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）：该项目开发了用于识别，隔离和表征的新工具，从生产者有机体中聚酮化合物合成酶。我们专注于海洋鞭毛藻（Dinophyceae）的抗癌聚酮化合物，这是一类单细胞海洋真核生物，它们合成了包括冈田酸，brevetoxin和phidinolides在内的多种生物活性聚酮化合物。这些聚酮化合物的生物合成很难在遗传学上表征，因为鞭毛藻的大核基因组包含具有多个内含子含量的高基因重复。这些并发症加剧了以下事实：鞭毛藻经常含有细菌性内膜和外养生。我们提出了新型蛋白质方法的应用，用于使用模型系统（二烷基化的生物合成）在二苯烷sp中的生物合成的一般研究中的一般研究。两栖类药物是一大型宏观IDE，其中几个分子表现出有希望的抗癌活性。这套方法已通过操纵其载体蛋白（CP）结构域进行设计，以可视化，分离和操纵聚酮化合物合成酶。使用这些技术，将裂解液中的合酶酶用荧光或亲和力标签标记，并识别和纯化。将检查诸如可光透明报道链接，酶4'-磷酸耐氨酸裂解和磷酸乙基转移酶抑制剂之类的方法，以作为在其天然apo和holo-State中提供纯合酶的一种手段。将对纯化的合成酶进行多种下游测定，以识别单个负载域和模块化组织。这里介绍的方法构成了一个新的蛋白质组学工具平台，用于研究来自模块化合酶的天然产物生物合成酶，包括聚酮化合物和非核糖体肽合酶及其杂种。这些工具将进一步了解天然产品途径，并指导代谢工程以产生具有治疗意义的分子。