Collaborative Research: Molecular Genetic and Metabolic Analyses to Characterize Terpenoid Indole Alkaloid Pathways

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Collaborative Research: Molecular Genetic and Metabolic Analyses to Characterize Terpenoid Indole Alkaloid PathwaysSusan I. GibsonUniversity of Minnesota-Twin CitiesCBET-0729625Expertise in biochemical engineering and plant molecular biology will be combined to take an interdisciplinary approach to addressing some of the fundamental issues facing successful engineering of terpenoid indole alkaloids (TIAs) and other complex metabolic pathways. To increase basic understanding of TIA pathways, improved methods of metabolite analysis will be developed and implemented. In addition, metabolic engineering, particularly of the terpenoid pathway and of TIA transcription factors, will be used to further explore regulation of these pathways. The Specific Aims to be carried out in this work are to: (1) generate and characterize transgenic C. roseus hairy root lines overexpressing strategic combinations of terpenoid and indole pathway metabolic genes, (2) characterize the effects of overexpressing transcriptional activators of TIA genes and of decreasing expression of transcriptional repressors of TIA genes on TIA metabolism, and (3) develop the methodology for metabolic profiling of TIAs and pathway intermediates and apply this technology to the analysis of the transgenic hairy root lines developed.Intellectual merit: The proposed experiments will significantly increase understanding of TIA and related pathways. In particular, these experiments will help identify and quantify the metabolic intermediates for different steps in the pathways. These experiments will also provide critical information about the regulation of these pathways by examining the effects of manipulating expression of different combinations of TIA biosynthetic or regulatory genes. Broader impacts: The tools and knowledge to be gained from this work represent a necessary prerequisite to engineering TIA and other pathways to produce higher levels of commercially important pharmaceuticals, thereby helping to lower the costs of prescription drugs. The proposed work will also provide interdisciplinary training for undergraduate and graduate students, with significant efforts being made to include members of underrepresented groups.