Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution
© Yonemoto et al.; licensee BioMed Central Ltd. 2013
Received: 20 February 2013
Accepted: 26 June 2013
Published: 2 July 2013
Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.
We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.
Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.
KeywordsHydrogenase Cyanobacteria Iron-sulfur Cluster Alteromonas Macleodii “Deep Ecotype”
Hydrogen produced photobiologically from sunlight and water is a potential future biofuel and is an alternative to traditional carbon-based fuels [1–4]. Enzymes that produce hydrogen include nitrogenases and hydrogenases, the latter comprising the unrelated families of [Fe-Fe] hydrogenases, Fe-only hydrogenases, and [NiFe] hydrogenase enzymes . Cyanobacteria such as Synechococcus elongatus are ideal host strains for a biological system that converts solar energy to hydrogen because they are genetically tractable and easily cultured . Because cyanobacteria also perform oxygenic photosynthesis, the oxygen produced will inactivate most hydrogenases [7, 8]. Therefore, installing a hydrogenase that is less sensitive to oxygen is a primary design constraint of this system. Among the hydrogenase families, oxygen tolerance has been described only among the [NiFe] hydrogenases, although not all [NiFe] hydrogenases are oxygen tolerant [9–11]. There is some evidence the native cyanobacterial bidirectional hydrogenase may be capable of hydrogen production in the presence of oxygen , but the energetic consequences of using NADPH as an electron donor makes it less attractive.
We previously reported the discovery and characterization of a novel hydrogenase isolated from Sargasso Sea environmental DNA with high identity to the stable [NiFe] uptake hydrogenase from Thiocapsa roseopersicina. A closely-related sequence was later identified in the Alteromonas macleodii ‘ Deep ecotype’ genome (hereafter referred to as the A. macleodii hydrogenase), and was characterized as active in the presence of 2% oxygen . The DNA encoding the enzyme was then cloned and used to express active enzyme in both Escherichia coli and S. elongatus. Expressing active A. macleodii hydrogenase in both E. coli and cyanobacteria requires the co-expression of at least nine additional maturation factors to properly assembly an active enzyme . Due to the complex maturation requirements of [NiFe] hydrogenases, only a few other examples exist of heterologous expression in E. coli[18–20]. The dual expression system is ideal for rapidly generating variant hydrogenases using powerful E. coli genetic tools, quickly screening for desirable properties in E. coli, and confirming the persistence of the property in the final target cyanobacterial host.
At the medial Fe-S cluster, uptake [NiFe] hydrogenases have conserved amino acid residues deviating from the canonical 4-cysteine ligation characteristic of Fe-S cubane clusters. In A. macleodii and almost all members of the uptake NiFe hydrogenase family, the medial cluster features a proline in the place of a ligating cysteine resulting in the installation of a 3Fe-4S cluster instead of a 4Fe-4S cluster . Interestingly, the closely related small subunits of the H2-sensing regulatory hydrogenases, which show almost no hydrogen evolution, and the NiFeSe hydrogenases, which show more hydrogen evolution activity , both feature cysteinyl ligations at the homologous amino acid position [24, 30]. The unrelated small subunit of the F420-reducing hydrogenase, which also features 3 4Fe-4S clusters, has a 4Fe-4S cluster for its medial cluster . In A. macleodii hydrogenase, and uptake hydrogenases in general, the cluster distal to the active site is a 4Fe-4S cluster featuring an unusual iron-histidinyl ligation that is conserved in most [NiFe] hydrogenases. While its function is unknown, the unusual ligation may be related to intramolecular electron transfer [23, 32].
In Desulfovibrio fructosovorans, substitution of the proline residue associated with the medial cluster to cysteine (Df HynA P238C) resulted in approximately 30% reduction of hydrogen uptake activity and a nearly two-fold increase in evolution activity . Separately, when the histidine residue at the distal cluster was changed to cysteine (Df HynA H184C), hydrogen uptake activity was nearly abolished while evolution activity was only 50% reduced . We produced enzyme variants featuring substitutions in the A. macleodii hydrogenase at sites homologous to the D. fructosovorans uptake hydrogenase substitutions. The variants of the A. macleodii enzyme (HynSL - NCBI accession codes: YP_004425481.1, YP_004425482.1) featuring the corresponding substitutions based on homology (HynS H230C, P285C1) were created both individually and combined in an effort to find an enzyme with improved hydrogen evolution activity for production of hydrogen in cyanobacteria.
Taking advantage of our previously reported dual-species system, these modified enzymes were first screened in E. coli to assess their enzymatic properties. Initial screens with E. coli extracts indicated that the doubly-substituted enzyme had the highest hydrogen evolving activity. Purification of the enzyme indicated that this activity was intrinsic to the enzyme variant, and not due to enzyme titer or host factors persistent in crude lysate. Finally, we moved the expression system to the cyanobacterium S. elongatus, which recapitulated the relative activities of the enzymes, validating our general strategy of using E. coli as a rapid-turnover model system.
