Development and application of a functional metagenomic antibiotic discovery pipeline

：本建议中概述的工作旨在建立一个 新的交钥匙功能宏基因组抗生素发现平台--从生物合成的富集 从宏基因组文库的多样性到从富集文库中鉴定新的抗生素。 不幸的是，在大多数测序的细菌中，只有不到2%的基因组用于次级代谢。 因此，推而广之，预计在任何eDNA文库中发现的克隆中只有一小部分包含基因 参与小分子生物合成。宏基因组文库作为生物活性小分子来源的效用 分子将大大改善从新的方法能够富集克隆含有功能 生物合成系统尽管天然产物中使用的酶种类繁多， 在生物合成中，功能冗余的酶通常存在于不同的基因簇中。这些冗余 酶代表了常见的生物合成线索，通过这些线索，我提出了eDNA文库，自然贫乏， 基因簇内容，可以富集含有生物合成基因簇的克隆。在第一个目标中， 建议使用天然产物调节的转录因子（例如，TetR同源物）和互补 选择性富集BAC文库中含有天然产物克隆的功能冗余生物合成基因 生物合成基因簇在具体目标2中，我们将鉴定具有抗菌活性的新型抗生素， 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.