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Pramod P Wangikar - One of the best experts on this subject based on the ideXlab platform.
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detection of phase shifts in Batch fermentation via statistical analysis of the online measurements a case study with <B>RifamycinB> B fermentation
Journal of Biotechnology, 2007Co-Authors: Xuantien Doan, Prashant M Bapat, Rajagopalan Srinivasan, Pramod P WangikarAbstract:Industrial production of antiBiotics, Biopharmaceuticals and enzymes is typically carried out via a Batch or fed-Batch fermentation process. These processes go through various phases Based on sequential suBstrate uptake, growth and product formation, which require monitoring due to the potential Batch-to-Batch variaBility. The phase shifts can Be identified directly By measuring the concentrations of suBstrates and products or By morphological examinations under microscope. However, such measurements are cumBersome to oBtain. We present a method to identify phase transitions in Batch fermentation using readily availaBle online measurements. Our approach is Based on Dynamic Principal Component Analysis (DPCA), a multivariate statistical approach that can model the dynamics of non-stationary processes. Phase-transitions in fermentation produce distinct patterns in the DPCA scores, which can Be identified as singular points. We illustrate the application of the method to detect transitions such as the onset of exponential growth phase, suBstrate exhaustion and suBstrate switching for <B>RifamycinB> B fermentation Batches. Further, we analyze the loading vectors of DPCA model to illustrate the mechanism By which the statistical model accounts for process dynamics. The approach can Be readily applied to other industrially important processes and may have implications in online monitoring of fermentation Batches in a production facility.
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hierarchical amino acid utilization and its influence on fermentation dynamics <B>RifamycinB> B fermentation using amycolatopsis mediterranei s699 a case study
Microbial Cell Factories, 2006Co-Authors: Prashant M Bapat, Sujata V. Sohoni, Pramod P WangikarAbstract:Background Industrial fermentation typically uses complex nitrogen suBstrates which consist of mixture of amino acids. The uptake of amino acids is known to Be mediated By several amino acid transporters with certain preferences. However, models to predict this preferential uptake are not availaBle. We present the stoichiometry for the utilization of amino acids as a sole carBon and nitrogen suBstrate or along with glucose as an additional carBon source. In the former case, the excess nitrogen provided By the amino acids is excreted By the organism in the form of ammonia. We have developed a cyBernetic model to predict the sequence and kinetics of uptake of amino acids. The model is Based on the assumption that the growth on a specific suBstrate is dependent on key enzyme(s) responsiBle for the uptake and assimilation of the suBstrates. These enzymes may Be regulated By mechanisms of nitrogen cataBolite repression. The model hypothesizes that the organism is an optimal strategist and invests resources for the uptake of a suBstrate that are proportional to the returns.
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A cyBernetic model to predict the effect of freely availaBle nitrogen suBstrate on <B>RifamycinB> B production in complex media
Applied Microbiology and Biotechnology, 2006Co-Authors: Prashant M Bapat, Sujata V. Sohoni, Tessa A. Moses, Pramod P WangikarAbstract:It is well-known that secondary metaBolite production is repressed By excess nitrogen suBstrate availaBle in the fermentation media. Although the nitrogen cataBolite repression has Been known, quantitative process models have not Been reported to represent this phenomenon in complex medium. In this paper, we present a cyBernetic model for <B>RifamycinB> B production via Amycolatopsis mediterranei S699 in complex medium, which is typically used in industry. Nitrogen suBstrate is assumed to Be present in two forms in the medium; availaBle nitrogen ( S _ANS) such as free amino acids and unavailaBle nitrogen ( S _UNS) such as peptides and proteins. The model assumes that an induciBle enzyme catalyzes the conversion of S _UNS to S _ANS. Although S _ANS is required for growth and product formation, high concentrations were found to inhiBit <B>RifamycinB> production. To experimentally validate the model, five different organic nitrogen sources were used that differ in the ratio of S _ANS/ S _UNS. The model successfully predicts higher <B>RifamycinB> B productivity for nitrogen sources that contain lower initial S _ANS. The higher productivity is attriButed to the sustained availaBility of S _ANS at low concentration via conversion of S _UNS to S _ANS, thereBy minimizing the effects of nitrogen cataBolite repression on <B>RifamycinB> production. The model can have applications in model-Based optimization of suBstrate feeding recipe and in monitoring and control of fed Batch processes.
