Random Shear Shuttle BAC Libraries for Antimicrobial Discovery from Soil Metageno

描述(申请人提供)：在我们抵御细菌病原体的武器库中，社会上需要新的抗生素化合物，其中许多对现有抗生素的抗药性越来越强。具有潜在新作用机制的新抗生素结构的最佳可能来源是在自然环境中，特别是具有最大微生物生命多样性的土壤中。这项研究提案推进了元基因组学，即从整个微生物群落中克隆DNA，以发现新的抗生素并确定临床开发的最佳候选药物。Lucigen公司和奥本大学的科学家们正在联合四项关键技术突破，这些突破将共同产生下一代元基因组库，这是一种具有极大潜力发现抗生素的资源。具体地说，拟议的研究将使用1)用于从土壤微生物中分离和纯化高分子量基因组DNA的改进方法；2)用于增强克隆DNA表达的新的广泛宿主范围的穿梭载体；3)随机剪切克隆方法以产生非常大的插入片段(&gt；100kb)；以及4)快速和改进的筛选方法以在元基因组文库中识别产生抗生素的克隆。第一阶段的主要目标是利用上述技术生产概念验证的下一代元基因组文库，并对该文库进行细菌和酵母测试菌株的筛选，以产生一系列产生抗生素的克隆。第二阶段将在第一阶段成功的基础上，通过从多个环境样本中构建额外的元基因组文库，筛选这些文库的抗菌活性，以及最重要的是，表征在第一阶段和第二阶段中确定的抗菌剂，以确定临床开发的最佳候选药物。候选铅将具有新颖的化学结构，对多种细菌病原体(如MRSA)具有高效力，对真核细胞的毒性最小。拟议研究所需的每一种不同技术都已被证明是单独有效的；因此，这些不同方法的合成具有很高的成功几率，也代表着抗生素发现科学的重大进步。此外，本研究建立的文库是一种有价值的基因组资源，可以在后续研究中筛选其他生物活性化合物(例如，具有抗癌或抗病毒活性的化合物)。 公共卫生相关性：在现代医学史上，使用抗生素治疗细菌疾病是一个成功的故事，但仍然需要找到能够治疗细菌感染的新抗生素，特别是由多重耐药病原体引起的感染。这项研究将结合四项不同的技术突破，通过直接采集和表达微生物的遗传途径，从自然环境(例如土壤)的微生物中发现抗生素，而不需要在实验室培养不同的微生物。通过这种方式，这项技术将获得由多种微生物产生的抗生素，其中许多微生物是科学上未知的，并将识别用于治疗细菌疾病的最佳新型抗生素化合物。

DESCRIPTION (provided by applicant): There is societal need for new antibiotic compounds in our arsenal of defenses against bacterial pathogens, many of which are increasingly resistant to existing antibiotics. The best possible source for new antibiotic structures with potentially novel mechanisms of action is within natural environments, particularly soils, which have the greatest diversity of microbial life. This research proposal advances the science of metagenomics, the cloning of DNA from entire microbial communities, to discover novel antibiotics and identify the best lead candidates for clinical development. Scientists at the Lucigen Corporation and at Auburn University are uniting four key technological breakthroughs that together will result in the next generation of metagenomic libraries, a resource with greatly enhanced potential for antibiotic discovery. Specifically, the proposed research will identify antibiotic compounds using 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from soil microorganisms; 2) a novel broad host range shuttle vector for enhanced expression of cloned DNAs; 3) a random shear cloning method to produce very large insert sizes (>100 kb); and 4) a rapid and improved screening method to identify antibiotic-producing clones within a metagenomic library. The primary Phase I objectives are to produce the proof-of-concept next generation metagenomic library using the above technologies and to screen this library against bacterial and yeast tester strains to generate a collection of antibiotic- producing clones. Phase II will build upon the success of Phase I by constructing additional metagenomic libraries from multiple environmental samples, screening these libraries for antimicrobial activity, and, most importantly, characterizing the antimicrobial agents identified in Phase I and Phase II to determine the best lead candidates for clinical development. Lead candidates will have novel chemical structures, have high potency against multiple bacterial pathogens (e.g., MRSA), and minimal toxicity for eukaryotic cells. Each of the different technologies necessary for the proposed research has been proven effective separately; therefore, the synthesis of these different methods has a high probability of success and also represents a significant advancement for the science of antibiotic discovery. Furthermore, the libraries produced from this research are a valuable genomic resource that may be screened for other bioactive compounds (e.g., with anticancer or antiviral activities) in subsequent research. PUBLIC HEALTH RELEVANCE: The use of antibiotics to treat bacterial disease has been a success story in the history of modern medicine, and yet there is still a need to identify new antibiotics that can treat bacterial infections, particularly ones caused by multi-drug resistant pathogens. This research will combine four different technological breakthroughs to enable antibiotic discovery from microorganisms in natural environments (e.g., soils) by harvesting and expressing their genetic pathways directly, without the need to cultivate the different microorganisms in a laboratory. In this way, this technology will access the antibiotics produced by a great diversity of microorganisms, many of which are unknown to science, and will identify the best novel antibiotic compounds for use in treating bacterial disease.