title: Nonribosomal biosynthesis of vancomycin-type antibiotics: a heptapeptide backbone and eight peptide synthetase modules.
abstract: During analysis of the recently identified gene cluster for the glycopeptide antibiotic balhimycin, produced by Amycolatopsis mediterranei DSM 5908, novel genes were identified and characterized in detail. The gene products of four of the identified genes (bpsA, bpsB, bpsC and bpsD) are nonribosomal peptide synthetases (NRPSs); one (Orf1-protein) shows similarities to small proteins associated with several NRPSs without an assigned function. BpsA and BpsB are composed of three modules each (modules 1-6), BpsC of one module (module 7) and BpsD of a minimal module (module 8). Thus, the balhimycin gene cluster encodes eight modules, whereas its biosynthetic product is a heptapeptide. Non-producing mutants were created by a gene disruption of bpsB, an in-frame deletion of bpsC and a gene replacement of bpsD. After establishment of a gene complementation system for Amycolatopsis strains, the replacement mutant of bpsD was complemented, demonstrating for the first time that BpsD, encoding the eighth module, is indeed involved in balhimycin biosynthesis. After feeding with beta-hydroxytyrosine the capability of the bpsD mutant to produce balhimycin was restored, demonstrating the participation of BpsD in the biosynthesis of this amino acid. The specificity of four of the eight adenylation domains was determined by ATP/PP(i) exchange assays: modules 4 and 5 activated L-4-hydroxyphenylglycine, module 6 activated beta-hydroxytyrosine and module 7 activated L-3,5-dihydroxyphenylglycine, which is in accordance with the sequence of the non-proteogenic amino acids 4 to 7 of the balhimycin backbone.
authors.affiliation: Eberhard-Karls-Universität Tübingen, Mikrobiologie/ Biotechnologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Recktenwald
authors.affiliation: Eberhard-Karls-Universität Tübingen, Mikrobiologie/ Biotechnologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
authors.forename: Riham
authors.initials: R
authors.lastname: Shawky
authors.affiliation: Eberhard-Karls-Universität Tübingen, Mikrobiologie/ Biotechnologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
authors.forename: Oliver
authors.initials: O
authors.lastname: Puk
authors.affiliation: Technische Universität Berlin, Max-Volmer-Institut, Fachgebiet Biochemie und Molekulare Biologie, Franklinstr. 29, D-10587 Berlin-Charlottenburg, Germany2.
authors.forename: Frank
authors.initials: F
authors.lastname: Pfennig
authors.affiliation: Technische Universität Berlin, Max-Volmer-Institut, Fachgebiet Biochemie und Molekulare Biologie, Franklinstr. 29, D-10587 Berlin-Charlottenburg, Germany2.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Keller
authors.affiliation: Eberhard-Karls-Universität Tübingen, Mikrobiologie/ Biotechnologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wohlleben
authors.affiliation: Eberhard-Karls-Universität Tübingen, Mikrobiologie/ Biotechnologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pelzer
mesh_terms: D000192:Actinomycetales / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D016366:Open Reading Frames; D010453:Peptide Synthases / Q000378:metabolism*; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism*; D013379:Substrate Specificity; D014640:Vancomycin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D009842:Oligopeptides; C087127:balhimycin; D014640:Vancomycin; D010453:Peptide Synthases