BE1027221B1 - Monomeric polypeptide exhibiting hydrogenase activity, in particular recombinant monomeric polypeptide exhibiting hydrogenase activity - Google Patents

Abstract

The present invention relates to a monomeric polypeptide comprising a single subunit comprising the active site of a protein of [NiFe] -hydrogenase type, said monomeric polypeptide exhibiting 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, on a host cell including this monomeric polypeptide and on 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 encouraged the emergence of clean and renewable technologies. Hydrogen (Hz) is considered a promising renewable energy carrier due to its high energy content and the lack of pollution when used in fuel cells. Hz production 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 biotechnology production methods Hz, 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 which catalyze the chemical reaction: 2 H * + 2e ’+ Ha. The reaction is reversible and its direction

? BE2019 / 5783 depends on the redox potential of the components capable of interacting with the enzyme. In the presence of Hz 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 Hz. Hydrogenases are widely used among microorganisms because many of them use hydrogen as a carrier 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 (Pt 9): 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.

[FeFe] -hydrogenases are monomeric enzymes with a highly conserved catalytic center. It is a bi-nuclear iron site linked to a [4Fe-45] center by a cysteine bridge. The non-protein ligands, cyanide (CN) and carbon monoxide (CO), are bonded 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 electron transfer 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 Hz molecules. Three chaperone proteins named HydE, HydF and HydG are known to be necessary for the correct assembly of [FeFe] -hydrogenases (Vignais et al.

2001. Classification and phylogeny of hydrogenases. FEMS Microbiol Rev 25: 455-501). These enzymes are 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 [FeFe] -hydrogenases thermodynamically promote the reoxidation of the cofactor (ferredoxin or NADH) and thus generate H2. These enzymes are therefore generally involved in the elimination of excess redox power in the cell in order to avoid, for example, a cessation of 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-Like1-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 hetero-multimer contain several medial and distal FeS centers, which conduct electrons between the active site and the physiological electron donor or acceptor. The group [4Fe-45] located near the active site is considered to be 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 heteromimer. Indeed, the maturation of [NiFe] - hydrogenases follows a complex pathway involving at least seven helper proteins (HypA-F and an endopeptidase) (Vignais and Billoud. 2007.

Occurrence, classification, and biological function of hydrogenases: an overview.

Chem Rev 107: 4206-4272). Another characteristic of the active site of [NiFe] - hydrogenases is the strong affinity for hydrogen. These enzymes therefore act mainly by consuming H2 in the host microorganism, even if some [NiFe] -hydrogenases have a good capacity for hydrogen production. [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 is in good agreement with the groups derived from physiological functions (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

> BE2019 / 5783 production of hydrogen at 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 O2 because the enzyme is irreversibly damaged after exposure to small concentrations of O2 (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 damage caused by oxygen. In addition, [NiFe] - hydrogenases are reversibly inactivated by O2. Some microorganisms, such as Ra / stonia 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 catalytic activity, that is, 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 proteins responsible for the interaction in vivo with the substrate (NADH, flavodoxin or reduced ferredoxin). HoxFU contain the medial and distal FeS centers carrying electrons to HoxYH. The function of HoxE is unclear, but it may be an anchoring subunit in the membrane.

Mutational analysis of the maturation pathway identified seven essential maturation factors, called HypA, HypB, HypC, HypD, HypE, HypF and HoxW (Hoffmann et al. 2006. Mutagenesis of hydrogenase accessory genes of Synechocystis sp.

PCC 6803. Additional homologues 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). During the assembly of HoxHY, the HoxH subunit is treated with the specific protease HoxW, then 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 have been 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 enzyme, sensitive to oxygen, is therefore inactive under aerobic conditions.

The precise physiological function is still debated, but HoxEFUYH is said to function as a safety valve that dissipates excess electrons under unfavorable redox conditions, 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 HoxEFUYH in the presence of oxygen is complete and almost instantaneous.

However, HoxEFUYH 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, Mcintosh et al. 2011. The [NiFe] -hydrogenase of the cyanobacterium Synechocystis sp.

PCC 6803 works bidirectionally with a bias to H2 production.

J Am Chem Soc 133: 11308-11319). Several purification protocols are available (Schmitz et al. 2002. HoxE - a subunit specific 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 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). The ability to efficiently catalyze the production of hydrogen, and with limited sensitivity to oxygen, has 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 recombinant production systems. 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. In fact, 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 difficult (English et al. 2009. Recombinant and in vitro expression systems for hydrogenases: new frontiers in basic and applied studies for biological and synthetic H2 production. Dalton Trans 9970-9978).

First, the difficulty arises from the complexity and specificity of the active site [NiFe] assembly process, which 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 a precise sequence is necessary to achieve enzymatic activity, which can be difficult to control and ensure 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 [NiFe] -hydrogenase has been produced in E. coli (Kim et al. 2011. Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe] - hydrogenase in Escherichia coli. J Biotechnol 155: 312-319, Maier et al. 2015. Identification, cloning and heterologous expression of active [NiFe] -hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J Biotechnol 199: 1-8, Schiffels et al . 2013. An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe] -hydrogenase from Cupriavidus necator in Escherichia coli. PLoS One 8: e68812, Weyman et al. 2011. Genetic analysis of the Alteromonas macleodii [ NiFe] -hydrogenase FEMS Microbiol Lett 322: 180-187).

In particular, the work of Sun and his collaborators (Sun et al. 2010. Heterologous expression and maturation of an NADP-dependent [NiFe] - hydrogenase: a key enzyme in biofuel production. PLoS One 5: e10526) allowed the expression of [NiFe] -hydrogenase from Pyrococcus furiosus in anoxia in E. coli by means of 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, PFO892, PF0893 and PF0894. After purification, this tetrameric recombinant enzyme was found to be functionally similar to the native enzyme purified from P. furiosus.

HoxEFUYH 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 HoxEFUYH is due to inhibition of the activity of endogenous H2-consuming hydrogenases 1 and 2 in E. coli.

We should also mention Wells and his collaborators who introduced the HoxEFUYH 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.

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 site

H BE2019 / 5783 active of a [NiFe] -hydrogenase-type protein, said monomeric polypeptide exhibiting hydrogenase activity.

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.

Preferably, according to the invention, said monomeric polypeptide consists of a single subunit comprising the active site of a protein of the [NiFe] -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' at least partially freeing from the obstacles hindering the use of [NiFe] -hydrogenases for hydrogen production, while guaranteeing a hydrogenase activity of at least 0.05 umol Hz. min ”. mg ”.

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 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 allows to increase the reproducibility during the process of obtaining the [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 the 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. Heterologous expression and maturation of an NADP-dependent [NiFe] -hydrogenase: a key enzyme in biofuel production. PLoS One 5: e10526, Wells et al. 2011. Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase. Metab Eng 13: 445-453). The 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 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 mass activity of the enzyme (number of catalytically active entities per mg of total protein) being thus obtained.

