Random Shear Shuttle BAC Libraries for Antimicrobial Discovery from Soil Metageno

描述（由申请人提供）：每年有10万美国人死于无法治疗的细菌感染。对多种多重耐药病原体有效的新型抗生素化合物的社会效益将是显著的。具有潜在新作用机制的新抗生素结构的最佳来源是在自然环境中，特别是在微生物生命多样性最大的土壤中。这项研究计划推动了宏基因组学的发展，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。在第一阶段的研究中，Lucigen公司和奥本大学的科学家们联合了四项关键技术突破，共同形成了下一代宏基因组库。该文库结合了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)一种新的宽宿主范围穿梭载体，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尺寸（> 100kb）；4)在宏基因组文库中鉴定产生抗生素的克隆的快速和改进的筛选方法。I期产生的文库与耐甲氧西林金黄色葡萄球菌（MRSA）临床分离物进行了筛选，鉴定出28个产生抗MRSA化合物的宏基因组克隆。对其中12个抗mrsa克隆进行测序分析，发现它们具有非常大的插入长度（平均113.5 kb）和前所未有的遗传多样性。此外，其中一个克隆被发现产生一种新的化学代谢物。这些结果比以前的努力效率高10-100倍。在第二阶段，将构建一个大型宏基因组文库，并广泛筛选对四种多重耐药病原体的抗菌活性。我们希望利用这种方法发现数百种新的化学实体，并利用一种新的MRSA体内试验来评估对多种细菌病原体具有高效能的候选药物的有效性。这些技术代表了抗生素发现科学的重要进步。此外，本研究产生的文库是一种宝贵的基因组资源，可用于筛选其他生物活性化合物（如抗癌、抗真菌或抗病毒活性）。

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).