EP4058567A1

EP4058567A1 - Monomer polypeptide having hydrogenase activity, in particular recombinant monomer polypeptide having hydrogenase activity

Info

Publication number: EP4058567A1
Application number: EP20803204.5A
Authority: EP
Inventors: Damien GODAUX; Philippe LORGE; Bart GHYSELS; Nathalie Job; Fabrice FRANCK; Giuseppe CALDARELLA; Pierre CARDOL; Claire REMACLE
Original assignee: H2win SA
Current assignee: H2win SA
Priority date: 2019-11-13
Filing date: 2020-11-12
Publication date: 2022-09-21
Also published as: WO2021094465A1; CA3160285A1; JP2023502034A; US20230011570A1; CN114901814A

Abstract

The invention relates to a monomer polypeptide comprising a single subunit comprising the active site of a [NiFe]-hydrogenase protein, said monomer polypeptide having hydrogenase activity.

Description

Monomeric polypeptide exhibiting hydrogenase activity, in particular recombinant monomeric polypeptide exhibiting hydrogenase activity

The present invention relates to a monomeric polypeptide exhibiting hydrogenase activity, in particular a recombinant monomeric polypeptide exhibiting hydrogenase activity, a host cell including this monomeric polypeptide and a host cell including a polynucleotide encoding this monomeric polypeptide. The present invention also relates to a process for obtaining a monomeric polypeptide exhibiting a hydrogenase activity, in particular a process for obtaining a recombinant monomeric polypeptide exhibiting a hydrogenase activity and to the use of such a monomeric polypeptide. , in particular on the use of such a recombinant monomeric polypeptide.

The decreasing availability of fossil energy sources and the fear of dramatic climate change have fostered the emergence of clean and renewable technologies. Hydrogen (H2) is considered a promising renewable energy carrier due to its high energy content and the lack of pollution when used in fuel cells. The production of dT½ is currently carried out by chemical extraction of fossil hydrocarbons, electrolysis of water or pyrolysis of biomass. However, the lack of both non-polluting and commercially viable production systems is a major limitation. The use of biotechnological methods of producing H2, also called bio-hydrogen, could be a solution. The amount of research on the production of bio-hydrogen has increased dramatically over the past decade. This includes microbial production mainly through the process of fermentation in bacteria and photosynthesis in microalgae, but also the enzymatic generation of hydrogen by "electro-enzymology". All of these methods rely on special enzymes called hydrogenases.

Hydrogenases are metallo-enzymes that catalyze the reaction chemical: 2 H ⁺ + 2e <® H2. This enzyme-dependent hydrogen production or consumption reaction is defined as hydrogenase activity. The reaction is reversible and its direction depends on the redox potential of the components capable of interacting with the enzyme. In the presence of H2 and an electron acceptor, a hydrogenase consumes hydrogen. In the presence of a low potential electron donor, a hydrogenase uses protons as electron acceptors and produces IΉ2. Hydrogenases are widely used among microorganisms because many of them use hydrogen as a vector or source of energy. These enzymes have many representatives in the field of bacteria and archaea and are also present in some unicellular eukaryotic organisms. Despite their diversity in many respects (host, size, quaternary structure, electron donors or acceptors), hydrogenases are divided into three phylogenetically distinct classes: [Fe] - hydrogenases, [FeFe] -hydrogenases and [NiFe ] -hydrogenases. Each class is characterized by a distinctive metallic composition of the active site. Physiologically, these enzymes have been shown to function as a valve for excess electrons.

The catalytic center of [Fe] -hydrogenases does not contain any FeS or Ni center and has therefore been named "iron-sulfur center free hydrogenase" or [Fe] - hydrogenases (Shima and Thauer. 2007. A third type of hydrogenase catalyzing H2 activation 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-sulfur 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). Associated with a specific cofactor, these enzymes have very different catalytic properties from other types of hydrogenases. Indeed, they do not catalyze the reversible reaction of H2 production (Vignais and Billoud. 2007. Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272). For this reason, as well as their poor distribution, this class of hydrogenases has been little studied. The vast majority of known hydrogenases belong to the other two classes.

[FeFej-hydrogenases are monomeric enzymes with a highly conserved catalytic center. It is a bi-nuclear iron site linked to a [4Fe-4S] center by a cysteine bridge. The non-protein ligands, cyanide (CN) and carbon monoxide (CO), are bound to iron atoms in the bi-nuclear center. Iron atoms also share two sulfur ligands (Nicolet et al. 2000. A novel 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). Additional domains host several FeS centers and ensure the transfer of electrons between the external electron source and the active site integrated in these monomeric proteins. In addition, a hydrophobic channel connects the surface to the active site and provides access for protons and exit for H2 molecules. Three chaperone proteins named HydE, HydF and HydG are known to be necessary for the correct assembly of [FeFej-hydrogenases (Vignais et al. 2001. Classification and phylogeny of hydrogenases. FEMS Microbiol Rev 25: 455-501). These enzymes are present in anaerobic prokaryotes (genera Clostridium or Desulfovibrio), but also in some lower eukaryotes such as anaerobic fungi or unicellular microalgae (genera Chlorella or Chlamydomonas) (Vignais and Billoud. 2007. Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272). The [FeFej-hydrogenases thermodynamically promote the reoxidation of the cofactor (ferredoxin or NADH) and thus generate IΉ2. These enzymes are therefore generally involved in the elimination of an excess of reducing equivalents in the cell in order to avoid, for example, stopping the fermentation. They are also capable of interacting with the photosynthetic chain of a small group of microalgae to oxidize the photosynthetic chain and thus activate carbon fixation after anoxic incubation (Ghysels et al. 2013. Function of the chloroplast hydrogenase in the microalga Chlamydomonas : the role of hydrogenase and State transitions 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-Likel-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii. Plant Physiol 168: 648-658).

[NiFe] -hydrogenases form globular heteromultimers (Volbeda et al. 1995. Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373: 580-587). The bi-metallic active site [NiFe] is located in the large subunit where it is coordinated by four cysteines and three non-protein ligands, one CO molecule and two CN, linked to the iron atom (Happe et al. 1997. Biological activation of hydrogen. 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 heteromimer contain several medial and distal FeS centers, which conduct electrons between the active site and the physiological electron donor or acceptor. The [4Fe-4S] group located near the active site is considered essential for the activity (Albracht. 1994. Nickel hydrogenases: in search of the active site. Biochim Biophys Acta 1188: 167-204). In addition to structural genes, there are several accessory genes involved in the processing and insertion of Ni, Fe, CO and CN into the active site of these heterometers. Indeed, the maturation of [NiFe] -hydrogenases follows a complex pathway involving at least seven helper proteins (HypA-F and one endopeptidase) (Vignais and Billoud. 2007. Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272). Another characteristic of the active site of [NiFe] -hydrogenases is the strong affinity for hydrogen. These enzymes therefore act mainly by consuming IΉ2 in the host microorganism, even if some [NiFe] -hydrogenases have a good capacity for producing hydrogen. [NiFe] -hydrogenases are enzymes quite widespread among prokaryotes with many representatives in bacteria and archaea. The classification of [NiFe] -hydrogenases is based on the amino acid sequence alignments of the different subunits and divides [NiFe] -hydrogenases into four groups. Remarkably, this classification agrees well with the groups derived from the functions physiological (Vignais and Billoud. 2007. Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107: 4206-4272).

The extreme sensitivity of hydrogenases to oxygen both in vitro and in vivo is a problem when considering these enzymes for the production of hydrogen on an industrial level. Oxygen binds as a ligand to the active site, accepts electrons, and is reduced to reactive oxygen species (ROS) trapped in the enzyme. This can lead to permanent damage when the ROS survives long enough to attack the vulnerable catalytic center. [FeFe] -hydrogenases show severe sensitivity to GO2 because the enzyme is irreversibly damaged after exposure to small concentrations of 02 (Ghirardi et al. 1997. Oxygen sensitivity of algal H2- production. Appl Biochem Biotechnol 63 -65: 141 -151). However, [NiFe] -hydrogenases are described as being more resistant than [FeFe] -hydrogenases to oxygen damage. In addition, the [NiFe] -hydrogenases are reversibly inactivated by O2. Some microorganisms, such as Ralstonia sp., Have even developed an active site tolerant to oxygen, and are able to 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 towards oxygen. J Biol Inorg Chem 9: 616- 626). These various characteristics make [NiFe] -hydrogenases better candidates for industrially viable technological use.

HoxEFUYH is a well-characterized [NiFe] -hydrogenase present in the cyanobacterium Synechocystis sp. PCC6803. HoxEFUYH is a [NiFe] - pentameric and cytoplasmic hydrogenase. HoxY and HoxH form the “hydrogenase” part while HoxE, HoxF and HoxU constitute the part in contact with 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 the catalytic activity, that is to say the subunit comprising the active site of [NiFe] -hydrogenase. HoxH contains the conserved residues for the bonding of nickel and iron atoms. HoxY contains the proximal group [4Fe-4S] near the NiFe catalytic center. HoxF and HoxU are iron-sulfur type proteins responsible for in vivo interaction with the substrate (NADH, flavodoxin or reduced ferredoxin). HoxFU contain the medial and distal FeS centers carrying electrons to FloxYFI. The function of FloxE is unclear, but it may be a membrane anchor subunit. Mutational analysis of the maturation pathway identified seven essential maturation factors, called FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW (Floffmann et al. 2006. Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803. Additional homologs of hypA and hypB are not active in hydrogenase maturation. FEBS J 273: 4516-4527). A HoxEFUYH maturation model has been proposed (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 PCC6803. Int J Mol Sci 15: 19938-19951). The HoxH subunit is treated with the specific protease HoxW and the [Ni-Fe] site is added to the catalytic subunit by the HypABCDEF complex. The complete genome of Synechocystis sp. PCC6803 has been sequenced. The HoxEFUYH genes were identified as being grouped together in an octacistronic operon, unlike the hypABCDEF genes dispersed in the chromosome of Synechocystis. 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 PCC6803. J Bacteriol 194: 5423-5433 ). It is regulated by various environmental conditions, such as the availability of hydrogen, light, nitrates, nickel, oxygen or sulfur (Oliveira and Lindblad. 2009. Transcriptional regulation of the cyanobacterial bidirectional Hox-hydrogenase. Dalton Trans 9990- 9996). It is important to note that the hox genes are expressed constitutively in the presence of oxygen (Kiss et al. 2009. Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803. J Biotechnol 142: 31-37), but that the The enzyme, sensitive to oxygen, is therefore inactive under aerobic conditions. The precise physiological function is still the subject of debate, but HoxEFUYH would function as a safety valve which dissipates excess electrons under redox conditions. unfavorable, thus maintaining an adequate oxidation / reduction balance in the cell during fermentation or photosynthesis (Carrieri et al. 2011. The role of the bidirectional hydrogenase in cyanobacteria. Bioresour Technol 102: 8368-8377).

There are a large number of studies on HoxEFUYH. Many characteristics make this enzyme a good candidate for the production of bio hydrogen. First, this [NiFe] -hydrogenase exhibits a bias in favor of proton reduction (Mclntosh 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). The operon, and hence the enzyme, is weakly expressed in Synechocystis sp. PCC6803 in aerobic condition (Kiss et al. 2009. Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803. J Biotechnol 142: 31-37). The inactivation of FloxEFUYFI in the presence of oxygen is total and almost instantaneous. However, FloxEFUYFI can be reactivated quickly (delays of the order of a minute) in redox condition (for example by reduction with hydrogen and / or by elimination of oxygen) (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, Mclntosh 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). Several purification protocols (Schmitz et al. 2002. HoxE-a subunit specify for the pentameric 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 of implementation in electrochemistry (Mclntosh 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) are available. The ability to efficiently catalyze the production of hydrogen, and with limited sensitivity to oxygen, allowed HoxEFUYH to be identified as a good candidate.

A common and efficient method for producing a large amount of a protein of interest is the recombinant production of that protein within a heterologous host. This biotechnological process involves the introduction and expression of genes of interest in the genome of the host organism in order to produce a large amount of the protein of interest, with an excellent degree of purity. There are several systems of recombinant production. The prokaryotic system remains the fastest and easiest system to produce a protein of interest, the eukaryotic system being slower and more complicated to implement. Each organism has its own advantages and disadvantages. There is not yet a universally applicable system of expression. It is very difficult to predict which host will work best for a particular protein or for a particular end use. Escherichia coli (E. coli) is the reference organism for recombinant production. Indeed, this bacterium is very well known from the point of view of genetic and physiological engineering, with for example: optimized cell (high productivity, use of codons, inhibition of endogenous proteases), optimized culture medium, short doubling time, low contamination, availability of many commercial vectors, industrial scale-up, high production yield.

The production and engineering of recombinant hydrogenases, like metalloproteins in general, has had limited success. The literature provides examples of [FeFe] -hydrogenases expressed heterologously in E. 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 requiring a limited number of processing factors.

The heterologous production of [NiFe] -hydrogenases is described as difficile (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).

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

Second, the correct folding of each of the hetero-multimeric subunits is required, which is particularly difficult to control and ensure in heterologous production.

Third, the correct assembly of each subunit in the hetero-multimeric complex is mandatory. Poor folding can lead to aggregation and therefore to a decrease 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 an accurate sequence is necessary to achieve enzymatic activity, which can be difficult to control and secure in a heterologous host.

All this explains why [NiFe] -hydrogenases are not always active when produced by heterologous recombination. However, there are several cases in the literature where an active [NiFej-hydrogenase has been produced in E. coli (Kim et al. 2011. Production of biohydrogen by heterologous expression of oxygen-tolerant Flydrogenovibrio marinus [NiFej-hydrogenase in Escherichia coli. J Biotechnol 155: 312-319, Maier et al. 2015. Identification, cloning and heterologous expression of active [NiFej-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 [NiFej-hydrogenase from Cupriavidus necator in Escherichia coli. PLoS One 8: e68812, Weyman et al. 2011. Genetic analysis of the Alteromonas macleodii [NiFej-hydrogenase. FEMS Microbiol Lett 322: 180-187).

