Intracellular screens discovery:natural products*(RMI)

描述（由申请人提供）：尚未培养的细菌是发现天然产物的丰富潜在资源。在大多数环境中，容易培养的细菌占总群落的不到1%，土壤可能是最具生物多样性的土壤之一，因为它是每克4000到40000个物种的家园。土壤的生物多样性与其化学多样性相匹配：土壤是比任何其他自然栖息地更多的抗生素和其他药用化合物的来源，但实际上所有的化学成分都来自于土壤群落中容易培养的成员。我们已经开发了功能宏基因组学，对细菌组合进行基因组分析，作为获取土壤微生物群落生物和化学潜力的一种手段。功能宏基因组学需要直接从土壤中提取DNA，将其克隆到细菌宿主中，并筛选产生的文库以进行感兴趣的活动。迄今为止，功能宏基因组分析的主要限制是在具有许多克隆的文库中活性克隆的频率较低。低频率可能是由于(1)携带土壤DNA的细菌中外源基因的低表达和(2)当前筛选的不敏感。在提出的研究中，我们的目标是制定通用策略来解决这两个限制，这将广泛适用于任何环境的不同功能的功能宏基因组分析。我们打算在四种不同细菌的代表中开发表达系统：γ -变形菌门（大肠杆菌），α -变形菌门（农杆菌），厚壁菌门（枯草芽孢杆菌）和放线菌门（色链霉菌）。为了筛选在这些宿主中维持的文库，我们将设计和实施细胞内筛选，其中宏基因组DNA位于同一细胞内，作为检测多种天然产物的生物传感器。这些屏幕是敏感和快速的，可以很容易地应用到包含数百万克隆的库。这项研究将导致通用表达系统，这将有助于宏基因组分析和其他依赖于异源基因表达的技术。

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.