A derivative of the glycopeptide A40926 produced by inactivation of the beta-hydroxylase gene in Nonomuraea sp. ATCC39727

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the beta-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the beta-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free beta-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.     

The border sequence of the balhimycin biosynthesis gene cluster from Amycolatopsis balhimycina contains bbr, encoding a StrR-like pathway-specific regulator

Balhimycin, produced by the actinomycete Amycolatopsis balhimycina DSM5908, is a glycopeptide antibiotic highly similar to vancomycin, the antibiotic of 'last resort' used for the treatment of resistant Gram-positive pathogenic bacteria. Partial sequence of the balhimycin biosynthesis gene cluster was previously reported. In this work, cosmids which overlap the region of the characterized gene cluster were isolated and sequenced. At the 'left' end of the cluster, genes were identified which are involved in balhimycin biosynthesis, transport, resistance and regulation. The 'right' end border is defined by a putative 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (dahp) gene. The proximate gene is similar to a type I polyketide synthase gene of the rifamycin producer Amycolatopsis mediterranei indicating that another biosynthesis gene cluster might be located directly next to the balhimycin gene cluster. The newly identified StrR-like pathway-specific regulator, Bbr, was characterized to be a DNA-binding protein and may have a role in balhimycin biosynthesis. Purified N-terminally His-tagged Bbr shows specific DNA-binding to five promoter regions within the gene cluster. By in silico analysis and by comparison of the DNA sequences binding Bbr, conserved inverted repeat sequences for the Bbr-binding site are proposed. The putative Bbr consensus sequence differs from that published for StrR.     

Improved production of A40926 by Nonomuraea sp. through deletion of a pathway-specific acetyltransferase

Nonomuraea strain ATCC 39727 produces the glycopeptide A40926, used for manufacturing dalbavancin, currently in advanced clinical trials. From the gene cluster involved in A40926 biosynthesis, a strain deleted in dbv23 was constructed. This mutant can produce only the glycopeptides lacking the O-linked acetyl residue at position 6 of the mannose moiety, while, under identical fermentation conditions, the wild-type strain produces mostly glycopeptides carrying an acetylated mannose. Furthermore, the total amount of glycopeptides produced by the mutant strain was found to be approximately twice that of the wild type. The reduced level of glycopeptides observed in the wild-type strain may be due to an inhibitory effect exerted by the acetylated compound on the biosynthesis of A40926. Indeed, spiking production cultures with ≥1 μg/ml of the acetylated glycopeptide inhibited A40926 production in the mutant strain.     

Characterization of VanY(n) , a novel d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance in Nonomuraea sp. ATCC 39727

VanY(n) is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC?39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase?III of clinical development. VanY(n) (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanY(n) was successfully expressed as a soluble and active protein in Escherichia?coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn(2+) and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanY(n) as in VanY, VanX and VanXY Zn(2+) -dependent d,d-carboxypeptidases and d-Ala-d-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanY(n) shows d,d-carboxypeptidase and d,d-dipeptidase activity, but lacks d,d-carboxyesterase ability on d-Ala-d-Lac-terminating peptides. VanY(n) belongs to the metallo-d,d-carboxypeptidase family, but it is inhibited by β-lactams. Its characterization provides new insights into the evolution and transfer of resistance determinants from environmental glycopeptide-producing actinomycetes (such as Nonomuraea sp.) to glycopeptide-resistant pathogens (enterococci and staphylococci). It may also contribute to an early warning system for emerging resistance mechanisms following the introduction into clinics of a second-generation glycopeptide such as dalbavancin. Database The nucleotide sequence of vanY(n) is available in the GenBank data base under accession number CAD91202.     

