# Patent

## Title
Method of producing hydrogen from Thermococcus spp

## Abstract

The present invention relates to novel hydrogenases isolated from novel hyperthermophilic strains belonging to Thermococcus spp., genes encoding the hydrogenases, and methods of producing hydrogen using strains having the genes. According to the hydrogen production methods of the invention, a large amount of hydrogen can be produced merely by culturing the strains in specific culture conditions. Thus, the methods of the invention have advantages in that they are more economic and efficient than existing hydrogen production methods and can produce hydrogen even at high temperature.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY
This application is a divisional application of application Ser. No. 12/746,090, filed Jul. 8, 2010, now U.S. Pat. No. 8,597,926, which is a National Stage entry from International Application No. PCT/KR2009/005060, filed Sep. 7, 2009, which claims priority to Korean Patent Application Nos. 10-2008-0087794, filed Sep. 5, 2008, and 10-2008-0087806, filed Sep. 5, 2008, entire contents of which are incorporated herein by reference.
BACKGROUND
1. Technical Field
The present invention relates to novel hydrogenases isolated from novel strains belonging to the genus Thermococcus, genes encoding the same, and methods of producing hydrogen using strains having the genes.
2. Background Art
Hydrogen energy is receiving attention as a next-generation energy source that can substitute for fossil fuels, because its calorific value per unit weight is at least three times higher than that of petroleum oils, while it does not emit substances that can adversely affect the environment, such as carbon dioxide, NOx and SOx.
Conventional processes for producing hydrogen include electrolysis of water, and the thermal-cracking or steam reforming of natural gas or naphtha. However, these production processes have a problem in that they require fossil fuels to provide high-temperature and high-pressure conditions. Also, these methods generate mixed gases containing carbon monoxide, and thus require a difficult process of removing carbon monoxide from the mixed gases.
On the other hand, biological methods of producing hydrogen using microorganisms have advantages in that it is not needed to make high-temperature and high-pressure conditions by introducing separate energy and in that the produced gases contain no carbon monoxide. Such biological hydrogen production methods can be broadly classified into methods utilizing photosynthetic microorganisms and methods utilizing non-photosynthetic microorganisms (mainly anaerobic microorganisms). Examples of the former methods include a method described in Korean Patent Registration No. 10-0680624, entitled “A method of producing hydrogen using the photosynthetic bacteria Rhodobacter sphaeroides strain having high hydrogen productivity at high salt concentration.
However, the technology of culturing photosynthetic bacteria at high concentration using light as an energy source is not yet sufficiently developed, and prior photosynthetic bacteria have a shortcoming in that substrate inhibition is severe when a substrate of high partial pressure exists. Also, these bacteria have a problem in that their hydrogen production capacity can be maintained only in the presence of light.
Accordingly, attempts to produce hydrogen using microorganisms that can produce hydrogen using organic carbon have been continuously made, and examples thereof include Korean Patent Registration No. 10-0315663, entitled “Citrobacter sp. Y19 and production of hydrogen using the same”, and Korean Patent Registration No. 10-0315662, entitled “Rhodopseudomonas palustris P4 and production of hydrogen using the same”.
The present inventors previously filed a patent application relating to novel proteins isolated from novel hyperthermophilic Thermococcus onnurineus NA1 (accession number KCTC 10859BP) and genes encoding the same on Sep. 5, 2008 (Korean Patent Application No. 10-2008-0087794), and the present invention particularly relates to genes related to hydrogen production among the proteins and genes disclosed in the patent application. The present inventors have carried out experiments on the hydrogen production capacity of the above-described strain and, as a result, have found that the strain produces a large amount of hydrogen even in a high-temperature environment, and have also found novel hydrogenases which are highly expressed, particularly in culture conditions supplemented with carbon monoxide (CO) or formate, thereby completing the present invention.
SUMMARY
It is an object of the present invention to provide hydrogenases isolated from hyperthermophilic Thermococcus spp. which can produce hydrogen even in a high-temperature environment, genes encoding the same, and methods of efficiently producing hydrogen using strains having the genes.
To achieve the above object, the present invention provides hydrogenases isolated from the Thermococcus spp. strain capable of producing hydrogen in aerobic culture conditions, and genes encoding the same. Also, the present invention provides a method of producing hydrogen by culturing the strain, and a method of producing hydrogen using the genes.
In a first aspect, the present invention provides hydrogenases which are produced by the novel hyperthermophilic strain Thermococcus onnurineus NA1 (accession number: KCTC 10859BP). T. onnurineus NA1 has eight novel hydrogenase gene clusters, and the amino acid sequences of hydrogenases belonging thereto are shown in SEQ ID NO. 1 to SEQ ID NO: 8.
In a second aspect, the present invention provides genes encoding said amino acid sequences. The genes are preferably, but not limited to, genes of SEQ ID NO. 12 to SEQ ID NO: 19 (the amino acid sequences of SEQ ID NO. 1 to SEQ ID NO: 8 correspond to the genes of SEQ ID NO. 12 to SEQ ID NO: 19, respectively).
In a third aspect, the present invention provides a method of producing hydrogen by culturing Thermococcus spp. The method comprises the steps of: 1) preparing a medium in a culture vessel; 2) culturing Thermococcus spp. in the culture vessel; 3) extracting hydrogen from the culture vessel. The Thermococcus spp. is preferably Thermococcus onnurineus NA1 (accession number: KCTC 10859BP).
In addition, the medium may be a medium supplemented with one or more selected from the group consisting of carbon monoxide, formate and starch. The culturing may be carried out at a high temperature of 80° C. in an anaerobic condition.
In a fourth aspect, the present invention provides a dehydrogenase comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 11.
In a fifth aspect, the present invention provides a gene encoding the dehydrogenase. Preferably, the gene has a base sequence selected from SEQ ID NO: 20 to SEQ ID NO: 22 (the amino acids of SEQ ID NOs: 9 to 11 correspond to SEQ ID NOs: 20 to 22, respectively).
In a sixth aspect, the present invention provides a recombinant vector comprising genes that are organized in a CODH-MCH-MNH3 hydrogenase cluster in T. onnurineus NA1, wherein the genes are all operably linked. Preferably, the genes include, but are not limited to, genes of SEQ ID NO: 21 (CODH dehydrogenase) and SEQ ID NO: 16 (MCH hydrogenase). In addition, the present invention provides a host cell transformed with the recombinant vector.
Also, the present invention provides a method of producing hydrogen using said transformant, the method comprising the steps of: preparing a medium in a culture vessel; feeding carbon monoxide into a gas phase of the culture vessel; culturing said transformant in the culture vessel; and extracting hydrogen from the culture vessel.
In a seventh aspect, the present invention provides a recombinant vector comprising genes that are organized in a FDH2-MFH2-MNH2 hydrogenase cluster in T. onnurineus NA1, wherein the genes are all operably linked. Preferably, the genes include, but are not limited to, genes of SEQ ID NO: 22 (FDH2 dehydrogenase) and SEQ ID NO: 18 (MFH2 hydrogenase). In addition, the present invention provides a host cell transformed with the recombinant vector.
Also, the present invention provides a method of producing hydrogen using said transformant, the method comprising the steps of: preparing a formate-containing medium in a culture vessel; culturing said transformant in the culture vessel; and extracting hydrogen from the culture vessel.
In an eighth aspect, the present invention provides a recombinant vector comprising genes that are organized in a FDH1-MFH1-MNH1 hydrogenase cluster in T. onnurineus NA1, wherein the genes are all operably linked. Preferably, the genes include, but are not limited to, genes of SEQ ID NO: 20 (FDH1 dehydrogenase) and SEQ ID NO: 13 (MFH1 hydrogenase). In addition, the present invention provides a host cell transformed with the recombinant vector.
Also, the present invention provides a method of producing hydrogen using said transformant, the method comprising the steps of: preparing a starch-containing medium in a culture vessel; culturing said transformant in the culture vessel; and extracting hydrogen from the culture vessel.
The hydrogen production methods according to the present invention have advantages in that they do not require high-temperature and high-pressure conditions, unlike the prior chemical hydrogen production methods, can generate hydrogen in ambient temperature and atmospheric pressure conditions, and do not generate harmful byproducts. Also, the methods of the present invention have advantages in that they can produce high-purity hydrogen at high efficiency compared to the prior art methods of producing hydrogen using microorganisms and can produce hydrogen even in high-temperature conditions.
Accordingly, the present invention has an economic advantage in that it allows high-temperature carbon monoxide discharged from petroleum purification processes and the like to be used directly for hydrogen production without a separate cooling process after capturing the carbon monoxide. Also, the present invention is useful for air conditioning.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B present a Venn diagram showing the shared and unique portions of the proteome of four Thermococcales strains, T. onnurineus NA1 (NA1), T. kodakaraensis, P. furiosus, and P. abyssi. The protein sets for the strains were obtained from the RefSeq collection in NCBI.
FIG. 2A is a representative map of eight hydrogenase gene clusters in T. onnurineus NA1. A, B, C, and D: membrane-bound hydrogenases and cytoplasmic NiFe-hydrogenases. S1, S2, and S3: T. onnurineus NA1. Genes were colored according to COG functional categories. TON—0051-0055 represents SEQ ID NOs: 1 to 5; TON—0486-0498 represents SEQ ID NOs: 35 to 47; TON—0533-0544 represents SEQ ID NOs: 48 to 59; TON—1583-1595 represents SEQ ID NOs: 100 to 112; TON—0261-0289 represents SEQ ID NOs: 6 to 34; TON—1016-1031 represents SEQ ID NOs: 60 to 75; and TON—1559-1582 represents SEQ ID NOs: 76 to 99.
FIG. 2B shows the gene organization of three hydrogenase gene clusters (fdh1-mfh1-mnh1, fdh2-mfh2-mnh2 and codh-mch-mnh) having a 3-module gene cluster on the genome of T. onnurineus NA1. Genes belonging to the same subclusters were indicated by the same color.
FIG. 3 shows the distribution and conservation patterns of hydrogenase gene clusters in 31 archaeal genomes. Blue brackets (the first bracket, the third bracket, the fifth bracket from the bottom) encompass CDSs showing low similarities (<25%) to any CDSs from 31 archaeal genomes. The black brackets indicate CDSs similar to hydrogenase 4 from P. abyssi. The 31 archaeal genes are as follows:











