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.     

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.     

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.     

High-yield production of lipoglycopeptide antibiotic A40926 using a mutant strain Nonomuraea sp. DP-13 in optimized medium

The lipoglycopeptide antibiotic A40926 produced by Nonomuraea sp. is a complex of structurally related components differing in the fatty acid moiety. Besides showing an intrinsic antibacterial activity, A40926 is the precursor of the semisynthetic antibiotic Dalvance. In this work, A40926 production by a mutant strain Nonomuraea sp. DP-13 was investigated. It was found that A40926 production was markedly promoted by using poorly assimilated carbon source maltodextrin and nitrogen source soybean meal. Addition of Cu²⁺ resulted in a stimulation of A40926 production, but Co²⁺ had an inhibitory effect. L-Leucine addition greatly improved total A40926 production and modified the complex composition toward factor B0. An optimized production medium IM-3 was developed and a maximum A40926 production of 1096 mg/L was obtained in the 10-L fermenter. This was the highest A40926 productivity so far reported.     

Methods for the genetic manipulation of <Emphasis Type="Italic">Nonomuraea</Emphasis> sp. ATCC 39727

Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes. This microorganism produces the teicoplanin-like glycopeptide A40926, which is the starting material for the synthesis of the second-generation glycopeptide dalbavancin. Notwithstanding the strain’s pharmaceutical relevance, the lack or poor efficiency of genetic tools to manipulate Nonomuraea sp. ATCC 39727 has hampered strain and product improvement. Here we report the development of gene transfer systems based on protoplast transformation and intergeneric conjugation from Escherichia coli. Efficiency of transformation and conjugation, followed by site specific or homologous recombination with the Nonomuraea sp. genome, were determined using the integrative plasmid pSET152 (5.7 kb), and the Supercos1 derivative cosmid A40ΔY (30 kb). To our knowledge, this is the first report of the transformation of protoplasts of Nonomuraea sp. ATCC 39727, even though the improved procedure for intergeneric conjugation makes it the method of choice for introducing large segments of DNA into Nonomuraea sp. ATCC 39727.     

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.     

Factors influencing cell fatty acid composition and A40926 antibiotic complex production in <Emphasis Type="Italic">Nonomuraea</Emphasis> sp. ATCC 39727

A40926 is a glycopeptide antibiotic complex consisting of several structurally related factors. It is produced by fermentation of Nonomuraea sp. ATCC 39727 and the complex components differ in the structure of the fatty acid moiety linked to the aminoglucuronic acid unit. In previous work, we observed that the production of single factors in glycopeptide antibiotic complexes could be selectively enhanced by the addition of suitable precursors to the culture medium. In this contribution, we examine the effects of branched amino acid addition to fermentation of Nonomuraea sp. in a chemically defined minimal medium. The changes in the composition of cell fatty acids correlate to the fatty acid distribution within the A40926 complex in diverse cultivation conditions. Nonomuraea sp. prefers isobutyric, butyric and propionic acids as initiators of fatty acid biosynthesis. The relative amount of the produced fatty acids is significantly influenced by the availability of intermediates or final products from the amino acid catabolic pathways. Antibiotic complex composition closely reflects the cell fatty acid pattern, in agreement with the assumption that the antibiotic fatty acid moieties are synthesized by shortening the chain of cell fatty acids.     

A two-dimensional electrophoresis and mass spectrometry protein analysis of the antibiotic producer Nonomuraea sp. ATCC 39727 in different growth conditions

Nonomuraea sp. ATCC 39727 is an aerobic actinomycete, industrially important as a producer of the glycopeptide A40926, which is used as a precursor of the semi-synthetic antibiotic dalbavancin. Previous studies showed that the production of A40926 is depressed by calcium, but promoted by l-glutamine or l-asparagine. In this study, the protein expression changes of Nonomuraea sp. ATCC 39727 in these two different growth and antibiotic-production conditions have been investigated by two-dimensional electrophoresis and mass spectrometry (MS) analysis. Few protein spots show statistically significant expression changes, and, among this group of proteins, malate dehydrogenase (MDH) shows a significant decrease in the overproduction condition. The decrease of MDH is of particular interest because it is the first described significant change in the expression levels of enzymes of the central metabolism related with A40926 overproduction.     

Synthesis and antibacterial activity of alkyl derivatives of the glycopeptide antibiotic A40926 and their amides

New derivatives of the glycopeptide antibiotic A40926 were synthesized and evaluated for antimicrobial activity against VRE. Deacylated A40926 was obtained by microbial transformation of the parent antibiotic with the use of Actinoplanes teichomyceticus ATCC 31121. Regioselective synthesis of alkylated derivatives of Deacyl A40926 was carried out using lipophilic aliphatic and aromatic halides or aldehydes. Further modification of the two carboxylic acids was performed to increase antibiotic activity. Poor antimicrobial activity was observed for the derivatives obtained by lipophilic mono- or dialkylation of the amino groups present on the molecule, while simultaneous condensation of both carboxylic groups, in hydrophobic derivatives, with dibasic amines led to a strong increase in antibiotic activity.     

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.     

