# Patent

## Title
Process and genes for expression and overexpression of active [FeFe] hydrogenases

## Abstract

A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

## Background
This application claims the benefit of the Feb. 28, 2005 filing date of provisional application No. 60/656,957.


CONTRACTUAL ORIGIN OF THE INVENTION
The United States Government has rights in this invention pursuant to Contract No. DE-AC36-99GO10337 between the United States Department of Energy and the Midwest Research Institute.


TECHNICAL FIELD
The invention relates to the use of genes to provide expression and over-expression of any active [FeFe]-hydrogenases, expressed in any suitable host, using an [FeFe]-hydrogenase assembly of genes from a suitable organism.
BACKGROUND ART
Hydrogen has enormous potential to serve as a non-polluting fuel, thereby alleviating the environmental and political concerns associated with fossil energy utilization.
Among the most efficient H2-generating catalysts known are the [FeFe]-hydrogenase enzymes found in numerous microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. The use of Chlamydomonas reinhardtii, also known as green algae, to produce hydrogen from water has been recognized for more than 60 years. The reaction that produces hydrogen is catalyzed by the reversible hydrogenase, an enzyme that is induced in the cells after exposure to a short period of anaerobiosis. This activity is rapidly lost as soon as light is turned on, due to immediate inactivation of the reversible hydrogenase by photosynthetically generated O2 
Ghirardi et al. in Biological Systems For Hydrogen Photoproduction (FY 2004 Progress Report) disclose a method for generating algal hydrogenase mutants with higher O2 tolerance to function with aerobic H2 production systems, which further optimize H2 photoproduction using an algal production system. It generates a recombinant alga expressing an [FeFe]hydrogenase that displays increased tolerance to O2 due to closure of the pathways by which O2 accesses the catalytic site of the enzyme.
T. Happe et al. in Differential Regulation Of The Fe-hydrogenase During Anaerobic Adaptation In The Green Alga Chlamydomonas reinhardtii Eur. J. Biochem. 269, 1022-1032 (2002) disclose using the suppression subtractive hybridization (SSH) approach, wherein the differential expression of genes under anaerobiosis was analyzed. A PCR fragment with similarity to the genes of bacterial Fe-hydrogenases was isolated and used to screen an anaerobic cDNA expression library of C. reinhardtii. The cDNA sequence of HydA contains a 1494-bp ORF encoding a protein with an apparent molecular mass of 53.1 kDa. The transcription of the hydrogenase gene is very rapidly induced during anaerobic adaptation of the cells. The deduced amino-acid sequence corresponds to two polypeptide sequences determined by sequence analysis of the isolated native protein. The Fe-hydrogenase contains a short transit peptide of 56 amino acids, which routes the hydrogenase to the chloroplast stroma. The isolated protein belongs to the class of Fe-hydrogenases. All four cysteine residues and 12 other amino acids, which are strictly conserved in the active site (H-cluster) of Fe-hydrogenases, have been identified. The N-terminus of the C. reinhardtii protein is markedly truncated compared to other non algal Fe-hydrogenases. Further conserved cysteines that coordinate additional Fe—S-cluster in other Fe-hydrogenases are missing. Ferredoxin PetF, the natural electron donor, links the hydrogenase from C. reinhardtii to the photosynthetic electron transport chain. The hydrogenase enables the survival of the green algae under anaerobic conditions by transferring the electrons from reducing equivalents to the enzyme.
Isolation and characterization of a second [FeFe]-hydrogenase gene from the green alga, Chlamydomonas reinhardtii, wherein a HydA2 gene which encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C. reinhardtii. HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of [FeFe]-hydrogenases disclosed by Forestier et al in Expression Of Two [Fe]-Hydrogenases In Chlamydomonas reinhardtii Under Anaerobic Conditions, Eur. J. Biochem. 270, 2750-2758 (2003). It is demonstrated that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures. Further, the expression levels of both transcripts are regulated by incubation conditions, such as the length of anaerobiosis, the readdition of O2, the presence of acetate, and/or the absence of nutrients such as sulfate during growth. Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C. reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein. Homology-based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H2-gas channel. The major differences between HydA1, HydA2 and CpI are the absence of the N-terminal Fe—S centers and the existence of extra sequences in the algal enzymes.
It is disclosed that Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes in Iron-Dependent Hydrogenases of Entamoeba histolytica and Giardia lamblia: Activity of the Recombinant Entamoebic Enzyme and Evidence for Lateral Gene Transfer Biol. Bull. 204: 1-9. (February 2003). A recombinant E. histolytica short Fe-hydrogenase was prepared and its activity is measured in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic sequences, and RT-PCR showed that cultured entamoebas and giardias transcribe short Fe-hydrogenase mRNAs. A second E. histolytica gene, which encoded a long Fe-hydrogenase, was identified from shotgun genomic sequences. Phylogenetic analyses suggested that the short Fe-hydrogenase genes of entamoeba and diplomonads share a common ancestor, while the long Fe-hydrogenase gene of entamoeba appears to have been laterally transferred from a bacterium. These results are discussed in the context of competing ideas for the origins of genes encoding fermentation enzymes of these protists.
U.S. Patent Application No. 2004/02009256 discloses methods and compositions for engineering microbes to generate hydrogen. Some methods of the invention involve recoding of hydrogenase genes followed by subjecting the recoded genes to annealing-based recombination methods. The invention further provides methods of mating organisms that are transformed with recoded and recombined hydrogenase genes with other organisms containing different genome sequences.
A need exists in the art of H2-generating catalysts of [FeFe]-hydrogenase enzymes, which are found in numerous microorganisms (including C. reinhardtii) to identify the genes essential for formation of active algal [FeFe]-hydrogenase enzymes, due to the fact that expression of an algal [FeFe] hydrogenase structural gene without the co-expression of C. reinhardtii genes results in the accumulation of an inactive [FeFe]-hydrogenase.
Further still, a need exists in the art of H2— generating catalysts of [FeFe]-hydrogenase enzymes to provide co-expression of the C. reinhardtii genes and an algal [FeFe] hydrogenase structural gene in E. coli to produce synthesis of an active [FeFe]-hydrogenase in this bacterium, which lacks a native [FeFe]-hydrogenase.
