Analysis of ethanologenic E.coli strain GLBRCE1 during fermentation of alkaline-pretreated corn stover hydrolysate
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PROVIDER: PRJNA150853 | ENA |
REPOSITORIES: ENA
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Project description:The physiology of ethanologenic Escherichia coli grown anaerobically in alkaline-pretreated plant hydrolysates is not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pre-treated by ammonia fiber expansion. Despite the high sugar content (~6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses throughout the different stages of growth. During the exponential and transition phases of growth, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate media. However, after the majority of amino acids were depleted from the media cells entered stationary phase and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin and ethanol stress collectively limits growth, sugar utilization rates and ethanol yields in alkaline-pretreated lignocellulosic hydrolysates. 38 samples in total. 24 samples were derived from biological replicate fermentations of alkaline-pretreated cornstover hydrolysate (12 datapoint time-series per fermentation). The remaining samples were obtained from fermentations conducted in defined media (Glucose Minimal Media (GMM, n=7), Synthetic Hydrolysate media (SynH, n=7)).
2012-06-17 | E-GEOD-35049 | biostudies-arrayexpress
Project description:The physiology of ethanologenic Escherichia coli grown anaerobically in alkaline-pretreated plant hydrolysates is not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pre-treated by ammonia fiber expansion. Despite the high sugar content (~6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses throughout the different stages of growth. During the exponential and transition phases of growth, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate media. However, after the majority of amino acids were depleted from the media cells entered stationary phase and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin and ethanol stress collectively limits growth, sugar utilization rates and ethanol yields in alkaline-pretreated lignocellulosic hydrolysates.
2012-06-18 | GSE35049 | GEO
Project description:The fermentation of Zymomonas mobilis 2032 was conducted in 6% and 9% glucan-loading AFEX-pretreated corn stover hydrolysates, as well as in 7% glucan-loading AFEX-pretreated switchgrass hydrolysate, and RNA samples were collected at both glucose and xylose growth stages. Here we present a comprehensive RNA-Seq data set at both growth stages.
2022-04-24 | GSE201229 | GEO
Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 4 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH), synthetic hydrolysate with added lignotoxins (SynH_LT), or synthetic hydrolysate containing acid or amide lignotoxins only (SynH_Acids_Amides). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. Two biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 6 time points, corresponding to early exponential (Exp1), Mid-exponential (Exp2), Late-exponential (Exp3), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.
2014-07-18 | E-GEOD-58977 | biostudies-arrayexpress
Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 3 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH) and syntetic hydrolysate with added lignotoxins (SynH_LT). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. 3 biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.
2014-07-18 | E-GEOD-58806 | biostudies-arrayexpress
Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 3 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH) and syntetic hydrolysate with added lignotoxins (SynH_LT). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. 3 biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.
2014-07-18 | E-GEOD-59397 | biostudies-arrayexpress
Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 or GLBRCE1 lacking the plasmid-borne PET cassette (GLBRCE1_pBBR) was grown in AFEX corn stover hydrolysate (ACSH) Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH, and cultures were diluted into ACSH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. One biological replicate was grown in each medium. RNA samples were obtained at 3 time points, corresponding to exponential (Exp), transitional (Trans), and stationary (Stat) growth phases.
2014-07-18 | E-GEOD-58902 | biostudies-arrayexpress
Project description:Multistage Transcriptomics Data Set of Zymomonas mobilis 2032 During Fermentation in 6% and 9% Glucan-Loading AFEX-Pretreated Corn Stover Hydrolysates and 7% Glucan-Loading AFEX-Pretreated Switchgrass Hydrolysate
| PRJNA830472 | ENA
Project description:Here, we explored natural variation in stress tolerance and in transcriptomic responses to synthetic hydrolysate, mimicking chemically pretreated plant material, to dissect the physiological effects hydrolysate components. Using six different Saccharomyces cerevisiae strains that together maximized phenotypic and genetic diversity, we explored transcriptomic differences between resistant and sensitive strains. We identified both common and strain-specific responses. Comparing responses of resistant and sensitive strains provided insights about primary cellular targets of hydrolysate toxins, implicating cell wall structure, protein and DNA stability, energy stores and redox balance. Importantly, we uncovered lower expression of thiamine genes while in the presence of toxins, which we argue are most likely an indirect effect that increases sensitivity. We also demonstrate synergistic interactions between the nutrient composition, osmolarity, pH, and classes of hydrolysate toxins. Together, this work provides a platform for further dissecting hydrolysate toxins and strain responses.
2016-04-01 | GSE77505 | GEO
Project description:Here, we explored natural variation in stress tolerance and in transcriptomic responses to synthetic hydrolysate, mimicking chemically pretreated plant material, to dissect the physiological effects hydrolysate components. Using six different Saccharomyces cerevisiae strains that together maximized phenotypic and genetic diversity, we explored transcriptomic differences between resistant and sensitive strains. We identified both common and strain-specific responses. Comparing responses of resistant and sensitive strains provided insights about primary cellular targets of hydrolysate toxins, implicating cell wall structure, protein and DNA stability, energy stores and redox balance. Importantly, we uncovered lower expression of thiamine genes while in the presence of toxins, which we argue are most likely an indirect effect that increases sensitivity. We also demonstrate synergistic interactions between the nutrient composition, osmolarity, pH, and classes of hydrolysate toxins. Together, this work provides a platform for further dissecting hydrolysate toxins and strain responses. RNA-seq and transcriptome analysis of six S. cerevisiae natural isolates having different resistant to lignocellulosic hydrolysate. Two biological replicate cell samples (collected on different days) were harvested for RNAseq analysis. Strains were grown in YPD, synthetic hydrolysate without toxins (SynH -HTs), and synthetic hydrolysate with toxins (SynH). Cells were grown for at least three generations to log phase (OD600 ~0.5) and collected by centrifugation.
2016-04-01 | E-GEOD-77505 | biostudies-arrayexpress