Discovery and Functional Identification of 2,3-Oxidosqualene Cyclases and Cytochrome P450s in Triterpenoid Metabolic Pathways of Actinidia eriantha.

Actinidia eriantha Benth, known as the "king of fruits", is rich in triterpenoid compounds, particularly ursane-type and oleanane-type triterpenic acids. These secondary metabolites have been widely applied in medicine, cosmetics, agriculture, and other fields. To date, key enzyme genes involved in triterpenoid metabolic pathways in A. eriantha remain unexplored. This study employed transcriptome sequencing analysis combined with synthetic biology approaches involving heterologous expression in yeast to identify crucial genes responsible for the biosynthesis of triterpenoid components in A. eriantha: Two 2,3-oxidosqualene cyclases (AeOSC2 and AeOSC3) were characterized to catalyze the formation of major triterpene scaffolds, α-amyrin [precursor of ursolic acid (UA)], β-amyrin [precursor of oleanolic acid (OA)], and ψ-taraxasterol, and two cytochrome P450s (AeCYP716A8 and AeCYP716A9) mediating three-step oxidation at the C-28 position of ursane-type and oleanane-type triterpene scaffolds to form UA, OA, and intermediate oxidation products. We successfully reconstructed the biosynthetic pathway of ursane- and oleanane-type triterpenoids from A. eriantha in a heterologous yeast host and elucidated the two-step enzymatic reactions involved in triterpenoid biosynthesis. These findings lay the foundation for further understanding the biosynthesis of key active components in A. eriantha.