In particular, the work of Sun and his collaborators (Sun et al. 2010. Fleterologous expression and maturation of an NADP-dependent [NiFej-hydrogenase: a key enzyme in biofuel production. PLoS One 5: e10526) the expression of [NiFe] -hydrogenase from Pyrococcus furiosus in anoxia in E. 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. made it possible to obtain a tetrameric recombinant enzyme of [NiFe] -hydrogenase type comprising the four subunits called PF0891, PF0892, PF0893 and PF0894. After purification, this tetrameric recombinant enzyme was found to be functionally similar to the native enzyme purified from P. furiosus.

FloxEFUYFI being a prokaryotic protein of the cyanobacterium Synechocystis, the E. coli system is suitable for its recombinant production. This production in E. coli has already been carried out successfully. In this sense, Maeda and his colleagues have shown an in vivo increase in hydrogen production in E. coli cells expressing, in anoxic condition, the cyanobacterial HoxEFUYH enzyme (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). Such increased hydrogen production in the presence of FloxEFUYFI is due to inhibition of the activity of endogenous Fte-consuming hydrogenases 1 and 2 in E. coli.

We should also mention Wells and his collaborators who introduced the FloxEFUYFI genes and these associated maturation factors into E. 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 the production of hydrogen, in anoxia, both in vivo and in vitro via HoxEFUYH in a host null for endogenous hydrogenases. They indicate coupling with host electron transfer systems such as fermentation and show the potential of HoxEFUYH in metabolic engineering to improve hydrogen production yields.

In the state of the art, there is no disclosure of a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFej-hydrogenase type, in particular of an isolated monomer obtained from of a complex of [NiFej-hydrogenase type, exhibiting by itself an activity hydrogenase, even though the documents D2 (Hornhardt et al. 1986. Characterization of a native subunit of the NAD-linked hydrogenase isolated from 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.

Document D2 describes the characterization of a native subunit of [NiFe] -hydrogenase from Alcaligenes eutrophus H16. This peptide represents the catalytic subunit of [NiFe] -hydrogenase and is described as totally inactive with NAD as redox mediator, but showing very low residual activity with methylene blue, ferricyanide and cytochrome C.

Document D3 suggests the existence of [NiFe] - hydrogenase type monomers with hydrogenase activity. As examples, document D3 mentions, citing document D2, [NiFe] -hydrogenase from Alcaligenes eutrophus, and also [NiFe] -hydrogenase-1 from Escherishia coli.

However, subsequent published studies, including the studies described in 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 Escherichia coli is dependent on the coexpression of the iron-sulfur cluster-containing small subunit. Arch Microbiol 193: 893-903), have shown that the conclusions of the studies in documents D2 and D3 are erroneous.

The study described in document D1 demonstrated that the subunit containing the active site of the [NiFe] -hydrogenase of Alcaligenes eutrophus H16 on its own did not exhibit any hydrogenase-type activity, and this with several redox mediators, in particular the benzyl viologen. Document D1 mentions that the smallest entity of Alcaligenes eutrophus [NiFe] -hydrogenase capable of having hydrogenase activity consists of the large subunit containing the center [NiFe] and the small subunit with a minimum of an FeS center. Similarly, the studies presented in documents D4 and D5 oppose the teaching of document D3 by showing that the catalytic subunit alone of hydrogenase-1 [NiFe] of Escherishia coli does not exhibit any hydrogenase activity. The majority of the work carried out on this subject, including the studies of documents D1, D4 and D5 consider that the center [4Fe-4S] located in the small subunit is essential and indispensable for the hydrogenase activity of the large subunit. [NiFe] -hydrogenase unit (Albracht. 1994. Nickel hydrogenases: in search of the active site. Biochim Biophys Acta 1188: 167-204). In addition, structural analyzes of [NiFe] -hydrogenases have largely demonstrated the absence of an FeS center in the catalytic subunit containing the active site (Lubitz et al. 2014. Hydrogenases. Chem Rev 114: 4081-4148),

Thus, the results described previously in document D2 can only be explained by contamination of the catalytic subunit by the small subunit, as evidenced by the demonstration of an FeS center in the preparations allowing the weak hydrogenase activity. measured.

In conclusion, all the work carried out which constitutes the state of the art consider that the reduction in the number of subunits is neither favorable to the activity nor to the stability of the hydrogenases and that the subunit containing the NiFe center, isolated from multimeric complexes of NiFe-type hydrogenases, does not exhibit hydrogenase activity on its own.

Unfortunately, as emerges in particular from the state of the art mentioned above, there remain several drawbacks which considerably slow down the use of [NiFe] -hydrogenases, and in particular the use of HoxEFUYH, for a bio-production of hydrogen. commercially profitable.

To date, there is therefore a real need to overcome the obstacles slowing down the use of [NiFe] -hydrogenases for the production of hydrogen since, as indicated above, the [NiFe] -hydrogenases undeniably have a high potential. for the production of hydrogen.

To solve these problems, it is provided according to the invention, a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, said monomeric polypeptide exhibiting enzymatic activity, in particular enzymatic activity of hydrogenase type, more particularly catalytic activity, more particularly catalytic activity of hydrogenase type.

Preferably, according to the invention, said monomeric polypeptide comprises a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type.

Preferably, according to the invention, said monomeric polypeptide consists of a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type.

In other words, preferably, there is provided according to the invention a monomeric polypeptide comprising a single subunit comprising only the active site of a protein of [NiFe] -hydrogenase type, said monomeric polypeptide alone exhibiting a enzymatic activity, in particular an enzymatic activity of hydrogenase type, more particularly a catalytic activity, more particularly still a catalytic activity of hydrogenase type.

In the context of the present invention, it has been demonstrated that such a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type and exhibiting a hydrogenase activity makes it possible to s' freeing at least in part from the obstacles slowing down the use of [NiFe] -hydrogenases for hydrogen production, this while guaranteeing a hydrogenase activity of at least 0.01 mitioI H2. min ^-1 . ^_1 mg of enzyme, preferably at least 0.05 mitioI H2. min ^-1 . rng ^-1 enzyme.

Indeed, since it is according to the invention a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, the difficulties encountered with [NiFe] - hydrogenases, in particular to ensure the assembly of the active site, to ensure a adequate folding of each of the subunits involved and to ensure correct assembly of each subunit in a heteromimeric complex, are greatly reduced or even eliminated. This makes it possible to increase the reproducibility during the process of obtaining [NiFe] -hydrogenase since only one subunit is involved. Indeed, the production of a single monomeric polypeptide greatly simplifies the complex folding process in comparison with the dimeric, tetrameric and pentameric enzymes for which a correct assembly of the active site, of each of the subunits and finally of the entire enzyme. is particularly difficult to control and guarantee, which constitutes an obstacle to their use. According to the invention, adequate folding of a single subunit is necessary and no assembly between different subunits is required to form a heteromimeric complex.

Furthermore, it has been demonstrated that the process for the manufacture of such a monomeric polypeptide according to the invention can be completely carried out in aerobic condition (no precaution required with respect to oxygen during the stages of expression and of purification), which eliminates the problems encountered with the manufacturing processes for dimeric, tetrameric and pentameric [NiFe] -hydrogenases recombinant encountered in the state of the art and for which the manufacturing processes are carried out in anoxia. As mentioned above, even if the accumulation of biomass is carried out in the presence of oxygen, the production phase of the recombinant hydrogenase is itself carried out in anoxia according to the methods known from the state of the art (Sun et al. al. 2010. Fleterologous 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 absence of oxygen is also reported (anoxic box, sodium dithionite in the buffers) during the various chromatographic stages of the purification. Maintaining such a level of anoxia during the process obviously involves an increase in the costs associated with the recombinant production, which is solved by the present invention. Advantageously according to the invention, the size of the monomeric polypeptide according to the invention is markedly smaller in comparison with the heteromimer, an increase in the activity by mass of the enzyme (number of catalytically active entities per mg of total protein) being thus obtained.

Better integration and densification of the enzyme catalyst is also achievable, for example, during electrochemical processing of the monomeric polypeptide according to the invention, such as, for example, in a fuel cell.

In addition, the three-dimensional structure of a monomeric polypeptide according to the invention can be easily modeled, in particular to determine the residues exposed at the surface of the protein, for example to facilitate the orientation as well as the adsorption with respect to an interface, by example compared to a carbon electrode. This characteristic advantageously makes it possible, for example, to improve the stability of the link between the interface and the enzymatic catalyst, but also to optimize the direct transfer of electrons between the interface and the active site. Energy efficiency is therefore improved by the absence of additional redox relays, which limits energy losses.

In the context of the present invention, it has also been demonstrated that the monomeric polypeptide is indeed active and capable of producing by catalysis of H2 (for example via the standard test for in-vitro production of FL by hydrogenases using the reduced methyl viologen as redox mediator) as well as consumption by catalysis of H2 (for example via the standard test of in-vitro consumption of H2 by hydrogenases using oxidized benzyl viologen as redox mediator), without the presence of Proximal FeS center present in the small-subunit and described as essential, nor for that matter of any other redox relay. This is quite surprising since such a thorough simplification of the heteromimeric enzyme has never made it possible to demonstrate the hydrogenase activity in the state of the art.

All the more advantageously, whereas only a limited production of H2 is currently obtained with the known recombinant [NiFej-hydrogenases (Sun et al. 2010. Fleterologous expression and maturation of an NADP- depend on [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), it has been shown that the monomeric polypeptide according to the invention exhibits a hydrogenase activity at least equivalent or even greater than those obtained with the known (recombinant) [NiFe] -hydrogenases.

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

For example, according to the invention, said monomeric polypeptide can be derived from / isolated from a prokaryote. Examples include, but are not limited to a member of the genus Escherichia (eg, Escherichia coli), a member of the genus Desulfovibrio (eg, Desulfovibrio gigas), a member of the genus Hydrogenophilus (eg, Hydrogenophilus thermoluteolus), a member of the genus Desulfomicrobium (such as, for example, Desulfomicrobium baculatum), Synechocystis (such as, for example, Synechocystis sp. PCC6803), a member of the genus Phormidium (such as, for example, Phormidium ambiguum) or a member of the genus Spirulina (such as, for example, Spirulina platensis). For example, said monomeric polypeptide can be derived from a cell or a microbe or can be produced in vitro or in vivo.

According to one embodiment according to the invention, the monomeric polypeptide is recombinant or heterologous.

Advantageously, according to the invention, the monomeric polypeptide is purified.

Preferably, according to the invention, the monomeric polypeptide has a truncated or non-truncated amino acid sequence exhibiting at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, plus more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the acid sequence amino acids of SEQ ID NO: 2 and / or relative to the amino acid sequence of SEQ ID NO: 4.

Even more preferably, according to the invention, the monomeric polypeptide has a truncated or non-truncated amino acid sequence exhibiting at least 81% identity, at least 82% identity, at least 83% identity, at least 84 % identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% d 'identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity , at least 97% identity, at least 98% identity, at least 99% identity, relative to the amino acid sequence of SEQ ID NO: 2 and / or relative to the amino acid sequence of SEQ ID NO: 4.

Advantageously, according to the invention, said monomeric polypeptide is characterized in that said subunit comprising the active site of a protein of [NiFe] -hydrogenase type is the HoxH subunit of the protein of [NiFe] -hydrogenase type. FloxEFUYFI in / from Synechocystis sp. PCC6803.

Preferably, according to the invention, said monomeric polypeptide exhibits a hydrogenase activity of at least 0.01 mitioI H2. min ^-1 . ^_1 mg of enzyme, preferably at least 0.05 mitioI H2. min ^-1 . rng ^-1 enzyme, preferably at least 10 mitioI H2. m ¹ . rng ^-1 enzyme.

The present invention also relates to a host cell including a monomeric polypeptide according to the invention, said monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, said monomeric polypeptide exhibiting hydrogenase activity. .

Preferably, the present invention relates to a host cell including a monomeric polypeptide according to the invention, the subunit comprising the active site of a protein of [NiFe] -hydrogenase type is the subunit HoxH of the [NiFe] -hydrogenase-type protein HoxEFUYH in / from Synechocystis sp. PCC6803.

By way of example, according to the invention, the host cell, in particular for the expression of said monomeric polypeptide, perhaps a host bacterial cell of the genus Escherichia (such as for example Escherichia coll), of the genus Bacillus (such as for example Bacillus subtilis), of the genus Streptomyces (such as for example Streptomyces coelicolor), of the genus Synéchocystis (such as for example Synéchocystis sp. PCC6803), of the genus Synéchococcus (such as for example Synéchococcus WFI8102), or any other prokaryotic cell, a eukaryotic cell for example of the genus Chlamydomonas (such as, for example, Chlamydomonas reinhardtii), of the genus Saccharomyces (such as, for example, Saccharomyces cerevisiae), of the Pichia type (such as, for example, Pichia pastoris), or another type of eukaryotic cell.

According to the invention, said unrelated monomeric polypeptide in said host cell may itself but not necessarily be derived from the expression of an unwanted gene in an expression vector, for example in a plasmid inserted into said host cell.

Advantageously, according to the invention, said monomeric polypeptide induced in said host cell has a truncated or non-truncated amino acid sequence exhibiting at least 20% identity, more preferably at least 40% identity, more preferably still at least 60%. identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to to the amino acid sequence of SEQ ID NO: 2 and / or to the amino acid sequence of SEQ ID NO: 4.

Preferably, according to the invention, said host cell can include one or more maturation factors of said [NiFe] -hydrogenase type protein, preferably one or more maturation factors of said [NiFe] -hydrogenase type protein selected from , the group consisting of the maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW a) whose respective amino acid sequences each have at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, respectively with respect to the sequences in amino acids SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; or b) encoded together by a concatenary nucleotide sequence exhibiting at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the nucleotide sequence SEQ ID NO: 19, which codes for all of these ripening factors.

