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1.
Appl Microbiol Biotechnol ; 102(13): 5635-5643, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29728728

RESUMO

The glycopeptide antibiotic A82846B (chloroeremomycin) produced by Amycolatopsis orientalis is the precursor of the semi-synthetic antibiotic oritavancin. However, during the industrial production of A82846B, two major impurities, A82846A (63.6%) and A82846C (12%) which are structurally similar to A82846B (24.4%), are also produced. In this study, to improve the ratio of A82846B to A and C, the genes encoding halogenase in A82846B and vancomycin synthesis were integrated into A. orientalis SIPI18099 to test their halogenation ability, respectively. The results indicated that chal from the A82846B biosynthesis pathway was more efficient in reducing A and C factors. Moreover, by increasing the chal copy number, the proportion of A and C were gradually reduced while the titer and proportion of A82846B were improved. In a scaled-up industrial process, the proportion of A and C were decreased to 11.6% and 0.2% in the recombinant strain A.orientalis chal-3 with three gene copies of chal and the titers of A82846B (2.2 g/L) has increased by 2.8-folds compared to 780 mg/L produced by the parental strain, suggesting that the recombinant strain was suitable for the industrial production of A82846B with lower impurities.


Assuntos
Actinomycetales/enzimologia , Actinomycetales/genética , Microbiologia Industrial/métodos , Vancomicina/análogos & derivados , Vias Biossintéticas/genética , Família Multigênica , Vancomicina/biossíntese
2.
J Org Chem ; 83(13): 7309-7317, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29806454

RESUMO

We report a general method for synthesizing diverse d-Tyr analogues, one of the constituents of the antibiotic vancomycin, using a Negishi cross-coupling protocol. Several analogues were incorporated into the vancomycin substrate-peptide and reacted with the biosynthetic enzymes OxyB and OxyA, which install the characteristic aromatic cross-links. We find that even small structural perturbations are not accepted by OxyA. The same modifications, however, enhance the catalytic capabilities of OxyB leading to the formation of a new macrocycle within the vancomycin framework.


Assuntos
Antibacterianos/biossíntese , Tirosina/metabolismo , Vancomicina/biossíntese , Antibacterianos/química , Catálise , Sistema Enzimático do Citocromo P-450/química , Especificidade por Substrato , Vancomicina/química
3.
Angew Chem Int Ed Engl ; 57(27): 8048-8052, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29697176

RESUMO

The bioactivity of vancomycin is enabled by three aromatic crosslinks, the biosynthesis of which has been an active area of investigation for two decades. Two cytochrome P450 enzymes, OxyB and OxyA, have been shown to introduce bisaryl ether linkages with the help of a so-called X-domain. The final crosslink, however, a biaryl bond thought to be installed by OxyC, has remained elusive. We report the in vitro reconstitution of the OxyC reaction and formation of the first carbon-carbon crosslink in any glycopeptide antibiotic. Using a cascade sequence, in which the peptide substrate was incubated with the Oxy enzymes in turn, we completed the chemoenzymatic synthesis of a vancomycin aglycone variant. This approach was also used to generate a new analogue carrying a thioamide linkage at residue 4, a precursor to the amidine derivative, which is effective against vancomycin-resistant pathogens. Our results set the stage for creating therapeutic vancomycin derivatives by using the native metalloenzymes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Vancomicina/biossíntese , Biocatálise , Ciclização , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Isoquinolinas/química , Isoquinolinas/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Especificidade por Substrato , Vancomicina/análogos & derivados
4.
Lett Appl Microbiol ; 63(3): 222-8, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27432613