No.
Gene
















1
Aeropyrum_pernix


2
Pyrobaculum_aerophilum


3
Sulfolobus_acidocaldarius_DSM_639


4
Sulfolobus_solfataricus


5
Sulfolobus_tokodaii


6
Haloarcula_marismortui_ATCC_43049


7
Natronomonas_pharaonis


8
Halobacterium_sp


9
Haloquadratum_walsbyi


10
Methanococcoides_burtonii_DSM_6242


11
Picrophilus_torridus_DSM_9790


12
Trhermoplasma_acidophilum


13
Thermoplasma_volcanium


14
Methanosaeta_thermophila_PT


15
Pyrobaculum_islandicum_DSM_4184


16
Thermofilum_pendens_Hrk_5


17
Pyrococcus_abyssi


18
Pyrococcus_furiosus


19
Pyrococcus_horikoshii


20
Thermococcus_kodakaraensis_KOD1


21
Archaeogiobus_fulgidus


22
Methanosarcina_barkeri_fusaro


23
Methanosarcina_mazei


24
Methanosarcina_acetivorans


25
Methanospirillum_hungatei_JF-1


26
Methanobacterium_thermoautotrophicum


27
Methanococcus_jannaschii


28
Methanococcus_maripaludis_S2


29
Methanosphaera_stadtmanae


30
Methanopyrus_kandleri


31
Nanoarchaeum_equitans









FIGS. 4 and 5 show a comparison of the a subunits of CODH and F420 hydrogenase proteins. FIG. 4 is the phylogenetic tree of CODH, and FIG. 5 is the phylogenetic tree of the α subunit of F420 hydrogenase. Homologues of the proteins on the phylogenetic trees were obtained from the NCBI nr database.
FIGS. 6A through 6C show the growth profile of T. onnurineus NA1 depending on CO. T. onnurineus NA1 was grown in medium 1 supplemented with CO (lane 2; triangles), sulfur (lane 3; squares) or both (lane 4; triangles down). Controls without supplement (lane 1; circles) and culture in YPS medium (C) were included. DAPI-stained cells were directly counted on filters by fluorescence microscopy. FIG. 6A: Effect of medium composition at various concentrations of yeast extract (YE). FIG. 6B: Growth curves of T. onnurineus NA1 in medium 1 with other supplements. FIG. 6C: Analysis of the transcription of the CODH gene.
FIG. 7A shows the growth and hydrogen production of T. onnurineus NA1 in YPS and FIG. 7B shows the growth and hydrogen production of T. onnurineus NA1 in CO-containing medium. Open circles: growth; and closed circles: hydrogen production.
FIG. 8A shows the microarray analysis of the expression of hydrogenase gene clusters in T. onnurineus NA1 in YPS and FIG. 8B shows RT-PCR analysis of the expression of hydrogenase gene clusters in T. onnurineus NA1 in CO-containing medium. More particularly, FIG. 8A shows the microarray analysis of eight hydrogenase gene clusters in T. onnurineus NA1. Hierarchical clustering of the mRNA changes in CO was compared with one in an YPS growth condition as a control. Up-regulation and down-regulation were indicated by red and green, respectively. Growth conditions were indicated on top of the clustering picture. At the right side of the clustering picture, ORFs of each of codh-mch-mnh3, fdh1-mfh1-mnh1 or fdh2-mfh2-mnh2 were indicated as bars. YE: yeast extract. FIG. 8B shows the results of quantitative RT-PCR analysis in CO or YPS conditions, carried out for each of the large subunits of mbh (TON—1593), mbx (TON 0489), frh (TON—1560), sulf I (TON—0534), mch (TON—1023), mfh2 (TON—1569) and mfh1 (TON—0276) hydrogenases. The chaperonin-encoding gene (cha) was used as a control to normalize expression levels.
FIGS. 9A and 9B show the targeted gene disruption of the large subunit of each of mch (TON—1023) and mfh2 (TON—1569) hydrogenases. FIG. 9A shows the gene organization of each of codh-mch-mnh3 and fdh2-mfh2-mnh2 clusters in T. onnurineus NA1. Pgdh: the 5′-upstream promoter region of the glutamate dehydrogenase gene of T. kodakaraensis KOD1; and hmgPfu: the 3-hydroxy-methylglutaryl coenzyme A reductase gene of Pyrococcus furiosus. FIG. 9B shows the identification of gene disruption by PCR. The left panel shows PCR amplification with primers for the overexpression cassette (Pgdh-hmgPfu) region. The right panel shows PCR amplification with primers for the large subunit of each of mchTNA1 and mfh2TNA1 hydrogenases. M: size marker (1 kb ladder); W: wild type; lanes 1 and 2: mutant strains.
FIG. 10A shows the growth and hydrogen production of a ΔmchTNA1 mutant strain in YPS. Open circles: growth; closed circles: hydrogen production. FIG. 10B shows the growth and hydrogen production of a Δmfh2TNA1 mutant strain in YPS. FIG. 10C shows the growth and hydrogen production of a ΔmchTNA1 mutant strain in CO-containing medium. FIG. 10D shows the growth and hydrogen production of a Δmfh2TNA1 mutant strain in CO-containing medium.



