Carotenogenic Enzyme Complexes and Metabolic Channeling in Synechocystis PCC6803

类胡萝卜素是植物和蓝藻中光合器官的基本成分。这些脂溶性的黄色、橙色和红色色素可以驱散多余的光能，并防止被激发态叶绿素分子致敏的潜在致命的光氧化。通向类胡萝卜素和叶绿素的途径是密不可分的：哪里有叶绿素，哪里就一定有类胡萝卜素。这个项目正在研究植物和蓝藻决定在其光合膜中积累的各种类胡萝卜素的数量和具体身份的机制。代谢“通道”在假定的类胡萝卜素途径的酶复合物将在聚囊藻PCC6803中进行检查，这是一种蓝藻，其整个基因组序列已经确定。为了便于分离和纯化，一些聚囊藻类胡萝卜素途径酶在其C端被“表位标记”。包含纯化的类胡萝卜素酶复合物的多肽将通过质谱法鉴定。使用识别表位标签的抗血清确定标记酶的亚细胞定位。遗传方法将用于鉴定与类胡萝卜素途径酶相互作用的其他多肽。类胡萝卜素不足导致的基因表达变化将被检查以确定参与胡萝卜素生成的调控回路。研究人员的长期目标是全面了解类胡萝卜素酶复合物的结构和功能，以及聚囊藻PCC6803（高等植物和藻类中类胡萝卜素生物合成和光合作用的模式生物）中这一途径的调控。对植物和蓝藻调节类胡萝卜素途径的机制的进一步了解有望促进类胡萝卜素途径的代谢工程，以提高植物性食物的营养价值。类胡萝卜素除了具有光合作用的基本功能外，还能使水果和花朵呈现出诱人的黄色、橙色或红色，为产生诱人的水果香气和花香提供原料，并且是人类饮食中维生素A的主要来源。研究生和本科生的多元化组合，包括少数民族，将参与并在这个项目的过程中接受高级培训。

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.