GENOMIC SEQUENCING OF UNCULTURED MICROORGANISMS

分子系统发育技术已经确定，绝大多数的 地球上的微生物还未被培养。因此，这些微生物构成了一个重要的 编码新酶和代谢途径的新基因的未开发资源。 目前还没有一个自由生活的未培养的微生物的基因组测序。 我们建议访问特定未培养微生物的基因组， 从环境样本中提取的基因组DNA的复杂混合物。同相 1、我们将确定选择环境样本的复杂性，基于 16 S核糖体RNA（rRNA）基因。发现微生物在遗传学上是遥远的 将从已知的栽培生物中选择一种作为“基因组”的测试物种。 走路。使用16 S rRNA基因作为起点， 将对16 S rRNA基因进行测序。这一新进程将使整个 特定未培养微生物的基因组进行PCR扩增和测序-- 直接从微生物基因组DNA的复杂混合物。通过测序多个 生物体平行，我们将能够确定十到 来自不同门的20种未培养的微生物。在第二和第三阶段， 当我们继续遍历基因组时，通量测序工作将开始 这些未被培养的微生物。具有生物技术产品潜力的基因区域将 通过与已知基因的同源性来鉴定，并且那些被发现是 高度趋异将在大肠杆菌或酵母中表达， 活动确定未培养微生物的第一个完整序列 还应该提供关于如何成功培养这些生物体的见解 在未来 拟定商业应用： 我们将对未培养生物的基因组进行排序， 基因和酶的研究。酶来自于从未 研究应该为工业过程提供新的催化剂，包括 制造药品。代谢研究基于研究这些新的 基因组也可能为培养不可培养的物种带来新的见解。

Molecular phylogenetic techniques have determined that the vast majority of microbes on earth remain uncultivated. Thus, these microbes constitute a significant untapped resource for new genes encoding novel enzymes and metabolic pathways. There is currently no sequenced genome from a free-living uncultivated microbe. We propose to access the genomes of specific uncultured microorganisms from complex mixtures of genomic DNA extracted from environmental samples. In Phase 1, we will determine the complexity of select environmental samples, based on the 16S ribosomal RNA (rRNA) gene. Microbes found to be phylogenetically distant from known cultivated organisms will be chosen as test species for 'genome walking'. Using the 16S rRNA gene as the starting point, genomic regions flanking the 16S rRNA gene will be sequenced. This new process will allow for the entire genome of specific uncultured microbes to be PCR amplified and sequenced -- directly from complex mixtures of microbial genomic DNA. By sequencing multiple organisms in parallel, we will be able to determine the genomic sequences of ten to twenty uncultured microbes from several diverse phyla. In Phases 2 and 3, a high throughput sequencing effort will begin as we continue to walk through the genomes of these uncultivated microbes. Gene regions with potential for biotech products will be identified by homology with known genes, and those genes that are found to be highly divergent will be expressed in Escherichia coli or yeast and assayed for novel activities. Determining the first complete sequences of uncultivated microorganisms should also provide insight into how these organisms might be successfully cultured in the future. PROPOSED COMMERCIAL APPLICATION: We will sequence the genomes of uncultured organisms that shall yield information on genes and enzymes previously inaccessable. Enzymes from taxa that have never been studied should provide new catalysts for industrial processes, including the manufacture of pharmaceuticals. Metabolic studies based on studying these new genomes may also lead to new insights into culturing the unculturable.