: The work outlined in this proposal is designed to establish a new turnkey functional metagenomic antibiotic discovery platform -- from the enrichment of biosynthetic diversity in metagenomic libraries through to the identification of novel antibiotics from enriched libraries. Unfortunately, in most sequenced bacteria less than 2% of the genome is devoted to secondary metabolism. Thus, by extension, only a small fraction of the clones found in any eDNA library is expected to contain genes involved in small molecule biosynthesis. The utility of metagenomic libraries as sources of bioactive small molecules would improve greatly from new methods capable of enriching for clones containing functional biosynthetic systems. While an incredibly diverse collection of enzymes is used in natural product biosynthesis, functionally redundant enzymes are commonly found in diverse gene clusters. These redundant enzymes represent common biosynthetic threads through which I propose that eDNA libraries, naturally poor in gene cluster content, can be enriched for clones containing biosynthetic gene clusters. In the first aim we propose to use natural product regulated transcription factors (e.g., TetR homologs) and complementation of functionally redundant biosynthetic genes to selectively enrich BAC libraries for clones containing natural product biosynthetic gene clusters. In specific Aim 2, we will identify novel antibiotics with activities against antibiotic resistant bacterial pathogens.