Harnessing two UDP-glycosyltransferases from Saponaria officinalis toward heterologous production of endosomal escape enhancer saponins in Gypsophila elegans hairy roots.

Soapwort (Saponaria officinalis) synthesizes a vast repertoire of specialized metabolites with bioactive properties. Among those, a specific type of highly glycosylated oleanane-derived triterpenoid saponins has potential applications in the pharmaceutical industry due to their anticancer activity and enhancement of endosomal escape for targeted therapeutics. Saponarioside A and saponarioside B are representative of this group and the major bioactive saponins in soapwort. Recently, the enzymes involved in the biosynthetic pathway of saponarioside B have been characterized. SO1861, another soapwort saponin known for its endosomal escape properties, shares common structural features with these saponins, suggesting saponariosides may represent SO1861 precursors. However, the late-pathway biosynthetic steps converting saponariosides into SO1861 remain unknown. Here, we report the identification and function of the uridine diphosphate (UDP)-glycosyltransferase (UGT), UGT73EU1, and uncover an additional function of the previously characterized UGT73M2, both involved in the late biosynthetic pathway leading to SO1861 and its structural derivatives. We demonstrate their activity and utility through in vitro assays with recombinant UGT proteins produced in a cell-free tobacco system. Furthermore, overexpression of UGT73EU1 in hairy root cultures of Gypsophila elegans, a close relative of Saponaria, led to the accumulation of triterpenoid saponins typically not produced by this species. Our work showcases the potential of producing high-value saponins in plants through genetic engineering approaches.