Sustainable Bioconversion of Methanol: A Renewable Employing Novel Alcohol Oxidase and Pyruvate Aldolase

Methanol is an ideal one-carbon (C1) feedstock for bioconversion into multicarbon value-added compounds. Biocatalytic approaches to methanol conversion provide sustainable and environmentally friendly alternatives to conventional methods. This process is facilitated by methanol-oxidizing enzymes, including alcohol oxidase (AOx). Here, we report an AOx from <i>Pestalotiopsis fici</i> (PfAOx) with the highest methanol oxidation activity and efficient heterologous expression compared to other AOxs. To investigate the bioconversion of a multicarbon compound (C4 chemical, 2-keto-4-hydroxybutyrate, 2-KHB) from cost-effective methanol, we developed a one-pot enzyme system including PfAOx and pyruvate aldolase from <i>Deinococcus radiodurans</i> (DrADL) with catalase from <i>Bos taurus</i> (BtCAT, commercially available enzyme) to remove toxic H₂O₂. The optimal reaction conditions for 2-KHB production using PfAOx, DrADL, and BtCAT were determined as pH 8.0, 35 °C, 0.9 mg mL^-1 PfAOx, 0.3 mg mL^-1 DrADL, 1.5 mg mL^-1 BtCAT, 150 mM methanol, 100 mM pyruvate, and 5 mM Mg²⁺ with shaking at 200 rpm. Under these reaction conditions, 88.8 mM (10.4 g L^-1) of 2-KHB was produced for 75 min, representing a 74.0-fold higher yield compared to previously reported 2-KHB production systems from methanol and pyruvate. This study demonstrates a promising multi-enzyme cascade approach for the bioconversion of methanol into valuable compounds.