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Purpose Glucose is one of the most important carbon and energy source for heterotrophic growth in all living organisms. However, glucose has been reported as a poor substrate to support the growth of hyperthermophilic archaea belonging to the order Thermococcales. To enhance glucose-assisted growth of Thermococcus onnurineus NA1, adaptive evolution process was applied. In an effort for industrial applications, glucose-adapted cells were further tested for H2 producing potential using food processing waste as a promising zero-value substrate containing polysaccharides composed of glucose. Methods Adaptive evolution of T. onnurineus NA1 was performed by transferring cells to fresh medium containing glucose until cell growth increased. Genome sequencing was conducted to identify genetic changes in adapted cells. H2 production in the parent strain and glucose-adapted cells was analyzed using either glucose or potato peel waste as substrate. Results The glucose-adapted cells, WG-100T, had 10.8-fold and 14.7-fold increases in cell density and glucose consumption, respectively, compared to the parent strain. Genome sequencing of WG-100T revealed a total of 17 genomic changes in genes, including those encoding transcription factors and several proteins involved in various transport systems. WG-100T produced H2 using potato peel waste through simultaneous saccharification and fermentation. Conclusion This study showed that the performance of the Thermococcales strain was improved by adaptive evolution, resulting in faster use of glucose. In addition, it was shown that the use of a hyperthermophile made it possible to produce biohydrogen without pretreatment of food processing waste for saccharification. Graphical Abstract

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.

The Korean rockfish, Sebastes schlegeli, is most commonly farmed in sea cages along the coast of Korea; however, detailed information on intestinal microbiota regarding this fish is not readily available. In this study, comparison of the seasonal changes of microbial communities in the intestine between farmed and wild through the amplicon sequencing approach was conducted. The composition of major species in the intestine of this fish was very simple compared to that of other marine fish species, with members affiliated with the family Vibrionaceae hyper-dominating and comprising on average 97.6% of microbiota. However, the composition at the genus or species level and the pattern of seasonal changes of diversity indices showed significant differences between farmed and wild fish. In the farmed fish, Photobacterium phophoreum was most dominant throughout the year, accounting for 58.8% of the total. Aliivibrio fisherii and/or Aliivibrio finisterrensis also were dominant in the fall to winter but substituted by Photobacterium damselae during spring to summer. In the wild fish, on the other hand, opportunistic pathogens in the genera Aliivibrio or Vibrio were dominant in most of the samples. The analysis of shared species between gut microbiome, feed microbiota, and seawater microbiota indicated that the intestinal microbial diversity of farmed fish was affected more by microbiota of seawater than that of feed in spring and winter seasons. Additionally, the proportion of potential pathogenic Vibrio spp. in the gut showed a negative correlation with plasma glucose levels of the host. This study and following studies will be helpful in understanding the interaction between microbiome hosts and the development of techniques to enhance production of healthy Korean rockfish.

In this study, we performed a kinetic analysis of CO-dependent H2 production by metabolically engineered Thermococcus onnurineus NA1, MC01 in terms of the cell activity as well as mass transfer rate. We conducted continuous cultures with varying dilution rate, CO flow rate, or agitation speed. Despite oscillations in cell density, the cultures reached steady states at all operating conditions. As the dilution rate increased from 0.1 to 0.3 h−1, specific activity of H2 production (SAHP) and volumetric cell production rate were linearly increased. Also, the SAHP remained almost constant at the fixed dilution rate of 0.3 h−1 even though the CO transfer rate was changed by adjusting the CO flow rate or the agitation speed. This relationship could be expressed as a typical Luedeking-Piret model, implying that high cell density culture with a sufficient growth rate is essential to obtain higher H2 productivity. On the other hand, more elevated CO transfer at the same dilution rate improved H2 production rate (HPR) by the increase of the cell density, not in the rise of SAHP. Through the continuous culture, 108 mmol/g-cell/h and 121 mmol/L/h of SAHP and HPR, respectively, could be achieved at a dilution rate of 0.3 h−1 with CO supply rate of 0.07 vvm and agitation speed of 700 rpm. Considering high H2 production activity and long-term stability of the strain over 1,000 h, MC01 is confirmed to have an outstanding potential for CO-dependent H2 production.

