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Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor
- PMID: 37693387
- PMCID: PMC10491207
- DOI: 10.1101/2023.08.29.555398
Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor
Update in
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Shifting redox reaction equilibria on demand using an orthogonal redox cofactor.Nat Chem Biol. 2024 Nov;20(11):1535-1546. doi: 10.1038/s41589-024-01702-5. Epub 2024 Aug 13. Nat Chem Biol. 2024. PMID: 39138383
Abstract
Natural 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.
Keywords: Escherichia coli; Nicotinamide mononucleotide; biomimetic cofactor; butanediol; cell-free biomanufacturing; noncanonical redox cofactor; water-forming NADH oxidase (NOX).
Conflict of interest statement
The authors declare no competing financial interests.
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References
-
- Cresko J.; Rightor E.; Carpenter A.; Peretti K.; Elliott N.; Nimbalkar S.; Morrow W. R.; Hasanbeigi A.; Hedman B.; Supekar S.; McMillan C.; Hoffmeister A.; Whitlock A.; Lgogo T.; Walzberg J.; D’Alessandro C.; Anderson S.; Atnoorkar S.; Upsani S.; King P.; Grgich J.; Ovard L.; Foist R.; Conner A.; Meshek M.; Hicks A.; Dollinger C.; Liddell H. Industrial Decarbonization Roadmap; 2022.
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