Engineering Nicotinamide Adenine Dinucleotide Oxidase for Regeneration of Oxidized Non‐natural Cofactor

Nicotinamide adenine dinucleotide (NAD⁺) and its reduced form NADH are universal redox cofactors, and thus manipulating NAD(H) supply often leads to unpredictable outcome because of metabolic crosstalk. To overcome intrinsic limitations associated to natural cofactors, this study previously introduces nicotinamide cytosine dinucleotide (NCD⁺) as a non-natural cofactor for redox biochemistry. While several enzymes have been devised to generate NCDH as driving force at the expense of cheap chemicals for reductive metabolic reactions, it remains inaccessible to generate NCD⁺ for oxidative reactions. In this study, it engineers an H₂O-forming NADH oxidase (EfNOX) from Enterococcus faecalis to favor NCDH. Compared to the wild-type enzyme, the best mutant NADH oxidase (NOX)-KRGT oxidizes NCDH with 14- and 107-fold higher catalytic efficiency and selectivity, respectively. Docking analysis shows that those mutations acquired a narrower cofactor binding cavity and positively charged environment contributing to the preference toward NCDH. Coupling NOX mutants with NCD-favoring phosphite dehydrogenase mutant enables Escherichia coli BW14329 to utilize phosphite as sole phosphorus source for growth. This work provides a traceless and effective tool to convert NCDH into NCD⁺, which should greatly expand our capacity in developing NCD-linked redox subsystems and further facilitate the implementation of non-natural cofactors in chemical biology and synthetic biology.