Carotenogenic Enzyme Complexes and Metabolic Channeling in Synechocystis PCC6803

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Carotenoids are essential constituents of the photosynthetic apparatus in plants and cyanobacteria. These lipid-soluble yellow, orange and red pigments dissipate excess light energy and protect against potentially lethal photo-oxidation sensitized by excited state molecules of chlorophyll. The pathways leading to carotenoids and chlorophylls are inextricably linked: wherever there is chlorophyll, there must also be carotenoids. The mechanisms by which plants and cyanobacteria determine the amounts and specify the identities of the various carotenoids that accumulate in their photosynthetic membranes are being investigated in this project. Metabolic "channeling" in putative enzyme complexes of the carotenoid pathway will be examined in Synechocystis PCC6803, a cyanobacterium for which the entire genome sequence has been determined. Several Synechocystis carotenoid pathway enzymes have been "epitope tagged" at their C terminal ends in order to facilitate their isolation and purification. Polypeptides that comprise the purified carotenoid enzyme complexes will be identified by mass spectrometry. Subcellular localization of the tagged enzymes is being ascertained using an antiserum that recognizes the epitope tag. A genetic approach will be employed to identify other polypeptides that interact with carotenoid pathway enzymes. Changes in gene expression that result from an insufficiency of carotenoids will be examined to define the regulatory circuits involved in carotenogenesis. The long term goal of the investigators is to achieve a comprehensive understanding of the architecture and function of carotenoid enzyme complexes and the regulation of this pathway in Synechocystis PCC6803, a model organism for carotenoid biosynthesis and photosynthesis in higher plants and algae.An improved understanding of the mechanisms by which plants and cyanobacteria regulate the carotenoid pathway is expected to facilitate metabolic engineering of the carotenoid pathway to improve the nutritional value of plant foods. In addition to their essential functions in photosynthesis, carotenoid pigments impart an attractive yellow, orange or red colors to fruits and flowers, provide the raw material for the production of alluring fruit aromas and floral fragrances, and serve as the major source of vitamin A in the human diet. A diverse combination of graduate and undergraduate students, including minorities will participate and receive advanced training in the course of this project.