Co-Authors:
Groves, R.A., Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
Hagel, J.M., Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
Zhang, Y., Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada
Kilpatrick, K., Department of Biology, Western University, London, ON, Canada, Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food, London, ON, Canada
Levy, A., Robert H. Smith Institute of Plant Science and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot, Israel, Department of Aromatic, Medicinal and Spice Crops, Newe ya'Ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay, Israel
Marsolais, F., Department of Biology, Western University, London, ON, Canada, Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food, London, ON, Canada
Lewinsohn, E., Department of Aromatic, Medicinal and Spice Crops, Newe ya'Ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay, Israel
Sensen, C.W., Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada
Facchini, P.J., Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
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. © 2015 Groves et al.
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