Construction of pyruvate producing strain with intact pyruvate dehydrogenase and genome-wide transcription analysis

  • Maohua Yang
  • Xiang Zhang
Original Paper


To obtain strain YP211 with a high tendency for accumulating pyruvate, central metabolic pathways were modified in Escherichia coli MG1655. Specifically, seven genes (ldhA, pflB, pta-ackA, poxB, ppc, frdBC) were knocked out sequentially and full pyruvate dehydrogenase was retained. In batch fermentation with M9 medium, pyruvate yield and production rate reached 0.63 g/g glucose and 1.89 g/(1 h), respectively. Meanwhile, the production of acetate, succinate, and other carboxylates was effectively controlled. To understand the physiological observations, we further completed genome-wide transcription analysis of wild-type and YP211. As the acetic acid pathways were blocked, the pathways of convertion of pyruvate to phosphoenol pyruvate and acetyl CoA were enhanced. The transcription of pck, as an alternative gene for ppc, was increased by 2.6 times. So even if gene ppc was inactivated, the tricarboxylic acid pathway was still enhanced in YP211. In order to balance intracellular NADH/NAD+, oxidative phosphorylation and flagellar assembly system were also up-regulated significantly.

Graphical Abstract

Biochemical pathways involved in pyruvate accumulation in YP211 (a). Transcriptional differences of genes related to pyruvate metabolism between strain YP211 and E. coli wild-type (b).


Pyruvate Escherichia coli Transcription analysis Oxidation–Reduction Flagellar assembly 



The work was supported by the National High Technology Research and Development Program of China, No. 2014AA021900, and the Foundation (No. 2015IM002) of Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology (Tianjin University of Science & Technology).

Supplementary material

11274_2016_2202_MOESM1_ESM.docx (47 kb)
Supplementary material 1 (DOCX 46 KB)


  1. Causey T, Shanmugam K, Yomano L, Ingram L (2004) Engineering Escherichia coli for efficient conversion of glucose to pyruvate. Proc Natl Acad Sci USA 101:2235–2240CrossRefGoogle Scholar
  2. Chang Y, Wang A, Jr J (1994) Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by therpoS (katF) gene. Mol Microbiol 11:1019–1028CrossRefGoogle Scholar
  3. Chang D, Shin S, Rhee J, Pan J (1999) Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival. J Bacteriol 181:6656–6663Google Scholar
  4. Koebmann B, Westerhoff H, Snoep J, Dan N, Jensen P (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J Bacteriol 184:3909–3916CrossRefGoogle Scholar
  5. Lee PC, Schmidt-Dannert C (2002) Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl Microbiol Biotechnol 60:1–11CrossRefGoogle Scholar
  6. Li Y, Chen J, Lun S (2001) Biotechnological production of pyruvic acid. Appl Microbiol Biotechnol 57:451–459CrossRefGoogle Scholar
  7. Li Y, Hugenholtz J, Chen J, Lun S (2002) Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy. Appl Microbiol Biotechnol 60:101–106CrossRefGoogle Scholar
  8. Liu L, Xu Q, Li Y, Shi Z, Zhu Y, Du G, Chen J (2007) Enhancement of pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata. Biotechnol Bioeng 97:825–832CrossRefGoogle Scholar
  9. Liu S, Zhang L, Mao J, Ding Z, Shi G (2015) Metabolic engineering of Escherichia coli for the production of phenylpyruvate derivatives. Metab Eng 32:55–65CrossRefGoogle Scholar
  10. Liu Y, Yang M, Chen J, Yan D, Cheng W, Wang Y, Thygesen A, Chen R, Xing J, Wang Q, Ma Y (2016) PCR-based seamless genome editing with high efficiency and fidelity in Escherichia coli. PLoS ONE doi:  10.1371/journal.pone.0149762 Google Scholar
  11. Shi A, Zhu X, Lu J, Zhang X, Ma Y (2013) Activating transhydrogenase and NAD kinase in combination for improving isobutanol production. Metab Eng 16:1–10CrossRefGoogle Scholar
  12. Tomar A, Eiteman M, Altman E (2003) The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli. Appl Microbiol Biotechnol 62:76–82CrossRefGoogle Scholar
  13. Vemuri G, Altman E, Sangurdekar D, Khodursky A, Eiteman M (2006) Overflow metabolism in Escherichia coli during steady-state growth: transcriptional regulation and effect of the redox ratio. Appl Environ Microb 72:3653–3661CrossRefGoogle Scholar
  14. Wada M, Narita K, Yokota A (2007) Alanine production in an H+-ATPase and lactate dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase. Appl Microbiol Biotechnol 76:819–825CrossRefGoogle Scholar
  15. Wang Z, Gao C, Wang Q, Liang Q, Qi Q (2012) Production of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering. Biochem Eng J 67:126–131CrossRefGoogle Scholar
  16. Wendisch V, Bott M, Eikmanns B (2001) Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids. Curr Opin Microbiol 9:268–274CrossRefGoogle Scholar
  17. Xu P, Qiu J, Gao C, Ma C (2008) Biotechnological routes to pyruvate production. J Biosci Bioeng 105:169–175CrossRefGoogle Scholar
  18. Yang S, Chen X, Xu N, Liu L, Chen J (2014) Urea enhances cell growth and pyruvate production in Torulopsis glabrata. Biotechnol Prog 30:19–27CrossRefGoogle Scholar
  19. Yokota A, Shimizu H, Terasawa Y, Takaoka N, Tomita F (1994) Pyruvic acid production by a lipoic acid auxotroph of Escherichia coliW1485. Appl Microbiol Biotechnol 41:638–643CrossRefGoogle Scholar
  20. Yokota A, Henmi M, Takaoka N, Hayashi C, Takezawa Y, Fukumori Y, Tomita F (1997) Enhancement of glucose metabolism in a pyruvic Acid-hyperproducing Escherichia coli mutant defective in F1-ATPase Activity. J Ferment Bioeng 83:132–138CrossRefGoogle Scholar
  21. Zhou J, Huang L, Liu L, Chen J (2009) Enhancement of pyruvate productivity by inducible expression of a F0F1-ATPase inhibitor INH1 in Torulopsis glabrata CCTCC M202019. J Biotechnol 144:120–126CrossRefGoogle Scholar
  22. Zhu Y, Eiteman M, Altman R, Altman E (2008) High glycolytic flux improves pyruvate production by a metabolically engineered Escherichia coli strain. Appl Environ Microb 74:6649–6655CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Institute of Process EngineeringChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Institute of Agro-food Science and TechnologyShandong Academy of Agricultural SciencesJi’nanPeople’s Republic of China

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