Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l -Lysine Production

Abstract

ABSTRACT A sufficient supply of NADPH is a critical factor in l -lysine production by Corynebacterium glutamicum . Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans , which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP + to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l -lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l -lysine producer was evaluated. The amounts of l -lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l -lysine production was attributed to GapN. These results demonstrate effective l -lysine production by C. glutamicum with an additional source of NADPH during glycolysis.