Deciphering the biosynthetic pathway of triterpene saponins in Prunella vulgaris

SUMMARY The traditional Chinese medicinal plant Prunella vulgaris contains numerous triterpene saponin metabolites, notably ursolic and oleanolic acid saponins, which have significant pharmacological values. Despite their importance, the genes responsible for synthesizing these triterpene saponins in P. vulgaris remain unidentified. This study used a comprehensive screening methodology, combining phylogenetic analysis, gene expression assessment, metabolome–transcriptome correlation and co‐expression analysis, to identify candidate genes involved in triterpene saponins biosynthesis. Nine candidate genes – two OSCs, three CYP716s and four UGT73s – were precisely identified from large gene families comprising hundreds of members. These genes were subjected to heterologous expression and functional characterization, with enzymatic activity assays confirming their roles in the biosynthetic pathway, aligning with bioinformatics predictions. Analysis revealed that these genes originated from a whole‐genome duplication (WGD) event in P. vulgaris , highlighting the potential importance of WGD for plant metabolism. This study addresses the knowledge gap in the biosynthesis of triterpene saponins in P. vulgaris , establishing a theoretical foundation for industrial production via synthetic biology. Additionally, we present an efficient methodological protocol that integrates evolutionary principles and bioinformatics techniques in metabolite biosynthesis research. This approach holds significant value for studies focused on unraveling various biosynthetic pathways.