CaOSC5-CaOSC7 module governs metabolic partitioning between asiaticoside and asiaticoside B in Centella asiatica

Asiaticoside and asiaticoside B, bioactive triterpenoid saponins from Centella asiatica (C. asiatica), exhibit distinct structural, pharmacological, and accumulation profiles. Asiaticoside (ursane-type, α-amyrin-derived) possesses skin-lightening and wound-healing properties, whereas asiaticoside B (oleanane-type, β-amyrin-derived) displays antitumor activity. Their accumulation varies significantly across different C. asiatica sources. To elucidate the basis for this differential accumulation, we analyzed 26 wild C. asiatica accessions from diverse Chinese regions. HPLC revealed substantial variation, with accession A38 accumulating higher levels of asiaticoside and A41 accumulating more asiaticoside B. Integrated omics and biochemical analyses identified CaOSC5 and CaOSC7 as key oxidosqualene cyclases (OSCs). CaOSC7 produced both α-amyrin and β-amyrin (in a 7:3 ratio), while CaOSC5 exclusively synthesized β-amyrin. AlphaFold3 predictions combined with mutagenesis pinpointed critical residues (CaOSC5: V263, K526, G424, G489, V613, S676, H680, Y742) responsible for their functional divergence. Dominant CaOSC5 expression in A41 favored asiaticoside B accumulation, whereas dominant CaOSC7 expression in A38 promoted asiaticoside accumulation. Thus, CaOSC5 and CaOSC7 form a core genetic module regulating metabolic flux balance through their differential activities and expression patterns. Correlation analysis of saponins and OSC transcription levels across 26 wild populations further confirmed that CaOSC5 and CaOSC7 expression positively correlates with corresponding asiaticoside B and asiaticoside accumulation, highlighting their marker-assisted breeding for high-asiaticoside B varieties. This study elucidates the molecular basis for the genetic regulation of asiaticoside and asiaticoside B biosynthesis in C. asiatica, providing a foundation for targeted OSC metabolic engineering.