An anaerobic, rod-shaped, mesophilic, chemolithoautotrophic, sulfate-reducing bacterial strain IOR2T was isolated from a newly found deep-sea hydrothermal vent (OVF, Onnuri Vent Field) area in the central Indian Ocean ridge (11°24'88″ S 66°25'42″ E, 2021 m water depth). The 16S rRNA gene sequence analysis revealed that the strain IOR2T was most closely related to Desulfovibrio senegalensis BLaC1T (96.7%). However, it showed low similarity with the members of the family Desulfovibrionaceae, such as Desulfovibrio tunisiensis RB22T (94.0%), D. brasiliensis LVform1T (93.9%), D. halophilus DSM 5663T (93.7%), and Pseudodesulfovibrio aespoeensis Aspo-2T (93.2%). The strain IOR2T could grow at 23-42°C (optimum 37°C), pH 5.0-8.0 (optimum pH 7.0) and with 0.5-6.5% (optimum 3.0%) NaCl. The strain could use lactate, pyruvate, H2, and glycerol as electron donors and sulfate, thiosulfate, and sulfite as electron acceptors. The major fatty acids of the strain IOR2T were iso-C15:0, iso-C17:0, ante-iso-C15:0, and summed feature 9 (C16:0 methyl/iso-C17:1ω9c). Both the strains IOR2T and BLaC1T could grow with CO2 and H2 as the sole sources of carbon and energy, respectively. Genomic evidence for the Wood-Ljungdahl pathway in both the strains reflects chemolithoautotrophic growth. The DNA G + C content of the strain IOR2T and BLaC1T was 58.1-60.5 mol%. Based on the results of the phylogenetic and physiologic studies, Paradesulfovibrio onnuriensis gen. nov., sp. nov. with the type strain IOR2T (= KCTC 15845T = MCCC 1K04559T) was proposed to be a member of the family Desulfovibrionaceae. We have also proposed the reclassification of D. senegalensis as Paradesulfovibrio senegalensis comb. nov.

The genome of the hyperthermophilic archaeon

Hydrogen has been receiving considerable attention as a clean energy carrier. Thermococcus onnurineus NA1, a hyperthermophilic archaeon, is capable of producing hydrogen using carbon monoxide (CO) in a growth-associated manner, providing a promising platform to upcycle industrial waste gases. In order to improve cost-effectiveness of the biological hydrogen production from carbon monoxide (CO), we developed formulation of a low-cost medium for T. onnurineus 156T by substituting fishmeal and sea salt for yeast extract (YE) and reagentgrade salts, respectively. In serum bottle cultures, hydrogen production by the 156T strain was less than 30% in fishmeal-containing media when compared to that of the YE-containing MM1 medium. However, hydrogen production was increased substantially by adaptive evolution in the fishmeal-sea salt (FMSS) medium. In bioreactor experiments, the resulting strain 156T-FA showed shorter lag phase and 70% higher maximum hydrogen production rate in FMSS medium than the parent strain 156T. Our results showed that the H2 production cost was reduced by ca. 95%, when YE and chemical salts were replaced by fishmeal and sea salt, respectively.

The loss of imprinting of MEST has been linked to certain types of cancer by promoter switching. However, MEST-mediated regulation of tumorigenicity and metastasis are yet to be understood. Herein, we reported that MEST is a key regulator of IL-6/JAK/STAT3/Twist-1 signal pathway-mediated tumor metastasis. Enhanced MEST expression is significantly associated with pathogenesis of breast cancer patients. Also, MEST induces metastatic potential of breast cancer through induction of the EMT-TFs-mediated EMT program. Moreover, MEST leads to Twist-1 induction by STAT3 activation and subsequently enables the induction of activation of the EMT program via the induction of STAT3 nuclear translocation. Furthermore, the c-terminal region of MEST was essential for STAT3 activation via the induction of JAK2/STAT3 complex formation. Finally, MEST is required for metastasis in an experimental metastasis model. These observations suggest that MEST is a promising target for intervention to prevent tumor metastasis.

