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1.
Appl Environ Microbiol ; 81(19): 6621-36, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26187956

RESUMO

Pristinamycin production in Streptomyces pristinaespiralis Pr11 is tightly regulated by an interplay between different repressors and activators. A γ-butyrolactone receptor gene (spbR), two TetR repressor genes (papR3 and papR5), three SARP (Streptomyces antibiotic regulatory protein) genes (papR1, papR2, and papR4), and a response regulator gene (papR6) are carried on the large 210-kb pristinamycin biosynthetic gene region of Streptomyces pristinaespiralis Pr11. A detailed investigation of all pristinamycin regulators revealed insight into a complex signaling cascade, which is responsible for the fine-tuned regulation of pristinamycin production in S. pristinaespiralis.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pristinamicina/biossíntese , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Streptomyces/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Mol Biol (Mosk) ; 49(2): 289-96, 2015.
Artigo em Russo | MEDLINE | ID: mdl-26065256

RESUMO

In order to enhance pristinamycin production, six homologous ptr genes from high pristinamycin-producing strains of Streptomyces pristinaespiralis were selected for DNA shuffling, and the reason for the altered activities of the shuffled ptr gene was speculated by sequence alignment. The highest pristinamycin yield of 0.12 g/L was achieved with a sixfold increase in strain sps16 obtained by DNA shuffling when compared to ancestral strain ATCC 25486. Sequence analysis of theptr gene variant from the sps16 strain indicated that five mutations (H16P, N63D, T75P, Q107R, and P435A) were introduced into the gene, two of them (N63D and T75P) located in the second of the 14 transmembrane segments (TMS). Prediction of the secondary structure of the gene product indicated that mutations at the N-terminus resulted in the shortening of the corresponding α-helix, while the mutation at the C-terminus lengthened the helix. In conclusion, combination of DNA shuffling with genome shuffling is an effective breeding strategy for increasing the antibiotic yield by directed evolution of target genes.


Assuntos
Proteínas de Bactérias/genética , Embaralhamento de DNA , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Proteínas de Membrana/genética , Pristinamicina/biossíntese , Streptomyces/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Membrana/metabolismo , Estrutura Terciária de Proteína , Streptomyces/metabolismo
3.
Appl Microbiol Biotechnol ; 99(17): 7151-64, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25957493

RESUMO

In this study, using a transposon-based strategy, two novel regulatory genes were identified as being involved in the biosynthesis of both pristinamycin I (PI) and II (PII) in Streptomyces pristinaespiralis, including a TetR-family regulatory gene atrA-p (SSDG_00466) and an orphan histidine kinase gene SSDG_02492. The mechanism by which AtrA-p exerted a positive role in pristinamycin production was elucidated. We showed that deletion of atrA-p resulted in a delayed production of both PI and PII as well as reduced PII production. Transcriptional analysis integrated with electrophoretic mobility shift assays (EMSAs) demonstrated that AtrA-p played a positive role in pristinamycin production by directly activating the transcription of two cluster-situated regulatory genes, spbR and papR5, which encode a γ-butyrolactone receptor protein and a TetR-family repressor, respectively. The precise AtrA-p-binding sites upstream of these two targets were determined, which allowed the identification of a relatively conserved binding motif comprising two 5-nt inverted repeats separated by a variable 5-nt sequence (5'-GGAAT-n5-ATTCC-3') possibly required for the regulation of AtrA-like regulators in Streptomyces. Base substitutions of the AtrA-p-binding sites on the genome caused similar decreases in spbR and papR5 transcription as those observed in ∆atrA-p. Taken together, herein, a novel mechanism for AtrA-dependent regulation of antibiotic biosynthesis was revealed in S. pristinaespiralis, which is distinct from those of its homologs, AtrA-c from Streptomyces coelicolor, AtrA-g from Streptomyces griseus, and AtrA from Streptomyces roseosporus that perform their effects in antibiotic biosynthesis directly via pathway-specific activator genes or the biosynthetic structural genes.


