Production of 3-hydroxypropionate using a novel malonyl-CoA-mediated biosynthetic pathway in genetically engineered: E. coli strain

Article abstract of DOI:10.1039/c9gc02286d

3-Hydroxypropionic acid (3-HP) is a promising platform chemical with a wide range of applications. The traditional chemical synthesis of 3-HP is well-established, but the resource limitations, high price and toxicity of the used raw materials do not meet the new sustainable development goals. Accordingly, the microbial synthesis of 3-HP by fermentation will become a promising and attractive route mainly due to its environmentally friendly production, use of renewable resources, and sustainable development. Herein, to biosynthesize 3-HP directly from malonate, a novel malonyl-CoA-mediated biosynthetic pathway was successfully developed as follows. Firstly, various transporters involved in malonate transportation was systematically investigated and screened. Secondly, to biosynthesize 3-HP, an original strategy was employed by heterologously co-expressing the mutant of malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus and malonyl-CoA synthetase (MatB) from Rhodopseudomonas palustris in the Escherichia coli C43 (DE3) strain, which was screened from three different MatB enzymes. Finally, to further enhance the production of 3-HP, native transhydrogenase (PntAB) and NAD kinase (YfjB) genes were expressed to increase the NADPH supply in E. coli. The final genetically modified strain SGN78 showed a significant improvement in malonate utilization and produced 1.20 ± 0.08 g L^-1 of 3-HP in the flask culture. Thus, this work demonstrates the production of 3-HP in E. coli with the shortest route for the biosynthesis of 3-HP, which involved only three steps from the substrate. Also, it opens a path for the biosynthesis of 3-HP and other malonyl-CoA-based valuable chemicals directly from malonate in E. coli.

Full text of DOI:10.1039/c9gc02286d

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Production of 3-hydroxypropionate using a novel
malonyl-CoA-mediated biosynthetic pathway in
genetically engineered E. coli strain†
Cite this: Green Chem., 2019, 21,
6103
Bo Liang,‡a,b Guannan Sun,‡a,b Zhaobao Wang,a,b Jian Xiao c and
a,b
Jianming Yang
*
3-Hydroxypropionic acid (3-HP) is a promising platform chemical with a wide range of applications. The
traditional chemical synthesis of 3-HP is well-established, but the resource limitations, high price and tox-
icity of the used raw materials do not meet the new sustainable development goals. Accordingly, the
microbial synthesis of 3-HP by fermentation will become a promising and attractive route mainly due to
its environmentally friendly production, use of renewable resources, and sustainable development.
Herein, to biosynthesize 3-HP directly from malonate, a novel malonyl-CoA-mediated biosynthetic
pathway was successfully developed as follows. Firstly, various transporters involved in malonate transpor-
tation was systematically investigated and screened. Secondly, to biosynthesize 3-HP, an original strategy
was employed by heterologously co-expressing the mutant of malonyl-CoA reductase (MCR) from
Chloroflexus aurantiacus and malonyl-CoA synthetase (MatB) from Rhodopseudomonas palustris in the
Escherichia coli C43 (DE3) strain, which was screened from three different MatB enzymes. Finally, to
further enhance the production of 3-HP, native transhydrogenase (PntAB) and NAD kinase (YfjB) genes
were expressed to increase the NADPH supply in E. coli. The final genetically modified strain SGN78
showed a significant improvement in malonate utilization and produced 1.20 0.08 g L−1 of 3-HP in the
flask culture. Thus, this work demonstrates the production of 3-HP in E. coli with the shortest route for
the biosynthesis of 3-HP, which involved only three steps from the substrate. Also, it opens a path for the
biosynthesis of 3-HP and other malonyl-CoA-based valuable chemicals directly from malonate in E. coli.
Received 6th July 2019,
Accepted 2nd October 2019
DOI: 10.1039/c9gc02286d
rsc.li/greenchem
sodium cyanide. For the β-propiolactone route, the raw
materials ketene and methanol are highly toxic, and the inter-
1. Introduction
3-Hydroxypropionic acid (3-HP) is an important platform com- mediate β-propiolactone is carcinogenic. In addition, 3-HP can
pound, which is ranked in the list of the top 12 most potential also be produced directly via the oxidation of acrylic acid or
chemicals by the US Department of Energy due to its excellent PDO.3,5 However, due to the rapid increase in the price of
performance and broad application prospect.1,2 Traditionally, acrylic acid and PDO in recent years, they have become econ-
3-HP can be derived from the hydrolysis of β-propiolactone or omically unfavorable.3 Thus, the traditional chemical synthesis
3-hydroxypropionitrile. However, the major disadvantages of of 3-HP is well-established, but the resource limitations, high
these routes are the high toxicity of the synthesis process.3,4 price and toxicity of the used raw materials do not meet the
The 3-hydroxypropionitrile route needs the toxic and noxious new sustainable development goals.6 Thus, compared with
conventional chemical synthesis, the microbial synthesis of
3-HP by fermentation will become a promising and attractive
route mainly for its many advantages, including cheaper sub-
strates, nontoxic raw materials, mild reaction conditions and
aEnergy-rich Compounds Production by Photosynthetic Carbon Fixation Research
Center of Qingdao Agricultural University, People’s Republic of China.
E-mail: yjming888@126.com
bShandong Key Lab of Applied Mycology, College of Life Sciences,
Qingdao Agricultural University, Qingdao, Shandong Province,
People’s Republic of China
cCollege of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University,
Qingdao, Shandong Province, People’s Republic of China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9gc02286d
simple operation, which are compatible with sustainable devel-
opment, and the reduced amounts of byproducts help reduce
the cost of production.
Currently, the fermentation of 3-HP mainly focuses on
various organisms using glucose and glycerol as substrates,
including Escherichia coli,7–9 yeast,6,10,11 Klebsiella sp.12,13
Corynebacterium glutamicum,14 Synechococcus sp.15,16 and
‡These authors contributed equally to this work.
