# Patent

## Title
Hydrogen production

## Abstract

The enzyme, iron hydrogenase (HydA), has industrial applications for the production of hydrogen, specifically, for catalyzing the reversible reduction of protons to molecular hydrogen. The present invention relates to the isolation of a nucleic acid sequence from the algae Scenedesmus obliquus, Chlamydomonas reinhardtii, and Chlorella fusca that encodes iron hydrogenase. The invention further discloses the genomic nucleic acid, c-DNA and the protein sequences for HydA. The genes and gene products may be used in a photosynthetic process for hydrogen production which includes growing a microorganism containing the gene coding for HydA in a culture medium under illuminated conditions sufficient to accumulate an endogenous substrate; depleting a nutrient selected from the group consisting of sulfur, iron, and manganese from the medium; then allowing the culture to become anaerobic by consumption of an endogenous or exogenous substrate in the light.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Ser. No. 60/269,872, which was filed with the U.S. Patent and Trademark Office on Feb. 16, 2001.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the isolation of a nucleic acid sequences that encode an enzyme that catalyzes the transfer of electrons to protons for the production of molecular hydrogen, and more particularly to iron hydrogenase and genes encoding for the iron hydrogenase in microscopic organisms known as unicellular green algae.
2. Prior Art
Molecular hydrogen is currently being considered as a candidate for replacing or supplementing fossil fuels and as a source of clean energy. A potential method for producing hydrogen on a commercial scale is the photobiological production of hydrogen by eukaryotic organisms. Green algae respond to anaerobic stress by switching the oxidative pathway to a fermentative metabolism. The fermentation of organic compounds and residual photosynthetic electron trasport in the green algae are associated with hydrogen evolution. The key enzyme hydrogenase, which is synthesized only after an anaerobic adaptation, catalyzes the reversible reduction of protons to molecular hydrogen. This method is capable of generating renewable hydrogen fuel from light and water, which are among nature's most plentiful resources.
The ability of green algae, such as Chlamydomonas reinhardtii, to produce hydrogen from water has been recognized for over 55 years. This reaction is catalyzed by a reversible hydrogenase, an enzyme that is induced in the cells after exposure to a short period of anaerobiosis. However, the activity of the hydrogenase is rapidly lost when cells are illuminated because of the immediate inactivation of the reversible hydrogenase by photosynthetically generated O2.
Methods have been devised to circumvent the hydrogenase inactivation problem. U.S. Pat. No. 4,532,210 discloses the biological production of hydrogen in an algal culture using an alternating light and dark cycle. The process comprises alternating a step for cultivating the alga in water under aerobic conditions in the presence of light to accumulate photosynthetic products (starch) in the alga, and a step for cultivating the alga in water under microaerobic conditions in the dark to decompose the accumulated material by photosynthesis to evolve hydrogen. This method uses a nitrogen gas purge technique to remove oxygen from the culture.
U.S. Pat. No. 4,442,211 discloses that the efficiency of a process for producing hydrogen, by subjecting algae in an aqueous phase to light irradiation, is increased by culturing algae which has been bleached during a first period of irradiation in a culture medium in an aerobic atmosphere until it has regained color and then subjecting this algae to a second period of irradiation wherein hydrogen is produced at an enhanced rate. A reaction cell is used wherein light irradiates the culture in an environment which is substantially free of CO2 and atmospheric O2. This environment is maintained by passing an inert gas (e.g. helium) through the cell to remove all hydrogen and oxygen generated by the splitting of water molecules in the aqueous medium. Although continuous purging of H2-producing cultures with inert gases has allowed for the sustained production of H2, such purging is expensive and impractical for large-scale mass cultures of algae. In view of the foregoing, there remains a need for a microorganism that produces a hydrogenase enzyme suitable for use in a sustainable process of photosynthetic hydrogen production.
SUMMARY
Accordingly, it is an object of the present invention to provide a gene encoding for hydrogenase and a method for using the gene product for the microbial production of molecular hydrogen. Specifically, the invention provides isolated nucleic acid sequences encoding a stable hydrogenase enzyme (HydA) that will catalyze the reduction of protons to form molecular hydrogen.
Another object of the present invention is to provide isolated nucleic acid sequences encoding a protein that catalyzes the reduction of protons to form molecular hydrogen comprising SEQ. ID. NO. 1. SEQ. ID. NO. 1 comprises a nucleic acid sequence that encodes Scenedesmus obliquus HydA.
It is yet a further object of the present invention to provide isolated nucleic acid sequences encoding a protein that catalyzes the reduction of protons to form molecular hydrogen comprising SEQ. ID. NO. 2. SEQ. ID. NO. 2 comprises a nucleic acid sequence that encodes Chlamydomonas reinhardtii HydA.
A further object of the present invention is to provide fragments of the nucleic acid sequence comprising SEQ. ID. NO.1 or SEQ. ID. NO.2, encoding iron hydrogenase, that code for products that maintain the biological activity necessary to catalyze the transfer of electrons to protons in a process for producing molecular hydrogen. Such fragments can be either recombinant or synthetic or a combination thereof.
The features of the invention believed to be novel are set forth with particularity in the appended claims. However the invention itself, both as to organization and method of operation, together with further objects and advantages thereof may be best be understood by reference to the following description taken in conjunction with the accompanying drawings in which:



