Molecular Analysis of Modular Polyketide Synthases

描述(申请人提供)：聚酮合成酶(PKS)使用多步骤过程来产生结构复杂的天然产品，具有广泛的制药和农业应用。在过去的十年里，人们对混合PKS的产生产生了极大的兴趣并取得了显着的成功。这些代谢系统很有吸引力，因为它们具有产生新的天然产物的潜力，具有包括药物发现和开发在内的有价值的应用。尽管如此，这一努力中一个重要且尚未解决的问题是，混合模块化PKS系统要么催化效率低下，要么干脆不起作用。正在进行的天然产物生物合成基因簇的发现，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的文库，生物活性分析可以用来鉴定那些具有显著催化活性的文库。我们还开发了一种新的化学酶方法来产生新的聚酮结构，既可以利用天然的也可以利用杂交的PKS。该项目将利用这些最新的进展，并展示如何将它们应用和组合起来，以产生新的具有生物活性的大环内酯类抗生素。这项工作将基于匹克罗霉素PKS(Pik PKS)，它生成酮内酯大内酯核心分子(一类用于治疗多重耐药病原体的抗生素)。这四个目标是：1)化学酶法合成新的大环内酯类产物；2)松弛酮酰基载体蛋白合成酶结构域的特异性，并进化其迭代特性；3)开发利用不同底物进行有效引发的新方法；以及4)高效和选择性地改变延伸单元的特异性。总体而言，该项目将提供可应用于其他PKSS系统的新技术和遗传工具，包括催化受损的杂交PKSS，并将代表着朝着将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迈出的重要一步。

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.