title: From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028.
abstract: Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods. Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound. The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes Tü6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.
authors.affiliation: Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-Universität Freiburg, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Greule
authors.affiliation: Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-Universität Freiburg, Germany.
authors.forename: Songya
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biology, Universität Siegen.
authors.forename: Thomas
authors.initials: T
authors.lastname: Paululat
authors.affiliation: Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-Universität Freiburg, Germany; andreas.bechthold@pharmazie.uni-freiburg.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bechthold
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001688:Biological Products / Q000737:chemistry*; D006038:Glyoxylates / Q000737:chemistry*; D005810:Multigene Family; D013302:Streptomyces / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001688:Biological Products; D006038:Glyoxylates; C111268:polyketomycin