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 Pestalotiopsis fici (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 Deinococcus radiodurans (DrADL) with catalase from Bos taurus (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.