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journal homepage: www.elsevier.com/locate/meteng
Magnesium starvation improves production of
malonyl-CoA-derivedmetabolites in Escherichia coli
Kento Tokuyamaa, Yoshihiro Toyaa, Fumio Matsudaa, Brady F.
Cressb, Mattheos A.G. Koffasb,c,Hiroshi Shimizua,⁎
a Department of Bioinformatic Engineering, Graduate School of
Information Science and Technology, Osaka University, 1-5
Yamadaoka, Suita, Osaka 565-0871, JapanbDepartment of Chemical and
Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY,
USAc Department of Biological Sciences, Rensselaer Polytechnic
Institute, Troy, NY, USA
A R T I C L E I N F O
Keywords:Nutrient starvationMetabolic profiling
A B S T R A C T
Starvation of essential nutrients, such as nitrogen, sulfur,
magnesium, and phosphorus, leads cells into stationaryphase and
potentially enhances target metabolite production because cells do
not consume carbon for thebiomass synthesis. The overall metabolic
behavior changes depend on the type of nutrient starvation
inEscherichia coli. In the present study, we determined the optimum
nutrient starvation type for producing mal-onyl-CoA-derived
metabolites such as 3-hydroxypropionic acid (3HP) and naringenin in
E. coli. For 3HP pro-duction, high production titer (2.3 or 2.0 mM)
and high specific production rate (0.14 or 0.28mmol gCDW−1
h−1) was observed under sulfur or magnesium starvation, whereas
almost no 3HP production was detectedunder nitrogen or phosphorus
starvation. Metabolic profiling analysis revealed that the
intracellular malonyl-CoA concentration was significantly increased
under the 3HP producing conditions. This accumulation
shouldcontribute to the 3HP production because malonyl-CoA is a
precursor of 3HP. Strong positive correlation(r=0.95) between
intracellular concentrations of ATP and malonyl-CoA indicates that
the ATP level is im-portant for malonyl-CoA synthesis due to the
ATP requirement by acetyl-CoA carboxylase. For
naringeninproduction, magnesium starvation led to the highest
production titer (144 ± 15 μM) and specific productivity(127 ± 21
μmol gCDW−1). These results demonstrated that magnesium starvation
is a useful approach toimprove the metabolic state of strains
engineered for the production of malonyl-CoA derivatives.
Extensive efforts in metabolic engineering and synthetic
biologyhave led to the development of microbial processes for
heterologousmetabolites production. In recent years, the microbial
process for theproduction of malonyl-CoA derivatives has been
studied extensively(Xu et al., 2014). A target metabolite,
3-hydroxypropionic acid (3HP)can be produced from malonyl-CoA and
has a wide variety of com-mercial applications (Werpy and Petersen,
2004; Tokuyama et al.,2014; Rathnasingh et al., 2012; Liu et al.,
2013; Cheng et al., 2016;Jiang et al., 2009; Kumar et al., 2013;
Kim et al., 2014; Borodina et al.,2015; Lan et al., 2015; Liu et
Plant-derived flavonoids have been widely used in functional
foods,nutraceuticals, and cosmetics in global market (Panche et
al., 2016),and some them are potential candidates as lead compounds
for onsetdiseases, such as Alzheimer's disease and cancers
(Baptista et al., 2014;Ibrahim et al., 2014). The microbial
productions have attracted
increased attention due to lack of availability of plant sources
for fla-vonoids production, a lot of metabolic engineering studies
have beenreported to produce flavonoids in microorganisms (Xiu et
al., 2017;Jones et al., 2016a; Xu et al., 2011; Leonard et al.,
2007; Raman et al.,2014; Pandey et al., 2016). Naringenin is a
potential immunomodulator(Zeng et al., 2018), and has
neuro-protective effect on permanent is-chemic damage (Bai et al.,
2014). The compound is synthesized from p-coumaric acid as a
precursor using malonyl-CoA. In previous reports,malonyl-CoA
availability was considered as a major bottleneck,
becausemalonyl-CoA is used as a precursor of biomass components and
itsconcentration is tightly controlled in the cells. Some previous
studieshave focused on the optimization of de novo biosynthesis of
malonyl-CoA using genetic engineering strategies (Zha et al., 2009;
Xu et al.,2011; Johnson et al., 2017).
Bio-production during stationary phase is also an effective
approachto enhance availability of intermediates related to biomass
components,by decreasing the demand for cell growth (Toya et al.,
https://doi.org/10.1016/j.ymben.2018.12.002Received 13 August
2018; Received in revised form 4 December 2018; Accepted 5 December
⁎ Corresponding author.E-mail address: firstname.lastname@example.org
Metabolic Engineering 52 (2019) 215–223
Available online 06 December 20181096-7176/ © 2018 International
Metabolic Engineering Society. Published by Elsevier Inc. All
nutrient starvation is a cost-effective way to lead the cells
into sta-tionary phase, and shift overall metabolic activity
through regulatorymachinery, for adapting to the environmental
perturbation (Chubukovand Sauer, 2014). There are various essential
nutrients, such as ni-trogen, sulfur, magnesium, and phosphorus,
for Escherichia coli. It hasbeen reported that the metabolic
behavior changes depending on thetype of nutrient starvation in E.
coli (Chubukov and Sauer, 2014). Liet al. (2016) successfully
demonstrated that sulfur starvation was sui-table to produce
acetyl-CoA derived mevalonate and tyrosine. Masudaet al. (2017)
applied 13C-metabolic flux analysis on the mevalonateproducing E.
coli, and showed that the sulfur starvation improvedacetyl-CoA
availability compared to the growing cells. Since
acetyl-CoAcarboxylase is the sole pathway of de novo synthesis of
malonyl-CoA,nutrient starvation potentially improves malonyl-CoA
availability cou-pled with the increased acetyl-CoA supply.
