[Cofactor preference engineering of meso-diaminopimelate dehydrogenase and its application in d-phenylalanine synthesis].

d-phenylalanine is an important d-amino acid with a wide range of applications in the pharmaceutical and chemical industries. meso-diaminopimelate dehydrogenase (DAPDH) catalyzes the asymmetric reductive amination of α-keto acids to produce d-amino acids with high optical purity, serving as a key enzymatic module for constructing multi-enzyme cascade systems. However, the strict dependence of wild-type DAPDH on NADPH results in high cofactor costs, limiting its industrial application. To address this issue, this study focused on a previously engineered yet still NADPH-preferring type I DAPDH from Corynebacterium glutamicum (CgDAPDH). Based on structural analysis of the protein, a three-pronged engineering strategy involving steric hindrance reduction, local electrostatic potential modulation, and conformational flexibility enhancement was proposed. Through site-directed and combinatorial mutagenesis of residues at the cofactor-binding pocket entrance (Ser35, Arg36, Arg37) and conserved regions (Ser68, Thr88, Asp120), a mutant S35E/R36E/R37A/S68T/T88A was obtained. This mutant exhibited a catalytic efficiency (kcat/Km) of 274.87 mmol/(L·s) toward NADH, representing a 16.9-fold improvement over the starting enzyme, while achieving comparable catalytic competence with both NADH and NADPH. This mutant was coupled with formate dehydrogenase (FDH) to construct an NADH self-regeneration system for whole-cell catalytic reductive amination using phenylpyruvate as the substrate. After 6 h of reaction, the titer of d -phenylalanine reached 36.22 g/L with a conversion rate of 90.12%. When this system was applied to three-enzyme cascade employing l-phenylalanine as the substrate, the d-phenylalanine titer reached 16.25 g/L, which was comparable to that obtained with a glucose dehydrogenase/NADPH regeneration system (17.21 g/L). This study demonstrates that cofactor engineering can alleviate the NADPH dependence of DAPDH and achieve efficient conversion of phenylpyruvate to d-phenylalanine, thereby providing a new enzymatic module and a technical route for cost-effective biomanufacturing of d-amino acids.