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Stéphane Delaunay - One of the best experts on this subject based on the ideXlab platform.
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Decoupling of oxygen transfer and power dissipation for the study of the production of Pristinamycins by Streptomyces pristinaespiralis in shaking flasks
Biochemical Engineering Journal, 2012Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, Philippe Marchal, W. Klöckner, J. Büchs, Stéphane DelaunayAbstract:Streptomyces pristinaespiralis is a filamentous bacterium used in the pharmaceutical industry for the production of Pristinamycins. In previous works, it was shown that the occurrence of production and the antibiotics concentration could be related to gas-liquid transfer and power dissipation in shaking flasks. Nevertheless, in standard cultures, both mechanisms are coupled. It is then a difficult task to assign a precise physiological response to either oxygen transfer or power dissipation. The aim of the present study was to decouple the oxygen transfer coefficient (k(L)a) and the power dissipation per unit of volume (Ply) to study their respective impact on Pristinamycin production. During cultures in flasks, the rotation diameter of the shaking table was changed to modify the k(L)a but not the power dissipation P/V. The influence of operating conditions with P/V ranging from 0.55 to 10.3 kW m(-3) and k(L)a ranging from 30 to 490 h(-1) have been determined on the microbial kinetics and also on the pellet diameters. The final biomass concentration and the release of antibiotics were then related to k(L)a, whereas the final Pristinamycins concentrations, as well as the bacterial pellet diameter were mainly correlated to P/V independently of the k(L)a. The change in the pellet diameter could be the crucial parameter for the Pristinamycins production as it might influence the nutriment transfer inside the pellets and the ratio of active cells in each pellet.
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Oxygen supply controls the onset of Pristinamycins production by Streptomyces pristinaespiralis in shaking flasks.
Biotechnology and bioengineering, 2011Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, W. Klöckner, J. Büchs, D. Ullisch, Stéphane DelaunayAbstract:Antibiotics are secondary metabolites, gener- ally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics pro- duction are still not clearly established. In a previous study, the induction of Pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/ V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and Pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of Pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, Pristinamycins pro- duction was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L � 1 h � 1 . This sug- gested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce Pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting. Biotechnol. Bioeng. 2011;108: 2151-2161. 2011 Wiley Periodicals, Inc.
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Oxygen Supply Controls the Onset of Pristinamycins Production by Streptomyces pristinaespiralis in Shaking Flasks
Biotechnology and Bioengineering, 2011Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, W. Klöckner, J. Büchs, D. Ullisch, Stéphane DelaunayAbstract:Antibiotics are secondary metabolites, generally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics production are still not clearly established. In a previous study, the induction of Pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and Pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of Pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, Pristinamycins production was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L(-1) h(-1). This suggested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce Pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting.
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Intracellular pH of Streptomyces pristinaespiralis is correlated to the sequential use of carbon sources during the Pristinamycins-producing process
Enzyme and Microbial Technology, 2004Co-Authors: Philippe F.x Corvini, Stéphane Delaunay, Frédéric Maujean, Emmanuel Rondags, H. Vivier, Jean-louis Goergen, Pierre GermainAbstract:This work assesses, with epifluorescence microscopy and an image analysis method, glucose and amino acid metabolisms as carbon sources during Pristinamycin batch production process in correlation with intracellular pH-variations. At the start of the culture, results suggested preferential consumption of glutamate as carbon source over glucose and arginine. Besides, sequential use of glutamate and arginine as nitrogen sources has been observed, glutamate being assimilated before arginine. Once the glutamate is exhausted, glucose and arginine begin to become metabolised. Shortly before Pristinamycin excretion, a massive lactate and succinate excretion occurs. Excretion of Pristinamycins and organic acids and glucose and amino-acid consumption were shown to be correlated to measured intracellular pH-fluctuations, so that many cytoplasmic pH-variations can be explained by this study.
Charles J Thompson - One of the best experts on this subject based on the ideXlab platform.
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Novel Pristinamycin-responsive expression systems for plant cells.