Redirection of acyl donor metabolic flux for lipopeptide A40926B0 biosynthesis

The metabolic flux of fatty acyl-CoAs determines lipopeptide biosynthesis efficiency, because acyl donor competition often occurs from polyketide biosynthesis and homologous pathways. We used A40926B0 as a model to investigate this mechanism. The lipopeptide A40926B0 with a fatty acyl group is the active precursor of dalbavancin, which is considered as a new lipoglycopeptide antibiotic. The biosynthetic pathway of fatty acyl-CoAs in the A40926B0 producer Nonomuraea gerenzanensis L70 was efficiently engineered using endogenous replicon CRISPR (erCRISPR). A polyketide pathway and straight-chain fatty acid biosynthesis were identified as major competitors in the malonyl-CoA pool. Therefore, we modified both pathways to concentrate acyl donors for the production of the desired compound. Combined with multiple engineering approaches, including blockage of an acetylation side reaction, overexpression of acetyl-CoA carboxylase, duplication of the dbv gene cluster and optimization of the fermentation parameters, the final strain produced 702.4 mg l^-1 of A40926B0, a 2.66-fold increase, and the ratio was increased from 36.2% to 81.5%. Additionally, an efficient erCRISPR-Cas9 editing system based on an endogenous replicon was specifically developed for L70, which increased conjugation efficiency by 660% and gene-editing efficiency was up to 90%. Our strategy of redirecting acyl donor metabolic flux can be widely adopted for the metabolic engineering of lipopeptide biosynthesis.     

Synthetic analogs of anoplin show improved antimicrobial activities

We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin‐resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin‐resistant Enterococcus faecium (ATCC 700221) (VRE), and Candida albicans (ATCC 200955). The anoplin analogs contain substitutions in amino acid positions 2, 3, 5, 6, 8, 9, and 10. We use these peptides to study the effect of altering the charge and hydrophobicity of anoplin on activity against red blood cells and microorganisms. We find that increasing the charge and/or hydrophobicity improves antimicrobial activity and increases hemolytic activity. For each strain tested, we identify at least six anoplin analogs with an improved therapeutic index compared with anoplin, the only exception being Enterococcus faecium, against which only few compounds are more specific than anoplin. Both 2Nal⁶ and Cha⁶ show improved therapeutic index against all strains tested. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome

A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli–Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividans∷NmESAC50 and S. lividans∷NmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT–PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividans∷NmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.     

Some 3-Amino and 3-Substituted-amino Derivatives of α4-Norpyridoxol

Some analogs of α⁴-norpyridoxol in which 3-hydroxyl group is replaced by amino and substituted-amino groups have been prepared and evaluated for anticoccidal activity. 3-Aroylamino analogs of α⁴-norpyridoxol have some coccidiostatic effect towards Eimeria tenella.     

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.     

Substrate-mediated Fidelity Mechanism Ensures Accurate Decoding of Proline Codons

Aminoacyl-tRNA synthetases attach specific amino acids to cognate tRNAs. Prolyl-tRNA synthetases are known to mischarge tRNA^Pro with the smaller amino acid alanine and with cysteine, which is the same size as proline. Quality control in proline codon translation is partly ensured by an editing domain (INS) present in most bacterial prolyl-tRNA synthetases that hydrolyzes smaller Ala-tRNA^Pro and excludes Pro-tRNA^Pro. In contrast, Cys-tRNA^Pro is cleared by a freestanding INS domain homolog, YbaK. Here, we have investigated the molecular mechanism of catalysis and substrate recognition by Hemophilus influenzae YbaK using site-directed mutagenesis, enzymatic assays of isosteric substrates and functional group analogs, and computational modeling. These studies together with mass spectrometric characterization of the YbaK-catalyzed reaction products support a novel substrate-assisted mechanism of Cys-tRNA^Pro deacylation that prevents nonspecific Pro-tRNA^Pro hydrolysis. Collectively, we propose that the INS and YbaK domains co-evolved distinct mechanisms involving steric exclusion and thiol-specific chemistry, respectively, to ensure accurate decoding of proline codons.     

Dicarboxylic acid analogs of Gramicidin A: Dimerization kinetics and single channel properties

Summary According to the model of Urry, the cation-permeable gramicidin channel is a dimeric helix formed by association of two peptide monomers linked at their amino ends. In this paper the channel properties of gramicidin analogs are described which have been obtained by chemical modification at the coupling site of the two half-channels. In these analogs the amino terminal-CHO group is replaced by-CO(CH₂) _n COOH(n=2, 3, 4, 5, 6). All analogs form conducting channels in black lipid membranes with the same general properties as found for gramicidin A. The observation that the channel-forming activity decreases with increasing pH is consistent with the notion that the half-channels are linked at the amino terminus. The channel lifetime of the different analogs varies between 2 msec and 50 sec, the longest lifetime being found for the compound withn=3. The single-channel conductance is always smaller than that of gramicidin A, but the reduction of depends on the nature of the permeable ion. Ion specificity was studied at 1m electrolyte by measuring the conductance for different permeable ions (Na⁺, K⁺, Cs⁺). The conductance ratio(Cs⁺)/(Na⁺) was found to vary between 2 and 10.5 for the different analogs.