Shifting redox reaction equilibria on demand using an orthogonal redox cofactor

Area of Science:

• Biotechnology and Synthetic Biology
• Enzyme Engineering
• Metabolic Engineering

Background:

• Nature utilizes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) as primary redox cofactors, with distinct reduction potentials driving catabolism and anabolism.
• Biomanufacturing requires flexible control over redox reaction direction, independent of native metabolic pathways.

Purpose of the Study:

• To establish nicotinamide mononucleotide (NMN+) as a noncanonical cofactor orthogonal to NAD(P)+ for decoupled redox control.
• To develop a toolkit for modulating the NMN+/NMNH ratio, enabling novel biocatalytic applications.

Main Methods:

• Engineered a reduced NMN+ (NMNH)-specific oxidase (Nox Ortho) and an NMN+-specific glucose dehydrogenase (GDH Ortho).
• Applied enzyme engineering and modeling principles to create NMN(H)-orthogonal biocatalysts with high cofactor specificity.
• Assembled engineered enzymes for producing 2,3-butanediol in cell-free systems and Escherichia coli.

Main Results:

• Developed NMN(H)-orthogonal biocatalysts exhibiting a ~10^3-10^6-fold cofactor specificity switch from NAD(P)+ to NMN+.
• Successfully produced stereo-pure 2,3-butanediol using the engineered system, demonstrating decoupled redox control.
• Established a robust toolkit for precise manipulation of the NMNH:NMN+ ratio.

Conclusions:

• Nicotinamide mononucleotide (NMN+) serves as a versatile, noncanonical cofactor for orthogonal redox control in biomanufacturing.
• The developed NMN(H)-orthogonal biocatalysts and toolkit offer unprecedented flexibility in designing synthetic metabolic pathways.
• This approach decouples redox ratios from native NAD(H) and NADP(H) pools, paving the way for advanced bioproduction strategies.