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Methylobacterium sp.17 To date, the malonyl-CoA pathway is periplasmic (TRAP) transporters involving malonate transport
generally recognized as the shortest route for the biosynthesis have been identified in Rhodobacter capsulatus and
of 3-HP, which is composed of three modules, including the Sinorhizobium meliloti.34,35 TRAP transporters are a family of
fermentation production of acetyl-CoA in cells, production of substrate-binding protein (SBP)-dependent secondary trans-
malonyl-CoA from acetyl-CoA by acetyl-CoA carboxylase (ACC) porters in prokaryotes, composed of three subunits, the sub-
and biosynthesis of 3-HP by malonyl-CoA reductase (MCR) strate-binding protein (SBP), and the large and small-trans-
from malonyl-CoA. However, the key intermediate malonyl- membrane domains (TMDs).36 TRAP transporters are not only
CoA is the essential precursor for the synthesis of polyketones involved in C4-dicarboxylate transportation, but also are essen-
and fatty acids in cells,18,19 and a portion of acetyl-CoA enters the tial for the uptake of sialic acid and N-acetylneuraminic acid.37
tricarboxylic acid cycle (TCA) for providing energy and metabolic However, the ability of all the above transporters to take in
intermediates. Thus, great efforts have been made to improve exogenous malonate into E. coli has not been identified.
the expression level and activity of ACC, including pulling carbon
In this study, the feasibility of the production of 3-HP using
into the cytosolic pool, reducing the consumption of acetyl-CoA malonate as the substrate was demonstrated. We expressed the
and constructing new acetyl-CoA pathways.6 However, the avail- malonate metabolic pathway for 3-HP production by introdu-
ability of malonyl-CoA is limiting for the production of 3-HP due cing the transporters MatB and MCR into E. coli. We observed
to the highly regulated nature of ACC.7,15 Therefore, it is malonate transport and the production of 3-HP directly from
assumed that the supply of malonyl-CoA can be enhanced by malonate (Fig. 1). Finally, our resultant strain accumulated
using a substrate that is not necessary for cell growth.
1.20 0.08 g L−1 3-HP using glucose as the sole carbon source
Despite the extensive research on malonyl-CoA production and malonate as the substrate in shaking flask cultivation.
by ACC,20 evidence is available in literature showing that These results demonstrate the in vivo functioning of the meta-
malonyl-CoA synthetase (MatB) also has the ability to produce bolic pathway composed of the malonate transporters MatB
malonyl-CoA directly from malonic acid, largely shortening the and MCR, and the effect of carbon flux enhancement by
route for the synthesis of malonyl-CoA. MatB (EC 6.2.1.14) bypassing ACC. To the best of our knowledge, this is the first
belongs to the AMP-forming acyl-CoA synthetase protein report of a technique for the efficient production of the plat-
family (PFAM 00501) (Pfam: the protein families database) and
the ANL superfamily, which contains aryl-CoA synthetases, the
adenylation domains of nonribosomal peptide synthetases,
and firefly luciferase.21 MatB catalyzes the conversion of malo-
nate and coenzyme A to malonyl-CoA with the hydrolysis of
adenosine triphosphate (ATP) into AMP and PPi via a ping-
pong mechanism. MatB exists extensively in bacteria, mamma-
lian mitochondria and eukaryotic organisms.22,23 Among
them, prokaryotic MatB has been widely studied, mainly
originating from Bradyrhizobium japonicum,24 Rhizobium
leguminosarum bv. Trifolii,25 Streptomyces coelicolor,26
Rhodopseudomonas palustris27,28 and Rhizobium japonicum.29
The physiological function of MatB is the degradation of
malonic acid in organisms, which is a classic competitive
inhibitor of succinate dehydrogenase (SDH), a component of
the TCA cycle and complex II of the electron transport chain.23
As a common organic acid in plant root exudate, malonic acid
has been listed as one of the top 30 chemicals that can be pro-
duced from biomass by the US Department of Energy,1 and it
has been used as a building block chemical to produce diverse
valuable compounds.30 In recent years, the biosynthesis of
fatty acids, polyketides and flavonoids from malonic acid has
been realized.31 For example, flavonoid production was
improved 7-fold through the introduction of the malonate
assimilation operon into Streptomyces venezuelae.32 However,
the utilization of malonate to produce malonyl-CoA-based
valuable bio-products in E. coli has rarely been reported.
To date, several types of malonate transporters have been
Fig. 1 3-HP synthetic pathway in E. coli using malonate as the sub-
strate. The pathway used in herein is noted by red arrows. MatB:
malonyl-CoA synthetase; MCR: malonyl-CoA reductase; PntAB: transhy-
drogenase; and YfjB: NAD kinase. The expression of multiple genes was
reported, including a putative malonate carrier protein (MatC)
in R. leguminosarum bv trifolii25 and classified malonate/
sodium symporter MdcF from Klebsiella pneumoniae.33 In
addition to the above transporters, tripartite ATP-independent conducted using plasmids and chromosomal integration.
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form chemical 3-HP from malonate. This novel malonyl-CoA- pSGN-27 (pMAL-c2x-matPQM) and pSGN-28 (pMAL-c2x-
mediated biosynthetic pathway can realize the shortest route dctPQM), all the above genes were amplified by PCR using
for the bioproduction of 3-HP, which is characterized by easy- primers matC-F/matC-R2, mdcF-F/mdcF-R2, matPQM-F/
to-control process, cost-effectiveness and low by-products, pro- matPQM-R2 and dctPQM-F/dctPQM-R2, respectively, and separ-
viding an attractive and eco-friendly substitute over the exist- ately cloned into the BamH I/Sal I sites of pMAL-c2x.
ing chemical synthetic routes.
2.2.2 Construction of plasmids expressing MatB. Three
genes encoding MatB from R. palustris (GenBank No.
CAE25665.1) (namely RpMatB), B. japonicum (GenBank No.
AAF28840.1) (namely BjMatB) and Arabidopsis thaliana
(GenBank No. OAP02278.1) (namely AtMatB) were synthesized
by Generay Biotech Co., Ltd (Shanghai, China) with codon
optimization for E. coli. These three MatB genes were ampli-
fied from cloning vector by PCR using primers RpmatB-F/
RpmatB-R, BjmatB-F/BjmatB-R and AtmatB-F/AtmatB-R, and
inserted into the BamH I and Hind III sites of pACYCDuet-1 to
generate plasmids pSGN-01 (pACYCDuet-RpmatB), pSGN-15
(pACYCDuet-BjmatB) and pSGN-16 (pACYCDuet-AtmatB),
respectively.
2.2.3 Construction of plasmids for co-expressing MatB and
MCR. Two types of genes encoding the MCR mutant
N940V/K1106W/S1114R were synthesized from Generay
Biotech Co., Ltd (Shanghai, China), including MCR with a
linker (GGGGS) between the N-terminal domain and
C-terminal domain and MCR without any redundant amino
acid between the N-terminal domain and C-terminal domain.
