Chemoenzymatic synthesis of macrolactones utilizing PolyketideSynthases (PKSs) for the generation of novel macrolide antibiotics

建议书摘要 调节大量天然产物构建的巨型合成酶代表了其中一些最 自然界中的复杂分子机器。在Sherman组中，聚酮合成酶(PKS)是来自 一个多学科的视角。PKS负责多种次生代谢物的生物合成 经济和治疗方面的重要性，包括抗生素、抗癌剂和免疫调节剂。抗生素 根据世界卫生组织(WHO)的说法，耐药性是全球卫生的最大威胁之一。这个 疾病控制和预防中心(CDC)显示，仅在美国，它就导致200多万人死亡 每年有23,000人感染和死亡。据估计，这些令人震惊的数字每隔一年就会继续递增 据估计，2050年全球将有1000万人死亡。出于这些原因，我们有动力使用PKSS 促进大环内酯类新型抗生素的设计和生成，促进开发 新的、有效的治疗方法。 PKS的不同子集产生对大环内酯类生产至关重要的大环环系统，包括 产生吡克罗霉素(Pik)、红霉素(Debs)和泰乐菌素(TYL)微生物的途径。在这 项目，我将侧重于使用合成方法来促进这些化合物的组装和 他们的类似物使用生物催化和酶工程。不同聚酮链延伸的合成 与后期生物合成机制(如糖基转移酶，P450)结合的中间体 单加氧酶)促进了对新分子的有效访问，这些新分子对 仅使用合成方法生成。PKS酶为选择性催化KEY提供了一种有效的方法 聚酮链上的转化以产生大内酯，这些大内酯随后可以转化为新的 大环内酯类抗生素。 谢尔曼实验室之前的工作表明，将PKS模块应用于 各种大内酯的产生取决于Pik硫代酯酶(TE)结构域的选择性。这些发现 建议TE在处理非天然底物以产生新奇的过程中起到守门人的作用 大循环。在拟议的研究中，我计划(1)设计和合成非天然底物来探索PKS 对底物加载、延长和环化的选择性和耐受性，以产生奇- 成员环大内酯，(2)追求TE定向进化方法，以提高总周转率，以及 扩大底物范围以产生新的大内酯产品，(3)应用化学酶法合成 不同的大环内酯类化合物，并确定其对人类细菌病原体的生物活性。这些努力将 是开发新的大环内酯类抗生素以控制和克服人类新出现的耐药性的关键 细菌病原体和提高治疗参数这类重要的抗感染药物。

Proposal Summary The megasynthases that mediate construction of a vast array of natural products represent some of the most complex molecular machines in Nature. In the Sherman group, polyketide synthases (PKSs) are of interest from a multi-disciplinary perspective. PKSs are responsible for the biosynthesis of diverse secondary metabolites of economic and therapeutic importance including antibiotics, anticancer agents and immune-modulators. Antibiotic resistance is one of the biggest threats of global health according to the World Health Organization (WHO). The Centers for Disease Control and Prevention (CDC) showed that in the US alone, it causes more than 2 million infections and 23,000 deaths a year. These alarming numbers are estimated to continue incrementally every year, with 10 million estimated deaths worldwide in 2050. For these reasons, we are motivated to utilize PKSs to facilitate the design and generation of novel antibiotics from the macrolides class to improve the development of new, effective therapeutics. A diverse subset of PKSs generate macrocyclic ring systems that are essential for macrolide production, include pathways from the Pikromycin (Pik), Erythromycin (DEBS) and Tylosin (Tyl) producing microorganisms. In this project, I will be focusing on the use of synthetic approaches to facilitate assembly of these compounds and their analogs using biocatalysis and enzyme engineering. The synthesis of diverse polyketide chain elongation intermediates in conjunction with late-stage biosynthetic machinery (e.g. glycosyltransferases, P450 monooxygenases) facilitates efficient access to a repertoire of novel molecules, which are challenging to generate using synthetic methods alone. PKS enzymes provide a powerful method to selectively catalyze key transformations on polyketide chains to generate macrolactones, which can be subsequently converted to novel macrolide antibiotics. Previous work in the Sherman lab has revealed that the primary hurdle to applying PKS modules for the production of diverse macrolactones hinges on the selectivity of the Pik thioesterase (TE) domain. These findings suggested that the TE functions as a gatekeeper in the processing of unnatural substrates to generate novel macrocycles. In the proposed research, I plan to (1) Design and synthesize unnatural substrates to explore PKS selectivity and tolerance toward substrate loading, elongation, and cyclization for the generation of odd- membered ring macrolactones, (2) Pursue a TE directed evolution approach for improved total turnover, and expansion of substrate scope to generate new macrolactone products, (3) Apply chemoenzymatic synthesis for diverse macrolides and determine their bioactivity profile against human bacterial pathogens. These efforts will be crucial to developing new macrolide antibiotics to control and overcome emerging resistance in human bacterial pathogens and to improve therapeutic parameters in this important class of anti-infective agents.