A) Multiple sequences alignment of bldA gene from S. asterosporus DSM 41452 with

its orthologous; (B) The genome sequence comparison of the upstream intergenic region of adpA

between S. asterosporus DSM 41452 and S. calvus ........................................................................ 84

Figure 3. 2. The genome comparison between S. asterosporus DSM 41452 and S. avermitilis (A), S.

coelicolor A3(2) ............................................................................................................................... 85

18

Figure 3. 3. (A) PCR verification of the upstream intergenic region of adpA in S. asterosporus DSM

41452 and S. calvus; (B) Morphological development of S. asterosporus DSM 41452 and its mutants

........................................................................................................................................................ 86

Figure 3. 4. The phylogenetic relationship of S. asterosporus DSM 41452 with other strains based

on 16S rRNA gene sequences.. ....................................................................................................... 87

Figure 3. 5. OrthoMCL analysis of strain S. asterosporus DSM 41452, S. coelicolor A3(2), S.

avermitilis, and Kutzneria albida. .................................................................................................... 88

Figure 4. 1. Schematics representing the basic sample labeling principle of SILAC proteomics

approach. ........................................................................................................................................ 92

Figure 4. 2. Effects of exogenous AdpA overexpression on the morphology of S. asterosporus DSM

41452 ............................................................................................................................................ 100

Figure 4. 3. The secondary metabolite profiles of strains S. asterosporus DSM 41452::pTESa-adpAsc,

S. asterosporus DSM 41452::pTESa-adpAgh and S. asterosporus DSM 41452::pTESa ................. 101

Figure 4. 4. Multiple protein sequence alignment of AdpA from S. asterosporus DSM 41452 and the

homologous proteins .................................................................................................................... 102

Figure 4. 5. Inactivation of the arginine biosynthetic gene in strains S. asterosporus DSM

41452::pSET152 and S. asterosporus DSM 41452::pSET152-adpAgh(TTA) by insertion of plasmid

pKGLP2-InArg into the bacterial genome via single crossover ...................................................... 104

Figure 4. 6. Inactivation of the Lysine biosynthetic gene in strains S. asterosporus DSM

41452::pSET152 and S. asterosporus DSM 41452::pSET152-adpAgh(TTA) by inserting plasmid

pLERE-Inlys into the bacterial genome via single cross-over ........................................................ 105

Figure 4. 7. Phenotypes of S. asterosporus DSM 41452 and its mutant on the minimal (MM1)

media……………………………………………………………………………………………………………………………………….104

Figure 4. 8. (A) Scatter plot representing the correlation of two biological replicates measured by

mass spectrometry. (B) Histograms of log2 transformed protein intensities representing the

distribution of proteome differences of AdpA mutant strain compared to the WT strain in two

biological replicates. ...................................................................................................................... 106

Figure 4. 9. Heat map of expressed proteins that were up- and down-regulated in the biological

replicates of S. asterosporus DSM 41452 :: pSET152AdpA relative to the parental strain. .......... 108

Figure 5. 1. The chemical structure of WS9326A and its derivatives ............................................ 126

Figure 5. 2. MS/MS spectra of WS9326F(A) and WS9326G (B) produced by S. asterosporus DSM

41452; (C) Partial H NMR Spectrum comparison between WS9326F and WS9326G ................... 128

Figure 5. 3. HPLC chromatogram of FDAA derivative of WS9326A and the corresponding standard

amino acids ................................................................................................................................... 129

Figure 5. 4. HPLC profiles of S. asterosporus DSM 41452 wildtype and its mutant strains S.

asterosporus DSM 41452::pUC19Δ3100spec ................................................................................ 130

19

Figure 5. 5. Postulated chemical structure of SY11 base on key NMR signals .............................. 130

Figure 5. 6. (A) ESI-MS/MS fragmentation of SY11; (B) ESI-MS/MS fragmentation of SY12. ....... 131

Figure 5. 7. H NMR spectrum comparison between SY11 and SY12, R represents the unknown

moiety. .......................................................................................................................................... 132

Figure 5. 8. Drug response phenotypes for Plasmodium falciparum Dd2 (A), HB3 (B) and 3D7 (C)

strains. ........................................................................................................................................... 133

Figure 5. 9. Organization comparison of the WS9326A gene clusters in S. asterosporus DSM 41452

and Streptomyces calvus ............................................................................................................... 134

Figure 5. 10. Inactivation of the gene orf(-1) and sas1 in S. asterosporus DSM 41452 via single

crossover.. ..................................................................................................................................... 135

Figure 5. 11. NRPS domain organization are shown in the order for the WS9326A biosynthetic

assembly line. ................................................................................................................................ 136

Figure 5. 12. Inactivation of the gene sas16 and sas13 in S. asterosporus DSM 41452 via single

crossover. ...................................................................................................................................... 141

Figure 5. 13. The HPLC chromatogram of the ethyl acetate extracts of the culture broth of the

Wildtype strain, the mutant strain S. asterosporus DSM 41452::pKC1132-SAS13 and S. asterosporus

DSM 41452::pKC1132-SAS16. ....................................................................................................... 141

Figure 5. 14. (A) Plasmid diagram of pKGLP2-GusA-SAS16::aac3(IV); (B) Schematic representation

of the in-frame deletion of sas16 in S. asterosporus DSM 41452 ................................................. 143

Figure 5. 15. (A) The PCR verification of the sas16 deletion mutant; (B) The LC/MS extracted ion

chromatogram(EICs) for [M-H]- ions corresponding to WS9326A, WS9326B, SY11, SY12 in organic

extracts of S. asterosporus DSM 41452ΔSAS16. ........................................................................... 143

Figure 5. 16. (A)Plasmid diagram of pTESa-SAS16; (B) The digestion result of plasmid pTESa-SAS16

by KpnI and EcoRI; C) The LC/MS extracted ion chromatogram(EICs) for [M-H]- ions corresponding

to WS9326A, WS9326B, SY11, SY12 in organic extracts of S. asterosporus DSM

41452ΔSAS16::pTESa-SAS16. ........................................................................................................ 144

Figure 5. 17. (A) Diagram of plasmid for MTase domain deletion and (B) the schematics

representing the NRPS domain organization in the WT strain and the ΔMTase mutant strain. ... 145

Figure 5. 18. Schematics representing the construction (A) and PCR verification (B) of the MTase

encoding gene deletion mutant strain. ......................................................................................... 147

Figure 5. 19. HPLC chromatograms of S. asterosporus DSM 41452 and its mutant S. asterosporus

DSM 41452 ΔMTase ...................................................................................................................... 147

Figure 5. 20. SDS-PAGE analysis of MTase domain expression test and manual Ni-NTA purification.

...................................................................................................................................................... 149

Figure 5. 21. HPLC-MS analysis (Extracted ion chromatogram) of compounds WS9326A, B, D, E, F,

G, SY11 and SY12 from the cultures of the wildtype S. asterosporus DSM 41452 and its mutant S.

20

asterosporus DSM 41452:: pUC19Δ3100spec ............................................................................... 155

Figure 5. 22. Comparison of the genes organization for the cinnamoyl side chain biosynthesis in the

SAS and SKY gene clusters ............................................................................................................ 156

Figure 5. 23. Comparative HPLC Profiles analysis of metabolites from the culture of Streptomyces

calvus and strain S. asterosporus DSM 41452. .............................................................................. 156

Figure 5. 24. UV/Vis spectrum and HR-ESIMS spectrum of WS9326F. .......................................... 156

Figure 5. 25. UV/Vis spectrum and HR-ESIMS spectrum of WS9326G. ......................................... 157

Figure 5. 26. ESI-MS/MS fragmentation of WS9326A. .................................................................. 157

Figure 5. 27. ESI-MS/MS fragmentation of WS9326B. .................................................................. 157

Figure 5. 28. ESI-MS/MS fragmentation of WS9326D. .................................................................. 158

Figure 5. 29. ESI-MS/MS fragmentation of WS9326E. .................................................................. 158

Figure 5. 30. H NMR spectrum of WS9326A. ................................................................................ 159

Figure 5. 31. C NMR spectrum of WS9326A. ................................................................................ 159

Figure 5. 32. H NMR spectrum of WS9326F. ................................................................................. 160

Figure 5. 33. C NMR spectrum of WS9326F. ................................................................................. 160

Figure 5. 34. HSQC spectrum of WS9326F. ................................................................................... 161

Figure 5. 35. HMBC spectrum of WS9326F. .................................................................................. 161

Figure 6. 1. Chemical structures of NRPS containing dehydrogenated amino acid ..................... 164

Figure 6. 2. Possible mechanism of the dehydrogenation in amino acid residues ...................... 165

Figure 6. 3. Phylogenetic bootstrap consensus tree of Sas16 with other P450s. ......................... 170

Figure 6. 4. Protein Sequence comparison of Sas16 with OxyB and OxyC. ................................... 172

Figure 6. 5. Clustal Omage alignment of the amino acid sequences of Sas16 with other

characterized Cytochrome P450 monooxygenases from secondary metabolites biosynthetic

pathways. ...................................................................................................................................... 176

Figure 6. 6. (A) Diagram of plasmid pET28-SAS16; (B) Cultivation method optimization of Sas16

expression. ................................................................................................................................... 177

Figure 6. 7. (A) FPLC Chromatogram of Ni-NTA his-tag purification of Sas16 from lysed E. coli BL21

star (DE3) :: pET28-SAS16; (B) SDS-PAGE analysis of the fraction from Ni-NTA column. .............. 178

Figure 6. 8. (A) SDS-PAGE analysis of fractions from gel filtration eluted from a Sephadex G-25

column containing Sas16; (B) Crystals of Sas16 from S. asterosporus DSM 41452. . .................... 179

Figure 6. 9. The overall protein structure of Cytochrome Sas16. . ............................................... 180

Figure 6. 10. Close-up view of Sas16 showing critical catalytic residues interacting with the heme

propionate groups with hydrogen bonding .................................................................................. 181

Figure 6. 11. The hydrogen bonding interactions between residues from different secondary

structural elements enforcing the geometry of the active site pocket. ........................................ 182

Figure 6. 12. Secondary structure comparison of Sas16 with other P450 homologous proteins..

21

...................................................................................................................................................... 183

Figure 6. 13. (A) Schematics of carbon monoxide (CO) spectrum of cytochrome P450; (B)

Absorption spectra for Sas16 and its ferrous–carbon monoxide complex. .................................. 184

Figure 6. 14. (A) The diagram of the heme-iron center inside the active site of cytochrome P450;

(B) Typical UV difference spectrum of catalytic substrate binding to the cytochrome P450 OxyD,

representing the heme iron state change due to the ligand binding. ........................................... 186

Figure 6. 15. UV spectrum changes after titration of the corresponding substrate into the P450

protein solution. ............................................................................................................................ 187

Figure 6. 16. The schematics represents the Sas16 catalytic reaction system .............................. 188

Figure 6. 17. The electron transfer system for cytochrome P450 enzyme based on ferredoxin

reductase and ferredoxin .............................................................................................................. 189

Figure 6. 18. SDS-PAGE gel showing the fractions containing PuR and PuxB eluted from the weak

anion exchanger and gel filtration column ................................................................................... 190

Figure 6. 19. (A) Plasmid diagram of pET-Trx-PCP constructed for PCP domain expression; (B)

Plasmid diagram of pET-Trx-A-NMT-PCP; (C) Schematic representation of protein expression vector

pET-Trx with fusion partner thioredoxin; (D) The SDS-PAGE gel showing the fractions containing the

PCP-Trx fusion protein eluted from the gel filtration.. .................................................................. 191

Figure 6. 20. Phosphopantetheinyl transferase (PPTase)-catalyzed 4’-phosphopantetheinyl (Ppant)

group transfer to a conserved Ser residue in peptidyl carrier proteins (PCP) or acyl carrier proteins

(ACP). ............................................................................................................................................. 193

Figure 6. 21. SDS-PAGE analysis of the fractions from manual Ni-NTA column for sfp protein

purification .................................................................................................................................... 194

Figure 6. 22. Scheme of synthesis of tyrosine-PCP conjugate and possible reaction products

catalyzed by Sas16......................................................................................................................... 195

Figure 6. 23 HPLC chromatogram of Sas16 assay………………………………………………………………211

Figure 6. 24. Scheme of modified Ppant ejection assay, and possible Ppant fragment generated in

the reaction ................................................................................................................................... 197

Figure 6. 25. Sequence alignment of the PCP domain of NRPS module 2 encoded by sas17 from

WS9326A gene cluster with its homologues................................................................................. 197

Figure 6. 26. (A) Schematics of plasmid pET28-SAS13; (B) Agarose gel verification of plasmid pET28-

SAS13 ............................................................................................................................................ 198

Figure 6. 27. SDS-PAGE analysis of Sas13 expression test and manual Ni-NTA purification……… 199

Figure 6.28. (A) Postulated biosynthetic mechanism of dehydroxytyrosine in WS9326As; (B)

Schematic of A domain substrate preference test base on Malachite Green Phosphatase Assay.

...................................................................................................................................................... 200

Figure 6. 29. (A) Schematics of plasmid pET28a-A domain; (B) Agarose gel verification of plasmid

22

pET28-SAS13 by restriction enzyme digestion; (C) SDS-PAGE of manual Ni-NTA column fraction for

A domain purification.................................................................................................................... 202

List of Tables

Table 1. 1. The general feature of sequenced Streptomyces genome and other kinds species ..... 31

Table 2. 1. Chemicals and Antibiotics .............................................................................................. 56

Table 2. 2. Antibiotic Stock Solution and Working Concentrations ................................................. 57

Table 2. 3. Enzymes and Kits ........................................................................................................... 58

Table 2. 4. Media for cultivation of Streptomyces strains ............................................................... 58

Table 2. 5. Software and Bioinformatics Tools ................................................................................ 60

Table 2. 6. Buffers and solution used for plasmid isolation from E. coli ......................................... 61

Table 2. 7. Buffers for isolation of genomic DNA from Streptomyces strains .................................. 62

Table 2. 8. Buffers for DNA gel electrophoresis ............................................................................... 62

Table 2. 9. Buffers and Solutions for SDS-PAGE and Coomassie staining ........................................ 63

Table 2. 10. Buffer for protein samples preparation of SILAC ......................................................... 63

Table 2. 11. Buffers for protein purification .................................................................................... 64

Table 2. 12. Buffers for Sas16 enzymatic assay ............................................................................... 65

Table 2. 13. Stock solutions for blue/white selection ..................................................................... 65

Table 2. 14. Buffer and solutions used for the Malachite Green phosphatase assay ..................... 65

Table 2. 15. Components for PCR reaction system ......................................................................... 67

Table 2. 16. Conditions for a typical PCR reaction cycles ................................................................ 68

Table 2. 17. Composition for typical restriction reactions. ............................................................. 69

Table 3. 1. Primers fragments used in this study ............................................................................ 74

Table 3. 2. Plasmid information ....................................................................................................... 74

Table 3. 3. General features of the chromosome of S. asterosporus DSM 41452........................... 76

Table 3. 4. Assignment of 4047 genes of S. asterosporus DSM 41452 to the functional groups of the

actNOG subset of the eggNOG database ........................................................................................ 78

Table 3. 5. Secondary metabolites gene clusters (BGC) identified in S. asterosporus DSM 41452..79

Table 3. 6. ORFs associated with the Nucleocidin biosynthetic cluster in S. asterosporus DSM 41452

........................................................................................................................................................ 82

Table 4. 1. Primers fragments used in this study ............................................................................ 93

Table 4. 2. Plasmid information ....................................................................................................... 94

Table 4. 3. Strain constructed and used in this study ...................................................................... 94

Table 4. 4. Proteins up- and downregulated in S. asterosporus AdpA mutant.............................. 112

Table 5. 1. Primers fragments used in this study .......................................................................... 118

Table 5. 2. Plasmid information ..................................................................................................... 120

23

Table 5. 3. Proposed Functions of Open Reading Frames of WS9326A Biosynthesis Gene Cluster in

S. asterosporus DSM 41452 .......................................................................................................... 138

Table 5. 4. Predicted highly conserved core motifs of A domain binding pockets in NRPSs within the

SAS cluster. .................................................................................................................................... 152

Table 5. 5. List of putative biosynthesis genes involved in the biosynthesis of the side chain of the

WS9326As and their homologues in the biosynthesis gene cluster of Skyllamycin ..................... 153

Table 5. 6. Summary of NMR Data for WS9326A and WS9326F inDMSO-d6. ............................... 153

Table 6. 1. Primers fragments used in this study .......................................................................... 165

Table 6. 2. Plasmid information ..................................................................................................... 166

Table 6. 3. Strain constructed and used in this study .................................................................... 166

Table 6. 4. Crystal parameters and data-collection statistics for the crystal of Sas16 .................. 179

24

List of abbreviations

Symbol Full name

°C degree celsius

2D two dimensional

6×His hexahistidines

a.a. amino acid

aac(3)IV apramycin resistance gene

ACP acyl carrier protein

amp ampicillin resistance gene

APS ammonium persulfate

ATP adenosine triphosphate

attP attachment site on plasmid for phage integration

BLAST basic logical alignment search tool

bla carbenicillin/ampicillin resistance gene

bp base pair

ca. (preceding a data or amount) circa

CDCl3 deuterated chloroform

cre gene encoding Cre recombinase

Cml chloramphenicol resistance gene

COGs Clusters of Orthologous Groups

CV Column volumn

Da Dalton

DAD diode array detector

DMSO dimethyl sulfoxide

DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen

DNA deoxyribonucleic acid

dNTP deoxyribonucleoside 5´-triphosphates

dsDNA double-stranded deoxyribonucleic acid

DTT 1,4-dithiothreitol

E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

ESI electrospray ionization

ermE constitutive promoter in streptomycetes

eV electron volt

FAD Flavin adenine dinucleotide

FMN Flavin mononucleotide

25

g gram

h hour

HAc acetic acid

HCl hydrochloric acid

HMBC heteronuclear multiple-bond correlation

HPLC high performance liquid chromatography

hph hygromycin resistance gene

HSQC heteronuclear single-quantum coherence

Hz hertz

IPTG isopropyl-β-thiogalactoside

int phage integrase gene

lacZ gene encoding -galactosidase for blue/white selection

J coupling constant

k kilo

KAc potassium acetate

kb kilobase

kDa kilodalton

KR ketoreductase

KS ketosynthase

L liter

LC-MS liquid chromatography-mass spectrometry

M molar

m milli-

m/z mass-to-charge ratio

min minute

MS mass spectroscopy

MW molecular weight

n nano

NaAc sodium acetate

NaOH sodium hydroxide

Ni-NTA nickel-nitrilotriacetic acid

NMR nuclear magnetic resonance

ORF open reading frame

oriT origin of transfer

ori origin of replication

PCP peptidyl carrier protein

PCR polymerase chain reaction

PKS polyketide synthase

pSG5rep a temperature-sensitive replicon in streptomycetes

26

RNA ribonuclear acid

RNase ribonuclease

RP reverse phase

rpm rotation per minute

RT room temperature

S. Streptomyces

SDS sodium dodecyl sulphate

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SM Secondary metabolites

ssDNA single-stranded deoxyribonucleic acid

TEMED N,N,N´,N´-tetramethylethylenediamine

TES N-Tris-(hydroxymethyl)-methyl-2-aminoethanesulfonic acid

Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol

tfd phage terminator sequence

tipA thiostrepton-inducible promoter

tsr thiostreptone resistance-conferring gene

UV ultraviolet

WT wild-type

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

X-Gluc 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt

Chapter 1. Introduction and Background

1.1 Streptomyces and its AdpA regulon System

Streptomyces is a kind of filamentous growth, spore propagation, Gram-positive bacteria

(Figure 1.1). They belong to prokaryotic with a similar hype diameter like bacteria, and own

type-I cell wall in which the main component is peptidoglycan containing the LL-form

diaminopimelic acid. The cell is sensitive to lysozyme and antibiotics, and its optimal growth

pH is slightly alkaline (Embley and Stackebrandt 1994). They are the largest genus of

Actinobacteria phylum, which distribute ubiquitously in the terrestrial and marine

environments (Barka et al. 2016). As a bunch of important industrial strains for producing

antibiotic, currently the well-studied Streptomyces include Streptomyces coelicolor A3(2)

(Bentley et al. 2002), Streptomyces avermitilis (Ikeda et al. 2003), Streptomyces griseus

(Ohnishi et al. 2008), and so on.

As just noted above, one notable feature of Streptomyces is the complex, fungal-like life cycle.

During its complex development life cycle (Figure 1. 1), Streptomyces undergoes an unique

27

morphological and physiological differentiation (Ohnishi et al. 2005). At their initial stage, a

free spore germinates under favorable conditions and grows to form substrate mycelium by

extension and branching. After 2 or 3 days, substrate mycelium grows up into the air to form

the aerial hyphae, and then the distal parts of the aerial mycelium undergo cell division to

develop a chain of spore. Ultimately, the matured spores are released to continue the

following generation. Amongst, during the switch phase from substrate mycelium to aerial

hyphae, the production of secondary metabolites such as antibiotic is initiated. In other

words, the sporulation of Streptomyces accompany with the production of antibiotics (Chater

2006).

A B

Figure 1. 1. (A) Typical colony of Streptomyces. Image from http://www.bacteriainphotos.com/Streptomyces

%20coelicolor%20%20colony.html; (B) The Characteristic life cycle of Streptomyces, the image is adapted from

(Ohnishi et al. 2005).

It was reported previously that bald mutants of Streptomyces are deficient in the biosynthesis

of specific secondary metabolites (Ohnishi et al. 2005). At least 20 reported gene defects will

lead to loss of aerial mycelium formation and cause the bald phenotype, those relevant genes

were systematically designated as bld series of gene including bldA, bldB, bldC, bldD, bldH,

bldG, bldI, bldJ, bldK, bldM and bldN, etc (Chater 2001). All of those bld genes belong to a

complex extracellular signaling cascade which regulate the formation of aerial mycelium in

Streptomyces (Takano et al. 2003; Chater 2006).

During the growth and development of Streptomyces, AdpA (known as BldH in S. coelicolor)

is a central transcriptional regulator in the A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-

butyrolactone) regulatory cascade. It plays a very crucial role in morphological differentiation

and secondary metabolite production in several Streptomyces species (Pan et al. 2009). AdpA

was first discovered in the strain Streptomyces griseus, it belongs to the AraC/XylS family in

Streptomyces, which consists of two domains, a ThiJ/PfpI/DJ-1-like dimerization domain at its

N-terminus and a DNA-binding domain including two HTH motifs (HTH-1 and HTH-2) at its C-

28

terminal part. Its consensus AdpA-binding sequence is 5ʹ-TGGCSNGWWY-3ʹ (S, G or C; W, A or

T; Y, T or C; N, any nucleotide) (Ohnishi et al. 2005).

A B

Figure 1. 2. Schematics representing the transcription mechanism of adpA triggered by A-factor, figure is adapted

from (Ohnishi et al. 2005). Figure A: the transcription of adpA is initially blocked. The binding of ArpA with the

promoter of adpA obstructs the binding of RNA polymerase to the promoter of adpA. Figure B: the gene

expression of adpA is turn on. When the concentration of A-factor reaches a certain threshold, the binding of A-

factor with ArpA will released it from the promoter region of adpA.

In the system of Streptomyces griseus, AdpA is the target of an A-factor receptor called ArpA,

which is the sensor of pleiotropic S. griseus-specific regulatory molecule. When the

concentration of A-factor inside the cell near a critical threshold, it will trigger the binding

behavior with ArpA, then ArpA will be released from the adpA promoter region. Generally, A-

factor as a microbial hormone, exert its pleiotropic regulatory effects in S. griseus entirely by

regulating the transcription of adpA gene (Figure 1. 2).

So far AdpA gene is the only one found in all Streptomyces that always contain a TTA codon

(Gessner et al. 2015). In Streptomyces, gene bldA is responsible for encoding the rare tRNA

molecule (Leu-tRNAUUA) that is necessary for the translation of mRNA UUA codons (Hackl and

Bechthold 2015), thereby the abundance of bldA tRNA to some extent determine whether

adpA gene will be expressed and work properly (Figure 1.3).

29

Figure 1. 3. The AdpA regulatory cascade leading to the morphological development and secondary metabolite

production in Streptomyces griseus and other Streptomyces, figure is adapted from (Ohnishi et al. 2005). Note:

unless otherwise stated, the network represents the genes relationship in S. griseus; Arrows suggests positive

regulatory effects; perpendicular lines suggest negative regulatory effects.

AdpA regulon is probably the most significant one in Streptomyces, many researches show

that the expression of AdpA is implicated with the secondary metabolism and morphological

differentiation in numerous Streptomyces species (Figure 1. 3) (Dyson 2011). For example, in

Streptomyces griseus, AdpA influence the expression of more than 1000 genes, transcriptome

analysis suggested that there are more than 500 genes being directly controlled by AdpA (Higo

et al. 2012), including gene adsA which encode an extracytoplasmic function (ECF) sigma

factor of RNA polymerase (Yamazaki et al. 2000); gene sgmA which encode a

metalloendopeptidase (Kato et al. 2002); ssgA (SCO3926 in S. coelicolor A3(2)) which influence

the hyphal development by stimulating septum formation (van Wezel et al. 2000), and the

pathway-specific activator gene strR, orf1 and griR in S. griseus for the biosynthesis of

streptomycin, polyketide and Grixazone, etc (Ohnishi et al. 2005). Streptomyces coelicolor

A3(2) likewise contains a remarkable number of genes regulated by AdpA, however the

specific target genes are not completely consistent in those two species (Wolański et al. 2011).

In contrast with S. griseus, the transcription of AdpA in S. coelicolor A3(2) does not depend on

the butyrolactone regulatory cascade system, and AdpA has been proven to be essential for

actinorhodin production, but not for undecylprodigiosin biosynthesis (Nguyen et al. 2003; Yu

30

et al. 2014). Wolański etal found that AdpA not only can directly influence the expression of

several genes, also particularly inhibit the chromosome replication at the initiation stage in S.

coelicolor (Wolański et al. 2012) (Wolański et al. 2011).

Moreover, many investigations on AdpA suggest that the regulatory network of AdpA deviate

in different Streptomyces species. In S. chattanoogensis, the AdpA homolog was proven to

indirectly activate natamycin biosynthesis, and the transcription of adpA was not affected by

the butyrolactone system in S. chattanoogensis (Du et al. 2011), in this strain gene

mutagenesis results revealed that AdpA homolog is essential for both nikkomycin biosynthesis

and morphological differentiation, in addition, transcriptional analysis demonstrated that

gene sanG, a specific activator for nikkomycin biosynthesis, is regulated by the expression of

AdpA-L in S. ansochromogenes (Pan et al. 2009). In their recent report, this AdpA homologue

was confirmed to repress oviedomycin biosynthesis by regulating the cluster-situated

regulators (OvmZ and OvmW) in the same strain (Xu et al. 2017).

1.2 Streptomyces Genome Features

Since the first Streptomyces module strain, Streptomyces coelicolor A3 (2) was sequenced by

the Sanger institute in 2002, which announced the era coming of research on Streptomyces

through genomics (Bentley et al. 2002). Since then, more and more important Streptomyces

strain has been sequenced, one of them is the most notably industrial strain of avermectins

producer: Streptomyces avermitilis. (Ikeda et al. 2003). So far, there are 106 complete genomic

sequences deposited in the Genbank database, at least 125 draft sequence maps available in

the Genbank database up to September, 2017. It's predictable that in the future more and

more Streptomyces will be sequenced for their irresistible charm.

The chromosome of Streptomyces shows a linear topology with complex structure, and some

of them own relatively bigger genome than other prokaryotes. The genome size of S. coelicolor

A3 (2) is 8,667,507bp, containing 7825 open reading frames (ORFs) (Table 1. 1). Its size is two

times bigger than the one of E. coli K-12, smaller than the genome of eukaryotic

Saccharomyces cerevisiae (approximately 12 Mb, 16 chromosomes). Another Streptomyces

species such as S. avermitilis even own a 9Mb genome.

One of the notable features of Streptomyces is their genome containing high guanine-plus-

cytosine (G+C) content. Due to the composition difference between the leading strand and the

lagging strand (normally there are more G and T on the leading strand, more A and C on the

lagging strand), which will cause a shift for breaking the base frequency, those shifts are named

31

as GC-skew. The corresponding GC skew is mapped based on the calculation of G-C/G+C value.

This value shows positive on the leading strand, and negative on the lagging strand. Thereby

it clearly exhibits the start and stop point of the leading strand and lagging strand. For

Streptomyces, the G+C skew inversion is usually used as a signal showing the locus of the origin

of replication (oriC) (Bentley et al. 2002).

Table 1. 1. The general feature of sequenced Streptomyces genome and other kinds species

Species Length

(bp)

Avg G+C

content

(%)

No. of

protein-

coding

genes

Genome

topology

Coding

density

(%)

No. of

rRNA

genes

No. of

tRNA

genes

No. Of

SM gene

clusters

S. griseus IFO

13350 a

8,545,929 72.2 7,138 Linear 88.1 6 66 34

S. coelicolor

A3(2) b

8,667,507 72.1 7,825 Linear 88.9 6 63 24

S. avermitilis

MA-4680 c

9,025,608 70.7 7,583 Linear 86.3 6 68 38

K. albida DSM

43870 d

9,874,926 70.6 8,822 Circular 88.49 9 47 46

S. albus J1074

e

6,841,649 73.3 5832 Linear 86.8 7 66 22

E. coli K-12 f 4,639,221 50.8 4288 Circular 87.8 7 86 -

S. cerevisiae g 12,156,677 38.4 5885 linear 70 140 275 -

Note: aS. griseus IFO 13350(Ohnishi et al. 2008); bS. coelicolor A3(2)(Bentley et al. 2002); cS. avermitilis MA-

4680(Ikeda et al. 2014); dKutzneria albida DSM 43870 (Rebets et al. 2014); eS. albus J1074(Myronovskyi et al. 2014);

fEscherichia coli K-12 (Blattner et al. 1997); gSaccharomyces cerevisiae (Goffeau et al. 1996; Förster et al. 2003), it

contains 16 linear chromosomes;

In terms of the chromosomal composition of Streptomyces, the Streptomyces chromosome

consists of a 6.5 Mb “core region” located in the middle of the genome, and a 1.5 Mb “left

arm” and 2.3 Mb “right arm” positioned at the terminus. These features are especially evident

in the genomes of S. coelicolor A3(2) and Streptomyces avermitilis.

Genome comparison between S. coelicolor A3(2) and S. avermitilis revealed that the core

conserved region of the genome (SAV1652-7142 in S. avermitilis and SCO1196-6804 in S.

coelicolor A3(2), respectively) were distributed with most of essential genes which are

responsible for important cellular function in the bacteria around the oriC site (Ikeda et al.

2003). In addition, bioinformatic analysis demonstrated that the regions near both telomeres

are less conserved. The presence of terminal inverted repeats (TIRs) at the terminus of

Streptomyces chromosome make the terminal part of chromosome become less conserved.

Those TIRs could go through deletion and expansion during the process of genetic

32

development of Streptomyces, which result in the unequal size of those “arm region” and

prompt the gene transfer in different strains. Furthermore, those structural disability

happening at the chromosome terminal portion don’t make significant influence on the

normal physiological metabolism of Streptomyces (Ohnishi et al. 2005). By systematic deletion

of nonessential genes around the chromosome terminus of Streptomyces avermitilis,

genomically minimized S. avermitilis SUKA22 was constructed as a robust and versatile model

host for heterologous expression of secondary metabolite biosynthesis (Komatsu et al. 2010).

These findings demonstrated the structural variability of the telomeric and the subtelomeric

region on the chromosome of Streptomyces may be unique to linear bacterial chromosome

like Streptomyces, and it plays an important role during the strain evolution.

It is worth mentioning that more than half of the secondary metabolites related genes were

found in the subtelomeric regions in the form of gene cluster. The genome of S. coelicolor

A3(2) contains 23 gene clusters, accounting for 5% of the total genes; S. avermitilis contain 30

gene clusters, accounting for 6.6% of the protein-coding gene. Those genes are believed to be

acquired by microorganism through horizontal gene transfer (Barlow 2009; Jiang et al. 2017).

1.3 Proteomics for Streptomyces

The whole regulatory network plays a very important role in the control of primary and

secondary metabolites in Streptomyces (Liu et al. 2013). Regulatory genes take a large

proportion in the genome of Streptomyces. For instance, Streptomyces coelicolor A3 (2)

contains 965 regulation-related encoding genes which account for 12.3% of the whole

genome.

Due to the presence of the complicated regulatory network in Streptomyces, searching

effective method to monitor the dynamic changes of gene expression inside the strain cells

become indispensable (Hesketh et al. 2002). System biology methods such as transcriptomics

and proteomics are very effective methods to understand Streptomyces differentiation in

various kinds of developmental condition (Hwang et al. 2014).

Nowadays, the rapid advance of genomics greatly facilitates the development of those omics.

Particularly, proteomics as a high throughput technique based on mass spectrometry, show

significant technique advantage. Proteomics approach can detect the expressed proteins and

their catalytic product which are often no observable under lab conditions. Moreover, Soft

ionization technique and multiple series fragmentation make it more efficient and sensitive

to determine the target protein. In addition, proteomics data even can provide unique

33

information about dynamic protein-protein interaction, post-translational modification of

proteins and biomolecules in trace amounts in biological samples (Lee et al. 2006).

Streptomyces biology has been studied using proteomics approaches in various cellular

contexts. Main researches based on proteomics focus on their study on the expression

difference of specific protein in the cell under specific condition, which were used to reveal

the hidden genome-wide interactions among genes. For example, through proteomics

analysis, Manteca et al validated that genes for primary metabolism such as TCA cycle, energy

production, and lipid metabolism, were activated during MI phase, while genes involved in

the biosynthesis of secondary metabolism got higher expression at MII phase than MI phase,

etc (Manteca et al. 2010). By integrating two proteomics methods (SILAC and iTRAQ), a

dynamic analysis of turnover rates have been accomplished to detect the degradation rates

of 115 intracellular proteins from mRNA transcription to protein degradation during metabolic

shift phase (Jayapal et al. 2010).

1.4 Antibiotics Discovery and Their Action Mechanism

Since half century ago, Alexander Fleming discovered Penicillin from fungi, more and more

potent antimicrobials have been discovered and developed during the period from the 1940s

to 1960s, at that time human being went through a golden era of antibiotic discovery. Some

of the antibiotics discovered at that time or their derivatives are still being used in our current

clinic therapy (Figure 1. 4). Among those microorganisms, Streptomyces plays an important

role in antibiotic development during this process. According to literature report, nowadays

more than 80% antibiotics are originated from Streptomyces (de Lima Procópio et al. 2012).

34

Figure 1. 4. Antibiotics discovered from microorganisms.

Today the battle of human with bacteria are still proceeding. The rapid emergence of

antibiotic resistance has been challenging the patient and the scientists (O’Neill 2016).

Another disadvantage is that conventional platform for antibiotic discovery have been

upgraded slowly due to various kinds of technical limitations over the last decades. Searching

new effective antibiotic drug has becoming increasingly tough (Lewis 2013).

As a Chinese proverb saying that heaven always leaves a way out. Since the world entering

the new century, the advent of gene sequencing technique promotes the rapid progress of

the molecular biotechnology, which shown that microorganisms still have huge potential

await to be exploited. Moreover, by means of genomic bioinformatics analysis, it becomes

more and more systematic to investigate the microorganism. In recent years, more and more

genomics-based strategies for antibiotic discovery were applied include genome mining (Mao

et al. 2015; Yan et al. 2016; Adamek et al. 2017), proteomic-based approach for secondary

metabolism investigation (Bumpus et al. 2009; Chen et al. 2011), activation of cryptic gene

cluster (Luo et al. 2013), etc. One representative approach I want to mention here is metabolic

engineering due to its significant advantages. By introduction of rational genetic modifications

in a specific organism, the metabolic profile and the biosynthetic capability can be changed to

produce ‘non-natural” natural product (Pickens et al. 2011; Bian et al. 2017; Billingsley et al.

2017). In addition, metabolomic mining base on mass spectrometry (Hou et al. 2012) are

making great efforts to alleviate the dereplication of secondary metabolites, which contribute

the screening of novel natural product. Certainly, we should not forget to emphasize the

HNH2N

COOH OO

N

S

COOHIsopenicillin N

HN

N

O

NH

N

S

S

O

HO

O

HN

OH OH

NH

N

NS

NH

O

O

O

HN

NH2O

S

N

ONH

S

N

OHN

O

NH

OHN

O

O

N

OH

NH

O

H

OH

Thiostrepton

OHOH

OCOOH

OHOH

OH

OHOHOHO

O OOH

H2NHO

H

Amphotericin B

O

O

OH

HOHO

NH

Streptomycin OO

O

O

OHO

OH

NH

O

OH OH

OHO

NN

N

Rifampicin

NH HN

HN

NH

O

O

O

O

HN

HN

O

HN

O

H2N

HO H

NH2

NH2

NH

OHN

O

NH

HN

O

R

O

NH2 NH2

OH

H

R =

R =Polymyxin B1

Polymyxin B2

NH

OHN

NH

O NH

NH OHN

O

NH2

O

O

O

O

NH

HN

NH

O

O O

O

OHO

HO

HOOC

H2N

HN

NH

O

O

O

NH

NH

O

CONH2

COOH

HOOC

Daptomycin

O

N

NH2

NH2

N

H2N

NH2OH

OHHO

CH3OHOHC

H3C

HN

OO

HN

OH

NH

O

HN

NH

OHN

HN

O

O

NH O

NH HN

HN

O O

O NH2

OH

O

HNNH

HN

OTeixobactin

35

“uncultured bacteria”. As a source of antibiotics that has not been developed on a large scale,

uncultivable microorganisms account for approximately 99% of all species in the

environments, they are gradually exhibiting the astonishing potentiality. In 2015, a new cell

wall inhibitor Teixobactin, was isolated from Gram-negative uncultured bacteria by cultivation

in situ through a special diffusion chambers. It can inhibit cell wall synthesis of Gram-positive

bacteria by binding to the conserved motif of lipid II (precursor of peptidoglycan) and lipid III

(precursor of cell wall teichoic acid)(Ling et al. 2015).

In fact, from a positive point of view, during this long-term battle with bacteria, although

human being paid a heavy price, Pharmacologist were able to research the bacterial

pathogenesis and decipher the activity mechanisms of antibiotics. Antibiotics can be classified

into several major groups based on their action mode, mainly including cell wall/membrane

inhibitor, nucleic acid (DNA and RNA) biosynthesis inhibitor, and protein synthesis inhibitor,

etc.

The largest group antibiotics serves as cell wall biosynthesis inhibitors, the representative

compounds include penicllins, cephalosporins and vancomycin. By interfering with the

biosynthesis of cell wall, the inhibitors can selectively kill or inhibit bacterial organisms. For

instance, the pharmacophore of β-lactam antibiotic molecules is the cyclic amide ring which

is an analog of the terminal peptidoglycan, the basic components of cell wall. β-lactam kill the

bacteria by blocking the cross-linking of peptidoglycan units, further block the biosynthesis of

cell wall (Kohanski et al. 2010). Cell membranes are also important barriers for both eukaryotic

and prokaryotic cells defense. Most clinically used inhibitors of cell membrane include

Daptomycin and Polymixins, and so on.

By contrast, Nucleic acid (DNA and RNA) biosynthesis inhibitors represented by

fluroquinolones and sulfonamides interfere the essential process of DNA or RNA synthesis

regular function. Protein synthesis inhibitors such as tetracyclines, macrolides and

aminoglycosides they are able to block the essential protein synthesis in bacteria. No matter

of Nucleic acid inhibitor or protein synthesis inhibitor, their action consequently leads to the

disruption of the normal cellular metabolism and the death of the organism (Kohanski et al.

2010).

Base on the bioactivity fingerprinting of various kinds of antibiotics, Wong et al developed an

innovative antibiotic screening strategy using antibiotic mode of action profile (BioMAP)

(Wong et al. 2012), which has been verified to be a efficacious approach for discovery of new

antibiotics. Certainly, there are many more other antibiotic action mechanism, here I am not

going to discuss detailed content.

36

1.5 Natural Product Biosynthesis Mechanism

Natural product own abundant chemical structural diversity which to some extent enrich their

biodiversity. Interestingly, no matter what kind of natural product, all of their molecular

skeletons are assembled by special “building block”. Furthermore, even the compounds which

are classified as totally different type, they can be assembled using same set of building blocks

like compounds Lovastatin, Avermectins, Erythromycin and Tetracycline (Figure 1.5), they are

assembled through different biosynthetic mechanism, but all of them are built up based on

the normal building block such as acetyl-CoA, propionyl-CoA, malonyl-CoA, methylmalonyl-

CoA, etc. For NRPS and alkaloid kinds of natural product like Vancomycin and Penicillin, their

biosynthetic precursors are various amino acid and their variants, for terpene-derived natural

products like Artemisinin, their biosynthesis precursor normally origin from isoprene subunit

(Figure 1. 5).

O

OH

H

H

O O

O

Artemisinin

O

O

CH3

HO

H3C

OH

CH3

O

O

O

OHH3C

H3CCH3

O

OCH3

CH3OH

CH3

OHO

N

H3CCH3

CH3

Erythromycin A

HO

OH

O HOHO

O

NH2

O

OH

N

Tetracycline

NH

HN

NH

HN

NH

HN

HN

O

HO

HO

O OHOH

O

O

O

O

OHO

O

O

NH2

O

HO Cl

ClO

OH

OHOH

OO

H2N

HO

Vancomycin

O

N

SHN

OO

O

OH

L-Cysteine

D-ValinePenicillin G

O

O O

O

OH

O

O

OH

O

O

O

O

OHO

Avermectin B1a

O

OH

OHO

O

Lovastatin

OH

O

O

OO

NH

OO

O

HOO

O

O O

OH

Taxol (Paclitaxel)

N

H3CO

H3CO

H3CO

H3CO

Papaverine

Figure 1. 5. Examples of natural product assembled by diverse building blocks.

All secondary metabolites are derived from regular primary metabolites. By the fundamental

photosynthesis, glycolysis and tricarboxylic acid cycle, the living organisms are able to produce

abundant energy and primary metabolites for further complex biosynthesis and life activity

(Dewick 2002). Primary metabolism is essential for all organisms, by way of generating

indispensable elements and energy to maintain their survival. In the meanwhile, they

remarkable influence secondary metabolism by regulating the supply of the precursor (Rokem

et al. 2007). Through primary metabolism, nutritional substrates such as glucose, fructose,

37

glutamate are consumed by plant or microorganism, as a result, abundant energy and primary

intermediates are generated (Figure 1.6).

Figure 1. 6. A schematic overview of the origin of the precursor building blocks for secondary metabolism. This

figure was adapted from (Dewick 2002; Hwang et al. 2014; King et al. 2016). Some steps are omitted for clarity.

During the process of primary metabolism, there are mainly 12 kinds of precursor metabolites

being generated, and they are subsequently converted into various secondary metabolites.

Those intermediates include glucose 6-phosphate, fructose 6-phosphate, erythrose 4-

phosphate, ribose 5-phosphate, glyceraldehyde 3-phosphate, 3-phosphate glycerate,

phosphoenolpyruvate, pyruvate, acetyl-CoA, oxaloacetate, 2-oxoglutarate and succinyl-CoA

(Figure 1. 6) (Rokem et al. 2007).

The most important building blocks utilized in the natural product biosynthesis are acetyl CoA

(Dewick 2002) (Figure 1. 6). Acetyl-CoA derived from the breakdown of carbohydrates through

glycolysis and the decomposition of fatty acids through β-oxidation. Afterwards acetyl-CoA

enters the Krebs cycle, where it will be converted as the origin of some amino acids. Acetyl-

CoA itself is the precursor of polyketide compound (Figure 1. 6). In addition, three molecules

of acetyl-CoA will be assembled as a mevalonic acid which is the base molecule for the

biosynthesis of a vast of terpenoid and steroid through mevalonate pathway. Another

alternative pathway for terpenoid and steroid metabolism is called deoxyxylulose phosphate

38

pathway which employ pyruvic acid (pyruvate) and glyceraldehyde 3-phosphate as the

precursor(Hwang et al. 2014).

Most of nitrogen-containing natural product such as peptides and alkaloids are derived from

amino acid. Among them the aromatic amino acids like phenylalanine, tyrosine, and

tryptophan are the products generated from the shikimate pathway. Those aromatic amino

acids can be integrated as the chemical skeleton of natural product after special catalytic

modification. In addition, during the process of glycolysis and Krebs cycle, many intermediates

are used to construct various kinds of amino acids, which can be used as the building blocks

for the assembly of NRPS kinds of compound like Vancomycin. Beside from

phosphoenolpyruvate from glycolysis pathway, erythrose 4-phophate from pentose

phosphate pathway also can be convert to intermediate shikimic acid (Figure 1. 6).

In all the prokaryotes and eukaryotes, the successful crosstalk between primary and

secondary metabolism need the participation of cofactors such as coenzyme A, flavin, ATP,

NADH, NADPH, FAD, metal ion, etc. Those cofactors serve as a helper molecule that assist

various kinds of enzyme to exert their biological activity, which play a very important role for

controlling the metabolism inside organism. For example, in Streptomyces coelicolor A3(2),

more than 21% metabolism process need the participation of ATP, which is often used as the

Gibbs free energy input to promote the biosynthesis of antibiotics (Bentley et al. 2002).

NADPH as electron acceptor, is also frequently involved into all sorts of natural product

biosynthesis (Rokem et al. 2007).

Generally, the chemical structural of secondary metabolites (SM) can be grouped as

polyketides, alkaloids, NRPS derivatives, and terpenoids according to their different

biosynthesis pathway. Two large classes of SM produced by Streptomyces are polyketide and

nonribosomal peptide which displays a wide variety of structural and physiological functions,

and will be described in great detail in the next section.

1.5.1 Polyketides

Polyketide kind of natural products are widely produced by various kinds of prokaryotic and

eukaryotic as secondary metabolites. As one of the largest family of natural product,

polyketides show a wide range of biological activity, such as antibiotics Erythromycin,

Enterocin, and Azalomycins, antitumor agents Geldanamycin, antiparasitic agent Avermectin

and cholesterol lowering agent lovastatin, etc (Figure 1. 7).

39

Figure 1. 7. Polyketides with diverse structure and function from natural product.

Polyketide biosynthesis are performed in a way similar to the process of fatty acid biosynthesis

(FAS). Fatty acids are integrated by complex polyketide synthetase (PKSs) through repetitive

decarboxylative claisen condensations of extender units derived from malonyl-CoA with an

activated starter unit. For FAS, the typical precursor as the starter unit and extender unit are

acetyl moiety and malonyl-CoA. By contrast, PKSs can utilize broader range of biosynthetic

building blocks such as acetyl-CoA, ethyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-,

methylmalonyl-, and ethylmalonyl-CoA, etc (Figure 1.6) (Hertweck 2009). Another typical

feature of fatty acid biosynthesis is the fully reduced carbon chain and its defined length. In

comparison, polyketides usually have diverse degrees of reduction at their carbon chain

(Hopwood and Sherman 1990).

Based on the architecture of polyketide and the action mode of the enzymatic catalysis for

the structure assembly, polyketide synthases can be classified into four groups: type I PKSs,

iterative type I PKSs, type II PKSs, and type III PKSs (Figure 1. 8 and Figure 1. 9).

S

N

O

O

OH OO

Epothilone A (anticancer)

OH

OHO

OOMe O

OH

OH3C

HONH2

CH2OH

O

Doxorubicin (antitumor)

O

NH

O

OH

O

O

O

O

OO

NH2

Geldanamycin (antitumor)

O

O

O

OH

OH

O

HO O

HO

H

OH

Enterocin (antibiotic)

O

HO

OHO

O

OH

OHOH

OH

OH

OH

OH OO

HOOC

HO

Azalomycins F3a (antimicrobial)

NH

HN

NH2

O

OH OH

OO

HO

Rishirilide A (2-macroglobulininhibitor)

O

OH

OR1

O

HN

O COOH

OHO

OOCH3

OO

OCH3

O

O

OH

O

OR2O

Ganefromycin a: R1= PhCH2CO; R2= H b: R1= H; R2= PhCH2CO (antibiotic )

O

O

CH3

HO

H3C

OH

CH3

O

O

O

OHH3C

H3CCH3

O

OCH3

CH3OH

CH3

OHO

N

H3CCH3

CH3

Erythromycin A (antibiotic)

O

O O

O

OH

O

O

OH

O

O

O

O

OHO

Avermectin B1a (antiparastics)

O

OH

OHO

O

Lovastatin (cholesterol lowering agent )

40

Figure 1. 8. The type I PKSs modular, 6-deoxyerythronolide-B-synthase (DEBS) for erythromycin biosynthesis.

Type I polyketide synthases commonly present in bacterial system, which consist of linear

organized multiple modules that contain covalently fused domains with different function.

Every module is responsible for one cycle of elongation, in which each domain performs one

enzymatic reaction in the process of polyketide chain assembly (Hertweck 2009).

The backbone part of type I PKSs module is constituted by three core domains: an acyl

transferase (AT) domain which is responsible for the selection and activation of acyl-CoA

substrate, then transfers the active substrate to the phosphopantetheinyl arm of the acyl

carrier proteins (ACP) domain (Figure 1. 8), where a thioester bond is formed to tether the

elongating polyketide chain; then the ketosynthase (KS) domain will catalyze the decarboxylic

condensation reaction between the extender unit and the preceding module. After several

cycles of elongation, the matured polyketide chain is transferred to a thioesterase (TE)

domain, where the total polyketide chain is finally released or cyclized. However, the final

structures of each extension unit and matured molecule are determined by some optional

domains with tailoring function in the PKS synthetase. Some commonly appeared optional

domains include ketoreductase (KR), dehydratase (DH), enoylreductase (ER), and

epimerization (E) domain (Shen 2003).

Type-I PKSs typically follow the principle of collinearity during their assembly process. So,

based on the sequence of the PKSs encoding gene, it’s possible to make a reasonable

prediction about the structure of the metabolites. Now there are many online software such

as Antismash (Weber 2014), PRISM (Skinnider et al. 2017) which can help make the automatic

online analysis base on corresponding gene sequence.

One well-studied example of type-I polyketide module is 6-deoxyerythronolide B synthases

(DEBS) which are responsible for the assembly of 6-deoxyerythronolide B as the aglycon part

of antibiotic erythromycin (Figure 1. 8). DEBSs consist of six successive modules encoded by

AT

P C P

load ing m odule 2

TEKS AT

K R AC P

m odule 3

K S ATAC P

m odu le 4

KS

D H

ATKRKR

AC P

m odule 5

KS A TK R

A C P

SS S

S

S

O

OO

m odule 1

K S ATK R

AC P

SOO

ER

KS A T

K R

AC P

m odule 6

O HO H

O HO H

O H

O

O H

O H

O

O H

O H

O H

O

O

O H

O H

O H

O

O H

O

S

O O H

O H

O H

O

O

6-dEB

D E BS 1 D E BS 2 D E BS 3

41

three genes DEBS1, DEBS2, and DEBS3 as well as a loading module. The first module (DEBS1)

is preceded by a loading domain for the selection of the starter unit (propionyl-CoA), and the

last module (DEBS3) is followed by a thioesterase (TE) domain for product release and

cyclization (Figure 1. 8).

Figure 1. 9. Examples of iterative PKSs involved in the biosynthesis of lovastatin (fungal iterative type I PKS),

doxorubicin (bacterial type II PKS), and chalcone (plant Type III PKS, chalcone synthase), figure adapted from

(Hertweck 2009).

Type I PKSs in fungal system serve as an iterative mode. The most representative example of

fungal iterative type I PKSs is lovastatin synthases (Figure 1. 9). The single module of lovastatin

nonaketide synthase is used iteratively to assembly the final polyketide scaffold (Campbell

and Vederas 2010).

In contrast with type I PKSs, type II PKSs system so far is only found in bacteria. The size of

type II PKSs is relatively smaller, because it only consists of a minimal set of iteratively used

enzymes, generally called “minimal PKS”, which is used to catalyze the iterative

decarboxylative condensation of malonyl-CoA extender units with an acyl starter unit (Figure

1. 9). The “minimal PKS” comprises of two ketosynthease units (KSα and KSβ) and an ACP

domain. Normally, genes encoding these three proteins are grouped together, and show a

typical KSα/KSβ/ACP architecture. In type II PKS complexes, the chain length is largely

controlled by the KSβ subunit which is also named as chain length factor (CLF) (Shen 2003).

After assembly by the minimal PKSs, the resulting linear product poly-β-keto chain is then

subjected to a series of modifications by ketoreductase, cyclase, aromatase, oxygenase and

so on, to yield the final aromatic compounds.

The most well-studied type III PKSs is the enzymes for the biosynthesis of chalcones (CHS).

Type III PKSs is multifunctional, which serves to select the starter unit, govern the polyketide

42

assembly, and catalyze the specific cyclization reaction. In the case of chalcone synthase (CHS),

the synthetase generate the complex aromatic polyketide chalcone through series of Claisen

condensation using a cinnamoyl-CoA as starter unit and three malonyl-CoA as extender unit

(Figure 1. 9) (Hertweck 2009).

1.5.2. NRPS Derivatives

In addition to polyketide, many pharmaceutically important natural product medicines are

nonribosomally biosynthesized by complex multienzyme called non-ribosomal peptide

synthetases (NRPS). To data, there are more than 20 marketed non-ribosomal peptide drugs,

including antibacterials (vancomycin, penicillin, bacitracin, gramicidin and daptomycin),

antifungals (fengycin), biosurfactants (surfactin A), antitumor drug (bleomycin), and

immunosuppressants (cyclosporine), etc (Sussmuth and Mainz 2017) (Figure 1. 10).

From the perspective of microorganism, the production of NRPS compounds play an

important role in their cell defense and competition in the surrounding environment. In some

case, NRPS compound secreted by bacteria serves as a weapon to inhibit the growth of other

competitors in their ecological niche, which is the most direct function of NRPS produced by

bacteria. For example, diketopiperazine-type peptide gliotoxin, a selective virulence factors,

produced by Aspergillus fumigatus can suppress the organism defense ability and possibly

cause invasive pathogenicity (Cramer et al. 2006). Some NRPS molecules are produced to

assist microorganisms maintaining their physiological development. The Iron (Fe) is

recognized as a physiological requirement for bacterial development (Weber et al. 2006),

under Fe ion limited environment, bacteria, cyanobacteria and fungus will be induced to

produce abundant iron ion chelating agent siderophore, in order to capture enough Fe ion for

life maintenance (Sharma and Johri 2003; Haas et al. 2008). In the case of cyanophycin, a

nonribosomal peptide produced by cyanobacteria Cyanothece sp. ATCC 51142, it accumulates

in the cytoplasm of cyanobacteria, and be considered to be a dynamic reservoir of nitrogen in

the organism (Picossi et al. 2004) (Figure 1. 10).

43

Figure 1. 10. Representative NRPS-derived natural products produced by microorganisms.

NRPS kinds of compound is biosynthesized by non-ribosomal peptide synthetase which is a

large multifunctional enzyme modularly organized by successive modules covalently fused by

discrete catalytic domains. A complete NRPSs consists of different modules arranged in a

special spatial arrangement. The core domains inside a characteristic module include

adenylation (A) domain, condensation (C) domain, and peptidyl carrier protein (PCP) domain.

In the NRPS assembly line, one module performs the integration of one single amino acid to

the peptide chain. The chain extension is performed by a series of reactions (Figure 1.11).

Firstly, the adenylation (A) domain select the substrate from the “substrate pool”, and activate

it as aminoacyl-AMP under the help of ATP (Figure 1. 11A); Secondly, the aminoacyl-AMP is

transferred to connect with the terminal thiol group of the 4’-phosphopantetheine prosthetic

group in the peptidyl carrier protein (PCP) domain, forming an aminoacyl-S-PCP complex

(Figure 1.11B); Thirdly, the aminoacyl-S-PCP will be shuttled to the condensation (C) domain,

where a nucleophilic reaction will be catalyzed between the aminoacyl-S-PCP complex

(acceptor substrate) and the peptidyl of peptidyl-S-complex (donor substrate) from previous

module, generating the new extended peptidyl-S-complex (Figure 1.11C); Fourthly, the C-

terminal module usually is a thioesterase (TE) domain which are responsible for releasing the

matured oligopeptide from the NRPS machinery and often mediating the macrocyclization

44

during this release step (Mootz et al. 2002) (Figure 1.11D). Cyclicpeptide has better stability

against hydrolysis catalyzed by proteases and peptidase, which is a main consideration in the

development of NRPS-derived medicines (Felnagle et al. 2008; Agrawal et al. 2016).

A

B

C

D

Figure 1. 11. Architecture of NRPS synthetase and the biosynthesis mechanism of NRPS compound. The figures

are adapted from (Sieber and Marahiel, 2005).

A1 PCP

module 2

SH

module 1

C A2 PCP

SH

C

N

NN

N

NH2

O

OH

OPO

O-

O

P

O-

O

OH

OP-O

O-

O

OH

R

H2N

O

N

NN

N

NH2

O

OH

OPO

O-

O

OH

R

H2N

O

Mg2+PPi

A

ATPamino acid aminoacyl adenylate

A1 PCP

module 2

SH

module 1

C A2 PCP

SH

C

O

R1

NH2

O

A1 PCP

module 2

S

module 1

C A2 PCP

S

C

AMPO

R2

NH2

OAMP

aminoacyl adenylate aminoacyl adenylate

AR2

NH2

O

R2

NH2

O-2 AMP

P C P

m o d u le 2

S

m o d u le 1

C P C P

SC

R 1

H 2 N O

-2 A M P

R 2

N H 2

O

d o n o r s u b s tra te

a c c e p to r s u b s tra te

C A 1 P C P

m o d u le 2m o d u le 1

C A 2 P C P

S

CCb a

R 2H N

OR 1

O

N H 2

S H

A1 PCP

module 2module 1

C A2 PCP

S

TEC

R2HN

ORn

O

NH

SHOH

H2N R1

O

n

TE

A1 PCP

module 2module 1

C A2 PCP

SH

TEC

SHO

R2HN

ORn

O

NH

H2N R1

O

n

H2O

A. hydrolysis

B. cyclization

A.

B.

NH

HN

O

HN

NH

H2NOOCO

HN

-OOC

HO OH

OH

O

O

O

NH

NH3+

O

OH

HOOH

Cl

OH

HO

linear product (Vancomycin precursor, etal )

HN

OO

O

NH

HN

ONH

COOH

O

R

O

NH OH

O

NO

HNO

NH

OHN

ONH

O

OH

HNO

NH

O

OHHO

Cyclic product (Skyllmycin, etal)

45

In many case, except those “core domain” for the NRPS backbone assembly, abundant

optional domains widely present in NRPS module, which increase the structural diversity of

NRPS by installing additional modification on the backbone of the peptide, including

epimerization (E) domain, formylation (F) domain, methylation (M) domain, heterocyclization

(Cy) domain, reduction (R) domain, and oxidation (Ox) domains, etc (Sieber and Marahiel,

2005). Many new domains have been discovered as NRPS optional domain recently,

researcher found that in the echinomycin NRPSs, a MbtH-like domain serves as an auxiliary

protein of a A-PCP didomain to mediate the formation of β-hydroxytryptophan residue

catalyzed by cytochrome P450 hydroxylase (Zhang et al. 2013). In the case of Vancomycin

biosynthesis, an unusual X-domain as a P450 recruitment domain, is responsible for recruiting

a specific cytochrome P450 enzyme to the last NRPS module and catalyze the crosslinking of

the aromatic side chain in glycopeptide (Peschke et al. 2016).

Base on the biosynthetic logic of NRPSs, NRPS compound can be basically divided into three

groups: type A (linear-) NRPSs, its peptide chain extension follows the principle of collinearity.

In this mode, each module arranges their core domain in the order C-A-PCP, and each module

only work one time and integrates one building block on the assembly line. Isopenicillin is

shown as a representative type A NRPSs in Figure 1. 12A (Finking and Marahiel 2004).

The machinery of type B (Iterative-) NRPS biosynthesis resembles the mechanism of type II

PKS biosynthesis. Iterative NRPSs can repeatedly use their modules or domains during the

assembly of one NRPS compound. For instance, the iron-chelating siderophore Enterobactin

produced by Escherichia coli, is a cyclic trimer of dihydroxybenzoylserine. In its biosynthesis,

three peptide synthetases EntE, EntB, and EntF are responsible for the whole backbone

assembly of Enterobactin. Among them, EntF consists of one iterative (C-A-PCP) module which

were used three time to generate the intermediates for the chain extension (Zhou et al. 2007)

(Figure 1. 12B).

In contrast to the standard (C-A-PCP) module architecture of linear NRPSs, one significant

feature of Type C (nonlinear-) NRPSs is the unusual arrangement of those core domain. (Mootz

et al. 2002) . In myxochelin synthetase, the module 2 encoded by mxcG contains the domain

organization C-A-PCP-R, in which only one C domain is responsible for the condensation of

both amino groups of the activated lysine residue. PCP domain of MxcF transfer the activated

dihydroxybenzoyl group to this C domain which carry out both reactions (Li et al. 2008) (Figure

1. 12C).

46

A

A1 PCP

module 2

S

module 1

C PCP

S

A2 A3 PCP

S

module 3

CE Te

O

Te

HO

O

H2N

O

OH

O

NH2

O

HN SH

O

NH

O

OH

O

NH2

O

HN SH

O

NH

O

OH

O

NH2

O

HN SH

O

acvA

HN

H2N

COOH

O

O

N

SCOOH

Isopenicillin N

B

A1PCP

S

module 1

A2 PCP

S

module 2

CTe

OH

Te

entE entB

O

OH

OH

entF

O

H2N

OH

A2 PCP

S

module 2

CTe

OH

Te

entF

O

NH

HO

O

OH

OH

A2 PCP

SH

module 2

CTe

O

Te

entF

O

NH

HO

O

OH

OH

A2 PCP

S

module 2

CTe

O

Te

entF

O

NHHOO

OH

OH

O

NH

HO

O

OH

OH

A2 PCP

S

module 2

CTe O

Te

entF

OHN

OO

HO OH

O NH

HO O

OH

OHO

NH

HO

O

OH

OH

A2 PCP

SH

module 2

CTe O

Te

entF

OHN

OO

HO OH

O NH

O O

OH

OHO

NH

HO

O

OH

OH

O O

ONH

O OH

HN

O

OH

NHO

HO

O

O

O

OH

HO

OH

Enterobactin

C

A1 PCP

module 2

S

module 1

C PCP

S

A2 R

O

MxcE MxcF

NH2

OH2N

HO

HO

IC

MxcG

HNO

HO

HO

NH O

OH

OH

HO

Myxochelin A

cycle 1

cycle 2

Figure 1. 12. Examples of different NRPS assembly method. (A) Biosynthesis mode of linear NRPS (Type A)

exemplified by isopenicillin N; (B) Biosynthesis mode of iterative NRPS (Type B) exemplified by Enterobactin; (C)

Biosynthesis mode of non-linear NRPS (Type C) exemplified by Myxochelin.

The building blocks for the biosynthesis of NRPS are mainly from the 20 proteinogenic amino

acids. As we have discussed at the section 1.5, amino acids are produced as the intermediates

of primary metabolism in organism. In addition, due to the presence of some tailoring enzyme

targeted amino acids, a significant number of amino acids are modified through various kinds

47

of catalytic modification (Süssmuth and Mainz 2017). For NRPS, those nonproteinogenic

amino acids play a crucial role to enlarge the chemical and biological diversity of nonribosomal

peptides (Walsh et al. 2001).

The most common nonproteinogenic amino acids in NRPS compound could be the

hydroxylated and methylated amino acids such as compound skyllmycin, cyclosporin etc. For

instance, the β-hydroxylated tyrosine, leucine, and phenylalanine amino residues in

Skyllmycin, intriguingly those hydroxylation of three different amino acids are catalyzed by

only one cytochrome P450 monooxygenase P450sky (Uhlmann et al. 2013; Walsh et al. 2013).

Another large group of nonproteinogenic amino acids are the amino acids with N-based side

chain, which are selected as the precursor existing in many NRPS compounds. For example, in

the biosynthesis of Kutznerides, five nonproteinogenic amino acid building blocks were

integrated into the assembly line. One amino acid with N-based side chain, the chlorinated

piperazate residues is originated from L-glutamate and L-glutamine which go through

oxidization, dehydrogenation, and halogenation to yield the final product. Another special

nonproteinogenic amino acid in Kutznerides is the methylcyclopropyl-glycine (MeCPGly). It is

originated from L-Isoleucine and L-allo-Isoleucine, which undergoes halogenation,

dehydrogenation and subsequent rearrangement of the halogenated aliphatic amino acid in

succession to give the cyclopropyl variant MeCPGly (Figure 1. 13B) (Fujimori et al. 2007).

Except the basic structural alteration, asymmetric center transformation of normal amino acid

is also a way of generating nonproteinogenic amino acid. For example, the stereoisomer L-

allo-isoleucine (L-allo-Ile) in desotamides produced by Streptomyces scopuliridis SCSIO ZJ46,

marformycins produced by Streptomyces drozdowiczii SCSIO 10141 (Li et al. 2016); and the L-

allo-Threonine residue in WS9326As produced by Streptomyces calvus(Johnston et al. 2015).

Li Qin (2016) reported that the biosynthesis of L-allo-isoleucine were catalyzed by a group of

aminotransferase/isomerase enzymes pair, DsaD/DsaE in desotamides and MfnO/MfnH in

marformycins(Li et al. 2016) (Figure 1. 13C).

In addition, N-terminal acylation as very common modification at N-terminus of NRPS present

in diverse group of nonribosomal peptides such as Daptomycin, CDA, Skyllmycin, Telomycin,

Surfactin, Echinocandins, Thalassospiramide, etc (Süssmuth and Mainz 2017).

48

A

B

NHHN

HN O

O

NHO

NH

HN

O

O

O

O

R1

NH

desotamide

l-allo-Isoleucine

OH

O

NH2

(s)

(s)

l-isoleucine (l-Ile)

DsaD/DsaE or MfnO/MfnH

OH

O

NH2

(s)

l-allo-isoleucine

(R)

Figure 1. 13. Examples of nonproteinogenic amino acid biosynthesis in NRPS natural product.

1.5.3 Tailoring Enzymes

The structure variation of PKS and NRPS compound could be generated due to the variety of

the starter units and extender units, by the varying the chain length of backbone, or by the

diverse cyclization mode (Hertweck 2009). Furthermore, the post-tailoring modification also

show remarkable influence on the structural diversity of natural product. Tailoring enzymes

plays a vital role of modifying various kinds of secondary metabolites. The class of tailoring

enzymes found in the natural product biosynthesis mainly include oxidoreductase,

methyltransferase (MTs) (Velkov et al. 2011), glycosyltransferase (GTs) (McCranie and

Bachmann, 2014), prenyltransferase (Ma et al. 2017), halogenase (Neumann et al. 2008), etc

(Figure 1. 14).

HNN

OO

OHO

HO

HN O

NH

O

OMe

NNH

HN

OO

O

Cl

ClOH

ClKutzneride 2

NH2

O OH

L-lle orL-allo-lle

KtzBKtzC KtzD

KtzC

NH2

O

S

KtzC

NH2

O

S

Cl

KtzA

-H2

KtzC

NH2

O

S

Cl

-HCl

KtzC

NHO

S

+H2

KtzC

NH2

O

S

KtzF

MecPGly

H2N

OHO

O

OH

L-Glu orL-GlnH2N

OHO

NH2

L-ornithine

KtzI(FAD oxygenase)

NH

NH

O

OH

NH

NH

O

OH

-H2

NH

NH

O

OH

KtzQ/KtzR

Cl

chlorinated piperazate

49

Figure 1. 14. Natural product with post-tailoring modifications, modified moieties are labelled in red.

Methyltransferase (MTs) is a group of alkyl group-transferring enzyme which are responsible

for transferring activated methyl group from S-adenosylmethinonine (SAM) to the oxygen,

nitrogen, carbon-atom of the premature intermediates. Methylation promote the lipophilicity

of a molecule and eliminate the hydrogen-bond donor sites. In the case of Cyclosporin A, N-

methylation maintains the biological activity of Cyclosporin. In addition, N-methylation of

specific amide in the Cyclosporin backbone is critical for the recognition by the acceptor site

of the downstream condensation domain (Velkov et al. 2011).

Glycosylation is widely occurrence and important step in the natural product post-

modification. Changes in the structure of a sugar moiety of a glycosylated compound

contribute to its bioactivity, target selectivity, and pharmacokinetic properties. Special

catalytic mechanism of GTs makes them own variable substrates including the macrolides,

aromatic polyketide, peptide, the aminoglycosides, nucleosides, steroids, and many others

(Zhang and Bechthold 2016) (McCranie and Bachmann 2014). Avermectins, the most famous

glycosylated macrolides (Figure 1. 14), are 16-membered macrocyclic lactones produced by

Streptomyces avermitilis. GTs AveBI catalyzes a tandem glycosylation in a stepwise manner.

Those glycosylation greatly enhance the potency and activity of avermectin derivatives (Ikeda

et al. 1999).

Besides the diversity of catalytic function, some tailoring enzymes that have long attracted

the attention of chemists are their high reactivity and substrate promiscuity, which make

them possible to catalyze and achieve some chemically impossible structure transformatioin

(Friedrich and Hahn 2015). One impressive example is polyketide antibiotic Enterocin, it is first

assembled by a versatile type II PKS system, then undergo various series of post-modification

50

catalyzed by a group of tailoring enzymes. After backbone integration by the minimal PKS in

the system, the linear intermediate is rearranged by EncM, a FAD-dependent multifunctional

enzyme. It first serves as an oxygenase to catalyze a Favorskii–type carbon-carbon bond

rearrangement, the presumed substrate is a linear C-9 reduced octaketide which is oxidized

at C12 to form a trione intermediate. Subsequently, EncM catalyzes two aldol condensations

between C-6 and C-11, C-7 and C-14 to form the precursor of Enterocin, desmethy-5-

deoxyenterocin (Figure 1. 15) (Teufel et al. 2013).

Favorskii- like oxidative rearrangement

S-EncC

O

EncA,EncB, EncC,FabD

7x malonyl-CoAPh

S-EncC

OH O OH

HO

O O O O

Ph

OHO

OHO

O

S-Enz

O

O

EncM

Enterocin

NADPH

benzoyl-CoA (Ph-CoA)

type II PKS

(FAD-dependent)

OH

Ph

OHO

OO

O

S-Enz

O

O

OH

O2

H2O

O

Ph

OO

OO

O

S-Enz

O

O

OH OHPh

OH14

O

11

OH

7

6

O

OHO

OH

O OH

Ph

OHO

OHO

OO

OH

O OH

EncKSAM

EcRFerredoxin,ferredoxin-NADP-reductase

desmethyl-5-deoxyEnterocin

Figure 1. 15. Special tailoring enzyme catalyzing Favorskiise rearrangement.

As the most famous enzyme family involved in the tailoring modification of numerous natural

product, cytochrome P450 enzyme can be categorized as monooxygenase, hydroxylase,

oxidoreductase and so forth based on their diverse catalytic activity. At the next section I will

confine the scope and make an in-depth description around those amazing tailoring enzymes.

1.5.4 Cytochrome P450 enzyme

Cytochrome P450 (CYP) enzymes are kind of protein containing heme-thiolate, they are well

known for their typical absorbance peak at 450nm in their reduced state after integrating with

carbon monoxide. CYP are ubiquitous in prokaryotic and eukaryotic organism. To date, more

than 80,000 genes encoding P450 have been published on the GenBank database. In addition,

with the rapid development of the structural biology, more and more P450 crystal structures

have been elucidated. P450cam (CYP101A1) from the strain pseudomonas putida is the first

P450 structure elucidated by Poulos in 1985 with the resolution of 2.6 Å (Poulos and Raag

1992). Now more than 750 P450 protein structures have been deposited in Protein data bank

51

(PDB), and those structures significantly facilitate the understanding of people on catalytic

mechanism of P450.

The core part of the P450 were generally formed by four conserved helices (D, E, I, and L helix)

bundles, which construct a triangular prism shape with predominant α-helical secondary

structure. The structural flexibility of the B-C/F-G loop and the spatial conformation of F-/ G-

helix influence the entry of substrate into the P450 active catalytic pocket and the release of

catalytic product (Podust and Sherman 2012).

The homology of different P450 enzymes could be as low as 30%, however their three-

dimensional structure is highly conservative, especially the conserved helix (D, E, I, and L

helix). Generally, in P450 protein there are three positions that are strictly conserved: the

absolutely invariant amino acid residue cysteine at the conserved Cys-ligand loop which

contains the P450 signature sequence FxxGxHxCxG; another two invariant residues (Glu and

Arg) which composed the ExxR motif in the K-helix; and the amino acid residues at the I-helix

which are mainly involved in the oxygen molecule activation in the process of catalysis (Rudolf

et al. 2017). The heme group in P450 protein is composed of a porphyrin ring complex

harboring an iron atom, and it’s bound to the protein through the absolutely conserved

cysteine residue (Figure 1. 16).

A B

Figure 1. 16. (A) The overall structure of P450 protein (exemplified by Orf6* (CYP165D3)); (B) Top view of heme

group. The iron atom is bound via a cysteinyl sulfur with P450 protein.

Most of the reactions catalyzed by cytochrome P450 require the participation of the redox

partner proteins which are responsible for transferring two electrons from the cofactor

NAD(P)H to P450 (Renault et al. 2014). Depend on the category of the redox partner protein

utilized in the catalytic reaction, the P450 enzyme can be classified into five different groups:

Class I cytochrome P450 require the redox partner protein system consisting of a ferredoxin

52

(Fdx) containing a Fe2S2 cluster and a ferredoxin reductase (FdR) harboring a FAD cofactor,

this kind of system normally only present in bacteria and the mitochondria in fungus; Class II

redox partner protein of P450 is a cytochrome P450 reductase (CPR) which contain FAD/FMN

as cofactor, normally exist in eukaryotic, present in the form of membrane protein. Class III

P450 enzyme naturally merged together with its CPR system such as the P450BM3 from

Bacillus megaterium (Munro et al. 2002). Class IV P450 naturally combined with FMN/Fe2S2

redox partner protein such as P450Rhf from Rhodococcus sp. NCIMB 9784 (Roberts et al.

2002). Class V P450 protein can utilize NAD(P)H directly without the help of redox partner

protein (Zhong et al. 2016).

Figure 1. 17. The catalytic cycle of cytochrome P450 enzymes, Figure adapted from (Zhang and Li 2017)(Podust

and Sherman 2012).

Here we make a detailed demonstration about P450’s catalytic mechanism exemplified by a

P450 hydroxylase (Figure 1. 17). In this reaction, one oxygen atom will be integrated into the

reaction substrate in the form of hydroxyl moiety, another oxygen atom need be transferred

into water form. And during this process, the two electrons will be transferred from cofactor

NAD(P)H by the redox partner protein to the heme group (Figure 1.17). The active substrate

first enters into the active site of P450 close to the heme group. The substrate binding will

53

cause the water ligand displacement which will change the spin state of heme iron from low-

spin to high-spin. Then, the ferric heme iron (Fe3+) is reduced to Fe2+ after obtaining an

electron from the redox partner protein, and the later further form oxyferrous (Fe2+-O2)

complex by binding a molecular oxygen. Thirdly, a ferric hydroperoxyl (Fe3+-OOH) complex

(Compound 0) is generated after ferrous dioxy complex (Fe2+-O2) acquiring one more electron

and a proton. Afterwards, the peroxo group (Fe3+-OOH) rapidly undergoes a second

protonation to form the highly reactive ferryl-oxo intermediate referred to as P450 Compound

1 (Fe4+=O, porphyrine π-cation racial), in the meanwhile one molecule of water is released.

Subsequently, one hydrogen atom is abstracted from the Compound I complex, the

hydroxylated product is generated through recombination, finally the product dissociated

from the active site, the enzyme will return to the initial ferric state. In addition, under the

presence of peroxide, such as H2O2, the first three catalytic steps can be bypassed through a

route called peroxide shunt pathway (Figure 1. 17) (Denisov et al. 2005).

As a kind of versatile enzyme, P450s are able to catalyze a wide array of reaction involved in

the biosynthesis of natural product, moreover, P450 catalytic reaction often shows significant

substrate stereo- and regioselectivity. Among them, hydroxylation and oxidation are the most

well-studied reactions catalyzed by P450 (Figure 1.18) (Rudolf et al. 2017). In the biosynthesis

of erythromycin, CYP450 monooxygenase EryF and EryK perform the hydroxylation reaction

at C-6 of the 14-membered ring macrolactone 6-deoxyerythronolide B and the C-12 of the

macrolactone intermediate erythromin D, respectively (Weber et al. 1991; Stassi et al. 1993).

In the biosynthesis of epothilone A and B, P450 EpoK is responsible for the epoxidation at the

C12-C13 (Ogura et al. 2004). Some multifunctional P450 enzymes can exert their catalytic

activity with a broader range of substrate. In the biosynthesis of Tirandamycin, P450s TamI

first catalyzes the hydroxylation at the C-10 of Tirandamycin C to from Tirandamycin E. The

latter is oxidized into Tirandamycin D by flavin monooxygenase TamL. Then CYP450 TamI will

take Tirandamycin D as catalysis substrate again and transform it to Tirandamycin A by

epoxidation at C-11/C-12, and further to Tirandamycin B by hydroxylation at C-18 (Carlson et

al. 2010). During the industrial production of Artemisinic acid, an important intermediate of

antimalarial drug Artemisinic, cytochrome P450 CYP71AV1 catalyzes three successive

oxidative reactions at the C12 position of precursor amorpha-4,11-diene to yield Artemisinic

acid. By engineering this enzyme, Keasling etal (Ro et al. 2006) have been able to realize the

large-scale production of Artemisinic acid in yeast with 100mg/L (Figure 1. 18).

54

Figure 1. 18. Examples of P450 enzymes with diverse function involved in the biosynthesis of natural product,

red labelled parts show the moieties of compounds modified by corresponding P450 enzymes.

In addition, more and more researches on P450 have demonstrated that they own powerful

ability of catalyzing many unusual reactions in nature product biosynthesis, including

decarboxylation(Grant et al. 2016), nitration (Barry et al. 2012), C-C bond formation (Makino

et al. 2007), heterocyclization (Richter et al. 2008), aryl and phenol coupling (Zerbe et al. 2002;

Pylypenko et al. 2003), oxidative rearrangement of carbon skeleton (Cheng et al. 2010) and

C-C bond cleavage (Cryle and Schlichting 2008), etc (Figure 1.18). Due to the limited page in

this introduction part, I wouldn’t go through more detailed here about all types of P450

function.

1.6 Research Aims

Currently, exploiting natural product from Streptomyces is still an effective method for

discovery of active small molecule for clinic use. Streptomyces asterosporus DSM 41452

arouse our research interests due to its special physiological features, and its powerful

capability of producing secondary metabolites. In this thesis, our research on strain S.

asterosporus DSM 41452 will mainly focus on the four points below:

(1) Genomic analysis on S. asterosporus DSM 41452, explaining its special physiological

characteristics as a natural non-sporulating Streptomyces, and exploiting its potential of

producing secondary metabolites;

(2) Proteomics research on the regulatory network of S. asterosporus DSM 41452, validating

the feasibility of adopting SILAC proteomics analysis approach in Streptomyces system;

55

(3) In terms of its secondary metabolites, as a new WS9326A producer, S. asterosporus DSM

41452 enable us to exploit more novel active derivatives. Through genomic sequencing and

gene mutagenesis, the WS9326A gene cluster is determined and annotated in this strain, in

addition, it provides us the opportunity to study and reveal the detailed biosynthetic

machinery of WS9326A;

(4) Studies on the specific catalytic function of a cytochrome P450 enzyme Sas16 by structural

biology and biochemical research methods.

56

Chapter 2. General Materials and Methods

2.1 Chemicals and Antibiotics

Table 2. 1. Chemicals and Antibiotics

Component Source

1,4-Dithiothreitol (DTT) Carl Roth GmbH

5-Bromo-4-chlor-3-indolyl-β-D-galactopyranoside (X-gal) AppilChem (Darmstadt, Germany)

5-Bromo-4-chloro-3-indolyl β-D-glucuronide (X-gluc) Sigma-Aldrich (Deisenhofen, Germany)

Acetone Carl Roth GmbH

Acetonitrile Carl Roth GmbH

Acrylamide Carl Roth GmbH

Agar Carl Roth GmbH

Agarose Carl Roth GmbH

Ammonium persulfate (APS) Carl Roth GmbH

Bromophenol blue Carl Roth GmbH

Cobalt(II) Chloride Hexahydrate Carl Roth GmbH

Calcium chloride Carl Roth GmbH

Calcium carbonate Carl Roth GmbH

Chloroform Carl Roth GmbH

Coomassie Brilliant Blue G250 Carl Roth GmbH

Dichloroform Carl Roth GmbH

Dipotassium phosphate Carl Roth GmbH

Dimethyl sulfoxide (DMSO) Carl Roth GmbH

D-mannitol Carl Roth GmbH

Dithiothreitol (DTT) Carl Roth GmbH

DNase I New England Biolabs GmbH

Ethanol Carl Roth GmbH

Ethidium bromide Carl Roth GmbH

Ethyl acetate Carl Roth GmbH

Ethylene diamine tetra acetic acid (EDTA) Roth (Karlsruhe, Germany)

Glacial acetic acid Carl Roth GmbH

Glucose Carl Roth GmbH

Glycerol Carl Roth GmbH

GelRed™10,000X stock solution GoldBio

Hydrochloric acid Carl Roth GmbH

Isopropanol Carl Roth GmbH

Isopropyl-β-thiogalactoside (IPTG) Carl Roth GmbH

Iodoacetamide Carl Roth GmbH

Imidazole Carl Roth GmbH

LB medium Carl Roth GmbH

57

L- Arabinose Carl Roth GmbH

L-Valin Carl Roth GmbH

Malt extract Carl Roth GmbH

Methanol Carl Roth GmbH

Maltose Carl Roth GmbH

Magnesium chloride Carl Roth GmbH

Monopotassium phosphate Carl Roth GmbH

Methylhydrazine Carl Roth GmbH

Ni-NTA Agarose QIAGEN GmbH

N,N,N’,N’-Tetramethylethylenediamine (TEMED) Carl Roth GmbH

N-Tris-(Hydroxymethyl)-methyl-2-aminoethane sulfonic acid

(TES)

Carl Roth GmbH

Peptone Becton-Difco, Heidelberg, Germany

Phenol/Chloroform/Isoamylalkohol (25:24:1) Carl Roth GmbH

Phenylmethylsulfonyl fluoride (PMSF) Sigma-Aldrich (Deisenhofen, Germany)

Protein Marker VI (10-245) prestained AppliChem GmbH

Rotiphorese Gel 30% (M/V) (37,5:1) Carl Roth GmbH

Sodium chloride Carl Roth GmbH

Sodium hydroxide Carl Roth GmbH

Saccharose Suedzucker (Mannheim, Germany)

Sodium dodecyl sulfate (SDS) Carl Roth GmbH

Soybean flour W. Schoenenberger GmbH (Magstadt,

Germany)

Tris(hydroxymethyl)aminomethane (Tris base) Carl Roth GmbH

Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) Carl Roth GmbH

Tryptic soy broth (TSB) Carl Roth GmbH

Trypton Becton-Difco, Heidelberg, Germany

Trypsin Carl Roth GmbH

Yeast extract Carl Roth GmbH

WS9326K (Acyl- 1Thr-2Tyr-3Leu-4Phe-5Thr-6Asn) GL Biochem(Shanghai) Ltd

WS9326L (Acyl- 1Thr-2Tyr-3Leu-4Phe-5Thr) GL Biochem(Shanghai) Ltd

Table 2. 2. Antibiotic Stock Solution and Working Concentrations

Antibiotic Abbre

viation

Solvent Stock

Concentration

(mg/mL)

Working

Concentration

(μg/mL)

Adding (ul) in

100mL medium

Source

Kanamycin Kana H2O 30 30 100ul in liquid;

100ul in solid

Roth

Ampicillin Amp 50%

EtOH

100 100 100ul in liquid;

100ul in solid

Sigma

Apramycin Apra H2O 100 50 50ul in liquid;

25ul in solid

Fluka

58

Note: The aqueous solutions were sterilized by filtration through 0.22 μm filter. All antibiotics were dissolved at

stock concentration and stored at -20℃.

2.2 Enzymes and Kits

Table 2. 3. Enzymes and Kits

Enzyme and kits Source

Marfey’s reagent (Number 48895) Thermo Scientific

Lysozyme Fluka (Taufkirchen, Germany)

primers for PCR Eurofins MWG Operon (Ebersberg, Germany)

RNAse A Qiagen

dNTP mixer

1 kb DNA ladder

Proteinase K

Restriction endonucleases

T4-DNA-Ligase

New England Biolabs

Phusion-polymerase (5 U/ul)

Pfu-Polymerase (5 U/μL)

Pfu-Polymerase reaction buffer (10x)

Taq-Polymerase (5 U/μL)

Taq-Polymerase reaction buffer

Lab-made

Wizard SV Gel and PCR Clean-up System

Pure Yield Plasmid Midiprep System

Wizard SV Minipreps DNA Purification System

Promega (Mannheim, Germany)

Malachite Green Phosphatase Assay Kit Echelon Biosciences Inc. Salt Lake City, USA

2.3 Media

Table 2. 4. Media for cultivation of Streptomyces and E. coli strains

Medium Components Note

LB-medium (for E. coli) LB medium 20 g/L pH 7.2

Chloramphenicol Cam 100%

EtOH

30 10 33ul in liquid;

33ul in solid

AppliChem

Hygromycin Hyg H2O 100 100 100ul in liquid;

50ul in solid

Roth

Phosphomycin Phosp

ho

H2O 400 200 50ul in liquid;

50ul in solid

Roth

Spectinomycin Spec H2O 100 100 50ul in liquid;

100ul in solid

Sigma

Thiostrepton Thio DMSO 50 5 10ul in liquid;

10ul in solid

AppliChem

59

Distilled water 1000 mL

Autoinduction medium (for E. coli) Tryptone

Yeast extract

Glycerol

KH2PO4

K2HPO4

12g/L

24g/L

0.5%

12.54g/L

15g/L

pH 7.0

HA-medium (for Streptomyces) Glucose

Yeast extract

Malt extract

Tap water

4 g

4 g

10 g

1000 mL

pH 7.2

MS-medium (for Streptomyces) Soybean flour

D-Mannitol

Tap water

MgCl2

20 g

20 g

990 mL

10 mM

pH 7.2, autoclave MgCl2

separately and add at

the time of use.