To generate plasmid pSGN-34 (pRSFDuet-mcr) and pSGN-35
(pRSFDuet-mcr(linker)), the MCR encoding genes were ampli-
fied from cloning vector by PCR using primers mcr-F1/mcr-R1,
and inserted into the Bgl II and Xho I sites of pRSFDuet-1.
2. Materials and methods
2.1 Bacterial strains and growth conditions
All PCR primers for gene amplification and strains used in
this study are listed in Tables S1 and S2.† E. coli DH5α was
used for recombinant DNA manipulation, while E. coli C43
(DE3) was the host for the expression of enzymes. E. coli
strains were grown in Luria–Bertani (LB) medium at 37 °C
with shaking. For 3-HP production, the different strains were
cultivated in shake-flask conditions with medium containing
20 g L−1 glucose, 4.5 g L−1 K2HPO4·3H2O, 9.0 g L−1 (NH4)2SO4,
3.0 g L−1 citric acid monohydrate, 3.0 g L−1 trisodium citrate,
5.7 g L−1 KCl, 0.226 g L−1 FeSO4·7H2O, 0.5 g L−1 MgSO4,
and 0.1 mL trace element solution, which included
(NH4)6Mo7O24·4H2O 3.7 g L−1, ZnSO4·7H2O 2.9 g L−1, H3BO4
24.7 g L−1, CuSO4·5H2O 2.5 g L−1, and MnCl2·4H2O 15.8 g L−1
Where appropriate, antibiotics were added as follows: ampi-
cillin (Amp, 100 µg mL−1), kanamycin (Kan, 50 µg mL−1) and
chloramphenicol (Cm, 34 µg mL−1).
.
2.2 Construction of plasmids
2.2.1 Construction of plasmids for expressing malonate Then the RpmatB encoding gene was amplified from cloning
transporters. The genes encoding MatC from R. leguminosarum vector by PCR using primers matB-F1/matB-R1, and inserted
bv trifolii (GenBank No. ACI53872.1), MdcF from K. pneumoniae into the BamH I site of the above plasmids to generate
(GenBank No. AAC45458.1), TRAP dicarboxylate transporter pSGN-40 (pRSFDuet-RpmatB-mcr) and pSGN-41 (pRSFDuet-
from S. meliloti (consisting of DctM, DctQ and DctP subunits, RpmatB-mcr(linker)). The plasmid pSGN-36 (pRSFDuet-mcr
responding to GenBank No. ABR63913.1, AAK64740.1 and (linker)-RpmatB) was constructed by cloning the MCR encod-
ABR63911.1, respectively) (namely MatPQM) and TRAP dicar- ing gene into the BamH I and Sal I sites of pRSFDuet-1 with
boxylate transporter from R. capsulatus (consisting of DctM, primers mcr-F2/mcr-R2 and then cloning RpmatB into the Bgl
DctQ and DctP subunits, responding to GenBank No. II site with primers matB-F2/matB-R2.
CAA45387.1, CAA45386.1 and CAA45385.1, respectively) (namely
2.2.4 Construction of plasmids expressing PntAB. The
DctPQM) were codon-optimized for E. coli and synthesized by gene encoding PntAB was amplified from the E. coli DH5α
Generay Biotech Co., Ltd (Shanghai, China). MatC, MdcF, genome by PCR using primers pntAB-F/pntAB-R, and then the
MatPQM and DctPQM encoding genes were amplified from PntAB gene was inserted into the Hind III site of pSGN-41
cloning vector by PCR using primers matC-F/matC-R, mdcF-F/ (pRSFDuet-RpmatB-mcr(linker)) to generate plasmid pSGN-42
mdcF-R, matPQM-F/matPQM-R and dctPQM-F/dctPQM-R, (pRSFDuet-RpmatB-PntAB-mcr(linker)) using the ClonExpress
respectively, and separately inserted into the BamH I/Hind III Ultra One Step Cloning Kit (Vazyme Biotech Co., Ltd, China).
sites of pET-28a(+) to generate plasmids pSGN-10 (pET28-
2.2.5 Chromosomal integration of YfjB gene. The inte-
matC), pSGN-18 (pET28-mdcF), pSGN-20 (pET28-matPQM), gration was carried out using suicide vector pRE112.7 The yfjB
pSGN-21 (pET28-dctPQM), respectively. To construct plasmids gene was cloned from E. coli DH5α by PCR with primers yfjB-F/
pSGN-69 (pBAD24-matC), pSGN-70 (pBAD24-mdcF), pSGN-68 yfjB-R. The promoter of glyceraldehyde phosphate dehydrogen-
(pBAD24-matPQM) and pSGN-72 (pBAD24-dctPQM), all the ase (Pgapdh) and yfjB gene was inserted into the PstI sites of
above genes were amplified by PCR using primers matC-F1/ pRE112-ΔprpR to generate the plasmid pRE112-yfjB, which
matC-R1, mdcF-F1/mdcF-R1, matPQM-F1/matPQM-R1 and was used to mediate the allelic exchange to generate SGN34.
dctPQM-F1/dctPQM-R1 respectively, and separately cloned into
2.3 Identification of malonate transportation function in E. coli
the Pst I sites of pBAD24 using the ClonExpress Ultra One Step
Cloning Kit (Vazyme Biotech Co., Ltd, China). For the obtained E. coli C43 (DE3), SGN10, SGN18, SGN20, SGN21, SGN22,
vectors pSGN-22 (pMAL-c2x-matC), pSGN-25 (pMAL-c2x-mdcF), SGN25, SGN27, SGN28, SGN68, SGN69, SGN70 and SGN72
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strains were separately cultured in 100 mL LB/IPTG or arabi- Protein purity was analyzed by SDS-PAGE, and protein concen-
nose/malonate medium with Kan or Amp at 30 °C for enzyme tration was determined via the Bradford assay.
production and malonate transportation. After 16 h shaking,
the cell cultures were collected for the following experiments.
