Selective Enrichment of Environmental DNA for Antibiotic Producing Gene Clusters

描述(申请人提供)：由土壤细菌合成的非核糖体多肽(NRP)和聚酮(PK)代谢物是临床有用化合物的重要来源，包括许多抗生素、抗肿瘤和免疫抑制化合物；然而，传统的基于培养的天然产物分离方法无法在实验室条件下检测99%顽强生长的细菌的代谢物。这一“未培养的大多数”的基因组代表着尚未探索的生物合成多样性的最大池之一，这种资源可以通过直接从土壤中克隆环境衍生基因组DNA(EDNA)并在异源宿主中表达恢复的基因簇来评估其生产有用代谢物的能力。虽然以高通量的方式筛选Edna文库是可取的，但Edna文库的巨大规模(107个成员)和产生代谢物的基因丰度低使得过去很难做到这一点。这一建议旨在通过开发一系列细菌宿主来促进小分子药物的发现工作，这些宿主可用于快速丰富包含生物合成簇的克隆的EDNA文库，然后筛选这些集中的亚文库来生产抗菌代谢物。先前的工作表明，EDNA文库对PPTase缺陷菌株的遗传互补导致了集中的亚库，其NRP/PK编码基因比任何其他已发表的DNA池都丰富。由于选择性策略依赖于NRP编码的铁载体生物合成的互补，这是一个高度保守的铁获取系统，几乎任何细菌菌株都可以转化为浓缩宿主。在目标1中，学员将总共创建两个 通过对天然PPTase和非NRP铁载体基因的靶向基因缺失，富集链霉菌、伯克霍尔德氏菌和假单胞菌家族的革兰氏阴性和两个革兰氏阳性宿主。在AIM2中，学员将使用能够在革兰氏阳性(PWeb436)或革兰氏阴性(PJWC1)细菌宿主中复制的穿梭载体创建EDNA文库，目标是每年创建两个文库，或每年创建四个宿主-文库对。每个文库-宿主对都将通过在低铁培养基上进行选择来丰富NRP/PK生物合成基因。在目标3中，受训者将使用重叠分析和浓缩培养物抽提物的现场分析来筛选抗菌活性文库。具有抗菌活性的克隆将被分离出来，并用于鉴定它们编码的抗菌代谢产物。

DESCRIPTION (provided by applicant): Non-ribosomosal peptide (NRP) and polyketide (PK) metabolites synthesized by soil-dwelling bacteria are an important source of clinically useful compounds including many antibiotic, antitumor, and immunosuppressant compounds; however, the classic, culture-based approach to natural product isolation is unable to examine the metabolites of the 99% of bacteria that are recalcitrant to growth in laboratory conditions. The genomes of this "uncultured majority" represent one of the largest pools of unexplored biosynthetic diversity, a resource that can be evaluated for the production of useful metabolites by cloning environmentally-derived genomic DNA (eDNA) directly from soil and expressing the recovered gene clusters in heterologous hosts. While it would be desirable to screen eDNA libraries in a high-throughput manner, the large size of eDNA libraries (107 members) and the low abundance of metabolite-producing genes have made it difficult to do so in the past. This proposal aims to facilitate small molecule drug discovery efforts by developing a series of bacterial hosts that can be used to rapidly enrich eDNA libraries for clones containing biosynthetic clusters and then to screen those focused sublibraries for the production of antibacterial metabolites. Previous work has shown that genetic complementation of phosphopantetheinyl transferase (PPTase) deficient bacterial strains by eDNA libraries results in focused sublibraries that are richer in NRP/PK- encoding genes than any other published pool of DNA. Because the selective strategy is dependent upon complementation of NRP-encoded siderophore biosynthesis, a highly conserved iron-acquisition system, almost any bacterial strain can be transformed into an enrichment host. In Aim 1, the trainee will create a total of two Gram-negative and two Gram-positive enrichment hosts belonging to the Streptomyces, Burkholderia, and Pseudomonad families by using targeted gene deletions of the native PPTase and non-NRP-siderophore genes. In Aim2, the trainee will create eDNA libraries using shuttle vectors capable of replicating in either Gram-positive (pWeb436) or Gram-negative (pJWC1) bacterial hosts, aiming for the creation of two libraries per year, or four host-library pairs per year. Each library-host pair will be enriched for NRP/PK biosynthetic genes by selection on low-iron media. In Aim 3, the trainee will screen the libraries for antibiotic activity using overlay assays, and spot-assays with concentrated culture extracts. Clones exhibiting antibiotic activity will be isolated and used to characterize the antibacterial metabolites they encode.