title: Overproduction of Ristomycin A by activation of a silent gene cluster in Amycolatopsis japonicum MG417-CF17.
abstract: The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is Amycolatopsis. However, Amycolatopsis japonicum does not produce an antibiotic under standard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina (bbrAba), into A. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of A. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum/pRM4-bbrAba synthesizes ristomycin A.
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Spohn
authors.affiliation: German Centre for Infection Research (DZIF), Partner Site Tuebingen, Tuebingen, Germany Pharmaceutical Institute, Department of Pharmaceutical Biology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Kirchner
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kulik
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Jochim
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Wolf
authors.affiliation: Department of Physiology I, University of Tuebingen, Tuebingen, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Muenzer
authors.affiliation: Department of Cardiology and Cardiovascular Medicine, University of Tuebingen, Tuebingen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Borst
authors.affiliation: German Centre for Infection Research (DZIF), Partner Site Tuebingen, Tuebingen, Germany Pharmaceutical Institute, Department of Pharmaceutical Biology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Gross
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germany German Centre for Infection Research (DZIF), Partner Site Tuebingen, Tuebingen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wohlleben
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germany German Centre for Infection Research (DZIF), Partner Site Tuebingen, Tuebingen, Germany evi.stegmann@biotech.uni-tuebingen.de.
authors.forename: Evi
authors.initials: E
authors.lastname: Stegmann
mesh_terms: D000192:Actinomycetales / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D005810:Multigene Family / Q000235:genetics*; D012310:Ristocetin / Q000378:metabolism*; D014640:Vancomycin / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D012310:Ristocetin; C087127:balhimycin; C016719:ristocetin A; D014640:Vancomycin
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