Identification and characterization of PpUGT91BP1 as a trillin synthase from Paris polyphylla

Polyphyllins are the active ingredients of the medicinal plant Paris polyphylla. The biosynthesis of different types of polyphyllins all requires the catalysis of glycosyltransferases. Even though significant efforts have been made to identify PpUGTs capable of catalyzing the initial glycosylation reaction, the specific glycosyltransferases responsible for the synthesis of trillin have not been reported in P. polyphylla. Here, we identified a new trillin synthase, named PpUGT91BP1, which was highly expressed in the rhizome. Importantly, PpUGT91BP1 could specifically glycosylate diosgenin but not pennogenin. To improve its catalytic efficiency, we introduced random mutations through error-prone PCR and conducted an activity-based screening. Three mutants with significantly enhanced trillin synthase activity were identified. Finally, we successfully reconstituted trillin biosynthesis in Nicotiana benthamiana, achieving a yield of 3.69 mg/g of plant dry weight using the mutant PpUGT91BP1. Taken together, our results deepen the understanding of the PpUGT91 family's role in polyphyllin biosynthesis in P. polyphylla, facilitating rational selection of better P. polyphylla cultivars and guiding future studies in the metabolic engineering of polyphllins.