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of S. obliquus HydA genomic and cDNA structures.
FIG. 2 is a comparison of the S. obliquus-derived iron hydrogenase amino acid sequence with HydA sequences derived from other organisms.
FIG. 3 is a schematic diagram showing the conserved cysteine residues and other important amino acids of the H cluster
FIG. 4(A) is a schematic view of the structure of S. obliquus HydA.
FIG. 4(B) is a schematic view of the S. obliquus electron donor ferredoxin.
FIG. 5(A) is a schematic map of the cDNA and the genomic DNA region of HydA from C. reinhardtii.showing the structural features of the HydA cDNA. Coding regions are marked as large arrows with the transit peptide shown in black.lines indicate 5′ and 3′ URTs.
FIG. 5(B) is a schematic map of the cDNA and the genomic DNA region of HydA from C.reinhardtii.showing the structural features of the HydA cDNA. In FIG. 3 (B), the mosaic structure of hydA is illustrated by gray (exons) and white (introns) boxes. The RNA and DNA probes that were used for the blotting experiments are noted on the Figure.
FIG. 6 shown the nucleotide sequence of the hydA cDNA and the deduced amino acid sequence of the hydrogenase from C. reinhardtii. 
FIG. 7 is a schematic diagram showing the light-dependent photoevolution of hydrogen in green algae.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
The isolation, purification and biochemical and genetic characterization of a novel iron hydrogenase from S. obliquus and C. reinhardtii and C. fuscus is disclosed.
I. Scenedesmus obliquus 
S. obliquus Algal Strains and Growth Conditions
Wild-type S. obliquus Kützing 276-6 was obtained originally from the culture collection of algae at the University of Göttingen Cells were cultured photoheterotrophically in batch cultures at 25° C. under continuous irradiance of 150 μmol photons per square meter per second. For anaerobic adaptation, 4-liter cultures were bubbled with air supplemented with 5% CO2. After harvesting the cells in the mid-exponential exponential stage of growth, the pellet was resuspended in fresh Tris acetate phosphate (TAP) medium. The algae were anaerobically adapted by flushing the culture with argon in the dark.
Hydrogen Evolution Assay
The in vitro hydrogenase activity was measured by using a Hewlett Packard (HP 5890, Series II) gas chromatograph, equipped with a thermal conductivity detector and a molecular sieve column. Methyl viologen reduced by sodium dithionite was used as an electron donor. 1 unit is defined as the amount of hydrogenase evolving 1 mmol of molecular hydrogen (H2) per minute at 25° C.
The in vivo activity in the presence of different inhibitors of the photosynthetic electron flow was determined as described by Happe et al., in: European Journal of Biochemistry, 214, 475-481 (1993). After anaerobic adaptation, algal cells were harvested, diluted in fresh Tris acetate phosphate medium, and transferred to sealed tubes. Inhibitors were added 1 hour before H2 evolving activity was measured. Cells were broken by sonification. Thylakoid membranes and photosynthetic transport chain remained intact as demonstrated by oxygen polarography. Ferrodoxin of both C. reinhardtii and S. obliquus was isolated according to the method of Schmitter et al. (Eur. J.l of Biochem., 172, pages 405-412 (1988)).
Rapid Amplification of cDNA Ends-Polymerase Chain Reaction (RACE-PCR)
RACE-PCR was performed with the Clontech SMARTTMRACE cDNA Amplification Kit (Clontech Laboratories, Palo Alto, Calif.) according to the manufacturer's recommendations, except for modification of the PCR and hybridization conditions. Starting material consisted of 1 μg of mRNA from anaerobically adapted cells. The reverse transcription reaction was carried out with a Moloney murine leukemia virus reverse transcriptase in two separate reaction tubes containing either the 5′ or the 3′ RACE-PCR specific primer from the kit. The cDNA of each sample served as template for the following PCR. For the 5′-RACE-PCR, a Universal Primer Mix (UPM) and the antisense primer, Sc7, were used. The amplification of the 3′-cDNA end was performed with a UPM and the sense primer Sc6. To obtain more distinct PCR signals, the PCR was repeated for both reactions with nested universal primers and designed primers (inverse Sc6 and inverse Sc7, respectively) using a dilution of the products of the first PCR as template.
Primer Extension
RACE-PCR was also implemented to map the transcription initiation site of the hydA mRNA. A gene-specific primer (Sc17) was used to carry out the first strand cDNA synthesis with the Superscript II reverse transcriptase (Life Technologies, Rockville, Md., USA) and 200 ng of mRNA as template. PCR was performed using either Sc12 or Sc27 and the SMARTTM specific adapter primer UPM. Two different DNA fragments of 234 bp and 183 bp were amplified under standard PCR conditions. Both fragments were cloned into the pGEMTMT-Easy vector (Promega, Madison, Wis., USA) and sequenced using primers from the polylinker of the vector.
Genome Walking with Genomic DNA
Applying the Clontech Walker Kit (Clontech Laboratories), genomic libraries from S. obliquus were generated by digestion with different blunt-end cutting endonucleases (NaeI DraI, PvuIIm, HincII and EcORV) and by adapter ligation at the ends of the resulting DNA fragments. These libraries were utilized as independent templates in five different PCR reactions. Two gene-specific primers (Sc27, Sc35) derived from the hydA cDNA sequence of S. Obliquus were used in combination with a kit adapter primer (AP1) in a first PCR reaction. Subsequently, 1 μl of the first PCR served as a template in a secondary PCR, applying two nested gene-specific primers (i-Sc10, Sc32) along with a nested kit adapter primer (AP2). The resulting products were cloned into pGEMTMT-Easy and sequenced. Sequencing was performed by the dideoxy termination method (see, for example: Sanger et al., Proc. Natl. Acad. Sci., U.S,A., 74, 5463-5467 (1977)).
Purification of the Fe-Hydrogenase
40-liter cultures of Scenedesmus obliquus were grown heterotrophically. After centrifugation (10 min, 5000×g) the pellet we re-suspended in 200 ml TAP medium. The cells were anaerobically adapted by flushing the solution with argon for 1 hour in the dark. All further purification steps were performed in an anaerobic chamber (Coylab, Ann Arbor, Mich., USA). The cells were disrupted in a 50 mM Tris/HCl, buffer pH 8.0, 10 mM sodium dithionite by vortexing 3 min with glass beads. The further purification steps were made as described hereinbelow for the isolation of the Fe-hydrogenase of Chlamydomonas reinhardti. Automated Edman degradation of the N-terminal site of the protein was performed with an Applied Biosystem model 477A sequencer with online analysator model 120 A.
RNA Blot Hybridization
Total RNA of S. Obliquus was isolated according to the method described by Johanningmeier et al. (J. Biol. Chem. 259, 13541-13549 (1984)). Equal amounts (20 μg) were separated electrophoretically on 1.2% agarose gels containing formaldehyde. The RNA was transferred onto nylon membranes (Hybond+, Amersham) and hybridized with RNA probes labeled DIG-dUPT using in vitro transcription methodology. A 1.3 kb EcOR1 cDNA fragment was used to detect transcripts with a hydA gene, while a DIG-dUPT-labeled cDNA encoding constitutively expressed plastocyanin, was used as a control. Hybridization reactions were carried out using protocols supplied by the manufacturer (Roche Diagnostics, Mannheim, Germany).
Sequence Analysis Software
Nucleic acid and protein sequences were analyzed with the programs Sci Ed Central (Scientific Educational Software) and ClustalW. The Blast server of the National Center for Biotechnology Information (Bethesda, Md.) was used for database searches.
Recombinant Expression in E. coli 
The hydA open reading frame was amplified by PCR using the primer pair Sc29 and Ac30 containing flanking NdeI-BamHI sites. The PCR product was cloned into the pGEMTMT-Easy vector. After digestion with NdeI-BamHI, the hydA gene was cloned into the corresponding site of the pET9a expression vector (Promega), producing pLF29.2. The insert of pLF29.2 was sequenced confirming that the fragment contained the exact full coding region of the hydrogenase without transit peptide. E. coli strain BL21(DE3)pLysS was transformed with pLF29.2. Expression was induced with 1 mM isopropyl-thio-β-D-galactoside at an OD600 of 0.3. Pelleted cells were re-suspended in lysis buffer (100 mM Tris/HCl; 4 mM EDTA; 16% Glycine; 2% SDS; 2% Mercaptoethanol; 0.05% Bromophenolblue; 8 M Urea). After heating, the protein extract was separated by 10% SDS-PAGE and blotted onto a PVDF membrane. Western blot analyses were performed using antisera against the Fe-hydrogenase of Chlamydomonas reinhardtii at 1:1000 dilution.
Results for Scenedesmus obliquus 
Induction of Hydrogenase Activity and Purification of the Fe-hydrogenase Protein
Anaerobic adaptation is the most efficient way to induce hydrogenase activity in Scenedesmus obliquus. Bubbling the alga culture in the dark with argon led to a dramatic increase (10-fold) of hydrogenase activity during the first 2 hours. The enzyme of S. obliquus was purified to homogeneity by successive column chromatography. Since the enzyme is irreversibly inactivated by very low levels of oxygen, all purification steps were performed under strictly anaerobic conditions and in the presence of reducing agents (dithionite). The purification scheme resulted in a 5200-fold purification of HydA with 5% recovery (data not shown). The most powerful step for purifying the protein was a Q-Sepharose high performance column chromatography with pH gradient elution. Gel infiltration chromatography of hydrogenase on a calibrated Superdex-75 column resulted in a single activity peak corresponding to a molecular mass of 45 kDa. The monomeric structure of the enzyme could also be shown on a SDS polyacrylamide gel after Coomassie-blue staining (data not shown). The N-terminal sequence of HydA was determined by Edman degradation. The protein sequence (AGPTAECDRPPAPAPKAXHWQ) is, except for two amino acids, identical to the amino acid sequence deduced from the DNA data (AGPTAECDCPPAPAPKAPHWQ). In the course of the purification procedure there was no indication of a second hydrogenase in S. obliquus because the hydrogenase activity was never separated into distinct fractions. Biochemical data show a high similarity of HydA to the Fe-hydrogenase from C. reinhardtii (Table 1). The enzymes have a high temperature optimum of about 60° C., are strongly inhibited by O2 and CO, and catalyze the H2-evolution with a typical high specific activity. Experiments with inhibitors of translators on ribosomes (data not shown) and analysis of the gene structure show that HydA from S. obliquus is translated in the cytoplasm and transported to the chloroplast.







TABLE I







Biochemical data comparison of purified iron hydrogenases from


C. reinhardtii and S. obliquus










C. Reinhardtii
S. obliquus





Size
49 kDa
44.5 kDa


Specific activity
935 U/mg protein
700 U/mg protein


Temperature optimum
60° C.
60° C.


pH optimum
6.9
7.3


Localization
chloroplast stroma
chloroplast


Coding site
nuclear
nuclear


pI value
5.3
5.17


KM value (MV)
830 μM
800 μM


KM value (ferredoxin)
35 μM
Not determined









Ferredoxin is the Natural Electron Donor of the Fe-Hydrogenase

Hydrogenase activity was determined in intact and broken cells after anaerobic adaptation. The integrity of the photosynthetic electron transport in the sonified cell preparation was demonstrated by the rate of oxygen evolution (154 μmole O2/mg Chl×h). This rate corresponds to 85% of the oxygen evolution measure with intact Scenedesmus cells.
In S. obliquus, the hydrogen evolution is linked to the photosynthetic electron transport chain through PSI. As shown in Table II, the cells were still able to photoproduce hydrogen when electron flow on the PSII was blocked by DCMU. In contrast, addition of DCMIB resulted in inhibition of the H2-production, thus giving evidence of the involvement of PSI in the supply of electrons to hydrogenase. With reference to Table II, after anaerobic adaptation, cells were harvested, diluted in fresh, TAP medium, and incubated with inhibitors as described herein. α-PetF-antibody was raised against spinach ferredoxin. In Table II, DCMU=3-(3,4-dichlorophenyl)-1,1-dimethylurea; DBMIB=2,5-dibromo-3-methyl-6 isopropyl-p-benzochinone; Sulfo-DSPD=sulfodisalicylidinepropanediamin; DCPIP=2,6-dichlorophenolindophenol. The electron transport from PSI to ferredoxin was inhibited using the artificial electron acceptor DCPIP. In this reaction, DCPIP is reduced instead of ferredoxin and electron transfer to hydrogenase is interrupted.







