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 genes bgtfA 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.

FIG. 1

Chemical structures of the glycopeptide antibiotics balhimycin (A. mediterranei DSM5908), vancomycin (A. orientalis C329.4), and A82846B (A. orientalis A82846).

FIG. 2

Integration of the plasmid pSP1.5Pst into the balhimycin gene cluster and Southern hybridization. (A) The gene organization of a part of the balhimycin biosynthetic cluster in the A. mediterranei wild type is shown. Downstream of peptide synthetase genes (′bps), the oxygenase genes (oxyA to -C), a halogenase gene (bhaA), the glycosyltransferase genes (bgtfA to -C), and orfX′ are located. (B) Construction of the gene inactivation mutant SP1-1 obtained after integration of plasmid pSP1.5Pst into the chromosome by a single-crossover event. The relevant PstI and SphI restriction sites are indicated. The size of the expected hybridizing SphI fragments with the 1,459-bp ′oxyA-oxyB′ (A′-B′) PstI fragment as a probe is drawn below the clusters. (C) Genomic DNA of the A. mediterranei wild type (lane 1) and of the oxygenase mutants SP1-1 (lane 2) and SP1-3 (lane 3) and DNA of the plasmid pSP1.5Pst (lane 4) were digested with SphI and probed with the 1,459-bp ′oxyA-oxyB′ (A′-B′) PstI fragment in a Southern hybridization. Lane M, DIG-labeled DNA Molecular Weight Marker VII (Boehringer). The sizes (in base pairs) of the characteristic fragments are shown.

FIG. 3

Reconstructed total-ion chromatogram of a liquid chromatography-MS run of crude culture broth of SP1-1 mutant (13.7 min) (A) and wild type (balhimycin, 11.6 min; desvancosamine-balhimycin, 12.2 min) (B). The corresponding mass spectrum of panel A shows two peptides, SP969 and SP1134, coeluting at 13.7 min. Neither peptide mass was detected in the wild type. Rel. int., relative intensity.

FIG. 4

Isotopically resolved mass spectra of HD mutants. The mass distribution of the isotopic patterns indicates the presence of two covalently bound chlorine atoms in the molecules. The characteristic mass differences of 16 Da (HD1-HD3 and HD2-HD4) and 14 Da (HD1-HD2 and HD3-HD4) between the derivatives suggest hydroxylation and methylation as major structural differences. Rel. int., relative intensity.

FIG. 5

MS analysis of peptide SP969. (A) Isotopically resolved mass spectrum. The observed isotopic pattern is consistent with the theoretical, calculated isotopic pattern (bars) for a molecule containing two chlorine atoms. (B) The daughter-ion spectrum reveals the partial sequence Leu-Xxx-Asn-Hpg-Hpg-Yyy. Rel. int., relative intensity.

FIG. 6

Proposed structure for the linear peptide SP969 deduced from the experimental data of MS and amino acid analysis.