Proteomic methodologies for polyketide biosynthesis(RMI)

描述(由申请人提供)：该项目开发了新的工具，用于从生产者生物体中鉴定、分离和表征聚酮合成酶。我们专注于从海洋甲藻(甲藻)中提取的抗癌多酮，这是一类单细胞海洋真核生物，可以合成包括冈田酸、短杆菌毒素和两性内酯在内的各种生物活性多酮。这些多酮类化合物的生物合成很难从遗传学上进行表征，这是因为甲藻的大核基因组含有高度的基因重复和多个内含子。甲藻通常含有细菌内外共生体，这一事实加剧了这些并发症。我们提出了一套新的蛋白质合成方法应用于甲藻聚酮生物合成的一般研究，使用的是一个模型系统，即两性内酯的生物合成。两面针内酯是一大类大分子内酯，其中几个分子显示出良好的抗癌活性。这套方法被设计成通过操纵聚酮合成酶的载体蛋白(CP)结构域来可视化、分离和操纵聚酮合成酶。使用这些技术，将用荧光或亲和标签标记、鉴定和提纯Amphidina裂解物中的合成酶。例如光解报告连接、酶促4‘-磷酸天冬氨酸裂解和磷酸天冬氨酸基转移酶抑制剂等方法将被作为一种手段来提供天然脱氧核糖核酸和全息态的纯合酶。将对纯化的合酶进行各种下游检测，以确定个别的负载域和模块化结构。这里介绍的方法学构成了一个新的蛋白质组学工具平台，用于研究来自模块合成酶的天然产物生物合成酶，包括聚酮和非核糖体肽合成酶及其杂交物。这些工具将允许进一步了解自然产物的途径，并指导代谢工程产生具有治疗意义的重要分子。

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.