RESUMO

UNLABELLED: Recently, many in vitro studies have reported that MbtH-like proteins are very necessary for the adenylation of amino acid by adenylating enzymes present in the biosynthetic machineries of nonribosomal peptides (NRPs). However, in vivo studies on mbtH-like genes are somewhat controversial since their mutants still produce the target compounds. Here, we report unambiguous evidence of the crucial role of MbtH-like protein in the biosynthesis of NRP based on in vivo study of vancomycin producer, Amycolatopsis orientalis. Deletion of mbtH-like gene (vcm11) in the vancomycin biosynthetic gene cluster completely abolished production of vancomycin and its complementation strain showed almost full recovery of vancomycin production. As a result, we propose that the mbtH-like gene is a good genetic engineering target to increase the yield of NRP, as verified by increased vancomycin production (by 60 and 80%) upon overexpression of cognate (Vcm11) as well as noncognate (CloY) MbtH-like proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Elucidation and application of biosynthetic machineries of bioactive compounds containing amino acids such as antibiotics, immunosuppressants and siderphores etc. are significant for the production and development of drugs. Here, we observed an apparent increase in the yield of vancomycin, a type of NRP, upon overexpression of MbtH-like protein in Amycolatopsis orientalis. Our result is the first example of increased NRP(s) yield following overexpression of mbtH-like genes to develop the strain for economic production and elucidate the role of MbtH-like protein in vivo for combinatorial biosynthesis.


Assuntos
Actinomycetales/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Vancomicina/biossíntese , Actinomycetales/genética , Antibacterianos/metabolismo , Bacillus subtilis/efeitos dos fármacos , Família Multigênica/genética , Vancomicina/farmacologia
5.
Microb Drug Resist ; 22(6): 499-509, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27420548

RESUMO

In enterococci and in Streptomyces coelicolor, a glycopeptide nonproducer, the glycopeptide resistance genes vanHAX are colocalized with vanRS. The two-component system (TCS) VanRS activates vanHAX transcription upon sensing the presence of glycopeptides. Amycolatopsis balhimycina, the producer of the vancomycin-like glycopeptide balhimycin, also possesses vanHAXAb genes. The genes for the VanRS-like TCS VnlRSAb, together with the carboxypeptidase gene vanYAb, are part of the balhimycin biosynthetic gene cluster, which is located 2 Mb separate from the vanHAXAb. The deletion of vnlRSAb did not affect glycopeptide resistance or balhimycin production. In the A. balhimycina vnlRAb deletion mutant, the vanHAXAb genes were expressed at the same level as in the wild type, and peptidoglycan (PG) analyses proved the synthesis of resistant PG precursors. Whereas vanHAXAb expression in A. balhimycina does not depend on VnlRAb, a VnlRAb-depending regulation of vanYAb was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and RNA-seq analyses. Although VnlRAb does not regulate the vanHAXAb genes in A. balhimycina, its heterologous expression in the glycopeptide-sensitive S. coelicolor ΔvanRSSc deletion mutant restored glycopeptide resistance. VnlRAb activates the vanHAXSc genes even in the absence of VanS. In addition, expression of vnlRAb increases actinorhodin production and influences morphological differentiation in S. coelicolor.


Assuntos
Actinomycetales/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Streptomyces coelicolor/genética , Transcrição Genética , Actinomycetales/metabolismo , Antraquinonas/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Parede Celular/química , Parede Celular/metabolismo , Mapeamento Cromossômico , Deleção de Genes , Genoma Bacteriano , Família Multigênica , Peptidoglicano/biossíntese , Peptidoglicano/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Streptomyces coelicolor/metabolismo , Vancomicina/análogos & derivados , Vancomicina/biossíntese , Resistência a Vancomicina/genética
6.
Nature ; 521(7550): 105-9, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25686610

RESUMO

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/biossíntese , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína , Teicoplanina/análogos & derivados , Teicoplanina/biossíntese , Teicoplanina/química , Teicoplanina/metabolismo , Vancomicina/biossíntese
7.
J Basic Microbiol ; 55(2): 247-54, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25384460

RESUMO

Many strains of Amycolatopsis, such as Amycolatopsis orientalis, A. balhimycina, and A. mediterranei, are important antibiotic producers. Three indigenous plasmids, pMEA100, pMEA300, and pA387, found in this genus have been sequenced. However, only some vectors based on pA387 have been widely applied in Amycolatopsis research. An indigenous plasmid, pXL100, was isolated from the vancomycin producer A. orientalis HCCB10007. Sequence analysis of pXL100 revealed its total length to be 33,499 bp and GC content to be 68.9%. A 2830-bp fragment containing three ORFs has been identified as essential for replication in A. orientalis, but it has no significant similarity to any known replicons. A vector, pLYZW7-3, was constructed based on the pXL100 replicon and could be transferred into A. mediterranei and A. orientalis by electroporation or conjugation with high frequency. A mutant with a disrupted gene was successfully complemented with the pLYZW7-3 vector, indicating that the vector is potentially useful in Amycolatopsis research.


