MONOMER POLYPEPTIDE HAVING HYDROGENASE ACTIVITY, IN PARTICULAR RECOMBINANT MONOMER POLYPEPTIDE HAVING HYDROGENASE ACTIVITY

Abstract

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

Claims

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

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

4. Monomeric polypeptide according to claim 1, characterized in that it is purified.

5. Monomeric polypeptide according to claim 1, the truncated or non-truncated amino acid sequence of which has at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably at least 60% identity, more preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 99% identity, with respect to the amino acid sequence of SEQ ID NO: 2 and/or with respect to the amino acid sequence of SEQ ID NO: 4.

6. Monomeric polypeptide according to claim 1, characterized in that said subunit comprising the active site of a protein of the [NiFe]-hydrogenase type is the FloxFI subunit of the protein of the [NiFe]-hydrogenase FloxEFUYFI protein in/from Synechocystis sp. PCC6803.

7. Monomeric polypeptide according to claim 1, characterized in that it has a hydrogenase activity of at least 0.01 mitil H.sub.2.Math. min −1.Math.Rng −1, preferably at least 0.05 mitil H.sub.2.Math.min −1.Math.mgJ of enzyme, preferably at least 10 mitil H2.Math.min −1.Math.mgJ of enzyme.

8. A host cell comprising a monomeric polypeptide according to claim 1.

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

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

11. A host cell comprising a polynucleotide encoding a monomeric polypeptide according to claim 1.

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

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

14. A method for obtaining a monomeric polypeptide having hydrogenase activity according to claim 1, said method comprising the following steps: a step of genetic modification, performed in-vivo or in-vitro, of an entity comprising genetic material, for example a host cell or an expression vector, to obtain a genetically modified entity, for example a genetically modified host cell or a genetically modified expression vector; a step of incubating said genetically modified entity, for example of said genetically modified host cell or said genetically modified expression vector, to obtain a monomeric polypeptide comprising a single subunit comprising the active site of a protein of the [NiFe]-hydrogenase type, said monomeric polypeptide having hydrogenase activity.

15. Method for obtaining a monomeric polypeptide having hydrogenase activity according to claim 14, characterized in that said step of genetic modification, carried out in-vivo or in vitro, comprises: a) a genetic modification of a host cell and/or an expression vector by including an exogenous polynucleotide of which at least a portion encodes a monomeric polypeptide comprising a single subunit comprising the active site of a protein of the [NiFe]-hydrogenase type, to obtain a genetically modified host cell and/or a genetically modified expression vector to be incubated in said incubation step performed according to incubation conditions for ensuring expression of said exogenous polynucleotide to produce said monomeric polypeptide; or b) inducing at least one genetic mutation of a host cell to obtain a genetically modified host cell to be incubated in said incubation step performed according to incubation conditions to produce said monomeric polypeptide.

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

17. A method for obtaining a monomeric polypeptide having hydrogenase activity according to claim 15, characterized in that said step of genetic modification of said host cell consists of an inclusion in said host cell of said exogenous polynucleotide of which at least a portion encodes said monomeric polypeptide whose truncated or non-truncated amino acid sequence has at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably at least 60% identity, more preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 99% identity, relative to the amino acid sequence of SEQ ID NO: 2 and/or with respect to the amino acid sequence of SEQ ID NO: 4.

18. The method for obtaining a monomeric polypeptide having hydrogenase activity according to claim 15, characterized in that said step of genetic modification of said host cell further comprises including in said host cell at least one maturation factor of said [NiFe]-hydrogenase-like protein, said at least one maturation factor being endogenous to the host cell and/or exogenous to the host cell, preferably the inclusion of at least one maturation factor of said [NiFe]-hydrogenase protein selected from the group consisting of maturation factors FlypA, HypB, FlypC, HypD, HypE, HypF and FloxW a) whose respective amino acid sequences each have at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably at least 60% identity, more preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 99% identity, respectively relative to the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; or b) encoded together by a concatenated nucleotide sequence having at least 15% identity, preferably at least 20% identity, more preferably at least 40% identity, more preferably at least 60% identity, more preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 99% identity, with respect to the nucleotide sequence SEQ ID NO: 19, which code for all of these maturation factors.

19. A method for obtaining a monomeric polypeptide having a hydrogenase activity according to claim 18, characterized in that said at least one maturation factor is derived from the expression of at least one gene invariant in an expression vector, said expression vector being inactivated in said host cell.

20. Process for obtaining a monomeric polypeptide having hydrogenase activity according to claim 14, characterized in that it comprises a subsequent step of isolating and/or purifying said monomeric polypeptide.

21. (canceled)

Description

[0125] Other characteristics, details and advantages of the invention will emerge from the examples given below, not by way of limitation and with reference to the appended figures.

[0126] FIG. 1 illustrates the map of plasmid pET-26b(+) (5360 bp).

[0127] FIG. 2 illustrates the agarose gel analysis of the digestion of the expression vector pET26b(+)+HoxH by the NdeI and BIpI restriction enzymes.

[0128] FIG. 3 illustrates the map of plasmid pACYCDuet-1 (4008 bp).

[0129] FIG. 4 illustrates the agarose gel analysis of the digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW by the NcoI and HindIII restriction enzymes.

[0130] FIG. 5 illustrates the agarose gel analysis of the digestion of the expression vector pET26b(+)+HoxH, derived from the plasmid DNA of an E. coli colony, by the NdeI and BIpI restriction enzymes.

[0131] FIG. 6 illustrates the agarose gel analysis of the digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW, derived from the plasmid DNA of an E. coli colony, by the NcoI and HindIII restriction enzymes.

[0132] FIG. 7 illustrates the method of purification of the HoxH recombinant protein of interest. NA, sample not absorbed on the Ni-NTA affinity column. Wash, sample eluted with the wash buffer containing 10 mM Imidazole. 50 mM, sample eluted at a concentration of 50 mM imidazole. 100 mM, sample eluted at a concentration of 100 mM imidazole. 150 mM, sample eluted at a concentration 150 mM imidazole. 200 mM, sample eluted at a concentration of 200 mM imidazole. 250 mM, sample eluted at a concentration of 250 mM imidazole.

[0133] FIG. 8 illustrates the SDS-PAGE analysis of the protein composition of various fractions collected during the affinity chromatography. Load, supernatant applied to the Ni-NTA affinity column and derived from the lysis of the recombinant E. coli cells; NA, sample not absorbed on the Ni-NTA affinity column. Wash, sample eluted with the wash buffer containing 10 mM Imidazole. M, molecular weight marker. 50 mM, sample eluted at a concentration of 50 mM imidazole. 100 mM, sample eluted at a concentration of 100 mM imidazole. 150 mM, sample eluted at a concentration of 150 mM imidazole. 200 mM, sample eluted at a concentration of 200 mM imidazole. 250 mM, sample eluted at a concentration of 250 mM imidazole.

[0134] FIG. 9 illustrates the immuno-detection analysis (Western blot) of the presence of HoxH in various fractions collected during the affinity chromatography. M, molecular weight marker. Load, supernatant applied to the Ni-NTA affinity column and derived from the lysis of the recombinant E. coli cells. NA, sample not absorbed on the Ni-NTA affinity column; 50 mM, sample eluted at a concentration of 50 mM imidazole. 200 mM, sample eluted at a concentration of 200 mM imidazole.

[0135] FIG. 10 is a schematic representation of the structure of HoxEFUYH, [NiFe]-hydrogenase from Synechocystis sp. PCC 6803. HoxE, 19 KDa and 1 FeS center; HoxF, 57.5 KDa, 2 FeS centers and a FMN center; HoxU, 26 KDa and 4 FeS centers; HoxY, 20 KDa and a FeS center; HoxH, 53 KDa and the active site.

[0136] FIG. 11 is a schematic representation (shaded arrow) of the electron transfer and the expected interactions with NADPH and methyl-viologen (MV) in HoxEFUYH, [NiFe]-hydrogenase from Synechocystis sp. PCC 6803.

[0137] FIG. 12 is a schematic representation where the question mark raises the question whether or not the active site of the HoxH recombinant protein can accept electrons directly from MV and therefore produce hydrogen in the absence of additional redox relays.

