Identification and functional validation of a beta-amyrin synthase for Clinopodiside A biosynthesis in medicinal...

Clinopodium chinense (Benth.) Kuntze is the botanical source of the traditional Chinese medicine "Duanxueliu", whose pharmacological activities are largely attributed to the oleanane-type triterpenoid saponin Clinopodiside A. Beta-amyrin synthase (bAS) catalyzes the first committed step in oleanane-type triterpenoids biosynthesis. However, the genetic basis underlying Clinopodiside A biosynthesis remains largely unknown, particularly in non-model medicinal plants. Here, we quantified Clinopodiside A contents and analyzed transcriptomes across ten tissues and developmental stages of C. chinense. Weighted gene co‑expression network analysis (WGCNA) identified the MEgreen module, which was strongly correlated with Clinopodiside A accumulation (r = 0.89, p < 0.001), and contained a candidate bAS gene, designated CcbAS. The full-length coding sequence of CcbAS is 2289 bp and encodes a protein of 763 amino acids. High-Pressure Liquid Chromatography (HPLC) and quantitative real-time PCR (qPCR) analyses revealed highly consistent tissue-specific and developmental patterns between CcbAS expression and Clinopodiside A accumulation. Subcellular localization in Nicotiana benthamiana showed that CcbAS is localized in the cytoplasm and associated with the plasma membrane. To overcome the lack of stable transformation systems, we established two rapid, complementary Agrobacterium-based platforms (transient overexpression and VIGS) in C. chinense. Functional validation demonstrated that CcbAS overexpression increased Clinopodiside A accumulation by 2.18‑fold, while VIGS‑mediated silencing reduced it by 53.06%. These findings identify CcbAS as a key gene involved in Clinopodiside A biosynthesis and establish an efficient platform for rapid functional characterization of biosynthetic genes in C. chinense, providing a foundation for pathway elucidation and metabolic engineering.