According to the invention, said at least one maturation factor may be endogenous to the host cell and / or exogenous to the host cell.

Advantageously, for a host cell according to the invention, said monomeric polypeptide and / or said at least one maturation factor is / are derived from the expression of at least one undue gene in an expression vector, said expression vector being induced in said host cell.

The present invention also relates to a host cell including a polynucleotide encoding a monomeric polypeptide according to the invention comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, said monomeric polypeptide exhibiting hydrogenase activity. .

Preferably, the present invention relates to a host cell including a polynucleotide encoding a monomeric polypeptide whose sub- unit comprising the active site of a [NiFe] -hydrogenase-like protein is the HoxH subunit of the [NiFe] -hydrogenase-like protein FloxEFUYFI in / from Synechocystis sp. PCC6803.

Preferably, according to the invention, said polynucleotide encoding a monomeric polypeptide exhibits a nucleotide sequence having at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% d identity, respectively with respect to the sequences, with respect to the nucleotide sequences SEQ ID NO: 1 and / or SEQ ID NO: 3.

By way of example, according to the invention, the host cell including a polynucleotide encoding a monomeric polypeptide according to the invention, and being used in particular for the expression of said monomeric polypeptide, can be a host bacterial cell of the genus Escherichia (such as for example Escherichia coli), of the genus Bacillus (such as for example Bacillus subtilis), of the genus Streptomyces (for example Streptomyces coelicolor), a photosynthetic bacterial cell of the genus Synechocystis (such as for example Synéchocystis sp. PCC6803) or Synéchococcus (as for example Synechococcus WFI8102) or any other prokaryotic cell, a eukaryotic cell for example of the genus Chlamydomonas (for example Chlamydomonas reinhardtii), of the genus Saccharomyces (for example Saccharomyces cerevisiae), of the Pichia type (for example Pichia pastoris), or another eukaryotic cell type.

According to the invention, said polynucleotide induced in said host cell may itself but not necessarily be induced in an expression vector, for example in a plasmid, inserted into said host cell.

Advantageously, according to the invention, said monomeric polypeptide encoded by said induced polynucleotide in said host cell has a truncated or non-truncated amino acid sequence exhibiting at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% of identity, more preferably still at least 99% identity, relative to the amino acid sequence of SEQ ID NO: 2 and / or relative to the amino acid sequence of SEQ ID NO: 4.

Preferably, according to the invention, said host cell including a polynucleotide encoding a monomeric polypeptide can include one or more maturation factors of said [NiFe] -hydrogenase type protein, preferably one or more maturation factors of said protein of type [NiFe] - hydrogenase chosen from the group consisting of the maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW a) whose respective amino acid sequences each have at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity , more preferably still at least 95% identity, more preferably still at least 99% identity, respectively relative to the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID

NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; or b) encoded together by a concatenary nucleotide sequence exhibiting at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the nucleotide sequence SEQ ID NO: 19, which codes for all of these ripening factors.

According to the invention, said at least one maturation factor can be endogenous to the host cell and / or exogenous to the host cell.

The present invention also relates to a process for obtaining, for example in a host cell (for example in E. coli), a monomeric polypeptide exhibiting hydrogenase activity according to the invention, said process comprising the following steps:

• modification of an expression vector, for example of a plasmid, by including therein an exogenous polynucleotide of which at least a part codes for a monomeric polypeptide comprising a single subunit comprising the active site of a protein of type [ NiFe] -hydrogenase; and

Incubation of said modified expression vector under incubation conditions making it possible to ensure expression of said exogenous polynucleotide to produce a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] - hydrogenase type, said monomeric polypeptide exhibiting hydrogenase activity.

The method according to the invention makes it possible to produce a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type.

Those skilled in the art are of course able to define the required and adequate incubation conditions.

According to the invention, during the step of genetically modifying a host cell, the polynucleotide may itself but not necessarily be included in an expression vector, for example in a plasmid.

Advantageously, according to the invention, said step of modifying said expression vector consists of an inclusion in said expression vector of said exogenous polynucleotide, at least part of which codes for said polypeptide. monomer in which the amino acid sequence, truncated or not, has at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity , more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the amino acid sequence of SEQ ID NO: 2 and / or relative to the amino acid sequence of SEQ ID NO: 4.

Preferably, according to the invention, said step of modifying said expression vector may comprise the inclusion in said expression vector of one or more factors for maturation of said protein of [NiFe] - hydrogenase type, preferably one or more maturation factors of said [NiFe] -hydrogenase type protein chosen from the group consisting of the maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW a) whose respective acid sequences amines each have at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% d 'identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, respectively relative to the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; or b) encoded together by a concatenary nucleotide sequence exhibiting at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the nucleotide sequence SEQ ID NO: 19, which codes for all of these ripening factors. According to the invention, during the step of modifying said expression vector, a sequence encoding one or more maturation factors of said [NiFe] -hydrogenase type protein can itself but not necessarily be included in a vector. expression, for example in a plasmid.

Advantageously, the method according to the invention comprises a subsequent step of isolation and / or purification of said monomeric polypeptide.

In particular and preferably, the invention relates to a process for obtaining a monomeric polypeptide exhibiting hydrogenase activity according to the invention, said process comprising the following steps:

• a step of genetic modification, carried out in-vivo or in-vitro, of an entity comprising genetic material, for example of a host cell or of an expression vector, to obtain a genetically modified entity, for example a genetically modified host cell or a genetically modified expression vector;

A step of incubation of said genetically modified entity, for example of said genetically modified host cell or of said genetically expression vector, to obtain a monomeric polypeptide comprising a single subunit comprising the active site of a protein of type [ NiFe] -hydrogenase, said monomeric polypeptide exhibiting hydrogenase activity.

Preferably, according to the invention, said step of genetic modification, carried out in-vivo or in-vitro, consists of: a) a genetic modification of a host cell and / or of an expression vector by including therein a polynucleotide exogenous at least one part of which codes for a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, to obtain a genetically modified host cell and / or a vector of genetically modified expression to be incubated during said incubation step carried out under incubation conditions making it possible to ensure expression of said exogenous polynucleotide to produce said monomeric polypeptide; or b) by inducing at least one genetic mutation in the genetic material of a host cell to obtain a genetically modified host cell to be incubated during said incubation step carried out under incubation conditions to produce said polypeptide monomeric.

For example, according to the invention, said induction of at least one genetic mutation is carried out by homologous recombination, a method well known to those skilled in the art.

Advantageously, according to the invention, said step of genetic modification of said host cell by inclusion of an exogenous polynucleotide consists of an inclusion in said host cell of an expression vector, in particular of a modified expression vector, including said exogenous polynucleotide.

Preferably, according to the invention, said step of genetic modification of said host cell consists of an inclusion in said host cell of said exogenous polynucleotide, at least part of which codes for said monomeric polypeptide, the amino acid sequence of which is or is not present at least. 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the amino acid sequence of SEQ ID NO: 2 and / or relative to to the amino acid sequence of SEQ ID NO: 4.

Advantageously, according to the invention, said step of genetic modification of said host cell further comprises the inclusion in said host cell of at least one maturation factor of said [NiFe] -hydrogenase type protein, said at least one maturation factor being endogenous to the host cell and / or exogenous to the host cell, preferably the inclusion of at least a maturation factor of said [NiFe] -hydrogenase type protein chosen from the group consisting of maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and HoxW a) whose respective amino acid sequences each have at least 15 % identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least

80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, respectively relative to the amino acid sequences SEQ ID NO : 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID

NO: 18; or b) encoded together by a concatenary nucleotide sequence exhibiting at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the nucleotide sequence SEQ ID NO: 19, which codes for all of these ripening factors.

Preferably, according to the invention, said at least one maturation factor is derived from the expression of at least one undue gene in an expression vector, said expression vector being induced in said host cell.

Preferably, the process for obtaining a polypeptide monomer exhibiting a hydrogenase activity according to the invention gives rise to the production of a monomeric polypeptide of which the subunit comprising the active site of a protein of [NiFe] -hydrogenase type is the HoxH subunit of the protein of type [NiFe] -hydrogenase FloxEFUYFI in Synechocystis sp. PCC6803.

The present invention also relates to a use of a monomeric polypeptide according to the invention comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, said monomeric polypeptide exhibiting hydrogenase activity and being present or not in a cell, to produce or consume hydrogen by incubating said monomeric polypeptide under incubation conditions making it possible to ensure production or consumption of hydrogen.

The present invention also relates to a use of a monomeric polypeptide according to the invention or of a monomeric polypeptide obtained according to the process according to the invention comprising a single subunit comprising the active site of a protein of [NiFe] type. -hydrogenase, said monomeric polypeptide exhibiting hydrogenase activity and being present or not in a cell, for coating a surface, in particular for coating a surface with an electrical conductor, for example for coating a surface of an anode or a cathode.

Definitions

By the term “polypeptide”, it is understood, within the meaning of the present invention, a single chain composed of a minimum of two amino acids linked together by a peptide bond between the carboxylic group of an amino acid and the amine group. of the following amino acid. The term "polypeptide" also includes molecules which contain more than one polypeptide, the latter linked together, for example by disulfide bridges, or polypeptide complexes, the latter linked together, for example covalently or not. -covalent, and forming a multimer (for example a dimer, a trimer, a quadrimer, a pentamer). A polypeptide can also contain non-protein ligands, such as for example inorganic iron (Fe), nickel (Ni), an iron-sulfur center (FeS), or other organic ligand such as for example carbon monoxide (CO) , cyanide (CN) or a flavin. The terms peptide, polypeptide, enzyme, subunit or protein are all included within the definition of polypeptide and these terms can be used interchangeably. The definition of "polypeptide" does not take into account the length of said polypeptide or the way in which said polypeptide is produced.

By the term “recombinant polypeptide” or “heterologous polypeptide”, it is understood, within the meaning of the present invention, a polypeptide which is not naturally present in the environment and / or which is not naturally present in the cell. host used for its production, in particular in a host cell, and the production of which is carried out by recombinant techniques, for example by adding genetic material to a host cell.

By the terms “monomeric polypeptide”, it is understood, within the meaning of the present invention, a single chain composed of a minimum of two amino acids linked together by a peptide bond between the carboxylic group of an amino acid and the group. amine of the next amino acid. As opposed to the term "polypeptide", the term "monomeric polypeptide" includes only molecules which contain only one polypeptide, that is, without interaction with any other polypeptide. For example, it is in no case a multimer (for example a dimer, a trimer, a quadrimer, a pentamer, ...). A monomeric polypeptide can also contain non-protein ligands, such as for example inorganic iron (Fe), nickel (Ni), iron-sulfur centers (FeS), or other organic ligand such as for example carbon monoxide (CO ), cyanide (CN) or a flavin. The definition of "monomeric polypeptide" does not take into account the length of said polypeptide or the way in which said polypeptide is produced.

By the terms “recombinant monomeric polypeptide” or “heterologous monomeric polypeptide”, it is understood, within the meaning of the present invention, that the monomeric polypeptide is not naturally present in the environment, in particular in a host cell, and of which the production is carried out by recombinant techniques, for example by adding genetic material to a host cell.

By the terms “monomeric polypeptide exhibiting hydrogenase activity”, it is understood, within the meaning of the present invention, that the polypeptide monomeric is able to catalyze the reversible reaction H2 <® 2H ⁺ + 2e. This definition of hydrogenase activity is not related to the experimental conditions but only to the production or consumption of hydrogen through the enzymatic activity of hydrogenase.

By the terms "active site", it is understood, within the meaning of the present invention, the part of the polypeptide which, when the tertiary structure is formed, is responsible for the catalytic activity of said polypeptide, for example of the hydrogenase activity. The part of the polypeptide may comprise, for example, several portions of the amino acid sequence and / or the linkage with one or more non-protein ligands.

By the term “polynucleotide”, it is understood, within the meaning of the present invention, a single chain composed of a minimum of two nucleotides linked together, for example by a covalent bond, regardless of whether they are ribonucleotides. or deoxynucleotides. This therefore includes RNAs and DNAs, single stranded or double stranded. The definition of “polynucleotide” does not take into account the length of said polynucleotide, nor of the function, nor of the form, nor of the way in which said polynucleotide is produced. A polynucleotide can be, for example, a plasmid, part of a plasmid, a gene or a gene fragment.

By the terms "exogenous polynucleotide", it is understood, within the meaning of the present invention, a polynucleotide which is not normally present in the host cell. For example, the exogenous polynucleotide can be a coding sequence for a polypeptide which is not naturally present in the host cell or a plasmid.

By the term "concatenated", it is understood, within the meaning of the present invention, a concatenation of several sequences in order to form only one, for example of polynucleotide sequences or of polypeptide sequences.

By the term “plasmid”, it is understood, within the meaning of the present invention, a double-stranded DNA molecule distinct from chromosomal DNA, exogenous, capable of autonomous replication, by virtue of its own origin of replication. The plasmid can also contain other sequences of interest, such as by example a gene encoding a selection factor (resistance to an antibiotic, etc.), a multiple cloning site allows the addition of polynucleotide, and / or sequences for regulating transcription. The terms "plasmid" or "expression vector" are interchangeable.

By the term “host cell”, it is understood, within the meaning of the present invention, a cell which has undergone a modification. This modification can for example be the introduction of an exogenous polynucleotide into the cell, for example via a plasmid.

By the terms "maturation factor", it is understood, within the meaning of the present invention, any molecule, biological or not, which participates in the formation of the structure of another biological molecule, for example of a protein.

By the terms “endogenous maturation factor”, it is understood, within the meaning of the present invention, any molecule, biological or not, which participates in the formation of the structure of another biological molecule, for example of a protein. , and which is naturally present in the host cell, that is to say without genetic modification of said host cell.

