Abstract
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).