DETAILED DESCRIPTION
In a first aspect, the present invention provides hydrogenases which are produced by the novel hyperthermophilic strain Thermococcus onnurineus NA1 (accession number: KCTC 10859BP) that produces hydrogen in anaerobic conditions. The strain was isolated from a deep-sea hydrothermal vent area at the PACMANUS field in the East Manus Basin. The isolated strain was deposited in the Korean Collection for Type Cultures (KCTC) at the Korean Research Institute of Bioscience and Biotechnology (KRIBB) on Oct. 7, 2005 and assigned accession number KCTC 10859BP on Oct. 20, 2005. The characteristics and culture methods of the strain are described in Korean Patent Application No. 10-2007-0127255 on which the present invention is based.
T. onnurineus NA1 has eight novel hydrogenase gene clusters. The hydrogenases are key enzymes related to the metabolism of molecular hydrogen (H2) and act as catalysts in the following reversible reaction: 2H++2e−H2. Preferably, the hydrogenases belonging to the above-described clusters provide proteins and functional equivalents thereof comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8. As used herein, the term “functional equivalent” is intended to include amino acid sequence variants having amino acid substitutions in some or all of the proteins, or amino acid additions or deletions in some of the proteins. The amino acid substitutions are preferably conservative substitutions. Examples of the conservative substitutions of naturally occurring amino acids are as follow: aliphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (Ile, Leu, and Val), aromatic amino acids (Phe, Tyr, and Tip), acidic amino acids (Asp, and Glu), basic amino acids (H is, Lys, Arg, Gln, and Asn), and sulfur-containing amino acids (Cys, and Met). The deletions of amino acids are preferably located in a region which is not directly involved in the activity of the hydrogenases.
In a second aspect, the present invention provides genes encoding the above-described amino acid sequences. The genes are preferably, but not limited to, genes of SEQ ID NOs: 12 to 19 (the amino acid sequences of SEQ ID NOs: 1 to 8 correspond to the genes of SEQ ID NOs: 12 to 19, respectively).
In a third aspect, the present invention provides a method of producing hydrogen by culturing Thermococcus spp. The method comprises the steps of 1) preparing a medium in a culture vessel; 2) culturing Thermococcus spp. in the culture vessel; and 3) extracting hydrogen from the culture vessel. The Thermococcus spp. is preferably Thermococcus onnurineus NA1 (accession number: KCTC 10859BP).
In addition, the medium may be a medium supplemented with one or more selected from the group consisting of carbon monoxide, formate and starch. Also, the culturing may be carried out at a high temperature of 80° C. in anaerobic conditions.
In a fourth aspect, the present invention provides a dehydrogenase comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 11. The dehydrogenase, Fdh1(SEQ ID NO: 20), Fdh2(SEQ ID NO: 22) and CODH(SEQ ID NO: 21), respectively may be carried out by cluster with hydrogenase MFH1, MFH2 and MCH hydrogenase.
In a fifth aspect, the present invention provides a gene encoding said dehydrogenase. Preferably, the gene is selected from genes of SEQ ID NO: 20 to SEQ ID NO: 22 (the amino acid sequences of SEQ ID NOs: 9 to 11 correspond to the genes of SEQ ID NOs: 20 to 22, respectively).
In a sixth aspect, the present invention provides a recombinant vector comprising genes that are organized in a CODH-MCH-MNH3 hydrogenase cluster in T. onnurineus NA1, wherein the genes are all operably linked. Preferably, the genes include, but are not limited to, genes of SEQ ID NO: 21 (CODH dehydrogenase) and SEQ ID NO: 16 (MCH hydrogenase). As used herein, the term “vector” means a nucleic acid molecule that can carry another nucleic acid bound thereto.
As an expression vector which can synthesize a protein encoded by a recombinant gene carried by said vector, a plasmid, cosmid or phage may be used A preferred vector is a vector that can self-replicate and express a nucleic acid bound thereto.
In addition, the present invention provides a host cell transformed with the recombinant vector. The recombinant vector can be used to transform cells such as prokaryotic, fungal, plant and animal cells so as to prepare transformed cells which can produce hydrogen at high efficiency. As used herein, the term “transformation” means that foreign DNA or RNA is absorbed into cells to change the genotype of the cells. A public announded transformation method along each cell can be used to make the host cell.
Also, the present invention provides a method of producing hydrogen using said transformant, the method comprising the step of: preparing a medium in a culture vessel; feeding carbon monoxide into a gas phase of the culture medium; culturing said transformant in the culture vessel; and extracting hydrogen from the culture vessel.
In a seventh aspect, the present invention provides a recombinant vector comprising genes that are organized in a FDH2-MFH2-MNH2 hydrogenase cluster in T. onnurineus NA1, wherein the genes are all operably linked. Preferably, the genes include, but are not limited to, genes of SEQ ID NO: 22 (FDH2 dehydrogenase) and SEQ ID NO: 18 (MFH2 hydrogenase). In addition, the present invention provides a host cell transformed with the recombinant vector.
Particulars regarding the “vector”, “transformation” and “host cell” are as described in the above sixth aspect.
Also, the present invention provides a method of producing hydrogen using said transformant, the method comprising the steps of: preparing a formate-containing medium in a culture vessel; culturing said transformant in the culture vessel; and extracting hydrogen from the culture vessel.
In an eighth aspect, the present invention provides a recombinant vector comprising genes that are organized in a FDH1-MFH1-MNH1 hydrogenase cluster in T. onnurineus NA1, wherein the genes are all operably linked. Preferably, the genes include, but are not limited to, genes of SEQ ID NO: 20 (FDH1 dehydrogenase) and SEQ ID NO: 13 (MFH1 hydrogenase). In addition, the present invention provides a host cell transformed with the recombinant vector.
Particulars regarding the “vector”, “transformation” and “host cell” are as described in the above sixth aspect.
Also, the present invention provides a method of producing hydrogen using said transformant, the method comprising the steps of: preparing a starch-containing medium in a culture vessel; culturing said transformant in the culture vessel; and extracting hydrogen from the culture vessel.
Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
Example 1
Analysis of Hydrogenase Genes of Thermococcus onnurineus NA1 Strain
(1) Test Methods
1) Culture Conditions
For routine cultivation, cells were grown anaerobically at 80° C. in a yeast extract-peptone-sulfur (YPS) medium (Holden et al. 2001). Physiological tests were performed using modified medium 1 (Sokolova, T. G., C. Jeanthon, N. A Kostrikina, N. A. Chernyh, A. V. Lebedinsky, E. Stackebrandt, and E. A. Bonch-Osmolovskaya. 2004. The first evidence of anaerobic CO oxidation coupled with H2 production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Extremophiles 8:317-323) supplemented with 1 ml of a trace element mixture, 1 ml of vitamin solution (Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbio l. Rev. 43:260-296), NaCl (30 g g/l), and yeast extract (0.5 g/l). The pH was adjusted to 8.0 using NaOH. The anaerobically prepared medium was dispensed into 25-ml serum bottles, and the gas phase (15 ml) was charged with N2/C02 (80:20; 1 bar) or 100% CO. When the cells were cultured with formate or starch, 10 g/L of sodium formate (Sigma) or 5 g/L of soluble starch (Sigma) was added to the medium before autoclaving. All the cultures for physiological tests were all carried out at 80° C. for 2 days.
2) Gene Sequencing
The genome sequence of T. onnurineus NA1 was determined by whole-genome shotgun sequencing and pyrosequencing. For capillary sequencing, a 2-kb to 3-kb insert library (11,028 clones), 40-kb insert library (1,870 clones), and 35-kb insert library (288 clones) were constructed and sequenced using an ABI 3730XL sequencer (Applied Biosystems, CA). For pyrosequencing, 581,990 fragments of DNA were sequenced using a GS-20 sequencer (454 Life Sciences). The contigs generated by both sequencers were combined, and closure of the sequencing gap was performed by clone walking and PCR sequencing. ORFs and RNA genes were predicted through a combination of Glimmer 3.0 (University of Maryland), GSFinder and RBSFinder, followed by a manual ORF fitting process. After all the ORFs had been determined, further analysis of the protein sequence was performed by BLASTP searches against the nonredundant protein sequences of the National Center for Biotechnology Information (NCBI), Kyoto Encyclopedia of Genes and Genomes (KEGG), and COG (clusters of orthologous groups of proteins) databases (Tatusova, R. L., D. A. Natale, I. V. Garkavtsev, T. A. Tatusova, U. T. Shankavaram, B. S. Rao, B. Kiryutin, M. Y. Galperin, N. D. Fedorova, and E. V. Koonin. 2001. The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 29:22-28). tRNAScan-SE was used for the tRNA predictions (Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955-964).
3) Protein Analysis
T. onnurineus NA1 cells were suspended in 100 mM Tris-HCl buffer (pH 6.8) containing 4% sodium dodecyl sulfate (SDS) and 4 mM EDTA and boiled for 10 min, followed by centrifugation at 22,000 g for 20 min. The cell lysate was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 30 fractions were obtained based on molecular size. They were then in-gel digested using trypsin (Promega, USA) (Kim, Y. H., K. Cho, S. H. Yun, J. Y. Kim, K. H. Kwon, J. S. Yoo, and S. I. Kim. 2006. Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 by combined proteomic approach: 2-DE/MS and cleavable ICAT analysis. Proteomics 6:1301-1318), and the tryptic digests were dissolved in 0.5% trifluoroacetic acid solution to be analyzed by mass spectrometry (Thermo Finnigan LTQ IT). The identities of peptides were determined using the Sequest program (Thermo Finnigan, San Jose, Calif.).
4) Total RNA Isolation and RT-PCR Analysis
A 50-ml culture of T. onnurineus NA1 was grown to mid-exponential growth phase in modified medium 1 supplemented with various concentrations of yeast extract under the gas phase of N2/CO2 (80:20, 1 bar) or 100% CO. Cells were harvested by centrifugation at 6,000×g for 30 min. The pellet was resuspended in 50 μl of 50 mM Tris-HCl buffer (pH 7.5) supplemented with 500 μl of Trizol reagent (Invitrogen, Carlsbad, Calif.). The cells were lysed by freezing and thawing, and then the samples were extracted with 200 μl of chloroform. The aqueous phase containing total RNA was further processed by ethanol precipitation and then resuspended in distilled water. RNA concentration and integrity were determined by measuring the absorbance at 260 and 280 nm, as well as by 0.8% agarose gel analysis. Reverse transcription and PCR amplification were
carried out using SuperScriptTM II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The following two sets of primers were used for amplification of CODH (carbon monoxide dehydrogenase) and Hsp60 (chaperonin) as controls:







(SEQ ID NO: 23)


CODH gene (forward, 5′-GGACCATGTAGAATCGAYCCGTTY-3′


and 


 


(SEQ ID NO: 24))


reverse, 5′-TTCRTTTCCGGTACAGCA-3′; 


and


 


(SEQ ID NO: 25) 


Hsp60 gene (forward, 5′-ATGGCACAGCTTAGTGGACAG-3′ 


and


 


(SEQ ID NO: 26))


reverse, 5′-CAAGGATTTCCTGGGCTTTCTC-3′.






5) Computer Analysis
The homology search of amino acid sequences was performed using the BLAST program against the non-redundant protein database of the NCBI. A motif search for proteins having the L1 signal (C[GS][ILV]C[AGNS]xxH, wherein x indicates any amino acid) of the group 4 hydrogenase was performed using the ProteinFinder program (Ensoltek, Korea) against the non-redundant protein database of the NCBI. Multiple sequence alignment for proteins was performed using the ClustalW program (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680), and a phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (Mega 4.1) software (Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596-1599). The phylogenetic tree of 16S rRNA sequences was made using pre-aligned sequences derived from the Ribosomal database site.
6) Generation of Signature Logos
Logo representations are used to visualize the information content associated with each position of a given motif shared by related sequences. In the graphical representation, the overall height of each position is correlated to the conservation at that position (expressed in bits), whereas the relative sizes of the symbols within a position indicate their relative frequencies. Logo analyses were performed at the Berkeley Structural Genomics Center.
(2) Analysis Result
1) General Features of T. onnurineus NA1 Gene
To provide some insight into factors that contributed to the apparent successful competition of Thermococcus spp. in hydrothermal vent fields, the genome sequence of T. onnurineus NA1 was determined by combining random whole-genome shotgun sequencing with pyrosequencing. As a result, it was revealed that T. onnurineus NA1 has a single circular chromosome (1,847,607 bp) without any exochromosomal elements, and a total of 1,976 coding DNA sequences (CDSs) were identified (Table 1 and FIGS. 1A and 1B). Of these, 1,104 CDSs (55.8%) were annotatable by homology and domain searches, but the function of the residual 872 CDSs could not be predicted from the primary structure. When protein similarity was searched on a genome-wide scale, 82.7% of the T. onnurineus NA1 proteins showed similarity to those of other members of the Thermococcales.







TABLE 1







General features of the T. onnurineus NA1 genome and


T. kodakaraensis KOD1 and Pyrococcus strains













NA1
KOD1
P. abyssi
P. horikoshii
P. furiosus
















Genome size (bp)
1,847,607
2,088,737
1,765,118
1,738,505
1,908,256


Protein-coding regions
90.1%
92.1%
91.1%
91.2%
92.5%


GC content
51.0%
52.0%
44.7%
41.9%
40.8%


CDSsa
1976
2306
1784
2064
2065


tRNAs
46
46
46
46
46


rRNAs
5S(x2),
5S(x2),
5S(x2),
5S(x2),
5S(x2),



7S, 16S, 32S
7S, 16S, 32S
7S, 16S, 32S
7S, 16S, 32S
7S, 16S, 32S





aThe protein sets for the strains were obtained from the RefSeq collection in NCBI.






