Switching the Cofactor Preference of Formate Dehydrogenase to Develop an NADPH-Dependent Biocatalytic System for Synthesizing Chiral Amino Acids

Efficient formate dehydrogenase (FDH)-based cofactor regeneration systems are widely used for biocatalytic processes due to their ready availability, low reduction potential, and production of only benign byproducts. However, FDHs are usually specific to NAD⁺, and NADPH regeneration with formate is challenging. Herein, an FDH with a preference for NAD⁺ from Azospirillum palustre (ApFDH) was selected owing to its high activity. By static and dynamic structural analyses, a beneficial substitution, D222Q, was identified for cofactor-preference switching. However, its total activity was substantially decreased by 90% owing to the activity-specificity trade-off. Subsequently, a semirational library was designed and screened, which yielded a variant ApFDH^{D222Q+A199G+H380S} with satisfactory activity and NADP⁺ specificity. Our analysis of dynamical cross-correlations revealed a substitution combination that brought balance to the dynamical correlation network. This combination successfully overcame the activity-specificity-stability trade-off and resulted in a beneficial outcome. The substitution combination (D222Q-A199G/H380S-C256A/C146S) enabled the simultaneous improvement of activity, specificity, and stability and was successfully applied to other 17 FDHs. Finally, by employing engineered ApFDH, an NADPH regeneration system was developed, optimized, and utilized for the asymmetric biosynthesis of l-phosphinothricin.