Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon
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ABSTRACT:
ORGANISM(S): Thermococcus onnurineus NA1
PROVIDER: GSE64036 | GEO | 2015/04/28
SECONDARY ACCESSION(S): PRJNA269922
REPOSITORIES: GEO
Similar Datasets
Project description:A strong promoter increases transcription of the genes of interest and enhances the production of various valuable substances. For a hyperthermophilic archaeon Thermococcus onnurineus NA1, which can produce H2 by carbon monoxide oxidation, we searched for a novel endogenous strong promoter by transcriptome analysis using high-throughput RNA sequencing. Based on the relative transcript abundance, we selected one promoter to encode a hypothetical gene, of which homologs were found only in several Thermococcales strains. This promoter, PTN0510, was introduced into the front of CO-responsible hydrogenase gene cluster encoding a carbon monoxide dehydrogenase (CODH), a hydrogenase and a Na+/H+ antiporter. In the resulting mutant strain, KS0510, transcription and translation level of the gene cluster increased by 4- to 14-folds and 1.5- to 1.9-folds, respectively, in comparison with those of wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of wild-type strain. The PTN0510 was identified to be much stronger than the well-known two strong promoters, gdh and slp promoters from Thermococcus strains, through RT-qPCR and western blotting analyses and kinetics of H2 production. In this study, we demonstrated that the RNA-seq approach is a good strategy to mine a novel strong promoter of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce valuable metabolites through a metabolic engineering. RNA expreesion profile of T. onnurineus NA1 genes in CO-containing media, in duplicate, using Illumina HiSeq 2500
2015-04-28 | E-GEOD-64036 | biostudies-arrayexpress
Project description:A hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on a variety of CO-containing feed stocks such as by-product gas generated from steel-mill process. In this study we applied a long-term adaptive evolution to enhance H2 productivity. Through serial transfer of cell cultures with carbon monoxide (CO) as an energy source, we observed physiological changes in cell density, CO consumption rate and H2 production rate. To understand the underlying mechanism for the changes, we performed systems analysis of genomic, transcriptomic and epigenomic data. Genomic analysis of an evolved strain, designated as 156T, revealed that single or multiple bases were substituted, deleted or inserted in the sequence of the parental strain. A single point mutation in a putative transcriptional regulator (TON_1525) seemed to play a pivotal role in changing cellular phenotypes by increasing the expression level of genes of a CO dehydrogenase-hydrogenase gene cluster. Additionally, a mutation in an aromatic amino acid permease (TON_0820) contributed to increasing cell growth. Transcriptomic analysis revealed that genes belonging to the categories of transcription, translation and energy metabolism in archaeal Clusters of Orthologous Genes (arCOGs) were significantly changed. In particular, genes involved in energy conservation via CO oxidation were highly upregulated. Epigenomic analysis suggested that methylation change might be a way of gene regulation in a hyperthermophilic archaeon. The evolved 156T strain showed highly enhanced hydrogen productivity with CO at high flow rates of 800 ml min-1 and above, indicating the adaptation rendered the strain less sensitive to high CO. The 156T strain was demonstrated to be appropriate for H2 production by using synthetic gas obtained by coal gasification. This study is the first example to show that evolutionary engineering is very effective in enhancing H2 productivity of a hyperthermophilic archaeon on CO
2017-02-09 | GSE73031 | 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 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
Project description:Diverse aerobic bacteria use atmospheric hydrogen (H2) and carbon monoxide (CO) as energy sources to support growth and survival. Though recently discovered, trace gas oxidation is now recognised as a globally significant process that serves as the main sink in the biogeochemical H2 cycle and sustains microbial biodiversity in oligotrophic ecosystems. While trace gas oxidation has been reported in nine phyla of bacteria, it was not known whether archaea also use atmospheric H2. Here we show that a thermoacidophilic archaeon, Acidianus brierleyi (Thermoproteota), constitutively consumes H2 and CO to sub-atmospheric levels. Oxidation occurred during both growth and survival across a wide range of temperatures (10 to 70°C). Genomic analysis demonstrated that A. brierleyi encodes a canonical carbon monoxide dehydrogenase and, unexpectedly, four distinct [NiFe]-hydrogenases from subgroups not known to mediate aerobic H2 uptake. Quantitative proteomic analyses showed that A. brierleyi differentially produced these enzymes in response to electron donor and acceptor availability. A previously unidentified group 1 [NiFe]-hydrogenase, with a unique genetic arrangement, is constitutively expressed and upregulated during stationary phase and aerobic hydrogenotrophic growth. Another archaeon, Metallosphaera sedula, was also found to oxidize atmospheric H2. These results suggest that trace gas oxidation is a common trait of aerobic archaea, which likely plays a role in their survival and niche expansion, including during dispersal through temperate environments. These findings also demonstrate that atmospheric H2 consumption is a cross-domain phenomenon, suggesting an ancient origin of this trait, and identify previously unknown microbial and enzymatic sinks of atmospheric H2 and CO.
