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. 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 NADPH-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 (kcat/Km) 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.