In the present study, we determined the optimum nutrient
starva-tion condition for producing malonyl-CoA derivatives, such
as 3HP andthe plant-derived flavonoid naringenin, in E. coli. The
stationary phasecultures of 3HP-producing E. coli strain on M9
medium lacking ni-trogen, sulfur, magnesium, or phosphorus, showed
significant differ-ences in 3HP productivities depending on the
type of nutrient starva-tion. The cause of the differences in 3HP
productivity following nutrientstarvation was investigated by
metabolic profiling analysis of the cen-tral carbon metabolism.
2. Materials and methods
2.1. Strains and plasmids
All strains and plasmids used in this study are listed in Table
1. E.coli strain DH5α was used for plasmid construction and E. coli
strainMG1655(DE3) was used as production host for 3HP. Malonyl-CoA
re-ductase (MCR) gene was separated into N-terminal region and
C-terminal region, to enhance its enzymatic activity (Liu et al.,
2013), andintroduced into the host strain. The N-terminal region of
MCR (mcrN,amino acids 1–549) and the C-terminal region (mcrC, amino
acids550–1219) were derived from Chloroflexus aurantiacus. These
gene re-gions were codon-optimized for expression in E. coli and
synthesizedwith NdeI and XhoI sites in pMK-RQ by GeneArtR (Life
technologies).T7 expression vector pETM6 was used to construct MCR
expressionplasmid (Xu et al., 2012). McrN and mcrC were assembled
in pETM6vector as a pseudo-operon following the protocol described
by Xu et al.(2012). NdeI-mcrN-XhoI and NdeI-mcrC-XhoI fragments
from pMK-RQwere cloned into the same restriction sites in pETM6
(pETM6-mcrN andpETM6-mcrC). The AvrII-mcrC-SalI fragment from
pETM6-mcrC wasligated with SpeI/SalI-digested pETM6-mcrN
(pETM6-mcrNC). Nar-ingenin producing E. coli (FlavoOpt) was
previously constructed usingBL21star(DE3) that was transformed with
a plasmid vector carrying the
naringenin biosynthetic genes (Jones et al., 2016b).
2.2. Batch culture protocol for 3HP production
Lennox medium (10 g L−1 tryptone, 5 g L−1 yeast extract, 5 g
NaCl, 1 g L−1 glucose), standard M9 medium, and specific
nutrient-freeM9 medium were used for culture experiments. Complete
mediumcompositions are provided in Table S1 in the Supplemental
materials.All cultures were supplemented with 0.08 g L−1 ampicillin
to maintainthe pETM6 plasmid. Strain E. coli MG1655(DE3)/pETM6
mcrNC wasgrown aerobically at 37 °C overnight in a test tube
containing 3mL ofLennox medium. The culture was diluted into 500mL
baffled Erlen-meyer flasks containing 200mL of standard M9 medium
with an initialoptical density at 600 nm (OD600) of 0.05 and cells
were grown aero-bically at 37 °C in a shaking incubator at 150 rpm
(BR-43FL, Taitec,Japan). For expression of MCR, 1.0mM isopropyl
β-D-1-thiogalacto-pyranoside (IPTG), 0.04 g L−1 biotin and 20mM
NaHCO3 were addedat mid log phase. After 20 h of cultivation, cells
were collected bycentrifugation at 2900×g for 10min at 25 °C and
inoculated into200mL baffled Erlenmeyer flasks containing 50mL of
nitrogen-free,phosphorus-free, sulfur-free or magnesium-free M9
medium with aninitial OD600 of 2.0. Cells were cultured at 37 °C in
a shaking incubatorat 150 rpm (BR-43FL, TAITEC, Saitama,
2.3. Batch culture protocol for naringenin production
Standard M9 medium and specific nutrient-limited M9 mediumwere
used for culture experiments. Complete medium compositions
areprovided in Table S2 in the Supplemental materials. All cultures
weresupplemented with 0.08 g L−1 ampicillin to maintain the
plasmid. The strain E. coli BL21star(DE3)/pFlavoopt was grown
over-night at 37 °C in a 50-mL falcon tube containing 5mL of
standard M9medium. After 16 h, the overnight cultures were serially
diluted in 0.9%NaCl and then 50 μL of the diluted cells were
inoculated in poly-propylene 48-well plates (5 mL, VWR) containing
5mL of standard M9medium or nutrient-limited M9 medium. The culture
was inoculated atdilution ratios of 1:20, 1:30, 1:40, 1:60, 1:80,
1:120, 1:160, and 1:240from the overnight culture. The cells were
allowed to grow aerobicallyat 37 °C for 6 h before induction with
1mM IPTG and 10mg/L biotin.As a substrate, 0.61mM p-coumaric acid
was added at induction, and30 °C was used as the induction
2.4. Analytical methods
Cell optical density was measured at 600 nm by using UV-mini
1240(Shimadzu, Kyoto, Japan) for 3HP-producing E. coli, and using
BioTekSynergy 4 microplate reader (BioTek, USA) for
naringenin-producing E.coli. For determining cell dry weight (CDW)
of 3HP-producing strain
Table 1E. coli strains and plasmids used in this study.