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structured kinetic model to represent the utilization of multiple suBstrates in complex media during <B>RifamycinB> B fermentation
Biotechnology and Bioengineering, 2006Co-Authors: Prashant M Bapat, Sharad Bhartiya, K V Venkatesh, Pramod P WangikarAbstract:Industrial fermentations typically use media that are Balanced with multiple suBstitutaBle suBstrates including complex carBon and nitrogen source. Yet, much of the modeling effort to date has mainly focused on defined media. Here, we present a structured model that accounts for growth and product formation kinetics of <B>RifamycinB> B fermentation in a multi-suBstrate complex medium. The phenomenological model considers the organism to Be an optimal strategist with an in-Built mechanism that regulates the sequential and simultaneous uptake of the suBstrate comBinations. This regulatory process is modeled By assuming that the uptake of a suBstrate depends on the level of a key enzyme or a set of enzymes, which may Be induciBle. Further, the fraction of flux through a given metaBolic Branch is estimated using a simple multi-variaBle constrained optimization. The model has the typical form of Monod equation with terms incorporating multiple limiting suBstrates and suBstrate inhiBition. Several Batch runs were set up with varying initial suBstrate concentrations to estimate the kinetic parameters for the <B>RifamycinB> overproducer strain Amycolatopsis mediterranei S699. Glucose and ammonium sulfate (AMS) demonstrated significant suBstrate inhiBition toward growth as well as product formation. The model correctly predicts the experimentally oBserved regulated simultaneous uptake of the suBstitutaBle suBstrate comBinations under different fermentation conditions. The modeling results may have applications in the optimization and control of <B>RifamycinB> B fermentation while the modeling strategy presented here would Be applicaBle to other industrially important fermentations. © 2005 Wiley Periodicals, Inc.
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structured kinetic model to represent the utilization of multiple suBstrates in complex media during <B>RifamycinB> B fermentation
Biotechnology and Bioengineering, 2006Co-Authors: Prashant M Bapat, Sharad Bhartiya, K V Venkatesh, Pramod P WangikarAbstract:Industrial fermentations typically use media that are Balanced with multiple suBstitutaBle suBstrates including complex carBon and nitrogen source. Yet, much of the modeling effort to date has mainly focused on defined media. Here, we present a structured model that accounts for growth and product formation kinetics of <B>RifamycinB> B fermentation in a multi-suBstrate complex medium. The phenomenological model considers the organism to Be an optimal strategist with an in-Built mechanism that regulates the sequential and simultaneous uptake of the suBstrate comBinations. This regulatory process is modeled By assuming that the uptake of a suBstrate depends on the level of a key enzyme or a set of enzymes, which may Be induciBle. Further, the fraction of flux through a given metaBolic Branch is estimated using a simple multi-variaBle constrained optimization. The model has the typical form of Monod equation with terms incorporating multiple limiting suBstrates and suBstrate inhiBition. Several Batch runs were set up with varying initial suBstrate concentrations to estimate the kinetic parameters for the <B>RifamycinB> overproducer strain Amycolatopsis mediterranei S699. Glucose and ammonium sulfate (AMS) demonstrated significant suBstrate inhiBition toward growth as well as product formation. The model correctly predicts the experimentally oBserved regulated simultaneous uptake of the suBstitutaBle suBstrate comBinations under different fermentation conditions. The modeling results may have applications in the optimization and control of <B>RifamycinB> B fermentation while the modeling strategy presented here would Be applicaBle to other industrially important fermentations.
Prashant M Bapat - One of the best experts on this subject based on the ideXlab platform.
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detection of phase shifts in Batch fermentation via statistical analysis of the online measurements a case study with <B>RifamycinB> B fermentation
Journal of Biotechnology, 2007Co-Authors: Xuantien Doan, Prashant M Bapat, Rajagopalan Srinivasan, Pramod P WangikarAbstract:Industrial production of antiBiotics, Biopharmaceuticals and enzymes is typically carried out via a Batch or fed-Batch fermentation process. These processes go through various phases Based on sequential suBstrate uptake, growth and product formation, which require monitoring due to the potential Batch-to-Batch variaBility. The phase shifts can Be identified directly By measuring the concentrations of suBstrates and products or By morphological examinations under microscope. However, such measurements are cumBersome to oBtain. We present a method to identify phase transitions in Batch fermentation using readily availaBle online measurements. Our approach is Based on Dynamic Principal Component Analysis (DPCA), a multivariate statistical approach that can model the dynamics of non-stationary processes. Phase-transitions in fermentation produce distinct patterns in the DPCA scores, which can Be identified as singular points. We illustrate the application of the method to detect transitions such as the onset of exponential growth phase, suBstrate exhaustion and suBstrate switching for <B>RifamycinB> B fermentation Batches. Further, we analyze the loading vectors of DPCA model to illustrate the mechanism By which the statistical model accounts for process dynamics. The approach can Be readily applied to other industrially important processes and may have implications in online monitoring of fermentation Batches in a production facility.
