Monomeric polypeptides having hydrogenase activity, in particular recombinant monomeric polypeptides having hydrogenase activity

Abstract

The present invention relates to a monomeric polypeptide comprising a single subunit, wherein the single subunit comprises an active site of a [NiFe]-hydrogenase protein, and the monomeric polypeptide has hydrogenase activity.

Description

Technical Field

[0001] The present invention relates to a monomeric polypeptide having hydrogenase activity, in particular to a recombinant monomeric polypeptide having hydrogenase activity, a host cell comprising the monomeric polypeptide, and a host cell comprising a polynucleotide encoding the monomeric polypeptide. The present invention also relates to a method for preparing the monomeric polypeptide having hydrogenase activity, in particular to a method for obtaining a recombinant monomeric polypeptide having hydrogenase activity, and to the application of the monomeric polypeptide, in particular to the application of the recombinant monomeric polypeptide. Background Art

[0002] The reduction in fossil energy supplies and concerns about drastic climate change have led to the emergence of clean and renewable technologies. Hydrogen (H2) is considered a carrier to promote renewable energy due to its high energy content and the absence of greenhouse gases and nanoparticles when used in fuel cells. Currently, H2 is produced by chemical extraction of fossil hydrocarbons, electrolysis of water or pyrolysis of biomass. However, the main limitation of H2 is the lack of pollution-free and commercially viable production systems. Methods for producing hydrogen using biotechnology, also known as biohydrogen, may be a solution. The number of studies on biological hydrogen production has increased significantly in the past decade. This includes microbial production, mainly through bacterial fermentation and microalgae photosynthesis processes, but also enzymatic production of hydrogen through "electroenzymology". All of these methods rely on specific enzymes called hydrogenases.

[0003] Hydrogenases are metalloenzymes that catalyze the chemical reactions: This enzyme-dependent reaction of producing or consuming hydrogen is defined as hydrogenase activity. The reaction is reversible and its direction depends on the redox potential of the component that can interact with the enzyme. In the presence of H2 and an electron acceptor, hydrogenases consume hydrogen. In the presence of a low-potential electron donor, hydrogenases utilize protons as electron acceptors and produce H2. Many microorganisms use hydrogen as an energy carrier or source, so hydrogenases are widespread in microorganisms. These enzymes have many representatives in the bacterial and archaeal domains and are also found in some unicellular eukaryotic organisms. Although hydrogenases are diverse in many aspects (host, size, quaternary structure, electron donor or acceptor), they can be phylogenetically divided into three different classes: [Fe]-hydrogenases, [FeFe]-hydrogenases, and [NiFe]-hydrogenases. Each class is characterized by a unique metal component in the active site. Physiologically, these enzymes have been shown to act as valves for excess electrons.

[0004] The catalytic center of [Fe]-hydrogenase does not contain any FeS or Ni center, so it is named "hydrogenase without [FeS] center" or [Fe]-hydrogenase (Shima and Thauer, 2007, A third type of hydrogenase catalyzing H2activation, Chem Rec 7: 37-46). [Fe]-hydrogenases are limited to certain methanogenic microorganisms (Pilak et al., 2006, The crystal structure of the apoenzyme of the iron-sulphur cluster-free hydrogenase. J Mol Biol 358: 798-809), where they are essential for growth during nickel deficiency (Thauer, 1998, Biochemistry of methanogenesis: a tribute to Marjory Stephenson. 1998 Marjory Stephenson Prize Lecture. Microbiology 144: 2377-2406). These enzymes are associated with specific cofactors and have catalytic properties that are very different from other types of hydrogenases. In fact, these enzymes do not catalyze the reversible H2 generation reaction (Vignais and Billoud, 2007, Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272). For this reason, and also because of their low distribution, this type of hydrogenase has been little studied. The vast majority of known hydrogenases belong to the other two categories.

[0005] [FeFe]-hydrogenase is a highly conserved monomeric enzyme in the catalytic center. It is a binuclear iron site that binds to the [4Fe-4S] center through a cysteine ​​bridge. Non-protein ligands, cyanide (CN) and carbon monoxide (CO) bind to the iron atom in the binuclear center. The iron atom also shares two sulfur ligands (Nicolet et al., 2000, Anovel FeS cluster in Fe-only hydrogenases.Trends Biochem Sci 25:138-143; Nicolet et al., 2002, Fe-only hydrogenases:structure, function and evolution J Inorg Biochem 91:1-8). Other domains have multiple FeS centers and provide electron transfer chains between external electron sources and active sites embedded in these monomeric proteins. In addition, hydrophobic channels connect the surface to the active site and provide channels for protons, providing outlets for H2 molecules. It is known that three chaperone proteins named HydE, HydF and HydG are essential for the correct assembly of [FeFe]-hydrogenases (Vignais et al., 2001, Classification and phylogeny of hydrogenases. FEMS Microbiol Rev 25: 455-501). These enzymes are found in anaerobic prokaryotes (Clostridium or Desulfovibrio) and also in some lower eukaryotes, such as anaerobic fungi or unicellular microalgae (Chlorella or Chlamydomonas) (Vignais and Billoud, 2007, Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272). [FeFe]-hydrogenases thermodynamically promote the reoxidation of cofactors (ferredoxin or NADH) to generate H2. Therefore, these enzymes are usually involved in eliminating excess reducing equivalents in cells to avoid, for example, stopping fermentation.They are also able to interact with a restricted set of microalgae's photosynthetic chains, oxidizing them and thereby activating carbon fixation after anoxic incubation (Ghysels et al., 2013, Function of the chloroplasthydrogenase in the microalga Chlamydomonas: the role of hydrogenase and statetransitions during photosynthetic activation in anaerobiosis. PLoS One 8: e64161; Godaux et al., 2015, Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii. Plant Physiol 168: 648-658).

[0006] [NiFe]-hydrogenase forms a globular heteropolymer (Volbeda et al., 1995, Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373: 580-587). The bimetallic [NiFe] active site is located in the large subunit, coordinated by four cysteines and three non-protein ligands (one CO molecule and two CN) to the iron atom (Happe et al., 1997, Biological activation of hydrogen. Nature 385: 126; Nature 385: 126, Pierik et al., 1999, Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases. NiFe(CN)2CO, Biology's way to activate H2. J Biol Chem 274: 3331-3337). The other subunits of the heteropolymer contain several medial and distal FeS centers that conduct electrons between the active site and the physiological electron donor or acceptor. The [4Fe-4S] group located near the active site is considered to be the key to activity (Albracht, 1994, Nickel hydrogenases: in search of the active site. Biochim Biophys Acta 1188: 167-204). In addition to the structural genes, there are several auxiliary genes involved in the maturation of nickel, iron, CO and CN and the active sites of these heteropolymers. In fact, the maturation of [NiFe]-hydrogenase follows a complex pathway involving at least seven auxiliary proteins (HypA-F and an endopeptidase) (Vignais and Billoud, 2007, Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272). Another feature of the [NiFe]-hydrogenase active site is its strong affinity for hydrogen. Therefore, even though some [NiFe]-hydrogenases have a good ability to produce hydrogen, these enzymes mainly function by consuming H2 from the host microorganism. [NiFe]-hydrogenases are very common enzymes in prokaryotes, for example, there are many representatives in bacteria and archaea. Classification of [NiFe]-hydrogenases Based on the amino acid sequence alignment of different subunits, [NiFe]-hydrogenases are divided into four categories.It is noteworthy that this classification is very consistent with the classification based on physiological function (Vignais and Billoud, 2007, Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272).

[0007] When considering these enzymes for hydrogen production at an industrial level, the extreme sensitivity of hydrogenases to oxygen in vitro and in vivo is a problem. Oxygen binds to the active site as a ligand, accepts electrons, and is reduced to reactive oxygen species (ROS) trapped in the enzyme. When reactive oxygen species survive long enough to attack the fragile catalytic center, permanent damage may result. [FeFe]-hydrogenases are extremely sensitive to O2 because [FeFe]-hydrogenases are irreversibly damaged after exposure to low concentrations of O2 (Ghirardi et al., 1997, Oxygen sensitivity of algal H2-production. Appl Biochem Biotechnol 63-65: 141-151). However, [NiFe]-hydrogenases are more resistant to oxygen-induced damage than [FeFe]-hydrogenases. In addition, the inactivation of [NiFe]-hydrogenases by O2 is reversible. Some microorganisms, such as Ralstonia solanacearum, have even developed an oxygen-tolerant active site that can oxidize hydrogen even in the presence of air (Van der Linden et al., 2004, The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity toward oxygen. J Biol Inorg Chem 9: 616-626). These different properties make [NiFe]-hydrogenases more suitable candidates for industrially viable technologies.

[0008] HoxEFUYH is a well-characterized [NiFe]-hydrogenase found in Synechocystis sp PCC 6803 of the cyanobacteria. HoxEFUYH is a pentameric cytoplasmic [NiFe]-hydrogenase. HoxY and HoxH constitute the "hydrogenase" part, while HoxE, HoxF and HoxU constitute the part that contacts the redox cofactor (Carrieri et al., 2011, The role of the bidirectional hydrogenase in cyanobacteria. Bioresour Technol 102: 8368-8377). HoxH is the subunit responsible for catalytic activity, i.e., the subunit containing the active site of [NiFe]-hydrogenase. HoxH contains conserved residues bonded to nickel and iron atoms. HoxY contains a proximal [4Fe-4S] group close to the nickel-iron catalytic center. HoxF and HoxU are iron-sulfur proteins responsible for interacting with substrates (NADH, flavin redox proteins or reduced iron redox proteins) in vivo. HoxFU contains medial and distal FeS centers that transfer electrons to HoxYH. The function of HoxE is unclear, but it may be a membrane-anchored subunit. Mutational analysis of the maturation pathway identified seven essential maturation factors, called HypA, HypB, HypC, HypD, HypE, HypF and HoxW (Hoffmann et al., 2006, Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC6803. Additional homologs of hypA and hypB are not active in hydrogenase maturation. FEBS J 273: 4516-4527). A maturation model of HoxEFUYH has been proposed in the prior art (Carrieri et al., 2011, The role of the bidirectional hydrogenase in cyanobacteria. Bioresour Technol 102:8368-8377; Cassier Chauvat et al., 2014, Advances in the function and regulation of hydrogenase in the cyanobacterium Synechocystis PCC 6803. Int J Mol Sci 15:19938-19951).The HoxH subunit is processed by the HoxW specific protease, and the [nickel-iron] site is added to the catalytic subunit by the HypABCDEF complex. The whole genome of Synechocystis PCC 6803 has been sequenced. Unlike the hypABCDEF genes dispersed in the chromosome of Synechocystis, the HoxEFUYH genes were identified as clustered in an eight cistron operon. The promoter of the hoxEFUYH operon is not very active (Dutheil et al., 2012, The AbrB2 autorepressor, expressed from an atypical promoter, represses the hydrogenase operon to regulate hydrogen production in Synechocystis strain PCC 6803. J Bacteriol 194: 5423-5433). It is regulated by various environmental conditions, such as the presence of hydrogen, light, nitrate, nickel, oxygen or sulfur (Oliveira and Lindblad, 2009, Transcriptional regulation of the cyanobacterial bidirectional Hox-hydrogenase. Dalton Trans 9990-9996). It is noteworthy that hox genes are constitutively expressed under aerobic conditions (Kiss et al., 2009, Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803. J Biotechnol 142: 31-37), and this oxygen-sensitive enzyme is inactive under aerobic conditions. The exact physiological function remains controversial, but HoxEFUYH would act as a safety valve, dissipating excess electrons under adverse redox conditions, thus maintaining the proper redox balance within the cell during fermentation or photosynthesis (Carrieri et al., 2011, The role of the bidirectional hydrogenase in cyanobacteria. Bioresour Technol 102:8368-8377).

[0009] There are a lot of studies on HoxEFUYH. Many characteristics make this enzyme a good candidate enzyme for biological hydrogen production. First, this [NiFe]-hydrogenase has a bias for proton reduction (McIntosh et al., 2011, The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp.PCC 6803 works bidirectionally with a bias to H2 production. J Am Chem Soc 133: 11308-11319). Under aerobic conditions, operons and enzymes are weakly expressed in Synechocystis sp. PCC 6803 (Kiss et al., 2009, Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803. J Biotechnol 142: 31-37). In the presence of oxygen, HoxEFUYH is completely inactivated and is almost instantaneously inactivated. However, under redox conditions (e.g., by hydrogen reduction and / or by elimination of oxygen), HoxEFUYH can be rapidly reactivated (with a delay of approximately one minute) (Appel et al., 2000, The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis. Arch Microbiol 173:333-338; Germer et al., 2009, Overexpression, isolation, and spectroscopic characterization of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC 6803. J Biol Chem 284:36462-36472; McIntosh et al., 2011, The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with abias to H2 production. J Am Chem Soc 133:11308-11319).Several purification schemes (Schmitz et al., 2002, HoxE--a subunit specific for the petmeric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria. Biochim Biophys Acta 1554:66-74, Germer et al., 2009, Overexpression, isolation, and spectroscopic characterization of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC 6803. J Biol Chem 284:36462-36472) and implementations in electrochemistry (McIntosh et al., 2011, The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with abias to H2 production. J Am Chem Soc 133:11308-11319) are available. HoxEFUYH was identified as a good candidate due to its efficient catalytic hydrogen evolution and limited sensitivity to oxygen.

[0010] A common and efficient method for preparing large quantities of a target protein is the recombinant production of said protein in a heterologous host. This biotechnological process involves the introduction and expression of the target gene into the genome of the host organism in order to prepare large quantities of the target protein with extremely high purity. There are several recombinant production systems. The prokaryotic system remains the fastest and simplest system for preparing the target protein, while the eukaryotic system is slower and more complicated. Each organism has its own advantages and disadvantages. There is currently no universally applicable expression system. It is difficult to predict which host is best for a specific protein or a specific end use. Escherichia coli (E. coli) is the reference organism for recombinant production. In fact, this bacterium is well known from the point of view of genetic and physiological engineering, for example: optimized cells (high productivity, codon usage, inhibition of endogenous proteases), optimized culture medium, short doubling time, low contamination, availability of many commercial vectors, industrial scale-up, high yield.

[0011] As with metalloproteins, the production and engineering of recombinant hydrogenases have also achieved limited success. The literature provides examples of heterologously expressed [FeFe]-hydrogenases in Escherichia coli (King et al., 2006, Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system. J Bacteriol 188: 2163-2172; Yacoby et al., 2012, Optimized expression and purification for high-activity preparations of algal [FeFe]-hydrogenase PLoS One 7: e35886; Kuchenreuther et al., 2009, Tyrosine, cysteine, and S-adenosyl methionine stimulate in vitro [FeFe] hydrogenase activation. PLoS One 4: e7565). [FeFe]-hydrogenases are monomeric enzymes that require a limited number of maturation factors.

[0012] Heterologous production of [NiFe]-hydrogenase is considered difficult (English et al., 2009, Recombinant and in vitro expression systems for hydrogenases: new frontiers in basic and applied studies for biological and synthetic H2 production. Dalton Trans 9970-9978).

[0013] First, the difficulty stems from the complexity and specificity of the [NiFe] active site assembly process, which theoretically requires at least seven maturation factors for functional assembly.

[0014] Second, each subunit of the heteromultimer needs to be folded correctly, which is particularly difficult to control and ensure during heterologous production.

[0015] Third, each subunit in the heteropolymer complex must be assembled correctly. Misfolding can lead to aggregation, resulting in a reduction in the amount of active enzyme (Singh et al., 2015, Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact 14: 41). Obtaining enzyme activity requires obtaining an accurate sequence, which may be difficult to control and ensure in a heterologous host.

[0016] All of these explain why not all heterologous recombinantly produced [NiFe]-hydrogenases are active. However, there are several examples in the literature showing the production of active [NiFe]-hydrogenases in Escherichia coli (Kim et al., 2011, Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibriomarinus [NiFe]-hydrogenase in Escherichia coli J Biotechnol 155:312-319; Maier et al., 2015, Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J Biotechnol 199:1-8; Schiffels et al., 2013, An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli. PLoS One 8:e68812; Weyman et al., 2011, Genetic analysis of the Alteromonas macleodii[NiFe]-hydrogenase.FEMS MicrobiolLett 322:180-187).

[0017] In particular, the work of Sun et al. (Sun et al., 2010, Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production. PLoS One 5: e10526) enabled the expression of a [NiFe]-hydrogenase from a thermophilic archaeon in anaerobic Escherichia coli via four expression vectors, allowing the co-expression of 13 heterologous genes (four structural genes and nine maturation factors). More specifically, the work of Sun et al. enabled the generation of a tetrameric recombinant [NiFe]-hydrogenase-like enzyme comprising four subunits named PF0891, PF0892, PF0893 and PF0894. After purification, the tetrameric recombinant enzyme was found to be functionally similar to the native enzyme purified from a thermophilic archaeon.

[0018] Since HoxEFUYH is a prokaryotic protein of the cyanobacteria Synechocystis sp., the E. coli system is suitable for its recombinant production. Its production in E. coli has been successfully carried out. In this sense, Maeda et al. showed that under anaerobic conditions, the hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH enzyme increased (Maeda et al., 2007, Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803. BMC Biotechnol 7: 25). This increase in hydrogen production in the presence of HoxEFUYH is due to the inhibition of the activity of endogenous hydrogen-consuming hydrogenases 1 and 2 in E. coli.

[0019] We should also mention Wells et al., who introduced the HoxEFUYH gene and these associated maturation factors into Escherichia coli (Wells et al., 2011, Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase. Metab Eng 13: 445-453). This work demonstrated that endogenous hydrogenases produce hydrogen in vitro and in vivo via HoxEFUYH in a null host under anaerobic conditions. They demonstrate coupling with host electron transfer systems such as fermentation and show the potential of HoxEFUYH for improving hydrogen production in metabolic engineering.

[0020] In the prior art, there is no disclosure of a monomeric polypeptide comprising a single subunit comprising the active site of a [NiFe]-hydrogenase protein, especially an isolated monomer derived from a [NiFe]-hydrogenase complex, having its own hydrogenase activity, although documents D2 (Hornhardt et al., 1986, Characterization of a native subunit of the NAD-linked hydrogenase isolatedfrom a mutant of Alcaligenes eutrophus H16. Biochimie, Masson, Paris, FR, vol. 68(1), pages 15-24) and D3 (Przybyla et al., 1992, Structure-function relationships among the nickel-containing hydrogenases. FEMS Microbiol Rev 8:109-135) seem to suggest this is the case.