Strains or plasmids Description Source
StrainsDH5α F－, Φ80d lacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1,
endA1, hsdR17(rK－ mK+), phoA, supE44, λ－, thi－1, gyrA96,
relA1MG1655(DE3) F−, λ−, rph-1, λ (DE3[lacI lacUV5-T7 gene l
indl sam7 nin5]) (Tokuyama et al., 2014)MG1655(DE3)/pETM6 mcrNC
MG1655(DE3) transformed with pETM6 mcrNC This
studyBL21star(DE3)/pFlavoOpt BL21star(DE3) transformed with
pFlavoOpt (Jones et al., 2016b)PlasmidspETM6 lacI; expression
vector; T7 promoter; T7 terminetor; ColE1-ori; Ampr (Xu et al.,
2012)pMK-RQ mcrN Synthesized NdeI-mcrN-XhoI; ColE1-ori; Kanr This
studypMK-RQ mcrC Synthesized NdeI-mcrC-XhoI; ColE1-ori; Kanr This
studypETM6 mcrN pETM6 carrying artificially synthesized mcrN; Ampr
This studypETM6 mcrC pETM6 carrying artificially synthesized mcrC;
Ampr This studypETM6 mcrNC pETM6 carrying artificially synthesized
mcrNC; Ampr This studypFlavoopt pETM6 carrying naringenin
biosynthetic genes (At4CL, PhCHS,CmCHI); Ampr (Jones et al.,
K. Tokuyama et al. Metabolic Engineering 52 (2019) 215–223
under each starvation condition, a consistent amount of cells
[OD600× culture volume (mL) =100] was harvested by 0.5-μm pore size
filter(PTFE-type membrane, Advantec, Tokyo, Japan) at 20 h for
nitrogenstarvation, 8 h for phosphorus or sulfur starvation, and 4
h for magne-sium starvation. These samples were dried at 60 °C
until constantweight. For determining CDW of naringenin-producing
strain, 40mL ofculture was collected by centrifugation at 3000×g
for 10min at roomtemperature using a BD Falcon 50mL conical tube.
After removing thesupernatant, the conical tube was dried at 60 °C
until constant weight.Based on the subtraction of the initial
weight of the filter or conicaltube, the conversion coefficients
between OD600 and CDW were cal-culated.
Concentrations of glycerol, 3HP, succinate, lactate, acetate,
formate,and ethanol present in the supernatant of the culture
broths of 3HP-producing E. coli were determined by an HPLC system
(HPLCProminence, Shimadzu) equipped with an Aminex HPX-87H
column(Bio-Rad, Hercules, CA, U.S.A.), a UV/Vis detector (SPD-20A),
and arefractive index detector (RID-10A). The column temperature
was set to65 °C, and 1.5 mM H2SO4 was used as the mobile phase with
a flow rateof 0.6 mLmin−1. The flow cell temperature of the
refractive index de-tector was set to 35 °C. The supernatant of the
culture broth was ob-tained by centrifugation at 21,500×g for 5min
at 4 °C, and then filteredthrough a Millex HV 0.45-μm filter (Merck
KGaA, Germany). The su-pernatant of naringenin-producing E. coli
was analyzed for quantifica-tion of naringenin as described
previously (Xiu et al., 2017).
2.5. Collection of intermediate metabolites
Culture broth of 3HP-producing E. coli was sampled rapidly
andfiltered through a 0.5-μm pore size filter (PTFE-type
membrane,Advantec, Tokyo, Japan). Cells on the filter were
immediately im-mersed in 1.6 mL methanol (−80 °C) containing 5 μM
cycloleucine asan internal standard for the quantitation by gas
chromatography-massspectrometry (GC/MS) and kept at −80 °C until
extraction. For thepreparation of isotope-labeled standards to
quantify intracellular ab-solute metabolite concentration by liquid
chromatography-tandemmass spectrometry (LC-MS/MS), 25mL culture
broth of MG1655(DE3)strain grown on a standard M9 medium containing
4 g L− 1 [U-13C]glucose was collected at mid-log phase and
stationary phase. Followingaddition of 1.6mL of chloroform (−30 °C)
and 640 μL of Milli-Q water(4 °C) and vortexing for 1min, the
mixture was centrifuged at 3700×gfor 20min at 4 °C. The aqueous
layer was dispensed into sevenEppendorf tubes as 250 μL in each and
dried using a SpeedVac SPD1010(Thermo Fisher Scientific, Waltham,
MA, USA) at room temperature. Todetermine the concentrations of the
13C-labeled intermediates, thedried extract was dissolved in 50 μL
of standard solution containingknown concentrations of unlabeled
metabolites. For analysis ofMG1655(DE3)/pETM6 mcrNC, 10mL culture
broth was collected at20 h for nitrogen starvation, 8 h for
phosphorus or sulfur starvation, and4 h for magnesium starvation.
