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Evy Battaglia - One of the best experts on this subject based on the ideXlab platform.
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A novel L-Arabinose-responsive regulator discovered in the rice-blast fungus Pyricularia oryzae (Magnaporthe oryzae)
FEBS Letters, 2016Co-Authors: Sylvia Klaubauf, Miaomiao Zhou, Marc-henri Lebrun, Evy BattagliaAbstract:In this study we identified the L-Arabinose-responsive regulator of Pyricularia oryzae that regulates L-Arabinose release and catabolism. Previously we identified the Zn2Cys6 transcription factor (TF), AraR, that has this role in the Trichocomaceae family (Eurotiales), but is absent in other fungi. Candidate Zn2Cys6 TF genes were selected according to their transcript profiles on L-Arabinose. Deletion mutants of these genes were screened for their growth phenotype on L-Arabinose. One mutant, named Delta ara1, was further analyzed. Our analysis demonstrated that Ara1 from P. oryzae is the functional analog of AraR from A. niger, while there is no significant sequence similarity between them.
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analysis of regulation of pentose utilisation in aspergillus niger reveals evolutionary adaptations in eurotiales
Studies in Mycology, 2011Co-Authors: Evy Battaglia, G. Jerre Van Veluw, L Visser, A. Nijssen, Han A B Wosten, R P De VriesAbstract:Aspergilli are commonly found in soil and on decaying plant material. D-xylose and L-Arabinose are highly abundant components of plant biomass. They are released from polysaccharides by fungi using a set of extracellular enzymes and subsequently converted intracellularly through the pentose catabolic pathway (PCP). In this study, the L-Arabinose responsive transcriptional activator (AraR) is identified in Aspergillus niger and was shown to control the L-Arabinose catabolic pathway as well as expression of genes encoding extracellular L-Arabinose releasing enzymes. AraR interacts with the D-xylose-responsive transcriptional activator XlnR in the regulation of the pentose catabolic pathway, but not with respect to release of L-Arabinose and D-xylose. AraR was only identified in the Eurotiales, more specifically in the family Trichocomaceae and appears to have originated from a gene duplication event (from XlnR) after this order or family split from the other filamentous ascomycetes. XlnR is present in all filamentous ascomycetes with the exception of members of the Onygenales. Since the Onygenales and Eurotiales are both part of the subclass Eurotiomycetidae, this indicates that strong adaptation of the regulation of pentose utilisation has occurred at this evolutionary node. In Eurotiales a unique two-component regulatory system for pentose release and metabolism has evolved, while the regulatory system was lost in the Onygenales. The observed evolutionary changes (in Eurotiomycetidae) mainly affect the regulatory system as in contrast, homologues for most genes of the L-Arabinose/D-xylose catabolic pathway are present in all the filamentous fungi, irrespective of the presence of XlnR and/or AraR.
Antonius J A Van Maris - One of the best experts on this subject based on the ideXlab platform.
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metabolome transcriptome and metabolic flux analysis of arabinose fermentation by engineered saccharomyces cerevisiae
Metabolic Engineering, 2010Co-Authors: Wouter H Wisselink, Barbara Crimi, Jack T Pronk, Chiara Cipollina, Bart Oud, Joseph J Heijnen, Antonius J A Van MarisAbstract:One of the challenges in strain improvement by evolutionary engineering is to subsequently determine the molecular basis of the improved properties that were enriched from the natural genetic variation during the selective conditions. This study focuses on Saccharomyces cerevisiae IMS0002 which, after metabolic and evolutionary engineering, ferments the pentose sugar arabinose. Glucose- and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor were subjected to transcriptome analysis, intracellular metabolite measurements and metabolic flux analysis. Increased expression of the GAL-regulon and deletion of GAL2 in IMS0002 confirmed that the galactose transporter is essential for growth on arabinose. Elevated intracellular concentrations of pentose-phosphate-pathway intermediates and upregulation of TKL2 and YGR043c (encoding transketolase and transaldolase isoenzymes) suggested an involvement of these genes in flux-controlling reactions in arabinose fermentation. Indeed, deletion of these genes in IMS0002 caused a 21% reduction of the maximum specific growth rate on arabinose.