The effects of specific growth rate and specific nutrient uptake rate on morphological differentiation of

To date, NAD(P)H, ferredoxin, and coenzyme F(420) have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoded hydrogenase, a homolog of the F(420)-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pulldown assays. Electrons derived from H(2) oxidation by the frhAGB-encoded hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was 7-fold less efficient than when NADPH was the electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoded hydrogenase utilizing a protein as an electron acceptor. IMPORTANCE This study has importance in that TrxR can use H(2) as an electron donor with the aid of the frhAGB-encoded hydrogenase as well as NAD(P)H in T. onnurineus NA1. Further studies are needed to explore the physiological significance of this protein. This study also has importance as a significant step toward understanding the functionality of the frhAGB-encoded hydrogenase in a nonmethanogen; the hydrogenase can transfer electrons derived from oxidation of H(2) to a protein target by direct contact without the involvement of an electron carrier, which is distinct from the mechanism of its homologs, F(420)-reducing hydrogenases of methanogens.

Volume 86, no. 6, AEM02630-19, 2020, [https://doi.org/10.1128/AEM.02630-19][1]. Page 9: The following reference should read as shown. 11. Susanti D, Loganathan U, Mukhopadhyay B. 2016. A novel F420-dependent thioredoxin reductase gated by low potential FAD: a tool for redox regulation in an

To date, NAD(P)H, ferredoxin, and coenzyme F420 have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoded hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pulldown assays. Electrons derived from H2 oxidation by the frhAGB-encoded hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was 7-fold less efficient than when NADPH was the electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoded hydrogenase utilizing a protein as an electron acceptor.IMPORTANCE This study has importance in that TrxR can use H2 as an electron donor with the aid of the frhAGB-encoded hydrogenase as well as NAD(P)H in T. onnurineus NA1. Further studies are needed to explore the physiological significance of this protein. This study also has importance as a significant step toward understanding the functionality of the frhAGB-encoded hydrogenase in a nonmethanogen; the hydrogenase can transfer electrons derived from oxidation of H2 to a protein target by direct contact without the involvement of an electron carrier, which is distinct from the mechanism of its homologs, F420-reducing hydrogenases of methanogens.

Here, we developed a pressurized bioreactor system that increase carbon monoxide (CO) transfer efficiency in order to enhance the hydrogen productivity in the microbial water gas shift reaction by Thermococcus onnurineus NA1. The effects of CO pressure on the hydrogen production rate, CO consumption rate and the cell growth were investigated using small scale stainless steel bottles at various CO partial pressures. It was found that CO solubility increased by applying pressure can affect hydrogen production positively as long as the increased toxicity of CO is endurable to cells. The hydrogen productivity increased to some extent with CO pressure, but decreased drastically at the pressure higher than 4 bar. On the other hand, the effect of pressure itself on the cell's activity was not as significant as that of CO solubility increase. In the experiments at various system pressures with identical CO partial pressure of 1 bar, more than 80% of the cell activity remains even at total pressure of 10 bar. Also, it was important to determine the appropriate time to increase pressure for preventing excess CO in the reactor. Based on these results, a fermentation strategy for the pressurized system was designed and applied to a 5 L bioreactor with the continuous supply of the gas containing 60% CO. When the pressure was introduced to the bioreactor up to 4 bar at CO limitation condition, the unprecedented high productivity (360 mmol L−1 h−1) could be obtained.

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.

The F420-reducing hydrogenase of methanogens functions in methanogenesis by providing reduced coenzyme F420 (F420H2) as an electron donor. In non-methanogens, however, their physiological function has not been identified yet. In this study, we constructed an ΔfrhA mutant, whose frhA gene encoding the hydrogenase α subunit was deleted, in the non-methanogenic Thermococcus onnurineus NA1 as a model organism. There was no significant difference in the formate-dependent growth between the mutant and the wild-type strains. Interestingly, the mutation in the frhA gene affected the expression of genes involved in various cellular functions such as H2 oxidation, chemotactic signal transduction, and carbon monoxide (CO) metabolism. Among these genes, the CO oxidation gene cluster, enabling CO-dependent growth and H2 production, showed a 2.8- to 7.0-fold upregulation by microarray-based whole transcriptome expression profiling. The levels of proteins produced by this gene cluster were also significantly increased not only under the formate condition but also under the CO condition. In a controlled bioreactor, where 100% CO was continuously fed, the ΔfrhA mutant exhibited significant increases in cell growth (2.8-fold) and H2 production (3.4-fold). These findings strongly imply that this hydrogenase is functional in non-methanogens and is related to various cellular metabolic processes through an unidentified mechanism. An understanding of the mechanism by which the frhA gene deletion affected the expression of other genes will provide insights that can be applied to the development of strategies for the enhancement of H2 production using CO as a substrate.