In the art of H2-generating catalysts of [FeFe]-hydrogenase enzymes, there is yet another need to demonstrate and provide a process to over-express active [FeFe]-hydrogenase in a stable, recombinant E. coli system, and to assemble and insert an H-cluster into C. reinhardtii [Fe]-hydrogenase using the C. acetobutylicum HydE, HydF and HydG proteins to accomplish this activation of non-cognate [FeFe]-hydrogenases—and not limit to the [FeFe]-hydrogenase assembly genes from C. acetobutylicum, the structural genes from C. acetobutylicum or C. reinhardtii, or use of E. coli as an expression host, but to accomplish the expression of any [FeFe]-hydrogenase, expressed in any suitable host, using [FeFe]-hydrogenase assembly genes from any suitable organism.
DISCLOSURE OF THE INVENTION
One object of the present invention is to identify the genes essential for formation of active algal [FeFe]-hydrogenase enzymes, given that expression of an algal [FeFe]-hydrogenase structural gene without the co-expression of C. reinhardtii genes results in the accumulation of an inactive [FeFe]-hydrogenase.
Another object of the present invention is to provide a process in which the co-expression of C. reinhardtii genes and the algal [FeFe]-hydrogenase structural gene can be used for assembly of an active algal [FeFe]-hydrogenase in C. reinhardtii. 
An object further still of the present invention is to provide co-expression of the C. reinhardtii genes and an algal [FeFe]-hydrogenase structural gene in E. coli to produce synthesis of an active [FeFe]-hydrogenase in this bacterium, which lacks a native [Fe]-hydrogenase.
Yet another object of the present invention is to demonstrate and provide a process to over-express active [FeFe]-hydrogenase in a stable, recombinant E. coli system and to assemble and insert the H-cluster into C. reinhardtii [FeFe]-hydrogenase using the C. acetobutylicum HydE, HydF and HydG proteins to accomplish this activation of non-cognate [FeFe]-hydrogenases—and not limit to the [FeFe]-hydrogenase assembly genes from C. acetobutylicum, the structural genes from C. acetobutylicum or C. reinhardtii, or use of E. coli as an expression host, but to accomplish the expression of any [FeFe]-hydrogenase, expressed in any suitable host, using [FeFe]-hydrogenase assembly genes from any suitable organism.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows initial characterization of the C. reinhardtii hydEF-1 mutant, wherein (A) colonies of C. reinhardtii insertional mutants growing on TAP agar plates (left) and photo-production of H2, following anaerobic induction, detected visually on the chemochromic sensor (right) as a dark blue spot. Colony four (bottom, left) failed to produce sufficient H2 for colorimetric detection; (B) shows rates of algal photosynthesis and respiration; and (C) shows initial rates of H2 photo-production from cultures anaerobically-induced in the dark. Only the WT produced H2.
FIG. 2 shows gene disruption in the hydEF-1 mutant and complementation of the mutant phenotype; wherein (A) shows a schematic of the organization of the HydEF and HydG genes in the C. reinhardtii genome (top). Exons are shown as rectangles, filled gray or black for HydG and HydEF, respectively. The 5′ UTRs are depicted as block arrows ending at the respective ATG start codons. Stop codons are also shown as asterisks followed by the 3′ UTRs. The location of the arg7 gene insertion in HydEF is shown with the inverted triangle. The portion of the HydEF gene to the right of the triangle is deleted in the HydEF-1 mutant. The putative promoter region is shown as a white rectangle. An expanded schematic of the promoter region followed by the first two exons is shown below; (B) shows photo-production of H2 measured in (1-r) the parental strain (CC425), the hydEF-1 mutant and the hydEF-1 mutant complemented with the HydEF gene (assayed four hours after dark anaerobic induction). Average deviations from the mean are indicated; and (C) shows Southern blotting of the same clones shown in (B). Genomic DNA was digested with Nco1, blotted, and probed using a DNA sequence that had been deleted in the hydEF-1 mutant.
FIG. 3 shows putative homologues of C. reinhardtii HydA1, HydEF (both the HydE and HydF domains, respectively) and HydG found in organisms with sequenced genomes. The gene identification, percent identical amino acids (% I), E values, homology length and homology rank to the C. reinhardtii proteins are shown. Organization of the respective genes within the genomes of the organism is also included.
FIG. 4 shows Northern blots of the (A) HydA1, (B) HydA2 (C) HydG and (D) HydEF transcripts isolated from CC425 or hydEF-1 cultures. Anaerobic induction times of 0, 0.5 or 4 hours are indicated. RNA from WT and hydEF-1 cultures were electrophoresed, blotted and probed together in Northern blot experiments. The ribosomal 23S RNA band is shown as a loading control below each Northern blot. In (E), the Western blot is of partially purified protein extracts from aerobic and anaerobically induced samples. The blot was probed with an antibody designed to recognize both C. reinhardtii HydA1 and HydA2.
FIG. 5 shows hydrogen-production rares from purified HydA1 heterologously expressed E. coli either alone or co-expressed with the indicated Hyd proteins. Hydrogen production was measured using the methyl viologen-based assay. The data shown represent the average of four independent experiments, and average deviations from the mean are shown.
FIG. 6 Shows alignments of C. reinhardtii (A and B) HydEF and (C) HydG amino acid sequences with the corresponding putative homologues found in other organisms. The amino acid sequences were derived from the C. reinhardtii cDNAs. Regions of identical amino acids are shown in black, and regions of similar amino acids are shaded in gray. Shown in (A and B) are HydE homologues followed by the C. reinhardtii linker region, and lastly by HydF homologues. Organisms shown in the alignment include: Thermotoga maritima, Clostridium thermocellum, Clostridium tetani, Desulfovibrio desulfuricans, Shewanella oneidensis, Bacteroides thetaiotaomicron, Clostridium perfringens and Clostridium acetobutylicum. 
FIG. 7 shows plasmid constructs for T7-promoter expression of [FeFe]-hydrogenase maturation and structural genes. (A) Backbone pCaE2 was used to co-express HydE with either CaHydA (pCaAE) (depicted in the figure), CaHydB (pCaBE) or CaHydAΔN (pCaAΔNE) from C. acetobutylicum; or CpHyd, (pCpAE) or CpHydAΔN (pCpAΔNE) from C. pasteurianum. (B) Backbone pCaE1 was used to co-express HydE with either C. reinhardtii CrHydA1 (pECr1) (depicted in the figure) or CrHydA2 (pECr2). (C) pCaFG co-expresses C. acetobutylicum HydF and HydG. (D) pCaHydA expresses C. acetobutylicum HydA. (E) pCaE1 (HydE at the NcoIBamHI sites of MCS1) and pCaE2 (HydE at the NdeI-BglII sites of MCS2). (F) pCaF expresses C. acetobutylicum HydF. (G) pCaG expresses C. acetobutylicum HydG.
FIG. 8. shows a Western blot analysis of purified, StrepII-tagged [FeFe]-hydrogenases. Lane 1, C. acetobutylicum HydA (1.5 μg, 65 Kd); lane 2, C. acetobutylicum HydAΔN (2 μg, 43 Kd); lane 3, C. acetobutylicum HydB (5 μg, 50 Kd); lane 4, C. reinhardtii HydA1 (2.25 μg, 49 Kd); lane 5 C. reinhardtii HydA2 (1 μg, 49 Kd); lane 6, molecular weight markers, 75, 50 and 35 Kd.
FIG. 9 is a schematic representation of aligned sequences of [FeFe]-hydrogenases used in this study (Ca=C. acetobutylicum, Cp=C. pasteuriaiium, Cr=C. reinhardtii). The top diagram represents the relative location of conserved F-cluster binding domains (cross-hatched lines), and the H-cluster binding motifs HC1 (TSCCP) and HC2 (MACPGGC) (dark bars) found in soluble [FeFe]-hydrogenases. The arrow heads indicate the N-termini of conserved F-cluster-binding domains deleted in C. acetobutylicum and C. pasteurianum HydAΔN constructs.



