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Rafael P. Mellado - One of the best experts on this subject based on the ideXlab platform.
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Four thiol-oxidoreductases involved in the formation of disulphide bonds in the Streptomyces Lividans TK21 secretory proteins
Microbial cell factories, 2019Co-Authors: Sonia Gullón, Silvia Marín, Rafael P. MelladoAbstract:Background Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces Lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction.
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Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces Lividans
PloS one, 2015Co-Authors: Sonia Gullón, Silvia Marín, Rafael P. MelladoAbstract:Streptomyces Lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces Lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. Lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients’ depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. Lividans as a protein production host.
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Summing up particular features of protein secretion in Streptomyces Lividans
World Journal of Microbiology and Biotechnology, 2011Co-Authors: Rafael P. MelladoAbstract:In recent years much attention has been given to the identification and characterisation of the key elements of the secretory machinery of Streptomyces Lividans , a non-pathogenic filamentous Gram-positive soil bacterium, whose metabolism is relatively well characterised and capable of secreting large amounts of proteins when grown in laboratory conditions. The relevance of S. Lividans from a commercial standpoint is due to its potential usefulness for the overproduction of secretory homologous and heterologous proteins of interest. Therefore, this review focuses on the knowledge already obtained on the S . Lividans secretion pathways.
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Influence of a Streptomyces Lividans SecG functional analogue on protein secretion.
International microbiology : the official journal of the Spanish Society for Microbiology, 2008Co-Authors: Carmen Palomino, Rafael P. MelladoAbstract:The membrane protein complex translocase mediates the translocation of bacterial proteins. In this complex, the SecY, SecE, and SecG proteins constitute an integral membrane domain. Sequence comparison revealed a potential secG-like gene in the gram-positive soil bacterium Streptomyces Lividans. Chromosomal deletion of this gene resulted in a sporulation defect and an overall deficiency in secretion. The SecG-depleted strain was able to overproduce and secrete alpha-amylase, but the appearance of the oversynthesized protein outside the cell was delayed compared to the protein produced by the wildtype strain. SecG deficiency was found to result in more pronounced effects in S. Lividans than in Bacillus subtilis or Escherichia coli.
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Streptomyces Lividans as a host for the production and secretion of Escherichia coli TEM ββ‐lactamase
Letters in applied microbiology, 1999Co-Authors: C. Isiegas, V. Parro, Rafael P. MelladoAbstract:The regulatory region and the region coding for the signal peptide of an extracellular agarase have been used to synthesize and secrete the heterologous Escherichia coli TEM β-lactamase in Streptomyces Lividans. The transcriptional regulation of the chimeric gene, and the secretion pattern of the chimeric gene product, coincided with those of the agarase gene. The negative glucose effect on the secretion of the protein was reverted when the recombinant bacterium was grown in the chemostat under phosphate limiting conditions.
Rolf Morosoli - One of the best experts on this subject based on the ideXlab platform.
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Effect of protease mutations on the production of xylanases in Streptomyces Lividans.
Canadian journal of microbiology, 2007Co-Authors: Éliana AriasÉ. Arias, Rolf MorosoliAbstract:Three protease mutants — 7 (tap–), 12 (tap–, ssp–), and 17 (multiple mutations) — of Streptomyces Lividans were tested for their influence on protein secretion. Streptomyces Lividans grown in xylan...
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Effect of carbon source, growth and temperature on the expression of the sec genes of Streptomyces Lividans 1326
Canadian journal of microbiology, 1999Co-Authors: Rolf Morosoli, Stéphane Ostiguy, Claude DupontAbstract:The mRNA level in sec genes of Streptomyces Lividans was studied as a function of growth temperature, glucose effect, and growth using two different carbon sources. Glucose and xylan, a complex hemicellulose, were used as carbon sources for the growth of S. Lividans. For both substrates, the mRNA levels of secA, secD, secE, secF, and secY genes were almost constant during the early and log phases, but showed a marked decrease at the beginning of the stationary phase followed by a full recovery of mRNA level in the late stationary phase. This indicates that the sec genes are actively transcribed during the differentiation process. The mRNA level in xylan was generally from 1.5- to 2-fold that in glucose. At growth temperatures of 28°C, 34°C, or 40°C, there was no significant difference in the sec gene mRNA levels.Key words: Streptomyces Lividans, sec genes, glucose repression, growth-phase dependent expression.
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Cloning and sequencing of the secY homolog from Streptomyces Lividans 1326.
