Region-specific engineering of a bifunctional alcohol dehydrogenase enables efficient single-enzyme sequential...

Efficient biocatalytic oxidation of primary alcohols to carboxylic acids is attractive for sustainable synthesis, but complete alcohol-to-acid conversion usually requires coordinated control of sequential oxidation steps and continuous regeneration of oxidized cofactors. Here, we report a region-specific engineering strategy to improve GtADH2, a bifunctional NAD⁺-dependent alcohol dehydrogenase capable of catalyzing single-enzyme sequential oxidation of terminal hydroxy fatty acids. Guided by a GtADH2-12-hydroxydodecanoic acid-NAD⁺ ternary complex model, the substrate-binding pocket and NAD⁺-binding region were separately engineered and then combinatorially integrated. The resulting variant M4-1 (N147T/N8Q/S274D/D268Q) exhibited a 10.2-fold increase in activity and an approximately 17.3-fold improvement in catalytic efficiency toward 12-hydroxydodecanoic acid compared with the wild type. Kinetic analysis showed that M4-1 improved both substrate utilization and NAD⁺-dependent catalytic efficiency, while molecular dynamics simulations supported enhanced NAD⁺ stabilization and more favorable hydride-transfer geometry. To sustain sequential oxidation under low cofactor loading, M4-1 was integrated with a flavinium-assisted NAD⁺ recycling module. Using only 0.2 mM NAD⁺, the M4-1/FocH system converted 2 mM 12-hydroxydodecanoic acid to 1.96 mM dodecanedioic acid within 2 h. M4-1 also showed improved activity toward terminal hydroxy fatty acids with different chain lengths. This work demonstrates that region-specific engineering of substrate- and cofactor-associated regions, combined with flavinium-assisted cofactor recycling, provides an effective single-enzyme platform for NAD⁺-dependent alcohol-to-acid oxidation.