ME: Collaborative Research: Metabolic Engineering of Hairy Roots for Alkaloid Production

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The objective of this project is to improve the productivity of indole alkaloids in a well-characterized model plant tissue culture system, C. roseus. The overall goals are to determine the metabolic effects of genetic manipulations of some key enzymes in the indole and non-mevalonate terpenoid pathways on flux through the indole alkaloid synthesis pathways, with special emphasis on the interplay between primary and secondary metabolism on the final alkaloid productivity. The specific objectives are to: 1) construct and characterize transgenic hairy root lines overexpressing key enzymes, such as ORCA3, G10H, DXS, DXR and AS-beta under the control of a recently characterized inducible promoter system; 2) develop methods for NMR- based metabolic flux maps; 3) perform metabolic characterization of these first generationtransgenic lines; 4) develop techniques for co-transformation that allow the introduction ofmultiple genes; 5) construct and characterize a second generation of transgenic lines expressing strategic combinations of genes as determined from the results of the first generation transgenic lines above.