[0138] FIG. 13 illustrates the highlighting of the hydrogenase activity by the production of hydrogen which results from the addition of the HoxH recombinant protein to a reagent containing methyl-viologen previously reduced by sodium dithionite in the absence of oxygen. HoxH is able to take the electrons of the methyl-viologen previously reduced to combine them with protons (present in the buffer) in order to produce hydrogen according to the equation H.sub.2.Math.2H.sup.++2e.sup.− which represents the hydrogenase-catalyzed reaction. 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 HoxH recombinant protein is made, moment concomitant with the increase in the level of dissolved hydrogen detected by the micro-sensor.

[0139] FIG. 14 illustrates the highlighting of the hydrogenase activity by the reduction of benzyl viologen which results from the addition of the HoxH recombinant protein to a reagent containing benzyl viologen (BV) in the presence of H.sub.2. HoxH is able to take electrons from hydrogen to transfer them to a redox mediator, for example benzyl viologen, at the same time as the production of protons according to the equation H.sub.2.Math.2H.sup.++2e.sup.− which represents the hydrogenase-catalyzed reaction. The level of hydrogen consumed is equivalent to the level of reduced benzyl viologen, a level that can be measured continuously by spectrophotometry at a wavelength of 578 nm.

[0140] FIG. 15 illustrates the highlighting of the hydrogenase activity by the production of hydrogen which results from the addition of the HoxH recombinant protein, produced in the absence of the HupABCDEFHoxW maturation factors exogenous to E. coli, to a reagent containing methyl-viologen previously reduced by sodium dithionite in the absence of oxygen. HoxH is able to take electrons of the previously reduced methyl-viologen to combine them with protons (present in the buffer) in order to produce hydrogen according to the equation H.sub.2.Math.2H.sup.++2e.sup.− which represents the hydrogenase-catalyzed reaction.

EXAMPLES

[0141] 1. Construction of a HoxH Expression Vector

[0142] 1.1. “Simple” Sequence of HoxH

[0143] HoxH corresponds to the large subunit comprising the active site of HoxEFUYH [NiFe]-hydrogenase 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 (1425 bp) of HoxH is as follows and is named SEQ ID NO:1 in the context of the present invention:

TABLE-US-00001 atgtctaaaaccattgttatcgatcccgttacccggattgaaggccatgccaaaatctccattttcctcaa cgaccagggcaacgtagatgatgttcgtttccatgtggtggagtatcggggttttgaaaaattttgcgaa ggtcgtcccatgtgggaaatggctggtattaccgcccgtatttgcggcatttgtccggttagccatctgct ctgtgcggctaaaaccggggataagttactggcggtgcaaatccctccagccggggaaaaactgc gccgtttaatgaatttagggcaaattacccaatcccacgccctaagttttttccatctcagcagtcctgatt ttctgcttggttgggacagtgatcccgctactcgcaatgtgtttggtttaattgctgctgaccccgatttagc tagggcaggtattcggttacggcaatttggccaaacggtaattgaacttttgggagctaaaaaaatcc actctgcttggtcagtgcccggtggagtccgatcgccgttgtcggaagaaggcagacaatggattgtg gaccgtttaccagaagcaaaagaaaccgtttatttagccttaaatttgtttaaaaatatgttggaccgctt ccaaacagaagtggcagaatttggcaaatttccctccctatttatgggcttagttgggaaaaataatga atgggaacattatggcggctccctgcggtttaccgacagtgaaggcaatattgtcgcggacaatctca gtgaagataattacgctgattttattggtgaatcggtggaaaaatggtcctatttaaaatttccctactaca aatctctgggttatcccgatggcatttatcgggttggtccccttgcccgccttaatgtttgtcatcacattgg caccccggaagcagaccaagaattagaagaatatcggcaacgggctggaggtgtggccacgtcc tctttcttttatcattacgcccgcttggtggaaattcttgcctgtttagaagccatcgaattgttaatggctga ccctgatattttgtccaaaaattgtcgagctaaggcagaaattaattgtaccgaagcggtgggagtga gcgaagcaccccggggtactttattccaccattacaagatagatgaagatggtctaattaagaaagt gaatttgatcattgccacgggcaacaataacttagccatgaataaaaccgtggcccaaattgccaaa cactacattcgcaatcatgatgtgcaagaagggtttttaaaccgggtggaagcgggtattcgttgttatg atccctgccttagttgttctacccatgcagcgggacaaatgccattgatgatcgatttagttaaccctcag ggggaactaattaagtccatccagcgggattaa

[0144] The amino acid sequence (474 aa) of HoxH is as follows and is named SEQ ID NO:2 in the context of the present invention:

TABLE-US-00002 MSKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEKFCEGRP MWEMAGITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLRRLMNLG QITQSHALSFFHLSSPDFLLGWDSDPATRNVFGLIAADPDLARAGIRLR QFGQTVIELLGAKKIHSAWSVPGGVRSPLSEEGRQWIVDRLPEAKETV YLALNLFKNMLDRFQTEVAEFGKFPSLFMGLVGKNNEWEHYGGSLRFT DSEGNIVADNLSEDNYADFIGESVEKWSYLKFPYYKSLGYPDGIYRVGP LARLNVCHHIGTPEADQELEEYRQRAGGVATSSFFYHYARLVEILACLE AIELLMADPDILSKNCRAKAEINCTEAVGVSEAPRGTLFHHYKIDEDGLI KKVNLIIATGNNNLAMNKTVAQIAKHYIRNHDVQEGFLNRVEAGIRCYDP CLSCSTHAAGQMPLMIDLVNPQGELIKSIQRD

[0145] FIG. 1 shows the map of the plasmid pET26b(+) used to construct the HoxH expression vector by inserting SEQ ID NO:3 into pET26b(+). pET26b(+) is a 5360 bp plasmid possessing 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 T7 transcription terminator. It also contains the lacl gene encoding a transcription repressor. This repression of transcription can be lifted by adding IPTG.

[0146] 1.2. “Optimized” Sequence of HoxH

[0147] Optionally, according to an embodiment of to the invention, the sequences SEQ ID NO:1 and SEQ ID NO:2 are optimized for codon usage in E. coli. In particular, the NdeI (catatg) and BIpI (gctnagc) restriction sites are added respectively at the beginning and at the end of the nucleotide sequence for cloning in the plasmid pET26b(+) and the sequence caccaccaccaccatcac (underlined below) is also added (sequence encoding the poly-histidine tag close to the N-terminal end of the protein).

[0148] The optimized nucleotide sequence (1453 bp) is as follows and is named SEQ ID NO:3 in the context of the present invention:

TABLE-US-00003 catatgagccaccaccaccaccatcacaaaaccatcgtcatcgacccagtcacccgcatcgaagg ccacgccaaaattagcatttttctgaacgaccagggcaacgtcgacgacgtccgctttcacgttgttga ataccgtggcttcgaaaaattttgtgaaggtcgtccgatgtgggaaatggccggtatcacggcacgta tttgtggaatttgtccggtgagccatctgctgtgtgccgcaaaaaccggagataaactgctggcagtgc agattccgccggcaggtgaaaaactgcgtcgtctgatgaatctgggtcagattacacagtcgcatgc actgtctttctttcatctgagtagcccagattttctgctggggtgggatagcgacccggcaacacgtaat gtgtttggtctgattgcggctgatccggatctggcgcgtgccggtattcgtctgcgtcagtttggtcagac agttattgagctgctgggggcgaaaaagattcatagtgcatggtctgtgccgggtggtgttcgtagtcc gctgagtgaagaaggtcgtcagtggattgttgatcgtctgccggaggcaaaagaaacggtctatctg gcactgaatctgtttaaaaatatgctggatcgtttccagacagaagttgcagaatttggaaaatttccgt cactgtttatgggtctggttggtaaaaataatgaatgggaacactatggtggtagcctgcgtttcacgga ctctgaaggtaatattgttgcggataatctgagcgaagacaattatgcagattttatcggtgaaagtgtg gaaaaatggagctatctgaaatttccgtattacaaaagcctgggctatccggatgggatctaccgtgtt ggaccgctggcacgtctgaacgtttgtcatcatattggtaccccggaagcagatcaggaactggaag aatatcgtcagcgtgcgggtggtgttgcgactagcagctttttttatcattatgcacgtctggttgaaattct ggcctgtctggaggcaattgaactgctgatggcagatcctgatattctgtctaaaaattgtcgtgcaaa agcagaaattaactgtaccgaggcagttggtgttagtgaggcgccgcgtggtaccctgtttcatcacta taaaattgacgaagatggtctgattaaaaaggttaatctgattatcgcaaccggtaacaataatctggc aatgaataaaaccgttgcacagattgcaaaacactacattcgcaaccacgatgttcaggaagggttt ctgaatcgtgtagaagccggcattcgctgttatgatccgtgtctgagctgtagcacccatgcagcaggt cagatgcctctgatgattgacctggttaatccgcagggtgagctgattaaaagcattcagcgtgattaa gctgagc