2) Hydrogenase Clusters
An extraordinary proportion of hydrogenases and related proteins was detected in the T. onnurineus NA1 genome (5.5%), reflecting enhanced conservation or recycling of reducing potentials in association with oxidoreductases, including CO dehydrogenase and formate dehydrogenases.
Hydrogenases can be divided into the following three major groups based on their catalytic metal center: [NiFe]-hydrogenases, [FeFe]-hydsrogenases, and [Fe]-hydrogenases. Based on the unique functional center conserved in each of the hydrogenase groups, it is considered that all hydrogenases in T. onnurineus NA1, except for one hydrogenase, belong to [NiFe]-hydrogenases. According to the hydrogenase classification system of Vignais et al., the [NiFe]-hydrogenases in T. onnurineus NA1 belong to group 3 (one F420-reducing hydrogenase and two NADP-reducing hydrogenases) or group 4 (four membrane-associated hydrogenases) (Silva, P. J., van den Ban, E. C., Wassink, H., Haaker, H., de Castro, B., Robb, F. T. and Hagen, W. R. (2000) Enzymes of hydrogen metabolism in Pyrococcus furiosus. Eur. J. Biochem. 267, 6541-6551). The hydrogenases belonging to group 4 were termed “energy-converting hydrogenases” (Ech) and are widespread among bacteria and archaea.
To understand the molecular basis of hydrogenase metabolism, hydrogenase gene clusters were comparatively analyzed. The hydrogenases were phylogenetically analyzed and, as a result, the group 4 hydrogenases could be divided into two subgroups, 4a and 4b, and a pair of motif patterns common to all the sequences of subgroup 4b could be found.
As shown in FIG. 2, three additional hydrogenase clusters (FDH1-MFH1-MNH1 (Hyg4-I, S1:TON—0279-0274, MFH1: SEQ ID NO: 2), CODH-MCH-MNH3 (Hyg4-II, S2:TON—1021-1024, MCH: SEQ ID NO: 5) and FDH2-MFH2-MNH2 (Hyg4-III, S3:TON—1565-1571, MFH2: SEQ ID NO: 7)) and Frh (TON—1559-1562, SEQ ID NO: 6) were found in the T. onnurineus NA1 genome along with the two membrane-bound hydrogenases (Mbh (TON—1590-1595, SEQ ID NO: 8) and Mbx (TON—0489-0486, SEQ ID NO: 3)) and two cytoplasmic NiFe-hydrogenases (Sulf-I (TON—0533-0544, SEQ ID NO: 4) and Sulf-II (TON—0051-0055, SEQ ID NO: 1) reported in Pyrococcus spp. and T. kodakaraensis KOD1. Gene cluster analysis of hydrogenases with CDSs from 31 archaeal genomes clearly showed that FDH1-MFH1-MNH1 (Hyg4-I), CODH-MCH-MNH3 (Hyg4-II) and FDH2-MFH2-MNH2 (Hyg4-III) were unique in primary sequence, showing low similarities to hydrogenase 4 components from P. abyssi and R. rubrum (FIG. 3). Similarly to the additional hydrogenases, the FDH2-MFH2-MNH2 (Hyg4-III) cluster (TON—1559-1582, SEQ ID NOs: 76 to 99) included the α/β/γ subunits of F420 hydrogenase (TON—1559-1561, SEQ ID NOs: 76 to 78) in the genome. The subunits of F420 hydrogenase had unique primary sequences, showing similarities to the coenzyme F420-reducing hydrogenase (YP—001097176) from Methanococcus maripaludis (33%) and coenzyme F420-reducing hydrogenase (NP—987940) from M. maripaludis S2 (33%) (FIG. 3 and FIG. 5). No F420-hydrogenase homologues from the Thermococcales have been reported.
3) Construction of 3-Module Gene Clusters
It was found that each of the three Ech hydrogenases (MFH1, MFH2, and MCH) belonging the group 4 of [NiFe]-hydrogenases was a portion of large 17- or 18-gene clusters (fdh1-mfh1-mnh1, fdh2-mfh2-mnh2 and codh-mch-mnh3) consisting of TON—266-TON—282, TON—1563-TON—1580 and TON—1016-TON—1031 ORFs (open reading frames) (FIG. 2B). The ORFs in the clusters can be divided into three subclusters. The first part encodes oxidoreductase such as formate dehydrogenases (Fdh1 (SEQ ID NO: 9) or Fdh2 (SEQ ID NO: 11)) or carbon monoxide dehydrogenase (Codh (SEQ ID NO: 10)). The second part encodes multimeric membrane-bound hydrogenases (MFH1, MFH2 or MCH) having 5-7 subunits. The last part encodes cation/proton antiporters such as Na+/H+ antiporter. Such 3-module gene clusters have not yet been reported.
Example 2
Analysis of Gas Composition
(1) Analysis Method
Hydrogen gas was measured using a gas chromatograph HP 5890 series II (Hewlett Packard) equipped with an HP-PLOT Molesieve column (Agilent) and a TCD detector. Argon was used as a carrier gas. To quantify hydrogen gas, a gas calibration standard (Supleco) containing 1% (w/w) of each of components (CO, CO2, H2, CH4 and O2) in nitrogen was used.
(2) Production of Hydrogen Using Various Substances
In order to examine whether a number of hydrogenases cause T. onnurineus NA1 to efficiently produce hydrogen in various environments, hydrogen production rate was analyzed using various energy sources (Table 2). As a result, the NA1 strain could produce hydrogen using starch, CO and formate even under sulfur-free conditions. Particularly, CO and formate were very good energy sources for efficiently producing hydrogen.







TABLE 2







Comparison of hydrogen production of T. onnurineus


NA1 under various conditions










Medium
Hydrogen production (mmol/L)







M + CO
30.7



M + Formate
49.7



M + Starch
15.6







M: modified medium 1






The hydrogen productivity of the NA1 strain in batch culture is similar to those obtained in the continuous culture of T. kodakaraensis KOD1 and Pyrococcus furiosus. Hyperthermophilic archaea have various advantages in that they show a specific production rate higher than the hydrogen production by mesophilic bacterial fermentation or photobacteria in spite of their low volumetric productivity and produce high-purity hydrogen. The high hydrogen production rates described herein can be much improved through the optimization of culture conditions and treatment processes and metabolic engineering.
Example 3
CO-Dependent H2 Production by Thermococcus onnurineus NA1: Identification of CO-Responsive Hydrogenases
(1) CODH Gene Cluster and Carboxydotrophic Growth
As described above, it was found that T. onnurineus NA1 possessed a unique gene cluster (CODH-MCH-MNH3) that was comprised of a putative transcriptional regulator (TON—1016), a CODH accessory protein (CooC, TON—1019), a CODH catalytic subunit (CooS, TON—1018), and an electron transfer protein (CooF, TON—1017), along with a hydrogenase (mch, TON—1021-1024, SEQ ID NO: 5) (FIG. 2B). CooS (TON—1018), a central enzyme in microbial carbon monoxide (CO) metabolism, showed significant similarities with CODHs from CO-oxidizing methanogenic archaea such as CODH (AAM06652) from Methanosarcina acetivorans C2A (60%) and CODH (AAM29817) from Methanosarcina mazei Go1 (59%) (FIGS. 3 and 4). It seemed a monofunctional CODH (Bonam, D., L. Lehman, G P Roberts, and P. W. Ludden., 1989, Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: effects of CO and oxygen on synthesis and activity. J. Bacteriol. 171:3102-3107; and Wu, M. Q. Ren, A. S. Durkin, S. C. Daugherty, L. M Brinkac, R. J. Dodson, R. Madupu, S. A. Sullivan, J. F. Kolonay, W. C. Nelson, L. J. Tallon, K. M. Jones, L. E. Ulrich, J. M. Gonzalez, I. B. Zhulin, F. T. Robb, and J. A. Eisen. 2005, Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901. PLoS Genet. 1:e65), lacking the acetyl coenzyme A synthesis/cleavage activity of the bifunctional CODH/acetyl coenzyme A synthetase enzyme. According to Fox et al. (Fox, J. D., R. L. Kerby, G. P. Roberts, and P. W. Ludden, 1996, Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme. J. Bacteriol. 178:1515-1524), the monofunctional CODH/hydrogenase complex from Rhodospirillum rubrum participated in CO-driven proton respiration, whereby energy is conserved in the form of a proton gradient generated across the cell membrane. In this sense, to address the issue that the CODH cluster could play a similar role in energy conservation by oxidizing CO, the present inventors tested whether T. onnurineus NA1 could utilize CO. As a result, it was found that the strain, indeed, was able to grow better in medium 1 under a CO atmosphere, in both the absence and the presence of sulfur, than in the basal medium (FIGS. 6A and 6B), even though the growth yield was still lower than that in the YPS medium (FIG. 6A). The growth under CO atmosphere was associated with the transcription of the CooS gene, indicating that the gene could be induced by the presence of CO (FIG. 6C). It is noteworthy that the addition of sulfur decreased the transcriptional level of the CooS gene. These results support the hypothesis that T. onnurineus NA1 generates energy from CO. Hereinafter, specific test methods and results for verifying the hypothesis will be described.
(2) Test Methods
1) Culture Conditions
T. onnurineus NA1 was anaerobically cultured in a yeast extract-peptone-sulfur (YPS) medium at 80° C. To examine the growth characteristics of a mutant strain, modified medium 1 supplemented with 30.0 g/L of NaCl was used as a basal medium (Uffen, R. L., 1976, Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate. Proc. Natl. Acad. Sci. USA 73:3298-3302). The medium was autoclaved, and then 1.0 ml/L of vitamin solution (Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296) and 0.5 g/L of yeast extract were added to modified medium 1 in an aerobic chamber. The pH was adjusted to 8.0 by adding 1N NaOH to the basal medium. 30 ml of the prepared medium was dispensed into 150-ml serum bottles, and the gas phase (120 ml) was changed to 100% CO (105 Pa). All the cultures for physiological tests were carried out at 80° C. in anaerobic conditions for 24 hours, and the tests were carried out in duplicate.
2) RNA Extraction and Microarray Design
Cultures were centrifuged at 4° C. at 3,500×g for 10 min, and the total RNA was extracted from the cultures with TRIZOL reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, Calif.). The total RNA samples were quantitatively and qualitatively analyzed by a NanoDrop spectrophotometer (ND-1000, Thermo Scientific) and electrophoresis. The microarray used in this experiment was a Roche NimbleGen microarray. Briefly, a total of 31,994 unique 60-mer oligonucleotides were designed and synthesized in situ using photo-deprotection chemistry. Each unique oligonucleotide was repeated twice on the array (a total of  ̃72,000 features).
3) cDNA Synthesis and Hybridization Conditions
A microarray test was carried out according to the manufacturer's protocol. Each total RNA sample (5 μg) was labeled with Cy5-dCTP (Amersharm, Piscataway, N.J.) by a reverse transcription reaction using reverse transcriptase, SuperScript II (Invitrogen, Carlsbad, Calif.). Then, the labeled cDNA mixture was concentrated using ethanol precipitation. 30 μl of the concentrated Cy5-labeled cDNAs were suspended in a hybridization solution (GenoCheck, Korea). The labeled cDNAs were located on the microarray, and then covered by a MAUI Mixer X4 hybridization chamber (BioMicro Systems, Salt Lake City, Utah). The slides were hybridized using MAUI 12-bay systems (BioMicro Systems, Salt Lake City, Utah) at 42° C. for 12 hours. The hybridized slides were washed at room temperature in 2×SSC, 0.1% SDS for 2 min, 1×SSC for 3 min, and then 0.2×SSC for 2 min. The slides were centrifuged at 1,000×g for 20 sec, followed by drying.
4) Slide Scanning, Normalization, and Data Analysis
Arrays were scanned using a GenePix 4000B scanner (Molecular Devices Corporation, Union City, Calif.), and the data were extracted using NimbleScan 2.4 software. Array normalization was performed using a median polish and quantile normalization method (Amaratunga, D., and J. Cabrera. 2001. Statistical analysis of viral microchip data. J. Am. Stat. Assoc. 96:1161-1170). Normalized expression values for the individual probes were used to obtain expression values for a given ORF using the RMA (robust multi-array average) method previously described by Irizarry et al. (Karl, D. M. 1995. The microbiology of deep-sea hydrothermal vents. CRC Press, Boca Raton, Fla.). Finally, n-fold change ratios (R) were calculated using the RMA-processed expression values (RMA calls) obtained for a particular gene in a sample. Data analysis was performed using GeneSpring GX 7.3.1 (Agilent Technologies, CA). Fold change filters included the requirement that the genes be present in at least 200% of controls for up-regulated genes and lower than 50% of controls for down-regulated genes. The data were clustered into groups of genes that behave similarly in experiments using GeneSpring GX 7.3.1 (Agilent technologies, CA). An algorithm based on the Euclidean distance and average linkage was used to separate the gene of similar patterns.
5) Quantitative RT-PCR
Gene specific primers were designed from the genome sequence of T. onnurineus NA1 (Genbank accession number CP000855). The primer sequences are shown in Table 3 below.
cDNAs were synthesized from 350 ng of total RNA using reverse transcriptase, SuperScrip II (Invitrogen, Carlsbad, Calif.), according to the manufacturer's protocol. PCR reactions were performed with rTaq (Takara) DNA polymerase using a T1 thermocycler (Biometra). The reactions were performed in 50 μl of a mixture containing first-strand reaction cDNA, 10 pmol primers, 250 μM dNTPs and manufacturer's buffer. Also, the PCR amplification reactions were performed in the following conditions: denaturation of 2 min for 94° C.; and then 25 cycles of denaturation (30 sec at 94° C.), annealing (30 sec at 56° C.) and extension (1 min at 72° C.). The PCR products were analyzed by 0.8% agarose gel electrophoresis. The expression level was measured using GelPro32.EXE v4.6 (Media Cybernetics, Inc.). A Chaperonin-encoding gene, named “cha”, was used as a control to normalize expression levels.







