Hyperthermophilic Archaeon Thermococcus Onnurineus NA1 Research Articles

An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Development of natural seawater-based continuous biohydrogen production process using the hyperthermophilic archaeon Thermococcus onnurineus NA1

Previously, we reported that the hyperthermophilic archaeon, Thermococcus onnurineus NA1 could produce hydrogen from various resources including carbon monoxide (CO) with high productivity. In this study, a high-performance and cost-effective fermentation process was developed as a prerequisite for the industrial implementation of H2 production from CO using the strain. Firstly, an economical medium was formulated on the basis of natural seawater. Cysteine as a reducing agent was found to be an important factor to consider. Secondly, three variables, initial concentration of yeast extract, CO supply rate and agitation rate, were statistically optimized in batch culture by applying a response surface methodology. Notably, biomass productivity of 0.39 g/L/h could be observed and cell density reached 14 optical units at 600 nm, which is exceptionally high among members of the order Thermococcales. Finally, continuous cultures were tested at various dilution rates with new medium formulation and optimized culture conditions. As a result, the steady state of H2 production and cell growth were stably maintained, yielding H2 production rate of 471.5 mmol/L/h at a dilution rate of 0.1 h−1, which is one of the highest values at ambient CO pressure to date. Our study shows that T. onnurineus NA1 has outstanding potential as an economical and high performance industrial strain suitable for long-term processes for CO-dependent H2 production.

A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1.

The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H2 production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP+ as electron acceptors, respectively. While most FDHs exhibited NAD+-dependent formate oxidation activity, the Fdh3 of T. onnurineus NA1 showed a strong preference for NADP+ over NAD+ as a cofactor. The catalytic efficiency (kcat/Km) of Fdh3 for NADP+ was measured to be 5,281 mM−1 s−1, which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg204 and Arg205 of Fdh3B may contribute to the stabilization of the 2′-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO2 reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin.

Enhanced H2 production by deletion of the Tfx family DNA-binding protein in the hyperthermophilic archaeon Thermococcus onnurineus NA1

A gene encoding an archaeal transcription regulator (TON_1525) homologous to the Tfx family DNA-binding protein has been identified to affect gene expression of carbon monoxide metabolism in the hyperthermophilic archaeon Thermococcus onnurineus NA1. To broadly understand gene regulation by the TON_1525 gene, a gene deletion mutant (Δ1525) was constructed and transcriptome changes were investigated. A total of 351 genes were found to be differentially expressed in the Δ1525 mutant. The expression levels of the hydrogenase genes were significantly changed; mbh (membrane-bound hydrogenase) and mch (membrane-bound carbon monoxide-dependent hydrogenase) genes were increased, and mfh2 (membrane-bound formate-dependent hydrogenase) and soluble hydrogenase genes were decreased. The Δ1525 mutant was cultured in a medium supplemented with maltodextrin or carbon monoxide to investigate whether changes in gene expression lead to physiological changes. The maximum H2 production rates of the Δ1525 mutant were significantly enhanced on both substrates compared to the wild-type strain. These results indicate that the TON_1525 gene can affect the expression of various genes including mbh and mch, consequently regulating the metabolism of T. onnurineus NA1. This study expands our understanding of the functional role of the Tfx family DNA-binding proteins.

In vitro assembly of thermostable Csm complex in CRISPR-Cas type III/A system.

The CRISPR-Cas system is the prokaryotic immune response that destroys invading foreign nucleic acids. Based on the architecture and distinct mechanism of targeting, the CRISPR-Cas system is classified into six types (I-VI). The Csm complex belongs to the type III system and consists of five subunits (Cas10 and Csm2-5) and a crRNA. The Csm complex targets RNA and RNA-dependent single-strand DNA. Here, we present a protocol for in vitro reconstitution of a Csm complex from a hyperthermophilic archaeon Thermococcus onnurineus NA1 (ToCsm complex). The method consists of coexpression and copurification of the subunits, in vitro synthesis of the crRNA and assembly of the ToCsm complex. Purification with heat treatment and affinity and size exclusion chromatography resulted in homogeneous Cas10/Csm4 and Csm2/Csm5 binary complexes, while in vitro transcription with the T7 promoter enabled synthesis of the crRNA. Addition of each component in the presence of the crRNA with a molar ratio of Cas10/Csm4:Csm3:Csm2/Csm5:crRNA=1:3:2:1 yielded an assembled functional Csm complex. This protocol for reconstitution of the Csm complex is presumably applicable to other thermostable effector complexes, which would allow biochemical, structural, or functional studies of the CRISPR-Cas type III/A system in vitro.

