Proteomic methodologies for polyketide biosynthesis(RMI)

描述（由申请人提供）：本项目开发用于从生产生物中鉴定、分离和表征聚酮合成酶的新工具。我们的研究重点是从海洋鞭毛藻（鞭毛藻科）中提取的抗癌聚酮，鞭毛藻是一类单细胞海洋真核生物，可合成多种生物活性聚酮，包括冈田酸、草毒素和两栖素内酯。这些多酮的生物合成一直难以遗传表征，因为鞭毛藻的大核基因组含有高基因重复和多个内含子含量。这些并发症加剧的事实，鞭毛藻往往窝藏细菌的内和外共生体。我们提出了一套新的蛋白质方法应用于双鞭毛藻中聚酮生物合成的一般研究，使用一个模型系统，在Amphidinium sp.中amphidinolides的生物合成。amphidinolides是一类大分子，其中一些分子显示出有抗癌活性。这套方法已被设计成可视化，分离，并通过操纵其载体蛋白（CP）结构域来操纵聚酮合成酶。利用这些技术，将用荧光或亲和标签标记，鉴定和纯化Amphidinium裂解物中的合酶。诸如光可切割报告键、酶促4'-磷酸蜂氨酸切割和磷酸蜂氨酸转移酶抑制剂等方法将作为提供天然载子和全空状态的纯合成酶的手段进行研究。将对纯化的合成酶进行各种下游分析，以确定单个负载域和模块化组织。本文介绍的方法构成了一个新的蛋白质组学工具平台，用于研究模块化合成酶的天然产物生物合成酶，包括聚酮酶和非核糖体肽合成酶及其杂交种。这些工具将允许进一步了解自然产物途径，并指导代谢工程产生治疗重要的分子。

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.