Genetic analysis of the balhimycin (vancomycin-type) oxygenase genes.
In the balhimycin biosynthesis three oxygenases OxyA, OxyB and OxyC are responsible for the oxidative phenol coupling reactions, which lead to the ring-closures between the aromatic amino acid side chains in the heptapeptide aglycone. These ring-closures constrain the peptide backbone into the cup-shaped conformation that is required for binding to the Lys-D-Ala-D-Ala-terminus of the cell wall precursor peptide and represent one of the essential features of glycopeptide antibiotics. In the balhimycin biosynthetic gene cluster the oxygenase genes oxyA, oxyB and oxyC have been identified downstream of the peptide synthetase genes. Reverse transcription (RT)-PCR analyses revealed that these oxygenase genes in Amycolatopsis balhimycina are co-transcribed. Non-polar mutants (NPoxyA, DeltaoxyB and DeltaoxyC) were constructed, cultivated in production medium and assayed for the presence of glycopeptides and glycopeptide precursors by HPLC-ESI-MS. The mutant NPoxyA produces mainly monocyclic, the mutant DeltaoxyB linear and the mutant DeltaoxyC bicyclic peptides. These results definitely confirm the sequence of the three oxidative ring-closing steps (OxyB-OxyA-OxyC). The heterologous complementation of the mutant strains with the corresponding oxygenase genes from the vancomycin producer A. orientalis restored the production of balhimycin, which proves the functional equivalence of the oxygenases from the balhimycin and vancomycin producer. For the first time it is now possible to combine the genetic data obtained from the balhimycin producer with the biochemical and structural data obtained from the vancomycin producer.
Stegmann
Evi
E
Eberhard-Karls-Universität Tübingen, Fakultät Biologie, Mikrobiologisches Institut, Mikrobiologie/Biotechnologie, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
Pelzer
Stefan
S
Bischoff
Daniel
D
Puk
Oliver
O
Stockert
Sigrid
S
Butz
Diane
D
Zerbe
Katja
K
Robinson
John
J
Süssmuth
Roderich D
RD
Wohlleben
Wolfgang
W
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D020868:Gene Silencing; D006020:Glycopeptides / Q000737:chemistry; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014640:Vancomycin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry
D006020:Glycopeptides; C087127:balhimycin; D014640:Vancomycin; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases