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Wolfgang Hillen - One of the best experts on this subject based on the ideXlab platform.
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contribution of glucose kinase to glucose repression of Xylose Utilization in bacillus megaterium
Journal of Bacteriology, 1997Co-Authors: C Spath, Alexandra Kraus, Wolfgang HillenAbstract:The glk gene from Bacillus megaterium, which encodes glucose kinase, was isolated and analyzed. Disruption by a transcriptional glk-luxAB fusion indicated that glk is the only glucose kinase gene in that strain but did not affect growth of that mutant on glucose. Determination of luciferase activity under various growth conditions revealed constitutive transcription of glk. Expression of a xylA-lacZ fusion was repressed by glucose in the strain with the glk disruption about twofold less efficiently than in the wild type. The potential contribution of glk expression to glucose repression is discussed.
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Regulation of Xylose Utilization in Bacillus licheniformis: Xyl repressor-xyl-operator interaction studied by DNA modification protection and interference.
Molecular microbiology, 1994Co-Authors: Andrea Scheler, Wolfgang HillenAbstract:Xylose Utilization in Bacillus licheniformis is inducible by Xylose. We establish here that the Xyl repressor recognizes and binds an xyl operator sequence located 12 nucleotides downstream from the transcription start site of the xyl operon. DNA-retardation experiments employing xyl regulatory DNA and soluble protein extracts indicate complex formation in the presence of Xyl repressor. Two repressor-operator complexes are distinguished by different gel mobilities. They yield the same in situ copper-phenanthroline footprint. This result suggests that a single xyl operator may be bound by different oligomers of Xyl repressor. Methylation and hydroxyl radical cleavage protection of the xyl operator by Xyl repressor binding and ethylation interference of Xyl repressor binding to the xyl operator reveals symmetrical interaction of the repressor with two half sites of the operator, which show palindromic symmetry and are located on the same side of the B-form DNA structure.
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using fusions with luxab from vibrio harveyi mav to quantify induction and catabolite repression of the xyl operon in staphylococcus carnosus tm300
Fems Microbiology Letters, 1993Co-Authors: Christine Sizemore, Walter Geiβdorfer, Wolfgang HillenAbstract:The luxA,B genes from the Gram-negative marine bacterium Vibrio harveyi MAV were used in Staphylococcus carnosus TM300 as a reporter system for regulated expression of Xylose Utilization. The luciferase genes were fused to the xyl operon from Staphylococcus xylosus C2a. Expression of bioluminescence was induced through addition of Xylose and repressed in the presence of glucose. A method to quantitate bioluminescence directly from the culture is described.
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Regulation of Staphylococcus xylosus Xylose Utilization genes at the molecular level.
Journal of bacteriology, 1992Co-Authors: Christine Sizemore, Friedrich Gotz, B Wieland, Wolfgang HillenAbstract:We have investigated the regulation of the operon encoding Xylose Utilization in Staphylococcus xylosus C2a and Staphylococcus carnosus TM300. For in vivo studies, transcriptional fusions of the xylAB regulatory region to the lipase gene from Staphylococcus hyicus were constructed. Repression of lipase activity depended on a functional xylR gene and an xyl operator palindrome downstream of the promoter, while induction was obtained in the presence of Xylose. Inactivation of either xylR or the xyl operator led to constitutive expression in the absence of Xylose. Crude protein extracts from xylR+ staphylococci led to gel mobility shifts of the xyl regulatory DNA in the absence but not in the presence of Xylose. A copper-phenanthroline footprint of the shifted band revealed protection of 28 phosphodiesters from cleavage in each strand of the xyl operator. Thus, the Xyl repressor covers the DNA over more than 2.5 helical turns. Glucose repression of the xyl operon occurs at the level of transcription and is independent of a functional xylR gene. A potential cis-active sequence element for glucose repression is discussed on the basis of sequence similarities to respective elements from bacilli.
