Iterative computational bioprospecting of hydroxymethylfurfural oxidases combining molecular simulation and machine learning

The enzymatic oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) is a key step in the sustainable production of polyethylene furanoate (PEF), yet the final oxidation of 5-formyl-2-furancarboxylic acid (FFCA) to FDCA remains the principal bottleneck for industrial application. Although hydroxymethylfurfural oxidases (HMFOs) can catalyze this transformation, naturally occurring enzymes with high FFCA-oxidizing activity remain largely unexplored. Here, we present an iterative computational bioprospecting campaign to identify natural HMFOs with improved activity toward this rate-limiting reaction. Across four successive rounds, the computational workflow evolved from conservative homology-based screening to large-scale sequence mining assisted by machine learning, with each iteration redesigned according to the principal limitations identified through molecular simulations and experimental characterization. This adaptive strategy led to the discovery of several natural HMFOs with substantially improved FFCA oxidation activity, including candidates whose performance under the tested conditions approached that of the engineered benchmark 8BxHMFO. Beyond the identification of promising biocatalysts, the study demonstrates the value of continuous integration of computational prediction and experimental feedback for refining enzyme discovery campaigns, and provides practical insights into balancing catalytic prioritization, sequence diversity, developability, and computational scalability in future simulation-guided bioprospecting efforts.