Engineering corynebacterium glutamicum for the production of pyruvate

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. glutamicum ΔaceE Δpqo, 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 ( ΔC-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 ΔaceE Δpqo ΔldhA ΔC-T ilvN produced about 190 mM pyruvate with a YP/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 ΔaceE Δpqo ΔldhA ΔC-T ilvN ΔalaT ΔavtA 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. © Springer-Verlag 2012.