Analytical Metagenomics Paradigm for Structure Based Screening

描述（申请人提供）：每年有10万美国人因无法治疗的细菌感染而死亡。有效对抗多种多重耐药病原体的新抗生素化合物的社会效益将是显著的。具有潜在新型作用机制的新型抗生素支架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。我们最近的研究推进了宏基因组学的科学，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。来自学术界和Lucigen公司的科学家团队联合了四项关键技术突破，共同促成了下一代宏基因组文库。该文库结合了1）用于从土壤微生物中分离和纯化高分子量基因组DNA的改进的方法; 2）用于增强克隆DNA表达的新的宽宿主范围穿梭载体; 3）产生非常大的插入片段大小（>100 kb）的随机剪切克隆方法;和4）鉴定宏基因组文库内的产芽孢杆菌克隆的快速和改进的筛选方法。针对耐甲氧西林金黄色葡萄球菌（MRSA）的临床分离株筛选文库，导致鉴定出28个产生抗MRSA化合物的宏基因组克隆，命中率为1/685。通过测序分析了这些抗MRSA克隆中的12个，发现它们具有非常大的插入片段大小（平均113.5 kb）和以前没有遇到过的新的遗传多样性。此外，其中一个克隆被发现产生一种新的代谢产物。这些结果比以前的工作效率高10-100倍。然而，现在的主要瓶颈是阐明这些化合物的结构。传统技术通常需要许多人年的努力来阐明单个结构。本第一阶段研究提案致力于将这一水平提高约10倍。事实上，我们建议通过筛选结构从下一代宏基因组文库中发现新的小分子。如果成功的话，天然产物发现的速度可以比基于功能的筛选快许多倍。 公共卫生相关性：在对抗微生物传染病的斗争中，由于抗生素耐药性的发展以及我们无法找到替代药物，我们正在节节败退。生命损失和治疗负担是对美国公民的重大公共卫生威胁。这项拟议中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.