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Title: Cloning of Casbene Synthases from Euphorbiaceae Plants and Implications for Diterpene Biosynthesis in This Family

item KIRBY, JAMES - University Of California
item NISHIMOTO, MINOBU - University Of California
item PARK, JUNGWON - University Of California
item WITHERS, SYDNOR - University Of California
item NOWROOZI, FARNAZ - University Of California
item BEHRENDT, DOMINIK - University Of California
item GARCIA RUTLEDGE, ELIZABETH - University Of California
item FORTMAN, JEFFREY - University Of California
item JOHNSON, HOLLY - University Of California
item Anderson, James
item KEASLING, JAY - University Of California

Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2010
Publication Date: 9/1/2010
Citation: Kirby, J., Nishimoto, M., Park, J.G., Withers, S.T., Nowroozi, F., Behrendt, D., Garcia Rutledge, E.J., Fortman, J.L., Johnson, H.E., Anderson, J.V., Keasling, J.D. 2010. Cloning of Casbene and Neocembrene Synthases from Euphorbiaceae Plants and Expression in Saccaromyces cerevisiae. Phytochemistry. 71:1466-1473. DOI:10.1016/j.phytochem.2010.06.001.

Interpretive Summary: Plants in the Euphorbiaceae family produce naturally occurring compounds which may have therapeutic activity. In this study, genes obtained from Euphorbiaceae plants such as castor bean and leafy spurge were used in genetically engineered systems to produce compounds that could be of medical benefit to patients with HIV and cancer.

Technical Abstract: A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton we consider it likely that casbene is the precursor to the majority of diterpenes in the Euphorbiaceae.