TABLE II







Effects of different photosynthetic inhibitors on hydrogenase activity











Hydrogenase activity




Units/mg chlorophyll







Intact cells (control)
0.11 



+ DCMU (10−5 M)
0.10 



+ DBMIB (10−5 M)
0.005



Broken cells
0.1 



+ DCMU (10−5 M)
0.11 



+ DBMIB (10−5 M)
0.006



+ DCPIP (10−4 M)
0.003



+ sulfo-DSPD (10−4 M)
0.003



+ α-PetF-antibody (1:1000)
0.008










Hydrogenase activity was dramatically reduced (up to 30-fold) by the ferredoxin antagonist sulfo-DSPD (Table II). Similar results were achieved with α-PetF-antibodies that specifically recognize the ferredoxin protein. In both cases, the hydrogenase enzyme cannot evolve hydrogen, thus demonstrating the role of ferredoxin as the obligatory donor for the hydrogenase reaction.
The electron transfer properties of different plant-type ferredoxins were measured in vitro with dithionite as a reducing reagent. The ferredoxin proteins of spinach, C. reinhardtii and S. obliquus were comparable regarding their capability to reduce purified S. obliquus hydrogenase. In this assay, H2-evolving activities of 420, 390 and 350 U/mg protein with S. obliquus, C. reinhardtii and spinach ferredoxin, respectively, were observed. No hydrogen production could be measured with other possible electron donors like cytochrome and NADPH. In D. desulfuricans, the Fe-hydrogenase has been reported to catalyze both hydrogen production and uptake with low potential multiheme cytochromes such as cytochrome c3.
Molecular Characterization of hdyA Encoding a Fe-Hydrogenase
In order to isolate the gene encoding a Fe-hydrogenase in S. obliquus, polyA+ RNA was isolated from cell cultures after one hour of anaerobic adaptation. Isolated RNA was transcribed and amplified by RT-PCR using oligonucleotides derived from conserved regions within the C. reinhardtii HydA gene (Happe, unpublished results). The complete cDNA clone of 2609 bp was obtained by 5′- and 3′-RACE PCR. It contains an open reading frame of 1344 bp encoding a polypetide of 448 amino acids (FIG. 1) followed by an extensive 3′ UTR of about 1100 bp. The coding region of S. obliquus hydA exhibits features common to other green algae such as high GC content (64.2%) and a characteristic putative polyadenylation signal, TGTAA, 15 bp: upstream of the polyA+ sequence.
In order to examine the exon-intron structure and the promoter region of the hydA gene, about 5 kb of the genomic DNA from S. obliquus were sequenced. The gene comprises 5 introns with a total size of 1310 bp (FIG. 1) whose 5′- and 3′-end contain typical plant splice donor and acceptor sites that follow the GT/AG rule. In FIG. 1, which is a schematic representation of S. obliquus hydA genomic and cDNA structures, the coding region of the hydA cDNA is illustrated as a large arrow with the transit peptide shown in black. The untranslated 59 and 39 sequences are marked as lines. The arrows below indicate the sequencing strategy; each arrow represents an independent sequence determination. TSP, transcription start point; ATG, start codon. The mosaic structure of hydA is indicated by gray (exons) and white (introns) boxes. The S2 probe and different restriction enzymes that were used in the Southern blot experiments are indicated on the figure.
A genomic southern blot was probed with a 750 bp PCR fragment to determine the copy number of the hydA gene. Single bands were observed in lanes with samples digested with HincII, EcORV and NdeI and a double band in the lane containing genomic DNA digested with SacI. The band migration positions matched the sizes predicted from the sequence of the hydA gene, indicating that HydA is encoded by a single copy gene. The same hybridization pattern was observed even under low stringency conditions (hybridization temperature 50° C.; data not shown). The transcription start position was determined by primer extension using RACE-PCR and was found 139 bp upstream of the ATG start codon. Several primers within 100 bp of the 5′-end of the known hydA cDNA were designed to confirm the accuracy of the transcription initiation site. All of the sequenced PCR clones had the same 5′-ends at position +1. As described for other green algae genes, a highly conserved TATA box element upstream of the transcription start point is absent (see, for example: C. D. Siflow in: The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp 25-40, (J. D. Goldschmidt-Clermont et al., eds.) Kluwer Academic Publishers, Dorecht, The Netherlands (1998)). However, the TACATAT motive at position −25 in a GC rich region shows similarities to other TATA motives in C. reinhardtii and therefore might be involved in gene expression.
HydA is a Novel Type of Fe-Hydrogenase
The polypeptide derived from the cDNA sequence has a length of 448 amino acids and a predicted molecular mass of 48.5 kDa (44.5 kDa excluding the transit peptide); consequently HydA is the smallest hydrogenase protein known so far. The N-terminus of HydA is basic and contains numerous hydroxylated amino acids and a Val-X-Ala motive at position 35, a characteristic feature of chloroplast transit peptides. The processed HydA protein is compared with four bacterial and two eukaryotic Fe-hydrogenases as shown in FIG. 2. FIG. 2 is a comparison of the S. obliquus-derived iron hydrogenase amino acid sequence with HydA sequences derived from other organisms. In FIG. 2, the protein alignment was done by using the Vector NTI program (InforMax). White letters with black background indicate amino acids identical to the HydA protein. Black letters with gray background indicate conserved changes of the amino acids. S. o., S. obliquus (this work); M. e., Megasphaera elsdenii; D. d., D. desulricans; T. v., Trichomonas vaginalis; C. p., C. pasteurianum; T. m., T. maritima; N. o., N. ovalis. 
The homology in the carboxy-terminal region of all proteins is quite striking. For example, the S. obliquus HydA protein shows 44% identity and 57% similarity to the C. pasteurianum Fe-hydrogenase. The H-cluster in S. obliquus might be coordinated by four cysteine residues at positions 120, 175, 335, and 340. Other strictly conserved amino acid structures such as FTSCCPGW (334-350), TGGVMEAALR (474-483) and MACPGGCXXGGGQP (586-589) probably define a pocket surrounding the active center as shown by the structural data of C. pasteurianum and D. desulfuricans. On the other hand, the N-terminal region is completely different from all other Fe-hydrogenases. The protein sequences of the other enzymes comprise at least two [4Fe-4S] ferredoxin-like domains (called “f-cluster”) which are necessary for the electron transport from the electron donor to the catalytic center. The Fe-hydrogenases of C. pasteurianum, Thermotoga maritima and Nyctotherus ovalis contain an extra [4fe-4S] cluster and one [2Fe-2S] center. This N-terminal domain with the F-cluster or other [Fe-S] centers is completely lacking in HydA of S. obliquus. This indicates that there is a direct electron transport pathway from the exogenous donor to the H-cluster.
To verify that the isolated cDNA encodes a Fe-hydrogenase, the hydA clone was expressed in the heterologous system E. coli. One band of recombinant HydA was observed on SDS-PAGE at approximately 44 kDa, in agreement with the molecular mass of the polypetide predicted from the cDNA sequence. Antibodies raised against the HydA protein of the C. reinhardtii, which cross-react with other Fe-hydrogenases but not with NiFe-hydrogenases (data not shown), were applied in Western blot analysis. One distinct signal with the over-expressed HydA protein of S. obliquus was obtained. The lysate of induced E. coli cells exhibited no hydrogenase activity. This result corresponds to observations by Voordouw et al. (Eur. J. Biochem., 162, 31-36 (1987)) and Stokkermans et al. (FEMS Microbiol. Lett., 49, 217-222 (1989)) who also detected no H2-production of recombinant Fe-hydrogenases in E. coli cells. The reason for that might be that the bacterial cells do not have the ability to assemble the special H-cluster of the Fe-hydrogenases.
Rapid Induction of hydA mRNA During Anaerobic Adaptation
The regulation of the hydA gene expression was examined by Northern blot analysis and reverse transcription-PCR (RT-PCR). Aerobically grown cells of S. obliquus did not show a hydrogenase activity. Total RNA and mRNA were isolated from cells which were induced by argon bubbling for 0, 1 and 4 hours. Northern blot analysis and RT-PCR demonstrated that the hydA gene is expressed after anaerobic adaptation. There is a very weak signal without adaptation (t=0), but strong signals of the transcript could be detected after anaerobic induction. The full length of the hydA cDNA clone was confirmed by the transcript signal (2.6 kb) on the Northern blot.
Discussion
In green algae, the occurrence of a hydrogen metabolism induced by anaerobic conditions is well established. Despite the great interest in hydrogen evolution for practical applications (“biophotolysis”), the hydrogenase genes from green algae have heretofore not been isolated. The hydA gene and the HydA protein of Scenedemus obliquus presented herein belong to the class of Fe-hydrogenases.
Fe-hydrogenases have been isolated only from certain anaerobic bacteria and some anaerobic eukaryotes as well as from the anaerobically adapted green alga C. reinhardtii (T. Happe et al., Eur. J. Biochem. 214, 475-481 (1993)). The enzymes are found to exist in monomeric, dimeric and multimeric forms; however, in eukaryotes, only monomeric proteins have been isolated.
