Engineering Corynebacterium glutamicum for the production of pyruvate.
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.
Institute of Microbiology and Biotechnology, University of Ulm, Ulm, Germany.
Stefan
S
Wieschalka
Bastian
B
Blombach
Bernhard J
BJ
Eikmanns
D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D019289:Pyruvic Acid / Q000378:metabolism*; D014633:Valine / Q000378:metabolism
D019289:Pyruvic Acid; D014633:Valine; D005947:Glucose