Engineering a Non-Canonical Redox Cofactor System for Biocatalysis

Cofactors such as NAD(P)⁺ are essential reagents in cell-free biomanufacturing. However, they represent a substantial cost in purified enzyme-based biocatalysis. In crude lysate-based biocatalysis, although it is suggested that NAD(P)⁺ present in the crude lysate may replace the need for cofactor supplementation to reduce cost, numerous NAD(P)⁺-dependent enzymes also present in the crude lysate may mediate byproduct formation and reducing power dissipation. Here we report the development of a non-canonical redox cofactor system based on nicotinamide mononucleotide (NMN⁺). NMN⁺ is a simpler and potentially less costly analog of NAD(P)⁺. And we showed that it deliver reducing power in an orthogonal manner to NAD(P)⁺. The enzyme that supplies reducing power in the system is a computationally designed glucose dehydrogenase (GDH) with a 10⁷-fold cofactor specificity switch towards NMN⁺ over NAD(P)⁺. We demonstrated that this system can support diverse redox chemistries in vitro with high total turnover number (~39,000), including reduction of activated C=C double bonds, C≡C triple bonds, and nitro groups, as well as supplying electrons to cytochrome P450. Importantly, we also demonstrated that the protein design principles discovered here are translatable to engineering other key enzymes in cell-free biocatalysis such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in glycolysis, phosphite dehydrogenase, and alcohol dehydrogenase. Furthermore, we established a high-throughput selection platform to enable the engineering of NMN⁺-dependent enzymes in directed evolution. Overall, these results suggest that the non-canonical cofactors may have potential as viable alternatives in cell-free biomanufacturing.