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Abstract Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN⁺) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN⁺-based reducing power. This is based on an orthogonal, NMN⁺-dependent glycolytic pathway inEscherichia coliwhich can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN⁺. With a throughput of >10⁶variants per iteration, the growth selection discovers aLactobacillus pentosusNADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes. 

Abstract Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase inEscherichia colito exclusively rely on the reduced NMN⁺(NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA⁺), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)⁺. 

Abstract Background Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes. Results In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN + ), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN + biosynthetic pathways in E. coli . Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN + , a 130-fold increase over the cell’s basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN + accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor. Conclusion These results further the understanding of effective production and integration of NMN + into E. coli . This may enable the implementation of NMN + -directed biocatalysis without the need for exogenous cofactor supply.