For the determination of kinetic parameters, the purified
enzyme was incubated with various concentrations of malo-
2.3.1 Transcription analysis of transporter genes. A portion nate ranging from 40 to 1200 μM at 37 °C. The assay mixture
of the collected cells was disrupted by lysozyme. Total RNA was contained 100 mM K3PO4 buffer (pH 7.0), 0.2 mM CoA,
isolated using the TRIzol reagent method (Takara Company, 0.4 mM ATP, and 2 mM MgCl2. The concentration of purified
Japan). The RNA concentration was quantified using
a
enzyme was 0.052 μM for RpMatB, 0.104 μM for BjMatB and
NanoDrop ND1000 (Thermo Scientific, Wilmington, DE, USA). 0.087 μM for AtMatB. The reaction was stopped by adding an
Transporter encoding gene expression analysis was performed equal volume of methanol. The concentrations of malonyl-CoA
using quantitative reverse transcription PCR (qRT–PCR). 16S were determined by HPLC with a ThermoFisher Ultimate 3000
rRNA was used as the internal standard.38 The primers used using a C18 column (250 mm × 4.6 mm, 5 μm). The mobile
for the assays are indicated in Table S3.† The cDNA synthesis phase consisted of solvent A (100 mM potassium phosphate,
kit was used for reverse transcription (Takara Company, pH = 5.2) and solvent B (acetonitrile). The gradient elution pro-
Japan). ChamQ Universal SYBR qPCR Master Mix (Vazyme cedure was as follows: 0 to 5 min, 1.2% of B; 5 to 45 min, and
Biotech Co., Ltd, China) and qTOWER3 Real-Time PCR a linear gradient from 1.2% to 12% of B. The flow rate was
Thermal Cycler (Jena, Germany) were used for all quantitative 0.5 mL min−1 at a temperature of 25 °C. The amount of
assays. The comparative CT method was used for relative malonyl-CoA was detected by a UV detector with a wavelength
quantification of gene expression. The relative quantification of 245 nm.39 The kinetic constants were calculated by fitting
for each of the mRNA levels was calculated using the ΔCT the initial rate data into the Michaelis–Menten equation using
method, which is the difference between the CT of the gene of the Graph-Pad Prism 5 software (GraphPad Software, Inc., USA,
interest and that of the reference gene.
http://www.graphpad.com).
2.3.2 Protein expression analysis of transporter genes.
Another portion of collected cells was disrupted by sonication,
2.5 Synthesis of 3-HP using malonate as substrate
and the crude extracts were centrifuged, and the supernatants Plasmids pSGN-36(pRSFDuet-mcr(linker)-RpmatB), pSGN-40
were used for western blot analysis. Protein samples were elec- (pRSFDuet-RpmatB-mcr) and pSGN-41(pRSFDuet-RpmatB-mcr
troblotted onto PVDF membrane and allowed to incubate with (linker)) were separately co-transformed with pSGN-68
Anti-His Tag Mouse Monoclonal Antibody (Solarbio, China). (pBAD24-matPQM) in E. coli C43 (DE3) to generate strains
Subsequently, the samples were probed with Antimouse IgG SGN73, SGN74 and SGN75, respectively. Plasmids pSGN-42
secondary antibody conjugated with HRP. After washing with (pRSFDuet-RpmatB-pntAB-mcr(linker)) and pSGN-68(pBAD24-
PBST, BCIP/DAB (Solarbio, China) was added to detect matPQM) were co-transformed into E. coli C43 (DE3)::yfjB to
antigen–antibody conjugates. The reaction was quenched with make strain SGN78. The strains were grown in fermentation
distilled water.
medium, as described above, supplemented with ampicillin
2.3.3 Malonate transportation function analysis of trans- (100 μg mL−1) and kanamycin (30 μg mL−1) at 200 rpm and
porter genes in E. coli. The concentration of malonate in the 37 °C to an optical density at 600 nm (OD600) value of 0.6, and
supernatants of the cell cultures was detected by HPLC with then induced with IPTG (final concentration of 0.6 mM) as
Sepax Carbomix H-NP10 column (300 mm × 7.8 mm, 10 μm). well as arabinose (final concentration of 0.1%) at 30 °C for
The mobile phase was 2.5 mM H2SO4, and the flow rate was 16 h. Simultaneously, substrate malonate was added to the
0.6 mL min−1 at a temperature of 55 °C. The amount of malo- culture to synthesize 3-HP. The supernatants obtained by the
nate was detected by a UV detector with a wavelength of centrifugation of the culture samples at 10 000g for 10 min
210 nm.
were filtered through a 0.22 μm filter and eluted through a C18
column (250 mm × 4.6 mm, 5 μm) with methanol–10 mM
H3PO4 (5 : 95, v/v) as the mobile phase. The flow rate and the
detection wavelength were set to 1.5 mL min−1 and UV
2.4 Protein purification and determination of kinetic
parameters of MatB
SGN01, SGN15 and SGN16 were separately cultured in LB/Cm/ 210 nm, respectively.9 The product was characterized by direct
IPTG medium at 20 °C for enzyme production. After 16 h comparison with standard 3-HP (Sigma-Aldrich, USA). The
shaking, the cells were disrupted by sonication, and the super- peak area was converted to 3-HP concentration by comparison
natants were collected by centrifugation (13 000 rpm, 40 min) with a standard curve plotted with a set of known concen-
at 4 °C. The sample was loaded onto an Ni–NTA column pre- tration of 3-HP.
equilibrated with resuspended buffer (20 mM PBS pH 7.4,
300 mM NaCl). Then the column was washed with washing
2.6 Optimization of fermentation process
buffer (20 mM PBS pH 7.4, 300 mM NaCl, 15 mM imidazole,
2.6.1 Effect of organic nitrogen source. The shake-flask cul-
and 1 mM β-mercaptoethanol). The bound protein was eluted tures were incubated in different organic nitrogen sources (BE
with elution buffer (20 mM PBS pH 7.4, 300 mM NaCl, (So), beef extract from Solarbio; BE(Si), beef extract from
300 mM imidazole, and 1 mM β-mercaptoethanol). Finally, the Sinopharm; T, tryptone; and YE, yeast extract) at 20 °C for 16 h
protein was dialyzed, concentrated and stored at −80 °C. with the concentration of IPTG and arabinose of 0.5 mM and
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0.2%, respectively. Simultaneously, the substrate malonate was 1 mL crude extract. The incubation time for the assays for
added to the culture to synthesize 3-HP, and the 3-HP products MatB activity was 30 min. The reaction was stopped by adding
were calculated. an equal volume of methanol. The concentrations of malonyl-
2.6.2 Effect of induction temperature. The E. coli strain CoA were determined by HPLC.
was inoculated in 50 mL of fermentation medium with the
The reaction mixture for the determination of MCR activity
most suitable organic nitrogen source and cultured at 37 °C (500 µl) contained 100 mM Tris-HCl (pH 7.8), 2 mM MgCl2,
with shaking at 180 rpm. When the OD600 of the bacterial 0.4 mM NADPH, and 1 mL (each) crude extract. The reaction
culture reached 0.6, the shake-flask cultures were incubated at was started by the addition of 0.15 mM malonyl-CoA and incu-
different induction temperatures (16 °C, 20 °C, 30 °C, 34 °C bated at 37 °C for 30 min. Then the reaction was stopped by
and 37 °C) for 16 h with the concentration of IPTG and arabi- the addition of an equal volume of acetonitrile. After centrifu-
nose of 0.5 mM and 0.2%, respectively. Simultaneously, the gation, the supernatant (3-HP) was analyzed by HPLC.40
substrate malonate was added to the culture to synthesize
2.8 Determination of NADPH levels
3-HP, and the 3-HP products were assayed.