The HydA protein of S. obliquus is synthesized in the cytoplasm. The first 35 residues M1 to A35) of the amino acid sequence derived from the cDNA sequence are supposed to function as a short transit peptide which routes the nuclear encoded protein to the chloroplast. Several positively charged amino acids which describe a typical feature for algal transit peptides are found in HydA. The three terminal residues of the signal sequence, Val-X-Ala, constitute the consensus for stromal peptidases.
The hydrogenase of S. obliquus represents a novel type of Fe-hydrogenase. The monomeric enzyme of 448 amino acids and a calculated molecular mass of 44.5 kDA for the processed protein is the smallest Fe-hydrogenase isolate so far. The protein sequence consists of an unusual N-terminal domain and a large carboxyterminal domain containing the catalytic site. The structurally important C-terminus of the S. obliquus HydA sequence is very similar to that of other Fe-hydrogenases. Four cysteine residues at positions C120, C175, C336 and C340 coordinate the special [6Fe] cluster (H-cluster) of the active site. A number of addition residues define the environment of the catalytic center. Peters et al. postulated twelve amino acids in C. pasteurianum to form a hydrophobic pocket around the cofactor (Science, 282, 1853-1858 (1998)). Ten residues are strictly conserved while two amino acids vary within the Fe-hydrogenase family (S232, I268, in C. pasteurianum, A119, T155 in T. vaginalis and A44, T80 in S. obliquus). A small insertion of 16 amino acids is noted in S. obliquus but this addition occurs in an external loop of the protein and probably has no special function.
Until now, all Fe-hydrogenases possess a ferredoxin-like domain in the N-terminus coordinating two [4Fe4S] clusters (FS4A, FS4B, as shown in FIG. 3. FIG. 3 is a schematic diagram showing the conserved cysteine residues and other important amino acids of the H cluster. In FIG. 3, the protein is illustrated as a large gray arrow. Small arrows indicate parallelograms which demonstrate conserved amino acids in the protein. Cysteines participating at the coordination of the [Fe-S] clusters are gray, whereas identical amino acids are black. An insertion of 16 amino acids in the S. obliquus protein is illustrated as a spotted bar. FS4 indicates the [4Fe-4S] cluster; and FS2 indicates the [2Fe-2S] cluster.
The iron sulfur cluster facilitates the transfer of electrons between external electron donors or acceptors and the H-cluster. The N-terminus of the S. obliquus protein is strongly reduced compared to other Fe-hydrogenases and no conserved cysteines are found. Therefore it is postulated that all accessory Fe-S clusters (FS2, FS4A, FS4B, FS4C) are missing. No indication of a second subunit has been observed during purification of the protein.
In contrast to earlier observations in S. obliquus, the present inventor could neither detect the postulated two subunits of a potential NiFe-hydrogenase, nor could he find a Ni-dependency related to the hydrogenase activity. Francis reported about two forms of hydrogenases in S. obliquus (Photosynthetica, 23, 43-48 (1989)), but although the present inventor used the same alga strain and identical adaptation conditions, a second hydrogenase activity was not detected during the purification steps.
Physiological studies by others have shown that the hydrogen evolution is coupled to the light reaction of the photosynthesis. In contrast to earlier observations in S. obliquus, the measurment of PSII independent H2-production was not influenced by DCMU. The electrons required for H2-evolution come from redox equivalents of the fermentative metabolism and are supplied into the photosynthetic electron transport chain via the plastochinone pool.
For the first time, it is demonstrated that the ferredoxin PetF functions as the in vivo electron donor of the Fe-hydrogenase from S. obliquus. Hydrogenase activity can be specifically blocked by addition of the ferredoxin antagonist sulfo-DSPD (A. Trebst, J Methods Enzymol., 69, 675-715 (1980)) and antibodies raised against the PetF protein. In vitro, a hydrogen evolution by HydA was only measured with plant-type [2Fw-2S] ferredoxins like PetF of S. obliquus, C. reinhardtii and spinach as electron mediators. Bacterial Fe-hydrogenases are known to be reduced by [4Fe4S] ferredoxins and do not accept electrons from plant-type proteins (J. M. Moulis et al., Biochemistry, 34, 16781-16788 (1995)).
The analysis of the 3D-structure of the Fe-hydrogenase from C. pasteurianum (CpI) gave evidence that the interaction with external electron donors might occur at the accessory [Fe-S] clusters in the N-terminal domain (J. W. Peters, Curr. Opin. Struct. Biol., 9, 670-676 (1999)). Based on the X-ray structure of CpI (N. Guex et al., Trends Biochem. Sci., 24, 364-367 (1999), the Fe-hydrogenase of S. obliquus was modeled. FIG. 4 is a Schematic view of the structures of S. obliquus HydA (A), and the electron donor ferredoxin (B). The figure shows the α carbons and the side chains of charged residues that might be important for the electron transfer reaction or the interaction between HydA and the ferredoxin from S. vacuolatus. The 16-amino acid insertion of the hydrogenase appears as external loop and is distinguished as a dotted line. The amino acid sequence of the mature HydA protein (His 19-Tyr 404) was submitted to the SWISS-MODEL server. The present inventor generated a model of HydA with the known three-dimensional structure of the iron hydrogenase from C. pasteurianum as template, sharing 57% sequence identity with the submitted sequence. The Protein Data Bank file was visualized by the Swiss-PDB viewer !(J. M. Moulis, Biochemistry, 34, 16781-16788 (1995)). As shown in FIG. 4, a region of positive surface potential is observed within HydA based on a local concentration of basic residues. In contrast to the docking position of ferredoxin in CpI, these charged amino acids in the S. obliquus Fe-hydrogenase are located within the C-terminal domain, forming a niche for electron donor fixation.
The known alga ferredoxin proteins exhibit high degrees of sequence identity (over 85%) and the charged amino acids are strictly conserved. The petF sequence of S. obliquus is unknown, but very recently the X-ray model of the ferredoxin from another Scenedesmus species (Scenedesmus vacuolatus) was published (M. T. Bes et al., Structure, 7, 1201-1213 (1999)). The structure revealed negatively charged amino acids like aspartate and glutamate near the [2Fe-2S] cluster. The [Fe-S] center and the H-cluster of the hydrogenase probably come into close proximity through electrostatic interactions. This geometry is consistent with efficient electron transfer among these prosthetic groups.
As already shown in various studies, a correlation exists between the duration of time of the anaerobic adaptation and increase of hydrogen production. RT-PCR and Northern blot analyses with mRNA of aerobic and anaerobically adapted cells from S. obliquus showed an increased level of hydA transcript after one hour of induction. Correspondingly, hydrogen evolution was only measured after a short time anaerobic adaptation. These results suggest that the expression of the hydA gene is regulated at the transcriptional level. The small amount of transcript that was detected at t=0 may be due to transcript analysis induced by micro-anaerobic conditions during the RNA isolation procedure. Alternatively, a low level of hydA transcript might be constitutively present in the cell and is only drastically increased after anaerobic adaptation.
The foregoing discloses a monomeric enzyme, iron hydrogenase (HydA), having a molecular mass of 44.5 kDa. (exclusive of the transit peptide associated therewith) derived from Scenedesmus obliquus. The polypeptide derived from the cDNA sequence, set forth herein as SEQ. ID. NO. 4, has a length of 448 amino acids and is the smallest hydrogenase described to date. The nucleic acid sequence coding HydA in Scenedesmus obliquus is set forth in SEQ. ID. NO. 1 appended hereto, and the cDNA sequence is set forth as SEQ. ID. NO. 7.
In addition to the unicellular green algae Scenedesmus obliquus, discussed above, other algae within the order of Chlorophyta (e.g. Chlamydomonas reinhardtii and Chlorella fusca) contain a gene (hydA) coding for a novel iron-hydrogenase enzyme (HydA) as will be discussed below. This gene, through its encoded enzyme, catalyzes the synthesis of molecular hydrogen from protons and high potential energy electrons, and releases significant amounts of hydrogen gas, which is a valuable and clean source of energy. As with Scenedesmus obliquus, the process of H2-production entails the utilization of sunlight and the oxidation of water or organic substrate in photosynthesis to generate reduced ferredoxin, which is the carrier of the high potential energy electrons. The isolation, sequencing and characterization of the hydA genomic DNA, cDNA, precursor and mature iron-hydrogenase of two additional photosynthetic eukaryotes which may be used for hydrogen gas production is disclosed below.
II. Chlamydomonas reinhardtii 
C. reinhardtii Algal Strains and Growth Conditions
Wild-type C. reinhardtii 137c(mt+) strain was originally obtained from the Chlamydomonas Culture Collection at Duke University. The stain was cultured photoheterotrophically in batch cultures at 25° C. under continuous irradiance of 150 μmol photons per square meter per second. Cultures containing TAP (Tris acetate phosphate) medium were flushed vigorously with air supplemented with 5% CO2. Cells were collected by centrifugation (8 minutes @ 5000 g) in the mid-exponential growth stage (1−2×106 cells per ml). After harvesting the cells in the id-exponential stage of growth, the pellet was resuspended in 0.02 vol. of fresh TAP medium. The algae were anaerobically adapted by flushing the culture with argon in the dark.