Assuntos
Actinomycetales/genética , Vetores Genéticos , Plasmídeos , Actinomycetales/metabolismo , Sequência de Bases , Conjugação Genética , Replicação do DNA , DNA Bacteriano/genética , Eletroporação , Dados de Sequência Molecular , Fases de Leitura Aberta , Replicon , Análise de Sequência de DNA , Vancomicina/biossíntese
8.
Antimicrob Agents Chemother ; 58(11): 6454-61, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25136027

RESUMO

National treatment guidelines for invasive methicillin-resistant Staphylococcus aureus (MRSA) infections recommend targeting a vancomycin 24-h area under the concentration-time curve (AUC0-24)-to-MIC ratio of >400. The range of vancomycin trough concentrations that best predicts an AUC0-24 of >400 in neonates is not known. This understanding would help clarify target trough concentrations in neonates when treating MRSA. A retrospective chart review from a level III neonatal intensive care unit was performed to identify neonates treated with vancomycin over a 5-year period. Vancomycin concentrations and clinical covariates were utilized to develop a one-compartment population pharmacokinetic model and examine the relationships between trough and AUC0-24 in the study neonates. Monte Carlo simulations were performed to examine the effect of dose, postmenstrual age (PMA), and serum creatinine level on trough and AUC0-24 achievement. A total of 1,702 vancomycin concentrations from 249 neonates were available for analysis. The median (interquartile range) PMA was 39 weeks (32 to 42 weeks) and weight was 2.9 kg (1.6 to 3.7 kg). Vancomycin clearance was predicted by weight, PMA, and serum creatinine level. At a trough of 10 mg/liter, 89% of the study neonates had an AUC0-24 of >400. Monte Carlo simulations demonstrated that troughs ranging from 7 to 11 mg/liter were highly predictive of an AUC0-24 of >400 across a range of PMA, serum creatinine levels, and vancomycin doses. However, a trough of ≥10 mg/liter was not readily achieved in most simulated subgroups using routine starting doses. Higher starting doses frequently resulted in troughs of >20 mg/liter. A vancomycin trough of ∼10 mg/liter is likely adequate for most neonates with invasive MRSA infections based on considerations of the AUC0-24. Due to pharmacokinetic and clinical heterogeneity in neonates, consistently achieving this target vancomycin exposure with routine starting doses is difficult. More robust clinical dosing support tools are needed to help clinicians with dose individualization.


Assuntos
Antibacterianos/farmacocinética , Área Sob a Curva , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Vancomicina/farmacocinética , Antibacterianos/biossíntese , Antibacterianos/uso terapêutico , Peso Corporal , Creatinina/sangue , Relação Dose-Resposta a Droga , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Masculino , Testes de Sensibilidade Microbiana , Método de Monte Carlo , Estudos Retrospectivos , Infecções Estafilocócicas/microbiologia , Vancomicina/biossíntese , Vancomicina/uso terapêutico
9.
BMC Genomics ; 15: 363, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24884615

RESUMO

BACKGROUND: Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus. RESULTS: The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred. CONCLUSIONS: This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.


Assuntos
Actinomycetales/genética , Antibacterianos/metabolismo , Genoma Bacteriano , Vancomicina/biossíntese , Actinomycetales/classificação , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Hibridização Genômica Comparativa , Sequenciamento de Nucleotídeos em Larga Escala , Redes e Vias Metabólicas , Dados de Sequência Molecular , Família Multigênica , Fosfolipídeos/biossíntese , Alinhamento de Sequência , Análise de Sequência de DNA , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/química , Vancomicina/farmacologia
10.
Org Biomol Chem ; 12(30): 5574-7, 2014 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-24756572

