Characterization of oxidosqualene cyclases involved in pentacyclic triterpene biosynthesis in Korean chestnut (Castanea crenata)

Plant triterpenoids are secondary metabolites with high chemical diversity and interesting biological properties. Chestnuts are deciduous trees in the genus Castanea, and their nuts have been used as an important food. In this work, we identified various types of triterpenes, such as α-amyrin, β-amyrin, lupeol, and friedelin, in the leaves and/or stem bark of Korean chestnut (Castanea crenata). Triterpene biosynthesis occurs by the cyclization of 2,3-oxidosqualene to triterpenes, catalyzed by oxidosqualene cyclases (OSCs). A total of 65 putative OSC sequences were obtained from the leaf transcriptome data of C. crenata plants using PacBio sequencing. We selected 5 putative OSC unigenes, named CcOSC1-5, for functional characterization of genes involved in triterpene biosynthesis. Functional characterization of the CcOSC1-5 genes by heterologous expression in erg7 mutant yeast revealed that both CcOSC1 and CcOSC2 had a similar function, encoding multifunctional triterpene synthases producing mainly β-amyrin and a small amount of α-amyrin and lupeol. CcOSC3 encodes mixed amyrin synthase, which mainly produces β-amyrin and a small amount of α-amyrin. CcOSC4 produced mainly α-amyrin and lupeol and a small amount of β-amyrin. CcOSC5 encodes an enzyme for lupeol production as a single product. In conclusion, we identified various triterpenes and functionally characterized the triterpene synthase genes that participate in β-amyrin, α-amyrin, and lupeol biosynthesis in C. crenata.