NL19 (for Streptomyces) Soybean flour

D-Mannitol

Tap water

20.0 g

20.0 g

1000 mL

pH 7.2

SG-medium (for Streptomyces) Soy peptone

Glucose

L-Valine

CaCO3

CoCl2-

solution(1mg/mL)

Tap water

10.0 g

20.0 g

2.34 g

2.0 g

1 mL

1000 mL

pH 7.2

Minimal medium (MM1) (for

Streptomyces)

maltose

glutamic acid

K2HPO4

NaCl

Na2SO4

MgSO4

ZnSO4

CaCl2

Trace elements

solution

10g

8.9g

4g

2.5g

2.5g

0.45g

10mg

7.5mg

1mL

Note

Trace elements solution ZnCl2

FeCl3.6H2O

CuCl2.2H2O

MnCl2.4H2O

Na2B4O6.10H2O

(NH4)6Mo7O24.4H2O

Distilled water

40 mg

200 mg

10 mg

10mg

10 mg

10 mg

1000 mL

Note

TSB-Medium (for Streptomyces) Tryptic Soy Broth 30 g pH 7.2

60

Tap water 1000 mL

Note: The pH value of the media was adjusted by 1 M HCl or 1 M NaOH solution before autoclave. Supplementary

components such as trace elements solution need to be autoclaved separately and added into the sterile media at

the time of use. For preparing solid agar plates, 21 g/L agar was added in the media before autoclave. Afterwards,

every Petri dish was made with 20mL media for E.coli cultivation or 30mL for Streptomyces cultivation. The well-

mixed media solution was autoclaved for 20 min at 121 ℃ (15 psi). Liquid media were stored at room temperature

and solid agar plates were stored in refrigerator at 4 ℃. Unless otherwise stated, the media were prepared with

distilled water.

2.4 Software and Bioinformatics Tools

Table 2. 5. Software and Bioinformatics Tools

Name Basic information and Links

Circos Canada's Michael Smith Genome Sciences Centre (Krzywinski et al. 2009),

http://circos.ca/intro/genomic_data/

Mauve The Darling lab at the University of Technology Sydney (Darling et al. 2004),

http://darlinglab.org/mauve/mauve.htmL

Clustal Omega Tool for multiple sequence alignment for DNA or proteins, European

Bioinformatics Institute, https://www.ebi.ac.uk/Tools/msa/clustalo/

ANTISMASH A software for identification, annotation and analysis of secondary metabolite

biosynthesis gene clusters in bacterial (Medema et al. 2011),

https://antismash.secondarymetabolites.org/#!/start

FastQC A quality control tool for high throughput sequence data (Andrews 2010),

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

RAST An online Server for rapid annotations using Subsystems Technology (Aziz et al.

2008), http://rast.nmpdr.org/

Artemis a DNA sequence viewer and annotation tool (Rutherford et al. 2000),

http://www.sanger.ac.uk/Software/Artemis

Perseus A computational platform for comprehensive analysis of proteomics data,

http://www.perseus-framework.org

MaxQuant A quantitative proteomics software package designed for analyzing large-scale

mass-spectrometric data sets, http://www.biochem.mpg.de/5111795/maxquant

BLAST

Basic Local Alignment Search Tool, an algorithm for comparing primary biological

sequence information,

http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome

ChembioDraw Ultra 8.0 A versatile software for chemical structure drawing and analysis, Cambridge soft

Cambridge, UK

ChemStation Rev.

A.09.03

A Software for control and analysis of LC/MS, Agilent Technologies, Inc. USA

Clone Manager

Professional Suite 8

A Software for DNA sequence analysis, Scientific & Educational Software,

Durham, NC, USA

61

Conserved domain

search

A tool for the annotation of functional units in proteins, NCBI,

http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

MestReNova A Program for the analysis of NMR data, Mestrelab Research, Santiago de

Compostela, Spain

Pfam (Protein Families) A tool for searching conserved domains in proteins, http://pfam.sanger.ac.uk/

SEARCHPKS A software for detection and analysis of polyketide synthase (PKS) domains in a

polypeptide sequence, National Institute of Immunology, New Delhi, Indian,

http://www.nii.res.in/searchpks.htmL

Softberry An online gene and protein analysis tool,

http://linux1.softberry.com/berry.phtmL

StreptomeDB 2.0 an extended resource of natural products produced by Streptomyces (Klementz

et al. 2015),

http://www.pharmaceutical-bioinformatics.org/streptomedb2/

ARTS Antibiotic Resistant Target Seeker (Alanjary et al. 2017),

http://arts.ziemertlab.com/index

Pymol A molecular visualization system on an open-source foundation, maintained and

distributed by Schrödinger, https://pymol.org/2/

Espript 3 A program which extracts the protein sequence similarities and secondary

structure information from aligned sequences (Robert and Gouet 2014),

http://espript.ibcp.fr/ESPript/ESPript/

MEGA7 A software suite for analyzing DNA and protein sequence data,

http://www.megasoftware.net/

GraphPad prism 7 A software for analyze, graph and present scientific data,

https://www.graphpad.com/scientific-software/prism/

2.5 Buffers and Solution

2.5.1 Buffers for plasmid isolation from E. coli

Table 2. 6. Buffers and solution used for plasmid isolation from E. coli

Name Component Note

P1 buffer Tris

EDTA

RNAse A

50 mM

10 mM

100 μg/mL

pH 7.8, add RNAse A before use. Store at

4 ℃

P2 buffer NaOH

SDS

200 mM

1% (m/V)

Preparing 2M NaOH and 10% SDS stock

solution separately, freshly prepare

working concentration when use

P3 buffer KOAc 3 M Adjust pH 5.2 by acetic acid, store at 4 ℃

Ethanol solution Ethanol 70% (v/v) Store at -20℃

Isopropanol

solution

Isopropanol 100% Store at -20℃

Note: Unless otherwise stated, the buffers were prepared with distilled water and stored at room temperature.

62

2.5.2 Buffers for isolation of genomic DNA from Streptomyces

Table 2. 7. Buffers for isolation of genomic DNA from Streptomyces strains

Puffer Component Note

SET-buffer Tris-HCl

EDTA

NaCl

20 mM

25 mM

75 mM

pH 8

Lysozyme solution Lysozyme 50 mg/mL Dissolved in SET buffer

RNase A solution RNase A 10mg/mL Dissolved in ddH2O

Proteinase K solution Proteinase K 20 mg/ML Dissolved in SET buffer

SDS solution SDS 10%

NaCl solution NaCl 5 M

2.5.3 Buffers for DNA gel electrophoresis

Table 2. 8. Buffers for DNA gel electrophoresis

Buffer Components Note

50 x TAE Tris base

EDTA (0.5 M, pH 8.0)

Glacial acetic acid

2M

0.05M

52.5 mL

Adjust the pH to 8.0 with

glacial acetic acid.

Loading buffer Glycerol

Bromophenol blue

30% (w/V)

0.25% (w/V)

Store at 4 ℃

Agarose 0.7% (m/V) Agarose

TAE-buffer (1x)

7 g

1000 mL

Dissolve the agarose thorough

in the microwave oven, then

store at 55 ℃.

Gel Red buffer GelRed™ 10,000X stock solution

Water

1μL

3500 μL

Store at room temperature

Methylene Blue

buffer

Methylene Blue

Water

4g

100mL

Working concentration is 0.2%

(m/v), Store at room

temperature

63

2.5.4 Buffers and solutions for protein gel electrophoresis (SDS-PAGE)

Table 2. 9. Buffers and Solutions for SDS-PAGE and Coomassie staining

Buffer/Solution Components Note

Stacking gel (4%) Acrylamide Solution

1.0M Tris-HCl (pH 6.8)

10% (w/V) SDS

Distilled water

10% (w/V) APS

TEMED

0.5 mL

0.625 mL

0.4 % V

1.375 mL

25 μL

2.5 μL

Mixed all the components

sufficiently.

Resolving gel (10%) Acrylamide Solution

1.5M Tris-HCl (pH 8.8)

10% (w/V) SDS

Distilled water

10% (w/V) APS

TEMED

2.5mL

1.25mL

0.4%

1.25mL

50μL

5μL

Mixed all the components

sufficiently.

10 x Running buffer Tris base

Glycine

SDS

Distilled water

30g

144g

10g

1000mL

Stored at room temperature

4 x Sample loading

buffer(40mL)

Distilled water

0.5M Tris, pH6.8

50%Glycerol

10% SDS

0.5 Bromophenol blue

16mL

5mL

8mL

8mL

2mL

Add 25μL β-mercapto- ethanol to

975 μL sample buffer prior to use

Destained Solution(1L) Ethanol

Acetic acid

Distilled water

450mL

100mL

450mL

Coomassie Brilliant

Blue G-250 solution

Coomassie Brilliant Blue

G-250

Acetic acid

Methanol

Distilled water

0.25%(w/V)

10 % (V/V)

45 % (V/V)

45 % (V/V)

2.5.5 Buffer for protein samples preparation of SILAC

Table 2. 10. Buffer for protein samples preparation of SILAC

Buffer Components Note

PBS solution NaCl

KCl

Na2HPO4

KH2PO4

8 g/L

0.2g/L

1.42 g/L

0.24 g/L

64

lysis buffer Tris

SDS

100 mM

4%

pH 7.6; supplemented with

protease inhibitor

LaemmLi sample buffer

(6X)

10% (w/v) SDS

Bromophenol

Glycerol

1 M Tris-Cl (pH 6.8)

DTT

1.2 g

6 mg

4.7 mL

1.2 mL

0.93g

This ingredient is for 10mL

volume. Stored at -20˚C

ABC buffer Ammonium

bicarbonate

100 mM pH 7.5

2% TFA Trifluracid

ddH2O

400ul

20mL

Buffer A HAc 0.5% in ddH2O

Buffer B Acetonitril

HAc

80%

0.5%

in ddH2O

EtOH Ethanol 100% in ddH2O

Buffer A* Acetonitril

TFA

3%

0.3%

in ddH2O

Buffer A*/A Acetonitril

Buffer A*

Buffer A

<1%

30%

70%

in ddH2O

2.5.6 Buffers for protein purification

Table 2. 11. Buffers for protein purification

Buffer Component Note

Buffer A Tris-HCl

NaCl

Imidazol

50 mM (pH 8.0)

300 mM

10-15 mM

Stored at 4℃

Buffer B Tris-HCl

NaCl

Imidazol

50 mM (pH 8.0)

300 mM

250 mM

Stored at 4 ℃

Lysis buffer Tris-HCl

NaCl

Lysozyme

50 mM (pH 8.0)

300 mM

4 mg/mL

Stored at 4 ℃

Storage buffer Tris-HCl

Glycerol

20 mM (pH 7.5)

15%

Stored at 4 ℃

Wash buffer Tris-HCl

NaCl

Imidazol

50 mM (pH 8.0)

300 mM

20-30 mM

Stored at 4 ℃

Buffer T Tris-HCl

DTT

50mM (pH 7.4)

1mM

Stored at 4 ℃

65

2.5.7 Buffers for Sas16 enzymatic assay

Table 2. 12. Buffers for Sas16 enzymatic assay

Buffer Component

Loading buffer (for tyrosine-CoA

conjugate synthesis)

HEPES buffer

NaCl

MgCl2

50mM, pH 7.0

50mM

10mM

HEPES buffer HEPES

NaCl

25 mM, pH 7.0

50 mM

Digestion buffer Sodium deoxycholate

NH4HCO3

1% (w/V)

50mM

2.5.8 Solutions for blue/white selection of E. coli

Table 2. 13. Stock solutions for blue/white selection

2.5.9 Buffer and solutions used for the Malachite Green phosphatase

assay

Table 2. 14. Buffer and solutions used for the Malachite Green phosphatase assay

Buffer/solution composition Notes

Amino acid solutions 10mM All the amino acids are dissolved in

ddH2O. Tyrosine is dissolved in

0.1N NaOH

ATP 0.5-5mM Stock solution

Inorganic phosphate 0.1mM From a stock solution of 1mM

present in the Malachite Green

Phosphatase Assay Kit.

Mixed solution

Tris-HCl 50mM pH 7.5

MgCl2 10mM

glycerol 10%(v/V)

DTT 1mM

Gel filtration Buffer Tris-HCl

NaCl

50mM (pH 7.4)

150mM

Stored at 4 ℃

Solution Component Note

IPTG solution IPTG 100 mM Sterilize by filtering, store at -20 ℃. Add 20 μL for each

plate.

X-Gal solution X-Gal 100 mg/L Dissolved in DMSO, store at 20 ℃, keep away from light.

Add 40 μL for each plate.

66

amino acid 0.5mM

inorganic pyrophosphatase 0.4U/mL

2.6 General Methods

2.6.1 Cultivation of strains Streptomyces and E. coli

For solid incubation, Streptomyces strains were generally grown on the TSB agar petri plates

at 28 ℃. E. coli strains were grown on LB agar plate at 37 ℃. For liquid cultivation,

Streptomyces strains were generally grown in HA or TSB liquid medium in baffled Erlenmeyer

flasks containing a steel spring at 28 ℃, 180 rpm for 2 to 3 days. E. coli strains were cultivated

in liquid LB medium at 37℃, 180 rpm, cultivation time depending on the experiments. For

inoculation, 1% fresh liquid cultured Streptomyces in TSB liquid medium or 0.2 mL mycelium

suspension in 25% saccharose stock was added into productive medium. For intergeneric

conjugation and isolation of genomic DNA, Streptomyces were cultured in liquid TSB medium.

E. coli strains were cultured in liquid LB medium at 37℃, 180 rpm overnight. Selective

antibiotics were supplemented into the medium with suitable concentration when necessary.

MS agar plates were used for conjugation.

2.6.2 Plasmid Isolation from E. coli

Kits “Wizard SV Minipreps DNA Purification System” and “Pure Yield Plasmid Midiprep

System” from Promega was used to extracted plasmid from E. coli following the manufacture’s

protocol. If use alkaline lysis, 2-10 mL of E. coli overnight culture was pelleted by centrifugation

(14, 000 rpm, room temperature (RT), 1 min). The cell pellets were thoroughly suspended in

200 μL P1 buffer by vortex. After that, 200 μL P2 buffer was added to the suspension and mix

gently by inversion until the solution show low turbidity, following the supplement of 200 μL

P3 buffer, then the solution was incubated on ice for 5 min. After centrifugation (14, 000 rpm,

RT, 10 min), the supernatant was moved into a new Eppendorf tube. Then the DNA was

precipitated by adding 500 μL ice-cold isopropanol. The DNA pellet was collected through

centrifugation (14, 000 rpm, RT, 10 min), then it was washed twice with 500 μL 70% ethanol

and air-dried for 15 min. For long term storage, the plasmid DNA was dissolved in 30-100 μL

TE buffer and stored at -20 ℃. The concentration of extracted plasmid was measured by

NanoDrop 2000 (Thermo Scientific™).

67

2.6.3 Genomic DNA Extraction of Streptomyces

Genomic DNA from Streptomyces was isolated as described in the PHD dissertation of Anja

Greule (Greule 2016). After 24 hours cultivation in TSB medium, 2mL bacterial culture was

harvested by centrifugation (14,000 rpm, 4 ℃, 5 min). The cell pellets were collected and

washed with nuclease free water several times, then resuspended in 500 μL SET buffer

supplemented with lysozyme (50mg/mL, in SET buffer) and 2 μL RNaseA (10mg/mL) by vortex.

The suspension was incubated at 37 ℃ for 30 min, inverted occasionally. 14ul proteinase K

(20mg/mL, in SET buffer) and 50ul 10% SDS buffer were added into the system following with

incubation at 55 ℃ for 1 h. After adding 200ul NaCl (5M) solution and 500ul chloroform,

mixing it 2 min, the lysate was centrifuged 10 min, 14000rpm at 4 ℃. Then the supernatant

was moved into new Eppi, adding 500ul 100% isopropanol and mixing by inversion, the pellet

was collected by centrifugation for 10 min, 14000rpm at 4 ℃. Then it was pelleted twice after

resuspending by 500ul 70% ethanol.

Finally, the organic solution was air dried totally at room temperature for 30min. The genomic

DNA was dissolved using 50-100ul sterilized nuclease free water, stored at -20℃. The

concentration of extracted genomic DNA was measured by Thermo Scientific™ NanoDrop

2000.

2.6.4 PCR Amplification

PCR amplifications were performed using the high-fidelity PCR system according to the

manufacturer’s instruction (Roche). All primers were designed using primer premier 5.0 or

Clone Manager Professional Suite 8, and synthesized by Eurofins genomics. The long PCR

primers (see section 5.2.1) for gene in-frame deletion were designed following the protocol

of PCR targeting system (Gust et al. 2003). All buffer, solution, and enzymes used all listed in

section 2.5. PCR amplification was normally performed in 20ul or 50ul reaction volume. The

components for PCR reaction system and amplification conditions are summarized in Table 2.

15 and Table 2. 16.

Table 2. 15. Components for PCR reaction system

Components Final concentration Note

Polymerase reaction buffer 1 x 10 x or 5 x

dNTP-mix 0.2 mM each

Primer 1 20 pmol

Primer 2 20 pmol

Template DNA 250 ng (1 μL)

68

Table 2. 16. Conditions for a typical PCR reaction cycles

For PCR amplification reaction, the denaturation temperature was chosen according to the

different DNA polymerases (95 °C for Taq, 98 °C for Pfu and Fhusion polymerase). Annealing

temperature was chosen depending on the melting temperature of primers. Normally the

elongation temperature was set up to 72 °C with Taq DNA polymerase and to 68 °C with high-

fidelity polymerase. The elongation time was calculated based on the length of PCR products

and the working efficiency of the applied polymerase.

2.6.5 DNA fragment purification by agarose gel electrophoresis

DNA fragment analysis and purification were carried out through agarose gel electrophoresis

with 0.7% -1.2% (w/V) concentration of agarose gel. The buffer used for electrophoresis is 1x

TAE buffer. The DNA samples mixed with 1/6 volume DNA loading dye and the 1 kb DNA ladder

from Promega were individually loaded into the wells on the gel using pipette. The

electrophoresis was performed at 80-120 voltage for approximately 0.5-1.5h. After isolation

on the gel, the gel was stained with gel red staining solution and detected under the UV light

at 365 nm wavelength. The size of the DNA fragment was determined by comparing with DNA

ladder.

For DNA fragment purification, the agarose gel band containing the target DNA fragment was

cut out of the gel as a slice, then is was immersed in the membrane binding solution from the

kit of “Wizard SV Gel and PCR Clean-Up System”, then it was dissolved totally by heating at 55

°C for 10 min. In the further steps, the DNA fragment were eluted from the column following

the kit protocol.

DMSO 10% Used for GC-rich templates

Polymerase 5 U (1μL) Taq or Pfu (5 U/μL)

H2O Add to total volume of 20 or 50 μL Using 20μL for analytic purpose

Steps Temperature Time Cycles (n times x)

Initial

denaturation

95 ℃ 5 min 1 x

Denaturation 95 ℃ 45 s 1 x

Annealing 50-75 ℃ 1 min (25-35) x

Elongation 72 ℃ 15s-5 min (calculated based on polymerase)

For Taq: 30s-1 min/kb, For Pfu: 1-2 min/kb

For Fhusion 15-30s/kb

1 x

Final elongation 72 ℃ 10min 1 x

69

2.6.6 Plasmid construction

DNA restriction with endonucleases was firstly performed according to the manufacturer’s

instructions. For a typical analytic digestion, a total volume of 20 μL was used, while for

preparative digestion, a total volume of 50-100 μL was used. Unless otherwise stated, the

restriction reaction was carried out at 37 ℃, then DNA ligation reaction was carried out using

T4-DNA ligase at RT for 1-2h or at 16 ℃ overnight. The ligation system contains 1 U T4-DNA

ligase, 1x ligase buffer and appropriate insert and vector, with a total volume of 10 μL. The

composition for typical restriction reactions are summarized in Table 2. 17.

Table 2. 17. Composition for typical restriction reactions.

2.6.7 DNA Transformation into E. coli

Two kinds of method were adapted for DNA transformation in this dissertation: CaCl2-

mediated heat shock transformation and electroporation transformation.

For preparation of the CaCl2-competent cells, 0.5 mL overnight cultured E. coli was inoculated

into 50 mL fresh LB liquid medium, the latter was incubated at 37 ℃, 180 rpm until its OD600

reached roughly 0.6 (2-3 hours for DH5α, BL21 star(DE3), BL21 DE(3) pLysS and XL-1 blue, 3-5

hours for ET 12567). The cell pellets were harvested by centrifugation (5,000 rpm, 4 ℃, 10

min), then resuspended in 40 mL ice-cold 0.1 M CaCl2 and centrifugated again (5,000 rpm, 4

℃, 10 min) to collect the pellets which will be resuspended in 20 mL ice-cold 0.1M CaCl2 and

incubated on ice for 30 min. Afterwards, the cell pellets were collected by centrifugation

(5,000 rpm, 4 ℃, 10 min), and suspended in 2 mL buffer (0.1 M CaCl2, 15% glycerol). These

prepared competent cells can be used immediately or stored at -80 ℃ in 100 μL aliquots in

the 1.5 mL Eppendorf tube. For transformation, 1-2 μL plasmid or 5-10 μL ligation product was

added to the tube containing 100 μL CaCl2-competent cells then incubated on ice for 30 min.

Afterwards the tube was put into the water bath at 42 ℃ for 90 seconds and immediately

cooled down on ice for 5 min. Subsequently, 800 μL fresh LB medium was added into the tube

Name Analytic digestion Preparative digestion (N means

amount of reaction tubes)

BSA (100 ×) 0.2 μL 0.5 μL x N

DNA 2.0 μL 1-2μg x N

10 × restriction buffer 2.0 μL 5.0 μL x N

Enzyme 1 0.3 μL 0.5 μL x N

Enzyme 2 (optional) 0.3 μL 0.5 μL x N

Total 20 μL (add ddH2O) 50 μL x N

70

and incubated at 37 ℃ for 1 hour. After that the cell pellets were collected by centrifugation

(7,000 rpm, 3 min), 400 μL supernatant was discarded and the cell pellet was resuspended

using the rest of LB medium. Then those cells were coated on the LB agar plate supplemented

appropriate antibiotic. Colonies with correct antibiotic resistance could grow up on the plate

after incubation for approximately 16 h.

For preparation of the electrocompetent cells, 0.2 mL overnight cultured E. coli was inoculated

into 20 mL fresh LB medium, the latter was incubated at 37 ℃, 180 rpm until its OD600 reached

roughly 0.6 (2-3 hours for DH5α, BL21 star(DE3), BL21 DE(3) pLysS and XL-1 blue, 3-4 hours for

ET 12567). The cell pellets were harvested by centrifugation (7,000 rpm, 4 ℃, 10 min), and

subsequently washed with 40 mL 10% ice-cold glycerol twice. Afterwards the pellets were

collected and resuspended in 2 mL 10% ice-cold glycerol. These prepared competent cells can

be used immediately or stored at -80 ℃ in 100 μL aliquots in the 1.5 mL eppendorf tube. For

the electroporation transformation, 100 ng plasmid DNA or PCR fragment was added into the

fresh prepared electrocompetent cells then it was incubated on ice for about 1 min.

Subsequently, the cell suspension with DNA were transferred into a 0.2 cm ice-cold

electroporation cuvette. Electroporation was carried out using a BioRad Gene Pulser with the

parameter set as: 1.8 Ω voltage and 5 ms time constant. After electroporation, 800 μL fresh

LB medium was added into the cuvette, the cells were suspended by pipetting, then

transferred into a sterile 1.5 mL tube and incubated at the 37 ℃ for 1 hour. The cells were

harvested by centrifugation (5,000 rpm, 3 min). 400 μL supernatant was discarded and the

cell pellet was resuspended using the rest of LB medium. Then those cells were coated on the

LB agar plate supplemented appropriate antibiotic. Colonies with correct antibiotic resistance

could grow up on the plate after incubation for approximately 16 hours.

2.6.8 Plasmid from E. coli to Streptomyces by intergeneric conjugation

During our study, the intergeneric conjugal transfer of plasmids from E. coli to Streptomyces

were performed according to the modified method mentioned in the dissertation of Irene

Santillana Larraona (Larraona 2015). plasmids harboring the oriT from the IncP plasmid RP4

were firstly transformed into methylation- defective E. coli strain ET12567(dam-13::Tn9 dcm-

6 hsdM Cmr)/pUZ8002(a derivative of RK2 with a mutation in oriT), and then it was transferred

to Streptomyces acceptor strain with the help of the non-transmissible pUZ8002 plasmid

which will encode the tra functions required for the mobilization of the conjugative plasmid

(Paranthaman and Dharmalingam 2003; Hopwood 2011).

71

10mL culture of E. coli ET12567/pUZ8002 harboring the conjugative plasmid was grown to an

OD600 of 0.4-0.6. The cells pellets were collected by centrifugation, washed three times by

fresh LB medium, and resuspended in 500 μL of LB medium. In the meanwhile, 2 mL overnight

culture of Streptomyces strain was washed three times by fresh LB medium, and diluted 1:100

and 1:1000 in TSB medium sequentially. Then the E. coli cells and diluted Streptomyces cells

were mixed in a 1.5 mL Eppendorf tube. The supernatant was discarded after centrifugation

(7,000 rpm, 3 min), the pellet was resuspended with 400 μL of TSB and plated on the MS agar

plates. Then the plates were incubated at 28 ℃ for 12-16 hours. After that, the plates were

overlaid with 1 mL sterile H2O containing 30ul phosphomycin (200mg/mL) and the antibiotic

for plasmid selection (concentration depends on the antibiotic used). Then the plates were

incubated at 28 ℃ for maximum 7 days waiting for appearance of the correct exconjugants.

2.6.9 Gene disruption by single crossover

In this thesis we use the conventional gene inactivation method base on single crossover

(Larraona 2015; Greule 2016). In this method, the target gene was disrupted by integration of

a suicide plasmid through the recombination of homologous gene fragment. For this purpose,

we firstly ligated the internal gene fragment of the target gene into suicide vector pKC1132.

Then the engineered plasmid pKC1132 was introduced into Streptomyces strain through

intergeneric conjugation. Once single crossover happened, the plasmid would be integrated

into the chromosome of the target strain through homologous recombination, which would

make the target gene truncated and loss of function (Figure 2.1A). In addition, due to the

presence of the apramycin-resistant marker on the pKC1132 plasmid, the mutant strain also

acquired apramycin resistance. Hence the correct mutant strain with the integrated gene

inactivation plasmid enable to grow under apramycin resistance screening. Detail

experimental information see the following chapter 4 and 5.

72

A B

Figure 2. 1. Schematic representation of gene inactivation via single crossover (A) and in-frame gene deletion

via double crossover method (B).

2.6.10 Targeted Gene deletion by double crossover method

PCR targeting system is much more efficient than the traditional gene replacement strategy

in Streptomyces app. In this thesis, the in-frame gene deletion by double crossover was

performed following the protocol of PCR targeting system (Gust et al. 2003). Through

homologous recombination mediated by λ RED (E. coli BW25113/pIJ790) technique, suicide

vector pKGLP2-GusA was engineered to the plasmid for in-frame gene deletion, which

contained the gene cassette: 1500bp up-arm sequence of the target gene, flanking with a

loxP-aac3(IV)-loxP cassette, and 1500bp down-arm sequence of target gene (Figure 2.1B).

Then this gene deletion plasmid was conjugated into Streptomyces. Through two times

successful recombination of the up-arm sequence and the down arm sequence with their

respective identical sequence on the chromosome of the Streptomyces, the gene of interest

was replaced by the 1045 bp cassette of loxP-aac3(IV)-loxP. Afterwards, the resistance marker

was removed through the loxP site-specific recombination mediated by Cre recombinase

(Fedoryshyn et al. 2008) (Figure 2.1B). Detailed experimental information sees section 5.3.4.3

and 5.3.4.5 in Chapter 5.

2.6.11 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE)

All the buffers for the SDS-PAGE were listed in Table 2.5.6. 10% polyacrylamide resolving gel

and polyacrylamide 4% stacking gel were prepared as the SDS-PAGE Polyacrylamide gel. Mini-

PROTEAN Electrophoresis System (BIO-RAD, Germany) was used to perform the gel

electrophoresis.

73

The protein samples were mixed with 4 x sample loading buffer and denatured by heating at

95 °C for 10 min. The denatured protein samples were centrifuged at 14000 rpm for 3 min,

then 10 µL of the supernatants were loaded into the well of stacking gel. Protein markers were

always loaded into one of the lane on the gel. The gel electrophoresis was carried out at 120V

voltage for 1-1.5 h, SDS-PAGE running was stopped when the downmost line of the protein

marker almost reached the bottom of the gel. After electrophoresis was completed, the gel

was gently moved into a tray and stained with staining buffer by heating few minutes in the

microwave oven and shaking for 30 min on shaker. Subsequently, the stained gel was rinsed

with water and stripping buffer several times until obvious protein band were shown on the

gel. The size of the proteins could be estimated by comparing with the protein markers.

Chapter 3. The complete genome sequence of Streptomyces

asterosporus DSM 41452

3.1 Background

Streptomyces asterosporus DSM 41452, a strain with bald phenotype, is deficient in the

formation of aerial mycelium and spores. The strain was recently discovered as a high-amount

producer of WS9326A and its derivatives. These cyclodepsipeptides were firstly isolated from

Streptomyces violaceoniger no.9326 in 1993 as potent tachykinin antagonist (Shigematsu et

al. 1993). Its derivative WS9326E exhibited inhibitory activity against B. malayi asparaginyl-

tRNA synthetase (BmAsnRS) (Yu et al. 2012). In addition, as the potent receptor antagonists

in the agr/fsr system, WS9326A and WS9326B exhibited significant therapeutic potential

against the cyclic peptide-mediated quorum sensing of the Gram-positive pathogens(Desouky

et al. 2015).

A notable feature of Streptomyces is the complex, fungal-like life cycle (Ohnishi et al. 2005).

During its sporulation stage, the biosynthesis of many secondary metabolites is activated. It

was reported previously that bald mutants of Streptomyces are deficient in the biosynthesis

of specific secondary metabolites (Ohnishi et al. 2005). As we have described in the Chapter

1, AdpA is a central transcriptional regulator in the A-factor regulatory cascade. It plays a very

crucial role in morphological differentiation and secondary metabolite production in

Streptomyces species (Pan et al. 2009). One component of the AdpA regulon is bldA which

encodes a rare tRNA molecule (Leu-tRNAUUA) that is necessary for the translation of mRNA

UUA codons (Gessner et al. 2015; Hackl and Bechthold 2015). We previously reported that

the defective bldA gene prevents the generation of aerial hyphae and the formation of

secondary metabolites in Streptomyces calvus by inhibiting the expression of the TTA-

74

containing adpA gene (Hackl and Bechthold 2015). Non-sporulation mutants fail to generate

the aerial mycelium due to different reason. To date at least 20 reported genes are involved

in the aerial mycelium formation (Takano et al. 2003).

In this study, we present the complete genome sequence of S. asterosporus DSM 41452.

Detailed genome sequence comparison was performed, in addition, by complementation of

functional bldA and adpA gene in this strain, we were able to decipher the non-sporulation

mechanism in S. asterosporus DSM 41452. Considering the global regulatory role of AdpA, we

expect this work will provide worthy insights into the regulatory machinery of Streptomyces.

3.2 Materials and Methods

3.2.1 Primers fragments used in this study

Table 3. 1. Primers fragments used in this study

3.2.2 Plasmid information

Table 3. 2. Plasmid information

Name Sequence

Primer for amplifying the entire adpA gene with its upstream 48bp region from the Streptomyces calvus

genome, and to construct plasmid pTESa-AdpA (S.calvus)

KpnI-radpA F ATAGGTACCCAACCGAGGAGCCGCGACCAC

EcoRI-radpA R ATAGAATTCTCACGGCGCGCTGCGCTG

Primers for amplifying 16s rRNA in S. asterosporus DSM 41452

pA AGAGTTTGATCCTGGCTCAG

pH AAGGAGGTGATCCAGCCGCA

Primers for amplifying intergenic region between adpA and the upstream uspA gene

UspA-F GGCTGTCTCGGGGTGGTGATCCTTTGAAC

EcoRI-radpA R ATAGAATTCTCACGGCGCGCTGCGCTG

Name Description Reference

Plasmids

pTESa-adpAsc Integrative vector carrying adpA gene from S.

asterosporusDSM 41452, based on phage ϕC31 integration

system, Aprar

This study

pTESa-bldA Integrative vector carrying bldA gene from S. coelicolor A3(2),

based on phage ϕC31 integration system, Aprar

(Kalan et al.

2013)

75

3.2.3 Genomic DNA preparation and whole-genome sequencing

The gene encoding 16S rRNA was amplified using two universal primers (pA and pH) (Kim et

al. 1998). The sequencing result revealed a high sequence similarity (100%, 1465/1466)

between S. asterosporus NBRC 15872 and Streptomyces calvus NBRC 13200 as the closest

homologous strain. S. asterosporus DSM 41452 was cultivated in 20 mL TSB medium at 28 ℃

for 2 days on a rotary shaker at 180 r·min−1. Cells were pelleted by centrifugation and genomic

DNA extracted using the methods described by Kim et al (Kim et al. 1998). The whole genome

was sequenced using a combination of Illumina Hiseq and Pacific Bioscience SMRT (PacBio

RSII) sequencing platform, with 601-fold average genome coverage. The genome sequencing,

assembly and basic bioinformatic analysis of Streptomyces asterosporus DSM 41452 were

performed by Biozeron sequencing company.

3.2.4 Genome assembly and annotation

A total of 33, 359, 358 reads (average length 100bp) from Illumina sequencing data were

assembled de novo by the SOAPdenovo (v2.04) method (Koren et al. 2012). The PacBio

sequencing data was corrected by mapping the Illumina sequencing reads on BLASR (Basic

Local Alignment with Successive Refinement), and then assembled by the Celera Assembler

(http://wgs-assembler.sourceforge.net). After generating a reliable scaffold, correction of

sequencing reads was performed again based on the Illumina data. Sequencing quality control

on raw sequence data was checked by online software FastQC

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ).

Putative protein-coding sequences were predicted based on results from GLIMMER 3.02

(https://ccb.jhu.edu/software/glimmer/). Additional analysis was carried out using the

UniProt database (http://www.uniprot.org/), the RAST database (https://rast.nmpdr.org/),

Clusters of Orthologous Groups (Huerta-Cepas, Szklarczyk et al. 2015), and KEGG

(http://www.genome.jp/kegg/). rRNA and tRNA genes were predicted with RNAmmer-1.2

(Lagesen et al. 2007) and tRNA scan-SE V1.3.1 (Lowe and Chan 2016). Genome-wide

collinearity analysis was performed using Mauve (Darling et al. 2004). Secondary metabolites

analysis was done with antiSMASH database (Blin et al. 2013), followed by careful manual

pTESa pSET152 derivatives; attP flanked by loxP site, ermEp1

promoter flanked by tfd terminator sequences, Aprar

(Herrmann et al.

2012)

76

correction. Insertable elements were predicted with ISfinder (http://www-is.biotoul.fr)

(Siguier et al. 2006).

3.3 Results and Discussion

3.3.1 General genome features

The main features of the S. asterosporus DSM 41452 chromosome are summarized in Scheme

3.1 and Table 3. 3. Compared with other Streptomyces strains, with 7,766,581 bp, S.

asterosporus DSM 41452 has a relatively small genome (S. avermitilis: 9,025,608 bp, S.

coelicolor: 8,667,507 bp, without plasmids). The genome contains 6,782 predicted protei-

coding genes. The complete genome sequence indicates a single linear chromosome. This

single chromosome contains 9 rRNA operons (16s-23s-5s) and 67 tRNA genes. The average

G+C content of the chromosome is 72.49% (Table 3. 3).

Table 3. 3. General features of the chromosome of S. asterosporus DSM 41452

Species S. asterosporus DSM 41452

Length (bp) 7,766,581

Average G+C content (%) 72.49

Number of protein-coding genes 6,782

Average ORF size (bp) 998

Coding density (%) 86.5

Number of rRNA (16S-23S-5S) operons 9

Number of tRNA genes 67

Number of Secondary metabolite gene clusters 28

77

Scheme 3. 1. Genomic overview of S. asterosporus DSM 41452. Circus A: Locations of predicted secondary

metabolite gene cluster (green) and rRNAs (5S-16S-23S, red); Circus B: G+C-content; Circus C: G+C-skew; Circus

D: Location of transposable genetic elements.

We identified the putative origin of replication (oriC) as a 1,273 bp non-coding sequence at

the position 3598337-3599609 on the chromosome. This region is flanked by the gene dnaA

(RAST gene ID: fig|1.39.peg.3068), encoding a chromosomal replication initiator protein and

dnaN, encoding a DNA polymerase III beta subunit. As expected, the oriC region includes

classic 19 DnaA box-like sequences [TT(G/A)TCCACA], and shows significant similarity of the

genome sequence around oriC with other Streptomyces species. The GC-skew inversion

observed between the left and right arm of the chromosome supports this prediction (Scheme

3. 1). In addition, the locus of oriC shows symmetry and situated roughly in the middle of the

genome (approximately 0.36Mb away from the center toward the right end) of S. asterosporus

DSM 41452.

78

Table 3. 4. Assignment of 4047 genes of S. asterosporus DSM 41452 to the functional groups of the actNOG

subset of the eggNOG database

Function Type Category Number

information storage

and processing (725)

A RNA processing and modification 1

B Chromatin structure and dynamics 1

J Translation, ribosomal structure and biogenesis 208

K Transcription 384

L Replication, recombination and repair 131

cellular processes

and signaling (879)

D Cell cycle control, cell division, chromosome

partitioning

47

M Cell wall/membrane/envelope biogenesis 229

N Cell motility 11

O Posttranslational modification, protein turnover,

chaperones

155

T Signal transduction mechanisms 225

U Intracellular trafficking, secretion, and vesicular

transport

35

V Defense mechanisms 140

Y Nuclear structure 0

Z Cytoskeleton 0

W Extracellular structures 5

X Mobilome: prophages, transposons 32

Metabolism (1880) E Amino acid transport and metabolism 379

F Nucleotide transport and metabolism 102

G Carbohydrate transport and metabolism 401

79

H Coenzyme transport and metabolism 215

I Lipid transport and metabolism 196

P Inorganic ion transport and metabolism 202

Q Secondary metabolites biosynthesis, transport and

catabolism

152

C Energy production and conversion 233

poorly characterized

(563)

R General function prediction only 415

S Function unknown 148

The COG protein functional annotation and classification of S. asterosporus DSM 41452 were

performed with BLASTP (using protein BLAST with an exception value cut-off 0.001) based on

string database (string database v9.05) (Table 3. 4). The COG annotation results show that

4047 out of 6782 genes (60.3%) have at least one biological function assignment, with some

genes assigned to more than one category. Among those genes with functional assignments,

the proteins associated with primary metabolism are the most abundant group, 1,880

proteins (46.4%) including 379 genes related with amino acid transport and metabolism, 401

genes associated with carbohydrate metabolism, 152 genes participating in secondary

metabolites biosynthesis, transport and catabolism. 879 of the annotated proteins are related

with cellular process and signaling, 725 proteins are assigned to be involved in the information

storage and processing. In addition, there are 563 proteins with poor characterization due to

their unknown functions.

3.3.2 Gene clusters related with Secondary metabolism

Genome analysis by antiSMASH (Version 4.0.0rc1) reveal 28 gene clusters potentially involved

in secondary metabolism, including the gene clusters of the compounds annimycin and

WS9326A which have been isolated from this strain. General information about the secondary

metabolism gene clusters in S. asterosporus DSM 41452 are summarized in Table 3. 5.

Table 3. 5. Secondary metabolites gene clusters (BGC) identified in S. asterosporus DSM 41452 (antiSMASH 3.0)

Gene cluster BGC type BGC position Most similar known cluster

1 t1pks 153548-242834 Candicidin biosynthetic gene cluster (90% of

genes show similarity)

2 t1pks- nrps 322164-401091 Albachelin biosynthetic gene cluster (80% of

genes show similarity)

80

3 Terpene 429743-452386 Carotenoid biosynthetic gene cluster (36% of

genes show similarity)

4 t2pks 674439-716949 Spore pigment biosynthetic gene cluster (83%

of genes show similarity)

5 Terpene 791816-818621 Hopene biosynthetic gene cluster (92% of

genes show similarity)

6 Lassopeptide 1178209-1200802 -

7 amglyccycl-cf

saccharide

1209085-1240027 Cetoniacytone A biosynthetic gene cluster

(9% of genes show similarity)

8 Siderophore 1275939-1289099 Grincamycinbiosyntheticgenecluster (8% of

genes show similarity)

9 Terpene 1456838-1478995 Geosmain synthase

10 Bacteriocin 1522956-1534381 -

11 Lassopeptide 1616191-1638712 SSV-2083 biosynthetic gene cluster (62% of

genes show similarity)

12 t1pks-otherks 1699373-1767949 A33853 biosynthetic gene cluster (100% of

genes show similarity)

13 Siderophore 2004622-2016611 -

14 lantipeptide-nrps 2420235-2494800 Tetrocarcin A biosynthetic gene cluster (4% of

genes show similarity)

15 Terpene 2628240-2649173 Albaflavenone biosynthetic gene cluster

(100% of genes show similarity)

16 Butyrolactone 3627022-3638015 Coelimycin biosynthetic gene cluster (8% of

genes show similarity)

17 Phenazine 4077990-4098458 Istamycin biosynthetic gene cluster (4% of

genes show similarity)

18 Siderophore 4944035-4955808 Desferrioxamine B biosynthetic gene cluster

(100% of genes show similarity)

19 Lassopeptide 4979828-5002415 GE81112 biosynthetic gene cluster (7% of

genes show similarity)

20 Melanin 5040525-5071594 Melanin biosynthetic gene cluster (80% of

genes show similarity)

21 Ladderane 5548177-5630048 WS9326As biosynthetic gene cluster (100% of

genes show similarity)

22 Ectoine 6117097-6127496 Ectoine biosynthetic gene cluster (100% of

genes show similarity)

23 t1pks-nrps 6664067-6721291 Antimycin biosynthetic gene cluster (100% of

genes show similarity)

24 t1pks 6740672-6810773 4-Z-annimycin biosynthetic gene cluster

(100% of genes show similarity)

81

25 Nrps 7037447-7112235 Stenothricin biosynthetic gene cluster (13% of

genes show similarity)

26 Other 7239742-7280483 -

27 bacteriocin-t1pks-

terpene

7372052-7457845 Informatipeptin biosynthetic gene cluster

(57% of genes show similarity)

28 t1pks 7582112-7688485 Candicidin biosynthetic gene cluster (33% of

genes show similarity)

The most representative type of SM biosynthesis gene clusters within the S. asterosporus DSM

41452 genome are the one encoding polyketide synthases. Genome analysis revealed that

there are 4 type I PKS synthetases and 1 type II PKS (Table 3. 5). Two PKS clusters Cluster 1

(Location: 153549 - 242834 nt) and cluster 28 (Location: 7582113 - 7688485 nt) have very high

similarity with genes encoding type I PKS Candicidin synthase, interestingly, those two genes

distribute at two different telomerale position on the chromosome, it is possible that one

Candicidin gene cluster was separated into two parts resulting from the genome assembly.