Metabolites were extracted by additionof 1.6 mL of chloroform, 640
μL of Milli-Q water dissolving the twodried samples of the isotope
labeled extract. Dried samples were ob-tained by the same procedure
2.6. LC-MS/MS analysis
LC-MS/MS analysis (LC: Agilent 1100 series, Agilent
Technologies,Santa Clara, CA, USA; MS/MS: API 2000, AB SCIEX, MA,
USA) wasperformed under the following conditions (Nishino et al.,
2015):column, ProteCol-P C18 HQ103 (2.1× 150mm, particle size of 3
µm);mobile phase, 10mM tributylamine/15mM acetic acid in water (A)
andmethanol (B); flow rate: 0.2mLmin−1; gradient curve, 100% A:0% B
at0min, 100% A:0% B at 8min, 10% A:90% B at 24min, 100% A:0% B
at24.1 min, and 100% A:0% B at 30min; injection volume, 3 μL;
columntemperature, 35 °C; mode of mass analysis, negative ion mode;
nebu-lizer flow, 55 psi; dry gas flow rate, 10 Lmin−1; sheath gas
11 Lmin−1; dry gas temperature, 300 °C; sheath gas
temperature,380 °C; capillary voltage, 3.5 kV. The dried sample was
dissolved in50 μL of Milli-Q water. The peak of each target
metabolite was identi-fied by comparing its chromatographic
behavior with that of an au-thentic standard. Peak area was
determined using the software Analyst(version 1.6.2, AB SCIEX).
Quantitation was performed based on the MSdata using the
intracellular concentration of each metabolite (mmol L-cell−1)
determined using the ratio of the 13C peak area to the 12C
peakarea. The ratio of aqueous volume to cellular dry weight is
0.00177 L/gCDW for E. coli (Neidhardt et al., 1996).
2.7. GC/MS analysis
GC/MS analysis (GC/MS: GCMS-QP2010 Ultra, Shimadzu, Japan)was
performed under the following conditions (Tsugawa et al., 2011):The
column was a 30m×0.25mm i.d. fused silica capillary columncoated
with 0.25 µm CP-SIL 8 CB low bleed/MS (Agilent). The frontinlet
temperature was 230 °C. The helium gas flow rate through thecolumn
was 1mLmin−1. The column temperature was held at 80 °C for2min
isothermally and then raised by 15 °Cmin−1 to 330 °C and heldthere
for 6min isothermally. The transfer line and ion-source
tem-peratures were 250 °C and 200 °C, respectively. The dried
extract wasdissolved in 50 μL methoxyamine hydrochloride
(20mgmL-pyr-idine−1) and incubated at 30 °C for 90min. For
trimethyl silylation,25 μL of N-methyl-N-(trimethylsilyl)
trifluoroacetamide was added andincubated at 37 °C for 30min. After
2 h cooling and centrifugation for5min at 20,400×g, 1 μL of
derivatized supernatants were injected withsplit injection ratio of
2.8. Metabolome data analysis
Metabolite peak areas were obtained by analyzing LC-MS/MS
orGC/MS data with each instrument's software. For the quantitation
byLC-MS/MS, the intracellular concentration of each metabolite was
de-termined using the ratio of the 13C peak area to the 12C peak
area. Forthe quantitation by GC/MS, the intracellular concentration
of eachmetabolite was determined with internal standard method
using cy-cloleucine as an internal standard. The correlation
analysis betweenmetabolites concentrations and culture profile was
performed by R3.2.4. software (http://www.r-project.com) and
3.1. Comparison of 3HP production under different nutrient
3HP-producing E. coli of MG1655(DE3)/pETM6 mcrNC was
con-structed by over-expression of codon optimized mcrNC gene
derivedfrom C. aurantiacus using pETM6 vector in MG1655(DE3). The
strainwas aerobically cultivated overnight in standard M9 medium.
Cellpellets were obtained by centrifugation and resuspended in
nitrogen-free or phosphorus-free or sulfur-free or magnesium-free
M9 mediumwith an initial OD600 of 2.0. The strain continuously
consumed glucosewith no cell growth in all starved conditions until
glucose was com-pletely depleted (Fig. 1). The conversion
coefficients between OD600and CDW for magnesium, sulfur,
phosphorus, and nitrogen starvationwere 0.33 ± 0.02, 0.32 ± 0.00,
0.32 ± 0.00, and 0.33 ± 0.02gCDW L−1 OD600−1, respectively. No
significant difference was ob-served among the conditions. The
culture results are summarized inTable 2. High-level 3HP production
by the strain was observed undersulfur starvation (titer and yield
were 2.3 ± 0.1mM and 6.0 ± 0.1C-mol%) or magnesium starvation
(titer and yield were 2.0 ± 0.0mMand 5.7 ± 0.1C-mol%), whereas
almost no 3HP production was de-tected under nitrogen starvation
(titer and yield were 0.1 ± 0.0mMand 0.2 ± 0.0C-mol%) or phosphorus
starvation (not detected).Acetate was produced as a major byproduct
in all the cultures. The
K. Tokuyama et al. Metabolic Engineering 52 (2019) 215–223
highest production of acetate was confirmed under phosphorus
star-vation (15.6 ± 0.3mM). Ethanol, lactate, and succinate were
not de-tected in all the cultures. Total carbon recovery was lowest
under ni-trogen starvation (28.2 ± 0.8C-mol%). A part of consumed
glucoseunder nitrogen starvation might be used for glycogen
synthesis (Holmeand Palmstierna, 1956).
