Identification and Analysis of the Balhimycin Biosynthetic Gene Cluster and Its Use for Manipulating Glycopeptide Biosynthesis in Amycolatopsis mediterranei DSM5908

ABSTRACT Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genesbgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

[1]  A. D. McLachlan,et al.  Codon preference and its use in identifying protein coding regions in long DNA sequences , 1982, Nucleic Acids Res..

[2]  J. Blumbach,et al.  Balhimycin, a new glycopeptide antibiotic with an unusual hydrated 3-amino-4-oxoaldopyranose sugar moiety , 1994 .

[3]  P. A. Harder,et al.  Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes , 1991, Molecular microbiology.

[4]  R. Nagarajan.  Structure-activity relationships of vancomycin-type glycopeptide antibiotics. , 1993, The Journal of antibiotics.

[5]  M. Staver,et al.  Identification of a Saccharopolyspora erythraea gene required for the final hydroxylation step in erythromycin biosynthesis , 1993, Journal of bacteriology.

[6]  D. Lipman,et al.  Improved tools for biological sequence comparison. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. Arthur,et al.  Quantitative analysis of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide‐resistant enterococci , 1996, Molecular microbiology.

[8]  F Wright,et al.  Codon usage in the G+C-rich Streptomyces genome. , 1992, Gene.

[9]  R. Yao,et al.  Glycopeptides: Classification, Occurrence, and Discovery , 1994 .

[10]  R. H. Baltz,et al.  A gene cloning system for 'Streptomyces toyocaensis'. , 1996, Microbiology.

[11]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[12]  W R Strohl,et al.  Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. , 1992, Nucleic acids research.

[13]  S. R. Nadkarni,et al.  Balhimycin, a new glycopeptide antibiotic produced by Amycolatopsis sp. Y-86,21022. Taxonomy, production, isolation and biological activity. , 1994, The Journal of antibiotics.

[14]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[15]  G. Vriend,et al.  Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints. , 1990, Journal of molecular biology.

[16]  D. Hill,et al.  Functions Encoded by Pyrrolnitrin Biosynthetic Genes from Pseudomonas fluorescens , 1998, Journal of bacteriology.

[17]  W. Wohlleben,et al.  Cloning and analysis of a peptide synthetase gene of the balhimycin producer Amycolatopsis mediterranei DSM5908 and development of a gene disruption/replacement system. , 1997, Journal of biotechnology.

[18]  D. Hill,et al.  NADH‐Dependent Halogenases Are More Likely To Be Involved in Halometaolite Biosynthesis Than Haloperoxidases , 1997 .

[19]  David J. States,et al.  Identification of protein coding regions by database similarity search , 1993, Nature Genetics.

[20]  C. Hsieh,et al.  Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus , 1995, Journal of bacteriology.

[21]  C. Hutchinson,et al.  Genetic analysis of erythromycin production in Streptomyces erythreus , 1985, Journal of bacteriology.

[22]  W. Bullock.  XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. , 1987 .

[23]  C R Hutchinson,et al.  Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production. , 1997, Microbiology.

[24]  K. O'Brien,et al.  Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. , 1992, Gene.

[25]  Steven J. M. Jones,et al.  Sequencing and analysis of genes involved in the biosynthesis of a vancomycin group antibiotic. , 1998, Chemistry & biology.

[26]  Roderic D. M. Page,et al.  TreeView: an application to display phylogenetic trees on personal computers , 1996, Comput. Appl. Biosci..

[27]  R. Thompson,et al.  Semisynthetic glycopeptide antibiotics derived from LY264826 active against vancomycin-resistant enterococci , 1996, Antimicrobial agents and chemotherapy.

[28]  C. Richard Hutchinson,et al.  The Streptomyces peucetius dpsY anddnrX Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis , 1998, Journal of bacteriology.

[29]  M. Limbert,et al.  New 4-oxovancosamine-containing glycopeptide antibiotics from Amycolatopsis sp. Y-86,21022. , 1996, The Journal of antibiotics.

[30]  R. Thompson,et al.  Production of hybrid glycopeptide antibiotics in vitro and in Streptomyces toyocaensis. , 1997, Chemistry & biology.

[31]  B. Shen,et al.  Triple hydroxylation of tetracenomycin A2 to tetracenomycin C in Streptomyces glaucescens. Overexpression of the tcmG gene in Streptomyces lividans and characterization of the tetracenomycin A2 oxygenase. , 1994, The Journal of biological chemistry.

[32]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[33]  J. McAlpine,et al.  An erythromycin derivative produced by targeted gene disruption in Saccharopolyspora erythraea. , 1991, Science.

[34]  A. Kirschning,et al.  Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57 , 1997, Journal of bacteriology.

[35]  R. H. Baltz,et al.  Efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmids , 1987, Journal of bacteriology.

[36]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[37]  D. Hopwood.  Genetic manipulation of Streptomyces : a laboratory manual , 1985 .

[38]  P. Terpstra,et al.  Prediction of the Occurrence of the ADP-binding βαβ-fold in Proteins, Using an Amino Acid Sequence Fingerprint , 1986 .

[39]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[40]  P. Leadlay,et al.  Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. , 1996, Gene.

[41]  R. Gaylord.  unpublished results , 1985 .