Overcoming NADPH product inhibition improves D-sorbitol conversion to L-sorbose.

Gluconobacter oxydans sorbitol dehydrogenase (GoSLDH) exhibits a higher catalytic efficiency than other L-sorbose producing enzymes. During the reaction catalysed by GoSLDH, NADP is reduced to NADPH and D-sorbitol is oxidized to L-sorbose. However, GoSLDH activity is inhibited by the NADPH (K = 100 μM) formed during the enzymatic reaction. Therefore, Escherichia coli producing both GoSLDH for D-sorbitol oxidation and LreNOX (NAD(P)H oxidase from Lactobacillus reuteri) for NADP regeneration was generated and used for L-sorbose production. Whole cell biocatalysts with the LreNOX cofactor recycling system showed a high conversion rate (92%) of D-sorbitol to L-sorbose in the presence of low concentration of NADP (0.5 mM). By alleviating NADPH accumulation during the catalytic reactions, E. coli exhibited 23-fold higher conversion rate of D-sorbitol than E. coli. L-Sorbose production by E. coli reached 4.1 g/L after 40 min, which was 20.5-fold higher than that of E. coli. We also constructed G. oxydans and G. oxydans strains, and they exhibited 1.2- and 2.9-fold higher conversion rates than the wild-type G. oxydans KCTC 1091. The results indicate that overcoming NADPH product inhibition using LreNOX improves chemical production in NADP-dependent enzymatic reactions.