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novel evolutionary engineering approach for accelerated utilization of glucose xylose and arabinose mixtures by engineered saccharomyces cerevisiae strains
Applied and Environmental Microbiology, 2009Co-Authors: Wouter H Wisselink, Maurice J Toirkens, Jack T Pronk, Antonius J A Van MarisAbstract:Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of D-xylose and L-Arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g–1 of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel evolutionary engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter–1 glucose, 15 g liter–1 xylose, and 15 g liter–1 arabinose.
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engineering of saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of l arabinose
Applied and Environmental Microbiology, 2007Co-Authors: Wouter H Wisselink, Johannes P. Van Dijken, Maurice J Toirkens, M Del Rosario Franco Berriel, Aaron Adriaan Winkler, Jack T Pronk, Antonius J A Van MarisAbstract:For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as L-Arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of L-Arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of L-Arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the L-Arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h–1 g [dry weight]–1) and ethanol production (0.29 g h–1 g [dry weight]–1) and a high ethanol yield (0.43 g g–1) during anaerobic growth on L-Arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.
Wouter H Wisselink - One of the best experts on this subject based on the ideXlab platform.
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the penicillium chrysogenum transporter pcarat enables high affinity glucose insensitive l arabinose transport in saccharomyces cerevisiae
Biotechnology for Biofuels, 2018Co-Authors: Jasmine M Bracher, Arnold J. M. Driessen, Maarten D Verhoeven, Wouter H Wisselink, Barbara Crimi, Jeroen G Nijland, Paul KlaassenAbstract:L-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of L-Arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in L-Arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in L-Arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on L-Arabinose upon expression in an engineered L-Arabinose-fermenting S. cerevisiae strain in which the endogenous L-Arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity L-Arabinose-proton symporter that does not transport d-xylose or d-glucose. An L-Arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in L-Arabinose-limited chemostat cultures than a congenic strain in which L-Arabinose import depended on Gal2 (4.2 × 10−3 and 1.8 g L−1, respectively). Inhibition of L-Arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, L-Arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L−1 L-Arabinose and 20 g L−1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on L-Arabinose transport via Gal2. Its high affinity and specificity for L-Arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.
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metabolome transcriptome and metabolic flux analysis of arabinose fermentation by engineered saccharomyces cerevisiae
Metabolic Engineering, 2010Co-Authors: Wouter H Wisselink, Barbara Crimi, Jack T Pronk, Chiara Cipollina, Bart Oud, Joseph J Heijnen, Antonius J A Van MarisAbstract:One of the challenges in strain improvement by evolutionary engineering is to subsequently determine the molecular basis of the improved properties that were enriched from the natural genetic variation during the selective conditions. This study focuses on Saccharomyces cerevisiae IMS0002 which, after metabolic and evolutionary engineering, ferments the pentose sugar arabinose. Glucose- and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor were subjected to transcriptome analysis, intracellular metabolite measurements and metabolic flux analysis. Increased expression of the GAL-regulon and deletion of GAL2 in IMS0002 confirmed that the galactose transporter is essential for growth on arabinose. Elevated intracellular concentrations of pentose-phosphate-pathway intermediates and upregulation of TKL2 and YGR043c (encoding transketolase and transaldolase isoenzymes) suggested an involvement of these genes in flux-controlling reactions in arabinose fermentation. Indeed, deletion of these genes in IMS0002 caused a 21% reduction of the maximum specific growth rate on arabinose.
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novel evolutionary engineering approach for accelerated utilization of glucose xylose and arabinose mixtures by engineered saccharomyces cerevisiae strains
Applied and Environmental Microbiology, 2009Co-Authors: Wouter H Wisselink, Maurice J Toirkens, Jack T Pronk, Antonius J A Van MarisAbstract:Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of D-xylose and L-Arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g–1 of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel evolutionary engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter–1 glucose, 15 g liter–1 xylose, and 15 g liter–1 arabinose.
