Protein Engineering of PhUGT, a Donor Promiscuous Glycosyltransferase, for the Improved Enzymatic Synthesis of Antioxidant Quercetin 3---Acetylgalactosamine.

Quercetin 3---acetylgalactosamine (Q3GalNAc), a derivative of dietary hyperoside, had never been enzymatically synthesized due to the lack of well-identified -acetylgalactosamine-transferase (GalNAc-T). Herein, PhUGT, an identified flavonoid 3--galactosyltransferase from , was demonstrated to display quercetin GalNAc-T activity, transferring a -acetylgalactosamine (GalNAc) from UDP--acetylgalactosamine (UDP-GalNAc) to the 3-OH of quercetin to form Q3GalNAc with a low conversion of 11.7% at 40 °C for 2 h. Protein engineering was thus performed, and the resultant PhUGT variant F368T got an enhanced conversion of 75.5% toward UDP-GalNAc. The enzymatically synthesized Q3GalNAc exhibited a comparable antioxidant activity with other quercetin 3--glycosides. Further studies revealed that PhUGT was a donor promiscuous glycosyltransferase (GT), recognizing seven sugar donors. This finding overturned a previous notion that PhUGT exclusively recognized UDP-galactose (UDP-Gal). The reason why PhUGT was mistaken for a UDP-Gal-specific GT was demonstrated to be a shorter reaction time, in which many quercetin 3--glycosides, except hyperoside, could not be effectively synthesized. The fact that the microbial cell factory expressing PhUGT could yield an array of Q3Gs further confirmed the donor promiscuity of PhUGT. This study laid a foundation for the scale production of Q3GalNAc and provided a potent biocatalyst capable of glycodiversifying quercetin as well.