Better integration and therefore densification of the enzymatic catalyst can also be achieved, for example, during electrochemical use 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 relative to an interface, by example compared to a carbon electrode. This characteristic advantageously makes it possible, for example, to improve the stability of the bond 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 in 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 suitable for the production by HZ catalysis (standard test for in-vitro production of H2 by hydrogenases using methyl-viologen as redox mediator; electrochemical implementation), without the presence of the proximal FeS center or any other redox relay. This is quite surprising since such a far-reaching simplification of the heteromimeric enzyme has never been proposed or suggested by the state of the art. On the contrary, all of the work carried out and therefore all of the state of the art considers that the center [4Fe-45] located near the active site is essential and indispensable for the hydrogenase activity (Albracht. 1994 . Nickel hydrogenases: in search of the active site. Biochim Biophys Acta 1188: 167-204) but also that the reduction in the number of subunits is neither favorable to the activity nor to the stability of the hydrogenase. All the more advantageously, whereas only a limited production of H2 is currently obtained with the known recombinant [NiFe] -hydrogenases (Sun et al. 2010. Heterologous expression and maturation of an NADP-dependent [NiFe] -hydrogenase : a key enzyme in biofuel production. PLoS One 5: e10526, Schiffels et al. 2013. An innovative cloning platform enables large- scale production and maturation of an oxygen-tolerant [NiFe] -hydrogenase from Cupriavidus necator in Escherichia coli. PLoS One 8: e68812, Maier et al. 2015. Identification, cloning and heterologous expression of active [NiFe] -hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J Biotechnol 199: 1-8), it has been shown that the monomeric polypeptide according to the invention has 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.

By way of 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, Synéchocystis 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. less 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.

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. Preferably, according to the invention, said monomeric polypeptide exhibits a hydrogenase activity of at least 0.05 µmol Hz. min ”. mg ”, preferably at least 10 µmol Ha. min *. mg ”.

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 a hydrogenase activity. .

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 coli, of the genus Bacillus (such as for example Bacillus subtilis ), of the genus Streptomyces (such as for example Streptomyces coelicolor), of the genus Synechocystis (for example Synéchocystis sp. PCC6803), of the genus Synéchococcus (such as for example Synéchococcus WH8102), or any other prokaryotic cell, a eukaryotic cell for example of the genus Chlamydomonas (such as for example Chlamydomonas reinhardti), 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 monomeric polypeptide included in said host cell may itself but not necessarily be derived from the expression of a gene included in an expression vector, for example in a plasmid inserted into said host cell.

Advantageously, according to the invention, said monomeric polypeptide included 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 for said [NiFe] -hydrogenase type protein, preferably one or more maturation factors for said [NiFe] -hydrogenase type protein chosen from, group consisting of the maturation factors HypA, HypB, HypC, 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% of 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 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.

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 a hydrogenase activity. .

Preferably, according to the invention, said polynucleotide encoding a monomeric polypeptide has 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% identity, respectively relative to the sequences, relative 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 WH8102) 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 included in said host cell may itself but not necessarily be included 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 polynucleotide included in said host cell has a truncated or non-truncated amino acid sequence having 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% of 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 factors for maturation of said protein of [NiFe] -hydrogenase type, preferably one or more factors for maturation of said protein of [NiFe] -hydrogenase type chosen from the group consisting of the maturation factors HypA, HypB, HypC, 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 concatenated 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.

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 a 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 the [NiFe] type - hydrogenase; and e incubating said modified expression vector under incubation conditions allowing 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 modifying an expression vector, 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 one part of which codes for said monomeric polypeptide, the amino acid sequence of which is truncated or not present in the less 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 maturation factors HypA, HypB, HypC, HypD, HypE, HypF and HoxW 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 concatenated 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 the maturation factor may itself but not necessarily be included in an expression vector, 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.

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 incubation of 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 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, 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 group amine of the next amino acid.

The term "polypeptide" also includes molecules which contain more than one polypeptide, the latter linked together, for example by disulfide bonds, 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, in particular in a host cell, and whose production 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 that contain only one polypeptide, that is, without interaction with other polypeptides, regardless of the nature of the link. 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 monomeric polypeptide is capable of catalyzing the reversible reaction H2> 2H * + 2e.

By the terms "active site" is meant, 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 the hydrogenase activity. The portion 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, thanks to its own origin of replication, and not essential. to the survival of the cell. The plasmid can also contain other sequences of interest, such as for example a gene encoding a selection factor (resistance to an antibiotic, etc.), a multiple cloning site allows the addition of polynucleotide, and / or sequences regulation of 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 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 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 [NiFe] -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 purification, such as for example affinity chromatography or a molecular sieve. For example, a polynucleotide, a polypeptide, or H2 can be purified. Preferably, a substance is purified when it represents at least 60% relative to the other components which are associated with it, preferentially 75% relative to the other components which are associated with it, preferentially 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% with respect 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 sequences of nucleotides or amino acids must however remain the same.

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 expression vector pET26b (+) + HoxH 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 the pACYCDuet-1 + HypABCDEFHoxW expression vector by the restriction enzymes Ncol and Hindlll.

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 plasmid DNA from 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 the 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 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 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 or not the active site of the recombinant protein HoxH can accept electrons directly from the MV and therefore produce hydrogen in the absence of redox relay additional.

Figure 13 illustrates the production of hydrogen resulting from the addition of the recombinant protein HoxH to a reagent containing methyl viologen previously reduced with sodium dithionite in the absence of oxygen. 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 made, a moment concomitant with the increase in the level of dissolved hydrogen detected by the micro-sensor.

Examples

1. Construction of a HoxH expression vector

1.1. "Single" sequence of HoxH HoxH corresponds to the large subunit comprising the active site of [NiFe] -hydrogenase HoxEFUYH in Synechocystis sp. PCC6803. The accession number of the protein 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 named SEQ ID NO: 1 in the context of the present invention:

atgtctaaaaccattgttatcgatcccgttacccggattgaaggccatgccaaaatctccattitcctcaacgacc agggcaacgtagatgatgttcgtttccatgtggtggagtatcggggttttgaaaaattttgcgaaggtcgtcccat gtgggaaatggctggtattaccgcccgtatttgeggcatttgtccggttagccatctgctctgtgcggctaaaacc ggggataagttactggcggtgcaaatccctccagccggggaaaaactgcgccgtttaatgaatttagggcaa attacccaatcccacgccctaagttttttccatctcagcagtcctgattttctgettggttgggacagtgatcccgcta ctcgcaatgtgtttggtttaattgctgctgaccccgatttagctagggcaggtattcggttacggcaatttggccaa acggtaattgaacttttgggagctaaaaaaatccactctgcttggtcagtgcccggtggagtccgatcgccgttg tcggaagaaggcagacaatggattgtggaccgtttaccagaagcaaaagaaaccgtttatitagccttaaattt gtttaaaaatatgttggaccgcttccaaacagaagtggcagaatttggcaaatttccctccctatttatgggcttag ttgggaaaaataatgaatgggaacattatggcggctccctgcggtttaccgacagtgaaggcaatattgtcgcg gacaatctcagtgaagataattacgctgattttattggtgaatcggtggaaaaatggtcctatttaaaatttcccta ctacaaatctctgggttatcccgatggcatttatcgggttggtccccttgcccgccttaatgtttgtcatcacattggc at accccggaagcagaccaagaattagaagaatatcggcaacgggctggaggtgtggccacgtcctctttetttt cattacgcccgcttggtggaaattcttgcctgtttagaagccatcgaattgttaatggctgaccctgatattttgtc caaaaattgtcgagctaaggcagaaattaattgtaccgaagcggtgggagtgagcgaagcaccccggggta ctttattccaccattacaagatagatgaagatggtctaattaagaaagtgaatttgatcattgccacgggcaaca ataacttagccatgaataaaaccgtggcccaaattgccaaacactacattcgcaatcatgatgtgcaagaag ggtttttaaaccgggtggaagcgggtattcgttgttatgatccctgccttagttgttctacccatgcagcgggacaa atgccattgatgatcgatttagttaaccctcagggggaactaattaagtccatccagcgggattaa The amino acid sequence (474 aa) of HoxH is as follows and is called SEQ ID NO: 2 in the context of the present invention:

MSKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEKFCEGRPMWE - MAGITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLRRLMNLGQITQSHAL SFFHLSSPDFLLGWDSDPATRNVFGLIAADPDLARAGIRLRQFGQTVIELLGAK KIHSAWSVPGGVRSPLSEEGRQWIVDRLPEAKETVYLALNLFKNMLDRFGQTE VAEFGKFPSLFMGLVGKNNEWEHYGGSLRFTDSEGNIVADNLSEDNYADFIG ESVEKWSYLKFPYYKSLGYPDGIYRVGPLARLNVCHHIGTPEADQELEEYRQR AGGVATSSFFYHYARLVEILACLEAIELLMADPDILSKNCRAKAEINCTEAVGVS EAPRGTLFHHYKIDEDGLIKKVNLIATGNNNLAMNKTVAQIAKHYIRNHDVQEG

FLNRVEAGIRCYDPCLSCSTHAAGQMPLMIDLVNPQGELIKSIQRD FIG. 1 represents the map of the plasmid pET26b (+) used to construct the HoxH expression vector by insertion of SEQ ID NO: 3 in pET26b (+). pET26b (+) is a 5360bp plasmid with an origin of replication for E. coli, a kanamycin resistance gene, a multiple cloning site (MCS) containing numerous restriction sites, the T7 promoter and the transcription terminator 77. It also contains the gene / ac / encoding 40 for a transcription repressor. This repression of transcription can be removed by adding IPTG.

1.2. “Optimized” HoxH Sequence Optionally, according to one embodiment according to the invention, the sequences SEQ ID NO: 1 and SEQ ID NO: 2 are optimized for use of codons in E. coli. In particular, the Ndel (catatg) and BlpI (gcinagc) 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 polyhistidine tag near the N-terminal end of the protein). The optimized nucleotide sequence (1453bp) is as follows and is called SEQ ID NO: 3 in the context of the present invention: catatgagccaccaccaccaccatcacaaaaccatcgtcatcgacccagtcacccgcatcgaaggccacg ccaaaattagcatttttctgaacgaccagggcaacgtcgacgacgtccgctttcacgttgttgaataccgtggctt cgaaaaattttgtgaaggtcgtccgatgtgggaaatggccggtatcacggcacgtatttgtggaatttgtccggt gagccatctgctgtgtgccgcaaaaaccggagataaactgctggcagtgcagattccgccggcaggtgaaa aactgcgtcgtctgatgaatctgggtcagattacacagtcgcatgcactgtctttctttcatctgagtagcccagatt ttctgctggggtgggatagcgacccggcaacacgtaatgtgtttggtctgattgcggctgatccggatctggcgec gtgccggtattcgtctgcgtcagtttggtcagacagttattgagctgctgggggcgaaaaagattcatagtgcatg gtctgtgccgggtggtgttcgtagtccgctgagtgaagaaggtcgtcagtggattgttgatcgtctgccggaggc aaaagaaacggtctatctggcactgaatctgtttaaaaatatgctggatcgtttccagacagaagttgcagaatt tggaaaatttccgtcactgtttatgggtctggttggtaaaaataatgaatgggaacactatggtggtagcctgcgtt tcacggactctgaaggtaatattgttgcggataatctgagcgaagacaattatgcagattttatcggtgaaagtgt ggaaaaatggagctatctgaaatttccgtattaca aaagcctgggctatccggatgggatctaccgtgttggac cgctggcacgtctgaacgtttgtcatcatattggtaccccggaagcagatcaggaactggaagaatatcgtca gcgtgcgggtggtgttgcgactagcagctttttttatcattatgcacgtctggttgaaattctggcctgtctggaggc aattgaactgctgatggcagatcctgatattctgtctaaaaattgtcgtgcaaaagcagaaattaactgtaccga ggcagttggtgttagtgaggcgccgcgtggtaccctgtttcatcactataaaattgacgaagatggtctgattaaa aaggttaatctgattatcgcaaccggtaacaataatctggcaatgaataaaaccgttgcacagattgcaaaac actacattcgcaaccacgatgttcaggaagggtttctgaatcgtgtagaagccggcattcgctgttatgatccgt gtctgagctgtagcacccatgcagcaggtcagatgcctctgatgattgacctggttaatccgcagggtgagctg attaaaagcattcagcgtgattaagcigage The amino acid sequence (480 aa) is optimized and the following is called SEQ ID NO: 4 in the context of the present invention:

MSHHHHHHKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEKFCEG RPMWEMAGITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLRRLMNLGQIT QSHALSFFHLSSPDFLLGWDSDPATRNVFGLIAADPDLARAGIRLRQFGQTVIE LLGAKKIHSAWSVPGGVRSPLSEEGRQWIVDRLPEAKETVYLALNLFKNMLDR FQTEVAEFGKFPSLFMGLVGKNNEWEHYGGSLRFTDSEGNIVADNLSEDNYA DFIGESVEKWSYLKFPYYKSLGYPDGIYRVGPLARLNVCHHIGTPEADQELEE YRQRAGGVATSSFFYHYARLVEILACLEAIELLMADPDILSKNCRAKAEINCTEA VGVSEAPRGTLFHHYKIDEDGLIKKVNLIIATGNNNLAMNKTVAQIAKHYIRNHD

VQEGFLNRVEAGIRCYDPCLSCSTHAAGQMPLMIDLVNPQGELIKSIQRD Plasmid pET26b (+) is used to construct the HoxH expression vector by insertion of 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 maturation factors are considered within the scope of the present invention: HypA, HypB, HypC, HypD, HypE, HypF and HoxW.