Biotechnology and bioengineering, 2001Co-Authors: Alexander D. Frey, Charles J Thompson, Markus Rimann, James E. Bailey, Pauli Kallio, Martin FusseneggerAbstract:Novel gene regulation systems were designed for plant cells responsive to the streptogramin antibiotic Pristinamycin. The Pristinamycin-repressible plant gene regulation concept (PIPpOFF) is based on a transcriptional activator (PIT) which consists of the Pip protein, the repressor of the Pristinamycin resistance operon of Streptomyces coelicolor, fused to the VP16 transactivation domain of the Herpes simplex virus. PIT mediates Pristinamycin-repressible activation of a synthetic plant promoter (P(pPIR)) in tobacco cells consisting of a nine Pip-binding site-containing artificial operator (PIR3) placed upstream of a TATA-box derived from the cauliflower mosaic virus 35S promoter (P(CaMV35S)). Pristinamycin interferes with induction by negatively regulating the DNA-binding capacity of the Pip moiety of PIT. A second, streptogramin-inducible plant gene regulation system (PIPpON) was constructed by combining Pip expression with a plant-specific Pristinamycin-inducible promoter (P(pPIRON)). P(pPIRON) consists of a PIR3 module cloned downstream of the strong constitutive plant promoter P(CaMV35S). As in the native Streptomyces configuration, Pip binds to its cognate sequence within P(pPIRON) in the absence of regulating antibiotic and silences the chimeric plant promoter. Upon addition of Pristinamycin, Pip is released from the PIR3 operator and full P(CaMV35S)-driven expression of desired plant genes is induced. The PIPpOFF and PIPpON systems performed well in Nicotiana tabacum suspension cultures and promise to provide an attractive extension of existing plant gene regulation technology for basic plant research or biopharmaceutical manufacturing using plant tissue culture.
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a transcriptional regulator of a Pristinamycin resistance gene in streptomyces coelicolor
Journal of Biological Chemistry, 2001Co-Authors: Marc Folcher, Rowan P Morris, Glenn E Dale, Khadidja Salahbeyhocini, Patrick H Viollier, Charles J ThompsonAbstract:Abstract Pip is aPristinamycin-induced transcriptional regulatorprotein detected in many Streptomyces species by its ability to specifically bind sequence motifs within the promoter of a Streptomyces pristinaespiralis multidrug resistance gene (ptr). To investigate the possible role of Pip in regulating multidrug resistance, it was purified from a genetically characterized species, Streptomyces coelicolor, utilizing an affinity matrix of the ptr promoter conjugated to magnetic beads. Reverse genetics identified the corresponding locus and confirmed that it encoded Pip, a protein belonging to the TetR family of procaryotic transcriptional repressors. Pip binding motifs were located upstream of the adjacent gene pep, encoding a major facilitator antiporter homologous to ptr. In vivo analysis of antibiotic susceptibility profiles demonstrated that pep conferred elevated levels of resistance only to Pristinamycin I (PI), a streptogramin B antibiotic having clinical importance. Purified recombinant Pip was a dimer (in the presence or absence of PI) and displayed a high affinity for its palindromic binding motifs within theptr promoter and the upstream region of pep. The Pip/ptr promoter complex was dissociated by PI but not by any of the other nonstreptogramin antibiotics that were described previously as transcriptional inducers. These procaryotic regulatory elements served as the basis for the development of systems allowing repression or induction of cloned genes in mammalian and plant cells in response to streptogramin antibiotics (including Pristinamycin, virginiamycin, and Synercid®).
