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Antonius J A Van Maris - One of the best experts on this subject based on the ideXlab platform.
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metabolic engineering strategies for optimizing acetate reduction ethanol yield and Osmotolerance in saccharomyces cerevisiae
Biotechnology for Biofuels, 2017Co-Authors: Ioannis Papapetridis, Antonius J A Van Maris, Marlous Van Dijk, Jack T PronkAbstract:Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining Osmotolerance. GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L−1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and Osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.
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evolutionary engineering of a glycerol 3 phosphate dehydrogenase negative acetate reducing saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations
Microbial Biotechnology, 2014Co-Authors: Victor Guadalupemedina, Jack T Pronk, Robert Mans, Bart Oud, Benjamin Metz, Charlotte M Van Der Graaf, Antonius J A Van MarisAbstract:Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S.?cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve Osmotolerance of a Gpd– mhpF-expressing S.?cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1?M glucose, at a specific growth rate of 0.12?h?1. The evolved strain produced glycerol at low concentrations (0.64?±?0.33?g?l?1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that Osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.
Roy D. Sleator - One of the best experts on this subject based on the ideXlab platform.
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a genomic analysis of Osmotolerance in staphylococcus aureus
Gene, 2021Co-Authors: Dylan Casey, Roy D. SleatorAbstract:A key phenotypic characteristic of the Gram-positive bacterial pathogen, Staphylococcus aureus, is its ability to grow in low aw environments. A homology transfer based approach, using the well characterised osmotic stress response systems of Bacillus subtilis and Escherichia coli, was used to identify putative Osmotolerance loci in Staphylococcus aureus ST772-MRSA-V. A total of 17 distinct putative hyper and hypo-osmotic stress response systems, comprising 78 genes, were identified. The ST772-MRSA-V genome exhibits significant degeneracy in terms of the osmotic stress response; with three copies of opuD, two copies each of nhaK and mrp/mnh, and five copies of opp. Furthermore, regulation of Osmotolerance in ST772-MRSA-V appears to be mediated at the transcriptional, translational, and post-translational levels.
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The role of the Cronobacter sakazakii ProP C-terminal coiled coil domain in Osmotolerance.
Gut pathogens, 2014Co-Authors: Audrey Feeney, Christopher D Johnston, Alan Lucid, Jim O'mahony, Aidan Coffey, Brigid Lucey, Roy D. SleatorAbstract:We investigate the role of the C-terminal coiled coil of the secondary proline porter ProP in contributing to Cronobacter sakazakii Osmotolerance. The extended C-terminal domain of ProP1 (encoded by ESA_02131) was spliced onto the truncated C-terminal end of ProP2 (encoded by ESA_01706); creating a chimeric protein (ProPc) which exhibits increased Osmotolerance relative to the wild type. It appears that the C-terminal coiled coil domain tunes ProP at low osmolality, whereas ProP transporters lacking the coiled coil domain are more active at a higher osmolality range.
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Analysis of the role of the Cronobacter sakazakii ProP homologues in Osmotolerance.
Gut pathogens, 2014Co-Authors: Audrey Feeney, Christopher D Johnston, Jim O'mahony, Aidan Coffey, Rodney Govender, Roy D. SleatorAbstract:Bacteria respond to elevated osmolality by the accumulation of a range of low molecular weight molecules, known as compatible solutes (owing to their compatibility with the cells' normal physiology at high internal concentrations). The neonatal pathogen Cronobacter sakazakii is uniquely osmotolerant, surviving in powdered infant formula (PIF) which typically has a water activity (aw) of 0.2 – inhospitable to most micro-organisms. Mortality rates of up to 80% in infected infants have been recorded making C. sakazakii a serious cause for concern. In silico analysis of the C. sakazakii BAA-894 genome revealed seven copies of the osmolyte uptake system ProP. Herein, we test the physiological role of each of these homologues following heterologous expression against an osmosensitive Escherichia coli host.
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An in silico analysis of Osmotolerance in the emerging gastrointestinal pathogen Cronobacter sakazakii.
Bioengineered bugs, 2011Co-Authors: Audrey Feeney, Roy D. SleatorAbstract:Up to 80% of infants infected with Cronobacter sakazakii die within days of birth, making this emerging gastrointestinal pathogen a serious cause for concern. The mode of transmission most often associated with C. sakazakii infection is powdered infant formula (PIF), which typically has a water activity (aw) of ca 0.2 – inhospitable to most bacterial pathogens. In the current study a comparative genomic approach was used to investigate the distinctive ability of this pathogen to survive and persist in such low aW conditions. A comprehensive review of the mechanisms involved in bacterial osmoadaptation was followed by an exhaustive homology transfer based approach to identify putative Osmotolerance loci in the C. sakazakii genome. In total 53 osmotoleance loci were identified, including both hyper- and hypo-osmotic stress response systems, helping to construct a concise overview of the C. sakazakii Osmotolerance response. Interestingly, while C. sakazakii contains homologues of all the principal osmotolera...
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A novel role for the LisRK two-component regulatory system in listerial Osmotolerance
Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases, 2005Co-Authors: Roy D. Sleator, Colin HillAbstract:Understanding how pathogenic bacteria sense and respond to environmental signals, including those involved in triggering virulence gene expression, is a fundamental biological goal. It is now known that the two-component regulatory system LisRK has a novel role in osmosensing and osmoregulation in the intracellular foodborne pathogen Listeria monocytogenes. Furthermore, htrA, a gene linked to Osmotolerance and virulence potential in L. monocytogenes, is now known to be under the transcriptional control of LisRK.
