Combinatorial Biosynthesis of Polyketides

描述(申请人提供)：聚酮是一种复杂的天然产物，富含土壤细菌。从不到10,000个已知的聚酮化合物中，已经衍生出了大量的治疗剂，覆盖了每个重要的治疗领域。聚酮合成酶(PKS)由一组(模块)催化结构域组成，通过顺序作用构建长碳链，合成复杂的聚酮类化合物。关于PKS模块或结构域有效相互作用的规则的基本知识是存在的，但这种理解水平已经否定了许多改造所需聚酮的尝试。这项建议的长期目标是克服这一问题，并确定如何通过以有效的方式组合异源模块来构建几乎所有的PKS，从而构建任何聚酮。它的新颖性在于以半经验的方法分析了成百上千个模块组合，并选择了那些运行良好的模块组合。这是由于我们最近开发的高通量长基因合成技术，以及通用PKS模块的设计，每个模块和结构域边界都包含相同的限制位点，从而实现了组合生物合成的“积木”方法。合理的PKS工程的知识库将为生产所需的治疗药物带来巨大的好处，并使发酵衍生结构在化学合成和制造中用作中间体。这项第一阶段提案的具体目标是(1)测试具有&gt；200组合的2-模块混合PKS系统的功能，并确定那些产生聚酮产品的组合；(2)测试与生物调节系统中重叠模块获得的结果的连通性，以合理地创建更大的聚酮链。所产生的技术将在第二阶段研究中得到扩展和完善，用于生产来自植物、动物和微生物来源的天然产品，如抗生素、抗癌药物和其他治疗剂，以及用于制造当前感兴趣的聚酮的有用的合成起始材料。

DESCRIPTION (provided by applicant): Polyketides are complex natural products that are abundant in soil bacteria. From fewer than 10,000 known polyketides, a large number of therapeutic agents have been derived, covering every important therapeutic area. Complex polyketides are synthesized by polyketide synthase (PKS) consisting of sets (modules) of catalytic domains acting sequentially to build long carbon chains. Rudimentary knowledge of the rules governing the ability of PKS modules or domains to interact productively exists but the level of understanding has negated many attempts to engineer desired polyketides. The long-term aim of this proposal is to overcome this problem and to determining how to construct almost any PKS, and thus any polyketide, by combining heterologous modules in a productive fashion. Its novelty lies in the analysis of hundreds or thousands of module combinations in a semi-empirical approach and selecting those that work well. This is enabled by our recent development of high-throughput long gene synthesis technology, and a design of generic PKS modules that each contains the same restriction sites at module and domain boundaries, enabling a "building block" approach to combinatorial biosynthesis. The knowledge base for rational PKS engineering will bring enormous benefits to the manufacture of desired therapeutics as well as the availability of fermentation-derived structures to be used as intermediates in chemical synthesis and manufacturing. The specific aims of this Phase I proposal are (1) to test functionality of 2-module hybrid PKS systems with >200 combinations and identify those that produce polyketide product and (2) to test the connectivity of the results obtained with overlapping modules in the biomodular system to rationally create larger polyketide chains. The resulting technology will be expanded and perfected in Phase II research for the production of natural products derived from plant, animal, and microbial sources, such as antibiotics, anticancer drugs, and other therapeutic agents, and useful synthetic starting materials for manufacturing polyketides of current interest.