Assuntos
Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Pristinamicina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Sítios de Ligação , Elementos de DNA Transponíveis , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Mutagênese Insercional , Regiões Promotoras Genéticas , Ligação Proteica
4.
J Bacteriol ; 197(12): 2062-71, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25868645

RESUMO

UNLABELLED: Pristinamycin I (PI), produced by Streptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two proteinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of streptogramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is understood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded by SSDG_03033) was identified as playing a positive role in PI biosynthesis. Its homologue, PaaR, from Corynebacterium glutamicum serves as a transcriptional repressor of the paa genes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator as PaaR. Deletion of paaR led to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identical to the function of its homologue from C. glutamicum, PaaR is also involved in the suppression of paa expression. Given that phenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway of L-phenylglycine (L-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription of paa genes in a ΔpaaR mutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolic flux toward L-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI production of a ΔpaaR mutant was restored by L-Phg supplementation as well as by deletion of the paaABCDE operon in the ΔpaaR mutant. Altogether, this study provides new insights into the regulation of PI biosynthesis by S. pristinaespiralis. IMPORTANCE: A better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues for Streptomyces strain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription of paa genes involved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) by affecting the supply of one of seven amino acid precursors, L-phenylglycine, in Streptomyces pristinaespiralis. To our knowledge, this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis in Streptomyces strains. Considering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, it could be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Reguladores/fisiologia , Pristinamicina/biossíntese , Streptomyces/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Proteínas de Bactérias/genética , Deleção de Genes , Glicina/análogos & derivados , Glicina/metabolismo , Estrutura Molecular , Pristinamicina/química , Pristinamicina/metabolismo , Transcrição Gênica
5.
Int J Med Microbiol ; 304(1): 44-50, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24119565

RESUMO

Streptogramins are potent drugs against numerous highly resistant pathogens and therefore are used as antibiotics of last-resort human therapy. They consist of a mixture of two different types of chemical substances - the group A streptogramins, which are polyunsaturated macrolactones, and the group B streptogramins, representing cyclic hexadepsipeptides. Streptogramins are unique in their mode of action: each component alone exhibits a moderate bacteriostatic activity by binding to the bacterial 50S ribosomal subunit and thereby blocking translation, whereas the synergic combination of both substances is up to hundred fold more effective than the single compounds, resulting in a bactericidal activity. The streptogramin biosynthetic genes are organized as large antibiotic superclusters. These clusters harbour numerous regulatory genes, which encode different types of regulators that together form a complex hierarchical signalling system, which governs the regulation of streptogramin biosynthesis. Resistance is also regulated by this cascade. However, whereas resistance against streptogramins is quite well understood in diverse pathogenic organisms, only little is known about how the natural producer strains protect themselves against these toxic compounds. Here, we give an overview about the recent advances in streptogramin investigations with a main focus on the best-studied representatives, pristinamycin and virginiamycin. We concentrate on the biosynthesis of these compounds, their regulation and resistance determinants as well as their application in medicine and food industry.


Assuntos
Antibacterianos/farmacologia , Vias Biossintéticas/genética , Farmacorresistência Bacteriana , Viabilidade Microbiana/efeitos dos fármacos , Pristinamicina/farmacologia , Virginiamicina/farmacologia , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/uso terapêutico , Sinergismo Farmacológico , Indústria Alimentícia , Humanos , Pristinamicina/biossíntese , Pristinamicina/química , Pristinamicina/uso terapêutico , Virginiamicina/biossíntese , Virginiamicina/química , Virginiamicina/uso terapêutico
6.
Bioprocess Biosyst Eng ; 36(3): 259-72, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22923137

RESUMO

Streptomyces are filamentous bacteria which are widely used industrially for the production of therapeutic biomolecules, especially antibiotics. Bioreactor operating conditions may impact the physiological response of Streptomyces especially agitation and aeration as they influence hydromechanical stress, oxygen and nutrient transfer. The understanding of the coupling between physiological response and bioreactor hydrodynamics lies on a simultaneous description of the flow and transfers encountered by the bacteria and of the microbial response in terms of growth, consumption, morphology, production or intracellular signals. This article reviews the experimental and numerical works dedicated to the study of the coupling between bioreactor hydrodynamics and antibiotics producing Streptomyces. In a first part, the description of hydrodynamics used in these works is presented and then the main relations used. In a second part, the assumptions made in these works are discussed and put into emphasize. Lastly, the various Streptomyces physiological responses observed are detailed and compared.


Assuntos
Reatores Biológicos , Biotecnologia/métodos , Streptomyces/fisiologia , Antibacterianos/biossíntese , Fermentação , Hidrodinâmica , Oxigênio/química , Pristinamicina/biossíntese , Reologia , Streptomyces/metabolismo , Estresse Mecânico
7.
Curr Microbiol ; 65(6): 792-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22991052

