Shifting redox reaction equilibria on demand using an orthogonal redox cofactor.

Nature's two redox cofactors, nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ), are held at different reduction potentials, driving catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction decoupled from catabolism and anabolism. We established nicotinamide mononucleotide (NMN + ) as a noncanonical cofactor orthogonal to NAD(P) + . Here we present the development of Nox Ortho, a reduced NMN + (NMNH)-specific oxidase, that completes the toolkit to modulate NMNH:NMN + ratio together with an NMN + -specific glucose dehydrogenase (GDH Ortho). The design principle discovered from Nox Ortho engineering and modeling is facilely translated onto six different enzymes to create NMN(H)-orthogonal biocatalysts with a consistent ~10 3 -10 6 -fold cofactor specificity switch from NAD(P) + to NMN + . We assemble these enzymes to produce stereo-pure 2,3-butanediol in cell-free systems and in Escherichia coli, enabled by NMN(H)'s distinct redox ratio firmly set by its designated driving forces, decoupled from both NAD(H) and NADP(H).