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molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the Pristinamycin producing organism streptomyces pristinaespiralis
Molecular Microbiology, 1995Co-Authors: Veronique Blanc, Marc Folcher, Khadidja Salahbey, Charles J ThompsonAbstract:A multidrug resistance gene (mdr) has been cloned from Streptomyces pristinaespiralis, a producer of two antibiotics having synergistic activities together known as Pristinamycin. This gene, ptr, provides resistance not only to two structurally dissimilar compounds (Pristinamycin I, PI; Pristinamycin II, PII) and the natural Pristinamycin mixture but also to rifampicin. Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analyzed. It contained an open reading frame of 1506 bp which encoded a putative membrane protein with 14 hydrophobic domains, and showed sequence similarity to a superfamily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic compounds. Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosynthetic gene clusters in actinomycetes. In vitro coupled transcription-translation experiments were used to identify the ptr gene product. Analysis of the upstream region did not reveal a divergently transcribed repressor gene, as is the case for several related resistance determinants involved in antibiotic transport, suggesting that ptr is regulated by a different mechanism. Transcriptional analyses of this gene, carried out in both S. pristinaespiralis and Streptomyces lividans, indicated the same transcriptional start point and predicted -10 and -35 hexamers which were somewhat similar to Streptomyces vegetative-type promoters.
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Molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the Pristinamycin‐producing organism, Streptomyces pristinaespiralis
Molecular microbiology, 1995Co-Authors: Veronique Blanc, Marc Folcher, Khadidja Salah-bey, Charles J ThompsonAbstract:A multidrug resistance gene (mdr) has been cloned from Streptomyces pristinaespiralis, a producer of two antibiotics having synergistic activities together known as Pristinamycin. This gene, ptr, provides resistance not only to two structurally dissimilar compounds (Pristinamycin I, PI; Pristinamycin II, PII) and the natural Pristinamycin mixture but also to rifampicin. Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analyzed. It contained an open reading frame of 1506 bp which encoded a putative membrane protein with 14 hydrophobic domains, and showed sequence similarity to a superfamily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic compounds. Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosynthetic gene clusters in actinomycetes. In vitro coupled transcription-translation experiments were used to identify the ptr gene product. Analysis of the upstream region did not reveal a divergently transcribed repressor gene, as is the case for several related resistance determinants involved in antibiotic transport, suggesting that ptr is regulated by a different mechanism. Transcriptional analyses of this gene, carried out in both S. pristinaespiralis and Streptomyces lividans, indicated the same transcriptional start point and predicted -10 and -35 hexamers which were somewhat similar to Streptomyces vegetative-type promoters.
Jean-louis Goergen - One of the best experts on this subject based on the ideXlab platform.
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Decoupling of oxygen transfer and power dissipation for the study of the production of Pristinamycins by Streptomyces pristinaespiralis in shaking flasks
Biochemical Engineering Journal, 2012Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, Philippe Marchal, W. Klöckner, J. Büchs, Stéphane DelaunayAbstract:Streptomyces pristinaespiralis is a filamentous bacterium used in the pharmaceutical industry for the production of Pristinamycins. In previous works, it was shown that the occurrence of production and the antibiotics concentration could be related to gas-liquid transfer and power dissipation in shaking flasks. Nevertheless, in standard cultures, both mechanisms are coupled. It is then a difficult task to assign a precise physiological response to either oxygen transfer or power dissipation. The aim of the present study was to decouple the oxygen transfer coefficient (k(L)a) and the power dissipation per unit of volume (Ply) to study their respective impact on Pristinamycin production. During cultures in flasks, the rotation diameter of the shaking table was changed to modify the k(L)a but not the power dissipation P/V. The influence of operating conditions with P/V ranging from 0.55 to 10.3 kW m(-3) and k(L)a ranging from 30 to 490 h(-1) have been determined on the microbial kinetics and also on the pellet diameters. The final biomass concentration and the release of antibiotics were then related to k(L)a, whereas the final Pristinamycins concentrations, as well as the bacterial pellet diameter were mainly correlated to P/V independently of the k(L)a. The change in the pellet diameter could be the crucial parameter for the Pristinamycins production as it might influence the nutriment transfer inside the pellets and the ratio of active cells in each pellet.
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Oxygen supply controls the onset of Pristinamycins production by Streptomyces pristinaespiralis in shaking flasks.
