A new UDP-glycosyltransferase for rare ginsenoside biosynthesis from Gynostemma pentaphyllum (Thunb.)

Heterologous biosynthesis of ginsenosides, which possess remarkable therapeutic potential as drug candidates, is currently a research hotspot. The insufficient mining of UDP-glycosyltransferases (UGTs), which are key downstream enzymes in their biosynthetic pathway, limits the variety and yield of ginsenosides that can be bio-produced. As the only medicinal plant outside the Araliaceae family currently discovered to contain ginsenosides, the enzymes involved in ginsenoside synthesis in the Cucurbitaceae plant Gynostemma pentaphyllum (Thunb.) have great development value. In this study, a new glycosyltransferase was mined from G. pentaphyllum and was classified into the UGT74 family based on sequence homology, named GpUGT74A1. GpUGT74A1 exhibits relatively low sequence homology compared to other UGTs that have been reported. Although GpUGT74A1 was almost entirely insoluble when cloned and expressed in Escherichia coli, its soluble expression was successfully achieved by fusion with the MBP solubility tag. In vitro enzyme activity experiments found that it could catalyze the C-20 glycosylation of ginsenosides PPD, PPT, and Rh₂ to produce ginsenosides CK, F₁, and F₂, respectively. This study further enriched the sequence of plant-derived glycosyltransferase genes and provided new candidate elements for the heterologous synthesis of rare ginsenosides.