Transcriptomic responses of Clostridium (Ruminiclostridium) thermocellum ATCC27405 and ethanol tolerant strains to ethanol stress
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ABSTRACT:
ORGANISM(S): Acetivibrio thermocellus ATCC 27405
PROVIDER: GSE77089 | GEO | 2018/03/31
REPOSITORIES: GEO
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Project description:Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production. However, C. thermocellum is relatively sensitive to ethanol compared to yeast. Previous studies have investigated the membrane and protein composition of wild-type and ethanol tolerant strains, but relatively little is known about the genome changes associated with the ethanol tolerant C. thermocellum strain. In this study, C. thermocellum cultures were grown to mid-exponential phase and then either shocked with the supplementation of ethanol to a final concentration of 3.95 g/L (equal to 0.5% [v/v]) or were untreated. Samples were taken pre-shock and 2, 12, 30, 60, 120, 240 min post-shock for multiple systems biology analyses. The addition of ethanol dramatically reduced the C. thermocellum growth and the final cell density was approximately half of the control fermentations, with concomitant reductions in substrate consumption in the treated cultures. The response of C. thermocellum to ethanol was dynamic and involved more than six hundred genes that were significantly and differentially expressed between the different conditions over time and every functional category was represented. Cellobiose was accumulated within the ethanol-shocked C. thermocellum cells, as well as the sugar phosphates such as fructose-6-P and cellobiose-6-P. The comparison and correlation among intracellular metabolites, proteomic and transcriptomics profiles as well as the ethanol effects on cellulosome, hydrogenase glycolysis and nitrogen metabolism are discussed, which led us to propose that C. thermocellum may utilize the nitrogen metabolism to bypass the arrested carbon metabolism in responding to ethanol stress shock, and the nitrogen metabolic pathway and redox balance may be the key target for improving ethanol tolerance and production in C. thermocellum.
2012-08-16 | GSE25236 | GEO
Project description:The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. To gain insights into the C. thermocellum genes, using an updated version of the C. thermocellum ATCC 27405 genome annotation, that are required for specific growth on the cellulosic feedstocks of either pretreated switchgrass or Populus, duplicate fermentations were conducted with a 5 g/L solid substrate loading. High quality RNA was extracted using a method we report for C. thermocellum grown on solid substrates. Transcriptome profiles were obtained at two time points during actively growing fermentations (12 h and 37 h post inoculation). A comparison of two transcriptomic analytical techniques, microarray and RNAseq, was performed and the data analyzed for statistical significance. When thresholds for genes passing significance of FDR>0.05 were applied, microarray (2351 genes) had a greater number of significant genes relative to RNA-seq (280 genes when normalized by KDMM). When a 2-fold difference in expression threshold was applied, seventy-three genes were significantly differentially expressed in common between the two techniques. We identified genes differentially expressed when C. thermocellum ATC 27405 was grown on the two biomass substrates, with two putative efflux/transport systems highly differentially regulated (>5-fold). This study has revealed consistency between these two transcriptomics analytical platforms that gives confidence in our switch from the DNA microarray platform to an RNAseq based platform for routine transcriptomics analyses.
2013-05-16 | GSE47010 | GEO
Project description:The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. To gain insights into the C. thermocellum genes, using an updated version of the C. thermocellum ATCC 27405 genome annotation, that are required for specific growth on the cellulosic feedstocks of either pretreated switchgrass or Populus, duplicate fermentations were conducted with a 5 g/L solid substrate loading. High quality RNA was extracted using a method we report for C. thermocellum grown on solid substrates. Transcriptome profiles were obtained at two time points during actively growing fermentations (12 h and 37 h post inoculation). A comparison of two transcriptomic analytical techniques, microarray and RNAseq, was performed and the data analyzed for statistical significance. When thresholds for genes passing significance of FDR>0.05 were applied, microarray (2351 genes) had a greater number of significant genes relative to RNA-seq (280 genes when normalized by KDMM). When a 2-fold difference in expression threshold was applied, seventy-three genes were significantly differentially expressed in common between the two techniques. We identified genes differentially expressed when C. thermocellum ATC 27405 was grown on the two biomass substrates, with two putative efflux/transport systems highly differentially regulated (>5-fold). This study has revealed consistency between these two transcriptomics analytical platforms that gives confidence in our switch from the DNA microarray platform to an RNAseq based platform for routine transcriptomics analyses. To gain insights into the C. thermocellum genes, using an updated version of the C. thermocellum ATCC 27405 genome annotation, that are required for specific growth on the cellulosic feedstocks of either pretreated switchgrass or Populus, duplicate fermentations were conducted with a 5 g/L solid substrate loading. High quality RNA was extracted using a method we report for C. thermocellum grown on solid substrates. Transcriptome profiles were obtained at two time points during actively growing fermentations (12 h and 37 h post inoculation). A comparison of two transcriptomic analytical techniques, microarray and RNAseq, was performed and the data analyzed for statistical significance.