Adaptive evolution of a hyperthermophilic archaeon pinpoints a formate transporter as a critical factor for the growth enhancement on formate

Previously, we reported that the hyperthermophilic archaeon Thermococcus onnurineus NA1 could grow on formate and produce H2. Formate conversion to hydrogen was mediated by a formate-hydrogen lyase complex and was indeed a part of chemiosmotic coupling to ATP generation. In this study, we employed an adaptation approach to enhance the cell growth on formate and investigated molecular changes. As serial transfer continued on formate-containing medium at the serum vial, cell growth, H2 production and formate consumption increased remarkably. The 156 times transferred-strain, WTF-156T, was demonstrated to enhance H2 production using formate in a bioreactor. The whole-genome sequencing of the WTF-156T strain revealed eleven mutations. While no mutation was found among the genes encoding formate hydrogen lyase, a point mutation (G154A) was identified in a formate transporter (TON_1573). The TON_1573 (A52T) mutation, when introduced into the parent strain, conferred increase in formate consumption and H2 production. Another adaptive passage, carried out by culturing repeatedly in a bioreactor, resulted in a strain, which has a mutation in TON_1573 (C155A) causing amino acid change, A52E. These results implicate that substitution of A52 residue of a formate transporter might be a critical factor to ensure the increase in formate uptake and cell growth.

Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased. The underlying mechanism for those physiological changes could be explained by using multi-omics approaches including genomic, transcriptomic and epigenomic analyses. A putative transcriptional regulator was newly identified to regulate the expression levels of genes related to CO oxidation. Transcriptome analysis revealed significant changes in the transcript levels of genes belonging to the categories of transcription, translation and energy metabolism. Our study presents the first genome-scale methylation pattern of hyperthermophilic archaea. Adaptive evolution led to highly enhanced H2 productivity at high CO flow rates using synthesis gas produced from coal gasification.

Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle.

A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.

Proteomic Insights into Sulfur Metabolism in the Hydrogen-Producing Hyperthermophilic Archaeon Thermococcus onnurineus NA1.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H2 when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H2-evolving substrate culture conditions. Using label-free nano-UPLC-MSE-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins) was found to be significantly up-regulated (≥1.5-fold) under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe–S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H2-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.

Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon.

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 from 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, P TN0510 , 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 the wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of the wild-type strain. The P TN0510 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 novel strong promoters of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce valuable products such as enzymes and metabolites through metabolic engineering.

Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon.

Thermococcus onnurineus NA1 is known to grow by the anaerobic oxidation of formate to CO2 and H2, a reaction that operates near thermodynamic equilibrium. Here we demonstrate that this reaction is coupled to ATP synthesis by a transmembrane ion current. Formate oxidation leads to H(+) translocation across the cytoplasmic membrane that then drives Na(+) translocation. The ion-translocating electron transfer system is rather simple, consisting of only a formate dehydrogenase module, a membrane-bound hydrogenase module, and a multisubunit Na(+)/H(+) antiporter module. The electrochemical Na(+) gradient established then drives ATP synthesis. These data give a mechanistic explanation for chemiosmotic energy conservation coupled to formate oxidation to CO2 and H2. Because it is discussed that the membrane-bound hydrogenase with the Na(+)/H(+) antiporter module are ancestors of complex I of mitochondrial and bacterial electron transport these data also shed light on the evolution of ion transport in complex I-like electron transport chains.

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.

Comparison of CO-dependent H2 production with strong promoters in Thermococcus onnurineus NA1

To overproduce biotechnologically valuable products, the expression level of target genes has been modulated by using strong promoters. In a hyperthermophilic archaeon Thermococcus onnurineus NA1, two promoters, P(TN0413) and P(TN0157), which drive expression of the genes encoding the S-layer protein and glutamate dehydrogenase were inserted in front of a gene cluster encoding a carbon monoxide dehydrogenase, a hydrogenase and a Na⁺/H⁺ antiporter. Two promoters exhibited strong activity by increasing the transcription and translation levels of the gene cluster in the mutant strains by 2.5- to 49-folds and 1.4- to 3.3-folds, respectively, than the native promoter in the wild-type strain. While KS0413 with P(TN0413) promoter exhibited 2.7 to 4.7 times higher transcript level than KS0157 with P(TN0157) promoter, the levels of proteins were a little different between them. The biomass concentrations and H₂ production rates of two mutants were 2- to 3-fold higher than those of the wild-type strain in a bioreactor where CO was supplied at a flow rate of 120 ml min⁻¹. Two mutants showed differential response to the higher CO flow rate, 240 ml min⁻¹, in terms of growth pattern and product formation, indicating two promoters were regulated by culture conditions. The results demonstrate that not only promoter strength but also product-forming conditions should be considered in promoter engineering.

