Engineering Corynebacterium glutamicum for the production of pyruvate
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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. glutamicum △aceE △pqo, 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. glutamicum △aceE △pqo △ldhA △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. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA 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.
KeywordsCorynebacterium glutamicum Pyruvate production Pyruvate dehydrogenase complex Pyruvate:quinone oxidoreductase NAD+-dependent l-lactate dehydrogenase Acetohydroxyacid synthase Transaminases Industrial biotechnology
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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