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hierarchical amino acid utilization and its influence on fermentation dynamics <B>RifamycinB> B fermentation using amycolatopsis mediterranei s699 a case study
Microbial Cell Factories, 2006Co-Authors: Prashant M Bapat, Sujata V. Sohoni, Pramod P WangikarAbstract:Background Industrial fermentation typically uses complex nitrogen suBstrates which consist of mixture of amino acids. The uptake of amino acids is known to Be mediated By several amino acid transporters with certain preferences. However, models to predict this preferential uptake are not availaBle. We present the stoichiometry for the utilization of amino acids as a sole carBon and nitrogen suBstrate or along with glucose as an additional carBon source. In the former case, the excess nitrogen provided By the amino acids is excreted By the organism in the form of ammonia. We have developed a cyBernetic model to predict the sequence and kinetics of uptake of amino acids. The model is Based on the assumption that the growth on a specific suBstrate is dependent on key enzyme(s) responsiBle for the uptake and assimilation of the suBstrates. These enzymes may Be regulated By mechanisms of nitrogen cataBolite repression. The model hypothesizes that the organism is an optimal strategist and invests resources for the uptake of a suBstrate that are proportional to the returns.
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A cyBernetic model to predict the effect of freely availaBle nitrogen suBstrate on <B>RifamycinB> B production in complex media
Applied Microbiology and Biotechnology, 2006Co-Authors: Prashant M Bapat, Sujata V. Sohoni, Tessa A. Moses, Pramod P WangikarAbstract:It is well-known that secondary metaBolite production is repressed By excess nitrogen suBstrate availaBle in the fermentation media. Although the nitrogen cataBolite repression has Been known, quantitative process models have not Been reported to represent this phenomenon in complex medium. In this paper, we present a cyBernetic model for <B>RifamycinB> B production via Amycolatopsis mediterranei S699 in complex medium, which is typically used in industry. Nitrogen suBstrate is assumed to Be present in two forms in the medium; availaBle nitrogen ( S _ANS) such as free amino acids and unavailaBle nitrogen ( S _UNS) such as peptides and proteins. The model assumes that an induciBle enzyme catalyzes the conversion of S _UNS to S _ANS. Although S _ANS is required for growth and product formation, high concentrations were found to inhiBit <B>RifamycinB> production. To experimentally validate the model, five different organic nitrogen sources were used that differ in the ratio of S _ANS/ S _UNS. The model successfully predicts higher <B>RifamycinB> B productivity for nitrogen sources that contain lower initial S _ANS. The higher productivity is attriButed to the sustained availaBility of S _ANS at low concentration via conversion of S _UNS to S _ANS, thereBy minimizing the effects of nitrogen cataBolite repression on <B>RifamycinB> production. The model can have applications in model-Based optimization of suBstrate feeding recipe and in monitoring and control of fed Batch processes.
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structured kinetic model to represent the utilization of multiple suBstrates in complex media during <B>RifamycinB> B fermentation
Biotechnology and Bioengineering, 2006Co-Authors: Prashant M Bapat, Sharad Bhartiya, K V Venkatesh, Pramod P WangikarAbstract:Industrial fermentations typically use media that are Balanced with multiple suBstitutaBle suBstrates including complex carBon and nitrogen source. Yet, much of the modeling effort to date has mainly focused on defined media. Here, we present a structured model that accounts for growth and product formation kinetics of <B>RifamycinB> B fermentation in a multi-suBstrate complex medium. The phenomenological model considers the organism to Be an optimal strategist with an in-Built mechanism that regulates the sequential and simultaneous uptake of the suBstrate comBinations. This regulatory process is modeled By assuming that the uptake of a suBstrate depends on the level of a key enzyme or a set of enzymes, which may Be induciBle. Further, the fraction of flux through a given metaBolic Branch is estimated using a simple multi-variaBle constrained optimization. The model has the typical form of Monod equation with terms incorporating multiple limiting suBstrates and suBstrate inhiBition. Several Batch runs were set up with varying initial suBstrate concentrations to estimate the kinetic parameters for the <B>RifamycinB> overproducer strain Amycolatopsis mediterranei S699. Glucose and ammonium sulfate (AMS) demonstrated significant suBstrate inhiBition toward growth as well as product formation. The model correctly predicts the experimentally oBserved regulated simultaneous uptake of the suBstitutaBle suBstrate comBinations under different fermentation conditions. The modeling results may have applications in the optimization and control of <B>RifamycinB> B fermentation while the modeling strategy presented here would Be applicaBle to other industrially important fermentations. © 2005 Wiley Periodicals, Inc.
