Targeted optimization of central carbon metabolism for engineering succinate production in Escherichia coli
© The Author(s). 2016
Received: 4 April 2016
Accepted: 15 June 2016
Published: 24 June 2016
Succinate is a kind of industrially important C4 platform chemical for synthesis of high value added products. Due to the economical and environmental advantages, considerable efforts on metabolic engineering and synthetic biology have been invested for bio-based production of succinate. Precursor phosphoenolpyruvate (PEP) is consumed for transport and phosphorylation of glucose, and large amounts of byproducts are produced, which are the crucial obstacles preventing the improvement of succinate production. In this study, instead of deleting genes involved in the formation of lactate, acetate and formate, we optimized the central carbon metabolism by targeting at metabolic node PEP to improve succinate production and decrease accumulation of byproducts in engineered E. coli.
By deleting ptsG, ppc, pykA, maeA and maeB, we constructed the initial succinate-producing strain to achieve succinate yield of 0.22 mol/mol glucose, which was 2.1-fold higher than that of the parent strain. Then, by targeting at both reductive TCA arm and PEP carboxylation, we deleted sdh and co-overexpressed pck and ecaA, which led to a significant improvement in succinate yield of 1.13 mol/mol glucose. After fine-tuning of pykF expression by anti-pykF sRNA, yields of lactate and acetate were decreased by 43.48 and 38.09 %, respectively. The anaerobic stoichiometric model on metabolic network showed that the carbon fraction to succinate of engineered strains was significantly increased at the expense of decreased fluxes to lactate and acetate. In batch fermentation, the optimized strain BKS15 produced succinate with specific productivity of 5.89 mmol gDCW−1 h−1.
This report successfully optimizes succinate production by targeting at PEP of the central carbon metabolism. Co-overexpressing pck-ecaA, deleting sdh and finely tuning pykF expression are efficient strategies for improving succinate production and minimizing accumulation of lactate and acetate in metabolically engineered E. coli.
Succinate, an important member of C4-dicarboxylic acid family, has been widely used in agricultural, food, pharmaceutical, cosmetic, textile and fine chemicals industries [1, 2]. Meanwhile, succinate has received considerable attention to synthesize various valuable molecules such as 1,4-butanediol, tetrahydrofuran, γ-butyrolactone and adipic acid . Petrochemistry-based succinate production requires various metal catalysts and discharges organic wastes, which make petrochemical processes costly and not environmental friendly. Bio-based succinate production is a promising and green process as it uses renewable bioresources as substrates and fixes greenhouse gas CO2 . Therefore, the concomitant economical and environmental advantages stimulate the efforts to engineer microorganisms for efficient succinate production.
Succinate can be naturally produced by many strict anaerobic bacteria and facultative anaerobes. Escherichia coli is most widely studied for succinate production due to its convenience for genetic manipulation and fast growth with flexible nutrient requirements . However, the wild E. coli strain prefers to produce lactate and acetate as major products with a small amount of succinate in mixed-acid fermentation under anaerobic conditions . Efforts of metabolic engineering and adaptive evolution have been made to obtain succinate-producing E. coli. Inactivation of genes accounting for biosyntheses of those byproducts was first pursued to produce succinate as the predominant fermentation product. However, the mutant E. coli strains deficient in ldhA (coding lactate dehydrogenase) and pflB (coding pyruvate-formate lyase), adhE (coding alcohol dehydrogenase) and pta (coding phosphotransacetylase) or their combinations were unable to anaerobically grow on glucose media and the titer and yield of succinate were relatively low. For example, the mutant E.coli strain NZN111 deficient in ldhA and pflB only produced minor amount of succinate . Evolutionary engineering of strain NZN111 led to spontaneous chromosomal mutant strain AFP111, which was able to ferment glucose anaerobically and produced higher succinate yield, as well as higher acetate . Similarly, by combining metabolic engineering and evolution of over 2000 generations screened on glucose minimal medium, E. coli strain KJ073 with deletions of ldhA, adhE, ackA (coding acetate kinase), focA (coding formate channel), pflB, mgsA (coding methylglyoxal synthase) and poxB (coding pyruvate oxidase) was capable of producing high succinate yield, but significant amounts of acetate and malate were also produced .