2013-05-16 | E-GEOD-47010 | biostudies-arrayexpress
Project description:The yeast Saccharomyces cerevisiae is well known for its high ethanol production performances. An original fermentation process that allows the yeast S. cerevisiae to produce in less than 45 h more than 150 g/l ethanol (i.e. 18.9°GL) was set up in our laboratory [1]. Under this condition, the yeast cells induce a dynamic process to adapt to increased ethanol concentration by a mechanism that is likely different to the stress response triggered by sudden ethanol addition to exponentially growing cells [2]. Kinetic analysis of the growth curve identified two main phases: a growth phase that ended up at 90 g/l ethanol and then an uncoupling phase during which non-growing cells kept producing ethanol. This latter phase is also characterized with an increased loss of viability. In order to investigate on a genome scale the expression changes occurring during this process, gene expression was quantified using DNA chips technology at six different time-points during fed-batch fermentation. [1] Alfenore et al, Appl. Microbiol. Biotechnol. 60 : 67-72, 2002. [2] Alexandre H. et al., FEBS Lett. 498(1) : 98-103, 2001.
2009-09-01 | GSE15493 | GEO
Project description:Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production. However, C. thermocellum is relatively sensitive to ethanol compared to yeast. Previous studies have investigated the membrane and protein composition of wild-type and ethanol tolerant strains, but relatively little is known about the genome changes associated with the ethanol tolerant C. thermocellum strain. In this study, C. thermocellum cultures were grown to mid-exponential phase and then either shocked with the supplementation of ethanol to a final concentration of 3.95 g/L (equal to 0.5% [v/v]) or were untreated. Samples were taken pre-shock and 2, 12, 30, 60, 120, 240 min post-shock for multiple systems biology analyses. The addition of ethanol dramatically reduced the C. thermocellum growth and the final cell density was approximately half of the control fermentations, with concomitant reductions in substrate consumption in the treated cultures. The response of C. thermocellum to ethanol was dynamic and involved more than six hundred genes that were significantly and differentially expressed between the different conditions over time and every functional category was represented. Cellobiose was accumulated within the ethanol-shocked C. thermocellum cells, as well as the sugar phosphates such as fructose-6-P and cellobiose-6-P. The comparison and correlation among intracellular metabolites, proteomic and transcriptomics profiles as well as the ethanol effects on cellulosome, hydrogenase glycolysis and nitrogen metabolism are discussed, which led us to propose that C. thermocellum may utilize the nitrogen metabolism to bypass the arrested carbon metabolism in responding to ethanol stress shock, and the nitrogen metabolic pathway and redox balance may be the key target for improving ethanol tolerance and production in C. thermocellum. A thirty array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different time points of 0, 12, 30, 60, 120, and 240 min post-inoculation with 3.95 g/L [0.5% (v/v)] treatment compred to that of control without ethanol supplementation. Two biological replicates for treatment and control condition.
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Project description:The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst for the conversion of lignocellulosic biomass into ethanol. The microorganism expresses enzymes for both cellulose solubilization and fermentation to produce lignocellulosic ethanol making it a good candidate for industrial biofuel production. Intolerance to stresses routinely encountered during industrial fermentations may hinder the commercial development of this organism. A recently published study by Yang et al., (2012) characterized the physiological and regulatory response of C. thermocellum to ethanol supplementation. Significant changes in nitrogen metabolism and an accumulation of carbon sources were identified, revealing potential targets for metabolic engineering. In the current study, the response of C. thermocellum to heat and furfural shock were compared with the known effects of ethanol shock. Improved tolerance to these stresses are desirable traits for C. thermocellum and further understanding of the effects that these particular stresses have on the organism are the focus of this work. A forty one array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different time points of 10, 30, 60, and 120 min post-treatment with 3.95 g.L-1 ethanol, 4 g.L-1 furfural or 68°C treatment compred to that of control without treatment. At least two biological replicates were performed for each treatment and control condition.
