Functional Characterization of UGT90A2 and Its Application in One-Pot Synthesis of Quercetin 3,4'-di-O-β-d-glucoside

Glycosyltransferases enable efficient and environmentally sustainable tailoring of plant natural products into O-glycosides with improved solubility and bioavailability. However, the hydroxyl-site preference of Arabidopsis clade C UDP-glycosyltransferases remains poorly defined, limiting their application in O-glycoside synthesis. Here, UGT90A2, as a representative C-clade UGT, was expressed and shown to efficiently catalyze the glycosylation of quercetin at the 3'-, 4'-, and 7-OH positions. Targeted engineering of the conserved residues K16 and G17 in the active site redirected the preference to the 4'-OH of quercetin, exclusively yielding quercetin 4'-O-β-d-glucoside. Coupling the double mutant with UGT72B1, which glycosylates quercetin 4'-O-β-d-glucoside into quercetin 3,4'-di-O-β-d-glucoside, and sucrose synthase for in situ UDP-Glc regeneration enabled complete glycosylation, producing 1.09 g·L^-1 of quercetin 3,4'-di-O-β-d-glucoside. This study provides an efficient synthetic route for quercetin 3,4'-di-O-β-d-glucoside with industrial production potential and supports its application as a nutritional supplement, plant growth regulator, and UV protector in agriculture.