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Jean-marc Daran - One of the best experts on this subject based on the ideXlab platform.
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Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach
BMC Systems Biology, 2011Co-Authors: Rutger D. Douma, Joana M. Batista, Kai M. Touw, Jan A. K. W. Kiel, Arjen M. Krikken, Zheng Zhao, Tânia Veiga, Paul Klaassen, Roel A. L. Bovenberg, Jean-marc DaranAbstract:Background In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called Strain Degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study Degeneration of penicillin production in a high producing Penicillium chrysogenum Strain during prolonged ethanol-limited chemostat cultivations.
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Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach
BMC Systems Biology, 2011Co-Authors: Rutger D. Douma, Joana M. Batista, Kai M. Touw, Jan A. K. W. Kiel, Arjen M. Krikken, Zheng Zhao, Tânia Veiga, Paul Klaassen, Roel A. L. Bovenberg, Jean-marc DaranAbstract:Background In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called Strain Degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study Degeneration of penicillin production in a high producing Penicillium chrysogenum Strain during prolonged ethanol-limited chemostat cultivations. Results During these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production. Conclusions Our findings indicate that the observed Degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during Degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.
Philippe Soucaille - One of the best experts on this subject based on the ideXlab platform.
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amyp a reporter gene to study Strain Degeneration in clostridium acetobutylicum atcc 824
Fems Microbiology Letters, 2002Co-Authors: Fabrice Sabathe, Christian Croux, Emmanuel Cornillot, Philippe SoucailleAbstract:Abstract Clostridium acetobutylicum produces an extracellular α-amylase when grown on glucose as the sole carbon source. This enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified. The 2283-bp amyP gene encodes a 83 013-Da mature protein with an N-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the Bacillus α-amylases. Transcriptional analysis revealed that amyP is transcribed in solventogenic but not in acidogenic chemostat cultures. These results are in agreement with the extracellular α-amylase activities indicating that the expression of amyP is regulated at the transcriptional level. amyP is located on the pSOL1 megaplasmid that carries all the genes involved in the final steps of solvent formation. Degeneration of C. acetobutylicum has been associated to the loss of pSOL1. We demonstrate here that amyP can be used as a reporter system to quantitatively follow this phenomenon.
Eva R Kashket - One of the best experts on this subject based on the ideXlab platform.
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A New Insertion Sequence, ISCb1, from Clostridium beijerinckii NCIMB 8052
Journal of Molecular Microbiology and Biotechnology, 2000Co-Authors: Hemachandra Liyanage, Eva R Kashket, Paul Holcroft, Victoria J. Evans, Stefanie Keis, Shane R Wilkinson, Michael YoungAbstract:The NCIMB 8052 Strain of Clostridium beijerinckii contains nine copies of a novel insertion sequence, ISCb1, belonging to the IS4 family. The 1764 bp element has 18 bp inverted repeats at its extremities, and generates 11 bp target repeats upon insertion. It contains a 1365 bp ORF whose predicted product (455 amino acids) resembles bacterial transposases. The highly conserved DD(35)E motif is present, as are signatures characteristic of the N3 and C1 domains of bacterial transposases. Codon usage of the ORF is somewhat different from that of other C. beijerinckii genes, suggesting that ISCb1 may have been acquired from another organism by horizontal gene transfer in the evolutionary past. One ISCb1 copy lies close to the site of insertion of Tn1545 in a mutant Strain, C10, which shows a reduced tendency to degenerate (i.e. loss of the potential to form solvents) compared with the wild type. In the C10 Strain, the characteristic pattern of DNA fragments detected by an IS-specific probe was altered, but this was due to the Tn1545 insertion itself, rather than an ISCb1-mediated genome re-arrangement. There is currently no evidence that the element is involved in Strain Degeneration, since 12 independently isolated spontaneous mutants that had lost the ability to form solvents had the same ISCb1 profile as that of the wild type Strain. The element is apparently restricted to a series of closely related solvent-forming clostridia.