Metabolic targets of the central carbon metabolism have been used to improve succinate production in E. coli. In order to enhance carbon flux to succinate, formation of oxaloacetate (OAA) from pyruvate or phosphoenolpyruvate (PEP) was chosen as metabolic target. Heterologous expressions of pyc (coding pyruvate carboyxlase, PYC) from Rhizobium etli  or from Lactococcus lactis [11, 12], pck (coding PEP carboxykinase, PCK) from Actinobacillus succinogenes [13, 14] and overexpression of native ppc (coding PEP carboxylase, PPC)  were shown to increase succinate production in recombinant E. coli strains. Subtle co-overexpression of both ppc and pck genes regulated by promoters with different strengths improved succinate production . To increase NADH availability in succinate-producing E. coli, several genes involved in redox reactions were identified to improve cell growth impairment under microaerobic conditions . Heterologous NAD+-dependent formate dehydrogenase gene fdh of Candida boidinii or native nicotinate phosphoribosyltransferase gene pncB were co-overexpressed with Lactococcus lactis pyc gene to achieve the redox and ATP balance [18, 19]. Activation of pentose phosphate pathway, transhydrogenase and pyruvate dehydrogenase were identified for improved succinate production by increasing reducing power supplement . To enhance glucose utilization in E. coli strain deficient in PEP carbohydrate phosphotransferase system (PTS), native galP (coding D-galactose transporter) and glk (coding glucokinase) were co-overexpressed or modulated to facilitate succinate production . Zymomonas mobilis glf gene (coding glucose facilitator, Glf) was more efficient than E. coli galP gene due to the higher transport velocity and lower energetic cost of Glf . In addition, C4-dicarboxylic acid transporter genes were also activated to decrease the feedback effects through accelerating succinate export [23, 24].
Results and discussion
Initial construction for succinate production
In succinate metabolic pathway, the carboxylation of PEP catalyzed by PPC or PCK is a rate-limiting step committed to succinate production. ATP is essentially consumed for PPC catalyzing the formation of OAA from PEP . On the contrary, one molecule ATP is generated from carboxylation of one molecule PEP catalyzed by PCK. The deletion of pck gene in E. coli remarkably inhibited succinate production as well as the cell growth , indicating that PCK might be more efficient than PPC. In addition, the function of PCK was partially inhibited by PPC under anaerobic fermentation [13, 14]. Thus, we deleted ppc gene to enhance energy supplement and activate PCK. Furthermore, both PEP and malate would convert to pyruvate, which is smoothly turned into byproducts lactate, acetate and formate via the decarboxylation, dehydrogenation, and pyruvate-formate lyase, respectively. Formate is further split into carbon dioxide and water by formate dehydrogenase, while lactate and acetate accumulate in fermentation broth. Since the substrate specificity of malic enzymes for malate is 6-fold higher than that for pyruvate, malic enzymes encoded by maeA and maeB tend to catalyze the decarboxylation of malate to pyruvate . The formation of pyruvate and its derivative byproducts strongly compete with succinate production for PEP and malate. Inactivation of pykA and pykF has been shown to be effective in inhibiting the conversion of PEP to pyruvate . Consequently, in order to inhibit the formation of pyruvate from PEP and malate, we deleted pykA, maeA and maeB genes. Unfortunately, compared to strain BKS4, strain BKS8 with deletion of pykA, ppc, maeA and maeB did neither significantly attenuate the accumulation of lactate and acetate, nor increase the succinate yield (Fig. 2). The low expression level of pck gene in wild-type E. coli could result in the insufficient metabolic flux to OAA , and pykF might be more active than pykA in the formation of pyruvate from PEP. It suggested that pck and pykF genes could be the potential targets. Therefore, using initial strain BKS8, we further optimize these two targets of succinate metabolic pathway to improve succinate production.
Combined optimization of targeting at TCA cycle and carboxylation of PEP to increase succinate production
Succinate, an essential intermediate of TCA cycle, cannot be efficiently accumulated in E. coli fermentation. In order to increase succinate production, we optimized succinate metabolic pathway by preventing the backflow of succinate to fumarate, activating glyoxylate shunt bypass to decrease the requirement of reducing power, and co-overexpressing pck-ecaA to fix CO2 more efficiently.
