Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis
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Casbene is a precursor to phorbol esters and down-regulating casbene synthase effectively reduces phorbol ester biosynthesis. Seed-specific reduction of phorbol ester (PE) helps develop Jatropha seed cake for animal nutrition.
Phorbol esters (PEs) are diterpenoids present in some Euphorbiaceae family members like Jatropha curcas L. (Jatropha), a tropical shrub yielding high-quality oil suitable as feedstock for biodiesel and bio jet fuel. Jatropha seed contains up to 40 % of oil and can produce oil together with cake containing high-quality proteins. However, skin-irritating and cancer-promoting PEs make Jatropha cake meal unsuitable for animal nutrition and also raise some safety and environmental concerns on its planting and processing. Two casbene synthase gene (JcCASA163 and JcCASD168) homologues were cloned from Jatropha genome and both genes were highly expressed during seed development. In vitro functional analysis proved casbene synthase activity of JcCASA163 in converting geranylgeranyl diphosphate into casbene which has been speculated to be the precursor to PEs. A seed-specific promoter driving inverted repeats for RNAi interference targeting at either JcCASA163 or both genes could effectively down-regulate casbene synthase gene expression with concurrent marked reduction of PE level (by as much as 85 %) in seeds with no pleiotropic effects observed. Such engineered low PE in seed was heritable and co-segregated with the transgene. Our work implicated casbene synthase in Jatropha PE biosynthesis and provided evidence for casbene being the precursor for PEs. The success in reducing seed PE content through down-regulation of casbene synthase demonstrates the feasibility of intercepting PE biosynthesis in Jatropha seed to help address safety concerns on Jatropha plantation and seed processing and facilitate use of its seed protein for animal nutrition.
KeywordsPhorbol ester Detoxification Jatropha curcas Animal nutrition Casbene synthase RNAi gene silencing
This project was supported by JOil-Temasek Life Sciences Laboratory Jatropha Biotechnology Program. We thank Yi CX for providing Jatropha seeds for transformation and various technical help.
Compliance with ethical standards
Conflict of interest
This project was supported by JOil-TLL Jatropha Biotechnology Program. A patent relating to Jatropha casbene synthase gene has been filed by Temasek Life Sciences Laboratory and JOil (S) Pte Ltd has a license agreement.
- GEXSI (2008) Global Market Study on JatrophaGoogle Scholar
- Ginwal HS, Rawat PS, Srivastava RL (2004) Seed source variation in growth performance and oil yield of Jatropha curcas Linn in central India. Silvae Genet 53:186–192Google Scholar
- Heller J (1996) Physic nut, Jatropha curcas. IPGRIGoogle Scholar
- Jennings SM, Tsay YH, Fisch TM, Robinson GW (1991) Molecular-Cloning and Characterization of the Yeast Gene for Squalene Synthetase. Proc Natl Acad Sci USA 88:6038–6042Google Scholar
- Makkar HPS, Becker K (1997) Jatropha curcas toxicity: identification of toxic principle(s). In: Barland T, Barr AC (eds) Toxin plants and other natural toxicants. CAB International, New York, pp 554–558Google Scholar
- Nakano Y, Ohtani M, Polsri W, TUsami T, Sambongi K, Demura T (2012) Characterization of the casbene synthase homolog from Jatropha (Jatropha curcas L.) Plant Biotechnol 29:185–189Google Scholar
- NIH (2007) Phorbol esters, safety data sheet. National Institute of HealthGoogle Scholar
- Qu J, Mao H-Z, Chen W et al (2012) Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid. Biotechnol Biofuels 5Google Scholar
- Yi C, Reddy C, Varghese K et al (2014) A new jatropha curcas variety (JO S2) with improved seed productivity. Sustainability 6:4355–4368Google Scholar