Oleanane-type triterpenoid saponins are the primary medicinal components of Eleutherococcus senticosus. During saponin biosynthesis in E. senticosus, various members of the 2,3-oxidosqualene cyclase (OSC) gene family can direct 2,3-oxidosqualene into triterpene saponin and sterol synthesis pathways. However, the precise molecular mechanism underlying this phenomenon remains unclear. We initially screened for β-amyrin synthase 1 (bAS1) and cycloartenol synthase 1 (CAS1) among 10 EsOSC genes using genome-wide identification and correlation analysis. Subcellular localization, catalytic experiments, and in vivo transient overexpression demonstrated that EsbAS1 and EsCAS1 catalyze the formation of the triterpene skeleton β-amyrin and sterol precursor cycloartenol exclusively in the cytoplasm, enhancing and inhibiting the in vivo biosynthesis of oleanane-type saponins, respectively. Results from site-directed mutagenesis and molecular docking indicated that W-WY and Y-WH triplets characterized the active sites of EsbAS1 and EsCAS1, respectively. GUS (β-glucuronidase) staining and electrophoretic mobility shift assay (EMSA) experiments on the promoter region revealed that various colored light quality, DNA methylation, and five transcription factors (EsNAC047, EsNAC098, EsWRKY40, EsMYB4, and EsERF66) regulated the expression of EsbAS1 and EsCAS1. This study provides preliminary insights into the molecular mechanisms by which EsbAS1 and EsCAS1 regulate saponin synthesis in E. senticosus.

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