Cluster 24 (Location: 6740673 - 6810773 nt) was annotated as a type I PKS synthetase

responsible for the biosynthesis of annimycin; Cluster 12 (Location: 1699374 - 1767949 nt)

show 100% of genes similarity with the A33853 biosynthetic gene cluster.

There are two PKS-NRPS hybrid synthetases related to the biosynthesis of compound

Antimycin (cluster 23, 100% gene cluster similarity, Location: 6664068-6721291nt) and

Albachelin (cluster 2, 80% of genes show similarity, location: 322164-401091nt).

In addition, five gene clusters for terpenoid biosynthesis were found in S. asterosporus DSM

41452. Among them, cluster 3 (Location: 429744 - 452386 nt) show 45% similarity with the

gene cluster for Calicheamicin biosynthesis. Cluster 5 (Location: 791817 - 818621 nt) contain

the genes showing 92% similarity with the hopene biosynthetic genes. Cluster 9

(Location:1456839-1478995nt) are annotated as geosmin synthase (WP_011030632, 88.9 %

identity) base on the best-known BLAST hit. Cluster 12 (Location: 2628240-2649173nt) was

predicted as Albaflavenone biosynthetic gene cluster (100% of genes show similarity) by

BLAST analysis. Based on antiSMASH analysis, several other types of SM could be potentially

produced by S. asterosporus DSM 41452, including Kanamycin and Gamma-butyrolactone,

etc.

Interestingly, the putative gene cluster of Nucleocidin (Table 3. 6), which was recently

rediscovered from Streptomyces calvus (Kalan, Gessner et al. 2013), is also found in S.

asterosporus DSM 41452 with a sequence similarity of 99.4%. Nucleocidin (Figure 1.14) is a

nucleoside kind of antibiotic and exhibits broad antibacterial activity against gram-positive

and negative bacteria. In the chemical structure of Nucleocidin, a fluorine is covalently bound

82

to the 4’-C of the adenosine through a C-F bond. It’s another chemical feature is the unique

sulfonamide group at the ribose-C5’ position (Morton, Lancaster et al. 1969).

Table 3. 6. ORFs associated with the Nucleocidin biosynthetic cluster in S. asterosporus DSM 41452

Gene Homologue in

S. calvus

Size(a.a.) Predicted function Identity [%]

Gene cluster for the formation of phosphoadenosine phosphosulfate (PAPS)

fig|1.39.peg.1209 ORF2331 565 putative sulfite reductase

[Streptomyces calvus]

100%

fig|1.39.peg.1210 ORF2333 236 putative PAPS reductase

[Streptomyces calvus]

100%

fig|1.39.peg.1211 nucB 178 putative adenylylsulfate kinase

[Streptomyces calvus]

100%

fig|1.39.peg.1212 nucA 311 putative sulfate adenylyltransferase

subunit 2 [Streptomyces calvus]

100%

fig|1.39.peg.1213 nucW 444 putative sulfate adenylyltransferase

subunit 1 [Streptomyces calvus]

100%

fig|1.39.peg.1214 ORF2341 367 putative sulfate ABC transporter

periplasmic binding protein

component [Streptomyces calvus]

100%

fig|1.39.peg.1215 ORF2342 262 putative sulfate ABC transporter ATP

binding protein component

[Streptomyces calvus]

100%

fig|1.39.peg.1216 ORF2345 296 putative sulfate ABC transporter

permease [Streptomyces calvus]

100%

Main cluster

fig|1.39.peg.156 ORF171 332 putative oxidoreductase

[Streptomyces calvus]

100%

fig|1.39.peg.157 nucU 452 NucU [Streptomyces calvus] 100%

fig|1.39.peg.158 ORF173 275 hypothetical protein [Streptomyces

calvus]

100%

fig|1.39.peg.159 ORF174 212 putative phosphoglycerate mutase

[Streptomyces calvus]

99%

83

fig|1.39.peg.160 43 _ _

fig|1.39.peg.161 ORF178 1047 putative transcriptional regulatory

protein [Streptomyces calvus]

100%

fig|1.39.peg.162 ORF181 137 putative aminoglycoside

phosphotransferase [Streptomyces

calvus]

99%

fig|1.39.peg.163 nucR 470 NucR [Streptomyces calvus] 100%

fig|1.39.peg.164 nucM 140 NucM [Streptomyces calvus] 100%

fig|1.39.peg.165 nucG 473 NucG [Streptomyces calvus] 100%

fig|1.39.peg.166 nucN 331 NucN [Streptomyces calvus] 100%

fig|1.39.peg.167 nucI 389 NucI [Streptomyces calvus] 100%

fig|1.39.peg.168 63 _ _

fig|1.39.peg.169 ORF191 312 putative StrR-like transcriptional

regulator [Streptomyces calvus]

99%

fig|1.39.peg.170 nucJ 560 NucJ [Streptomyces calvus] 100%

fig|1.39.peg.171 nucK 359 NucK [Streptomyces calvus] 100%

fig|1.39.peg.172 nucL 255 NucL [Streptomyces calvus] 100%

fig|1.39.peg.173 nucQ 158 NucQ [Streptomyces calvus] 100%

fig|1.39.peg.174 nucP 661 NucP [Streptomyces calvus] 100%

fig|1.39.peg.175 nucO 460 NucO [Streptomyces calvus] 100%

fig|1.39.peg.176 nucV 194 NucV [Streptomyces calvus] 100%

fig|1.39.peg.177 ORF203 198 putative histidine kinase

[Streptomyces calvus]

100%

fig|1.39.peg.178 ORF 206 273 putative nucleoside phosphorylase

[Streptomyces calvus]

100%

fig|1.39.peg.179 ORF 208 894 putative lycopene cyclase

[Streptomyces calvus]

99%

84

fig|1.39.peg.180 ORF 210 305 putative glycosyltransferase

[Streptomyces calvus]

100%

3.3.3 bldA and adpA gene in S. asterosporus DSM 41452

In order to find out the genetic reason causing the bald phenotype of this strain, genome

sequence alignment was used to compare S. asterosporus DSM 41452 with other

Streptomyces species. The result of bldA sequence alignments between S. asterosporus DSM

41452 and Streptomyces calvus, Streptomyces lividans (CP009124.1), Streptomyces coelicolor

(Y00209.1), Streptomyces iranensis(LK022848.1), Streptomyces rapamycinicus (CP006567.1),

Streptomyces scabiei (FN554889.1), Streptomyces pratensis (CP002475.1) (Figure 3. 1) shows

that the bldA gene in S. asterosporus DSM 41452 is functional, and will not encode the

misfolded Leu-tRNAUUA molecule as the incorrect gene encoding happened in S. calvus(Kalan

et al. 2013). Hence, we hypothesized that the unusual phenotype of S. asterosporus DSM

41452 is caused by a different mechanism.

A

B

Figure 3. 1. (A) Multiple sequences alignment of bldA gene from S. asterosporus DSM 41452, Streptomyces

calvus, Streptomyces lividans (CP009124.1), Streptomyces coelicolor (Y00209.1), Streptomyces iranensis

(LK022848.1), Streptomyces rapamycinicus(CP006567.1), Streptomyces scabiei(FN554889.1), and Streptomyces

pratensis(CP002475.1); Red triangle indicates the mutation point in S. calvus. (B) The genome sequence

comparison of the upstream intergenic region of adpA between S. asterosporus DSM 41452 and S. calvus.

Analysis of the promoter sequence upstream to adpA gene revealed an insertional element

(901bp) located between the promoter and adpA gene in the genome of S. asterosporus DSM

41452 (Figure 3. 1B). This insertional element gene is present at two positions on the genome

(position 1: from 4942447 to 4943347; position 2: from 5075579 to 5076479). Moreover, this

transposase-encoding gene is also present in many other Streptomyces species. This gene

fragment shows 73% sequence identity with the gene encoding a putative IS1647-like

transposase (sequence ID: AP009493.1) in the strain Streptomyces griseus subsp. griseus

85

NBRC 13350. It is worth noting that the associated gene also is located on two different

position in the genome of Streptomyces griseus subsp. griseus NBRC 13350 (position 1: from

122084 to 122662; position 2: from 8423268 to 8423846). While in S. coelicolor A3(2) and S.

avermitilis most of the transposon genes are located at the arm regions (especially at the sub-

TIR regions) (Bentley et al. 2002; Ikeda et al. 2003), the transposon genes are found

throughout the chromosome of S. asterosporus DSM 41452. Among the 42 transposase-

encoding sequences, most of them were categorized into 2 families: family IS481- and IS5-like

elements. High degree of horizontal gene transfer can be observed at the right region of oriC

which contains multiple insertions of mobile elements.

Figure 3. 2. The genome comparison between S. asterosporus DSM 41452 and S. avermitilis, S. coelicolor A3(2),

respectively by Mauve 2.2.0. The color blocks are referred to Locally Collinear Blocks (LCBs), which represent

homologous conserved regions without internal rearrangement among the compared sequence. The minimum

LCB weight value was used for those genome alignments. The red lines at the terminus of each chromosome

represent the genome boundaries. The colored blue and red bands were used to connect the corresponding

LCBs, depicting the location and orientation of the corresponding gene sequences in those two genomes. The

red and blue band show the different LCBs arrangement on those two genomes, also exhibiting their

chromosome structure difference to some extent.

The alignment between S. asterosporus DSM 41452 and other chromosomes of Streptomyces

(Figure 3. 2) revealed that most of the conserved genes of S. asterosporus DSM 41452 are

located in the center of the chromosomes, which is consistent to the reported core region of

the Streptomyces coelicolor A3(2) (Ikeda et al. 2003). The homologous conserved regions on

the chromosome of S. avermitilis and S. asterosporus DSM 41452 showed highly conserved

linearity as well as gene arrangement. By contrast, most of the conserved genomic regions

around the oriC locus show a structural asymmetry between the S. asterosporus DSM 41452

and S. coelicolor A3(2) (Figure 3. 2). In addition, under the condition of minimum weight

86

setting, 414 Locally Collinear Blocks (LCBs) were generated by comparing the genome of S.

asterosporus DSM 41452 and S. coelicolor A3(2), which is higher than 324 LCBs generated by

comparing S. asterosporus DSM 41452 with S. avermitilis, demonstrating that the closer

evolutionary relationship between S. asterosprous DSM 41452 and S. coelicolor A3(2). This

result is in accordance with the following 16s rRNA based phylogenetic analysis below.

3.3.4 Function verification of adpA gene

A B

Figure 3. 3. (A) PCR verification of the upstream intergenic region of adpA in S. asterosporus DSM 41452 and S.

calvus; Note: Lane 1, 2, and 3 (numbering from left to right); Lane 1 represents the PCR fragment (2200bp)

amplified from the genome of S. calvus; Lane 2 represents the PCR fragment (3102 bp) amplified from the

genome of S. asterosporus DSM 41452; Lane 3 represents the Marker. (B) Morphological development of S.

asterosporus DSM 41452 and its mutants S. asterosporus DSM 41452:: pTES-bldA, S. asterosporus DSM 41452 ::

pTES and S. asterosporus DSM 41452 :: pTES-adpAsc

In S. calvus, gene uspA is located upstream of adpA, the size of the intergenic region between

both genes is 967bp. In contrast, the intergenic region size is 1,867 bp in the genome of S.

asterosporus DSM 41452. Difference in the length of intergenic region was confirmed by PCR

(Figure 3. 3A). For more detailed analysis of the adpA genes in S. asterosporus DSM 41452, a

sequence containing the adpA gene with its native promoter region from S. calvus was cloned

into the E. coli-Streptomyces shuttle plasmids pTES, in which the adpA gene was under the

control of the strong constitutive ermE* promoter. The resulting pTES-adpASC is an integrative

plasmid based on ϕC31 integrase which constitutively express the adpA gene from ermE*

promoter. The mutant strain S. asterosporus DSM 41452::pTES-adpASC restored the

sporulation after 5 days incubation on the SG solid medium (Figure 3. 3B).

87

3.3.5 Phylogenetic and orthologous analysis

A

B

Figure 3. 4. The phylogenetic relationship of S. asterosporus DSM 41452 with other strains based on 16S rRNA

gene sequences. The 16S rRNA phylogram (A) was built based on the neighbor joining method, bootstrap

confidence value was obtained using 1000 resamplings. E. coli was chosen as an outgroup strain. The

phylogenetic tree (B) was constructed based on the selected species belonging to the genus Streptomyces. The

sequence alignment was performed in Clustal Omega, the figure of phylogenetic tree was reconstructed by

MEGA5.

Streptomyces asterosporus DSM 41452 (ex Krasil'nikov 1970) firstly was reported in 1986 by

Preobrazhenskaya, etc (Gause et al. 1983; Elferink 1997). An unsupervised nucleotide BLAST

analysis base on the 16S rRNA gene from S. asterosporus DSM 41452 with the 16S rRNA gene

from different actinobacteria was performed to determine their phylogenetic relationships,

among them, Escherichia coli, Sporosacina polymorpha, and Streptobacillus moniliformis were

chosen as out-group (Figure 3. 4A). The analysis clearly showed that S. asterosporus DSM

41452 is distinct from the genus Actinosynnema mirum and kutzneria albida, and closer

related to representatives of Streptomyces coelicolor. The highest similarity was observed

between 16S rRNA of S. griseus, S. albus, S. avermitilis, and S. coelicolor, all of which belong

to the Streptomyces family. Inside Streptomyces species S. asterosporus DSM 41452 keep

88

closest kinship with (Figure 3. 4B) S. asterosporus NBRC 15872, Streptomyces calvus NBRC

13200, Streptomyces virens NBRC B-24331, and those species form a branch clade distinct

from other Streptomyces species.

Figure 3. 5. OrthoMCL analysis of strain S. asterosporus DSM 41452, S. coelicolor A3(2), S. avermitilis, and

Kutzneria albida. The number of shared and genome-specific homologous genes are summarized as a Venn

diagram, and the proteins were clustered with the OrthoMCL parameters: e-value 1e-5; identity 50%; coverage

50%; score 40; MCL Markov clustering inflation index 1.5.

Based on a OrthoMCL clustering analysis of the genomic content between S. asterosporus

DSM 41452 and other actinomycetes (Kutzneria albida, Streptomyces avermitilis, and

Streptomyces coelicolor), shared proteins in Venn diagram were defined as the reciprocal

best-hit proteins with a threshold of 50% identity and 70% length coverage by the BLAST

algorithm.

The coding-gene sequences (CDSs) in S. asterosporus DSM 41452 were clustered into 5,433

families, 1892 (34.8%) gene families were found to be commonly shared with the other three

Streptomyces. S. asterosporus DSM 41452 shares the highest number of orthologs with the

Streptomyces coelicolor (4068), less with Streptomyces avermitilis (3745), while only share

2329 orthologs with a rare Actinobacteria Kutzneria albida. This molecular phylogeny analysis

fully corresponds to and supports our result of 16S rRNA phylogram between different

Streptomyces species.

In contrast to other actinomycetes, S. asterosporus DSM 41452 contains the least proportion

of strain-specific gene which only account for 19.5% of its protein encoding genes, and those

genes may be associated with its unique biological characteristics. Those reduced genetic

redundancy might contribute us to figure out the essential genes for Streptomyces species

and shed insight into their evolutionary history.

In addition, we clustered all annotated proteins of S. asterosporus DSM 41452 with the ones

from other three representative actinobacteria strains: S. coelicolor A3(2), S. avermitilis, and

89

kutzneria albida using pair-wise BLASTP program with a threshold of 60% identity and 70%

length coverage. 1419 single copy genes were found to be commonly present in all those four

actinomycetes. Interestingly, comparing with the previous report (Ohnishi et al. 2008), there

are 3039 proteins are present among those three Streptomyces species: S. coelicolor A3(2), S.

avermitilis, and S. griseus. In our case, the less number of shared single copy gene in those

four actinomycetes could be caused by the taxonomically far distant between Streptomyces

species and kutzneria albida, which belong to the genus of bacteria in Phylum Actinobacteria.

3.4 Conclusion

S. asterosporus DSM 41452 is a producer of WS9326A and its derivatives which belong to a

group of natural products with potent tachykinin antagonist activity (Shigematsu et al. 1993).

In this study, we present the complete genome sequence of a natural non-sporulation strain:

S. asterosporus DSM 41452. The genome reveals a single 7,837,567 bp linear chromosome

with 6782 annotated protein-coding sequences (CDSs). The sequencing results show that this

strain own abundant gene clusters for secondary metabolites, more than 28 natural product

gene clusters were detected in the genome, most interestingly, it contains the gene cluster of

the antibiotic Nucleocidin (Figure 1. 19). Nucleocidin was firstly isolated as an anti-

trypanosome antibiotic from Streptomyces calvus by scientists (Morton et al. 1969), but since

then lots of subsequent efforts to restore this molecule have failed (Maguire et al. 1993;

O’Hagan and B. Harper 1999). Interestingly, in 2015, David etal (Zhu et al. 2015) successfully

restored the production of Nucleocidin in a bldA mutant of S. calvus. Considering the close

kinship of those two strains, and the exist of Nucleocidin gene cluster in S. asterosporus DSM

41452, we tend to believe that S. asterosporus DSM 41452 own the capability of producing

Nucleocidin, the detailed secondary metabolites analysis is undergoing.

S. asterosporus DSM 41452 is a bald strain with wrinkled and shiny aerial surface. By

comparative genome analysis, a transposon gene was discovered to insert between the region

of ribosomal binding site (RBS) and gene adpA in S. asterosporus DSM 41452 which prevents

the transcription of adpA. We propose that, the baldness phenotype of S. asterosporus DSM

41452 is caused by this unusual genotype. The following gene complementation experiments

proven this machinery. In conclusion, the genome sequence of S. asterosporus DSM 41452

provides an interesting insight into the genetic of Streptomyces species with non-sporulation

phenotype.

90

In addition, our research illustrated a new mechanism resulting in the morphological defect

of baldness mutant in Streptomyces, which also provide a new alternative approach of waking

up the expression of “silent” gene cluster in Streptomyces species.

S. asterosporus DSM 41452 owns relatively less amounts of strain-specific gene, which may

reflect some its special evolutionary history to a certain extent. Those reduced genetic

redundancy might contribute to outline the essential gene for Streptomyces in the future

research. The complete genome sequence of S. asterosporus DSM 41452 has been uploaded

and deposited in the GenBank database with accession number [CP022310]. We anticipate

that the complete genome information contributes to the application development of S.

asterosporus DSM 41452 as an industrial strain in the future.

91

Chapter 4. Comparative Proteomic Analysis of Streptomyces

asterosporus DSM 41452

4.1 Introduction

As it has been described in the section 1.1 that Streptomyces is characterized by a set of

complicated development system (Ohnishi et al. 2005), and many studies have suggested that

the regulatory network of AdpA vary from Streptomyces species to species, and the

representatives have been exemplified. In the case of S. asterosporus DSM 41452, it not only

owns the potential ability of producing various kinds of secondary metabolites, but also

exhibits characteristic ‘’baldness” phenotype of its wildtype strain. In Chapter 3, we

successfully verified that the promoter region of native adpA in this strain was disrupted by

an insertional element gene located between the promoter and adpA gene in the genome of

S. asterosporus DSM 41452. However, our understanding of the regulatory mechanism in this

strain was still limited, we were interested to know whether this kind of genetic defect will

cause some other influence on strain’s normal growth and development. From other

perspective, AdpA is assumed to own the largest regulon in bacteria (Higo et al. 2012).

However, the exact pleiotropic regulatory network of AdpA in a native non-sporulating strain

remains poorly understood so far.

Proteomics is an efficient method to investigate the cellular physiology and metabolism of an

organism(Mallick and Kuster 2010), and it’s also an attractive approach of screening potential

engineering targets for strain improvement in various cell types (Jayapal et al. 2010; Manteca,

Jung et al. 2010; Manteca, Sanchez et al. 2010). The throughput and the detection limits of

proteomics have been increasingly improved with the advancement of genomic sequencing

technology and bioinformatics (Hwang et al. 2014). However, the traditional proteomics

method usually base on the technique combination of the protein fractionation by two-

dimensional polyacrylamide gel electrophoresis (2D-PAGE) and the target protein

identification of by mass spectrometry (Choi et al. 2010; Ye et al. 2014). The limitations of

these methods are the limited reproducibility and the low resolution of discerning the identity

of proteins. Therefore, it is necessary to employ more automated and sensitive detection

method. Mass spectrometry based approaches such as isobaric tags for relative and absolute

quantitation (iTRAQ) and the stable isotope labeling with amino acids in cell culture (SILAC)

are adequate to this purpose and widely used in quantitative proteomics to solve these issues

(Ong et al. 2002; Wiese et al. 2007).

92

Stable isotope labelling by amino acids in cell culture (SILAC) is a high-throughput and high-

accuracy approach base on mass spectrometry. SILAC could be utilized to examine proteome

changes in various states and compare the discrepancy among different cells. Due to the

advantages of this approach, SILAC has been widely used in various organisms, including

mammalian cells, plant cells, yeast, and in bacterial cells as Escherichia coli and Bacillus subtilis

(Mann 2006; Soufi et al. 2010), even in Streptomyces (Jayapal et al. 2010).

In comparison to other proteomics methods, SILAC method utilize a “bottom-up” proteomics

approach (Zhang et al. 2013). In this method, target protein is firstly proteolytic digested, then

the generated peptides are identified and characterized base on their amino acid detected by

mass spectrometry (Aebersold and Mann 2003). Moreover, SILAC adopts metabolic amino

acids labeling technique in cell culture, which significantly improves the accuracy of mass

spectrometric analysis. Figure 4. 1 shows concisely the basic sample labeling principle and

procedure of SILAC proteomics. Two sets of strains are firstly labeled during their growth by

light medium with normal arginine and lysine (Arg-0 and Lys-0) and heavy medium with

arginine and lysine isotope (Arg-10 and Lys-8), respectively. Through metabolism the labelled

isotope amino acids are incorporated into proteins, which subsequently results in a mass shift

of the corresponding peptides, and this mass shift can be detected by mass spectrometer as

indicated in Figure 4. 1. When both samples are combined with equal concentration, the ratio

intensity of signal peak in the mass spectrum reflects the relative abundance of labeled

protein in both samples.

Figure 4. 1. Schematics representing the basic sample labeling principle of SILAC proteomics approach.

Proteomics has become a very important systematic method to investigate the physiological

metabolism of Streptomyces. Nevertheless, SILAC proteomics method has not been as widely

utilized as other proteomics approach in Streptomyces system. In 2010, Jayapal etal adopted

93

SILAC and iTRAC combined proteomics method to investigate the dynamic turnover of

intracellular proteins in Streptomyces (Jayapal et al. 2010). In our current research, SILAC-

based comparative proteomic approach was employed to analyze the characteristics of

dynamic proteomics in S. asterosporus DSM 41452. It is the first time that the AdpA regulon

in a native non-sporulating Streptomyces was profiled. In this study, more than 1200 proteins

were identified, from which 52 regulated proteins of Streptomyces asterosporus DSM 41452

showed a significantly altered level relative to the wild-type strain. In addition, our analysis

shows that the AdpA regulatory network in S. asterosporus DSM 41452 is not limited to the

proteins involved in secondary metabolism and sporulation development, but also

participates in the primary metabolism, nitrogen metabolism, nutrient utilization, and stress

response et al. This analysis suggested that SILAC could be efficiently applied in Streptomyces

proteomics. These results could provide valuable information for understanding the

developmental mechanisms in Streptomyces development in the phase of sporulation.

4.2 Materials and Methods

4.2.1 Primers fragments used in this study

Table 4. 1. Primers fragments used in this study

Name Sequence

Primers for inactivation of gene fig|1.39.peg.1705 (RAST Gene ID) encoding DapB homologue in the

biosynthesis pathway of lysine (lys)

IndapB-F CAAGCTGGAGACCCTCGCCGA

IndapB-R GGCATGAAGCTGCTGTGGTGCA

Primer for verification of gene disruption in the mutant S. asterosporus:: pLERE-Inlys

InlysF CAAGCTGGAGACCCTCGCCGA

aadVF ATGAGGGAAGCGGTGATCGCCG

Primer for inactivation of gene fig|1.39.peg.5620 (RAST Gene ID) encoding Argininosuccinate synthase (EC

6.3.4.5) in the biosynthesis pathway of Arginine (Arg)

InArg2-F ATCGTCAAGCACCTCGTCGCC

InArg2-R CACCTCGCGGGACTTGATGC

Primer for verification of gene disruption in the mutant S. asterosporus::pKGLP2-InArg

InArgF ATCGTCAAGCACCTCGTCGCC

HygR TCAGCCAATCGACTGGCGA

94

4.2.2 Plasmid information

Table 4. 2. Plasmid information

4.2.3 Strain constructed and used in this study

Table 4. 3. Strain constructed and used in this study

Name Description Reference

pBluescript

SK(-)

Cloning vector, Ampr, lacZ’(α-complementation), f1(-)-origin, Carbr Stratagene

pSET152 Integrative vector for actinomycetes; based on phage ϕC31

integration system, aac(3)IV

(Bierman et al. 1992)

pTESa pSET152 derivatives; attP flanked by loxP site, ermEp1 promoter

flanked by tfd terminator sequences

(Herrmann et al. 2012)

pKC1132 Conjugative vector, Non-replicative in Streptomyces, Aprar (Bierman et al. 1992)

pSET152-

adpAgh(TTA)

pSET152 carrying adpAgh gene from strain Streptomyces

ghanaensis with TTA codon, along with its -500bp upstream region

(Makitrynskyy et al.

2013)

pSET152-

adpAgh(CTG)

pSET152 carrying adpAgh gene from strain Streptomyces

ghanaensis without CTG codon, along with its -500bp upstream

region

(Makitrynskyy et al.

2013)

pTESa-adpAgh Integrative vector pTESa carrying adpA gene from Streptomyces

ghanaensis, based on phage ϕC31 integration system, constitutive

promoter ermE*, Aprar

(Makitrynskyy et al.

2013)

pTESa-adpAsc Integrative vector pTESa carrying adpA gene from S. asterosporus

DSM 41452, based on phage ϕC31 integration system, constitutive

promoter ermE*, Aprar

This study

pLERE-spec Cloning vector containing amp, aadA, and oriT flanked by two

loxLE sites, and two loxRE sites, Specr

(Herrmann et al. 2012)

pKGLP2 pKCLP2 derivative with a gusA gene, replicative vector in E. coli,

suicide vector in Streptomyces, Hygrr

(Myronovskyi et al.

2011)

pLERE-Inlys Vector for inactivation of gene fig|1.39.peg.1705 (RAST Gene ID),

based on pLERE-spec, Specr

This study

pKGLP2-InArg Vector for disruption of gene fig|1.39.peg.5620 (RAST Gene ID),

based on pKGLP2, Hygrr

This study

Strains Relevant characteristics Reference

E. coli DH5α General cloning host Invitrogen

E. coli XL1 blue blue-white color screening for plasmid recombination Invitrogen

E. coli ET12567 (pUZ8002) Methylation-deficient E. coli strain for conjugation Invitrogen

S. asterosporus DSM 41452 Wild type strain, WS9326A producer This study

95

4.2.4 Bacterial strain and culture condition

The wildtype strain S. asterosporus DSM 41452 was purchased from Leibniz Institute DSMZ-

German Collection of Microorganisms and cell Cultures (DSMZ), Germany. The cultivation

conditions, transformation and conjugation methods followed the general protocols for

Streptomyces and E. coli as described in chapter 2.

4.2.5 Functional adpA overexpression in S. asterosporus DSM 41452

The construction of plasmid pTESa-adpAsc has been described in the chapter 3, other two

plasmids pSET152-adpAgh(TTA) and pSET152-adpAgh(CTG) were kindly provided by Dr.

Roman Makitrynskyy. Those plasmids firstly were individually transformed into E. coli ET

12567(pUZ8002). Then through intergeneric conjugation, those plasmids were introduced

into S. asterosporus DSM 41452 to yield mutants S. asterosporus DSM 41452::pSET152-

adpAgh(TTA), S. asterosporus DSM 41452::pSET152-adpAgh(CTG) and S. asterosporus DSM

41452::pSET152. The resultant exconjugants were screened on the solid MS medium

supplemented with selective antibiotics. The correct exconjugants carrying corresponding

plasmid were screened for resistance against apramycin (50 ug/mL).

4.2.6 Construction of Arginine and Lysine auxotrophic mutant strain of S.

asterosporus DSM 41452

Base on bioinformatic analysis, gene fig|1.39.peg.1705 (RAST Gene ID) in S. asterosporus DSM

41452 encode a homologue of DapB which is involved in the lysine biosynthesis DAP pathway

in Streptomyces. A 555 bp internal gene fragment of gene fig|1.39.peg.1705 was amplified

S. asterosporus DSM

41452::pSET152

S. asterosporus DSM 41452 strain carrying plasmid

pSET152

This study

S. asterosporus DSM

41452::pSET152-adpAgh(TTA)

S. asterosporus DSM 41452 carrying plasmid pSET152-

adpAgh(TTA)

This study

S. asterosporus DSM

41452::pSET152-AdpAgh(CTG)

S. asterosporus DSM 41452 carrying plasmid pSET152-

AdpAgh(CTG)

This study

S. asterosporus SILAC1 S. asterosporus DSM 41452:pSET152::pLERE-

Inlys::pKGLP2-InArg

This study

S. asterosporus SILAC2 S. asterosporus DSM 41452::pSET152-

adpAgh(TTA)::pLERE-Inlys:: pKGLP2-InArg

This study

96

from the genome of the wildtype strain S. asterosporus DSM 41452 by PCR using primer

IndapB-F and IndapB-R. The PCR product was ligated into EcoRV-digested pBluescripts to yield

the plasmid pBSK-partial-lys-DapB. Then the internal fragment of gene fig|1.39.peg.1705 was

digested at HindIII/BamHI restriction site, and then was cloned into vector pLERE-spec to

afford the suicide plasmid pLERE-Inlys for the disruption of gene fig|1.39.peg.1705.

Bioinformatic analysis shown that gene fig|1.39.peg.5620 (RAST Gene ID) in S. asterosporus

DSM 41452 encode the homolog of argininosuccinate synthetase (EC.6.3.4.5) which belong to

the Arginine biosynthesis pathway in Streptomyces. In order to construct the inactivation

plasmid of gene fig|1.39.peg.5620, a 519bp internal gene fragment of gene fig|1.39.peg.5620

was amplified from the genome of the wildtype strain S. asterosporus DSM 41452 by PCR using

primer InArg2-F and InArg2-R. The PCR product was ligated into EcoRV-digested pBluescripts

to yield plasmid pBSK-partial-Arg. Then the internal fragment was digested at HindIII/BamHI

restriction site and was cloned into a suicide vector pKGLP2 to afford the plasmid pKGLP2-

InArg.

After that, those two plasmids pLERE-Inlys and pKGLP2-InArg were introduced into E. coli ET

12567 (pUZ8002), then they were introduced into strain S. asterosporus DSM 41452::pSET152

by intergeneric conjugation to yield the lysine and Arginine auxotrophic mutant S.

asterosporus DSM 41452::pSET152::pLERE-Inlys::pKGLP2-InArg which was designated as S.

asterosporus SILAC1. In addition, plasmids pLERE-Inlys and pKGLP2-InArg were introduced

into S. asterosporus DSM 41452::pSET152-adpAgh(TTA) to construct its lysine and Arginine

auxotrophic mutant. By transformation and conjugation, those two plasmids were

homologous recombined onto the genome of S. asterosporus DSM 41452::pSET152-

adpAgh(TTA) to yield mutant S. asterosporus DSM 41452::pSET152-adpAgh(TTA)::pLERE-

Inlys::pKGLP2-InArg which was designated as S. asterosporus SILAC2.

The correct exconjugants were screened on solid MS medium supplemented with

spectinomycin-resistance (100 ug/ml) and hygromycin-resistance (100 ug/mL) selective

antibiotics. Furthermore, the exconjugants were tested and verified for target gene disruption

by colony PCR using primer pairs InlysF/aadVF and InArgF/HygR.

4.2.7 Bacterial culture for SILAC test

Strain cultivation were carried out on the 20 mL minimal medium MM1, all media were

supplemented with excessive amount of proline (1M, 400 uL/20 mL), 60ul arginine [L-arginine-

97

HCl in PBS solution, stock concentration 84g/L] and 34ul lysine [lysine-HCl in PBS solution,

stock concentration, 146g/L]. Their final concentration is 20 mg/L proline, 25 mg/L L-arginine

(Arg-0) and 25 mg/L L-lysine (Lys-0) for light labeled (L) medium; 20 mg/L proline, 25 mg/L L-

[13C6,15N4] arginine (Arg-10) and 25 mg/L L-[13C6,15N2] lysine (Lys-8) for heavy labeled (H)

medium.

200ul seed culture of strain S. asterosporus SILAC1 and strain S. asterosporus SILAC2 were

inoculated into 20mL MM1 liquid medium by 1: 1000 inoculation ratio, respectively. After 24

h culture, strain S. asterosporus SILAC1 was individually inoculated into fresh MM1 liquid

medium supplemented with heavy labelled Arginine and lysine. By contrast, strain S.

asterosporus SILAC2 was cultured in the MM1 liquid medium supplemented with light labeled

arginine and lysine. In the second-time biological replicates, the labels of those two strains

were reversed. Strain S. asterosporus SILAC2 was cultured in the heavy labeled (Arg-10, Lys-8)

MM1 medium, and S. asterosporus SILAC1 was cultured in the light labeled (Arg-0, Lys-0)

medium. All of the strains were incubated in at 28°C, 180rpm for 72 hours. Afterwards the

strains were harvested by centrifugation, the cell pellets were collected for protein sample

preparation.

4.2.8 Protein sample preparation for LC-MS/MS analysis

The harvested cells were resuspended in lysis buffer supplemented with protease inhibitor.

Then it was lysed by sonication, the cell debris was removed after a centrifugation at 14,000g

for 30 min. The resulting supernatant was collected for proteomics analysis. The protein

concentration was measured by Nanodrop (Thermo Scientific). The ratio of total protein from

parental strain and AdpA overexpression mutant were kept at the moderately equal amount.

Each 100 µg of labelled bacterial proteins was mixed into one sample, of which the volumes

were calculated depending on the proteins concentration. Protein mixtures were

supplemented with 6 x Laemmli sample buffer and 1mM dithiothreitol (DTT), subsequently

those mixtures were incubated at 97°C for 10 min. When the mixtures cooled down to room

temperature, iodoaceramide (IAA) was added in with a final concentration of 5.5 mM. Then

the mixtures were kept in dark at room temperature for 30 min. After that, the sample

mixtures were loaded into the SDS-PAGE (4-12% Bis-Tris mini gradient gel, Bio-Rad) lane.

After SDS-PAGE, the gel was stained with Coomassie blue, and each gel lanes were cut into 10

slices with equal size. In-gel digestion (Shevchenko et al. 2006) method was employed, each

gel slice was separately cut into 1-2mm2 pieces and then suspended in 150ul ABC buffer. All

fractions were incubated at room temperature for 10min under vigorous shaking. Then the

98

gel pieces were washed 3 times by ABC buffer and ethanol, alternately.

After that, 70 µl of trypsin solution was added into the Eppendorf tube to submerge the gel

pieces totally, the samples were then incubated overnight at 37˚C. 50 µl of 2% TFA was added

into the trypsin digested gel pieces to quench the reaction.

150 µl ethanol were added to the samples, which were then incubated at room temperature

for 10min. After centrifugation, supernatants containing the peptide were transferred to a

new microcentrifuge tube and peptides were concentrated to 50 µl by SpeedVec (Savant

SPD131DDA, Thermo Scientific), following dilution with 50ul Buffer A* and 150ul buffer A, and

then the samples were further processed by stage tips (Dumit et al. 2014). The columns of the

stage tips were washed by 100 µl buffer A twice in advance, then stage tips were loaded with

the peptide sample, washed with buffer A and eluted by 50 µl buffer B with a centrifugation

at 4000 rpm for 3 min. The peptide samples were concentrated by SpeedVec and dissolved

with 15 µl buffer A*/A. Then the resulting peptide mixtures are ready for the measurement

by HPLC-MS spectrometer.

4.2.9 Mass Spectrometry Measurement

Proteomics measurement were performed at the Center for Biological Systems Analysis (CF

Proteomics) in Freiburg University. Tryptic peptides were analyzed using an Agilent 1200

nanoflow-HPLC (Agilent Technologies GmbH, Waldbronn, Germany) combined with a LTQ

Orbitrap XL mass spectrometer (ThermoFisher Scientific, Bremen, Germany). 7 uL of volume

samples were loaded into the HPLC-column tips (fused silica, 75 m inner diameter, 20 cm of

length) which were self-packed with Reprosil-Pur 120 C18-AQ 3µm resin (Dr. Maisch GmbH,

Ammerbuch, Germany). Peptide samples were separated using a linear gradient method

(from 10% to 30% buffer B, flow rate of 250 nl/min).

The parameter of mass spectrometer was set as the described in paper (Dumit et al. 2014).

The mass spectrometer worked in a data-dependent acquisition mode to automatically

measure MS (max. of 1x10 ions) and MS/MS scan. Each MS scan was followed by a maximum

of five MS/MS scans in the linear ion trap using normalized collision energy of 35% and a target

value of 5,000. The data are acquired by MS and MS/MS scan with a Orbitrap resolution of

60,000 at a range from 370 to 2000 m/z.

Parent ions with one charge states and unassigned charge states were excluded

from fragmentation for MS/MS scans. Other MS parameters were set as follow: no sheath and

auxiliary gas flow; spray voltage is 2.3 kV; ion transfer tube temperature is 125°C.

99

4.2.10 Protein Identification

The MS raw data files were calculated through MaxQuant software version 1.4.1.2 (Cox and

Mann 2008) which performs mass peak and SILAC-pair detection, generates peak lists

of peptides with mass error corrected and result of database searching. The Andromeda was

used as the database search engine (Cox et al. 2011) and has been integrated into MaxQuant.

Protein identification were performed by searching the raw data files against the

corresponding protein FASTA files of S. asterosporus DSM 41452 (accession number:

CP022310), S. coelicolor A3(2) (accession number: PRJNA242), and S. avermitilis (accession

number: PRJNA277389).

The SILAC labeling parameter was set as a multiplicity of two (Light: Arg-0 and Lys-0, Heavy:

Arg-10 and Lys-8). Three miss cleavages were acceptable, enzyme specificity was trypsin/P,

and the MS/MS tolerance was set to 0.5 Da. The average mass precision of identified peptides

was in general less than 1 ppm after recalibration.

Peptide lists were further used to identify and relatively quantify proteins by MaxQuant

with the following parameters: the false discovery rates (FDR) was

set to 0.01, maximum peptide posterior error probability (PEP) was set to 0.1, minimum

peptide length was set to 7, minimum number peptides for identification and quantitation

of proteins was set to two of which one must be unique, and identified proteins have

been re-quantified. The “match-between-run” option (2 min) was used (Dumit et al. 2014).

The software Perseus (version 1.4.0.8) was employed for the data analysis and visualization,

including the log2 transformation of the protein ratios, the generations of the histograms for

the change ratios of proteome, and the heatmap representations (Cox and Mann 2012). For

constructing heatmaps, the SILAC protein ratios were hierarchically clustered using Euclidian

Distance as matrix (log2-transformed and z-score normalized). To address the biological

significance of the proteins, Gene Ontology (GO) and terms

were retrieved and tested for enrichment compared to the remainder of the dataset base on

the default settings with a minimum significance of p<0.05.

4.3 Results and Discussion

4.3.1 Complementation of the functional adpA gene in S. asterosporus

DSM 41452

Our previous research has established that the AdpA gene is defective in S. asterosporus DSM

41452, and the dysfunction of AdpA is caused by the insertion of a transposon gene at the

100

promoter region. A gene complementation of native adpA gene with its promoter region

cloned from strain Streptomyces calvus restored the sporulation in S. asterosporus DSM 41452

(see chapter 3, section 3.3.4).

In AdpA regulon, bldA gene encodes the rare tRNA molecule (Leu-tRNAUUA) which is required

for the translation of the mRNA with UUA codon (Takano et al. 2003; Kalan et al. 2013). So as

to validate the function of bldA gene in strain S. asterosporus DSM 41452, two kinds of

exogenous adpAgh genes from Streptomyces ghanaensis (Makitrynskyy et al. 2013) were

introduced into the wildtype strain. Plasmid pSET152-adpAgh(CTG) containing adpAgh gene

without TTA codon and pSET152-adpAgh(TTA) containing adpAgh gene with TTA were

individually conjugated into S. asterosporus DSM 41452 to yield the corresponding mutant

strains S. asterosporus DSM 41452::pSET152-adpAgh(CTG) and S. asterosporus DSM

41452::pSET152-adpAgh(TTA). Subsequently, strain S. asterosporus DSM 41452::pSET152

accompanied by those two new mutant strains were spread on solid MS plates for the

morphological observation. The plates were incubated at 28 °C for 5-7 days.

Figure 4. 2. Effects of exogenous AdpA overexpression on the morphology of S. asterosporus DSM 41452. Note:

strains grown on the MS agar plate for 7 days.

As shown in Figure 4. 2, after 7 days incubation, the AdpA overexpression mutant strains S.

asterosporus DSM 41452::pSET152-adpAgh(CTG), S. asterosporus DSM 41452::pSET152-

adpAgh(TTA) showed clear aerial hyphae on the surface of the solid plate, while strain S.

asterosporus harboring the control plasmid pSET152 still displayed the bald phenotype(Figure

4.2). It is indicated that the bldA gene in S. asterosporus DSM 41452 is functional. In addition,

integrative vector pTESa-adpAsc carrying the adpA gene from S. asterosporus DSM 41452 and

plasmid pTESa-adpAgh harboring the adpA gene from S. ghanaensis were introduced into S.

asterosporus DSM 41452, respectively, which can trigger the sporulation as well (Figure 4.2).

