Multi-omics and functional analyses in Aesculus wilsonii elucidate the biosynthetic pathways of moretane- and oleanane-type triterpenoids

Aesculus wilsonii, a medicinal tree widely used in traditional Chinese medicine, is rich in aescin and other structurally diverse triterpenoids; however, the biosynthetic basis underlying this chemical diversity remains poorly understood. In this study, we combined integrated multi-omics analyses with functional characterization to elucidate triterpenoid biosynthesis in A. wilsonii. Metabolomic profiling annotated 135 triterpenoids, which were classified into nine distinct skeleton types, including one previously uncharacterized scaffold. A near telomere-to-telomere genome assembly, together with seven transcriptomes, enabled comprehensive analyses of genome organization and evolution and led to the identification of four triterpenoid biosynthetic gene clusters (TBGC-1 to TBGC-4). Comparative genomics and co-expression analyses uncovered A. wilsonii-specific cytochrome P450 (CYP) genes. Functional characterization of seven CYPs in yeast, together with β-amyrin synthase and a CYP reductase, revealed that two CYP716A enzymes from TBGC-2 catalyze distinct oxidative reactions of oleanane-type triterpenoids. AwCYP716A1278 converts β-amyrin to 21β-hydroxyl-β-amyrin, whereas AwCYP716A277 produced 28-hydroxyl-β-amyrin and oleanolic acid. Molecular docking and mutational analyses identified key amino acid residues that determine product specificity. In addition, functional characterization of a neofunctionalized oxidosqualene cyclase, AwOSC13 from TBGC-4, revealed a previously unknown biosynthetic pathway leading to hop-17(21)-en-3β-ol and an uncharacterized triterpenoid. Structural elucidation using NMR and mass spectrometry identified this compound as moretenol. Heterologous expression of AwOSC13 in tobacco successfully reconstituted this pathway in planta. Together, these findings demonstrate how biosynthetic gene clusters and enzyme diversification shape triterpenoid metabolism in A. wilsonii and provide valuable genomic and biochemical resources for the discovery and engineering of bioactive plant natural products.