Increasing available NADH supply during succinic acid production by Corynebacterium glutamicum.

Abstract

A critical factor in the biotechnological production of succinic acid with Corynebacterium glutamicum is the sufficient supply of NADH. It is conceivable that cofactor availability and the proportion of cofactor in the active form may play an important role in dictating the succinic acid yield. PntAB genes from Escherichia coli can directly catalyze the reversible hydride transfer and adjust the dynamic balance between NADP(H) and NAD(H). Hence, we studied the physiological effect of coenzyme systems by expressing the membrane-bound transhydrogenase pntAB genes. We have shown experimentally that the pntAB genes could function as an alternative source of NADH. In an anaerobic fermentation with C. glutamicum NC-3-pntAB, a 16% higher succinic acid yield and a 57% higher production from glucose were obtained by pntAB expression. Moreover, the formation of by-products was significantly decreased. The concomitant increase in the consumption of intracellular NADPH from 0.6 to 1.2 mmol/g CDW and the increased NADH/NAD(+) ratio resulted from introduction of pntAB, suggesting that the membrane-bound transhydrogenase converted excess NADPH to NADH for succinic acid production. Finally, we explored whether the transhydrogenase had different effects on the succinic acid formation on different carbon sources. The succinic acid yield was increased in the presence of pntAB by 16% on glucose, 7% on sucrose, and without large influence on fructose and xylose. The results of this study demonstrated that the effectiveness of cofactor manipulation could be a promising strategy applied in metabolic engineering.