New Glycosyltransferases in Panax notoginseng Perfect Main Ginsenosides Biosynthetic Pathways

Ginsenosides, the main bioactive ingredients of the Panax genus, are dammarane or oleanane triterpenoids with glycosylated modifications at C3/C6/C20 hydroxyls or C28 carboxyl, and their diverse glycosylation pattern has attracted great attention. However, the biosynthesis of some important saponins is still unclear. In this study, six UGTs were characterized, two of which were novel. PnUGT71A3 catalyzes not only the C6 hydroxyl glycosylation of protopanaxatriol (PPT) and F1 to form Rh1 and Rg1, respectively, but also the C20 hydroxyl glycosylation of protopanaxadiol (PPD)-type Rg3 to generate Rd. Especially, PnUGT94M1 is UDP-β-l-rhamnose (UDP-Rha)-dependent, regioselectively catalyzing the C2' hydroxyl rhamnosylation of C6 glucose of the PPT-type ginsenosides Rg1 and Rh1 to generate ginsenosides Re and Rg2, respectively. Site-directed mutagenesis showed that His21, Asp120, Ser363, and Pro372 are key residues, and the triple mutant (G344S/G345S/L346T) highly improved the activity toward Rg1 and Rh1. The findings in this study, perfect main ginsenosides biosynthetic pathways in the Panax genus, expand the biocatalyst toolbox for ginsenoside production and show that the PSPG motif is one of the options to modify UGTs to improve their activities.