Glyoxylate shunt bypass could recover the metabolic flux of the oxidative TCA arm and acetyl-CoA of pyruvate metabolism with less reducing power used, and might contribute to succinate production. The aceBAK operon coding isocitrate lyase, malate synthase and isocitrate dehydrogenase kinase is responsible for the glyoxylate shunt bypass. The transcription of the aceBAK operon is tightly repressed by transcription factor IclR, but induced by inactivating iclR gene . Thus, the deletion of iclR gene resulted in strain BKS10. As shown in Fig. 3, the titer and yield of succinate in strain BKS10 was not apparently increased. It was likely that the gene expression involved in glyoxylate bypass are complex and regulated by multiple factors  and deletion of iclR was not sufficient for activating glyoxylate shunt bypass . Conversion of PEP to OAA in succinate metabolic pathway is net carbon integrated via CO2 fixation catalyzed by PCK. In fact, the active substrate for PCK is not CO2, but the chemically less reactive bicarbonate anion (HCO3 −) . Thus, CaCO3, MgCO3 or NaHCO3 were often added to the culture media. CO2 is more permeable across cell membrane than HCO3 −, but the hydration reaction rate of CO2 to HCO3 − is relatively slow. There might not be enough HCO3 − spontaneously made in vivo to access succinate production. Carbonic anhydrase encoded by ecaA gene catalyzes the hydration of intracellular CO2 to HCO3 −. Expression of ecaA gene of cyanobacterium Anabaena in E. coli led to an obvious increase in succinate production [38, 39]. Thus, the ecaA gene was co-expressed with pck in strain BKS10, generating strain BKS11. Compared to strain BKS10, combinatorial expression of pck-ecaA in strain BKS11 resulted in a 2.2-fold increase in succinate yield (1.16 mol/mol glucose) (p < 0.01) and a 1.2-fold increase in succinate titer (18.17 mM) (p < 0.01) (Fig. 3).
Fine tuning of pykF expression to improve succinate production
Using AUG to nucleotide +24 of the pykF mRNA as the binding sequence and selecting E. coli micC as the scaffold, anti-pykF sRNA working sequence was designed (Fig. 4a). We used two kinds of plasmids with different copy number and tested the inhibitory effects of anti-pykF sRNA on the accumulation of lactate and acetate in strain BKS12 with overexpression of pck gene. When anti-pykF sRNA was expressed on the high-copy-number plasmid pRSF and under the control of T7 promoter, no obvious changes were observed in the yields of succinate, lactate and acetate (Fig. 4b). Then, we constructed the low-copy-number plasmid pBldg-anti-pykF with a pY15A origin of replication, and expression of anti-pykF was controlled under lacUV5 promoter. The metabolite analysis of engineered strain BKS14 showed that the yields of lactate and acetate were decreased by 55.77 % (p < 0.01) and 47.73 % (p < 0.01), respectively, and the yield of succinate was increased by 23.38 % (p < 0.05) compared to BKS12(Fig 4b).
We further tested whether the expression of anti-pykF under the control of lacUV5 promoter in strain BKS11 would improve succinate production and attenuate accumulation of byproducts. pBldg-anti-pykF was transformed into strain BKS11, generating strain BKS15. Compared to strain BKS11, the low expression of anti-pykF in strain BKS15 led to the decrease of 43.48 % (p < 0.05) and 38.09 % (p < 0.01) in the yields of lactate and acetate, respectively (Fig 4c). Although succinate yield of strain BKS15 was not improved, succinate titer was increased by 13.43 % (p < 0.05) (Fig. 4d). The results showed that the down-regulated formation of pyruvate by expressing anti-pykF would enhance the metabolic flux from PEP to succinate.
Distribution of intracellular metabolic flux
As shown in Fig. 5, metabolic modifications led to the fact that fluxes to OAA (V5), malate (V12), fumarate (V13), succinate (V15 and V16) were significantly increased and that fluxes to pyruvate (V4), lactate (V6), and acetate (V7 + V10) were remarkably decreased from strains BW25113(DE3) to BKS15. The results indicated that our strategies favored the improvement of succinate production and the decrease of byproduct accumulation.