TABLE 3 







Oligonucleotides used in this Example









Gene name
Sense (5′→3′)
Anti-sense (5′→3′)










Primers used in RT-PCR









mbh
Cacgacataggctacgacacgg (SEQ ID NO: 27)
Ctggcttaactcctaggtcagg (SEQ ID NO: 28)


mbx
Gcgattcggtatgataccggac (SEQ ID NO: 29)
Ccatccttcgccgaagagctcg (SEQ ID NO: 30)


frh
Gtaagctcgacgagtacgacgtg (SEQ ID NO: 31)
Gcaccacaacctatgcagaggcc (SEQ ID NO: 32)


sulfI
Gcagtacgaggaagtcgagggg (SEQ ID NO: 33)
Gagggcctcgtcgataaggtcg (SEQ ID NO: 34)


mch
Ctaccggacgattggccagaagg (SEQ ID NO: 35)
Ccttatatactgtgctctctccg (SEQ ID NO: 36)


mth1
Gcgaccggtacggcaaccttcg (SEQ ID NO: 37)
Cttgtcagtcatgacgtagtgg (SEQ ID NO: 38)


mth2
Gacccgaggttcacctcgatagc (SEQ ID NO: 39)
Gcagacctggtcgtaggttagcc (SEQ ID NO: 40)


 







Primers used in gene disruption









Flk-mch
cgttgtctttgcccttggggcagggatatatc
ggcaattgcttggactgccgaaaagccaatggc



(SEQ ID NO: 41)
(SEQ ID NO: 42)


Flk-mfh1
gaagaaatcgcagagggcgcctatgactatcag
gctcctcgcttactcaagcgttggacaaatgg



(SEQ ID NO: 43)
(SEQ ID NO: 44)


Flk-mfh2
ggactgctcttcctgtcgacgggctcaatattc
ggacgcacttaaagtcggcgtagccctttgcc



(SEQ ID NO: 45)
(SEQ ID NO: 46)


Ivs-mch
aatttaccaccccaccactcccaaaatccaac
aatggggaggctgaaactactgggcaaggc



(SEQ ID NO: 47)
(SEQ ID NO: 48)


Ivs-mfh1
tggcccaggcgatttccttcaccgacagg
aattcaccaccccaccagcgctattatcagg



(SEQ ID NO: 49)
(SEQ ID NO: 50)


Ivs_mfh2
gagcaccacctcaccatcccagggaagctatc
gatggccgtgacgctgaagtaccccttcgtga



(SEQ ID NO: 51)
(SEQ ID NO: 52)


 







Primers used in identification of gene disruption









Pgdh-hmgPfu
gaacggtagttttcgacaaaagacg
gctcaccagccaaaaccgcaccagc



(SEQ ID NO: 53)
(SEQ ID NO: 54)


mchTNA1
gcaatgtaccacatattcaactgcgatac
ccgataccgagtttgaatggaggaatctc



(SEQ ID NO: 55)
(SEQ ID NO: 56)


mfh1TNA1
tcaggccacccccttgcccttctgt
atggagtgcagcgtgtgtgcgggtg



(SEQ ID NO: 57)
(SEQ ID NO: 58)


mfh2TNA1
atgtctgaagttatcaagtttaacg
tgaggcctttatggagagcttgttg



(SEQ ID NO: 59)
(SEQ ID NO: 60)









6) Targeted Gene Disruption
To analyze the function of hydrogenases in vivo of T. onnurineus, insertional inactivation mutants of the large subunit of mch or mfh2 hydrogenase were constructed using a gene disruption system used for the hyperthermophilic archaeon T. kodakaraensis KOD1 (Sapra, R., K. Bagramyan, and M. W. W. Adams, 2003, A simple energy-conserving system: Proton reduction coupled to proton translocation. Proc. Natl. Acad. Sci. USA 100:7545-7550). Specifically, DNA fragments comprising the flanking region of the large subunits ((TON—023 and TON—1569) of each of mch and mfh2 hydrogenases were amplified from the genomic DNA of T. onnurineus NA1 using primer sets (Table 3) for Flk-mch or Flk-mfh2. Each of the amplified fragments was ligated into pUC118 digested with HincII. Next, a template (Flk-mch_pUC118 or Flk-mfh2_pUC118 recombinant plasmid) was subjected to inverse PCR using a set of primers (Ivs-mch or Ivs-mfh2) (Table 3), and then ligated into a Pgdh-hmgPfu cassette (Sapra, R., K. Bagramyan, and M. W. W. Adams. 2003. A simple energy-conserving system: Proton reduction coupled to proton translocation. Proc. Natl. Acad. Sci. USA 100:7545-7550). The ligated product was transformed into Escherichia coli DH5α cells. Recombinant plasmids (mch::Pgdh-hmgPfu and mfh2::Pgdh-hmgPfu) were prepared with the plasmid mini kit (Qiagen, Hilden, Germany). Finally, the plasmids were transformed into T. onnurineus NA1 using a slight modification of the method of Sato et al. (Sato, T., T. Fukui, H. Atomi, and T. Imanaka. 2003. Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 185:210-220., Sato, T., T. Fukui, H. Atomi, and T. Imanaka. 2005. Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis. Appl. Environ. Microbiol. 71:3889-3899). The transformants were screened in ASW-YT-S medium in the presence of 10 μM simvastatin (Matsumi, R., K. Manabe, T. Fukui, H. Atomi, and T. Imanaka. 2007. Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance. J. Bacteriol. 189:2683-2691), and the candidate groups thought that the target gene was deleted therefrom could be confirmed by examining whether the Pgdh-hmgPfu cassette was present in the target region.
7) Kinetics on Growth and Hydrogen Production
Growth was observed directly by the eye. Samples were diluted in sterile water containing sea salt (30.0 g/L), formalin (2.5%) and 4′-6′-diamidino-2-phenylindole (0.01%) (Sato, T., T. Fukui, H. Atomi, and T. Imanaka. 2005. Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis. Appl. Environ. Microbiol. 71:3889-3899). The diluted samples were filtered through a black polycarbonate filter (pore size: 0.2 μm; Whatman), and then analyzed by an optical phase contrast microscope (Zeiss Axioplan). Hydrogen gas was measured using a gas chromatograph HP 5890 series II (Hewlett Packard) equipped with a HP-PLOT Molesieve column (Agilent) and a TCD detector. Argon was used as a gas carrier. The oven temperature was 40° C. 10 μl of a gas sample for analysis was taken with a gas-tight syringe through a butyl rubber plug. The measurement of the detected hydrogen gas was calculated by comparing the peak area with a calibration curve obtained by regression analysis using a standard gas containing 40% hydrogen in nitrogen.
(3) Test Results
1) In Silico Analysis of T. Onnurineus NA1 Hydrogenase
The previous genomic analysis of T. onnurineus NA1 showed the presence of eight hydrogenase gene clusters (Porter, K. G. and Y. S. Feig. 1980. The use of DAPI for identifying and counting microflora. Limnol. Oceanogr. 25:943-948), which include five membrane-bound [NiFe]-hydrogenases (Mbh, TON—1583-1595; Mbx, TON—0486-0498; Mfh1, TON—0273-0278; Mfh2, TON—1566-1573; and Mch, TON—1021-1024), and three cytoplasmic [NiFe]-hydrogenases (Fru, TON—1559-1562; Sulf I, TON—0533-0544; and Sulf II, TON—0051-0055). Through the comparative analysis of hydrogenase gene clusters and the Thermococcales strains whose genome sequencing has been completed, it could be seen that clusters homology to Mth1, Mfh2 and Mch clusters were very rare and were found in Thermococcales strains whose genome sequences have recently been determined, such as T. barophilus MP (Mfh1 and Mch homologues), Thermococcus sp. AM4 (Mfh1 and Mch homologues) (Unfinished sequence, GenBank accession number ABXN00000000), and T. gammatolerans (Mth1 and Mfh2 homologues) (GenBank accession number CP001398). The sequencing of fdh1-mfh1-mnh1 (termed “Hyg4-I” in the paper of Lee, H. S., S. G. Kang, S. S. Bae, J. K. Lim, Y. Cho, Y. J. Kim, J. H. Jeon, S.-S. Cha, K. K. Kwon, H.-T. Kim, C.-J. Park, H.-W. Lee, S. I. Kim, J. Chun, R. R. Colwell, S.-J. Kim, and J.-H. Lee. 2008. The complete genome sequence of Thermococcus onnurineus NA1 reveals a mixed heterotrophic and carboxydotrophic metabolism. J. Bacteriol. 190:7491-7499), fdh2-mfh2-mnh2 (termed “Hyg4-III”) and codh-mch-mnh3 (termed “Hyg4-II”) clusters in T. onnurineus NA1 showed that each of the clusters included oxidoreductases such as formate dehydrogenase (FDH) and CO dehydrogenase (Codh). Particularly, carboxydotrophic metabolism resulting from growth in a CO-containing atmosphere suggests the functional role of Codh-Mch-Mnh3 that provides energy in hydrogen production pathways by oxidizing CO.
2) Expression of Hydrogenase Genes Under CO-Driven Growth Conditions
A test for determining whether T. onnurineus NA1 can produce hydrogen while growing in a CO-containing atmosphere was carried out. As shown in FIGS. 7A and 7B, in the YPS medium, hydrogen production could not be detected, but in medium 1 supplemented with CO, the total hydrogen gas and the cell number increased with an increase in culture time.
In order to examine which of hydrogenases are involved in hydrogen production during carboxydotrophic growth, the expression levels of hydrogenase genes in a CO-containing growth condition or a complex medium (YPS) were analyzed. As shown in Tables 4 and 5 and FIG. 8A, the expression levels of some ORFs (10 of 16 ORFs) in the codh-mch-mnh3 cluster were up-regulated at least two-fold in the CO-containing growth condition compared to the YPS. In addition, the expression levels of several ORFs (TON—1563, and TON—1569-1571) in the fdh2-mfh2-mnh2 cluster were also up-regulated in a CO-containing growth condition containing 1 g of yeast extract. The expression levels of ORFs in the codh-mch-mnh3 cluster varied depending on the amount of yeast extract, suggesting that the yeast extract has a correlation with the inhibition or activation of catabolism in CO-driven metabolism (Tables 4 and 5). On the other hand, the expression of other hydrogenase gene clusters did not greatly change, whereas the expression of the genes (20 genes of 29 ORFs) in the fdh1-mfh1-mnh1 cluster was down-regulated. Quantitative RT-PCR data for the large subunit of each of hydrogenases were also consistent with the microarray data. The expression of the large subunits (TON—1023 and TON—1569) of mch and mfh2 hydrogenases was increased at least two-fold (FIG. 8B), whereas the expression of the large subunit (TON—0276) of mfh1 hydrogenase was inhibited and the expression of other large subunits (mbh, mbx, frh, and sulfl) was maintained constant in both conditions. Such results suggest that the codh-mch-mnh3 or fdh2-mfh2-mnh2 clusters can be derived by CO and involved in hydrogen production processes associated with carboxydotrophic metabolism.