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structured kinetic model to represent the utilization of multiple suBstrates in complex media during <B>RifamycinB> B fermentation
Biotechnology and Bioengineering, 2006Co-Authors: Prashant M Bapat, Sharad Bhartiya, K V Venkatesh, Pramod P WangikarAbstract:Industrial fermentations typically use media that are Balanced with multiple suBstitutaBle suBstrates including complex carBon and nitrogen source. Yet, much of the modeling effort to date has mainly focused on defined media. Here, we present a structured model that accounts for growth and product formation kinetics of <B>RifamycinB> B fermentation in a multi-suBstrate complex medium. The phenomenological model considers the organism to Be an optimal strategist with an in-Built mechanism that regulates the sequential and simultaneous uptake of the suBstrate comBinations. This regulatory process is modeled By assuming that the uptake of a suBstrate depends on the level of a key enzyme or a set of enzymes, which may Be induciBle. Further, the fraction of flux through a given metaBolic Branch is estimated using a simple multi-variaBle constrained optimization. The model has the typical form of Monod equation with terms incorporating multiple limiting suBstrates and suBstrate inhiBition. Several Batch runs were set up with varying initial suBstrate concentrations to estimate the kinetic parameters for the <B>RifamycinB> overproducer strain Amycolatopsis mediterranei S699. Glucose and ammonium sulfate (AMS) demonstrated significant suBstrate inhiBition toward growth as well as product formation. The model correctly predicts the experimentally oBserved regulated simultaneous uptake of the suBstitutaBle suBstrate comBinations under different fermentation conditions. The modeling results may have applications in the optimization and control of <B>RifamycinB> B fermentation while the modeling strategy presented here would Be applicaBle to other industrially important fermentations.
Heinz G. Floss - One of the best experts on this subject based on the ideXlab platform.
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identification of tailoring genes involved in the modification of the polyketide BackBone of <B>RifamycinB> B By amycolatopsis mediterranei s699
Microbiology, 2005Co-Authors: Jun Xu, Taifo Mahmud, Heinz G. FlossAbstract:<B>RifamycinB> B Biosynthesis By Amycolatopsis mediterranei S699 involves a numBer of unusual modification reactions in the formation of the unique polyketide BackBone and decoration of the molecule. A numBer of genes Believed to Be involved in the tailoring of <B>RifamycinB> B were investigated and the results confirmed that the formation of the naphthalene ring moiety of <B>RifamycinB> takes place during the polyketide chain extension and is catalysed By Rif-Orf19, a 3-(3-hydroxyphenyl)propionate hydroxylase-like protein. The cytochrome P450-dependent monooxygenase encoded By rif-orf5 is required for the conversion of the Δ12, 29 olefinic Bond in the polyketide BackBone of <B>RifamycinB> W into the ketal moiety of <B>RifamycinB> B. Furthermore, Rif-Orf3 may Be involved in the regulation of <B>RifamycinB> B production, as its knock-out mutant produced aBout 40 % more <B>RifamycinB> B than the wild-type. The work also revealed that many of the genes located in the cluster are not involved in <B>RifamycinB> Biosynthesis, But might Be evolutionary remnants carried over from an ancestral lineage.
