Identification of triterpenes and functional characterization of oxidosqualene cyclases involved in triterpene biosynthesis in lettuce (Lactuca sativa)

Abstract

Lettuce (Lactuca sativa) is a member of the family Asteraceae and is most often used for green salads. Triterpenes are the largest class of natural compounds in plants and have beneficial health effects. Here, we identified various triterpene esters (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, β-amyrin acetates, and germanicol acetate) and free triterpenes (α-amyrin, β-amyrin, taraxerol, and taraxasterol) in both the leaves and roots of lettuce. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene. None of the OSC genes involved in triterpene biosynthesis in lettuce have been characterized. Five putative lettuce OSC genes (LsOSC1, LsOSC2, LsOSC3, LsOSC4, and LsOSC5) were selected from a transcriptome database. These five genes were functionally characterized via heterologous expression in yeast. The first two enzymes were multifunctional triterpene synthase and the last three genes were monofunctional. Transgenic yeast expressing LsOSC1 produced five triterpenes, namely, taraxasterol, Ψ-taraxasterol, α-amyrin, β-amyrin, and dammarenediol-II. Yeast expressing LsOSC2 produced baurenol and Ψ-taraxasterol. LsOSC3, LsOSC4, and LsOSC5 expression led to β-amyrin, taraxerol, and lupeol production, respectively. Transcriptional activity assessment of the five genes revealed that all the OSC genes were more actively transcribed in roots than in leaves, and LsOSC5 among the five OSC genes showed the highest expression in both the leaves and the roots. In conclusion, we identified structurally diverse free triterpenes and triterpene esters in lettuce plants and characterized five OSC genes, which are key enzymes involved in the biosynthesis of diverse triterpenes in lettuce.