SBIR-STTR Award

Polyketides are a structurally diverse group of compounds that include many anti-microbial agents. Most known bloactive polyketides of microbial origin are made by actinomycetes, which are high GC Gram-positive bacteria frequenting both terrestrial and marine environments. Genes dedicated to the synthesis of a particular Type II polyketide are invariably clustered on the actinomycete chromosome and undoubtedly share a common evolutionary origin. Current cloning strategies using DNA hybridization have been limited to known microorganisms. In order to discover novel polyketide biosynthetic pathways from the majority of uncultured microorganisms, we propose an ultra-high-throughput (UHTP) screening technology that is based on Gel MicroDrop (GMD) in situ hybridization coupled with FACS (Fluorescence Activated Cell Sorting) screening. This technology, which we have called large insert FACS biopanning, will be combined with large insert cloning to capture Escherichia coli clones carrying potential anti-microbial PKS pathways from environmental large insert DNA libraries. In Phase I we will establish the proof of principle by biopanning a known PKS Il pathway from a genomic library and optimize conditions by using a spiked environmental library. The work undertaken in Phase I will provide the groundwork for the isolation of novel Type II PKS pathways from environmental libraries in Phase II. This technology will provide a powerful new approach to the discovery of novel PKS pathways and ultimately novel anti-microbial compounds from nature

Polyketides are a large group of important bioactive compounds that include many anti-microbial agents. Most bioactive polyketides of microbial origin are made by actinomycetes, which are high GC Gram-positive bacteria frequenting both terrestrial and marine environments. Genes dedicated to the synthesis of a particular Type II polyketide are invariably clustered on the actinomycete chromosome and undoubtedly share a common evolutionary origin. In order to discover novel polyketide biosynthetic pathways from the majority of uncultured microorganisms present in the environment, all of Diversa's large insert fosmid libraries will be screened for Type II polyketide synthase genes using the large insert FACS biopanning technology developed in Phase I. Diversa's fosmid library collection will cover approximately 13950 genomes. Putative polyketide pathways will be transferred to our proprietary expression host Streptomyces diversa and extracts from the resulting clones will be analyzed for anti-microbial activity. Extracts of active clones will be chemically characterized and the corresponding fosmids will be sequenced to identify pathways that encode potentially novel compounds. S. diversa clones apparently producing new anti-microbial polyketides will be used to produce sufficient quantities of the compounds for further pre-clinical analysis (e.g. range of activity, toxicity, and mechanism of action).

Thesaurus Terms:
antiinfective agent, drug design /synthesis /production, genetic library, genetic screening, polyketide synthase, protein biosynthesis Escherichia coli, drug discovery /isolation, drug screening /evaluation Streptomyces, biotechnology, flow cytometry, high throughput technology, in situ hybridization, molecular cloning, nucleic acid sequence