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isolation and characterization of 27 o demethyl<B>RifamycinB> sv methyltransferase provides new insights into the post pks modification steps during the Biosynthesis of the antituBercular drug <B>RifamycinB> B By amycolatopsis mediterranei s699
Archives of Biochemistry and Biophysics, 2003Co-Authors: Jun Xu, Taifo Mahmud, Heinz G. FlossAbstract:ABstract The gene rif orf14 in the <B>RifamycinB> Biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antituBercular drug <B>RifamycinB> B, encodes a protein of 272 amino acids identified as an AdoMet: 27- O -demethyl<B>RifamycinB> SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no <B>RifamycinB> B, But accumulates 27- O -demethyl<B>RifamycinB> SV (DMRSV) as the major new metaBolite, together with a small quantity of 27- O -demethyl-25- O -desacetyl<B>RifamycinB> SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to <B>RifamycinB> SV, But not that of DMDARSV or <B>RifamycinB> W. 27- O -Demethyl<B>RifamycinB> S was methylated poorly, if at all, By the enzyme to produce <B>RifamycinB> S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca 2+ or Mg 2+ inhiBits the enzyme activity slightly, Zn 2+ , Ni 2+ , and Co 2+ are strongly inhiBitory. The K m values for DMRSV and S -adenosyl- l -methionine (AdoMet) are 18.0 and 19.3 μM, respectively, and the K cat is 87 s −1 . The results indicate that DMRSV is a direct precursor of <B>RifamycinB> SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that <B>RifamycinB> S is not the precursor of <B>RifamycinB> SV in <B>RifamycinB> B Biosynthesis, But rather an oxidative shunt-product.
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mutational analysis and reconstituted expression of the Biosynthetic genes involved in the formation of 3 amino 5 hydroxyBenzoic acid the starter unit of <B>RifamycinB> Biosynthesis in amycolatopsis mediterranei s699
Journal of Biological Chemistry, 2001Co-Authors: Tin-wein Yu, Eckhard Leistner, Rolf Müller, Xiaohong Zhang, Michael Muller, Gerald Draeger, Heinz G. FlossAbstract:ABstract To investigate a novel Branch of the shikimate Biosynthesis pathway operating in the formation of 3-amino-5-hydroxyBenzoic acid (AHBA), the unique Biosynthetic precursor of <B>RifamycinB> and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated inAmycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined By incuBating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL,-M, and -N genes were shown to Be involved in the step(s) from either phosphoenolpyruvate/d-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-d-araBino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemBle enzymes involved in the shikimate Biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Muller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998)Chem. Biol. 5, 69–79). Mutants of the rifH and-J genes produced <B>RifamycinB> B at 1% and 10%, respectively, of the yields of the wild type; inactivation of therifG gene did not affect <B>RifamycinB> production significantly. Finally, coexpressing the rifG–N and-J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomicrifG–N and -J mutations on <B>RifamycinB> production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for <B>RifamycinB> Biosynthesis By A. mediterranei.
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thioesterases and the premature termination of polyketide chain elongation in <B>RifamycinB> B Biosynthesis By amycolatopsis mediterranei s699
The Journal of Antibiotics, 2000Co-Authors: Yukiko Doikatayama, Chayong Choi, Tin-wein Yu, Heinz G. Floss, C R HutchinsonAbstract:The role of two thioesterase genes in the premature release of polyketide synthase intermediates during <B>RifamycinB> Biosynthesis in the Amycolatopsis mediterranei S699 strain was investigated. Creation of an in-frame deletion in the rifR gene led to a 30-60% decrease in the production of Both <B>RifamycinB> B By the S699 strain or a series of tetra- to decaketide shunt products of polyketide chain assemBly By the rifF strain. Since a similar percentage decrease was seen in Both genetic Backgrounds, we conclude that the RifR thioesterase 2 is not involved in premature release of the carBon chain assemBly intermediates. Similarly, fusion of the Saccharopolyspora erythraea DEB S3 thioesterase 1 domain to the C-terminus of the RifE PKS suBunit did not result in a noticeaBle increase in the amount of the undecaketide intermediate formed nor in the amounts of the tetra- to decaketide shunt products. Hence, premature release of the carBon chain assemBly intermediates is an unusual property of the Rif PKS itself.
