Engineering of cofactor preference and catalytic activity of methanol dehydrogenase by growth-coupled directed evolution

Methanol, produced from carbon dioxide, natural gas, and biomass, has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties. Nicotinamide adenine dinucleotide (NAD⁺)-dependent methanol dehydrogenase (MDH) catalyzes the oxidation of methanol to formaldehyde via NADH generation, providing a highly active C1 intermediate and reducing power for subsequent biosynthesis. However, the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization, especially in nicotinamide adenine dinucleotide phosphate (NADP⁺)-dependent biosynthesis. Herein, we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM 2334. NADH or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs, establishing a positive correlation between the cell growth rate and MDH activity. Using this principle, MDH mutants exhibiting a 20-fold improvement in catalytic efficiency (k_cat/K_m) and a 90-fold cofactor specificity switch from NAD⁺ to NADP⁺ without a decrease in specific enzyme activity, were efficiently screened from random and semi-rationally designed libraries. We envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)⁺-dependent enzymes.