Targeted optimization of central carbon metabolism for engineering succinate production in Escherichia coli

BackgroundSuccinate 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.ResultsBy 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.ConclusionsThis 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.

[1]  Tao Chen,et al.  Redirection electron flow to high coupling efficiency of terminal oxidase to enhance riboflavin biosynthesis , 2006, Applied Microbiology and Biotechnology.

[2]  Ryan T Gill,et al.  Genes restoring redox balance in fermentation-deficient E. coli NZN111. , 2009, Metabolic engineering.

[3]  Xueli Zhang,et al.  Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization , 2011, Applied Microbiology and Biotechnology.

[4]  Michael Bott,et al.  Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum , 2011, Microbial biotechnology.

[5]  M. Jiang,et al.  Enhancement of succinate production by metabolically engineered Escherichia coli with co-expression of nicotinic acid phosphoribosyltransferase and pyruvate carboxylase , 2013, Applied Microbiology and Biotechnology.

[6]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. S. Millard,et al.  A novel fermentation pathway in anEscherichia coli mutant producing succinic acid, acetic acid, and ethanol , 1998, Applied biochemistry and biotechnology.

[8]  Wim Soetaert,et al.  Influence of C4-dicarboxylic acid transporters on succinate production , 2011 .

[9]  G. Bennett,et al.  Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate. , 2005, Biotechnology and bioengineering.

[10]  J. Chen,et al.  Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. , 2014, Metabolic engineering.

[11]  David P. Clark,et al.  The IdhA Gene Encoding the Fermentative Lactate Dehydrogenase of Escherichia Coli , 1997 .

[12]  L. Stols,et al.  Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant , 1997, Applied and environmental microbiology.

[13]  Gerrit Eggink,et al.  Metabolic Engineering of TCA Cycle for Production of Chemicals. , 2016, Trends in biotechnology.

[14]  D. Clark,et al.  The fermentation pathways of Escherichia coli. , 1989, FEMS microbiology reviews.

[15]  Y. Kai,et al.  Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. , 2003, Archives of biochemistry and biophysics.

[16]  V. Hatzimanikatis,et al.  Manipulating redox and ATP balancing for improved production of succinate in E. coli. , 2011, Metabolic engineering.

[17]  G. Bennett,et al.  Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. , 2005, Metabolic engineering.

[18]  D. Clark,et al.  The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. , 1997, Microbiology.

[19]  C. Vieille,et al.  Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli , 2004, Applied and Environmental Microbiology.

[20]  Y. Jang,et al.  Bio‐based production of C2–C6 platform chemicals , 2012, Biotechnology and bioengineering.

[21]  P. Cheng,et al.  Anti-obesity effects of gut microbiota are associated with lactic acid bacteria , 2013, Applied Microbiology and Biotechnology.

[22]  Fine-tuning of ecaA and pepc gene expression increases succinic acid production in Escherichia coli , 2015, Applied Microbiology and Biotechnology.

[23]  Tao Chen,et al.  Directed pathway evolution of the glyoxylate shunt in Escherichia coli for improved aerobic succinate production from glycerol , 2013, Journal of Industrial Microbiology & Biotechnology.

[24]  Xuebing Zhao,et al.  Biotechnological production of succinic acid: current state and perspectives , 2012 .

[25]  Ka-Yiu San,et al.  Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production. , 2011, Journal of biotechnology.

[26]  Recruiting alternative glucose utilization pathways for improving succinate production , 2013, Applied Microbiology and Biotechnology.

[27]  V. Debabov,et al.  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 , 2013, Biotechnology Letters.

[28]  M. Eiteman,et al.  Expression of Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli without Affecting Glucose Uptake , 1998, Biotechnology Letters.

[29]  Activating C4-dicarboxylate transporters DcuB and DcuC for improving succinate production , 2014, Applied Microbiology and Biotechnology.

[30]  Xueli Zhang,et al.  Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate , 2008, Biotechnology and bioengineering.

[31]  M. A. Eiteman,et al.  Effects of Growth Mode and Pyruvate Carboxylase on Succinic Acid Production by Metabolically Engineered Strains of Escherichia coli , 2002, Applied and Environmental Microbiology.

[32]  J. Park,et al.  Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs , 2013, Nature Biotechnology.

[33]  A. Kondo,et al.  Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives. , 2016, Metabolic engineering.

[34]  C. Wittmann,et al.  Top value platform chemicals: bio-based production of organic acids. , 2015, Current opinion in biotechnology.

[35]  Wim Soetaert,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) , 2011, BMC Microbiology.

[36]  Jing Chen,et al.  Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production , 2013, Applied and Environmental Microbiology.

[37]  G. Bennett,et al.  Succinate production in Escherichia coli , 2012, Biotechnology journal.

[38]  Tao Chen,et al.  Expression of Galactose Permease and Pyruvate Carboxylase in Escherichia coli ptsG Mutant Increases the Growth Rate and Succinate Yield under Anaerobic Conditions , 2006, Biotechnology Letters.

[39]  Z. Su,et al.  High-level succinic acid production and yield by lactose-induced expression of phosphoenolpyruvate carboxylase in ptsG mutant Escherichia coli , 2010, Applied Microbiology and Biotechnology.

[40]  K. Shimizu.  Metabolic Regulation of a Bacterial Cell System with Emphasis on Escherichia coli Metabolism , 2013, ISRN biochemistry.

[41]  Z. Su,et al.  Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli , 2009 .

[42]  G. Bennett,et al.  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. , 2013, Metabolic engineering.

[43]  R. Gunsalus,et al.  Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr , 1995, Molecular microbiology.

[44]  David P. Clark,et al.  Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose byEscherichia coli , 2001, Applied and Environmental Microbiology.

[45]  Xueli Zhang,et al.  Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli , 2009, Proceedings of the National Academy of Sciences.

[46]  J. Qiao,et al.  Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway. , 2013, Metabolic engineering.

[47]  Zhengxiang Wang,et al.  Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production. , 2013, Biotechnology advances.