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）：在我们的细菌病原体防御武器库中，社会需要新的抗生素化合物，其中许多对现有抗生素的耐药性越来越强。具有潜在新型作用机制的新型抗生素结构的最佳可能来源是自然环境，特别是土壤，其中微生物生命的多样性最大。这项研究提案推进了宏基因组学的科学，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候选药物。Lucigen公司和奥本大学的科学家正在联合四项关键技术突破，这些突破将共同产生下一代宏基因组文库，这是一种具有极大增强的抗生素发现潜力的资源。具体而言，拟议的研究将使用以下方法鉴定抗生素化合物：1）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表达的新型宽宿主范围穿梭载体; 3）产生非常大插入片段（>100 kb）的随机剪切克隆方法;和4）一种快速且改进的筛选方法来鉴定宏基因组库中产生抗生素的克隆。第I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，并针对细菌和酵母试验菌株筛选该文库以产生抗生素生产克隆的集合。第二阶段将在第一阶段成功的基础上，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确定的抗菌剂，以确定临床开发的最佳候选药物。先导候选物将具有新颖的化学结构，对多种细菌病原体（例如，MRSA），并且对真核细胞的毒性最小。所提出的研究所需的每种不同技术都已被证明是有效的;因此，这些不同方法的合成具有很高的成功概率，也代表了抗生素发现科学的重大进步。此外，从该研究产生的文库是有价值的基因组资源，其可以筛选其他生物活性化合物（例如，具有抗癌或抗病毒活性）。 公共卫生关系：使用抗生素治疗细菌性疾病在现代医学史上是一个成功的故事，但仍然需要确定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。这项研究将结合联合收割机四种不同的技术突破，使抗生素发现从微生物在自然环境中（例如，土壤），而不需要在实验室中培养不同的微生物。通过这种方式，该技术将获得由多种微生物产生的抗生素，其中许多是科学未知的，并将确定用于治疗细菌性疾病的最佳新型抗生素化合物。