Results and discussion
Heterologous expression for [NiFe] hydrogenase in E. coli
Evolution activity assay
Uptake activity assay
To test the effect of our substitutions on hydrogen uptake, we performed a benzyl viologen hydrogen uptake assay on E. coli lysates expressing the wild type A. macleodii [NiFe] hydrogenase, and variants featuring the substitutions H230C, P285C, and H230C/P285C. In this reaction the hydrogenase catalyzes the dissociation of hydrogen to protons and electrons. The electrons are transferred to benzyl viologen, causing a color change detectable by spectrophotometry at 555 nm. Lysates containing the H230C and P285C enzymes each had slightly increased uptake activity relative to WT; H230C had nearly twice the uptake activity as WT while P285C had slightly less activity than H230C. The hydrogen uptake activity of lysate containing the doubly-substituted enzyme was similar to WT and within the range of experimental error (Figure 3B).
Evolution assay on purified enzyme
Nonetheless, we found that purification afforded a more than 100-fold improved specific activity for both wild type and doubly-substituted enzymes. Furthermore, after parallel purification the doubly-substituted, strep-tagged enzyme still exhibited three-fold more activity than the wild type, strep-tagged enzyme, consistent with our observations using unpurified E. coli lysates in the hydrogen evolution assay (Figure 2A).
Thermal and oxygen tolerance assays
Relative performance in other bacterial strains
By substituting two residues in the small subunit of A. macleodii [NiFe] uptake hydrogenase, we have increased its hydrogen evolution activity. To our knowledge this is the first reported instance of this doubly-substituted enzyme analyzed in the context of hydrogen evolution. In the homologous doubly-substituted enzyme from D. fructosovorans constructed by Dementin and co-workers , activity in the evolving direction was not reported. Comparing the homologous substitutions in A. macleodii and D. fructosovorans hydrogenases, we observe qualitatively different structure-activity relationships. Thus, the effect of these substitutions may not be generalizable across different members of the enzyme family. Indeed, the inverse experiment, changing a medial 4Fe-4S cluster to a 3Fe-4S cluster in the Methanococcus janaschii F420-reducing hydrogenase, resulted in severely impaired uptake, inconsistent with the loose pattern otherwise observed in uptake hydrogenases . As such, others seeking to modify [NiFe] hydrogenase Fe-S cluster ligations should consider testing both single substitutions as well as the double substitution that are homologous to those discussed here when seeking an enzyme suitable for their needs.
The doubly-substituted A. macleodii hydrogenase we present here is an attractive launching point for future engineering of hydrogenases redesigned for use in photoautotrophs. The two substitutions have minimal effects on the thermotolerance of the enzyme, as measured by loss of activity after the harsh treatment of 85°C for 1 hour. Treatment at lower temperatures or shorter times did not cause appreciable loss of activity in either doubly-substituted or wild type enzymes (data not shown). Our observations suggest that the substitutions have not created severe structural changes, likely allowing further modification of the enzyme without disrupting function. Furthermore, the ability to function in the presence of partial oxygen, a critical property of photoautotrophic hydrogen production systems, appears to be unaffected and may be able to be improved by further modification.
Finally, because A. macleodii hydrogenase can be expressed as active enzyme in both E. coli and in the photoautotrophic cyanobacterium S. elongatus, our strategy moving forward is to use expression in E. coli as a preliminary model system to rapidly generate and test promising modifications of the enzyme prior to experimentation in S. elongatus, which has a longer turnaround time for genetic manipulations. The H230C/P285C enzyme demonstrated the same qualitative performance over the wild type enzyme in extracts prepared from both S. elongatus and E. coli, validating this strategy. Initial attempts at in vivo activity were performed in both host species aerobically and anaerobically, but did not yield measurable hydrogen production. To that end, further modifications we are pursuing to achieve this important result include: improving the oxygen tolerance by engineering a substitution leading to a 4Fe-3S proximal Fe-S cluster that is not natively present in our enzyme [37–40], and connecting the hydrogenase to cyanobacterial electron carriers, such as ferredoxin.