[0021] Document D2 describes the characterization of the native subunit of the [NiFe]-hydrogenase from Alcaligenes eutrophus H16. The peptide representing the catalytic subunit of the [NiFe]-hydrogenase was completely inactive towards NAD as a redox mediator, but had very low residual activity towards methylene blue, ferrocyanide and cytochrome c.

[0022] Document D3 shows that there are [NiFe]-hydrogenase monomers with hydrogenase activity. Document D3 cites Document D2 as an example, mentioning [NiFe]-hydrogenase of Alcaligenes eutrophus, and [NiFe]-hydrogenase-1 of Escherichia coli.

[0023] However, subsequently published studies, including those described in documents D1 (Massanz et al., 1998, Subforms and in vitro reconstitution of the NAD-reducing hydrogenase of Alcaligenes eutrophus. J Bacteriol 180: 1023-1029), D4 (Senger et al., 2017, Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis. J Biol Chem 292: 11670-11681) and D5 (Pinske et al., 2011, Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichiacoli is dependent on the coexpression of the iron-sulfur cluster-containing small subunit. Arch Microbiol 193: 893-903), indicated that the conclusions of the studies in documents D2 and D3 were wrong.

[0024] The studies described in document D1 show that the active site containing subunits of [NiFe]-hydrogenase from Alcaligenes eutrophus H16 alone has no hydrogenase-like activity towards several redox mediators, especially benzyl viologen. Document D1 mentions that the minimal entity of [NiFe]-hydrogenase from Alcaligenes eutrophus, which consists of a large subunit containing a [NiFe] center and a small subunit with a minimal FeS center, may have hydrogenase activity. Similarly, the studies in documents D4 and D5 contradict the teaching of document D3 because they show that the single catalytic subunit of [NiFe]-hydrogenase-1 from Escherichia coli has no hydrogenase activity.

[0025] Most of the work on this topic, including studies in documents D1, D4, and D5, suggests that the [4Fe-4S] center located in the small subunit is essential for the hydrogenase activity of the [NiFe]-hydrogenase large subunit (Albracht, 1994, Nickel hydrogenases: in search of the active site. Biochim Biophys Acta 1188: 167-204). In addition, structural analysis of [NiFe]-hydrogenases has largely shown that there is no FeS center in the catalytic subunit containing the active site (Lubitz et al., 2014, Hydrogenases. Chem Rev 114: 4081-4148).

[0026] Therefore, the results described in D2 can only be explained by contamination of the catalytic subunit by the small subunit, as evidenced by the highlighting of FeS centers in preparations that allow the measurement of low hydrogenase activities.

[0027] In summary, all prior art believes that the reduction in the number of subunits is not conducive to the activity or stability of hydrogenase, and that the subunits containing the nickel-iron center separated from the multimeric complex of nickel-iron hydrogenase have no hydrogenase activity themselves.

[0028] However, it is obvious from the above-mentioned prior art that there are still some drawbacks, which greatly hinder the application of [NiFe]-hydrogenase, especially HoxEFUYH, in commercially viable biohydrogen production.

[0029] Therefore, there is a real need to overcome the difficulties of [NiFe]-hydrogenase in hydrogen production so far, because, as mentioned above, [NiFe]-hydrogenase undoubtedly has a high potential for hydrogen production. Summary of the invention

[0030] In order to solve the above problems, the present invention provides a monomeric polypeptide, which comprises a single subunit, and the single subunit comprises the active site of a [NiFe]-hydrogenase protein. The monomeric polypeptide has enzymatic activity, especially hydrogenase enzyme activity, more specifically catalytic activity, and more specifically a catalytic activity similar to hydrogenase.

[0031] Preferably, according to the present invention, the monomeric polypeptide comprises a single subunit, and the single subunit comprises the active site of a [NiFe]-hydrogenase protein.

[0032] Preferably, according to the present invention, the monomeric polypeptide consists of a single subunit, which contains the active site of the [NiFe]-hydrogenase protein.

[0033] Preferably, according to the present invention, the monomeric polypeptide is made of a single subunit, which contains the active site of the [NiFe]-hydrogenase protein.

[0034] In other words, preferably, the present invention provides a monomeric polypeptide, which includes a single subunit, which only contains the active site of the [NiFe]-hydrogenase protein, and the monomeric polypeptide itself has enzymatic activity, especially hydrogenase enzyme activity, more specifically catalytic activity, more specifically a hydrogenase-like catalytic activity.

[0035] In the context of the present invention, it has been emphasized that the monomeric polypeptide comprises a single subunit, the single subunit comprises the active site of the [NiFe]-hydrogenase protein, and the monomeric polypeptide has hydrogenase activity, allowing the release of at least a portion of the substance that hinders the production of hydrogen by the [NiFe]-hydrogenase to ensure that the hydrogenase activity is at least 0.01 μmol H2·min -1 mg -1 enzyme, preferably at least 0.05 μmol H2·min -1 mg -1 Enzyme.

[0036] In fact, since the monomeric polypeptide according to the invention comprises a single subunit, which contains the active site of the [NiFe]-hydrogenase protein, the difficulties encountered by the [NiFe]-hydrogenase in ensuring the full folding of each relevant subunit and in ensuring the correct assembly of each subunit in the heteropolymer complex, in particular ensuring the assembly of the active site, can be greatly reduced or even eliminated. Since the monomeric polypeptide of the present invention only involves one subunit, the reproducibility of obtaining the [NiFe]-hydrogenase can be improved. In fact, the production of a single monomeric polypeptide greatly simplifies the complex folding process compared to dimeric, tetrameric and pentameric enzymes, the active sites of which, each subunit and ultimately the correct assembly of the entire enzyme are particularly difficult to control and ensure, which hinders the use of dimeric, tetrameric and pentameric enzymes. According to the present invention, the correct folding of the single subunit is necessary, and the heteropolymer complex can be formed without the need for assembly between different subunits.

[0037] In addition, the preparation method of the monomeric polypeptide of the present invention can be carried out completely under aerobic conditions (no need to prevent oxygen during expression and purification), which avoids the problems encountered in the prior art in preparing recombinant dimers, tetramers and pentamers under anoxic conditions. As mentioned above, even if the accumulation of biomass is carried out in the presence of oxygen, the production stage of the recombinant hydrogenase itself is carried out under anoxic conditions according to methods known in the prior art (Sun et al., 2010, Heterologous expression and maturation of an NADP-dependent [NiFe] -hydrogenase: a key enzyme in biofuel production. PLoS One 5: e10526; Wells et al., 2011, Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase. Metab Eng 13: 445-453). The purification of each chromatographic step also requires anoxic conditions (anoxic box, sodium dithionite in the buffer). Maintaining anoxic conditions during this period obviously involves an increase in the cost of recombinant production, and the present invention solves this problem.

[0038] Advantageously, the monomeric polypeptides of the invention are significantly smaller compared to heteromultimers, resulting in an increased mass activity of the enzyme (number of catalytically active entities per 1 mg of total protein).

[0039] The monomeric polypeptides of the invention may also enable better polymerization and densification of enzyme catalysts, for example, in electrochemical embodiments such as fuel cells.

[0040] In addition, the three-dimensional structure of the monomeric polypeptide of the present invention is easy to model to determine the residues exposed on the protein surface, for example, to facilitate orientation and adsorption relative to the interface (e.g., relative to the carbon electrode). This property can advantageously improve the stability of the connection between the interface and the enzyme catalyst, while also optimizing the direct electron transfer between the interface and the active site. Therefore, in the absence of other redox relays, energy efficiency can be improved, thereby limiting energy losses.

[0041] In the context of the present invention, it was also demonstrated that the monomeric polypeptide is indeed active and suitable for catalytic H2 production (e.g., by a standard in vitro H2 production assay for hydrogenases using reduced methyl viologen as a redox mediator) as well as catalytic H2 consumption (e.g., by a standard in vitro H2 consumption assay for hydrogenases using oxidized benzyl viologen as a redox mediator), without the presence of the proximal FeS center described as essential in the small subunit, nor any other redox relays. This is very surprising, since, in the prior art, such simplified heteropolymeric enzymes have never been shown to have outstanding hydrogenase activity.

[0042] More advantageously, although the currently known recombinant [NiFe]-hydrogenases can only obtain limited H2 (Sun et al., 2010, Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production. PLoS One 5:e10526; Schiffels et al., 2013, An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli. PLoS One 8:e68812; Maier et al., 2015, Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp.SG in Escherichia coli. J Biotechnol 199: 1-8), indicating that the monomeric polypeptides of the invention have hydrogenase activity that is at least equivalent to or even greater than the activity obtained using known (recombinant) [NiFe]-hydrogenases.

[0043] According to one embodiment of the present invention, the monomeric polypeptide is isolated from a natural environment, in particular, isolated from a natural [NiFe]-hydrogenase protein.

[0044] For example, the monomeric polypeptides of the present invention can be derived / isolated from prokaryotes. Examples include, but are not limited to, Escherichia coli (e.g., E. coli), Desulfovibrio (e.g., Desulfovibrio giganteus), Hydrogenophilus (e.g., Hydrogenophilus thermoluteolus), Desulfobacterium (e.g., Neutrophil sulfate-reducing bacteria), Synechocystis (e.g., Synechocystis PCC 6803), Phormidium (e.g., Phormidium dubium), or Spirulina (e.g., Spirulina platensis). For example, the monomeric polypeptides can be derived from cells or microorganisms, or can be produced in vitro or in vivo.

[0045] According to one embodiment of the present invention, the monomeric polypeptide is recombinant or heterologous.

[0046] Preferably, according to the present invention, the monomeric polypeptide is purified.

[0047] Preferably, according to the present invention, the truncated or non-truncated amino acid sequence of the monomeric polypeptide has no less than 15% identity with the amino acid sequence of SEQ ID NO:2 and / or the amino acid sequence of SEQ ID NO:4, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity.

[0048] More preferably, according to the present invention, the truncated or non-truncated amino acid sequence of the monomeric polypeptide has no less than 81%, no less than 82%, no less than 83%, no less than 84%, no less than 85%, no less than 86%, no less than 87%, no less than 88%, no less than 89%, no less than 90%, no less than 91%, no less than 92%, no less than 93%, no less than 94%, no less than 95%, no less than 96%, no less than 97%, no less than 98%, no less than 99% identity with the amino acid sequence of SEQ ID NO: 2 and / or the amino acid sequence of SEQ ID NO: 4.

[0049] Preferably, according to the present invention, the monomeric polypeptide is characterized in that the subunit comprising the active site of the [NiFe]-hydrogenase protein is the HoxH subunit of the HoxEFUYH [NiFe]-hydrogenase protein in / from Synechocystis CCP6803.

[0050] Preferably, according to the present invention, the hydrogenase activity of the monomeric polypeptide is not less than 0.01 μmol H2·min -1 mg -1 enzyme, preferably not less than 0.05 μmol H2·min-1 mg -1 enzyme, preferably not less than 10 μmol H2·min -1 mg -1 Enzyme.

[0051] The present invention also relates to a host cell comprising a monomeric polypeptide of the present invention, wherein the monomeric polypeptide comprises a single subunit, wherein the single subunit comprises an active site of a [NiFe]-hydrogenase protein, and wherein the monomeric polypeptide has hydrogenase activity.

[0052] Preferably, the present invention relates to a host cell comprising a monomeric polypeptide of the present invention, wherein a single subunit of the monomeric polypeptide comprises the active site of a [NiFe]-hydrogenase protein, and the single subunit is the HoxH subunit of the HoxEFUYH[NiFe]-hydrogenase protein in Synechocystis PCC 6803.

[0053] For example, according to the present invention, the host cell, in particular the host cell for expressing the monomeric polypeptide, can be Escherichia coli (such as Escherichia coli), Bacillus (such as Bacillus subtilis), Streptomyces (such as Streptomyces coelicolor), Synechocystis (such as Synechocystis PCC 6803), Synechococcus (such as Synechococcus WH8102) or any other prokaryotic cell, Chlamydomonas (such as Chlamydomonas reinhardtii), Saccharomyces (such as Saccharomyces cerevisiae), Pichia pastoris (such as Methanol yeast) or other types of eukaryotic cells.

[0054] According to the present invention, the monomeric polypeptide contained in the host cell itself may (but not necessarily) result from the expression of a gene in an expression vector, such as the expression of a gene inserted into a plasmid of the host cell.

[0055] Preferably, according to the present invention, the truncated or non-truncated amino acid sequence of the monomeric polypeptide comprised in the host cell has no less than 20% identity with the amino acid sequence of SEQ ID NO:2 and / or the amino acid sequence of SEQ ID NO:4, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity.

[0056] Preferably, according to the present invention, the host cell may include at least one maturation factor of the [NiFe]-hydrogenase protein, and the at least one maturation factor of the [NiFe]-hydrogenase protein is preferably selected from HypA, HypB, HypC, HypD, HypE, HypF and HoxW.

[0057] a) the amino acid sequences of HypA, HypB, HypC, HypD, HypE, HypF and HoxW have no less than 15% identity, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity with the amino acid sequences of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively; or

[0058] b) the HypA, HypB, HypC, HypD, HypE, HypF and HoxW are jointly encoded by a tandem nucleotide sequence, which has no less than 15% identity, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity with the nucleotide sequence of SEQ ID NO: 19 encoding all the maturation factors.

[0059] According to the present invention, the at least one maturation factor may be a host cell endogenous maturation factor and / or a host cell exogenous maturation factor.

[0060] Preferably, the host cell of the present invention comprises an expression vector, wherein at least one gene in the expression vector expresses the monomeric polypeptide and / or the at least one maturation factor.

[0061] The present invention also relates to a host cell, which comprises a polynucleotide encoding a monomeric polypeptide of the present invention, wherein the monomeric polypeptide comprises a single subunit, wherein the single subunit comprises an active site of a [NiFe]-hydrogenase protein, and wherein the monomeric polypeptide has hydrogenase activity.

[0062] Preferably, the present invention relates to a host cell, comprising a polynucleotide encoding a monomeric polypeptide, a single subunit of which comprises an active site of a [NiFe]-hydrogenase protein, and the single subunit is the HoxH subunit of the HoxEFUYH[NiFe]-hydrogenase protein in Synechocystis sp. PCC6803.

[0063] Preferably, according to the present invention, the nucleotide sequence of the polynucleotide encoding the monomeric polypeptide has no less than 15% identity with the nucleotide sequence of SEQ ID NO: 1 and / or the nucleotide sequence of SEQ ID NO: 3, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity.

[0064] For example, according to the present invention, a host cell comprising a polynucleotide encoding a monomeric polypeptide of the present invention, particularly for expressing the monomeric polypeptide, can be a host bacterial cell of the genus Escherichia coli (e.g., Escherichia coli), the genus Bacillus (e.g., Bacillus subtilis) and the genus Streptomyces (e.g., Streptomyces coelicolor), a photosynthetic bacterial cell of the genus Synechocystis (e.g., Synechocystis PCC 6803) or the genus Synechococcus (e.g., Synechococcus WH8102) or any other prokaryotic cell, a genus Chlamydomonas (e.g., Chlamydomonas reinhardtii), a genus Saccharomyces (e.g., Saccharomyces cerevisiae), a Pichia pastoris (e.g., Methanol yeast) or other types of eukaryotic cells.

[0065] According to the present invention, the polynucleotide comprised in the host cell may itself, but need not necessarily, be comprised in an expression vector such as a plasmid that is inserted into the host cell.

[0066] Preferably, according to the present invention, the truncated or non-truncated amino acid sequence of the monomeric polypeptide encoded by the polynucleotide in the host cell has no less than 20% identity with the amino acid sequence of SEQ ID NO:2 and / or the amino acid sequence of SEQ ID NO:4, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity.

[0067] Preferably, according to the present invention, the host cell comprising a polynucleotide encoding a monomeric polypeptide may include at least one maturation factor of the [NiFe]-hydrogenase protein, and the at least one maturation factor of the [NiFe]-hydrogenase protein is preferably selected from HypA, HypB, HypC, HypD, HypE, HypF and HoxW.

[0068] a) the amino acid sequences of HypA, HypB, HypC, HypD, HypE, HypF and HoxW have no less than 15% identity, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity with the amino acid sequences of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively; or

[0069] b) the HypA, HypB, HypC, HypD, HypE, HypF and HoxW are jointly encoded by a tandem nucleotide sequence, which has no less than 15% identity, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity with the nucleotide sequence of SEQ ID NO: 19 encoding all the maturation factors.

[0070] According to the present invention, the at least one maturation factor may be a host cell endogenous maturation factor and / or a host cell exogenous maturation factor.

[0071] Preferably, the host cell of the present invention comprises an expression vector, wherein at least one gene in the expression vector expresses the monomeric polypeptide and / or the at least one maturation factor.

[0072] The present invention also relates to a method for preparing a monomeric polypeptide having hydrogenase activity, for example in a host cell (for example in E. coli), the method comprising the following steps:

[0073] Modification of an expression vector such as a plasmid by introducing into the expression vector an exogenous polynucleotide, at least a portion of which encodes the monomeric polypeptide, wherein the monomeric polypeptide comprises a single subunit, and the single subunit comprises the active site of a [NiFe]-hydrogenase protein; and

[0074] Incubating the modified expression vector according to incubation conditions, wherein the incubation conditions are capable of maintaining expression of the exogenous polynucleotide to produce the monomeric polypeptide, wherein the monomeric polypeptide includes a single subunit, and the single subunit includes the active site of the [NiFe]-hydrogenase protein.

[0075] The method according to the invention enables the production of single subunits comprising the active site of [NiFe]-hydrogenase proteins.

[0076] Of course, one skilled in the art will be able to adjust the desired and appropriate incubation conditions.

[0077] According to the present invention, in the step of transforming the host cell, the polynucleotide itself may be, but not necessarily, included in an expression vector such as a plasmid.

[0078] Preferably, according to the present invention, the step of modifying the expression vector comprises introducing the exogenous polynucleotide into the expression vector, at least a portion of the exogenous polynucleotide encoding the monomeric polypeptide, and the truncated or non-truncated amino acid sequence of the monomeric polypeptide has no less than 20% identity with the amino acid sequence of SEQ ID NO:2 and / or the amino acid sequence of SEQ ID NO:4, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity.

[0079] Preferably, according to the present invention, the step of modifying the expression vector may include inserting one or more maturation factors of the [NiFe]-hydrogenase protein into the expression vector, preferably comprising one or more maturation factors of the [NiFe]-hydrogenase protein, and the at least one maturation factor is preferably selected from HypA, HypB, HypC, HypD, HypE, HypF and HoxW.

