Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-alcohol oxidase

Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.