Specific glucose uptake rate was lowest under nitrogen
starvation(0.28 ± 0.02mmol gCDW−1 h−1) and highest under
magnesiumstarvation (2.1 ± 0.06mmol gCDW−1 h−1). This result is
consistentwith the characteristics of wild type E. coli under
nutrition starvationconditions (Chubukov and Sauer, 2014).
3.2. Metabolic profiling analysis of 3HP-producing E. coli
The metabolic states under the four different stationary phase
cul-tivations were compared by metabolome-analysis with GC/MS and
LC-MS/MS. The concentrations of 42 intracellular metabolites
includingamino acids, sugar phosphates, and organic acids were
determined(Fig. 2). Correlation analysis of all metabolite
concentrations and thespecific rate was performed to evaluate
intracellular metabolic stateand identify key factors for 3HP
production (Fig. 3).
The specific 3HP production rate had a strong correlation(r >
0.85) with ATP, malonyl-CoA, intracellular 3HP, and
glycolysismetabolites, such as glucose 6-phosphate,
phosphoenolpyruvate (PEP). Because malonyl-CoA is a precursor
of3HP, the higher accumulation of malonyl-CoA under magnesium
orsulfur starvation condition contributed to the enhanced 3HP
produc-tion. The high ATP concentration under magnesium or sulfur
starvationcould contribute to the malonyl-CoA supply because
malonyl-CoA issynthesized from acetyl-CoA and CO2 using ATP by
acetyl-CoA car-boxylase. Strong positive correlation between ATP
and malonyl-CoA(r=0.95), as well as between 3HP production rate and
malonyl-CoA(r=0.84), also indicated that the ATP level is important
to producemalonyl-CoA by acetyl-CoA carboxylase (Fig. 3A, C).
Intracellular 3HPconcentrations also had a strong correlation with
the 3HP productionrate (r=0.92, Fig. 3B), and was higher than their
extracellular con-centrations under all starvation conditions. This
result suggested thatthe transport of 3HP could be a limiting step
for 3HP production duringstationary phase.
The specific glucose uptake rate had a strong correlation (r
> 0.85)with PEP and methionine. PEP accumulation contributed to
increase inthe specific 3HP production rate coupled with the
specific glucose up-take rate, because PEP is a substrate for the
phosphotransferase system(PTS) of glucose transport (Chubukov and
Sauer, 2014) (Fig. 3D). Al-though it has been reported that a
strong correlation exists betweenfructose-1,6-bisphosphate (FBP)
and the glycolytic flux in growing E.coli cells (Kochanowski et
al., 2013), no such correlation between FBPlevels and the specific
glucose uptake rate (r < 0.01) was observed
Fig. 1. Time-course of OD600, glucose, 3HP,and acetate levels in
the 3HP-producing E. coliunder the starvation conditions. (A)
nitrogenstarvation, (B) phosphorus starvation, (C)sulfur
starvation, and (D) magnesium starva-tion. Error bars represent
standard deviation oftriplicate experiments. Almost all the
errorbars in this figure are smaller than the symbols.
Table 2Summary of the culture results of 3-hydroxypropionic acid
producing E. coli strain.
Nitrogen starvation Phosphorus starvation Sulfur starvation
Consumed glucose (mM) 17.8 ± 0.4 17.1 ± 0.1 19.1 ± 1.0 17.2 ±
0.13HP titer (mM) 0.1 ± 0.0 0.0 ± 0.0 2.3 ± 0.1 2.0 ± 0.0Glucose
uptake rate (mmol gCDW-1 h-1) 0.28 ± 0.02 1.04 ± 0.04 0.73 ± 0.03
2.1 ± 0.063HP production rate (mmol gCDW-1 h-1) 0.00 ± 0.00 0.00 ±
0.00 0.14 ± 0.01 0.28 ± 0.01Acetate production rate (mmol gCDW-1
h-1) 0.22 ± 0.02 0.75 ± 0.03 0.54 ± 0.02 1.54 ± 0.063HP yield
(C-mol%) 0.2 ± 0.0 0.0 ± 0.0 6.0 ± 0.1 5.7 ± 0.1Acetate yield
(C-mol%) 22.9 ± 0.7 30.4 ± 0.4 25.0 ± 0.5 27.7 ± 0.6Biomass yield
(C-mol%) 5.0 ± 0.2 17.5 ± 1.3 8.5 ± 0.3 18.2 ± 1.1Total carbon
recovery (C-mol%) 28.2 ± 0.8 47.9 ± 1.7 39.5 ± 0.2 51.6 ± 0.9
K. Tokuyama et al. Metabolic Engineering 52 (2019) 215–223
Fig. 2. Metabolic profiling of central metabolic pathway. Each
panel represents the absolute metabolites concentrations of
MG1655(DE3)/pETM6 mcrNC undernitrogen starvation, phosphorus
starvation, sulfur starvation and magnesium starvation. Error bars
represent standard deviation of triplicate culture experiments.