By the terms “exogenous maturation factor”, it is understood, within the meaning of the present invention, any molecule, biological or not, which participates in the formation of the structure of another biological molecule, for example of a protein. , and which is not naturally present in the host cell, that is to say that a genetic modification of said host cell is necessary in order to add said exogenous maturation factor thereto, for example by adding a vector d 'expression.

By the term "isolated", it is understood, within the meaning of the present invention, any molecule which has been removed from its natural environment, for example which has been removed from a [natural NiFej-hydrogenase.

By the term “purified”, it is understood within the meaning of the present invention, any molecule which has been lonely by means of biochemical techniques. This definition does not take into account the way in which the molecule is produced, for example naturally or by recombination, chemically or enzymatically synthesized, nor the biochemical techniques used for the production. purification, such as affinity chromatography or molecular sieve. For example, a polynucleotide, a polypeptide or IΉ2 can be purified. Preferably, a substance is purified when it represents at least 60% relative to the other components which are associated with it, preferably 75% relative to the other components which are associated with it, preferably 90% relative to the other components which are associated with it. associates.

By the term “apparent homogeneity”, it is understood, within the meaning of the present invention, any purified molecule representing at least 90% relative to the other components which are associated with it.

By the term “identity”, it is understood, within the present invention, a structural similarity between two polynucleotides or two polypeptides. Structural similarity is determined by an alignment between the two sequences, which alignment optimizes the number of identical nucleotides or the number of identical amino acids along the sequence. Holes in one or both sequences are allowed in order to optimize alignment and therefore structural similarity. The nucleotide or amino acid sequences must however remain the same.

By the terms "genetic material", it is understood, within the meaning of the present invention, the genome of an entity and more precisely all the nucleic acids of this entity, coding and non-coding sequences included.

By the terms "genetic mutation", it is understood, within the meaning of the present invention, an accidental or induced modification of the genetic material of an entity, for example of a host cell or of an expression vector.

By the terms “entity comprising genetic material”, it is understood, within the meaning of the present invention, an entity which comprises genetic material according to the definition given above, for example a host cell or an expression vector such as 'a plasmid.

By the terms “genetically modified entity”, it is understood, within the meaning of the present invention, an entity comprising genetic material according to the definition given above and which has undergone a modification of its genetic material, for example a genetic mutation or the introduction of an exogenous polynucleotide in the cell, such as, for example, via a plasmid.

By the terms “genetically modified host cell”, it is understood, within the meaning of the present invention, a host cell according to the definition given above and which has undergone a modification of its genetic material, for example a genetic mutation or the introduction of an exogenous polynucleotide into the cell, such as for example via a plasmid.

By the terms “genetically modified expression vector”, it is understood, within the meaning of the present invention, an expression vector or a plasmid according to the definition given above and which has undergone a modification of its genetic material, by example the addition of a sequence of interest, such as for example a gene encoding a particular polypeptide.

Other characteristics, details and advantages of the invention will emerge from the examples given below, without limitation and with reference to the appended figures.

Figure 1 illustrates the map of plasmid pET-26b (+) (5360bp).

Figure 2 illustrates the agarose gel analysis of the digestion of the pET26b (+) + HoxH expression vector by the restriction enzymes Ndel and Blpl.

Figure 3 illustrates the map of the plasmid pACYCDuet-1 (4008bp).

Figure 4 illustrates the agarose gel analysis of the digestion of pACYCDuet-1 + HypABCDEFHoxW expression vector by restriction enzymes Ncol and Hindi II.

Figure 5 illustrates the agarose gel analysis of the digestion of the expression vector pET26b (+) + HoxH, derived from plasmid DNA from a colony of E. coli, by restriction enzymes Ndel and Blpl.

Figure 6 illustrates the agarose gel analysis of the digestion of the expression vector pACYCDuet-1 + HypABCDEFHoxW, derived from the plasmid DNA of a colony of E. coli, by the restriction enzymes Ncol and Hindlll.

FIG. 7 illustrates the method of purification of the recombinant protein of interest HoxH. NA, sample not absorbed on the Ni-NTA affinity column. Washing, sample eluted with the washing buffer containing 10 mM in Imidazole. 50 mM, sample eluted at a concentration of 50 mM in imidazole. 100 mM, sample eluted at a concentration of 100 mM in imidazole. 150 mM, sample eluted at a concentration of 150 mM in imidazole. 200 mM, sample eluted at a concentration of 200 mM in imidazole. 250 mM, sample eluted at a concentration of 250 mM in imidazole.

Figure 8 illustrates the SDS-PAGE analysis of the protein composition of various fractions collected during affinity chromatography. Load, supernatant applied to the Ni-NTA affinity column and obtained from the lysis of recombinant E. coli cells; NA, sample not absorbed on Ni-NTA affinity column. Washing, sample eluted with the washing buffer containing 10 mM in Imidazole. M, molecular weight marker. 50 mM, sample eluted at a concentration of 50 mM in imidazole. 100 mM, sample eluted at a concentration of 10OmM in imidazole. 150 mM, sample eluted at a concentration of 150 mM in imidazole. 200 mM, sample eluted at a concentration of 200 mM in imidazole. 250 mM, sample eluted at a concentration of 250 mM in imidazole.

Figure 9 illustrates the analysis by immunodetection (Western blot) of the presence of HoxH in various fractions collected during affinity chromatography. M, molecular weight marker. Load, supernatant applied to the Ni-NTA affinity column and obtained from the lysis of recombinant E. coli cells. NA, sample not absorbed on Ni-NTA affinity column; 50 mM, sample eluted at a concentration of 50 mM in imidazole. 200 mM, sample eluted at a concentration of 200 mM in imidazole.

Figure 10 is a schematic representation of the structure of HoxEFUYH, [NiFe] -hydrogenase from Synechocystis sp. PCC 6803. HoxE, 19KDa and 1 FeS center; HoxF, 57.5KDa, 2 FeS centers and one FMN center; HoxU, 26KDa and 4 FeS centers; HoxY, 20KDa and an FeS center; HoxH, 53KDa and the active site.

Figure 11 is a schematic representation (gray arrow) of electron transfer and expected interactions with NADPH and methylviologen (MV) in HoxEFUYH, [NiFe] -hydrogenase from Synechocystis sp. PCC 6803.

Figure 12 is a schematic representation where the question mark asks whether the active site of the recombinant protein HoxH may or may not accept electrons directly from the MV and therefore produce hydrogen in the absence of additional redox relays.

Figure 13 illustrates the demonstration of the hydrogenase activity by the production of hydrogen which results from the addition of the recombinant protein HoxH to a reagent containing methyl-viologen previously reduced with sodium dithionite in the absence of oxygen. . HoxH is able to take electrons from previously reduced methyl viologen to combine them with protons (present in the buffer) to produce hydrogen according to the equation H2 <® 2H ⁺ + 2e which represents the reaction catalyzed by l hydrogenase. The level of hydrogen dissolved in the reagent is continuously measured using a micro-sensor (Unisense®, Denmark). The arrow shows the moment when the addition of the recombinant HoxH protein is carried out, a moment concomitant with the increase in the level of dissolved hydrogen detected by the micro-sensor.

FIG. 14 illustrates the demonstration of the hydrogenase activity by the reduction of benzyl viologen which results from the addition of the recombinant protein HoxH to a reagent containing benzyl viologen (BV) in the presence of H2. HoxH is able to take electrons from hydrogen to transfer them to a redox mediator, for example benzyl viologen, at the same time as the production of protons according to the equation H2 <® 2H ⁺ + 2e which represents the reaction catalyzed by hydrogenase. The level of hydrogen consumed is equivalent to the level of reduced benzyl viologen, a level which can be measured continuously by spectrophotometry at a wavelength of 578 nm.

FIG. 15 illustrates the demonstration of the hydrogenase activity by the production of hydrogen which results from the addition of the recombinant protein HoxH, produced in the absence of the HupABCDEFHoxW maturation factors exogenous to E. coli, to a reagent containing methyl-viologen previously reduced with sodium dithionite in the absence of oxygen. HoxH is able to take electrons from previously reduced methyl viologen to combine them with protons (present in the buffer) to produce hydrogen according to the equation H2 <® 2H ⁺ + 2e which represents the reaction catalyzed by l hydrogenase. Examples

1. Construction of a HoxH expression vector 1.1. HoxH "simple" sequence

HoxH corresponds to the large subunit comprising the active site of [NiFe] -hydrogenase HoxEFUYH in Synechocystis sp. PCC6803. The protein's accession number in Genbank is BAA18091.1. The theoretical isoelectric point of HoxH is 5.86 for a theoretical molecular mass of 52996.53 Daltons. The nucleotide sequence (1425bp) of HoxH is as follows and is called SEQ ID NO: 1 in the context of the present invention: atgtctaaaaccattgttatcgatcccgttacccggattgaaggccatgccaaaatctccattttcctcaacgaccagg gcaacgtagatgatgttcgtttccatgtggtggagtatcggggttttgaaaaattttgcgaaggtcgtcccatgtggga aatggctggtattaccgcccgtatttgcggcatttgtccggttagccatctgctctgtgcggctaaaaccggggataag ttactggcggtgcaaatccctccagccggggaaaaactgcgccgtttaatgaatttagggcaaattacccaatccc acgccctaagttttttccatctcagcagtcctgattttctgcttggttgggacagtgatcccgctactcgcaatgtgtttggt ttaattgctgctgaccccgatttagctagggcaggtattcggttacggcaatttggccaaacggtaattgaacttttggg agctaaaaaaatccactctgcttggtcagtgcccggtggagtccgatcgccgttgtcggaagaaggcagacaatg gattgtggaccgtttaccagaagcaaaagaaaccgtttatttagccttaaatttgtttaaaaatatgttggaccgcttcc aaacagaagtggcagaatttggcaaatttccctccctatttatgggcttagttgggaaaaataatgaatgggaacatt atggcggctccctgcggtttaccgacagtgaaggcaatattgtcgcggacaatctcagtgaagataattacgctgat tttattggtgaatcggtggaaaaatggtcctatttaaaatttccctactacaaatctctgggttatcccgatggcat ttatc gggttggtccccttgcccgccttaatgtttgtcatcacattggcaccccggaagcagaccaagaattagaagaatat cggcaacgggctggaggtgtggccacgtcctctttcttttatcattacgcccgcttggtggaaattcttgcctgtttagaa gccatcgaattgttaatggctgaccctgatattttgtccaaaaattgtcgagctaaggcagaaattaattgtaccgaag cggtgggagtgagcgaagcaccccggggtactttattccaccattacaagatagatgaagatggtctaattaagaa agtgaatttgatcattgccacgggcaacaataacttagccatgaataaaaccgtggcccaaattgccaaacactac attcgcaatcatgatgtgcaagaagggtttttaaaccgggtggaagcgggtattcgttgttatgatccctgccttagttgt tctacccatgcagcgggacaaatgccattgatgatcgatttagttaaccctcagggggaactaattaagtccatcca gcgggattaa

The amino acid sequence (474 aa) of HoxH is as follows and is named SEQ ID NO: 2 in the context of the present invention: MSKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEKFCEGRPMWEMA GITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLRRLMNLGQITQSHALSFFHL SSPDFLLGWDSDPATRNVFGLIAADPDLARAGIRLRQFGQTVIELLGAKKIHSAWS VPGGVRSPLSEEGRQWIVDRLPEAKETVYLALNLFKNMLDRFQTEVAEFGKFPSL FMGLVGKNNEWEHYGGSLRFTDSEGNIVADNLSEDNYADFIGESVEKWSYLKFP YYKSLGYPDGIYRVGPLARLNVCHHIGTPEADQELEEYRQRAGGVATSSFFYHYA RLVEILACLEAIELLMADPDILSKNCRAKAEINCTEAVGVSEAPRGTLFHHYKIDED GLIKKVNLIIATGNNNLAMNKTVAQIAKHYIRNHDVQEGFLNRVEAGIRCYDPCLSC STHAAGQMPLMIDLVNPQGELIKSIQRD

Figure 1 shows the map of the plasmid pET26b (+) used to construct the FloxFI expression vector by insertion of SEQ ID NO: 3 into pET26b (+). pET26b (+) is a 5360bp plasmid possessing an origin of replication for E. coli, a kanamycin resistance gene, a multiple cloning site (MCS) containing numerous restriction sites, the promoter 77 and the transcription terminator T7. It also contains the lad gene encoding a transcription repressor. This repression of transcription can be removed by adding IPTG.