101

Figure 4. 3. The secondary metabolite profiles of strains S. asterosporus DSM 41452::pTESa-adpAsc, S.

asterosporus DSM 41452::pTESa-adpAgh and S. asterosporus DSM 41452::pTESa; The peak marked by arrow

represents WS9326A.

To evaluate the influence of AdpA on the secondary metabolism of S. asterosporus DSM

41452, AdpA mutants S. asterosporus DSM 41452::pTESa-adpAsc and S. asterosporus DSM

41452::pTESa-adpAgh were cultivated in the SG production medium for 4 days, their

harvested strain broth was individually extracted with ethyl acetate for analysis by LC-MS

(Figure 4. 3). As control, S. asterosporus DSM 41452::pTESa with the empty vector was

cultivated and analyzed in the same method. As shown in figure 4.3, the HPLC chromatograms

of secondary metabolite of mutant strains with the exogenous adpA gene (adpAsc and

adpAgh) don’t show significant difference compared with the chromatogram of the parental

strains.

4.3.2 In silico analysis of AdpA in S. asterosporus DSM 41452

Protein AdpA in S. asterosporus DSM 41452 consists of 409 amino acids, which are identical

with the AdpA in S. calvus, in addition it exhibits 86% identity with its homolog in S.

griseus(Ohnishi et al. 2005), and 89% sequence identity with the AdpA in S. ghanaensis, which

have been proven to regulate the production of moenomycin directly in Streptomyces

ghanaensis (Makitrynskyy et al. 2013).

102

Figure 4. 4. Multiple protein sequence alignment of AdpA from S. asterosporus DSM 41452 and the homologous

proteins from S. calvus, S. ghanaensis, S. griseus, S. filamentosus, S. chattanoogensis, S. azureus, and S.

toyocaensis; Non-conserved residues are colored gray, highly conserved residues are labeled with color (Figure

generated by Mview); Sequence in the parentheses represent the conserved motif.

The protein sequence alignment of AdpA from S. asterosporus DSM 41452 and its homologs

from other strains revealed the presence of a highly conserved helix-turn-helix(HTH) DNA-

binding domain at its C-terminal portion and a ThiJ/PfpI/DJ-1-like dimerization domain at its

N-terminus (Figure 4.4). The main region of conservation between these aligned AdpA

proteins share more than 95% identity. Notably, the amino acids at the AraC/XylS-type DNA-

binding domain are the most conserved among AdpA homologs (mostly 100% identity). By

contrast, the ThiJ/PfpI/DJ-1-like domain of AdpA proteins are less conserved.

Our previous AdpA complementation experiment shown that the exogenous AdpA (S.

ghanaensis) own the ability of restoring the sporulation in S. asterosporus DSM 41452. It is

striking that the conserved residues of AdpA (S. asterosporus DSM 41452) and its homolog in

103

S. ghanaensis are highly identical (Figure 4. 4). Only three exceptions are Ser87, Glu96 and

Glu100 located at ThiJ/PfpI/DJ-1-like domain. These combined findings suggested that all

species of Streptomyces share the highly identical AdpA-binding consensus sequence, and

those sequence variations between AdpA (S. asterosporus DSM 41452) and AdpA (S.

ghanaensis) are not sufficient to make significant difference of protein function.

Furthermore, in order to predict probable gene targets influenced directly by AdpA, and reveal

its regulon in S. asterosporus DSM 41452, we carried out in silico analysis of the entire S.

asterosporus DSM 41452 genome through bioinformatic analysis by PREDetector (Hiard et al.

2007). The AdpA-binding consensus sequence 5'-TGGCSNGWWY-3' was adapted to probe the

possible binding region on the S. asterosporus DSM 41452 genome. The AdpA-binding sites

region were confined between positions -300 bp and + 60 bp with reference to the

transcriptional start point of the target genes (Yamazaki et al. 2004). The screening result

shown that there are at least 810 predicted AdpA-binding sites on the chromosome of S.

asterosporus DSM 41452. The low DNA-binding specificity of AdpA enables it bind to many

sites on the genome, which facilitate its regulation on many other genes.

4.3.3 Construction of Arginine and Lysine auxotrophic mutant of S.

asterosporus DSM 41452

Typical SILAC approach relies on the integration of non-radioactive labeled amino acid (lysine

and arginine) into proteins through metabolic labelling technique (Mann 2006). In our SILAC

labeling experiments, two differential types of protein labeling were performed: the natural

amino acids L-arginine (Arg-0) and L-lysine (Lys-0) were utilized for light labelled protein, the

labelled L-[13C6,15N4] arginine (Arg-10) and L-[13C6,15N2] lysine (Lys-8) were utilized for heavy

labelled protein. In the end, the total proteins were digested by trypsin to generate the

labelled peptide mixture.

In order to increase the labelling efficiency of the proteins for SILAC experiment, and minimize

the intracellular amino acid interconversion, we decided to inactivate the biosynthesis of

endogenous arginine and lysine in strains S. asterosporus DSM 41452::pSET152 and S.

asterosporus DSM 41452::pSET152-adpA(gh). For this propose, gene fig|1.39.peg.5620(RAST

ID) encoding argininosuccinate synthetase(EC.6.3.4.5) which belong to the arginine

biosynthesis pathway was chosen to be disrupted in both of those strains. The target gene

disruptions were performed via homologous recombination. For vector construction, an

internal gene fragment of gene fig|1.39.peg.5620 was amplified and cloned into suicide vector

104

pKGLP2 to generate the plasmid pKGLP2-InArg (Figure 4. 5A), detailed description shown at

section 4.2.6. In addition, gene fig|1.39.peg.1705(RAST ID) encoding DapB (EC 1.17.1.8)

homolog involved in the lysine biosynthesis DAP pathway was chosen to be disrupted in both

of those strains, a partial gene fragment of fig|1.39.peg.1705 was amplified and cloned into

suicide vector pLERE-spec to form the plasmid pLERE-Inlys (Figure 4. 6A). Subsequently,

through intergeneric conjugation, plasmids pKGLP2-InArg and pLERE-Inlys were integrated

into the bacterial genome of S. asterosporus DSM 41452::pSET152-adpA(gh) and S.

asterosporus DSM 41452::pSET152, resulting in the lysine and arginine deficient mutant

strains, S. asterosporus SILAC1 and S. asterosporus SILAC2.

Correct transconjugants with hygromycin, spectinomycin resistance and Gus sensitivity

(Myronovskyi et al. 2011) were selected on solid MS medium supplemented with selective

antibiotics. Verification of the resulting mutant strains of S. asterosporus SILAC1 and S.

asterosporus SILAC2 were achieved by PCR amplification of the inserted fragment, which

clearly showed that the accordingly plasmid has been integrated into the correct position of

the bacterial genome (Figure 4. 5B and Figure 4. 6B).

A B

Figure 4. 5. Inactivation of the arginine biosynthetic gene in strains S. asterosporus DSM 41452::pSET152 and S.

asterosporus DSM 41452::pSET152-adpAgh(TTA) by insertion of plasmid pKGLP2-InArg into the bacterial genome

via single crossover; (A) schematic representation of the plasmid pKGLP2-InArg; (B) Verification of the resulting

mutants, showing amplification of an PCR fragment with size of around 2.9kb (using the primers InArgF and

HygR) in mutant S. asterosporus SILAC1 (lanes 1) and S. asterosporus SILAC2 (lane 4) in comparison with the

parental strain (lane 2 and lane 5), lane 3 and 6 represent 1kb marker.

105

A B

Figure 4. 6. Inactivation of the Lysine biosynthetic gene in strains S. asterosporus DSM 41452::pSET152 and S.

asterosporus DSM 41452::pSET152-adpAgh(TTA) by inserting plasmid pLERE-Inlys into the bacterial genome via

single cross-over; (A) schematic representation of the pLERE-Inlys plasmid; (B) Verification of the resulting

mutants, showing amplification of an PCR fragment with size around 1.5kb (using the primers InlysF and aadVF)

in mutant S. asterosporus SILAC1 (lanes 2) and S. asterosporus SILAC2 (lane 5) in comparison with the parental

strain (lane 1 and lane 6), lane 3 and 4 represent 1kb marker.

To confirm the chromosome stability of the mutant and exclude out the possibility of losing

selection marker caused by the gene recombination afterward, the mutant strains firstly were

cultured in the medium supplemented with spectinomycin and hygromycin antibiotic for

selection. During its exponential growth period the mycelium culture was inoculated into new

fresh medium (by the ratio of 1:100) without antibiotics supplementation. During the process

of inoculation, the mycelium was collect and washed by the fresh medium without any

antibiotics, repeat three times. After several times generation cultivation in the relaxed

culture condition, the offspring strain was inoculated on the solid plate supplemented with

antibiotic again, the strain’s single colony showed clear antibiotic resistance against

spectinomycin and hygromycin. Moreover, we verified the presence of the insertional

antibiotic marker tag on their genome by PCR amplification.

After gene disruption of the lysine and arginine biosynthesis gene (fig|1.39.peg.1705 and

fig|1.39.peg.5620), the growth and developmental rate of the mutate strains (S. asterosporus

SILAC1 and S. asterosporus SILAC2) were significantly delayed (Figure 4.7). Theoretically, the

arginine and lysine defect strain doesn’t survive on the minimum medium. This phenomenon

could be explained by the presence of other alternative lysine and arginine biosynthesis

pathway or alternative homologous enzyme.

106

Figure 4. 7. Phenotypes of S. asterosporus DSM 41452 and its mutant on the minimal (MM1) media agar plates

with 7 days incubation at 28°C. Note of strains: S. asterosporus DSM :: pSET152-adpAgh (TTA) (A); S. asterosporus

DSM :: pSET152 (B); S. asterosporus SILAC1 (C); S. asterosporus SILAC2 (D).

4.3.4 Statistical analysis of proteomics data

A B

Figure 4. 8. (A) Scatter plot representing the correlation of two biological replicates measured by mass

spectrometry. (B) Histograms of log2 transformed protein intensities representing the distribution of proteome

differences of AdpA mutant strain compared to the WT strain in two biological replicates.

For detailed investigation of AdpA regulon in S. asterosporus DSM 41452, demonstrating the

underlying molecular mechanism causing the growth deficiency in those native adpA defect

strain, the relative transcriptional abundance values between the parental strain S.

asterosporus SILAC1 and its AdpA mutant S. asterosporus SILAC2 were compared.

Strain S. asterosporus SILAC1 were grown in MM1 medium including the “light” forms of lysine

and arginine, while the AdpA mutant S. asterosporus SILAC2 were grown in the “heavy” forms

of lysine and arginine. For a second biological replicate, the parental strain SILAC1 and AdpA

mutant SILAC2 were cultured in the media with reversely labeled arginine and lysine. Cell

pellets were harvested when bacterial growth reached the early stationary stage. Heavy-

labelled protein sample was mixed with corresponding light-labeled sample in 1:1 ratio based

on total protein concentration relative to the light labeled counterpart.

The relative changes at proteome level were determined by the calculation of the heavy/light

(H/L) ratios for each mutant strain using MaxQuant. Two independent biological replicates at

two strain developmental stages (3 days and 4 days) were processed.

The relative abundance of regulated proteins was estimated and normalized to log2. To

optimize the data acquisition, statistically significant abundance ratio of detected protein was

set at a p value <0.05. Protein expression difference with two-fold increase or decrease ((log2)

>1 or <-1) were employed as the threshold to highlight the protein being significantly affected.

The positive values correspond to the up-regulated proteins in AdpA mutant, and the negative

value corresponds to the downregulated proteins in the AdpA mutant.

107

The two biological replicates from 3 days culture exhibited high correlation with regression

coefficients of 0.48 (Figure 4.8A). The corresponding expression changes of proteomics

showed a clear Gaussian distribution, which further suggest that the protein changes behave

as expected and prove the reliability of the experimental data (Figure 4.8B). In contrast, the

data reproducibility between two biological replicates from 4 days culture was not good

enough. Therefore, the correlation coefficient R2 of those two biological replicates from 4 days

culture was too low to reliably characterize the proteome, and this phenomenon could be

caused by the cell autolysis happening in the organism.

108

4.3.5 Proteomic analysis of the effects of AdpA in S. asterosporus DSM

41452

Figure 4. 9. Heat map of detected proteins that were up- (red) and down- (green) regulated in the biological

replicates of the AdpA mutant relative to the parental strain.

Comprehensive protein expression in the AdpA mutant strain was compared to that in the

native AdpA-defect parental strain. Totally, there are more than 1200 proteins (17.6% of S.

asterosporus DSM 41452 proteome) were identified from the peptides MS/MS spectra in two

Exp-1 Exp-2

A

B

C

D

E

109

biological replicates. Among those proteins, the result of statistical analysis demonstrated

that there are 52 proteins identified as with significant abundance difference of their levels in

those two samples (sample from parental strain as control and sample from AdpA mutant

strain) (Figure 4.9 and Table 4.4), furthermore, those proteins were detected in both biological

replicates.

These significantly expressed proteins were classified base on their functions annotated by

COGs database using protein BLAST with an expectation value threshold 0.001. The main COG

groups include “Information storage and processing”, “Cellular process and signaling”,

“Metabolism”, “General function prediction only” and “Function unknown” (Table 4.4).

Among those significantly regulated proteins, 31 proteins were up-regulated in the AdpA

mutant (ratio showing protein level deference higher than 1).

Not surprisingly, in the mutant protein AdpA was upregulated more than 16-fold, suggesting

that remarkable difference of protein expression indeed present in the AdpA mutant and the

parental strain.

A group of sporulation-related proteins were detected as up-regulated protein in the AdpA

mutant strain. Particularly, fig|1.39.peg.515, a homologue of sporulation-control protein

Spo0M, was upregulated up to 16-fold approximately. It shows 100% sequence identity with

SGR5704 in S. griseus. SGR5704 is an orthologue of the well-studied sporulation control

protein Spo0M in Bacillus subtilis (Birkó et al. 2009). Another protein fig|1.39.peg.408 was

upregulated 2 times, it is a homolog of stage II sporulation protein anti-sigma-B factor

antagonist, and shows 87% sequence identity with SCO7325 in S. coelicolor A3(2).

Among those significantly upregulated proteins, fig|1.39.peg.216 was predicted to be

involved in information storage and processing (Table 4.4). It was upregulated 16-fold in the

AdpA mutant. A sequence similarity search shown that fig|1.39.peg.216 is an ortholog of

protein SGR_3492 (75% identity) from S. griseus. In addition, it shows 40% identity with

SCO7465 in S. coelicolor A3(2), in which SCO7465 was predicted to encode a multi-component

regulatory system involved in the resistance to oxidative stress of bacteria.

The expression of 3 proteins related to “cellular processing and signaling” were up-regulated

by AdpA, including fig|1.39.peg.6192, fig|1.39.peg.1288, and fig|1.39.peg.2166.

fig|1.39.peg.6192, a putative DnaK suppressor protein, was upregulated by 8-fold in AdpA

mutant, which show 69% identity with SCO6164 from S. coelicolor A3(2). fig|1.39.peg.2166

was upregulated by 8-fold in the mutant. It is a cAMP-binding protein, and shows 81% identity

with eshA of S. avermitilis, 74% identity with SGR_2264 in S. griseus, and 88% identity with

SCO5249 of S. coelicolor A3(2). Protein fig|1.39.peg.1288 with unidentified function was

110

upregulated by 9-fold in the mutant strain.

It is also worth noting that nine of metabolism-associated proteins were significantly

upregulated in the AdpA mutant, including fig|1.39.peg.218, fig|1.39.peg.4800,

fig|1.39.peg.253, fig|1.39.peg.4326, etc.

Protein fig|1.39.peg.218 was upregulated by 8-fold in the mutant strain, it is a putative

cytochrome P450 protein which shows 84% identity with SGR_4392 of S. griseus, and 85%

identity with SCO0584 of S. coelicolor A3(2). fig|1.39.peg.4800, a putative cytochrome P450,

was upregulated by 8-fold in the mutant and show 82% identity with SCO0584 in S. coelicolor

A3(2). fig|1.39.peg.253, a non-ribosomal peptide synthetase, was upregulated by 8-fold in the

mutant strain and show 43% identity with SGR_3265 from S. griseus, 44% identity with

SCO3230 (CDA peptide synthetase I) in S. coelicolor A3(2). fig|1.39.peg.4326 was upregulated

by 4-fold in the mutant, which encodes a putative asparagine synthetase and shows 27%

identity with SGR_3903 from S. griseus, 28% identity with SCO4115 of S. coelicolor A3(2).

Protein fig|1.39.peg.5158 was upregulated by 4-fold in the mutant, it is a putative

methyltransferase and shows 62% identity with SGR_5540 from S. griseus, 84% identity with

SCO1993 of S. coelicolor A3(2). fig|1.39.peg.1991, a putative putative glycogen

phosphorylase, was upregulated by 4-fold in the mutant. It shows 76% identity with glgP from

S. griseus, 87% identity with SCO5444 of S. coelicolor A3(2). fig|1.39.peg.4616 was

upregulated by 8-fold in the mutant, it is a putative ferredoxin reductase and show 88%

identity with fprD of S. avermitilis, 79% identity with SGR_5065 from S. griseus, 84% identity

with SCO2469 of S. coelicolor A3(2). fig|1.39.peg.6254, a Argininosuccinate lyase (EC 4.3.2.1),

was upregulated by 8-fold in the mutant. It shows 84% identity with SCO0993 of S. coelicolor

A3(2). fig|1.39.peg.2374 was upregulated by 8-fold in the mutant, it is a putative α-1,2-

mannosidase and shows 40% identity with SGR_1503 of S. griseus, 46% identity with SCO6004

of S. coelicolor.

In the AdpA mutant, four significantly up-regulated proteins are categorized in the group of

“General function prediction only”, including fig|1.39.peg.5560, fig|1.39.peg.6013,

fig|1.39.peg.6441, and fig|1.39.peg.217. Protein fig|1.39.peg.5560 was upregulated by 8-fold

in the mutant, it belongs to a multi-component regulatory system-10 which contains a

roadblock/LC7 domain, which shows 68% identity with rarB of S. griseus and 79% identity with

SCO1629 of S. coelicolor A3(2). fig|1.39.peg.217, a putative ATP/GTP-binding protein, was

upregulated by 8-fold in the mutant, which shows 74% identity with SGR_2267 of S. griseus

and 83% identity with SCO5247 of S. coelicolor A3(2). fig|1.39.peg.6013 and fig|1.39.peg.6441

both were upregulated by 8-fold in the mutant strain, their functions are unknown.

111

Additionally, there are 12 unclassified and unidentified proteins were significantly regulated

in AdpA mutant (Table 4.4).

In addition to the significantly upregulated protein, there are 18 downregulated proteins

showing significant abundance difference (log2 value lower than -1) in comparison of AdpA

mutant with the parental strain (Table 4.4). Of them, fig|1.39.peg.2698 is the only one

grouped into “information storage and processing”, which was downregulated by 3-fold in the

AdpA mutant. It encodes a putative alanine acetyltransferase, and shows 64% identity with

SCO4311 in S. coelicolor A3(2).

Among those downregulated, ten proteins were predicted to be involved in the “metabolism”.

Of them, fig|1.39.peg.1839 is a putative nitrogen regulatory protein P-II. It was

downregulated by 8-fold in the AdpA mutant, and its homolog in S. avermitilis is glnB1 (99%

sequence identity) related with the regulation of nitrogen assimilation and metabolism. In

addition, its homologue in S. coelicolor A3(2) is SCO5584 (99% sequence identity) which

belongs to the GlnR regulon and plays a crucial role in the regulation of nitrogen metabolism

(Lewis, Shahi et al. 2011). Another protein fig|1.39.peg.4933 is glutamine synthetase (100%

identity with glnA2 in S. avermitilis) was downregulated by 4-fold in the AdpA mutant, it was

also predicted to belong to the GlnR regulon.

There are seven down-regulated proteins classified into “amino acid transport and

metabolism”. It is interesting to find that four proteins are all involved in the biosynthesis of

arginine, including fig|1.39.peg.5611 (argC homolog, 94% identity), fig|1.39.peg.5614 (argD

homolog, 88% identity), fig|1.39.peg.5613 (argB homolog, 96% identity), and

fig|1.39.peg.1402 (argF homolog, 89% identity). They are shown to be less abundant in the

AdpA mutant than in the parental strain. In addition, fig|1.39.peg.5710, a carbamoyl

phosphate synthase, was downregulated by 4-fold, it shows 92% sequence identity with carA

of S. coelicolor A3(2).

In term of the biosynthesis ability of AdpA mutant, it is interesting to observe the decreased

expression abundance of three “secondary metabolites biosynthesis” related proteins.

fig|1.39.peg.594, a 3-oxoacyl-ACP reductase involved into the fatty acid biosynthesis, was

downregulated by 8 times. It shows 100% sequence identity with SCO1346 in S. coelicolor

A3(2). fig|1.39.peg.6652 showing 43% identity with dioxygenase SCO7507 in S. coelicolor

A3(2) was deregulated by 8-fold. fig|1.39.peg.5707, a putative aspartate

carbamoyltransferase, was downregulated by 4-fold in the AdpA mutant strain. It shows 93%

identity with pyrB in S. avermitilis which is involved into the fructose and mannose

metabolism. In addition, 7 down-regulated proteins in the AdpA overexpression mutant were

112

classified within the group of general function prediction only (Table 4. 1).

Table 4. 4. Proteins up- and downregulated in S. asterosporus AdpA mutant

Protein RAST ID SCO1

Orthologue

(Identity%)

SGR2

Orthologue

(Identity%)

Putative Function Fold Change3

(Log2-

transformed)

COG

Category4

fig|1.39.peg.216 SCO7465

(49)

SGR_3492

(75)

multi-component

regulatory system-2

3.72 I

fig|1.39.peg.4286 SCO2792

(88)

adpA (85) Transcriptional

regulator, AraC family

3.94 I

fig|1.39.peg.6192 SCO6164

(69)

- putative DnaK

suppressor protein

3.44 T

fig|1.39.peg.1288 - - TPR domain protein,

putative component of

TonB system

2.69 T

fig|1.39.peg.408 SCO3029

(90)

lldP (89) anti-sigma F factor

antagonist (spoIIAA-2)

1.17 T

fig|1.39.peg.2166 SCO5249

(88)

SGR_2264

(74)

cAMP-binding proteins -

catabolite gene

activator and regulatory

subunit of cAMP-

dependent protein

kinases

3.51 T

fig|1.39.peg.218 SCO6310

(51)

SGR_3494

(69)

hypothetical protein 2.80 M

fig|1.39.peg.4800 SCO0584

(85)

SGR_4392

(84)

putative cytochrome

P450

3.05 M

fig|1.39.peg.253 SCO3230

(44)

SGR_3265

(43)

Siderophore

biosynthesis non-

ribosomal peptide

synthetase modules

3.33 M

fig|1.39.peg.4326 SCO4115

(28)

SGR_3903

(27)

Asparagine synthetase

[glutamine-hydrolyzing]

(EC 6.3.5.4)

2.48 M

fig|1.39.peg.5158 SCO1993

(84)

SGR_5540

(62)

hypothetical protein 2.26 M

fig|1.39.peg.1991 SCO5444

(87)

glgP (76) Glycogen

phosphorylase (EC

2.4.1.1)

2.44 M

fig|1.39.peg.4616 SCO2469

(84)

SGR_5065

(79)

Ferredoxin reductase 2.92 M

fig|1.39.peg.6254 SCO0993

(84)

SGR_6230

(56)

Argininosuccinate lyase

(EC 4.3.2.1)

3.62 M

113

fig|1.39.peg.2374 SCO6004

(46)

SGR_1503

(40)

Alpha-1,2-mannosidase 2.91 M

fig|1.39.peg.5560 SCO1629

(79)

rarB (68) multi-component

regulatory system-10,

containing

roadblock/LC7 domain

3.23 G

fig|1.39.peg.6013 SCO1155

(93)

SGR_335

(78)

hypothetical protein 3.32 G

fig|1.39.peg.6441 SCO0587

(39)

rarB (45) hypothetical protein 3.37 G

fig|1.39.peg.217 SCO5247

(83)

SGR_2267

(74)

Putative ATP/GTP-

binding protein

2.79 G

fig|1.39.peg.515 SCO2001

(69)

SGR_5529

(55)

Sporulation control

protein Spo0M

3.72 G

fig|1.39.peg.215 SCO7464

(55)

SGR_3491

(73)

FIG01127630:

hypothetical protein

2.63 G

fig|1.39.peg.3867 SCO0525

(58)

SGR_6103

(54)

hypothetical protein 2.82 G

fig|1.39.peg.1820 SCO5605

(82)

- hypothetical protein 3.49 G

fig|1.39.peg.2066 SCO5389

(95)

SGR_2148

(92)

hypothetical protein 2.53 G

fig|1.39.peg.4783 - - hypothetical protein 3.59 G

fig|1.39.peg.1719 SCO5725

(87)

SGR_1787

(34)

hypothetical protein 2.68 G

fig|1.39.peg.1406 SCO5275

(31)

SGR_5768

(36)

hypothetical protein 3.13 G

fig|1.39.peg.4785 - - hypothetical protein 2.89 G

fig|1.39.peg.6290 - - hypothetical protein 3.57 G

fig|1.39.peg.2936 SCO4051

(75)

- hypothetical protein of

Cupin superfamily

3.08 G

fig|1.39.peg.2929 SCO4070

(80)

SGR_3861

(78)

hypothetical protein

SCD25.06

2.42 G

fig|1.39.peg.911 SCO6493

(75)

SGR_1147

(71)

Lactoylglutathione lyase

and related lyases

1.75 G

fig|1.39.peg.8 SCO6616

(38)

SGR_624

(38)

putative secreted

protein

3.97 G

fig|1.39.peg.2698 SCO4311

(64)

SGR_5631

(42)

Ribosomal-protein-

alanine

acetyltransferase (EC

2.3.1.128)

-1.76 I

114

fig|1.39.peg.1838 GlnD (98) glnD (98) [Protein-PII]

uridylyltransferase (EC

2.7.7.59)

-2.79 T

fig|1.39.peg.1839 SCO5584

(99)

SGR_1894

(98)

Nitrogen regulatory

protein P-II

-3.35 M

fig|1.39.peg.5611 argC (94) SGR_5960

(94)

N-acetyl-gamma-

glutamyl-phosphate

reductase (EC 1.2.1.38)

-3.84 M

fig|1.39.peg.5614 argD (88) argD (87) Acetylornithine

aminotransferase (EC

2.6.1.11)

-4.25 M

fig|1.39.peg.5613 argB (96) argB (95) Acetylglutamate kinase

(EC 2.7.2.8)

-3.53 M

fig|1.39.peg.1402 argF (89) arcB (88) Ornithine

carbamoyltransferase

(EC 2.1.3.3)

-2.81 M

fig|1.39.peg.4933 SCO2210

(92)

SGR_5302

(38)

Glutamine synthetase

type II, eukaryotic (EC

6.3.1.2)

-1.79 M

fig|1.39.peg.5710 CarA (92) CarA (90) Carbamoyl-phosphate

synthase small chain (EC

6.3.5.5)

-1.60 M

fig|1.39.peg.594 SCO1346

(37)

SGR_4223

(36)

short chain

dehydrogenase/reducta

se family

-2.93 M

fig|1.39.peg.6652 SCO7507

(43)

SGR_2991

(40)

Alpha-ketoglutarate-

dependent taurine

dioxygenase (EC

1.14.11.17)

-3.53 M

fig|1.39.peg.5707 pyrB (93) pyrB (94) Aspartate

carbamoyltransferase

(EC 2.1.3.2)

-1.97 M

fig|1.39.peg.5934 SCO1250

(82)

SGR_6276

(76)

hypothetical protein -2.79 G

fig|1.39.peg.6428 SCO0795

(80)

SGR_265

(64)

conserved hypothetical

protein SCF43.06

-3.58 G

fig|1.39.peg.1565 SCO1159

(37)

SGR_6127

(31)

hypothetical protein -2.46 G

fig|1.39.peg.2806 SCO4186

(77)

SGR_3976

(63)

Enhanced intracellular

survival protein

-2.16 G

fig|1.39.peg.4728 SCO2346

(88)

SGR_5163

(71)

hydrolase -2.41 G

115

fig|1.39.peg.5633 SCO1560

(88)

SGR_5163

(45)

Putative phosphatase

YfbT

-2.40 G

fig|1.39.peg.1409 SCO5973

(88)

SGR_1574

(82)

Serine/threonine

protein phosphatase (EC

3.1.3.16)

-1.73 G

1SCO refers to Streptomyces coelicolor A3(2); 2SGR refers to Streptomyces griseus; 3Average fold change was

calculated based on the gene expression difference in S. asterosporus DSM 41452 adpA mutant relative to the

parental strain in biological replicate experiments; the protein ratios were hierarchically clustered using Euclidian

Distance as matrix (log2-transformed and z-score normalized); 4COG category: I represents proteins involved in

information storage and processing; T represents proteins involved in cellular processing and signaling; M

represents proteins involved in metabolism; G represents proteins involved in general function prediction only and

proteins with unknown function;

4.4 Conclusion and Outlook

S. asterosporus DSM 41452 is a potential industrial producer of WS9326A, Annimycin, and

antibiotic nucleocidin, which owns a huge research and development value. It is non-

sporulated during its natural growth. In chapter 3, we have illustrated that the baldness

phenotype of this strain was caused by the presence of a defect native adpA gene, which is

disrupted by a transposon. Upon complementation with a functional adpA gene (either adpA

from S. calvus or from S. ghanaensis), the mutant strain restored the sporulation immediately,

but without any secondary metabolites change. It is apparent that AdpA expression is tightly

connected with the morphological differentiation. In addition, through introduction of

exogenous adpAgh (with and without TTA codon by an integrative plasmid pSET152) genes

from S. ghanaensis, the mutant strains always keep the ability of sporulation, suggesting that

the native bldA gene in S. asterospours DSM 41452 is functional.

In silico bioinformatic analysis results display that the high sequence identity of gene adpA in

different Streptomyces. Moreover, multiple sequence alignments show that AdpAsa in S.

asterosporus DSM 41452 recognize and bind the same sequence as AdpAgh in S. ghanaensis,

because they share completely conserved the amino acid sequences forming the DNA-binding

domain.

To the best of our knowledge, it’s the first report about the characterization of the AdpA

regulon in a native non-sporulation Streptomyces, moreover, it’s first comparative proteomic

analysis base on SILAC method to reveal the gene network under the regulation of AdpA, the

proteomes of the parental strain and the AdpA mutant were relatively quantified and

compared. More than 1200 proteins were detected by mass spectrometry in two biological

116

replicates. The comparative proteomics analysis between the WT and the AdpA mutant

revealed that AdpA significantly affect the expression of 52 proteins (P-value < 0.05). Among

them, 33 proteins were upregulated and 19 proteins were downregulated. Of those

upregulated proteins, including the proteins involved in sporulation in Streptomyces, such as

the homolog of Spo0M fig|1.39.peg.515 and fig|1.39.peg.408, a homolog of the stage II

sporulation protein anti sigma-B factor antagonist. Furthermore, in silico analysis suggests

that the present of the AdpA binding site upstream of gene fig|1.39.peg.515, thus this gene

was predicted to be AdpA-dependent genes whose expression is directly activated by AdpA.

Among those significantly down-regulated proteins, of particular interest is a nitrogen

regulatory protein fig|1.39.peg.1839 and proteins fig|1.39.peg.4933, fig|1.39.peg.5611,

fig|1.39.peg.5614, fig|1.39.peg.1402 and fig|1.39.peg.5613, which exhibit strongly

relationship with nitrogen metabolism in Streptomyces. These results suggest that the AdpA

in S. asterosporus DSM 41452 could exert its influence on the morphological differentiation

and primary nitrogen metabolism in a specific manner.

Taken all together, our results clearly demonstrated that AdpA is not essential for the normal

growth and secondary metabolism of Streptomyces species, however it highly influence the

morphological development. We expect that this preliminary research will be contributed to

enlighten and motivate further study on the AdpA regulon in Streptomyces. More

importantly, we validated that SILAC proteomics method is a viable and efficient when applied

in Streptomyces system, and it is much more precise and highly reproducible alternative in

comparison with the conventional methods. The intracellular proteins were not completely

labelled with heavy arginine and lysine even when the culture was started using a very small

quantity of biomass(spores) in the labeled medium, which was most likely due to the synthesis

of the arginine by S. asterosporus DSM 41452 through endogenous mechanisms.

Further systematic researches on those proteins in this AdpA network are necessary, and it

might provide a sight to profile the detailed regulatory mechanism in Streptomyces. The

follow-up study will mainly focus on those highly regulated proteins by AdpA in S. asterosporus

DSM 41452. Transcriptional analysis will be applied to unveil whether those detected proteins

in AdpA regulon are constitutively expressed or not in the SG productive medium for the

native non-sporulating strain, to further validate the feasibility of the SILAC proteomics

method in Streptomyces.

117

Chapter 5. Research on the secondary metabolites of

Streptomyces asterosporus DSM 41452 and the biosynthesis of

WS9326As

5.1 Background

Nonribosomal peptide synthetases (NRPSs) represents one of the most important classes of

enzymes involved in natural product biosynthesis (Sussmuth and Mainz 2017). Many

pharmaceutically important antibiotics are biosynthesized by NRPSs (Winn et al. 2016).

Important NRPS derived antibiotics have been discovered from various kinds of

microorganisms (Finking and Marahiel 2004), including vancomycin produced by

Amycolatopsis orientalis (Barna and Williams 1984; Woithe et al. 2007), and daptomycin

produced by Streptomyces roseosporus (Baltz et al. 2005). NRPS natural products display a

wide range of chemical modifications that deviate from standard peptides, including

halogenation, hydroxylation, N-methylation, α/β-dehydrogenation (Süssmuth and Mainz

2017).

The cyclodepsipeptide WS9326A is notable for its unusual spectrum of bioactivities and its

production by a number of Streptomyces strains. The molecule was first isolated from

Streptomyces violaceusniger sp. 9326 by researchers at Fujisawa Pharmaceutical Co. on the

basis of its novel activity as a tachykinin receptor agonist (Hayashi et al. 1992; Shigematsu et

al. 1993). A total chemical synthesis confirmed the structure of WS9326A and probed the

relationship of the N-acyl group to bioactivity (Shigematsu et al. 1997). Subsequently,

WS9326A and a series of congeners (notably WS9326D) were isolated from Streptomyces sp.

9078 based on inhibition of an asparaginyl-tRNA synthetase from the filarial nematode

parasite Brugia malayl (Yu et al. 2012). More recently, WS9326A was found to be a

transcriptional inhibitor of pfoA gene regulated by the VirSR two-component system in

Clostridium perfringens, while WS9326B was observed to reduce the toxicity of Staphylococcus

aureus to human corneal epithelial cells (Desouky et al. 2015).

WS9326A exhibits an interesting chemical structure. The core NRPS backbone structure

consists of four standard amino acids (L-Thr, L-Leu, L-Asn, L-Ser) and three non-proteinogenic

amino acids (E-2,3-dehydrotyrosine, D-Phe, and L-allo-threonine). The E-2,3-dehydrotyrosine

(ΔTyr) residue is notable for not having been observed previously in NRPS derived peptides.

Amino acid residues with α,β-dehydrogenation have been observed in many other important

118

NRPS compound like Telomycin from Streptomyces canus ATCC 12646 (Fu et al. 2015), calcium-

dependent antibiotic (CDA), from Streptomyces coelicolor A3(2) (Hojati et al. 2002), and

Splenocin from Streptomyces sp. CNQ431 (Chang et al. 2015), along with different mechanisms

to form the α, β-alkene. The enzymatic mechanism to form the E-2,3-dehydrotyrosine in

WS9326A is still unknown.

Another prominent feature of WS9326A is the N-acyl group, consisting of a N-terminal

cinnamoyl moiety are also found in depsipeptide Skyllamycin (Pohle et al. 2011),

pepticinnamins (Omura et al. 1993), and the dimeric peptides Mohangamide A and B(Bae, Kim

et al. 2015). Structure and bioactivity relationships have shown that the Z-pentenyl-cinnamoyl

moiety is essential for activity (Shigematsu et al. 1997). Previous biosynthetic studies indicated

that this polyketide chain may arise from a complex biosynthetic pathway (Pohle et al. 2011),

but the exact mechanism has not been deciphered to date.

We were inspired to study S. asterosporus DSM 41452 in detail upon observing a surprising

biosynthetic kinship with Streptomyces calvus ATCC13382. The genome sequence of S.

asterosporus DSM 41452 revealed gene clusters with very high sequence identity to the

WS9326A (Johnston et al. 2015) and Annimycin clusters (Kalan et al. 2013) found in S. calvus.

Indeed, analysis of culture extracts of S. asterosporus DSM 41452 revealed the presence of a

NRPS compound WS9326A, two new WS9326A derivatives have been isolated from the

culture broth of this strain, the gene cluster of WS9326A was characterized through

bioinformatic analysis and genetic mutagenesis. It has also been observed that the production

of WS9326A in S. calvus is inversely correlated to the production of Annimycin (Kalan et al.

2013). Therefore, we decided to disrupt the Annimycin gene cluster in S. asterosporus DSM

41452. While titer of WS9326A in the resulting S. asterosporus mutant (S. asterosporus DSM

41452::pUC19Δ3100spec) was slightly improved. Moreover, we were surprised to observe the

production of two new WS9326A derivatives.

5.2 Materials and Methods

5.2.1 Primers fragments used in this study

Table 5. 1. Primers fragments used in this study

Name Sequence

Primer for vector construction of pKC1132-InAnn3

InAnn3-F CCGCTGAACGTCATGTCGACCGC

InAnn3-R CGTTCGGACCGCGCACCACGA

Primer for vector construction of pKC1132-orf(-1)

119

Orf(-1)-F GTCGGTGACGCCGTCGCCC

Orf(-1)-R GAGATGTGCAGCTTCTCCGCGATG

Primer for vector construction of pKC1132-SAS1

LuxR2-F CGCCATGACGCGTCCACCGTG

LuxR2-R GGTGCGGACGCTGCATCCCAGAC

Primer for vector construction of pKC1132-SAS16

P450S-F ATATaagcttGCGCCGCAGGGAACTGCTCGAC

P450S-R ATATggatccTGGTGGACGCCGTAGCCGAAC

Primer for vector construction of pKC1132-SAS13

SAS13F ATATaagcttCCGTGACGCGGCTTCGTGCT

SAS13R ATATtctagaGCACCTCGCCGAAGAAGCGG

PCR verification for gene ann3 disruption

Vann3-F GTACTTCGGGGTGCTGGCGGCC

Apra-R AGCTCAGCCAATCGACTGGCGAG

PCR verification for gene orf3100 disruption

V3100-F TGGTCTCCGCGGGCAGGAGCCAGAA

Vaad-R GTCGATCGTGGCTGGCTCGAAGATAC

PCR verification for gene orf(-1) disruption

VluxR1-F ACGGAGATCCGGGTGAGCCTGCG

Apra-R AGCTCAGCCAATCGACTGGCGAG

PCR verification for gene sas1 disruption

VluxR2-F AATCCGCCGACGGAGGAGGACACC

Apra-R AGCTCAGCCAATCGACTGGCGAG

PCR verification for gene sas16 disruption

Vsas16-F AGTCATGGGTCTGTCCACTCCAGTA

Apra-R AGCTCAGCCAATCGACTGGCGAG

PCR verification for gene sas13 disruption

Vsas13-F CGCACTCCGGGGACGTGGTGACC

Apra-R AGCTCAGCCAATCGACTGGCGAG

Primers for in-frame deletion of sas16 by λ Red-mediated recombination

SAS16F ATATaagctcCCGACTACACCGGCATCCTC 3'

SAS16R ATATgaattcGCTCGTCGTCCACCGTGTC 3'

SAS16-ApraF CGCAAGAAATGACCTCAGCTCAGATATAGGGGTAACGTCATGGATATCTCTAGATACCG

SAS16-ApraR GATGCAGCGGTCGCGTTCGGCGTGAACCTTCATGGCGCCTAAACAAAAGCTGGAGCTC

Primers for in-frame deletion of gene coding the N-methyltransferase domain in SAS17 by λ Red-mediated

recombination

Nmet4800bpF GACCTGGTCGGCTTCCTCGT

Nmet4800bpR GTCGGCGCGGTAGGTGAAG

Nmet-ApraF GAGAACTTCGCCGGCTGGCACAGCAGTTACGACGGCTCGGTGGATATCTCTAGATACCG

Nmet-ApraR CAGGCCAGGACGGGCACCGAGGCGAGGGAGACGGCTTCCGCAACAAAAGCTGGAGCTC

Primer for vector construction of pET28a-Nmet and pET24-Nmet

NmetF ATAcatatgGTGCTGCCCGAGGAGGAGATGC

120

5.2.2 Plasmid information

Table 5. 2. Plasmid information

NmetR ATActcgagCGCCGGGCGCTTGTGCA

Primer for vector construction of pET-Trx-Nmet

pET-Trx- NmetF ccatggTGCTGCCCGAGGAGGAGATGC

pET-Trx- NmetR ctcgagCGCCGGGCGCTTGTGCA

Name Description Reference

pIJ790 λ-Red plasmid, temperature sensitive replicon, Cmr (Gust et al. 2003)

pBluescript SK(-) Cloning vector, lacZ’(α-complementation), Ampr Stratagene

pKC1132 Conjugative vector, Non-replicative in Streptomyces, Aprar (Bierman et al.

1992)

pKC1132-orf(-1) Vector for gene disruption of orf(-1), based on pKC1132, Aprar This study

pKC1132-SAS1 Vector for gene disruption of sas1, based on pKC1132, Aprar This study

pKC1132-SAS16 Vector for gene disruption of sas16, based on pKC1132, Aprar This study

pKC1132-SAS13 Vector for gene disruption of sas13, based on pKC1132, Aprar This study

pUC19 Cloning and sequencing vector, Ampr (Yanischperron et

al. 1985)

pUC19Δ3100spec Vector for gene disruption of ann5, based on pUC19, aadA from

pLERE-Spec-oriT, Specr

(Kalan et al. 2013)

pUZ8002 Helper plasmid for conjugating plasmid containing the oriT

sequence, RK2-derived (IncP-1α group), tra1 and tra2 region,

Kanar

(Hopwood et al.

1985)

pBSK-SAS16 Plasmid containing gene sas16 for subcloning, Ampr This study

pKGLP2-GusA pKCLP2 derivative with gusA gene, Hygrr (Herrmann et al.

2012)

pKGLP2-GusA-

Nmet::aac3(IV)

Vector for double crossover of NMTase encoding gene in

Streptomyces, Aprar , Hygrr

This study

pKGLP2-GusA-

SAS16::aac3(IV)

Vector for double crossover of gene sas16 in Streptomyces,

Aprar , Hygrr

This study

pTESa-SAS16 an integrative plasmids pTESa base on φ31-based integrase,

Aprar

This study

pTESa pSET152 derivatives; attP flanked by loxP site, ermEp1

promoter flanked by tfd terminator sequences, Aprar

(Herrmann et al.

