Molecular Analysis of Modular Polyketide Synthases

聚酮合成酶(PKS)通过多步反应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， 对混合PKS的产生产生了重大的兴趣和显著的成功。这些新陈代谢系统 有吸引力，因为它们有潜力产生新的天然产品，具有有价值的应用，包括药物 发现和发展。尽管如此，这一努力中一个重大而悬而未决的问题是 重要的发现是，混合模块化PKS系统要么催化效率低下，要么干脆没有活性。 正在进行的天然产物生物合成基因簇的发现最近提供了新的 用于产生更高效的杂交PKS的遗传工具。遗传分析方法的最新进展 对大的生物合成基因簇的操纵现在允许快速生成杂交系统的文库， 生物活性测定可以用来鉴定那些具有显著催化活性的化合物。我们还有 开发了一种新的化学酶方法来产生既可利用又可利用的新型聚酮结构 原生和混合PKSS。该项目将利用这些最新的进展，并展示它们如何 被应用和组合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 关于产生酮内酯大内酯核心分子的吡克罗霉素PKS(Pik PKS) 可用于治疗多重耐药病原体的抗生素)。 这四个具体目标如下：1)化学酶法合成新的大环内酯类产物；2) 放宽酮酰基载体蛋白合成酶结构域的专一性和进化迭代特性；3) 开发利用不同底物进行高效引发的新方法；以及4)高效和选择性地 改变延伸单元的专一性。总体而言，该项目将提供新的技术和基因工具， 适用于其他PKSS系统，包括催化受损的混合PKSS，并将代表一种 向使用组合生物合成和化学酶的最终目标迈出的重要一步 用于实际药物开发的合成。

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.