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Organization, promoter analysis and transcriptional regulation of the Staphylococcus xylosus Xylose Utilization operon
Molecular and General Genetics MGG, 1991Co-Authors: Christine Sizemore, Eberhard Buchner, Thomas Rygus, Claudia Witke, Friedrich Gotz, Wolfgang HillenAbstract:The Staphylococcus xylosus xyl genes were cloned in Staphylococcus carnosus by complementation to Xylose Utilization. Xylose isomerase assays under inducing (Xylose present) and non-inducing (Xylose absent) conditions indicated the presence of a regulated xylA gene on the recombinant plasmid. The nucleotide sequence (4520 bases) revealed three open reading frames with the same polarity. They were identified by sequence homologies as xylR , encoding the Xyl repressor, xylA , encoding Xylose isomerase and xylB , encoding xylulokinase. Primer extension analyses indicated constitutive transcription of xylR and Xylose-inducible transcription of xylA . Promoter consensus sequences were found upstream of both transcriptional start sites. A transcriptional terminator between xylR and xylA separates the different transcriptional units. Potential regulatory elements were identified by sequence analysis and suggest a repressor-operator mechanism for the regulation of xylAB expression.
Fengwu Bai - One of the best experts on this subject based on the ideXlab platform.
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Improving Xylose Utilization of Saccharomyces cerevisiae by Expressing the MIG1 Mutant from the Self-Flocculating Yeast SPSC01
Protein and peptide letters, 2018Co-Authors: Xin-qing Zhao, Chen-guang Liu, Fengwu BaiAbstract:Background The major carbohydrate components of lignocellulosic biomass are cellulose and hemicelluloses. Saccharomyces cerevisiae cannot efficiently utilize Xylose derived upon the hydrolysis of hemicelluloses. Although engineering the yeast with Xylose metabolic pathway has been intensively studied, challenges are still ahead for developing robust strains for lignocellulosic bioethanol production. Objective The main objective of this study was to reveal the role of the MIG1 mutant isolated from the self-flocculating S. cerevisiae SPSC01 in Xylose Utilization, glucose repression and ethanol fermentation by S. cerevisiae. Methods The MIG1 mutant was amplified from S. cerevisiae SPSC01 by PCR and MIG1- overexpression-cassette was transformed into S. cerevisiae S288c and Xylose-metabolizing strain YB-2625-T through homologous recombination. Yeast growth was measured by colony assay on plates with or without Xylose supplementation. Then Xylose Utilization and ethanol production were further evaluated through flask fermentation when mixed sugars of glucose and Xylose at 3:1 and 2:1, respectively, were supplied. Fermentation products were detected by HPLC, and activities of Xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) were also measured. The transcription of genes regulated by the expression of the MIG1 mutant was analyzed by RTqPCR. Evolutionary relationship of various MIG1s was developed by gene sequencing and sequence alignment. Results No difference was observed for S288c growing with Xylose when it was engineered with the overexpression or deletion of its native MIG1, but its growth was enhanced when overexpressing the MIG1 mutant from SPSC01. The submerged culture of YB-2625-T MIG1-SPSC engineered with Xylose-metabolic pathway and the MIG1 mutant indicated that xylitol accumulation was decreased, and consequently, more biomass was accumulated. Furthermore, improved activities of the key enzymes such as XR, XDH and XK were detected in YB-2625-T MIG1-SPSC. Evolutionary analysis of MIG1s amplified from S. cerevisiae strains commonly used for ethanol production revealed a close relationship of SPSC01 and YB-2625. Conclusion Our results demonstrated the effect of the overexpression of the MIG1 mutant from SPSC01 on Xylose Utilization of S. cerevisiae. This study could be an alternative strategy for engineering S. cerevisiae with improved Xylose Utilization.
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Association of improved oxidative stress tolerance and alleviation of glucose repression with superior Xylose-Utilization capability by a natural isolate of Saccharomyces cerevisiae.