Comparative analysis and insights into the evolution of gene clusters for glycopeptide antibiotic biosynthesis

The bal, cep, dbv, sta and tcp gene clusters specify the biosynthesis of the glycopeptide antibiotics balhimycin, chloroeremomycin, A40926, A47934 and teicoplanin, respectively. These structurally related compounds share a similar mechanism of action in their inhibition of bacterial cell wall formation. Comparative sequence analysis was performed on the five gene clusters. Extensive conserved synteny was observed between the bal and cep clusters, which direct the synthesis of very similar compounds but originate from two different species of the genus Amycolatopsis. All other cluster pairs show a limited degree of conserved synteny, involving biosynthetically functional gene cassettes: these include those involved in the synthesis of the carbon backbone of two non-proteinogenic amino acids; in the linkage of amino acids 1–3 and 4–7 in the heptapeptide; and in the formation of the aromatic cross-links. Furthermore, these segments of conserved synteny are often preceded by conserved intergenic regions. Phylogenetic analysis of protein families shows several instances in which relatedness in the chemical structure of the glycopeptides is not reflected in the extent of the relationship of the corresponding polypeptides. Coherent branchings are observed for all polypeptides encoded by the syntenous gene cassettes. These results suggest that the acquisition of distinct, functional genetic elements has played a significant role in the evolution of glycopeptide gene clusters, giving them a mosaic structure. In addition, the synthesis of the structurally similar compounds A40926 and teicoplanin appears as the result of convergent evolution.     

Teicoplanin biosynthesis genes in Actinoplanes teichomyceticus

The genetic determinants for the biosynthesis of the glycopeptide antibiotic teicoplanin were identified. In order to isolate the corresponding gene cluster, oligonucleotides derived from highly conserved motifs in peptide synthetases were used. These synthetic probes, and gene fragments derived from the balhimycin gene cluster of Amycolatopsis mediterranei, led to the identification of the likely teicoplanin gene cluster centered on a region of ca. 110 kb from the genome of Actinoplanes teichomyceticus, the teicoplanin producer. Partial nucleotide sequences identified partial ORFs likely to encode two glycosyltransferases, three P-450 monooxygenases and one ABC transporter. The corresponding genes have been found in other glycopeptide gene clusters. Furthermore, upstream to the peptide synthetase region a segment was identified with a remarkable similarity to the vanHAX operon, conferring resistance to glycopeptides in enterococci. Thus, in contrast to the other glycopeptide producers thus far analyzed, in A. teichomyceticusthe genes for teicoplanin biosynthesis are closely linked to homologs of glycopeptide resistance commonly found in vancomycin-resistant enterococci.     

Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds

In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.     

The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production

The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.     

The ABC transporter Tba of <Emphasis Type="Italic">Amycolatopsis balhimycina</Emphasis> is required for efficient export of the glycopeptide antibiotic balhimycin

All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His₆-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.     

Understanding and manipulating glycopeptide pathways: the example of the dalbavancin precursor A40926

Glycopeptide antibiotics represent an important class of microbial compounds produced by several genera of actinomycetes. The emergence of resistance to glycopeptides among enterococci and staphylococci has prompted the search for second-generation drugs of this class and semi-synthetic derivatives are currently under clinical trials. Dalbavancin is obtained by chemical modification of the natural glycopeptide A40926, produced by a Nonomuraea sp. Recently, there has been considerable progress in the elucidation of biosynthesis of glycopeptide antibiotics; several gene clusters have been characterized, thus providing an understanding of the biosynthesis of these chemically complex molecules. Furthermore, such investigations have yielded the first glycopeptide derivatives produced by genetic or enzymatic intervention. We have isolated and characterized the dbv clusters, involved in the formation of the glycopeptides A40926. The development of a gene-transfer system for Nonomuraea sp. has allowed the manipulation of the A40926 pathway. New derivatives were obtained by inactivating selected dbv genes. In addition, our data suggest differences in the biosynthetic routes for heptapeptide formation between the vancomycin and the teicoplanin families of glycopeptides.     

