Polyketides are a large group of important bioactive compounds that include many anti-tumor agents. Most known 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. 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 based on Gel MicroDrop (GMD) in situ hybridization coupled with FACS (Fluorescence Activated Cell Sorting) screening. This technology, which we have termed large insert FACS biopanning, will be combined with large insert cloning to capture Escherichia coil clones carrying potential anti-tumor PKS pathways from environmental large insert DNA libraries. In Phase I we will establish proof-of-principle by biopanning a known PKS II pathway from a genomic library and optimize conditions 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-tumor compounds from nature.
Thesaurus Terms: cell sorting, flow cytometry, in situ hybridization, method development, molecular cloning, polyketide synthase Streptomyces, genetic library biotechnology