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2009-07-02 | GSE16892 | GEO
Project description:Clostridium thermocellum is a leading candidate organism for implementing a consolidated bioprocessing (CBP) strategy for biofuel production due to its native ability to rapidly consume cellulose and its existing ethanol production pathway. C. thermocellum converts cellulose and cellobiose to lactate, formate, acetate, H2, ethanol, amino acids, and other products. Elimination of the pathways leading to products such as H2 could redirect carbon flux towards ethanol production. Rather than delete each hydrogenase individually, we targeted a hydrogenase maturase gene (hydG), which is involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes by assembling the active site. This functionally inactivated all three Fe-Fe hydrogenases simultaneously, as they were unable to make active enzymes. In the ∆hydG mutant, the [NiFe] hydrogenase-encoding ech was also deleted to obtain a mutant that functionally lacks all hydrogenase. The ethanol yield increased nearly 2-fold in ∆hydG∆ech compared to wild type, and H2 production was below the detection limit. Interestingly, ∆hydG and ∆hydG∆ech exhibited improved growth in the presence of acetate in the medium. Transcriptomic and proteomic analysis reveal that genes related to sulfate transport and metabolism were up-regulated in the presence of added acetate in ∆hydG, resulting in altered sulfur metabolism. Further genomic analysis of this strain revealed a mutation in the bi-functional alcohol/aldehyde dehydrogenase adhE gene, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities, whereas the wild type strain can only utilize NADH. This is the exact same adhE mutation found in ethanol-tolerant C. thermocellum strain E50C, but ∆hydG∆ech is not more ethanol tolerant than the wild type. Targeting protein post-translational modification is a promising new approach to target multiple enzymes simultaneously for metabolic engineering.
2014-01-15 | GSE54082 | GEO
Project description:Clostridium thermocellum is a leading candidate organism for implementing a consolidated bioprocessing (CBP) strategy for biofuel production due to its native ability to rapidly consume cellulose and its existing ethanol production pathway. C. thermocellum converts cellulose and cellobiose to lactate, formate, acetate, H2, ethanol, amino acids, and other products. Elimination of the pathways leading to products such as H2 could redirect carbon flux towards ethanol production. Rather than delete each hydrogenase individually, we targeted a hydrogenase maturase gene (hydG), which is involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes by assembling the active site. This functionally inactivated all three Fe-Fe hydrogenases simultaneously, as they were unable to make active enzymes. In the ∆hydG mutant, the [NiFe] hydrogenase-encoding ech was also deleted to obtain a mutant that functionally lacks all hydrogenase. The ethanol yield increased nearly 2-fold in ∆hydG∆ech compared to wild type, and H2 production was below the detection limit. Interestingly, ∆hydG and ∆hydG∆ech exhibited improved growth in the presence of acetate in the medium. Transcriptomic and proteomic analysis reveal that genes related to sulfate transport and metabolism were up-regulated in the presence of added acetate in ∆hydG, resulting in altered sulfur metabolism. Further genomic analysis of this strain revealed a mutation in the bi-functional alcohol/aldehyde dehydrogenase adhE gene, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities, whereas the wild type strain can only utilize NADH. This is the exact same adhE mutation found in ethanol-tolerant C. thermocellum strain E50C, but ∆hydG∆ech is not more ethanol tolerant than the wild type. Targeting protein post-translational modification is a promising new approach to target multiple enzymes simultaneously for metabolic engineering. A seventeen array study using total RNA recovered from fermentation cultures of three strains (parental, ∆hydG and ∆hydG∆ech) of Clostridium thermocellum DSM1313. Cells were harvested at an OD 0.4-0.5 from cultures grown in the presence of additional 5mM acetate and compared to untreated controls. At least two biological replicates were performed for each treatment and control condition.
2014-01-15 | E-GEOD-54082 | biostudies-arrayexpress