Split ratios of fluxes to OAA, PYR, lactate, acetate and succinate
Fraction of PEP to OAA (V5/V3)
Fraction of PYR production (V4/V3)
Fraction of lactate production ( V6/V3)
Fraction of acetate production (V7 + V10)/V3
Fraction of succinate production (V16/V3)
3.08 ± 0.02 %
96.92 ± 0.02 %
23.46 ± 0.71 %
77.25 ± 2.66 %
2.61 ± 0.02 %
8.53 ± 0.03 %
91.47 ± 0.02 %
19.43 ± 0.48 %
73.70 ± 1.56 %
8.29 ± 0.01 %
12.39 ± 0.45 %
86.93 ± 1.43 %
19.95 ± 0.14 %
66.74 ± 0.08 %
12.61 ± 0.47 %
13.66 ± 0.22 %
86.34 ± 1.10 %
18.28 ± 0.44 %
67.84 ± 2.89 %
13.66 ± 0.22 %
45.94 ± 0.73 %
53.87 ± 0.72 %
16.97 ± 0.59 %
36.90 ± 0.32 %
55.54 ± 0.98 %
52.31 ± 0.83 %
47.69 ± 0.67 %
14.88 ± 0.76 %
29.91 ± 0.70 %
67.20 ± 0.78 %
In strain BKS11, 45.94 % of PEP was converted to OAA (V5/V3), 2.4-fold higher than that of strain BKS10 (p < 0.01). As a result, the fraction of the metabolic flux to succinate (V16/V3) increased from 13.66 % in strain BKS10 to 55.54 % in strain BKS11 (Table 1) (p < 0.01). Meanwhile, strain BKS11 showed lower acetic fluxes ((V7 + V10)/V3). This indicated that co-overexpression of pck-ecaA could significantly enhanced the metabolic flux of PEP to OAA, and simultaneously inhibit other metabolic branches. Compared to strain BKS11, the fractions of the metabolic flux to lactate (V6/V3) and acetate ((V7 + V10)/V3) of strain BKS15 decreased by 12.32 % (p < 0.05) and 18.94 % (p < 0.01), respectively (Table 1), indicating that expression of anti-pykF attenuated the accumulation of lactate and acetate. At last, with a series of metabolic modifications, compared to strain BW25113(DE3), the final fraction of the metabolic flux to succinate in BKS15 was increased by 24.8 fold (p < 0.01) and those to lactate and acetate were decreased by 36.57 % (p < 0.01) and 61.28 % (p < 0.01), respectively.
Anaerobic batch fermentation for succinate production
Parameters of succinate production by engineered E. coli strians during anaerobic fermentation
Growth rate (h−1)
Yield (mol/mol of glucose)
Specific productivity (mmol gDCW−1 h−1)
Productivity (mmol L−1 h−1)
0.071 ± 0.002
8.65 ± 0.73
12.06 ± 0.70
27.13 ± 2.56
0.31 ± 0.02
0.43 ± 0.02
0.98 ± 0.09
1.09 ± 0.06
1.47 ± 0.20
3.31 ± 0.13
0.12 ± 0.01
0.17 ± 0.01
0.39 ± 0.04
0.052 ± 0.003**
25.51 ± 1.79**
7.82 ± 0.63**
23.52 ± 1.53
0.92 ± 0.06**
0.28 ± 0.02**
0.85 ± 0.05
3.96 ± 0.13**
1.18 ± 0.04*
3.54 ± 0.08
0.36 ± 0.03**
0.11 ± 0.01
0.34 ± 0.02
0.043 ± 0.002*
30.12 ± 3.31
6.55 ± 0.33*
13.22 ± 1.64**
1.08 ± 0.11
0.24 ± 0.01*
0.48 ± 0.06**
5.89 ± 0.41**
1.20 ± 0.07
2.42 ± 0.19**
0.43 ± 0.05
0.09 ± 0.01
0.19 ± 0.02**
In this paper, PEP was selected as optimized target for increased succinate production and attenuated accumulation of byproducts in engineered E. coli under anaerobic conditions. By deleting ptsG, pykA, ppc and maeAB genes, we have designed and constructed initial succinate-producing E. coli strain. The succinate metabolic pathway was then enhanced with deletion of sdh and co-overexpression of pck-ecaA, resulting in succinate production of 25.51 mM. By introducing artificial sRNA of anti-pykF, the titer of succinate in the final optimized strain BKS15 was 30.12 mM with remarkable decrease in lactate and acetate. Metabolic flux analysis and fermentation kinetics showed that our optimization strategy could efficiently enhance the central carbon flux to succinate and decrease to byproducts. Recently, the progress in metabolic engineering suggested that limitation of cellular ATP supply and redox unbalance can be alleviated for improving succinate production in E. coli . Combination of our strategies with those targets would further develop high succinate-producing microorganisms.