2012)

pLERECJ Carrying aac(3)IV flanked by loxP-sites, Ampr, Aprar (Makitrynskyy et

al. 2013)

pUWLCre pUWLoriT derivative carrying cre under ermEp, Hygrr, Tsrr (Fedoryshyn et al.

2008)

pKC1132-InAnn3 Vector for gene disruption of ann3, based om pKC1132, Aprar This study

121

5.2.3 Strain constructed and used in this study

5.2.4 Genome sequencing and bioinformatic Analysis

The complete genome of S. asterosporus DSM 41452 was sequenced using the Illumina

HiSeq2000 technology, assembled and annotated by Shanghai Majorbio Biopharm

pET24-Nmet Vector for protein expression of MTase domain, based on

pET24, kanar

This study

pET-Trx-Nmet Vector for protein expression of MTase domain, based on pET-

Trx-1c, kanar

This study

pBSK-Nmet Plasmid containing MTase encoding gene for subcloning, Ampr This study

pET28-Nmet Vector for protein expression of MTase domain, based on

pET28a(+), kanar

This study

Strain Relevant characteristics Reference

E. coli DH5α General cloning host Invitrogen

E. coli ET12567(pUZ8002) Methylation-deficient E. coli strain for

conjugation with the helper plasmid

Invitrogen

E. coli BW25113 Host for DNA recombination (Makitrynskyy et

al. 2013)

S. asterosporus DSM 41452 Wild type strain of WS9326A producer DSMZ

S. asterosporus DSM

41452::pUC19Δ3100spec

S. asterosporus DSM 41452 strain containing

plasmid pUC19Δ3100spec, Specr

This study

S. asterosporus DSM 41452::pKC1132-

orf(-1)

Gene inactivation of orf(-1) in the WT strain,

Aprar

This study

S. asterosporus DSM 41452::pKC1132-

SAS1

Gene inactivation of sas1 in the WT strain,

Aprar

This study

S. asterosporus DSM 41452::pKC1132-

SAS16

Gene inactivation of sas16 in the WT strain,

Aprar

This study

S. asterosporus DSM 41452::pKC1132-

SAS13

Gene inactivation of sas13 in the WT strain,

Aprar

This study

S. asterosporus DSM 41452::pKC1132-

InAnn3

Gene inactivation of ann3 in the WT strain,

Aprar

This study

S. asterosporus DSM 41452 ΔSAS16 Gene sas16 knockout in the WT strain, Aprar This study

S. asterosporus DSM

41452ΔSAS16::pTESa-SAS16

Sas16 overexpression in the mutant S.

asterosporus DSM 41452 ΔSAS16, Aprar

This study

S. asterosporus DSM 41452 ΔMTase In-frame deletion of gene encoding MTase in

the WT strain, Aprar

This study

122

Technology Co, Ltd. (Shanghai, China). Prediction of the gene clusters was performed using

antiSMASH (http://antismash.secondarymetabolites.org/). A large DNA fragment without any

gaps was found to contain the putative SAS gene cluster. The orfs were determined by

application of the FramePlot 4.0 beta program (http://nocardia.nih.go.jp/fp4/). Protein

sequences were compared with BLAST programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Domain organization and substrate specificities for NRPSs were predicted by PKS/NRPS

analysis software (http://nrps.igs.umaryland.edu/).

5.2.5 Generation of gene sas13 disruption mutant in S. asterosporus DSM

41452

A 701bp internal fragment of gene sas13 was amplified by PCR using the chromosome of S.

asterosporus DSM 41452 as template using the following primers SAS13F and SAS13R. The

PCR product was ligated into the EcoRV-digested pBluescript SK (-) to yield pBSK-SAS13. The

insert was excised as an HindIII/XbaI fragment and subcloned into vector pKC1132 to generate

suicide plasmid pKC1132-SAS13. After conjugation of this plasmid into S. asterosporus DSM

41452, an apramycin-resistant mutant named S. asteroporous :: pKC1132-SAS13 was

obtained. The correct transconjugants carrying plasmid were screened for resistance against

apramycin (50 ug/ml). apramycin-sensitive colonies were tested for target gene disruption by

colony PCR using primer Vsas13-F and Apra-R.

5.2.6 Generation of gene sas16 disruption mutant in S. asterosporus DSM

41452

An 840bp internal fragment of gene sas16 was amplified by PCR using the chromosome of S.

asterosporus DSM 41452 as template using the following primers P450S-F and P450S-R. The

PCR product was ligated into the EcoRV-digested pBluescript SK(-) to yield pBSK-SAS16. The

insert was excised as a HindIII/XbaI fragment and subcloned into vector pKC1132 to generate

plasmid pKC1132-SAS16. After conjugation of this plasmid into S. asterosporus DSM 41452,

an apramycin-resistant mutant named S. asteroporous DSM 41452 :: pKC1132-SAS16 was

obtained. The correct transconjugants carrying plasmid were screened for resistance against

apramycin (50ug/ml). apramycin-sensitive colonies were tested for target gene disruption by

colony PCR using primer Vsas16-F and Apra-R.

123

5.2.7 Strain information, Fermentation, Extraction

The wildtype strain S. asterosporus DSM 41452 was purchased from Leibniz Institute DSMZ-

German Collection of Microorganisms and cell Cultures, Germany. The initial cultures were

maintained on the Tryptic soy broth (TSB) solid medium [Bacto™ Tryptone (Pancreatic Digest

of Casein) 17g, Bacto Soytone (Peptic Digest of Soybean Meal) 3g, Glucose 2.5g, Sodium

Chloride 5g, Dipotassium Hydrogen Phosphate 2.5g, Tap water 1000mL, pH 7.3]. A small loop

of spores growing on a TSB solid plate was inoculated into a 250 mL Erlenmeyer flask

containing 75 mL liquid productive SG medium (Soy peptone 10.0 g, Glucose 20.0 g, L-Valine

2.34 g, CaCO3 2.0 g, CoCl2-solution 1mg/mL 1 mL, tap water 1000 mL, pH7.2) and cultured at

28 ℃ for 3 days on a rotary shaker at 180 r·min−1. Then, 10 mL of the preculture (at a volume

ratio of 1:100) was inoculated into a 500 mL Erlenmeyer flask (40 flasks) containing 150 mL of

the SG medium, then incubated for 4 days, 180rpm, 28℃. The fermentation broth (6L) was

filtered through high speed centrifugation (8000 rpm, 10min, 22℃), yielding the supernatant

and cell pellet. Then the supernatant was extracted by double volume ethyl acetate using a

separating funnel, then this organic solvent was evaporated under reduced pressure. The

mycelium also was extracted by acetone, which was evaporate under reduced pressure. The

crude extract from the supernatant and the cell pellet were combined.

5.2.8 Isolation of Compound WS9326A, B, D, E, F, G

The crude extract (~5g) was dissolved in 15 mL MeOH and fractionated by reversed-phase (RP)

C18 liquid chromatography (Oasis® HLB 20 / 35cc), the starting elution solvent is 5% methanol,

then the column was eluted using a stepwise gradient MeOH (30%, 40%, 50%, 60%, 70%, 80,

90% and 100%). Fractions afforded from SPE column were analyzed by LC-MS. Among those

fractions, fractions (Nr.22-27) were subsequently subjected to a further purification by a semi-

preparative HPLC (Agilent Technologies), equipped with a Waters ZORBAX SB-C18 column (9.4

x 100 mm, 5 µm) and a X-Bridge C8 guard column (9.4 x 50 mm, 3.5 µm), the fraction was

eluted by an isocratic method (60% CH3CN-40% H2O, each solvent contains 0.5% acetic acid;

flow rate 1 mL/min), to yield compounds WS9326A (10mg), WS9326B (4.9mg), WS9326D

(10.9mg), WS9326E (2.4mg), WS9326F (9.4mg), and WS9326G (4.8mg).

5.2.9 Sample analysis by HPLC-MS

The organic phase was evaporated, resuspended in MeOH(1mL), and filtered through syringe

filters (LLG, PVDF, 0.45um) prior to LC-MS analysis. HPLC-MS analysis was performed on an

Agilent 1100 series LC/MS system with electrospray ionization (ESI) and detection in the

positive and negative modes. The LC system was equipped with a Zorbax Eclipse XDB-C18

124

column (3.5 μm particle size; 100 mm by 4.6 mm; Agilent) and a Zorbax XDB-C18 guard column

(5-μm particle size; 12.5 mm by 4.6 mm; Agilent), maintained at room temperature. Detection

wavelengths of the diode array detector were 254/360 nm, 480/800 nm, 360/580 nm, and

430/600 nm. The mobile system consisted of solvent A (acetic acid [0.5%, vol/vol] in

acetonitrile) and solvent B (acetic acid [0.5%, vol/vol]). A 10 μL aliquot of the MeOH-soluble

extract was injected for analysis each sample, a gradient elution method was used (A: CH3CN

with 0.1% HAc; B: H2O with 0.1% HAc; 5% A over 4 min, 5–95% A from 4 to 20 min, 95% A from

20 to 22 min, 95-5% A from 22 to 23 min, and 5% A from 23 to 30 min; 0.5mL/min). MSD

settings during the LC gradient were as follows: Acquisition—mass range m/z 150–1000, MS

scan rate 1s-1, MS/MS scan rate 2s-1, fixed collision energy 20 eV; ion source drying gas

temperature 350 °C, drying gas flow 10 L/min; Nebulizer pressure 35 psig; ion source mode

API-ES; capillary voltage 3000; The MS detector was autotuned using Agilent tuning solution

in positive and positive mode before measurement. LC (DAD) and MS data were analyzed with

ChemStation software (Agilent).

5.2.10 NMR methods and General instrument for structural

characterization

Nuclear magnetic resonance (NMR) was employed to elucidate the structures of Compound

WS9326A, B, D, E, F, G. The 1D NMR spectra [1H NMR (400 MHz) and 13C NMR (100 MHz)] and

2D NMR spectra [1H/1H-COSY (correlation spectroscopy), HSQC (Heteronuclear Single

Quantum Coherence), and HMBC (Heteronuclear Multiple Bond Correlation)] of these

compounds were measured on a Varian VNMR-S 600-MHz spectrometer in 150 μl DMSO-d6

at T = 35°C or 25°C. Residual solvent signals were used as an internal standard (DMSO-d6: δH

= 2.5ppm, δC = 39.5 ppm). For WS9326F, WS9326G, SY11 and SY12, their high-resolution

electron spray ionization mass spectra (HR-ESI-MS) were measured on a LTQ Orbitrap XL

(Thermo Scientific).

5.2.11 Structure information of compound 1-6

WS9326A (1): White amorphous powder; UV(MeOH) λmax 231 nm, 288 nm; ESI-MS m/z [M-

H]- 1035, [M+Cl]-1071, (calcd. for C54H68N8O13, 1036.49); 1H-NMR (400 MHz, DMSO-d6) and 13C-

NMR (100 MHz, DMSO-d6) data are shown in Table 5.6.

WS9326B (2): White amorphous powder; UV(MeOH) λmax 212 nm, 290 nm; ESI-MS m/z [M-

H]-1037, [M+Cl]-1073, HRESI-MS m/z [M+H]+ 1039.0229 (calcd. for C54H70N8O13, 1038.51);

125

WS9326D (3): White amorphous powder; UV(MeOH) λmax 209 nm, 289 nm; ESI-MS m/z [M-

H]-852.4(calcd. for C47H59N5O10, 853.43);

WS9326E (4): White amorphous powder; UV(MeOH) λmax 222 nm, 291 nm; ESI-MS m/z [M-

H]-838.4(calcd. for C46H57N5O10, 839.41);

WS9326F (5): White amorphous powder; UV(MeOH) λmax 210 nm, 290 nm; ESI-MS m/z [M-

H]- 966.4, HRESI-MS m/z [M+H]+ 968.4750 (calcd. for C51H65N7O12, 967.47); 1H-NMR (400 MHz,

DMSO-d6) and 13C-NMR (100 MHz, DMSO-d6) data are shown in Table 5.6.

WS9326G (6): White amorphous powder; UV(MeOH) λmax 225 nm, 293 nm; ESI-MS m/z [M-

H]- 952.4, HRESI-MS m/z [M+H]+ 954.4644(calcd. for C50H63N7O12, 953.45);

5.2.12 Antiparasite assay method and materials

Asexual, blood-stage parasites were cultured in vitro using standard conditions(Trager and

Jensen 1976). Briefly, parasites were maintained in 2% human O+ erythrocytes (Interstate

Blood Bank, Memphis, TN) in RPMI-1640 medium (Life Technologies, Grand Island, NY)

supplemented with 0.5% Albumax (Life Technologies), 24 mmol/L sodium bicarbonate, and

10 μg/mL gentamycin. Tissue culture flasks were incubated at 37 °C under a gas mixture of 5%

CO2, 5% O2, and 90% N2. Cultures were screened for mycoplasma using the Universal

Mycoplasma Detection Kit (ATCC, Manassas, VA).

In vitro drug responses were measured using 72-hr SYBR Green staining assays as described

previously with minor modifications(Desjardins, Canfield et al. 1979; Smilkstein, Sriwilaijaroen

et al. 2004). Parasites were diluted to 0.5% final parasitemia with 2% final hematocrit. The

diluted parasite culture (100 μL) was added to duplicate test wells in a 96-well plate containing

100 μL of the drug tested. IC50 values were determined by nonlinear regression analysis using

Prism 5.0 software (GraphPad Software, San Diego, CA). Drug assays were performed on three

independent occasions.

5.3 Results and Discussion

5.3.1 Chemical structure Elucidation of WS9326A derivatives from S.

asterosporus DSM 41452

Recently we report that the strain Streptomyces calvus produce a kind of NRPS derivatives

WS9326A, further analysis of other sporulation defective strain S. asterosporus DSM 41452,

we found that it can produce much more amount of WS9326As than that of Streptomyces

126

calvus. From 10L fermentation of S. asterosporus DSM 41452, four known despipeptides

WS9326A (1), WS9326B (2), WS9326D (3), WS9326E (4) were isolated (Figure 5. 1). The NMR

and MS/MS spectroscopic data for WS9326A (1) was consistent with previously reported

spectra (Appendix, Table 5. 6) (Yu et al. 2012), and its configuration was confirmed further by

Marfey’s method (Marfey 1984). Compounds WS9326B (2), WS9326D (3) and WS9326E (4)

were assigned based on their MS/MS spectra (Figure 5. 26, Figure 5. 27, Figure 5. 28 and Figure

5. 29).

HN

OO

O

N

OH

HN

ONHO

O

NH

O

NH

OH

OHN

OH2N

HO

O

HN

OO

O

N

OH

HN

ONHO

O

NH

O

NH

OH

OHN

OH2N

HO

O

WS9326A (1) WS9326B (2)

WS9326E (4)

HN

OHO

O

N

OH

HN

ONHO

O

NH

O

OH

HO

CH3

WS9326D (3)

HN

OHO

O

N

OH

HN

ONHO

O

NH

O

OH

HO

HN

OHO

O

N

OH

HN

ONHO

O

NH

O

NH

OH

OHO

OH2N

HN

OHO

O

N

OH

HN

ONHO

O

NH

O

NH

OH

OHO

OH2N

WS9326F (5) WS9326G (6)

Figure 5. 1. The chemical structure of WS9326A and its derivatives (WS9326B, WS9326D, WS9326E, WS9326F,

WS9326G)

Furthermore, we observed two additional compounds by LC-ESI-MS with m/z values of 966.4

(compound 5) and 952.4 (compound 6) (Figure 5. 24, Figure 5. 25). Both compounds were

isolated for subsequent NMR and MS/MS spectroscopic analysis to reveal two new WS9326A

analogues: WS9326F (5) and WS9326G (6).

WS9326F (5) was obtained as white amorphous powder. The molecular ion observed for 5 by

HRESI-MS corresponded to a molecule with the formula C51H65N7O12 and 23 degrees of

unsaturation (obsvd: m/z = 968.4750 [M+H]+, calcd for C51H66N7O12, m/z = 968.4764) The

structure of 5 (Figure 1) was determined by careful comparison with the NMR spectroscopic

data obtained from WS9326A (Yu et al. 2012). The 1H NMR (400 MHz, DMSO-d6) spectrum of

5 (Table 5. 6) exhibited significant signal characteristics of WS9326A, including the presence of

five α-amino methines protons at δ 4.29 (1H, m), 4.49 (1H, m), 4.62 (1H, m), 4.23 (1H, m) and

3.17 (1H, m) ppm, which correlated with five sp3 α-amino methine carbons at δ 52.4 (1Thr),

49.6 (3Leu), 54.6 (4Phe), 58.7 (5Thr) and 48.8 (6Asn) ppm, respectively, in the HSQC spectrum.

Based on the HMQC spectrum, six sets of methyl protons (δ 0.79, 1.05, 2.87, 0.70, 0.63 and

127

0.92 ppm) were assigned to the corresponding carbon atoms at δ 14.2 (Acyl-C14), 20.3 (1Thr),

34.8 (2N-methyl-ΔTyr), 22.6 (3Leu), 23.3 (3Leu) and 22.7 (5Thr), respectively. In addition, signals

corresponding to 8 carbonyl carbons at δ 165.6, 170.8, 165.2, 171.8, 170.6, 170.2 and 172.0

ppm were observed in the 13C NMR spectrum (100 MHz, DMSO-d6) which further indicate the

presence of 6 amino acid residues in 5. Shared MS/MS fragmentation patterns (Figure 5. 2)

were observed in the spectra of WS9326A, WS9326D and 5, corresponding to the peptides 2N-

methyl-ΔTyr-3Leu-4Phe (436.35 Da), 2N-methyl-ΔTyr-3Leu-4Phe-5Thr (537.40 Da), and Acyl-1Thr-

2N-methyl-ΔTyr-3Leu-4Phe (735.50 Da). In contrast, two unique m/z values (652.34 Da and

950.56 Da) were identified in the MS/MS spectrum of 5. Accordingly, 5 was designated as a

new WS9326A analog with the structure Acyl-1Thr-2N-methyl-ΔTyr-3Leu-4Phe-5Thr-6Asn. As 5

is a truncated analog of WS9326A, it is predicted to share the same amino acid configuration

with WS9326A.

WS9326G (6) was also obtained as white amorphous powder. The molecular ion observed by

HRESI-MS for 6 corresponded to a molecular formula of C50H63N7O12 (obsvd: m/z = 954.4644

[M+H]+ (calcd for C50H64N7O12, 954.4607). This reveals that 6 is 14 Da smaller than 5 which

corresponds to the absence of a methyl group. The chemical structure of 6 (Figure 5. 1) was

elucidated by comparing the MS/MS fragmentation spectrum with that obtained from 5

(Figure 5. 2). In addition to the mutual MS/MS mass fragments of 289.19 Da, 436.31 Da and

652.39 Da. Three unique masses are observed for 6: 822.51 Da (Acyl-1Ser-2N-methyl-ΔTyr-3Leu-

4Phe-5Thr), 721.47 Da (Acyl-1Ser-2N-methyl-ΔTyr-3Leu-4Phe), and 574.38 Da (Acyl-1Ser-2N-

methyl-ΔTyr-3Leu). These results demonstrate that 6 shares a similar chemical structure with

5, exception of a methyl group located within the N-terminus. Furthermore, compared to 5,

the 1H NMR spectrum of 6 (Figure 5. 2) reveals only five methyl proton signals (δ 0.79, 1.04,

2.87, 0.70, and 0.63 ppm). This agrees with the presence of an N-terminal Ser in 6 versus Thr

in 5. Therefore, 6 was proven to be a new WS9326A analog with the chemical backbone: Acyl-

1Ser-2NmetTyr-3Leu-4Phe-5Thr-6Asn, and its absolute configuration also was assigned as that

resembling WS9326A.

A

128

B

C

Figure 5. 2. MS/MS spectra of WS9326F(A) and WS9326G (B) produced by S. asterosporus DSM 41452; (C) Partial

H NMR Spectrum comparison between WS9326F and WS9326G

The Marfey’s method was carried out following the previous protocol (Marfey 1984), and the

Marfey’s reagent was purchased from Thermo Scientific (Number 48895). The amino acid

standards involved in the assembly of WS9326A were obtained commercially from Carl Roth

or Sigma-Aldrich. Each of them (1 mg) were dissolved in 100 ul H2O, respectively. Then the

amino acids were derivatized by adding 1 M NaHCO3 (40 µl) and 1% FDAA (in acetone, 200 µl).

The reaction mixture was heated at 40°C for 1 hour, then the mixture was neutralized with 2

M HCl after cooling at room temperature. The derivatives were then dried and dissolved in

CH3OH, then analyzed by HPLC-MS (ESI+ mode, Zorbax Eclipse XDB-C18 column,100 x 4.6 mm),

a gradient elution method was used (A: CH3CN with 0.1% HAc; B: H2O with 0.1% HAc; 10%-

40%-90% CH3CN in 50 min, 0.5 ml/min at 25°C, detection at wavelengths of 270 nm, and 340

129

nm, m/z range from 100-2000). WS9326A (0.5 mg) was hydrolyzed with 2 M HCl (2.0 mL) at

45°C for 2 hours. The solution was evaporated to dryness, and derivatized with Marfey’s

reagent.

The retention times of amino acid standards derivatized with Marfey’s reagent are as follows:

21.71 min (L-threonine FDAA derivative), 19.48 min (L-asparagine FDAA derivative), 41.92 min

(L-leucine FDAA derivative), 21.87 min (L-allo-threonine FDAA derivative), 20.31 min (L-serine

FDAA derivative), 44.55 min (D-phenylalanine FDAA derivative) (Figure 5. 2). The derivatives

of WS9326A acid hydrolysates were analyzed by LC-MS. The resulting amino acid FDAA

derivatives from WS9326A hydrolysates shown similar HPLC profile as the FDAA derivatives of

standard amino acids.

Figure 5. 3. HPLC chromatogram of FDAA derivative of WS9326A and the corresponding standard amino acids

(number representing the retention time).

5.3.2 Discovery of two new WS9326A analogs by disrupting Annimycin

production in S. asterosporus DSM 41452

Except the new derivatives of WS9326A (WS9326G and WS9326F) from S. asterosporus DSM

41452, in addition, from mutant S. asterosporus DSM 41452::pUC19Δ3100spec we were

surprised to observe the production of two new peaks with the m/z values of [M+H]+ 1135.5

and 1137.5 (Figure 5. 1), they were named as SY11 and SY12.

130

Figure 5. 4. HPLC profiles of S. asterosporus DSM 41452 wildtype and its mutant strains S. asterosporus DSM

41452::pUC19Δ3100spec (under 254nm wavelength). Note: labeled peaks refer to WS9326A (1), SY11 (2) and

SY12 (3).

For purification of compound SY11 and SY12, 6 L cell culture of S. asterosporus DSM

41452::pUC19Δ3100spec was cultivated, and crude extract was obtained using the same

fermentation and extraction methods mentioned in the above methods and materials section

5.2.7. Approximately 1g crude extract was subjected to an open silica gel column (20g) eluted

by mobile solution system (CH2Cl2: MeOH 0:100-20:100), the resulting fractions were analyzed

by LC-MS. The fractions containing target mass signal were collected and further purified

through semi-preparative HPLC, the compounds were eluted out with 60% acetonitrile with

isocratic method. Finally, roughly 15mg SY11 and 10mg SY12 were collected for NMR test.

According to the 1D NMR (1H NMR and 13C NMR) and 2D NMR (H-H COSY, HMBC, HSQC and

TOCSY) spectra (related NMR spectra not being attached), we have elucidated partial molecule

fragment of SY11 as shown Figure 5. 5. However, due to the serious signal overlapping and the

absence of the key correlation, its complete chemical structure has been determined yet. The

cyclopeptide part of SY11 and SY12 were verified base on MS/MS fragmentation analysis.

RHN

OO

O

N

OH

HN

O

NHO

O

NH

O

NH

OH

OHN

OH2N

HO

O

50.94.40

2.42

8.25

55.34.35

8.42

61.03.25

57.24.27

67.94.12

0.5

7.65

55.84.36 2.71

9.14

53.74.02

1.27

0.85

5.88

128.5

2.94

9.21

72.94.90

1.10

52.55.14

7.58

4.63

132.0

132.0

169.4

Acyl group

165.8

Figure 5. 5. Postulated chemical structure of compound SY11 base on key NMR data; some key NMR signals are

shown (key TOCSY correlations are marked in bold lines, key HMBC correlations are represented by arrow).

131

SY11 share similar MS/MS fragmentation patterns (Figure 5. 6) with WS9326A, corresponding

to the peptide signals: 839.1 Da (1Thr-2N-methyl-ΔTyr-3Leu-4Phe-5Thr-6Asn-7Ser), 738.3Da (2N-

methyl-ΔTyr-3Leu-4Phe-5Thr-6Asn-7Ser), 651.2Da (2N-methyl-ΔTyr-3Leu-4Phe-5Thr-6Asn), 537.1

Da (2N-methyl-ΔTyr-3Leu-4Phe-5Thr), and 436.1Da (2N-methyl-ΔTyr-3Leu-4Phe). In contrast, on

the MS/MS spectrum, SY12 show similar fragmentation m/z values with WS9326B, such as

741.3 (2N-methyl-Tyr-3Leu-4Phe-5Thr-6Asn-7Ser), 653.3 (2N-methyl-Tyr-3Leu-4Phe-5Thr-6Asn),

539.1 (2N-methyl-Tyr-3Leu-4Phe-5Thr), and 437.9 (2N-methyl-Tyr-3Leu-4Phe).

A

B

Figure 5. 6. (A) ESI-MS/MS fragmentation of SY11; (B) ESI-MS/MS fragmentation of SY12.

In addition, H NMR spectrum comparison between SY11 and SY12 clearly shows a unique β-

olefinic proton signal of the dehydrotyrosine residue present at δ 5.88 (1H, s) on the H NMR

spectrum of SY11, however absent on the H NMR spectrum of SY12 (Figure 5. 7). Moreover,

according to their HRESI-MS analysis results, compound SY11 (HRESI-MS m/z [M+H]+

132

1135.5360) and SY12 (HRESI-MS m/z [M+H]+ 1137.5594) shows 2 Da mass difference in term

of their molecular weight, which further demonstrates that the structural difference between

those SY11 and SY12 could result from the reduction of double bond at the N-methyl-

dehydrotyrosine residue in SY11.

Figure 5. 7. H NMR spectrum comparison between SY11 and SY12, R represents the unknown organic moiety.

To sum up, based on HRESI-MS, ESI-MS/MS and NMR data analysis, SY11 and SY12 were

designated as new WS9326A derivatives. SY11 has the similar cyclopeptide scaffold like

WS9326A, SY12 has an similar cyclopeptide scaffold like WS9326B. Their molecular weights

show that there is a special chemical moiety attached onto WS9326A’s cyclopeptide scaffold

(Thr-Tyr-Leu-Phe-Thr-Asn-Ser), and this modification appears at the polyketide side chain at

the N-terminus of the cyclic peptide. The comprehensive investigation on the structure of SY11

and SY12 will be carried out.

5.3.3 Antiparasitic activity assay of WS9326As

According to a previous report, WS9326A and its congeners display a surprising range of

bioactivities, from tachykinin antagonism (Shigematsu et al. 1993) to antifilarial activity (Yu et

al. 2012), this promoted us to further investigate the potency of the newly identified WS9326A

analogues SY11 and SY12, along with an annimycin analogue SY10. In an antimalarial assay,

we evaluated in three Plasmodium falciparum cell-lines (Dd2, HB3 and 3D7), which possess

variant drug-resistance phenotypes. Artemether (ATM) was used as positive control.

we screen the compounds against all three lines with a maximum concentration of 20 µM with

serial dilutions (in each experiment performed in duplicate). All those compounds were

133

soluble in DMSO, we use 20mM stock concentrations to minimize the amount of DMSO in the

assay.

The figures 5.8 show the assay results. Annimycin analogue SY10 (Chemical structure not

shown) had modest activity at the highest concentration tested (2.5 µM) with approximately

30% inhibitory activity against the three cell-lines tested, including the multidrug resistant Dd2

isolate. In contrast, WS9326A and its derivatives SY11 and SY12 did not demonstrate

significant antimalarial activity at the concentrations tested (Figure 5. 8). In addition, series of

antibacterial test shown that WS9326A and all its derivatives don’t have antibiotic activity

towards E. coli and Bacillus subtilis.

5.3.4 Characterization of the WS9326A gene cluster in S. asterosporus

DSM 41452.

WS9326A has also been identified in cultures of Streptomyces calvus ATCC1338. While the

gene cluster encoding WS9326A was identified in this study (Johnston et al. 2015), the specific

functions of individual genes were not examined.

To find the corresponding biosynthesis gene cluster, the genome of S. asterosporus DSM 41452

was completely sequenced. The cluster is very similar to the predicted WS9326A cluster

identified in the S. calvus ATCC13882 genome (Johnston et al. 2015). Analysis of genome

sequence of S. asterosporus DSM 41452 revealed the entire sets of genes required for

WS9326A molecule assembly clustered in a region of chromosome approximately 3.5 Mbp

Figure 5. 8. Drug response phenotypes for

Plasmodium falciparum Dd2 (A), HB3 (B) and 3D7

(C) strains. Artemether (ATM) is shown as a

positive antimalarial control. The results shown

are the average of three independent

experiments conducted in duplicate per

concentration, shown as the mean and standard

error. Figures were prepared by Dr. Richard

Eastman.

134

away from the oriC, located at the subtelomeric region. The WS9326A gene cluster is 60.3 kb

long and consists of 40 open reading frames (Figure 5. 9 and Table 5. 3). The 40 genes can be

classified in subcategories according to their functions in the biosynthesis of WS9326A.

Figure 5. 9. Organization comparison of the WS9326A gene clusters in S. asterosporus DSM 41452 and

Streptomyces calvus

5.3.4.1 Gene disruption of sas1 and orf(-1)

To attempt to define the boundaries of the WS9326A gene cluster in S. asterosporus DSM

41452, gene disruption was performed by single crossover.

The boundary gene sas1 at the C-terminus of the gene cluster encodes a protein sharing 48%

identity with a two-component sensor histidine kinase from S. olivaceus (Accession number

WP_052410686.1). For disrupting this gene, a partial 515 bp fragment of gene sas1 was

amplified by PCR using the genomic DNA of S. asterosporus DSM 41452 as template with

primers LuxR2-F and LuxR2-R. The PCR product was ligated into the EcoRV-digested pBluescript

SK (-) to yield pBSK-SAS1. After restriction enzyme digestion by HindIII and XbaI the

corresponding fragment was cloned into the plasmid pKC1132 to yield plasmid pKC1132-

SAS1(Figure 5. 9). Then pKC1132-SAS1 was introduced into S. asterosporus DSM 41452 by

intergeneric conjugation. The correct exconjugants carrying plasmid were screened for

resistance against apramycin (50 ug/ml). Apramycin-sensitive colonies were tested for target

gene disruption by colony PCR using primers VluxR2-F and Apra-R (Table 5.1)(Figure 5. 9).

Analysis of the culture extract by HPLC-ESI/MS showed that the production of WS9326As was

disrupted in this mutant, suggesting the sas1 is involved in the biosynthesis of WS9326A in this

strain (Figure 5. 9).

At the 5’ end of this gene cluster, gene orf(-1) shows 91% identity with its homologous gene

(Accession number WP_040907349.1) encoding a transcriptional regulator from

Streptomyces griseoflavus. For verifying the function of gene orf(-1), the mutant strain with

deficient orf(-1) was constructed. A 442bp internal fragment of gene orf(-1) was amplified by

PCR using the genomic DNA of S. asterosporus DSM 41452 as template with primer pair Orf(-

1)-F and Orf(-1)-R. The PCR product was ligated into the EcoRV-digested pBluescript SK (-) to

yield pBSK-orf(-1). After restriction enzyme digestion by HindIII and XbaI, the corresponding

fragment was cloned into plasmid pKC1132 to yield plasmid pKC1132-orf(-1) (Figure 5. 9).

135

pKC1132-orf(-1) was introduced into S. asterosporus DSM 41452 by intergeneric conjugation.

An apramycin-resistant (50ug/ml) mutant was isolated. Apramycin-sensitive colonies were

tested for target gene disruption by colony PCR using primers VluxR1-F and Apra-R (Table 5.

1) (Figure 5. 9).

The corresponding mutant strain S. asterosporus DSM 41452::pKC1132-orf(-1) was cultured

in the SG medium, the resulting culture extract was analyzed by HPLC-ESI/MS, the result

showed that this mutant still keep the capability of producing WS9326As (Figure 5. 9),

indicating that orf(-1) is not involved in WS9326A biosynthesis. Taken together, our results

strongly supported the hypothesis that genes sas1-sas40 are responsible for the biosynthesis

of WS9326A and its analogs.

A B

C

Figure 5. 10. Inactivation of the gene orf(-1) and sas1 in S. asterosporus DSM 41452 via single crossover. (A)

Schematic representation of plasmid pKC1132-SAS1, the agarose gel exhibits the verification of resulting mutant,

showing amplification of a 2.6 kb PCR fragment (using the primers VluxR2-F and Apra-R) in mutant (lane 1), in

comparison with the wild type (lane 2); (B) Schematic representation of plasmid pKC1132-orf(-1), the agarose

gel exhibits the verification of resulting mutant, showing amplification of a 2.8 kb PCR fragment (using the

primers VluxR1-F and Apra-R) in mutant (lane 1), in comparison with the wild type (lane 2); (C)The HPLC

chromatogram of the ethyl acetate extracts of the culture broth of the wildtype strain, mutants S. asterosporus

DSM 41452::pKC1132-orf(-1) and S. asterosporus DSM 41452::pKC1132-SAS1. The peak of WS9326A is marked

with an asterisk.

136

Figure 5. 11. NRPS domain organization are shown in the order for the WS9326A biosynthetic assembly line.

Domain notation: C, condensation; A, adenylation; PCP, peptidyl carrier protein; E, epimerization; NMT, N-

methyltransferase; TE, thioesterase.

The core part of the gene cluster contains 5 genes encoding nonribosomal peptide

synthetases (Sas17, Sas18, Sas19, Sas22 and Sas23) responsible for the molecular backbone

assembly of NRPS (Figure 5. 11). As a PKS-NRPS hybridization, genes sas27-sas37 were

predicted to be involved in the biosynthesis of the polyketide chain and attachment at the N-

terminal of the start amino acid serine or threonine. The remaining gene apart from several

genes of unknown function are identified as encoding tailoring enzymes, transporter, and

regulatory proteins.

For the biosynthesis of peptide backbone of WS9326A, the first module encoded by a 7703 bp

large gene sas17, a dimodule synthetase, consists of two sets of adenylation domain(A),

condensation(C), and peptidyl carrier protein(PCP) domains, the first C-A-PCP system

responsible for loading of the start unit serine or threonine, another set of C-A-PCP domain

system plus one N-methyltransferase domain(MTase) responsible for loading a modified

nonproteinogenic amino acid N-methyldehydrotyroine. The directly downstream adjacent

gene sas18 also encodes a large dimodule nonribosomal peptide synthetases (C-A-PCP-C-A-

PCP-E), in which the first set of C-A-PCP domain system responsible for the selectivity and

loading of the leucine unit, and the second module for the incorporation of D-Phe (Table 5. 4).

The epimerization of the second module might catalyze the epimerization of L-Phe into its D-

stereoisomer. The downstream adjacent gene sas19 which encodes a set of modules C-A-PCP-

137

C-A-PCP-TE was hypothesized for the assembly of the next extender unit threonine and

asparagine, but actually it was used to catalyze the assembly of the last one amino acid serine

and also for the release and cyclization from the assembly line.

A predicted type II thioesterase is encoded by sas20. Although the function of Sas20 in the

biosynthesis of WS9326A is unknown, type II thioesterases are well known to serve editing

functions in PKS and NRPS systems (Kotowska, Pawlik. 2014). Sas20 may also mediate release

of the linear peptides WS9326D, E, F, and G from the trans-acting domains Sas22 and Sas23.

Those two trans-acting A-PCP didomains encoded by genes sas22 and sas23 are predicted to

interact with the condensation domain in module 7 encoded by sas19 in trans to catalyze the

incorporation of the threonine and Asparagine residue on the assembly line. We proposed

that the first condensation domain in module Sas19 may be involved to catalyze the

condensation reaction.

Within the putative SAS gene cluster 18 genes were predicted to be involved in the

biosynthesis of the PKS-derived acyl side chain. Very similar genes were also found in the

Skyllmycin gene cluster (Pohle et al. 2011) (Table 5. 5)(Figure 5. 11). In contrast to the

Skyllmycin gene cluster, sky11 encoding a putative carboxyltransferase, is absent in our cluster.

Thus malonyl-CoA as the starting building block is most likely derived from the primary

metabolism in our strain. Further bioinformatic analysis of the genes within this locus revealed

that six genes (sas7, sas8, sas30, sas31, sas32, and sas33) were predicted to encode 3-oxoacyl-

ACP synthases. These might be involved in a series of condensation reactions with acetyl-CoA

and malonyl-CoA units to form the N-acyl C14-polyene before aromatic ring formation occurs

(Pohle et al. 2011). The terminal C12-C13 double bond in this C14-polyene intermediate might

be further reduced by a reductase encoded by sas21, which show significant homology (96%

amino acid identity) to an oxidoreductase from S. griseoflavus. The configurations of the C14-

acyl group in WS9326A and the C12-acyl group in skyllamycin are identical (2E, 10Z) according

to NMR spectroscopic data (Table 5. 6). Moreover, the double bond configuration at C4, C6,

and C8 may be important for aromatization during biosynthesis. The required configuration

conversion is most likely to be introduced by the gene product of sas27 encoding an isomerase,

which shares 62% amino acid identity with Sky27 and 57% amino acid identity with Has16

involved in haoxinamide biosynthesis (Pohle et al. 2011). The final aromatization to form the

benzene ring could be catalyzed by either the putative oxidoreductase Sas24 or the phytoene

dehydrogenase Sas28.

138

Three predicted regulatory genes were found in the 3’-region of the SAS gene cluster. Including

the essential biosynthetic gene sas1, encoding a two-component sensor histidine kinase, the

gene sas2 shows homology to the LuxR transcriptional regulator found in S. canus

[WP_059209681.1], and sas3 was predicted to encode a LysR transcription factor. In addition,

three transporter genes (sas38, sas39 and sas40) are located at the 5’ end of the SAS cluster.

According to the recent report given by Ju et al (Li et al. 2016), the putative two-component

sensor histidine kinase encoded by sas1 could interact with the isomerase encoded by sas27,

thereby enabling the epimerization of the Thr to form L-allo-Thr. The gene sas4 was predicted

to encode a MbtH-like protein, which shares 46% identical amino acids with PA2412 involved

in pyoverdine biosynthesis in Pseudomonas aeruginosa. We postulated that Sas4 is involved

in the NRPS assembly by interacting with the corresponding A domain(Zhang et al. 2010).

Table 5. 3. Proposed Functions of Open Reading Frames of WS9326A Biosynthesis Gene Cluster in S. asterosporus

DSM 41452

Protein Size (a.a.) Homologous Protein and

origin

Identity [%] predicted function

Orf(+1) 220 SDD75964.1 [Streptomyces

emeiensis]

87% DedA family membrane protein

Sas1 407 Cal1 [Streptomyces calvus] 99% Sensor histidine

kinase

Sas2 211 Cal2 [Streptomyces calvus] 100% LuxR transcriptional

regulator

Sas3 79 Cal3 [Streptomyces calvus] 99% LysR family transcriptional

regulator

Sas4 72 Cal4 [Streptomyces calvus] 99% MbtH domain protein

Sas5 248 Cal5 [Streptomyces calvus] 99% Thioesteraase

Sas6 293 Cal6 [Streptomyces calvus] 99% Hypothetical protein

Sas7 334 Cal7 [Streptomyces calvus] 99% 3-oxoacyl-ACP synthase II

Sas8 413 Cal8 [Streptomyces calvus] 99% 3-oxoacyl-ACP synthase II

Sas9 83 Cal9 [Streptomyces calvus] 99% polyketide-8 synthase ACP

Sas10 123 Cal10 [Streptomyces calvus] 100% hypothetical protein

Sas11 136 Cal11 [Streptomyces calvus] 100% hypothetical protein

Sas12 140 Cal12 [Streptomyces calvus] 99% translation initiation factor IF-2

Sas13 314 Cal13 [Streptomyces calvus] 99% 3-hydroxyacyl-ACP

dehydratase

Sas14 257 Cal14 [Streptomyces calvus] 98% hypothetical protein

Sas15 65 Cal15 [Streptomyces calvus] 100% ferredoxin

Sas16 407 Cal16 [Streptomyces calvus] 100% cytochrome P450

139

Sas17 2567 Cal17 [Streptomyces calvus] 99% NRPS(C-A-PCP-C-A-MTase-

PCP)

Sas18 2588 Cal18 [Streptomyces calvus] 99% NRPS(C-A-PCP-C-A-PCP-E)

Sas19 3389 Cal19 [Streptomyces calvus] 99% NRPS(C-PCP-C-A-PCP-C-A-

PCP-TE)

Sas20 233 Cal20 [Streptomyces calvus] 100% Thioesterase

Sas21 274 Cal21 [Streptomyces calvus] 100% Oxidoreductase

Sas22 974 Cal22 [Streptomyces calvus] 99% NRPS(A-PCP)

Sas23 601 Cal23 [Streptomyces calvus] 99% NRPS(A-PCP)

Sas24 400 Cal24 [Streptomyces calvus] 99% ferredoxin reductase or FAD-

dependent oxidoreductase

Sas25 264 Cal25 [Streptomyces calvus] 99% alpha/beta hydrolase

Sas26 333 Cal26 [Streptomyces calvus] 99% Acyl-CoA thioesterase

Sas27 241 Cal27 [Streptomyces calvus] 99% isomerase

Sas28 574 Cal28 [Streptomyces calvus] 99% Dehydrogenase or reductase

Sas29 86 Cal29 [Streptomyces calvus] 99% Acyl carrier protein

Sas30 416 Cal30 [Streptomyces calvus] 99% 3-oxoacyl-ACP synthase

Sas31 379 Cal31 [Streptomyces calvus] 99% 3-oxoacyl-ACP synthase

Sas32 314 Cal32 [Streptomyces calvus] 99% 3-oxoacyl-ACP synthase

Sas33 371 Cal33 [Streptomyces calvus] 99% 3-oxoacyl-ACP synthase

Sas34 87 Cal34 [Streptomyces calvus] 100% polyketide-synthase ACP

Sas35 133 Cal35 [Streptomyces calvus] 99% 3-oxoacyl-ACP dehydratase

Sas36 159 Cal36 [Streptomyces calvus] 99% 3-oxoacyl-ACP dehydratase

Sas37 248 Cal37 [Streptomyces calvus] 100% beta-ketoacyl-ACP reductase

Sas38 178 Cal38 [Streptomyces calvus] 100% tRNA synthetase

Sas39 318 Cal39 [Streptomyces calvus] 99% ABC transporter

Sas40 280 Cal40 [Streptomyces calvus] 100% multidrug ABC transporter

permease

Orf(-1) 319 WP_040907349.1

[Streptomyces griseoflavus]

91% transcriptional regulator

Orf(-2) 358 KES07412.1 [Streptomyces

toyocaensis]

81% polyprenyl diphosphate synthase

Orf(-3) 717 WP_004931928.1

[Streptomyces griseoflavus]

93% putative drug exporter

5.3.4.2 Gene disruption of sas13 and sas16

The molecular structure of WS9326A contain three nonproteinogenic amino acids: A N-

methyl-(E)-dehyrotyrosine, a Phenylalanine with D-configuration and a Threonine with L-allo-

configuration. By bioinformatic analysis, within the WS9326a biosynthesis gene cluster, genes

sas13, sas15 and sas16 were predicted to be involved in the formation of N-methyl-(E)-

140

dehyrotyrosine residue. According to a BLAST analysis, Sas16 (407 amino acids) has 97% amino

acid identity to the cytochrome P450-SU2 (WP_004931872.1) in the strain of streptomyces

griseoflavus, but its function is unknown. It also shares 40% identical amino acids with an

epothilone b hydroxylase from Streptomycres sp. AA4 (Accession no. EFL04897.1), 41%

sequence identity with SceE (Accession no. ANH11400.1) and SceD (Accession no.

