Design Glycosyltransferases with High Glycosyl Transfer Efficiency to Efficiently Produce Isoquercetin from Quercetin.

Isoquercetin is a bioactive flavonoid whose biosynthesis from quercetin depends on glycosyltransferases (GTs); however, wild-type GTs exhibit a limited catalytic efficiency. Here, we combined virtual screening, molecular docking, and experimental validation to identify and engineer the GTs. From about 1000 homologous sequences screened via protein BLAST and deep learning-based kcat prediction, the glycosyltransferase MiCGT from Mangifera indica was selected for rational design. After molecular docking and phylogenetic analysis, the engineered variant PCAA (MiCGTS121P/M148C/K253A/S281A) exhibited a 103-fold higher activity than wild-type MiCGT. To enable cost-effective production, sucrose synthase GmSUS was coupled with PCAA to regenerate UDP-glucose during quercetin glycosylation, yielding 3.91 mM isoquercetin with a 78.1% conversion rate. This work overcomes a major bottleneck in isoquercetin biosynthesis and offers a practical strategy for its application in the food industry.