Biotechnology and bioengineering, 2011Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, W. Klöckner, J. Büchs, D. Ullisch, Stéphane DelaunayAbstract:Antibiotics are secondary metabolites, gener- ally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics pro- duction are still not clearly established. In a previous study, the induction of Pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/ V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and Pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of Pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, Pristinamycins pro- duction was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L � 1 h � 1 . This sug- gested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce Pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting. Biotechnol. Bioeng. 2011;108: 2151-2161. 2011 Wiley Periodicals, Inc.
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Oxygen Supply Controls the Onset of Pristinamycins Production by Streptomyces pristinaespiralis in Shaking Flasks
Biotechnology and Bioengineering, 2011Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, W. Klöckner, J. Büchs, D. Ullisch, Stéphane DelaunayAbstract:Antibiotics are secondary metabolites, generally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics production are still not clearly established. In a previous study, the induction of Pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and Pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of Pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, Pristinamycins production was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L(-1) h(-1). This suggested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce Pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting.
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Intracellular pH of Streptomyces pristinaespiralis is correlated to the sequential use of carbon sources during the Pristinamycins-producing process
Enzyme and Microbial Technology, 2004Co-Authors: Philippe F.x Corvini, Stéphane Delaunay, Frédéric Maujean, Emmanuel Rondags, H. Vivier, Jean-louis Goergen, Pierre GermainAbstract:This work assesses, with epifluorescence microscopy and an image analysis method, glucose and amino acid metabolisms as carbon sources during Pristinamycin batch production process in correlation with intracellular pH-variations. At the start of the culture, results suggested preferential consumption of glutamate as carbon source over glucose and arginine. Besides, sequential use of glutamate and arginine as nitrogen sources has been observed, glutamate being assimilated before arginine. Once the glutamate is exhausted, glucose and arginine begin to become metabolised. Shortly before Pristinamycin excretion, a massive lactate and succinate excretion occurs. Excretion of Pristinamycins and organic acids and glucose and amino-acid consumption were shown to be correlated to measured intracellular pH-fluctuations, so that many cytoplasmic pH-variations can be explained by this study.
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Intracellular pH determination of Pristinamycin-producing Streptomyces pristinaespiralis by image analysis
Microbiology, 2000Co-Authors: Philippe F.x Corvini, Emmanuel Rondags, H. Vivier, Jean-louis Goergen, H. Gautier, Pierre GermainAbstract:Intracellular pH (pHi) is an essential parameter in the regulation of intracellular processes. Thus, its measurement might provide clues regarding the physiological state of cells cultivated in vitro. pHi of the filamentous, Pristinamycin-producing Streptomyces pristinaespiralis was determined by epifluorescence microscopy and image analysis using the pH-sensitive fluorescent probe BCECF-AM [2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. Staining cell culture samples (OD660=1) of S. pristinaespiralis with 20 μM BCECF at 28 °C for 30 min yielded a green/red fluorescence ratio (R 527/600) that correlated with the pHi of the cells for values ranging from 6·5 to 8·5. When S. pristinaespiralis was cultivated in Pristinamycin-producing conditions (in batch mode, with a constant external pH of 6·8), the measured pHi varied between 6·3 and 8·7. In fact, pHi correlated with the excretion of Pristinamycins and glucose consumption during the production process.
Nasir Mehmood - One of the best experts on this subject based on the ideXlab platform.
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Decoupling of oxygen transfer and power dissipation for the study of the production of Pristinamycins by Streptomyces pristinaespiralis in shaking flasks
Biochemical Engineering Journal, 2012Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, Philippe Marchal, W. Klöckner, J. Büchs, Stéphane DelaunayAbstract:Streptomyces pristinaespiralis is a filamentous bacterium used in the pharmaceutical industry for the production of Pristinamycins. In previous works, it was shown that the occurrence of production and the antibiotics concentration could be related to gas-liquid transfer and power dissipation in shaking flasks. Nevertheless, in standard cultures, both mechanisms are coupled. It is then a difficult task to assign a precise physiological response to either oxygen transfer or power dissipation. The aim of the present study was to decouple the oxygen transfer coefficient (k(L)a) and the power dissipation per unit of volume (Ply) to study their respective impact on Pristinamycin production. During cultures in flasks, the rotation diameter of the shaking table was changed to modify the k(L)a but not the power dissipation P/V. The influence of operating conditions with P/V ranging from 0.55 to 10.3 kW m(-3) and k(L)a ranging from 30 to 490 h(-1) have been determined on the microbial kinetics and also on the pellet diameters. The final biomass concentration and the release of antibiotics were then related to k(L)a, whereas the final Pristinamycins concentrations, as well as the bacterial pellet diameter were mainly correlated to P/V independently of the k(L)a. The change in the pellet diameter could be the crucial parameter for the Pristinamycins production as it might influence the nutriment transfer inside the pellets and the ratio of active cells in each pellet.
