Engineering a Formate Dehydrogenase for NADPH Regeneration**

Abstract Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio‐transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD + ‐dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP + ‐utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis ( Cd FDH), which showed no strict NAD + preference by a structure‐guided rational/semi‐rational design. A combinatorial mutant Cd FDH‐M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75‐fold intensification of catalytic efficiency ( k cat / K m ). Moreover, Cd FDH‐M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH‐required biocatalytic transformations.