[0080] a) the amino acid sequences of HypA, HypB, HypC, HypD, HypE, HypF and HoxW have no less than 15% identity, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity with the amino acid sequences of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively; or b) the HypA, HypB, HypC, HypD, HypE, HypF and HoxW are jointly encoded by a tandem nucleotide sequence, and the tandem nucleotide sequence is identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively. The nucleotide sequence of NO:19 has an identity of not less than 15%, preferably an identity of not less than 20%, more preferably an identity of not less than 40%, more preferably an identity of not less than 60%, more preferably an identity of not less than 80%, more preferably an identity of not less than 90%, more preferably an identity of not less than 95%, and more preferably an identity of not less than 99%.

[0081] According to the present invention, in the step of modifying the expression vector, the sequence encoding one or more maturation factors of the [NiFe]-hydrogenase protein itself can be but not necessarily included in an expression vector such as a plasmid.

[0082] Preferably, the method of the present invention comprises a subsequent step of isolating and / or purifying said monomeric polypeptide.

[0083] Specifically and preferably, the present invention relates to a method for preparing a monomeric polypeptide having hydrogenase activity according to the present invention, the method comprising the following steps:

[0084] · subjecting an entity such as a host cell or an expression vector comprising genetic material to a transgenic step in vivo or in vitro to obtain a transgenic entity such as a transgenic host cell or a transgenic expression vector;

[0085] A step of culturing the transgenic entity, such as the transgenic host cell or the transgenic expression vector, to obtain a monomeric polypeptide comprising a single subunit, the single subunit comprising the active site of a [NiFe]-hydrogenase-like protein, the monomeric polypeptide having hydrogenase activity.

[0086] Preferably, according to the present invention, the genetic modification step performed in vivo or in vitro comprises:

[0087] a) transferring an exogenous polynucleotide into a host cell and / or an expression vector through transgenesis to obtain a transgenic host cell and / or a transgenic expression vector, incubating the transgenic host cell and / or the transgenic expression vector, wherein at least a portion of the exogenous polynucleotide encodes the monomeric polypeptide, the monomeric polypeptide comprises a single subunit, and the single subunit comprises an active site of a [NiFe]-hydrogenase protein, and the incubation conditions are such that the expression of the exogenous polynucleotide can be maintained to produce the monomeric polypeptide; or

[0088] b) inducing at least one gene mutation in the genetic material of the host cell to obtain a transgenic host cell, and incubating the transgenic host cell under conditions that enable the production of the monomeric polypeptide.

[0089] Of course, one skilled in the art will be able to adjust the desired and appropriate incubation conditions.

[0090] For example, according to the present invention, the at least one gene mutation is induced by homologous recombination, using methods well known to those skilled in the art.

[0091] Preferably, according to the present invention, the step of transferring the exogenous polynucleotide into the host cell via transgenesis comprises transferring an expression vector into the host cell, especially a modified expression vector comprising the exogenous polynucleotide.

[0092] Preferably, according to the present invention, the genetic modification step of the host cell comprises transferring the exogenous polynucleotide into the host cell, at least a portion of the exogenous polynucleotide encodes the monomeric polypeptide, and the truncated or non-truncated amino acid sequence of the monomeric polypeptide has no less than 15% identity with the amino acid sequence of SEQ ID NO:2 and / or the amino acid sequence of SEQ ID NO:4, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity.

[0093] Preferably, according to the present invention, the genetic modification step of the host cell further comprises transferring at least one maturation factor of the [NiFe]-hydrogenase protein into the host cell, wherein the at least one maturation factor is endogenous and / or exogenous to the host cell, and the at least one maturation factor is preferably selected from HypA, HypB, HypC, HypD, HypE, HypF and HoxW,

[0094] a) the amino acid sequences of HypA, HypB, HypC, HypD, HypE, HypF and HoxW have no less than 15% identity, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity with the amino acid sequences of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively; or

[0095] b) the HypA, HypB, HypC, HypD, HypE, HypF and HoxW are jointly encoded by a tandem nucleotide sequence, which has no less than 15% identity, preferably no less than 20% identity, more preferably no less than 40% identity, more preferably no less than 60% identity, more preferably no less than 80% identity, more preferably no less than 90% identity, more preferably no less than 95% identity, more preferably no less than 99% identity with the nucleotide sequence of SEQ ID NO: 19 encoding all the maturation factors.

[0096] Preferably, according to the present invention, the host cell comprises an expression vector, at least one gene in the expression vector expresses the at least one maturation factor.

[0097] Preferably, the method for preparing a monomeric polypeptide having hydrogenase activity according to the present invention makes the monomeric polypeptide include a single subunit, wherein the single subunit comprises the active site of a [NiFe]-hydrogenase protein, and the single subunit is the HoxH subunit of the HoxEFUYH[NiFe]-hydrogenase protein in Synechocystis PCC6803.

[0098] The present invention also relates to the use of a monomeric polypeptide according to the present invention, comprising a single subunit, comprising the active site of a [NiFe]-hydrogenase protein, wherein the monomeric polypeptide has hydrogenase activity and is present or absent in the cell depending on the incubation conditions, and hydrogen is produced or consumed by incubating the monomeric polypeptide to ensure the production or consumption of hydrogen.

[0099] The present invention also relates to the use of a monomeric polypeptide according to the present invention or a monomeric polypeptide prepared by a method according to the present invention, wherein the monomeric polypeptide comprises a single subunit, wherein the single subunit comprises the active site of a [NiFe]-hydrogenase protein, wherein the monomeric polypeptide has hydrogenase activity and is present or absent in a cell, for coating a surface, in particular coating the surface of an electrical conductor such as an anode or cathode. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 A map of plasmid pET-26b(+) (5360 bp) is shown.

[0101] Figure 2 Agarose gel analysis of NdeI and BlpI restriction enzyme digestion of the expression vector pET26b(+)+HoxH is shown.

[0102] Figure 3 A map of plasmid pACYCDuet-1 (4008 bp) is shown.

[0103] Figure 4 Agarose gel analysis of NcoI and HindIII restriction endonuclease digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW is shown.

[0104] Figure 5 Agarose gel analysis of NdeI and BlpI restriction enzyme digestion of the expression vector pET26b(+)+HoxH derived from E. coli colony plasmid DNA is shown.

[0105] Figure 6 Agarose gel analysis of NcoI and HindIII restriction endonuclease digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW derived from E. coli colony plasmid DNA is shown.

[0106] Figure 7 The method for purifying the target HoxH recombinant protein is shown. NA, sample not adsorbed by Ni-NTA affinity column. Eluent: sample eluted with elution buffer containing 10 mM imidazole; 50 mM: sample eluted with 50 mM imidazole; 100 mM: sample eluted with 100 mM imidazole; 150 mM: sample eluted with 150 mM imidazole; 200 mM: sample eluted with 200 mM imidazole; 250 mM: sample eluted with 250 mM imidazole.

[0107] Figure 8SDS-PAGE analysis showing the protein composition of various fractions collected during affinity chromatography. Load: supernatant extracted from lysis of recombinant E. coli cells and applied to Ni-NTA affinity column; NA, sample not adsorbed by Ni-NTA affinity column. Eluent: sample eluted with elution buffer containing 10 mM imidazole. 50 mM: sample eluted with 50 mM imidazole concentration; 100 mM: sample eluted with 100 mM imidazole concentration; 150 mM: sample eluted with 150 mM imidazole concentration; 200 mM: sample eluted with 200 mM imidazole concentration; 250 mM: sample eluted with 250 mM imidazole concentration.

[0108] Fig. 9 Western blot showing the presence of HoxH in various fractions collected during affinity chromatography. M: molecular weight marker; Load: supernatant extracted from lysis of recombinant E. coli cells and applied to Ni-NTA affinity column; NA: sample not adsorbed by Ni-NTA affinity column; 50 mM: sample eluted with 50 mM imidazole concentration; 200 mM: sample eluted with 200 mM imidazole concentration.

[0109] Fig.10 Schematic diagram of the structure of HoxEFUYH and [NiFe]-hydrogenase in Synechocystis PCC 6803. HoxE, 19KDa and 1 FeS center; HoxF, 57.5KDa, 2 FeS centers and 1 FMN center; HoxU, 26KDa and 4 FeS centers; HoxY, 20KDa and FeS center; HoxH, 53KDa and active site.

[0110] Fig.11 Schematic diagram of the electron transfer and expected interactions between HoxEFUYH, [NiFe]-hydrogenase of Synechocystis PCC 6803, and NADPH and methyl viologen (MV) (shaded arrows).

[0111] Fig.12 A schematic diagram in which the question mark raises the question of whether the active site of the HoxH recombinant protein can directly accept electrons from the MV to produce hydrogen without an additional redox relay.

[0112] Fig.13 The hydrogen gas produced by adding HoxH recombinant protein to a reagent containing methyl viologen reduced by sodium dithionite, which has been subjected to anaerobic conditions, is shown, thereby indicating hydrogenase activity. HoxH is able to combine electrons from the previously reduced methyl viologen with protons (present in the buffer), thereby generating hydrogen according to the equation representing the hydrogenase catalyzed reaction. Generate hydrogen. Using a micro sensor ( Denmark) continuously measures the dissolved hydrogen content in the reagent. The arrows show the time of addition of HoxH recombinant protein, which causes an increase in dissolved hydrogen levels detected by the microsensor.

[0113] Fig.14 The figure shows the hydrogenase activity represented by the amount of reduction of benzyl viologen (BV) when HoxH recombinant protein was added to a reagent containing benzyl viologen (BV) in the presence of H2. HoxH is able to take electrons from hydrogen and transfer them to a redox mediator, such as benzyl viologen, according to the equation representing the hydrogenase catalyzed reaction Protons are generated. The amount of hydrogen consumed corresponds to the amount of benzyl viologen reduced, which can be continuously detected by spectrophotometry at a wavelength of 578 nm.

[0114] Fig.15 The figure shows the generation of hydrogen gas, indicative of hydrogenase activity, by adding HoxH recombinant protein produced in the absence of exogenous HupABCDEFHoxW maturation factors from E. coli to a reagent containing methyl viologen reduced by sodium dithionite under anaerobic conditions. HoxH is able to combine electrons from the previously reduced methyl viologen with protons (present in the buffer) to produce hydrogen according to the equation representing the hydrogenase catalytic reaction Generates hydrogen. DETAILED DESCRIPTION

[0115] definition

[0116] In the present invention, the term "polypeptide" is a single chain consisting of at least two amino acids, which are connected by a peptide bond between the carboxyl group of the amino acid and the amine group of the next amino acid. The term "polypeptide" also includes molecules containing multiple polypeptides linked together by disulfide bonds, or polypeptide complexes that form polymers (e.g., dimers, trimers, tetramers, pentamers) by, for example, non-covalent or covalent linkages. Polypeptides can also contain non-protein ligands, such as inorganic iron (Fe), nickel (Ni), iron sulfur centers (FeS) or other organic ligands, such as carbon monoxide (CO), cyanide (CN) or flavin. The definition of polypeptide includes terms such as peptide, polypeptide, enzyme, subunit or protein, which can be interchangeable. The definition of "polypeptide" does not take into account the length of the polypeptide or the way in which the polypeptide is produced.

[0117] In the present invention, the term "recombinant polypeptide" or "heterologous polypeptide" is a polypeptide that does not naturally occur in the environment and / or in the host cell used for its production, particularly in a host cell, and its production is carried out by recombinant technology, for example, by adding genetic material to the host cell.

[0118] In the present invention, the term "monomer polypeptide" is a single chain consisting of at least two amino acids, which are connected by a peptide bond between the carboxyl group of the amino acid and the amine group of the next amino acid. In contrast to the term "polypeptide", the term "monomer polypeptide" only includes molecules that only contain one polypeptide, i.e., molecules that do not interact with any other polypeptide. For example, it is by no means a polymer (e.g., a dimer, trimer, tetramer, pentamer, etc.). Monomer polypeptides can also contain non-protein ligands, such as inorganic iron (Fe), nickel (Ni), iron-sulfur centers (FeS), or other organic ligands, such as carbon monoxide (CO), cyanide (CN) or flavin. The definition of "monomer polypeptide" does not take into account the length of the polypeptide, nor the way in which the polypeptide is produced.

[0119] In the present invention, the term "recombinant monomeric polypeptide" or "heterologous monomeric polypeptide" is a monomeric polypeptide that does not naturally occur in the environment (particularly in a host cell) and is produced by recombinant technology (eg by adding genetic material to a host cell).

[0120] In the present invention, the term "monomer polypeptide having hydrogenase activity" refers to the monomer polypeptide capable of catalyzing a reversible reaction The definition of hydrogenase activity is independent of the experimental conditions and only relates to the production or consumption of hydrogen by the hydrogenase activity.

[0121] In the present invention, the term "active site" is the polypeptide portion that is responsible for the catalytic activity of the polypeptide, such as hydrogenase activity, when forming a tertiary structure. The polypeptide portion may include, for example, several parts of the amino acid sequence and / or a binding to one or more non-protein ligands.

[0122] In the present invention, the term "polynucleotide" is a single strand consisting of at least two nucleotides linked together, for example, by covalent bonds, which are ribonucleotides or deoxynucleotides. Thus, this includes single-stranded or double-stranded RNA and DNA. The definition of "polynucleotide" does not take into account the length, function, shape, and manner of producing the polynucleotide. For example, a polynucleotide can be a plasmid, a portion of a plasmid, a gene, or a gene fragment.

[0123] In the present invention, the term "exogenous polynucleotide" is a polynucleotide that is not normally present in the host cell. For example, the exogenous polynucleotide may be a sequence encoding a polypeptide that is not naturally present in the host cell or plasmid.

[0124] In the present invention, the term "tandem" refers to the extension of multiple sequences in order to form only one sequence, such as the extension of a polynucleotide sequence or a polypeptide sequence.

[0125] In the present invention, the term "plasmid" is a double-stranded DNA molecule that is different from exogenous chromosomal DNA and can replicate autonomously due to its own replication source. The plasmid can also include target sequences, such as genes encoding selection factors (resistance to antibiotics, etc.), multiple cloning sites and / or transcription regulatory sequences that can insert polynucleotides. The terms "plasmid" or "expression vector" are interchangeable.

[0126] In the present invention, the term "host cell" refers to a cell that has been transformed. For example, such transformation may be the transfer of exogenous polynucleotides into cells via plasmids.

[0127] In the present invention, the term "maturation factor" is any biological or non-biological molecule that participates in the formation of the structure of another biological molecule (eg, a protein).

[0128] In the present invention, the term "endogenous maturation factor" is any biological or non-biological molecule that participates in the formation of the structure of another biological molecule (such as a protein) and exists naturally in the host cell, that is, the host cell has not been genetically modified.

[0129] In the present invention, the term "exogenous maturation factor" refers to any biological or non-biological molecule that participates in the formation of the structure of another biological molecule (such as a protein) and does not naturally exist in the host cell, that is, the host cell needs to be genetically modified (for example, by introducing an expression vector) to transfer the exogenous maturation factor into it.

[0130] In the present invention, the term "isolated" refers to removing any molecule from its natural environment, such as removing a molecule from native [NiFe]-hydrogenase.

[0131] In the present invention, the term "purification" refers to the separation of any molecule by biochemical techniques. This definition does not take into account how the molecule is produced, such as naturally or by recombinant, chemical or enzymatic synthesis, nor does it take into account the biochemical techniques used for purification, such as affinity chromatography or molecular sieves. For example, purifying polynucleotides, polypeptides or H2. Preferably, a substance is purified when it accounts for at least 60%, preferably 75%, and preferably 90% of the other components with which it is associated.

[0132] In the present invention, the term "substantially homogeneous" means that the purified molecule accounts for at least 90% of the other components with which it is associated.

[0133] In the present invention, the term "identity" refers to the structural similarity between two polynucleotides or two polypeptides. The structural similarity is determined by the alignment between the two sequences, which optimizes the number of identical nucleotides or identical amino acids in the sequence. For better alignment, gaps can be present in one or both sequences to optimize the structural similarity. However, the sequence of nucleotides or amino acids must remain unchanged.

[0134] In the present invention, the term "genetic material" refers to the genome of an entity, more precisely to all nucleic acids of said entity, including coding and non-coding sequences.

[0135] In the present invention, the term "gene mutation" refers to an accidental or induced modification of the genetic material of an entity (eg, a host cell or an expression vector).

[0136] In the present invention, the term "an entity comprising genetic material" refers to an entity comprising genetic material according to the above definition, such as a host cell or an expression vector such as a plasmid.

[0137] In the present invention, the term "transgenic entity" refers to an entity comprising genetic material according to the above definition, and the entity has undergone modification of the genetic material, such as gene mutation or introduction of exogenous polynucleotides into the cell (such as via a plasmid).

[0138] In the present invention, the term "transgenic host cell" refers to a host cell according to the above definition, which has undergone modification of genetic material, such as gene mutation or introduction of exogenous polynucleotides into the cell (such as via a plasmid).

[0139] In the present invention, the term "transgenic expression vector" refers to an expression vector or plasmid according to the above definition, which has been modified with genetic material, such as the insertion of a target sequence, such as a gene encoding a specific polypeptide.

[0140] The following embodiments (with reference to the accompanying drawings) are intended to illustrate other characteristics, details and advantages of the invention without limiting the invention.

[0141] Example

[0142] 1. Construction of HoxH expression vector

[0143] 1.1 The “simple” sequence of HoxH

[0144] HoxH is the large subunit of the HoxEFUYH[NiFe]-hydrogenase active site in Synechocystis sp. PCC 6803. The accession number of this protein in Genbank is BAA18091.1. The theoretical isoelectric point of HoxH is 5.86 and the theoretical molecular weight is 52996.53 Daltons. The nucleotide sequence of HoxH (1425 bp) is shown below and is named SEQ ID NO: 1 in the context of the present invention:

[0145]

[0146] The amino acid sequence of HoxH (474aa) is shown below, and is designated as SEQ ID NO: 2 in the context of the present invention:

[0147] MSKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEKFCEGRPMWEMAGITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLRRLMNLGQITQSHALSFFHLSSPDFL LGWDSDPATRNVFGLIAADPDLARAGIRLRQFGQTVIELLGAKKIHSAWSVPGGVRSPLSEEGRQWIVDRLPEAKETVYLALNLFKNMLDRFQTEVAEFGKFPSLFMGLVGKNNEWEHY GGSLRFTDSEGNIVADNLSEDNYADFIGESVEKWSYLKFPYYKSLGYPDGIYRVGPLARLNVCHHIGTPEADQELEEYRQRAGGVATSSFFYHYARLVEILACLEAIELLMADPDILS KNCRAKAEINCTEAVGVSEAPRGTLFHHYKIDEDGLIKKVNLIIATGNNNLAMNKTVAQIAKHYIRNHDVQEGFLNRVEAGIRCYDPCLSCSTHAAGQMPLMIDLVNPQGELIKSIQRD

[0148] Figure 1 The illustrative plates of plasmid pET26b(+) are shown, and the HoxH expression vector is constructed by inserting SEQ ID NO:3 into pET26b(+). pET26b(+) is a 5360bp plasmid with an E. coli origin of replication, a kanamycin resistance gene, a multiple cloning site (MCS) containing many restriction sites, a T7 promoter, and a T7 transcription terminator. It also contains the lacI gene encoding a transcriptional repressor. This transcriptional repression can be relieved by adding IPTG.

