Characterization and Engineering of Multifunctional Glycosyltransferase HtUGT73EW3 for the Highly Efficient Synthesis of Flavonoid Mono/di-<i>O</i>-glycosides

Glycosylation modification is an effective way to improve the solubility, stability, and bioavailability of flavonoids. In this study, a multifunctional flavonoid glycosyltransferase HtUGT73EW3 was identified from <i>Helleborus thibetanus</i>. HtUGT73EW3 exhibited multisite selectivity for 3-, 6-, 7-, 2'-, 3'-, and 4'-OH of flavonoids and showed potent 3/3'-, 3/4'-, and 7/4'-di-<i>O</i>-glycosylation activity. HtUGT73EW3 was able to glycosylate structurally diverse flavonoid aglycones and monoglycosides, and showed efficient glycosylation capacity toward flavonoid structures modified with functional groups at the C-3, C-7, C-8, and C-4' positions. Notably, the mutation of Gln85 to Leu greatly enhanced its catalytic activity, enabling not only the conversion of steroids and terpenoids, but also the improved utilization of UDP-sugars. Furthermore, the Q85L and I94A variants were found to catalyze specific 7,4'- and 3,4'-di-<i>O</i>-glycosylation, respectively. A cost-effective one-pot synthetic reaction was established by coupling AtSuSy and HtUGT73EW3, and the gram-scale synthesis of flavonoid 4'-<i>O</i>-glucoside and 3,4'-/7,4'-di-<i>O</i>-glucoside was achieved by a fed-batch strategy.