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

利用聚酮化合物合成酶 (PKS) 化学酶法合成大环内酯，以生成新型大环内酯抗生素

• 批准号: 10674817
• 负责人: Maria Luisa Adrover-Castellano
• 金额: $ 1.78万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-02 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10674817
• 关键词: Address; Amides; Anabolism; Anti-Infective Agents; Antibiotic Resistance; Antibiotics; Antineoplastic Agents; Area; Biochemical; Biological Assay; Catalytic Domain; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chimeric Proteins; Clinical; Complex; Cyclization; Cytochrome P450; Deoxy Sugars; Development; Directed Molecular Evolution; Economics; Engineering; Environment; Enzymes; Erythromycin; Esters; Evaluation; Family; Gatekeeping; Generations; Genetic Transcription; Health; Human; Hydroxylation; Immunomodulators; In Vitro; Industry; Infection; Macrolides; Mediating; Metabolic Biotransformation; Methods; Mixed Function Oxygenases; Molecular; Molecular Machines; Natural Products; Nature; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Positioning Attribute; Process; Production; Property; Proteins; Rare Earth Metals; Reagent; Research; Resistance; Ribosomes; Series; Synthesis Chemistry; System; Testing; Therapeutic; Tylosin; Variant; Work; World Health Organization; analog; appendage; aqueous; catalyst; cost; design; flexibility; global health; glycosylation; glycosyltransferase; high throughput screening; improved; interest; member; microorganism; multidisciplinary; mutant; novel; novel therapeutics; oxidation; pathogenic bacteria; pathogenic microbe; picromycin; polyketide synthase; polyketides; scaffold; secondary metabolite; small molecule; synthetic enzyme; therapeutically effective; translation assay; virtual; wasting

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.

提案摘要 介导大量天然产品的构造的巨型酶是最多的 自然界中的复杂分子机。在谢尔曼组中，聚酮化合物合酶（PKSS）来自 多学科的观点。 PKS负责多种二级代谢物的生物合成 经济和治疗重要性，包括抗生素，抗癌药和免疫调节剂。抗生素 根据世界卫生组织（WHO）的数据，抵抗是全球健康的最大威胁之一。这 疾病控制与预防中心（CDC）表明，仅在美国，它就会导致超过200万 每年感染和23,000人死亡。这些令人震惊的数字估计会逐步继续 一年，2050年全球估计死亡人数为1000万。由于这些原因，我们有动力利用PKSS 为了促进大环内酯类类的新型抗生素的设计和产生以改善发展 新的，有效的治疗学。 PKSS的各种子集生成大环内酯类生产必不可少的大环环系统，包括 pikromycin（PIK），红霉素（DEB）和泰糖苷（Tyl）产生微生物的途径。在这个 项目，我将重点介绍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。多酮链链伸长的合成 中间体与后期生物合成机械（例如糖基转移酶，P450 单加氧酶）有助于有效地获得新分子的曲目，这对 仅使用合成方法生成。 PKS酶提供了一种有力催化钥匙的强大方法 聚酮化合物链的转换以生成大分子，随后可以转换为新颖 大环内酯类抗生素。 谢尔曼实验室的先前工作表明，应用PKS模块的主要障碍 多种大乳酸酯的产生取决于PIK硫酯酶（TE）结构域的选择性。这些发现 建议TE在处理不自然底物的处理中充当守门人以生成新颖的 大环。在拟议的研究中，我计划（1）设计和合成不自然的底物以探索PKS 对产生奇数的选择性和对底物载荷，伸长和环化的耐受性 成员环大管，（2）采用TE定向进化方法，以改善总营业额，并且 底物范围的扩展以生成新的大乳酸酯产品，（3）将化学酶合成应用 各种大环内酯类药物，并确定其针对人类细菌病原体的生物活性特征。这些努力会 对于开发新的大环内酯类抗生素以控制和克服人类的耐药性至关重要 细菌病原体并改善这种重要的抗感染剂中的治疗参数。