Fig. 3. Scatter plot with correlation analysisresults. Red solid
line indicates regression linewith strong correlation coefficient.
(Red) ni-trogen starvation, (Orange) phosphorus star-vation,
(Green) sulfur starvation and (Blue)magnesium starvation. (For
interpretation ofthe references to color in this figure legend,
thereader is referred to the web version of thisarticle.)
K. Tokuyama et al. Metabolic Engineering 52 (2019) 215–223
under the starvation conditions (Fig. 3E).The accumulation
levels of almost all intracellular free amino acids
showed the same tendency depending on the nutrient starvation
con-dition. Intracellular free amino acids under nitrogen
starvation were thelowest of all starvation conditions. Sulfur
starvation showed higheramount of accumulation of free amino acids
except for methionine.These results suggested that E. coli cells
consumed all of free aminoacids under nitrogen starvation, or
methionine under sulfur starvation,as a source of nitrogen or
3.3. Application of nutrient starvation for flavonoid
To demonstrate the effect of sulfur or magnesium starvation on
theother target metabolite derived from malonyl-CoA, the nutrient
star-vation conditions were implemented for naringenin production
usingan engineered E. coli strain constructed in previous study
(Jones et al.,2016b). Naringenin was chosen for two reasons. First,
naringenin isknown as a pharmacologically useful plant flavonoid
molecule (Wanget al., 2016). Second, several reports have
demonstrated that malonyl-CoA supply is a major bottleneck for the
naringenin production(Leonard et al., 2008; Fowler et al., 2009; Wu
et al., 2014). One mo-lecule of naringenin is synthesized from one
molecule of p-coumaricacid using three molecules of
The strain of BL21star(DE3)/pFlavoOpt has an IPTG-inducible
ex-pression vector containing biosynthetic genes for naringenin
produc-tion. The engineered strain optimized for the expression of
the nar-ingenin biosynthetic genes, was cultivated using standard
minimal M9medium and nutrient starved minimal M9 media. Because
productivityof the flavonoid production is sensitive to the
induction time point(Jones et al., 2016a), cells from stationary
phase culture broth onstandard M9 medium were serially diluted and
inoculated into a freshmedium, and IPTG was added at 6 h or 7 h.
The culture results areshown in Fig. 4. The highest production
titer of 144 ± 15 μM wasobserved under magnesium starvation with
IPTG induction at mid-logphase, whereas the maximum titer was 117 ±
7 μM on standard M9medium. Maximal productivity was also higher
under magnesiumstarvation culture (127 ± 21 μmol gCDW−1) than in
standard condi-tion (41 ± 0.5 μmol gCDW−1). These results indicated
that magne-sium starvation improved the carbon flux toward
malonyl-CoA synth-esis, and led to increase in naringenin
production. On the other hand,sulfur starvation was not suitable
for naringenin production(23 ± 0.4 μM maximum titer), because the
cell growth under sulfurstarvation condition was drastically
decreased after the induction (seeFig. S1). The productivity under
sulfur starvation was slightly improvedcompared with the standard
M9 condition (61 ± 2 μmol gCDW−1).
To investigate whether the nutrient starvation approach is
affectedby type of carbon sources, the naringenin production under
the mag-nesium starvation was also evaluated with xylose or
glycerol minimalmedium with IPTG induction after 6 h. Here again,
productivities of naringenin were higher under magnesium
starvationthan in the control condition (Fig. S2). These results
demonstrated thatthe effect of magnesium starvation is independent
of the carbon sourcetype, and is useful for the development of the
Essential nutrient starvation is one of the approaches for
bio-pro-duction in stationary phase. Nitrogen starvation is widely
used for in-hibiting cell growth and successfully enhancing
productivity of varioustargets, such as poly(3-hydroxybutyrate) and
butanol in stationaryphase (Wang and Lee, 1997; Al-Shorgani et al.,
2012). However, theseproductivities in stationary phase might
change with different nutrientstarvation because the metabolic
state is dependent on the specificnutrient limitation. In the
present study, we focused on the influence ofspecific nutrients
starvations on metabolic activities and investigatedthe optimum
nutrient starvation condition for producing malonyl-CoAderived
metabolites in E. coli.
High-level 3HP production was observed under sulfur or
magne-sium starvation, whereas almost no 3HP production was
detected undernitrogen or phosphorus starvation. Increased ATP
concentration shouldenhance the malonyl-CoA supply and lead to
production of 3HP undersulfur or magnesium starvation. Culture
experiments with the nar-ingenin-producing E. coli indicated that
magnesium starvation leads tothe highest production titer (144 ± 15
μM) and specific productivity(127 ± 21 μmol gCDW−1). These results
demonstrated that magne-sium starvation is a useful approach to
activate metabolic state for theproduction of malonyl-CoA
Specific substrate uptake rate is an important factor for 3HP
pro-duction because the specific glucose uptake rate in stationary
phasetends to be lower than in exponential growth phase (Chubukov
andSauer, 2014). E. coli producing 3HP under nitrogen starvation
showedthe lowest glucose uptake rate. This could be explained by
accumula-tion of α-ketoglutarate (αKG), because αKG acts as an
allosteric in-hibitor for PTS. The αKG concentration under nitrogen
starvation(4.1 mM) was higher than the Ki value of PTS (1.3 mM).
