Directed evolution of shikimate dehydrogenase to improve non-natural cofactor preference

The shikimate dehydrogenase (SDH)-catalyzed reduction of 3-dehydroshikimate represents a crucial step in the shikimate pathway for aromatic amino acids and related metabolites. As SDH catalyzed reaction depends on reduced nicotinamide adenine dinucleotide phosphate, it is considered as an attractive target for cofactor engineering to regulate the metabolic flux of the shikimate pathway. In this study, we reprogrammed the cofactor preference of Escherichia coli SDH (EcSDH) to create mutants that utilize the non-natural cofactor nicotinamide cytosine dinucleotide (NCD). Our best-performing mutant, EcSDH_NAD-M173N/S193E, displayed a remarkable 2.1 × 10⁴-fold switch in cofactor preference from NADP to NCD. Results demonstrated that the altered cofactor preference was primarily contributed by steric constraints within the cofactor-binding pocket and molecular interactions with the cytosine moiety. The EcSDH mutant efficiently catalyzed the reduction of 3-dehydroshikimate to shikimate by using NCDH produced in situ by NCD-dependent formate dehydrogenase. This work offers a new biopart to develop synthetic metabolism for the production of shikimate pathway-derived compounds.