Biosynthesis of phycoerythrins, photoysnthetic light harvesting proteins in cyanobacteria

蓝藻中藻红蛋白、光合光捕获蛋白的生物合成

• 批准号: 1244339
• 负责人: Wendy Schluchter
• 金额: $ 62.33万
• 依托单位: University of New Orleans
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1244339&HistoricalAwards=false
• 关键词: Biosynthesis; phycoerythrins; photoysnthetic; light; harvesting

Intellectual Merit: Small unicellular cyanobacteria such as Synechococcus RS9916 are estimated to contribute 25-30% of the total marine productivity. Synechococcus is likely to be one of the most ubiquitous phytoplanktonic organisms in the world's oceans and numerically the second most abundant marine phototroph in this ecosystem. This ubiquity and abundance is in large part due to its remarkable phenotypic plasticity in its use of a diverse set of photosynthetic pigments, which is sustained by a complex set of genes and regulatory systems that can be laterally transferred between lineages. Much of this diversity occurs within the protein Phycoerythrin (PE), which has five phycoerythrobilin (PEB) or phycourobilin (PUB) chromophores attached at six cysteine residues. These posttranslational modifications are catalyzed by bilin lyase/isomerase enzymes. As blue light is the dominating wavelength present in the oligotrophic open ocean, strains capable of a process called Type IV chromatic acclimation (CA4) have adapted by increasing the PUB content on their PE to take advantage of the light filtering through the water by a novel mechanism: induction of a bilin lyase/isomerase. The research in this project will contribute to defining the function of these PE bilin lyase/isomerase enzymes that are critical to the success and adaptation of this diverse group of photosynthetic prokaryotes. A better understanding of this novel type of chromatic acclimation (CA4) will be achieved. The first objective is to characterize the biosynthesis of a "simple" PE in Fremyella diplosiphon. Six genes with similarity to known bilin lyase enzymes are upregulated with phycoerythrin structural genes. The second objective involves characterizing the function of bilin lyases specific for PEI and PEII in Synechococcus RS9916, focusing on the characterization of lyases responsible for CA4 changes. Analyses of PE produced in the bilin lyase knockout mutants will be performed, and recombinant protein expression in E. coli to test for bilin ligation and isomerization activity will also be used. Not only will these studies contribute to understanding how these ecologically important organisms acclimate to thrive in the ocean, they will also biochemically characterize enzymes which will have very important potential biotechnological and cell biology applications for the use of phycoerythrins as fluorescent tags.Broader impacts: The PI has a strong track record of training undergraduates and graduate students, especially members of under-represented groups in science, and these efforts will continue during this granting period. The PI will incorporate her research into a course called Biological Research Tools where students learn basic molecular cloning and protein expression/purification of phycobiliproteins in E. coli. At least two undergraduates (over the course of the grant) will be selected to go to the laboratory of Dr. David Kehoe at Indiana University (after working in the Schluchter lab for a year first) for 6-8 weeks in the summer in order to learn techniques to generate clean deletions and to study gene regulation related to the CA4 collaboration project. The student is likely to be a member of an under-represented group (50% of the PI's past undergraduates have been minorities), and the purpose of these exchanges is to expose these students to a larger, research university.

智力优势：小型单细胞蓝藻，如Synechococcus RS9916，估计占海洋总生产力的25 - 30%。聚球藻可能是世界海洋中最普遍存在的植物营养生物之一，也是海洋生态系统中数量第二多的海洋光养生物。这种普遍性和丰富性在很大程度上是由于其在使用各种光合色素方面具有显着的表型可塑性，这是由一组复杂的基因和调控系统维持的，这些基因和调控系统可以在谱系之间横向转移。这种多样性的大部分发生在蛋白质藻红蛋白（PE）中，其具有五个连接在六个半胱氨酸残基上的藻红胆素（PEB）或藻胆素（PUB）发色团。 这些翻译后修饰由胆色素裂解酶/异构酶催化。 由于蓝光是贫营养开放海洋中存在的主要波长，能够进行IV型色适应（CA 4）过程的菌株通过增加其PE上的PUB含量来适应，以利用通过一种新机制过滤水的光：诱导胆色素裂解酶/异构酶。本项目的研究将有助于确定这些PE胆色素裂解酶/异构酶的功能，这些酶对这一多样化的光合原核生物群体的成功和适应至关重要。 更好地了解这种新类型的色驯化（CA4）将实现。第一个目标是表征在Fremyella diplosiphon中的"简单" PE的生物合成。 六个基因与已知的胆色素裂解酶的相似性上调藻红蛋白结构基因。第二个目标涉及表征聚球藻RS9916中PEI和PEII特异性的胆色素裂解酶的功能，侧重于表征负责CA4变化的裂解酶。将对胆色素裂解酶敲除突变体中产生的PE进行分析，并在E.还将使用大肠杆菌来测试胆色素连接和异构化活性。 这些研究不仅将有助于了解这些具有重要生态意义的生物如何适应环境，以便在海洋中茁壮成长，而且还将对酶进行生物化学表征，这些酶对于将藻红蛋白用作荧光标记具有非常重要的潜在生物技术和细胞生物学应用。PI在培训本科生和研究生方面有着良好的记录，特别是在科学领域代表性不足的群体，在这段拨款期内，这些工作会继续进行。PI将把她的研究纳入一门名为生物研究工具的课程，学生将在该课程中学习基本的分子克隆和藻胆蛋白的蛋白表达/纯化。杆菌 至少有两名本科生（在资助期间）将被选中前往印第安纳州大学的大卫凯霍博士的实验室（在Schluchter实验室工作一年后），在夏季进行6 - 8周的学习，以学习产生干净缺失的技术，并研究与CA4合作项目相关的基因调控。 学生很可能是一个代表性不足的群体的成员（PI过去的本科生中有50%是少数民族），这些交流的目的是让这些学生接触到一个更大的研究型大学。