[0149] 1.2 “Optimized” sequence of HoxH

[0150] Optionally, according to an embodiment of the present invention, the sequences SEQ ID NO: 1 and SEQ ID NO: 2 are optimized for codon usage in E. coli. Specifically, NdeI (catatg) and BlpI (gctnagc) restriction sites are added at the beginning and end of the nucleotide sequence cloned in plasmid pET26b (+), respectively, and the sequence caccacaccatcac (underlined below) (a sequence encoding a polyhistidine tag near the N-terminus of the protein) is also added.

[0151] The optimized nucleotide sequence (1453 bp) is shown below, and is named as SEQ ID NO: 3 in the context of the present invention:

[0152] catatgagc caccaccaccaccatcac

[0153] The optimized amino acid sequence (480aa) is shown below, and is designated as SEQ ID NO: 4 in the context of the present invention:

[0154] MSHHHHHHKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEKFCEGRPMWEMAGITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLRRLMNLGQITQSHALSFFHLSS PDFLLGWDSDPATRNVFGLIAADPDLARAGIRLRQFGQTVIELLGAKKIHSAWSVPGGVRSPLSEEGRQWIVDRLPEAKETVYLALNLFKNMLDRFQTEVAEFGKFPSLFMGLVGKNNEW EHYGGSLRFTDSEGNIVADNLSEDNYADFIGESVEKWSYLKFPYYKSLGYPDGIYRVGPLARLNVCHHIGTPEADQELEEYRQRAGGVATSSFFYHYARLVEILACLEAIELLMADPDIL SKNCRAKAEINCTEAVGVSEAPRGTLFHHYKIDEDGLIKKVNLIIATGNNNLAMNKTVAQIAKHYIRNHDVQEGFLNRVEAGIRCYDPCLSCSTHAAGQMPLMIDLVNPQGELIKSIQRD

[0155] Plasmid pET26b(+) was used to construct a HoxH expression vector by inserting SEQ ID NO: 3 into pET26b(+).

[0156] Figure 2 The digestion analysis of the expression vector pET26b(+)+HoxH (SEQ ID NO:3) by NdeI and BlpI restriction endonucleases is shown. Two DNA fragments of about 5300 bp (linearized pET26b(+)) and about 1500 bp (HoxH sequence excised from plasmid pET26b(+)) are highlighted. This confirms the presence of the target HoxH sequence in the expression vector pET26b(+).

[0157] 2. Construction of an expression vector for at least one maturation factor

[0158] In the context of the present invention, at least the following maturation factors are considered: HypA, HypB, HypC, HypD, HypE, HypF and HoxW.

[0159] HypA is the expression / formation protein of HoxEFUYH[NiFe]-hydrogenase in Synechocystis sp. PCC 6803. The accession number of the protein in Genbank is BAA18357.1. The theoretical isoelectric point of HypA is 4.94 and the theoretical molecular weight is 12773.47 Daltons. The nucleotide sequence (342 bp) of HypA is shown below and is designated as SEQ ID NO:5 in the context of the present invention:

[0160] atgcacgaagttagtctgatggagcaaactttggcgatcgccattgcccaggcggaagaccatggagccagccaaatccatcgtttaaccctgcgggtggggcaacagtctggggtggtggccgatgccctacggtttgcgtttgaagtggtgcgacaaaataccatggcc gccgaggcgagattggaaattgaagaaattcccgttacctgtcgttgccaacactgccacgaaaattttcagccagaggattggatttaccgctgtccccactgcgaccagattagccaaacagtaatggatggcaaacagttggaactagcatccctagaactgagttga

[0161] The amino acid sequence of HypA (113aa) is shown below, designated as SEQ ID NO:6 in the context of the present invention:

[0162] MHEVSLMEQTLAIAIAQAEDHGASQIHRLTLRVGQQSGVVADALRFAFEVVRQNTMAAEARLEIEEIPVTCRCQHCHENFQPEDWIYRCPHCDQISQTVMDGKQLELASLELS

[0163] HypB is the expression / formation protein of HoxEFUYH[NiFe]-hydrogenase in Synechocystis sp. PCC 6803. The accession number of the protein in Genbank is BAA18312.1. The theoretical isoelectric point of HyB is 5.75 and the theoretical molecular weight is 31242.97 Daltons. The nucleotide sequence of HypB (858 bp) is shown below and is designated as SEQ ID NO:7 in the context of the present invention:

[0164] atgtgccaaaactgcggttgtagtgcggtgggaaccgttgcccatagccaccatcaccatggcgatggaaattttgcccacagccatgatgaccatgaccagcaagaacatcatcaccaccatggcaactacagcaaaagtccaagtcagcagactgtgaccattgaacccgatcgccagtccattgccattggccaaggcattctcagcaagaatgaccgcctagcggaaaggaatcggggctatttccaggctaagggcttactggtgatgaatttcctctcttctcccggagccggtaaaactgctctgatcgaaaaaatggtcggcgatcgacaaaaagaccatcccaccgccgtcattgtgggggatttagccaccgataacgatgcccaacgtctccgcagtgccggggcgatcgccattcaggtcaccacaggaaatatttgccatctggaagcggaaatggtggccaaggcggcccaaaagttagatttagacaatatcgatcaattgatcattgaaaatgttggtaatttggtttgccccaccacctatgatctaggggaagatttacgggtcgtattattttccgtcacagaaggggaggataaaccccttaaatatcccgccaccttcaaatcagcccaggttattttagtcaccaaacaggacattgccgccgcagtggattttgatgcagagctggcttggcaaaacctacggcaagtggccccccaagcccaaatttttgcagtgtctgcccgcacggggaaaggattgcagtcctggtatgagtatttggatcaatggcaactccaacactattcgccgttggttgatccagcattggcctaa

[0165] The amino acid sequence of HypB (285 aa) is shown below and is designated SEQ ID NO:8 in the context of the present invention:

[0166] MCQNCGCSAVGTVAHSHHHHGDGNFAHSHDDHDQQEHHHHHGNYSKSPSQQTVTIEPDRQSIAIGQGILSKNDRLAERNRGYFQAKGLLVMNFLSSPGAGKTALIEKMVGDRQKDHPTAVIVGDLATDNDAQRLRSAGAIAI QVTTGNICHLEAEMVAKAAQKLDLDNIDQLIIENVGNLVCPTTYDLGEDLRVVLFSVTEGEDKPLKYPATFKSAQVILVTKQDIAAAVDFDAELAWQNLRQVAPQAQIFAVSARTGKGLQSWYEYLDQWQLQHYSPLVDPALA

[0167] HypC is the expression / formation protein of HoxEFUYH[NiFe]-hydrogenase in Synechocystis sp. PCC 6803. The accession number of the protein in Genbank is BAA18180.1. The theoretical isoelectric point of HypC is 4.20 and the theoretical molecular weight is 7987.42 Daltons. The nucleotide sequence (231 bp) of HypC is shown below and is designated as SEQ ID NO:9 in the context of the present invention:

[0168] Atgtgtctagccctacctggccaggttgtcagtttaatgcccaactccgatcccctgttatgacgggaaaggttagctttgggggcatcattaaaaccattagccttgcctacg tacccgaggttaaggtggggggattacgtgattgtccatgtgggctttgccattagcattgtggacgaagaggcggcccaggaaactttgatagacttggcagaaatggggagtttaa

[0169] The amino acid sequence of HypC (76aa) is shown below, designated as SEQ ID NO: 10 in the context of the present invention:

[0170] MCLALPGQVVSLMPNSDPLLLTGKVSFGGIIKTISLAYVPEVKVGDYVIVHVGFAISIVDEEAAQETLIDLAEMGV

[0171] HypD is the expression / formation protein of HoxEFUYH[NiFe]-hydrogenase in Synechocystis sp. PCC 6803. The accession number of the protein in Genbank is BAA16622.1. The theoretical isoelectric point of HypD is 6.31 and the theoretical molecular weight is 40632.94 Daltons. The nucleotide sequence (1125 bp) of HypD is shown below and is designated as SEQ ID NO: 11 in the context of the present invention:

[0172]

[0173] The amino acid sequence of HypD (374aa) is shown below, and is designated as SEQ ID NO: 12 in the context of the present invention:

[0174] MKYVDEYRDAQAVAHYRQAIAREITKPWTLMEICGGQTHSIVKYGLDALLPKNLTLIHGPGCPVCVTPMELIDQALWLAKQPEIIFCSFGDMLRVPGSGADLLSIKAQGGDVRIVYSPLDCLAIARENPNREVVFFGVGFETTAPATAMTLHQARAQGISNFSLLCAHVLVPPAMEALLGNPNSLVQ GFLAAGHVCTVTGERAYQHIAEKYQVPIVITGFEPVDIMQGIFACVRQLESGQFTCNNQYRRSVQPQGNAHAQKIIDQVFEPVDRHWRGLGLIPASGLGLRPAFAPWDAAVKFANLLQTMAPTMGETVCISGEILQGQRKPSDCPAFGTICTPEQPLGAPMVSSEGACAAYYRYRQQLPEPVGAARV

[0175] HypE is the expression / formation protein of HoxEFUYH[NiFe]-hydrogenase in Synechocystis sp. PCC 6803. The accession number of the protein in Genbank is BAA17478.1. The theoretical isoelectric point of HypE is 4.93 and the theoretical molecular weight is 36425.60 Daltons. The nucleotide sequence (1038 bp) of HypE is shown below and is designated as SEQ ID NO: 13 in the context of the present invention:

[0176]

[0177] The amino acid sequence of HypE (345 aa) is shown below, designated as SEQ ID NO: 14 in the context of the present invention:

[0178] MNLVCPVPLDRYPQVLLAHGGGGKLSQQLLKQIFLPAFGASETGSHDAAVFTANQSSLAFTTDSYVINPLFFPGGDIGSLAVHGTVNDLAMAGATPRYISVGFILEEGLPMETLWRVAQSLGQAAQNCGVEILTGDTKVVDRGKGDGIFINTSGIGSLDHQQTIHPNQVQVG DRLILSGDLGRHGMAIMAVRQGLEFETTIESDSAPVHREVQALLSAGIPIHCLRDLTRGGLASAVNEIAQTSGVTMALRETLIPVEAEVQAACELLGFDPLYVANEGRFLAIVPPEAEQKTVEILQTFHPQATAIGTVTGKSAQTLGLVSLESSIGAPRLLDMISGEQLPRIC

[0179] HypF is the expression / formation protein of HoxEFUYH[NiFe]-hydrogenase in Synechocystis sp. PCC 6803. The accession number of the protein in Genbank is BAA10154.1. The theoretical isoelectric point of HypF is 8.19 and the theoretical molecular weight is 85358.25 Daltons. The nucleotide sequence of HypF (2304 bp) is shown below and is designated as SEQ ID NO: 15 in the context of the present invention:

[0180]

[0181] The amino acid sequence of HypF (767 aa) is shown below and is designated SEQ ID NO:16 in the context of the present invention:

[0182] mlktvaiqvqgrvqgvgfrpfvytlaqemglngwvnnstqgatvvitadekaiadfterltktlpppglieqlaveqlplesftnftirpssdgpktasilpdlstcsacltelfdpsdrrylypfincthcgprytiiealpydrcrttmarfrqctdcereykqpgdrrfhaqpnacprcgpqlafwnrqgqviaeanealnfavdnlkvgniiaikglggfhlccdatdfeaveklrlrkhrpdkplavmygnlgqivehyqpnnlevellqsaaapivllnkkkqlilveniapgnprvgvmlaytplhhlllkklkkpmvatsgnlageqicidnidaltrlqniadgflvhdrpivcpvddsvvqivagkplflrrargyapqpitlpkptqkkllamgghykntvaiakqnqayvsqhlgdlnsaptyqnfeeaiahlsqlydfspqeivadlhpdyfshqyaenqalpvtfvqhhyahilavmaehgvmeesvlgiawdgtgygmdgtiwggeflkitqgtwqriahlqpfhllgnqqaikyphrialallwptfgddfsadslgnwlnfnngfknkinsrlnqdlnnknlrqlwqrgqapltssmgrlfdgiatliglinevtfegqaaialeaqimpnlteeyypltlnnkekklavdwrplikaittedrsktnliatkfhnslvnliitiaqqqgiekvalgggcfqncyllastitalkkagfsplwprelppndgaicmgqllakiqarqyic

[0183] HoxW is a hypothetical protein in Synechocystis PCC 6803. The accession number of the protein in Genbank is BAA17680.1. The theoretical isoelectric point of HoxW is 4.93 and the theoretical molecular weight is 17129.53 Daltons. The nucleotide sequence of HoxW (474 ​​bp) is shown below and is designated as SEQ ID NO: 17 in the context of the present invention:

[0184] atgccaggccaatccaccaagtccactttaatcatcggttacggcaataccctgcggggggacgacggcgtggggcgttacctagcggaagaaattgctcagcaaaactggccccatt gtggagttattccacccatcaactcaccccagaattggccgaggcgatcgccgctgtggaccgggtaattttcattgatgcccaactgcaggaatcagcaaacgaaccatcggtggaa gttgtggccttaaaaaccctggaacccaacgaactgtcaggggatttggggcaccggggtaatcccagggaactcttgaccctggctaaaattctctacggcgttgaggtaaaggctt ggtgggtgttgattccggccttcacctttgattatggagagaaattgtctcccctgaccgcccgggcccaagccgaagccttagcccagatccgccccttggtattgggggagagataa

[0185] The amino acid sequence of HoxW (157 aa) is shown below, and is designated as SEQ ID NO: 18 in the context of the present invention:

[0186] MPGQSTKSTLIIGYGNTLRGDDGVGRYLAEEIAQQNWPHCGVISTHQLTPELAEAIAAVDRVIFIDAQLQESANEPSVEVVALKTLEPNELSGDLGHRGNPRELLTLAKILYGVEVKAWWVLIPAFTFDYGEKLSPLTARAQAEALAQIRPLVLGER

[0187] Optionally, according to an embodiment of the present invention, all maturation factors HypA, HypB, HypC, HypD, HypE, HypF and HoxW are assembled in the form of a tandem nucleotide sequence (6515 bp). The nucleotide sequence includes an NcoI restriction site (CCATGG) at the beginning of the sequence and an AvrII restriction site (CCTAGG) at the end of the sequence for cloning in a pACYCDuet-1 plasmid. The tandem nucleotide sequence is as follows, and is named SEQ ID NO: 19 in the context of the present invention:

[0188]

[0189] Figure 3 The collection of illustrative plates of plasmid pACYCDuet-1 is shown, and the plasmid is used to construct at least one expression vector of maturation factor HypA, HypB, HypC, HypD, HypE, HypE, HypF and HoxW by inserting SEQ ID NO:5 and / or SEQ-ID NO:7 and / or SEQ ID NO:9 and / or SEQ ID NO:11 and / or SEQ ID NO:13 and / or SEQ ID NO:15 and / or SEQ ID NO:17 and / or SEQ ID NO:19 in pACYCDuet-1. pACYCDuet-1 is a 4008bp plasmid with an E. coli replication origin, a chloramphenicol resistance gene, two multiple cloning sites (MCS) comprising multiple restriction sites, a T7 promoter and a T7 transcription terminator. It also comprises the lacI gene encoding a transcriptional repressor. This transcriptional repression can be relieved by adding IPTG.

[0190] Figure 4 Analysis of the NcoI and HindIII restriction endonuclease digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW (tandem sequence SEQ ID NO: 19) is shown. As expected for the enzyme digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW, two bands of DNA fragments at about 6000 bp and about 4000 bp were shown. This confirms the presence of the sequence HypABCDEFHoxW in the expression vector pACYCDuet-1.

[0191] 3. Transfer the expression vectors pET26b(+)+HoxH and pACYCDuet-1+HypABCDEFHoxW into E. coli

[0192] The competent cells of Escherichia coli (DE3) BL21 were used to recombinantly express the HoxH subunit of the HoxEFUYH[NiFe]-hydrogenase of Synechocystis PCC 6803. The cells usually produce recombinant proteins under the control of the T7 promoter.

[0193] According to the conventional heat shock method well known to those skilled in the art, two expression vectors "pET26b (+) + HoxH" and "pACYCDuet-1 + HypABCDEFHoxW" were co-transformed into these cells. Transformants with dual resistance to kanamycin (50 μg / mL) and chloramphenicol (25 μg / mL) were stored on agar medium at 4°C.

[0194] 4. Verify whether the target DNA sequence exists in E. coli

[0195] Some colonies may possess two plasmids that confer resistance to the selected marker used, but lack the target DNA sequences, i.e., HoxH and HypABCDEFHoxW. Therefore, it is very important to verify the presence of these target DNA sequences. The present invention performs an experiment well known to those skilled in the art, which comprises extracting plasmid DNA from the colonies and then digesting the plasmid DNA with the restriction endonuclease used during the insertion of the target DNA sequence in the plasmid.

[0196] Therefore, plasmid DNA was extracted from colonies with dual resistance to kanamycin and chloramphenicol according to protocols well known to those skilled in the art.

[0197] The digestion was then performed according to protocols well known to those skilled in the art. The digestion of the expression vector pET26b(+)+HoxH by NdeI and BlpI restriction endonucleases produced two DNA fragments, namely the linearized plasmid pET26b(+) at 5300 bp and the HoxH DNA fragment at 1500 bp (see Figure 5 The expression vector pACYCDuet-1+HypABCDEFHoxW was digested by NcoI and HindIII restriction endonucleases to produce two DNA fragments, namely the linearized plasmid pACYCDuet-1 at 4000 bp and the HypABCDEFHoxW DNA fragment at 6500 bp (see Figure 6 ). This confirmed the presence of HoxH and HypABCDEFHoxW sequences in colonies expressing the pET26b(+) and PACYCDuet-1 vectors, respectively.