TABLE 4







Expression levels of ORFs of each of hydrogenase gene clusters in


CO-containing growth condition compared to those in YPS medium











Gene cluster

M + CO + 0.5 g
M + CO + 1 g
M + CO + 3 g


and ORFs
Annotation
YE/YPS
YE/YPS
YE/YPS














sulfII hydrogenase






cluster


TON_0051
hypothetical protein
1.273306
3.1454
2.569546


TON_0052
hydrogenase subunit alpha
1.085002
0.652365
0.550585


TON_0053
Sulfhydrogenase II, delta chain
0.896129
1.898148
1.337585


TON_0054
hydrogenase subunit gamma
0.606088
0.517724
0.630549


TON_0055
sulfhydrogenase II, beta chain
0.84527
1.44889
1.452254


fdh1-mfh1-mnh1


cluster


TON_0261
hypA hydrogenase expression/formation protein
0.658409
0.78969
0.746964


TON_0262
ATPase involved in chromosome partitioning, ParA/MinD
0.640277
0.776389
0.619674



family, Mrp homolo


TON_0263
hydrogenase maturation protease HycI
0.775719
1.531337
1.090123


TON_0264
molybdopterin-guanine dinucleotide biosynthesis protein A
0.538102
0.957335
0.826924


TON_0265
Nucleotidyltransferase, putative
0.421398
0.554433
0.503557


TON_0266
component F or formate hydrogen lyase
0.38336
0.338185
0.333459


TON_0267
Putative integral membrane protein, DUF68 family
0.296945
1.215972
0.838087


TON_0268
Putative integral membrane protein, DUF67 family
0.648077
0.424896
0.386235


TON_0269
Multisubunit Na+/H+ antiporter, putative MnhB subunit
0.591411
0.571996
0.600235


TON_0270
hypothetical protein
0.827927
0.85379
0.581906


TON_0271
Na+/H+ antiporter subunit
0.410417
0.328062
0.51901


TON_0272
Na+/H+ antiporter MnhF subunit, putative
0.194663
0.411647
0.331549


TON_0273
hypothetical protein
0.269222
0.652722
0.482705


TON_0274
component I or formate hydrogen lyase
0.385799
0.627629
0.614487


TON_0275
formate hydrogen lyase subunit 6
0.313283
0.621333
0.511819


TON_0276
component G or formate hydrogen lyase
0.275138
0.724228
0.692331


TON_0277
component C or formate hydrogen lyase
0.460646
0.357836
0.387126


TON_0278
component B or formate hydrogen lyase
0.720779
0.623875
0.510273


TON_0279
NADH ubiquinone oxidoreductase
0.80929
0.515429
0.54289


TON_0280
oxidoreductase iron-sulfur protein
0.3913
0.690322
0.647104


TON_0281
fdhA formate dehydrogenase, alpha subunit
0.584576
0.446401
0.44946


TON_0282
putative transcriptional regulator
1.340444
1.195043
0.883294


TON_0283
hypC hydrogenase expression/formation protein
1.007318
1.853137
2.021914


TON_0284
367aa long hypothetical hydrogenase expression/formation
1.036497
1.385466
1.182663



protein hypD


TON_0285
hypD hydrogenase expression/formation protein
0.904687
1.066448
0.758981


TON_0286
hydrogenase maturation protein HypF
0.685606
0.778307
0.719973


TON_0287
hydrogenase expression/formation protein HypE
0.851301
1.399569
1.257059


TON_0288
hypothetical protein
1.004008
1.964442
2.27641


TON_0289
cysteine desulfurase
0.666768
1.534991
1.148004


mbx hydrogenase


cluster


TON_0486
4Fe—4S cluster-binding subunit
0.821271
0.718277
0.504388


TON_0487
nuoD NADH dehydrogenase I, subunit D
0.606823
0.730138
0.517528


TON_0488
NADH dehydrogenase subunit
0.703506
0.618352
0.49939


TON_0489
nuoB NADH dehydrogenase I, subunit B
0.797159
0.618863
0.547462


TON_0490
NADH dehydrogenase subunit
0.372425
0.469467
0.278163


TON_0491
617aa long hypothetical protein
0.459201
0.532164
0.388201


TON_0492
MbxH subunit
0.862715
0.484349
0.423606


TON_0493
Multisubunit Na+/H+ antiporter, putative
0.70216
1.591408
1.138149


TON_0494
Multisubunit Na+/H+ antiporter, putative MnhB subunit
0.765286
0.716126
1.711304


TON_0495
MbxD subunit
0.658086
0.817339
0.749402


TON_0496
MbxC subunit
0.609819
1.200655
0.995067


TON_0497
MbxB subunit
0.860304
0.889029
0.810902


TON_0498
Multisubunit Na+/H+ antiporter, putative
0.675578
0.564247
0.483617


sulfI hydrogenase


cluster


TON_0533
hydrogenase-specific maturation endopeptidase
0.977803
0.516234
0.73084


TON_0534
cytosolic NiFe-hydrogenase, alpha subunit
1.283309
1.334781
1.071667


TON_0535
cytosolic NiFe-hydrogenase, delta subunit
0.652099
1.080516
0.800955


TON_0536
hydrogenase (gamma subunit)
1.100959
1.192121
0.759086


TON_0537
Sulfhydrogenase beta subunit
1.025726
1.458113
1.160718


TON_0538
probable formate transporter
0.989789
1.113613
0.960108


TON_0539
probable formate dehydrogenase, alpha subunit
1.15S997
1.791123
1.314525


TON_0540
oxidoreductase iron-sulfur protein
0.987581
1.525167
0.82483


TON_0541
4Fe—4S cluster-binding protein
1.112648
1.320761
0.855465


TON_0542
glutamate synthase beta chain-related oxidoreductase
0.842213
1.602828
0.936949


TON_0543
4Fe—4S cluster-binding protein
0.9185
1.257283
1.019564


TON_0544
alcohol dehydrogenase
0.87473
0.419323
0.605934


codh-mch-mnh3


cluster


TON_1016
putative transcriptional regulator, ModE family
1.18798
0.634394
0.731218


TON_1017
4Fe—4S ferredoxin, iron-sulfur binding domain
1.713085
4.46107
3.621394


TON_1018
carbon-monoxide dehydrogenase, catalytic subunit
1.474238
2.204944
1.785437


TON_1019
putative ATP-binding protein
0.824732
2.016924
0.974466


TON_1020
hypothetical protein
3.375546
9.366047
5.425704


TON_1021
component B or format hydrogen lyase
1.112538
1.711122
1.250867


TON_1022
respiratory-chain NADH dehydrogenase, subunit 1
0.877363
1.428883
0.57715


TON_1023
component G or format hydrogen lyase
1.927395
6.118015
4.270288


TON_1024
NADH dehydrogenase (ubiquinone), 20 kDa subunit
1.816315
3.578841
2.009973


TON_1025
Na+/H+ antiporter MnhF subunit, putative
2.443938
3.389037
1.333503


TON_1026
Na+/H+ antiporter subunit
1.337719
0.605839
0.623499


TON_1027
hypothetical protein
0.991048
1.43866
0.778446


TON_1028
Multisubunit Na+/H+ antiporter, MnhB subunit
1.019583
1.203407
0.836452


TON_1029
Putative integral membrane protein, DUF67 family
2.648286
2.171138
1.123898


TON_1030
Putative integral membrane protein, DUF68 family
3.779798
4.956998
1.592196