2024-02-26 | PXD040286 | Pride
Project description:We used Chlamydomonas microarray v2.0 to compare the time course expression profiles of two Chlamydomonas reinhardtii strains: wild-type WT and the high hydrogen producing mutant Stm6Glc4 during sulfur starvation induced hydrogen production. Major cellular reorganizations in photosynthetic apparatus, sulfur and carbon metabolism upon H2 production were confirmed as common to both strains. More importantly, our results pointed out factors which lead to the higher hydrogen production in the mutant including higher light sensitivity and lower competitions with hydrogenase by alternative electron sinks. Under S-starvation induced H2 producing conditions the induction of LHCSR3, a chlorophyll binding protein involving in non photochemical quenching, was significantly lower in Stm6Glc4 resulting in significant higher photodamage to photosystem II. Consequently, Stm6Glc4 had a shorter aerobic phase, consumed less starch reserves, and produced H2 earlier at higher rates than WT. We also showed that the loss of mitochondrial DNA-binding protein MOC1 in both knockdown and knockout mutant resulted in higher light sensitivity and improved H2 yield. Furthermore, by comparing our data with previously published ‘omics’ data, we were able to identify genes that responded specifically to either sulfur starvation, anaerobiosis or hydrogen production as well as to provide a more complete picture of S-deprived H2 production in the green alga C. reinhardtii. A total of 33 microarray hydridizations were performed covering samples taken during the course of S deprivation induced H2 producction. The samples included 4 time points in the high hydrogen producing mutant Stm6Glc4 (taken at 16, 28, 52 and 76h) and 6 time points in the wildtype CC-406 (taken at 16, 28, 52, 68, 92, 116h). Samples from each time point were compared directly with the sample taken prior to S starvation from the corresponding strain. Three biological replicates were tested at each time point.
2011-11-30 | E-GEOD-30252 | biostudies-arrayexpress
Project description:We used Chlamydomonas microarray v2.0 to compare the time course expression profiles of two Chlamydomonas reinhardtii strains: wild-type WT and the high hydrogen producing mutant Stm6Glc4 during sulfur starvation induced hydrogen production. Major cellular reorganizations in photosynthetic apparatus, sulfur and carbon metabolism upon H2 production were confirmed as common to both strains. More importantly, our results pointed out factors which lead to the higher hydrogen production in the mutant including higher light sensitivity and lower competitions with hydrogenase by alternative electron sinks. Under S-starvation induced H2 producing conditions the induction of LHCSR3, a chlorophyll binding protein involving in non photochemical quenching, was significantly lower in Stm6Glc4 resulting in significant higher photodamage to photosystem II. Consequently, Stm6Glc4 had a shorter aerobic phase, consumed less starch reserves, and produced H2 earlier at higher rates than WT. We also showed that the loss of mitochondrial DNA-binding protein MOC1 in both knockdown and knockout mutant resulted in higher light sensitivity and improved H2 yield. Furthermore, by comparing our data with previously published ‘omics’ data, we were able to identify genes that responded specifically to either sulfur starvation, anaerobiosis or hydrogen production as well as to provide a more complete picture of S-deprived H2 production in the green alga C. reinhardtii.
2011-11-30 | GSE30252 | GEO
Project description:Investigating the role of carbon monoxide and a CO sensor protein CooA in the physiology of Desulfovibrio vulgaris Hildenborough using whole genome expression analysis Comparison of whole genome expression changes in the wild type and a strain deleted for CooA (DVU2097) in the presence and absence of carbon monoxide
2012-07-30 | E-GEOD-36770 | biostudies-arrayexpress
Project description:Acetate is a simple carboxylic acid that is synthesized in various microorganisms. Although acetate toxicity and tolerance have been studied in many microorganisms, little is known about the effects of exogenous acetate on the cell growth of acetogenic bacteria. In this study, we report the phenotypic changes that occurred in the acetogenic bacterium Clostridium sp. AWRP as a result of an adaptive laboratory evolution under acetate challenge. When compared with the wild-type strain, the acetate-adapted strain displayed a tolerance to acetate up to 10 g L-1 and higher biomass yields in batch cultures, although the metabolite profiles greatly varied depending on culture conditions. Interestingly, genome sequencing revealed that the adapted strain harbored three point mutations in the genes encoding an electron-bifurcating hydrogenase, which is crucial to its autotrophic growth on CO2 + H2, in addition to one in the dnaK gene. Transcriptome analysis revealed the global change in the gene expression profile of the acetate-adapted strain. Strikingly, most genes involved in CO2-fixing Wood-Ljungdahl pathway and auxiliary pathways for energy conservation (e.g., Rnf complex, Nfn, etc.) were significantly down-regulated. In addition, we observed that a couple of metabolic pathways associated with dissimilation of nucleosides and carbohydrates were significantly up-regulated in the acetate-adapted strain as well as several amino acid biosynthetic pathways, indicating that the strain might increase its fitness by utilizing organic substrates in response to the down-regulation of carbon fixation. Further investigation into the carbon fixation degeneration of the acetate-adapted strain will provide practical implications in CO2 + H2 fermentation using acetogenic bacteria for long-term continuous fermentation. The transcriptome profiles of the wild-type Clostridium sp. AWRP and its acetate-tolerant derivative 46T-a were compared.
2022-06-26 | GSE206757 | GEO