Protein disulfide oxidoreductases are redox enzymes that catalyze thiol-disulfide exchange reactions. These enzymes include thioredoxins, glutaredoxins, protein disulfide isomerases, disulfide bond formation A (DsbA) proteins, and Pyrococcus furiosus protein disulfide oxidoreductase (PfPDO) homologues. In the genome of a hyperthermophilic archaeon, Thermococcus onnurineus NA1, the genes encoding one PfPDO homologue (TON_0319, Pdo) and three more thioredoxin- or glutaredoxin-like proteins (TON_0470, TON_0472, TON_0834) were identified. All except TON_0470 were recombinantly expressed and purified. Three purified proteins were reduced by a thioredoxin reductase (TrxR), indicating that each protein can form redox complex with TrxR. SurR, a transcription factor involved in the sulfur response, was tested for a protein target of a TrxR-redoxin system and only Pdo was identified to be capable of catalyzing the reduction of SurR. Electromobility shift assay demonstrated that SurR reduced by the TrxR-Pdo system could bind to the DNA probe with the SurR-binding motif, GTTttgAAC. In this study, we present the TrxR-Pdo couple as a redox-regulator for SurR in T. onnurineus NA1.

To develop a thermophilic cell factory system that uses CO gas, we attempted to engineer a hyperthermophilic carboxydotrophic hydrogenic archaeon Thermococcus onnurineus NA1 to be capable of producing thermophilic enzymes along with hydrogen (H2). The mutant strains 156T-AM and 156T-POL were constructed to have another copy of a gene encoding α-amylase or DNA polymerase, respectively, and exhibited growth rates and H2 production rates distinct from those of the parental strain, 156T, in gas fermentation using 100% CO or coal-gasified syngas. Purified α-amylase displayed starch-hydrolyzing activity, and whole-cell extracts of 156T-AM showed saccharifying activity for potato peel waste. PCR amplification was used to demonstrate that purified DNA polymerase was free from bacterial DNA contamination, in contrast to commercial bacteria-made enzymes. This study demonstrated that this archaeal strain could coproduce enzymes and H2 using CO-containing gas, providing a basis for cell factories to upcycle industrial waste gas.

To date, NAD(P)H, ferredoxin, and coenzyme F

A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic archaeon, designated as strain IOH1T, was isolated from a new deep-sea hydrothermal vent (Onnuri Vent Field) area in the Central Indian Ocean ridge. Strain IOH1T showed > 99% 16S rRNA gene sequence similarity with Thermococcus celericrescens TS2T (99.4%) and T. siculi DSM 12349T (99.2%). Additional three species T. barossii SHCK-94T (99.0%), T. celer Vu13T (98.8%), and T. piezophilus (98.6%) showed > 98.6% of 16S rRNA gene sequence similarity, however, the maximum OrthoANI value is 89.8% for the genome of T. celericrescens TS2T. Strain IOH1T cells are coccoid, 1.2-1.8 μm in diameter, and motile by flagella. Growth was at 70-82°C (optimum 80°C), pH 5.4-8.0 (optimum pH 6.0) with 2-4% (optimum 3%) NaCl. Growth of strain IOH1T was enhanced by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon sources, and elemental sulfur as an electron acceptor; clearly different from those of related species T. celecrescens DSM 17994T and T. siculi DSM 12349T. Strain IOH1T, T. celercrescence DSM 17994T, and T. siculi DSM 12349T reduced soluble Fe(III)-citrate present in the medium, whereas the amount of total cellular proteins increased with the concomitant accumulation of Fe(II). We determined a circular chromosome of 2,234 kb with an extra-chromosomal archaeal plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA G + C content was 54.9 mol%. Based on physiological properties, phylogenetic, and genome analysis, we proposed that strain IOH1T (= KCTC 15844T = JCM 39077T) is assigned to a new species in the genus Thermococcus and named Thermococcus indicus sp. nov.