RESUMO

Vancomycin is an important nosocomial antibiotic containing a glycosylated, cross-linked and doubly chlorinated heptapeptide backbone. During the biosynthesis of the vancomycin aglycone, two ß-hydroxytyrosine (Bht) residues are inserted at positions-2 and -6 into the heptapeptide backbone by a non-ribosomal peptide synthetase. A single flavin-dependent chlorinase (VhaA) is responsible for chlorinating both Bht residues at some ill-defined point in the assembly process. We show here using in vitro assays that VhaA is able to introduce a chlorine atom into each aromatic ring of both Bht residues at positions-2 and -6 of a peptide carrier protein-bound hexapeptide. The results suggest that VhaA can recognize and chlorinate two quite different sites within a linear hexapeptide intermediate during vancomycin biosynthesis.


Assuntos
Halogenação , Oligopeptídeos/metabolismo , Oxirredutases/metabolismo , Proteínas/metabolismo , Vancomicina/biossíntese , Cromatografia Líquida de Alta Pressão , Oligopeptídeos/química , Peptídeo Sintases/metabolismo , Vancomicina/química
11.
J Ind Microbiol Biotechnol ; 40(2): 235-44, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23184174

RESUMO

Secondary metabolites such as antibiotics are typically produced by actinomycetes as a response to growth limiting stress conditions. Several studies have shown that secondary metabolite production is correlated with changes observed in actinomycete pellet morphology. Therefore, we investigated the correlation between the production of balhimycin and the spatio-temporal distribution of live and dead cells in pellets of Amycolatopsis balhimycina in submerged cultures. To this end, we used laser scanning confocal microscopy to analyze pellets from balhimycin producing and nonproducing media containing 0.2 and 1.0 g l(-1) of potassium di-hydrogen phosphate, respectively. We observed a substantially higher fraction of live cells in pellets from cultures yielding larger amounts of balhimycin. Moreover, in media that resulted in no balhimycin production, the pellets exhibit an initial death phase which commences from the centre of the pellet and extends in the radial direction. A second growth phase was observed in these pellets, where live mycelia are seen to appear in the dead core of the pellets. This secondary growth was absent in pellets from media producing higher amounts of balhimycin. These results suggest that distribution of live and dead cells and its correlation with antibiotic production in the non-sporulating A. balhimycina differs markedly than that observed in Streptomycetes.


Assuntos
Actinomycetales/citologia , Actinomycetales/metabolismo , Antibacterianos/biossíntese , Reatores Biológicos , Vancomicina/análogos & derivados , Actinomycetales/efeitos dos fármacos , Actinomycetales/isolamento & purificação , Biomassa , Meios de Cultura/química , Meios de Cultura/farmacologia , Viabilidade Microbiana , Fosfatos/farmacologia , Compostos de Potássio/farmacologia , Fatores de Tempo , Vancomicina/biossíntese
12.
J Ind Microbiol Biotechnol ; 39(1): 27-35, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21643706

RESUMO

Actinomycetes, a class of filamentous bacteria, are an important source of several industrially relevant secondary metabolites. Several environmental factors including the media composition affect both biomass growth and product formation. Likewise, several studies have shown that environmental factors cause changes in cellular morphology. However, the relationship between morphology and product formation is not well understood. In this study, we first characterized the effect of varying concentrations of phosphate and ammonia in defined media on pellet morphology for an actinomycete Amycolatopsis balhimycina DSM 5908, which produces balhimycin, a glycopeptide antibiotic. Our results show that higher balhimycin productivity is correlated with the following morphological features: (1) higher pellet fraction in the biomass, (2) small elongated pellets, and (3) shorter filaments in hyphal growth in the periphery of the pellets. The correlation between morphology and product formation was also observed in industrially relevant complex media. Although balhimycin production starts after 72 h with maximum production around 168 h, the morphological changes in pellets are observed as early as 24 h after commencing of the batch. Therefore, morphology may be used as an early predictor of the end-of-batch productivity. We argue that a similar strategy can be developed for other strains where morphological indicators may be used as a batch monitoring tool.


