Random Shear Shuttle BAC Libraries for Antimicrobial Discovery from Soil Metageno

用于从土壤 Metageno 中发现抗菌剂的随机剪切穿梭 BAC 文库

• 批准号: 7801784
• 负责人: Chengcang Charles Wu
• 金额: $ 16.47万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7801784
• 关键词: Anti-Bacterial Agents; Antibiotics; Antifungal Agents; Antiviral Agents; Bacterial Infections; Biological Assay; Chemical Structure; Chemicals; Clinical; Cloning; Collection; Copyright; Custom; DNA; Development; Environment; Escherichia coli; Eukaryotic Cell; Genetic; Genomics; Genotype; Harvest; Laboratories; Lead; Libraries; Life; Mediating; Metagenomics; Methodology; Methods; Modern History (Medicine); Molecular Weight; Multi-Drug Resistance; Pathway interactions; Phase; Probability; Protocols documentation; Pseudomonas putida; Recombinants; Relative (related person); Research; Research Proposals; Resistance; Resources; Sales; Sampling; Science; Scientist; Screening procedure; Services; Shuttle Vectors; Small Business Innovation Research Grant; Soil; Source; Structure; Technology; Testing; Time; Toxic effect; United States National Institutes of Health; Universities; Yeasts; antimicrobial; antimicrobial drug; base; commercialization; denaturing gradient gel electrophoresis; expression cloning; improved; innovation; methicillin resistant Staphylococcus aureus; microbial; microbial community; microorganism; next generation; novel; pathogen; public health relevance; small molecule; soil sampling; success; vector

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.

描述（由申请人提供）：社会需要新的抗生素化合物来防御细菌病原体，其中许多病原体对现有抗生素的耐药性越来越强。具有潜在新颖作用机制的新型抗生素结构的最佳来源是自然环境，特别是微生物生命多样性最丰富的土壤。这项研究提案推进了宏基因组学科学，即从整个微生物群落中克隆 DNA，以发现新型抗生素并确定临床开发的最佳先导候选药物。 Lucigen 公司和奥本大学的科学家正在将四项关键技术突破结合起来，这些突破将产生下一代宏基因组库，这是一种大大增强抗生素发现潜力的资源。具体来说，拟议的研究将使用以下方法鉴定抗生素化合物：1）改进的方法，用于从土壤微生物中分离和纯化高分子量基因组 DNA； 2) 一种新型的广泛宿主范围的穿梭载体，用于增强克隆 DNA 的表达； 3) 随机剪切克隆方法，产生非常大的插入片段（>100 kb）； 4) 一种快速且改进的筛选方法，用于鉴定宏基因组文库中产生抗生素的克隆。第一阶段的主要目标是使用上述技术产生概念验证的下一代宏基因组文库，并针对细菌和酵母测试菌株筛选该文库，以生成一系列产生抗生素的克隆。第二阶段将建立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宏基因组文库，筛选这些文库的抗菌活性，最重要的是，表征第一阶段和第二阶段中确定的抗菌药物，以确定最佳的先导候选药物。临床开发。主要候选药物将具有新颖的化学结构，对多种细菌病原体（例如 MRSA）具有高效能，并且对真核细胞的毒性最小。拟议研究所需的每项不同技术均已分别被证明是有效的；因此，这些不同方法的合成成功的可能性很高，也代表了抗生素发现科学的重大进步。此外，本研究产生的文库是宝贵的基因组资源，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）。 公共卫生相关性：使用抗生素治疗细菌性疾病是现代医学史上的一个成功故事，但仍然需要找到可以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细菌感染。这项研究将结合四种不同的技术突破，通过直接收获和表达微生物的遗传途径，从自然环境（例如土壤）中的微生物中发现抗生素，而无需在实验室中培养不同的微生物。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未知的，并将鉴定出用于治疗细菌性疾病的最佳新型抗生素化合物。