The structure of TON1937 from archaeon Thermococcus onnurineus NA1 reveals a eukaryotic HEAT-like architecture

The members of the ARM/HEAT repeat-containing protein superfamily in eukaryotes have been known to mediate protein–protein interactions by using their concave surface. However, little is known about the ARM/HEAT repeat proteins in prokaryotes. Here we report the crystal structure of TON1937, a hypothetical protein from the hyperthermophilic archaeon Thermococcus onnurineus NA1. The structure reveals a crescent-shaped molecule composed of a double layer of α-helices with seven anti-parallel α-helical repeats. A structure-based sequence alignment of the α-helical repeats identified a conserved pattern of hydrophobic or aliphatic residues reminiscent of the consensus sequence of eukaryotic HEAT repeats. The individual repeats of TON1937 also share high structural similarity with the canonical eukaryotic HEAT repeats. In addition, the concave surface of TON1937 is proposed to be its potential binding interface based on this structural comparison and its surface properties. These observations lead us to speculate that the archaeal HEAT-like repeats of TON1937 have evolved to engage in protein–protein interactions in the same manner as eukaryotic HEAT repeats.

Hydrogen production from C1 compounds by a novel marine hyperthermophilic archaeon Thermococcus onnurineus NA1

A novel marine hyperthermophile, Thermococcus onnurineus NA1, was found to grow on C1 carbon compounds, such as formate and carbon monoxide (CO), and produce hydrogen (H2). In the present study, the growth and H2 production of NA1 were examined to determine its potential as H2 producer. NA1 showed relatively high specific growth rates, 0.48 h−1 and 0.40 h−1 with CO (20%, v/v) and formate (100 mM), respectively, when cultivated in batch mode in a minimal salt medium fortified with 1.0 g L−1 yeast extract. On the other hand, cell growth in both cases stopped at approximately 6 h and the final cell densities were extremely low at 18.2 and 12.1 mg protein L−1 with CO and formate, respectively. The maximum final cell density could be improved greatly to 36.0 mg protein L−1 by optimizing CO content (50%, v/v) and yeast extract concentration (4.0 g L−1), but it was still very low. During the cell growth, formate and CO were used as energy source rather than carbon source. In the resting cell experiments, NA1 exhibited remarkably high H2 production activities as 385.0 and 207.5 μmol mg protein−1 h−1 for CO and formate, respectively. When formate (100 mM) or CO (100%, v/v) was added repeatedly at 30–35 h intervals, NA1 showed consistent H2 production for 3 cycles with a yield of approximately 1.0 mol H2 mol−1 for both CO and formate. This study suggests that T. onnurineus NA1 has a high H2 production potential from formate or CO but a method for achieving a high cell density culture is needed.

Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification

In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.

Crystallization and Preliminary X-Ray Studies of TON_0559, a Putative Member of the Haloacid Dehalogenase (HAD) Superfamily from Thermococcus onnurineus NA1

To elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism of the enzymes belonging to the haloacid dehalogenase (HAD) superfamily, TON_0559, a putative HAD subfamily protein from a hyperthermophilic archaeon Thermococcus onnurineus NA1, was expressed, purified and crystallized. X-ray diffraction data were collected to 2.0 A resolution. The space group is C2, with unit cell parameters a = 121.2 A, b = 62.9 A, c = 37.5 A and beta= 106.5 degrees .

Characterization of a dUTPase from the Hyperthermophilic Archaeon Thermococcus onnurineus NA1 and Its Application in Polymerase Chain Reaction Amplification

Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 (TNA1) revealed the presence of a 471-bp open reading frame with 93% similarity to the dUTPase from Pyrococcus furiosus. The dUTPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dUTP at about a 10-fold higher rate than dCTP. The protein behaved as a dimer in gel filtration chromatography, even though it contains five motifs that are conserved in all homotrimeric dUTPases. The dUTPase showed optimum activity at 80 degrees C and pH 8.0, and it was highly thermostable with a half-life (t (1/2)) of 170 min at 95 degrees C. The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl(2), KCl, (NH(4))(2)SO(4), and Triton X-100 concentrations, although it was reduced by bovine serum albumin. Addition of the dUTPase to polymerase chain reactions (PCRs) run with TNA1 DNA polymerase significantly increased product yield, overcoming the inhibitory effect of dUTP. Further, addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase. This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases, including Pfu and KOD.