Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor

Area of Science:

• Biochemistry
• Synthetic Biology
• Metabolic Engineering

Background:

• Cellular metabolism relies on NAD+ and NADP+ redox cofactors, limiting flexible control of reaction direction.
• Existing cell-free systems face cost limitations with NADP+ for large-scale applications.
• Nicotinamide mononucleotide (NMN+) offers a cost-effective, noncanonical alternative for redox reactions.

Approach:

• Engineered six butanediol dehydrogenases to be NMN(H)-orthogonal biocatalysts.
• Achieved a 10^3-10^6 fold cofactor specificity switch from NAD(P)+ to NMN+.
• Developed Nox Ortho, an NMNH-specific water-forming oxidase, to modulate the NMN(H)/NMN+ ratio.

Key Points:

• Demonstrated parallel engineering of enzymes for NMN+-specific biocatalysis.
• Successfully produced chiral-pure 2,3-butanediol isomers in vitro and in E. coli.
• Established independent control of the NMN(H)/NMN+ redox ratio, decoupled from NAD(H) and NADP(H).

Conclusions:

• NMN+ serves as a versatile and cost-effective redox cofactor for biocatalysis.
• Enzyme engineering enables precise cofactor specificity switching for targeted metabolic control.
• This toolkit provides flexible redox manipulation for cell-free and in vivo applications.