Identification of a key glycosyltransferase and de novo biosynthesis of rare oleanane-type pseudoginsenoside RT1 in Saccharomyces cerevisiae

Objective Pseudoginsenoside RT1 (RT1) is a rare oleanane-type ginsenoside from Panax japonicus with promising pharmacological potential, but its application is restricted by low natural abundance and inefficient extraction. This study aimed to identify the glycosylation steps associated with RT1 biosynthesis and to establish an engineered yeast strain for its de novo production. Methods Candidate uridine diphosphate glycosyltransferase ( UGT ) genes were screened by co-expression analysis using PjbAS1 and PjbAS2 as bait genes and further prioritized by phylogenetic analysis. PjUGT67 was cloned from P. japonicus and functionally characterized in Escherichia coli . Its enzymatic properties, substrate specificity, donor preference, and subcellular localization were investigated. To reconstruct RT1 biosynthesis, the relevant pathway genes were stepwise introduced into Saccharomyces cerevisiae , together with a heterologous uridine diphosphate xylose (UDP-Xyl) supply module, followed by strain optimization and stability evaluation. Results Pj UGT67 was identified as a UGT capable of catalyzing the conversion of chikusetsusaponin IVa (IVa) to RT1. The enzyme showed optimal activity at 35 ℃ and pH 7.0, was predominantly localized in the cytoplasm, displayed narrow acceptor specificity, and utilized both UDP-Xyl and uridine diphosphate glucose (UDP-Glc) as glycosyl donors. In addition, Pj CSLM and Pj C28UGT were verified to catalyze the conversion of oleanolic acid (OA) to calenduloside E (CE) and CE to IVa, respectively, thereby defining the sequential glycosylation route from OA to RT1. By integrating OA biosynthesis, sequential glycosylation, and UDP-Xyl supply in yeast, an engineered strain RT1-02 was constructed and achieved de novo RT1 production at 17.34 mg/L. Serial passaging further confirmed the genetic and production stability of the engineered strain. Conclusion This study provides biochemical evidence for the previously unresolved xylosylation step of RT1, identifies and characterizes Pj UGT67 as a functional UDP-xylosyltransferase involved in the terminal glycosylation step, and establishes a yeast-based route for de novo RT1 production. These findings provide new enzymatic resources and a useful basis for the sustainable biosynthesis and future engineering of OA-type ginsenosides.