1.2. HoxH "optimized" sequence

Optionally, according to an embodiment according to the invention, the sequences SEQ ID NO: 1 and SEQ ID NO: 2 are optimized for the use of codons in E. coli. In particular, the Ndel (catatg) and Blpl (gctnagc) restriction sites are added respectively at the start and at the end of the nucleotide sequence for cloning into the plasmid pET26b (+) and the caccaccaccatcac sequence (underlined below) is also added (sequence encoding the poly-histidine tag near the N-terminal end of the protein). The optimized nucleotide sequence (1453bp) is as follows and is named SEQ ID NO: 3 in the context of the present invention: cafafqaqccaccaccaccaccatcacaaaaccatcqtcatcqacccaqtcacccqcatcqaaqqccacqcca aaattagcatttttctgaacgaccagggcaacgtcgacgacgtccgctttcacgttgttgaataccgtggcttcgaaa aattttgtgaaggtcgtccgatgtgggaaatggccggtatcacggcacgtatttgtggaatttgtccggtgagccatct gctgtgtgccgcaaaaaccggagataaactgctggcagtgcagattccgccggcaggtgaaaaactgcgtcgtct gatgaatctgggtcagattacacagtcgcatgcactgtctttctttcatctgagtagcccagattttctgctggggtggg atagcgacccggcaacacgtaatgtgtttggtctgattgcggctgatccggatctggcgcgtgccggtattcgtctgc gtcagtttggtcagacagttattgagctgctgggggcgaaaaagattcatagtgcatggtctgtgccgggtggtgttc gtagtccgctgagtgaagaaggtcgtcagtggattgttgatcgtctgccggaggcaaaagaaacggtctatctggc actgaatctgtttaaaaatatgctggatcgtttccagacagaagttgcagaatttggaaaatttccgtcactgtttatgg gtctggttggtaaaaataatgaatgggaacactatggtggtagcctgcgtttcacggactctgaaggtaatattgttgc ggataatctgagcgaagacaattatgcagattttatcggtgaaagtgtggaaaaatggagctatctgaaatttccgta ttacaaaagcctgggctatccggatgggatctaccgtgttggaccgctggcacgtctgaacgtttgtcatcatattggt accccggaagcagatcaggaactggaagaatatcgtcagcgtgcgggtggtgttg cgactagcagctttttttatca ttatgcacgtctggttgaaattctggcctgtctggaggcaattgaactgctgatggcagatcctgatattctgtctaaaa attgtcgtgcaaaagcagaaattaactgtaccgaggcagttggtgttagtgaggcgccgcgtggtaccctgtttcatc actataaaattgacgaagatggtctgattaaaaaggttaatctgattatcgcaaccggtaacaataatctggcaatg aataaaaccgttgcacagattgcaaaacactacattcgcaaccacgatgttcaggaagggtttctgaatcgtgtag aagccggcattcgctgttatgatccgtgtctgagctgtagcacccatgcagcaggtcagatgcctctgatgattgacc tggttaatccgcagggtgagctgattaaaagcattcagcgtgattaagcigagc

The optimized amino acid sequence (480 aa) is as follows and is named SEQ ID NO: 4 in the context of the present invention:

MSHHHHHHKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEKFCEGRP MWEMAGITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLRRLMNLGQITQSHA LSFFHLSSPDFLLGWDSDPATRNVFGLIAADPDLARAGIRLRQFGQTVIELLGAKKI HSAWSVPGGVRSPLSEEGRQWIVDRLPEAKETVYLALNLFKNMLDRFQTEVAEF GKFPSLFMGLVGKNNEWEHYGGSLRFTDSEGNIVADNLSEDNYADFIGESVEKW SYLKFPYYKSLGYPDGIYRVGPLARLNVCHHIGTPEADQELEEYRQRAGGVATSS FFYHYARLVEILACLEAIELLMADPDILSKNCRAKAEINCTEAVGVSEAPRGTLFHH YKIDEDGLIKKVNLIIATGNNNLAMNKTVAQIAKHYIRNHDVQEGFLNRVEAGIRCY DPCLSCSTHAAGQMPLMIDLVNPQGELIKSIQRD

Plasmid pET26b (+) is used to construct the HoxH expression vector by inserting SEQ ID NO: 3 into pET26b (+).

FIG. 2 shows the analysis of the digestion of the expression vector pET26b (+) + HoxH (SEQ ID NO: 3) by the restriction enzymes Ndel and Blpl. Two DNA fragments at approximately 5300 bp (linearized pET26b (+)) and at approximately 1500 bp (HoxH sequence excised from the plasmid pET26b (+)) are demonstrated. This confirms the presence of the HoxH sequence of interest in the expression vector pET26b (+).

2. Construction of the expression vector of at least one maturation factor

At least the following ripening factors are considered within the scope of the present invention: HypA, HypB, HypC, HypD, HypE, HypF and HoxW.

HypA is a FloxEFUYFI [NiFe] hydrogenase expression / formation protein in Synéchocystis sp. PCC6803. The protein's accession number in Genbank is BAA18357.1. The theoretical isoelectric point of HypA is 4.94 for a theoretical molecular mass: 12,773.47 Daltons. The nucleotide sequence (342bp) of Hypa is as follows and is called SEQ ID NO: 5 in the context of the present invention: atgcacgaagttagtctgatggagcaaactttggcgatcgccattgcccaggcggaagaccatggagccagcca aatccatcgtttaaccctgcgggtggggcaacagtctggggtggtggccgatgccctacggtttgcgtttgaagtggt gcgacaaaataccatggccgccgaggcgagattggaaattgaagaaattcccgttacctgtcgttgccaacactg ccacgaaaattttcagccagaggattggatttaccgctgtccccactgcgaccagattagccaaacagtaatggat ggcaaacagttggaactagcatccctagaactgagttga

The amino acid sequence (113 aa) of HypA is as follows and is named SEQ ID NO: 6 in the context of the present invention:

MHEVSLMEQTLAIAIAQAEDHGASQIHRLTLRVGQQSGVVADALRFAFEVVRQNT

MAAEARLEIEEIPVTCRCQHCHENFQPEDWIYRCPHCDQISQTVMDGKQLELASL

ELS

HypB is a [NiFe] - HoxEFUYH hydrogenase expression / formation protein in Synechocystis sp. PCC6803. The accession number of the protein in Genbank is BAA18312.1. The theoretical isoelectric point of HyB is 5.75 for a theoretical molecular mass: 31,242.97 Daltons. The nucleotide sequence (858bp) of HYPB is as follows and is called SEQ ID NO: 7 in the context of the present invention: atgtgccaaaactgcggttgtagtgcggtgggaaccgttgcccatagccaccatcaccatggcgatggaaattttgc ccacagccatgatgaccatgaccagcaagaacatcatcaccaccatggcaactacagcaaaagtccaagtcag cagactgtgaccattgaacccgatcgccagtccattgccattggccaaggcattctcagcaagaatgaccgcctag cggaaaggaatcggggctatttccaggctaagggcttactggtgatgaatttcctctcttctcccggagccggtaaaa ctgctctgatcgaaaaaatggtcggcgatcgacaaaaagaccatcccaccgccgtcattgtgggggatttagcca ccgataacgatgcccaacgtctccgcagtgccggggcgatcgccattcaggtcaccacaggaaatatttgccatct ggaagcggaaatggtggccaaggcggcccaaaagttagatttagacaatatcgatcaattgatcattgaaaatgtt ggtaatttggtttgccccaccacctatgatctaggggaagatttacgggtcgtattattttccgtcacagaaggggagg ataaaccccttaaatatcccgccaccttcaaatcagcccaggttattttagtcaccaaacaggacattgccgccgca gtggattttgatgcagagctggcttggcaaaacctacggcaagtggccccccaagcccaaatttttgcagtgtctgc ccgcacggggaaaggattgcagtcctggtatgagtatttggatcaatggcaactccaacactattcgccgttggttg atccagcattggcctaa

The amino acid sequence (285 aa) of HypB is as follows and is named SEQ ID NO: 8 in the context of the present invention:

MCQNCGCSAVGTVAHSHHHHGDGNFAHSHDDHDQQEHHHHHGNYSKSPSQQ TVTIEPDRQSIAIGQGILSKNDRLAERNRGYFQAKGLLVMNFLSSPGAGKTALIEK MVGDRQKDHPTAVIVGDLATDNDAQRLRSAGAIAIQVTTGNICHLEAEMVAKAAQ KLDLDNIDQLIIENVGNLVCPTTYDLGEDLRVVLFSVTEGEDKPLKYPATFKSAQVI LVTKQDIAAAVDFDAELAWQNLRQVAPQAQIFAVSARTGKGLQSWYEYLDQWQL QHYSPLVDPALA

FlypC is an expression / formation protein of [NiFe] - FloxEFUYFI hydrogenase in Synéchocystis sp. PCC6803. The accession number of the protein in Genbank is BAA18180.1. The theoretical isoelectric point of HypC is 4.20 for a theoretical molecular mass: 7987.42 Daltons. The nucleotide sequence (231 bp) of HypC is as follows and is named SEQ ID NO: 9 in the context of the present invention: atgtgtctagccctacctggccaggttgtcagtttaatgcccaactccgatcccctgttactgacgggaaaggttagct ttgggggcatcattaaaaccattagccttgcctacgtacccgaggttaaggtgggggattacgtgattgtccatgtgg gctttgccattagcattgtggacgaagaggcggcccaggaaactttgatagacttggcagaaatgggagtttaa The amino acid sequence (76 aa) of HypC is as follows and is called SEQ ID NO: 10 in the context of the present invention:

MCLALPGQVVSLMPNSDPLLLTGKVSFGGIIKTISLAYVPEVKVGDYVIVHVGFAISI

VDEEAAQETLIDLAEMGV

FlypD is a FloxEFUYFI [NiFe] hydrogenase expression / formation protein in Synechocystis sp. PCC6803. The protein's accession number in Genbank is BAA16622.1. The theoretical isoelectric point of HypD is 6.31 for a theoretical molecular mass of 40,632.94 Daltons. The nucleotide sequence (1125bp) of HYPD is as follows and is called SEQ ID NO: 11 in the context of the present invention: atgaaatacgttgatgaatatcgggatgcccaggcggtggcccattaccgtcaggcgatcgccagggagataacc aaaccttggacgctgatggagatttgcggcggccagacccacagcattgtcaaatatggcttggatgctttgttgccg aagaatttgactctgatccatggtcccggctgtcctgtgtgcgtcactccgatggaattaattgaccaggctttgtggtta gctaagcaaccggagatcattttttgttcctttggcgatatgttgcgggtgcccggcagtggggcggatttgctgagca ttaaagcccagggcggcgatgtgcgcattgtctattctcctttggattgtttggcgatcgccagggagaatcctaatcg ggaagtggtatttttcggagtaggttttgaaactacagcccctgccacggccatgactctccaccaagctagggccc agggaattagcaatttcagtttactttgcgcccatgtattggtgcccccggctatggaggctttattaggcaatcccaatt ccctcgtgcagggctttttggcggcagggcatgtctgtacggtgaccggggaaagggcctatcaacatatcgctga aaaataccaagtacccattgtcatcactggctttgaacctgtggatattatgcagggcatctttgcctgtgtgcgccaa ctggagtcgggacaattcacctgcaacaatcaatatcggcgatcggtccaaccccagggcaatgcccatgctcag has aaaattattgaccaagtgtttgagccagtcgatcgccattggcggggtttgggattaattccggccagcggtttgggttt aggccagcatttgccccctgggatgccgcagttaaattcgccaatttattgcaaaccatggccccaacgatggga gaaacagtgtgtattagcggggaaattttacagggacaacggaagcccagcgattgtccagcctttggtactatctg caccccagaacaacccttgggggctcccatggtttcctcggaaggagcctgtgccgcctattaccgttatcgccaac aattaccggaaccagtgggagcggccagagtttag

The amino acid sequence (374 aa) of HypD is as follows and is named SEQ ID NO: 12 in the context of the present invention: MKYVDEYRDAQAVAHYRQAIAREITKPWTLMEICGGQTHSIVKYGLDALLPKNLTL IHGPGCPVCVTPMELIDQALWLAKQPEIIFCSFGDMLRVPGSGADLLSIKAQGGDV RIVYSPLDCLAIARENPNREVVFFGVGFETTAPATAMTLHQARAQGISNFSLLCAH VLVPPAMEALLGNPNSLVQGFLAAGHVCTVTGERAYQHIAEKYQVPIVITGFEPVD IMQGIFACVRQLESGQFTCNNQYRRSVQPQGNAHAQKIIDQVFEPVDRHWRGLG LIPASGLGLRPAFAPWDAAVKFANLLQTMAPTMGETVCISGEILQGQRKPSDCPA FGTICTPEQPLGAPMVSSEGACAAYYRYRQQLPEPVGAARV

FlypE is a FloxEFUYFI [NiFe] hydrogenase expression / formation protein in Synechocystis sp. PCC6803. The protein's accession number in Genbank is BAA17478.1. The theoretical isoelectric point of HypE is 4.93 for a theoretical molecular mass of 36425.60 Daltons. The nucleotide sequence (1038bp) of hype is as follows and is called SEQ ID NO: 13 in the context of the present invention: gtgaacttagtctgtcccgttccccttgatcgttatccccaggtactgttagcccacggcggcggcggtaagttgagcc aacaattacttaagcaaatttttttaccggcctttggcgcttctgaaacgggtagtcatgatgcggcggtttttactgcca accaaagttctttagctttcaccaccgactcctatgtgatcaatcccctcttttttcctgggggcgatattggttctttggca gtccacggcaccgttaatgacctagccatggccggcgcaacccctcgctatatcagcgttggttttatcctcgaaga aggattgcccatggagaccctctggcgggtggcccaatccctagggcaagcggcccaaaactgtggggtggaa attcttaccggtgataccaaagtggtggaccggggtaagggagacggcattttcatcaacaccagcggcattggtt ccctcgaccatcaacaaactatccatcccaatcaggtacaggtaggcgatcgcctaattttgagcggtgatttggga cgtcatggcatggccattatggcagtgcgccaaggattagaatttgaaaccaccattgaaagtgattcggccccggt tcacagagaagtgcaggcattattgtcggcagggatcccaatccattgtctgcgggatttaaccagggggggatta gccagtgcggttaatgaaattgcccaaacttccggggtaaccatggctttacgagaaacgttaatcccggtggagg ccgaagtacaagccgcctgtgaactgttgggttttgaccccctctatgtggccaatgagggaagattcctggccattg tgcccccg gaagcagaacagaagaccgtggaaattttgcaaactttccatccccaagctacggcgatcggtaca gtaacaggcaaaagtgcacaaaccttggggttagtcagtttggaaagttccattggtgccccccggttgctagacat gatcagtggggagcaattacccttag

The amino acid sequence (345 aa) of HypE is as follows and is named SEQ ID N0: 14 in the context of the present invention: MNLVCPVPLDRYPQVLLAHGGGGGKLSQQLLKQIFLPAFGASETGSHDAAVFTAN QSSLAFTTDSYVINPLFFPGGDIGSLAVGFEGTVNDILE METLWRVAQSLGQAAQNCGVEILTGDTKVVDRGKGDGIFINTSGIGSLDHQQTIH PNQVQVGDRLILSGDLGRHGMAIMAVRQGLEFETTIESDSAPVHREVQALLSAGI PIHCLRDLTRGGLASAVNEIAQTSGVTMALRETLIPVEAEVQAACELLGFDPLYVA NEGRFLAIVPPEAEQKTVEILQTFHPQATAIGTVTGKSAQTLGLVSLESSIGAPRLL DMISGEQLPRIC

