Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives

Harnessing fungal lignocellulose-degrading enzymes offers sustainable pathways for converting biomass into value-added chemicals. Glyoxal oxidases (GLOX), copper metalloenzymes involved in lignin degradation, can catalyze the oxidation of several furan derivatives into 2,5-furandicarboxylic acid (FDCA), a bioplastic precursor. However, natural GLOX enzymes display limited catalytic activity, necessitating optimization via protein engineering. This study applied directed evolution to the Trametes versicolor glyoxal oxidase gene (tvglox), generating mutants via error-prone PCR and evaluating activity using a high-throughput, colorimetric, agar-based HRP-ABTS assay. In addition, both episomal and genome-integrated expression systems in Pichia pastoris were assessed. While no activity was detected from episomal expression variants, 2.5% of the genome-integrated mutants, along with wild-type controls, exhibited activity on methylglyoxal. Notably, three mutants displayed enhanced activity towards 5-hydroxy-2-furaldehyde carboxylic acid (FFCA), suggesting improved biocatalytic potential for FDCA synthesis. This study represents the first comparison of episomal and genome-integrated GLOX expression in P. pastoris, and highlights a rapid, cost-effective screening platform for furan-active GLOX variants.