BEST MODE FOR CARRYING OUT THE INVENTION
Experimental Procedures Strains
An insertional mutagenesis library was generated by transforming C. reinhardtii strain CC425 (cw15, sr-u-60, arg7-8, mt+) with the pJD67 plasmid (Davies, J. P. et al. [1999] Plant Cell 11, 1179-1190) provided by Professor Anastasios Melis (University of California, Berkeley, Calif.).
Hydrogen and Oxygen Assays
Chemochromic screening was performed using colonies growing on Tris-acetate-phosphate (TAP) agar plates. Hydrogenase activity was induced anaerobically in the dark, and H2 photo-production was monitored the following day. Rates of photosynthesis, respiration and H2 photoproduction in liquid cultures were determined (M. Forestier et al. [2003] Eur. J. Biochem. 270, 2750-2758).
Hydrogen was assayed from the headspace of anaerobically-sealed cultures using a Varian model 3700 gas chromatograph (GC). For the methyl viologen (MV) assay of hydrogenase activity, cells were removed and added to an equal volume of anaerobic 2×MV solution (100 mM MV (oxidized), 50 mM Tris, pH 8.0 and 0.2% Triton X-100) in a sealed anaerobic vial. Degassed sodium dithionite was added to a final concentration of 4 mM to initiate H2 production from reduced MV.
Anaerobic Induction of Liquid Cell Suspensions
C. reinhardtii cultures were grown on TAP medium to  ̃20 μg/ml total chlorophyll, centrifuged at 2500 g for 5 minutes and resuspended in 1/10th volume of induction buffer (AIB), containing 50 mM potassium phosphate pH 7.0 and 3 mM MgCl2 [27]. Samples were placed in vials that were wrapped with aluminum foil to exclude light, sealed with a rubber septum, flushed with argon for 15 minutes and incubated anaerobically in the dark at room temperature.
Southern and Northern Blot Analysis
Southern blotting experiments were performed using standard methodology. Genomic DNA was extracted and purified using a DNeasy Plant Mini Kit (Qiagen). Northern blot analysis was performed using 10 μg of total RNA for each sample as previously described. Probes were labeled using α-32dCTP (ICN) and the rediprime II DNA random-prime labeling system (Amersham Pharmacia Biotech).
Western Blot Analysis
After four hours of anaerobic induction, cells were lysed under anaerobic conditions. Aerobic control samples were lysed immediately after resuspension in AIB. Cells were disrupted with gentle rocking in lysis buffer (50 mM Tris, pH 8.5, and 0.25% Triton X-100) for thirty minutes, and the cellular extract was centrifuged for 10 minutes at 10,000 g. The hydrogenase protein was partially purified from induced and non-induced cells under strictly anaerobic conditions by loading the lysed supernatant, containing the hydrogenase activity, onto a Q-sepharose fast-flow column (Pharmacia). The column was washed once with 2 column volumes of wash buffer (50 mM Tris, pH 8.5, 100 mM KCl) and eluted with two column volumes of elution buffer (50 mM Tris, pH8.5, 250 mM KCl). Approximately 85% of the hydrogenase activity detected in the crude lysate from induced WT cultures was recovered in the partially purified fraction. Protein samples were concentrated using an Amicon protein-concentration cell and a YM10 membrane. Equal amounts of protein (A280) were loaded and separated using standard SDS-PAGE methodologies. Western blotting was performed using a BioRad Mini-Protean III electrophoresis and blotting apparatus. The primary hydrogenase antibody was derived from a synthetic peptide (DKAKRQAALYNL) containing a sequence common to both the HydA1 and HydA2 proteins and was generated commercially in rabbits (Sigma GenoSys). The secondary antibody was obtained commercially as an alkaline phosphatase conjugate (BioRad), and standard chemochromic detection techniques were utilized for hydrogenase detection.
Gene Identification
DNA regions flanking the insertion site of pJD67 were determined using genome walking. DNA downstream of the insertion site was amplified using the PCR methods outlined in the Universal GenomeWalker Kit and in the Advantage-GC Genomic PCR mix, both from Clontech. Coding sequences for both the HydEF and HydG proteins were obtained by sequencing the cDNA corresponding to both genes. The cDNA constructs were obtained from the Kazusa DNA Research Institute (http://www.kazusa.or.jp/). All DNA products were sequenced by the University of California, Davis.
Complementation
A BAC clone containing the HydEF and HydG genes was obtained from the Clemson University Genetics center. The genomic HydEF gene was obtained by KpnI digestion of the BAC clone, and the insert, containing the full-length HydEF gene with promoter and termination sequences, was cloned into the KpnI site of pSP124S (from Saul Purton, University College, London). The resulting plasmid, pMP101, contains the HydEF gene and the Bler gene used for antibiotic selection. The pMP101 plasmid was linearized by digestion with SwaI and transformed into the hydEF-1 mutant using the glass bead method of Kindle (K. L. Kindle [1990] Proc. Natl. Acad. Sci. USA 87, 1228-1232). Controls, using cells only or 1 μg of pSP124S (V. Lumbreras et al. Plant j., 441-448), were also used.
Heterologous Expression and Purification
Expression of active C. reinhardtii HydA1 was achieved by cloning the HydEF and HydG cDNA constructs into E. coli expression plasmids driven by the T7 promoter. The two genes were cloned into the pACYC Duet expression plasmid (Novagen). Additional control plasmids containing only the HydEF or HydG genes were also cloned into the pACYC plasmid. The C. reinhardtii HydA1 gene was cloned into pETBlue-1, and a Strep-Tactin affinity tag (Strep-Tag II) was added to its C-terminus for affinity purification of HydA1. Plasmids were co-transformed into E. coli B1-21 (DE3) cells (Novagen). The presence of appropriate plasmids was verified by restriction analysis and sequencing. Expression and purification of tagged HydA1 was done as follows: An inoculum from an overnight culture of transformed BL21 (DE3) was grown in L-broth containing appropriate antibiotics. Cells were grown until the OD600 reached 0.5-0.7, and isopropyl-beta-D-thiogalactopyranoside (IPTG) (Novagen) was added to 1.0 mM. After a 1-h aerobic induction, cultures were made anaerobic by purging with argon for five hours. Cells were harvested, then disrupted on ice by sonication. HydA1-StrepTag II was purified using Strep-Tactin Sepharose (IBA) and assayed for hydrogenase activity using MV.
Expression Cloning of C. acetobutylicum HydA, HydE, HydF and HydG—
