The meso‑diaminopimelate dehydrogenase (DapDH) is crucial for efficiently biosynthesizing L-lysine. In this study, a NADH-dependent DapDH was created and was used to increase L-lysine production in Corynebacterium glutamicum. To do this, the critical amino acid residues for switching cofactor preference in DapDH from C. glutamicum (CgDapDH) were investigated by bioinformatics analysis and alanine-scanning mutagenesis and indicated that three residues (Tyr11, Arg36 and Arg37) showed the critical roles in changing the cofactor preference. Subsequently, these residues were mutated by site-saturation mutagenesis and indicated that variants CgDapDHY11K, CgDapDHR36E and CgDapDHR37E showed an increased activity using NAD+ as cofactor. Moreover, the best variant CgDapDHY11K/R36E with enhanced affinity for NAD+ (Km = 2.3 ± 0.3 mM) and specific activity (406 ± 28 mU/(mg protein)) was obtained after combinatorial mutagenesis. The results of L-lysine fermentation showed that the resultant strain XQ-7 with overexpression of CgDapDHY11K/R36E increased the final titer (from 186.7 ± 5.6 g/L at 72 h to 191.5 ± 10.7 g/L at 60 h), carbon yield (from 0.53 g/(g glucose) to 0.61 g/(g glucose)) and average space-time yield (from 2.59 g/(L∙h) to 3.19 g/(L∙h)) compared with the original strain XQ-6 in fed-batch fermentation. This is the first report of an artificial NADH-dependent CgDapDH that increases L-lysine production because of decreasing the requirement of NADPH.
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