Ginsenoside accumulation and enzyme functional characterization of zingibroside R1 and ginsenoside Ro biosynthesis in Panax zingiberensis.

Panax zingiberensis is rich in oleanane-type ginsenosides and has gained significant attention as a kind of valuable traditional Chinese medicine. However, the biosynthesis of ginsenosides in P. zingiberensis, particularly the downstream glycosylation pathway, remains largely unexplored. The accumulations of ginsenoside Rg₁ (G-Rg₁), ginsenoside Rb₁ (G-Rb₁), ginsenoside Ro (G-Ro), chikusetsusaponin IVa (C-IVa), and chikusetsusaponin IV (C-IV) were quantified in different tissues of 2-year-old and 4-year-old P. zingiberensis. The results indicated that these ginsenosides primarily accumulated in the underground part, with higher concentrations found in the biennial of P. zingiberensis. Transcriptome sequencing revealed that genes related to ginsenoside accumulation were differentially expressed in different tissues in different years of P. zingiberensis. Weighted correlation network analysis (WGCNA) also identified some genes extremely related to ginsenoside synthesis. Notably, we identified PzUGT2, a key gene in the downstream glycosylation step of ginsenoside biosynthesis. In vitro enzymatic assays demonstrated that it catalyzes the reaction of oleanolic acid 3-O-β-D-glucuronide and C-IVa with UDP-Glc to form zingiberoside R₁ and G-Ro, respectively. The critical amino acid residues involved in this catalytic process were further characterized through molecular docking studies. This research elucidates the mechanisms of ginsenoside biosynthesis and accumulation in P. zingiberensis, highlighting two key glycosylation steps within the downstream pathway of ginsenoside biosynthesis.