title: An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35.
abstract: Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca(2+) ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate.
  
  
  
  The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.|Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.
authors.forename: Tina
authors.initials: T
authors.lastname: Netzker
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Schroeckh
authors.affiliation: Isomerase Therapeutics, Ltd., Cambridge, United Kingdom.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Gregory
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.|Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.
authors.forename: Michal
authors.initials: M
authors.lastname: Flak
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.|Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.
authors.forename: Mario K C
authors.initials: MKC
authors.lastname: Krespach
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Leadlay
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany axel.brakhage@leibniz-hki.de.|Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.
authors.forename: Axel A
authors.initials: AA
authors.lastname: Brakhage
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D018014:Gene Transfer Techniques; D010957:Plasmids / Q000378:metabolism; D020123:Sirolimus / Q000378:metabolism*; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D020123:Sirolimus