[0198] 5. Recombinant production of HoxH in E. coli and affinity chromatography purification

[0199] To obtain a recombinant form of the HoxH subunit of the HoxEFUYH[NiFe]-hydrogenase from Synechocystis sp. PCC 6803, four Erlenmeyer flasks with a volume of 250 mL were inoculated with colonies of E. coli containing two expression vectors. The medium used was 2XYT medium (16 g tryptone, 10 g yeast extract, 5 g NaCl per liter), supplemented with 100 μM FeAmCi, 50 μM NiSO4, 50 μM cysteine, 50 μg / mL kanamycin and 25 μg / mL chloramphenicol. At an optical density (DO600nm) of 1.2, 0.2 mM IPTG was added to the culture to induce the recombinant production of the HoxH subunit at 18°C ​​and agitation (stirring speed) of 200 rpm. The production time at 18°C ​​was 20 h. The cells were then collected by centrifugation (15 min at 4500 rpm) and the purification process was then started.

[0200] A purification method was established to confirm the production of recombinant forms of the HoxH subunit of the HoxEFUYH[NiFe]-hydrogenase from Synechocystis sp. PCC 6803. Figure 7 ) HoxH is purified to significant homogeneity in a single affinity chromatography step. The method involves immobilized metal affinity chromatography (IMAC). The chelating agent is nitrilotriacetic acid (NTA), which allows the stationary phase to bind to metal ions. The immobilized metal is nickel (Ni). This chromatography method can separate proteins containing polyhistidine tags with great specificity. After 20 hours of recombinant production of HoxH at 18°C, 1L of E. coli culture was collected by centrifugation (15min at 4500rpm). The cells were resuspended in 25mL of lysis buffer (20mM sodium phosphate buffer pH 7.5, 300mM KCl+2μL endonuclease (benzonase)+50μL MgCl21M+1 pellet consisting of a protease inhibitor cocktail without EDTA) and then lysed using a French press. The supernatant (25 mL) was recovered by centrifugation (15 min at 15000 rpm), filtered (0.45 μm), and applied to a Ni-NTA column (1 mL) previously equilibrated in elution buffer (20 mM sodium phosphate buffer, pH 7.5, 300 mM KCl + 10 mM imidazole). The column was then washed with 35 mL of elution buffer (20 mM sodium phosphate buffer, pH 7.5, 300 mM KCl + 10 mM imidazole) until the absorbance at 280 nm (representing protein concentration) dropped to zero. Then, elution was performed through 5 concentration gradients with increasing imidazole concentrations, namely 50 mM, 100 mM, 150 mM, 200 mM and 250 mM (see Figure 7 ).

[0201] SDS-PAGE analysis of the various fractions obtained during chromatography showed a major band of approximately 54 kDa in the fractions eluted with 100 mM, 150 mM, 200 mM, and 250 mM imidazole concentrations, which is the expected size of the HoxH subunit (see Figure 8 HoxH also showed excellent purity, as no additional bands were seen in the fractions eluted at 150 mM, 200 mM, and 250 mM imidazole concentrations. It can be concluded that HoxH has been purified to significant homogeneity in a single affinity chromatography step.

[0202] In order to confirm that it is indeed a recombinant form of the HoxH subunit containing the active site of the HoxEFUYH[NiFe]-hydrogenase of Synechocystis sp. PCC 6803, the present invention used a polyhistidine anti-tag antibody for immunodetection (see Fig. 9). A molecular weight marker containing a polyhistidine-tagged protein was added as a positive control. The HoxH recombinant protein was well detected in the load, i.e. the supernatant obtained from E. coli cell lysis and applied to the Ni-NTA affinity column. The HoxH recombinant protein was also present in the fraction that was not absorbed by the Ni-NTA column. This indicates that the loading capacity of the Ni-NTA column was exceeded and part of the HoxH recombinant protein could not bind to it. No signal appeared in the "50 mM elution fraction". At this low imidazole concentration, the HoxH recombinant protein still adhered to the Ni-NTA column. Finally, by immunodetection, the presence of the HoxH recombinant protein was confirmed in the 200 mM fraction at the expected size, i.e. close to 54 kDa. This clearly confirmed that the single affinity chromatography step purified the HoxH recombinant protein. When some of the HoxH recombinant protein appeared as bands in the immunodetection, some low-intensity bands also appeared at molecular weights below 54 kDa. This may be the HoxH recombinant protein that was partially degraded at the C-terminus. SDS-PAGE analysis did not show the presence of degraded HoxH recombinant protein bands, indicating that the proportion of degraded HoxH recombinant protein seemed to be minimal. It is worth noting that immunodetection is a very sensitive method that can detect very small amounts of protein.

[0203] 6. Detection of hydrogenase activity in HoxH recombinant protein

[0204] The ability to recombinantly express [NiFe]-hydrogenase provides a wide range of possibilities for generating mutant forms with properties that differ significantly from those of the native enzyme. Fig.10 The structure of HoxEFUYH, [NiFe]-hydrogenase from Synechocystis PCC 6803 is shown. NADPH is a cofactor for HoxEFUYH in Synechocystis PCC 6803. In a standard in vitro activity assay for [NiFe]-hydrogenase, methyl viologen (MV) was used as a redox mediator. Fig.11 Schematic diagram of electron transfer and expected interactions between HoxEFUYH and NADPH and / or MV (shaded arrows).

[0205] It is generally known to those skilled in the art that MV can transfer electrons directly to one or more FeS centers by bypassing FMN. Fig.12 As shown, it is currently unclear whether the single HoxH subunit containing the active site of the HoxEFUYH[NiFe]-hydrogenase from Synechocystis PCC 6803 can directly accept electrons from MVs to produce hydrogen without an additional redox relay.

[0206] In the context of the present invention, to test this possibility, a standard in vitro activity assay of [NiFe]-hydrogenase was performed in the presence of HoxH recombinant protein and MVs that had been reduced with sodium dithionite (see Fig.13 ). HoxH is able to take electrons from the reduced methyl viologen and combine them with protons in the buffer, thereby generating a hydrogenase-catalyzed reaction according to the equation Hydrogen is produced. According to protocols well known to those skilled in the art, the standard in vitro activity assay of the [NiFe]-hydrogenase includes the use of a 2 mL flask sealed by an airtight and nitrogen degassed septum. 100 mM sodium dithionite and 10 mM MV are dissolved in 10 mM phosphate buffer at pH 6.8 and nitrogen degassed to obtain a reaction mixture. 1 mL of the reaction mixture is added to the flask using a syringe. 200 μg of HoxH recombinant protein is then added to the reaction mixture. After the addition of the HoxH recombinant protein, hydrogen production begins ( Fig.13 The time indicated by the arrow in the middle). The protein content was determined by the Coomassie brilliant blue method (Bradford, 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254). The hydrogen production was continuously monitored using a calibrated hydrogen microsensor (Unisense, Aarhus, Denmark).

[0207] The amount of HoxH recombinant protein added (unit: mg mL -1 ) and the hydrogen evolution rate (μmol H2·min -1 ) is known, therefore, the activity of the HoxH recombinant protein can be calculated. For example, the specific activity can be 0.1 μmol H2·min -1 mg -1 Enzyme.

[0208] Surprisingly, the single catalytic HoxH subunit containing the active site of the HoxEFUYH[NiFe]-hydrogenase of Synechocystis sp. PCC 6803 can catalyze the reduction of protons, accepting electrons from a redox mediator such as methyl viologen, without the need for an additional FeS center as a redox relay, as has been emphasized in the context of the present invention.

[0209] In the context of the present invention, a standard in vitro activity assay of [NiFe]-hydrogenase in the presence of HoxH recombinant protein, benzyl viologen and hydrogen was also performed (see Fig.14 ). HoxH is able to take electrons from hydrogen, donate them to benzyl viologen, and according to the equation representing the hydrogenase catalyzed reaction Protons are produced. According to a protocol well known to those skilled in the art, the standard in vitro activity test of the [NiFe]-hydrogenase includes the use of a 2 mL flask sealed by a gas-tight and hydrogen-saturated diaphragm. 40 μmol of benzyl viologen was dissolved in 50 mM Tris buffer at pH 7.6 and degassed to obtain a reaction mixture. 2 mL of the reaction mixture was added to the flask using a syringe. The test was performed at 40 ° C. 340 μg of HoxH recombinant protein was then added to the reaction mixture. After the addition of HoxH recombinant protein, hydrogen consumption and reduction of benzyl viologen began. The protein content was determined by the Coomassie Brilliant Blue method (Bradford, 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254). The reduction of benzyl viologen was monitored spectrophotometrically at a wavelength of 578 nm. The molar extinction coefficient is 8600 M -1 cm -1 .

[0210] The amount of HoxH recombinant protein added (unit: mg mL -1 ) and hydrogen consumption rate (μmol H2·min -1 ) is known, therefore, the activity of the HoxH recombinant protein can be calculated, which is equivalent to the reduction rate of benzyl viologen upon addition of a specific amount of the recombinant protein. For example, the activity can be expressed as μmol H2·min -1 mg -1 Enzyme.

[0211] Surprisingly, a single catalytic HoxH subunit containing the active site of the HoxEFUYH[NiFe]-hydrogenase of Synechocystis sp. PCC 6803 can catalyze the oxidation of hydrogen, generating protons and donating electrons to redox mediators such as benzyl viologen, without the need for an additional FeS center as a redox relay, as has been emphasized in the context of the present invention.

[0212] In the absence of expression of the other subunits of the HoxEFUYH pentamer, the problems inherent in the prior art are at least partially solved: the HoxH recombinant protein is able to catalyze the reduction of protons by itself, accepting electrons from a redox mediator (e.g., methyl viologen) without the need for an additional FeS center as a redox relay. The HoxH protein itself is also able to catalyze the oxidation of H2, reducing a redox mediator (e.g., benzyl viologen) without the need for an additional FeS center as a redox relay. This special feature demonstrates the great advantage of the present invention, especially the recombinant production of a catalytically active single subunit having a [NiFe]-hydrogenase active site.

[0213] 7. Recombinant production, affinity chromatography purification and detection of hydrogenase activity in HoxH recombinant proteins produced in E. coli in the absence of exogenous maturation factors

[0214] In a manner similar to Example 3 above, the expression vector pET26b (+) + HoxH was transformed into Escherichia coli (competent cells BL21 (DE3)) by a conventional heat shock method well known to those skilled in the art, and this was performed without the expression vector pACYCDUET-1 + HypABCDEFHoxW. The transformant was resistant to kanamycin (50 μg / mL).

[0215] In a manner similar to that described in Example 4 above, the presence of the target HoxH DNA sequence was verified by a series of experiments well known to those skilled in the art, namely, extraction of plasmid DNA and enzyme digestion.

[0216] HoxH protein was recombinantly produced in E. coli in the absence of plasmid pACYCDUET-1+HupABCDEFHoxW encoding exogenous maturation factor HupABCDEFHoxW in a manner similar to that described in Example 5. HoxH protein was then purified by affinity chromatography in a manner similar to that described in Example 5.

[0217] In a manner similar to Example 6 above, the hydrogenase activity of HoxH proteins recombinantly produced in E. coli in the absence of exogenous maturation factors was tested according to protocols involving MV and well known to those skilled in the art.

[0218] Therefore, in the context of the present invention, a standard in vitro activity assay for [NiFe]-hydrogenase was performed in the presence of HoxH recombinant protein and MVs that had been reduced with sodium dithionite (see Fig.15 ). HoxH is able to take electrons from the reduced methyl viologen and combine them with protons in the buffer, thereby generating a hydrogenase-catalyzed reaction according to the equation Hydrogen is produced. According to protocols well known to those skilled in the art, the standard in vitro activity assay of the [NiFe]-hydrogenase includes the use of a 2 mL flask sealed by an airtight and nitrogen degassed septum. 100 mM sodium dithionite and 10 mM MV are dissolved in 10 mM phosphate buffer at pH 6.8 and degassed with nitrogen to obtain a reaction mixture. 1 mL of the reaction mixture is added to the flask using a syringe. 750 μg of HoxH recombinant protein is then added to the reaction mixture. After the addition of the HoxH recombinant protein, hydrogen production begins ( Fig.15 The time indicated by the arrow in the middle). The protein content was determined by the Coomassie brilliant blue method (Bradford, 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254). The hydrogen production was continuously monitored using a calibrated hydrogen microsensor (Unisense, Aarhus, Denmark).

[0219] The amount of HoxH recombinant protein added (unit: mg mL -1 ) and the hydrogen evolution rate (μmol H2·min -1 ) is known, therefore, the activity of the HoxH recombinant protein can be calculated. For example, the specific activity can be 0.1 μmol H2·min -1 mg -1 Enzyme.

[0220] In the context of the present invention, it has been highlighted that a single HoxH catalytic subunit containing the active site of the HoxEFUYH[NiFe]-hydrogenase of Synechocystis sp. PCC 6803 can catalyze the reduction of protons, accepting electrons from redox mediators such as methyl viologen, without the intervention of the exogenous maturation factors HupABCDEFHoxW of E. coli.

[0221] The present invention has been described in conjunction with specific embodiments, which are merely illustrative and should not be considered as limiting the present invention. Generally speaking, it should be clear to those skilled in the art that the present invention is not limited to the examples shown and / or described above.

[0222] The use of the verbs "comprise", "include" or any other variations and conjugations thereof shall not in any way exclude the presence of other elements than those stated above.

