Development and application of a functional metagenomic antibiotic discovery pipeline

：本提案中概述的工作旨在建立一个 新的交钥匙功能元基因组抗生素发现平台--从生物合成的丰富 从丰富的文库中鉴定新的抗生素，到元基因组文库的多样性。 不幸的是，在大多数测序细菌中，只有不到2%的基因组用于次生代谢。 因此，推而广之，在任何Edna文库中发现的克隆中，只有一小部分预计包含基因 参与小分子生物合成的。元基因组文库作为生物活性小分子物质来源的应用 分子将从新的方法中得到极大的改进，这些方法能够丰富含有功能的克隆 生物合成系统。虽然天然产品中使用了令人难以置信的多样化的酶集合 生物合成、功能冗余的酶普遍存在于不同的基因簇中。这些是多余的 酶是常见的生物合成线索，通过这些线索，我认为Edna文库天然缺乏 基因簇内容，可以丰富包含生物合成基因簇的克隆。在第一个目标中，我们 建议使用天然产物调节的转录因子(例如，TetR同源物)和互补 功能冗余生物合成基因选择性丰富含天然产物克隆的BAC文库 生物合成基因簇。在具体目标2中，我们将确定具有抗抗生素活性的新型抗生素 耐药细菌病原体。

: The work outlined in this proposal is designed to establish a new turnkey functional metagenomic antibiotic discovery platform -- from the enrichment of biosynthetic diversity in metagenomic libraries through to the identification of novel antibiotics from enriched libraries. Unfortunately, in most sequenced bacteria less than 2% of the genome is devoted to secondary metabolism. Thus, by extension, only a small fraction of the clones found in any eDNA library is expected to contain genes involved in small molecule biosynthesis. The utility of metagenomic libraries as sources of bioactive small molecules would improve greatly from new methods capable of enriching for clones containing functional biosynthetic systems. While an incredibly diverse collection of enzymes is used in natural product biosynthesis, functionally redundant enzymes are commonly found in diverse gene clusters. These redundant enzymes represent common biosynthetic threads through which I propose that eDNA libraries, naturally poor in gene cluster content, can be enriched for clones containing biosynthetic gene clusters. In the first aim we propose to use natural product regulated transcription factors (e.g., TetR homologs) and complementation of functionally redundant biosynthetic genes to selectively enrich BAC libraries for clones containing natural product biosynthetic gene clusters. In specific Aim 2, we will identify novel antibiotics with activities against antibiotic resistant bacterial pathogens.