ENGINEERED BIOSYNTHESIS OF NOVEL MACROLACTONE POLYKETIDE

1994年，基于我们将整个6-脱氧红细胞内酯B合成酶（DEBS）途径功能性转移到一种遗传友好（现在是基因组友好）的异源宿主中，该项目开始了。在过去五年中，我们的努力主要集中在两个方向上。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。同时，我们进行了一系列探索性研究，旨在揭示模块化聚酮合成酶（pks）的新特性。在接下来的5年里，我们建议继续我们的双轨方法，在越来越详细的水平上解剖模块化PKSs的特性，并开发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）定量进入的酰基链和单个模块的扩展剂单元特异性；（三）阐明影响这种特异性的确切结构特征；（iv）评估在模块中引入甲基转移等新化学物质的可行性；(v)理解相邻模块之间矢量链传递的机制原理；（六）阐明大环的形成是如何催化的，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，我们期望完善已建立的策略来操纵模块化PKSs的模内和模间化学，并开发新的策略。这些新一代方法很可能通过将基因工程与化学相结合来扩大分子多样性，并提高这些复杂化学物质的生产效率/产量。鉴于对基于天然产物的药物发现的重新出现的兴趣，我们的长期目标是使化学家和生物学家越来越容易获得聚酮类天然产物。

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.