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engineering of saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of l arabinose
Applied and Environmental Microbiology, 2007Co-Authors: Wouter H Wisselink, Johannes P. Van Dijken, Maurice J Toirkens, M Del Rosario Franco Berriel, Aaron Adriaan Winkler, Jack T Pronk, Antonius J A Van MarisAbstract:For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as L-Arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of L-Arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of L-Arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the L-Arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h–1 g [dry weight]–1) and ethanol production (0.29 g h–1 g [dry weight]–1) and a high ethanol yield (0.43 g g–1) during anaerobic growth on L-Arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.
Eckhard Boles - One of the best experts on this subject based on the ideXlab platform.
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codon optimized bacterial genes improve l arabinose fermentation in recombinant saccharomyces cerevisiae
Applied and Environmental Microbiology, 2008Co-Authors: Beate Wiedemann, Eckhard BolesAbstract:Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomyces cerevisiae, commonly used in bioethanol production, can utilize pentose sugars like L-Arabinose or d-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial L-Arabinose utilization pathway consisting of L-Arabinose isomerase from Bacillus subtilis and l-ribulokinase and l-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. L-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on L-Arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the L-Arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved L-Arabinose conversion rates. With this rational approach, the ethanol production rate from L-Arabinose could be increased more than 2.5-fold from 0.014 g ethanol h−1 (g dry weight)−1 to 0.036 g ethanol h−1 (g dry weight)−1 and the ethanol yield could be increased from 0.24 g ethanol (g consumed L-Arabinose)−1 to 0.39 g ethanol (g consumed L-Arabinose)−1. These improvements make up a new starting point for the construction of more-efficient industrial L-Arabinose-fermenting yeast strains by evolutionary engineering.
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Co-utilization of L-Arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains
Microbial Cell Factories, 2006Co-Authors: Kaisa Karhumaa, Beate Wiedemann, Eckhard Boles, Bärbel Hahn-hägerdal, Marie-francoise Gorwa-grauslundAbstract:Background Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-Arabinose, which are present in high amounts in lignocellulosic raw materials. Results We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-Arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-Arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. Conclusion Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae . In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.
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a modified saccharomyces cerevisiae strain that consumes l arabinose and produces ethanol
Applied and Environmental Microbiology, 2003Co-Authors: Jessica Becker, Eckhard BolesAbstract:Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-Arabinose for growth and to ferment it to ethanol. Expanding the substrate fermentation range of S. cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. After overexpression of a bacterial L-Arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-Arabinose-transporting yeast galactose permease, we were able to select an L-Arabinose-utilizing yeast strain by sequential transfer in L-Arabinose media. Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-Arabinose is a lowered activity of L-ribulokinase. Moreover, high L-Arabinose uptake rates and enhanced transaldolase activities favor utilization of L-Arabinose. With a doubling time of about 7.9 h in a medium with L-Arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight). h(-1) under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.
Sylvia Klaubauf - One of the best experts on this subject based on the ideXlab platform.
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A novel L-Arabinose-responsive regulator discovered in the rice-blast fungus Pyricularia oryzae (Magnaporthe oryzae)
FEBS Letters, 2016Co-Authors: Sylvia Klaubauf, Miaomiao Zhou, Marc-henri Lebrun, Evy BattagliaAbstract:In this study we identified the L-Arabinose-responsive regulator of Pyricularia oryzae that regulates L-Arabinose release and catabolism. Previously we identified the Zn2Cys6 transcription factor (TF), AraR, that has this role in the Trichocomaceae family (Eurotiales), but is absent in other fungi. Candidate Zn2Cys6 TF genes were selected according to their transcript profiles on L-Arabinose. Deletion mutants of these genes were screened for their growth phenotype on L-Arabinose. One mutant, named Delta ara1, was further analyzed. Our analysis demonstrated that Ara1 from P. oryzae is the functional analog of AraR from A. niger, while there is no significant sequence similarity between them.