Abstract

Paris species are renowned for the production of important medicinal steroidal saponins (e.g., polyphyllins). UDP-dependent glycosyltransferases (UGTs) play important roles in saponin biosynthesis. However, the glycosylation steps have not been fully elucidated. Here, we investigate four candidate UGTs (i.e., UGT91BP2, UGT703R1, UGT703R2 and UGT703R3) from the medicinal herb Paris polyphylla var. yunnanensis, followed by their phylogenetic, in vitro biochemical, and in planta functional characterization as saponin biosynthetic enzymes. These four recombinant PpUGTs expressed in Escherichia coli catalyze the glucosylation of diosgenin to produce diosgenin 3-O-glucoside (trillin); while UGT703R1, UGT703R2 and UGT703R3 glucosylate pennogenin to pennogenin 3-O-glucoside in vitro. Transient expression of these UGTs in Nicotiana benthamiana leaves, supplemented with diosgenin substrate, confirms their roles in trillin biosynthesis. Subcellular localization analysis in tobacco cells reveals their presence in both cytoplasm and nucleus. Gene transcript analysis reveals that the four PpUGTs exhibit higher expression in leaves and flowers than in stems, indicating distinct tissue-specific patterns. This work identifies key UGTs involved in polyphyllin biosynthesis, enriching the enzymatic toolbox involved in the glucosylation of diosgenin and pennogenin aglycones, and provides a valuable reference for future studies on overproduction of bioactive steroidal glycosides in heterologous hosts.