Structure-Guided Stepwise Engineering of Hydroxymethylfurfural Oxidase Enables HRP- and Cu-Free Cascade Oxidation to a Cefuroxime Side-Chain Precursor

Abstract 2-(Furan-2-yl)-2-oxoacetic acid (2-FOA) is a key precursor for constructing the C7 side chain of cefuroxime. Existing biocatalytic routes depend on a copper-dependent galactose oxidase system requiring horseradish peroxidase (HRP) for activation. To expand biocatalytic alternatives, we characterized FAD-dependent 5-hydroxymethylfurfural oxidase from Methylovorus sp. MP688 (MvHMFO), whose redox cycle eliminates demands for exogenous HRP and metal cofactors. However, MvHMFO showed low activity toward the non-natural substrate 2-furyl hydroxymethyl ketone (2-FHMK). Herein, we adopted a three-stage structure-guided stepwise engineering strategy by sequentially evaluating and combining substitutions from the catalytic core to peripheral loops, achieving cavity expansion and local hydration network reorganization. The optimal variant M4 showed 75.2-fold and 132.3-fold improvements in catalytic efficiency toward 2-FHMK and its aldehyde intermediate, respectively. Applying M4 in an elevated-temperature (40 °C) fed-batch process yielded 6.2 g/L of 2-FOA with a conversion of 88%. This work provides an HRP- and Cu-free HMFO platform for 2-FOA synthesis and an engineering framework for adapting oxidoreductases to non-natural substrates.