Identification and Characterization of Key Enzymes Involved in UDP-Sugar Donor Synthesis and Flavonoid Glycosylation from

Glycosylation, an effective strategy for enhancing the bioavailability and stability of dietary flavonoids, has primarily focused on developing enzyme resources from bacteria and plants. However, exploration of UDP glycosyltransferases (UGTs) from fungi remains insufficient. An in-depth study of fungal glycosyltransferases will enhance our understanding of the potential benefits of glycosylation in fungi and expand the range of available biocatalysts for glycosylation. In this study, we identified three key enzymes from Trametes hirsuta: UDP-glucose dehydrogenase (ThUGDH), UDP-xylose synthase (ThUXS), and UDP-flavonoid glycosyltransferase (Th3GT). These enzymes are involved in the synthesis of UDP sugar donors and the glycosylation of quercetin, efficiently converting it into UDP-GlcA, UDP-Xyl, quercetin-3-O-β-d-glucoside (Q3G), and quercetin-3-O-β-d-xylopyranoside (Q3X). Through mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, we confirmed the structures of these compounds. We employed a simplified purification process to obtain high-purity sugar donors and quercetin glycosides. Notably, glycosylation reduces the cytotoxicity of quercetin while improving its capacity to scavenge reactive oxygen species (ROS). In summary, this study not only enriches the knowledge framework of fungal UGTs but also provides valuable enzyme resources for industrial enzyme-catalyzed synthesis of UDP sugar donors and flavonoid glycosides.