Discovery of Antibiotics from Soil Microbiomes Using Metagenomics

项目摘要：耐药感染每年在美国导致超过6.5万人死亡， 识别能够对抗抗药性细菌病原体的新抗生素是非常迫切的。 需要的。细菌天然产物是一种良好的铅结构，具有悠久的历史 临床上有用的抗生素的开发，但不幸的是，从 近年来，培养的细菌数量急剧减少。同时，成本效益的出现 高通量测序和对细菌次生代谢物日益复杂的理解 在寻找新的天然产物方面，生物合成带来了两个重要的启示：第一， 大多数培养细菌的生物合成潜力，通过生物合成基因簇的数量来判断 在测序的基因组中观察到的，远远超过之前估计的；第二，细菌的数量 大多数环境中的物种至少比容易找到的物种数量多一百倍 有教养的。这些观察表明，传统的天然产品筛选方法依赖于 实验室从环境中随机培养细菌菌株，还没有完全实现 地球微生物群的生物合成潜力以及细菌方法的根本变化 因此，发现天然产物是必要的。因此，我的实验室试图开发出方法 允许从环境样本中发现天然产品，而不需要菌株分离和 有教养。我建议用来从元基因组文库中获取小分子的方法是 一般分为两大类：1)基于序列的元基因组学，它依赖于DNA序列 相似性以识别包含特定感兴趣基因的克隆和2)基于功能的元基因组学，其 依赖于在表型分析中随机、无偏见地筛选单个Edna克隆来识别克隆 产生生物活性代谢物。使用基于序列的方法，我们可以询问元基因组 新的基因簇(即新的抗生素)，以及编码临床上相关的同系物的簇 天然产物(即提高临床上重要的抗生素类的效用)。我们的功能性筛查 研究将集中在开发建立富含天然物质的元基因组文库的方法 生产基因簇，以促进对新型抗生素的更有效筛选。对于这个提案，我们将使用 我们现有的管道和改进的协议，当它们上线时，以识别新的天然产品 提高了对抗抗药性细菌病原体的活性。我们无疑才刚刚开始起步。 隐藏在环境细菌基因组中的表面。这里的工作将大大增加 获取隐藏在环境中的天然产品，并最终产生一系列新颖的 具有更好地治疗抗药性细菌病原体的潜力的天然抗生素。 好了！

Project summary: Drug-resistant infections kill more than 65,000 people in the United States per year and the identification of new antibiotics capable of combating antibiotic resistant bacterial pathogens is desperately needed. Bacterial natural products have a long proven history of being good lead structures for the development of clinically useful antibiotics but, unfortunately, the discovery of novel natural products from cultured bacteria has dramatically slowed in recent years. During the same time, the advent of cost effective high-throughput sequencing and an increasingly sophisticated understanding of bacterial secondary metabolite biosynthesis have led to two important revelations with respect to the search for new natural products: first, that the biosynthetic potential of most cultured bacteria, as judged by the number of biosynthetic gene clusters observed in sequenced genomes, is far greater than previously estimated; second, that the number of bacterial species in most environments is at least one hundred times greater than the number of species that is readily cultured. These observations indicate that conventional natural product screening approaches, which rely on laboratory culturing of random bacterial strains from the environment, have not come close to realizing the full biosynthetic potential of the earth's microbiome and that a fundamental change in the approach to bacterial natural products discovery is therefore needed. Consequently, my laboratory has sought to develop methods to allow natural product discovery from environmental samples, without the need for strain isolation and cultivation. The approaches I propose to use to harvest small molecules from metagenomic libraries are divided into two general categories: 1) Sequence-based metagenomics, which relies on DNA sequence similarity to identify clones containing a specific gene of interest and 2) Function-based metagenomics, which relies on the random, unbiased screening of individual eDNA clones in phenotypic assays to identify clones producing bioactive metabolites. Using sequence-based approaches we can interrogate metagenomes for novel gene clusters (i.e., new antibiotics), as well as clusters that encode congeners of clinically relevant natural product (i.e., improve the utility of clinically important classes of antibiotics). Our functional screening studies will focus on developing methods for creating metagenomic libraries that are enriched in natural product gene clusters to facilitate more efficient screening for novel antibiotics. For this proposal, we will use our existing pipelines and improved protocols, as they come on-line, to identify novel natural products with improved activities against antibiotic resistant bacterial pathogens. We have undoubtedly just begun to scratch the surface of what lies hidden in the genomes of environmental bacteria. The work here will greatly increase access to the natural products that remain hidden in the environment and ultimately yield a series of novel natural antibiotics with the potential to better treat antibiotic resistant bacterial pathogens. !