ENGINEERED BIOSYNTHESIS OF NOVEL MACROLACTONE POLYKETIDE

新型大分子内酯聚酮的工程生物合成

• 批准号: 6376140
• 负责人: CHAITAN KHOSLA
• 金额: $ 33.77万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-05 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6376140
• 关键词: Streptomyces; X ray crystallography; acyltransferase; chemical condensation; combinatorial chemistry; drug design /synthesis /production; drug discovery /isolation; enzyme complex; enzyme mechanism; enzyme structure; gene mutation; genetic library; genetic manipulation; macrolide antibiotics; microorganism metabolism; polyketide synthase; protein engineering; thioester

In 1994 this project was initiated based on our ability to functionally transfer the entire 6- deoxyerythronolide B synthase (DEBS) pathway into Streptomyces coelicolor, a genetics friendly (and now genomics-friendly) heterologous host. During the past 5 years our efforts have primarily been focused along two directions. On one hand we have sought to develop genetic and biochemical tools to study and manipulate DEBS with the aim of generating new "unnatural" natural products. At the same time, we have conducted a variety of exploratory studies aimed at uncovering novel properties of modular polyketide synthases (PKSs). Over the next 5 years, we propose to continue our twin-track approach of dissecting, at increasing level of detail, the properties of modular PKSs, and developing useful technology for combinatorial biosynthesis. Our plans include (i) the development and application of genetic screens for DEBS mutants with novel properties; (ii) quantification of the incoming acyl chain and the extender unit specificity of individual modules; (iii) elucidating the precise structural features that influence this specificity; (iv) evaluating the feasibility of introducing new chemistry such as methyltransfer into modules; (v) understanding the mechanistic principles underlying vectorial chain transfer between adjacent modules; (vi) elucidating how macrocycle formation is catalyzed, and the molecular recognition features associated with this amazing reaction; and (vii) (if possible) obtaining high-resolution X-ray crystal structures of DEBS and its variants. In the process we expect to refine established strategies for manipulating the intramodular and intermodular chemistry of modular PKSs, and also develop new ones. These next generation methods are likely to expand the molecular diversity attainable by interfacing genetic engineering with chemistry, and also increase the efficiency/yield with which these complex chemicals are made. Given the re-emerging interest in natural product based drug discovery, our long-term goal is to make polyketide natural products increasingly accessible to chemists and biologists alike.

1994 年，我们启动了该项目，因为我们能够将整个 6-脱氧赤酮内酯 B 合酶 (DEBS) 途径功能性转移到天蓝色链霉菌中，这是一种遗传学友好（现在是基因组学友好）的异源宿主。在过去的五年里，我们的努力主要集中在两个方向。一方面，我们寻求开发遗传和生化工具来研究和操纵 DEBS，目的是产生新的“非自然”天然产品。与此同时，我们进行了各种探索性研究，旨在揭示模块化聚酮合酶（PKS）的新特性。在接下来的 5 年里，我们建议继续采用双轨方法，以越来越详细的水平剖析模块化 PKS 的特性，并开发用于组合生物合成的有用技术。我们的计划包括 (i) 开发和应用具有新特性的 DEBS 突变体的遗传筛选； (ii) 引入酰基链的量化和各个模块的延伸单元特异性； (iii) 阐明影响这种特殊性的精确结构特征； (iv) 评估将甲基转移等新化学物质引入模块的可行性； (v) 了解相邻模块之间向量链传输的机械原理； (vi) 阐明如何催化大环形成，以及与这一惊人反应相关的分子识别特征； (vii)（如果可能）获得 DEBS 及其变体的高分辨率 X 射线晶体结构。在此过程中，我们期望完善用于操纵模块化 PKS 的模块内和模块间化学的既定策略，并开发新的策略。这些下一代方法可能会通过将基因工程与化学结合来扩大分子多样性，并提高制造这些复杂化学品的效率/产量。鉴于人们对基于天然产物的药物发现重新产生兴趣，我们的长期目标是让化学家和生物学家越来越容易获得聚酮化合物天然产物。