[0223] The use of the indefinite article "a", "an" or the definite article "the" to introduce one element does not exclude the presence of a plurality of such elements. Sequence Listing <110> H2WIN Co., Ltd. <120> Monomeric polypeptides having hydrogenase activity, in particular recombinant monomeric polypeptides having hydrogenase activity <130> T888EPprio <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 1425 <212> DNA <213> Synechocystis CCP6803 <400> 1 atgtctaaaa ccattgttat cgatcccgtt acccggattg aaggccatgc caaaatctcc 60 attttcctca acgaccaggg caacgtagat gatgttcgtt tccatgtggt ggagtatcgg 120 ggttttgaaa aattttgcga aggtcgtccc atgtgggaaa tggctggtat taccgcccgt 180 atttgcggca tttgtccggt tagccatctg ctctgtgcgg ctaaaaccgg ggataagtta 240 ctggcggtgc aaatccctcc agccggggaa aaactgcgcc gtttaatgaa tttagggcaa 300 attacccaat cccacgccct aagttttttc catctcagca gtcctgattt tctgcttggt 360 tgggacagtg atcccgctac tcgcaatgtg tttggtttaa ttgctgctga ccccgattta 420 gctagggcag gtattcggtt acggcaattt ggccaaacgg taattgaact tttggggagct 480 aaaaaaatcc actctgcttg gtcagtgccc ggtggagtcc gatcgccgtt gtcggaagaa 540 ggcagacaat ggattgtgga ccgtttacca gaagcaaaag aaaccgttta tttagcctta 600 aatttgttta aaaatatgtt ggaccgcttc caaacagaag tggcagaatt tggcaaattt 660 ccctccctat tttgggctt agttgggaaa aataatgaat gggaacatta tggcggctcc 720 ctgcggttta ccgacagtga aggcaatatt gtcgcggaca atctcagtga agataattac 780 gctgatttta ttggtgaatc gttggaaaaa tggtcctatt taaaatttcc ctactacaaa 840 tctctgggtt atcccgatgg catttatcgg gttggtcccc ttgcccgcct taatgtttgt 900 catcacattg gcaccccgga agcagaccaa gaattagaag aatatcggca acgggctgga 960 ggtgtggcca cgtcctcttt ctttatcat tacgcccgct tggtggaaat tcttgcctgt 1020 ttagaagcca tcgaattgtt aatggctgac cctgatattt tgtccaaaaa ttgtcgagct 1080 aaggcagaaa ttaattgtac cgaagcggtg ggagtgagcg aagcaccccg gggtacttta 1140 ttccaccatt acaagataga tgaagatggt ctaattaaga aagtgaattt gatcattgcc 1200 acggggcaaca ataacttagc catgataaa accgtggccc aaattgccaa acactacatt 1260 cgcaatcatg atgtgcaaga agggtttta aaccgggtgg aagcgggtat tcgttgttat 1320 gatccctgcc ttagttgttc tacccatgca gcgggacaa tgccattgat gatcgattta 1380 gttaaccctc aggggact aattaagtcc atccagcggg atta 1425 <210> 2 <211> 474 <212> PRT <213> CCP6803 <400> 2 Met Ser Lys Thr Ile Val Ile Asp Pro Val Thr Arg Ile Glu Gly His 1 5 10 15 Only Lys Ile Ser Ile Phe Leu Asn Asp Gln Gly Asn Val Asp Asp Val 20 25 30 Arg Phe His Val Val Glu Tyr Arg Gly Phe Glu Lys Phe Cys Glu Gly 35 40 45 Arg Pro Met Trp Glu Met Ala Gly Ile Thr Ala Arg Ile Cys Gly Ile 50 55 60 Cys Pro Will Be His Leu Leu Cys Ala Ala Lys Thr Gly Asp Lys Leu 65 70 75 80 Leu Ala Val Gln Ile Pro Pro Ala Gly Glu Lys Leu Arg Arg Leu Met 85 90 95 Asn Leu Gly Gln Ile Thr Gln Ser His Ala Leu Ser Phe Phe His Leu 100 105 110 Ser Ser Pro Asp Phe Leu Leu Gly Trp Asp Ser Asp Pro Ala Thr Arg 115 120 125 Asn Val Phe Gly Leu Ile Ala Ala Asp Pro Asp Leu Ala Arg Ala Gly 130 135 140 Ile Arg Leu Arg Gln Phe Gly Gln Thr Val Ile Glu Leu Leu Gly Ala 145 150 155 160 Lys Lys Ile His Ser Ala Trp Ser Val Pro Gly Gly Val Arg Ser Pro 165 170 175 Leu Ser Glu Glu Gly Arg Gln Trp Ile Val Asp Arg Leu Pro Glu Ala 180 185 190 Lys Glu Thr Val Tyr Leu Ala Leu Asn Leu Phe Lys Asn Met Leu Asp 195 200 205 Arg Phe Gln Thr Glu Val Ala Glu Phe Gly Lys Phe Pro Ser Leu Phe 210 215 220 Met Gly Leu Val Gly Lys Asn Asn Glu Trp Glu His Tyr Gly Gly Ser 225 230 235 240 Leu Arg Phe Thr Asp Ser Glu Gly Asn Ile Val Ala Asp Asn Leu Ser 245 250 255 Glu Asp Asn Tyr Ala Asp Phe Ile Gly Glu Ser Val Glu Lys Trp Ser 260 265 270 Tyr Leu Lys Phe Pro Tyr Tyr Lys Ser Leu Gly Tyr Pro Asp Gly Ile 275 280 285 Tyr Arg Val Gly Pro Leu Ala Arg Leu Asn Val Cys His His Ile Gly 290 295 300 Thr Pro Glu Ala Asp Gln Glu Leu Glu Glu Tyr Arg Gln Arg Ala Gly 305 310 315 320 Gly Val Ala Thr Ser Ser Phe Phe Tyr His Tyr Ala Arg Leu Val Glu 325 330 335 Ile Leu Ala Cys Leu Glu Ala Ile Glu Leu Leu Met Ala Asp Pro Asp 340 345 350 Ile Leu Ser Lys Asn Cys Arg Ala Lys Ala Glu Ile Asn Cys Thr Glu 355 360 365 Ala Val Gly Val Ser Glu Ala Pro Arg Gly Thr Leu Phe His His Tyr 370 375 380 Lys Ile Asp Glu Asp Gly Leu Ile Lys Lys Val Asn Leu Ile Ile Ala 385 390 395 400 Thr Gly Asn Asn Asn Leu Ala Met Asn Lys Thr Val Ala Gln Ile Ala 405 410 415 Lys His Tyr Ile Arg Asn His Asp Val Gln Glu Gly Phe Leu Asn Arg 420 425 430 Val Glu Ala Gly Ile Arg Cys Tyr Asp Pro Cys Leu Ser Cys Ser Thr 435 440 445 His Ala Ala Gly Gln Met Pro Leu Met Ile Asp Leu Val Asn Pro Gln 450 455 460 Gly Glu Leu Ile Lys Ser Ile Gln Arg Asp 465 470 <210> 3 <211> 1453 <212> DNA <213> Artificial sequence <220> <223> Optimized [NiFe]-hydrogenase HoxEFUYH <220> <221> misc_feature <222> (1)..(6) <223> Ndel restriction site <220> <221> misc_feature <222> (10)..(27) <223> Polyhistidine tag <220> <221> misc_feature <222> (1447)..(1453) <223> Blpl restriction site <400> 3 catatgagcc accaccacca ccatcacaaa accatcgtca tcgacccagt cacccgcatc 60 gaaggccacg ccaaaattag catttttctg aacgaccagg gcaacgtcga cgacgtccgc 120 tttcacgttg ttgaataccg tggcttcgaa aaattttgtg aaggtcgtcc gatgtgggaa 180 atggccggta tcacggcacg tatttgtgga atttgtccgg tgagccatct gctgtgtgcc 240 gcaaaaaccg gagataaact gctggcagtg cagattccgc cggcaggtga aaaactgcgt 300 cgtctgatga atctgggtca gattacacag tcgcatgcac tgtctttctt tcatctgagt 360 agcccagatt ttctgctggg gtgggatagc gacccggcaa cacgtaatgt gtttggtctg 420 attgcggctg atccggatct ggcgcgtgcc ggtattcgtc tgcgtcagtt tggtcagaca 480 gttattgagc tgctgggggc gaaaaagatt catagtgcat ggtctgtgcc gggtggtgtt 540 cgtagtccgc tgagtgaaga aggtcgtcag tggattgttg atcgtctgcc ggaggcaaaa 600 gaaacggtct atctggcact gaatctgttt aaaaatatgc tggatcgttt ccagacagaa 660 gttgcagaat ttggaaaatt tccgtcactg tttatgggtc tggttggtaa aaataatgaa 720 tgggaacact atggtggtag cctgcgtttc acggactctg aaggtaatat tgttgcggat 780 aatctgagcg aagacaatta tgcagatttt atcggtgaaa gtgtggaaaa atggagctat 840 ctgaaatttc cgtattacaa aagcctgggc tatccggatg ggatctaccg tgttggaccg 900 ctggcacgtc tgaacgtttg tcatcatatt ggtaccccgg aagcagatca ggaactggaa 960 gaatatcgtc agcgtgcggg tggtgttgcg actagcagct ttttttatca ttatgcacgt 1020 ctggttgaaa ttctggcctg tctggaggca attgaactgc tgatggcaga tcctgatatt 1080 ctgtctaaaa attgtcgtgc aaaagcagaa attaactgta ccgaggcagt tggtgttagt 1140 gaggcgccgc gtggtaccct gtttcatcac tataaaattg acgaagatgg tctgattaaa 1200 aaggttaatc tgattatcgc aaccggtaac aataatctgg caatgaataa aaccgttgca 1260 cagattgcaa aacactacat tcgcaaccac gatgttcagg aagggtttct gaatcgtgta 1320 gaagccggca ttcgctgtta tgatccgtgt ctgagctgta gcacccatgc agcaggtcag 1380 atgcctctga tgattgacct ggttaatccg cagggtgagc tgattaaaag cattcagcgt 1440 gattaagctg agc 1453 <210> 4 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> 480 aa <400> 4 Met Ser His His His His His His Lys Thr Ile Val Ile Asp Pro Val 1 5 10 15 Thr Arg Ile Glu Gly His Ala Lys Ile Ser Ile Phe Leu Asn Asp Gln 20 25 30 Gly Asn Val Asp Asp Val Arg Phe His Val Val Glu Tyr Arg Gly Phe 35 40 45 Glu Lys Phe Cys Glu Gly Arg Pro Met Trp Glu Met Ala Gly Ile Thr 50 55 60 Ala Arg Ile Cys Gly Ile Cys Pro Val Ser His Leu Leu Cys Ala Ala 65 70 75 80 Lys Thr Gly Asp Lys Leu Leu Ala Val Gln Ile Pro Pro Ala Gly Glu 85 90 95 Lys Leu Arg Arg Leu Met Asn Leu Gly Gln Ile Thr Gln Ser His Ala 100 105 110 Leu Ser Phe Phe His Leu Ser Ser Pro Asp Phe Leu Leu Gly Trp Asp 115 120 125 Ser Asp Pro Ala Thr Arg Asn Val Phe Gly Leu Ile Ala Ala Asp Pro 130 135 140 Asp Leu Ala Arg Ala Gly Ile Arg Leu Arg Gln Phe Gly Gln Thr Val 145 150 155 160 Ile Glu Leu Leu Gly Ala Lys Lys Ile His Ser Ala Trp Ser Val Pro 165 170 175 Gly Gly Val Arg Ser Pro Leu Ser Glu Glu Gly Arg Gln Trp Ile Val 180 185 190 Asp Arg Leu Pro Glu Ala Lys Glu Thr Val Tyr Leu Ala Leu Asn Leu 195 200 205 Phe Lys Asn Met Leu Asp Arg Phe Gln Thr Glu Val Ala Glu Phe Gly 210 215 220 Lys Phe Pro Ser Leu Phe Met Gly Leu Val Gly Lys Asn Asn Glu Trp 225 230 235 240 Glu His Tyr Gly Gly Ser Leu Arg Phe Thr Asp Ser Glu Gly Asn Ile 245 250 255 Val Ala Asp Asn Leu Ser Glu Asp Asn Tyr Ala Asp Phe Ile Gly Glu 260 265 270 Ser Val Glu Lys Trp Ser Tyr Leu Lys Phe Pro Tyr Tyr Lys Ser Leu 275 280 285 Gly Tyr Pro Asp Gly Ile Tyr Arg Val Gly Pro Leu Ala Arg Leu Asn 290 295 300 Val Cys His His Ile Gly Thr Pro Glu Ala Asp Gln Glu Leu Glu Glu 305 310 315 320 Tyr Arg Gln Arg Ala Gly Gly Val Ala Thr Ser Ser Phe Phe Tyr His 325 330 335 Tyr Ala Arg Leu Val Glu Ile Leu Ala Cys Leu Glu Ala Ile Glu Leu 340 345 350 Leu Met Ala Asp Pro Asp Ile Leu Ser Lys Asn Cys Arg Ala Lys Ala 355 360 365 Glu Ile Asn Cys Thr Glu Ala Val Gly Val Ser Glu Ala Pro Arg Gly 370 375 380 Thr Leu Phe His His Tyr Lys Ile Asp Glu Asp Gly Leu Ile Lys Lys 385 390 395 400 Val Asn Leu Ile Ile Ala Thr Gly Asn Asn Asn Leu Ala Met Asn Lys 405 410 415 Thr Val Ala Gln Ile Ala Lys His Tyr Ile Arg Asn His Asp Val Gln 420 425 430 Glu Gly Phe Leu Asn Arg Val Glu Ala Gly Ile Arg Cys Tyr Asp Pro 435 440 445 Cys Leu Ser Cys Ser Thr His Ala Ala Gly Gln Met Pro Leu 450 455 460 <210> 5 <211> 342 <212> DNA <213> Synechocystis sp. PCC6803 <400> 5 atgcacgaag ttagtctgat ggagcaaact ttggcgatcg ccattgccca ggcggaagac 60 catggagcca gccaaatcca tcgtttaacc ctgcgggtgg ggcaacagtc tggggtggtg 120 gccgatgccc tacggtttgc gtttgaagtg gtgcgacaaa ataccatggc cgccgaggcg 180 agattggaaa ttgaagaaat tcccgttacc tgtcgttgcc aacactgcca cgaaaatttt 240 cagccagagg attggattta ccgctgtccc cactgcgacc agattagcca aacagtaatg 300 gatggcaaac agttggaact agcatcccta gaactgagtt ga 342 <210> 6 <211> 113 <212> PRT <213> Synechocystis sp. PCC6803 <400> 6 Met His Glu Val Ser Leu Met Glu Gln Thr Leu Ala Ile Ala Ile Ala 1 5 10 15 Gln Ala Glu Asp His Gly Ala Ser Gln Ile His Arg Leu Thr Leu Arg 20 25 30 Val Gly Gln Gln Ser Gly Val Val Ala Asp Ala Leu Arg Phe Ala Phe 35 40 45 Glu Val Val Arg Gln Asn Thr Met Ala Ala Glu Ala Arg Leu Glu Ile 50 55 60 Glu Glu Ile Pro Val Thr Cys Arg Cys Gln His Cys His Glu Asn Phe 65 70 75 80 Gln Pro Glu Asp Trp With Tyr Arg Cys Pro His Cys Asp Gln With Ser 85 90 95 Gln Thr Val Met Asp Gly Lys Gln Leu Glu Leu Ala Ser Leu Glu Leu 100 105 110 Ser <210> 7 <211> 858 <212> DNA <213> PCC6803 <400> 7 atgtgccaaa actgcggttg tagtgcggtg ggaaccgttg cccatagcca ccatcaccat 60 ggcgatggaa attttgccca cagccatgat gaccatgacc agcaagaaca tcatcaccac 120 catggcaact acagcaaag tccagtcag cagactgtga ccattgaacc cgatcgccag 180 tccattgcca ttggccaagg cattctcagc aagaatgacc gcctagcgga aaggaatcgg 240 ggctatttcc aggctaaggg cttactggtg atgaatttcc tctcttctcc cggagccggt 300 aaaactgctc tgatcgaaaa aatggtcggc gatcgacaaa aagaccatcc caccgccgtc 360 attgtggggg atttagccac cgataacgat gcccaacgtc tccgcagtgc cggggcgatc 420 gccattcagg tcaccacagg aaatatttgc catctggaag cggaaatggt ggccaaggcg 480 gcccaaaagt tagatttaga caatatcgat caattgatca ttgaaaatgt tggtaatttg 540 gtttgcccca ccacctatga tctaggggaa gatttacggg tcgtattatt ttccgtcaca 600 gaaggggagg ataaacccct taaatatccc gccaccttca aatcagccca ggttatttta 660 gtcaccaaac aggacattgc cgccgcagtg gattttgatg cagagctggc ttggcaaaac 720 ctacggcaag tggcccccca agcccaaatt tttgcagtgt ctgcccgcac ggggaaagga 780 ttgcagtcct ggtatgagta tttggatcaa tggcaactcc aacactattc gccgttggtt 840 gatccagcat tggcctaa 858 <210> 8 <211> 285 <212> PRT <213> Synechocystis sp. PCC6803 <400> 8 Met Cys Gln Asn Cys Gly Cys Ser Ala Val Gly Thr Val Ala His Ser 1 5 10 15 His His His His Gly Asp Gly Asn Phe Ala His Ser His Asp Asp His 20 25 30 Asp Gln Gln Glu His His His His His Gly Asn Tyr Ser Lys Ser Pro 35 40 45 Ser Gln Gln Thr Val Thr Ile Glu Pro Asp Arg Gln Ser Ile Ala Ile 50 55 60 Gly Gln Gly Ile Leu Ser Lys Asn Asp Arg Leu Ala Glu Arg Asn Arg 65 70 75 80 Gly Tyr Phe Gln Ala Lys Gly Leu Leu Val Met Asn Phe Leu Ser Ser 85 90 95 Pro Gly Ala Gly Lys Thr Ala Leu Ile Glu Lys Met Val Gly Asp Arg 100 105 110 Gln Lys Asp His Pro Thr Ala Val Ile Val Gly Asp Leu Ala Thr Asp 115 120 125 Asn Asp Ala Gln Arg Leu Arg Ser Ala Gly Ala Ile Ala Ile Gln Val 130 135 140 Thr Thr Gly Asn Ile Cys His Leu Glu Ala Glu Met Val Ala Lys Ala 145 150 155 160 Ala Gln Lys Leu Asp Leu Asp Asn Ile Asp Gln Leu Ile Ile Glu Asn 165 170 175 Val Gly Asn Leu Val Cys Pro Thr Thr Tyr Asp Leu Gly Glu Asp Leu 180 185 190 Arg Val Val Leu Phe Ser Val Thr Glu Gly Glu Asp Lys Pro Leu Lys 195 200 205 Tyr Pro Ala Thr Phe Lys Ser Ala Gln Val Ile Leu Val Thr Lys Gln 210 215 220 Asp Ile Ala Ala Ala Val Asp Phe Asp Ala Glu Leu Ala Trp Gln Asn 225 230 235 240 Leu Arg Gln Val Ala Pro Gln Ala Gln Ile Phe Ala Val Ser Ala Arg 245 250 255 Thr Gly Lys Gly Leu Gln Ser Trp Tyr Glu Tyr Leu Asp Gln Trp Gln 260 265 270 Leu Gln His Tyr Ser Pro Leu Val Asp Pro Ala Leu Ala 275 280 285 <210> 9 <211> 231 <212> DNA <213> Synechocystis sp. PCC6803 <400> 9 atgtgtctag ccctacctgg ccaggttgtc agtttaatgc ccaactccga tcccctgtta 60 ctgacgggaa aggttagctt tgggggcatc attaaaacca ttagccttgc ctacgtaccc 120 gaggttaagg tgggggatta cgtgattgtc catgtgggct ttgccattag cattgtggac 180 gaagaggcgg cccaggaaac tttgatagac ttggcagaaa tgggagttta a 231 <210> 10 <211> 76 <212> PRT <213> Synechocystis sp. PCC6803 <400> 10 Met Cys Leu Ala Leu Pro Gly Gln Val Val Ser Leu Met Pro Asn Ser 1 5 10 15 Asp Pro Leu Leu Leu Thr Gly Lys Val Ser Phe Gly Gly Ile Ile Lys 20 25 30 Thr Ile Ser Leu Ala Tyr Val Pro Glu Val Lys Val Gly Asp Tyr Val 35 40 45 Ile Val His Val Gly Phe Ala Ile Ser Ile Val Asp Glu Glu Ala Ala 50 55 60 Gln Glu Thr Leu Ile Asp Leu Ala Glu Met Gly Val 65 70 75 <210> 11 <211> 1125 <212> DNA <213> Synechocystis sp. PCC6803 <400> 11 atgaaatacg ttgatgaata tcgggatgcc caggcggtgg cccattaccg tcaggcgatc 60 gccagggaga taaccaaacc ttggacgctg atggagattt gcggcggcca gacccacagc 120 attgtcaaat atggcttgga tgctttgttg ccgaagaatt tgactctgat ccatggtccc 180 ggctgtcctg tgtgcgtcac tccgatggaa ttaattgacc aggctttgtg gttagctaag 240 caaccggaga tcattttttg ttcctttggc gatatgttgc gggtgcccgg cagtggggcg 300 gatttgctga gcattaaagc ccagggcggc gatgtgcgca ttgtctattc tcctttggat 360 tgtttggcga tcgccaggga gaatcctaat cgggaagtgg tatttttcgg agtaggtttt 420 gaaactacag cccctgccac ggccatgact ctccaccaag ctagggccca gggaattagc 480 aatttcagtt tactttgcgc ccatgtattg gtgcccccgg ctatggaggc tttattaggc 540 aatcccaatt ccctcgtgca gggctttttg gcggcagggc atgtctgtac ggtgaccggg 600 gaaagggcct atcaacatat cgctgaaaaa taccaagtac ccattgtcat cactggcttt 660 gaacctgtgg atattatgca gggcatcttt gcctgtgtgc gccaactgga gtcgggacaa 720 ttcacctgca acaatcaata tcggcgatcg gtccaacccc agggcaatgc ccatgctcag 780 aaaattattg accaagtgtt tgagccagtc gatcgccatt ggcggggttt gggattaatt 840 ccggccagcg gtttgggttt aaggccagca tttgccccct gggatgccgc agttaaattc 900 gccaatttat tgcaaaccat ggccccaacg atgggagaaa cagtgtgtat tagcggggaa 960 attttacagg gacaacggaa gcccagcgat tgtccagcct ttggtactat ctgcacccca 1020 gaacaaccct tgggggctcc catggtttcc tcggaaggag cctgtgccgc ctattaccgt 1080 tatcgccaac aattaccgga accagtggga gcggccagag tttag 1125 <210> 12 <211> 374 <212> PRT <213> Synechocystis sp. PCC6803 <400> 12 Met Lys Tyr Val Asp Glu Tyr Arg Asp Ala Gln Ala Val Ala His Tyr 1 5 10 15 Arg Gln Ala Ile Ala Arg Glu Ile Thr Lys Pro Trp Thr Leu Met Glu 20 25 30 Ile Cys Gly Gly Gln Thr His Ser Ile Val Lys Tyr Gly Leu Asp Ala 35 40 45 Leu Leu Pro Lys Asn Leu Thr Leu Ile His Gly Pro Gly Cys Pro Val 50 55 60 Cys Val Thr Pro Met Glu Leu Ile Asp Gln Ala Leu Trp Leu Ala Lys 65 70 75 80 Gln Pro Glu Ile Ile Phe Cys Ser Phe Gly Asp Met Leu Arg Val Pro 85 90 95 Gly Ser Gly Ala Asp Leu Leu Ser Ile Lys Ala Gln Gly Gly Asp Val 100 105 110 Arg Ile Val Tyr Ser Pro Leu Asp Cys Leu Ala Ile Ala Arg Glu Asn 115 120 125 Pro Asn Arg Glu Val Val Phe Phe Gly Val Gly Phe Glu Thr Thr Ala 130 135 140 Pro Ala Thr Ala Met Thr Leu His Gln Ala Arg Ala Gln Gly Ile Ser 145 150 155 160 Asn Phe Ser Leu Leu Cys Ala His Val Leu Val Pro Pro Ala Met Glu 165 170 175 Ala Leu Leu Gly Asn Pro Asn Ser Leu Val Gln Gly Phe Leu Ala Ala 180 185 190 Gly His Val Cys Thr Val Thr Gly Glu Arg Ala Tyr Gln His Ile Ala 195 200 205 Glu Lys Tyr Gln Val Pro Ile Val Ile Thr Gly Phe Glu Pro Val Asp 210 215 220 Ile Met Gln Gly Ile Phe Ala Cys Val Arg Gln Leu Glu Ser Gly Gln 225 230 235 240 Phe Thr Cys Asn Asn Gln Tyr Arg Arg Ser Val Gln Pro Gln Gly Asn 245 250 255 Ala His Ala Gln Lys Ile Ile Asp Gln Val Phe Glu Pro Val Asp Arg 260 265 270 His Trp Arg Gly Leu Gly Leu Ile Pro Ala Ser Gly Leu Gly Leu Arg 275 280 285 Pro Ala Phe Ala Pro Trp Asp Ala Ala Val Lys Phe Ala Asn Leu Leu 290 295 300 Gln Thr Met Ala Pro Thr Met Gly Glu Thr Val Cys Ile Ser Gly Glu 305 310 315 320 Ile Leu Gln Gly Gln Arg Lys Pro Ser Asp Cys Pro Ala Phe Gly Thr 325 330 335 Ile Cys Thr Pro Glu Gln Pro Leu Gly Ala Pro Met Val Ser Ser Glu 340 345 350 Gly Ala Cys Ala Ala Tyr Tyr Arg Tyr Arg Gln Gln Leu Pro Glu Pro 355 360 365 Val Gly Ala Ala Arg Val 370 <210> 13 <211> 1038 <212> DNA <213> Synechocystis sp. PCC6803 <400> 13 gtgaacttag tctgtcccgt tccccttgat cgttatcccc aggtactgtt agcccacggc 60 ggcggcggta agttgagcca acaattactt aagcaaattt ttttaccggc ctttggcgct 120 tctgaaacgg gtagtcatga tgcggcggtt tttactgcca accaaagttc tttagctttc 180 accaccgact cctatgtgat caatcccctc ttttttcctg ggggcgatat tggttctttg 240 gcagtccacg gcaccgttaa tgacctagcc atggccggcg caacccctcg ctatatcagc 300 gttggtttta tcctcgaaga aggattgccc atggagaccc tctggcgggt ggcccaatcc 360 ctagggcaag cggcccaaaa ctgtggggtg gaaattctta ccggtgatac caaagtggtg 420 gaccggggta agggagacgg cattttcatc aacaccagcg gcattggttc cctcgaccat 480 caacaaacta tccatcccaa tcaggtacag gtaggcgatc gcctaatttt gagcggtgat 540 ttgggacgtc atggcatggc cattatggca gtgcgccaag gattagaatt tgaaaccacc 600 attgaaagtg attcggcccc ggttcacaga gaagtgcagg cattattgtc ggcagggatc 660 ccaatccatt gtctgcggga tttaaccagg gggggattag ccagtgcggt taatgaaatt 720 gcccaaactt ccggggtaac catggcttta cgagaaacgt taatcccggt ggaggccgaa 780 gtacaagccg cctgtgaact gttgggtttt gaccccctct atgtggccaa tgagggaaga 840 ttcctggcca ttgtgccccc ggaagcagaa cagaagaccg tggaaatttt gcaaactttc 900 catccccaag ctacggcgat cggtacagta acaggcaaaa gtgcacaaac cttggggtta 960 gtcagtttgg aaagttccat tggtgccccc cggttgctag acatgatcag