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

提案摘要 介导大量天然产物构建的巨型合成酶代表了一些最重要的合成酶。 自然界中复杂的分子机器。在 Sherman 小组中，聚酮合酶 (PKS) 引起了人们的兴趣 多学科的视角。 PKS 负责多种次级代谢产物的生物合成 经济和治疗重要性，包括抗生素、抗癌剂和免疫调节剂。抗生素 据世界卫生组织（WHO）称，耐药性是全球健康的最大威胁之一。的 疾病控制与预防中心 (CDC) 表明，仅在美国，它就导致了超过 200 万人 每年有 23,000 人感染并死亡。这些令人震惊的数字预计每年都会持续增加 年，预计 2050 年全球将有 1000 万人死亡。出于这些原因，我们有动力利用 PKS 促进大环内酯类新型抗生素的设计和生成，以改善开发 新的、有效的疗法。 PKS 的不同子集生成对大环内酯生产至关重要的大环系统，包括 来自产生匹克霉素 (Pik)、红霉素 (DEBS) 和泰乐菌素 (Tyl) 的微生物的途径。在这个 项目中，我将重点关注使用合成方法来促进这些化合物的组装和 它们的类似物使用生物催化和酶工程。多种聚酮化合物链延长的合成 与后期生物合成机器结合的中间体（例如糖基转移酶、P450 单加氧酶）有助于有效地获取一系列新分子，这对于 单独使用合成方法生成。 PKS 酶提供了一种强大的方法来选择性催化关键 聚酮化合物链上的转化生成大环内酯，随后可转化为新型大环内酯 大环内酯类抗生素。 Sherman 实验室之前的工作表明，将 PKS 模块应用于 多种大环内酯的产生取决于 Pik 硫酯酶 (TE) 结构域的选择性。这些发现 表明 TE 在处理非天然底物以产生新的 大环化合物。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 对底物负载、伸长和环化的选择性和耐受性，以产生奇数 元环大内酯，(2) 追求 TE 定向进化方法以提高总周转率，以及 扩大底物范围以生成新的大环内酯产物，（3）应用化学酶法合成 不同的大环内酯类化合物并确定它们针对人类细菌病原体的生物活性。这些努力将 对于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至关重要 细菌病原体并改善这一类重要抗感染药物的治疗参数。

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.