RESUMO

The mechanisms for the enhancement of pristinamycin production in the high-yielding recombinants of Streptomyces pristinaespiralis obtained by genome shuffling were investigated by quantitative real-time PCR (Q-PCR) and amplified fragment length polymorphism (AFLP) techniques. Q-PCR analysis showed that snaB and snbA involved, respectively, in the biosynthesis of pristinamycins II and I component had more extended high expression in the recombinant than that in the ancestor during fermentation process, indicating their expression changes might be key factors during the biosynthesis of the antibiotic. In addition, the antecedent establishment of the high self-resistance to pristinamycin, because ptr resistance gene started high-level expression ahead of the onset of the antibiotic production in the recombinant, might also lead to the increase of the antibiotics yield. AFLP analysis of these recombinants revealed genome variation of two novel genes, the homologs of AfsR regulatory gene and transposase gene, indicating these two gene variations were probably responsible for yield improvement of pristinamycin. This study provided several potential molecular clues for pristinamycin yield enhancement.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pristinamicina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Proteínas de Bactérias/genética , Biotecnologia/métodos , Embaralhamento de DNA , Fermentação , Genes Bacterianos , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Recombinação Genética , Streptomyces/crescimento & desenvolvimento
8.
Electron. j. biotechnol ; 15(4): 6-6, July 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-646956

RESUMO

Response surface methodology was used to optimize the fermentation conditions for the production of pristinamycin by immobilization of Streptomyces pristinaespiralis F213 in shaking flask cultivation. Seed medium volume, fermentation medium volume and shaking speed of seed culture were found to have significant effects on pristinamycin production by the Plackett-Burman design. The steepest ascent method was adopted to approach the vicinity of optimum space, followed by central composite design for further optimization. A quadratic model was built to fit the pristinamycin production. The optimum conditions were found to be seed medium volume of 29.5 ml, fermentation medium volume of 28.8 ml, and shaking speed of seed culture at 204 rpm. At the optimum conditions, a production of 213 mg/l was obtained, which was in agreement with the maximum predicted pristinamycin yield of 209 mg/l. This is the first report on pristinamycins production by immobilized S. pristinaespiralis using response surface methodology.


Assuntos
Fermentação , Pristinamicina/biossíntese , Streptomyces/metabolismo , Técnicas de Cultura
9.
J Microbiol Biotechnol ; 22(6): 793-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22573156

RESUMO

A gene related to high pristinamycin yield in Streptomyces pristinaespiralis was selected by amplified fragment length polymorphism (AFLP) and its functions were investigated by gene disruption. First, a 561 bp polymorphic sequence was acquired by AFLP from high-yield recombinants compared with the S. pristinaespiralis ancestor ATCC25486, indicating that this approach is an effective means of screening for valuable genes responsible for antibiotic yield. Then, a 2,127 bp open reading frame of a gene designated spy1 that overlaps with the above fragment was identified and its structure and biological functions were investigated. In silico analysis of spy1 encoding a deduced 708-amino-acid-long serine/threonine protein kinase showed that it only contains a catalytic domain in the N-terminal region, which is different from some known homologs. Gene inactivation of chromosomal spy1 indicated that it plays a pleiotropic regulatory function in pristinamycin production, with a positive correlation to pristinamycin I biosynthesis and a negative correlation to pristinamycin II biosynthesis.


Assuntos
Antibacterianos/biossíntese , Pristinamicina/biossíntese , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , DNA Bacteriano/análise , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Fases de Leitura Aberta , Análise de Sequência de DNA
10.
Bioprocess Biosyst Eng ; 35(4): 513-7, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21947672

RESUMO

Seven amino acids were tested as precursors to affect pristinamycin production by a mutant strain derived from Streptomyces pristinaespiralis ATCC25486. Of those, glycine was selected as the best precursor to facilitate both cell growth and pristinamycin production at the feeding time of 36-h incubation and the feeding rate of 0.75 g L(-1) flask culture. The optimized time and concentration of glycine feeding were applied to enlarged 3-L bioreactor fermentation with a resin added at the time of 20-h fermentation for in situ separation. As a result, a combination of the glycine feeding and the added resin resulted in the maximal pristinamycin yield of 616 mg L(-1) culture 12 h after glycine feeding. The yield from the combined treatment was 1.71-, 2.77- and 4.32-fold of those from the mere glycine and resin treatments and the control, respectively. Other parameters, including intracellular nucleic acid content, animo nitrogen content and pH level, during 72-h fermentation were also given in association with the pristinamycin yields in the different treatments. The results indicate that glycine feeding is an effective approach to enhance pristinamycin production in the culture of S. pristinaespiralis F213 with supplemented resin for in situ separation.