HypA is a [NiFe] - HoxEFUYH hydrogenase expression / formation protein in Synechocystis 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: atgcacgaagttagtctgatggagcaaactttggcgatcgccattgcccaggcggaagaccatggagccagc caaatccatcgtttaaccctgcgggtggggcaacagtctggggtggtggccgatgccctacggtitgcgtttgaa gtggtgcgacaaaataccatggccgccgaggcgagattggaaattgaagaaattcccgttacctgtegttgcc aacactgccacgaaaattttcagccagaggattggatttaccgctatecccactgcgaccagattagecaaac agtaatggatggcaaacagttggaactagcatccctagaactgagttga the amino acid sequence (113 aa) of Hypa is as follows and estnommée SEQ ID NO: 6 in the context of the present invention:

MHEVSLMEQTLAIAIAQAEDHGASQIHRLTLRVGQQSGVVADALRFAFEVVRQ NTMAAEARLEIEEIPVTCRCQHCHENFQPEDWIYRCPHCDQISQTVMDGKQL

ELASLELS HypB is a [NiFe] - HoxEFUYH hydrogenase expression / formation protein in Synechocystis sp. PCC6803. The protein's accession number 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: atgtgccaaaactgcggttgtagtgcggtgggaaccgttgcccatagccaccatcaccatggcgatggaaattt tgcccacagccatgatgaccatgaccagcaagaacatcatcaccaccatggcaactacagcaaaagtcca agtcagcagactgtgaccattgaacccgatcgccagtccattgccattggccaaggcattctcagcaagaatg accgcctagcggaaaggaatcggggctatttccaggctaagggcttactggtgatgaatttcctctcttctcccg gagccggtaaaactgctctgatcgaaaaaatggtcggcgatcgacaaaaagaccatcccaccgccgtcatt gtgggggatitagccaccgataacgatgcccaacgtctccgcagtgccggggcgatcgccattcaggtcacc acaggaaatatttgccatctggaagcggaaatggtggccaaggcggcccaaaagttagatttagacaatatc gatcaattgatcattgaaaatgttggtaatttggtttgccccaccacctatgatctaggggaagatttacgggtcgt attattttccgtcacagaaggggaggataaaccccttaaatatcccgccaccttcaaatcagcccaggttattitta gtcaccaaacaggacattgccgccgcagtggattttgatgcagagctggcttggcaaaacctacggcaagtg gccccccaagcccaaatttitgcagtgtctgcccgcacggggaaaggattgcagtcctggtatgagtatttggat caatggcaactccaacactattcgccgttggitgatccagcattggcctaa the 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:

MCQNCGCSAVGTVAHSHHHHGDGNFAHSHDDHDQQEHHHHHGNYSKSPS - QQTVTIEPDRQSIAIGQGILSKNDRLAERNRGYFQAKGLLVMNFLSSPGAGKT ALIEKMVGDRQKDHPTAVIVGDLATDNDAQRLRSAGAIAIQVTTGNICHLEAEM VAKAAQKLDLDNIDQLIENVGNLVCPTTYDLGEDLRVVLFSVTEGEDKPLKYP ATFKSAQVILVTKQDIAAAVDFDAELAWQNLRQVAPQAQIFAVSARTGKGLQS

WYEYLDQWQLQHYSPLVDPALA HypC is a [NiFe] - HoxEFUYH hydrogenase expression / formation protein in Synechocystis 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 (231bp) of HypC is as follows and is called SEQ ID NO: 9 in the context of the present invention: atgtgtctagccctacctggccaggttgtcagtttaatgcccaactccgatcccctgttactgacgggaaaggtta gctttgggggcatcattaaaaccattagccttgcctacgtacccgaggttaaggtgggggattacgtgattgtcc atgtgggctttgccattagcattgtggacgaagaggcggcccaggaaactttgatagacttggcagaaatggg agtttaa the amino acid sequence (76 aa) of HypC is as follows and is called SEQ ID NO: 10 within the scope of the present invention:

MCLALPGQVVSLMPNSDPLLLTGKVSFGGIIKTISLAYVPEVKVGDYVIVHVGF

AISIVDEEAAQETLIDLAEMGV HypD is a [NiFe] - HoxEFUYH 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: atgaaatacgttgatgaatatcgggatgcccaggcggtggcccattaccgtcaggcgatcgccagggagata accaaaccttggacgctgatggagatttgcggcggccagacccacagcattgtcaaatatggcttggatgcttt gttgccgaagaatttgactctgatccatggtcccggetgtectgtgtgegtcactccgatggaattaattgaccag gctttgtggttagctaagcaaccggagatcattttitgttcctttggcgatatgttgegggtgcccggcagtggggc ggatttgctgagcattaaagcccagggcggcgatgtgcgcattgtctattctcctttggattgtttggcgatcgcca gggagaatcctaatcgggaagtggtatttttcggagtaggttttgaaactacagcccctgccacggccatgact ctccaccaagctagggcccagggaattagcaatttcagtttactttgcgcccatgtattggtgcccccggctatg gaggctttattaggcaatcccaattccctcgtgcagggctttttggcggcagggcatgtctgtacggtgaccggg gaaagggcctatcaacatatcgctgaaaaataccaagtacccattgtcatcactggctttgaacctgtggatatt atgcagggcatctttgcctgtgtgcgccaactggagtcgggacaattcacctgcaacaatcaatatcggcgatc ggtccaaccccagggcaatgcccatgctcagaaaattattgaccaagtgtttgagccagtcgatcgccattgg cggggtttgggattaattccggccagcggtttgggtita aggccagcatttgccccctgggatgccgcagttaaa ttcgccaatttattgcaaaccatggccccaacgatgggagaaacagtgtgtattagcggggaaattttacaggg acaacggaagcccagcgattgtccagcctttggtactatctgcaccccagaacaacccttgggggetcccatg gtttcctcggaaggagcctgtgccgcctattaccgttatcgccaacaattaccggaaccagtgggagcggcca gagtttag The amino acid sequence (374 aa) of HYPD is as follows estnommée and SEQ ID NO: 12 in the context of the present invention:

MKYVDEYRDAQAVAHYRQAIAREITKPWTLMEICGGQTHSIVKYGLDALLPKN LTLIHGPGCPVCVTPMELIDQALWLAKOPEIFCSFGDMLRVPGSGADLLSIKA QGGDVRIVYSPLDCLAIARENPNREVVFFGVGFETTAPATAMTLHQARAQGIS NFSLLCAHVLVPPAMEALLGNPNSLVQGFLAAGHVCTVTGERAYQHIAEKYQV PIVITGFEPVDIMQGIFACVRQLESGQFTONNQYRRSVQPQGNAHAQKIIDQVF EPVDRHWRGLGLIPASGLGLRPAFAPWDAAVKFANLLQTMAPTMGETVCISG EILQGORKPSDCPAFGTICTPEQPLGAPMVSSEGACAAYYRYRQQLPEPVGA

ARV HypE is a [NiFe] - HoxEFUYH 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: gtgaacttagtctgtcccgttcccettgatcgttatccccaggtactgttagcccacggcggcggcggtaagttga gccaacaattacttaagcaaatttttttaccggcctttggcgcttctgaaacgggtagtcatgatgcggcggttttta ctgccaaccaaagttctttagctttcaccaccgactcctatgtgatcaatcccctcttitttcctgggggcgatattgg ttctttggcagtccacggcaccgttaatgacctagccatggccggcgcaacccctcgctatatcagcgttggtttt atcctcgaagaaggattgcccatggagaccctctggcgggtggcccaatccctagggcaagcggcccaaa actgtggggtggaaattcttaccggtgataccaaagtggtggaccggggtaagggagacggcattttcatcaa caccagcggcattggttccctcgaccatcaacaaactatccatcccaatcaggtacaggtaggcgatcgccta attttgagcggtgatttgggacgtcatggcatggccattatggcagtgcgccaaggattagaatttgaaaccacc attgaaagtgattcggccccggttcacagagaagtgcaggcattattgtcggcagggatcccaatccattgtct gcgggatttaaccagggggggattagccagtgcggttaatgaaattgcccaaacttccggggtaaccatggct ttacgagaaacgttaatcccggtggaggccgaagtacaagccgcctgtgaactgttgggttttgaccccctctat gtggccaatgagggaagattcctggccattgtgcccccg gaagcagaacagaagaccgtggaaattttgca aactttccatccccaagctacggcgatcggtacagtaacaggcaaaagtgcacaaaccttggggttagtcagt ttggaaagttccattggtgccccccggttgctagacatgatcagtggggagttgctagacatgatcagtggggagttgctagacatgatcagtggggagttgctagacatgatcagtggggag5 present in the following sequence of IDEgccttg is the following sequence of the IDEQtactcttgatcagtgggg5 34 is the sequence of the invention in the following sequence of IDEgccttgatgatcagtggg5 34 (14) in the following sequence of the IDGccttgatgatcagtggg5 14 (14) in the following sequence of the IDGccttgatgatcagtggg5 34 (14) in the ID 14) 14 (14) of the invention SEQ)

MNLVCPVPLDRYPQVLLAHGGGGKLSQQLLKQIFLPAFGASETGSHDAAVFT ANQSSLAFTTDSYVINPLFFPGGDIGSLAVHGTVNDLAMAGATPRYISVGFILE EGLPMETLWRVAQSLGQAAQNCGVEILTGDTKVVDRGKGDGIFINTSGIGSLD HQQTIHPNQVQVGDRLILSGDLGRHGMAIMAVRQGLEFETTIESDSAPVHREV QALLSAGIPIHCLRDLTRGGLASAVNEIAQTSGVTMALRETLIPVEAEVQAACEL LGFDPLYVANEGRFLAIVPPEAEQKTVEILQTFHPQATAIGTVTGKSAQTLGLV

SLESSIGAPRLLDMISGEQLPRIC HypF is a [NiFe] - HoxEFUYH hydrogenase expression / formation protein in Synechocystis sp. PCC6803. The protein's accession number in Genbank is BAA10154.1. The theoretical isoelectric point of HypF is 8.19 for a theoretical molecular mass of 85358.25 Daltons. The nucleotide sequence (2304bp) of HypF is as follows etestnommée SEQ ID NO: 15 in the context of the present invention: atgttaaaaaccgttgccatacaggtccagggaagggtgcaaggagtgggttttegtccctttgtttatacccttg cccaggaaatgggactgaatggttgggtgaataattccactcaaggagctaccgttgtcattaccgccgacga aaaggcgatcgccgactttacggagagattaacgaagacattacctccccctggtttgattgaacaattagccg ttgaacagttaccgctggaaagttttactaactttactatccgccccagtagtgatggccctaaaactgcgagtatt ttacccgatttatccacttgttccgcctgcttaacagaactatttgaccctagcgatcgccgttatctttacccctttatt aactgtacccattgcggtccccgctacaccattattgaagccctaccttacgaccgttgtcgtaccaccatggct aggtttcgccaatgtaccgactgtgaaagggaatataagcaaccaggcgatagacgcttccatgcccaacct aatgcctgtcctcgetgtggcccccaactggctttttggaaccgacaaggccaagtaattgcagaagcaaatg aagctttaaactitgctgtagataatttaaaagtcggcaatattatcgctattaaaggcttaggtggcttccatttgtg ttgtgatgccactgattttgaagctgtggaaaaattaagattaaggaaacatcgaccggataaacctttggcggt aatgtatggtaatcttggtcaaattgtggagcattaccaacctaataatctagaagttgaattgttacaaagtgcc gccgcccctattgtgttattaaacaaaaaaaa acaattaatittggtggaaaatattgccccaggcaacccccg agtcggcgtaatgttagcctatactcctttgcatcacttattactaaaaaaattaaagaaacccatggtagctacc agtggtaacttagctggggagcaaatttgcattgataatattgacgctttaacccggttacaaaatattgctgacg gttttctcgttcatgatcgcccgattgtttgtccagtggatgattccgttgtccaaatagtagctgggaagccattattt ttgcgtcgagcceggggttacgctcctcaacccattactttaccaaagcctactcaaaaaaaactattggcgat gggaggtcattataaaaatacagtggcgatcgccaaacaaaatcaagcttacgtcagccaacatttgggcga tttgaattctgctcccacctaccaaaattttgaagaagccattgcccatttaagccagctatacgatttctctcccca ggaaattgttgcagatttacaccctgattatticagtcatcaatatgctgaaaaccaagctttgcctgtcacttttgtg cagcatcactatgctcatattttagcggttatggcggaacatggagttatggaggagtccgtgttaggtattgettg ggatggcactggctacggcatggacggtactatttgggggggagaatitttaaaaatcacccaaggtacttggc agagaattgctcatctacaaccatitcatitattaggtaatcaacaagccattaaatatccccatcggattgctttg gcgttgttatggcccactittggtgatgatitttctgctgattctttaggaaattggttgaatticaataatgggtitaaaa acaagataaacagcaggttaaatcaggatctaaacaacaaaaatttacgtcaactttggcaacgagggcaa gcaccgctcacttcgagtatgggaagatt atttgacggtattgcgacactgataggattgattaacgaagtaactt ttgaaggtcaggcggccatagctctggaagctcagattatgccaaatttaactgaggagtattatcctttgactct aaacaacaaggaaaaaaaattagctgttgattggcgccccitaattaaagctataaccacagaagatagaa gcaaaactaacctaatagccactaaattccacaacagtitagtaaatttaattatcactattgcccaacagcagg gaatcgaaaaagttgctctggggggaggttgctttcaaaattgttatttgetigccagtaccattactgccctcaaa aaagctggtititctcctttgtggcccagagaactaccgcccaacgacggtgccatttgcatgggtcaactgttag ctaaaattcaggctcggcaatatatctgttaa 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: miktvaiqvqgrvagvafrpfvytlagemgIngwvnnstqgatvvitadekaiadfterltktlpppglieglaveq IplesfinftirpssdgpktasilpdIstcsacltelfdpsdrrylypfincthegprytiiealpydrcrttmarfrgctde ereykgpgdrrfhagpnacprcgpdlafwnrggqviaeanealnfavdnlkvgniiaikglggfnlccdatdfe aveklrirkhrpdkplavmygniggivehyqpnnlevellgsaaapivlinkkkd / lilveniapgnprvgvmlayt plhhlllkkikkpmvatsgnlagegicidnidaltriqniadgflvhdrpivcpvddsvvgivagkplflrrargyapq pitlpkptgkklla mgghykntvaiakgnqayvsghlgdinsaptygnfeeaiahlsqlydfspgqeivadlhpdy fshqyaengalpvtfvghhyahilavmaehgvmeesvigiawdgtgygmdgtiwggeflkitggtwqriahl qpfhllgnqgaikyphrialallwptfgddfsadsIgnwinfnngfknkinsriIngdinnknirglwqragapitss mgrlfdgiatliglinevtfegqaaialeagimpniteeyypltinnkekklavdwrplikaittedrsktnliatkfhns Ivnliitaqqqgiekvalgggefgneyllastitalkkagfsplwprelppndgaiemggllakiqarqyic 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: atgccaggccaatccaccaagtccactttaatcatcggttacggcaataccctgcggggggacgacggcatg gggcgttacctagcggaagaaattgctcagcaaaactggccccattgtggagttatttccacccatcaactcac cccagaattggccgaggcgatcgccgctgtggaccgggtaatittcaitgatgcccaactgcaggaatcagca aacgaaccatcggtggaagttgtggccttaaaaaccctggaacccaacgaactgtcaggggatttggggca ccggggtaatcccagggaactcttgaccctggctaaaattctctacggcgttgaggtaaaggcttggtgggtgtt gattccggccttcacctttgattatggagagaaattgtctcccctgaccgcccgggcccaagccgaagccttag cccagatccgccccttggtattgggggagagataa 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:

MPGQSTKSTLIIGYGNTLRGDDGVGRYLAEEIAQQNWPHCGVISTHQLTPELA EAIAAVDRVIFIDAQLQESANEPSVEVVALKTLEPNELSGDLGHRGNPRELLTL

AKILYGVEVKAWWVLIPAFTFDYGEKLSPLTARAQAEALAQIRPLVLGER Optionally, according to one embodiment according to the invention, all the maturation factors HypA, HypB, HypC, HypD, HypE, HypF and HoxW 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: ccatggcccacgaagttagcctgatggaacagacgctggccattgccattgcgcaggcggaagaccacgg ggcgagccaaattcaccgtttaacgctgcgcgttgggcagcagtcgggtgttgttgcagatgcattacgctttgc atttgaagttgttcgccagaacacaatggctgcagaagcacgtctggaaatcgaggaaattccggttacctgtc gttgtcagcattgtcatgaaaattttcagccggaggattggatatatagatgtccccattgtgaccagattagtcaa accgttatggacggcaaacagctggagttagcaagcctggaactgagctaagcatggaaaggaggtcgttat tatgtgccagaactgtgggtgtagcgcggttgggaccgttgcgcatagccaccatcaccacggggatggcaa ctttgcgcatagccatgacgaccacgaccagcaggagcaccaccaccaccacggtaactattcaaaatcac catcacagcagaccgtaaccatagaaccagacagacaaagcatagcaattggccaaggaattctgagcaa aaacgatcgtctggcagaacgcaaccgcggctacttccaggccaaaggtctgttagtaatgaatttcctgagc agcccgggagcaggcaaaaccgcactgatcgaaaaaatggttggtgatcgtcagaaagatcatccgaccg cagttattgttggtgatctggcaaccgataatgatgcacagcgtctgcgtagcgcaggtgcaattgcaattcagg ttaccaccggtaatatttgtcatctggaagcagaaatggttgcaaaagcagcacagaaactgga tctggataat attgatcagctgattattgaaaatgttggtaatctggtttgtccgaccacctatgatctgggtgaagatctgcgtgtt gttctgtttagcgttaccgaaggtgaagataaaccgctgaaatatccggcaacctttaaaagcgcacaggttatt ctggttaccaaacaggatattgcagcagcagttgattitgatgcagaactggcatggcagaatctgcgtcaggtt gcaccgcaggcacagatttttgcagttagcgcacgtaccggtaaaggtctgcagagctggtatgaatatctgg atcagtggcagctgcagcattatagcccgctggttgatccggcactggcataagagttgaaaggaggtttcctc catgtgcctggcgttaccggggcaggttgtttcgttaatgccgaactcggatccgctgttattaaccgggaaagtt agctttggtggtattattaaaaccattagcctggcgtatgttccggaagttaaagttggcgattatgttattgttcatgt tggttitgctatcagtattgttgatgaagaagcagcacaggagacactgattgatctggccgagatgggcgttta attcctaaaaggaggttttagccatgaagtacgttgacgaataccgcgacgcgcaggcagttgcccactaccg ccaggccattgcccgtgaaattaccaaaccgtggacgctgatggaaatttgtgggggccagacccatagcat cgttaaatatggtctggatgcattattaccgaaaaacttaaccttaatccacggtccgggttgtccggttigtgttac gccgatggaactgattgatcaggcattatggctggcaaaacagccggagattatttittgtagctttggtgatatg ctgcgcgtgccgggtagtggtgcagatctgctgagcattaaagcacaggggggagacgttcgtatagtttat TCT ccgttagattgtctggcgattgegcgtgaaaatccgaatcgtgaagttgttititttggtgtgggttitgaaactaccg ccccggcaaccgcaatgacactgcatcaggcacgggcccagggtattagcaattttagcttattatgtgcaca cgtgttagttccgccggcgatggaagctctgctgggtaacccgaatagcctggttcaagggtttttagcagcag gtcatgtttgtacggttaccggtgagcgggcgtatcagcatattgcagagaaatatcaggttccgatagttattac cggttitgaaccggttgatattatgcagggtatttttgcatgtgttegtcagctggagagcgggcagtttacatgtaa taatcagtaccggcggtcggttcagccgcagggtaacgcacatgcccagaaaattattgaccaggttitigaac cggtggatcgtcattggcgtggattaggtcttattceggcctcaggtttaggtttacgtccggcatttgcaccgtgg gacgcagcagttaaattcgcaaatctgttacagacaatggctccgacaatgggtgaaaccgtttgtatttctggce gaaattttacagggtcagcgcaaacctagtgattgtcctgcatttiggtaccatctgcaccccggaacaaccgct gggcgcccctatggttagcagtgaaggcgcttgtgccgcctattatcgttatcgtcagcaattaccggaaccggt tggtgccgcacgtgtttaattttgcaaaggaggtcctgccaatgaacctggtgtgtceggtgccgctggacegct acccgcaggttttactggcacacggggggggggggaagctgagtcagcagctgttaaaacagattittetgcc ggcgittggtgcatcagaaaccggtagccatgatgcagcagtttttaccgcaaatcagagcagcttagcattta caac agattcctatgttatcaatccgctgtttittcctggtggtgatattggtagtcttgcagttcatggaaccgttaat gatttagcaatggcaggtgcaacaccgcgttatattagcgttgggtttattctggaggagggtttaccgatggag acactttggcgtgttgcacaaagcctgggtcaggcagcacagaattgtggagtigaaatattaacaggtgatac caaagttgttgatcgtgggaagggagatggtattittattaatacatcgggtatcggtagtttagatcaccagcaa accattcatccgaatcaggttcaggttggtgatcgtctgattctgagtggggatttaggacggcatggtatggcaa ttatggcagttcgtcagggcctggaatttgaaacaaccattgaaagcgatagcgcaccggttcatcgtgaggtt