In summary, we have used an E. coli - cyanobacteria shuttle system to rapidly test candidate modifications of a heterologously-expressed A. macleodii [NiFe] hydrogenase in E. coli before transferring the most promising constructs to the slower-growing cyanobacteria. As a test of this strategy, we substituted the A. macleodii hydrogenase with modifications that in other systems have been shown to change the ligations of the medial and distal Fe-S clusters. When expressed in E. coli, we found that neither substitution alone improved enzyme activity; however, when combined, both substitutions increased hydrogen evolution activity approximately 4-fold relative to wild type, while hydrogen uptake remained unchanged. As we did not directly verify that these substitutions resulted in the corresponding alterations to the Fe-S clusters, we cannot completely rule out the possibility of more subtle effects on electron-transfer kinetics in the absence of the expected, new cysteinyl-iron bonds or the possibility that the native enzyme did not feature the expected Fe-S clusters. The effect of this combined modification was recapitulated with the enzymes in enriched form and with the enzymes expressed in cyanobacteria, suggesting that the enzymatic improvement is intrinsic to the enzyme and independent of extrinsic host factors. These results together validate our strategy of piloting hydrogenase modifications in the rapid-turnaround E. coli model system for use in cyanobacterial hosts.
Primers, full plasmid maps and sequences are provided in supplementary materials. Plasmid pIY003 was constructed by PCR-amplification of the hydrogenase operon from A. macleodii genomic DNA. Two amplicons spanning the operon were produced where the sequences had overlapping ends with each other and to the plasmid pTRC-NS1 (the backbone plasmid from ), which was linearized by double digest with the restriction enzymes BamHI and EcoRI. These three DNA fragments were assembled using Gibson isothermal assembly , and transformed into DH10B (Invitrogen) cells by electroporation. After screening clones by PCR, candidate constructs were sequence-verified by Sanger DNA sequencing. Plasmids pIY004, bearing the H230C substitution and pIY006, bearing the P285C substitution were constructed similarly, except using PCR products generated from primers containing the appropriate DNA nucleotide substitutions; plasmid pIY007, bearing the doubly-substituted enzyme, was constructed by repeating the procedure used to generate pIY006 except using pIY004 plasmid as a template.
Plasmid pIY033 was constructed by moving the hydrogenase operon into a pDEST-23 vector backbone using Gibson isothermal assembly followed by re-amplification of this operon (now including T7 promoter and terminator) and recloning into the pTRC-NS1 plasmid by Gibson isothermal assembly. Successful construction was verified by Sanger sequencing. pIY038 was constructed by PCR amplification of a segment of pIY007 around the hynS ORF followed by Gibson isothermal assembly into plasmid pIY033 which was linearized by double digest with the BamHI and AgeI restriction enzymes. Construction of plasmid pRC41-4 will be discussed elsewhere but the sequence is provided in the Supplemental Information; pCM012 was constructed by PCR amplification of the second half of the hynS ORF from pIY038, followed by Gibson isothermal assembly into pRC41-4 which was linearized by double digest with BamHI and AgeI restriction enzymes. Successful construction of pIY038 and pCM012 was verified by Sanger sequencing initiated with PCR primers used to amplify the modified region. Plasmids pIY085 and pIY086 were constructed similarly to pCM012, with pIY003 and pIY007 as templates, respectively, except using two sequential rounds of amplification with two reverse primers together generating sequence encoding the strep-tag epitope –RSAWSHPQFEK and the sequence bridging the C-terminal stop codon of hynS and the beginning of hynL up to the AgeI restriction site. Successful construction of pIY085 and pIY086 was verified by Sanger sequencing initiated with the PCR primers used to amplify the modified region.
A. macleodii Hydrogenase Expression in E. coli
All four plasmids pIY003, pIY004, pIY006, pIY007 were transformed into E. coli strain FTD147 cells, an MC4100(DE3) strain with the three active E coli hydrogenases removed . Plasmids pIY033 and pIY038 were transformed into BL21(DE3)ΔH4 cells . To grow E. coli cultures used to prepare extracts, 25 mL of autoinducer media  supplemented with 0.1 mM NiCl2 and 40 μg mL-1 spectinomycin was inoculated with 100 μL of a starter culture of the appropriate strain. Following inoculation, the expression culture was grown in an unbaffled 250 mL Erlenmeyer flask, rotating at 200 rpm, under ambient room atmosphere at 30°C overnight (~10-14 hrs). Under these conditions, the culture grew to a density in the approximate range of OD600 6–7. Piloting experiments showed that growth in baffled flasks resulted in higher specific activity but was avoided to maintain comparability with previous experiments . This was then harvested by centrifugation; the pellet was separated from the supernatant media and resuspended after addition of 0.8 mL lysis buffer (10 mM Tris, pH 7; 1 mM DTT; 0.5 mM EDTA). Lysis was performed by sonication on wet ice for one minute using a microprobe sonicator (Branson, Sonifier Model 250) at setting “4”, 40% duty cycle. The lysate was cleared by centrifugation for 10 minutes at 16,800 x g, at 4°C, and the supernatant was reserved as the “cleared lysate”. Protein content in the cleared lysate was measured with Bradford reagent (Bio-Rad) and comparison against a BSA (NEB) standard curve.
