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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.