[0149] The optimized amino acid sequence (480 aa) is as follows and is named SEQ ID NO:4 in the context of the present invention:

TABLE-US-00004 MSHHHHHHKTIVIDPVTRIEGHAKISIFLNDQGNVDDVRFHVVEYRGFEK FCEGRPMWEMAGITARICGICPVSHLLCAAKTGDKLLAVQIPPAGEKLR RLMNLGQITQSHALSFFHLSSPDFLLGWDSDPATRNVFGLIAADPDLAR AGIRLRQFGQTVIELLGAKKIHSAWSVPGGVRSPLSEEGRQWIVDRLPE AKETVYLALNLFKNMLDRFQTEVAEFGKFPSLFMGLVGKNNEWEHYG GSLRFTDSEGNIVADNLSEDNYADFIGESVEKWSYLKFPYYKSLGYPDG IYRVGPLARLNVCHHIGTPEADQELEEYRQRAGGVATSSFFYHYARLVE ILACLEAIELLMADPDILSKNCRAKAEINCTEAVGVSEAPRGTLFHHYKI DEDGLIKKVNLIIATGNNNLAMNKTVAQIAKHYIRNHDVQEGFLNRVEAG IRCYDPCLSCSTHAAGQMPLMIDLVNPQGELIKSIQRD

[0150] Plasmid pET26b(+) is used to construct the HoxH expression vector by inserting SEQ ID NO:3 into pET26b(+).

[0151] FIG. 2 shows the analysis of the digestion of the expression vector pET26b(+)+HoxH (SEQ ID NO:3) by the NdeI and BIpI restriction enzymes. Two DNA fragments at around 5300 bp (linearized pET26b(+)) and at around 1500 bp (HoxH sequence excised from plasmid pET26b(+)) are highlighted. This confirms the presence of the HoxH sequence of interest in the expression vector pET26b(+).

[0152] 2. Construction of the Expression Vector of at Least One Maturation Factor

[0153] At least the following maturation factors are considered in the context of the present invention: HypA, HypB, HypC, HypD, HypE, HypF and HoxW.

[0154] HypA is an expression/formation protein of HoxEFUYH [NiFe]-hydrogenase in Synechocystis sp. PCC6803. The accession number of the protein in Genbank is BAA18357.1. The theoretical isoelectric point of HypA is 4.94 for a theoretical molecular mass: 12773.47 Daltons. The nucleotide sequence (342 bp) of HypA is as follows and is named SEQ ID NO:5 in the context of the present invention:

TABLE-US-00005 atgcacgaagttagtctgatggagcaaactttggcgatcgccattgccc aggcggaagaccatggagccagccaaatccatcgtttaaccctgcgggt ggggcaacagtctggggtggtggccgatgccctacggtttgcgtttgaa gtggtgcgacaaaataccatggccgccgaggcgagattggaaattgaag aaattcccgttacctgtcgttgccaacactgccacgaaaattttcagcc agaggattggatttaccgctgtccccactgcgaccagattagccaaaca gtaatggatggcaaacagttggaactagcatccctagaactgagttga

[0155] The amino acid sequence (113 aa) of HypA is as follows and is named SEQ ID NO:6 in the context of the present invention:

TABLE-US-00006 MHEVSLMEQTLAIAIAQAEDHGASQIHRLTLRVGQQSGVADALRFAFE VVRQNTMAAEARLEIEEIPVTCRCQHCHENFQPEDWIYRCPHCDQISQ TVMDGKQLELASLELS

[0156] HypB is an expression/formation protein of HoxEFUYH [NiFe]-hydrogenase in Synechocystis sp. PCC6803. The accession number of the protein in Genbank is BAA18312.1. The theoretical isoelectric point of HyB is 5.75 for a theoretical molecular mass: 31242.97 Daltons. The nucleotide sequence (858 bp) of HypB is as follows and is named SEQ ID NO:7 in the context of the present invention:

TABLE-US-00007 atgtgccaaaactgcggttgtagtgcggtgggaaccgttgcccatagcc accatcaccatggcgatggaaattttgcccacagccatgatgaccatga ccagcaagaacatcatcaccaccatggcaactacagcaaaagtccaagt cagcagactgtgaccattgaacccgatcgccagtccattgccattggcc aaggcattctcagcaagaatgaccgcctagcggaaaggaatcggggcta tttccaggctaagggcttactggtgatgaatttcctctcttctcccgga gccggtaaaactgctctgatcgaaaaaatggtcggcgatcgacaaaaag accatcccaccgccgtcattgtgggggatttagccaccgataacgatgc ccaacgtctccgcagtgccggggcgatcgccattcaggtcaccacagga aatatttgccatctggaagcggaaatggtggccaaggcggcccaaaagt tagatttagacaatatcgatcaattgatcattgaaaatgttggtaattt ggtttgccccaccacctatgatctaggggaagatttacgggtcgtatta ttttccgtcacagaaggggaggataaaccccttaaatatcccgccacct tcaaatcagcccaggttattttagtcaccaaacaggacattgccgccgc agtggattttgatgcagagctggcttggcaaaacctacggcaagtggcc ccccaagcccaaatttttgcagtgtctgcccgcacggggaaaggattgc agtcctggtatgagtatttggatcaatggcaactccaacactattcgcc gttggttgatccagcattggcctaa

[0157] 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:

MCQNCGCSAVGTVAHSHHHHGDGNFAHSHDDHDQQEHHHHHGNYS

[0158]

TABLE-US-00008 KSPSQQTVTIEPDRQSIAIGQGILSKNDRLAERNRGYFQAKGLLVMNFL SSPGAGKTALIEKMVGDRQKDHPTAVIVGDLATDNDAQRLRSAGAIAIQ VTTGNICHLEAEMVAKAAQKLDLDNIDQLIIENVGNLVCPTTYDLGEDLR VVLFSVTEGEDKPLKYPATFKSAQVILVTKQDIAAAVDFDAELAWQNLR QVAPQAQIFAVSARTGKGLQSWYEYLDQWQLQHYSPLVDPALA

[0159] HypC is an expression/formation protein of HoxEFUYH [NiFe]-hydrogenase 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 (231 bp) of HypC is as follows and is named SEQ ID NO:9 in the context of the present invention:

TABLE-US-00009 atgtgtctagccctacctggccaggttgtcagtttaatgcccaactccg atcccctgttactgacgggaaaggttagctttgggggcatcattaaaac cattagccttgcctacgtacccgaggttaaggtgggggattacgtgatt gtccatgtgggctttgccattagcattgtggacgaagaggcggcccagg aaactttgatagacttggcagaaatgggagtttaa

[0160] The amino acid sequence (76 aa) of HypC is as follows and is named SEQ ID NO: 10 in the context of the present invention:

TABLE-US-00010 MCLALPGQVVSLMPNSDPLLLTGKVSFGGIIKTISLAYVPEVKVGDYVIV HVGFAISIVDEEAAQETLIDLAEMGV

[0161] HypD is an expression/formation protein of HoxEFUYH [NiFe]-hydrogenase in Synechocystis sp. PCC6803. The accession number of the protein in Genbank is BAA16622.1. The theoretical isoelectric point of HypD is 6.31 for a theoretical molecular mass of 40632.94 Daltons. The nucleotide sequence (1125 bp) of HypD is as follows and is named SEQ ID NO:11 in the context of the present invention:

TABLE-US-00011 atgaaatacgttgatgaatatcgggatgcccaggcggtggcccattacc gtcaggcgatcgccagggagataaccaaaccttggacgctgatggagat ttgcggcggccagacccacagcattgtcaaatatggcttggatgctttg ttgccgaagaatttgactctgatccatggtcccggctgtcctgtgtgcg tcactccgatggaattaattgaccaggctttgtggttagctaagcaacc ggagatcattttttgttcctttggcgatatgttgcgggtgcccggcagt ggggcggatttgctgagcattaaagcccagggcggcgatgtgcgcattg tctattctcctttggattgtttggcgatcgccagggagaatcctaatcg ggaagtggtatttttcggagtaggttttgaaactacagcccctgccacg gccatgactctccaccaagctagggcccagggaattagcaatttcagtt tactttgcgcccatgtattggtgcccccggctatggaggctttattagg caatcccaattccctcgtgcagggctttttggcggcagggcatgtctgt acggtgaccggggaaagggcctatcaacatatcgctgaaaaataccaag tacccattgtcatcactggctttgaacctgtggatattatgcagggcat ctttgcctgtgtgcgccaactggagtcgggacaattcacctgcaacaat caatatcggcgatcggtccaaccccagggcaatgcccatgctcagaaaa ttattgaccaagtgtttgagccagtcgatcgccattggcggggtttggg attaattccggccagcggtttgggtttaaggccagcatttgccccctgg gatgccgcagttaaattcgccaatttattgcaaaccatggccccaacga tgggagaaacagtgtgtattagcggggaaattttacagggacaacggaa gcccagcgattgtccagcctttggtactatctgcaccccagaacaaccc ttgggggctcccatggtttcctcggaaggagcctgtgccgcctattacc gttatcgccaacaattaccggaaccagtgggagcggccagagtttag

[0162] The amino acid sequence (374 aa) of HypD is as follows and is named SEQ ID NO:12 in the context of the present invention:

TABLE-US-00012 MKYVDEYRDAQAVAHYRQAIAREITKPWTLMEICGGQTHSIVKYGLDAL LPKNLTLIHGPGCPVCVTPMELIDQALWLAKQPEIIFCSFGDMLRVPGS GADLLSIKAQGGDVRIVYSPLDCLAIARENPNREVVFFGVGFETTAPATA MTLHQARAQGISNFSLLCAHVLVPPAMEALLGNPNSLVQGFLAAGHVC TVTGERAYQHIAEKYQVPIVITGFEPVDIMQGIFACVRQLESGQFTCNN QYRRSVQPQGNAHAQKIIDQVFEPVDRHWRGLGLIPASGLGLRPAFAP WDAAVKFANLLQTMAPTMGETVCISGEILQGQRKPSDCPAFGTICTPE QPLGAPMVSSEGACAAYYRYRQQLPEPVGAARV

[0163] HypE is an expression/formation protein of HoxEFUYH [NiFe]-hydrogenase in Synechocystis sp. PCC6803. The accession number of the protein 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 (1038 bp) of HypE is as follows and is named SEQ ID NO:13 in the context of the present invention:

TABLE-US-00013 gtgaacttagtctgtcccgttccccttgatcgttatccccaggtactgt tagcccacggcggcggcggtaagttgagccaacaattacttaagcaaat ttttttaccggcctttggcgcttctgaaacgggtagtcatgatgcggcg gtttttactgccaaccaaagttctttagctttcaccaccgactcctatg tgatcaatcccctcttttttcctgggggcgatattggttctttggcagt ccacggcaccgttaatgacctagccatggccggcgcaacccctcgctat atcagcgttggttttatcctcgaagaaggattgcccatggagaccctct ggcgggtggcccaatccctagggcaagcggcccaaaactgtggggtgga aattcttaccggtgataccaaagtggtggaccggggtaagggagacggc attttcatcaacaccagcggcattggttccctcgaccatcaacaaacta tccatcccaatcaggtacaggtaggcgatcgcctaattttgagcggtga tttgggacgtcatggcatggccattatggcagtgcgccaaggattagaa tttgaaaccaccattgaaagtgattcggccccggttcacagagaagtgc aggcattattgtcggcagggatcccaatccattgtctgcgggatttaac cagggggggattagccagtgcggttaatgaaattgcccaaacttccggg gtaaccatggctttacgagaaacgttaatcccggtggaggccgaagtac aagccgcctgtgaactgttgggttttgaccccctctatgtggccaatga gggaagattcctggccattgtgcccccggaagcagaacagaagaccgtg gaaattttgcaaactttccatccccaagctacggcgatcggtacagtaa caggcaaaagtgcacaaaccttggggttagtcagtttggaaagttccat tggtgccccccggttgctagacatgatcagtggggagcaattaccccgt atttgttag

[0164] The amino acid sequence (345 aa) of HypE is as follows and is named SEQ ID NO:14 in the context of the present invention:

TABLE-US-00014 MNLVCPVPLDRYPQVLLAHGGGGKLSQQLLKQIFLPAFGASETGSHDA AVFTANQSSLAFTTDSYVINPLFFPGGDIGSLAVHGTVNDLAMAGATPR YISVGFILEEGLPMETLWRVAQSLGQAAQNCGVEILTGDTKVVDRGKG DGIFINTSGIGSLDHQQTIHPNQVQVGDRLILSGDLGRHGMAIMAVRQG LEFETTIESDSAPVHREVQALLSAGIPIHCLRDLTRGGLASAVNEIAQT SGVTMALRETLIPVEAEVQAACELLGFDPLYVANEGRFLAIVPPEAEQK TVEILQTFHPQATAIGTVTGKSAQTLGLVSLESSIGAPRLLDMISGEQL PRIC

[0165] HypF is an expression/formation protein of HoxEFUYH [NiFe]-hydrogenase in Synechocystis sp. PCC6803. The accession number of the protein 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 (2304 bp) of HypF is as follows and is named SEQ ID NO: 15 in the context of the present invention:

TABLE-US-00015 atgttaaaaaccgttgccatacaggtccagggaagggtgcaaggagtgggttttcgtccctttgtttata cccttgcccaggaaatgggactgaatggttgggtgaataattccactcaaggagctaccgttgtcatta ccgccgacgaaaaggcgatcgccgactttacggagagattaacgaagacattacctccccctggttt gattgaacaattagccgttgaacagttaccgctggaaagttttactaactttactatccgccccagtagt gatggccctaaaactgcgagtattttacccgatttatccacttgttccgcctgcttaacagaactatttgac cctagcgatcgccgttatctttacccctttattaactgtacccattgcggtccccgctacaccattattgaa gccctaccttacgaccgttgtcgtaccaccatggctaggtttcgccaatgtaccgactgtgaaaggga atataagcaaccaggcgatagacgcttccatgcccaacctaatgcctgtcctcgctgtggcccccaa ctggctttttggaaccgacaaggccaagtaattgcagaagcaaatgaagctttaaactttgctgtagat aatttaaaagtcggcaatattatcgctattaaaggcttaggtggcttccatttgtgttgtgatgccactgatt ttgaagctgtggaaaaattaagattaaggaaacatcgaccggataaacctttggcggtaatgtatggt aatcttggtcaaattgtggagcattaccaacctaataatctagaagttgaattgttacaaagtgccgcc gcccctattgtgttattaaacaaaaaaaaacaattaattttggtggaaaatattgccccaggcaacccc cgagtcggcgtaatgttagcctatactcctttgcatcacttattactaaaaaaattaaagaaacccatgg tagctaccagtggtaacttagctggggagcaaatttgcattgataatattgacgctttaacccggttaca aaatattgctgacggttttctcgttcatgatcgcccgattgtttgtccagtggatgattccgttgtccaaata gtagctgggaagccattatttttgcgtcgagcccggggttacgctcctcaacccattactttaccaaagc ctactcaaaaaaaactattggcgatgggaggtcattataaaaatacagtggcgatcgccaaacaaa atcaagcttacgtcagccaacatttgggcgatttgaattctgctcccacctaccaaaattttgaagaag ccattgcccatttaagccagctatacgatttctctccccaggaaattgttgcagatttacaccctgattattt cagtcatcaatatgctgaaaaccaagctttgcctgtcacttttgtgcagcatcactatgctcatattttagc ggttatggcggaacatggagttatggaggagtccgtgttaggtattgcttgggatggcactggctacgg catggacggtactatttgggggggagaatttttaaaaatcacccaaggtacttggcagagaattgctc atctacaaccatttcatttattaggtaatcaacaagccattaaatatccccatcggattgctttggcgttgtt atggcccacttttggtgatgatttttctgctgattctttaggaaattggttgaatttcaataatgggtttaaaa acaagataaacagcaggttaaatcaggatctaaacaacaaaaatttacgtcaactttggcaacgag ggcaagcaccgctcacttcgagtatgggaagattatttgacggtattgcgacactgataggattgatta acgaagtaacttttgaaggtcaggcggccatagctctggaagctcagattatgccaaatttaactgag gagtattatcctttgactctaaacaacaaggaaaaaaaattagctgttgattggcgccccttaattaaa gctataaccacagaagatagaagcaaaactaacctaatagccactaaattccacaacagtttagta aatttaattatcactattgcccaacagcagggaatcgaaaaagttgctctggggggaggttgctttcaa aattgttatttgcttgccagtaccattactgccctcaaaaaagctggtttttctcctttgtggcccagagaa ctaccgcccaacgacggtgccatttgcatgggtcaactgttagctaaaattcaggctcggcaatatatc tgttaa