Hydrogen Evolution Assay
Hydrogenase activity of C. reinhardtii was determined in vitro with reduced methyl viologen using a gas chromatograph (Hewlett Packard 5890 {acute over (Å)} Series II, column: molecular Sieve 5 {acute over (Å)}, Mesh 60/80). The assay, containing in a final volume of 2 mL Pipes pH 6.8 (20 mM), Na2S2O4 (20 mM), MV (5 mM), was incubated anaerobically at 25° C. for 20 min. One unit is defined as he amount of hydrogenase evolving 1 μmol H2 per minute.
Purification of the Fe-hydrogenase and Amino Acid Sequence
Cells from a 40-L culture of C. reinhardtii were harvested by ultra filtration through an Amicon Ultrafiltration System DC 10 LA, with a hollow-fiber filter. The pellet was resuspended in 200 mL TAP medium. After anaerobic adaptation by flushing the solution with argon for 1 h in the dark, all steps were performed under strictly anaerobic conditions. The isolated Fe-hyrogenase was chemically cleaved by cyanogen brormide (CNBr). After separation of he CNBr fragments on an SDS polyacrylamide gel, four peptides were blotted onto a poly(vinylidene difluoride) membrane and were sequenced. Automated Edman degradation was performed with an Applied Biosystem model 477 A sequencer with online analysator model 120 A.
RNA Blot Hybridization
Total nucleic acids were isolated from algae grown under aerobic conditions and after anaerobic adaptation. Poly(A)+ RNA was isolated using the RNA Kit (Qiagen); 10 μg total RNA or 0.5 μg poly(A)+ RNA were separated on each lane of 1.2% agarose gels in formaldehyde. The RNA was transferred to n ion membranes (Hybond+, Amersham) and hybridized with RNA probes, which were labeled with digoxygenin (DIG)-dUTP by in vitro transcription. Transcripts of the hydA gene were detected using a 1.0-kb Sma I cDNA fragment. A DIG-dUTP labeled cDNA, which encodes the malate dehydrogenase, was used as a control for a constitutive express gene. FIG. 5(A) shows the structural features of the HydA cDNA. Coding regions are marked as large arrows with the transit peptide shown in black. Lines indicate 5′ and 3 URT's. In FIG. 5(B), the mosaic structure of HydA is illustrated by gray (exons) and white (introns) boxes. The RNA and DNA probes that were used for blotting are as noted on the figure.
Suppression Subtractive Hybridization (SSH)
SSH was performed with the Clontech P WR-select4 cDNA Subtraction Kit (Clontech Laboratories Inc., Palo Alto, Calif., USA.) according to the manufacturer's recommendations, except for modifications of th PCR and hybridization conditions. The mRNA was isolated from aerobically grown cells (driver) and from anaerobically adapted algae (tester). The driver and tester cDNAs were denatured separately for the first hybridization at 100° C. for 30 s and then incubated for 10 h a 68° C. For the second hybridization, driver cDNA was denatured at 100° C. for 30 s, then directly added to the pooled mix of the previous hybridization, and incubated at 68° C. for 20 h. Primary and secondary PCR conditions were altered to increase the specificity of the amplification. The PCR conditions with subtracted cDNA were as follows: 25 cycles each 94° C. for 30 s, 68° C. for 30 s, and 72° C. for 1 min. The subtracted cDNA was subjected to a second round of nested PCR, using the same PCR conditions with a decreased number of 15 cycles. Specific primers were used for the identification of the amplified HydA cDNA fragment. From the N-terminal amino-acid sequence, a degenerate oligonucleotide Hyd5 [5′-GCCGCCCC(GC)GC(GCT)GC(GCT)GA(AG)GC-3′] was synthesized, thing into account known C.reinhardtii amino-acid sequences. The second primer, Hyd2 (5′-CCAACCAGGGCAGCAGCTGGTGAA-3′), was deduced from the conservative amino acid sequence motif of Fe-hydrogenases FTSCCP.
PCR was performed using either Hyd5 or Hyd2 and the nested PCR primer 2R from the Clontech Subtraction Kit. The PCR conditions were as follows: 20 pmol per ml of each primer were used; 35 cycles (denaturing at 95° C. for 40s, annealing at 54° C. for 1 min, and extension at 72° C. for 1 min). The amplified cDNA fragments were cloned into the T overhang vector pGEM®-T Easy (Promega).
Screening of the cDNA Library, Cloning and Sequencing
A cDNA library was constructed using the Stratagene ZAP Express cDNA synthesis Kit (Stratagene, La Jolla, Calif., USA) with 5 μg mRNA of anaerobically adapted cells of C. reinhardtii. Double-stranded cDNA was ligated into the ZAP Express vector, packaged with the Gigapack Gold Kit, and transfected into Escherichia coli XL Blue MRF cells. The primary recombinant library contained 5×106 recombinant phages and was amplified according to the manufacturer's instructions.
A 366-bp PCR fragment was radiolabeled with [α-32P]dCTP using he random-primer method. Approximately 5×105 plaques were analyzed under stringent hybridization conditions, resulting in 20 positive signals. The pBK-CMV phagemid vector with the different cDNAs was excised and used as a template for PCR, which was performed by using Hyd2 and Hyd5 primers at an annealing temperature of 56° C. for 1 min. Four plasmids contained cDNA fragments that showed similarities to the 366-bp fragment. All cDNA fragments were partially sequenced, and the largest clone, pAK60, was completely sequenced. Sequencing was carried out by the dideoxy nucleotide triphosphate chain-termination method using the T7 sequencing Kit (Pharmacia Biotech). Both strands of genomic and cDNA of hydA were completely sequenced using a nested set of unidirectional deletions or hydA specific synthetic oligonucleotides. The sequences of the Fe-hydrogenase are available under accession number CREO 12098.
Primer extension experiments were performed as described previously by the present inventor (J. Biol. Chem., 276, 6125-6132 (2001)) using a 22-mer oligonucleotide (5′-AATAGGTGGTGCGATGAAGGAG-3′), which is complementary to the 5′ end of the hydA transcript.
Expression Studies in E. coli and Western Blot Analysis
The coding region of hydA was amplified by PCR The primers were identical to the cDNA sequences coding for he N-and the C-terminus of the mature protein plus several additional bases including NdeI and BamI restriction sites, respectively (underlined). The oligonucleotide sequences were: HydNde (5′-CATATGGCCGCACCCGCTGCGGAGGCGCCT-3′), HydBam (5′-CCGGATCC TCAAGCCTCTGGCGCTCCTCA-3′).
The hydA gene, corresponding to amino acids 57-497, was amplified, confirmed by sequences analysis and cloned into corresponding sites of he pET9a expression vector (Promega). The constructed plasmid was then ransformed into E. coli strain BL21(DE3). After induction with 1 mM isopropyl-thio-β-D-galactoside, the cells were resuspended in lysis buffer. Crude extracts from C. reinhardtii were isolated by harvesting cells after indicated anaerobic adaptation times. The pellet was resuspended in solubilization buffer and incubated with vigorous vortexing at RT for 30 min. The protein extracts from C. reinhardtii and E. coli were separated by 12% SDS/PAGE and blotted onto a poly(vinylidenedifluoride) membrane. Affinity-purified antibodies were diluted 1:200 and used for Western blot analyses.
Sequence Analysis and Protein Modeling
Nucleic acid and protein sequences were analyzed with the programs SCI ED CENTRAL (Scientific Educational Software) and CLUSTALW. The BLAST server (Altschul et al., Nucleic Acid Res., 25, 3389-3402 (1985)) of the National Center for Biotechnology Information (Bethseda, Md., USA) was used for database searches.
Isolation of cDNA Clones, which are Differentially Expressed During Anaerobic Adaptation
In order to amplify a part of the hydrogenase gene in a PCR reaction, degenerate oligonucleotides corresponding to conserved regions of known Fe-hydrogenases were used. All products of expected sizes were cloned and sequenced, but they showed no homologies to other hydrogenases (data not shown). Examinations were then focused on the process of anaerobic adaptation in C. reinhardtii, because the Fe-hydrogenase was only detected under these conditions. The present inventor isolated two different populations of mRNA and advantageously employed the SSH technique. Poly(A)+ RNA was isolated from aerobically grown C. reinhardtii cells and from a cell suspension flushed 15 min with argon. After cDNA synthesis, subtractive hybridization, and PCR experiments, the amplified PCR fragments were cloned and sequenced. Twenty different clones containing inserts of 184-438 bp were analyzed. In transcription analyses, 15 of them showed an increased signal under anaerobic conditions (data not shown). Database comparisons (using GenBank/EBI DataBank) confirmed that eight of these cDNA fragments are similar to genes encoding proteins of the cytoplasmic ribosome complex. The sequences of six clones did not correspond to any entries in the databases. Four of these novel clones showed differences in expression between aerobically grown and anaerobically adapted cultures. Another cDNA fragment indicated similarity to the 5′ region of the Fe-hydrogenase from bacteria.
Analysis of the hydA cDNA and Genomic Sequences
A cDNA expression library was constructed using poly(A)+ RNA from anaerobically adapted cells (15 min). Two oligonucleotides were generated on he basis of the cDNA fragment isolated by SSH and the N-terminal sequences of the purified hydrogenase. They were used to amplify a 366-bp cDNA fragment that showed 41% identity to the corresponding part of the Fe-hydrogenase of C. pasteurianum. The fiagment was labeled with [α-32P]dCTP and used to screen the cDNA library. Four independent cDNA clones with different sizes of 2.4-,1,9-, 1.7- and 1.6-kb were identified and sequenced. The nucleotide sequence of the largest clone, 2399-bp, revealed an ORF encoding a polypeptide of 497 amino acids. The cDNA also contained a 5′UTR (158-bp) and a longer 3′UTR (747-bp excluding the polyadenylated tail). Characteristic features of other C. renhardtii cDNA clones, e.g. a high average G/C content (62.1%)and a putative polyadenylation signal (TGTAA) 727-bp downstream of the stop codon were found. The transcription start position was confirmed by primer extension 158-bp upstream of the ATG start codon.