Biotechnology for biofuels, 2018Co-Authors: Cheng Cheng, Rui-qi Tang, Liang Xiong, Ronald E. Hector, Fengwu Bai, Xin-qing ZhaoAbstract:Saccharomyces cerevisiae wild strains generally have poor Xylose-Utilization capability, which is a major barrier for efficient bioconversion of lignocellulosic biomass. Laboratory adaption is commonly used to enhance Xylose Utilization of recombinant S. cerevisiae. Apparently, yeast cells could remodel the metabolic network for Xylose metabolism. However, it still remains unclear why natural isolates of S. cerevisiae poorly utilize Xylose. Here, we analyzed a unique S. cerevisiae natural isolate YB-2625 which has superior Xylose metabolism capability in the presence of mixed-sugar. Comparative transcriptomic analysis was performed using S. cerevisiae YB-2625 grown in a mixture of glucose and Xylose, and the model yeast strain S288C served as a control. Global gene transcription was compared at both the early mixed-sugar Utilization stage and the latter Xylose-Utilization stage. Genes involved in endogenous Xylose-assimilation (XYL2 and XKS1), gluconeogenesis, and TCA cycle showed higher transcription levels in S. cerevisiae YB-2625 at the Xylose-Utilization stage, when compared to the reference strain. On the other hand, transcription factor encoding genes involved in regulation of glucose repression (MIG1, MIG2, and MIG3) as well as HXK2 displayed decreased transcriptional levels in YB-2625, suggesting the alleviation of glucose repression of S. cerevisiae YB-2625. Notably, genes encoding antioxidant enzymes (CTT1, CTA1, SOD2, and PRX1) showed higher transcription levels in S. cerevisiae YB-2625 in the Xylose-Utilization stage than that of the reference strain. Consistently, catalase activity of YB-2625 was 1.9-fold higher than that of S. cerevisiae S288C during the Xylose-Utilization stage. As a result, intracellular reactive oxygen species levels of S. cerevisiae YB-2625 were 43.3 and 58.6% lower than that of S288C at both sugar Utilization stages. Overexpression of CTT1 and PRX1 in the recombinant strain S. cerevisiae YRH396 deriving from S. cerevisiae YB-2625 increased cell growth when Xylose was used as the sole carbon source, leading to 13.5 and 18.1%, respectively, more Xylose consumption. Enhanced oxidative stress tolerance and relief of glucose repression are proposed to be two major mechanisms for superior Xylose Utilization by S. cerevisiae YB-2625. The present study provides insights into the innate regulatory mechanisms underlying Xylose Utilization in wild-type S. cerevisiae, which benefits the rapid development of robust yeast strains for lignocellulosic biorefineries.
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synergistic effect of calcium and zinc on glucose Xylose Utilization and butanol tolerance of clostridium acetobutylicum
Fems Microbiology Letters, 2016Co-Authors: Chuang Xue, Fengwu Bai, Lijie Chen, Wenjie YuanAbstract:Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, Xylose and glucose/Xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/Xylose Utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved Xylose Utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best Xylose Utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.
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Synergistic effect of calcium and zinc on glucose/Xylose Utilization and butanol tolerance of Clostridium acetobutylicum.
FEMS microbiology letters, 2016Co-Authors: Chuang Xue, Lijie Chen, Wenjie Yuan, Fengwu BaiAbstract:Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, Xylose and glucose/Xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/Xylose Utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved Xylose Utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best Xylose Utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.
Aindrila Mukhopadhyay - One of the best experts on this subject based on the ideXlab platform.
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evolved hexose transporter enhances Xylose uptake and glucose Xylose co Utilization in saccharomyces cerevisiae
Scientific Reports, 2016Co-Authors: Amanda Reider Apel, Mario Ouellet, Heather Szmidtmiddleton, Jay D Keasling, Aindrila MukhopadhyayAbstract:Enhancing Xylose Utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing Utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous Xylose Utilization pathway, and imposed a laboratory evolution regime with Xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved Xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min(-1)•mg(-1)) that allows the S. cerevisiae strain to show significant growth with Xylose as the sole carbon source, as well as partial co-Utilization of glucose and Xylose in a mixed sugar cultivation.
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evolved hexose transporter enhances Xylose uptake and glucose Xylose co Utilization in saccharomyces cerevisiae
Scientific Reports, 2016Co-Authors: Amanda Reider Apel, Mario Ouellet, Heather Szmidtmiddleton, Jay D Keasling, Aindrila MukhopadhyayAbstract:Enhancing Xylose Utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing Utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous Xylose Utilization pathway, and imposed a laboratory evolution regime with Xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved Xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min(-1)•mg(-1)) that allows the S. cerevisiae strain to show significant growth with Xylose as the sole carbon source, as well as partial co-Utilization of glucose and Xylose in a mixed sugar cultivation.
Hung Lee - One of the best experts on this subject based on the ideXlab platform.
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Deletion of hxk1 gene results in derepression of Xylose Utilization in Scheffersomyces stipitis.
