Molecular Analysis of Modular Polyketide Synthases

聚酮酶（PKSs）是一种多步合成天然产物的酶 具有广泛的制药和农业应用。在过去的十年里， 在产生混合PKS方面的重大兴趣和显著成功。这些代谢系统是 由于它们具有产生具有有价值应用的新天然产物的潜力， 发现和发展。然而，在这一奋进中，一个重要的和未解决的问题是， 重要的发现是，混合模块化PKS系统要么催化效率低，要么根本不活跃。 天然产物生物合成基因簇的不断发现最近提供了新的生物合成基因簇。 遗传工具，用于产生更有效的混合PKS。遗传学方法的最新进展 大的生物合成基因簇的操作现在允许快速产生杂交系统的文库， 并且生物活性测定可用于鉴定具有显著催化活性的那些。我们还 开发了一种新的化学酶促方法，用于产生新的聚酮化合物结构， 天然和混合PKS。这个项目将利用这些最新的进展，并展示他们如何能够 应用和组合以产生新的生物活性大环内酯类抗生素。这项工作将基于 在吡克罗霉素PKS（Pik PKS）上，其产生酮内酯大环内酯核心分子（一类 可用于治疗多重耐药病原体的抗生素）。 具体目标如下：1）化学酶法合成新的大环内酯类化合物; 2） 放松酮脂酰酰基载体蛋白合酶结构域的特异性，并进化其迭代性质; 3） 开发利用不同底物有效引发的新方法;以及4）有效和选择性地 改变延伸单元特异性。总的来说，这个项目将提供新的技术和遗传工具， 将适用于其他PKS系统，包括催化受损的混合PKS，并将代表一个 向利用组合生物合成和化学酶的最终目标迈出了重要的一步 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.