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Applied Microbiology and Biotechnology

, Volume 94, Issue 2, pp 449–459 | Cite as

Engineering Corynebacterium glutamicum for the production of pyruvate

  • Stefan Wieschalka
  • Bastian Blombach
  • Bernhard J. EikmannsEmail author
Applied genetics and molecular biotechnology

Abstract

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicumaceEpqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD+-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicumaceEpqoldhA △C–T ilvN produced about 190 mM pyruvate with a Y P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicumaceEpqoldhA △C–T ilvNalaTavtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g (CDW) −1  h−1 (i.e., 0.08 g g(CDW) −1 h−1) in the production phase.

Keywords

Corynebacterium glutamicum Pyruvate production Pyruvate dehydrogenase complex Pyruvate:quinone oxidoreductase NAD+-dependent l-lactate dehydrogenase Acetohydroxyacid synthase Transaminases Industrial biotechnology 

Notes

Acknowledgements

We thank L. Eggeling and J. Marienhagen (both IBG-1, Research Center Jülich) for providing plasmids pK19mobsacB ΔalaT and pK19mobsacB ΔavtA, V.Wendisch (Faculty of Biology and CeBiTec, University of Bielefeld) for providing plasmid pK19mobsacB ΔldhA, and M. Oldiges (IBG-1, Research Center Jülich) for technical advice. The support of the Fachagentur Nachwachsende Rohstoffe (FNR) of the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (FNR grant 220-095-08A; BioProChemBB project, ERA-IB program) is gratefully acknowledged.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Stefan Wieschalka
    • 1
  • Bastian Blombach
    • 2
  • Bernhard J. Eikmanns
    • 1
    Email author
  1. 1.Institute of Microbiology and BiotechnologyUniversity of UlmUlmGermany
  2. 2.Institute of Biochemical EngineeringUniversity of StuttgartStuttgartGermany

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