A putative copper ion-sensing transcriptional regulator CopR (TON_0836) from Thermococcus onnurineus NA1 was characterized. The CopR protein consists of a winged helix-turn-helix DNA-binding domain in the amino-terminal region and a TRASH domain that is assumed to be involved in metal ion-sensing in the carboxyl-terminal region. The CopR protein was most strongly bound to a region between its own gene promoter and a counter directional promoter region for copper efflux system CopA. When the divalent metals such as nickel, cobalt, copper, and iron were present, the CopR protein was dissociated from the target promoters on electrophoretic mobility shift assay (EMSA). The highest sensible ion is copper which affected protein releasing under 10 µM concentrations. CopR recognizes a significant upstream region of TATA box on CopR own promoter and acts as a transcriptional repressor in an in vitro transcription assay. Through site-directed mutagenesis of the DNA-binding domain, R34M mutant protein completely lost the DNA-binding activity on target promoter. When the conserved cysteine residues in C144XXC147 motif 1 of the TRASH domain were mutated into glycine, the double cysteine residue mutant protein alone lost the copper-binding activity. Therefore, CopR is a copper-sensing transcriptional regulator and acts as a repressor for autoregulation and for a putative copper efflux system CopA of T. onnurineus NA1.

In this study, we developed a rapid and sensitive enzymatic assay for 2,3-butanediol (2,3-BDO) detection. The concentration of 2,3-BDO was determined by measuring the reduction of NADP(+) using Clostridium ljungdahlii 2,3-butanediol dehydrogenase (CL-Bdh). The enzymatic assay could detect as low as 0.01 mM of 2,3-BDO, while the high-performance liquid chromatography (HPLC) method required a much higher concentration than 0.15 mM. Gratifyingly, the developed method was 15 times more sensitive than the HPLC method. When the enzymatic assay was applied to high-throughput screening, the enzymatic assay detected 14 positive samples out of 23 tested, as compared to 8 by the HPLC method. These results suggest that the enzymatic assay is an effective screening method for the detection of 2,3-BDO-producing microbes in a microtiter plate-based format. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-1705-9) contains supplementary material, which is available to authorized users.

Strain IMCC1322 was isolated from a surface water sample from the East Sea of Korea. Based on 16S rRNA analysis, IMCC1322 was found to belong to the OCS28 sub-clade of SAR116. The cells appeared as short vibrioids in logarithmic-phase culture, and elongated spirals during incubation with mitomycin or in aged culture. Growth characteristics of strain IMCC1322 were further evaluated based on genomic information; proteorhodopsin (PR), carbon monoxide dehydrogenase, and dimethylsulfoniopropionate (DMSP)-utilizing enzymes. IMCC1322 PR was characterized as a functional retinylidene protein that acts as a light-driven proton pump in the cytoplasmic membrane. However, the PR-dependent phototrophic potential of strain IMCC1322 was only observed under CO-inhibited and nutrient-limited culture conditions. A DMSP-enhanced growth response was observed in addition to cultures grown on C1 compounds like methanol, formate, and methane sulfonate. Strain IMCC1322 cultivation analysis revealed biogeochemical processes characteristic of the SAR116 group, a dominant member of the microbial community in euphotic regions of the ocean. The polyphasic taxonomy of strain IMCC1322 is given as Candidatus Puniceispirillum marinum, and was confirmed by chemotaxonomic tests, in addition to 16S rRNA phylogeny and cultivation analyses.