Assuntos
Resinas Acrílicas/química , Reatores Biológicos/microbiologia , Glicina/administração & dosagem , Glicina/farmacocinética , Pristinamicina/biossíntese , Pristinamicina/isolamento & purificação , Streptomyces/metabolismo , Fermentação/efeitos dos fármacos , Fermentação/fisiologia , Streptomyces/efeitos dos fármacos
12.
Microb Biotechnol ; 4(2): 192-206, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21342465

RESUMO

Pristinamycin, produced by Streptomyces pristinaespiralis Pr11, is a streptogramin antibiotic consisting of two chemically unrelated compounds, pristinamycin I and pristinamycin II. The semi-synthetic derivatives of these compounds are used in human medicine as therapeutic agents against methicillin-resistant Staphylococcus aureus strains. Only the partial sequence of the pristinamycin biosynthetic gene cluster has been previously reported. To complete the sequence, overlapping cosmids were isolated from a S. pristinaespiralis Pr11 gene library and sequenced. The boundaries of the cluster were deduced, limiting the cluster size to approximately 210 kb. In the central region of the cluster, previously unknown pristinamycin biosynthetic genes were identified. Combining the current and previously identified sequence information, we propose that all essential pristinamycin biosynthetic genes are included in the 210 kb region. A pristinamycin biosynthetic pathway was established. Furthermore, the pristinamycin gene cluster was found to be interspersed by a cryptic secondary metabolite cluster, which probably codes for a glycosylated aromatic polyketide. Gene inactivation experiments revealed that this cluster has no influence on pristinamycin production. Overall, this work provides new insights into pristinamycin biosynthesis and the unique genetic organization of the pristinamycin gene region, which is the largest antibiotic 'supercluster' known so far.


Assuntos
Antibacterianos/biossíntese , Família Multigênica , Pristinamicina/biossíntese , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Streptomyces/classificação , Streptomyces/genética
13.
J Biotechnol ; 155(1): 63-7, 2011 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-21146568

RESUMO

Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid L-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added L-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed.


Assuntos
Genes Bacterianos , Glicina/análogos & derivados , Pristinamicina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Simulação por Computador , Glicina/biossíntese , Redes e Vias Metabólicas , Dados de Sequência Molecular , Mutagênese Insercional , Streptomyces/enzimologia
14.
Appl Biochem Biotechnol ; 160(6): 1853-64, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19629757

RESUMO

Improving pristinamycin production from Streptomyces pristinaespiralis was performed by introducing the resistance gene ptr followed by selection for enhanced tolerance to pristinamycin and fermentation test. To transfer ptr into S. pristinaespiralis, an effective method was established for the first time by using the intergeneric conjugation of DNA from Escherichia coli to S. pristinaespiralis. The procedure was optimized with heat treatment, spore concentration, optimum medium used in conjugation, concentration of MgCl(2), etc. With the optimized conditions, the conjugation frequency was up to 1.36 x 10(-3) exconjugants per recipient. The procedure was used to transfer the ptr gene into S. pristinaespiralis, resulting in 146 exconjugants. These exconjugants were screened on the pristinamycin-resistant plates, and then the fermentation test subsequently. Finally, two strains (SPR1 and SPR2) were obtained with a high yield of 0.11 and 0.15 g/l, respectively, which is about six to eight times more than that of wild-strain ATCC25486. The subculture experiments indicated that the hereditary character of the high-producing S. pristinaespiralis SPR1 and SPR2 was stable. Our work suggests that introducing resistance gene ptr into S. pristinaespiralis could be the way to improve the production of pristinamycin through the enhancement of antibiotic tolerance.


Assuntos
Conjugação Genética/genética , Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Pristinamicina/biossíntese , Streptomyces/genética , Clonagem Molecular , Conjugação Genética/efeitos dos fármacos , Meios de Cultura/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Fermentação/efeitos dos fármacos , Cloreto de Magnésio/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Esporos Bacterianos/citologia , Esporos Bacterianos/efeitos dos fármacos , Streptomyces/citologia , Streptomyces/efeitos dos fármacos , Temperatura
15.
Appl Biochem Biotechnol ; 144(2): 133-43, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18456945

RESUMO

The optimization of nutrient levels for the production of pristinamycins by Streptomyces pristinaespiralis CGMCC 0957 in submerged fermentation was carried out using the statistical methodologies based on the Plackett-Burman design, the steepest ascent method, and the central composite design (CCD). First, the Plackett-Burman design was applied to evaluate the influence of related nutrients in the medium. Soluble starch and MgSO4 x 7H2O were then identified as the most significant nutrients with a confidence level of 99%. Subsequently, the concentrations of the two nutrients were further optimized using response surface methodology of CCD, together with the steepest ascent method. Accordingly, a second-order polynomial regression model was finally fitted to the experimental data. By solving the regression equation from the model and analyzing the response surface, the optimal levels for soluble starch and MgSO4 x 7H2O were determined as 20.95 and 5.67g/L, respectively. Under the optimized medium, the yield of pristinamycins in the shake flask and 5-L bioreactor could reach 1.30 and 1.01 g/L, respectively, which is the highest yield reported in literature to date.


