Evolution of alcohol oxidase for improved methanol bioconversion and formaldehyde tolerance

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Synthetic Biology

Background:

• Methanol oxidation by alcohol oxidases (AOXs) is crucial for one-carbon (C1) bioconversion but is limited by low catalytic efficiency and formaldehyde toxicity.
• Developing robust AOXs is essential for advancing C1 chemical synthesis and sustainable biomanufacturing.

Purpose of the Study:

• To improve the catalytic efficiency and formaldehyde tolerance of alcohol oxidase from *Gloeophyllum trabeum* (GtAOX) for enhanced methanol bioconversion.
• To engineer a variant enzyme with superior performance characteristics for industrial applications.

Main Methods:

• Directed evolution was employed to generate mutant libraries of the *Gloeophyllum trabeum* alcohol oxidase.
• High-throughput screening was used to identify variants with improved methanol oxidation activity and formaldehyde tolerance.
• Molecular dynamics simulations were performed to elucidate the structural basis for enhanced enzyme performance.

Main Results:

• An engineered variant, GtAOXM3, was obtained with a sixfold increase in catalytic efficiency (7.7 s⁻¹ mM⁻¹).
• The GtAOXM3 variant demonstrated enhanced formaldehyde tolerance, thermostability, and methanol specificity compared to the wild-type enzyme.
• Incorporation of GtAOXM3 into multienzyme cascade systems facilitated efficient conversion of methanol to dihydroxyacetone (34.5 mM) and ethylene glycol (23.3 mM).

Conclusions:

• GtAOXM3 represents a significantly improved biocatalyst for methanol oxidation, overcoming key limitations of native AOXs.
• The engineered enzyme offers a cost-effective and efficient solution for methanol-based C1 biotransformation, paving the way for sustainable chemical production.
• This work highlights the power of directed evolution and computational simulations in designing enzymes for industrial biotechnology.