(19) & (11) EP 2 333 054 A2 (12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC (43) Date of publication: (51) Int Cl.: 15.06.2011 Bulletin 2011/24 C12N 9/04 (2006.01) C12N 15/53 (2006.01) C12P 3/00 (2006.01) (21) Application number: 09811732.8 (86) International application number: (22) Date of filing: 07.09.2009 PCT/KR2009/005060 (87) International publication number: WO 2010/027233 (11.03.2010 Gazette 2010/10) (84) Designated Contracting States: • KWON, Kae Kyoung AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Ansan-si HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL Gyeonggi-do 426-150 (KR) PT RO SE SI SK SM TR • CHA, Sun Shin Designated Extension States: Ansan-si AL BA RS Gyeonggi-do 425-020 (KR) • JEON, Jung Ho (30) Priority: 05.09.2008 KR 20080087794 Ansan-si 05.09.2008 KR 20080087806 Gyeonggi-do 426-170 (KR) • CHO, Yona (71) Applicant: Korea Ocean Research and Yongin-si Development Institute Gyeonggi-do 449-913 (KR) Ansan Si, Gyeonggi-do 426-744 (KR) • KIM, Yun Jae Ansan-si (72) Inventors: Gyeonggi-do 426-170 (KR) • LEE, Jung Hyun • BAE, Seung Seop Seongnam-si Ansan-si Gyeonggi-do 463-914 (KR) Gyeonggi-do 426-170 (KR) • KANG, Sung Gyun • LIM, Jae Kyu Ansan-si Siheung-si Gyeonggi-do 426-170 (KR) Gyeonggi-do 429-450 (KR) • LEE, Hyun Sook • JEONG, In Soon Ansan-si Changwon-si Gyeonggi-do 426-170 (KR) Gyeongsangnam-do 641-826 (KR) • KIM, Sang Jin Ansan-si (74) Representative: Viering, Jentschura & Partner Gyeonggi-do 426-751 (KR) Grillparzerstrasse 14 81675 München (DE) (54) NOVEL HYDROGENASE ISOLATED FROM THERMOCOCCUS SPP., GENES ENCODING THE SAME, AND METHODS FOR PRODUCING HYDROGEN USING MICROORGANISMS WITH THE GENES (57) The present invention relates to novel hydroge- in specific culture conditions. Thus, the methods of the nases isolated from novel hyperthermophilic strains be- invention have advantages in that they are more eco- longing to Thermococcus spp., genes encoding the hy- nomic and efficient than existing hydrogen production drogenases, and methods of producing hydrogen using methods and can produce hydrogen even at high tem- strains having the genes. According to the hydrogen pro- perature. duction methods of the invention, a large amount of hy- drogen can be produced merely by culturing the strains EP 2 333 054 A2 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 333 054 A2 2 EP 2 333 054 A2 Description BACKGROUND OF THE INVENTION 5 Technical Field [0001] The present invention relates to novel hydrogenases isolated from novel strains belonging to the genus Ther- mococcus, genes encoding the same, and methods of producing hydrogen using strains having the genes. 10 Background Art [0002] 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. 15 [0003] 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. [0004] On the other hand, biological methods of producing hydrogen using microorganisms have advantages in that 20 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 25 strain having high hydrogen productivity at high salt concentration. [0005] 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. 30 [0006] 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 "Rhodopseduomonas palustris P4 and production of hydrogen using the same". [0007] The present inventors previously filed a patent application relating to novel proteins isolated from novel hyper- 35 thermophilic Thermococcus onnurineus NA1 (accession number: KCTC 10859BP) and genes encoding the same on September 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 40 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 OF THE INVENTION 45 [0008] 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. [0009] 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 50 invention provides a method of producing hydrogen by culturing the strain, and a method of producing hydrogen using the genes. [0010] In a first aspect, the present invention provides hydrogenases which are produced by the novel hyperther- mophilic 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 55 ID NO. 1 to SEQ ID NO: 8. [0011] 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). 3 EP 2 333 054 A2 [0012] 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). 5 [0013] 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. [0014] 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. 10 [0015] 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). [0016] 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, 15 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. [0017] 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. 20 [0018] 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. [0019] Also, the present invention provides a method of producing hydrogen using said transformant, the method 25 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.