ANH11399.1) from the genome of Streptomyces sp. SD85 and 38% identical amino acids with

the cytochrome P450 hydroxylase from Saccharopolyspora erythraea NRRL 2338 (Accession

no. CAM02704.1), but all of their precise function remain unclear.

Moreover, Sas13 is predicted to be a 3-hydroxyacyl-ACP dehydratase (Streptomyces

griseoflavus Tu4000, 95% identity) base on the protein sequence alignment. Accordingly, we

hypothesized that the formation of the double bond in 2,3-dehydrotyrosine is formed by a

sequence of hydroxylation and dehydration catalyzed by Sas16 and Sas13, respectively.

In order to verify our postulation, we performed the gene disruption experiments by single

crossover. Genes sas16 and sas13 in the gene cluster of WS9326A was cloned and the

corresponding gene disruption vector (pKC1132-SAS16 and pKC1132-SAS13) were

constructed (Figure 5. 12). Both genes, sas16 and sas13, were disrupted in S. asterosporus

DSM 41452 by single crossover, respectively (section 5.2.5 and 5.2.6). The resulting mutant S.

asterosporus DSM 41452::pKC1132-SAS16 and S. asterosporus DSM 41452::pKC1132-SAS13

were screened on the basis of a apramycin sensitive and was further verified by PCR (section

5.2.5 and 5.2.6)(Figure 5. 12).

The validated defect mutant strains were fermented, the resulting broth were extracted with

ethyl acetate. HPLC-ESI/MS analysis (Figure 5. 13) of the ethyl acetate extracts revealed that

the sas16 inactivated mutant stop produce any WS9326A derivatives, in contrast the HPLC

analysis of the crude extracts showed that the sas13 disrupted mutant strain still keep the

capability of producing WS9326A. The gene disruption experiments demonstrated the gene

sas16 encoding a putative P450 cytochrome may be involved in the biosynthesis of WS9326A.

141

A

B

Figure 5. 12. Inactivation of the gene sas16 and sas13 in S. asterosporus DSM 41452 via single crossover. (A)

Schematic representation of plasmid pKC1132-SAS16 and the agarose gel exhibits the verification of resulting

mutant, showing amplification of a 3.2 kb PCR fragment (using the primers Vsas16-F and Apra-R) in mutant (lane

1), in comparison with the wild type (lane 2); (B) Schematic representation of plasmid pKC1132-SAS13 and the

agarose gel exhibits the verification of resulting mutant, showing amplification of a 3.1 kb PCR fragment (using

the primers Vsas13-F and Apra-R) in mutant (lane 1), in comparison with the wild type (lane 2).

Figure 5. 13. The HPLC chromatogram of the ethyl acetate extracts of the culture broth of the Wildtype strain,

the mutant strain S. asterosporus DSM 41452::pKC1132-SAS13 and S. asterosporus DSM 41452::pKC1132-SAS16.

Asterisk labelled peak refers to compound WS9326A.

142

5.3.4.3 Generation of gene Δsas16 mutant in S. asterosporus DSM 41452

For further confirmation of the exact function of gene sas16 in the biosynthesis of the methyl-

N-dehydroxytyrosine, and avoiding the possible deleterious upstream polar effects of the

single cross-over mutations, the targeted gene sas16 was deleted by replacing sas16 with an

antibiotic resistant marker gene.

In-frame gene deletions were performed following the REDirect protocol. Firstly, a 4472bp

DNA fragment including gene sas16 and its flanking regions was amplified from the genome

of S. asterosporus DSM 41452 by PCR using primers SAS16F and SAS16R. The PCR product was

ligated into the EcoRV-digested pBluescript SK (-) to yield pBSK-SAS16. The loxP-site-flanked

apramycin resistance cassette from plasmid pLERECJ was amplified with the pair of primer

SAS16-ApraF and SAS16-ApraR. The resulting amplicon was used to replace the coding

sequence of Sas16 in pBSK-SAS16 by gene recombination in E. coli BW25113 cell containing λ

RED plasmid pIJ790, yielding pBKS-SAS16::aac(3)IV. The latter was amplified using primers

SAS16F and SAS16R, the resulting fragment was cloned into the EcoRV-digested pKGLP2-GusA

to afford pKGLP2-GusA-SAS16::aac(3)IV (Figure 5. 14).

S. asterosporus DSM 41452 was conjugated with E. coli ET12567 (pUZ8002) harboring plasmid

pKGLP2-GusA-SAS16::aac(3)IV, the correct exconjugants carrying plasmid pKGLP2-GusA-

SAS16::aac(3)IV were screened for resistance against apramycin (50 ug/ml). To generate the

mutant strain containing a doube-crossover, initial conjugants were incubated at 28°C for 4

days and then screened for apramycin resistance and hygromycin sensitivity. Replacement of

sas16 with aac(3)IV in S. asterosporus DSM 41452 Δsas16:: aac(3)IV was confirmed by PCR

using primers SAS16F and SAS16R (Table 5. 1) (Figure 5. 14). The Cre recombinase expression

plasmid pUWLCre was then introduced into S. asterosporus DSM 41452 Δsas16::aac(3)IV to

eliminate aac(3)IV gene from its genome. The resulting exconjugants resistant to hygromycin

were incubated on MS solid medium plates and selected for the apramycin sensitivity. Then

the hygromycin resistance colony was cultured into the TSB medium at 37°C, and was

repeatedly passaged three times. Then hygromycin-sensitive colonies were tested for the loss

of plasmid pUWLCre. The correct in-frame excision of aac(3)IV gene from the S. asterosporus

DSM 41452 Δsas16 genome was confirmed by PCR using primers SAS16F and SAS16R (Table

5. 1) (Figure 5. 14).

143

A B

Figure 5. 14. (A) Plasmid diagram of pKGLP2-GusA-SAS16::aac3(IV); (B) Schematic representation of the in-frame

deletion of sas16 in S. asterosporus DSM 41452. The mutant Δsas16 was constructed by a double crossover

recombination between plasmid pKGLP2-GusA-SAS16::aac3(IV) and S. asterosporus DSM 41452 chromosome, a

1178 bp fragment containing sas16 gene was substituted with a 1045 bp DNA fragment containing loxp site and

aac(3)IV.

A B

Figure 5. 15. (A) The PCR verification of the sas16 deletion mutant, the general location of PCR amplified

fragment to verify the gene replacement are indicated by number; lane 1 refer to the PCR product using S.

asterosporus DSM 41452 chromosome as template (primers SAS16F and SAS16R), Lane 2 refer to the PCR

fragment using S. asterosporus DSM 41452 Δsas16 genome as template (primers SAS16F and SAS16R). (B) The

LC/MS extracted ion chromatogram(EICs) for [M-H]- ions corresponding to WS9326A, WS9326B, SY11, SY12 in

organic extracts of S. asterosporus DSM 41452ΔSAS16.

Subsequently, strains S. asterosporus DSM 41452ΔSAS16 was cultured in the productive SG

liquid medium. After three days cultivation, the harvested culture broth was extracted by

ethyl acetate, and the crude extract was dissolved in methanol. The secondary metabolites

profile of S. asterosporus DSM 41452ΔSAS16 was analyzed by LCMS, the resulting HPLC

chromatogram (Figure 5. 15) only shown the peaks representing WS9326B and SY12,

suggesting that the production of compounds WS9326A and SY11 were blocked in the sas16

defect mutant, however that doesn’t influence the production of WS9326B and SY12.

144

5.3.4.4 Complementation of S. asterosporus DSM 41452 ΔSAS16 strain with Sas16

In order to further verify the function of Sas16 in the biosynthesis of WS9326A, we decide to

complement a sas16 gene expression plasmid pTESa-SAS16 into the sas16 defect mutant S.

asterosporus DSM 41452 ΔSAS16 for complementation analysis.

To construct plasmid pTESa-SAS16, a 1272bp fragment carrying entire sas16 with its upstream

48bp region was amplified from S. asterosporus DSM 41452 genomic DNA using primers KpnI-

RBSp450-F and EcoRI-p450-R. The 1272bp fragment was firstly cloned into EcoRV-digested

pBluescript SK(-) to yield pBSK-SAS16. Then this pBSK-SAS16 was digested with KpnI and EcoRI,

and then DNA fragment was ligated to pTESa with a constitutive promoter ermEp, yielding

plasmid pTESa-SAS16 (Figure 5. 16). Then the plasmid was confirmed through restriction

digestion, S. asterosporus DSM 41452 exconjugants carrying the plasmid pTESa-SAS16 were

screened for resistance against apramycin (50 ug/ml) by spore conjugation from E. coli

ET12567 (pUZ8002) (Figure 5. 16).

A B C

Figure 5. 16. (A)Plasmid diagram of pTESa-SAS16; (B) The digestion result of plasmid pTESa-SAS16 by KpnI and

EcoRI; C) The LC/MS extracted ion chromatogram(EICs) for [M-H]- ions corresponding to WS9326A, WS9326B,

SY11, SY12 in organic extracts of S. asterosporus DSM 41452ΔSAS16::pTESa-SAS16.

Through LC-MS analysis on its secondary metabolites of mutant strain S. asterosporus DSM

41452ΔSAS16::pTESa-SAS16 (Figure 5. 16), we found that the extracted ion peaks of WS9326A

and SY11 show up again in the organic extracts of S. asterosporus DSM 41452ΔP450::pTESa-

SAS16. This result further confirmed that the gene sas16 in the gene cluster for the

biosynthesis of WS9326As is involved in the formation of the double bond.

5.3.4.5 Gene deletion of N-methyltransferase(MTase) encoding gene in Module 2 in S.

asteroporus DSM 41452

Many of the non-ribosomal peptides consists of N-, O- or C-methylated amino acids, those

methylation are helpful for the structural diversity and abundant bioactivity of natural product

from microorganism, as the case of cyclosporin exemplified at section 1.5.3. Typically, N-

145

methylation takes place before peptide bond formation where these intergral N-MTase

domain catalyze the transfer of the S-methyl group of S-Adenosyl methionine (AdoMet) to the

amide nitrogen of the thioiesterfied amino acid, releasing S-adenosyl-L-homocysteine as a

reaction product. N-methylation of the amino acid take places while the amino acid residue is

tethered to the 4‘-phosphopantetyheine arm of the peptidyl carrier protein(PCP) (Winn et al.

2016).

There are 6 genes encoding nonribosomal peptide synthetases (Sas17, Sas18, Sas19, Sas22,

and Sas23) for the peptide backbone assembly of WS9326A (Figure 5. 11). Among them SAS17

consists of two set of C-A-PCP modules, and the second module contains a N-

methyltransferase domain between the A and PCP domain. This module is responsible for the

formation of N-methyl-dehydrotyrosine in WS9326A. This nonproteinogenic amino acid N-

methyl-dehydrotyrosine residue was generated through two steps of chemical modification:

N-methylation catalyzed by the internal N-methyltransferase domain in module 2 of SAS17

and a α, β-dehydrogenation modified by the P450 cytochrome Sas16. Those unusual

continuous enzymatically catalytic modifications arouse our interests and some questions: (1)

Does the N-methylation modification of tyrosine take place prior to the dehydrogenation or

after? If after, does the bulky methyl group tethered at the N group influence the reaction

efficiency of dehydrogenation of tyrosine? (2) Whether the pre-tailoring N-methylation of

tyrosine in WS9326A is necessary for the substrate recognition and acceptor site binding of

the downstream C-domain? will it influence the biosynthesis assembly when the methyl motif

was deleted?

A B

C A PCP NMT

C A PCPC A PCP

C A PCP E

module 2module 1 module 3

module 4

C A PCPC A PCP

C A PCPC A PCP E

module 2module 1 module 3 module 4

SAS17 SAS18

SAS17 SAS18

Figure 5. 17. (A) Diagram of plasmid for MTase domain deletion and (B) the schematics representing the NRPS

domain organization in the WT strain and the ΔMTase mutant strain.

To answer those questions we are interested, we decided to remove this N-methyltransferase

domain encoding gene by in-frame gene deletion method. A 4860bp DNA fragment including

146

the domain encoding methyltransferase and its flanking region in gene sas17 was amplified

from the genome of S. asterosporus DSM 41452 by PCR using primers Nmet4800bpF and

Nmet4800bpR (Table 5. 1). The PCR product was ligated into the EcoRV-digested pBluescript

SK(-) to yield pBSK-Nmet. The loxP-site-flanked apramycin resistance cassette from plasmid

pLERECJ was amplified by PCR using primes Nmet-ApraF and Nmet-ApraR. The resulting

amplicon was used to replace the methyltransferase encoding sequence of pBSK-Nmet by

recombination in E. coli BW25113 cell containing λ RED plasmid pIJ790, yielding pBSK-

Nmet::aac(3)IV. The latter was amplified by PCR using primer Nmet4800bpF and

Nmet4800bpR. The resulting fragment was cloned into the EcoRV-digested pKGLP2-GusA to

generate pKGLP2-GusA-Nmet::aac(3)IV (Figure 5. 17).

The wildtype S. asterosporus DSM 41452 was conjugated with E. coli ET12567 (pUZ8002)

harboring plasmid pKGLP2-GusA-Nmet::aac(3)IV, then the correct transconjugants carrying

plasmid pKCLP2-gusA Nmet::aaa(3)IV were selected by antibiotic resistance screening against

apramycin (50 ug/ml). In order to generate the mutant strain with doube-crossover, initial

conjugants were incubated at 28°C for 4 days, and then screened for the apramycin resistance

and hygromycin sensitivity. Replacement of MTase domain encoding gene with aac(3)IV in S.

asterosporus DSM 41452 Δ MTase:: aac(3)IV was confirmed by PCR using primer

Nmet4800bpF and Nmet4800bpR(Figure 5. 18). The Cre recombinase expression plasmid

pUWLCre was then introduced into S. asterosporus DSM 41452ΔMTase::aac(3)IV to eliminate

aac(3)IV gene from its genome. The resulting conjugants resistant to hygromycin were

incubated on MS solid medium plates and selected for the apramycin sensitivity. Then the

hygromycin resistance colony was cultured into liquid TSB medium at 37°C, then it was

repeatedly passaged three times. Hygromycin-sensitive colonies were tested for target gene

deletion. The correct excision of aac(3)IV gene from the genome of S. asterosporus DSM 41452

ΔMTase genome was confirmed by PCR using primer Nmet4800bpF and Nmet4800bpR(Figure

5. 18).

147

A B

Figure 5. 18. Schematics representing the construction (A) and PCR verification (B) of the MTase encoding gene

deletion mutant strain. The mutant ΔMTase was constructed by a double crossover recombination between

plasmid pKGLP2-GusA-Nmet::aac3(IV) and S. asterosporus DSM 41452 chromosome, which substitutes a 656 bp

MTase encoding gene fragment with a 1045 bp DNA fragment containing loxp site and aac(3)IV. The general

location of PCR amplified fragment to verify the gene replacement are indicated by number; lane 1 refer to the

PCR product using S. asterosporus DSM 41452 chromosome as template (primers Nmet4800bpF and

Nmet4800bpR), Lane 2 refer to the PCR fragment using S. asterosporus DSM 41452 ΔMTase genome as template

(primers Nmet4800bpF and Nmet4800bpR).

Figure 5. 19. HPLC chromatograms of S. asterosporus DSM 41452 and its mutant S. asterosporus DSM 41452

ΔMTase (monitored at 254nm wavelength). The peak corresponding to WS9326A is marked with a star.

The crude extracts of S. asterosporus DSM 41452 ΔMTase was analyzed by HPLC-MS, the

resulting HPLC chromatogram (Figure 5. 19) shows that the production of WS9326A was

disrupted in the mutant strain. This implies that N-methylation of tyrosine is important for

downstream substrate recognition by the condensation domain.

The result of site-specific gene deletion of N-methyltransferase encoding gene in sas17 shown

that demethylation of tyrosine in the prematured peptides of WS9326A seems influence the

substrate recognization of the downstream condensation, or the other possible explanation

is that the gene deletion of N-methyltransferase encoding gene changes the intact

148

configuration of the corresponding nonribosomal peptide synthetases, the resulting

configuration change disrupts the normal enzymatic function.

5.3.4.6 N-MTase protein expression and purification

N-methyltransferase (MTase) domain in module 2 encoded by sas17 consists of 222 amino

acids, belongs to the typical S-adenosylmethionine-dependent methyltransferases (SAM or

AdoMet-MTase) superfamily. It shows 96% sequence coverage and 43% identity with protein

McnC (Accession number WP_012268171.1) from Microcystis aeruginosa, and high identity

with non-ribosomal peptide synthetase from S. toyocaensis (89%, WP_051858700.1) and S.

griseoflavus (92%, WP_004931873.1). In addition, this MTase domain show 37% identity with

the protein structure of Chain A in Methyltransferase Ccbj (PDB-Id 1: 4HGY) from

Streptomyces Caelestis (Bauer et al. 2014). SAM-dependent methyltransferase CcbJ catalyzes

the methylation of the N-atom of the proline moiety in compound celesticetin biosynthesis

(Bauer et al. 2014).

To further characterize the substrate specificity of the MTase domain, and demonstrate the

catalytic reaction order between methylation and dehydrogenation in the biosynthesis of N-

methyldehydrotyrosine in WS9326A, we decided to set up an in vitro enzyme assay against

this MTase. For this purpose, series of MTase domain recombinant vectors were constructed,

including plasmid pET28-Nmet and pET24-Nmet. The gene fragment encoding MTase domain

were amplified from S. asterosporus DSM 41452 by PCR using primers NmetF and NmetR. The

PCR product firstly was ligated into EcoRV-digested pUC19 plasmid to yield pBSK-Nmet. Then

the fragment was digested from pBSK-Nmet and ligated into pET28a(+) and pET24 to yield

pET28a-Nmet and pET24-Nmet, respectively. Afterwards, those plasmids were transferred

into different E. coli strains for condition optimization of protein expression. Unfortunately,

the protein expression test showed that the N-methyltransferase domain can’t be expressed

into soluble protein when pET28a and pET24 were used as expression plasmid, so as to

enhance the solubility of this MTase protein, we finally turn to the help of fusion protein

vector pET-Trx (detailed information sees section 6.2.2).

The gene fragment encoding this MTase domain was amplified from S. asterosporus DSM

41452 by PCR using primers pET-Trx-NmetF and pET-Trx-NmetR. The PCR product first was

ligated into EcoRV-digested pBluescript KS(-) plasmid to yield pBSK-Trx-Nmet. Then the

fragment was digested and cloned into pET-Trx to yield plasmid pET-Trx-Nmet. The

thioredoxin-MTase fusion protein sequence contains 351 amino acid residues, and the

calculated protein weigh is 38.02 KDa. The fusion protein sequence is shown below (the

149

sequence of thioredoxin is labelled in italic, the underlined part represents the sequence of

N-methyltransferase domain).

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGI

PTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGSENLYFQSAMVLPEEEMRAWRAATVERILDLRPKRVLEI

GVGAGLIMAPVAPHVELYWGADLSGTVIETLRRQTAADPVLADRTRFTAAPAHSLDGVPEGTFDTVVINSVAQYFP

SVDYLTEVIAKAFALLGDTGAVFLGDLRDLRLLRCMRAGVHRVAHPGDSPAAARAAVDRAVERETELLVDPGLFDTL

AGTLDGFGGVDIRIKQGAYDNELSRYRYDVVLHKRPALEHHHHHH

For optimizing the condition of protein expression, plasmid pET-Trx-Nmet was transformed

into strains including E. coli BL21 star(DE3), E. coli BL21(DE3) pLysS, E. coli BL21 Rosetta, and

E. coli BL21 Codon Plus RP(pL1SL2). Different cultivation conditions including temperature,

culture time, and addition amount of IPTG were optimized. As the SDS-PAGE (Figure 5. 20A)

analysis shown that a clear band with a size of about 38 KDa was detected in lane 2, in which

the protein sample was from the supernatant of the lysed E. coli BL21star(DE3) cell, and the

corresponding cultivation condition is 0.5mM IPTG supplementation for protein induction,

then incubation at 28°C overnight for protein expression.

A B

Figure 5. 20. SDS-PAGE analysis of N-MTase domain expression test and manual Ni-NTA purification. (A)

Cultivation method optimization of N-MTase domain expression. Samples from left to right lanes: supernatant

(1) and cell debris (4) from E. coli BL21 star(DE3):: pET21-Trx-Nmet cultured in Auto Induction Media induced by

IPTG with 0.5mM, incubated at 18°C overnight; supernatant (2) and cell debris (5) from E. coli BL21 star(DE3)::

pET21-Trx-Nmet cultured in LB medium induced by IPTG with 0.5mM, incubated at 18°C overnight; supernatant

(3) and cell debris (6) from E. coli BL21 star(DE3):: pET21-Trx-Nmet cultured in LB medium induced by IPTG with

0.5mM, incubated at 28°C overnight; supernatant (7) and cell debris (10) from E. coli BL21 Codon Plus

RP(pL1SL2):: pET21-Trx-Nmet cultured in Auto Induction Media induced by IPTG with 0.5mM, incubated at 18°C

overnight; supernatant (8) and cell debris (11) from E. coli BL21 Codon Plus RP(pL1SL2):: pET21-Trx-Nmet

cultured in LB medium induced by IPTG with 0.5mM, incubated at 18°C overnight; supernatant (9) and cell debris

(12) from E. coli BL21 Codon Plus RP(pL1SL2):: pET21-Trx-Nmet cultured in LB medium induced by IPTG with

0.5mM, incubated at 28°C overnight; (B) SDS-PAGE analysis of the fractions from Ni-NTA column for MTase

domain purification. Samples from left to right lanes: fraction eluted by buffer A; fraction eluted by buffer A with

10mM Imidazol; fraction eluted by buffer B.

150

For purifying this MTase domain, cell pellets from 1L strain culture were harvested and lysed

using French pressure. The resulting cell debris was removed by centrifugation. The

supernatant was subjected to a 2-ml Ni-NTA Superflow column that had been pre-equilibrated

in buffer A. The column first was eluted with 5 CV of buffer A, then with 1.5 CV of buffer B. All

fractions were collected and analyzed by SDS-PAGE (Figure 5. 20B). The SDS-PAGE analysis

result shown that fraction 3 eluted by buffer B exhibited a clear band with similar protein size

as the calculated.

5.4 Conclusion

In this chapter we focus this new potential strain S. asterosporus DSM 41452, which is capable

of producing a potent tachykinin receptor antagonist WS9326A and WS9326B with the high

yield up to 500 mg per liter. By various kinds of classic chromatography and spectroscopy

methods, we first isolated and characterized two new WS9326A derivatives, termed WS9326F,

WS9326G, with four known analogs WS9326A, WS9326B, WS9326D, WS9326E from S.

asterosporus DSM 41152. Among them the linearized WS9326F and WS9326G were

structurally elucidated base on NMR and MS/MS fragmentation. Both compounds are most

probably released from the assembly line following the hydrolysis mechanism.

We performed Marfey’s analysis of WS9326A and attempt to determine the stereochemical

configurations of all the amino acids. Unfortunately, were not able to determine the

configuration of the β-carbon of threonine due to the insufficient resolution of HPLC,

therefore we are not absolutely certain if we have L-allo-Thr in WS9326A. We now show the

structures of the compounds with explicit stereochemistry where this is known. We

designated the same configuration of WS9326A derivatives since they share the same

biosynthetic machinery.

Unexpectedly, from a annimycin-disrupted mutant S. asterosporus DSM

41152::pUC19Δ3100spec, we found two new WS9326A derivatives SY11 and SY12 with

molecular weight 1134 and 1136. Based on the data of NMR spectra and MS/MS

fragmentation analysis result, we were able to elucidate the cyclicpeptide scaffold of

compound SY11. Moreover, it is strongly demonstrated that the structure divergence

between SY11 and SY12 was resulted from the α, β-dehydrogenation happening at the

tyrosine residue as the structural difference between WS9326A and WS9326B. However, due

151

to the serious signal overlapping and the lack of key correlation, we failed to sketch their

complete structure temporarily. For solving the problem, next step we will turn to the help of

chemical derivatization.

From the perspective of biosynthesis, the occurrence of SY11 and SY12 suggest the high

tolerance and the plasticity of WS9326A nonribosomal peptide complex synthetases during

the molecular assembly. Another example which implied this feature is compound

mohangamide (structure see Figure 1.10). Mohangamides are the dimerized product of

WS9326A, they were recently isolated from strain Streptomyces sp. SNM55 (Bae et al. 2015).

The mohangamides are the largest dilactone-tethered, dimeric cyclic peptides discovered

from microorganism so far. According to literature reported (Bae et al. 2015), we postulated

that the dimerization could be catalyzed by a putative type II thioesterase encoded by sas20

in WS9326A gene cluster, unluckily, we haven’t found the presence of dimeric peptides in our

strain S. asterosporus DSM 41452. The explanation could attribute to the cultivation condition

(media, trace element, PH control, etc), or maybe mohangamides were biosynthesized via a

total different machinery. It’s interesting question awaiting to be answered in the future.

By means of genome sequencing, bioinformatics analysis and gene inactivation, we identified

and validated the corresponding WS9326A biosynthetic gene cluster in S. asterosporus DSM

41452. Gene deletion studies demonstrated a critical role of N-methylation in WS9326A

biosynthesis, Gene deletion of sas16 resulted in the abolishment of WS9326A, but the

resulting mutant strain S. asterosporus DSM 41452Δsas16 still keep the ability of producing

WS9326B where there is no dehydrogenation present on the N-methyl tyrosine residue, after

complementation, the production of WS9326A was restored, which demonstrated the role of

a P450 monooxygenase encoded by sas16 in forming a α, β-dehydrotyrosine group. Through

detailed MS analysis on the LC/MS extracted ion chromatogram (EICs), we didn’t find the [M-

H]- ion peak referring to the WS9326A derivative with β-hydroxy-tyrosine residue, so we

postulated that Sas16 are responsible for the dehydrogenation of the tyrosine amino acid in

the biosynthesis of WS9326As.

One derivative of WS9326A, WS9326D was reported to have anitfilarial activity (Yu et al. 2012),

our finding extends the range of activities of the WS9326A series to antimalarial activity (albeit

weak). In an antimalarial assay, except for an annimycin analogue SY10, none of WS9326A

congeners exhibited significant inhibitory activity against the three P. falciparum parasite lines

tested. There could be a number of reasons resulting in those nullity. In our active assay

system, tested compounds have to cross the erythrocyte plasma membrane to impact the

152

parasite resides inside the erythrocyte. WS9326As are very hydrophobic compounds, barely

soluble in aqueous solution. Thereby the membrane permeability of WS9326As could be a key

factor which lead to their low efficiency. The activity of WS9326A and its derivatives against

E. coli and B. subtilis also have been tested, unfortunately none of them shown significant

antibiotic activity.

In terms of the chemical structure, either the linearized WS9326A derivatives WS9326D,

WS9326E, WS9326F and WS9326G or the cyclized analogues WS9326A and SY11 they all

contain the dehydrotyrosine residue, suggesting that this dehydrogenation modification

might occurs prior to the release of matured peptide.

We expect these results set the stage for engineering S. asterosporus DSM 41452 for the

production of active novel WS9326A analogs in the future. The study on WS9326As may

contribute to develop and refine a new generation of analogues with significant antagonist

activity.

5.5 Appendix

Table 5. 4. Predicted highly conserved core motifs of A domain binding pockets in NRPSs within

the SAS cluster.

Domain Residues in the Binding Pocket Amino acid residues

235 236 239 278 299 301 322 330 Predicted in WS9326As

SAS17-A1 D F W N I G M V Thr Thr/Ser

SAS17-A2 D A S T T A A V Tyr Tyr

SAS18-A1 D A Y T W G A V Leu Leu

SAS18-A2 D A W T V A A V Phe Phe

SAS19-A1 D V W H L G X X Ser Ser

SAS19-A2 D V W H L S L V Ser Ser

SAS22-A D F W S V G M V Thr Thr

SAS23-A D L T K V G E V Asn Asn

Note: The analysis was carried out using NRPSpredictor and AntiSMASH.

153

Table 5. 5. List of putative biosynthesis genes involved in the biosynthesis of the side chain of

the WS9326As and their homologues in the biosynthesis gene cluster of Skyllamycin (Pohle et

al. 2011).

No. SAS Homologous gene in

Skyllamycin gene

cluster

Putative Protein Function Identity/Coverag

e (%)

1 sas21 --- Short chain dehydrogenase/reductase ---

2 sas24 --- Oxidoreductase ---

3 sas27 sky5 Isomerase 62/99

4 sas28 sky4 Phytoene

dehydrogenase/oxidoreductase

56/96

5 sas29 sky16 Acyl-carrier protein 71/96

6 sas30 sky17 3-oxoacyl-ACP synthase 77/96

7 sas31 sky18 3-oxoacyl-ACP synthase 62/100

8 sas32 sky19 3-oxoacyl-ACP synthase 54/99

9 sas33 sky22 3-oxoacyl-ACP synthase 53/98

10 sas34 sky23 Acyl-carrier protein 55/86

11 sas35 sky24 3-oxoacyl-ACP dehydratase 53/85

12 sas36 sky25 3-oxoacyl-ACP dehydratase 46/99

13 sas37 sky26 3-oxoacyl-ACP reductase 68/100

14 sas7 --- 3-oxoacyl-ACP synthase ---

15 sas8 --- 3-oxoacyl-ACP synthase ---

16 sas9 --- Acyl-carrier protein ---

17 sas25 sky20 Hydrolase ---

18 sas26 sky21 Thioesterase ---

19 --- sky11 Carboxyltransferase ---

20 --- sky13 ACP-acyltransferase ---

Table 5. 6. Summary of NMR Data for WS9326A and WS9326F in DMSO-d6.

position WS9326A WS9326F

δH δC δH δC HMBC

Acyl 1

2

3

4

5

6

7

8

6.69,1H,d(15.5)

7.42,1H,d(15.5)

7.20,1H,m

7.26,1H

7.27,1H

7.20,1H

165.7

123.0

127.7

133.5

126.5

127.2

128.8

130.1

6.88, 1H, d(15.1)

7.53, 1H, d(15.1)

-

-

-

-

165.6

128.5

137.4

165.6

154

9

10

11

12

13

14

6.50,1H,d(11.4)

5.83,1H,dt(11.4,7.4)

1.98,2H,m

1.36,2H,m

0.80,3H,t(7.2)

139.1

127.4

134.7

30.5

22.7

14.2

6.53, 1H, d(11.4)

5.79, 1H, dt(11.4, 7.4)

1.98, 2H, m

1.34, 2H, m

0.79, 3H, t(7.3)

127.1

134.5

30.4

22.1

14.2

30.4

127.1,

134.5,

22.1

134.5,

1Thr NH

α

β

γ

C=O

8.73,1H,d(9.4)

5.33,1H,t(9.8)

5.02,1H,dq(9.8,6.2)

1.15,3H,d(6.0)

53.6

73.9

17.3

169.4

-

4.29, 1H

3.69, 1H

1.05, 3H, brs

52.4

66.9

20.3

170.8

52.4, 66.9

2ΔM

eTyr

NMe

α

β

1

2,6

3,5

4

C=O

2.98,3H,s

6.13,1H,s

7.39,2H,d(8.6)

6.58,2H,d(8.6)

34.9

128.4

132.1

123.4

132.3

115.4

158.5

166.1

2.87, 3H,s

6.63, 1H, s

7.27, 2H, d(8.0)

6.65, 2H, d(8.0)

34.8

130.1

132.3

124.0

132.2

115.4

158.5

165.2

170.8

165.2,

132.2

130.1,

132.3,

158.5

124.0,

3Leu NH

α

β

γ

σ

C=O

9.25,1H.br.s

4.07,1H,m

1.26,2H,m

0.85,1H,m

0.76,3H

0.63,3H

54.1

39.7

24.0

22.6

23.3

172.5

-

4.49

1.23, 2H, m

0.81, 1H, m

0.70, 3H

0.65,3H

49.6

40.1

24.0

22.6

23.3

171.8

49.6, 40.1

4Phe NH

α

β

1

2,6

3,5

4

C=O

9.17,1H,d(8.0)

4.33,1H,m

3.28,1H,m

2.72,1H,m

7.33,2H,d

7.28,2H,d

56.5

36.8

139.1

129.4

128.4

127.2

170.6

-

4.62,1H, m

3.11, 1H, m

2.65, 1H, m

7.35, 2H, m

7.27, 2H, m

54.6

38.4

138.1

129.4

128.4

127.2

-

138.1

138.1,

127.2

5Thr NH

α

β

7.59,1H,d(9.8)

4.34,1H,m

4.28,1H,m

56.8

68.6

4.23, 1H, m

4.02, 1H, m

58.7

67.5

170.4

155

γ

OH

C=O

0.63,3H

5.18,1H,d(3.0)

22.7

170.4

0.92, 3H

-

22.7

170.4

6Asn NH

α

β

γ(C=O)

γ(NH2)

C=O

8.34,1H,d(7.3)

4.44,1H,m

2.45,2H

6.93,1H, 7.30,1H

51.3

37.2

171.6

172.0

-

3.17, 1H, m

2.45, 2H

-

48.8

37.2

171.2

-

48.8,

171.2

7Ser NH

α

β

OH

C=O

8.49,1H,d(9.5)

4.34,1H

3.15,1H

3.24,1H

4.79,1H,br

56.9

61.5

169.3

Note: Signal assignments based on the 1D and 2D NMR data.

Figure 5. 21. HPLC-MS analysis (Extracted ion chromatogram) of compounds WS9326A, B, D, E, F, G, SY11 and

SY12 from the cultures of the wildtype S. asterosporus DSM 41452 and its mutant S. asterosporus DSM 41452::

pUC19Δ3100spec

156

Figure 5. 22. Comparison of the genes organization for the cinnamoyl side chain biosynthesis in the SAS and

SKY gene clusters (highlighted in yellow).

Figure 5. 23. Comparative HPLC Profiles analysis of extracts from the cultures of S. calvus ATCC 13382 and S.

asterosporus DSM 41452. The peak of WS9326A is marked with an arrow.

Figure 5. 24. UV/Vis spectrum and HR-ESIMS spectrum of WS9326F.

222 288UV, 11.12min #3332

669.3209

968.4750

1936.9522

MM-s6_GC1_01_1465.d: +MS, 11.12min #13370

500

1000

Intens.

[mAU]

0.0

0.5

1.0

1.5

6x10

Intens.

250 500 750 1000 1250 1500 1750 2000 2250 m/z

200 250 300 350 400 450 500 550 Wavelength [nm]

157

Figure 5. 25. UV/Vis spectrum and HR-ESIMS spectrum of WS9326G.

Figure 5. 26. ESI-MS/MS fragmentation of WS9326A.

Figure 5. 27. ESI-MS/MS fragmentation of WS9326B.

218

288

UV, 10.92min #3272

669.3248

954.4644

1908.9351

MM-s5_GB8_01_1464.d: +MS, 10.92min #13130

200

400

600

Intens.

[mAU]

0.0

0.5

1.0

1.5

6x10

Intens.

250 500 750 1000 1250 1500 1750 2000 2250 m/z

200 250 300 350 400 450 500 550 Wavelength [nm]

158

Figure 5. 28. ESI-MS/MS fragmentation of WS9326D.

Figure 5. 29. ESI-MS/MS fragmentation of WS9326E.

159

Figure 5. 30. H NMR spectrum of WS9326A.

Figure 5. 31. C NMR spectrum of WS9326A.

160

Figure 5. 32. H NMR spectrum of WS9326F.

Figure 5. 33. C NMR spectrum of WS9326F.

161

Figure 5. 34. HSQC spectrum of WS9326F.

Figure 5. 35. HMBC spectrum of WS9326F.

162

Chapter 6. Biochemical characterization of Cytochrome P450

Sas16

6.1 Research Background

The double bond formation in polyketide is widely recognized to be catalyzed by dehydration

following a ketoreduction reaction (Keatinge-Clay 2012; He et al. 2014). Certainly, some

exceptions could happen in some special case. For example, TrdE, a glycoside hydrolase,

catalyzes the formation of the double bond in an unusual way during the Tirandanmycins

biosynthesis (Mo et al. 2012). In the case of phoslactomycin, the Δ2,3 double bond is installed

by a post-tailoring fashion catalyzed by PlmT2, a putative NAD-dependent

epimerase/dehydratase (Palaniappan et al. 2008).

In NRPS kinds of compound, the nonproteinogenic aromatic amino acid with α, β-

dehydrogenation also commonly present in a diverse range of natural product. So far, in

addition to WS9326As (including a α, β-dehydrotyrosine), other nonribosomal peptides

harboring α ,β-dehydroamino acid include: calcium-dependent lipopeptide antibiotics (CDA)

(including a 2’,3’-dehydrotryptophan) (Baltz et al. 2005), Telomycins (including a 2’,3’-

dehydrotryptophan) (Fu et al. 2015), Jahnellamides (including a 2’,3’-dehydrotryptophan)

(Plaza et al. 2013), Dityromycin (including a dehydrotyrosine) (Teshima et al. 1988),

Miuraenamides (Ojika et al. 2008; Yamazaki et al. 2015) and Tentoxin (including a α, β-

dehydrophenylalanine) (Li, Han et al. 2016).

Calcium-dependent-antibiotic (CDA) are produced by Streptomyces coelicolor A3(2), it belongs

to the family of nonribosomal lipopeptide antibiotics. In the biosynthesis of CDA, it comprises

a number of non-proteinogenic amino acids including a C-terminal (Z)-2’,3’-

163

dehydrotryptophan (Baltz et al. 2005). By isotope feeding labeled tryptophan derivative to the

Trp-His auxotrophic strain Streptomyces coelicolor WH101, Jason Mickledield etal (2007)

demonstrated that the function of (Z)-2’,3’-dehydrotryptophan residue of CDA was generated

through dehydrogenation from an (S)-tryptophanyl precursor. But due to the insufficient

information about the protein homology, the corresponding enzyme that catalyze the

formation of the Z-ΔTrp-residue of CDA is still unknown (Amir-Heidari and Micklefield 2007).

In the gene cluster of Telomycin, gene tem12 was predicted to be a medium chain

hydrogenase/dehydrogenase (MDR)/zinc-dependent alcohol dehydrogenase which was

related with the formation of the (Z)-2,3-dehydrotryptophan residue in Telomycin. However,

the in-frame gene deletion experiments didn’t provide supporting evidence (Fu et al. 2015).

Jahnellamides and Resomycin containing Z-2,3-dehydrotryptophan were isolated from the

myxobacterium Jahnella sp, in silico analysis of genome sequence proposed that the genes

orf8 and orf9 encode putative desaturases in Jahnellamide gene cluster which could be

responsible for the formation of the ΔTrp with similar mechanism as the tryptophan 2ˈ, 3ˈ -

oxidase from Chromobacterium violaceum (Plaza et al. 2013). Tentoxin is a cyclic tetrapeptide

produced by some Alternaria species, it is an inhibitor of the F₁-ATPase in chloroplasts. By

bioinformatic and targeted gene mutagenesis analysis, a NRPS gene (TES) and a cytochrome

P450 gene (TES1) were identified to be involved in the tentoxin biosynthesis in A. alternata.

Gene TES1 was predicted to be involved in the formation of nonproteinogenic residue

dehydrophenylalanine (Me-(Z)- ΔPhe) (Li, Han et al. 2016). Antibiotic Dityromycin was firstly

isolated from a soil-derived Streptomcyes sp. strain No. AM-2504, as a cyclic decapeptide, it

consists of various kinds of nonproteinogenic amino acids including a N-methyl-

dehydrotyrosine residue. so far there is no research about its biosynthetic machinery

(Teshima et al. 1988; Beau et al. 2012). Resormycin exhibiting antimicrobial activity against

phytopathogenic fungi, was isolated from Streptomyces platensis MJ953-SF5. In its chemical

structure, a residue of 4-chloro-3,5-dehydroxyphenylpropenoic acid is integrated into the

backbone, so far there is no report about its biosynthesis mechanism (Igarashi et al. 1997).

164

Figure 6. 1. Chemical structures of NRPS containing the dehydrogenated amino acid

Based on our previous research on the biosynthetic mechanism of WS9326A, gene sas16

encoding a Cytochrome P450 monooxygenase in WS9326A gene cluster from S. asterosporus

DSM 41452 was verified unambiguously to be involved in the formation of the

dehydrotyrosine. However, the direct evidence about the catalytic function of Sas16 was still

absent.

Initially, about the exact biosynthesis machinery of the dehydrotyrosine residue, we proposed

that it was attributed to the combination of a β-hydroxylation catalyzed by Sas16 and a

dehydration reaction catalyzed by Sas13 (Figure 6. 2). However, based on our subsequent

research data, the mutant strain with gene sas13 disruption didn’t influence the production

of WS9326A (see Chapter 5, Section 5.3.4). Moreover, WS9326A derivatives containing β-

hydroxylated tyrosine residue haven’t been detected to date. Therefore, we raised the

postulation of other three biosynthetic machineries which could contribute to the double

bond formation via a direct dehydrogenation reaction (Figure 6. 2). The postulated Route B

resemble the catalytic mechanism of the tryptophan 2ˈ, 3ˈ-oxidase from Chromobacterium

violaceum (Genet et al. 1995) and a tryptophan side chain oxidase II, a hemoprotein from

Pseudomonas (Takai et al. (1984), both of them enzymatically catalyze the dehydrogenation

of tryptophan. By contrast, routes C and D are involved in the formation of imines functional

group and the following proton rearrangement. In route C, Sas16 firstly catalyze the formation

of an imine intermediates, followed by an tautomerization reaction which finally result in the

formation of dehydrotyrosine. In Route D, the dehydrogenation reaction first generate an

165

indolyloazoline intermediate, then form the final product through isomerization (Amir-Heidari

et al. 2007).