TON_1031
component F or formate hydrogen lyase
1.030507
2.778588
1.982091


frh (F420 reducing


hydrogenase)


hydrogenase cluster


TON_1559
coenzyme F420 hydrogenase alpha subunit
0.695408
0.64447
0.726535


TON_1560
Coenzyme F420 hydrogenase gamma subunit
0.56769
0.715513
0.635236


TON_1561
4Fe—4S ferredoxin, iron-sulfur binding Nitrite/sulfite
0.824149
0.834127
0.807754



reductase


TON_1562
Formate dehydrogenase, subunit FdhD
0.908701
1.54082
1.468288


Fdh2-mfh2-mnh2


cluster


TON_1563
Probable formate dehydrogenase, alpha subunit
1.008259
3.037865
3.330042


TON_1564
4Fe—4S cluster-binding protein
1.178705
1.221574
1.445098


TON_1565
NADH ubiquinone oxidoreductase
0.811937
1.787894
2.088693


TON_1566
NADH dehydrogenase (quinone)
1.011779
0.736029
0.787627


TON_1567
component B or formate hydrogen lyase
1.091992
1.057076
1.184573


TON_1568
component C or formate hydrogen lyase
1.471267
1.365925
1.382301


TON_1569
component G or formate hydrogen lyase
1.204795
2.281342
1.780033


TON_1570
formate hydrogen lyase subunit 6
1.257649
2.427962
1.99559


TON_1571
component I or formate hydrogen lyase
1.030626
2.15871
1.806722


TON_1572
hypothetical protein
0.844948
0.504955
0.692272


TON_1573
probable formate transporter
1.451628
1.459657
1.158131


TON_1574
Na+/H+ antiporter MnhF subunit, putative
1.53015
1.602287
1.244315


TON_1575
Na+/H+ antiporter subunit
0.752784
0.646406
0.578467


TON_1576
hypothetical protein
1.168966
0.564328
0.60819


TON_1577
Multisubunit Na+/H+ antiporter, putative MnhB subunit
0.726511
0.655031
0.556386


TON_1578
Putative integral membrane protein, DUF67 family
0.989286
1.061599
0.900044


TON_1579
Putative integral membrane protein, DUF68 family
0.903028
0.812346
0.816319


TON_1580
component F or formate hydrogen lyase
1.015786
0.756283
0.529199


TON_1581
molybdopterin-guanine dinucleotide biosynthesis protein A
0.61468
1.708744
1.243367


TON_1582
hypothetical protein
0.753301
0.998059
0.952751


mbh hydrogenase


cluster


TON_1583
MbhB subunit
1.038442
0.519366
0.533773


TON_1584
MbhC subunit
0.947559
0.514932
0.5099


TON_1585
MbhD subunit
0.774604
1.015909
0.670497


TON_1586
MbhE subunit
1.101069
0.811262
0.659266


TON_1587
MbhF subunit
1.129566
0.811366
0.872572


TON_1588
MbhG subunit
1.027148
0.563686
0.510924


TON_1589
MbhH subunit
1.362042
0.948034
0.71984


TON_1590
MbhI subunit
1.334732
1.227655
0.904813


TON_1591
NiFe-hydrogenase small subunit
1.627315
1.158127
0.856929


TON_1592
NiFe-hydrogenase large subunit 1
0.940698
1.573248
0.956935


TON_1593
NiFe-hydrogenase large subunit 2
1.609483
2.286207
1.354849


TON_1594
MbhM subunit
0.982323
0.742211
0.458623


TON_1595
4Fe—4S cluster-binding subunit
1.589684
1.280298
0.491906





M, medium 1; CO, carbon monoxide; YE, yeast extract.













TABLE 5







Hierarchical clustering of 112 ORFs including hydrogenase clusters from T. onnurineus NA1















M + CO + 0.5 g
M + CO + 1 g
M + CO + 3 g


Gene cluster
ORFs
Annotation
YE/YPS
YE/YPS
YE/YPS















fdh1-mfh1-mnh1
TON_0272
Na+/H+ antiporter MnhF subunit, putative
0.194663
0.411647
0.331549


fdh1-mfh1-mnh1
TON_0267
Putative integral membrane protein, DUF68 family
0.296945
1.215972
0.838087


fdh1-mfh1-mnh1
TON_0273
hypothetical protein
0.269222
0.652722
0.482705


fdh1-mfh1-mnh1
TON_0275
formate hydrogen lyase subunit 6
0.313283
0.621333
0.511819


fdh1-mfh1-mnh1
TON_0276
component G or formate hydrogen lyase
0.275138
0.724228
0.692331


fdh1-mfh1-mnh1
TON_0274
component I or formate hydrogen lyase
0.385799
0.627629
0.614487


fdh1-mfh1-mnh1
TON_0280
oxidoreductase iron-sulfur protein
0.3913
0.690322
0.647104


fdh1-mfh1-mnh1
TON_0271
Na+/H+ antiporter subunit
0.410417
0.328062
0.51901


fdh1-mfh1-mnh1
TON_0266
component F or formate hydrogen lyase
0.38336
0.338185
0.333459


fdh1-mfh1-mnh1
TON_0277
component C or formate hydrogen lyase
0.460646
0.357836
0.387126


fdh1-mfh1-mnh1
TON_0268
Putative integral membrane protein, DUF67 family
0.648077
0.424896
0.386235


fdh1-mfh1-mnh1
TON_0281
fdhA formate dehydrogenase, alpha subunit
0.584576
0.446401
0.44946


fdh1-mfh1-mnh1
TON_0265
Nucleotidyltransferase, putative
0.421398
0.554433
0.503557


mbx
TON_0491
617aa long hypothetical protein
0.459201
0.532164
0.388201


mbx
TON_0490
NADH dehydrogenase subunit
0.372425
0.469467
0.278163


fdh1-mfh1-mnh1
TON_0261
hypA hydrogenase expression/formation protein
0.658409
0.78969
0.746964


mbx
TON_0495
MbxD subunit
0.658086
0.817339
0.749402


fdh1-mfh1-mnh1
TON_0286
hydrogenase maturation protein HypF
0.685606
0.778307
0.719973


mbx
TON_0494
Multisubunit Na+/H+ antiporter, putative MnhB subunit
0.765286
0.716126
0.711304


frh
TON_1559
coenzyme F420 hydrogenase alpha subunit
0.695408
0.64447
0.726535


mbx
TON_0487
nuoD NADH dehydrogenase I, subunit D
0.606823
0.730138
0.517528


fdh1-mfh1-mnh1
TON_0262
ATPase involved in chromosome partitioning, Mrp homolo
0.640277
0.776389
0.619674


frh
TON_1560
Coenzyme F420 hydrogenase gamma subunit
0.56769
0.715513
0.635236


sufII
TON_0054
hydrogenase subunit gamma
0.606088
0.517724
0.630549


fdh1-mfh1-mnh1
TON_0269
Multisubunit Na+/H+ antiporter, putative MnhB subunit
0.591411
0.571996
0.600235


mbx
TON_0486
4Fe—4S cluster-binding subunit
0.821271
0.718277
0.504388


fdh1-mfh1-mnh1
TON_0278
component B or formate hydrogen lyase
0.720779
0.623875
0.510273


mbx
TON_0488
NADH dehydrogenase subunit
0.703506
0.618352
0.49939


mbx
TON_0498
Multisubunit Na+/H+ antiporter, putative
0.675578
0.564247
0.483617


mbx
TON_0489
nuoB NADH dehydrogenase I, subunit B
0.797159
0.618863
0.547462


fdh2-mfh2-mnh2
TON_1575
Na+/H+ antiporter subunit
0.752784
0.646406
0.578467


fdh2-mfh2-mnh2
TON_1577
Multisubunit Na+/H+ antiporter, putative MnhB subunit
0.726511
0.655031
0.556386


sulfI
TON_0544
alcohol dehydrogenase
0.87473
0.419323
0.605934


sulfI
TON_0533
hydrogenase-specific maturation endopeptidase
0.977803
0.516234
0.73084


fdh2-mfh2-mnh2
TON_1572
hypothetical protein
0.844948
0.504955
0.692272


mbx
TON_0492
MbxH subunit
0.862715
0.484349
0.423606


fdh1-mfh1-mnh1
TON_0279
NADH ubiquinone oxidoreductase
0.80929
0.515429
0.54289


mbh
TON_1584
MbhC subunit
0.947559
0.514932
0.5099


mbh
TON_1583
MbhB subunit
1.038442
0.519366
0.533773


mbh
TON_1588
MbhG subunit
1.027148
0.563686
0.510924


sufII
TON_0052
hydrogenase subunit alpha
1.085002
0.652365
0.550585


fdh2-mfh2-mnh2
TON_1580
component F or formate hydrogen lyase
1.015786
0.756283
0.529199


mbh
TON_1594
MbhM subunit
0.982323
0.742211
0.458623


codh-mch-mnh3
TON_1016
putative transcriptional regulator, ModE family
1.187798
0.634394
0.731218


codh-mch-mnh3
TON_1026
Na+/H+ antiporter subunit
1.337719
0.605839
0.623499


fdh2-mfh2-mnh2
TON_1576
hypothetical protein
1.168966
0.564328
0.60819


codh-mch-mnh3
TON_1022
respiratory-chain NADH dehydrogenase, subunit 1
0.877363
1.428883
0.57715


fdh1-mfh1-mnh1
TON_0264
molybdopterin-guanine dinucleotide biosynthesis protein A
0.538102
0.957335
0.826924


sulfI
TON_0535
cytosolic NiFe-hydrogenase, delta subunit
0.652099
1.080516
0.800955


mbx
TON_0496
MbxC subunit
0.699819
1.200655
0.995067


fdh2-mfh2-mnh2
TON_1582
hypothetical protein
0.753301
0.998059
0.952751


fdh1-mfh1-mnh1
TON_0270
hypothetical protein
0.827927
0.85379
0.581906


fdh1-mfh1-mnh1
TON_0285
hypD hydrogenase expression/formation protein
0.904687
1.066448
0.758981


mbh
TON_1585
MbhD subunit
0.774604
1.015909
0.670497


mbx
TON_0497
MbxB subunit
0.860304
0.889029
0.810902


frh
TON_1561
4Fe—4S ferredoxin, iron-sulfur binding Nitrite/sulfite
0.824149
0.834127
0.807754




reductase


fdh2-mfh2-mnh2
TON_1579
Putative integral membrane protein, DUF68 family
0.903028
0.812346
0.816319


