The Experts below are selected from a list of 1386 Experts worldwide ranked by ideXlab platform
Craig A Townsend - One of the best experts on this subject based on the ideXlab platform.
-
rational strain improvement for enhanced clavulanic acid production by genetic engineering of the glycolytic pathway in Streptomyces Clavuligerus
Metabolic Engineering, 2006Co-Authors: Craig A TownsendAbstract:Clavulanic acid is a potent β-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of l-arginine and d-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, we genetically engineered the glycolytic pathway in Streptomyces Clavuligerus. Two genes (gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. Clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild type, suggesting that arginine became limiting for biosynthesis. This is the first reported application of genetic engineering to channel precursor flux to improve clavulanic acid production.
-
rational strain improvement for enhanced clavulanic acid production by genetic engineering of the glycolytic pathway in Streptomyces Clavuligerus
Metabolic Engineering, 2006Co-Authors: Craig A TownsendAbstract:Clavulanic acid is a potent beta-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of L-arginine and D-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, we genetically engineered the glycolytic pathway in Streptomyces Clavuligerus. Two genes (gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. Clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild type, suggesting that arginine became limiting for biosynthesis. This is the first reported application of genetic engineering to channel precursor flux to improve clavulanic acid production.
Bernhard O Palsson - One of the best experts on this subject based on the ideXlab platform.
-
Exploiting adaptive laboratory evolution of Streptomyces Clavuligerus for antibiotic discovery and overproduction. PLoS One 2012
2016Co-Authors: Pep Charusanti, Nicole L Fong, Harish Nagarajan, Alban R Pereira, William H Gerwick, Elisa A. Abate, Bernhard O PalssonAbstract:Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition-based adaptive laboratory evolution scheme is presented whereby an antibiotic-producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces Clavuligerus replicates were adaptively evolved against methicillin-resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the unevolved wild-type strain. Moreover, genome re-sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid, and acquired several single nucleotide polymorphisms in genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition-based adaptive laboratory evolution can constitute a platform to create mutants that overproduce know
-
exploiting adaptive laboratory evolution of Streptomyces Clavuligerus for antibiotic discovery and overproduction
PLOS ONE, 2012Co-Authors: Pep Charusanti, Nicole L Fong, Harish Nagarajan, Alban R Pereira, Elisa Abate, William H Gerwick, Bernhard O PalssonAbstract:Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition-based adaptive laboratory evolution scheme is presented whereby an antibiotic-producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces Clavuligerus replicates were adaptively evolved against methicillin-resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the unevolved wild-type strain. Moreover, genome re-sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid, and acquired several single nucleotide polymorphisms in genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition-based adaptive laboratory evolution can constitute a platform to create mutants that overproduce known antibiotics and possibly to discover new compounds as well.
Rigoberto Riosestepa - One of the best experts on this subject based on the ideXlab platform.
-
Streptomyces Clavuligerus shows a strong association between tca cycle intermediate accumulation and clavulanic acid biosynthesis
Applied Microbiology and Biotechnology, 2018Co-Authors: Howard Ramirezmalule, Stefan Junne, Mariano Nicolas Cruzbournazou, Peter Neubauer, Rigoberto RiosestepaAbstract:Clavulanic acid (CA) is produced by Streptomyces Clavuligerus (S. Clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. Clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. Clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. Clavuligerus.
-
an improved hplc dad method for clavulanic acid quantification in fermentation broths of Streptomyces Clavuligerus
Journal of Pharmaceutical and Biomedical Analysis, 2016Co-Authors: Howard Ramirezmalule, Stefan Junne, Peter Neubauer, Carlos Lopez, Julian Zapata, Alex Saez, Rigoberto RiosestepaAbstract:Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces Clavuligerus (Sc). It is a potent inhibitor of bacterial β-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate-imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400mg/L. The detection and quantification limits were 0.01 and 0.02mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate-imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.
Paloma Liras - One of the best experts on this subject based on the ideXlab platform.
-
Image_1_Activation of Secondary Metabolite Gene Clusters in Streptomyces Clavuligerus by the PimM Regulator of Streptomyces natalensis.pdf
2019Co-Authors: Yolanda Martínez-burgo, Javier Santos-aberturas, Antonio Rodríguez-garcía, Eva G. Barreales, José Rubén Tormo, Andrew W. Truman, Fernando Reyes, Jesús F. Aparicio, Paloma LirasAbstract:Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces Clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. Clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. Clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex.