Bacterial strains and plasmids
E. coli strains and plasmids used in this study
lacI q rrnB T14ΔlacZWJ16 hsdR514ΔaraBAD AH33
NBRP-E. coli at NIG
lacI q rrnB T14ΔlacZWJ16 hsdR514ΔaraBAD AH33dcm (DE3)
BW25113(DE3) harboring pCDF-pck
BW25113 harboring pCDF-pck
BL21(DE3) harboring pCDF-pck
BKS10 harboring pCDF-pck-ecaA
BKS8 harboring pCDF-pck
BKS8 harboring pCDF-pck and pRSF-anti-pykF
BKS8 harboring pCDF-pck and pBldg-anti-pykF
BKS11 harboring pBldg-anti-pykF
FRT(FLP recognition target) sites; CmR
(Datsenko and Wanner 2000)
Red recombinase expression vector; AmpR
(Datsenko and Wanner 2000)
FLP expression vector; AmpR,CmR
(Datsenko and Wanner 2000)
pBR322 ori with PT7; AmpR
CDF ori with PT7; StrR
RSF ori with PT7; KanR
p15A ori with PlacUV5; CmR
(Yao et al, 2013)
pCDFDute-1 with pck
pCDFDuet-1 with pck and ecaA
pRSF without RBS sequence
pRSFM1 with anti-pykF
pBldgbrick2 with anti-pykF
Construction of engineered strains and plasmids
Restriction endonucleases and T4 DNA ligase were purchased from Thermo Scientific (USA), High-Fidelity DNA polymerase used for PCR amplification was purchased from Transgene Biotech (Beijing, China). Appropriate restriction sites were added to 5′and 3′ ends of the primers and all primers used in this study were listed in Additional file 1: Table S4. All plasmids was constructed through the enzymatic digestion of PCR products and plasmids with appropriate restriction sites, followed by the ligation of the appropriate fragments. Clones bearing inserted gene were screened by PCR and recombinant plasmids were confirmed by DNA sequencing.
By using the lambda Red recombinase system , the gene coding for T7 RNA polymerase was inserted into the genome of E. coli BW25113. The DNA fragment containing 500 bp upstream of the ybhB gene, T7 RNA polymerase gene, chloramphenicol resistance cassette and 500 bp downstream of the ybhC gene was constructed. The detailed procedure was shown in Additional file 1: Figure S1 and the primers used were shown in Additional file 1: Table S4. This DNA fragment was then electrotransformed into E. coli BW25113 which expressed lambda Red system for homologous recombination. The positive clones were confirmed with primers F-ybhB and R-ybhC. Next, the chloramphenicol resistance cassette was removed with the help of pCP20 and its removal was confirmed with primers F-ybhB and R-ybhC. The function of T7 RNA polymerase in BW25113 (DE3) was verified by SDS-PAGE of BW25113 (DE3) carrying pCDF-pck, using BL21 (DE3) harboring pCDF-pck and BW25113 harboring pCDF-pck as positive and negative controls, respectively (Additional file 1: Figure S2).
All in-frame gene deletion strains were constructed in E. coli BW25113 (DE3) according to the procedure described previously  and confirmed by PCR. Briefly, for deleting ptsG as example, the DNA fragment containing the chloramphenicol resistance cassette for homologous recombination was amplified by PCR using F-ptsG-Q and R-ptsG-Q as primers and the plasmid pKD3 as the template. The DNA fragment was then electrotransformed into E. coli BW25113 (DE3) which expressed lambda Red system for homologous recombination. The replacement of ptsG gene was confirmed by PCR using the primers F-ptsG and R-ptsG and the removal of chloramphenicol resistance was confirmed with primers F-ptsG and R-ptsG listed in Additional file 1: Table S4. The same procedure was performed for deletions of pykA, ppc, maeA, maeB, sdh, and iclR.
For construction of pRSF-anti-pykF and pBldg-anti-pykF, the complementary sequence that spans to + 24 nucleotides of pykF coding mRNA was used as the binding sequence and was designed in the primer. In order to construct pRSF-anti-pykF, the sequence between RBS and terminator was removed from pRSFDuet-1 using primers F-RSF and R-RSF, followed by the ligation, resulting in pRSFM1. The scaffold micC with 24 bp binging sequence at the 5′ end  was amplified with primers F-RSF-anti-pykF and R-RSF-anti-pykF and cloned into the SpeI site of pRSFM1 (high-copy-number plasmid), and resulting in plasmid pRSF-anti-pykF. The correct construct pRSF-anti-pykF was screened by PCR using primers ACYCDuetUP1 and R-RSF-anti-pykF, and confirmed DNA sequencing. The DNA fragment containing 24 bp binding sequence and micC was amplified by PCR with primers F-Bldg-anti-pykF and R-Bldg-anti-pykF listed in Additional file 1: Table S4 using E. coli BW25113 genome as template. Then, PCR product was cloned into vector pBldgbrick2 (low-copy-number plasmid)  between HindIII and NcoI, resulting plasmid pBldg-anti-pykF. The plasmids with anti-pykF sequence were used to silence the expression of pykF gene.