FlypF is a FloxEFUYFI [NiFe] hydrogenase expression / formation protein in Synéchocystis sp. PCC6803. The accession number of the protein in Genbank is BAA10154.1. The theoretical isoelectric point of FlypF is 8.19 for a theoretical molecular mass of 85358.25 Daltons. The nucleotide sequence (2304bp) of HypF is as follows and is called SEQ ID NO: 15 in the context of the present invention: atgttaaaaaccgttgccatacaggtccagggaagggtgcaaggagtgggttttcgtccctttgtttatacccttgccc aggaaatgggactgaatggttgggtgaataattccactcaaggagctaccgttgtcattaccgccgacgaaaagg cgatcgccgactttacggagagattaacgaagacattacctccccctggtttgattgaacaattagccgttgaacagt taccgctggaaagttttactaactttactatccgccccagtagtgatggccctaaaactgcgagtattttacccgatttat ccacttgttccgcctgcttaacagaactatttgaccctagcgatcgccgttatctttacccctttattaactgtacccattg cggtccccgctacaccattattgaagccctaccttacgaccgttgtcgtaccaccatggctaggtttcgccaatgtacc gactgtgaaagggaatataagcaaccaggcgatagacgcttccatgcccaacctaatgcctgtcctcgctgtggc ccccaactggctttttggaaccgacaaggccaagtaattgcagaagcaaatgaagctttaaactttgctgtagataa tttaaaagtcggcaatattatcgctattaaaggcttaggtggcttccatttgtgttgtgatgccactgattttgaagctgtg gaaaaattaagattaaggaaacatcgaccggataaacctttggcggtaatgtatggtaatcttggtcaaattgtgga gcattaccaacctaataatctagaagttgaattgttacaaagtgccgccgcccctattgtgttattaaacaaaaaaa has acaattaattttggtggaaaatattgccccaggcaacccccgagtcggcgtaatgttagcctatactcctttgcatcac ttattactaaaaaaattaaagaaacccatggtagctaccagtggtaacttagctggggagcaaatttgcattgataat attgacgctttaacccggttacaaaatattgctgacggttttctcgttcatgatcgcccgattgtttgtccagtggatgattc cgttgtccaaatagtagctgggaagccattatttttgcgtcgagcccggggttacgctcctcaacccattactttacca aagcctactcaaaaaaaactattggcgatgggaggtcattataaaaatacagtggcgatcgccaaacaaaatca agcttacgtcagccaacatttgggcgatttgaattctgctcccacctaccaaaattttgaagaagccattgcccattta agccagctatacgatttctctccccaggaaattgttgcagatttacaccctgattatttcagtcatcaatatgctgaaaa ccaagctttgcctgtcacttttgtgcagcatcactatgctcatattttagcggttatggcggaacatggagttatggagg agtccgtgttaggtattgcttgggatggcactggctacggcatggacggtactatttgggggggagaatttttaaaaat cacccaaggtacttggcagagaattgctcatctacaaccatttcatttattaggtaatcaacaagccattaaatatccc catcggattgctttggcgttgttatggcccacttttggtgatgatttttctgctgattctttaggaaattggttgaatttcaata atgggtttaaaaacaagataaacagcaggttaaatcaggatctaaacaacaaaaatttacgtcaactttggcaac gagggcaagcaccgctcacttcgagtatgggaagat tatttgacggtattgcgacactgataggattgattaacgaa gtaacttttgaaggtcaggcggccatagctctggaagctcagattatgccaaatttaactgaggagtattatcctttga ctctaaacaacaaggaaaaaaaattagctgttgattggcgccccttaattaaagctataaccacagaagatagaa gcaaaactaacctaatagccactaaattccacaacagtttagtaaatttaattatcactattgcccaacagcaggga atcgaaaaagttgctctggggggaggttgctttcaaaattgttatttgcttgccagtaccattactgccctcaaaaaag ctggtttttctcctttgtggcccagagaactaccgcccaacgacggtgccatttgcatgggtcaactgttagctaaaatt caggctcggcaatatatctgttaa

The amino acid sequence (767 aa) of HypF is as follows and is called SEQ ID NO: 16 in the context of the present invention: mlktvaiqvqgrvqgvgfrpfvytlaqemglngwvnnstqgatvvitadekaiadfterltktlpppglieqlaveqlpl esftnftirpssdgpktasilpdlstcsacltelfdpsdrrylypfincthcgprytiiealpydrcrttmarfrqctdcereyk qpgdrrfhaqpnacprcgpqlafwnrqgqviaeanealnfavdnlkvgniiaikglggfhlccdatdfeaveklrlrkh rpdkplavmygnlgqivehyqpnnlevellqsaaapivllnkkkqlilveniapgnprvgvmlaytplhhlllkklkkp mvatsgnlageqicidnidaltrlqniadgflvhdrpivcpvddsvvqivagkplflrrargyapqpitlpkptqkkllam gghykntvaiakqnqayvsqhlgdlnsaptyqnfeeaiahlsqlydfspqeivadlhpdyfshqyaenqalpvtfv qhhyahilavmaehgvmeesvlgiawdgtgygmdgtiwggeflkitqgtwqriahlqpfhllgnqqaikyphrial allwptfgddfsadslgnwlnfnngfknkinsrlnqdlnnknlrqlwqrgqapltssmgrlfdgiatliglinevtfegqa aialeaqimpnlteeyypltlnnkekklavdwrplikaittedrsktnliatkfhnslvnliitiaqqqgiekvalgggcfqn cyllastitalkkagfsplwprelppndgaicmgqllakiqarqyic

HoxW is a hypothetical protein in Synechocystis sp. PCC6803. The accession number of the protein in Genbank is BAA17680.1. HoxW's theoretical isoelectric point is 4.93 for a theoretical molecular mass of 17129.53 Daltons. The nucleotide sequence (474bp) of HoxW is as follows and is called SEQ ID NO: 17 in the context of the present invention: atgccaggccaatccaccaagtccactttaatcatcggttacggcaataccctgcggggggacgacggcgtggg gcgttacctagcggaagaaattgctcagcaaaactggccccattgtggagttatttccacccatcaactcaccccag aattggccgaggcgatcgccgctgtggaccgggtaattttcattgatgcccaactgcaggaatcagcaaacgaac catcggtggaagttgtggccttaaaaaccctggaacccaacgaactgtcaggggatttggggcaccggggtaatc ccagggaactcttgaccctggctaaaattctctacggcgttgaggtaaaggcttggtgggtgttgattccggccttcac ctttgattatggagagaaattgtctcccctgaccgcccgggcccaagccgaagccttagcccagatccgccccttg gtattgggggagagataa The amino acid sequence (157 aa) of HoxW is as follows and is named SEQ ID NO: 18 in the context of the present invention:

MPGQSTKSTLIIGYGNTLRGDDGVGRYLAEEIAQQNWPHCGVISTHQLTPELAEAI AAVDRVIFIDAQLQESANEPSVEVVALKTLEPNELSGDLGHRGNPRELLTLAKILY GVEVKAWWVLIPAFTFDYGEQPLEKLSPLTARA

Optionally, according to one embodiment according to the invention, all of the FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW maturation factors are assembled in the form of a concatenary nucleotide sequence (6515bp). This nucleotide sequence comprises the Ncol restriction site (CCATGG) at the start of the sequence and the Avril restriction site (CCTAGG) at the end of the sequence to carry out the cloning in the plasmid pACYCDuet-1. This nucleotide sequence concaténaire is as follows and is called SEQ ID NO: 19 in the context of the present invention: ccaiggcccacgaagttagcctgatggaacagacgctggccattgccattgcgcaggcggaagaccacggggc gagccaaattcaccgtttaacgctgcgcgttgggcagcagtcgggtgttgttgcagatgcattacgctttgcatttgaa gttgttcgccagaacacaatggctgcagaagcacgtctggaaatcgaggaaattccggttacctgtcgttgtcagca ttgtcatgaaaattttcagccggaggattggatatatagatgtccccattgtgaccagattagtcaaaccgttatggac ggcaaacagctggagttagcaagcctggaactgagctaagcatggaaaggaggtcgttattatgtgccagaactg tgggtgtagcgcggttgggaccgttgcgcatagccaccatcaccacggggatggcaactttgcgcatagccatga cgaccacgaccagcaggagcaccaccaccaccacggtaactattcaaaatcaccatcacagcagaccgtaac catagaaccagacagacaaagcatagcaattggccaaggaattctgagcaaaaacgatcgtctggcagaacg caaccgcggctacttccaggccaaaggtctgttagtaatgaatttcctgagcagcccgggagcaggcaaaaccg cactgatcgaaaaaatggttggtgatcgtcagaaagatcatccgaccgcagttattgttggtgatctggcaaccgat aatgatgcacagcgtctgcgtagcgcaggtgcaattgcaattcaggttaccaccggtaatatttgtcatctggaagc agaaatggttgcaaaagcagcacagaaactgga tctggataatattgatcagctgattattgaaaatgttggtaatct ggtttgtccgaccacctatgatctgggtgaagatctgcgtgttgttctgtttagcgttaccgaaggtgaagataaaccg ctgaaatatccggcaacctttaaaagcgcacaggttattctggttaccaaacaggatattgcagcagcagttgatttt gatgcagaactggcatggcagaatctgcgtcaggttgcaccgcaggcacagatttttgcagttagcgcacgtaccg gtaaaggtctgcagagctggtatgaatatctggatcagtggcagctgcagcattatagcccgctggttgatccggca ctggcataagagttgaaaggaggtttcctccatgtgcctggcgttaccggggcaggttgtttcgttaatgccgaactc ggatccgctgttattaaccgggaaagttagctttggtggtattattaaaaccattagcctggcgtatgttccggaagtta aagttggcgattatgttattgttcatgttggttttgctatcagtattgttgatgaagaagcagcacaggagacactgattg atctggccgagatgggcgtttaattcctaaaaggaggttttagccatgaagtacgttgacgaataccgcgacgcgc aggcagttgcccactaccgccaggccattgcccgtgaaattaccaaaccgtggacgctgatggaaatttgtgggg gccagacccatagcatcgttaaatatggtctggatgcattattaccgaaaaacttaaccttaatccacggtccgggtt gtccggtttgtgttacgccgatggaactgattgatcaggcattatggctggcaaaacagccggagattattttttgtagc tttggtgatatgctgcgcgtgccgggtagtggtgcagatctgctgagcattaaagcacaggggggagacgttcgtat agtttattctccgttagattgtctggcgattgcgcgtgaaaatccgaatcgtgaagttgttttttttggtgtgggttttgaaac taccgccccggcaaccgcaatgacactgcatcaggcacgggcccagggtattagcaattttagcttattatgtgcac acgtgttagttccgccggcgatggaagctctgctgggtaacccgaatagcctggttcaagggtttttagcagcaggtc atgtttgtacggttaccggtgagcgggcgtatcagcatattgcagagaaatatcaggttccgatagttattaccggtttt gaaccggttgatattatgcagggtatttttgcatgtgttcgtcagctggagagcgggcagtttacatgtaataatcagta ccggcggtcggttcagccgcagggtaacgcacatgcccagaaaattattgaccaggtttttgaaccggtggatcgt cattggcgtggattaggtcttattccggcctcaggtttaggtttacgtccggcatttgcaccgtgggacgcagcagttaa attcgcaaatctgttacagacaatggctccgacaatgggtgaaaccgtttgtatttctggcgaaattttacagggtcag cgcaaacctagtgattgtcctgcatttggtaccatctgcacccc ggaacaaccgctgggcgcccctatggttagcag tgaaggcgcttgtgccgcctattatcgttatcgtcagcaattaccggaaccggttggtgccgcacgtgtttaattttgca aaggaggtcctgccaatgaacctggtgtgtccggtgccgctggaccgctacccgcaggttttactggcacacggg gggggggggaagctgagtcagcagctgttaaaacagatttttctgccggcgtttggtgcatcagaaaccggtagcc atgatgcagcagtttttaccgcaaatcagagcagcttagcatttacaacagattcctatgttatcaatccgctgttttttcc tggtggtgatattggtagtcttgcagttcatggaaccgttaatgatttagcaatggcaggtgcaacaccgcgttatatta gcgttgggtttattctggaggagggtttaccgatggagacactttggcgtgttgcacaaagcctgggtcaggcagca cagaattgtggagttgaaatattaacaggtgataccaaagttgttgatcgtgggaagggagatggtatttttattaata catcgggtatcggtagtttagatcaccagcaaaccattcatccgaatcaggttcaggttggtgatcgtctgattctgag tggggatttaggacggcatggtatggcaattatggcagttcgtcagggcctggaatttgaaacaaccattgaaagc gatagcgcaccggttcatcgtgaggttcaggctctgctgagcgcagggattccgattcactgtctgcgtgacttaaca cgtggtggtctggcaagcgccgtgaacgaaattgcacaaacctcaggtgttacaatggctctgcgtgaaaccttaat tccggttgaggcggaagttcaagccgcctgtgaactgctgggttttgatcctttatatgttgcgaacgaaggccgtttcc tggccattgttccgc cggaagccgaacagaaaaccgttgaaattctgcagacctttcacccgcaggcgaccgcaa ttggtaccgttaccggcaagagtgcacagaccttaggtctggttagcctggagagtagcataggtgccccacgtctg ttagatatgattagcggagaacaactgccacgtatttgttaagactccaaaggaggctagattaatgctgaaaaccg ttgccattcaggttcaggggcgcgttcagggggttggttttcggccgtttgtttacaccttagcccaggaaatgggtctg aatggctgggttaataactctacgcagggtgcaaccgttgttattaccgcagatgagaaagcaattgcagattttacc gaacgtctgaccaaaacactgccgccaccgggactgatcgaacaactggcagtggaacagctgccgctggaa agctttaccaactttaccattagaccgagtagcgatggtccgaaaaccgcaagcatcctgccagatctgagcacat gtagcgcctgtctgaccgaattatttgatcccagtgatcgtcgttatctgtacccttttattaattgtacccactgtggtcct cgctataccattattgaagcactgccttatgaccgttgtcgtaccacaatggctcgttttcgtcagtgtacggattgtgaa cgtgaatataagcagccgggggaccgccgttttcatgcacagccaaacgcgtgtccgcgttgtggtccgcagctg gcattctggaaccgtcagggtcaagttattgcagaagccaatgaagcactgaatttcgcagtagataatttaaaggt cggtaatattatcgcaatcaaaggtctgggtggttttcatttatgttgtgatgcaaccgattttgaagccgttgaaaaact gcgtttacgtaaacatcgcccggataagccgctggccgttatgtacggtaatctgggtcagattgttgagc attatca gccgaataatttagaagttgagctgctgcagagcgcagcagcacctattgttcttctgaataaaaagaaacagctg attctggttgaaaatattgcaccgggcaatccgcgtgtgggtgttatgctggcatataccccgttacatcacctgttactt aaaaagttaaagaagccgatggttgcaacctccggtaacttagcaggcgaacagatttgtattgacaatattgacg cactgacccgtttacaaaatattgccgacggctttctggttcacgatcgtccgattgtttgtccggttgacgatagtgttgt tcagattgtggcaggtaaaccgttatttttaagaagagcccgcggttatgcaccgcagccgattacccttcctaaacc cacccagaaaaagttattagcaatgggaggccattataaaaataccgttgcaattgcaaagcagaatcaggcata tgtaagccagcatttaggtgatttaaacagcgcaccaacctaccaaaatttcgaagaggcgatagcccatttatcac agctgtatgactttagtccccaggaaattgtcgcagatctgcatccggattactttagccatcagtacgcagaaaacc aagccctgccggtgacgtttgtacagcatcattatgcacatattctggcagttatggcagaacatggtgttatggaag aaagcgttttaggcattgcatgggatggcaccggttatggtatggatggtaccatttggggtggtgaatttctgaaaatt acgcaggggacctggcaaagaattgcacatctgcagccgtttcatctgttagggaatcagcaggcaattaaatatc cgcaccggattgcacttgctctgctgtggccgacattcggggacgattttagcgccgatagtctgggtaattggttaaa ttttaacaacggtttcaagaacaagatcaacagccgtttaaaccaagacttaaataataagaacctgagacaactg tggcagcgtgggcaggcaccgctgacctcgagcatgggcagattatttgatggtatcgcaacactgattggtctgat caatgaagtaacctttgaaggccaggcagcaattgcattagaggcacaaattatgc cgaatctgaccgaagaata ctatccgcttaccctgaataacaaagaaaaaaaactggcagttgattggcgtccgctgattaaagcaattaccacc gaagatcgtagcaaaaccaatctgattgcaaccaaatttcataatagcctggttaatctgattattaccattgcacag cagcagggtattgaaaaagttgcactgggtggtggttgttttcagaattgttatctgctggcaagcaccattaccgcac tgaaaaaagcaggttttagcccgctgtggccgcgtgaactgccgccgaatgatggtgcaatttgtatgggtcagctg ctggcaaaaattcaggcacgtcagtatatttgttaactcaacaaaggaggagctggttatgccgggtcagagcacc aaaagcaccctgattatcgggtacgggaacaccttacgtggggacgatggggtggggcgctacctggcagaag aaatagcacagcagaactggccgcactgtggtgttattagcacacatcagctgaccccggaactggccgaagca attgcagcagtggatagagtgatttttattgacgcccaactgcaggaaagtgcaaatgaaccgtcagttgaagttgtt gccctgaaaaccttagaacccaatgaattaagtggagatctgggtcatcgtggtaatccgcgtgagctgctgacctt agccaaaatattatatggtgttgaagtcaaagcgtggtgggttctgattccggcctttacctttgattatggtgagaaatt atcgcccttaacagcacgtgctcaggccgaagcactggcacagattcgtccgctggttctgggggaacgttaaccf agg