Previous studyreported that FBP concentration had a strong
correlation with theglycolytic flux in growing E. coli cells
(Kochanowski et al., 2013).However, no correlation between FBP
concentration and the specificglucose uptake rate was observed in
this study (r < 0.01, Fig. 3E). Theaccumulation of αKG under
nitrogen starvation could have hamperedthis correlation. Nitrogen
starvation is a challenge for improving theproduction rate because
αKG strongly inhibits glucose uptake and ATPsupply in TCA cycle
(Fig. 5A) (Doucette et al., 2011; Wright et al.,1967). In this
study, the specific glucose uptake rate had a strong cor-relation
with PEP (r=0.87). PEP is needed for glucose transport as
asubstrate of PTS and the accumulation of PEP contributed to
glucoseuptake into the cell. Increasing PEP accumulation would be
Fig. 4. Naringenin production under nutrient
starvationconditions. (A) Naringenin production versus cell
densityat induction time point using standard M9 and
nutrient-limited M9 (sulfur or magnesium starvation). (B)
Maximalproductivity of naringenin per final culture volume at24 h.
Cell density was calculated from OD600 using theexperimentally
determined conversion coefficient of 0.8gCDW L−1 OD600−1. Error
bars represent standard de-viation of triplicate experiments.
K. Tokuyama et al. Metabolic Engineering 52 (2019) 215–223
enhancing glucose uptake rate in stationary phase.Phosphorus
starvation decreased the intracellular concentration of
malonyl-CoA and ATP. A previous study reported that
phosphorusstarvation decreases flux of the TCA cycle with
activation of tran-scriptional regulator arcA (Marzan and Shimizu,
2011). The decrease influx of TCA cycle led to shortage of ATP and
malonyl-CoA (Fig. 5B).Sulfur starvation resulted in the highest ATP
level and caused 3HPproduction increase due to increased supply of
malonyl-CoA. E. coliuptakes sulfate as a source of sulfur and
produces cysteine with con-sumption of two molecules of ATP and one
molecule of NADPH persulfate molecule (Sekowska et al., 2000).
Higher accumulation ofmalonyl-CoA should be achieved by reducing
consumption of ATP viasulfur assimilation (Fig. 5C). Magnesium
starvation improved glucoseuptake rate and specific 3HP production
rate to the highest level in allof the cultures. Pyruvate kinase
converts PEP to pyruvate, and needsmagnesium in the active site of
the enzyme (Garfinkel and Garfinkel,1985; Kumar and Barth, 2010).
Magnesium starvation decreased theactivity of pyruvate kinase and
accumulated PEP, which led to in-creased specific glucose uptake
(Fig. 5D). Magnesium starvation in-creased PEP levels compared with
the other starvations. The sulfur ormagnesium starvation improved
production of malonyl-CoA-derivedmetabolites. Because these two
starvations increase ATP concentrationthrough different mechanisms
(Fig. 5), the 3HP productivity might befurther enhanced under the
double starvation of magnesium and sulfur.In a large scale process,
cells cannot be collected by centrifugation andtransferred to a
fresh medium, therefore, it should be led to the sta-tionary phase
by reducing the concentration of an essential nutrient inthe
initial medium. Since the cell growth stops when an essential
nu-trient is depleted, it must be difficult to simultaneously
deplete twoessential nutrients in industrial bio-production
process. However, theevaluations under such combinatorial
starvations would provide ussome interesting knowledge for the
Strong positive correlation (r= 0.945) between intracellular
con-centrations of ATP and malonyl-CoA indicates that the ATP level
isimportant for malonyl-CoA synthesis. However, there is difference
in3HP productivity among the conditions of sulfur or magnesium
star-vation despite similar intracellular ATP (Fig. 3A, and C).
This differencealso be related to the binding between Mg ion and
ATP. In many en-zymes including acetyl-CoA carboxylase (Guchhait et
al., 1974), ATPhas to bind with magnesium ion due to their
activity. The proportion ofMgATP must decrease under magnesium
starvation. Actually, it hasbeen reported that the proportion of
MgATP was 88% under standardconditions, whereas it was 70% under
magnesium-free conditions incytosol of sycamore cells (Gout et al.,
2014). Although the proportion ofMgATP was not measured in this
study, that under the magnesiumstarvation must be lower than under
sulfur starvation. However, Fig. 3Ashows that the 3HP production
under the magnesium starvation ishigher than that under the sulfur
starvation. This suggests that there is
no shortage of MgATP under the magnesium starvation. Since the
cellswere transfer to the magnesium-free medium after growing on
normalM9 medium, a certain amount of Mg would exist in the cells
under themagnesium starvation. A hypothesis can explain the reason
why 3HPproduction under magnesium starvation was higher than that
undersulfur starvation. The malonyl-CoA reductase required NADPH as
co-factor. Chubukov and Sauer (2014) have proposed a hypothesis
thatglycolytic flux was shuttled to the Entner-Doudoroff (ED)
pathwayunder magnesium starvations. Because a NADPH is generated
whenglucose is catabolized via the ED pathway, the activity of
malonyl-CoAreductase would be activated due to the enhanced NADPH
supply. Al-though the amount of NADPH under the magnesium
starvation in Fig. 2did not increase, it would be balanced at this
concentration becauseNADPH consumption also increased due to the
increasing 3HP pro-duction rate.