[0166] The amino acid sequence (767 aa) of HypF is as follows and is named SEQ ID NO: 16 in the context of the present invention:

TABLE-US-00016 mlktvaiqvqgrvqgvgfrpfvytlaqemglngwvnnstqgatvvitadekaiadfterltktlpppglie qlaveqlplesftnftirpssdgpktasilpdlstcsacltelfdpsdrrylypfincthcgprytiiealpydrc rttmarfrqctdcereykqpgdrrfhaqpnacprcgpqlafwnrqgqviaeanealnfavdnlkvgnii aikglggfhlccdatdfeaveklrlrkhrpdkplavmygnlgqivehyqpnnlevellqsaaapivllnk kkqlilveniapgnprvgvmlaytplhhlllkklkkpmvatsgnlageqicidnidaltrlqniadgflvhdr pivcpvddsvvqivagkplflrrargyapqpitlpkptqkkllamgghykntvaiakqnqayvsqhlgd lnsaptyqnfeeaiahlsqlydfspqeivadlhpdyfshqyaenqalpvtfvqhhyahilavmaehg vmeesvlgiawdgtgygmdgtiwggeflkitqgtwqriahlqpfhllgnqqaikyphrialallwptfgd dfsadslgnwlnfnngfknkinsrlnqdlnnknlrqlwqrgqapltssmgrlfdgiatliglinevtfegqa aialeaqimpnlteeyypltlnnkekklavdwrplikaittedrsktnliatkfhnslvnliitiaqqqgiekv algggcfqncyllastitalkkagfsplwprelppndgaicmgqllakiqarqyic

[0167] HoxW is a hypothetical protein in Synechocystis sp. PCC6803. The accession number of the protein in Genbank is BAA17680.1. The theoretical isoelectric point of HoxW is 4.93 for a theoretical molecular mass of 17129.53 Daltons. The nucleotide sequence (474 bp) of HoxW is as follows and is named SEQ ID NO:17 in the context of the present invention:

TABLE-US-00017 atgccaggccaatccaccaagtccactttaatcatcggttacggcaataccctgcggggggacgac ggcgtggggcgttacctagcggaagaaattgctcagcaaaactggccccattgtggagttatttccac ccatcaactcaccccagaattggccgaggcgatcgccgctgtggaccgggtaattttcattgatgccc aactgcaggaatcagcaaacgaaccatcggtggaagttgtggccttaaaaaccctggaacccaac gaactgtcaggggatttggggcaccggggtaatcccagggaactcttgaccctggctaaaattctcta cggcgttgaggtaaaggcttggtgggtgttgattccggccttcacctttgattatggagagaaattgtctc ccctgaccgcccgggcccaagccgaagccttagcccagatccgccccttggtattgggggagagat aa

[0168] 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:

TABLE-US-00018 MPGQSTKSTLIIGYGNTLRGDDGVGRYLAEEIAQQNWPHCGVISTHQLT PELAEAIAAVDRVIFIDAQLQESANEPSVEVVALKTLEPNELSGDLGHRG NPRELLTLAKILYGVEVKAWWVLIPAFTFDYGEKLSPLTARAQAEALAQI RPLVLGER

[0169] Optionally, according to an embodiment in accordance with the invention, all of the maturation factors HypA, HypB, HypC, HypD, HypE, HypF and HoxW are assembled in the form of a concatenary nucleotide sequence (6515 bp). This nucleotide sequence comprises the NcoI restriction site (CCATGG) at the beginning of the sequence and the AvrII restriction site (CCTAGG) at the end of the sequence to carry out the cloning in the pACYCDuet-1 plasmid. This concatenary nucleotide sequence is as follows and is named SEQ ID NO: 19 in the context of the present invention:

TABLE-US-00019 ccatggcccacgaagttagcctgatggaacagacgctggccattgccattgcgcaggcggaagac cacggggcgagccaaattcaccgtttaacgctgcgcgttgggcagcagtcgggtgttgttgcagatg cattacgctttgcatttgaagttgttcgccagaacacaatggctgcagaagcacgtctggaaatcgag gaaattccggttacctgtcgttgtcagcattgtcatgaaaattttcagccggaggattggatatatagatg tccccattgtgaccagattagtcaaaccgttatggacggcaaacagctggagttagcaagcctggaa ctgagctaagcatggaaaggaggtcgttattatgtgccagaactgtgggtgtagcgcggttgggacc gttgcgcatagccaccatcaccacggggatggcaactttgcgcatagccatgacgaccacgacca gcaggagcaccaccaccaccacggtaactattcaaaatcaccatcacagcagaccgtaaccata gaaccagacagacaaagcatagcaattggccaaggaattctgagcaaaaacgatcgtctggcag aacgcaaccgcggctacttccaggccaaaggtctgttagtaatgaatttcctgagcagcccgggagc aggcaaaaccgcactgatcgaaaaaatggttggtgatcgtcagaaagatcatccgaccgcagttatt gttggtgatctggcaaccgataatgatgcacagcgtctgcgtagcgcaggtgcaattgcaattcaggtt accaccggtaatatttgtcatctggaagcagaaatggttgcaaaagcagcacagaaactggatctg gataatattgatcagctgattattgaaaatgttggtaatctggtttgtccgaccacctatgatctgggtgaa gatctgcgtgttgttctgtttagcgttaccgaaggtgaagataaaccgctgaaatatccggcaaccttta aaagcgcacaggttattctggttaccaaacaggatattgcagcagcagttgattttgatgcagaactg gcatggcagaatctgcgtcaggttgcaccgcaggcacagatttttgcagttagcgcacgtaccggta aaggtctgcagagctggtatgaatatctggatcagtggcagctgcagcattatagcccgctggttgatc cggcactggcataagagttgaaaggaggtttcctccatgtgcctggcgttaccggggcaggttgtttcg ttaatgccgaactcggatccgctgttattaaccgggaaagttagctttggtggtattattaaaaccattag cctggcgtatgttccggaagttaaagttggcgattatgttattgttcatgttggttttgctatcagtattgttgat gaagaagcagcacaggagacactgattgatctggccgagatgggcgtttaattcctaaaaggaggt tttagccatgaagtacgttgacgaataccgcgacgcgcaggcagttgcccactaccgccaggccatt gcccgtgaaattaccaaaccgtggacgctgatggaaatttgtgggggccagacccatagcatcgtta aatatggtctggatgcattattaccgaaaaacttaaccttaatccacggtccgggttgtccggtttgtgtta cgccgatggaactgattgatcaggcattatggctggcaaaacagccggagattattttttgtagctttgg tgatatgctgcgcgtgccgggtagtggtgcagatctgctgagcattaaagcacaggggggagacgtt cgtatagtttattctccgttagattgtctggcgattgcgcgtgaaaatccgaatcgtgaagttgttttttttggt gtgggttttgaaactaccgccccggcaaccgcaatgacactgcatcaggcacgggcccagggtatt agcaattttagcttattatgtgcacacgtgttagttccgccggcgatggaagctctgctgggtaacccga atagcctggttcaagggtttttagcagcaggtcatgtttgtacggttaccggtgagcgggcgtatcagc atattgcagagaaatatcaggttccgatagttattaccggttttgaaccggttgatattatgcagggtatttt tgcatgtgttcgtcagctggagagcgggcagtttacatgtaataatcagtaccggcggtcggttcagcc gcagggtaacgcacatgcccagaaaattattgaccaggtttttgaaccggtggatcgtcattggcgtg gattaggtcttattccggcctcaggtttaggtttacgtccggcatttgcaccgtgggacgcagcagttaa attcgcaaatctgttacagacaatggctccgacaatgggtgaaaccgtttgtatttctggcgaaattttac agggtcagcgcaaacctagtgattgtcctgcatttggtaccatctgcaccccggaacaaccgctggg cgcccctatggttagcagtgaaggcgcttgtgccgcctattatcgttatcgtcagcaattaccggaacc ggttggtgccgcacgtgtttaattttgcaaaggaggtcctgccaatgaacctggtgtgtccggtgccgct ggaccgctacccgcaggttttactggcacacggggggggggggaagctgagtcagcagctgttaa aacagatttttctgccggcgtttggtgcatcagaaaccggtagccatgatgcagcagtttttaccgcaa atcagagcagcttagcatttacaacagattcctatgttatcaatccgctgttttttcctggtggtgatattggt agtcttgcagttcatggaaccgttaatgatttagcaatggcaggtgcaacaccgcgttatattagcgttg ggtttattctggaggagggtttaccgatggagacactttggcgtgttgcacaaagcctgggtcaggca gcacagaattgtggagttgaaatattaacaggtgataccaaagttgttgatcgtgggaagggagatg gtatttttattaatacatcgggtatcggtagtttagatcaccagcaaaccattcatccgaatcaggttcag gttggtgatcgtctgattctgagtggggatttaggacggcatggtatggcaattatggcagttcgtcagg gcctggaatttgaaacaaccattgaaagcgatagcgcaccggttcatcgtgaggttcaggctctgctg agcgcagggattccgattcactgtctgcgtgacttaacacgtggtggtctggcaagcgccgtgaacg aaattgcacaaacctcaggtgttacaatggctctgcgtgaaaccttaattccggttgaggcggaagttc aagccgcctgtgaactgctgggttttgatcctttatatgttgcgaacgaaggccgtttcctggccattgttc cgccggaagccgaacagaaaaccgttgaaattctgcagacctttcacccgcaggcgaccgcaatt ggtaccgttaccggcaagagtgcacagaccttaggtctggttagcctggagagtagcataggtgccc cacgtctgttagatatgattagcggagaacaactgccacgtatttgttaagactccaaaggaggctag attaatgctgaaaaccgttgccattcaggttcaggggcgcgttcagggggttggttttcggccgtttgttt acaccttagcccaggaaatgggtctgaatggctgggttaataactctacgcagggtgcaaccgttgtt attaccgcagatgagaaagcaattgcagattttaccgaacgtctgaccaaaacactgccgccaccg ggactgatcgaacaactggcagtggaacagctgccgctggaaagctttaccaactttaccattagac cgagtagcgatggtccgaaaaccgcaagcatcctgccagatctgagcacatgtagcgcctgtctga ccgaattatttgatcccagtgatcgtcgttatctgtacccttttattaattgtacccactgtggtcctcgctata ccattattgaagcactgccttatgaccgttgtcgtaccacaatggctcgttttcgtcagtgtacggattgtg aacgtgaatataagcagccgggggaccgccgttttcatgcacagccaaacgcgtgtccgcgttgtg gtccgcagctggcattctggaaccgtcagggtcaagttattgcagaagccaatgaagcactgaatttc gcagtagataatttaaaggtcggtaatattatcgcaatcaaaggtctgggtggttttcatttatgttgtgat gcaaccgattttgaagccgttgaaaaactgcgtttacgtaaacatcgcccggataagccgctggccg ttatgtacggtaatctgggtcagattgttgagcattatcagccgaataatttagaagttgagctgctgcag agcgcagcagcacctattgttcttctgaataaaaagaaacagctgattctggttgaaaatattgcaccg ggcaatccgcgtgtgggtgttatgctggcatataccccgttacatcacctgttacttaaaaagttaaaga agccgatggttgcaacctccggtaacttagcaggcgaacagatttgtattgacaatattgacgcactg acccgtttacaaaatattgccgacggctttctggttcacgatcgtccgattgtttgtccggttgacgatagt gttgttcagattgtggcaggtaaaccgttatttttaagaagagcccgcggttatgcaccgcagccgatta cccttcctaaacccacccagaaaaagttattagcaatgggaggccattataaaaataccgttgcaatt gcaaagcagaatcaggcatatgtaagccagcatttaggtgatttaaacagcgcaccaacctaccaa aatttcgaagaggcgatagcccatttatcacagctgtatgactttagtccccaggaaattgtcgcagat ctgcatccggattactttagccatcagtacgcagaaaaccaagccctgccggtgacgtttgtacagca tcattatgcacatattctggcagttatggcagaacatggtgttatggaagaaagcgttttaggcattgcat gggatggcaccggttatggtatggatggtaccatttggggtggtgaatttctgaaaattacgcagggg acctggcaaagaattgcacatctgcagccgtttcatctgttagggaatcagcaggcaattaaatatcc gcaccggattgcacttgctctgctgtggccgacattcggggacgattttagcgccgatagtctgggtaa ttggttaaattttaacaacggtttcaagaacaagatcaacagccgtttaaaccaagacttaaataataa gaacctgagacaactgtggcagcgtgggcaggcaccgctgacctcgagcatgggcagattatttga tggtatcgcaacactgattggtctgatcaatgaagtaacctttgaaggccaggcagcaattgcattag aggcacaaattatgccgaatctgaccgaagaatactatccgcttaccctgaataacaaagaaaaaa aactggcagttgattggcgtccgctgattaaagcaattaccaccgaagatcgtagcaaaaccaatct gattgcaaccaaatttcataatagcctggttaatctgattattaccattgcacagcagcagggtattgaa aaagttgcactgggtggtggttgttttcagaattgttatctgctggcaagcaccattaccgcactgaaaa aagcaggttttagcccgctgtggccgcgtgaactgccgccgaatgatggtgcaatttgtatgggtcag ctgctggcaaaaattcaggcacgtcagtatatttgttaactcaacaaaggaggagctggttatgccgg gtcagagcaccaaaagcaccctgattatcgggtacgggaacaccttacgtggggacgatggggtg gggcgctacctggcagaagaaatagcacagcagaactggccgcactgtggtgttattagcacacat cagctgaccccggaactggccgaagcaattgcagcagtggatagagtgatttttattgacgcccaac tgcaggaaagtgcaaatgaaccgtcagttgaagttgttgccctgaaaaccttagaacccaatgaatt aagtggagatctgggtcatcgtggtaatccgcgtgagctgctgaccttagccaaaatattatatggtgtt gaagtcaaagcgtggtgggttctgattccggcctttacctttgattatggtgagaaattatcgcccttaac agcacgtgctcaggccgaagcactggcacagattcgtccgctggttctgggggaacgttaacctag g

[0170] FIG. 3 represents the map of the pACYCDuet-1 plasmid used to construct the expression vector of at least one of the maturation factors HypA, HypB, HypC, HypD, HypE, HypF and HoxW by inserting SEQ ID NO:5 and/or SEQ ID NO:7 and/or SEQ ID NO:9 and/or SEQ ID NO:11 and/or SEQ ID NO:13 and/or SEQ ID NO:15 and/or SEQ ID NO:17 and/or SEQ ID NO:19 in pACYCDuet-1. pACYCDuet-1 is a 4008 bp plasmid, possessing an origin of replication for E. coli, a chloramphenicol resistance gene, two multiple cloning sites (MCS) containing numerous restriction sites, the T7 promoter and the T7 transcription terminator. It also contains the lacl gene encoding a transcription repressor. This repression of the transcription can be lifted by adding IPTG.