FIG. 5(A) is a schematic map of the cDNA and the genomic DNA region of HydA from C. reinhardtii.showing the structural features of the HydA cDNA. Coding regions are marked as large arrows with the transit peptide shown in black.lines indicate 5′ and 3′ URTs. FIG. 5(B) is a schematic map of the cDNA and the genomic DNA region of HydA from C. reinhardtii.showing the structural features of the HydA cDNA. In FIG. 5 (B), the mosaic structure of hydA is illustrated by gray (exons) and white (introns) boxes. The RNA and DNA probes that were used for the blotting experiments are noted on the Figure.
Approximately 5-kb of the hydA genomic region was determined. The coding sequence is interrupted by seven introns with sequences at their 5′ and 3′ ends, corresponding to the typical splicing sequences from eukaryotes as shown in FIG. 5B. The promoter region does not contain a putative TATA box or any other known transcription motifs. The sequence data were submitted to the GenBank/EBI DataBank under accession number CRE012098. In subsequent studies, parts of the cDNA sequence were determined by another group and deposited under accession number AF289201.
Southern hybridization experiments were performed at high stringency using a PCR fragment as probe. They showed the presence of one hybridizing signal of similar intensity in different digestions, suggesting that HydA is encoded by a single copy gene in the C. reinhardtii genome. The same hybridization pattern was observed even under low stringency conditions (hybridization temperature 50° C.; data not shown).
Characterization of the Fe-Hydrogenase HydA from C. reinhardtii 
The mature polypeptide consists of 441 amino acids with a calculated molecular mass of 47.5 kDa and a predicted isoelectric point of 5.6. The N-terminal 56 amino acids probably function as a transit peptide, because they show characteristics of polypeptides that route proteins into the chloroplast stroma. The stromal targeting domain is most likely cleaved by a stromal peptidase at the conserved cleavage motive Val-Ala-Cys-Ala. In addition to the detection of the protein using antibodies raised against the Fe-hydrogenase, the localization of the mature protein in the chloroplast stroma is indicated by a high content of hydroxylated and basic amino acids in the transit peptide sequence.
The deduced amino-acid sequence of he mature HydA polypeptide from C. reinhardtii shows 60% identity and 71% similarity to the Fe-hydrogenase of S.obliquus, Comparisons with NiFe-hydrogenases of bacteria (including the photosynthetic cyanobacteria) had obviously lower scores, e.g. 25% similarity with the NiFe-hydrogenase (HoxH) of Ralstonia eutropha. A conserved domain of about 300 amino acids is found in the C-terminal part of all Fe-hydrogenases. The sequences are highly conserved, especially in the region that is involved in the catalytic mechanism (H-cluster), indicating structural similarity between Fe-hydrogenases. Four cysteine residues at positions 114, 169, 361 and 365 might coordinate the H-cluster in C. reinhardtii. Twelve strictly conserved amino acids of HydA proteins probably define a binding pocket surrounding the active center as shown by structural data reported by others for C. pasteurianum and D. desulficans iron hydrogenase. All of them are present in the C. reinhardtii protein (Pro37, Ala38, Thr74, Ala78, Cys113, Pro138, Met167, Lys172, Glu175, Phe234, Val240 and Met359). An interesting insertion of 45 amino acids was only identified at the C-terminus of the C.reinhardtii polypeptide (position 285.329).
The N-terminal region of the green algae protein is much shorter and completely different than all known Fe-hydrogenases. Amino-acid sequence analyses have indicated that Fe-hydrogenases, in general, contain two [4Fe)4S ] clusters (F-cluster) in a ferredoxin-like domain. They might be involved in the transfer of electrons from the donor to the catalytic center. This N-terminal domain with the F-cluster or other conserved cysteines is completely missing in HydA of C. reinhardtii. A novel electron transport pathway is postulated from the exogenous donor (ferredoxin) directly to the H-cluster.
Protein Sequencing of the Enzyme and Recombinant Expression of HydA in E. coli 
To verify that the hydA ORF encodes the Fe-hydrogenase of C. reinhardtii, the enzyme was purified according to Happe and Naber (Eur. J. Biochem. 214,475-481 (1993)). The purified protein was able to evolve hydrogen, when incubated with reduced methyl viologen. After proteolytic digestion with cyanogen bromide, four bands of 4, 8, 9 and 11 kDa were detected after SDSIPAGE separation (data not shown). Two fragments (9 and 11 kDa) were sequenced by Edman degradation. FIG. 6 shows the nucleotide sequence of the hydA cDNA and the deduced amino acid sequence of the hydrogenase from C. reinhardtii. The two fragment sequences are identical with he deduced amino-acid sequence of hydA (sequences are shadowed in gray in FIG. 6). The fragment corresponding to the cDNA region between 158 and 1636 bp of hydA was NdeI-BamHI cloned into the expression vector pET9a. The heterologous expressed protein was detected using antibodies raised against the Fe-hydrogenase. Both the purified Fe-hydrogenase of C. reinhardtii and the overexpressed enzyme had the same size (47.5 kDa). No hydrogenase activity could be measured within the lysate of the induced E. coli cells. This result is in agreement with Stokkermans et al. and Voordouw et at. (ibid) who also detected no H2-production of the recombinant expressed Fe-hydrogenase from Desulfovibrio vulgaris in E. coli cells. An explanation might be the inability of E. coli to assemble the unique active site of the Fe-hydrogenases. It is known that E. coli has only three NiFe-hydrogenases with a different maturation system for the catalytic center.
FIG. 6 shows the nucleotide sequence of the hydA cDNA and the deduced amino acid sequence of the hydrogenase from C. reinhardtii. The sequence was submitted to the GenBank/EBI Data Bank under accession number CRE012098. A curved arrow marks the transcription start point. The ATG start codon and the TGA stop codon are drawn in boxes. Boldface letters indicate the cDNA sequence. Gray shadows mark amino acids corresponding to polypeptide sequences that were determined by sequencing the N-terminus of the protein. Black shadows mark the putative transit peptide, and the underlined amino acids indicate the putative cleavage site for the endopeptidase. Boldface double underlined letters indicate a signal for polyadenylation.
Induction of Gene Expression During Anaerobic Adaptation
In aerobically grown cells, neither hydrogenase activity nor protein can be identified by immunoblot analysis. However, HydA can be detected only 15 min after anaerobic adaptation. The expression of the hydA gene is probably regulated at the transcriptional level. Total RNA was isolated from cells that had been anaerobically adapted by flushing with argon for 0,15, and 30 min. Northern blot hybridization demonstrated that the hydA gene is expressed very rapidly after the beginning of anaerobic adaptation. No transcript could be detected before adaptation (t=0), but a significant signal occurred after just 15 min of anaerobiosis. The size of the transcripts (2.4 kb) confirmed the full-length of the isolated hydA cDNA fragment.
Differentially Expressed Genes During Anaerobic Adaptation
In the light, algae degrade cellular starch via glycolysis and hydrogen gas is evolved. It has been suggested that reducing equivalents from the glycolysis or the citric acid cycle can transfer their electrons to the photosynthetic electron transport chain (M. Gibbs et al., Plant Physiol. 82, 160166 (1986)). However, the molecular principles of the gene induction under anaerobic conditions in C. reinhardtii are poorly understood.
The present inventor has investigated the patterns of gene expression in aerobically grown and anaerobically adapted cells by isolating differentially expressed genes. The SSH method combines subtractive hybridization with PCR to generate a population of PCR fragments enriched with gene sequences that are only expressed under anaerobic conditions. Compared to other PCR-based cloning strategies, such as differential display, the great advantage of SSH is that fewer false positives are generated; 70% of the cloned fragments represented differentially expressed genes.
Among the 20 sequenced cDNA clones, three DNA fragments encoding the ribosomal S8 protein were found. Most of the other sequences (eight of 20) also corresponded to ribosomal protein sequences. This might indicate that the transcripts of the ribosomal protein genes (rps, rpl) accumulate under stress conditions. This is in good agreement.with Dumont et al. (Plant Sci., 89, 55-67 (1993)) who found that an accumulation of ribosomal proteins takes place under phosphate starvation. Moreover, two of the identified cDNAs encode for proteins, (aldolase, enolase), which are induced in other organisms by anaerobic stress. Anaerobic treatment of maize seedlings alters he profile of total protein synthesis. It is known that the induction of the anaerobic proteins is the result of an increased mRNA level. Maize (Zea mays L.) responds to anaerobic stress by redirecting the synthetic machinery towards the synthesis of some enzymes involved in glycolysis or sugar-phosphate metabolism.