Journal of industrial microbiology & biotechnology, 2015Co-Authors: Mehdi Dashtban, Xin Wen, Paramjit K. Bajwa, Hung LeeAbstract:A major problem in fermenting Xylose in lignocellulosic substrates is the presence of glucose and mannose which inhibit Xylose Utilization. Previous studies showed that catabolite repression in some yeasts is associated with hexokinases and that deletion of one of these gene(s) could result in derepressed mutant strain(s). In this study, the hxk1 encoding hexokinase 1 in Scheffersomyces stipitis was disrupted. The ∆hxk1 SS6 strain retained the ability to utilize the main hexoses and pentoses commonly found in lignocellulosic hydrolysates as efficiently as the wild-type (WT) strain. SS6 also fermented the dominant sugars to ethanol; however, on Xylose, the ∆hxk1 strain produced more xylitol and less ethanol than the WT. On mixed sugars, as expected the WT utilized glucose ahead of Xylose and Xylose Utilization did not commence until all the glucose was consumed. In contrast, the ∆hxk1 mutant showed derepression in that it started to utilize Xylose even when considerable glucose (about 1.72 %, w/v) remained in the medium. Similarly, mannose did not repress Xylose Utilization by the ∆hxk1 mutant and Xylose and mannose were simultaneously utilized. The results are of interest in efforts to engineer yeast strains capable of efficiently utilizing glucose and Xylose simultaneously for lignocellulosic biomass conversion.
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Research letterReversible inactivation of d-Xylose Utilization by d-glucose in the pentose-fermenting yeast Pachysolen tannophilus
FEMS Microbiology Letters, 1992Co-Authors: Hung LeeAbstract:A major problem in fermenting pentoses using lignocellulosic substrates is the presence of d-glucose which inhibits d-Xylose Utilization. We previously showed that d-glucose represses the induction of Xylose reductase and xylitol dehydrogenase activities, thereby inhibiting d-Xylose Utilization in Pachysolen tannophilus. The question arose whether d-glucose can also inactivate d-Xylose fermentation. P. tannophilus cells were grown on a defined d-Xylose-containing liquid medium. At about 40 h, d-glucose was added to a final concentration of 3% (w/v). This led to a rapid cessation of d-Xylose Utilization, which resumed after 10–12 h before d-glucose was completely consumed. This suggests that d-glucose inactivated existing d-Xylose catabolic enzymes and that inactivation was reversed at low d-glucose concentrations. This reversible inactivation was distinct from d-glucose repression. Addition of cycloheximide did not block the resumption of d-Xylose consumption, suggesting that reactivation was independent of protein synthesis.
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Reversible inactivation of d-Xylose Utilization by d-glucose in the pentose-fermenting yeast Pachysolen tannophilus
FEMS Microbiology Letters, 1992Co-Authors: Hung LeeAbstract:A major problem in fermenting pentoses using lignocellulosic substrates is the presence of d-glucose which inhibits d-Xylose Utilization. We previously showed that d-glucose represses the induction of Xylose reductase and xylitol dehydrogenase activities, thereby inhibiting d-Xylose Utilization in Pachysolen tannophilus. The question arose whether d-glucose can also inactivate d-Xylose fermentation. P. tannophilus cells were grown on a defined d-Xylose-containing liquid medium. At about 40 h, d-glucose was added to a final concentration of 3% (w/v). This led to a rapid cessation of d-Xylose Utilization, which resumed after 10–12 h before d-glucose was completely consumed. This suggests that d-glucose inactivated existing d-Xylose catabolic enzymes and that inactivation was reversed at low d-glucose concentrations. This reversible inactivation was distinct from d-glucose repression. Addition of cycloheximide did not block the resumption of d-Xylose consumption, suggesting that reactivation was independent of protein synthesis.
Chuang Xue - One of the best experts on this subject based on the ideXlab platform.
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synergistic effect of calcium and zinc on glucose Xylose Utilization and butanol tolerance of clostridium acetobutylicum
Fems Microbiology Letters, 2016Co-Authors: Chuang Xue, Fengwu Bai, Lijie Chen, Wenjie YuanAbstract:Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, Xylose and glucose/Xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/Xylose Utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved Xylose Utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best Xylose Utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.
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Synergistic effect of calcium and zinc on glucose/Xylose Utilization and butanol tolerance of Clostridium acetobutylicum.
FEMS microbiology letters, 2016Co-Authors: Chuang Xue, Lijie Chen, Wenjie Yuan, Fengwu BaiAbstract:Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, Xylose and glucose/Xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/Xylose Utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved Xylose Utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best Xylose Utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.