The C. acetobutylicum Hyd genes were isolated from purified genomic DNA (strain ATCC 824) by PCR amplification. Gene specific primers were based on the known sequence of HydA (Genbank accession no. AAB03723) (19, 20), and the sequences of HydE (Genbank accession no. CAC1631), HydF (Genbank accession no. CAC1651), HydG (Genbank accession no. CAC1356) and HydB (Genbank accession no. CAC3230) identified by tBLASTn homology searches of the C. acetobutylicum genome at NCBI using the C. reinhardtii HydEF, HydG and HydA2 peptide sequences. Gene-specific primers were designed to match the ends of each Hyd gene (IDT Technologies), and also to contain a suitable restriction site for expression cloning. Approximately 20 μg of genomic DNA were digested overnight with BamHI, and 200 ng were used as a template for PCR amplification reactions performed with KOD polymerase (Novagen). PCR fragments were gel purified, digested overnight with restriction enzymes and sub-cloned into the dual multiple cloning sites (MCS) of either plasmid pCDFDuet-1 (Novagen) (HydF and HydG) to form pCaFG, or pETDuet-1 (Novagen) (HydA Or HydB and HydE) to form pCaAE and 8pCaBE. The StrepII-tag sequence WSHPQFEK was added to the C-terminal end of [FeFe]-hydrogenase structural genes, HydA and HydB, during PCR amplification. The sequence and reading frame of each gene were confirmed by DNA sequencing (Davis Sequencing, LLC).
Expression Cloning of Other [FeFe]-Hydrogenase Genes
The [FeFe]-hydrogenase structural genes from C. reinhardtii, and Clostridium pasteurianum were cloned from purified genomic DNA as follows. To clone C. pasteurianum HydA, the ATCC strain 6013 was cultured anaerobically on reinforced clostridial media, and the genomic DNA was purified using the Qiagen DNAeasy Tissue Kit (Qiagen). Purified DNA (500 ng) was digested overnight with BamHI, and 100 ng were used in a PCR reaction with HydA specific oligonucleotides that contained a 5′-NcoI and 3′-BamHI site for in-frame cloning. Gene fragments were gel purified, digested with NcoI and BamHI, and cloned into MCS1 of pCaE2 to generate pCpIE.
The C. reinhardtii HydA1 and HydA2 cDNA's clones were used as templates for PCR amplification with oligonucleotides designed with 5′- and 3′-end restriction sites as described above. A StrepII-tag sequence WSHPQFEK was added to the 5′-end oligonucleotide of HydA1, and to the 3′-end oligonucleotide of HydA2. Gene fragments were isolated by gel electrophoresis, digested, and sub-cloned into MCS site 2 of pCaE1 (pETDuet-1 with HydE at MCS site 1) to form pECr1 (HydA1), and pECr2 (HydA2). The reading frames and gene sequences were confirmed by DNA sequencing.
[FeFe]-Hydrogenase Expression in E. coli—
For expression testing of constructs, plasmids that harbored a complete set of T7 regulated Hyd genes were co-transformed into the E. coli strain BL21 (DE3) (Novagen) with co-selection for Apr (pETDuet-1 clones), and Smr (pCDFDuet clones). Transformed cells were grown overnight in LB media (Sigma) plus antibiotics, and the next day were sub-cultured (1:50 dilution) into 115 ml of fresh LB media supplemented with antibiotics, and 100 μM Fe-Citrate. Cultures were grown aerobically at 37° C. on a rotary shaker at 250 rpm to an OD600 of 0.5-0.7. Isopropyl-beta-D-thiogalactopyranoside (IPTG) (Novagen) was added to a final concentration of 1.5 mM, and cultures shaken at room temperature at  ̃100 rpm to allow for pre-induction of Hyd expression prior to anaerobic induction. After 1 h, the cultures were transferred to a 120 ml serum vial, sealed with rubber septa, and sparged with argon at room temperature for a period of 3-5 h to achieve anaerobic conditions and induction of [FeFe]-hydrogenase biosynthesis. The expression of [FeFe]-hydrogenases for affinity purification was performed in minimal media. Transformed cells were cultured overnight in 5 ml of M63, supplemented with 0.5% glucose, 0.4% casein-hydrolysate, 100 ∝M Fe-citrate, 300 ∝cg/ml ampicillin, and 50 ∝g/ml streptomycin. Overnight-grown cultures were diluted 1:50 into 25 ml of fresh media, grown at 37° C. until the OD600 reached 0.5 and used to inoculate 1 L of M63 (without Fe-citrate). The 1 L culture was grown at 37° C. to an OD600 of 0.5. A 1 M solution of Fe-citrate was added to a final concentration of 100 μM, and the cultures incubated an additional 10 min at 37° C. IPTG (1.5 mM) was added and the cultures shaken at 100 rpm for 1 h at room temperature. Following the initial induction period, the cultures were transferred to a sealed 1 L flask and sparged with argon at room temperature overnight to induce biosynthesis of [FeFe]-hydrogenase.
Purification of Recombinant, StrepII-Tagged [FeFe]-Hydrogenases—
Purification steps were performed under anaerobic conditions. Cells expressing StrepII-tagged [FeFe]-hydrogenases, were collected by centrifugation at 6000×g for 10 minutes. The cell pellet was resuspended in break buffer (BB) (150 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM DTT, 1 mM Na-dithionite, 100 μM PMSF, 5% glycerol), and broken in a French press. Avidin was added at 3 nM to block binding of biotin and biotinylated proteins. The disrupted cell suspensions were centrifuged at 19,000×g for 30 min to pellet cell debris. The clarified crude extracts were passed over a Streptactin-Sepharose (IBA) affinity column, pre-equilibrated with buffer BB. Columns were washed with 3-5 column volumes of ice-cold BB, and the StrepII-tagged hydrogenases were eluted in BB containing 2.5 mM desthiobiotin.
SDS-PAGE and Western Blots of Heterologously-Expressed Proteins
For SDS-PAGE, protein samples were diluted in 1× SDS-PAGE loading buffer (Novagen), boiled for 10 min and cooled on ice. Samples were loaded onto a 12% SDS gel and run at 45 mA for 2 hours. Following electrophoresis, proteins were blotted onto PVDF membranes and detected using a streptactin-alkaline phosphatase conjugate detection kit (IBA).
[FeFe]-Hydrogenase Activity Assays—
Activities of purified [FeFe]-hydrogenases or of whole cell extracts were routinely measured as the production of H2 gas from reduced methyl viologen (MV). Activity assays of whole cells were performed in argon flushed, 13.5 ml sealed serum vials that contained 1 ml of an anaerobically prepared, 2× whole-cell reaction buffer (50 mM potassium phosphate, pH 7; 10 mM methyl viologen; 20 mM sodium dithionite; 6 mM NaOH; 0.2% Triton X-100) and 1 ml of cells. Assays of purified enzymes were performed on aliquots (25-50 □l) diluted to 1 ml in anaerobically prepared BB buffer in an argon flushed vial that also contained 1 ml of an anaerobically prepared, 2× enzyme-reaction buffer (50 mM potassium phosphate, pH 7; 10 mM methyl viologen; 20 mM sodium dithionite; 6 mM NaOH). All reactions were incubated at 37 □C. After incubation, 400 □l of headspace gas was removed with a gas-tight syringe and H2 levels measured by gas chromatography (Hewlett Packard, 5820).
Results
Mutant Characterization