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thioesterases and the premature termination of polyketide chain elongation in <B>RifamycinB> B Biosynthesis By amycolatopsis mediterranei s699
The Journal of Antibiotics, 2000Co-Authors: Yukiko Doikatayama, Chayong Choi, Heinz G. Floss, Yeo J Yoon, C R HutchinsonAbstract:The role of two thioesterase genes in the premature release of polyketide synthase intermediates during <B>RifamycinB> Biosynthesis in the Amycolatopsis mediterranei S699 strain was investigated. Creation of an in-frame deletion in the rifR gene led to a 30 approximately 60% decrease in the production of Both <B>RifamycinB> B By the S699 strain or a series of tetra- to decaketide shunt products of polyketide chain assemBly By the rifF strain. Since a similar percentage decrease was seen in Both genetic Backgrounds, we conclude that the RifR thioesterase 2 is not involved in premature release of the carBon chain assemBly intermediates. Similarly, fusion of the Saccharopolyspora erythraea DEBS3 thioesterase I domain to the C-terminus of the RifE PKS suBunit did not result in a noticeaBle increase in the amount of the undecaketide intermediate formed nor in the amounts of the tetra- to decaketide shunt products. Hence, premature release of the carBon chain assemBly intermediates is an unusual property of the Rif PKS itself.
Jin Wang - One of the best experts on this subject based on the ideXlab platform.
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a feedBack regulatory model for rifq mediated repression of <B>RifamycinB> export in amycolatopsis mediterranei
Microbial Cell Factories, 2018Co-Authors: Jingzhi Wang, Guoping Zhao, Jin WangAbstract:Due to the important role of <B>RifamycinB> in curing tuBerculosis infection, the study on <B>RifamycinB> has never Been stopped. Although RifZ, which locates within the <B>RifamycinB> Biosynthetic cluster, has recently Been characterized as a pathway-specific regulator for <B>RifamycinB> Biosynthesis, little is known aBout the regulation of <B>RifamycinB> export. In this work, we proved that the expression of the <B>RifamycinB> efflux pump (RifP) was regulated By RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on Bacterial growth, But resulted in improved <B>RifamycinB> production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic moBility shift assay and DNase I Footprinting assay, RifQ was found to directly Bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to Be upstream of the RifQ Binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, <B>RifamycinB> B (the end product of <B>RifamycinB> Biosynthesis) remarkaBly decreased the DNA Binding affinity of RifQ, which led to derepression of <B>RifamycinB> export, reducing the intracellular concentration of <B>RifamycinB> B as well as its toxicity against the host. Here, we proved that the export of <B>RifamycinB> B was repressed By RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could Be specifically relieved By <B>RifamycinB> B, the end product of <B>RifamycinB> Biosynthesis, Based on which a feedBack model was proposed for regulation of <B>RifamycinB> export. With the findings here, one could improve the antiBiotic yield By simply inactivating the negative regulator of the antiBiotic transporter.
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MOESM8 of A feedBack regulatory model for RifQ-mediated repression of <B>RifamycinB> export in Amycolatopsis mediterranei
2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:Additional file 8: Figure S8. Effect of <B>RifamycinB>s to the transcription of rifP. <B>RifamycinB> SV and <B>RifamycinB> B were added into the LYZL11 culture medium, respectively, and the rifP transcriptional level was measured at 24Â h after the addition of <B>RifamycinB>s. DMSO was used as a Blank control and rpoB was used as an internal control
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A feedBack regulatory model for RifQ-mediated repression of <B>RifamycinB> export in Amycolatopsis mediterranei
