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
