Generation of a library of recombineered novel polyketides and non-ribosomal peptides

由微生物如细菌和真菌制备的聚酮化合物天然产物代表了用于治疗危及生命的疾病的抗生素和抗癌药物的主要来源。它们还可作为移植手术后的免疫抑制剂、治疗发展中国家寄生虫病的药物以及用于作物保护的无害环境杀虫剂。尽管取得了历史性的成功，但聚酮化合物在新药开发中的应用仍然不足，部分原因是缺乏可重复和可扩展的开发过程，无法与现代制药和农业化学工业使用的其他技术竞争。该项目将帮助我们的工业合作伙伴Isomerase开发新的遗传方法，以修饰制造聚酮化合物天然产物的细菌机器（酶），并使我们更深入地了解导致其自然进化的过程。这些酶作为装配线与一个单独的“部分”或“域”执行组装过程中的每个化学步骤。为了产生完整的聚酮化合物，这些结构域被组装在重复模块中，所述重复模块使用来自细胞的简单前体，将它们键合在一起，然后修饰它们以产生复杂的最终分子。我们的建议将建立在DNA测序的最新进展，使长，重复的DNA片段能够准确，快速地测序。这对于构成编码聚酮合酶的基因的DNA的长而重复区域非常重要，聚酮合酶包括一些自然界已知的最大蛋白质。我们将对通过早期应用异构酶的“重组工程”技术产生的10种菌株的基因组进行测序。令人惊讶的是，这些产生了基于单一天然结构的结构多样的化合物库，并且仅来自一个实验。我们将分析重组的基因，以确定重组热点，并获得对重组工程的天然和诱导实例的理解。这也将有助于我们设计更好的合理实验，对聚酮酶进行离散改变，以产生特定的靶向化合物。用于治疗耐药细菌和新出现的病毒的感染剂，这些病毒代表着直接和令人担忧的公共卫生威胁。

Polyketide natural products made by microorganisms such as bacteria and fungi represent a major source of antibiotic and anticancer pharmaceuticals for the treatment of life threatening disease. They are also important as immunosuppressant agents after transplant surgery, as drugs for the treatment of parasitic diseases in developing countries and as environmentally benign insecticides for use in crop protection. Despite this historical success polyketides are underutilised for the discovery of new medicines, in part due to the perceived lack of a repeatable and scalable process for their development that can compete with other technologies used by the modern pharmaceutical and agrochemical industries.This project will aid our industrial partner Isomerase in the development of new genetic methods to modify bacterial machinery (enzymes) that make polyketide natural products, and provide us with a deeper understanding of the processes leading to their natural evolution. These enzymes act as assembly lines with an individual 'part' or 'domain' to perform each chemical step in the assembly process. In order to produce a complete polyketide these domains are assembled in repeating modules which use simple precursors from the cell, bond these together and then modify them in order to generate the complex final molecule. Our proposal will build on recent advances in DNA sequencing which enables long, repetitive stretches of DNA to be sequenced accurately and quickly. This is important for the long and repetitive regions of DNA making up the genes that encode polyketide synthase enzymes which include some of the largest proteins known to nature. We will sequence the genomes of 10 strains generated through early application of Isomerase's 'recombineering' technology. Amazingly these produce a structurally diverse library of compounds based on a single nautural structure and were derived from just a single experiment. We will analyse the recombineered genes in order to identify the recombination hotspots and derive an understanding of natural and induced examples of recombineering This will also aid us in devising better rational experiments for making discrete changes to polyketide synthases in order to produce specific targeted compounds.The compounds produced by this and subsequent experiments have the potential to be leads for the discovery of new medicines including anti-infective agents for the treatment of drug resistant bacteria and emerging viruses which represent immediate and alarming public health threats.