BMC, 2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:ABstract Background Due to the important role of <B>RifamycinB> in curing tuBerculosis infection, the study on <B>RifamycinB> has never Been stopped. Although RifZ, which locates within the <B>RifamycinB> Biosynthetic cluster, has recently Been characterized as a pathway-specific regulator for <B>RifamycinB> Biosynthesis, little is known aBout the regulation of <B>RifamycinB> export. Results In this work, we proved that the expression of the <B>RifamycinB> efflux pump (RifP) was regulated By RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on Bacterial growth, But resulted in improved <B>RifamycinB> production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic moBility shift assay and DNase I Footprinting assay, RifQ was found to directly Bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to Be upstream of the RifQ Binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, <B>RifamycinB> B (the end product of <B>RifamycinB> Biosynthesis) remarkaBly decreased the DNA Binding affinity of RifQ, which led to derepression of <B>RifamycinB> export, reducing the intracellular concentration of <B>RifamycinB> B as well as its toxicity against the host. Conclusions Here, we proved that the export of <B>RifamycinB> B was repressed By RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could Be specifically relieved By <B>RifamycinB> B, the end product of <B>RifamycinB> Biosynthesis, Based on which a feedBack model was proposed for regulation of <B>RifamycinB> export. With the findings here, one could improve the antiBiotic yield By simply inactivating the negative regulator of the antiBiotic transporter
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two genes rif15 and rif16 of the <B>RifamycinB> Biosynthetic gene cluster in amycolatopsis mediterranei likely encode a transketolase and a p450 monooxygenase respectively Both essential for the conversion of <B>RifamycinB> sv into B
Acta Biochimica et Biophysica Sinica, 2011Co-Authors: Hua Yuan, Xiaoming Ding, Yi Zhong, Jin Wang, Wei Zhao, Guoping ZhaoAbstract:Amycolatopsis mediterranei produces an important antiBiotic <B>RifamycinB>, the Biosynthesis of which involves many unusual modifications. Previous work suggested a putative P450 enzyme encoded By rif16 within the <B>RifamycinB> Biosynthetic gene cluster (rif ) was required for the conversion of the intermediate <B>RifamycinB> SV into the end product <B>RifamycinB> B. In this study, we genetically proved that a putative transketolase encoded By rif15 is another essential enzyme for this conversion. Expression of merely rif15 and rif16 in a rif cluster null mutant of A. mediterranei U32 was aBle to convert <B>RifamycinB> SV into B. However, this Rif15- and Rif16-mediated conversion was only detected in intact cells of A. meidterranei, But not in Streptomyce coelicolor or MycoBacterium smegmatis, suggesting that yet-characterized gene(s) in A. mediterranei other than those encoded By the rif cluster should Be involved in this process.
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complete genome sequence of the <B>RifamycinB> sv producing amycolatopsis mediterranei u32 revealed its genetic characteristics in phylogeny and metaBolism
Cell Research, 2010Co-Authors: Wei Zhao, Huajun Zheng, Gang Lu, Hua Yuan, Feng Xu, Yi Zhong, Jin Wang, Ying Wang, Zhi Hui ShaoAbstract:Amycolatopsis mediterranei is used for industry-scale production of <B>RifamycinB>, which plays a vital role in antimycoBacterial therapy. As the first sequenced genome of the genus Amycolatopsis, the chromosome of strain U32 comprising 10 236 715 Base pairs, is one of the largest prokaryotic genomes ever sequenced so far. Unlike the linear topology found in streptomycetes, this chromosome is circular, particularly similar to that of Saccharopolyspora erythraea and Nocardia farcinica, representing their close relationship in phylogeny and taxonomy. Although the predicted 9 228 protein-coding genes in the A. mediterranei genome shared the greatest numBer of orthologs with those of S. erythraea, it was unexpectedly followed By Streptomyces coelicolor rather than N. farcinica, indicating the distinct metaBolic characteristics evolved via adaptation to diverse ecological niches. Besides a core region analogous to that common in streptomycetes, a novel 'quasi-core' with typical core characteristics is defined within the non-core region, where 21 out of the total 26 gene clusters for secondary metaBolite production are located. The <B>RifamycinB> Biosynthesis gene cluster located in the core encodes a cytochrome P450 enzyme essential for the conversion of <B>RifamycinB> SV to B, revealed By comparing to the highly homologous cluster of the <B>RifamycinB> B-producing strain S699 and further confirmed By genetic complementation. The genomic information of A. mediterranei demonstrates a metaBolic network orchestrated not only for extensive utilization of various carBon sources and inorganic nitrogen compounds But also for effective funneling of metaBolic intermediates into the secondary antiBiotic synthesis process under the control of a seemingly complex regulatory mechanism.
Guoping Zhao - One of the best experts on this subject based on the ideXlab platform.