In C. reinhardtii, two [FeFe]-hydrogenase enzymes, HydA1 and HydA2, are known. In order to identify genes required for expression and activity of these enzymes, we used chemochromic H2 sensors4 to screen a random insertional mutagenesis library for clones incapable of photo-producing H2 following the required anaerobic induction. Mutants were generated by transforming the Arg7 gene into C. reinhardtii strain CC425, which is an arginine auxotroph. The Arg7 gene is randomly incorporated into the C. reinhardtii genome and disrupts small sections of wild-type (WT) genomic DNA. The mutant hydEF-1 was identified by its inability to produce detectable quantities of H2 as shown in FIG. 1A. The dark blue spots observed for the other five colonies from the same library are indicative of WT H2-production capacity.
The mutant hydEF-1 grew on minimal medium agar plates with CO2 as the sole carbon source, thereby demonstrating that the cells were photosynthetically competent. Furthermore, photosynthetic and respiratory rates of both the parental and hydEF-1 strains were measured in liquid media using a Clark-type electrode (FIG. 1B). Compared to the WT, the hydEF-1 mutant exhibited normal rates of respiration and photosynthetic O2 evolution. This demonstrates that the lack of H2 photo-production activity in hydEF-1 is not the consequence of a secondary metabolic, or photosynthetic electron transport defect, but rather is specific to the hydrogenase enzyme.
Hydrogenase activity in C. reinhardtii is induced by anaerobiosis achieved either in the dark by using an inert gas (or exogenous reductant) to purge O2 from sealed cultures or in the light by depriving sealed cultures of sulfur, which results in attenuated rates of photosynthetic O2 evolution. Hydrogen production, following dark anaerobic induction, was monitored from WT and hydEF-1 mutant cultures using several techniques: (1) initial rates of H2 photo-production (FIG. 1C) were assayed using a Clark-type electrode, (2) hydrogenase activity mediated by reduced MV was detected by GC, and (3) fermentative H2 production was assayed by GC analysis. In contrast to WT cultures, H2 production was not detected from hydEF-1 mutant cultures by any of these assays. Moreover, hydEF-1 mutant cultures that were induced anaerobically in the light, under conditions of sulfur deprivation, failed to produce any detectable H2. One-liter CC425 WT cultures consistently produced at least 70 ml of H2, over the course of several days, under identical conditions. We therefore concluded that the hydEF-1 mutant is unable to synthesize an active [Fe]-hydrogenase under all of our induction and assay conditions.
Identification of the HydEF and HydG Genes
To determine the genetic mutation responsible for the observed phenotype of the hydEF-1 mutant, we cloned and sequenced the genomic DNA flanking the mutagenizing Arg7 insert using a genome walking strategy. The gene disrupted by Arg7 insertion was determined by comparing the flanking WT sequence to the recently sequenced C. reinhardtii genome. The deleted gene in hydEF-1, denoted HydEF, and was shown to encode a protein with two unique domains. The N-terminal portion of the HydEF protein is homologous to a distinct group of proteins that to date are only found in prokaryotes containing [FeFe]-hydrogenases and belongs to a previously uncharacterized subset of the Radical SAM protein superfamily (H. J. Sofia et al [2001] Nucleic Acids Res. 1097-1106). The C-terminal portion of the HydEF protein contains a domain with predicted GTPase activity. This domain is homologous to a second distinct group of prokaryotic proteins, which are also unique to organisms that contain [FeFe]-hydrogenases. Directly adjacent to the disrupted HydEF gene in C. reinhardtii is a second gene, HydG, which is arranged in an order suggestive of divergent expression from the same promoter region. BLAST searches revealed that proteins homologous to HydG comprise a third set of unique proteins that also belong to the Radical SAM protein superfamily. As with HydE and HydF, the HydG homologues are only found in prokaryotes with [FeFe]-hydrogenases. The cDNAs corresponding to HydEF and HydG in C. reinhardtii were obtained and then sequenced to confirm the protein coding sequence of the two genes. A schematic indicating the genomic organization of the C. reinhardtii genes and the site of HydEF disruption is shown in FIG. 2A.
Strikingly, in the genomes of Bacteroides thetaiotaomicron, Desulfovibrio vulgaris, Desulfovibrio desulfuricans and Shewanella oneidensis, the HydE, HydF and HydG genes form putative operons with [FeFe]-hydrogenase structural genes (FIG. 3). However, the functions of HydE, HydF and HydG have not until now been assigned. As discussed below, our data indicate that these proteins are required for the assembly of active [FeFe]-hydrogenase, and therefore, we have named the C. reinhardtii genes, HydEF and HydG, according to the suggested hydrogenase nomenclature (P. M. Vignais et al. [2001] FEMS Microbiol. Rev. 25, 455-501). In C. reinhardtii, the HydEF gene is assigned the two letters E and F to correspond to the two distinct genes observed in prokaryotic organisms. FIG. 3 compares the C. reinhardtii HydEF and HydG protein homologies to prokaryotic organisms containing [FeFe]-hydrogenases. This figure also shows the organization of the HydE, HydF and HydG open reading frames in relationship to the putative [FeFe]-hydrogenase gene(s) within these organisms. Although the proposed [FeFe]-hydrogenase assembly genes observed in the previously mentioned organisms are found in putative operons along with the [Fe]-hydrogenase structural genes, these proposed assembly proteins within the majority of the organisms shown in FIG. 3 are found separated from the structural genes.
Complementation of the HydEF Gene
To link the observed loss of H2 production in the C. reinhardtii hydEF-1 mutant to disruption of HydEF, we used gene complementation. Genomic DNA, containing the WT HydEF gene, was obtained from a single BAC clone found in a library of C. reinhardtii genomic DNA. The BAC plasmid, containing the HydEF gene, was digested with appropriate restriction enzymes to generate a fragment predicted to contain only the full length HydEF genomic gene and its putative promoter. This insert was cloned into plasmid SP124S, which contains the Ble gene that confers resistance to the antibiotic zeocin. The hydEF-1 mutant was transformed with this construct, grown on TAP-agar plates containing zeocin, and clones with restored H2-production capacity were obtained as shown in FIG. 2B. Integration of the complementing gene and verification of the mutant background were confirmed by Southern blotting (FIG. 2C). The CC425 sample shows the WT band, which is absent in both the mutant and the complemented clone. The complemented clone shows two strong bands corresponding to multiple random integration of the transformed HydEF genomic fragment into the mutant genome, as well as a faint band that may represent integration of only a portion of the HydEF gene.