This study evaluated the effect of nutritional starvation on
3HP-producing E. coli with overexpression of malonyl-CoA reductase;
mag-nesium or sulfur starvation led to high-level 3HP production
yield(5.7C-mol% and 6.0C-mol%). In a previous report, a higher
yield of3HP was achieved at 18C-mol% by the batch cultivation of E.
coli withoverexpression of acetyl-CoA carboxylase derived from C.
glutamicum(Cheng et al., 2016). The additional overexpression of
acetyl-CoA car-boxylase should improve 3HP production during
stationary phase.Metabolic profiling analysis revealed that 3HP
transport system is alimiting step because intracellular
concentration of 3HP was higherthan that in extracellular. The 3HP
production could be enhanced byactivation of the transporter for
3HP. Since little is known about thetransport system of 3HP in E.
coli, it is important to identify thetransporter for 3HP.
The type of nutrient starvation should be selected for each
targetmetabolite in bio-production with stationary phase cells.
However, ef-fects of nutrient starvation for production of each
target metabolitewere largely unclear. In this study, metabolic
profiling analysis char-acterized the metabolic state during
stationary phase and suggested anoptimum nutrient starvation
condition for target metabolite produc-tion. For example, in
production of malonyl-CoA derivatives, such as3HP, fatty-acids or
flavonoids, it might be suitable to use sulfur ormagnesium
starvation because of accumulation of high amount of in-tracellular
malonyl-CoA. The naringenin-producing E. coli demon-strated that
sulfur or magnesium starvation increased the specific pro-ductivity
of naringenin (Fig. 4B). Furthermore, the production titerunder
magnesium starvation condition was higher than of standardminimal
medium without nutrient starvation, though magnesiumstarvation
decreased the final cell density. For further development
offermentation process of naringenin production, magnesium-limited
fedbatch technique is a possible way to maintain the metabolic
state forhigh production, and also improve cell densities (Willrodt
et al., 2016).Production of acetyl-CoA derivatives, such as
Fig. 5. Metabolic state of 3HP-producing E. coli with
malonyl-CoA reductase under several nutrient starvation conditions.
Gray arrow indicates possible low-activereactions revealed by the
metabolic profiling analysis.
K. Tokuyama et al. Metabolic Engineering 52 (2019) 215–223
isopropanol, might be increased using sulfur, magnesium, or
phos-phorus starvation because of accumulation of high amount of
in-tracellular acetyl-CoA. Amino acid production might be increased
usingsulfur starvation because the highest accumulation of amino
acids wasobserved under sulfur starvation. On the other hand,
sulfur starvationmust not be the best strategy to improve the
production of cysteine ormethionine because these amino acids are a
source of sulfur for cellgrowth. Li et al. demonstrated the
increased production of tyrosine andmevalonate in engineered E.
coli upon sulfur starvation (Li et al., 2016).As shown in our
results, evaluation of productivity of each metaboliteunder
different nutrient starvation conditions is important for
CRediT authorship contribution statement
Kento Tokuyama: Conceptualization, Data curation, Writing
-original draft, Writing - review & editing. Yoshihiro
Toya:Conceptualization, Writing - original draft, Writing - review
&editing. Fumio Matsuda: Conceptualization, Methodology,
Writing -original draft, Writing - review & editing. Brady F.
Cress:Conceptualization, Writing - original draft, Writing - review
&editing. Mattheos A.G. Koffas: Conceptualization, Writing -
originaldraft, Writing - review & editing. Hiroshi Shimizu:
Conceptualization,Writing - original draft, Writing - review &
This research was supported by a Grant-in-Aid for Japan Society
forthe Promotion of Science (JSPS) Fellows (15J06350) and “Program
forLeading Graduate Schools” of the Ministry of Education,
Culture,Sports, Science and Technology, Japan. This work was
partially sup-ported by a Japan Science and Technology Agency
(JST)-Mirai ProgramGrant Number JPMJMI17EJ, Japan, and a
Grant-in-Aid for ScientificResearch (B) No.16H04576, Japan. M.A.G.
would like to acknowledgefunding from NSF CBET award number
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K. Tokuyama et al. Metabolic Engineering 52 (2019) 215–223
Magnesium starvation improves production of malonyl-CoA-derived
metabolites in Escherichia coliIntroductionMaterials and
methodsStrains and plasmidsBatch culture protocol for 3HP
productionBatch culture protocol for naringenin
productionAnalytical methodsCollection of intermediate
metabolitesLC-MS/MS analysisGC/MS analysisMetabolome data
ResultsComparison of 3HP production under different nutrient
starvation conditionsMetabolic profiling analysis of 3HP-producing
E. coliApplication of nutrient starvation for flavonoid