title: Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis.
abstract: 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.
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. chunhong@tll.org.sg.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Li
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. ailing0909@gmail.com.|School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore. ailing0909@gmail.com.
authors.forename: Ailing
authors.initials: A
authors.lastname: Ng
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. lifen@tll.org.sg.
authors.forename: Lifen
authors.initials: L
authors.lastname: Xie
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. maohz@tll.org.sg.
authors.forename: Huizhu
authors.initials: H
authors.lastname: Mao
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. chengxiang.qiu@ndsu.edu.
authors.forename: Chengxiang
authors.initials: C
authors.lastname: Qiu
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. sri@tll.org.sg.
authors.forename: Ramachandran
authors.initials: R
authors.lastname: Srinivasan
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. yinzc@tll.org.sg.
authors.forename: Zhongchao
authors.initials: Z
authors.lastname: Yin
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Singapore. yhong@ntu.edu.sg.|JOil (S) Pte Ltd, 1 Research Link, Singapore, 117604, Singapore. yhong@ntu.edu.sg.|School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore. yhong@ntu.edu.sg.
authors.forename: Yan
authors.identifier: http://orcid.org/0000-0002-2954-8788
authors.initials: Y
authors.lastname: Hong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D056804:Biofuels; D015536:Down-Regulation; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering; D006801:Humans; D031286:Jatropha / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D009928:Organ Specificity; D010703:Phorbol Esters / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D034622:RNA Interference; D012639:Seeds / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D056804:Biofuels; D010703:Phorbol Esters; D010940:Plant Proteins; C044955:casbene synthetase; D019761:Phosphorus-Oxygen Lyases
keywords: Animal nutrition; Casbene synthase; Detoxification; Jatropha curcas; Phorbol ester; RNAi gene silencing