Mining of Highly Regioselective Carboxyl Glycosyltransferases and Their Application in Biosynthesizing Triterpenoid Glycosides

Pentacyclic triterpenoids represent important natural products with wide applications as food additives, sweeteners, and dietary supplements. Recently, carboxyl glycosyltransferases (CLGTs) have received extensive attention due to their special role in glycosylating pentacyclic triterpenoids at the carboxyl group for function enhancement. However, the understanding of CLGTs has been largely underexplored. In this study, three CLGTs UGT73BR4, UGT73CH11, and UGT73N3 were identified by a phylogeny-based mining strategy from Olea europaea, which exhibited high regioselectivity toward the carboxyl of 10 terpenoids. UGT73BR4 also recognized diverse nucleotide sugar donors to facilitate multiple ester glycoside biosynthesis. Then, the pH profile of UGT73BR4 was shifted to the acidic region through protein engineering, which allowed for construction of an efficient UDP-Glc regeneration system by coupling with sucrose synthase. With this enzyme cascade, betulinic acid was in vitro glycosylated to produce 28-O-glc-betulinic acid with a 95% theoretical UDP recycling efficiency. Finally, the in vivo biosynthesis of 28-O-glc-betulinic acid was achieved in engineered yeast.