Effects of overexpression of NAPRTase, NAMNAT, and NAD synthetase in the NAD(H) biosynthetic pathways on the NAD(H) pool, NADH/NAD+ ratio, and succinic acid production with different carbon sources by metabolically engineered Escherichia coli

• We constructed five recombinant Escherichia coli strains.
• Different levels of NAD(H) pool size have effect on succinic acid production.
• The high NAD(H) pool size can improve the glucose consumption and cell growth.
• The lowest NADH/NAD+ ratio was obtained in CA102 during anaerobic fermentations.
• Different reductive carbon sources can change the NADH/NAD+ ratio.

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To regulate NAD(H) pool size and NADH/NAD+ ratio, overexpression of the enzymes in the NAD(H) biosynthetic pathways in BA002 was investigated. The results clearly demonstrate that the increased NAD(H) pool size and the decreased NADH/NAD+ ratio improved the glucose consumption and cell growth, which improved succinic acid production. When the pncB and the nadD genes were co-overexpressed in CA102, the ratio of NADH/NAD+ was decreased from 0.60 to 0.12, and the concentration of NAD(H) was the highest among that of all the strains. Moreover, the dry cell weight (DCW), glucose consumption, and the concentration of succinic acid in CA102 were also the highest. Based on the sufficient NAD+ supply after gene modification in the NAD(H) biosynthetic pathways, reductive carbon sources with different amounts of NADH can further change the distribution of metabolites. When sorbitol was used as a carbon source in CA102, the byproducts were lower than those of glucose fermentation, and the yield of succinic acid was increased.