Identification of Multiple PlOSCs Involved in the Biosynthesis Pathway of Triterpenoids in Paeonia lactiflora

Triterpenoid saponins are important bioactive compounds synthesized through the isoprenoid pathway, in which 2,3-oxidosqualene serves as a precursor of triterpenoid saponins. In this study, we identified and characterized eight oxidosqualene cyclase (PlOSC) genes in Paeonia lactiflora using molecular cloning and bioinformatic analyses. Full-length cDNAs of PlOSCs (PlOSC1-PlOSC8) were cloned, and the protein sequences exhibited significant similarities to known cyclases, including β-amyrin synthase and cycloartenol synthase. Phylogenetic analysis revealed distinct groups of PlOSCs corresponding to lupeol, β-amyrin, and cycloartenol synthases. Sequence alignment confirmed the presence of highly conserved motifs, including the "SDCTAE" and "QW" motifs, which are crucial for cyclization and stability in PlOSCs. To determine the functional roles of PlOSCs, we conducted functional expression studies in Saccharomyces cerevisiae. The results showed that PlOSC3 and PlOSC6 are monofunctional β-amyrin synthases that produce β-amyrin in yeast culture, as confirmed through GC-MS analysis. Further investigation of PlOSC gene expression in various tissues indicated that PlOSC3 was predominantly expressed in roots, whereas PlOSC6 was highly expressed in leaves. Corresponding metabolite analyses revealed that triterpenoid accumulation was significantly higher in roots than in leaves, suggesting tissue-specific biosynthesis and accumulation patterns in triterpenoid biosynthesis. These findings contribute to our understanding of the regulation of triterpenoid biosynthesis in P. lactiflora and provide insights into the functional roles of OSCs in triterpenoid and nortriterpenoid formation.