Combinatorial Biosynthesis of Polyketides

描述（由申请人提供）：聚酮化合物是复杂的天然产物，在土壤细菌中含量丰富。从不到10，000种已知的聚酮化合物中，已经衍生出大量的治疗剂，涵盖了每个重要的治疗领域。复杂的聚酮化合物由聚酮化合物合成酶（PKS）合成，所述聚酮化合物合成酶由催化结构域的组（模块）顺序作用以构建长碳链组成。管理PKS模块或域的能力的规则的基本知识，以有效地相互作用存在，但理解的水平已经否定了许多试图工程所需的聚酮化合物。该建议的长期目标是克服这个问题，并确定如何通过以生产方式组合异源模块来构建几乎任何PKS，从而构建任何聚酮化合物。它的新奇在于以半经验的方法分析数百或数千个模块组合，并选择那些工作良好的模块。这是通过我们最近开发的高通量长基因合成技术和通用PKS模块的设计实现的，所述通用PKS模块各自在模块和结构域边界处包含相同的限制性位点，从而实现组合生物合成的“构建块”方法。合理PKS工程的知识基础将为所需治疗剂的生产以及发酵衍生结构的可用性带来巨大的好处，这些结构将用作化学合成和生产中的中间体。该I期提案的具体目的是（1）测试具有>200种组合的2-模块混合PKS系统的功能性，并鉴定产生聚酮化合物产物的那些组合，以及（2）测试生物模块系统中重叠模块获得的结果的连通性，以合理地产生更大的聚酮化合物链。由此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药物，以及用于制造当前感兴趣的聚酮化合物的有用合成起始材料。

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.