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: 8252358
• 负责人: XING-CONG LI
• 金额: $ 100万
• 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 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252358
• 关键词: Acinetobacter; American; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Antifungal Agents; Antiviral Agents; Applications Grants; Bacterial Infections; Biochemical; Biochemistry; Biological Assay; Biological Factors; Budgets; Businesses; Characteristics; Chemical Structure; Chemicals; Clinical; Cloning; Communicable Diseases; Custom; DNA; Data; Development; Economics; Environment; Escherichia coli; Evaluation; Fermentation; Genetic; Genetic Variation; Genomics; Goals; Health; Human; Incidence; Investments; Klebsiella; Lead; Libraries; Life; Medical; Medicine; Melilotus; Metagenomics; Methodology; Methods; Modeling; Molecular Weight; Multi-Drug Resistance; Mus; Nosocomial Infections; Pathway interactions; Perception; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Protocols documentation; Pseudomonas; Public Health; Research; Research Proposals; Resistance; Resources; Safety; Sales; Science; Scientist; Screening procedure; Sequence Analysis; Services; Shuttle Vectors; Site; Small Business Innovation Research Grant; Soil; Source; Stretching; Structure; Technology; Therapeutic; Time; Toxic effect; Universities; Work; antimicrobial; chemical genetics; cost; drug discovery; drug resistant bacteria; expression cloning; fight against; improved; in vivo; innovation; member; metagenome; methicillin resistant Staphylococcus aureus; microbial; microbial community; microorganism; mouse model; next generation; novel; pathogen; research and development; research clinical testing; small molecule; tool; vector; weapons

DESCRIPTION (provided by applicant): 100,000 Americans perish each year due to untreatable bacterial infections. The societal benefits of new antibiotic compounds that are effective against numerous multiple drug resistant pathogens would be significant. 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. During Phase I research scientists at Lucigen Corporation and Auburn University united four key technological breakthroughs that together resulted in the next generation metagenomic library. This library combined 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from soil microorganisms; 2) a new 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 library produced in Phase I was screened against a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the identification of 28 metagenomic clones that produce anti- MRSA compounds. 12 of these anti-MRSA clones were analyzed by sequencing and found to have very large insert sizes (average 113.5 kb) and novel genetic diversity not encountered before. Moreover, one of the clones was found to produce a novel chemical metabolite. These results are 10-100 fold more efficient than previous efforts. In Phase II a large metagenomic library will be constructed and extensively screened for antimicrobial activity against four multiple drug resistant pathogens. We expect to uncover hundreds of novel chemical entities using this approach, and lead candidates with high potency against multiple bacterial pathogens will be evaluated for efficacy using a novel in vivo assay of MRSA. These technologies represent an important 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., anticancer, antifungal or antiviral activities). PUBLIC HEALTH RELEVANCE: In the fight against microbial infectious disease we are losing ground due to the development of antibiotic resistance and our inability to find replacement drugs. The loss of life and the burden of treatment is a significant public health threat to American citizens. The proposed research unleashes a new set of tools for drug discovery that is 10-100 times more efficient than conventional technologies.

描述（由申请人提供）：由于细菌感染不足，每年有100,000名美国人每年灭亡。针对多种多种耐药病原体有效的新抗生素化合物的社会益处将是显着的。具有潜在新型作用机制的新抗生素结构的最佳来源是在自然环境中，尤其是土壤，这些土壤具有微生物寿命最大的多样性。这项研究建议推进了宏基因组学的科学，即从整个微生物群落的DNA克隆，以发现新型抗生素并确定临床开发的最佳铅候选者。在第一阶段，Lucigen Corporation和Auburn University的研究科学家联合了四个关键的技术突破，这共同导致了下一代的元基因组图书馆。该文库结合了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）一种新的广泛的寄主范围航天飞机向量，以增强克隆DNA的表达； 3）一种随机的剪切克隆方法，可产生非常大的插入尺寸（> 100 kb）； 4）一种快速而改进的筛选方法，以鉴定宏基因组文库中产生抗生素的克隆。对I期生产的文库进行了筛选，该库是针对金黄色葡萄球菌（MRSA）的临床分离物，从而鉴定出28种产生抗MRSA化合物的宏基因组克隆。通过测序分析了这些抗MRSA克隆中的12个，发现插入量很大（平均113.5 kb）和以前未遇到的新遗传多样性。此外，发现其中一个克隆会产生一种新型的化学代谢产物。这些结果比以前的工作效率更高10-100。在II期中，将构建一个大元基因组文库，并广泛筛选针对四种抗多种耐药病原体的抗菌活性。我们希望使用这种方法揭开数百种新型化学实体，并使用MRSA的新型体内测定法对多种细菌病原体的高效力进行效力。这些技术代表了抗生素发现科学的重要进步。此外，这项研究产生的文库是一种有价值的基因组资源，可以筛选其他生物活性化合物（例如抗癌，抗真菌或抗病毒药物活性）。 公共卫生相关性：在与微生物传染病作斗争中，由于抗生素耐药性的发展和无法找到替代药物的发展，我们正在失去立场。丧生和治疗负担是对美国公民的重大公共卫生威胁。拟议的研究释放了一套新的药物发现工具，其效率比传统技术高10​​-100倍。