fdh2-mfh2-mnh2
TON_1566
NADH dehydrogenase (quinone)
1.011779
0.736019
0.787627


mbh
TON_1587
MbhF subunit
1.129566
0.811366
0.872572


mbh
TON_1586
MbhE subunit
1.101069
0.811262
0.659266


sufII
TON_0051
hypothetical protein
1.273306
3.1454
2.569546


fdh2-mfh2-mnh2
TON_1563
Probable formate dehydrogenase, alpha subunit
1.008259
3.037865
3.330042


fdh1-mfh1-mnh1
TON_0283
hypC hydrogenase expression/formation protein
1.007318
1.853137
2.021914


fdh1-mfh1-mnh1
TON_0288
hypothetical protein
1.004008
1.964442
2.27641


fdh2-mfh2-mnh2
TON_1571
component I or formate hydrogen lyase
1.030636
2.15871
1.806722


fdh2-mfh2-mnh2
TON_1565
NADH ubiquinone oxidoreductase
0.811937
1.787894
2.088693


codh-mch-mnh3
TON_1031
component F or formate hydrogen lyase
1.030507
2.788568
1.982091


codh-mch-mnh3
TON_1018
carbon-monoxide dehydrogenase, catalytic subunit
1.474238
2.204944
1.785437


fdh2-mfh2-mnh2
TON_1569
component G or formate hydrogen lyase
1.204795
2.281342
1.760033


fdh2-mfh2-mnh2
TON_1570
formate hydrogen lyase subunit 6
1.257649
2.427962
1.99559


fdh1-mfh1-mnh1
TON_0287
hydrogenase expression/formation protein HypE
0.851301
1.399569
1.257059


sufII
TON_0055
sulfhydrogenase II, beta chain
0.84527
1.44889
1.452254


frh
TON_1562
Formate dehydrogenase, subunit FdhD
0.908701
1.54082
1.468288


sufII
TON_0053
Sulfhydrogenase II, delta chain
0.896129
1.898148
1.337585


sulfI
TON_0539
probable formate dehydrogenase, alpha subunit
1.158997
1.791123
1.314525


codh-mch-mnh3
TON_1021
component B or format hydrogen lyase
1.112538
1.711122
1.250867


fdh1-mfh1-mnh1
TON_0263
hydrogenase maturation protease HycI
0.775719
1.531337
1.090123


fdh1-mfh1-mnh1
TON_0289
cysteine desulfurase
0.666768
1.534991
1.148004


mbx
TON_0493
Multisubunit Na+/H+ antiporter, putative
0.70216
1.591408
1.138149


fdh2-mfh2-mnh2
TON_1581
molybdopterin-guanine dinucleotide biosynthesis protein A
0.61468
1.708744
1.243367


codh-mch-mnh3
TON_1019
putative ATP-binding protein
0.824732
2.016924
0.974466


sulfI
TON_0542
glutamate synthase beta chain-related oxidoreductase
0.842213
1.602828
0.936949


mbh
TON_1592
NiFe-hydrogenase large subunit 1
0.940698
1.573248
0.956935


fdh1-mfh1-mnh1
TON_0284
367aa long hypothetical hydrogenase protein hypD
1.036497
1.385466
1.152663


sulfI
TON_0537
Sulfhydrogenase beta subunit
1.025726
1.458113
1.160718


sulfI
TON_0534
cytosolic NiFe-hydrogenase, alpha subunit
1.283309
1.334781
1.071667


fdh2-mfh2-mnh2
TON_1564
4Fe—4S cluster-binding protein
1.178705
1.221574
1.445098


fdh2-mfh2-mnh2
TON_1567
component B or formate hydrogen lyase
1.091992
1.057076
1.184573


fdh2-mfh2-mnh2
TON_1568
component C or formate hydrogen lyase
1.471267
1.365925
1.382301


fdh2-mfh2-mnh2
TON_1573
probable formate transporter
1.451628
1.459657
1.158131


fdh2-mfh2-mnh2
TON_1574
Na+/H+ antiporter MnhF subunit, putative
1.53015
1.602287
1.244315


sulfI
TON_0541
4Fe—4S cluster-binding protein
1.112648
1.320761
0.855465


sulfI
TON_0536
hydrogenase (gamma subunit)
1.100959
1.192121
0.759086


codh-mch-mnh3
TON_1028
Multisubunit Na+/H+ antiporter, putative MnhB subunit
1.019583
1.203407
0.836452


sulfI
TON_0540
oxidoreductase iron-sulfur protein
0.987581
1.525167
0.82485


codh-mch-mnh3
TON_1027
hypothetical protein
0.991048
1.43866
0.778446


sulfI
TON_0543
4Fe—4S cluster-binding protein
0.9185
1.257283
1.019564


sulfI
TON_0538
probable formate transporter
0.989789
1.113613
0.960108


fdh2-mfh2-mnh2
TON_1578
Putative integral membrane protein, DUF67 family
0.989286
1.061599
0.900044


fdh1-mfh1-mnh1
TON_0282
putative transcriptional regulator
1.340444
1.195043
0.883294


mbh
TON_1590
MbhI subunit
1.334732
1.227655
0.904813


mbh
TON_1591
NiFe-hydrogenase small subunit
1.627315
1.158127
0.856929


mbh
TON_1589
MbhH subunit
1.362042
0.948034
0.71984


mbh
TON_1595
4Fe—4S cluster-binding subunit
1.589684
1.280298
0.491906


codh-mch-mnh3
TON_1030
Putative integral membrane protein, DUF68 family
3.779798
4.956998
1.592196


codh-mch-mnh3
TON_1024
NADH dehydrogenase (ubiquinone), 20 kDa subunit
1.816315
3.578841
2.009973


codh-mch-mnh3
TON_1025
Na+/H+ antiporter MnhF subunit, putative
2.443938
3.389037
1.333503


codh-mch-mnh3
TON_1029
Putative integral membrane protein, DUF67 family
2.648286
2.171138
1.123898


mbh
TON_1593
NiFe-hydrogenase large subunit 2
1.609483
2.286207
1.354849


codh-mch-mnh3
TON_1020
hypothetical protein
3.375546
9.366047
5.425704


codh-mch-mnh3
TON_1017
4Fe—4S ferredoxin, iron-sulfur binding domain
1.713085
4.46107
3.621394


codh-mch-mnh3
TON_1023
component G or formate hydrogen lyase
1.927395
6.118015
4.270288









3) Gene Disruption and Phenotype Analysis of Disruption Mutants
Transcriptome analysis suggests that the mch hydrpogenase clustered close to codh (FIG. 9A) can play an important role in carboxydotrophic hydrogenogenesis in T. onnurineus NA1. However, the up-regulation of the fdh2-mfh2-mnh2 cluster and the high copy number of mRNA of other clusters raise a question about whether codh-mch-mnh3 alone is involved in carboxydotrophic hydrogenogenesis or whether other hydrogenases can become alternative pathways for mch by forming complexes in combination with dehydrogenases or recycling electronic carriers such as FADH2 or NADH. Thus, the present inventors constructed disruption mutants of the large subunit of each of mch and mfh2 hydrogenases (Matsumi, R., K. Manabe, T. Fukui, H. Atomi, and T. Imanaka. 2007. Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance. J. Bacteriol. 189:2683-2691). The method of constructing the disruption mutants are shown in FIG. 9A. The large subunit of the Mch or Mth2 hydrogenase gene cluster was disrupted by insertional inactivation of the Pgdh-hmgPfu, cassette by homologous recombination in the targeted region and the resulting overexpression of the hmg-CoA gene. The targeted gene disruption was confirmed by examining the presence of the Pgdh-hmgPfu, cassette through PCR amplification after screening candidate groups in a YPS medium supplemented with 10 μM simvastatin (FIG. 9B). Pgdh-hmgPfu could be amplified in the disruption candidate groups (ΔmchTNA1 and Δmfh2TNA1), whereas the amplification of the large subunits of mch or mfh2 was failed. Such results show that the gene disruption system reported in T. kodakaraensis KOD1 (Sapra, R., K. Bagramyan, and M. W. W. Adams. 2003. A simple energy-conserving system: Proton reduction coupled to proton translocation. Proc. Natl. Acad. Sci. USA 100:7545-7550) can also be applied to T. onnurinues NA1.
Because the disruption mutants (ΔmchTNA1 and Δmfh2TNA1) could be obtained in the YPS medium, it can be seen that Mch or Mth2 is not essential for the metabolic consumption of the YPS medium. As can be seen in FIGS. 10A-10D, the changes in the growth and morphology of the mutant strains ΔmchTNA1 and Δmfh2TNA1 confirm that the genes are not necessarily essential in the YPS medium. In addition, the Δmfh2TNA1 strain was able to grow and produce hydrogen in the CO-containing growth condition at a level similar to the wild-type strain (FIGS. 7B and 10D). On the other hand, the ΔmchTNA1 mutant was not able to grow in the CO-containing growth condition and did not produce hydrogen in this condition (FIG. 10C). This indicates that the absence of the large subunit of Mch leads to complete disruption of the carboxydotrophic survival ability of T. onnurineus NA1 in the presence of CO. Putting these results together, it appears that, when CO is fed as a substrate, only Mch hydrogenase is involved in growth and hydrogen production.
As described above, the novel hydrogenases of the present invention can produce a large amount of hydrogen by responding specifically to various substrates such as carbon monoxide, formate or starch. According to the hydrogen production methods of the present invention, a large amount of hydrogen can be produced merely by culturing the above-described strain in specific culture conditions. Thus, the methods of the present invention have advantages in that they are more economic and efficient than existing hydrogen production methods and can produce hydrogen even at high temperature.

## Claims

1. A method of producing hydrogen from Thermococcus spp., the method comprising:
preparing a medium in a culture vessel;
culturing Thermococcus spp. in the culture vessel, wherein the Thermococcus spp. is Thermococcus onnurineus NA1 (accession number: KCTC 10859BP); and
extracting hydrogen from the culture vessel.

2. The method of claim 1, wherein the medium is a medium supplemented with one or more selected from the group consisting of carbon monoxide, formate and starch.

3. The method of claim 2, wherein the culturing is carried out at 80° C.

4. The method of claim 2, wherein the culturing is carried out in an anaerobic condition.

5. The method of claim 1, wherein the culturing is carried out at 80° C.

6. The method of claim 1, wherein the culturing is carried out in an anaerobic condition.

7. The method of claim 1, wherein the medium is a medium supplemented with one or more selected from the group consisting of carbon monoxide and formate.