caggctctgctgagcgcagggattccgattcactgtctgegtgacttaacacgtggtggtctggcaagcgccgt gaacgaaattgcacaaacctcaggtgttacaatggctctgegtgaaaccttaattccggttgaggcggaagttc aagccgcctgtgaactgctgggttitgatcctttatatgttgcgaacgaaggccgtttcetggccattgttcegccg gaagccgaacagaaaaccgttgaaattctgcagaccttticacccgcaggcgaccgcaattggtaccgttacc ggcaagagtgcacagaccttaggtctggttagcctggagagtagcataggtgccccacgtctgttagatatgat tagcggagaacaactgccacgtatttgttaagactccaaaggaggctagattaatgctgaaaaccgttgccatt caggttcaggggcgcgttcagggggttggtittcggccgtttgtttacaccttagcccaggaaatgggtctgaatg gctgggttaataac tctacgcagggtgcaaccgttgttattaccgcagatgagaaagcaattgcagattitaccg aacgtctgaccaaaacactgccgccaccgggactgatcgaacaactggcagtggaacagctgccgctgga aagctitaccaactttaccattagaccgagtagcgatggtccgaaaaccgcaagcatcctgccagatctgagc acatgtagcgcctgtctgaccgaattatttgatcccagtgatcgtcgttatctgtacccttttattaattgtacccactg tggtcctcgctataccattattgaagcactgccttatgaccgttgtcgtaccacaatggctcgttitegtcagtgtac ggattgtgaacgtgaatataagcagccgggggaccgcegttttcatgcacagccaaacgcgtgtccgcgttgt ggtccgcagctggcattctggaaccgtcagggtcaagttattgcagaagccaatgaagcactgaatitcgcag

10 tagataatttaaaggtcggtaatattatcgcaatcaaaggtctgggtggttttcatttatgttgtgatgcaaccgatttt gaagccgttgaaaaactgcgtttacgtaaacatcgcccggataagccgctggccgttatgtacggtaatctgg gtcagattgttgagcattatcagccgaataatttagaagttgagctgctgcagagcgcagcagcacctattgttct tctgaataaaaagaaacagctgattctggttgaaaatattgcaccgggcaatccgcgtgtgggtgttatgctgge atataccccgttacatcacctgttacttaaaaagttaaagaagccgatggttgcaacctccggtaacttagcagg cgaacagatttgtattgacaatattgacgcactgacccgtttacaaaatattgccgacggctttctggttcacgatc gtccgattgtttgtccggttgacgatagtgttgttcagattgtggcaggtaaaccgttatttttaagaagagcccgcg gttatgcaccgcagccgattacccttcctaaacccacccagaaaaagttattagcaatgggaggccattataa aaataccgttgcaattgcaaagcagaatcaggcatatgtaagccagcatttaggtgatttaaacagcgcacca acctaccaaaatttcgaagaggcgatagcccatttatcacagctgtatgactttagtccccaggaaattgtcgca gatctgcatccggattactttagccatcagtacgcagaaaaccaagccctgccggtgacgtttgtacagcatca ttatgcacatattctggcagttatggcagaacatggtgttatggaagaaagcgtittaggcattgcatgggatggc accggttatggtatggatggtaccatttggggtggtgaatttctgaaaattacgcaggggacctggcaaagaatt CCGA atctgcagccgtitcatctgttagggaatcagcaggcaattaaatatccgcaccggattgcacttgctctg ctgtggccgacattcggggacgattttagcgccgatagtctgggtaattggttaaattttaacaacggtttcaaga acaagatcaacagccgtttaaaccaagacttaaataataagaacctgagacaactgtggcagcgtgggcag gcaccgctgacctcgagcatgggcagattatttgatggtatcgcaacactgattggtctgatcaatgaagtaac ctttgaaggccaggcagcaattgcattagaggcacaaattatgccgaatctgaccgaagaatactatccgctt accctgaataacaaagaaaaaaaactggcagttgattggcgtccgctgattaaagcaattaccaccgaagat cgtagcaaaaccaatctgattgcaaccaaatttcataatagcctggttaatctgattattaccattgcacagcagc agggtattgaaaaagttgcactgggtggtggttgttttcagaattgttatctgctggcaagcaccattaccgcactg aaaaaagcaggttttagcccgctgtggccgcgtgaactgccgccgaatgatggtgcaatttgtatgggtcagct gctggcaaaaattcaggcacgtcagtatatttgttaactcaacaaaggaggagctggttatgccgggtcagag caccaaaagcaccctgattatcgggtacgggaacaccttacgtggggacgatggggtggggcgctacctgg cagaagaaatagcacagcagaactggccgcactgtggtgttattagcacacatcagctgaccccggaactg gccgaagcaattgcagcagtggatagagtgattittattgacgcccaactgcaggaaagtgcaaatgaaccgt cagttgaagttgttgccctgaaaaccttagaaccc aatgaattaagtggagatctgggtcatcgtggtaatccgc gtgagctgctgaccttagccaaaatattatatggtgttgaagtcaaagcgtggtgggttctgattccggcctttacc tttgattatggtgagaaattatcgcccttaacagcacgtgctcaggccgaagcactggcacagattcgtccgctg gttctgggggaacgttaacctagg 3 shows the map of plasmid pACYCDuet-1 used to construct the expression vector of at least one of Hypa maturation factors, HYPB, HypC, HYPD, HYPE, HypF HoxW and 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 terminator of transcription 77. It also contains the gene / ac / 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 pET26b (+) + HoxH and pACYCDuet-1 + HypABCDEFHoxW expression vectors in E. coli.

