Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids

Summary Apple ( Malus  ×  domestica ) accumulates bioactive ursane‐, oleanane‐, and lupane‐type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology‐based approach to identify and functionally characterize two new oxidosqualene cyclases (Md OSC 4 and Md OSC 5) and one cytochrome P450 ( CYP 716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. Md OSC 4 encodes a multifunctional oxidosqualene cyclase producing an oleanane‐type triterpene, putatively identified as germanicol, as well as β‐amyrin and lupeol, in the proportion 82 : 14 : 4. Md OSC 5 cyclizes 2,3‐oxidosqualene into lupeol and β‐amyrin at a ratio of 95 : 5. CYP 716A175 catalyses the C‐28 oxidation of α‐amyrin, β‐amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of Md OSC 1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of Md OSC 5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C‐28 oxidase were identified in Malus  ×  domestica . This study also suggests that Md OSC 1 and Md OSC 5 are key genes in apple fruit triterpene biosynthesis.