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a feedBack regulatory model for rifq mediated repression of <B>RifamycinB> export in amycolatopsis mediterranei
Microbial Cell Factories, 2018Co-Authors: Jingzhi Wang, Guoping Zhao, Jin WangAbstract:Due to the important role of <B>RifamycinB> in curing tuBerculosis infection, the study on <B>RifamycinB> has never Been stopped. Although RifZ, which locates within the <B>RifamycinB> Biosynthetic cluster, has recently Been characterized as a pathway-specific regulator for <B>RifamycinB> Biosynthesis, little is known aBout the regulation of <B>RifamycinB> export. In this work, we proved that the expression of the <B>RifamycinB> efflux pump (RifP) was regulated By RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on Bacterial growth, But resulted in improved <B>RifamycinB> production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic moBility shift assay and DNase I Footprinting assay, RifQ was found to directly Bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to Be upstream of the RifQ Binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, <B>RifamycinB> B (the end product of <B>RifamycinB> Biosynthesis) remarkaBly decreased the DNA Binding affinity of RifQ, which led to derepression of <B>RifamycinB> export, reducing the intracellular concentration of <B>RifamycinB> B as well as its toxicity against the host. Here, we proved that the export of <B>RifamycinB> B was repressed By RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could Be specifically relieved By <B>RifamycinB> B, the end product of <B>RifamycinB> Biosynthesis, Based on which a feedBack model was proposed for regulation of <B>RifamycinB> export. With the findings here, one could improve the antiBiotic yield By simply inactivating the negative regulator of the antiBiotic transporter.
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MOESM8 of A feedBack regulatory model for RifQ-mediated repression of <B>RifamycinB> export in Amycolatopsis mediterranei
2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:Additional file 8: Figure S8. Effect of <B>RifamycinB>s to the transcription of rifP. <B>RifamycinB> SV and <B>RifamycinB> B were added into the LYZL11 culture medium, respectively, and the rifP transcriptional level was measured at 24Â h after the addition of <B>RifamycinB>s. DMSO was used as a Blank control and rpoB was used as an internal control
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A feedBack regulatory model for RifQ-mediated repression of <B>RifamycinB> export in Amycolatopsis mediterranei
BMC, 2018Co-Authors: Chao Lei, Jingzhi Wang, Guoping Zhao, Yuanyuan Liu, Xinqiang Liu, Jin WangAbstract:ABstract Background Due to the important role of <B>RifamycinB> in curing tuBerculosis infection, the study on <B>RifamycinB> has never Been stopped. Although RifZ, which locates within the <B>RifamycinB> Biosynthetic cluster, has recently Been characterized as a pathway-specific regulator for <B>RifamycinB> Biosynthesis, little is known aBout the regulation of <B>RifamycinB> export. Results In this work, we proved that the expression of the <B>RifamycinB> efflux pump (RifP) was regulated By RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on Bacterial growth, But resulted in improved <B>RifamycinB> production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic moBility shift assay and DNase I Footprinting assay, RifQ was found to directly Bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to Be upstream of the RifQ Binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, <B>RifamycinB> B (the end product of <B>RifamycinB> Biosynthesis) remarkaBly decreased the DNA Binding affinity of RifQ, which led to derepression of <B>RifamycinB> export, reducing the intracellular concentration of <B>RifamycinB> B as well as its toxicity against the host. Conclusions Here, we proved that the export of <B>RifamycinB> B was repressed By RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could Be specifically relieved By <B>RifamycinB> B, the end product of <B>RifamycinB> Biosynthesis, Based on which a feedBack model was proposed for regulation of <B>RifamycinB> export. With the findings here, one could improve the antiBiotic yield By simply inactivating the negative regulator of the antiBiotic transporter
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two genes rif15 and rif16 of the <B>RifamycinB> Biosynthetic gene cluster in amycolatopsis mediterranei likely encode a transketolase and a p450 monooxygenase respectively Both essential for the conversion of <B>RifamycinB> sv into B
Acta Biochimica et Biophysica Sinica, 2011Co-Authors: Hua Yuan, Xiaoming Ding, Yi Zhong, Jin Wang, Wei Zhao, Guoping ZhaoAbstract:Amycolatopsis mediterranei produces an important antiBiotic <B>RifamycinB>, the Biosynthesis of which involves many unusual modifications. Previous work suggested a putative P450 enzyme encoded By rif16 within the <B>RifamycinB> Biosynthetic gene cluster (rif ) was required for the conversion of the intermediate <B>RifamycinB> SV into the end product <B>RifamycinB> B. In this study, we genetically proved that a putative transketolase encoded By rif15 is another essential enzyme for this conversion. Expression of merely rif15 and rif16 in a rif cluster null mutant of A. mediterranei U32 was aBle to convert <B>RifamycinB> SV into B. However, this Rif15- and Rif16-mediated conversion was only detected in intact cells of A. meidterranei, But not in Streptomyce coelicolor or MycoBacterium smegmatis, suggesting that yet-characterized gene(s) in A. mediterranei other than those encoded By the rif cluster should Be involved in this process.
