(117b) Orthogonal Glycolytic Pathway Enables Directed Evolution of Noncanonical Cofactor Oxidase

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN⁺) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. We established a self-sufficient growth selection to evolve enzymes that can utilize NMN⁺-based reducing power. This is based on an orthogonal, NMN⁺-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN⁺ (NMNH). With a throughput of >10⁶ variants per iteration, the growth selection discovered a NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Importantly, molecular modeling and experimental validation suggest that the mutations, instead of directly contacting NCBs, optimize the enzyme's global conformational dynamics to resemble that with the native cofactor bound. Thus, restoring the enzyme’s conformation states, through deep navigation of protein sequence space by high-throughput evolution, provides a universal route to engineer NCB-dependent enzymes.