[0171] FIG. 4 shows the analysis of the digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW (concatenary sequence SEQ ID NO: 19) by the NcoI and HindIII restriction enzymes. Two DNA fragments at around 6000 bp and at around 4000 bp are highlighted, 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.

[0172] 3. Introduction of the Expression Vectors pET26b(+)+HoxH and pACYCDuet-1+HypABCDEFHoxW in E. coli.

[0173] The competent cells (DE3) BL21 of E. coli were used for the recombinant expression of the HoxH subunit of HoxEFUYH [NiFe]-hydrogenase from Synechocystis sp. PCC6803. These cells are routinely used for the production of recombinant protein under the control of the T7 promoter.

[0174] 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 having double resistance to kanamycin (50 μg/ml) and to chloramphenicol (25 μg/ml) were stored on agar medium at 4° C.

[0175] 4. Verification of the Presence of DNA Sequences of Interest in E. Coll.

[0176] It is possible that some colonies possess the two plasmids allowing resistance to the selection markers used without however possessing the DNA sequences of interest, namely HoxH and HypABCDEFHoxW. Therefore, it is important to verify the presence of these DNA sequences of interest. An experiment, well known to those skilled in the art and consisting of an extraction of the plasmid DNA from the colony followed by an enzymatic digestion of this plasmid DNA by the restriction enzymes used during the insertion of the DNA sequences of interests in plasmids, has been carried out.

[0177] Thus, an extraction of plasmid DNA, according to a protocol well known to those skilled in the art, was carried out on a colony having the double resistance to kanamycin and to chloramphenicol.

[0178] Enzymatic digestion was then carried out, according to a protocol well known to those skilled in the art. The digestion of the expression vector pET26b(+)+HoxH by the NdeI and BIpI restriction enzymes gives two DNA fragments, respectively the linearized plasmid pET26b(+) at 5300 bp and the HoxH DNA fragment of interest at 1500 bp (see FIG. 5). The digestion of the expression vector pACYCDuet-1+HypABCDEFHoxW by the NcoI and HindII restriction enzymes gives two DNA fragments, respectively the linearized plasmid pACYCDuet-1 at 4000 bp and the HypABCDEFHoxW DNA fragment of interest at 6500 bp (see FIG. 6). This confirms the presence in the bacterial colony of the HoxH and HypABCDEFHoxW sequence, respectively in the expression vectors pET26b(+) and PACYCDuet-1.

[0179] 5. Recombinant Production and Purification by Affinity Chromatography of HoxH in E. Coll.

[0180] To obtain the recombinant form of the HoxH subunit of HoxEFUYH [NiFe]-hydrogenase from Synechocystis sp. PCC6803, an E. coli colony containing the two expression vectors was used to inoculate 4 erlenmeyer flasks with a volume of 250 m each. The culture medium used is the 2XYT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl per litre) supplemented with 100 μM FeAmCi, 50 μM NiSO.sub.4, 50 μM cysteine, 50 μg/ml kanamycin and 25 μg/ml chloramphenicol. At an optical density (DO 600 nm) of 1.2, 0.2 mM IPTG are added to the culture in order to induce the recombinant production of the HoxH subunit at 18° C. and under stirring (stirring speed) of 200 rpm. The production time is 20 h at 18° C. The cells are then collected by centrifugation (15 min at 4500 rpm) before starting the purification process.

[0181] A purification method was developed in order to confirm the production of the recombinant form of the HoxH subunit of HoxEFUYH [NiFe]-hydrogenase from Synechocystis sp. PCC6803. This method (see FIG. 7) purifies HoxH to apparent homogeneity in a single affinity chromatography step. The method involves immobilized metal affinity chromatography (IMAC). The chelating agent is nitrilotriacetic acid (NTA) which allows the binding between the immobile phase and the metal ion. The immobilized metal is nickel (Ni). This chromatography allows a very specific separation of proteins containing a poly-histidine tag. After 20 h of recombinant production of HoxH at 18° C., 1 liter of culture of E. coli is collected by centrifugation (15 min at 4500 rpm). The cells are re-suspended in 25 ml of lysis buffer (20 mM sodium phosphate buffer pH 7.5 300 mM KCl+2 μl benzonase+50 μl MgCl.sub.2 1M+1 pellet composed of a cocktail of proteases inhibitors without EDTA) and then lysed by the French press. The supernatant (25 ml) is recovered by centrifugation (15 min at 15000 rpm), filtered (0.45 μm) and applied to the Ni-NTA column (1 ml) previously equilibrated in the wash buffer (20 mM sodium phosphate buffer pH 7.5 300 mM KCl+10 mM imidazole). The column is then washed with 35 ml of wash 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 through 5 stages with increasing concentration of imidazole, respectively 50 mM, 100 mM, 150 mM, 200 mM and 250 mM) (see FIG. 7).

[0182] The SDS-PAGE analysis of the various fractions obtained during the chromatography shows a predominant band close to 54 kDa, which is the expected size for the HoxH subunit, in the fractions eluted at a concentration of 100 mM, 150 mM, 200 mM and 250 mM imidazole (see FIG. 8). HoxH also shows an excellent degree of purity since no additional band is viewable in the fractions eluted at concentrations of 150 mM, 200 mM and 250 mM imidazole. This allows us to conclude that HoxH has been purified to apparent homogeneity in a single affinity chromatography step.

[0183] In order to confirm that it is indeed the recombinant form of the HoxH subunit comprising the active site of HoxEFUYH [NiFe]-hydrogenase from Synechocystis sp. PCC6803, an immuno-detection using a poly-histidine anti-tag antibody was carried out (see FIG. 9). A molecular weight marker containing a protein marked with a poly-histidine tag was added to serve as a positive control. The HoxH recombinant protein is well detected in the load, that is to say the supernatant derived from the lysis of the cells of E. coli and applied to the Ni-NTA affinity column. The HoxH recombinant protein is also present in the fraction not absorbed by the Ni-NTA column. This indicates that the loading capacity of the Ni-NTA column is exceeded and that a proportion of the HoxH recombinant protein could not bind to it. No signal appears in the “50 mM elution fraction”. The HoxH recombinant protein remains attached to the Ni-NTA column at this low imidazole concentration. Finally, the presence of the HoxH recombinant protein is confirmed by immuno-detection in the 200 mM fraction at the expected size, that is to say close to 54 kDa. This unambiguously confirms the purification of the HoxH recombinant protein in a single affinity chromatography step. When the HoxH recombinant protein is highlighted by immuno-detection in a fraction, some bands of low intensity also appear at molecular weights below 54 kDa. It is probably the HoxH recombinant protein partially degraded at the C-terminal end. The proportion of degraded HoxH recombinant proteins seems minimal because the SDS-PAGE analysis does not show the presence of these bands. As a reminder, the immuno-detection is an extremely sensitive method highlighting very small quantities of protein.

[0184] 6. Highlighting of Hydrogenase Activity in the HoxH Recombinant Protein

[0185] The ability to recombinantly express [NiFe]-hydrogenases allows a wide range of possibilities with a view to producing mutant forms with very different properties from the native enzyme. FIG. 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 activity test of [NiFe]-hydrogenases. FIG. 11 is a schematic representation (shaded arrow) of the electron transfer and the expected interactions between HoxEFUYH and NADPH and/or MV.

[0186] It is generally acquired by those skilled in the art that MV can directly transmit electrons to one or more FeS centers by avoiding FMN. However, as shown in FIG. 12, it is currently not known whether the single HoxH subunit, containing the active site of HoxEFUYH [NiFe]-hydrogenases from Synechocystis sp. PCC 6803, can accept electrons directly from MV and therefore produce hydrogen in the absence of additional redox relay.

