Functional Identification of HhUGT74AG11-A Key Glycosyltransferase Involved in Biosynthesis of Oleanane-Type Saponins in Hedera helix

Hedera helix is a traditional medicinal plant. Its primary active ingredients are oleanane-type saponins, which have extensive pharmacological effects such as gastric mucosal protection, autophagy regulation actions, and antiviral properties. However, the glycosylation-modifying enzymes responsible for catalyzing oleanane-type saponin biosynthesis remain unidentified. Through transcriptome, cluster analysis, and PSPG structural domain, this study preliminarily screened four candidate UDP-glycosyltransferases (UGTs), including Unigene26859, Unigene31717, CL11391.Contig2, and CL144.Contig9. In in vitro enzymatic reactions, it has been observed that Unigene26859 (HhUGT74AG11) has the ability to facilitate the conversion of oleanolic acid, resulting in the production of oleanolic acid 28-O-glucopyranosyl ester. Moreover, HhUGT74AG11 exhibits extensive substrate hybridity and specific stereoselectivity and can transfer glycosyl donors to the C-28 site of various oleanane-type triterpenoids (hederagenin and calenduloside E) and the C-7 site of flavonoids (tectorigenin). Cluster analysis found that HhUGT74AG11 is clustered together with functionally identified genes AeUGT74AG6, CaUGT74AG2, and PgUGT74AE2, further verifying the possible reason for HhUGT74AG11 catalyzing substrate generalization. In this study, a novel glycosyltransferase, HhUGT74AG11, was characterized that plays a role in oleanane-type saponins biosynthesis in H. helix, providing a theoretical basis for the production of rare and valuable triterpenoid saponins.