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 a-(3)aceE a-(3)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 (a-C-3-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 a-(3)aceE a-(3)pqo a-(3)ldhA a-C-3-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 a-(3)aceE a-(3)pqo a-(3)ldhA a-C-3-T ilvN a-(3)alaT a-(3)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.