Proteomic methodologies for polyketide biosynthesis(RMI)

聚酮化合物生物合成（RMI）的蛋白质组学方法

• 批准号: 7125524
• 负责人: Michael D. Burkart
• 金额: $ 32.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125524
• 关键词: Dinoflagellates; NIH Roadmap Initiative tag; SDS polyacrylamide gel electrophoresis; antineoplastics; aquatic organism; biotechnology; biotherapeutic agent; enzyme activity; eukaryote; method development; microorganism culture; microorganism genetics; polyketide synthase; protein biosynthesis; protein purification; protein quantitation /detection; protein structure function; proteomics; saltwater environment; transport proteins; western blottings

DESCRIPTION (provided by applicant): This project develops new tools for the identification, isolation, and characterization of polyketide synthases from producer organisms. We focus on anticancer polyketides from marine dinoflagellates (dinophyceae), a class of single-cellular marine eukaryotes that synthesize an extraordinary variety of bioactive polyketides, including okadaic acid, brevetoxin and amphidinolides. The biosynthesis of these polyketides has been difficult to characterize genetically due to the fact that the large nuclear genomes of dinoflagellates contain high gene duplication with multiple intron content. These complications are intensified by the fact that dinoflagellates often harbor bacterial endo- and exo-symbionts. We propose the application of a novel suite of protein methods for the general study of polyketide biosynthesis in dinoflagellates using a model system, the biosynthesis of amphidinolides in Amphidinium sp. The amphidinolides are a large class of macro ides, of which several molecules demonstrate promising anti-cancer activity. This suite of methods has been engineered to visualize, isolate, and manipulate polyketide synthases through manipulation of their carrier protein (CP) domains. Using these techniques synthase enzymes in Amphidinium lysates will be tagged with fluorescent or affinity tags, identified, and purified. Methods such as photocleavable reporter linkages, enzymatic 4'-phosphopantetheine cleavage, and phosphopantetheinyltransferase inhibitors will be examined as a means to provide pure synthases in their natural apo- and holo- state. A variety of downstream assays will be conducted on the purified synthases to identify the individual loading domains and modular organization. The methodologies introduced here constitute a new platform of proteomic tools to investigate natural product biosynthetic enzymes from modular synthases, including polyketide and non-ribosomal peptide synthases and their hybrids. These tools will allow further understanding of natural product pathways and guide metabolic engineering to produce therapeutically important molecules.

描述（由申请人提供）：该项目开发了用于从生产生物体中鉴定、分离和表征聚酮化合物脱氢酶的新工具。我们专注于抗癌聚酮从海洋甲藻（甲藻），一类单细胞海洋真核生物，合成一个非凡的各种生物活性聚酮，包括冈田酸，短毒素和amphidinolides。这些聚酮化合物的生物合成一直难以表征遗传，由于事实上，大型核基因组的甲藻含有高基因重复与多个内含子的内容。这些并发症加剧的事实，腰鞭毛虫往往窝藏细菌内和外共生体。我们提出了一套新的蛋白质方法的应用程序的聚酮生物合成的甲藻使用的模型系统，生物合成的amphidinolides在Amphidinium sp.的amphidinolides是一个大类的macroides，其中几个分子表现出很有前途的抗癌活性的一般性研究。这套方法已经被设计成通过操纵其载体蛋白（CP）结构域来可视化、分离和操纵聚酮酶。使用这些技术，前沟藻裂解物中的合酶将用荧光或亲和标签标记，鉴定和纯化。将检查诸如可光裂解的报告物连接、酶促4 '-磷酸泛酰巯基乙胺裂解和磷酸泛酰巯基乙胺基转移酶抑制剂的方法，作为提供天然脱辅基和全态的纯辅酶A酶的手段。将对纯化的酶进行多种下游测定以鉴定各个加载结构域和模块化组织。这里介绍的方法构成了一个新的平台的蛋白质组学工具，以调查天然产物生物合成酶从模块化的糖苷酶，包括聚酮和非核糖体肽糖苷酶及其杂交。这些工具将允许进一步了解天然产物途径，并指导代谢工程生产治疗上重要的分子。