Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The molecular response of Clostridium thermocellum ATCC 27405 to ethanol shock

ABSTRACT: 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.

ORGANISM(S): Clostridium thermocellum ATCC 27405

SUBMITTER: Shihui YANG

PROVIDER: E-GEOD-25236 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress.

Yang Shihui S Giannone Richard J RJ Dice Lezlee L Yang Zamin K ZK Engle Nancy L NL Tschaplinski Timothy J TJ Hettich Robert L RL Brown Steven D SD

BMC genomics 20120723

<h4>Background</h4>Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis.<h4 ...[more]

PMID: 22823947

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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.

Project description:Background: Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium with a great potential to be a consolidated bioprocessing biocatalyst that can ferment cellulose to ethanol. To understand its physiology and genetic targets for future strain development, we have carried out several studies including ethanol-tolerant mutant resequencing and ethanol shock experiments. Methods: In this study, several approaches were applied to enrich mRNA for next-generation sequencing based RNA-Seq and the transcriptomic profiling of C. thermocellum ethanol shock responses was investigated using high-density tiling array and RNA-Seq. Correlations among different transcriptomic platforms of expression array, tiling array, and RNA-Seq were then compared, and data generated from systems biology studies were used for transcriptional architecture improvement. Results: Our results indicate that cloning-based Sanger sequencing can be used for mRNA enrichment ratio determination, and low-copy number plasmid performed better than high-copy one for cDNA library construction. In addition, high correlations were observed among same array platform using different analyses as well as those between two different array platforms of tiling and expression array when probe intensity was compared. Correlations between RNA-Seq and array results especially the one between RNA-Seq and tiling array are also high. Moreover, combining both RNA-Seq and microarray data the transcriptional architecture of C. thermocellum including the prediction and verification of novel transcript (gene), transcriptional start site (TSS), CAZyme genes, ncRNA, and operon were systematically updated and improved. Conclusions: RNA-seq has high correlation with array-based transcriptomic platforms and can provide additional information for transcriptional architecture and genome annotation improvement, which will facilitate future omics-based studies. A twelve array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different time points of 30, 60, and 120 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.

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.

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.

Project description:Roberts2010 - Genome-scale metabolic network of Clostridium thermocellum (iSR432) This model is described in the article: Genome-scale metabolic analysis of Clostridium thermocellum for bioethanol production. Roberts SB, Gowen CM, Brooks JP, Fong SS. BMC Syst Biol 2010; 4: 31 Abstract: BACKGROUND: Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. Clostridium thermocellum (ATCC 27405) is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous. RESULTS: Here we present a genome-scale model of C. thermocellum metabolism, iSR432, for the purpose of establishing a computational tool to study the metabolic network of C. thermocellum and facilitate efforts to engineer C. thermocellum for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the iSR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production. CONCLUSIONS: By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of C. thermocellum and establish a strong foundation for rational strain design. In addition, we are able to draw some important conclusions regarding the underlying metabolic mechanisms for observed behaviors of C. thermocellum and highlight remaining gaps in the existing genome annotations. This model is hosted on BioModels Database and identified by: MODEL1507180004. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Ruminiclostridium thermocellum DSM 1313 strain adhE*(EA) expression was studied along with ∆hydG and ∆hydG∆ech mutants strains deposited under GSE54082. All strains have been described in a study entitled Elimination of hydrogenase post-translational modification blocks H2 production and increases ethanol yield in Clostridium thermocellum. Biswas, et .al. Biotechnology for Biofuels 2015 8:20 Ruminiclostridium (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. This GEO study pertains to expression profiles generated for C. thermocellum DSM 1313 strain adhE*(EA)

Dataset Information

The molecular response of Clostridium thermocellum ATCC 27405 to ethanol shock

Publications

Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress.

Similar Datasets

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