Assuntos
Meios de Cultura , Microbiologia Industrial/métodos , Modelos Estatísticos , Pristinamicina/biossíntese , Streptomyces/metabolismo , Antibacterianos/biossíntese , Reatores Biológicos
16.
Biotechnol Lett ; 30(8): 1423-9, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18368295

RESUMO

Amplified fragment length polymorphism (AFLP) was used to analyze genomic variability between high pristinamycin-producing recombinants of Streptomyces pristinaespiralis produced by genome shuffling and their ancestral strain. The AFLP fingerprints obtained with two restriction enzyme combinations of ApaI/TaqI and PstI/SacII showed together that there was no major polymorphism (less than 10%) between these high yield recombinants and their ancestor. However, the unique polymorphic bands, which might be related to the yield increasing of pristinamycin, could be distinguished from all the recombinants. Clustering analysis further indicated that the recombinants with similar ability of pristinamycin production had similar genomic variability.


Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Variação Genética , Genoma Bacteriano/genética , Polimorfismo Genético , Pristinamicina/biossíntese , Streptomyces/genética , Análise por Conglomerados , DNA Bacteriano/genética , Fermentação , Filogenia
17.
Biotechnol Lett ; 28(22): 1811-5, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16912920

RESUMO

Batch fermentation by Streptomyces pristinaespiralis with the addition of adsorbent resins was used to increase the production of pristinamycin. In consideration of the adsorption capacity and the desorption ability, a polymeric resin, JD-1, was finally selected. The maximum production of pristinamycin in Erlenmeyer flasks went up to 1.13 from 0.4 g l(-1), by adding 12% (w/v) resin JD-1 into the culture broth at 20 h after inoculation. In a 3 l bioreactor, pristinamycin fermentation with the addition of 12% (w/v) resin JD-1 at 20 h after inoculation reached 0.8 g l(-1), which was a 1.25-fold increase over fermentation without resin.


Assuntos
Antibacterianos/biossíntese , Reatores Biológicos , Biotecnologia/métodos , Fermentação , Pristinamicina/biossíntese , Resinas Vegetais/química , Streptomyces/metabolismo , Adsorção , Antibacterianos/química , Bioquímica/métodos , Concentração de Íons de Hidrogênio , Mutação , Pristinamicina/química , Fatores de Tempo
18.
Microbiology (Reading) ; 147(Pt 9): 2447-2459, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11535785

RESUMO

Phosphate-limited synthetic culture media were designed to investigate the growth and the pristinamycin production of 'Streptomyces pristinaespiralis' using different nitrogen sources. During balanced growth, either mineral or organic nitrogen sources were readily utilized. However, glutamate and alanine were used as both nitrogen and carbon source, sparing the utilization of the primary carbon source, glucose. Valine was utilized only for its nitrogen and consequently 2-ketoisovalerate was excreted in the medium. Ammonium prevented the utilization of nitrate. Upon phosphate limitation, glycerol, originating from the breakdown of teichoic acids, was released, allowing the recovery of phosphate from the cell wall and the continuation of growth. Under such conditions, ammonium was excreted following the consumption of glutamate and alanine and was later reassimilated after exhaustion of the primary nitrogen source. The mode of utilization of valine prevented the production of pristinamycins due to excretion of 2-ketoisovalerate, one of their direct precursors. For other nitrogen sources, pristinamycin production was controlled by nitrogen catabolic regulation linked to the residual level of ammonium. In the case of nitrate, the negative regulation was alleviated by the absence of ammonium and production then occurred precociously. In the case of amino acids and ammonium, production was delayed until after exhaustion of amino acids and depletion of ammonium.


Assuntos
Antibacterianos/biossíntese , Nitrogênio/metabolismo , Pristinamicina/biossíntese , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Alanina/metabolismo , Cloreto de Amônio/metabolismo , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Cinética , Modelos Biológicos , Nitratos/metabolismo , Valina/metabolismo
19.
J Biol Chem ; 276(47): 44297-306, 2001 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-11557748

RESUMO

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias , Pristinamicina/biossíntese , Receptores de Superfície Celular/fisiologia , Streptomyces/metabolismo , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Streptomyces/genética
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