FIG. 3 represents the map of the plasmid pACYCDuet-1 used to construct the vector for the expression of at least one of the maturation factors HypA, HypB, HypC, HypD, HypE, HypF and HoxW by insertion of 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, possessing an origin of replication for E. coli, a chloramphenicol resistance gene, two multiple cloning sites (MCS) containing numerous restriction sites, the T7 promoter and the T7 transcription terminator . It also contains the lad gene encoding a transcription repressor. This repression of transcription can be removed by adding IPTG. FIG. 4 shows the analysis of the digestion of the expression vector pACYCDuet-1 + HypABCDEFHoxW (concatenary sequence SEQ ID NO: 19) by the restriction enzymes Ncol and Hindlll. Two DNA fragments at approximately 6000 bp and at approximately 4000 bp are demonstrated, as expected for such an enzymatic digestion of the expression vector pACYCDuet-1 + HypABCDEFHoxW. This confirms the presence of the sequence HypABCDEFHoxW in the expression vector pACYCDuet-1.

3. Introduction of expression vectors pET26b (+) + HoxH and pACYCDuet-1 + HypABCDEFHoxW in E. coli.

The competent BL21 (DE3) cells of E. coli were used for the recombinant expression of the HoxH subunit of [NiFe] -hydrogenase HoxEFUYH from Synechocystis sp. PCC6803. These cells are used routinely for the production of recombinant protein under the control of the T7 promoter.

The two expression vectors "pET26b (+) + HoxH" and

"PACYCDuet-1 + HypABCDEFHoxW" were introduced into these cells by co-transformation according to the traditional heat shock method well known to those skilled in the art. Transformants showing dual resistance to kanamycin (50 µg / ml) and chloramphenicol (25 µg / ml) were stored on agar medium at 4 ° C.

4. Verification of the presence of DNA sequences of interest in E. coli.

It is possible that certain colonies possess the two plasmids allowing resistance to the selection markers used without however possessing the DNA sequences of interest, namely HoxH and HypABCDEFHoxW. Therefore, it is important to verify the presence of these DNA sequences of interest. An experiment, well known to those skilled in the art and consisting of an extraction of the plasmid DNA from the colony followed by an enzymatic digestion of this plasmid DNA by the restriction enzymes used during the insertion DNA sequences of interest in plasmids, was produced.

Thus, an extraction of plasmid DNA, according to a protocol well known to those skilled in the art, was carried out on a colony exhibiting double resistance to kanamycin and to chloramphenicol.

The enzymatic digestion was then carried out, according to a protocol well known to those skilled in the art. Digestion of the expression vector pET26b (+) + HoxH by restriction enzymes Ndel and Blpl gives two DNA fragments, respectively the plasmid pET26b (+) linearized at 5300 bp and the DNA fragment of interest HoxH at 1500 bp (see figure 5). Digestion of the expression vector pACYCDuet-1 + HypABCDEFHoxW by restriction enzymes Ncol and Hindi II gives two DNA fragments, respectively the plasmid pACYCDuet-1 linearized at 4000 bp and the DNA fragment of interest HypABCDEFHoxW at 6500 bp ( see figure 6). This confirms the presence in the bacterial colony of the sequence HoxH and HypABCDEFHoxW, respectively in the expression vectors pET26b (+) and PACYCDuet-1.

5. Recombinant production and purification by affinity chromatography of HoxH in E. coli.

To obtain the recombinant form of the HoxH subunit of [NiFe] - HoxEFUYH hydrogenase from Synechocystis sp. PCC6803, a colony of E. coli containing the two expression vectors was used to inoculate 4 Erlenmeyer flasks with a volume of 250ml each. The culture medium used is 2XYT medium (16g of tryptone, 10g of yeast extract, 5g of NaCl per liter) supplemented with 100mM FeAmCi, 50mM NiSC, 50mM cysteine, 50mg / ml kanamycin and 25pg / ml chloramphenicol. At an optical density (OD 600nm) of 1.2, 0.2mM IPTG are added to the culture in order to induce the recombinant production of the HoxH subunit at 18 ° C and with stirring (stirring speed) of 200rpm. The production time is 20 hours at 18 ° C. The cells are then harvested by centrifugation (15min at 4500rpm) before starting the purification process.

A purification method has been developed to confirm the production of the recombinant form of the HoxH subunit of [NiFe] - HoxEFUYH hydrogenase from Synechocystis sp. PCC6803. This method (see FIG. 7) makes it possible to purify FloxFI to apparent 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 bond between the immobile phase and the metal ion. The immobilized metal is nickel (Ni). This chromatography allows a very specific separation of proteins containing a polyhistidine tag. After 20 h of recombinant production of HoxH at 18 ° C, 1 liter of E. coli is harvested by centrifugation (15min at 4500rpm). The cells are resuspended in 25ml of lysis buffer (20mM sodium phosphate buffer pH 7.5300mM KCI + 2mI benzonase + 50mI 1M MgCL + 1 pellet composed of a cocktail of protease inhibitors without EDTA) and then lysed with French press. The supernatant (25ml) is recovered by centrifugation (15min at 15000rpm), filtered (0.45miti) and applied to the Ni-NTA column (1 ml) previously equilibrated in the washing buffer (20mM sodium phosphate buffer pH 7.5 300mM KCl + 10mM imidazole). The column is then washed with 35 ml of washing buffer (20 mM sodium phosphate buffer pH 7.5 300 mM KCl + 10 mM imidazole) until the absorbance at 280 nm (representative of the protein concentration) falls back to zero. The elution is then carried out via 5 stages with increasing concentration of imidazole, respectively 50 mM, 100 mM, 150 mM, 200 mM and 250 mM) (see FIG. 7).

SDS-PAGE analysis of the various fractions obtained during the chromatography shows a predominant band close to 54kDa, which is the expected size for the HoxH subunit, in the fractions eluted at a concentration of 100mM, 150mM, 200mM and 250mM. in imidazole (see Figure 8). HoxH also shows an excellent degree of purity since no additional band is visible in the fractions eluted at concentrations of 150 mM, 200 mM and 250 mM of imidazole. This allows us to conclude that HoxH was purified to apparent homogeneity in a single affinity chromatography step. In order to confirm that this is indeed the recombinant form of the HoxH subunit comprising the active site of [NiFe] -hydrogenase HoxEFUYH from Synechocystis sp. PCC6803, immunodetection using an anti-polyhistidine tag antibody was performed (see Figure 9). A molecular weight marker containing a protein labeled with a polyhistidine tag was added to serve as a positive control. The recombinant FloxFI protein is well detected in the load, that is to say the supernatant obtained from the lysis of the cells of E. coli and applied to the Ni-NTA affinity column. The recombinant HoxH protein is also present in the fraction not absorbed by the Ni-NTA column. This indicates that the loading capacity of the Ni-NTA column is exceeded and that a proportion of the recombinant HoxH protein has not been able to bind to it. No signal appears in the “50 mM elution fraction”. The recombinant HoxH protein remains attached to the Ni-NTA column at this low concentration of imidazole. Finally, the presence of the recombinant HoxH protein is confirmed by immunodetection in the 200 mM fraction at the expected size, that is to say close to 54 kDa. This unambiguously confirms the purification of the recombinant HoxH protein in a single affinity chromatography step. When the recombinant HoxH protein is demonstrated by immunodetection in a fraction, a few low intensity bands also appear at molecular weights below 54kDa. This is probably the recombinant HoxH protein partially degraded at the C-terminus. The proportion of degraded HoxH recombinant proteins appears to be minimal because SDS-PAGE analysis does not show the presence of these bands. As a reminder, immunodetection is an extremely sensitive method demonstrating very small amounts of protein. 6. Demonstration of hydrogenase activity in the recombinant protein HoxH

The ability to recombinantly express [NiFej-hydrogenases allows a wide range of possibilities in the optics of producing mutant forms with very different properties from the native enzyme. Figure 10 shows the structure of HoxEFUYH, [NiFe] -hydrogenase from Synechocystis sp. PCC 6803. NADPH is the cofactor of HoxEFUYH in vivo in Synechocystis sp. PCC 6803. Methyl viologen (MV) is used as a redox mediator in the standard in vitro test for [NiFe] -hydrogenase activity. Figure 11 is a schematic representation (gray arrow) of electron transfer and expected interactions between HoxEFUYH and NADPH and / or MV.

It is generally understood by those skilled in the art that the MV can directly transmit electrons to one or more FeS centers bypassing the FMN. However, as shown in Figure 12, it is not known to date whether the only HoxH subunit, containing the active site of [NiFej-HoxEFUYH hydrogenases from Synechocystis sp. PCC 6803, can accept electrons directly from the MV and therefore produce hydrogen in the absence of an additional redox relay.

In the context of the present invention, to test this possibility, the standard test for in vitro activity of [NiFej-hydrogenases in the presence of the recombinant protein HoxH and of MV reduced by sodium dithionite was implemented (see figure 13). HoxH is able to take electrons from previously reduced methyl viologen to combine them with protons present in the buffer to produce hydrogen according to the equation H2 <® 2H ⁺ + 2e which represents the reaction catalyzed by hydrogenase . This standard test for in vitro activity of [NiFej-hydrogenases, according to a protocol well known to those skilled in the art, includes the use of a 2 ml flask closed with a septum, airtight and degassed in the air. 'nitrogen. 1 ml of reaction mixture, composed of 100 mM of sodium dithionite and 10 mM of MV dissolved in 10 mM phosphate buffer pH 6.8 and also degassed with nitrogen, is added to the flask using a syringe. 200 µg of recombinant HoxH proteins are also added to the reaction mixture. The production of hydrogen starts from the moment when the recombinant HoxH proteins are added (moment indicated by an arrow in FIG. 13). The protein content was determined by the Bradford 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). Hydrogen production is continuously monitored using a pre-calibrated hydrogen micro-sensor (Unisense, Aarhus, Denmark).

