ENGINEERED BIOSYNTHESIS OF NOVEL MACROLACTONE POLYKETIDE

该项目的长期目标是： L.为了了解酶的结构如何控制催化特异性， 模块化聚酮酶（PKS），一个多功能的 参与多种天然产物生物合成的酶; 2.利用这些知识来合成合理设计的 聚酮化合物;和 3.使用相同的信息来产生化学多样性 通过设计和构建突变PKS的基因文库， 具有不同的催化特异性。 这里提出的研究模型系统是脱氧核糖核酸 B合酶（DEBS; 10，283 aa），其负责 6-dEB（6-deoxyanalogrone，6-deoxyanalogrone B），14元大环内酯前体， 红霉素这种酶复合物由3种非常大的蛋白质组成， 包括29个推定的活性位点。最近，有人表示， 异源宿主使用适合于方便的遗传策略， 诱变大量的6-dEB和8，8a-脱氧齐墩果酸 （其与6dEB相同，除了乙酸酯起始物单元）是 从重组系统中分离。 利用上述表达系统，针对本建议的具体目标 期间包括： 1.一系列突变体的构建和分析，旨在阐明 模块化PKS内的催化自由度， 2.第二模块PKS（呋喃霉素合酶; FS）使用上述表达系统， 3.来自DEBS的模块（或其结构域）的组合表达和 FS和 4.体外结构和机制研究系统的开发 DEBS及其突变体的研究。 除了对结构-功能提供有价值的见解外， 模块化PKS内的关系，这里提出的研究也可以 对药物发现和改进领域产生影响。在 特别是，这项工作的结果将阐明。 模块化PKS内的生物合成自由度。同时对 在这些研究期间开发的工具应适用于 操作和分析来自放线菌的其他模块PKS， 不发达的基因工具反过来，这些概念和 技术进步可能会导致遗传学的发展 化学多样性的工程战略， 医药上重要的一类化合物。

The long-term goals of this project are: l. To understand how enzyme structure controls catalytic specificity in modular polyketide synthases (PKSs), a large family of multifunctional enzymes involved in biosynthesis of diverse natural products; 2. To use this knowledge for the biosynthesis of rationally designed polyketides; and 3. To use the same information for the generation of chemical diversity through the design and construction of genetic libraries of mutant PKSs with varying catalytic specificities. The model system for the research proposed here is the deoxyerythronolide B synthase (DEBS; 10,283 aa), which is responsible for the biosynthesis of 6 deoxyerythronolide B (6-dEB), the 14 membered macrolide precursor of erythromycin. This enzyme complex is made up of 3 very large proteins and includes 29 putative active sites. Recently, it has been expressed in a heterologous host using a genetic strategy amenable to convenient mutagenesis. Substantial quantities of 6-dEB and 8,8a-deoxyoleandolide (which is identical to 6dEB except for an acetate starter unit) were isolated from the recombinant system. Using the above expression system, the specific aims for this proposal period include: 1. Construction and analysis of a series of mutants aimed at elucidating the catalytic degrees of freedom within a modular PKS, 2. Heterologous expression of a second modular PKS (furanomycin synthase; FS) using the above expression system, 3. Combinatorial expression of modules (or domains thereof) from DEBS and FS, and 4. Development of an in vitro system for structural and mechanistic studies on DEBS and its mutants. In addition to providing valuable insights into structure-function relationships within modular PKSs, the research proposed here could also have an impact on the areas of drug discovery and improvement. In particular, the results of this work will shed light upon the. biosynthetic degrees of freedom within modular PKSs. At the same time, the tools developed during these studies should be applicable for the manipulation and analysis of other modular PKSs from actinomycetes with poorly developed genetic tools. In turn, these conceptual and technological advances could lead to the development of genetic engineering strategies for the generation of chemical diversity within a medicinally important class of compounds.