Figure 6. 2. Possible mechanism of the dehydrogenation in amino acid residues, figure adapted from (Amir-

Heidari et al. 2007).

For solving all the questions, Sas16 was heterologous expressed for in vitro enzymatic assay

to assess its biochemical role for the biosynthesis of N-methyl-dehydrotyrosine residue in

WS9326A. In addition, we elucidate the protein structure of Sas16 through structural biology

approach to reveal the underlying molecular basis for its catalytic activity.

6.2 Materials and Methods

6.2.1 Primers fragments used in this study

NH2

O

OH

HO

NH2

O

OH

NH

O

OH

HOHO

H2

NH2

O

OH

NH2

O

OH

HO HO

OH

SAS16

SAS

13

H2

H2O

NH

O

O

OH

N

O

O

OH

NH

O

O

OH

Route A

Route B

Route C

Route D

SAS16

SAS16

Hydroxylation

Dehydrogenation

Dehydrogenation

Dehydra

tion

H2SAS16

Dehydrogenation

Table 6. 1. Primers fragments used in this study

Name Sequence

Primer for vector construction of pET28-SAS16

P450pET-F ATAgaattcATGACCGACGCCGAGACG

P450pET-R TCActcgagCTACCAGCCGATCGTCAGCTT

Primer for vector construction of pET-Trx-PCP

pET-Trx-PCP-F CCATGGAGGAGACCATCGCCCGGATCTTC

pET-Trx-PCP-R CTCGAGGGCCGCCGCGGCGATGGC

Primer for vector construction of pET28-SAS13

00140pET-F ATACATATGACCGGGCCCGCC

00140pET-R ATACTCGAGTCAGGAGGCGGTGGTCAGC

Primers for vector construction of pET28-Adomain

A-F CATATGTCGTACGCCGAGCTGGAGGTGCGG

A-R TTAGACGGCCGCCGACGCGACCT

166

6.2.2 Plasmid information

6.2.3 Strain constructed and used in this study

Table 6. 3. Strain constructed and used in this study

Primer for vector construction of pET-Trx-A-NMT-PCP

For_A_NMT_PCP_NcoI ATTccatgGACACCCTGCCCGCCCTG

Rev_A_NMT_PCP_XhoI ATTctcgagAACGCTCCAATTCGATGCG

Table 6. 2. Plasmid information

Name Description Reference

pET28a(+) Protein expression vector carrying an N terminal His6-

Tag/thrombin/T7 promoter, f1 ori, pBR322 ori, kanar

Invitrogen

pET24b(+) C terminal His6-Tag, T7 promoter, f1 ori, pBR322 ori, kanar Invitrogen

pBluescript SK(-) Cloning vector, lacZ’(α-complementation), Ampr Stratagene

pUC19 Cloning and sequencing vector for E. coli, Ampr Invitrogen

pBSK-SAS16 Plasmid containing gene sas16 for subcloning, Ampr This study

pET28-SAS16 Vector for protein expression of Sas16, based on pET28(+),

kanar

This study

pET26 PuR Vector for protein expression of PuR, kanar (Yanischperron et al.

1985)

pET26 PuxB A105V Vector for protein expression of PuxB, kanar (Kalan et al. 2013)

pET21-sfp-R4-4 Vector for protein expression of sfp-R4-4, kanar (Hopwood et al. 1985)

pET-Trx_1c Vector with an internal hexahistidine tag and a protease

cleavage site between the fusion protein and the cloned

protein sequence Trx- PCP, T7 promoter

(Corsini et al. 2008)

pBSK-PCP Plasmid containing PCP encoding gene for subcloning,

based on pBluescript SK(-), Ampr

This study

pET-Trx-PCP Vector for protein expression of PCP domain, based on pET-

Trx-1c, kanar

This study

pET-Trx-A-NMT-PCP Vector for protein expression of A-NMT-PCP domain, based

on pET-Trx-1c, kanar

This study

pET28-A domain Vector for protein expression of A domain, based on

pET28(+), kanar

This study

Strain Relevant characteristics Reference

E. coli DH5α General cloning host Invitrogen

S. asterosporus DSM 41452 Wild type strain of WS9326A producer DSMZ

E. coli BL21 star(DE3) F-ompT hsdSB (rB-, mB

-) galdcmrne131 (DE3) Invitrogen

E. coli BL21(DE3) pLysS F-, ompT, hsdSB (rB-mB

-), gal, dcm, (DE3) pLysS (CamR) Invitrogen

167

6.2.4 Cloning of sas16 gene into pET28 vector

A 1224bp DNA fragment containing sas16 was amplified from the S. asterosporus DSM 41452

genomic DNA by PCR using oligonucleotides P450pET-F and P450pET-R (Table 6. 1). The PCR

product was firstly ligated into EcoRV-digested pUC19 plasmid to yield pUC19-SAS16. Then

the sas16 gene fragment was cleaved out of pUC19-SAS16 and ligated into pET28a(+) vector

to yield plasmid pET28-SAS16. The resulting plasmid pET28-SAS16 was sequenced through the

sas16 reading frame to ensure that there is no mutation happening in the protein encoding

sequence.

6.2.5 Purification of Sas16

Plasmid pET28-SAS16 was chemically transformed into E. coli BL21 star(DE3). 7ml transformed

E. coli BL21 star(DE3) with plasmid pET28-SAS16 were grown overnight at 37 °C in LB medium

supplemented by kanamycin (50 mg/liter) to provide a seed culture for Sas16 protein

expression. 700 ml cultures of LB with kanamycin (50 mg/liter) were inoculated with 1% (v/v)

of overnight culture and grown at 37 °C to an OD absorbance (600 nm) of 0.4, then the culture

temperature was reduced to 28 °C. Expression of the Sas16 was induced using 0.2mM IPTG.

After 6 hours culture, the cell pellet was collected by centrifugation, then the pellet was

resuspended in buffer A, and lysed using French press.

After centrifugation, the supernatant was subjected to a FPLC ÄKAT system with a 2 ml Ni-

NTA column that had been pre-equilibrated in buffer A. The column was eluted with 5 CV of

buffer A, then eluted with 1.5 CV of buffer B. A fraction with red color protein was collected

and concentrated by ultrafiltration (molecular mass cutoff 10,000 Da), then it was desalted

using a Sephadex G-25 column (200 mm × 40 mm) which previously had been preequilibrated

with gel filtration buffer.

For long term storage, the protein solutions were concentrated using an Amicon Ultra

centrifugal filter with a 5,000-molecular weight cut-off and divided it into aliquots, then the

E. coli BL21 Rosetta F- ompT hsdSB(rB- mB

-) gal dcm (DE3) pRARE (CamR) Invitrogen

E. coli BL21 Codon Plus

RP(pL1SL2)

Heterologous expression host, coexpressing

Streptomyces chaperonin genes

Prof. Peter F. Leadlay

(Cambridge University)

E. coli XL1 blue recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1,

lac [F´proAB, lacIqZDM15, Tn10 (tet)]

Stratagene

168

buffer was exchanged to buffer A with 15% glycerol, then flash frozen in liquid nitrogen before

being stored at −80 °C.

6.2.6 CO difference spectrum of Sas16

Two anaerobic cuvettes were firstly filled with buffer A, the baseline of buffer absorption was

recorded with a dual-beam spectrophotometer from 400 to 600 nm. Then the sample cuvette

was replaced with Sas16 protein solution in the same buffer, after that the cuvette was

degassed and the gas phase was replaced with oxygen-free CO.

Then an amount (a few microliters) of sodium dithionite (Na2S2O4) solution was injected into

the protein solution to reduce the hemoprotein, and spectra were recorded every 2 mins until

the hemoprotein was totally reduced.

6.2.7 Substrate binding study

The substrate solution of the tyrosine, cyclic WS9326B, and the linear WS9326K and WS9326L

(chemically synthesized by solid phase peptide synthesis, GL Biochem(Shanghai) Ltd) were

prepared with concentration 1mM in stocking buffer (20 mM Tris HCl and 500 mM NaCl).

The homogeneous protein Sas16 with concentration 65mg/mL was stocked in buffer A. Then

aliquots (500uL) of diluted Sas16 enzyme were divided into reference and sample cuvette,

after thermal equilibration at 30 ˚C, the baseline was recorded between 600 and 350mm

followed by sequential additions (25ul) of concentrated substrate solution. The possible

substrates were individually titrated into the Sas16 protein solution, and mix gently. The

corresponding absorbance spectrum were recorded using UV-visible spectrophotometer

under Shortwave NIR wavelengths from 200 to 1100 nm (USB-ISS-UV-VIS-2, Ocean Optics).

6.2.8 Crystallization and Data Collection

Protein crystallization and structural determination were performed in the lab of Prof. Dr.

Oliver Einsle at the institute of biochemistry in Freiburg University. The concentrated Sas16

protein (10 mg/ml) after size exclusion chromatography (Superdex S200 26/60) in buffer (20

mM Tris pH8.0, 150 mM NaCl) was used for setting up initial screens by using sitting drop

vapor-diffusion method at 20℃. Initial crystallization experiments were established by using

an automatic crystallization drop-set system (Oryx Nano, Douglas Instrument), drops of 0.6 ul

in total with different protein to reservoir ratios (33%, 50%, 67%) were set and equilibrated

again with reservoir solutions (50 ul).

Reddish rod-like crystals initial appeared after two weeks cultivation in the buffer with 25%

polyethylene glycol (PEG) 3350, 0.2 M magnesium chloride and 0.1 M HEPES pH 7.5. Then

169

these crystals were crushed for seeding experiment by using Oryx Nano. Large diamond shape

single crystals were obtained in condition with 4% polyethylene glycol 6000, 4% polyethylene

glycol 8000, 4% polyethylene glycol 10000, 0.1 M potassium thiocyanate, 0.1 M sodium

bromide and 0.1 M MES pH 6.5.

Crystals were mounted on cryoloops and flash frozen in liquid nitrogen prior to data collection.

Multiple datasets to 2.0 Å was collected at beamline X06DA at Swiss Light Source (Villigen,

Switzerland) with the PILATUS pixel detector (Dectris). The data was processed by using

iMosflm (Battye et al. 2011) and XDS package (Kabsch 2010), the crystal was assigned to the

space group P42212 with unit cell dimensions a=b=112.8 Å and C=146.2 Å. The asymmetric

unit contains two subunits, with a solvent content of 52.45%.

6.2.9 Structure Determination and Refinement

Crystal structure of Sas16 was determined by single-wavelength anomalous dispersion (SAD)

with AutoSol and AutoBuild in PHENIX suite (Adams, Afonine et al. 2010) using dataset

collected at the Fe X-ray absorption edge (K-edge, 7172 eV). Then the low resolution model

after AutoBuild was used for molecular replacement in MOLREP (Vagin and Teplyakov 2010)

of the higher resolution dataset. Refinement of the initial electron density was carried out

using cycles of the program REFMAC5 (Murshudov, Skubak et al. 2011) in the CCP4 Suite (Winn

et al. 2011), and model building was performed in COOT (Emsley and Cowtan 2004). The final

structure was refined to Rcryst=20% and Rfree=24% at resolution of 2.0 Å. The quality of the

structure was validated by MOLPROBITY (Chen et al. 2010). Data collection and refinement

statistics are shown in Table 6. 4.

6.2.10 Malachite Green Phosphatase Assay of A domain

In a non-ribosomal peptide biosynthesis machinery, with a specific amino acid is activated

and incorporated into the NRPS assembly by acetyltransferase (A) domain, while one

pyrophosphate (PPi) is released along with the ATP energy consumption. In the method of

Malachite Green Phosphatase Assay, the released PPi are converted by the pyrophosphatase

to orthophosphate (Pi), which binds with the Malachite Green reagent can form a chemical

complex with green color, and this reaction can be monitored with the colorimetric method

(Figure 6. 28)(McQuade et al. 2009).

The Malachite Green Phosphatase Assay was performed according to the protocol provided

by the kit manufacturer (Echelon Biosciences Inc. Salt Lake City, USA) and the dissertation of

Anja Greule. Firstly, a standard curve of Pi was created with several dilutions in ddH2O from a

170

stock solution of Pi (0.1mM). Milli-Q water was used as blank. Every 100ul these solutions

were mixed with 100ul malachite green reagent. After incubation for 15 minutes at 30°C, their

absorbance was measured at 620nm. Secondly, the mix solution of A domain was prepared

as described (Greule 2016). The purified A domain (0.5 μM) was supplemented with 0.5 mM

ATP and 1 mM various amino acids (L-alanine, L-arginine, L-asparagine, L-cysteine, L-glycine,

L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-

tyrosine, and L-valine) then incubated at room temperature for 30 min. Afterwards, 0.5 μL of

alkaline phosphatase (2000 U) was added in and incubated at room temperature for 15 min.

Then 10 μL DTT solution and 100 μL Malachite Green reagent were added. The mixture was

incubated for 30 min at room temperature and the accordingly absorption was

measured(620nm) by UV-Visible spectrophotometer. Sample without A domain and amino

acids was used as base line to substract the absorbance of the solution background. A sample

with A domain but without any amino acid was used as control.

6.3 Results and Discussion

6.3.1 Multiple sequence alignment of Sas16

Figure 6. 3. Phylogenetic bootstrap consensus tree of Sas16 with other P450s. The tree was developed with the

neighbor-joining method based on the protein sequence alignment made with the MUSCLE algorithm

171

implemented in MEGA 5.2. The aligned proteins include Epi-isozizaene 5-monooxygenase (Q9K498); CalO2, a

P450 monooxygenase (Q8KND6); BioI cytochrome P450 for Biotin biosynthesis (R9TUM1); Epothilone C/D

epoxidase (Q9KIZ4); Monooxygenase P450sky32 (F2YRY7); Cytochrome P450 hydroxylase SalD (F1C7Q5);

Quinaldate 3-hydroxylase TioI (Q333V7); Cytochrome P450 Sare_4553 (A8M6V4); Cytochrome P450 NikQ

(Q9L465); Nikkomycin biosynthesis protein SanQ (Q9EYL2); Putative cytochrome P450 (F8JKH2); Putative P450

monooxygenase (E9L1N0); P450 monooxygenase oxyD (G4V4S6); Veg29 (B7T1A1); Cytochrome P450

PCZA361.17 (O52802); Erythromycin C-12 hydroxylase (P48635); Putative cytochrome P450 AurH (Q70KH6);

Cytochrome P450 monooxygenase PikC (O87605); 6-deoxyerythronolide B hydroxylase EryF (Q00441);

Cytochrome P450 StaP (Q83WG3); Cytochrome P450 monooxygenase OleP (A0A1C4RKB5); Mycinamicin IV

hydroxylase/epoxidase MycG (Q59523); Cytochrome P450 PimD (Q9EW92); Cytochrome P450 OxyC (Q8RN03);

Cytochrome P450 OxyB (Q8RN04); Cytochrome P450 NysN (Q9L4W8); 20-oxo-5-O-mycaminosyltylactone 23-

monooxygenase TylH1 (Q9ZHQ1); Cytochrome P450 ChmHI (Q5SFA8); Mycinamicin VIII C21 methyl hydroxylase

MycCI (Q83WF5); Note: numbers in the parentheses refers to Uniprot accession number.

To determine the phylogenetic relationship of Sas16 with other P450s, in total thirty P450

enzymes were selected as reference sequences for phylogenetic analysis. The phylogenetic

tree was separated into two large clades (Clade I and II) (Figure 6. 3).

Sas16 was grouped into Clade I, this group contains ChmHI, a cytochrome P450 from

Streptomyces bikiniensis; MycCI, a Mycinamicin VIII C21 methyl hydroxylase from

Micromonospora griseorubida; TylH1, a 20-oxo-5-O-mycaminosyltylactone 23-

monooxygenase from S. fradiae; NysN, a cytochrome P450 from S. noursei; OxyB, a

cytochrome P450 from Amycolatopsis orientalis; OxyC, a cytochrome P450 from

Amycolatopsis orientalis. Amongst them, MycCI, ChmHI and TylH1 are hydroxylase, they are

individually responsible for the hydroxylation of macrolide antibiotics mycinamicin,

chalomycin and tylosin at specific position (Reeves et al. 2004; Ward et al. 2004; Anzai et al.

2008). P450 monooxygenase NysN is a oxidase which catalyze the oxidation of methyl group

at C-16 in nystatin A1 biosynthesis from S. noursei ATCC 11455 (Bruheim et al. 2004).

Pairwise Sequence Alignment by BLAST showed that Sas16 show 31% identity to OxyB and 33%

identity to OxyC in Clade I, 28% identity to OxyD (accession number: G4V4S6) and 25% identity

to P450sky (accession number: F2YRY7) in Clade II. Cytochrome P450 OxyB and OxyC are

responsible for catalyzing the first and the last oxidative phenol coupling reaction in the

biosynthesis of glycopeptide antibiotic Vancomycin, respectively (Zerbe et al. 2002; Pylypenko

et al. 2003). OxyD is involved into the hydroxylation of β-hydroxytyrosine residue in

Vancomycin biosynthesis (Cryle et al. 2010). P450sky catalyzes the β-hydroxylation of three

different amino acids (phenylalanine, tyrosine, and leucine) bound to the peptidyl carrier

protein (PCP) domain during the biosynthesis of Skyllamycin (Uhlmann et al. 2013). Those

172

alignment result is in accordance with the previous phylogenetic tree, suggesting that Sas16

show much closer phylogenetic relationship with OxyB and OxyC. It is likely that Sas16 may

work with the similar mechanism like other homologs (Zerbe et al. 2002; Pylypenko et al.

2003).

Figure 6. 4. Protein Sequence comparison of Sas16 with OxyB and OxyC. Identical residues are shown in red

boxes. Secondary structure elements of Sas16 are shown above the sequence alignment. Important Helix or

173

strands are labeled with parentheses. The sequence alignment was performed in Clustal Omega and the figure

was made by ESPript 3.

Multiple sequence alignment of Sas16 with OxyB and OxyC based on their core motifs and

secondary structure elements were carried out(Figure 6. 4). It is clearly showed that the

characteristic feature of P450 signature amino acid sequence FGxGxHxCxG exists in Sas16. In

addition, the substrate recognition sequences (Denisov et al. 2005) of Sas16 exhibit significant

similarity with OxyB and OxyC, including the B’-helix region (containing conserved sequence

DPPxHTRxR), the Helix-I region, and the cysteine loop region. The important variations of

substrate recognition sequence exist at the C-terminal protion of F-helix region (conserved

sequence CELxGxPxxDxxxF) and the following G-F loop until the initial part of the G-helix.

Another significant difference exists at the connecting region of K helix- β2 sheet.

For in-depth understanding the protein features of Sas16, the protein sequence alignment of

Sas16 with other ten P450 proteins was constructed by Clustal Omega (Figure 6. 5). The

alignment indicated that Sas16 shares the conserved nucleotide binding site with other

members of the P450 superfamily. The presence of the signature sequence FxxGxHxCxG, and

the conserved cysteine as the proximal thiolate iron ligand were confirmed. The alignment

result shown that the sequence differences of Sas16 with other homologues mainly occur at

the B-B1 loop region (from residue 67 to 78); the F-G loop region (from residue 178 to 189);

the N-terminal region of the I-helix (from residue 232 to 245); and the helix-K adjacent to the

β2-sheet region (from residue 293 to 295). Those regions in close proximity to the active site

of cytochrome P450 protein maybe will make influence on the substrate specificity of enzyme.

Sas16 MTDAE-----------TKMAKCPVAPHGWPNP-LLPEYDQLPEGRPLTQVT-MPSGSKAW

tr|F8JKH2|F8JKH2_STREN ----------MNTVTGVTTFPDLTDPAFWARDDSHAVLRELRRR-SPLWRLESEAEGPLW

tr|G4V4S6|G4V4S6_AMYOR --------------MQTTTAVDLGNPDLYTTLDRHTRWREFATEDAMVWSEPGSSPTGFW

tr|E9L1N0|E9L1N0_9ZZZZ --------------MQTTEALDLGNPDLYTTVDRHARWRELAAEDAMVWSEPGSSPTGFW

tr|O52802|O52802_AMYOR -------------MQTTTAVGDLGNPDLYTTLDRHARWRELAARDAMVWSEPGSSPTGFW

tr|B7T1A1|B7T1A1_9BACT -------------MQ-TTTAVDLGNPDLYTSLERHARWRELAARDAMVWSEPGSSPSGFW

tr|A8M6V4|A8M6V4_SALAI --------MPHGEPTLTLYANELCDPELYRQGNPEDLWRRMHAAAPVHEGA-FEG-RRFH

tr|Q9EYL2|Q9EYL2_9ACTO ------------------MRVDLSDPLLYRDSDPAPVWSRLRAEHPVHRNERANG-EHFW

tr|Q9L465|Q9L465_STRTE ------------------MRVDLSDPLLYRDSDPEPVWSRLRAEHPVYRNERANG-EHFW

tr|F1C7Q5|F1C7Q5_9ACTO MTATDAKDVGQVGSRNVASTIDLADPATFAGHDLTNFWQRLRDEEPIYWNPPTSGRRGFW

tr|Q333V7|Q333V7_9ACTO ----------MSSPTASTSALDLTDPTTFVRHDTHVFWAEVRDHNPVYWYQGREDRPGFW

* : ..

B-B1 loop region

Sas16 LVAQHDHIQRLLADN-RFSVEPHPTFPIRFPAPQELLDMIARDAKNLLVTMDPPRHTRVR

174

tr|F8JKH2|F8JKH2_STREN CVLSHALANEVLGDAARFSSERGSLL-------GTGRDRAPAGAGKMMALTDPPRHRDLR

tr|G4V4S6|G4V4S6_AMYOR SVFSHRACAAVLGPSAPFTSEYGMMI-------GFDREHPDTSGGQMMVVSEQDQHRRLR

tr|E9L1N0|E9L1N0_9ZZZZ SVFSHRACAAVLGPSAPFTSEYGMMI-------GFDRDHPDTSGGRMMVVSEKEPHRRLR

tr|O52802|O52802_AMYOR SVFSHRACAAVLAPSAPFTSEYGMMI-------GFDRDHPDKSGGQMMVVSEQEQHRKLR

tr|B7T1A1|B7T1A1_9BACT SVFSHRACAAVLAPSAPFTSEYGMMI-------GFDRDHPDQSGGQMMVVSEQDQHKKLR

tr|A8M6V4|A8M6V4_SALAI AVISHALISRMLKDPKGFSSERGMRL-------DQNPAATSLAAGKMLIITDPPRHGKIR

tr|Q9EYL2|Q9EYL2_9ACTO AVMTHGLCTDMLTDPRVFSSRNGMRL-------DSDPKVLAAAAGKMLNITDPPRHDKIR

tr|Q9L465|Q9L465_STRTE AVMTHGLCTDMLTDPRVFSSQNGMRL-------DSDPQVLAAAAGKMLNITDPPRHDKIR

tr|F1C7Q5|F1C7Q5_9ACTO VLSRHADILEVYRDDMTFTSERGNVL-------VTLLAGGDAGAGRMLAVTDGPRHTELR

tr|Q333V7|Q333V7_9ACTO VVSRYVDVVASYTDAARLSSARGTVL-------DVLLRGEDSAGGRMLAVTDRPRHRELR

: : :: : . .:: : * :*

Sas16 QMALPDFTIKAAEKLRPRMQDLIDYYLDKMEAEGAPADLVQALALPFPAQVICELAGIPE

tr|F8JKH2|F8JKH2_STREN GLVLPFFSKRKAAELGARVADLTRQVVRDALG-TARTDFVRDISTTVPLTVMCDLLGVPD

tr|G4V4S6|G4V4S6_AMYOR KLVGPLLSRAAARKLAERVRTEVRGVLDKVLD-GEVCDVAAAIGPRIPAAVVCEILGVPA

tr|E9L1N0|E9L1N0_9ZZZZ RLVGPLLSRAAARELTERVRTEVRGVLDQVLD-GGVCDVAAAIGPRIPAAVVCDILGVPA

tr|O52802|O52802_AMYOR RLVGPLLSRAAARKLSERVRTEVSGVLDQVLD-GGVCDVATAIGPRIPAAVVCEILGVPA

tr|B7T1A1|B7T1A1_9BACT RLVGPLLSRAAARKLSERVRTEVTGVLDQVLD-GAELDAATAIGPRIPAAVVCEILGVPA

tr|A8M6V4|A8M6V4_SALAI RIVNSVFTPRMVARLEENMRVTAAGIVDQAIE-EGECDF-TDVAARLPLSAICDMLGVPP

tr|Q9EYL2|Q9EYL2_9ACTO KVVSSAFTPRMVSRLEATMRKTAAEAIDEALA-AGECEF-TRVAQKLPVSVICDMLGVAP

tr|Q9L465|Q9L465_STRTE KVVSSAFTPRMVSRLEATMRETAAKAIDEALA-AGECEF-TRVAQKLPVSVICDMLGVAP

tr|F1C7Q5|F1C7Q5_9ACTO KLLLRALGPRVLGPVCRAVRANTRQMIGEAAA-NGECDFATDIASRIPMITISNLLGVPE

tr|Q333V7|Q333V7_9ACTO NVMLRAFSPRVLGRVVEQVHRRADELIRRVTG-TGAFDFATEVAESIPMGTICDLLSIPP

: : : : : : :. .* .:.:: .:

F-G loop region

Sas16 NDREIFTRNAAIMVGT-RHSYT-MEQKLAANEELMKYFAALVTEKQSNPTDDMLGNFIAR

tr|F8JKH2|F8JKH2_STREN EDRDHVVAMCDRAFLGDTPE-----ERSEAHQQLLPYLFALGLRRRTDPRDDIISQLVTH

tr|G4V4S6|G4V4S6_AMYOR EDEDMLIDLTNHAFGGEDELFD-GMTPRQAHTEILVYFDELIAARRERPGDDLVSTLLTD

tr|E9L1N0|E9L1N0_9ZZZZ EDQDMLIELTNHAFGGEDELYD-GMTPRQAHTEILVYFDELITARRERPGDDLVSTLVTD

tr|O52802|O52802_AMYOR EDEDMLIELTNHAFGGEDELFD-GMTPRQAHTEILVYFDELITARRERPADDLVSTLVTD

tr|B7T1A1|B7T1A1_9BACT EDEDVLIELTNHAFGGEDELFD-GMTPRQAHTEILVYFDELITARRERPGDDLVSALVTD

tr|A8M6V4|A8M6V4_SALAI EDWDFMLDRTMVAFGSGEAD---ELAMAEAHADILSYYEDLIRRRRREPREDVVTALVNG

tr|Q9EYL2|Q9EYL2_9ACTO ADWDFMVERTRFAWSSTALDEAEEARKIRAHTEILLHFQDLAAQRRREPQDDLMSALVCG

tr|Q9L465|Q9L465_STRTE ADWDFMVERTRFAWSSTALDEAEEARKVRAHTEILLHFQDLAAERRREPKDDLMSALVCG

tr|F1C7Q5|F1C7Q5_9ACTO ADRASLLKMTKTALSADDESIS-DTDSEMARNEILLYFQDFVEFRRKNPGEDVVSMLVNS

tr|Q333V7|Q333V7_9ACTO ADRPDLLRWNKNALSSDEADAN-LYAALEARNQILLYFMDLAEQRRASPGDDVISMIATA

* . *. ::: : : :: * :*:: :

N-terminal region of the I-helix

Sas16 AGKTDEFDHHGLTLMTKMLLLAGYEFIVNRIALGIQALVENPEQLAALRADLPGLMPKTV

tr|F8JKH2|F8JKH2_STREN EVDGRRLPLDEALLNCDNILVGGVQTVRHTSTMAMLALTRHPHAWQAMRAD-GYDPETGV

175

tr|G4V4S6|G4V4S6_AMYOR ----DELTIDDVVLNCDNVLIGGNETTRHAITGAVHAFATVPGLLARVRDG-DEDVDTVV

tr|E9L1N0|E9L1N0_9ZZZZ ----DGLTTDDVLLNCDNVLIGGNETTRHALTGAVHALATVPGLLTGLRDG-SADVDTVV

tr|O52802|O52802_AMYOR ----DELTIDDVLLNCDNVLIGGNETTRHAITGAVHALATVPGLLTGLQDG-SADVDTVV

tr|B7T1A1|B7T1A1_9BACT ----EALSIDDVLLNCDNVLIGGNETTRHAITGAVHALATVPGLLTGLQDG-SADVDTVV

tr|A8M6V4|A8M6V4_SALAI VVDGTKLTDEEIFLNCDGLISGGNETTRHATIGGFLALLDNPEQWETLRDD-PGLLPGAV

tr|Q9EYL2|Q9EYL2_9ACTO EIDGAPLTDQEILYNCDALVSGGNETTRHATVGGLLALIDNPDQWHRLRDE-PALMPSAI

tr|Q9L465|Q9L465_STRTE EIDGAPLTDQEILYNCDALVSGGNETTRHATVGGLLALIDNPDQWHRLRDE-PALMPSAI

tr|F1C7Q5|F1C7Q5_9ACTO SIDGVPLSDDDIVLNCYSLIIGGDETSRLTMIDSINTLAANPGQWRRLKEG-RCDIDKAV

tr|Q333V7|Q333V7_9ACTO TVGGEPLSIDDVALNCYSLILGGDESSRMSAICAVKAFADFPDQWRAVRDG-DVAIDTAV

: . :: .* : .. :: * :: :

helix-K adjacent to the β2-sheet region

Sas16 DEVLRYYSLVDEIIARVALEDVEIDGVTIKAGEGILVLKGLGDRDPSKYPNPDVFDIHRD

tr|F8JKH2|F8JKH2_STREN EELLRWTSVGL-HVLRTARHDTELAGHHIRAGDRVVVWTPAANRDEAEFHHPDDLLLDRT

tr|G4V4S6|G4V4S6_AMYOR DEVLRWTSPAM-HVLRVTTGEVTINGRDLAPGTPVVAWLPAANRDPAVFDDPDTFRPGRK

tr|E9L1N0|E9L1N0_9ZZZZ EEVLRWTSPAM-HVLRVTTGDVTVNGRDLPSGTPVVAWLPAANRDPAEFDDPDAFRPRRT

tr|O52802|O52802_AMYOR EEVLRWTSPAM-HVLRVSTDDVTINGQDLPAGTPVVAWLPAANRDPAEFDDPDTFLPGRK

tr|B7T1A1|B7T1A1_9BACT EELLRWTSPAM-HVLRVSTEDVTINGQDVPSGTPVVAWLPAANRDPAEFDDPDTFLAGRK

tr|A8M6V4|A8M6V4_SALAI QEILRYTSPAM-HVLRTAVAPTRIGEYALNPGDPVALWLSAGNRDPQVFADPDRFDITRS

tr|Q9EYL2|Q9EYL2_9ACTO QEIVRYTSPVM-HALRTATEDVEFGGELISAGDHVVAWLPSANRDEKVFDDPDRFDIGRE

tr|Q9L465|Q9L465_STRTE QEIVRYTSPVM-HALRTATEDVEFGGERISAGDHVVAWLPSANRDEKVFDDPDRFDIERE

tr|F1C7Q5|F1C7Q5_9ACTO DEVLRWASPSM-HFGRTAVRETVIHGERIQVDDIVTLWGASGNRDERAFKQPEVFDLGRV

tr|Q333V7|Q333V7_9ACTO EEVLRWSTPAM-HFARTATTDFELRGQQVRAGDIVTLWNLSANFDEREFDRPYRFEVGRT

:*::*: : *.: . : : .: * : * : *

Sas16 SRDHLAFGYGVHQCLGQHVARLMLEMCLTSLVERFPGLHLVEGDEPIEL--IDGLPPVHK

tr|F8JKH2|F8JKH2_STREN PNRHLAFGWGPHYCIGAPLARVELASLFAALTEAAEHVEVLEPPVPNRSIINFGLDALVV

tr|G4V4S6|G4V4S6_AMYOR PNRHIAFGHGMHHCLGSALARIELAVVVRELAERVSRVELAKEPAWLRAIVVQGYRELPV

tr|E9L1N0|E9L1N0_9ZZZZ PNRHITFGHGVHHCLGSALARIELSVVLRELAERVSRVELLDEPTWLRAVVVQGYRELRV

tr|O52802|O52802_AMYOR PNRHITFGHGMHHCLGSALARIELSVVLRVLAERVSRVELVKEPAWLRAIVVQGYAELSA

tr|B7T1A1|B7T1A1_9BACT PNRHITFGHGMHHCLGSALARIELAVLVQVLAERVSRVELLSEPEWLRAIVVQGYRGLPV

tr|A8M6V4|A8M6V4_SALAI PNPHLTFSTGAHYCLGSALATSELTVLFDRLLRRVDSAELTGPPRRTRSILIWGYDSVPV

tr|Q9EYL2|Q9EYL2_9ACTO PNRHLGFIQGNHYCIGSSLAKLELTVMFEELLARVEIAELAGQVRRLRSNLLWGFDSLPV

tr|Q9L465|Q9L465_STRTE PNRHLGFIQGNHYCIGSSLAKLELTVMFEELLARVEVAELAGQVRRLRSNLLWGFDSLPV

tr|F1C7Q5|F1C7Q5_9ACTO PNRHLSFGHGPHYCIGSYLAKVEISELLIALRDLILGFEVIGEPQRIRSNLLSGFSTMPV

tr|Q333V7|Q333V7_9ACTO PNKHLSFGHGPHFCLGAYLGRAELQALLTALVGTVSRIESAGSPRRVYSNFLNGHSSLPV

. *: * * * *:* :. : . * . * :

Sas16 LTIGW-----------

tr|F8JKH2|F8JKH2_STREN RLHPRGAAG-------

tr|G4V4S6|G4V4S6_AMYOR RFTGR-----------

176

tr|E9L1N0|E9L1N0_9ZZZZ RFTGR-----------

tr|O52802|O52802_AMYOR RFTGR-----------

tr|B7T1A1|B7T1A1_9BACT RFTGR-----------

tr|A8M6V4|A8M6V4_SALAI RLTAGSER--------

tr|Q9EYL2|Q9EYL2_9ACTO TFVPRA----------

tr|Q9L465|Q9L465_STRTE KFVPRA----------

tr|F1C7Q5|F1C7Q5_9ACTO RFDADRTGLASEAREG

tr|Q333V7|Q333V7_9ACTO AFTGR-----------

Figure 6. 5. Clustal Omage alignment of the amino acid sequences of Sas16 with other characterized Cytochrome

P450 monooxygenases from secondary metabolites biosynthetic pathways. Identical residues are indicated with

asterisks. The heme-binding site (FxxGxHxCxG) is indicated in light blue; the cysteine involved in heme

coordination is highlighted in bold and underlined. ExxR motif in the K-helix is highly conserved, which was

marked in grey. The aligned sequences include SCATT_p15680, a Putative cytochrome P450 from Streptomyces

cattleya (Genbank accession number: F8JKH2); OxyD, a P450 monooxygenase from Amycolatopsis orientalis

(Nocardia orientalis) (Genbank accession number: G4V4S6); CA878-31, a uncharacterized protein from

uncultured organism CA878 (Genbank accession number:); PCZA361.17 from Amycolatopsis orientalis (Genbank

accession number: O52802); Veg29, from uncultured soil bacterium (Genbank accession number: B7T1A1);

Sare_4553, a putative cytochrome P450 from Salinispora arenicola (strain CNS-205) (Genbank accession number:

A8M6V4); SanQ, protein involved in Nikkomycin biosynthesis from Streptomyces ansochromogenes (Genbank

accession number: Q9EYL2); NikQ, from Streptomyces tendae (Genbank accession number: Q9L465); SalD, a

cytochrome P450 hydroxylase from Salinispora pacifica (Genbank accession number: F1C7Q5), and TioI,

encoding Quinaldate 3-hydroxylase from Micromonospora sp. ML1 (Genbank accession number: Q333V7).

6.3.2 Vector Construction, Expression and Purification of Sas16

To construct the plasmid for Sas16 expression, sas16 gene was amplified from the genome of

S. asterosporus DSM 41452, then it was cloned into the recombinant expression vector

pET28a(+) with N-terminal hexahistidine-tag and a constitutive T7 Promoter. Detailed

experimental information is described in the section 6.2.4. Sas16 as a recombinant protein

has a calculated molecular weight of 45.5KDa. Protein expression cells E. coli BL21 star(DE3)

and E. coli BL21 Codon Plus RP(pL1SL2) were used for the method optimization of Sas16

expression. In the meanwhile, different additive amount of IPTG, culture temperature and

culture time were tested to screen the best cultivation condition. Figure 6. 6 shows the SDS-

PAGE gels of the Sas16 expression test with different cultivation condition.

177

A B

Figure 6. 6. (A) Diagram of plasmid pET28-SAS16; (B) Cultivation method optimization of Sas16 expression.

Samples from right to left lanes: Marker (M); supernatant (1) and cell debris (2) from E. coli BL21 Codon Plus

RP(pL1SL2)::pET28-SAS16 cultured in LB medium induced by IPTG with 0.1mM, incubated at 37°C for 4 hours;

supernatant (3) and cell debris (4) from E. coli BL21 star(DE3)::pET28-SAS16 cultured in LB medium induced by

IPTG with 0.1mM, incubated at 37°C for 4 hours; supernatant (5) and cell debris (6) from E. coli BL21

star(DE3)::pET28-SAS16 cultured in LB medium induced by IPTG with 0.1mM, incubated at 28°C overnight;

supernatant (7) and cell debris (8) from E. coli BL21 star(DE3)::pET28-SAS16 cultured in LB medium induced by

IPTG with 0.8mM, incubated at 28°C overnight; supernatant (9) and cell debris (10) from E. coli BL21 Codon Plus

RP(pL1SL2)::pET28-SAS16 cultured in LB medium induced by IPTG with 0.1mM, incubated at 20°C overnight; Red

arrow indicates the band of Sas16.

Finally, E. coli BL21 star(DE3) cell was chosen as the host for Sas16 protein expression. For

large-scale fermentation, E. coli BL21 star (DE3)::pET28-SAS16 cell were cultured in LB medium,

when the OD600 value reach 0.4, 0.2mM IPTG was supplemented into the medium, then the

strains were incubated at 28°C for 8 hours. After that, the harvested cell pellet was lysed using

French press, the soluble protein in supernatant was further purified by ÄKTA FPLC system

with Ni-NTA Superflow column. A red color fraction eluted from 2mL Ni-NTA column by 80%

buffer B, then the fraction was concentrated and further purified through gel-filtration

chromatography. The obtained soluble protein was measured with approximately 2mg/liter

by nanodrop. Figure 6. 7 shows the UV chromatogram (λ=280 nm) of the Sas16 protein

purification through Ni-NTA affinity column, and the corresponding SDS-PAGE analysis of the

collected fractions.

178

A B

Figure 6. 7. (A) FPLC Chromatogram of Ni-NTA his-tag purification of Sas16 from lysed E. coli BL21 star (DE3) ::

pET28-SAS16. Shown are the UV absorption at 280 nm during elution with Buffer B by gradient method, peak

marked by the red arrow represent the fraction containing Sas16; (B) SDS-PAGE analysis of the fraction from Ni-

NTA column.

6.3.3 Crystallization and Structure determination of Sas16

With the purpose of further understanding the biochemical property of Sas16, and

demonstrating the substrate structure-reactivity relationship, we decided to determine the

crystal structure of Sas16.

Sas16 protein sample for crystallization was purified as described in Section 6.2.4, and the

protein concentration is 67 mg/mL measured by BCA method. Prior to crystallization, the

Sas16 protein was further purified by gel filtration using a Superdex 75 gel filtration column

(16 mm x 31000 mm) equilibrated with buffer C (20 mM HEPES, pH 7.0, 150 mM NaCl). The

purified Sas16 is in dark red color, analytical size exclusion chromatography of Sas16 yielded

a single symmetric peak with a molecular mass of 55 KDa indicating the monomeric state of

Sas16 in solution.

The crystals of Sas16 were cultivated by the sitting-drop vapor diffusion method at 20°C under

aerobic conditions. The initial crystals of Sas16 were obtained on the condition with 0.1 M

HEPES, pH 7.5, 25% w/v polyethylene glycol 3350 and 0.2M magnesium chloride. Then the

cultivation conditions of crystallization were further optimized by variation of precipitant,

protein concentration, buffer, pH value and additive. Finally, a diamond-shaped crystal of

Sas16 (Figure 6. 8) with better quality, strong diffracting crystals was obtained in the reservoir

solution composed by 4% polyethylene glycol 6000, 4% polyethylene glycol 8000, 4%

polyethylene glycol 10000, 0.1 M potassium thiocyanate, 0.1 M sodium bromide and 0.1 M

MES pH 6.5 with the ratio 2:1 (4mg/mL Sas16 concentration).

179

A B

Figure 6. 8. (A) SDS-PAGE analysis of fractions from gel filtration eluted from a Sephadex G-25 column

(200mm×40mm) containing Sas16; (B) Crystals of Sas16 from S. asterosporus DSM 41452. This recombinant

protein was expressed and purified from E. coli BL21 star (DE3) :: pET28-SAS16, the protein was crystallized in

the reservoir solution 28% BMW, 0.1M Tris pH 8.5, 0.15M Ammonium acetate.

By X-ray diffraction data analysis, the crystal structure of Sas16 was determined to a resolution

of 2.0 Å using single-wavelength anomalous scattering from crystals. The crystallographic and

the refinement statistics are summarized in Table 6. 4.

Table 6. 4. Crystal parameters and data-collection statistics for the crystal of Sas16

Data set

Space group P 42 21 2

Cell constants a, b, c [Å]

α, β, γ [°]

112.8, 112.8, 146.2

90, 90, 90

Resolution limits [Å] 146.15 – 2.0 (2.05 – 2.0)

Completeness (%) 100 (100)

Unique reflections 64321

Multiplicity (%) 26.6 (28.1)

Rmerge* 0.236 (1.802)

Rp.i.m. 0.047 (0.345)

Mean I/σ(I) 13.1 (2.5)

CC1/2 0.998 (0.390)

Refinement statistics

Rcryst† 0.20

Rfree 0.24

r.m.s.d. bond lengths [Å] 0.0235

r.m.s.d. bond angels [°] 2.30

Average B-factor [Å2] 27

Note: *Rmerge = Σhkl [(Σi |Ii − I |)/Σi Ii]; †Rcryst = Σhkl ||Fobs| − |Fcalc||/Σhkl |Fobs|; RMSD: Root Mean Square Deviation

is the square root of the mean of the square of the distances between the matched atoms.

The crystal structure of Sas16 forms a dimer linked through a disulfide-bridge generated by

the cysteine-11 in each monomer (Figure 6. 9A). The overall monomeric structure of Sas16

adopts the typical triangular P450 protein folding which is composed of 14 α-helices and 10

180

β-sheets including the conserved four-helix bundle core part (I-, K-, L-, and C-helices). The

heme group in P450 is confined between those core helixes and the cysteine ligand loop