tggggagcaa 1020 ttaccccgta tttgttag 1038 <210> 14 <211> 345 <212> PRT <213> Synechocystis sp. PCC6803 <400> 14 Met Asn Leu Val Cys Pro Val Pro Leu Asp Arg Tyr Pro Gln Val Leu 1 5 10 15 Leu Ala His Gly Gly Gly Gly Lys Leu Ser Gln Gln Leu Leu Lys Gln 20 25 30 Ile Phe Leu Pro Ala Phe Gly Ala Ser Glu Thr Gly Ser His Asp Ala 35 40 45 Ala Val Phe Thr Ala Asn Gln Ser Ser Leu Ala Phe Thr Thr Asp Ser 50 55 60 Tyr Val Ile Asn Pro Leu Phe Phe Pro Gly Gly Asp Ile Gly Ser Leu 65 70 75 80 Ala Val His Gly Thr Val Asn Asp Leu Ala Met Ala Gly Ala Thr Pro 85 90 95 Arg Tyr Ile Ser Val Gly Phe Ile Leu Glu Glu Gly Leu Pro Met Glu 100 105 110 Thr Leu Trp Arg Val Ala Gln Ser Leu Gly Gln Ala Ala Gln Asn Cys 115 120 125 Gly Val Glu Ile Leu Thr Gly Asp Thr Lys Val Val Asp Arg Gly Lys 130 135 140 Gly Asp Gly Ile Phe Ile Asn Thr Ser Gly Ile Gly Ser Leu Asp His 145 150 155 160 Gln Gln Thr Ile His Pro Asn Gln Val Gln Val Gly Asp Arg Leu Ile 165 170 175 Leu Ser Gly Asp Leu Gly Arg His Gly Met Ala Ile Met Ala Val Arg 180 185 190 Gln Gly Leu Glu Phe Glu Thr Thr Ile Glu Ser Asp Ser Ala Pro Val 195 200 205 His Arg Glu Val Gln Ala Leu Leu Ser Ala Gly Ile Pro Ile His Cys 210 215 220 Leu Arg Asp Leu Thr Arg Gly Gly Leu Ala Ser Ala Val Asn Glu Ile 225 230 235 240 Ala Gln Thr Ser Gly Val Thr Met Ala Leu Arg Glu Thr Leu Ile Pro 245 250 255 Val Glu Ala Glu Val Gln Ala Ala Cys Glu Leu Leu Gly Phe Asp Pro 260 265 270 Leu Tyr Val Ala Asn Glu Gly Arg Phe Leu Ala Ile Val Pro Pro Glu 275 280 285 Ala Glu Gln Lys Thr Val Glu Ile Leu Gln Thr Phe His Pro Gln Ala 290 295 300 Thr Ala Ile Gly Thr Val Thr Gly Lys Ser Ala Gln Thr Leu Gly Leu 305 310 315 320 Val Ser Leu Glu Ser Ser Ile Gly Ala Pro Arg Leu Leu Asp Met Ile 325 330 335 Ser Gly Glu Gln Leu Pro Arg Ile Cys 340 345 <210> 15 <211> 2304 <212> DNA <213> Synechocystis sp. PCC6803 <400> 15 atgttaaaaa ccgttgccat acaggtccag ggaagggtgc aaggagtggg tttcgtccc 60 tttgtttata cccttgccca ggaatggga ctgaatggtt gggtgaataa ttccactcaa 120 ggagctaccg ttgtcattac cgccgacgaa aaggcgatcg ccgactttac ggagagatta 180 acgaagacat tacctcccc tggttgatt gacaattag ccgttgaca gttaccgctg 240 gaaagtttta ctaactttac tatccgcccc agtagtgatg gccctaaaac tgcgagtattt 300 ttacccgatt tatccacttg ttccgcctgc ttacagaac tattttgacc tagcgatcgc 360 cgttatcttt acccctttat taactgtacc cattgcggtc cccgctacac cattattgaa 420 gccctacctt acgaccgttg tcgtaccacc atggctaggt ttcgccaatg taccgactgt 480 gaaagggaat ataagcacc aggcgataga cgcttccatg cccacctaa tgcctgtcct 540 cgctgtggcc cccaactggc ttttggac cgacaaggcc aagtaattgc agaagcaat 600 gaagctttaa actttgctgt agataattta aaagtcggca atattacgc tattaaggc 660 ttaggtggct tccattgtg tgtgatgcc actgatttg aagctgtgga aaaattaaga 720 ttaaggaaac atcgaccgga taaacctttg gcggtaatgt atggtaatct tggtcaaatt 780 gtggagcatt accaacctaa taatctagaa gttgaattgt tacaaagtgc cgccgcccct 840 attgtgttat taaacaaaaa aaaacaatta atttggtgg aaaatattgc cccaggcaac 900 ccccgagtcg gcgtaatgtt agcctatact cctttgcatc acttattact aaaaaatta 960 aagaaaccca tggtagctac cagtggtaac ttagctgggg agcaaatttg cattgataat 1020 attgacgctt taacccggtt acaaaatatt gctgacggtt ttctcgttca tgatcgcccg 1080 attgtttgtc cagtggatga ttccgttgtc caaatagtag ctgggaagcc attatttttg 1140 cgtcgagccc ggggttacgc tcctcaaccc attactttac caaagcctac tcaaaaaaaa 1200 ctattggcga tgggaggtca ttataaaaat acagtggcga tcgccaaaca aaatcaagct 1260 tacgtcagcc aacatttggg cgatttgaat tctgctccca cctaccaaaa ttttgaagaa 1320 gccattgccc atttaagcca gctatacgat ttctctcccc aggaaattgt tgcagattta 1380 caccctgatt atttcagtca tcaatatgct gaaaaccaag ctttgcctgt cacttttgtg 1440 height height height height height height 1500 gtgttaggta ttgcttggga tggcactggc tacggcatgg acggtactat tggggggga 1560 gatttttaa aaatcacca aggtacttgg cagagaattg ctcatctaca accattcat 1620 ttattaggta atcacaagc cattaaatat cccatcgga ttgctttggc gttgttatgg 1680 cccactttg gtgatgattt ttctgctgat tctttagga attggttga ttcaataat 1740 gggtttaaa acagataaa cagcaggtta atcaggatc taacaca aaatttacgt 1800 caactttggc aacgagggca agcaccgctc acttcgagta tgggaagatt atttgacggt 1860 attgcgacac tgattaggatt gattaacgaa gtaacttg aaggtcaggc ggccatagct 1920 ctggaagctc agattgcc aaatttaact gaggagtatt atcctttgac tctaacac 1980 aaggaaaaaaattagctgt tgattggcgc cccttaatta aagctataac cacagagat 2040 agaagcaaa ctacctaat agccactaa ttccacaca gtttagtaaa tttaattatc 2100 actattgccc aacagcagggg aatcgaaaaa gttgctgg ggggaggttg ctttcaaat 2160 tgttatttgc ttgccagtac cattactgcc ctcaaaaaag ctggtttttc tcctttgtgg 2220 cccagagaac taccgcccaa cgacggtgcc atttgcatgg gtcaactgtt agctaaaatt 2280 caggctcggc aatatatctg ttaa 2304 <210> 16 <211> 767 <212> PRT <213> Synechocystis sp. PCC6803 <400> 16 Met Leu Lys Thr Val Ala Ile Gln Val Gln Gly Arg Val Gln Gly Val 1 5 10 15 Gly Phe Arg Pro Phe Val Tyr Thr Leu Ala Gln Glu Met Gly Leu Asn 20 25 30 Gly Trp Val Asn Asn Ser Thr Gln Gly Ala Thr Val Val Ile Thr Ala 35 40 45 Asp Glu Lys Ala Ile Ala Asp Phe Thr Glu Arg Leu Thr Lys Thr Leu 50 55 60 Pro Pro Pro Gly Leu Ile Glu Gln Leu Ala Val Glu Gln Leu Pro Leu 65 70 75 80 Glu Ser Phe Thr Asn Phe Thr Ile Arg Pro Ser Ser Asp Gly Pro Lys 85 90 95 Thr Ala Ser Ile Leu Pro Asp Leu Ser Thr Cys Ser Ala Cys Leu Thr 100 105 110 Glu Leu Phe Asp Pro Ser Asp Arg Arg Tyr Leu Tyr Pro Phe Ile Asn 115 120 125 Cys Thr His Cys Gly Pro Arg Tyr Thr Ile Ile Glu Ala Leu Pro Tyr 130 135 140 Asp Arg Cys Arg Thr Thr Met Ala Arg Phe Arg Gln Cys Thr Asp Cys 145 150 155 160 Glu Arg Glu Tyr Lys Gln Pro Gly Asp Arg Arg Phe His Ala Gln Pro 165 170 175 Asn Ala Cys Pro Arg Cys Gly Pro Gln Leu Ala Phe Trp Asn Arg Gln 180 185 190 Gly Gln Val Ile Ala Glu Ala Asn Glu Ala Leu Asn Phe Ala Val Asp 195 200 205 Asn Leu Lys Val Gly Asn Ile Ile Ala Ile Lys Gly Leu Gly Gly Phe 210 215 220 His Leu Cys Cys Asp Ala Thr Asp Phe Glu Ala Val Glu Lys Leu Arg 225 230 235 240 Leu Arg Lys His Arg Pro Asp Lys Pro Leu Ala Val Met Tyr Gly Asn 245 250 255 Leu Gly Gln Ile Val Glu His Tyr Gln Pro Asn Asn Leu Glu Val Glu 260 265 270 Leu Leu Gln Ser Ala Ala Ala Pro Ile Val Leu Leu Asn Lys Lys Lys 275 280 285 Gln Leu Ile Leu Val Glu Asn Ile Ala Pro Gly Asn Pro Arg Val Gly 290 295 300 Val Met Leu Ala Tyr Thr Pro Leu His His Leu Leu Leu Lys Lys Leu 305 310 315 320 Lys Lys Pro Met Val Ala Thr Ser Gly Asn Leu Ala Gly Glu Gln Ile 325 330 335 Cys Ile Asp Asn Ile Asp Ala Leu Thr Arg Leu Gln Asn Ile Ala Asp 340 345 350 Gly Phe Leu Val His Asp Arg Pro Ile Val Cys Pro Val Asp Asp Ser 355 360 365 Val Val Gln Ile Val Ala Gly Lys Pro Leu Phe Leu Arg Arg Ala Arg 370 375 380 Gly Tyr Ala Pro Gln Pro Ile Thr Leu Pro Lys Pro Thr Gln Lys Lys 385 390 395 400 Leu Leu Ala Met Gly Gly His Tyr Lys Asn Thr Val Ala Ile Ala Lys 405 410 415 Gln Asn Gln Ala Tyr Val Ser Gln His Leu Gly Asp Leu Asn Ser Ala 420 425 430 Pro Thr Tyr Gln Asn Phe Glu Glu Ala Ile Ala His Leu Ser Gln Leu 435 440 445 Tyr Asp Phe Ser Pro Gln Glu Ile Val Ala Asp Leu His Pro Asp Tyr 450 455 460 Phe Ser His Gln Tyr Ala Glu Asn Gln Ala Leu Pro Val Thr Phe Val 465 470 475 480 Gln His His Tyr Ala His Ile Leu Ala Val Met Ala Glu His Gly Val 485 490 495 Met Glu Glu Ser Val Leu Gly Ile Ala Trp Asp Gly Thr Gly Tyr Gly 500 505 510 Met Asp Gly Thr Ile Trp Gly Gly Glu Phe Leu Lys Ile Thr Gln Gly 515 520 525 Thr Trp Gln Arg Ile Ala His Leu Gln Pro Phe His Leu Leu Gly Asn 530 535 540 Gln Gln Ala Ile Lys Tyr Pro His Arg Ile Ala Leu Ala Leu Leu Trp 545 550 555 560 Pro Thr Phe Gly Asp Asp Phe Ser Ala Asp Ser Leu Gly Asn Trp Leu 565 570 575 Asn Phe Asn Asn Gly Phe Lys Asn Lys Ile Asn Ser Arg Leu Asn Gln 580 585 590 Asp Leu Asn Asn Lys Asn Leu Arg Gln Leu Trp Gln Arg Gly Gln Ala 595 600 605 Pro Leu Thr Ser Ser Met Gly Arg Leu Phe Asp Gly Ile Ala Thr Leu 610 615 620 Ile Gly Leu Ile Asn Glu Val Thr Phe Glu Gly Gln Ala Ala Ile Ala 625 630 635 640 Leu Glu Ala Gln Ile Met Pro Asn Leu Thr Glu Glu Tyr Tyr Pro Leu 645 650 655 Thr Leu Asn Asn Lys Glu Lys Lys Leu Ala Val Asp Trp Arg Pro Leu 660 665 670 Ile Lys Ala Ile Thr Thr Glu Asp Arg Ser Lys Thr Asn Leu Ile Ala 675 680 685 Thr Lys Phe His Asn Ser Leu Val Asn Leu Ile Ile Thr Ile Ala Gln 690 695 700 Gln Gln Gly Ile Glu Lys Val Ala Leu Gly Gly Gly Cys Phe Gln Asn 705 710 715 720 Cys Tyr Leu Leu Ala Ser Thr Ile Thr Ala Leu Lys Lys Ala Gly Phe 725 730 735 Ser Pro Leu Trp Pro Arg Glu Leu Pro Pro Asn Asp Gly Ala Ile Cys 740 745 750 Met Gly Gln Leu Leu Ala Lys Ile Gln Ala Arg Gln Tyr Ile Cys 755 760 765 <210> 17 <211> 474 <212> DNA <213> Synechocystis sp. PCC6803 <400> 17 atgccaggcc aatccaccaa gtccacttta atcatcggtt acggcaatac cctgcggggg 60 gacgacggcg tggggcgtta cctagcggaa gaaattgctc agcaaaactg gccccattgt 120 ggagttattt ccacccatca actcacccca gaattggccg aggcgatcgc cgctgtggac 180 cgggtaattt tcattgatgc ccaactgcag gaatcagcaa acgaaccatc ggtggaagtt 240 gtggccttaa aaaccctgga acccaacgaa ctgtcagggg atttggggca ccggggtaat 300 cccagggaac tcttgaccct ggctaaaatt ctctacggcg ttgaggtaaa ggcttggtgg 360 gtgttgattc cggccttcac ctttgattat ggagagaaat tgtctcccct gaccgcccgg 420 gcccaagccg aagccttagc ccagatccgc cccttggtat tgggggagag ataa 474 <210> 18 <211> 157 <212> PRT <213> Synechocystis sp. PCC6803 <400> 18 Met Pro Gly Gln Ser Thr Lys Ser Thr Leu Ile Ile Gly Tyr Gly Asn 1 5 10 15 Thr Leu Arg Gly Asp Asp Gly Val Gly Arg Tyr Leu Ala Glu Glu Ile 20 25 30 Ala Gln Gln Asn Trp Pro His Cys Gly Val Ile Ser Thr His Gln Leu 35 40 45 Thr Pro Glu Leu Ala Glu Ala Ile Ala Ala Val Asp Arg Val Ile Phe 50 55 60 Ile Asp Ala Gln Leu Gln Glu Ser Ala Asn Glu Pro Ser Val Glu Val 65 70 75 80 Val Ala Leu Lys Thr Leu Glu Pro Asn Glu Leu Ser Gly Asp Leu Gly 85 90 95 His Arg Gly Asn Pro Arg Glu Leu Leu Thr Leu Ala Lys Ile Leu Tyr 100 105 110 Gly Val Glu Val Lys Ala Trp Trp Val Leu Ile Pro Ala Phe Thr Phe 115 120 125 Asp Tyr Gly Glu Lys Leu Ser Pro Leu Thr Ala Arg Ala Gln Ala Glu 130 135 140 Ala Leu Ala Gln Ile Arg Pro Leu Val Leu Gly Glu Arg 145 150 155 <210> 19 <211> 6515 <212> DNA <213> Artificial sequence <220> <223> Optimized expression sequence <220> <221> misc_feature <222> (1)..(6) <223> NcoI restriction site <220> <221> misc_feature <222> (6510)..(6515) <223> AvrII restriction site <400> 19 ccatggccca cgaagttagc ctgatggaac agacgctggc cattgccatt gcgcaggcgg 60 aagaccacgg ggcgagccaa attcaccgtt taacgctgcg cgttgggcag cagtcgggtg 120 ttgttgcaga tgcattacgc tttgcatttg aagttgttcg ccagaacaca atggctgcag 180 aagcacgtct ggaaatcgag gaaattccgg ttacctgtcg ttgtcagcat tgtcatgaaa 240 attttcagcc ggaggattgg atatatagat gtccccattg tgaccagatt agtcaaaccg 300 ttatggacgg caaacagctg gagttagcaa gcctggaact gagctaagca tggaaaggag 360 gtcgttatta tgtgccagaa ctgtgggtgt agcgcggttg ggaccgttgc ccatagccac 420 catcaccacg gggatggcaa ctttgcgcat agccatgacg accacgacca caggagcac 480 caccaccacc acggtaacta ttcaaaatca ccatcacagc agaccgtaac catagaacca 540 gacagacaaa catagagcaat tggccaagga attctgagca aaaacgatcg tctggcagaa 600 cgcaaccgcg gctacttcca ggccaaaggt ctgttagtaa tgaatttcct gagcagcccg 660 ggagcaggca aaaccgcact gatcgaaaaa atggttggtg atcgtcagaa agatcatccg 720 accgcagtta ttgttggtga tctggcaacc gataatgatg cacagcgtct gcgtagcgca 780 ggtgcaattg caattcaggt taccaccggt aatatttgtc atctggaagc agaaatggtt 840 caaaagcag cacagaaact ggatctggat aatattgatc agctgattat tgaaaatgtt 900 ggtaatctgg tttgtccgac cacctatgat ctgggtgaag atctgcgtgt tgttctgttt 960 agcgttaccg aaggtgaaga taaaccgctg aaatatccgg caacctttaa aagcgcacag 1020 gttatctgg ttaccaaaca ggatattgca cagcagttg attttgatgc agaactggca 1080 tggcagaatc tgcgtcaggt tgcaccgcag gcacagattt ttgcagttag cgcacgtacc 1140 ggtaaaggtc tgcagagctg gtatgaatat ctggatcagt ggcagctgca gcattatagc 1200 ccgctggttg atccggcact ggcataagag ttgaaaggag gtttcctcca tgtgcctggc 1260 gttaccgggg caggttgttt cgttaatgcc gaactcggat ccgctgttat taaccgggaa 1320 agttagcttt ggtggtatta ttaaaaccat tagcctggcg tatgttccgg aagttaaagt 1380 tggcgattat gttattgttc atgttggttt tgctatcagt attgttgatg aagaagcagc 1440 acaggagaca ctgattgatc tggccgagat gggcgtttaa ttcctaaaag gaggttttag 1500 ccatgaagta cgttgacgaa taccgcgacg cgcaggcagt tgcccactac cgccaggcca 1560 ttgcccgtga aattaccaaa ccgtggacgc tgatggaaat ttgtgggggc cagacccata 1620 gcatcgttaa atatggtctg gatgcattat taccgaaaaa cttaacctta atccacggtc 1680 cgggttgtcc ggtttgtgtt acgccgatgg aactgattga tcaggcatta tggctggcaa 1740 aacagccgga gattattttt tgtagctttg gtgatatgct gcgcgtgccg ggtagtggtg 1800 cagatctgct gagcattaaa gcacaggggg gagacgttcg tatagtttat tctccgttag 1860 attgtctggc gattgcgcgt gaaaatccga atcgtgaagt tgtttttttt ggtgtgggtt 1920 ttgaaactac cgccccggca accgcaatga cactgcatca ggcacgggcc cagggtatta 1980 gcaattttag cttattatgt gcacacgtgt tagttccgcc ggcgatggaa gctctgctgg 2040 gtaacccgaa tagcctggtt caagggtttt tagcagcagg tcatgtttgt acggttaccg 2100 gtgagcgggc gtatcagcat attgcagaga aatatcaggt tccgatagtt attaccggtt 2160 ttgaaccggt tgatattatg cagggtattt ttgcatgtgt tcgtcagctg gagagcgggc 2220 agtttacatg tataatcag taccggcggt cggttcagcc gcagggtaac gcacatgccc 2280 agaaaattat tgaccaggtt tttgaaccgg tggatcgtca ttggcgtgga ttaggtctta 2340 ttccggcctc aggtttaggt ttacgtccgg catttgcacc gtgggacgca gcagttaaat 2400 tcgcaaatct gttacagaca atggctccga caatgggtga aaccgtttgt atttctggcg 2460 aaatttaca gggtcagcgc aaacctagtg attgtcctgc atttggtacc atctgcaccc 2520 cggaacaacc gctgggcgcc cctatggtta gcagtgaagg cgcttgtgcc gcctattatc 2580 gttatcgtca gcaattaccg gaaccggttg gtgccgcacg tgtttaattt tgcaaaggag 2640 gtcctgccaa tgaacctggt gtgtccggtg ccgctggacc gctacccgca ggttttactg 2700 gcacacgggg ggggggggaa gctgagtcag cagctgttaa aacagatttt tctgccggcg 2760 tttggtgcat cagaaaccgg tagccatgat gcagcagttt ttaccgcaaa tcagagcagc 2820 ttagcattta caacagattc ctatgttatc aatccgctgt ttttcctgg tggtgatatt 2880 ggtagtcttg cagttcatgg aaccgttaat gatttagcaa tggcaggtgc aacaccgcgt 2940 tatattagcg ttgggttat tctggaggag ggtttaccga tggagacact ttggcgtgtt 3000 gcacaaagcc tgggtcaggc agcacagaat tgtggagttg aaatattaac aggtgatacc 3060 aaagttgttg atcgtgggaa gggagatggt atttttatta atacatcggg tatcggtagt 3120 ttagatcacc agcaaaccat tcatccgaat caggttcagg ttggtgatcg tctgattctg 3180 agtggggatt taggacggca tggtatggca attatggcag ttcgtcaggg cctggaattt 3240 gaaacaacca ttgaaagcga tagcgcaccg gttcatcgtg aggttcaggc tctgctgagc 3300 gcagggattc cgattcactg tctgcgtgac ttaacacgtg gtggtctggc aagcgccgtg 3360 aacgaaattg cacaaacctc aggtgttaca atggctctgc gtgaaacctt aattccggtt 3420 gaggcggaag ttcaagccgc ctgtgaactg ctgggttttg atcctttata tgttgcgaac 3480 gaaggccgtt tcctggccat tgttccgccg gaagccgaac agaaaaccgt tgaaattctg 3540 cagacctttc acccgcaggc gaccgcaatt ggtaccgtta ccggcaagag tgcacagacc 3600 ttaggtctgg ttagcctgga gagtagcata ggtgccccac gtctgttaga tatgattagc 3660 ggagaacaac tgccacgtat ttgttaagac tccaaaggag gctagattaa tgctgaaaac 3720 cgttgccatt caggttcagg ggcgcgttca gggggttggt tttcggccgt ttgtttacac 3780 cttagcccag gaaatgggtc tgaatggctg ggttaataac tctacgcagg gtgcaaccgt 3840 tgttattacc gcagatgaga aagcaattgc agattttacc gaacgtctga ccaaaacact 3900 gccgccaccg ggactgatcg aacaactggc agtggaacag ctgccgctgg aaagctttac 3960 CAACttacc attagaccga gtagcgatgg tccgaaacc gcaagcatcc tgccagatct 4020 gagcacatgt agcgcctgtc tgaccgaatt atttgatccc agtgatcgtc gttatctgta 4080 cccttttatt aattgtacc actgtgtcc tcgctatacc attattgag cactgcctta 4140 tgaccgttgt cgtaccacaa tggctcgttt tcgtcagtgt acggattgtg aacgtgaata 4200 taagcagccg ggggaccgcc gtttcatgc acagccaaac gcgtgtccgc gttgtgtcc 4260 gcagctggca ttctggaacc gtcagggtca agttattgca gaagccaatg aagcactgaa 4320 tttcgcagta gataatttaa aggtcggtaa tattatcgca atcaaggtc tggtggttt 4380 tcatttatgt tgtgatgcaa ccgatttga agccgttgaa aactgcgtt tacgtaaca 4440 tcgcccggat aagccgctgg ccgttatgta cggtaatctg ggtcagatg tgagcatta 4500 tcagccgaat aatttagaag tgagctgct gcagagcgca gcagcaccta tgttctct 4560 gataaaaag aaacagctga ttctggttga aaatattgca ccggcaatc cgcgtgtggg 4620 tgttatgctg gcatataccc cgttacatca cctgttactt aaaagttaa agaagccgat 4680 ggttgcaacc tccggtaact tagcaggcga acagatttgt attgacaata ttgacgcact 4740 gaccgttta caaaatattg ccgacggctt tctggttcac gatcgtccga ttgtttgtcc 4800 ggttgacgat agtgttgttc agattgtggc aggtaaaccg ttatttttaa gaagagcccg 4860 cggttatgca ccgcagccga ttacccttcc taaacccacc cagaaaaagt tattagcaat 4920 gggaggccat tataaaaata ccgttgcaat tgcaaagcag aatcaggcat atgtaagcca 4980 gcatttaggt gatttaaaca gcgcaccaac ctaccaaaat ttcgaagagg cgatagccca 5040 tttatcacag ctgtatgact ttagtcccca ggaaattgtc gcagatctgc atccggatta 5100 ctttagccat cagtacgcag aaaaccaagc cctgccggtg acgtttgtac agcatcatta 5160 tgcacatatt ctggcagtta tggcagaaca tggtgttatg gaagaaagcg ttttaggcat 5220 tgcatgggat ggcaccggtt atggtatgga tggtaccatt tggggtggtg aatttctgaa 5280 aattacgcag gggacctggc aaagaattgc acatctgcag ccgtttcatc tgttagggaa 5340 tcagcaggca attaaattc cgcaccggat tgcacttgct ctgctgtggc cgacattcgg 5400 ggacgatttt agcgccgata gtctgggtaa ttggttaaat tttaacaacg gtttcaagaa 5460 caagatcaac agccgtttaa accaagactt aaataataag aacctgagac aactgtggca 5520 gcgtgggcag gcaccgctga cctcgagcat gggcagatta tttgatggta tcgcaacact 5580 gattggtctg atcaatgaag taacctttga aggccaggca gcaattgcat tagaggcaca 5640 aattatgccg aatctgaccg aagaatacta tccgcttacc ctgaataaca aagaaaaaaaaa 5700 actggcagtt gattggcgtc cgctgattaa agcaattacc accgaagatc gtagcaaaac 5760 caatctgatt gcaaccaaat ttcataatag cctggttaat ctgattatta ccattgcaca 5820 gcagcagggt attgaaaaag ttgcactggg tggtggttgt tttcagaatt gttatctgct 5880 ggcaagcacc attaccgcac tgaaaaaagc aggttttagc ccgctgtggc cgcgtgaact 5940 gccgccgaat gatggtgcaa tttgtatggg tcagctgctg gcaaaaattc aggcacgtca 6000 gtatatttgt taactcaaca aggaggagc tggttatgcc gggtcagagc accaaaagca 6060 ccctgattat cgggtacggg aacaccttac gtggggacga tggggtgggg cgctacctgg 6120 cagaagaaat agcacagcag aactggccgc actgtggtgt father catcagctga ccccggaact ggccgaagca attgcagcag tggataggt gattttttt gacgcccaac tgcaggaag tgcaaatgaa ccgtcagttg aagttgttgc cctgaaaacc ttagaaccca atgaattaag tggagatctg ggtcatcgtg gtaatccgcg tgagctgctg accttagcca 6420. aaatattata tggtgttgaa gtcaaagcgt ggtgggttct gattccggcc tttacctttg attatggtga gaattatcg cccttaacag cacgtgctca ggccgaagca ctggcacaga ttcgtccgct ggttctgggg ctagg 6515

