Derek Aspacio, Yulai Zhang, Youtian Cui, Edward King, William B Black, Sean Perea, Emma Luu, Justin B Siegel, Han Li
bioRxiv : the preprint server for biology 2023 Aug 30Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+ is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries. Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103 - 106 -fold cofactor specificity switch from NAD(P)+ to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and in E. coli cells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.
Derek Aspacio, Yulai Zhang, Youtian Cui, Edward King, William B Black, Sean Perea, Emma Luu, Justin B Siegel, Han Li. Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor. bioRxiv : the preprint server for biology. 2023 Aug 30
PMID: 37693387
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