Engineering Candida boidinii formate dehydrogenase for activity with NMN(H)

Multi-step enzymatic reaction cascades often involve cofactors that serve as electron donors/acceptors in addition to the primary substrates. The co-localization of cascades can lead to cross-talk and competition, which can be unfavorable for the production of a targeted product. Orthogonal pathways allow reactions of interest to operate independently from the metabolic reactions within a cell; non-canonical cofactor analogs have been explored as a means to create these orthogonal pathways. Here, we aimed to engineer the formate dehydrogenase from Candid boidinii (CbFDH) for activity with the non-canonical cofactor nicotinamide adenine mononucleotide (NMN(H)). We used PyRosetta and structural alignment to design mutations that enable CbFDH to use NMN ⁺ for the oxidation of formate. Although the suggested mutations did not result in enhanced activity with NMN ⁺ , we found that PyRosetta was able to easily design single mutations that disrupted all enzymatic activity.