The activity of the recombinant HoxH protein can therefore be calculated since the quantity of recombinant HoxH protein added (in mg.ml ^_1 ) is known as the rate of hydrogen evolution (pmol H2.min ^_1 ) for this specific quantity of recombinant protein added. This specific activity can, for example, be 0.1 mlH 2 min ^_1 mg ^_1 of enzyme.

Unexpectedly and surprisingly, the fact that the only HoxH catalytic subunit containing the active site of [NiFej-HoxEFUYH hydrogenase from Synechocystis sp. PCC6803 can catalyze the reduction of protons, accepting electrons from a redox mediator (eg, methyl viologen) without the intermediary of additional FeS centers serving as redox relays, has been demonstrated in the context of the present invention. .

In the context of the present invention, the standard test for the in vitro activity of [NiFej-hydrogenases in the presence of the recombinant protein HoxH, of benzyl viologen and of hydrogen was also implemented (see FIG. 14). HoxH is able to take electrons from hydrogen to give them to benzyl viologen, producing protons on the way according to the equation H2 <® 2H ⁺ + 2e which represents the reaction catalyzed by hydrogenase. This standard test for [NiFej-hydrogenase activity in vitro, according to a protocol well known to those skilled in the art, includes the use of a 2 ml flask closed by a septum, airtight and saturated with gas. hydrogen. 2 ml of reaction mixture, composed of 40 pmoles of benzyl viologen dissolved in 50 mM Tris buffer pH 7.6 and also degassed with hydrogen, is added to the flask using a syringe. The experiment is carried out at 40 ° C. 340 pg of recombinant HoxH proteins are also added to the reaction mixture. The consumption of hydrogen and therefore the reduction of benzyl viologen starts the moment the recombinant HoxH proteins are added. The protein content was determined by the Bradford 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 is monitored spectrophotometrically at a wavelength of 578nm. A molar extinction coefficient of 8,600 M ^_1 cm ^-1 was taken into account.

The activity of the recombinant HoxH protein can therefore be calculated since the quantity of recombinant HoxH protein added (in mg.ml ^-1 ) is known as the rate of hydrogen consumption (pmol H2.min ^_1 ), equivalent to the rate of reduction of benzyl viologen for that specific amount of recombinant protein added. This specific activity can, for example, be 0.1 mitioI H2.min ^_1 .mg ^{· 1} of enzyme.

Unexpectedly and surprisingly, the fact that the only HoxH catalytic subunit containing the active site of [NiFej-HoxEFUYH hydrogenase from Synechocystis sp. PCC6803 could catalyze the oxidation of hydrogen, producing protons and donating electrons to a redox mediator (eg, benzyl viologen) without the intermediary of additional FeS centers serving as redox relays, has been shown in the within the scope of the present invention.

In the absence of the expression of the other subunits of the pentamer HoxEFUYH, the problems inherent in the state of the art are at least partially solved: the recombinant protein HoxH is capable on its own of catalyzing the reduction of protons, accepting electrons from a redox mediator (eg methyl viologen) without the intermediary of additional FeS centers serving as redox relays. The HoxH protein is also capable on its own of catalyzing the oxidation of IΉ2, reducing a redox mediator (eg benzyl viologen) without the intermediary of an additional FeS center serving as a redox relay. This exceptional feature demonstrates the great advantage of the present invention, in particular the recombinant production of a single subunit possessing the active site of a [NiFej-hydrogenase and being catalytically active. 7. Recombinant production, purification by affinity chromatography and demonstration of the hydrogenase activity in the recombinant protein HoxH produced recombinantly in E. coli in the absence of exogenous maturation factors.

In a similar manner to Example 3 above, the expression vector pET26b (+) + HoxH is introduced into E. coli (competent cells BL21 (DE3)) by transformation according to the traditional method of heat shock well known from the a person skilled in the art, and that in the absence of the expression vector pACYCDUET-1 + HypABCDEFHoxW. Transformants show resistance to kanamycin (50 µg / ml).

Similarly to Example 4 above, the presence of the DNA sequence of interest HoxH was verified by the sequence of experiments well known to those skilled in the art which are the extraction of plasmid DNA. and enzymatic digestion.

Similar to Example 5 above, the HoxH protein was produced recombinantly in E. coli, but in the absence of the pACYCDUET-1 + HupABCDEFHoxW plasmid encoding the exogenous HupABCDEFHoxW processing factors. The HoxH protein was then purified by affinity chromatography, similar to Example 5 above.

Similar to Example 6 above, the hydrogenase activity of the HoxH protein produced recombinantly in E. coli in the absence of exogenous maturation factors was demonstrated according to the protocol involving the MV and well known to those skilled in the art

Thus, within the framework of the present invention was implemented the standard test for in vitro activity of [NiFe] -hydrogenases in the presence of the recombinant protein HoxH and of MV previously reduced by sodium dithionite (see FIG. 15). . HoxH is able to take electrons from previously reduced methyl viologen to combine them with protons present in the buffer to produce hydrogen according to the equation H2 <® 2H ⁺ + 2e which represents the reaction catalyzed by hydrogenase . This standard test for in vitro activity of [NiFe] - hydrogenases, according to a protocol well known to those skilled in the art, includes the use of a 2 ml flask closed by a septum, airtight and degassed with nitrogen. 1 ml of reaction mixture, composed of 10OmM sodium dithionite and 10mM MV dissolved in 10mM phosphate buffer pH 6.8 and also degassed with nitrogen, is added to the flask using a syringe. 750pg of recombinant HoxH proteins are also added to the reaction mixture. The production of hydrogen starts from the moment when the recombinant HoxH proteins are added (moment indicated by an arrow in FIG. 15). The protein content was determined by the Bradford 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). Hydrogen production is continuously monitored using a pre-calibrated hydrogen micro-sensor (Unisense, Aarhus, Denmark).

The activity of the recombinant HoxH protein can therefore be calculated since the quantity of recombinant HoxH protein added (in mg.ml ^_1 ) is known as the rate of hydrogen evolution (pmol H2.min ^_1 ) for this specific quantity of recombinant protein added. This specific activity can, for example, be 0.1 mitioI H2.min ¹ .mg ¹ of enzyme.

In the context of the present invention, it has been demonstrated that the only HoxH catalytic subunit containing the active site of [NiFe] - HoxEFUYH hydrogenase from Synechocystis sp. PCC6803 can catalyze the reduction of protons, accepting electrons from a redox mediator (eg, methyl viologen) without the intervention of HupABCDEFHoxW maturation factors exogenous to E. coli.

The present invention has been described in relation to specific embodiments, which have a purely illustrative value and should not be considered as limiting. In general, it will be obvious to those skilled in the art that the present invention is not limited to the examples illustrated and / or described above.

The use of the verbs "to understand", "to include", "to include", or any other variant, as well as their conjugations, can in no way exclude the presence of elements other than those mentioned.

The use of the indefinite article "a", "a", or of the definite article "the", "the" or "", to introduce an element does not exclude the presence of a plurality of these elements .

Claims

1. A monomeric polypeptide comprising a single subunit comprising the active site of a protein of the [NiFe] -hydrogenase type, said monomeric polypeptide exhibiting hydrogenase activity.

2. Monomeric polypeptide according to claim 1, characterized in that it is isolated from its natural environment, in particular isolated from a natural protein of the [NiFe] -hydrogenase type.

3. A monomeric polypeptide according to claim 1, characterized in that it is recombinant or heterologous.

4. Monomeric polypeptide according to any one of the preceding claims, characterized in that it is purified.

5. A monomeric polypeptide according to any one of the preceding claims, the amino acid sequence of which, whether or not truncated, has at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99 % identity, relative to the amino acid sequence of SEQ ID NO: 2 and / or relative to the amino acid sequence of SEQ

ID NO: 4.

6. A monomeric polypeptide according to any one of the preceding claims, characterized in that said subunit comprising the active site of a [NiFe] -hydrogenase type protein is the FloxFI subunit of the [NiFe] type protein. -hydrogenase FloxEFUYFI in / from Synechocystis sp. PCC6803.

7. A monomeric polypeptide according to any one of the preceding claims, characterized in that it exhibits a hydrogenase activity of at least 0.01 mioI H2. min ^-1 . rng ^-1 of enzyme, preferably at least 0.05 mitioI H2. min ^-1 . ^_1 mg of enzyme, preferably at least 10 mitioI H2. min ^-1 . ^_1 mg of enzyme.

8. A host cell including a monomeric polypeptide according to any one of claims 1 to 7.

9. Host cell according to claim 8, characterized in that it further includes at least one maturation factor of said [NiFe] type protein - hydrogenase, said at least one maturation factor being endogenous to the host cell and / or exogenous to the host cell, preferably at least one maturation factor of said [NiFe] -hydrogenase type protein chosen from the group consisting of the maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW a) including the respective amino acid sequences each have at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, respectively relative to the amino acid sequences SEQ ID NO : 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; or b) encoded together by a concatenated nucleotide sequence having at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the nucleotide sequence SEQ ID NO: 19, which codes for all of these maturation factors.

10. Host cell according to claim 8 or 9, characterized in that said monomeric polypeptide and / or said at least one maturation factor is / are derived from the expression of at least one undue gene in a vector d expression, said expression vector being induced in said host cell.

11. A host cell including a polynucleotide encoding a monomeric polypeptide according to any one of claims 1 to 7.

12. Host cell according to claim 11, characterized in that it further includes at least one maturation factor of said protein of [NiFe] - hydrogenase type, said at least one maturation factor being endogenous to the host cell and / or exogenous to the host cell, preferably at least one maturation factor of said [NiFe] -hydrogenase type protein chosen from the group consisting of the maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW a) including the respective amino acid sequences each have at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, respectively by relative to amino acid sequences SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; or b) encoded together by a concatenary nucleotide sequence exhibiting at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the nucleotide sequence SEQ ID NO: 19, which codes for all of these ripening factors.

13. Host cell according to claim 11 or 12, characterized in that said monomeric polypeptide and / or said at least one maturation factor is / are derived from the expression of at least one undue gene in a vector d. expression, said expression vector being induced in said host cell.

14. A process for obtaining a monomeric polypeptide exhibiting hydrogenase activity according to any one of claims 1 to 7, said process comprising the following steps:

• a step of genetic modification, carried out in-vivo or in-vitro, of an entity comprising genetic material, for example of a host cell or of an expression vector, to obtain a genetically modified entity, for example a genetically modified host cell or a genetically modified expression vector; A step of incubation of said genetically modified entity, for example of said genetically modified host cell or of said genetically expression vector, to obtain a monomeric polypeptide comprising a single subunit comprising the active site of a protein of type [ NiFe] -hydrogenase, said monomeric polypeptide exhibiting hydrogenase activity.

15. Process for obtaining a monomeric polypeptide exhibiting hydrogenase activity according to claim 14, characterized in that said step of genetic modification, carried out in-vivo or in-vitro, consists of: a) a genetic modification of a host cell and / or an expression vector by including therein an exogenous polynucleotide of which at least a part codes for a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, to obtain a genetically modified host cell and / or a genetically modified expression vector to be incubated during said incubation step carried out under incubation conditions making it possible to ensure expression of said exogenous polynucleotide to produce said monomeric polypeptide; or b) by inducing at least one genetic mutation of a host cell to obtain a genetically modified host cell to be incubated in said incubation step carried out under incubation conditions to produce said monomeric polypeptide.

16. A method of obtaining a monomeric polypeptide exhibiting hydrogenase activity according to claim 15, characterized in that said step of genetic modification of said host cell by inclusion of an exogenous polynucleotide consists of inclusion in said host cell of. an expression vector, in particular a modified expression vector, including said exogenous polynucleotide.

17. Process for obtaining a monomeric polypeptide exhibiting hydrogenase activity according to claim 15 or 16, characterized in that said step of genetic modification of said host cell consists of inclusion in said host cell of said exogenous polynucleotide, at least one of which is part codes for said monomeric polypeptide whose amino acid sequence, truncated or not, has at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60 % identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, by relative to the amino acid sequence of SEQ ID NO: 2 and / or relative to the amino acid sequence of SEQ ID NO: 4.

18. Process for obtaining a monomeric polypeptide exhibiting a hydrogenase activity according to any one of claims 15 to 17, characterized in that said step of genetic modification of said host cell further comprises the inclusion in said host cell of d 'at least one maturation factor of said [NiFe] -hydrogenase-type protein, said at least one maturation factor being endogenous to the host cell and / or exogenous to the host cell, preferably the inclusion of at least one maturation factor of said [NiFe] -hydrogenase type protein chosen from the group consisting of maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW a) whose respective amino acid sequences each have at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably in core at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, respectively relative to the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; or b) encoded together by a concatenary nucleotide sequence exhibiting at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably still at least 60% identity, more preferably still at least 80% identity, more preferably still at least 90% identity, more preferably still at least 95% identity, more preferably still at least 99% identity, relative to the nucleotide sequence SEQ ID NO: 19, which codes for all of these ripening factors.

19. Process for obtaining a monomeric polypeptide exhibiting a hydrogenase activity according to claim 18, characterized in that said at least one maturation factor is obtained from the expression of at least one undue gene in an expression vector. , said expression vector being induced in said host cell.

20. Process for obtaining a monomeric polypeptide exhibiting hydrogenase activity according to any one of claims 14 to 20, characterized in that it comprises a subsequent step of isolation and / or purification of said monomeric polypeptide.

21. Use of a monomeric polypeptide according to any one of claims 1 to 7 or of a monomeric polypeptide obtained according to the process according to claims 14 to 20, said monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, said monomeric polypeptide exhibiting hydrogenase activity and being present or not in a cell, to produce or consume hydrogen by incubation of said polypeptide monomeric under incubation conditions to ensure production or consumption of hydrogen or to coat a surface, in particular to coat a surface of an electrical conductor, for example to coat a surface of an anode or a cathode.