C. reinhardtii HydA Belongs to a New Class of Fehydrogenases
HydA of C. reinhardtii, the first isolated gene encoding a hydrogenase of a photosynthetic eukaryotic cell, represents a novel type of Fe-hydrogenases. Parts of the deduced amino-acid sequence of the cDNA correspond to the polypeptide sequence of the tryptic fragment (VPAPGSKFEELLKHRAAARA), and the N-terminus (AAPAAAEAPLSHVQQALAELAKPKD) from the purified native enzyme. Further evidence that the isolated cDNA encodes an Fe-hydrogenase is the fact that the recombinant HydA specifically reacts with the antibodies raised against the active enzyme. The amino-acid sequence of HydA shows only considerable similarity to Fe-hydrogenases but not to NiFe-hydrogenases. The Fe-hydrogenase family is one class of hydrogenases defined by Vignais et al. (FEMS Microbiol. Rev. 25, 455-501 (2001)). The enzymes have been identified in a small group of anaerobic microbes, where they often catalyze the reduction of protons with a high specific activity to yield hydrogen. Interestingly, Fe-hydrogenases were not found in cyanobacteria, the free-living ancestor of plastids, suggesting a noncyanobacterial origin for the algal hydrogenases.
The important structural features found among the amino-acid sequences of Fe-hydrogenases are also present in the C. reinhardtii hydrogenase sequence. A highly conserved domain of about 130 amino acids was detected in the C-terminal part of the protein. The designated active site domain consists of an atypical [Fe-S] cluster (H-cluster). In C. pasteurianum, the H-cluster contains six Fe atoms arranged as a [4Fe-4S] subcluster bridged to a [2Fe] subdluster by a single cysteinyl sulfur. The [4Fe-4S] subcluster is coordinated to the protein by four cysteine ligands, which have also been found in the amino acid sequence of C. reinhardiii. A number of mostly hydrophobic amino acid residues define the environment of the active site and might have a function in protecting the H-cluster from solvent access. In contrast to all Fe-hydrogenases, including HydA of S. obliquus, the enzyme of C. reinhardtii has an interesting additional 18 protein domain. A small insertion of 45 amino acids between residue Ser284 and Val330builds an external loop of the protein that might be involved in electrostatic binding of the natural electron donor ferredoxin (Happe et al., unpublished results)
In the N-terminus of other Fe-hydrogenases, further cysteine residues were found that bind accessory iron sulfur clusters. Others have shown that a ferredoxin homologous domain (F-cluster) coordinates two [4FeAS] clusters in all non-algal Fe-hydrogenases. An additional [4Fe-4S] cluster and one [2Fe-2S] center were detected within the Fe-hydrogenases of C. pasteurianum (Peters et al., Science, 282, 1853-1858 (1998)). Based on similarities of the primary sequences, the same cofactors are proposed for Thermotoga maritime (A. Akhmanova et al., Nature, 396,527-528 (1998)) and Nyctotherus ovalis (J. Meyer et al., Biochim. Biophys. Acta, 1412, 212-229 (1999)). The F-cluster is responsible for the electron transfer from the electron donor (mostly ferredoxin) to the active center. It has been suggested by Vignais et al. (FEMS Microbiol. Rev., 25, 455-501 (2001)) that the proteins containing two F-clusters are ancestors of the Fe-hydrogenases.
The N-terminus of the C. reinhardtii and S. obliquus proteins is strongly reduced, and conserved cysteines were also not found. Therefore it is suggested that all accessory [Fe-S] clusters are missing in the algal hydrogenases. The native protein of C. reinhardtii is located in the chloroplast stroma. The first 56 amino acids of the unprocessed enzyme probably function as a transit peptide, because they were not characterized in the purified hydrogenase and a putative peptidase cleavage site (Val-Ala-Cys-Ala) could be detected at the end of this fragment.
The natural electron donor of the hydrogenase in C. reinhardtii is the ferredoxin (PetF) of the photosynthetic electron ransport pathway. Measuring the H2-evolution, it has been found that the hydrogenase activity is directly linked to the 47.5 kDa subunit. As a second subunit necessary for hydrogenase activity has not been found, it is suggested that a direct electron transfer from PetF to HydA takes place. In vitro, a hydrogen evolution by HydA was only measured with plant-type [2Fe-2S] ferredoxins such as PetF of C. reinhardtii, S. obliquus and spinach as electron mediators (data not shown).
FIG. 7 is a schematic diagram illustrating the light-dependent photoevolution of hydrogen in green algae. The electrons for hydrogen evolution are fed into the photosynthetic electron transport chain either via PS II or via the plastoquinone pool after oxidation of reducing equivalents. The natural electron donor, PetF, transfers the electrons from PS I to the hydrogenase.
The most likely explanation for photosynthetic green algae retaining the anaerobically induced hydrogenases is that the enzymes ensure the survival of the ceus under anaerobic conditions. Melis et al. have shown that H2-evolution is the only mechanism available to the algae for generating sufficient amounts of ATP under S-depleted anaerobic conditions. It is known that C. reinhardtii is still able to photoproduce hydrogen when photosystem II is inhibited by DCMU, but no H2-evolution occurs after an addition of 2,5-dibromo-3-methyl-6-isopropyl-p-benzochinon (DBMIB; FIG. 7). Under anaerobic conditions, accumulated reducing equivalents from the fermentative metabolism cannot be oxidized via respiration, as the electron acceptor oxygen is missing. The NAD(P)H reductase protein complex has recently been isolated from plants, and inhibitor experiments have shown evidence of a membrane-bound, chloroplast-located reductase in C. reinhardtii (D. Godde et al., Arch Microbiol. 127, 245-252 (1980)). The light-dependent electron transport of the H2-evolution is driven by plastoquinone and photosystem I. The donor ferredoxin transfers electrons to the hydrogenase in a final step and molecular hydrogen is released (FIG. 7).
Regulation of hydA at the Transcriptional Level
Studies indicate that there is a correlation between the increase of hydrogen production and the anaerobic adaptation, which was documented by activity measurements (T. Happe et al., Eur. J. Biochem. 222, 769-775 (1994)), and immunoblots. It is likely that the induction of hydA is regulated on the level of transcription. It is observed that the amount of mRNA increased directly with the measured H2-evolution. In C. reinhardtii, a dramatic change in the hydrogenase transcript level occurs during the shift from an aerobic to an anaerobic atmosphere, which means that the transcription is regulated by the oxygen status of the cells. A very rapid increase of the hyd transcript was detected in the first 30 min of anaerobiosis. This quick increase of gene transcription is only reported for the cyc6 gene in C. reinhardtii (K. L. Hill et al., J. Biol. Chem. 266, 15060-15067 (1991)) and for the SAUR (Small Auxin-Up RNA) genes in plants (Y. Li et al., Plant Physiol. 106, 37-43 (1994)). Interestingly, the hyd, gene of S. obliquus is constitutively transcribed under aerobic conditions indicating another regulation system for the expression of the hydrogenase. At the moment, it is not clear if his effect rests upon a new synthesis or a higher stability of the hydA mRNA.
As with other nuclear genes, the promoter region of the hydA from C. reinhardtii contains no conserved TATA box or other motif similarities. As no defined motif structures in the promoter region of hydA have been found, further genetic analyses are necessary to investigate the rapid induction of hydA in C. reinhardtii. 
III. Chlorella fusca 
The isolation and molecular characterization of the Fe-hydrogenase from the unicellular green alga Chlorella fusca was also performed. Hydrogenase activity was observed in a culture of the unicellular green alga Chlorella fucsa after anaerobic incubation, but not in the related species Chlorella vulgaris. Specific PCR techniques lead to the isolation of the cDNA and the genomic DNA of a special type of [Fe]-hydrogenase in C. fusca. The functional Fe-hydrogenase was purified to homogeneity as described above, and its N-terminus was sequenced. The polypeptide sequence shows a high degree of identity with the amino acid sequence deduced from the respective cDNA region. Structural and biochemical analyses indicate that ferredoxin is the main physiological electron donor to this [Fe]-hydrogenase. The nucleotide sequence reported herein has been submitted to the GenBank/EBI Data Bank with accession number AJ 298227. The nucleic acid sequence of the genomic DNA (3,290 bp) of C. fusca is set forth in SEQ. ID. NO. 3, and the amino acid sequence of the precursor protein (436 amino acids) is set forth in SEQ. ID. NO. 6. The cDNA sequence is presented in SEQ. ID. NO. 9.
The transcription of the iron-hydrogenase is very rapidly induced during anaerobic adaptation of the green algae. Hydrogen photoproduction by the cells can be observed soon following this induction. The genomic, cDNA and polypeptide sequences of three representative green algae are offered as examples of the properties of the HydA gene and of the enzyme that it encodes. These genomic, cDNA and polypeptide sequences from three representative green algae are also offered as examples of the potential application of the hydA gene from Chlorophyta in the process of commercial hydrogen production:
Scenedesmus obliquus 