Assuntos
Actinomycetales/metabolismo , Antibacterianos/biossíntese , Vancomicina/análogos & derivados , Actinomycetales/citologia , Actinomycetales/crescimento & desenvolvimento , Sulfato de Amônio/farmacologia , Meios de Cultura/química , Fosfatos/farmacologia , Vancomicina/biossíntese
13.
J Biol Chem ; 286(42): 36281-90, 2011 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-21890635

RESUMO

MbtH-like proteins consist of ∼70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes.


Assuntos
Aminocumarinas/metabolismo , Proteínas de Bactérias/metabolismo , Nucleotidiltransferases/metabolismo , Streptomyces coelicolor/enzimologia , Tirosina/metabolismo , Vancomicina/biossíntese , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutação de Sentido Incorreto , Nucleotidiltransferases/genética , Streptomyces coelicolor/genética , Tirosina/genética
14.
Prep Biochem Biotechnol ; 41(1): 94-105, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21229467

RESUMO

In vivo pentose phosphate pathway (PPP) enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and transaldolase (TAL) activities as well as ATP- and ADP-level variations of Amycolatopsis orientalis were investigated with respect to glucose concentration and incubation period. G6PDH, 6PGDH, and TAL activities of A. orientalis reached maximum levels at 48 hr for all glucose concentrations used, after which the levels began to decline. G6PDH, 6PGDH, and TAL activities showed positive correlation with the glucose concentration up to 15 g/L, while further increases had an opposite effect. Intracellular ATP level showed a positive correlation with glucose concentrations, while ADP level increased up to 15 g/L. ATP concentration of A. orientalis increased rapidly at 48 hr of incubation, as was the case also for G6PDH, 6PGDH, and TAL activities, although the incubation period corresponding to maximum values of ADP shifted to 60 hr. Production of the glycopeptide antibiotic vancomycin increased with the increases in glucose concentrations up to 15 g/L, by showing coherence in the rates of oxidative and nonoxidative parts of the PPP.


Assuntos
Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Via de Pentose Fosfato/fisiologia , Fosfogluconato Desidrogenase/metabolismo , Transaldolase/metabolismo , Actinomycetales/enzimologia , Actinomycetales/crescimento & desenvolvimento , Difosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Antibacterianos/biossíntese , Gluconatos/metabolismo , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/análise , Fosfogluconato Desidrogenase/análise , Transaldolase/análise , Vancomicina/análise , Vancomicina/biossíntese
15.
J Antibiot (Tokyo) ; 64(1): 103-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21119677

RESUMO

Analogs of vancomycin and pseudo-vancomycin with new sugar attachments in mono- and di-saccharide form have been enzymatically synthesized by glycosylation with overexpressed glycosyltransferases, ß1,4-galactosyltransferase and α2,3-sialyl transferases. All four analogs, including galactose-containing derivatives (6 and 8) and galactose- and sialic acid-containing derivatives (7 and 9) were prepared and characterized by HPLC, LC-MS, NMR and MIC test.


Assuntos
Galactosiltransferases/metabolismo , Glicosiltransferases/metabolismo , Vancomicina/análogos & derivados , Vancomicina/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Cromatografia Líquida , Galactose/análogos & derivados , Galactose/química , Galactose/metabolismo , Galactose/farmacologia , Glicosilação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/farmacologia , Vancomicina/química , Vancomicina/farmacologia
16.
Microb Cell Fact ; 9: 95, 2010 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-21110849

RESUMO

BACKGROUND: Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted. RESULTS: Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38 ± 0.02 and 0.33 ± 0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map http://www.unipa.it/ampuglia/Abal-proteome-maps matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA). CONCLUSION: In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement.