2.6.3 Effect of inducer (IPTG and arabinose) concentration.
The shake-flask culture was incubated in different inducer
(IPTG) concentrations (0.1 mM, 0.25 mM, 0.5 mM and 1 mM)
and inducer (arabinose) concentrations (0.05%, 0.1%, 0.2%,
0.3% and 0.4%) at the above-optimized temperature for 16 h.
Simultaneously, the substrate malonate was added to the
culture to synthesize 3-HP, and the 3-HP products were
measured.
2.6.4 Effect of liquid volume. The E. coli strain was cul-
tured in 250 mL shake-flasks with different liquid volumes
(30 mL, 50 mL, 80 mL, 100 mL, and 120 mL) at 37 °C and 180
rpm. When the OD600 of the bacterial culture reached 0.6, the
shake-flask cultures were incubated at 30 °C for 16 h with the
concentration of IPTG and arabinose of 0.6 mM and 0.1%,
respectively. Simultaneously, the substrate malonate was
The E. coli SGN78 and SGN74 strains were cultured in 250 mL
shake-flasks with LB/Km/IPTG/arabinose medium at 30 °C and
180 rpm. The cells with 1 mL B1 of NADPH Kit (ChemPartner,
China) were broken on ice and incubated at 60 °C for 30 min.
Then the supernatants were collected by centrifugation (12 000
rpm, 10 min) at 4 °C. After centrifugation, solutions I, II, and
III were added and the NADPH concentration was monitored
at 450 nm using an Agilent, Cary 60 Bio UV-Visible
spectrophotometer.
3. Results and discussion
3.1 Transport of malonate by various malonate transporters
in E. coli
added to the culture to synthesize 3-HP, and the 3-HP products To identify suitable malonate transporters, we screened several
were detected. protein candidates. We used MatC from R. leguminosarum bv
2.6.5 Effect of induction opportunity. The E. coli strain-har- trifolii, MdcF from K. pneumoniae, TRAP dicarboxylate trans-
boring expression vectors were grown to an optical density at porter from S. meliloti and TRAP dicarboxylate transporter
600 nm (OD600) value of 0.4–1.0, and then induced with from R. capsulatus. We examined each malonate transporter in
IPTG (0.6 mM) and arabinose (0.1%) at 30 °C for 16 h. detail by determining their transcription, expression and func-
Simultaneously, the substrate malonate was added to the tion in E. coli cells. To elucidate the function of each transpor-
culture to synthesize 3-HP, and the 3-HP products were ter, an appropriate set of expression vectors was constructed
assayed.
for the matC, matPQM, dctPQM and mdcF genes under the
2.6.6 Effect of induction time. E. coli cell-carrying IPTG-inducible T7 and tac promoter, as well as the arabinose
expression vectors were grown at 200 rpm and 37 °C. When the araBAD promoter (Fig. 2A and Fig. S1A†). After induction with
culture reached an OD600 value of 0.6, IPTG (0.6 mM) and ara- inducers, malonate was added to the medium. After 16 h
binose (0.1%) were added to the culture for the induction of further culture at 30 °C, the cells and supernatant were separ-
recombinant protein expression, and then further cultivated at ately collected.
30 °C for 8 h, 12 h, 16 h, 24 h and 36 h. Simultaneously, the
Firstly, we assessed the expression of the target genes
substrate malonate was added to the culture to synthesize (Fig. 2C and Fig. S1C†). The results showed that the matC and
3-HP. The samples were taken regularly to determine 3-HP.
matPQM genes were obviously transcribed in E. coli when three
types of expression vectors were used. Varying results were
obtained when different plasmids were used to express MdcF.
Obvious transcription was observed only when the pBAD24
2.7 Determination of MCR and MatB activities of crude
extract
SGN73, SGN74 and SGN75 were separately cultured in LB/Km/ plasmid was used to express MdcF, although a slight transcrip-
IPTG/arabinose medium at 30 °C for enzyme production. After tion was obtained using tac promoter. Moreover, dctPQM
16 h shaking, the cells were disrupted by sonication, and the failed to be transcribed in E. coli regardless of the plasmid
supernatants were collected by centrifugation (13 000 rpm, used.
40 min) at 4 °C. The reaction mixtures (0.5 mL, 37 °C) were
Then, the whole-cell crude extract was obtained and ana-
prepared for product analysis. Each reaction contained lyzed by immunoblot assay. As shown in Fig. 2B and Fig. S1B,†
100 mM potassium phosphate buffer (pH 7.0), 40 mM sodium compared with the negative control, the strains harboring
malonate, 0.2 mM CoA, 0.4 mM ATP, and 2 mM MgCl2 and MatPQM and MatC have specific expression bands corres-
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Fig. 2 Identification of malonate transportation function in E. coli. (A) Construction of recombinant strains carrying malonate transport genes. (B)
Western blot analysis of expressed malonate transport proteins. (C) Transcription analysis of transporter genes. (D) Relative malonate transport
efficiency of tested transporters.
ponding to the MatPQM and MatC protein (about 106 kDa and three types of vector. Unfortunately, the TRAP system from
46 kDa), respectively. However, there was no obvious expressed R. capsulatus has no function in E. coli.
protein band in the result of the other strains even though the
A previous study suggested that the organization and
genes were codon-optimized and controlled by the same T7 arrangement of the genes involved in malonate transport vary
and tac promoters. When these malonate transporters were across different rhizobia. In R. leguminosarum bv. trifolii, only
expressed by arabinose inducer, besides strains SGN68 and the matC gene performs malonate transport.25 However, in
SGN69, strain SGN70 also produced considerable proteins S. meliloti, the three genes (MatP, MatQ and MatM) are respon-
(40 kDa). The observed transcription of MatPQM and MatC sible for malonate uptake.35 The amino acid residues of MatP,
genes under these three promoters as well as the protein MatQ and MatM exhibited similarity to the DctP, DctQ and
expression bands suggest that these two transporters were suc- DctM subunits of the C4-dicarboxylate TRAP transporter
cessfully produced in E. coli.
