(309g) Invited Talk: Engineering Redox Chemistry in Biocatalysis

In Natural metabolism, redox cofactors NAD and NADP are insulated from each other to enable individual control of catabolism and anabolism. However, in purified enzyme-based biomanufacturing, NADP is avoided due to its formidable cost and low stability. This leaves NAD as the only electron carrier which cannot efficiently support both oxidation and reduction simultaneously without forming a futile cycle. Secondly, in whole cell-based biomanufacturing, NAD(P)/H are used by numerous natural enzymes resulting in byproduct formation. Here, we demonstrate the utilization of a cost-effective, artificial cofactor, nicotinamide mononucleotide (NMN), to tackle both challenges. We engineered a full set of enzymes to reduce or oxidize NMN, respectively, as well as using NMN reducing power to perform diverse industrially important chemistries, and to shift redox equilibrium on demand. More importantly, we leveraged evolution to discover enzymes that can utilize NMN as well as other simpler artificial cofactors such as 1-benzylnicotinamide (BNA) and 1-Butylnicotinamide (BuNA). Surprisingly, the general design principles emerged from these efforts suggest that mutations are required to mimic the larger, natural cofactors NAD(P), in modulating the enzymes' conformational dynamics, rather than directly interacting with the artificial cofactors. In summary, although natural evolution has only explored NAD and NADP as two universal redox cofactors in all life, we demonstrate that an expanded toolkit of artificial redox cofactors can be developed in laboratory evolution for efficient biocatalysis.