title: Transcriptome profiling of khat (Catha edulis) and Ephedra sinica reveals gene candidates potentially involved in amphetamine-type alkaloid biosynthesis.
abstract: Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors: Ryan A
authors: RA
authors: Groves
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors: Jillian M
authors: JM
authors: Hagel
authors: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
authors: Ye
authors: Y
authors: Zhang
authors: Department of Biology, Western University, London, Ontario, Canada; Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food, London, Ontario, Canada.
authors: Korey
authors: K
authors: Kilpatrick
authors: The Robert H. Smith Institute of Plant Science and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot, 76100, Israel; Department of Aromatic, Medicinal and Spice Crops, Newe Ya'ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay, 30095, Israel.
authors: Asaf
authors: A
authors: Levy
authors: Department of Biology, Western University, London, Ontario, Canada; Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food, London, Ontario, Canada.
authors: Frédéric
authors: F
authors: Marsolais
authors: Department of Aromatic, Medicinal and Spice Crops, Newe Ya'ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay, 30095, Israel.
authors: Efraim
authors: E
authors: Lewinsohn
authors: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
authors: Christoph W
authors: CW
authors: Sensen
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors: Peter J
authors: PJ
authors: Facchini
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis*; D029754:Catha / Q000235:genetics* / Q000378:metabolism; D057225:Data Mining; D030541:Databases, Genetic; D029790:Ephedra sinica / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D017343:Genes, Plant; D059014:High-Throughput Nucleotide Sequencing; D010802:Phylogeny; D059467:Transcriptome
chemical_list: D000470:Alkaloids