[0187] In the context of the present invention, to test this possibility, the standard in vitro activity test of [NiFe]-hydrogenases in the presence of the HoxH recombinant protein and MV previously reduced by sodium dithionite is implemented (see FIG. 13). HoxH is able to take electrons from the previously reduced methyl-viologen to combine them with protons present in the buffer in order to produce hydrogen according to the equation H.sub.2.Math.2H.sup.++2e.sup.− which represents the hydrogenase-catalyzed reaction. This standard in vitro activity test of [NiFe]-hydrogenases, according to a protocol well known to those skilled in the art, includes the use of a 2 ml flask closed by an airtight and nitrogen degassed septum. 1 ml of reaction mixture, composed of 100 mM sodium dithionite and 10 mM MV dissolved in 10 mM phosphate buffer pH 6.8 and also nitrogen degassed, is added to the flask using a syringe. 200 μg of HoxH recombinant proteins are also added to the reaction mixture. The hydrogen production starts from the moment that the HoxH recombinant 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). The hydrogen production is continuously monitored using a previously calibrated hydrogen micro-sensor (Unisense, Aarhus, Denmark).

[0188] The activity of the HoxH recombinant protein can therefore be calculated since the quantity of HoxH recombinant protein added (in mg.Math.ml.sup.−1) is known, as is the speed of hydrogen evolution (μmol H.sub.2.min.sup.−1) for this specific quantity of recombinant protein added. This specific activity can, for example, be 0.1 μmol H.sub.2.min.sup.−1.mg.sup.−1 of enzyme.

[0189] Unexpectedly and surprisingly, the fact that the single catalytic HoxH subunit containing the active site of HoxEFUYH [NiFe]-hydrogenase from Synechocystis sp. PCC6803 can catalyze the reduction of protons, accepting electrons from a redox mediator (for example, methyl-viologen) without the intermediary of additional FeS centers serving as redox relays, has been highlighted in the context of the present invention.

[0190] In the context of the present invention, the standard in vitro activity test of [NiFe]-hydrogenases in the presence of the HoxH recombinant protein, benzyl viologen and hydrogen has also been implemented (see FIG. 14). HoxH is able to take electrons from hydrogen to donate them to benzyl viologen, producing protons on the way according to the equation H.sub.2.Math.2H.sup.++2e.sup.− which represents the hydrogenase-catalyzed reaction. This standard in vitro activity test of [NiFe]-hydrogenases, according to a protocol well known to those skilled in the art, includes the use of a 2 ml flask closed by an airtight and hydrogen gas saturated septum. 2 ml of reaction mixture, composed of 40 μmoles of benzyl viologen dissolved in 50 mM Tris buffer pH 7.6 and also hydrogen degassed, is added to the flask using a syringe. The experiment is carried out at 40° C. 340 μg of HoxH recombinant proteins are also added to the reaction mixture. The consumption of hydrogen and therefore the reduction of benzyl viologen starts from the moment that the HoxH recombinant proteins are added. The protein content was determined by the Bradford method (Bradford. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254). The reduction of benzyl viologen is monitored by spectrophotometry at a wavelength of 578 nm. A molar extinction coefficient of 8,600 M.sup.−1 cm.sup.−1 was taken into account.

[0191] The activity of the HoxH recombinant protein can therefore be calculated since the quantity of HoxH recombinant protein added (in mg.Math.ml.sup.−1) is known, as is the speed of hydrogen consumption (μmol H.sub.2.min.sup.−1), equivalent to the speed of reduction of benzyl viologen for that specific quantity of recombinant protein added. This specific activity can, for example, be 0.1 μmol H.sub.2.min.sup.−1.mg.sup.−1 of enzyme.

[0192] Unexpectedly and surprisingly, the fact that the single catalytic HoxH subunit containing the active site of HoxEFUYH [NiFe]-hydrogenase from Synechocystis sp. PCC6803 can catalyze the oxidation of hydrogen, producing protons and donating electrons to a redox mediator (for example, benzyl viologen) without the intermediary of additional FeS centers serving as redox relays, has been highlighted in the context of the present invention.

[0193] In the absence of the expression of the other subunits of the HoxEFUYH pentamer, the problems inherent in the prior art are solved at least in part: the HoxH recombinant protein is able on its own to catalyze the reduction of protons, accepting electrons from a redox mediator (for example methyl-viologen) without the intermediary of additional FeS centers serving as redox relays. The HoxH protein is also able on its own to catalyze the oxidation of H.sub.2, reducing a redox mediator (for example benzyl viologen) without the intermediary of an additional FeS center serving as a redox relay. This exceptional characteristic demonstrates the great advantage of the present invention, in particular the recombinant production of a single subunit possessing the active site of [NiFe]-hydrogenase and being catalytically active.

[0194] 7. Recombinant Production, Purification by Affinity Chromatography and Highlighting of Hydrogenase Activity in the HoxH Recombinant Protein Recombinantly Produced in E. coli in the Absence of Exogenous Maturation Factors

[0195] In a manner similar to example 3 above, the expression vector pET26b(+)+HoxH is introduced into E. coli (competent cells BL21 (DE3)) by transformation according to the traditional method of heat shock well known to the skilled in the art, and this in the absence of the expression vector pACYCDUET-1+HypABCDEFHoxW. The transformants have resistance to kanamycin (50 μg/ml).

[0196] In a manner similar to example 4 above, the presence of the HoxH DNA sequence of interest was verified by the chain of experiments well known to those skilled in the art, which are the extraction of plasmid DNA and the enzymatic digestion.

[0197] In a manner similar to example 5 above, the HoxH protein was recombinantly produced in E. coli, but in the absence of the plasmid pACYCDUET-1+HupABCDEFHoxW encoding the exogenous maturation factors HupABCDEFHoxW. The HoxH protein was then purified by affinity chromatography, in a manner similar to example 5 above.

[0198] In a manner similar to example 6 above, the hydrogenase activity of the HoxH protein recombinantly produced in E. coli in the absence of exogenous maturation factors was highlighted according to the protocol involving MV and well known to those skilled in the art

[0199] Thus, within the context of the present invention, the standard in vitro activity test of [NiFe]-hydrogenases in the presence of the HoxH recombinant protein and MV previously reduced by sodium dithionite was implemented (see FIG. 15). HoxH is able to take the electrons from the previously reduced methyl-viologen to combine them with protons present in the buffer in order to produce hydrogen according to the equation H.sub.2.Math.2H.sup.++2e.sup.− which represents the hydrogenase-catalyzed reaction. This standard in vitro activity test of [NiFe]-hydrogenases, according to a protocol well known to those skilled in the art, includes the use of a 2 ml flask closed by an airtight and nitrogen degassed septum. 1 ml of reaction mixture, composed of 100 mM sodium dithionite and 10 mM MV dissolved in 10 mM phosphate buffer pH 6.8 and also nitrogen degassed, is added to the flask using a syringe. 750 μg of HoxH recombinant proteins are also added to the reaction mixture. The hydrogen production starts from the moment that the HoxH recombinant proteins are added (moment indicated by an arrow in FIG. 15). The protein content was determined by the Bradford method (Bradford. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254). The hydrogen production is continuously monitored using a previously calibrated hydrogen micro-sensor (Unisense, Aarhus, Denmark).

[0200] The activity of the HoxH recombinant protein can therefore be calculated since the quantity of HoxH recombinant protein added (in mg.Math.ml.sup.−1) is known, as is the speed of hydrogen evolution (μmol H.sub.2.min.sup.−1) for this specific quantity of recombinant protein added. This specific activity can, for example, be 0.1 μmol H.sub.2.min.sup.−1.mg.sup.−1 of enzyme.

[0201] It has been highlighted, in the context of the present invention, the fact that the single HoxH catalytic subunit containing the active site of the HoxEFUYH [NiFe]-hydrogenase from Synechocystis sp. PCC6803 can catalyze the reduction of protons, accepting electrons from a redox mediator (for example, methyl-viologen) without the intervention of maturation factors HupABCDEFHoxW exogenous to E. coli.

[0202] The present invention has been described in relation to the specific embodiments, which have a purely illustrative value and should not be considered as limitative. In general, it will appear obvious to the person skilled in the art that the present invention is not limited to the examples illustrated and/or described above.

[0203] The use of the verbs “comprise”, “include”, or any other variant, as well as the conjugations thereof, can in no way exclude the presence of elements other than those mentioned.

[0204] The use of the indefinite article “a”, “an”, or of the definite article “the”, to introduce an element does not exclude the presence of a plurality of these elements.