Gene, 1996Co-Authors: Stéphane Ostiguy, François Shareck, Dieter Kluepfel, Michel Gilbert, Rolf MorosoliAbstract:Two conserved regions of SecY proteins from six Gram+ bacteria were exploited in a PCR-based strategy for isolating a secY homolog from Streptomyces Lividans (Sl). The nucleotide sequence of part of a 3.8-kb fragment showed that the secYhomolog is flanked, at the 5' end, by the gene encoding ribosomal protein L15 and, at the 3' end, by an adenylate kinase-encoding gene. The deduced gene product of secYwould have 437 amino acids (aa) and an Mr of 47 200. Sl SecY shows 89.5, 56.1, 42 and 40% identity to its homologs from Streptomyces scabies, Brevibacterium flavum, Bacillus subtilis and Escherichia coli, respectively. Promoterprobe analyses indicated that the secYgene probably contains its own promoter.
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Improved Production of Mannanase by Streptomyces Lividans.
Applied and environmental microbiology, 1996Co-Authors: Francoise Marga, Rolf Morosoli, C Ghakis, Claude Dupont, D KluepfelAbstract:Replacement of the natural promoter of the (beta)-mannanase gene of Streptomyces Lividans by lacp resulted in a 15-fold increase in enzyme production over that of the previously reported clone S. Lividans IAF36, a clone carrying multiple copies of manA, and a 350-fold increase over that of the wild-type strain S. Lividans 1326. In addition, the use of lacp in the shuttle vector pIAF199 allowed synthesis of the enzymes on carbon sources that did not contain mannan, such as xylan and whey, which offers interesting possibilities for industrial production of the enzyme.
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Purification and characterization of the CelB endoglucanase from Streptomyces Lividans 66 and DNA sequence of the encoding gene.
Applied and environmental microbiology, 1994Co-Authors: S Wittmann, François Shareck, D Kluepfel, Rolf MorosoliAbstract:Abstract The endoglucanase CelB isolated from culture filtrates of Streptomyces Lividans IAF9 has an M(r) of 36,000. With carboxymethyl cellulose as the substrate, the Vmax and Km values are 110 IU/mg of enzyme and 1.3 mg/ml, respectively. Comparison of primary amino acid sequences classifies CelB in the H family of cellulases.
Akihiko Kondo - One of the best experts on this subject based on the ideXlab platform.
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Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces Lividans as a host
Microbial cell factories, 2015Co-Authors: Shuhei Noda, Takuya Matsumoto, Tsutomu Tanaka, Akihiko KondoAbstract:Background Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces Lividans as a host.
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cinnamic acid production using Streptomyces Lividans expressing phenylalanine ammonia lyase
Journal of Industrial Microbiology & Biotechnology, 2011Co-Authors: Shuhei Noda, Chiaki Ogino, Tsutomu Tanaka, Takaya Miyazaki, Takanori Miyoshi, Michiru Miyake, Naoko Okai, Akihiko KondoAbstract:Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces Lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. Lividans has great potential to produce cinnamic acid as well as other aromatic compounds.
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over production of various secretory form proteins in Streptomyces Lividans
Protein Expression and Purification, 2010Co-Authors: Shuhei Noda, Chiaki Ogino, Tsutomu Tanaka, Nobuaki Shimizu, Yuko Ito, Akihiko KondoAbstract:Abstract Streptomyces Lividans is known to produce large amounts of proteins in culture supernatants. In this report, to expand the secretory expression system with a strong promoter derived from phospholipase D of Streptoverticillium cinnamoneum , we expressed three kinds of proteins: transglutaminase from Stv. cinnamoneum (StvcMTG) and β-1,4-endoglucanase and β-glucosidase from Thermobifida fusca YX . The StvcMTG gene was introduced into S. Lividans using the shuttle vector pUC702 for Escherichia coli and S. Lividans , and high level secretory production of StvcMTG (230 μg/ml in the culture supernatant) was achieved. The other prokaryotic proteins, β-1,4-endoglucanase and β-glucosidase, were also expressed in (His) 6 -tag fused form into culture supernatants and retained high activity. Furthermore, complete purification was achieved by conventional column or affinity column chromatography for each recombinant protein with 1 mg/ml over protein concentration. Three independent proteins were thus successfully expressed and purified, and we expect to use this system for the expression of other valuable heterologous proteins.