Analysis of Gene Expression and [FeFe]-Hydrogenase Accumulation
Northern blot analyses were then performed to determine (a) whether the observed loss of hydrogenase activity in the hydEF-1 mutant was due to disruption of HydA1 and/or HydA2 gene transcription and (b) if HydEF and HydG are co-expressed anaerobically with the hydrogenase genes. RNA aliquots were collected from aerobic WT and hydEF-1 mutant cultures, as well as from WT and hydEF-1 mutant cultures anaerobically induced in the dark for 0.5 and 4.0 hours. FIGS. 4A-D compare, respectively, the expression profiles of the HydA1, HydA2, HydG and HydEF genes from both CC425 parental WT and hydEF-1 mutant cultures. The data demonstrate that HydEF and HydG are anaerobically induced concomitantly with the HydA1 and HydA2 genes in WT cultures. Likewise, the HydA1, HydA2, and HydG transcripts are also induced anaerobically in the hydEF-1 mutant, and as expected, the HydEF transcript is absent. The presence of HydA1 and HydA2 transcripts in anaerobically induced hydEF-1 cultures clearly indicates that disruption of the HydEF gene does not affect hydrogenase transcription in any significant fashion and that the loss of H2 production in hydEF-1 cultures is not the consequence of a defect in hydrogenase gene transcription.
Western blots were then obtained to determine the consequence of HydEF gene disruption on hydrogenase protein levels (FIG. 4E). An antibody designed to recognize both C. reinhardtii HydA1 and HydA2 was used to probe for the presence of hydrogenase proteins. As expected, the partially purified WT sample (see Experimental Procedures) shows only a single anaerobically induced band with an electrophoretic mobility of approximately 47-48 kd, due to co-migration of the two hydrogenases. Although full length HydA1 and HydA2 hydrogenase enzymes from C. reinhardtii have predicted masses of 53.1 kd and 53.7 kd, respectively, HydA1 undergoes N-terminal proteolytic processing of a chloroplast transit peptide sequence, resulting in a mature 47.5 kd protein localized in the chloroplast (T. Happe et al. [1993] Eur. J. Biochem. 214, 475-481). The HydA2 protein is predicted to undergo similar processing, resulting in an estimated 47.3 kd mature protein (M. Forestier et al. [2003] Eur. J. Biochem. 270, 2750-2758). The Western data from anaerobically induced hydEF-1 cultures indicate that immunologically detectable enzyme is also found in hydEF-1 mutant cultures, despite the lack of detectable enzyme activity. The electrophoretic mobility of the hydrogenase band from hydEF-1 mutant cultures is shifted slightly lower relative to the WT band and is consistent with the electrophoretic mobility of unprocessed C. reinhardtii hydrogenase. In the case of [NiFe]-hydrogenases, proteolytic processing occurs after insertion of Ni, resulting in shifted Western bands relative to the unprocessed [NiFe]-enzyme (N. K. Menon et al [1991] J. Bacterol. 173, 4851-4861) (A. Jacobi et al. [1992] Arch. Microbiol. 158, 444-451) The presence of shifted bands in the anaerobically induced hydEF-1 protein extracts suggests that this might also occur in the case of C. reinhardtii [Fe]-hydrogenases lacking a fully assembled active site.
Heterologous Expression of C. reinhardtii HydA1 in E. coli. 
Additional evidence supporting the conclusion that the HydEF and HydG proteins are required for formation of an active [Fe]-hydrogenase is shown by the heterologous expression of active C. reinhardtii HydA1 protein in E. coli, a bacterium that lacks a native [FeFe]-hydrogenase. The HydA1 protein was expressed as a fusion protein containing a Strep-Tag II affinity sequence and purified from E. coli extracts. The expression of the HydA1 construct alone or co-expression of the HydA1 and HydEF, or HydA1 and HydG genes in E. coli all resulted in the expression of non-functional HydA1 protein after purification as shown in FIG. 5. However, the co-expression of C. reinhardtii HydA1 along with both HydEF and HydG in anaerobic E. coli cultures yielded an active HydA1 enzyme (FIG. 5). Since the expression system has yet to be optimized, the amount of active HydA1 obtained from independent experiments is low and varies significantly. Nevertheless, functional [FeFe]-hydrogenase was only obtained in the presence of all three expressed genes. It should be noted that some Radical SAM proteins act with extremely low turnover numbers, and may even be reactants and not catalysts (H. J. Sofia et al. [2001] Nucleic Acids Res. 29. 1097-1106).
Radical SAM Homology
The HydEF and HydG proteins belong to the Radical SAM (also known as the AdoMet radical) superfamily. These proteins participate in numerous biochemical reactions, including, but not limited to: sulfur insertion, radical formation, organic ring synthesis, and anaerobic oxidation. The HydG protein and the HydE domain of the C. reinhardtii HydEF protein both contain the signature Cys-X3-Cys-X2-Cys motif that is typically found within the Radical SAM protein superfamily (FIG. 6). This motif coordinates a redox active [4Fe4S] cluster under reducing conditions. The reactions performed by Radical SAM proteins are typically initiated by the generation of a free radical after the reductive cleavage of S-adenosylmethionine (SAM) at the [4Fe4S] cluster, which yields methionine and a 5′-deoxyadenosyl radical. This high-energy organic radical then abstracts a hydrogen atom from substrates unique to each Radical SAM protein.
Roles for HydEF and HydG in H-Cluster Assembly
Radical SAM proteins are frequently involved in the anaerobic synthesis of complex biomolecules and coordinate unusual [FeS] clusters that are often labile. These characteristics are consistent with the types of chemistries required to synthesize the unique ligands of the H-cluster and to assemble the [FeFe]-hydrogenase catalytic cluster. A recent classification of the Radical SAM superfamily suggests that the most distantly related proteins, including biotin synthase (BioB) and the nitrogenase accessory protein NifB, appear to be involved in S transfer. Remarkably, Fe and S originating from the metabolic product of NifB, the NifB-cofactor, ultimately become incorporated into the [FeMo]-cofactor of dinitrogenase (R. M. Allen [1995] J. Biol. Chem. 270, 26890-26896), another enzyme capable of H2 production. Thus, there is precedent for the involvement of a Radical SAM protein in the donation of Fe to the catalytic metal cluster of an [Fe]-metalloenzyme, and we propose that the HydE and/or HydG proteins play a similar role in the mobilization of Fe for assembly of the [FeFe]-hydrogenase H-cluster.
