Evolution of alcohol oxidase for improved methanol bioconversion and formaldehyde tolerance.

Methanol oxidation by alcohol oxidases (AOXs) is a key bottleneck in one-carbon (C1) bioconversion due to limited catalytic efficiency and poor formaldehyde tolerance. Here, we report the directed evolution of an alcohol oxidase from Gloeophyllum trabeum, yielding an optimized variant, GtAOXM3. The engineered enzyme exhibits a sixfold increase in catalytic efficiency (7.7 s-1 mM-1), together with enhanced formaldehyde tolerance, thermostability, and high methanol specificity. Molecular dynamics simulations suggest that increased global rigidity and cooperative residue dynamics contribute to the improved performance. When incorporated in multienzyme cascade systems, GtAOXM3 enables efficient conversion of methanol to the value-added chemicals dihydroxyacetone (34.5 mM) and ethylene glycol (23.3 mM). This work establishes GtAOXM3 as an efficient and cost-effective biocatalyst for methanol-based C1 biotransformation.