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clostridial Strain Degeneration
Fems Microbiology Reviews, 1995Co-Authors: Eva R KashketAbstract:Abstract Strain Degeneration, the loss of the capacity to produce solvents and form spores, typically occurs when Clostridium acetobutylicum and related clostridia are repeatedly subcultured in batch culture or grown in continuous culture, as opposed to being grown from germinated, heat-treated spores. Several mechanisms for Degeneration have been identified thus far. (i) Degeneration can be caused by excessive acidification of the culture during exponential growth. We present data interpreted to mean that C. beijerinckii (formerly C. acetobutylicum ) NCIMB 8052 cells ferment glucose to acetic and butyric acids at an uncontrolled rate, so that, during rapid growth, the rate of acid production can exceed the rate of induction of the solventogenic pathway enzymes. As a result, the medium pH drops to bactericical levels, and the cells cannot switch to solventogenesis and sporulation. The clostridia seem to be poised either to produce excess acids, or to initiate solventogenesis, depending on small differences in the rates of growth. (ii) We have isolated transposon-insertion mutants of C. beijerinckii NCIMB 8052 that are resistant to Degeneration, suggesting the involvement of a regulatory region of the clostridial chromosome. (iii) Involvement of a global regulatory gene has been inferred in C. beijerinckii NCIMB 8052 which degenerates irreversibly in chemostat culture. (iv) Impairment of butanol formation due to a defect in NADH generation has been reported in an oligosporogenous Strain which can revert to the non-degenerate phenotype. (v) In continuous culture, degenerate cells may be selected because they continue to divide, while the non-degenerate cells stop dividing and start differentiating.
Rutger D. Douma - One of the best experts on this subject based on the ideXlab platform.
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Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach
BMC Systems Biology, 2011Co-Authors: Rutger D. Douma, Joana M. Batista, Kai M. Touw, Jan A. K. W. Kiel, Arjen M. Krikken, Zheng Zhao, Tânia Veiga, Paul Klaassen, Roel A. L. Bovenberg, Jean-marc DaranAbstract:Background In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called Strain Degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study Degeneration of penicillin production in a high producing Penicillium chrysogenum Strain during prolonged ethanol-limited chemostat cultivations.
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Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach
BMC Systems Biology, 2011Co-Authors: Rutger D. Douma, Joana M. Batista, Kai M. Touw, Jan A. K. W. Kiel, Arjen M. Krikken, Zheng Zhao, Tânia Veiga, Paul Klaassen, Roel A. L. Bovenberg, Jean-marc DaranAbstract:Background In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called Strain Degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study Degeneration of penicillin production in a high producing Penicillium chrysogenum Strain during prolonged ethanol-limited chemostat cultivations. Results During these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production. Conclusions Our findings indicate that the observed Degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during Degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.
Hanspeter M Blaschek - One of the best experts on this subject based on the ideXlab platform.
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effect of acetate on molecular and physiological aspects of clostridium beijerinckii ncimb 8052 solvent production and Strain Degeneration
Applied and Environmental Microbiology, 1999Co-Authors: Chih Kuang Chen, Hanspeter M BlaschekAbstract:The addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production and also increase glucose utilization by Clostridium beijerinckii NCIMB 8052. RNA and enzyme analyses indicated that coenzyme A (CoA) transferase was highly expressed and has higher activity in C. beijerinckii NCIMB 8052 grown in MP2 medium containing added sodium acetate than in the microorganism grown without sodium acetate. RNA analysis suggested the existence of a sol operon and confirmed the presence of a ptb-buk operon in C. beijerinckii NCIMB 8052. In addition to CoA transferase, C. beijerinckii NCIMB 8052 grown in MP2 medium containing added acetate demonstrated higher acetate kinase- and butyrate kinase-specific activity than when the culture was grown in MP2 medium containing no added acetate. Southern blot analysis with chromosomal DNA isolated from solventogenic and degenerated C. beijerinckii NCIMB 8052 indicated that C. beijerinckii NCIMB 8052 Strain Degeneration does not involve loss of the CoA transferase genes. The addition of acetate to MP2 medium may induce the expression of the sol operon, which ensures solvent production and prevents Strain Degeneration in C. beijerinckii NCIMB 8052.