We have previously identified a sulfate methane transition zone (SMTZ) within the methane hydrate-bearing sediment in the Ulleung Basin, East Sea of Korea, and the presence of ANME-1b group in the sediment has been shown by phylogenetic analysis of a 16S rRNA gene. Herein, we describe taxonomic and functional profiling in the SMTZ sample by metagenomic analysis, comparing with that of surface sediment. Metagenomic sequences of 115 Mbp and 252 Mbp were obtained from SMTZ and surface sediments, respectively. The taxonomic profiling using BLASTX against the SEED within MG-RAST showed the prevalence of methanogens (19.1%), such as Methanosarcinales (12.0%) and Methanomicrobiales (4.1%) predominated within the SMTZ metagenome. A number of 185,200 SMTZ reads (38.9%) and 438,484 surface reads (62.5%) were assigned to functional categories, and methanogenesis-related reads were statistically significantly overrepresented in the SMTZ metagenome. However, the mapping analysis of metagenome reads to the reference genomes, most of the sequences of the SMTZ metagenome were mapped to ANME-1 draft genomes, rather than those of methanogens. Furthermore, the two copies of the methyl-coenzyme M reductase gene (mcrA) segments of the SMTZ metagenome were clustered with ANME-1b in the phylogenetic cluster. These results indicate that ANME-1b reads were miss-annotated to methanogens due to limitation of database. Many of key genes necessary for reverse methanogenesis were present in the SMTZ metagenome, except for N 5,N 10-methenyl-H4MPT reductase (mer) and CoB-CoM heterodisulfide reductase subunits D and E (hdrDE). These data suggest that the ANME-1b represents the primary player the anaerobic methane oxidation in the SMTZ, of the methane hydrate-bearing sediment at the Ulleung Basin, East Sea of Korea.

This project sought to conduct an economic feasibility study regarding the commercial production of bio-hydrogen by the marine hyperthermophilic archaeon, Thermococcus onnurineus NA1 using carbon monoxide-containing industrial off-gas. We carried out the economic evaluation of the biohydrogen production process using the raw material of steel mill by-product gas. The process parameter was as follows: H2 production rate was 5.6 L/L/h; the conversion of carbon monoxide was 60.7%. This project established an evaluation criterion for about 10,000 tonne/year. Inflation factors were considered as 3%. The operating costs were recalculated based on prices in 2014. The total investment required for development was covered 30% by capital and 70% by a loan. The operation cost for the 0.5-year test and integration, and the cost for the first three months in the 50% production period were considered as the working capital in the cost estimation. The costs required for the rental of office space, facilities, and other related costs from the construction through to full-scale production periods were considered as continuing expenses. Materials, energy, waste disposal and other charges were considered as the operating cost of the development system. Depreciation, tax, maintenance and repair, insurance, labor, interest rate charges, general and administrative costs, lubrication and miscellaneous expenses were also calculated. The hydrogen price was set at US$ 4.15/kg for the economic evaluation. As a result, the process was considered to be economical with the payback period of 6.3 years, NPV of 18 billion Won and IRR of 26.7%.

Vestimentiferan tubeworms acquire their symbionts through horizontal transmission from the surrounding environment. In the present study, we constructed a 16S rRNA gene clone library to investigate the phylogenetic relationship between diverse microbes in the sediment and symbiotic bacteria in the trophosome of the tubeworm, Lamellibrachia satsuma, from Kagoshima Bay, Japan. Two symbiotic bacterial phylotypes belonging to the classes γ- and e-Proteobacteria were found from this tubeworm trophosome. They were very closely related to the symbionts of several other marine invertebrates. The most predominant bacteria in the sediment were e-Proteobacteria. A broad diversity of bacteria belonged to non-proteobacterial phyla such as Planctomycetes, Acidobacteria, and Chloroflexi was observed. The presence of sulfur oxidizers (i.e., e-Proteobacteria and γ-Proteobacteria) and sulfur reducers (i.e., δ-Proteobacteria) may play a significant role in the sulfur cycle in these habitats and provide multiple sources of nutrition to the cold-seep communities. Closely related clones of e-Proteobacteria symbiont in the species level and of γ-Proteobacteria symbiont in the genus level were found in the surrounding sediment. The similarity of symbiont clones of L. satsuma with other symbionts and free-living bacteria suggests the possibility of opportunistic symbiosis in e-Proteobacteria and the co-evolution of γ-Proteobacteria having occurred after symbiosis with the tubeworms.