-
Table_5_Activation of Secondary Metabolite Gene Clusters in Streptomyces Clavuligerus by the PimM Regulator of Streptomyces natalensis.xlsx
2019Co-Authors: Yolanda Martínez-burgo, Javier Santos-aberturas, Antonio Rodríguez-garcía, Eva G. Barreales, José Rubén Tormo, Andrew W. Truman, Fernando Reyes, Jesús F. Aparicio, Paloma LirasAbstract:Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces Clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. Clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. Clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex.
-
Activation of Secondary Metabolite Gene Clusters in Streptomyces Clavuligerus by the PimM Regulator of Streptomyces natalensis
'Frontiers Media SA', 2019Co-Authors: Yolanda Martínez-burgo, Javier Santos-aberturas, Antonio Rodríguez-garcía, Eva G. Barreales, José Rubén Tormo, Andrew W. Truman, Fernando Reyes, Jesús F. Aparicio, Paloma LirasAbstract:Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces Clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. Clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. Clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex
-
characterization of dna binding sequences for ccar in the cephamycin clavulanic acid supercluster of Streptomyces Clavuligerus
Molecular Microbiology, 2011Co-Authors: Irene Santamarta, M T Lopezgarcia, A Kurt, N Nardiz, Ruben Alvarezalvarez, Rosario Perezredondo, Juan F Martin, Paloma LirasAbstract:RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces Clavuligerus ATCC 27064 and the mutant S. Clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.
-
Streptomyces Clavuligerus rela null mutants overproduce clavulanic acid and cephamycin c negative regulation of secondary metabolism by p ppgpp
Microbiology, 2008Co-Authors: Juan Pablo Gomezescribano, Juan F Martin, Andrew Hesketh, Maureen J Bibb, Paloma LirasAbstract:The (p)ppGpp synthetase gene, relA, of Streptomyces Clavuligerus was cloned, sequenced and shown to be located in a genomic region that is highly conserved in other Streptomyces species. relA-disrupted and relA-deleted mutants of S. Clavuligerus were constructed, and both were unable to form aerial mycelium or to sporulate, but regained these abilities when complemented with wild-type relA. Neither ppGpp nor pppGpp was detected in the S. Clavuligerus relA-deletion mutant. In contrast to another study, clavulanic acid and cephamycin C production increased markedly in the mutants compared to the wild-type strain; clavulanic acid production increased three- to fourfold, while that of cephamycin C increased about 2.5-fold. Complementation of the relA-null mutants with wild-type relA decreased antibiotic yields to approximately wild-type levels. Consistent with these observations, transcription of genes involved in clavulanic acid (ceaS2) or cephamycin C (cefD) production increased dramatically in the relA-deleted mutant when compared to the wild-type strain. These results are entirely consistent with the growth-associated production of both cephamycin C and clavulanic acid, and demonstrate, apparently for the first time, negative regulation of secondary metabolite biosynthesis by (p)ppGpp in a Streptomyces species of industrial interest.
Pep Charusanti - One of the best experts on this subject based on the ideXlab platform.
-
Exploiting adaptive laboratory evolution of Streptomyces Clavuligerus for antibiotic discovery and overproduction. PLoS One 2012
2016Co-Authors: Pep Charusanti, Nicole L Fong, Harish Nagarajan, Alban R Pereira, William H Gerwick, Elisa A. Abate, Bernhard O PalssonAbstract:Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition-based adaptive laboratory evolution scheme is presented whereby an antibiotic-producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces Clavuligerus replicates were adaptively evolved against methicillin-resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the unevolved wild-type strain. Moreover, genome re-sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid, and acquired several single nucleotide polymorphisms in genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition-based adaptive laboratory evolution can constitute a platform to create mutants that overproduce know
-
exploiting adaptive laboratory evolution of Streptomyces Clavuligerus for antibiotic discovery and overproduction
PLOS ONE, 2012Co-Authors: Pep Charusanti, Nicole L Fong, Harish Nagarajan, Alban R Pereira, Elisa Abate, William H Gerwick, Bernhard O PalssonAbstract:Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition-based adaptive laboratory evolution scheme is presented whereby an antibiotic-producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces Clavuligerus replicates were adaptively evolved against methicillin-resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the unevolved wild-type strain. Moreover, genome re-sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid, and acquired several single nucleotide polymorphisms in genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition-based adaptive laboratory evolution can constitute a platform to create mutants that overproduce known antibiotics and possibly to discover new compounds as well.