Dual phase fermentation mode was employed . For all engineered E. coli strains, a seed inoculum of 500 μL from an overnight 3 mL of LB culture was first inoculated at 37 °C in 250 mL shake flask containing 100 mL of liquid LB medium for aerobic growth. When the optical density (OD) reached 1.0, cells were induced with a final concentration of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and grown for another 3 h for recombinant protein expression. Then, bacterial cells were collected by centrifugation and resuspended in 150 mL shake flask containing 100 mL of fresh YM9 medium (1*M9 salts, 1 g/L yeast extract) at an initial OD of 1.0 for anaerobic fermentation. At that point, 5 g/L CaCO3, 2 g/L NaHCO3, 0.1 mM IPTG were added. Flasks were sealed with non-ventilated plugs. The cells were incubated at 37 °C on a shaker (150 rpm) and sample were collected at 40 h for analysis. For kinetic study, samples were collected at 0, 8, 16, 24, 34, 46, 58 and 70 h. Appropriate amounts of antibiotics (50 mg/L ampicillin, 30 mg/L streptomycin, 30 mg/L kanamycin) were added to media when needed.
Cell growth was monitored by measuring the optical density (OD) at 600 nm (UV-vis spectrophotometer) and was transformed into dry cell weight using the coefficient as: dry cell mass (g L−1) = 0.48*OD600 . The concentration of glucose was measured using SBA-90B biosensor (Biology Institute of ShanDong Academy of Science, China). The sample was centrifuged and the supernatant of fermentation sample was filtered through 0.2 μm syringe filter and metabolites were analyzed using an Waters 1515 differential HPLC system equipped with a Bio-Red HPX-87H HPLC column. 10 μL of sample was injected into the HPLC at column temperature of 65 °C and ran isocratically with 5 mM H2SO4 as mobile phase sat on a flow rate of 0.6 ml/min.
Metabolic flux analysis
The metabolic network was constructed based on engineered pathways in anaerobically grown E. coli (Fig. 1). This network included glycolysis, TCA cycle and glyoxylate bypass (Fig. 5). As an attempt to analyze the distribution of carbon source, the fluxes through each pathway in the metabolic network were designated by V1-V16. The simplified central metabolic reactions were described in detail in Additional file 1: Table S1. According to the law of mass conversation and the quasi-steady-state assumption, these metabolic flux relationships were constructed to simplify the computational process, and shown in Additional file 1: Table S2, in which V1, V6, V16, and V7 + V10 were measurable quantities while the others were the metabolic fluxes of the corresponding intermediates. In this study, Lingo software  was used to obtain the solutions to distribution of metabolic fluxes that were limited by the formulas in Additional file 1: Table S2.
The data are shown as mean values ± standard deviation (SD) of three replicates. The Student’s t test was used for all statistical analysis using SPSS 17.0. The p-value of < 0.05 and < 0.01 was considered statistically significant, more significant, respectively.
ATP, adenosine triphosphate; G3P, Glyceraldehyde 3-P; Glf, glucose facilitator; IPTG, isopropyl-β-D-thiogalactopyranoside; NADH, Nicotinamide adenine dinucleotide; OAA, oxaloacetate; PCK, PEP carboxykinase; PEP, phosphoenolpyruvate; PPC, PEP carboxylase; PTS, PEP carbohydrate phosphotransferase system; PYC, pyruvate carboxylase; PYR, pyruvate; SDH, succinate dehydrogenase
We acknowledge Professor Jian-Min Xing (Chinese Academy of Sciences) for providing ecaA gene. We also thank Associate Professor Tao Chen and Dr. Zhiwen Wang (Tianjin University) for their kind suggestions to this manuscript.
This work was supported by the National Basic Research Program of China (2011CBA00800), the National High-Tech R&D Program of China (2012AA02A701), the National Natural Science Foundation of China (31570087), and the Natural Science Foundation of Tianjin (13JCZDJC27600).
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article (and its additional file).