Acetogens have often been observed to be inhibited by CO above an inhibition threshold concentration. In this study, a two-stage culture consisting of carboxydotrophic archaea and homoacetogenic bacteria is found to be effective in converting industrial waste gas derived from a steel mill process. In the first stage, Thermococcus onnurineus could grow on the Linz-Donawitz converter gas (LDG) containing ca. 56% CO as a sole energy source, converting the CO into H2 and CO2. Then, in the second stage, Thermoanaerobacter kivui could grow on the off-gas from the first stage culture, consuming the H2 and CO in the off-gas completely and producing acetate as a main product. T. kivui alone could not grow on the LDG gas. This work represents the first demonstration of acetate production using steel mill waste gas by a two-stage culture of carboxydotrophic hydrogenogenic microbes and homoacetogenic bacteria.

The biosynthetic pathway of 2,3-butanediol (2,3-BDO) production from pyruvate under anaerobic conditions includes three enzymes: acetolactate synthase (ALS), acetolactate decarboxylase (ALDC), and acetoin reductase (AR). Recently, in anaerobic hyperthermophilic Pyrococcus furiosus, it has been reported that acetoin, a precursor of 2,3-BDO, is produced from pyruvate by ALS through a temperature-dependent metabolic switch. In this study, we first attempted to produce 2,3-BDO from Thermococcus onnurineus NA1 using a simple biosynthetic pathway by two enzymes (ALS and AR) at a high temperature. Two heterologous genes, acetolactate synthase (alsS) from Pyrococcus sp. NA2 and alcohol dehydrogenase (adh) from T. guaymacensis, were introduced and expressed in T. onnurineus NA1. The mutant strain produced approximately 3.3 mM 2,3-BDO at 80 °C. An acetyl-CoA synthetase IIIα (TON_1001) was further deleted to enhance 2,3-BDO production, and the mutant strain showed a 25% increase in the specific production of 2,3-BDO. Furthermore, when carbon monoxide (CO) gas was added as a reductant, specific production of 2,3-BDO increased by 45%. These results suggest a new biosynthetic pathway for 2,3-BDO and demonstrate the possibility of T. onnurineus NA1 as a platform strain for 2,3-BDO production at high temperatures.

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

Background The production of biohydrogen (H2) as a promising future fuel in anaerobic hyperthermophiles has attracted great attention because H2 formation is more thermodynamically feasible at elevated temperatures and fewer undesired side products are produced. However, these microbes require anoxic culture conditions for growth and H2 production, thereby necessitating costly and time-consuming physical or chemical methods to remove molecular oxygen (O2). Therefore, the development of an O2-tolerant strain would be useful for industrial applications. Results In this study, we found that the overexpression of frhAGB-encoding hydrogenase genes in Thermococcus onnurineus NA1, an obligate anaerobic archaeon and robust H2 producer, enhanced O2 tolerance. When the recombinant FO strain was exposed to levels of O2 up to 20% in the headspace of a sealed bottle, it showed significant growth. Whole transcriptome analysis of the FO strain revealed that several genes involved in the stress response such as chaperonin β subunit, universal stress protein, peroxiredoxin, and alkyl hydroperoxide reductase subunit C, were significantly up-regulated. The O2 tolerance of the FO strain enabled it to grow on formate and produce H2 under oxic conditions, where prior O2-removing steps were omitted, such as the addition of reducing agent Na2S, autoclaving, and inert gas purging. Conclusions Via the overexpression of frhAGB genes, the obligate anaerobic archaeon T. onnurineus NA1 gained the ability to overcome the inhibitory effect of O2. This O2-tolerant property of the strain may provide another advantage to this hyperthermophilic archaeon as a platform for biofuel H2 production. Electronic supplementary material The online version of this article (10.1186/s13068-019-1365-3) contains supplementary material, which is available to authorized users.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H-2 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 H-2-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, H-2-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.

