Analytical Metagenomics Paradigm for Structure Based Screening

描述(申请人提供)：每年有10万美国人死于无法治愈的细菌感染。对许多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型抗生素支架的最佳来源是在自然环境中，特别是拥有最多微生物生命的土壤中。我们最近的研究推动了元基因组学的科学发展，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。来自学术界和Lucigen公司的一个科学家团队联合了四项关键技术突破，共同产生了下一代元基因组文库。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)新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，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，以在元基因组库中鉴定产生抗生素的克隆。该文库针对耐甲氧西林金黄色葡萄球菌(MRSA)的临床分离株进行筛选，结果鉴定出28个产生抗MRSA化合物的元基因组克隆，命中率为1/685。对这些抗MRSA克隆中的12个进行了测序分析，发现它们具有非常大的插入片段(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能产生一种新的代谢物。这些结果比以前的努力效率高出10-100倍。然而，目前的主要瓶颈是阐明这些化合物的结构。传统技术往往需要花费很多人年的努力来阐明一个单一的结构。这个第一阶段的研究计划致力于将这一水平提高大约十倍。事实上，我们建议通过筛选结构，从下一代元基因组学文库中寻找新的小分子。如果成功，与基于功能的筛选相比，自然产品发现的速度可能会加快许多倍。 与公共卫生相关：在与微生物传染病的斗争中，由于抗生素耐药性的发展和我们无法找到替代药物，我们正在失去优势。生命损失和治疗负担是对美国公民的重大公共健康威胁。这项拟议的研究释放了一套新的药物发现工具，其效率是传统技术的10-100倍。

DESCRIPTION (provided by applicant): Each year, 100,000 Americans perish 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 scaffolds with potentially novel mechanisms of action is within natural environments, particularly soils, which have the greatest diversity of microbial life. Our recent research 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. A team of scientists from academia and Lucigen Corporation have 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 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, a hit rate of 1 in 685. Twelve 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 metabolite. These results are 10-100 fold more efficient than previous efforts. However, the primary bottleneck now is elucidating the structure of these compounds. Conventional technologies often require many man-years of effort to elucidate a single structure. This Phase I research proposal endeavors to improve that level by approximately ten-fold. In fact we propose to find new small molecules from this next generation metagenomics library by screening for structures. If successful the rate of natural product discovery could be accelerated many fold compared to functional based screens. 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.