Integrated metabolome and transcriptome analyses revealed key cytochrome P450 genes involved in the biosynthesis of oleanane-type saponins in Hedera helix L.

Abstract

Hedera helix L. is a traditional Chinese medicinal and industrial crop commonly used to treat coughs and upper respiratory tract diseases. Additionally, it can be utilized as insecticidal, mosquito repellent and biopesticide. Its primary components are pentacyclic triterpenoid saponins include oleanolic acid, hederagenin, hederacoside C, etc. Currently, cytochrome P450 (CYP450) has been shown to be closely associated with the structural diversification and functional modification of the triterpenoid. However, the research on H. helix is still shallow, especially the functional characterization of CYP450 gene in the stage of modifying pentacyclic triterpenoid skeleton. This study integrated analyzed transcriptome and the accumulation modes of the main metabolites of H. helix and screened six CYP450 candidate genes. RT-qPCR results showed that candidate genes exhibited tissue specificity and inducible expression specificity. Based on in vitro and in vivo validation, both HhCYP716A409 and HhCYP716S11 showed activity of oxidase in β-amyrin C-28, producing oleanolic acid by participating in the C-28 oxidization of β-amyrin. HhCYP72D57, HhCYP72A1140, and HhCYP72A1141 produced hederagenin by participating in the hydroxylation of oleanolic acid C-23. Additionally, HhCYP72D57, HhCYP72A1139, and HhCYP72A1141 were also involved in the hydroxylation of hederagenin C-16 to produce 16-OH hederagenin. This study confirms the pivotal roles of CYP716 and CYP72 families in oleanane-type triterpenoid biosynthesis and establishes a method to efficiently produce hederacoside C and derivatives, providing a genetic toolkit for metabolic engineering of H. helix to scale saponin production for pharmaceuticals, agrochemicals, or synthetic biology-driven design of novel triterpenoid biopesticides.