Based on the genomic analysis of Thermococcus pacificus P-4, we identified a putative GH1 β-glucosidase-encoding gene (Tpa-glu). The gene revealed a 1,464 bp encoding 487 amino acid residues, and the deduced amino acid residues exhibited 77% identity with Pyrococcus furiosus β-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherichia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. Tpa-Glu showed optimum activity at pH 7.5 and 75°C, and thermostability with a half life of 6 h at 90°C. Tpa-Glu exhibited hydrolyzing activity against various pNP-glycopyranosides, with k cat /K m values in the order of pNP-β-glucopyranoside, pNP-β-galactopyranoside, pNP-β-mannopyranoside, and pNP-β-xylopyranoside. In addition, the enzyme exhibited exo-hydrolyzing activity toward β-1,3-linked polysaccharide (laminarin) and β-1,3- and β-1,4-linked oligosaccharides. This is the first description of an enzyme from hyperthermophilic archaea that displays exo-hydrolyzing activity toward β-1,3-linked polysaccharides and could be applied in combination with β-1,3-endoglucanase for saccharification of laminarin.

Among four basic mechanisms for biological hydrogen (H2) production, dark fermentation has been considered to show the highest hydrogen evolution rate (HER). H2 production from one-carbon (C1) compounds such as formate and carbon monoxide (CO) is promising because formate is an efficient H2 carrier, and the utilization of CO-containing syngas or industrial waste gas may render the industrial biohydrogen production process cost-effective. A variety of microbes with the formate hydrogen lyase (FHL) system have been identified from phylogenetically diverse groups of archaea and bacteria, and numerous efforts have been undertaken to improve the HER for formate through strain optimization and bioprocess development. CO-dependent H2 production has been investigated to enhance the H2 productivity of various carboxydotrophs via an increase in CO gas-liquid mass transfer rates and the construction of genetically modified strains. Hydrogenogenic CO-conversion has been applied to syngas and by-product gas of the steel-mill process, and this low-cost feedstock has shown to be promising in the production of biomass and H2. Here, we focus on recent advances in the isolation of novel phylogenetic groups utilizing formate or CO, the remarkable genetic engineering that enhances H2 productivity, and the practical implementation of H2 production from C1 substrates.

In spite of their pivotal roles in transcriptional and post-transcriptional processes, the regulatory elements of archaeal genomes are not yet fully understood. Here, we determine the primary transcriptome of the H2-producing archaeon Thermococcus onnurineus NA1. We identified 1,082 purine-rich transcription initiation sites along with well-conserved TATA box, A-rich B recognition element (BRE), and promoter proximal element (PPE) motif in promoter regions, a high pyrimidine nucleotide content (T/C) at the −1 position, and Shine-Dalgarno (SD) motifs (GGDGRD) in 5′ untranslated regions (5′ UTRs). Along with differential transcript levels, 117 leaderless genes and 86 non-coding RNAs (ncRNAs) were identified, representing diverse cellular functions and potential regulatory functions under the different growth conditions. Interestingly, we observed low GC content in ncRNAs for RNA-based regulation via unstructured forms or interaction with other cellular components. Further comparative analysis of T. onnurineus upstream regulatory sequences with those of closely related archaeal genomes demonstrated that transcription of orthologous genes are initiated by highly conserved promoter sequences, however their upstream sequences for transcriptional and translational regulation are largely diverse. These results provide the genetic information of T. onnurineus for its future application in metabolic engineering.

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3'-5' exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.

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, PTN0413 and PTN0157, 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 PTN0413 promoter exhibited 2.7 to 4.7 times higher transcript level than KS0157 with PTN0157 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.

Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at 85℃. The melting temperature of TcCD was determined to be 93oC by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed α-cyclodextrin (α-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one α-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-α-CD, G1-β- CD, and G2-β-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and 6(2)-α-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β-lactam antibiotics. This result explains the weak β-lactamase activity of EstU1 compared with class C β-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum. Proteins 2013; 81:2045–2051. © 2013 Wiley Periodicals, Inc.

Hydrogenogenic CO oxidation (CO + H 2 O → CO 2 + H 2 ) has the potential for H 2 production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H 2 , has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H 2 , the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na + /H + antiporter and a 1.8-fold-higher specific activity for CO-dependent H 2 production than did the wild-type strain. The H 2 production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H 2 production rate of the engineered strain was severalfold higher than those of any other CO-dependent H 2 -producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H 2 production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.

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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.