(DctPQM) from R. capsulatus (24%, 25% and 31% identity),
To further investigate their functions, the amount of malo- respectively.35 It has been shown that the TRAP transporter
nate in the supernatants was determined. As depicted in uses an SBP that captures specific solutes with high affinity
Fig. 2D and Fig. S1D,† for the TRAP system from S. meliloti, and transfers them to their partner permease complex located
different expression plasmids led to obvious differences in in the bacterial inner membrane.36 The TRAP transporters
transport activity. Especially, when pBAD24 was used as the employed by S. meliloti may have such a high affinity that they
expression vector, the transport efficiency of the recombinant can acquire malonate effectively. To the best of our knowledge,
strain SGN68 reached the highest level, which was about three this is the first detailed investigation of the transporters
and twenty-four times higher than that of strains SGN20 and involved in malonate transport in E. coli.
SGN27, respectively. In contrast, MatC showed almost the
same transport activity when varying the expression vector.
3.2 Characterization of MatB
Similar to the results from the transcription analysis, quite Three types of MatB were purified and analyzed by SDS-PAGE
different activity was observed for cloning mdcF genes with analysis (Fig. 3A). Then, the kinetic analysis of MatB for malo-
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Fig. 3 Characterization of MatB. (A) SDS-PAGE analysis of the purified MatB. (B) Michaelis–Menten equation curves for three types of MatB. (C)
Sequence alignment of MatB from R. palustris (RpMatB), B. japonicum (BjMatB) and A. thaliana (AtMatB). The alignment was performed using
ClusterW and output was generated by ESPript.
in turnover rate (kcat) over the other two types of MatB.
Consequently, the kcat/KM value of RpMatB (2.42 0.22 min−1
μM−1) was 2.56- and 2.41-fold higher than that of BjMatB
Table 1 Kinetic analysis with malonate of MatB
Enzyme
KM (μM)
kcat (min−1
)
kcat/KM (min−1 μM−1
)
(0.68
0.07 min−1 μM−1) and AtMatB (0.71
0.08 min−1
RpMatB
BjMatB
AtMatB
146.8 19.26
299.0 50.52
386.9 70.78
355.3 13.76
204.4 13.19
275.3 18.67
2.42 0.22
0.68 0.07
0.71 0.08
μM−1), respectively. Our results were similar to that reported
earlier,27,41 suggesting that RpMatB has higher affinity for mal-
onate and higher catalyzing efficiency than the other two MatB
proteins. The amino acid sequence of RpMatB exhibits 82%
nate was performed. As shown in Fig. 3B and Table 1, the KM and 35% identity with BjMatB and AtMatB, respectively
value of RpMatB was 146.8 19.26 μM, which was half of that (Fig. 3C). Although the X-ray crystal structure of RpMatB has
of BjMatB and nearly a third of that of AtMatB. RpMatB had a been resolved, the mechanistic basis of higher activity remains
kcat of 355.30 13.76 min−1. This represents a minor increase hard to understand due to the lack of BjMatB and AtMatB
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protein structure information. Therefore, RpMatB was chosen enzyme with an apparent KM value of 41.7
2.2 µmol for
for the synthesis of 3-HP using malonate as the substrate in malonyl-CoA.40 The N-terminal and the C-terminal region of
our following study.
MCR were functionally distinct. MCR-C catalyzes the reduction
of malonyl-CoA to malonate semialdehyde, which can be
reduced to 3-HP by MCR-N. The first step is the rate-limiting
step. Thus, great efforts have been made to increase the
activity of MCR-C. Some mutants with increased activity have
been obtained in previous studies. Among them, the MCR
3.3 Biosynthesis of 3-HP in engineered strains
To realize the in vivo synthesis of 3-HP in the recombinant
E. coli strains, the co-expression of RpMatB and MCR was
investigated (Fig. 4A). MCR of C. aurantiacus is a bifunctional
Fig. 4 Biosynthesis of 3-HP in engineered strains. (A) Construction of 3-HP biosynthesis strains. (B) SDS-PAGE analysis of crude extract of the
engineered strains. (C) Biosynthesis of 3-HP using the engineered strains. (D) Relative activity of MatB of the engineered strains. (E) Relative activity of
MCR of the engineered strains.
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mutant N940V/K1106W/S1114R improved the catalytic Lower expression levels of pathway genes and the use of low-
efficiency by 14.2-fold over the wild-type.7 Moreover, the copy-number plasmid positively affect the production of 3-HP.8
enzyme activity increased when the N-terminal and C-terminal
Since E. coli host cells can biosynthesize malonyl-CoA from
regions of MCR were separated by fusing a flexible linker glucose, it is possible that 3-HP was produced using this
between the two enzymatic units.40 Hence, to enhance the portion of malonyl-CoA. Thus, the ability of cells only harbor-
production of 3HP, the MCR mutant N940V/K1106W/S1114R ing MCR to synthesize 3-HP from glucose was investigated. As
was employed, and the separation of N-terminal and shown in Fig. 4C, no 3-HP was detected when the cells only
C-terminal regions of MCR by
a linker (GGGGS) was expressed MCR. In contrast, a considerable yield of 3-HP was
attempted in this study. As depicted in Fig. 4B, it was found observed in the engineered strain harboring the whole metab-
that these two proteins levels in crude cell extract were olite pathway from malonate, including malonate transporter,
different, with varying cloning sites in the plasmid MatB and MCR. Thus, these results suggest that the total pro-
pRSFDuet-1. For RpMatB corresponding to 54 kDa, the strain duction of 3-HP originates from the additional added malo-
SGN73 cloning the RpMatB encoding gene in the second nate instead of glucose.
cloning site of the vector outperformed the strain SGN74
carrying the RpMatB encoding gene in the first cloning site.