GRZ, YZ and CSW conceived method and designed experiment; YZ, CSW, FFL and ZNL performed experiment; GRZ, YZ and CSW analyzed the data; YZ analyzed metabolic fluxes; YZ wrote the manuscript with help by GRZ. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Jang YS, Kim B, Shin JH, Choi YJ, Choi S, Song CW, et al. Bio-based production of C2–C6 platform chemicals. Biotechnol Bioeng. 2012;109(10):2437–59.View ArticleGoogle Scholar
- Becker J, Lange A, Fabarius J, Wittmann C. Top value platform chemicals: bio-based production of organic acids. Curr Opin Biotechnol. 2015;36:168–75.View ArticleGoogle Scholar
- Cheng KK, Zhao XB, Zeng J, Zhang JA. Biotechnological production of succinic acid: current state and perspectives. Biofuels Bioprod Bioref. 2012;6(3):302–18.View ArticleGoogle Scholar
- Vuoristo KS, Mars AE, Sanders JPM, Eggink G, Weusthuis RA. Metabolic engineering of TCA cycle for production of chemicals. Trends Biotechnol. 2015;34(3):191–7.View ArticleGoogle Scholar
- Chen X, Zhou L, Tian K, Kumar A, Singh S, Prior BA, et al. Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production. Biotechnol Adv. 2013;31(8):1200–23.View ArticleGoogle Scholar
- Clark DP. The fermentation pathways of Escherichia coli. FEMS Microbiol Rev. 1989;5(3):223–34.Google Scholar
- Bunch PK, Mat-Jan F, Lee N, Clark DP. The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology. 1997;143(1):187–95.View ArticleGoogle Scholar
- Donnelly MI, Millard CS, Clark DP, Chen MJ, Rathke JW. A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol. Appl Biochem Biotechnol. 1998;70-72:187–98.View ArticleGoogle Scholar
- Jantama K, Haupt MJ, Svoronos SA, Zhang XL, Moore JC, Shanmugam KT, et al. Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate. Biotechnol Bioeng. 2008;99(5):1140–53.View ArticleGoogle Scholar
- Vemuri GN, Eiteman MA, Altman E. Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol. 2002;68(4):1715–27.View ArticleGoogle Scholar
- Sanchez AM, Bennett GN, San KY. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab Eng. 2005;7:229–39.View ArticleGoogle Scholar
- Thakker C, Zhu JF, San KY, Bennett G. Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production. J Biotechnol. 2011;155(2):236–43.View ArticleGoogle Scholar
- Singh A, Soh KC, Hatzimanikatis V, Gill RT. Manipulating redox and ATP balancing for improved production of succinate in E. coli. Metab Eng. 2011;13(1):76–81.View ArticleGoogle Scholar
- Kim P, Laivenieks M, Vieille C, Zeikus JG. Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli. Appl Environ Microbiol. 2004;70(2):1238–41.View ArticleGoogle Scholar
- Wang D, Li Q, Mao Y, Xing JM, Su ZG. High-level succinic acid production and yield by lactose-induced expression of phosphoenolpyruvate carboxylase in ptsG mutant Escherichia coli. Appl Microbiol Biotechnol. 2010;87(6):2025–35.View ArticleGoogle Scholar
- Tan Z, Zhu X, Chen J, Li Q, Zhang X. Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in combination for improvement of succinate production. Appl Environ Microbiol. 2013;79(16):4838–44.View ArticleGoogle Scholar
- Singh A, Lynch MD, Gill RT. Genes restoring redox balance in fermentation-deficient E. coli NZN111. Metab Eng. 2009;11:347–54.View ArticleGoogle Scholar
- Balzer GJ, Thakker C, Bennett GN, San KY. Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD+-dependent formate dehydrogenase. Metab Eng. 2013;20(5):1–8.View ArticleGoogle Scholar
- Ma JF, Gou DM, Liang LY, Liu RM, Chen X, Zhang CQ, et al. Enhancement of succinate production by metabolically engineered Escherichia coli with co-expression of nicotinic acid phosphoribosyltransferase and pyruvate carboxylase. Appl Microbiol Biotechnol. 2013;97(15):6739–47.View ArticleGoogle Scholar
- Zhu X, Tan Z, Xu H, Chen J, Tang J, Zhang X. Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. Metab Eng. 2014;24:87–96.View ArticleGoogle Scholar
- Lu J, Tang J, Liu Y, Zhu X, Zhang T, Zhang X. Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization. Appl Microbiol Biotechnol. 2012;93(6):2455–62.View ArticleGoogle Scholar
- Tang J, Zhu X, Lu J, Liu P, Xu H, Tan Z, et al. Recruiting alternative glucose utilization pathways for improving succinate production. Appl Microbiol Biotechnol. 2013;97(6):2513–20.View ArticleGoogle Scholar