Claims

1. A monomeric polypeptide, comprising a single subunit, the single subunit comprising an active site of a [NiFe]-hydrogenase protein, the monomeric polypeptide having hydrogenase activity, wherein the subunit is a HoxH subunit of a HoxEFUYH [NiFe]-hydrogenase protein in / from Synechocystis CCP6803, and wherein the amino acid sequence of the monomeric polypeptide is SEQ ID NO:

4.

2. The monomeric polypeptide of claim 1, wherein the monomeric polypeptide is isolated from a natural environment.

3. The monomeric polypeptide of claim 1, wherein the monomeric polypeptide is recombinant or heterologous.

4. The monomeric polypeptide according to any one of the preceding claims, wherein the monomeric polypeptide is purified.

5. The monomeric polypeptide according to any one of claims 1 to 3, wherein The hydrogenase activity of the monomeric polypeptide is not less than 0.01 μmol H2·min -1 mg -1 Enzyme.

6. The monomeric polypeptide according to any one of claims 4, wherein The hydrogenase activity of the monomeric polypeptide is not less than 0.01 μmol H2·min -1 mg -1 Enzyme.

7. A host cell comprising the monomeric polypeptide according to any one of claims 1 to 6, wherein the host cell is Escherichia coli.

8. The host cell according to claim 7, wherein the host cell further comprises at least one maturation factor of the [NiFe]-hydrogenase protein, wherein the at least one maturation factor is endogenous and / or exogenous to the host cell.

9. The host cell according to claim 8, wherein at least one maturation factor of the [NiFe]-hydrogenase protein is selected from HypA, HypB, HypC, HypD, HypE, HypF and HoxW, and the amino acid sequences of HypA, HypB, HypC, HypD, HypE, HypF and HoxW are SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively, or the HypA, HypB, HypC, HypD, HypE, HypF and HoxW are jointly encoded by a tandem nucleotide sequence, and the tandem nucleotide sequence is shown in SEQ ID NO:

19.

10. The host cell according to any one of claims 7 to 9, wherein the host cell comprises an expression vector, at least one gene in the expression vector expressing the monomeric polypeptide and / or the at least one maturation factor.

11. A host cell comprising a polynucleotide encoding the monomeric polypeptide of any one of claims 1 to 6, wherein the host cell is Escherichia coli.

12. The host cell according to claim 11, wherein the host cell further comprises at least one maturation factor of the [NiFe]-hydrogenase protein, wherein the at least one maturation factor is endogenous and / or exogenous to the host cell.

13. The host cell according to claim 12, wherein at least one maturation factor of the [NiFe]-hydrogenase protein is selected from HypA, HypB, HypC, HypD, HypE, HypF and HoxW; the amino acid sequences of HypA, HypB, HypC, HypD, HypE, HypF and HoxW are SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively; or the HypA, HypB, HypC, HypD, HypE, HypF and HoxW are jointly encoded by a tandem nucleotide sequence, and the tandem nucleotide sequence is as shown in SEQ ID NO:

19.

14. The host cell according to any one of claims 11 to 13, wherein the host cell comprises an expression vector, at least one gene in the expression vector expressing the monomeric polypeptide and / or the at least one maturation factor.

15. A method for preparing a monomeric polypeptide having hydrogenase activity according to any one of claims 1 to 6, the method comprising the following steps: The exogenous polynucleotide shown in SEQ ID NO:3 is transferred into a host cell and / or an expression vector through transgenesis to obtain a transgenic host cell and / or a transgenic expression vector, and the transgenic host cell and / or the transgenic expression vector are incubated, at least a portion of the exogenous polynucleotide encodes the monomeric polypeptide, and the monomeric polypeptide is composed of a single subunit, and the single subunit contains the active site of a [NiFe]-hydrogenase protein. The incubation conditions are capable of maintaining the expression of the exogenous polynucleotide to produce the monomeric polypeptide, wherein the host cell is Escherichia coli.

16. The method for preparing a monomeric polypeptide having hydrogenase activity according to claim 15, wherein the genetic modification step of the host cell comprises transferring at least one maturation factor of the [NiFe]-hydrogenase protein into the host cell, and the at least one maturation factor is endogenous and / or exogenous to the host cell.

17. The method for preparing a monomeric polypeptide having hydrogenase activity according to claim 16, wherein the at least one maturation factor of the transferred [NiFe]-hydrogenase protein is selected from HypA, HypB, HypC, HypD, HypE, HypF and HoxW, and the amino acid sequences of HypA, HypB, HypC, HypD, HypE, HypF and HoxW are SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, respectively; or the HypA, HypB, HypC, HypD, HypE, HypF and HoxW are jointly encoded by a tandem nucleotide sequence, and the tandem nucleotide sequence is as shown in SEQ ID NO:

19.

18. The method for preparing a monomeric polypeptide having hydrogenase activity according to claim 16, wherein the host cell comprises an expression vector, and at least one gene in the expression vector expresses the at least one maturation factor.

19. The method for preparing a monomeric polypeptide having hydrogenase activity according to any one of claims 15 to 18, wherein the method comprises a subsequent step of isolating and / or purifying the monomeric polypeptide.

20. Use of the monomeric polypeptide according to any one of claims 1 to 6 or the monomeric polypeptide prepared by the preparation method according to any one of claims 15 to 19 as a polypeptide having hydrogenase activity in producing or consuming hydrogen.

Images