Intracellular screens discovery:natural products*(RMI)

细胞内筛选发现：天然产物*(RMI)

• 批准号: 7011055
• 负责人: JO E. HANDELSMAN
• 金额: $ 28.89万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7011055
• 关键词: bacterial DNA; bacterial genetics; biological products; biosensor device; biotechnology; biotherapeutic agent; drug discovery /isolation; fluorescence microscopy; gene expression; genetic promoter element; high throughput technology; host organism interaction; intracellular; microorganism culture; molecular cloning; pharmacokinetics; structural biology

DESCRIPTION (provided by applicant): The as-yet-uncultured bacteria are a rich potential resource for natural product discovery. In most environments, the readily cultured bacteria represent <1% of the total community, and soil is likely to be one of the most biologically diverse, as it is home to 4,000 to 40,000 species/g. Soil's biological diversity is matched by its chemical diversity: soil has been the source of more antibiotics and other medicinal compounds than any other natural habitat, but virtually all of the chemistry is derived from the readily cultured members of the soil community. We have developed functional metagenomics, the genomic analysis of assemblages of bacteria, as a means to access the biological and chemical potential of soil microbial communities. Functional metagenomics entails extracting DNA directly from soil, cloning it into a bacterial host, and screening the resulting libraries for activities of interest. The major limitation in functional metagenomic analysis to date has been the low frequency of active clones among libraries with many clones. The low frequency is likely due to (1) low expression of foreign genes in the bacteria harboring the soil DNA and (2) insensitivity of current screens. In the proposed research, we aim to develop universal strategies to address both of these limitations that will be broadly applicable to functional metagenomic analysis of any environment for diverse functions. We intend to develop expression systems in representatives of four diverse divisions of bacteria: Gamma-Proteobacteria (E. coli), alpha-Proteobacteria (Agrobacterium tumefaciens), Firmicutes (Bacillus subtilis), and Actinobacteria (Streptomyces coelicolor). To screen libraries maintained in each of these hosts, we will design and implement intracellular screens in which the metagenomic DNA is inside the same cell as a biosensor that detects diverse natural products. These screens are sensitive and rapid and can be applied easily to libraries containing millions of clones. This research will lead to universal expression systems that will be useful for metagenomic analysis and other techniques that rely on heterologous gene expression.

描述（由申请人提供）：尚未培养的细菌是自然产品发现的丰富潜在资源。在大多数环境中，易于培养的细菌占整体社区的<1％，土壤可能是生物学上最多样化的细菌，因为它是4,000至40,000种/g的家园。土壤的生物多样性与其化学多样性相匹配：与任何其他自然栖息地相比，土壤一直是抗生素和其他药物化合物的来源，但实际上所有的化学作用均来自土壤社区的易于培养的成员。我们已经开发了功能性宏基因组学，即细菌组合的基因组分析，以此来获得土壤微生物群落的生物学和化学潜力。功能性宏基因组需要直接从土壤中提取DNA，将其克隆到细菌宿主中，并筛选所得的文库以获取感兴趣的活动。迄今为止，功能性宏基因组分析的主要局限性是具有许多克隆的库中活动克隆的低频。低频率可能是由于（1）外国基因在携带土壤DNA的细菌中的低表达和（2）当前筛查的不敏感性。在拟议的研究中，我们旨在制定通用策略，以解决这两种局限性，这些局限性将广泛适用于对各种功能的任何环境的功能性元基因组分析。我们打算在细菌四种不同分裂的代表中开发表达系统：γ-蛋白杆菌（大肠杆菌），α-蛋白酶细菌（tumefaciens），富公司（枯草芽孢杆菌）和肠杆菌菌（Coelictocces coelicoloror）。为了在每个宿主中维护的屏幕库中​​，我们将设计和实施细胞内筛选，其中元基因组DNA与检测到不同天然产物的生物传感器在同一细胞内。这些屏幕是敏感和快速的，可以轻松地应用于包含数百万个克隆的文库。这项研究将导致通用表达系统，这些系统将用于宏基因组分析和其他依赖异源基因表达的技术。