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Oxygen supply controls the onset of Pristinamycins production by Streptomyces pristinaespiralis in shaking flasks.
Biotechnology and bioengineering, 2011Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, W. Klöckner, J. Büchs, D. Ullisch, Stéphane DelaunayAbstract:Antibiotics are secondary metabolites, gener- ally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics pro- duction are still not clearly established. In a previous study, the induction of Pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/ V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and Pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of Pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, Pristinamycins pro- duction was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L � 1 h � 1 . This sug- gested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce Pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting. Biotechnol. Bioeng. 2011;108: 2151-2161. 2011 Wiley Periodicals, Inc.
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Oxygen Supply Controls the Onset of Pristinamycins Production by Streptomyces pristinaespiralis in Shaking Flasks
Biotechnology and Bioengineering, 2011Co-Authors: Nasir Mehmood, Jean-louis Goergen, Eric Olmos, Fabrice Blanchard, W. Klöckner, J. Büchs, D. Ullisch, Stéphane DelaunayAbstract:Antibiotics are secondary metabolites, generally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics production are still not clearly established. In a previous study, the induction of Pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and Pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of Pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, Pristinamycins production was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L(-1) h(-1). This suggested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce Pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting.
Zhihua Jin - One of the best experts on this subject based on the ideXlab platform.
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Probing the molecular mechanisms for Pristinamycin yield enhancement in Streptomyces pristinaespiralis.
Current microbiology, 2012Co-Authors: Qingchao Jin, Li-jing Zhang, Zhihua Jin, Shan-jing YaoAbstract: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.
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Isolation and Functional Analysis of spy1 Responsible for Pristinamycin Yield in Streptomyces pristinaespiralis
Journal of microbiology and biotechnology, 2012Co-Authors: Qingchao Jin, Huali Yin, Xiaowei Hong, Zhihua JinAbstract: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.
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Glycine feeding improves Pristinamycin production during fermentation including resin for in situ separation.
Bioprocess and biosystems engineering, 2011Co-Authors: Li-jing Zhang, Zhihua Jin, Xiao-guang Chen, Qin-chao Jin, Ming-guang FengAbstract: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.
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Conjugal Transferring of Resistance Gene ptr for Improvement of Pristinamycin-Producing Streptomyces pristinaespiralis
Applied biochemistry and biotechnology, 2009Co-Authors: Zhihua Jin, Xin Jin, Qingchao JinAbstract: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.
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Improvement of Pristinamycin production by genome shuffling and medium optimization for Streptomyces pristinaespiralis
Biotechnology and Bioprocess Engineering, 2009Co-Authors: Zhihua Jin, Qingchao Jin, Peilin CenAbstract:To isolate an improved Pristinamycin producing strain of Streptomyces pristinaespiralis, the technique of Genome shuffling was used which resulted in a high-yield recombinant G 3-56 strain. Strain G 3-56 yielded 322 ± 17 mg/L of Pristinamycin which was 11.4-fold higher than that of the initial strain and 3.7-fold higher than strain UN-78 which previously had the highest yield of Pristinamycin. The genetic characteristics of the recombinant G 3-56 strain was stable as revealed by our subculture experiments. The optimal production medium was determined using the orthogonal matrix method. Under the optimal medium conditions, the maximum yield of Pristinamycin was 412 mg/L with about 1.24-fold higher than the original medium.