    
    
        Genomic DNA: 5,001 bp, SEQ. ID NO. 1 (attached hereto)
        cDNA: 2,636 bp SEQ. ID NO. 7 (attached)
        Precursor protein: 448 amino acids, 44.5 kD, SEQ. ID NO. 4 (attached hereto)
    
    


Chlamydomonas reinhardtii 


    
    
        Genomic DNA: 5,208 bp, SEQ. ID. NO. 2 (attached)
        cDNA: 2,399 bp, SEQ. ID. NO. 8 (attached)
        Precursor protein: 497 amino acids, 53.1 kD, SEQ. ID. NO. 5 (attached)
    
    


Chlorella fusca 


    
    
        Genomic DNA: 3,265 bp, SEQ. ID. NO. 3 (attached)
        cDNA: 2,421 bp, SEQ. ID NO. 9 (attached)
        Precursor protein: 436 amino acids, SEQ. ID. NO. 6 (attached)
    
    


This new class of iron-hydrogenases has a C-terminal portion and active site region (H-cluster) similar to that reported in non-photosynthetic prokaryotes (e.g. Clostridium pasteurianum). Cysteine residues and distinct other amino acids which are strictly conserved in the active site (H-cluster) of such non-photosynthetic prokaryotes are also conserved in the iron-hydrogenase of green algae. However, the N-terminal region of the green alga iron-hydrogenase is substantially different from that of HydA in non-photosynthetic prokaryotes, revealing novel and unobvious pathways of electron transport for photosynthetic hydrogen production in green algae.
Distinct iron-sulfur [Fe-S] centers, referred to as FS2, FS4C, FS4B and FS4A, are encountered in the N-terminal region of the iron-hydrogenase in non-photosynthetic prokaryotes, and are thought to be instrumental in the transport of high potential energy electrons from bacterial ferredoxins to the catalytic site of the H-cluster of the hydrogenase. These distinct FS2, FS4C, FS4B and FS4A [Fe-S] centers are missing from the HydA of green algae. A mature-protein folding-model of the green alga iron-hydrogenase and analysis of its structure revealed a protein region of positive surface potential, evidenced by the presence of basic amino acids, which are uniquely localized within the C-terminal domain and, therefore, near the catalytic site of the H-cluster. On the other hand, a model of the structure of green alga ferredoxin, which is different from prokaryotic ferredoxins, revealed negatively charged amino acids near the [2Fe-2S] electron donor site of this molecule. Structural analysis revealed that the [2Fe-2S] center of green algal ferredoxin and the H-cluster of the hydrogenase probably come into close proximity through electrostatic interactions. This molecular geometry is consistent with a direct and efficient electron transfer between these two prosthetic groups. Thus, the hydA gene of green algae encodes an iron-hydrogenase polypeptide with a novel structure, one that uniquely permits a direct coupling and efficient electron-transfer from a [2Fe-2S] photosynthetic ferredoxin to the active site of the H-cluster. In support of this conclusion, inhibitor experiments revealed that the PetF ferredoxin functions as a natural electron donor in green algae, linking the iron-hydrogenase with the photosynthetic electron transport chain in the chloroplast of these unicellular organisms.
In summary, a process, operable in a culture comprising unicellular green algae, is described whereby transcription of algal HydA genomic DNA, followed by translation of the resulting HRNA, followed by targeting of the precursor protein and import of the polypeptide into the chloroplast, followed by the mature iron-hydrogenase folding and catalysis, leads to hydrogen (H2) gas production.
Levels of HydA protein in the cells are very low, even under hydrogen production conditions, and this is a primary reason for the currently low yield of hydrogen production in green algae. It is suggested that genetic engineering of the green algae in order to overexpress the hydA gene would result in strains with far greater yields of hydrogen production, which is of obvious practical importance. Moreover, another use of the hydA genes is that they can be transformed then transferred into other photosynthetic and non-photosynthetic organisms that lack the ability to produce hydrogen. Such genetic transformation with the hydA gene(s) of green algae, will confer to a variety of organisms commercial utility for the production of hydrogen.
There are many species of unicellular green algae that may have variants of the HydA gene described herein for Scenedesmus obliquus, Chlamydomonas reinhardiii and Chlorella fusca. It is the intended to encompass within the scope of the present invention all green alga HydA genes and gene products that are similar to the unique HydA genes and HydA described herein. A HydA that is “similar” is meant to mean that the HydA has a 75% or greater homology in the amino acid sequence between an isolated HydA and a polypeptide selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

## Claims

1. An isolated nucleic acid sequence comprising SEQ. ID. NO. 2.

2. An isolated nucleic acid sequence comprising SEQ. ID. NO. 8.

3. A cell comprising an isolated nucleic acid sequence encoding a protein comprising SEQ. ID. NO. 5.