Selective Enrichment of Environmental DNA for Antibiotic Producing Gene Clusters

抗生素生产基因簇环境 DNA 的选择性富集

• 批准号: 8784469
• 负责人: Zachary Charlop-Powers
• 金额: $ 5.51万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8784469
• 关键词: 4' -phosphopantetheine; Acinetobacter; Actinobacteria class; Anabolism; Anti-Bacterial Agents; Antibiotics; Bacteria; Biological Assay; Biological Factors; Breathing; Burkholderia; Bypass; Cloning; Cosmids; DNA; DNA Library; Engineering; Ensure; Enterobacter; Enterococcus faecium; Environment; Enzyme Gene; Enzymes; Exhibits; Family; Gene Cluster; Gene Deletion; Genes; Genetic; Genetic Materials; Genome; Genomic DNA; Goals; Growth; Immunosuppressive Agents; Iron; Klebsiella pneumonia bacterium; Laboratories; Libraries; Mass Spectrum Analysis; Metagenomics; Methods; Molecular Weight; Organism; Organism Cloning; Partner in relationship; Peptides; Population; Process; Production; Proteobacteria; Pseudomonas aeruginosa; Pseudomonas putida; Publishing; Recombinants; Resources; Series; Shuttle Vectors; Siderophores; Soil; Source; Spottings; Staphylococcus aureus; Streptomyces; Streptomyces coelicolor; System; Taxon; Work; X-Ray Crystallography; aqueous; base; density; drug discovery; environmental enrichment for laboratory animals; functional group; interest; member; novel; pathogen; phosphopantetheinyl transferase; public health relevance; screening; small molecule; tool; vector

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.

描述（申请人提供）：土壤细菌合成的非核糖体肽（NRP）和聚酮（PK）代谢物是临床上有用的化合物的重要来源，包括许多抗生素、抗肿瘤和免疫抑制剂化合物;然而，经典的，基于培养的天然产物分离方法不能检测99%的在实验室条件下不适于生长的细菌的代谢物。这种“未培养的大多数”的基因组代表了未开发的生物合成多样性的最大池之一，可以通过直接从土壤中克隆环境来源的基因组DNA（eDNA）并在异源宿主中表达回收的基因簇来评估有用代谢物的生产。虽然期望以高通量方式筛选eDNA文库，但eDNA文库的大尺寸（107个成员）和产生代谢物的基因的低丰度使得过去难以这样做。该提案旨在通过开发一系列细菌宿主来促进小分子药物发现工作，这些细菌宿主可用于快速富集含有生物合成簇的克隆的eDNA文库，然后筛选那些集中的子文库以产生抗菌代谢物。 先前的工作已经表明，通过eDNA文库对磷酸泛酰巯基乙胺基转移酶（PPTase）缺陷型细菌菌株的遗传互补产生了集中的亚文库，其比任何其他公开的DNA库更富含NRP/PK编码基因。由于选择性策略依赖于NRP编码的铁载体生物合成的互补，这是一种高度保守的铁获取系统，几乎任何细菌菌株都可以转化为富集宿主。在目标1中，受训者将创建总共两个 革兰氏阴性和两个革兰氏阳性富集主机属于链霉菌，伯克霍尔德氏菌，和假单胞菌家族通过使用天然PPT酶和非NRP-铁载体基因的靶向基因缺失。在目标2中，学员将使用能够在革兰氏阳性（pWeb 436）或革兰氏阴性（pJWC 1）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-宿主对将通过在低铁培养基上选择来富集NRP/PK生物合成基因。在目标3中，受训者将使用覆盖测定和用浓缩培养物提取物的斑点测定来筛选文库的抗生素活性。将分离显示抗生素活性的克隆并用于表征它们编码的抗菌代谢物。