Multi-omics and functional analyses of Aesculus wilsonii uncover moretane- and oleanane-type triterpenoid biosynthesis

Aesculus wilsonii, a medicinal tree used in Traditional Chinese Medicine, is rich in aescin and other structurally diverse triterpenoids, yet the biosynthetic mechanism of this diversity remains poorly understood. In this study, we employed integrated omics analyses and functional characterization to elucidate triterpenoid biosynthesis in A. wilsonii. Metabolic profiling annotated 135 triterpenoids that were classified into nine skeleton types, including one previously uncharacterized scaffold. A near telomere-to-telomere genome assembly together with seven transcriptomes enabled comprehensive analysis of genome organization and evolution and determined four triterpenoid biosynthetic gene clusters (TBGC-1 to TBGC-4). Comparative genomics and co-expression analyses identified A. wilsonii-specific cytochrome P450 genes. The functional characterization of seven in yeast together with β-amyrin synthase and cytochrome P450 reductase revealed two CYP716A enzymes from TBGC-2 that catalyzed distinct oxidative reactions. AwCYP716A1278 converted 2,3-oxidosqualene to 21β-hydroxyl-β-amyrin, whereas AwCYP716A277 produced 28-hydroxyl-β-amyrin and oleanolic acid, two oleanane-type triterpenoids. Molecular docking and mutational analyses revealed amino acid residues critical for product specificity. Moreover, functional characterization of a neofunctionalized oxidosqualene cyclase, AwOSC13 from TBGC-4, uncovered a unknown pathway leading to hop-17(21)-en-3β-ol and an uncharacterized triterpenoid. Structural elucidation using NMR and MS identified this compound as moretenol. Heterologous expression of AwOSC13 in tobacco successfully reconstituted the pathway in planta. Together, these findings reveal how biosynthetic gene clusters and enzyme diversification shape triterpenoid metabolism in A. wilsonii and provide valuable resources for discovering and engineering bioactive plant natural products.