Production of the glycopeptide antibiotic A40926 by<Emphasis Type="Italic"> Nonomuraea</Emphasis> sp. ATCC 39727: influence of medium composition in batch fermentation

Nonomuraea sp. ATCC 39727 is a novel actinomycete species and the producer of A40926, a glycopeptide antibiotic structurally similar to teichoplanin. In the present study, a defined minimal medium was designed for Nonomuraea fermentation. The influence of initial phosphate, glucose and ammonium concentrations on antibiotic productivity was investigated in batch fermentation and the effect of glucose limitation was studied in fed-batch fermentation. It was found that low initial concentrations of phosphate and ammonium are beneficial for A40926 production and that productivity is not enhanced during glucose limitation. Furthermore, the initiation of A40926 production was not governed by residual ammonium and phosphate concentrations, although the level of these nutrients strongly influenced A40926 production rates and final titers. Electronic Publication     

Design of mineral medium for growth of <Emphasis Type="Italic">Actinomadura</Emphasis> sp. ATCC 39727, producer of the glycopeptide A40926: effects of calcium ions and nitrogen sources

Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin, with significant activity against Neisseria gonorrhoeae and precursor of the semi-synthetic antibiotic dalbavancin. In this study the production of A40926 by Actinomadura under a variety of growth conditions was investigated. The use of chemically defined mineral media allowed us to analyze the influence of carbon and nitrogen sources, phosphate, ammonium and calcium on the growth and the antibiotic productivity of Actinomadura. We confirm recent data [Gunnarsson et al. (2003) J Ind Microbiol Biotechnol 30:150–156] that low initial concentrations of phosphate and ammonium are beneficial for growth and A40926 production, and we provide new evidence that the production of A40926 is depressed by calcium, but promoted when l-glutamine or l-asparagine are used as nitrogen sources instead of ammonium salts.     

Increased glycopeptide production after overexpression of shikimate pathway genes being part of the balhimycin biosynthetic gene cluster

Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp), and a prephenate dehydrogenase (pdh) gene. In a metabolic engineering approach for increasing the precursor supply for balhimycin production, the dahp and pdh genes from the biosynthetic cluster were overexpressed both individually and together and the resulting strains were subjected to quantitative physiological characterisation. The constructed strains expressing an additional copy of the dahp gene and the strain carrying an extra copy of both dahp and pdh showed improved specific glycopeptide productivities by approximately a factor three, whereas the pdh overexpression strain showed a production profile similar to the wild type strain. In addition to the overexpression strains, corresponding deletion mutants, Δdahp and Δpdh, were constructed and characterised. Deletion of dahp resulted in significant reduction in balhimycin production whereas the Δpdh strain had production levels similar to the parent strain. Based on these results the relation between primary and secondary metabolism with regards to Dahp and Pdh is discussed.     

Genome-scale metabolic representation of Amycolatopsis balhimycina

Infection caused by methicillin-resistant Staphylococcus aureus (MRSA) is an increasing societal problem. Typically, glycopeptide antibiotics are used in the treatment of these infections. The most comprehensively studied glycopeptide antibiotic biosynthetic pathway is that of balhimycin biosynthesis in Amycolatopsis balhimycina. The balhimycin yield obtained by A. balhimycina is, however, low and there is therefore a need to improve balhimycin production. In this study, we performed genome sequencing, assembly and annotation analysis of A. balhimycina and further used these annotated data to reconstruct a genome-scale metabolic model for the organism. Here we generated an almost complete A. balhimycina genome sequence comprising 10,562,587 base pairs assembled into 2,153 contigs. The high GC-genome (～ 69%) includes 8,585 open reading frames (ORFs). We used our integrative toolbox called SEQTOR for functional annotation and then integrated annotated data with biochemical and physiological information available for this organism to reconstruct a genome-scale metabolic model of A. balhimycina. The resulting metabolic model contains 583 ORFs as protein encoding genes (7% of the predicted 8,585 ORFs), 407 EC numbers, 647 metabolites and 1,363 metabolic reactions. During the analysis of the metabolic model, linear, quadratic and evolutionary programming algorithms using flux balance analysis (FBA), minimization of metabolic adjustment (MOMA), and OptGene, respectively were applied as well as phenotypic behavior and improved balhimycin production were simulated. The A. balhimycina model shows a good agreement between in silico data and experimental data and also identifies key reactions associated with increased balhimycin production. The reconstruction of the genome-scale metabolic model of A. balhimycina serves as a basis for physiological characterization. The model allows a rational design of engineering strategies for increasing balhimycin production in A. balhimycina and glycopeptide production in general.