The competent BL21 (DE3) cells of E. coli were used for the recombinant expression of the HoxH subunit of HoxEFUYH [NiFe] -hydrogenase from Synechocystis sp. PCC6803. These cells are used routinely for the production of recombinant protein under the control of promoter 77.

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. The transformants exhibiting the double resistance to kanamycin (50 ug / ml) and to chloramphenicol (25 ug / 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 some colonies have the two plasmids allowing resistance to the selection markers used without having 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 of the DNA sequences of interest in plasmids, was carried out. 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 with the 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 Hindlll 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] -hydrogenase HoxEFUYH 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, 59 of NaCl per liter) supplemented with 100uM FeAmCi, 50uM NiSO4, 50uM cysteine, 50ug / ml kanamycin and 25ug / 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 Figure 7) allows HoxH to be purified 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 binds the immobile phase to 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 re-suspended in 25ml of lysis buffer (20mM sodium phosphate buffer pH 7.5 300mM KCI + 2ul benzonase + 50ul 1M MgCla + 1 pellet composed of a cocktail of protease inhibitors without EDTA) and then lysed by the press. by French.

The supernatant (25ml) is recovered by centrifugation (15min at 15000rpm), filtered (0.45um) and applied to the Ni-NTA column (1ml) previously equilibrated in the washing buffer (20mM sodium phosphate buffer pH 7.5 300mM KCI + 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) drops to zero.

The elution is then carried out via 5 stages with increasing concentration of imidazole, respectively 50mM, 100mM, 150mM, 200mM and 250mM) (see Figure 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 150mM, 200mM and 250mM 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 the [NiFe] -hydrogenase HoxEFUYH from Synechocystis sp.

PCC6803, an immunodetection using an antibody

A1 BE2019 / 5783 anti-poly-histidine tag 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 HoxH 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 has been exceeded and that a proportion of the recombinant HoxH protein has failed 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 bands of low intensity also appear at molecular weights below 54kDa. Presumably this is the recombinant HoxH protein partially degraded at the C-terminus. The proportion of degraded recombinant HoxH proteins seems minimal because SDS-PAGE analysis does not show the presence of these bands. As a reminder, immunodetection is an extremely sensitive method showing very small amounts of protein.

6. Demonstration of the hydrogenase activity in the recombinant protein HoxH The ability to express recombinantly [NiFe] - hydrogenases allows a wide range of possibilities with a view to 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 by avoiding the FMN. However, as shown in Figure 12, it is not known to date whether the only HoxH subunit, containing the active site of [NiFe] -hydrogenases HoxEFUYH 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 [NiFe] -hydrogenases in the presence of the recombinant protein HoxH and of MV was implemented (see FIG. 13). This standard test for [NiFe] -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 with a septum, airtight and degassed at nitrogen. 1ml of the reaction mixture, composed of 100mM 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. 200ug 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.m! *) Is known as the rate of hydrogen evolution (umol H2.min "t) for this quantity specific recombinant protein added This specific activity may, for example, be 0.1 µmol Ha.min * .mg ”.

Unexpectedly and surprisingly, the fact that the only HoxH catalytic subunit containing the active site of [NiFe] -hydrogenase HoxEFUYH 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 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 the protons. electrons from a redox mediator (eg methyl viologen) without the intermediary of additional FeS centers serving as redox relays. 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 [NiFe] - hydrogenase and being catalytically active.

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 "l" ", to introduce an element does not exclude the presence of a plurality of these elements.

Claims (15)

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. Monomeric polypeptide according to any one of the preceding claims, characterized in that it exhibits a hydrogenase activity of at least 0.05 µmol Hz. min ”. mg ”, preferably at least 10 umol Hz. min ”mg”. 7. A host cell including a monomeric polypeptide according to any one of claims 1 to 6. 8. Host cell according to claim 7, characterized in that it further includes at least one maturation factor of said [NiFe] type protein - hydrogenase, preferably at least one maturation factor of said [NiFe type protein. ] -hydrogenase chosen from the group consisting of maturation factors HypA, HypB, HypC, 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 concatenated 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. 9. A host cell including a polynucleotide encoding a monomeric polypeptide according to any one of claims 1 to 6. 10. Host cell according to claim 9, characterized in that it further includes at least one maturation factor of said [NiFe] type protein - hydrogenase, preferably at least one maturation factor of said [NiFe type protein. ] -hydrogenase chosen from the group consisting of maturation factors HypA, HypB, HypC, 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 concatenated 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. 11. A process for obtaining, for example in a host cell, a monomeric polypeptide exhibiting a hydrogenase activity according to any one of claims 1 to 6, said process comprising the following steps: modifying an expression vector , for example 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 [NiFe] - hydrogenase type; and e incubating said modified expression vector under incubation conditions allowing 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. 12.Process for obtaining a monomeric polypeptide exhibiting a hydrogenase activity according to claim 11, characterized in that said step of modifying said expression vector consists of an inclusion in said expression vector of said exogenous polynucleotide, at least one of which is part codes for said monomeric polypeptide, the amino acid sequence of which, 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. 13.Process for obtaining a monomeric polypeptide exhibiting a hydrogenase activity according to claim 11 or 12, characterized in that said step of modifying said expression vector further comprises the inclusion in said expression vector of at at least one maturation factor of said [NiFe] -hydrogenase type protein, preferably at least one maturation factor of said [NiFe] -hydrogenase type protein chosen from the group consisting of maturation factors HypA, HypB, HypC, 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% of 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 90% identity less 99% identity, respectively with respect 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 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. 14.Process for obtaining a monomeric polypeptide exhibiting hydrogenase activity according to any one of claims 11 to 13, characterized in that it comprises a subsequent step of isolation and / or purification of said monomeric polypeptide. 15.Use of a monomeric polypeptide according to any one of claims 1 to 6 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 according to incubation conditions allowing production or consumption of hydrogen or for coating a surface, in particular for coating a surface with an electrical conductor, for example to coat a surface of an anode or a cathode.