methyl-jasmonate and beta-amyrin

The medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane- and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated. Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil. CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of α-amyrin and β-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid. Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification.

Topics: Acetates; Cyclopentanes; Cytochrome P-450 Enzyme System; Expressed Sequence Tags; Gene Expression Regulation, Plant; Ocimum basilicum; Oleanolic Acid; Oxylipins; Plant Proteins; Triterpenes

The saponins of the model legume Medicago truncatula are glycosides of at least five different triterpene aglycones: soyasapogenol B, soyasapogenol E, medicagenic acid, hederagenin and bayogenin. These aglycones are most likely derived from beta-amyrin, a product of the cyclization of 2,3-oxidosqualene. Mining M. truncatula EST data sets led to the identification of sequences putatively encoding three early enzymes of triterpene aglycone formation: squalene synthase (SS), squalene epoxidase (SE), and beta-amyrin synthase (beta-AS). SS was functionally characterized by expression in Escherichia coli, two forms of SE by complementation of the yeast erg1 mutant, and beta-AS by expression in yeast. Beta-amyrin was the sole product of the cyclization of squalene epoxide by the recombinant M. truncatulabeta-AS, as judged by GC-MS and NMR. Transcripts encoding beta-AS, SS and one form of SE were strongly and co-ordinately induced, associated with accumulation of triterpenes, upon exposure of M. truncatula cell suspension cultures to methyl jasmonate. Sterol composition remained unaffected by jasmonate treatment. Molecular verification of induction of the triterpene pathway in a cell culture system provides a new tool for saponin pathway gene discovery by DNA array-based approaches.

Topics: Acetates; Amino Acid Sequence; Carbohydrate Sequence; Cells, Cultured; Cyclopentanes; Escherichia coli; Expressed Sequence Tags; Farnesyl-Diphosphate Farnesyltransferase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genomics; Intramolecular Transferases; Medicago; Molecular Sequence Data; Oleanolic Acid; Oxygenases; Oxylipins; Phylogeny; Phytosterols; Saponins; Signal Transduction; Squalene Monooxygenase; Triterpenes