Jack T Pronk - One of the best experts on this subject based on the ideXlab platform.
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metabolic engineering strategies for optimizing acetate reduction ethanol yield and Osmotolerance in saccharomyces cerevisiae
Biotechnology for Biofuels, 2017Co-Authors: Ioannis Papapetridis, Antonius J A Van Maris, Marlous Van Dijk, Jack T PronkAbstract:Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining Osmotolerance. GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L−1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and Osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.
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evolutionary engineering of a glycerol 3 phosphate dehydrogenase negative acetate reducing saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations
Microbial Biotechnology, 2014Co-Authors: Victor Guadalupemedina, Jack T Pronk, Robert Mans, Bart Oud, Benjamin Metz, Charlotte M Van Der Graaf, Antonius J A Van MarisAbstract:Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S.?cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve Osmotolerance of a Gpd– mhpF-expressing S.?cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1?M glucose, at a specific growth rate of 0.12?h?1. The evolved strain produced glycerol at low concentrations (0.64?±?0.33?g?l?1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that Osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.
Marlous Van Dijk - One of the best experts on this subject based on the ideXlab platform.
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metabolic engineering strategies for optimizing acetate reduction ethanol yield and Osmotolerance in saccharomyces cerevisiae
Biotechnology for Biofuels, 2017Co-Authors: Ioannis Papapetridis, Antonius J A Van Maris, Marlous Van Dijk, Jack T PronkAbstract:Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining Osmotolerance. GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L−1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and Osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.
Hana Sychrová - One of the best experts on this subject based on the ideXlab platform.
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stl1 transporter mediating the uptake of glycerol is not a weak point of saccharomyces kudriavzevii s low Osmotolerance
Letters in Applied Microbiology, 2019Co-Authors: Jana Zemančíková, Klara Papouskova, Roberto Pereztorrado, Amparo Querol, Hana SychrováAbstract:Saccharomyces kudriavzevii is a nonconventional and rather osmosensitive yeast with a high potential of use in fermentation processes. To elucidate the basis of its relative osmosensitivity, the role of the STL1 gene encoding a putative glycerol uptake system was studied. Under higher osmotic pressure, the addition of a low amount of glycerol to the growth medium improved the growth of S. kudriavzevii and the expression of the STL1 gene was highly induced. Deletion of this gene decreased the strain's ability to grow in the presence of higher concentrations of salts and other solutes. Moreover, the mutant had a disturbed homeostasis of intracellular pH. Expression of the SkSTL1 gene in Saccharomyces cerevisiae complemented the osmosensitivity of the S. cerevisiae hog1Δ stl1Δ mutant, and the gene's tagging with GFP localized its product to the plasma membrane. Altogether, a deficiency in glycerol uptake did not seem to be the reason for S. kudriavzevii's low Osmotolerance; its Stl1 transporter properly contributes to the regulation of intracellular pH and is crucial to its survival of osmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: An increasing demand for food products with benefits for human health turns the attention to less-exploited nonconventional yeasts with interesting traits not found in Saccharomyces cerevisiae. Among them, Saccharomyces kudriavzevii has good potential for aroma-compound production, fermentations and other biotechnological applications, but it is less adapted to stressful industrial conditions. This report studied S. kudriavzevii relative osmosensitivity and its capacity for active glycerol uptake. The results obtained (on the activity and physiological function of S. kudriavzevii glycerol transporter) may contribute to a further engineering of this species aiming to improve its Osmotolerance.
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Osmotolerance of Dekkera bruxellensis and the role of two Stl glycerol-proton symporters.
FEMS microbiology letters, 2018Co-Authors: Jana Zemančíková, Michala Dušková, Hana Elicharová, Klara Papouskova, Hana SychrováAbstract:Dekkera bruxellensis is important for lambic beer fermentation but is considered a spoilage yeast in wine fermentation. We compared two D. bruxellensis strains isolated from wine and found that they differ in some basic properties, including Osmotolerance. The genomes of both strains contain two highly similar copies of genes encoding putative glycerol-proton symporters from the STL family that are important for yeast Osmotolerance. Cloning of the two DbSTL genes and their expression in suitable osmosensitive Saccharomyces cerevisiae mutants revealed that both identified genes encode functional glycerol uptake systems, but only DbStl2 has the capacity to improve the Osmotolerance of S. cerevisiae cells.
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two glycerol uptake systems contribute to the high Osmotolerance of zygosaccharomyces rouxii
Molecular Microbiology, 2015Co-Authors: Michala Dušková, Celia Ferreira, Cândida Lucas, Hana SychrováAbstract:The help of Dr. P. Ergang with the real-time PCR experiments is gratefully acknowledged. We thank O. Zimmermannova for critical reading of the paper. This work was supported by the following grants: Grant Agency of the Czech Republic P503/ 10/0307, institutional concept RVO:6798582, Grant Agency of the Charles University 299611/2011/B-Bio/PrF, an Lifelong Learning Programme ERASMUS practical placement grant and by Fundo Europeu de Desenvolvimento Regional – Programa Operacional de Fatores de Competitividade – COMPETE and by national funds from Fundacao para a Ciencia e Tecnologia through the project PEstC/BIA/UI4050/ 2011.