3.4 Optimization of fermentation conditions
For MCR corresponding to 132 kDa, the linker between the Fermentation conditions are of great importance because they
C-terminal domain and N-terminal domain did not influence play an important role in the formation and yield of the target
the protein expression (SGN74 and SGN75). However, the product.49 Thus, optimization of the fermentation conditions
MCR protein level dramatically decreased when the gene was for engineered strains is a good way to improve product quality
cloned in the upstream site of the plasmid. For multi-gene and quantity. The one-factor-at-a-time method is a closed-
clusters, the upstream genes usually have higher expression ended system for fermentation processes. Based on the classi-
levels than the downstream genes.42 However, our results cal method of changing one independent variable while fixing
were an exceptional case, which were also observed in other all others,50,51 this method can be used to optimize medium
studies.43
components as well as process conditions. Herein, the three
The corresponding results of the MatB and MCR activities most important factors, organic nitrogen source, induction
of the crude extract are in good agreement with above proteins temperature and inducer concentration were optimized to
level assays. In detail, the strain SGN73 showed the highest enhance the production of 3-HP using the strain SGN74.
MatB activity, which was about 3 times higher than that of
3.4.1 Effect of organic nitrogen source on 3-HP production.
strains SGN74 and SGN75 (Fig. 4D). Also, the strain exhibited The nitrogen source in the medium plays a crucial role in
the maximum MCR activity when MCR with a linker encoding enhancing the production of the desired product.52 Thus, to
gene was inserted into the second cloning site of the vector investigate the effect of the organic nitrogen source on 3-HP
(Fig. 4E). Under shaking flask conditions (Fig. 4C), the strain production, four different organic nitrogen sources were
SGN74 was the most productive, reaching an observed titer of assessed at the 20 °C, with the concentration of IPTG and ara-
0.27 0.03 g L−1 3-HP when the RpMatB encoding gene was binose of 0.5 mM and 0.2%, respectively (Fig. 5A). Among the
inserted into the first cloning site of pRSFDuet-1 and the organic nitrogen supplements tested, the beef extract
linker existed between C-terminal domain and N-terminal (Sinopharm Chemical Reagent Co., Ltd, China) permitted a
domain of MCR. Collectively, all these results indicate that the significantly higher 3-HP production than the other organic
3-HP synthetic reaction from malonyl-CoA catalyzed by MCR is nitrogen sources. The highest concentration of 3-HP was
the rate-limiting step for the whole 3-HP synthetic pathway. 0.37 0.05 g L−1, which was about 4 times the lowest value
Compared with the strain SGN75, the production of 3-HP by observed. Similarly, the cell density reached the maximum
the strain SGN74 increased 1-fold, suggesting the separation of when the beef extract (Sinopharm Chemical Reagent Co., Ltd,
the C-terminal domain and N-terminal domain of MCR China) was used as the organic nitrogen source in the fermen-
indeed improved the enzymatic activity by facilitating the tation medium, suggesting that this nitrogen source enhanced
entrance of the substrate malonyl-CoA into the C-terminal the accumulation of 3-HP by accelerating cell growth.
domain active site.40 Our findings prove that the adjustment of
3.4.2 Effect of induction temperature on 3-HP production.
the protein expression level and balance of enzyme activities Low induction temperatures can increase the amount of active
are essential in the multistep pathway to improve production. recombinant enzymes because low temperatures reduce the
Some recent studies also focused on balancing the pathway inclusion bodies in engineered E. coli.53 However, successful
enzymes.44–47 For instance, to balance the enzymatic activities control of the cultivation temperature has to balance cell
of glycerol dehydratase (GDHt) and aldehyde dehydrogenase growth, enzyme expression and product formation.53 Hence,
(ALDH), varying-strength promoters and bicistronic ribosome in this study, to increase 3-HP production, the different induc-
binding sites (RBSs) were attempted. As a result, the accumu- tion temperatures of 16 °C, 20 °C, 30 °C, 34 °C and 37 °C were
lation of intermediate 3-hydroxypropionaldehyde (3-HPA) was investigated. As shown in Fig. 5B, the maximum 3-HP pro-
avoided and the production of 3-HP improved.48 In addition, duction was obtained at 30 °C, at 0.56 0.06 g L−1, which was
the production of 3-HP by the recombinant strain is highly about 1.4 times, 1.1 times and 1.4 times greater than that
dependent on the expression level of MCR-N and MCR-C.7 observed at 20 °C (0.39 0.03 g L−1), 34 °C (0.49 0.04 g L−1
)
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Fig. 5 Effects of fermentation source and culture conditions on 3-HP production by SGN74. (A) Effect of nitrogen source on 3-HP production: BE
(So), beef extract from Solarbio; BE(Si), beef extract from Sinopharm; T, tryptone; and YE, yeast extract. (B) Effect of temperature on 3-HP production.
(C) Effect of concentration of inducer IPTG on 3-HP production. (D) Effect of the concentration of arabinose on 3-HP production. Black line of filled
circles, OD600 value of strain SGN74. Black columns and black line of filled squares, 3-HP production under different fermentation conditions.
and 37 °C (0.40 0.02 g L−1), respectively. Hence, the optimum from strain to strain. Thus, to determine the optimal oxygen
induction temperature for 3-HP production was 30 °C.
demand, cells were cultured in different liquid volumes. It can
3.4.3 Effect of inducer concentration on 3-HP production. be seen from Fig. 5E that the dissolved oxygen has a signifi-
In general, exogenous gene expression gives rise to a metabolic cant effect on the growth of recombinant E. coli and the pro-
burden on cells, which can lead to reduced cell yields, growth duction of 3-HP. As the amount of liquid increased, the con-
rates, plasmid stability and product yield.54,55 Considering all centration of bacteria and 3-HP increased, and when the
the above factors, various IPTG and arabinose concentrations liquid volume reached 50 mL, the cell density of the cells and
ranging from 0.2 mM to 1 mM and 0.05% to 0.4%, respect- 3-HP production reached a maximum of 2.43 0.22 and 1.01
ively, were tested to optimize the inducer concentration at the 0.03 g L−1, respectively. Subsequently, the cell density and
above-optimized temperature of 30 °C. As shown in Fig. S2,† 3-HP titer began to decrease with a further increase in the
obvious increased MCR and MatB levels were observed with an liquid volume.
increase in the concentration of IPTG from 0.2 to 0.6.