The H-cluster also requires CN, CO and the putative di(thiomethyl)amine ligand. It is conceivable that the accessory proteins HydEF and/or HydG described are also responsible for biosynthesis and assembly of these products coordinated to Fe. Since CN and CO are among the most toxic compounds in biology, and likely do not exist freely within the cell, it would be necessary to synthesize these ligands at the site of H-cluster assembly. In the case of the [NiFe]-hydrogenases, strong evidence indicates that CN and CO are synthesized by the HypE and HypF proteins, using carbamoyl phosphate as a precursor to form a thiocarbamate. However, no homologues of the HypE and HypF proteins have been observed in C. reinhardtii, or in other organisms containing only [FeFe]-hydrogenases. This suggests an alternative pathway for CN and CO synthesis or an alternative means to form thiocarbamate. Radical SAM proteins utilize chemistries that include organic radical formation, persulfide formation, pyroxidal phosphate activation, thiocarbonyl formation, and amine migration, all or any one of which could be involved in the synthesis of the H-cluster organic ligands.
Homology alignments between C. reinhardtii HydEF and HydG relative to their prokaryotic homologues are shown in FIG. 6. In addition to the Radical SAM motifs, the HydG and HydF proteins have other conserved sequences with the potential to coordinate metal ions. These include a E(A/G)CXH and a (L/V)HC(G/A)(G/A)C motif near the C-terminus of the HydF domain, and a CT(A/G)CYR motif near the C-terminus of the HydG protein. All three of these motifs are strictly conserved in the [FeFe]-hydrogenase assembly proteins, but they are absent from other Radical SAM proteins, which suggests that these motifs are unique to the [FeFe]-hydrogenase accessory proteins. Several other conserved amino acids are found throughout the HydEF and HydG proteins; however, the elucidation of roles for these determinants and the potential metal-binding motifs in the assembly of [FeFe]-hydrogenase will likely have to await future investigation. It should also be noted that the HydF domain of the HydEF protein contains a putative GTPase domain, and the HypB protein, which also has GTPase activity, facilitates Ni incorporation into the active site of [NiFe]-hydrogenases. Interestingly, neither the HydEF or the HydG proteins are highly homologous to the TM1420 protein characterized from T. maritima (G. Pan et al. [2003] J. Biol. Inorg. Chem. 8, 469-474). The latter is only 8.5 kD long and does not contain a characteristic Radical SAM motif. This suggests that TM1420 may be unique to T. maritima, which has the most complex [FeFe]-hydrogenase characterized to date.
Heterologous Expression of Chlamydomonas [FeFe]-Hydrogenase
The heterologous expression of C. reinhardtii HydA1 in E. coli, demonstrates that only two C. reinhardtii gene products, HydEF and HydG (equivalent to three prokaryotic genes) are required for assembly of HydA 1; however, a minimum of seven accessory gene products are required for the formation of an active [NiFe]-hydrogenase enzyme (L. Casalot et al. [2001] Trends Microbiol. 9, 228-237). This is consistent with the prediction that the [FeFe]-hydrogenases may require fewer maturation proteins because these enzymes lack Ni (P. M. Vignais et al. [2001] FEMS Microbiol. Rev. 25, 455-501). The existence of entirely unique maturation proteins required for the assembly of [FeFe]-hydrogenase is consistent with the absence of a phylogenetic relationship between [NiFe] and [FeFe]-hydrogenases.
Previous attempts to express the CpI or DdH [FeFe]-hydrogenase enzymes in E. coli resulted in the synthesis of inactive proteins that were unable to evolve or uptake H2 gas (G. Voordouw et al. [1987] Eur. J. Biochem. 162, 31-36) (Y. Asada et al. [2000] Biochim. Biophys. Acta. 1490, 269-278). In contrast, transformation of the cyanobacterium, Synechococcus PCC7942, with the CpI [FeFe]-hydrogenase structural gene yielded strains that expressed an active [FeFe]-hydrogenase. Given that there is no biochemical or genetic evidence for the presence of an [FeFe]-hydrogenase in Synechoccocus PCC7942, it appears that accessory proteins responsible for assembling the Synechococcus [NiFe]-hydrogenases are flexible enough to also activate the CpI [FeFe]-hydrogenase enzyme. It is not clear why this is possible in Synechoccocus and not in E. coli, but these results emphasize the complex nature of hydrogenase expression and activation in different microorganisms.
Expression and Biosynthesis of C. acetobutylicum [FeFe]-Hydrogenase HydA in E. coli—
Although the purified algal [FeFe]-hydrogenase expressed in E. coli as described above was active, the instability of the expression plasmids made the transformants difficult to propagate, which resulted in low HydA1 expression levels. The DNA compositions of algal genes are highly GC-biased at 64% overall, and 90% at the third codon position. To address codon bias effects on gene stability and expression, we searched the sequenced genomes of various anaerobic microbes for homologues of HydEF and HydG to use as alternatives to the algal genes. The genome of C. acetobutylicum was found to possess HydE, HydF and HydG homologues in agreement with previous reports on characterization of a soluble, monomeric [FeFe]-hydrogenase (CaHydA) in this organism. Unlike the high GC content of the C. reinhardtii HydEF (70%) and HydG (65%) genes, the C. acetobutylicum genes were more AT-rich (GC-content; HydE, 32%; HydF, 33%; HydG, 35%) and thus were expected to be more stable, and better expressed in E. coli. The C. acetobutylicum HydE, HydF and HydG genes were PCR amplified and the products cloned into a set of T7 expression plasmids together with the HydA gene encoding [FeFe]-hydrogenase I (FIG. 7). Plasmids that harbored a complete set of C. acetobutylicum maturation and structural genes were transformed into E. coli strain BL21 (DE3) for IPTG-inducible expression. Compared to the plasmid-encoded C. reinhardtii HydEF and HydG genes, which were observed to undergo some rearrangements upon propagation in E. coli (unpublished results), the plasmid-encoded C. acetobutylicum HydE, HydF, and HydG genes did not exhibit any sequence alterations. The higher stability of the C. acetobutylicum genes resulted in greater numbers of transformed cells, and higher growth rates under expression conditions (data not shown).
When E. coli is cultured under anaerobic growth in the absence of fermentable sugars, the endogenous [NiFe]-hydrogenases, Hyd1, Hyd2 and Hyd3 are uninduced due to the lack of formate. Formate is a fermentative metabolite required for the transcriptional activation of the hyp and hyc operons encoding maturation and Hyd3 structural genes respectively. As a result, anaerobic growth of E. coli in the absence of formate results in basal levels of [NiFe]-hydrogenase activities in whole-cell extracts as shown in TABLE I, which also, shows evolution of activities of [FeFe] hydrogenases anaerobically coexpressed with the C. acetobutylicum maturation proteins in E. coli.










