Cloning and Characterization of Oxidosqualene Cyclases Involved in Taraxasterol, Taraxerol and Bauerenol Triterpene Biosynthesis in Taraxacum coreanum

Triterpenes, consisting of six isoprene units, are one of the largest classes of natural compounds in plants. The genus Taraxacum is in the family Asteraceae and is widely distributed in the Northern Hemisphere. Various triterpenes, especially taraxerol and taraxasterol, are present in Taraxacum plants. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene after the rearrangement of the triterpene skeleton. However, no functional characterization of the OSC genes involved in triterpene biosynthesis, except for a lupeol synthase in Taraxacum officinale, has been performed. Taraxacum coreanum, or Korean dandelion, grows in Korea and China. Putative OSC genes in T. coreanum plants were isolated by transcriptome analysis, and four of these (TcOSC1, TcOSC2, TcOSC3 and TcOSC4) were functionally characterized by heterologous expression in yeast. Both TcOSC1 and TcOSC2 were closely related to dammarenediol-II synthases. TcOSC3 and TcOSC4 were strongly grouped with β-amyrin synthases. Functional analysis revealed that TcOSC1 produced several triterpenes, including taraxasterol; Ψ-taraxasterol; α-, β- and δ-amyrin; and dammarenediol-II. TcOSC2 catalyzed the production of bauerenol and another unknown triterpene, TcOSC3 catalyzed the production of β-amyrin. TcOSC4 catalyzed the production of taraxerol. Moreover, we identified taraxasterol, ψ-taraxasterol, taraxerol, lupeol, δ-amyrin, α-amyrin, β-amyrin and bauerenol in the roots and leaves of T. coreanum. Our results suggest that TcOSC1, TcOSC2, TcOSC3 and TcOSC4 are key triterpene biosynthetic enzymes in T. coreanum. These enzymes are novel triterpene synthases involved in the production of taraxasterol, bauerenol and taraxerol.

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.

Functional genomics approach to the study of triterpene biosynthesis

The Arabidopsis thaliana genome-sequencing project has identified the presence of 13 oxidosqualene cyclase homologs in this plant. In addition to the already identified clones, namely, CAS1 cycloartenol synthase, LUP1 lupeol synthase, and YUP8H12R.43 multifunctional triterpene synthase, two new cDNAs of the putative oxidosqualene cyclase genes, F1019.4 and T30F21.16, were obtained by polymerase chain reaction (PCR) and functionally expressed in yeast. Liquid chromatography/mass spectrometry (LC/MS) analysis led to the identification of some of their reaction products. Interestingly, except for CAS1 for sterol biosynthesis of primary metabolism, so-far-obtained all triterpene synthases of this plant are multifunctional, producing more than one cyclization product. A feeding experiment of 13C-labeled acetate with LUP1 lupeol synthase transformant demonstrated the stereospecific water addition to lupenyl cation intermediate, yielding 3β,20 dihydroxylupane, which accounts for the multiproduct nature of this synthase.