3.4.5 Effect of induction opportunity on 3-HP production.
However, the amounts of expressed proteins decreased with a The induction opportunity is an important factor influencing
further increase in the concentration of IPTG. Based on the cell density and protein expression.56 If the induction opportu-
data shown in Fig. 5C and D, the yield of 3-HP reached a nity is too early, the expression of foreign proteins influences
maximum of 0.90
0.07 g L−1 when the concentrations of the physiological state of the cells, which is harmful to the
IPTG and arabinose were 0.6 mM and 0.1%, respectively. growth of engineered bacteria and protein expression. If the
These results for 3-HP production are consistent with that induction opportunity is too late, the bacteria are aging, the
from the SDS-PAGE analysis, indicating that inducers affect nutrients in the medium are scarce, and the metabolic by-
the synthesis of products by controlling the protein expression products increase, which are not conducive to protein
level. Moreover, the amounts of inducers have little effect on expression. The results of the cell growth and 3-HP production
cell growth.
by different induction opportunities are shown in Fig. 5F.
3.4.4 Effect of liquid volume on 3-HP production. Suitable When the inducers were added to the medium at the OD600 of
dissolved oxygen is a key factor in maintaining respiratory 0.6, the 3-HP production reached the maximum, suggesting
metabolism and product synthesis in aerobic microorganisms, that the optimum induction opportunity for 3-HP production
and the amount of oxygen required by microorganisms varies was 0.6.
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Table 2 Production of 3-HP through malonyl-CoA-mediated biosyn-
thetic pathway from different feedstocks
3.4.6 Effect of induction time on 3-HP production. E. coli
cytoplasm has various proteolytic enzymes. Exogenous pro-
teins are easily degraded by enzymes in the cytoplasm. Hence,
the induction time is directly related to 3-HP production, fer-
mentation cycle and cost. As shown in Fig. 5G, 3-HP pro-
duction reached the highest level when the induction time was
16 h. After that, the output remained stable. Hence, the
optimum induction time for 3-HP production was 16 h.
Based on the above data, the most suitable culture con-
ditions for 3-HP production using the engineered strain SGN74
were 30 °C for 16 h, 0.6 mM IPTG, 0.1% arabinose, induction
opportunity at OD600 of 0.6 and 50 mL fermentation medium
with beef extract (Sinopharm) as the organic nitrogen source.
Titer of shake-flask
Feedstock Organism
(g L−1
)
Ref.
Glucose
Glucose
CO2
Glucose
Glucose
Saccharomyces cerevisiae
Klebsiella pneumoniae
Synechocystis sp. PCC6803 0.83
0.46
1.8
59
60
16
7
10
17
E. coli
3.72 0.01
0.477 0.001
0.0698
Saccharomyces cerevisiae
Methanol Methylobacterium
extorquens
Schizosaccharomyces pombe 1.0
Glucose
11
Fatty acids E. coli
Malonate E. coli
5.38 0.53
1.20 0.08
8
This study
3.5 Improvement of NADPH supply for enhancing 3-HP
production
work has great potential. Thus, to further improve the 3-HP pro-
duction, a series of studies will be conducted in the following
study, including optimization of promoter and RBSs, genome
integration of function genes, and large-scale fermentation.
The N-terminal and the C-terminal regions of MCR are func-
tionally distinct, which catalyze the two-step reduction of
malonyl-CoA to 3-hydroxypropionate (3HP). Each step con-
sumes 1 mol NADPH, and accordingly, the production of
1 mol of 3-HP requires 2 mol of NADPH (Fig. 1). Thus, the
NADPH supply is an important factor influencing the
efficiency of the 3-HP biosynthetic pathway.
4. Conclusions
In this study, we successfully constructed a novel malonyl-CoA-
mediated biosynthetic pathway in an engineered E. coli strain,
which could produce high levels of 3-HP directly from
malonate. Using a multi-step metabolic engineering strategy
designed to increase the precursor and NADPH cofactor
supplies for 3-HP production, the final engineered strain pro-
duced 1.20 0.08 g L−1 of 3-HP in flask culture. Thus, our
work thus demonstrated the production of 3-HP in E. coli with
the shortest route for the biosynthesis of 3-HP, which involved
only three steps. Also, it opens new possibilities to produce
other malonyl-CoA-based valuable chemicals directly from
malonate in E. coli.
In E. coli, the three major sources of NADPH regeneration
are the pentose phosphate pathway (PPP), tricarboxylic acid cycle
(TCA) and membrane-bound transhydrogenase (PntAB).57 PntAB
is a membrane-bound proton translocating transhydrogenase,
which transfers a hydride from NADH to NADP+ with the con-
current production of NADPH and NAD+, powered by the proton
motive force.58 Thus, to recycle NAD+ for continuous transhydro-
genation, NAD+ needs to be converted to NADP+ (Fig. 1).
In this work, to enhance the NADPH supply in the cells,
NAD kinase (YfjB), which catalyzes the phosphorylation of
NAD+ to NADP+, was over-expressed together with transhydro-
genase in the engineered strain SGN74. Under shaking flask
conditions, the strain SGN78 with the overexpression of PntAB
and YfjB could accumulate 1.20
0.08 g L−1 3-HP, which
Conflicts of interest
resulted in about 33.33% increase of 3-HP titer produced by
the control strain SGN74. NADPH is tightly regulated in the
cell. Studies have been conducted to manipulate the levels of
NADPH in the cytosol. The results showed that the strain
There are no conflicts of interest to declare.
SGN78 produced 0.22
higher than that of strain SGN74 (0.17
0.02 nmol mL−1 NADPH, 31.14%
Acknowledgements
0.02 nmol mL−1),
indicating that PntAB and YfjB over-expression improved the
NADPH production, which in turn promoted the biosynthesis
of 3-HP. This result demonstrates that the co-expression of
PntAB and YfjB can lead to a synergistic effect on increasing
the NADPH supply and improving 3-HP production.
Table 2 shows recent studies on the bioproduction of 3-HP
through the malonyl-CoA-mediated biosynthetic pathway from
different feedstocks. For small-scale fermentation, the 3-HP
titer of strain SGN78 in the present study was much higher than
that of most engineered strains, demonstrating the efficient pro-
duction of 3-HP from malonate. E. coli recombinant strains
using glucose or fatty acids as substrates showed slightly higher
titers than that of strain SGN78, indicating that the present
This work was supported by the National Natural Science
Foundation of China (Grant No. 21572242), the Talents of
High Level Scientific Research Foundation (Grant No.
6631113326; 6651119011) of Qingdao Agricultural University.
Many thanks to Caroline Stone Harwood for deep discussion
and helpful suggestions.
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