- Beauprez JJ, Foulquié-Moreno MR, Maertens J, Horen EV, Bekers K, Baart GJE, et al. Influence of C4-dicarboxylic acid transporters on succinate production. Green Chem. 2011;13(8):2179–86.View ArticleGoogle Scholar
- Chen J, Zhu XN, Tan ZG, Xu HT, Tang JL, Xiao DG, et al. Activating C4-dicarboxylate transporters DcuB and DcuC for improving succinate production. Appl Microbiol Biotechnol. 2014;98(5):1–9.View ArticleGoogle Scholar
- Chatterjee R, Millard CS, Champion K, Clark DP, Donnelly MI. Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl Environ Microbiol. 2001;67(1):148–54.View ArticleGoogle Scholar
- Wang Q, Wu C, Chen T, Chen X, Zhao XM. Expression of galactose permease and pyruvate carboxylase in Escherichia coli ptsG mutant increases the growth rate and succinate yield under anaerobic conditions. Biotechnol Lett. 2006;28(2):89–93.View ArticleGoogle Scholar
- Zhang X, Jantama K, Moore JC, Jarboe LR, Shanmugam KT, Ingram LO. Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli. Proc Natl Acad Sci U S A. 2009;106(48):20180–5.View ArticleGoogle Scholar
- Stols L, Donnelly MI. Production of succinic acid through overexpression of NAD+-dependent malic enzyme in an Escherichia coli mutant. Appl Environ Microbiol. 1997;63(7):2695–701.Google Scholar
- Noda S, Shirai T, Oyama S, Kondo A. Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives. Metab Eng. 2016;33:119–29.View ArticleGoogle Scholar
- Park SJ, Tseng CP, Gunsalus RP. Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr. Mol Microbiol. 1995;15(3):473–82.View ArticleGoogle Scholar
- Li N, Zhang B, Chen T, Wang ZW, Tang YJ, Zhao XM. Directed pathway evolution of the glyoxylate shunt in Escherichia coli for improved aerobic succinate production from glycerol. J Ind Microbiol Biotechnol. 2013;40(12):1461–75.View ArticleGoogle Scholar
- Lin H, Bennett GN, San KY. Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate. Biotechnol Bioeng. 2005;89(2):148–56.View ArticleGoogle Scholar
- Litsanov B, Kabus A, Brocker M, Bott M. Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum. Microb Biotechnol. 2012;5(1):116–28.View ArticleGoogle Scholar
- Waegeman H, Beauprez J, Moens H, Maertens J, Mey MD, Foulquié-Moreno WR, et al. Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3). BMC Microbiol. 2011;11(15):70.View ArticleGoogle Scholar
- Shimizu K. Metabolic regulation of a bacterial cell system with emphasis on Escherichia coli metabolism. ISRN Biochem. 2013;6.Google Scholar
- Skorokhodova AY, Gulevich AY, Morzhakova AA, Shakulov RS, Debabov VG. Comparison of different approaches to activate the glyoxylate bypass in Escherichia coli K-12 for succinate biosynthesis during dual-phase fermentation in minimal glucose media. Biotechnol Lett. 2013;35(4):577–83.View ArticleGoogle Scholar
- Kai Y, Matsumura H, Izui K. Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. Arch Biochem Biophys. 2003;414(2):170–9.View ArticleGoogle Scholar
- Wang D, Li Q, Li WL, Xing JM, Su ZG. Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli. Enzyme Microb Technol. 2009;45(6):491–7.View ArticleGoogle Scholar
- Wang J, Qin DD, Zhang BY, Li Q, Li S, Zhou XH, et al. Fine-tuning of ecaA and pepc gene expression increases succinic acid production in Escherichia coli. Appl Microbiol Biotechnol. 2015;99(20):8575–86.View ArticleGoogle Scholar
- Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. Metabolic engineering of E. coli using synthetic small regulatory RNAs. Nat Biotechnol. 2013;31(2):170–4.View ArticleGoogle Scholar
- Thakker C, Martínez I, San KY, Bennett GN. Succinate production in Escherichia coli. Biotechnol J. 2012;7(2):213–24.View ArticleGoogle Scholar
- Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97:6640–5.View ArticleGoogle Scholar
- Yao YF, Wang CS, Qiao J, Zhao GR. Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway. Metab Eng. 2013;19(5):79–87.View ArticleGoogle Scholar
- Gokarn RR, Eiteman MA, Altman E. Expression of pyruvate carboxylase enhances succinate production in Escherichia coli without affecting glucose uptake. Biotechnol Lett. 1998;20(8):795–8.View ArticleGoogle Scholar
- Li XJ, Chen T, Chen X, Zhao XM. Redirection electron flow to high coupling efficiency of terminal oxidase to enhance riboflavin biosynthesis. Appl Microbiol Biotechnol. 2006;73(2):374–83.View ArticleGoogle Scholar