Recently, noncanonical redox cofactors have emerged as a low-cost alternative to natural redox cofactors in enzymatic biotransformation. However, due to the structurally deviant nature of most noncanonical cofactors, enzymes must first be engineered to utilize and regenerate this exclusive electron pool. In this work, we present a high-throughput, directed evolution platform to discover enzyme candidates from mutagenesis libraries with high activity towards the noncanonical redox cofactor nicotinamide mononucleotide, NMN+. By engineering a glutathione reductase to exclusively utilize NMNH, we were able to create a redox cofactor cycling system that can rescue growth when a mutagenesis candidate effectively reduces NMN+ under high oxidative stress. Utilizing this system, we engineered a thermo-stable phosphite dehydrogenase, PTDH, to readily reduce NMN+. Consistent with the strong response shown in the directed evolution platform, the best PTDH mutant exhibited an industrially relevant total turnover number of &gt;45,000 with NMN(H). Curiously, this PTDH mutant also exhibited new activity with a variety of other noncanonical cofactors. We envision this platform as high-throughput tool to expand the scope of accessible chemistries and productivity in NMN(H)-based biotransformation systems.

Directed evolution platform for engineering noncanonical redox cofactor-dependent enzymes

ACS Spring 2022

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