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: 9094548
• 负责人: Zachary Charlop-Powers
• 金额: $ 6.2万
• 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/9094548
• 关键词: 4' -phosphopantetheine; Acinetobacter; Actinobacteria class; Anabolism; Anti-Bacterial Agents; Antibiotics; Bacteria; Biological Assay; Breathing; Burkholderia; Bypass; Cloning; Cosmids; DNA; DNA Library; Engineering; Ensure; Enterobacter; Enterococcus faecium; Environment; Enzymes; Exhibits; Family; Gene Cluster; Gene Deletion; Genes; Genetic; Genetic Materials; Genome; Genomic DNA; Goals; Growth; Health; Immunosuppressive Agents; Iron; Klebsiella pneumonia bacterium; Laboratories; Libraries; Mass Spectrum Analysis; Metagenomics; Methods; Molecular Weight; Natural Products; 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; 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（由申请人提供）：土壤细菌合成的非核糖肽（NRP）和聚酮化合物（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检查 99% 在实验室条件下难以生长的细菌的代谢物。这个“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库之一，可以通过直接从土壤中克隆环境来源的基因组 DNA (eDNA) 并表达回收的基因簇来评估这种资源是否产生有用的代谢物在异源宿主中。虽然以高通量方式筛选 eDNA 文库是可取的，但 eDNA 文库规模大（107 个成员）和代谢物产生基因的低丰度使得过去很难做到这一点。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，然后筛选这些重点子文库以产生抗菌代谢物。 先前的工作表明，eDNA 文库对磷酸泛酰硫胺基转移酶 (PPTase) 缺陷的细菌菌株进行遗传互补，可产生比任何其他已发表的 DNA 库更富含 NRP/PK 编码基因的重点子文库。由于选择性策略依赖于 NRP 编码的铁载体生物合成（一种高度保守的铁获取系统）的互补，因此几乎任何细菌菌株都可以转化为富集宿主。在目标 1 中，学员将总共创建两个 通过使用天然 PPTase 和非 NRP 铁载体基因的定向基因删除，对属于链霉菌、伯克霍尔德氏菌和假单胞菌科的革兰氏阴性和两种革兰氏阳性富集宿主。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。年。通过在低铁培养基上进行选择，每个文库-宿主对都将富集 NRP/PK 生物合成基因。在目标 3 中，学员将使用叠加测定和浓缩培养物提取物的点测定来筛选文库的抗生素活性。将分离表现出抗生素活性的克隆并用于表征它们编码的抗菌代谢物。

项目成果

Aquatic environmental DNA detects seasonal fish abundance and habitat preference in an urban estuary.

• DOI: 10.1371/journal.pone.0175186
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Stoeckle MY;Soboleva L;Charlop-Powers Z
• 通讯作者: Charlop-Powers Z