Engineering a broad-spectrum glucose oxidase via substrate channel and linker design for enhanced lignocellulose bioconversion.

Glucose oxidase (GOD) is a widely used enzyme in biotechnology, yet its narrow substrate specificity limits its application in complex bioconversion processes such as agricultural waste valorization. In this study, we employed synthetic biology and protein engineering strategies to engineer a broad-spectrum glucose oxidase from Aureobasidium sp. (AreGOD). Initially, site-directed mutagenesis at N82, a key gatekeeper at the dimer interface, modulated substrate channel geometry, leading to increased catalytic activity towards various sugars, particularly stachyose and xylose. Furthermore, systematic linker engineering between the spore anchor protein CotG and AreGOD revealed that flexible linkers, particularly the (GGGGS)5 repeat (LK3), dramatically expanded the enzyme's substrate spectrum towards various mono-, di-, and oligosaccharides. The optimized spore-displayed AreGOD (CotG-LK3-AreGOD) exhibited strong synergistic effects with cellulase in wheat straw degradation, significantly enhancing the hydrolysis of cellulose, hemicellulose, and lignin. Our work demonstrates an effective and generalizable strategy for engineering substrate-promiscuous oxidases, highlighting the potential of integrative enzyme design for sustainable bioprocessing and agricultural biotechnology.