Assuntos
Actinomycetales/metabolismo , Antibacterianos/biossíntese , Proteoma/análise , Vancomicina/análogos & derivados , Actinomycetales/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Eletroforese em Gel Bidimensional , Ácidos Graxos/metabolismo , Glucose/farmacologia , Biossíntese de Proteínas , Vancomicina/biossíntese
17.
Prikl Biokhim Mikrobiol ; 46(5): 519-26, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21058501

RESUMO

The relationship between tricarboxylic acid (TCA) and glyoxalate cycle and the effect of their metabolites levels on the vancomycin production of Amycolatopsis orientalis were investigated in different concentration of glycerol (2.5-20 g/l). Intracellular glycerol levels increased with respect to increases in glycerol concentrations of the growth medium. Extracellular glycerol levels decreased slowly up to 24 h while uptake rates were increased during 36-48 h for 10 and 15 g/l and during 36-60 h at 20 g/l of glycerol. Intracellular citrate, alpha-ketoglutarate, fumarate levels increased up to 10 g/l glycerol concentration. However, intracellular succinate and malate levels were increased up to 15 g/l glycerol. Extracellular citrate, alpha-ketoglutarate, succinate and malate levels increased with respect to increases in glycerol concentration. The highest alpha-ketoglutarate dehydrogenase activity was determined at 15 g/l glycerol. Isocitrate lyase activity showed a positive correlation with the increases in glycerol concentration of the growth medium. Vancomycin production increased with the increases in glycerol concentration from 5 to 10 g/l. These results showed that A. orientalis grown in glycerol containing medium used glyoxalate shunt actively instead of TCA cycle which supports precursors of many amino acid which are effective on the antibiotic production.


Assuntos
Actinomycetales , Ciclo do Ácido Cítrico/efeitos dos fármacos , Glicerol/farmacologia , Glioxilatos/metabolismo , Solventes/farmacologia , Vancomicina/biossíntese , Actinomycetales/crescimento & desenvolvimento , Actinomycetales/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Solventes/metabolismo
18.
FEMS Microbiol Lett ; 306(1): 45-53, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20337711

RESUMO

Ferredoxins are required to supply electrons to the cytochrome P450 enzymes involved in cross-linking reactions during the biosynthesis of the glycopeptide antibiotics balhimycin and vancomycin. However, the biosynthetic gene clusters for these antibiotics contain no ferredoxin- or ferredoxin reductase-like genes. In a search for potential ferredoxin partners for these P450s, here, we report an in silico analysis of the draft genome sequence of the balhimycin producer Amycolatopsis balhimycina, which revealed 11 putative Fe-S-containing ferredoxin genes. We show that two members (balFd-V and balFd-VII), produced as native-like holo-[3Fe-4S] ferredoxins in Escherichia coli, could supply electrons to the P450 OxyB (CYP165B) from both A. balhimycina and the vancomycin producer Amycolatopsis orientalis, and support in vitro turnover of peptidyl carrier protein-bound peptide substrates into monocyclic cross-linked products. These results show that ferredoxins encoded in the antibiotic-producing strain can act in a degenerate manner in supporting the catalytic functions of glycopeptide biosynthetic P450 enzymes from the same as well as heterologous gene clusters.


Assuntos
Actinomycetales/enzimologia , Actinomycetales/genética , Antibacterianos/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Ferredoxinas/genética , Genoma Bacteriano , Glicopeptídeos/biossíntese , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Vancomicina/análogos & derivados , Vancomicina/biossíntese
19.
Chembiochem ; 11(2): 266-71, 2010 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-19998400

RESUMO

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel-chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with "haloperoxidase"/perhydrolase activity, it did not show any enzymatic activity with standard "haloperoxidase"/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p-nitrophenyl acetate, p-nitrophenyl phosphate and S-thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S-beta-hydroxytyrosyl-N-acetyl cysteamine thioester (beta-OH-Tyr-SNAC) with 15 times the efficiency of S-L-tyrosyl-N-acetyl cysteamine thioester (L-Tyr-SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of beta-OH-Tyr-S-PCP (PCP=peptidyl carrier protein) to free beta-hydroxytyrosine (beta-OH-Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP-bound beta-OH-Tyr and not a "haloperoxidase"/perhydrolase or nonspecific esterase.


Assuntos
Actinomycetales/enzimologia , Antibacterianos/biossíntese , Di-Hidroxifenilalanina/metabolismo , Tioléster Hidrolases/metabolismo , Vancomicina/análogos & derivados , Actinomycetales/genética , Antibacterianos/química , Biocatálise , Família Multigênica , Especificidade por Substrato , Tioléster Hidrolases/genética , Vancomicina/biossíntese , Vancomicina/química
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