TABLE I







Whole-cell
Affinity purified



[FeFe]
extractsa (nmol H2
(□mol H2


Organism
hydrogenase
ml−1 min−1)
mg−1 min−1)


















E. coli

0.35b
NDc


C. reinhardtii
HydA1
61
150


C. reinhardtii
HydA2
108
116.1


C. acetobutylicum
HydA
96
75.2



HydAΔN
6
31.6



HydB
13
8.6


C. pasteurianum
HydA
150
ND



HydAΔN
15
ND





aWhole cells solubilized with 0.1% Triton X-100.


bWhole cell activity in the absence of [FeFe]hydrogenase structural and maturation proteins.


cND, not determined.






The basal [NiFe]-hydrogenase activities under these growth conditions allows for the study of maturation and biosynthesis of recombinantly expressed [FeFe]-hydrogenases. As shown in TABLE 1, the extracts of anaerobically grown E. coli cells expressing the C. acetobutylicum maturation system with the CaHydA structural protein exhibited reduced-MV-catalyzed, H2-evolution activities fold greater than the activities in extracts of untransformed cells. These elevated hydrogenase activities are directly attributable to the high levels of plasmid-encoded C. acetobutylicum gene expression and the biosynthesis of CaHydA [FeFe]-hydrogenase.
The C. acetobutylicum maturation system produced mg-per-liter amounts of CaHydA, whereas our previous C. reinhardtii maturation system produced only μg-per-liter amounts of the C. reinhardtii HydA1. As shown in TABLE 1, the specific activity of reduced-MV catalyzed, H2-evolution by affinity-purified StrepII-tagged CaHydA was 75 μmol H2 mg−1 min−1, 7.5-fold higher than the value reported by Girbal et al. of CaI purified from C. acetobutylicum (L. Gerbal et al. Appl. Env Microbiol. 71, 2777-2781).
Biosynthesis of Heterologous [FeFe]-Hydrogenases by the C. acetobutylicum Maturation System—
Our interest in studying the biochemical and structural properties of the [FeFe]-hydrogenases found in the green algae C. reinhardtii prompted a test of the capability of the C. acetobutylicum to biosynthesize the algal enzymes. A characteristic of the algal [FeFe]-hydrogenase peptide sequences is the lack of accessory iron-sulfur-cluster domains auxiliary to the highly conserved H-cluster/catalytic domain (FIG. 9). This reduced structural complexity classifies the algal hydrogenases as the simplest yet characterized. In C. reinhardtii, the CrHydA1 [FeFe]-hydrogenase undergoes N-terminal processing as a result of translocation from the cytoplasm to the chloroplast stroma. Similar to CrHydA1, the N-terminal sequence of CrHydA2 also possesses signal sequence characteristics with a predicted cleavage site near amino acid position 61. For expression in E. coli, a truncated HydA2 was cloned into expression plasmid pCaE, which created an N-terminus at position 62 that corresponds to the predicted processed product. As shown in Table 1, the mature forms of both CrHydA1 and CrHydA2 were biosynthesized as active enzymes in E. coli. Following affinity purification the typical yields of these proteins ranged from 0.8 to 1.0 mg-per-liter-of-culture. The H2-production activities from reduced-MV of purified CrHydA1 and CrHydA2 were 150 and 116 μmol H2 mg−1 min−1 respectively. This measured activity for CrHydA1 purified from our E. coli expression system was 5 to 6-fold lower than the previously published activities of this enzyme purified from a recombinant, or native source. It has been established that under anaerobic conditions, C. reinhardtii utilizes reduced [2Fe2S]-ferredoxin as electron-donor to [FeFe]-hydrogenase for in vivo H2-production. Previous measurements of H2-evolution kinetics with partially purified C. reinhardtii hydrogenases and reduced C. reinhardtii [2Fe2S]-ferredoxin showed a Km of 10 μM. The Km of reduced spinach [2Fe2S]-ferredoxin for purified HydA2 in this study was measured at 31 μM. This value is similar to the previous reported value of 35 μM for purified HydA1 and reduced spinach ferredoxin, suggesting that HydA2 is capable of catalyzing in vivo H2-production in C. reinhardtii. 
In summary, two novel genes, HydEF and HydG, found in C. reinhardtii, are strictly conserved in organisms containing [Fe]-hydrogenases. The HydEF and HydG genes are transcribed anaerobically in parallel with the HydA1 and HydA2 [Fe]-hydrogenase genes in C. reinhardtii. Disruption of HydEF abolishes all H2 production, and although full-length hydrogenase protein is detected by Western blotting, no enzyme activity is observed. Hydrogen production is restored after complementation of the hydEF-1 mutant with WT genomic DNA containing the HydEF gene. Moreover, we report the first successful co-expression of the C. reinhardtii HydEF, HydG and HydA1 genes in E. coli, and the synthesis of an active [FeFe]-hydrogenase in this bacterium. The current study also identifies a new class of metallo-enzyme accessory proteins and assigns assembly function to two proteins belonging to a subset of the Radical SAM superfamily. Characterization of these [FeFe]-hydrogenase assembly proteins will greatly facilitate additional examination of the mechanism by which [Fe]-hydrogenases are synthesized in nature.
Our results clearly show that H-cluster biosynthesis is a highly conserved process. Together with recent structural data on [FeFe]-hydrogenases CpI (denoted CpHydA in this study) and DdH, our work supports early observations that various [FeFe]-hydrogenases possess essentially identical H-clusters. This is perhaps more apparent in the case of CaHydB, which has only low sequence identity with CaHydA (119%), but undergoes maturation by the same set of C. acetobutylicum proteins.
The efforts to develop biological alternatives to fossil fuels have helped stimulate an ongoing interest in the use of microorganisms as production sources for a number of energy carriers. The physiology of H2-producing organisms, and the hydrogenases that mediate H2 metabolism, have been intensely studied for use as large-scale H2-production sources. A greater understanding of how the hydrogenases are biosynthesized, and how their unique structures contribute to biochemical and metabolic function will assist in the continued development of both biological and bio-inspired H2-production systems.
While the invention has been described in detail with reference to preferred embodiments, it is to be understood that this description is by way of example only and not to be construed as limiting. Accordingly, numerous changes in the details of the embodiments of the invention and additional embodiments of the invention will be apparent to, and may be made by persons of ordinary skill in the art having reference to this description, and all such changes and additional embodiments are within the true scope of the spirit of the invention, as claimed hereafter.

## Claims

1. An E. coli cell that co-expresses exogenous genes encoding HydEF and HydG and also expresses a hydrogenase structural gene.

2. The E. coli cell of claim 1, wherein the HydEF and HydG genes are from a green algae.

3. The E. coli cell of claim 1, wherein the HydEF and HydG genes are from an organism of the genus Chlamydomonas.

4. The E. coli cell of claim 1, wherein the HydEF and HydG genes are from Chlamydomonas reinhardtii.

5. The E. coli cell of claim 1, wherein the hydrogenase structural gene is HydA1 or HydA2.

6. The E. coli cell of claim 5, wherein the HydA1 or HydA2 gene is from a green algae.

7. The E. coli cell of claim 5, wherein the HydA1 or HydA2 gene is from an organism of the genus Chlamydomonas.

8. The E. coli cell of claim 5, wherein the HydA1 or HydA2 gene is from Chlamydomonas reinhardtii.