Characterization of lactic acid and acetic acid mutants in batch fermentation
Ontology highlight
ABSTRACT:
ORGANISM(S): Acetivibrio thermocellus DSM 1313 Acetivibrio thermocellus ATCC 27405
PROVIDER: GSE27046 | GEO | 2012/03/01
SECONDARY ACCESSION(S): PRJNA137641
REPOSITORIES: GEO
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Project description:Four C. thermocellum DSM-1313 derived strains were assessed using metabolite and DNA microarray tools in order to better understand carbon and electron flow within this organism. C. thermocellum is able to ferment cellulose into its fermentation end products L-lactate, acetate, formate, hydrogen gas, and ethanol, with the latter being the desired end product to be used as biorenewable fuel. In addition to the parent strain (genotype: hpt spo0A), strains with either or both of the genes encoding lactate dehydrogenase (ldh) and phosphate acetyltransferase (pta) deleted were studied. The strains used are a parent strain (M1726: genotype: hpt spo0A), and strains with either the gene encoding lactate dehydrogenase (M1629: hpt spo0A ldh) or phosphate acetyltransferase (M1630: hpt spo0A pta) deleted, or with both genes deleted (M1725: hpt spo0A ldh pta). Controlled batch fermentations using cellobiose as sole carbon source were grown for each strain, and samples in mid-exponential phase and at the time of carbon depletion were examined by DNA microarray. Four strains were grown each as three independent biological replicates (fresh batch of media was made before each run). Per fermentation, two samples were taken for DNA microarray analysis as was determined by the optical density: mid-exponential was defined as O.D. 0.4 (measured by Dasgip probe); point of carbon depletion was defined by both the maximum O.D. reached and observation that no base was added to the fermentation to control pH. In total, 4 strains x 3 fermentation x 2 time points per fermentor = 24 arrays. Parent strain was used as reference strain.
2012-03-01 | E-GEOD-27046 | biostudies-arrayexpress
Project description:Differential RNA-Seq analyses to investigate the basis for metabolic inhibition of Clostridium thermocellum M1570 by xylose. The M1570 strain was developed in the C. thermocellum DSM 1313 Δhpt background strain. Lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta) genes are deleted (Argyros DA, Tripathi SA, Barrett TF, Rogers SR, Feinberg LF, Olson DG, Foden JM, Miller BB, Lynd LR, Hogsett DA, Caiazza NC: High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microbiol 2010, 77:8288-8294 )
2016-10-31 | GSE74918 | GEO
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)
2017-02-01 | GSE77273 | GEO
Project description:Clostridium thermocellum is a candidate for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Understanding how this organism regulates gene expression is of importance for developing a better fundamental understanding of this industrial relevant bacterium. We are primarily interested in gene regulation by three predicted LacI regulators. A previous report of one LacI regulator in C. thermocellum ATCC27405 (Cthe_2808) indicated this was a transcriptional repressor of neighboring genes with repressor activity relieved in the presence of laminaribiose (a β-1,3 disaccharide). The current work is aimed at understanding if a homolog of this LacI transcriptional regulator in C. thermocellum DSM1313 and two others putatively characterized as LacI regulators are in fact global regulatory proteins with extensive regulons or, if like the majority of LacI transcription factors, the regulation is limited to local genomic regions. Each of the three LacI regulators was deleted in C. thermocellum DSM1313. Genome re-sequencing was used to confirm the deletion and ensure nonspecific recombination events were avoided during the gene deletion strategy. Growth of each strain on cellobiose was unaffected by any of the gene deletions under pH controlled fermentations. Global gene expression patterns taken at mid-log phase for each strain identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly up-regulated (up to 9 fold) in the absence of each LacI regulator. Thus suggesting these were repressed under wild type conditions. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters with putative motifs ranging from 16-18 bp. Work is ongoing to confirm the specific binding motif recognized by the two previously un-characterized transcription factors and assess the occurrence of this motif in the C. thermocellum DSM1313 genome. The identification of LacI repressor activity on hemicellulose gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum. Four strains of C. thermocellum DSM1313 including a parental strain (Îhpt) and three others with deletion in lacI transcription factors were characterised by RNA-seq. The strains were grown in 1L bioreactors, cells (from 50 ml samples) were harvested at mid exponential, late exponential and 30 min after acid production halted during the transition to stationary phase. The growth studies were performed in triplicate and thirty six samples were analyzed by RNA-sequencing.
2015-10-31 | E-GEOD-68423 | biostudies-arrayexpress
Project description:Investigation of sulfur metabolism in Clostridium thermocellum DSM 1313 ∆hpt, to determine growth and gene expression when the organism is incubated with either the oxidized (i.e., sulfate) or the reduced and assimilated (i.e., cysteine) forms of sulfur. A sulfite reductase (∆hpt ∆SO3R) knockout mutant to limit sulfur assimilation was created to compare the resulting gene expression patterns by RNAseq transciptomics against the parental strain (∆hpt) when both are grown in the presence of sulfate. Additionally, we bypass the sulfate auxotrophy of the mutant by providing assimilated sulfur in the form of cysteine to determine whether growth is restored to normal and whether methionine can be biosynthesized by yet uncharacterized pathways in this organism.
2020-11-01 | GSE100047 | 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
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 to produce bioethanol directly from cellulosic biomass. However, few transcriptomic studies have been reported so far for C. thermocellum using biomass as carbon source. In this study, samples were taken from exponential and stationary phases of C. thermocellum cells growing in MTC media with pretreated switchgrass as carbon source, and transcriptomic profiling change of C. thermocellum during different growth phase was investigated using both expression array and tiling array. This study will help the understanding of gene expression of C. thermocellum using cellulosic biomass as carbon source and the knowledge will facilitate future metabolic engineering effort for strain improvement.
2014-05-08 | GSE26926 | GEO
Project description:In order to study the effect of lactate dehydrogenase (?ldh 1.2) mutant on the transcriptome DNA microarray technology was used. Stains were grown in a chemically defined medium (CDM-LAB) containing 1.1% of glucose as a carbon sources at 37?C on anaerobic condition to an optical density of 0.6.
2010-12-13 | E-MTAB-472 | biostudies-arrayexpress
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 Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production to produce bioethanol directly from cellulosic biomass. However, few transcriptomic studies have been reported so far for C. thermocellum using biomass as carbon source. In this study, samples were taken from exponential and stationary phases of C. thermocellum cells growing in MTC media with pretreated switchgrass as carbon source, and transcriptomic profiling change of C. thermocellum during different growth phase was investigated using both expression array and tiling array. This study will help the understanding of gene expression of C. thermocellum using cellulosic biomass as carbon source and the knowledge will facilitate future metabolic engineering effort for strain improvement. [HX12 expression array]: A eleven array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different growth phase of T2 and T3 with switchgrass as carbon source. Two biological replicates used for each phase. [3Plex tiling array]: A six array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different growth phase of T2 and T3 with switchgrass as carbon source. Two biological replicates used for each phase.
2014-05-08 | E-GEOD-26926 | biostudies-arrayexpress