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over expression system for secretory phospholipase d by Streptomyces Lividans
Applied Microbiology and Biotechnology, 2004Co-Authors: Chiaki Ogino, Akihiko Kondo, Masayuki Kanemasu, Y Hayashi, Nobuaki Shimizu, Shinji Tokuyama, Y Tahara, Shunichi Kuroda, Katsuyuki TanizawaAbstract:The structural gene for phospholipase D (PLD) of an actinomycete, Streptoverticillium cinnamoneum, together with its promoter region was introduced into Streptomyces Lividans using a shuttle vector—pUC702—for Escherichia coli and S. Lividans. The transformant was found to secrete a large amount of PLD (about 2.0×104 U/l, 42 mg/l) when cultured in a jar fermentor. Both an initial glucose concentration of 17.5 g/l and the feeding of carbon and nitrogen sources are effective for efficient secretion of PLD; under these culture conditions, the amount of PLD secreted reached a maximum level (about 5.5×104 U/l, 118 mg/l) after about 60 h. In contrast to the original producer, Stv. cinnamoneum, which secretes only a small amount of PLD (about 1.1×103 U/l, 2 mg/l) along with other extracellular proteins, this heterologous expression system is markedly more efficient in production of secretory PLD.
François Shareck - One of the best experts on this subject based on the ideXlab platform.
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Adaptation of the Highly Productive T7 Expression System to Streptomyces Lividans
Applied and environmental microbiology, 2009Co-Authors: François-xavier Lussier, François Denis, François ShareckAbstract:Streptomyces Lividans is a Gram-positive bacterium known for its remarkable secretion efficiency and low extracellular protease activity. In the present work, we adapted the highly productive T7 expression system to S. Lividans. A codon-optimized T7 RNA polymerase gene was chromosomally integrated, and a bifunctional T7 expression vector was constructed.
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Cloning and sequencing of the secY homolog from Streptomyces Lividans 1326.
Gene, 1996Co-Authors: Stéphane Ostiguy, François Shareck, Dieter Kluepfel, Michel Gilbert, Rolf MorosoliAbstract:Two conserved regions of SecY proteins from six Gram+ bacteria were exploited in a PCR-based strategy for isolating a secY homolog from Streptomyces Lividans (Sl). The nucleotide sequence of part of a 3.8-kb fragment showed that the secYhomolog is flanked, at the 5' end, by the gene encoding ribosomal protein L15 and, at the 3' end, by an adenylate kinase-encoding gene. The deduced gene product of secYwould have 437 amino acids (aa) and an Mr of 47 200. Sl SecY shows 89.5, 56.1, 42 and 40% identity to its homologs from Streptomyces scabies, Brevibacterium flavum, Bacillus subtilis and Escherichia coli, respectively. Promoterprobe analyses indicated that the secYgene probably contains its own promoter.
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Purification and characterization of the CelB endoglucanase from Streptomyces Lividans 66 and DNA sequence of the encoding gene.
Applied and environmental microbiology, 1994Co-Authors: S Wittmann, François Shareck, D Kluepfel, Rolf MorosoliAbstract:Abstract The endoglucanase CelB isolated from culture filtrates of Streptomyces Lividans IAF9 has an M(r) of 36,000. With carboxymethyl cellulose as the substrate, the Vmax and Km values are 110 IU/mg of enzyme and 1.3 mg/ml, respectively. Comparison of primary amino acid sequences classifies CelB in the H family of cellulases.
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Sequences of three genes specifying xylanases in Streptomyces Lividans.
Gene, 1991Co-Authors: François Shareck, Rolf Morosoli, Camille Roy, Yaguchi Makoto, Dieter KluepfelAbstract:The entire nucleotide (nt) sequences of three genes (xlnA, xlnB and xlnC) of Streptomyces Lividans encoding three distinct xylanases (Xln) have been determined. The nt sequences were confirmed by comparing the deduced amino acid (aa) sequences with the ones derived from the N-terminal aa sequences of the mature purified proteins. The N-terminus of the XlnA showed some homology with either the N-termini or the C-termini of eight other Xln and of two exo-glucanases. The N-terminus of XlnB is homologous to that of XlnC and to Xln of seven other microorganisms.
Sonia Gullón - One of the best experts on this subject based on the ideXlab platform.
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Four thiol-oxidoreductases involved in the formation of disulphide bonds in the Streptomyces Lividans TK21 secretory proteins
Microbial cell factories, 2019Co-Authors: Sonia Gullón, Silvia Marín, Rafael P. MelladoAbstract:Background Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces Lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction.
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Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces Lividans
PloS one, 2015Co-Authors: Sonia Gullón, Silvia Marín, Rafael P. MelladoAbstract:Streptomyces Lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces Lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. Lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients’ depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. Lividans as a protein production host.