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.

Ways of Assembling Complex Natural Products on Modular Nonribosomal Peptide Synthetases A list of abbreviations can be found at the end of the text.

“…Examples for linear NRPSs are the tyrocidine (1), [29] bacitracin (3), [30] surfactin (4), [31] actinomycin, [32,33] ACV (2; the penicillin and cephalosporin precursor), [34] cyclosporin, [35] pristinamycin, [36,37] fengycin, [38,39] and ergotamine [40] NRPSs and the glycopeptide antibiotics of the vancomycin family, chloroeremomycin, [41] balhimyicin, [42] and complestatin. [43] Figure 2 illustrates linear NRPSs in the example of the biosynthesis of the tripeptide ACV and the module organization of the tyrocidine and surfactin NRPSs.…”

Control of Fluxes Towards Antibiotics and the Role of Primary Metabolism in Production of Antibiotics

“…The glycopeptide antibiotics vancomycin and teichoplanin are currently used as the antibiotics of last resort against infections of multi-resistant gram-positive bacteria. Biosynthesis of these antibiotics includes synthesis of specific precursors, assembly of the heptapeptide backbone through the action of non-ribosomal peptide synthases (NRPS), crosslinking of the heptapeptide, glycosylation and halogenation.A partial understanding of this complex pathway has been achieved, primarily by the identification and analysis of the biosynthesis gene clusters of chloroeremomycin [80] and balhimycin [81], both compounds differing from vancomycin only in the glycosylation patterns ( Table 4).…”

Structure Elucidation and Chemical Synthesis of Microbial Metabolites

“…The target molecule of the group of W. Wohlleben and S. Pelzer is balhimycin, a vancomycin-type glycopeptide antibiotic, produced by Amycolatopsis mediterranei, from which the biosynthesis gene cluster was sequenced [68]. After identification of the biosynthesis gene cluster, in 1998 the first gene-dis- Figure 19.16: Structures of the linear biosynthesis intermediates of balhimycin, a vancomycin type tricyclic glyco-peptide antibiotic [19].…”

“…19.16). They give a first insight into the biosynthesis pathway of the glycopeptide antibiotics [19,68] (Fig. 19.17).…”