Molecular Analysis of Modular Polyketide Synthases

描述（由申请人提供）：聚酮合成酶（pks）使用多步骤过程生成结构复杂的天然产物，具有广泛的制药和农业应用。在过去的十年里，人们对混合型PKSs的产生产生了浓厚的兴趣，并取得了显著的成功。这些代谢系统是有吸引力的，因为它们有潜力产生新的天然产物，具有包括药物发现和开发在内的有价值的应用。然而，在这一努力中，一个重要且未解决的问题是，混合模块化PKS系统要么催化效率低下，要么就是不活跃。天然产物生物合成基因簇的不断发现，最近为产生更高效的杂交pks提供了新的遗传工具。大型生物合成基因簇的遗传操作方法的最新进展现在允许快速生成杂交系统文库，生物活性测定可用于识别具有显著催化活性的基因。我们还开发了一种新的化学酶方法来生成新的聚酮结构，可以利用原生和杂交pks。该项目将利用这些最新进展，并展示如何将它们应用和结合起来，以产生新的生物活性大环内酯类抗生素。这项工作将以产生酮内酯内酯核心分子的匹克霉素PKS （Pik PKS）为基础（一类用于治疗多重耐药病原体的抗生素）。具体目标如下：1)化学酶法合成大环内酯类新产品；2)放宽酮酰基酰基载体蛋白合成酶结构域的特异性，进化其迭代特性；3)开发利用不同底物的高效引发新方法；4)有效和选择性地改变扩展器单元特异性。总体而言，该项目将提供新的技术和遗传工具，可应用于其他PKSs系统，包括催化受损的杂交PKSs，并将代表着使用组合生物合成和化学酶合成用于实际药物开发的最终目标的重要一步。

DESCRIPTION (provided by applicant): Polyketide synthases (PKSs) use a multi-step process to generate structurally complex natural products with wide ranging pharmaceutical and agricultural applications. Over the past decade there has been significant interest and notable successes in the generation of hybrid PKSs. These metabolic systems are attractive due to their potential to generate new natural products with valuable applications including drug discovery and development. Nonetheless, a significant and unresolved problem in this endeavor has been the significant finding that hybrid modular PKS systems are either catalytically inefficient, or simply inactive. The ongoing discovery of natural product biosynthetic gene clusters has recently provided new sets of genetic tools for the generation of more efficient hybrid PKSs. Recent advances in methods for genetic manipulation of large biosynthetic gene clusters now permit rapid generation of libraries of hybrid systems, and bioactivity assays can be used to identify those with significant catalytic activity. We have also developed a new chemoenzymatic approach for generating novel polyketide structures that can utilize both native and hybrid PKSs. This project will capitalize on these recent advances and demonstrate how they can be applied and combined to generate new biologically active macrolide antibiotics. The work will be based on the pikromycin PKS (Pik PKS) that generates a ketolide macrolactone core molecule (a class of antibiotics useful for treatment of multidrug resistant pathogens). The four specific aims are as follows: 1) chemoenzymatic synthesis of new macrolide products; 2) relaxing the specificity, and evolving iterative properties of ketoacyl acyl carrier protein synthase domains; 3) developing new methods for efficient initiation utilizing different substrates; and 4) efficiently and selectively altering extender unit specificity. Overall this project will provide new techniques and genetic tools that can be applied to other PKSs systems, including catalytically impaired hybrid PKSs, and will represent an important step towards the ultimate goal of using of combinatorial biosynthesis and chemoenzymatic synthesis for practical drug development.