A. macleodii Hydrogenase Expression in S. elongatus
Plasmids pRC41-4 and pCM012 were mobilized into S. elongatus PCC 7942 strain PW416 by conjugative transfer from E. coli as previously described [16, 44]. Genomic integration into neutral site I (NSI) was verified by PCR. Cyanobacterial cultures were grown in 50 mL Bg11 medium in baffled 250 mL flasks to an OD730 of approximately 0.5. The cultures were induced by addition of IPTG to a final concentration of 0.25 mM, and additionally 0.5 μM NiCl2 was supplemented. Cells were grown for an additional 24 h, centrifuged, resuspended in 800 μL sonication buffer and sonicated as for E. coli. The lysates were cleared as described above for E. coli and the supernatant was reserved as the “cleared lysate”. In vitro hydrogenase evolution assays were conducted as described below.
Evolution activity assay
Cleared lysate (0.2 mL) was added to 1.7 mL of methyl viologen assay buffer containing 1.5 mL deionized water, 0.1 mL 40 mg mL-1 methyl viologen (Aldrich), and 0.1 mL of 500 mM potassium phosphate, pH 7.0 (Gibco). The combined samples in 13 mL gastight vials was sealed using rubber septa (Aldrich), and sparged under argon (Westair) for 20 minutes to remove oxygen. After sparging, 0.1 mL of 2 M sodium dithionite (Aldrich) was added by syringe anoxically. The resulting assay solution was a 1:10 dilution of lysate containing 25 mM potassium phosphate, 8 mM methyl viologen, and 100 mM sodium dithionite, pH ~7.0. E. coli samples were incubated for 2 hours at 30°C, followed by gas chromatography (CP-3800, Varian) using a Fused Silica Molsieve 5A column (CP7537, Varian) of 100 μL samples taken from the vial headspace. Previous experiments [13, 14] and piloting experiments for this experiment demonstrated time-dependent linearity of hydrogen evolution over the course of ~20 hours, suggesting that enzyme quality is unlikely to have been affected by the reaction conditions, including damage by oxidized dithionite products. S. elongatus samples were prepared similarly but were incubated at 30°C overnight before GC analysis. Hydrogen peaks were identified and integrated to quantify hydrogenase yield by comparison to a standard curve prepared with pure hydrogen from which a specific activity was calculated based on total protein in the lysate. For plasmids pIY003, pIY004, pIY006, and pIY007, the evolution activity was measured for four biological replicates with three technical replicates for each expression. For plasmids pIY033, pIY038, the evolution activity was measured for three technical replicates, and for pRC41-4, and pCM012, the evolution activity was measured for three biological replicates.
Uptake activity assay
Cleared lysate (0.1 mL) was added to 0.9 mL of benzyl viologen assay buffer containing 0.05 mL of benzyl viologen (65 mg mL-1) and 0.05 mL of 0.5 M potassium phosphate buffer, pH 7.0, and 0.8 mL deionized water. The combined assay sample was prepared in a 2 mL round-rim disposable UV cuvette (Brandtech) and sealed with a rubber septum (Aldrich), coated with a small amount of vacuum grease (Fisher). The cuvette was sparged for 20 minutes under 10% hydrogen balanced by nitrogen (Westair). Cuvettes were then transferred to a multichannel spectrophotometer and monitored at 555 nm at 30°C. For all four variants, four instances of expression were conducted with four replicates for each instance, such that three of these replicate experiments appeared to have self-consistent progress curves without prejudice to the data processing. Reaction rates were identified by fitting a linear regression to the data between OD555 = 1 to OD555 = 3.
Thermal exposure assay
The assay was conducted as the evolution assay, except cleared lysate was further incubated at 85°C for one hour and centrifuged a second time for 10 minutes at 16,800 x g and 4°C to clear additional insoluble debris prior to addition to MV buffer. Equivalent volumes of heat-treated and control-treated cleared lysate were added to assay buffer, and total hydrogen evolution activities without normalizing to protein concentration were used for subsequent calculations. Tolerance to thermal exposure was expressed by dividing the mean of three technical replicates of the denatured sample by the mean of three technical replicates of the undenatured sample. This process was subjected to three biological replicates; the geometric mean and geometric standard error are reported.
Oxygen exposure assay
The H-D exchange assay to test sensitivity to oxygen exposure was performed as follows: cleared lysate (0.1 mL) was added to 0.8 mL of mass spectrometry buffer containing 0.05 mL of 0.5 M potassium phosphate buffer, pH 7.0 (Gibco), and 0.75 mL deionized water. The samples were placed in 10 mL scintillation vials, and capped with new rubber septa. Vials were sparged for 10 minutes under pressurized argon. A polyimide-coated fused silica capillary, ID 50 μm OD 220 μm (Scientific Instrument Services) adapted for mass spectrometry was then inserted into the vial headspace followed by mass spectrometric monitoring (Omnistar, Pfeiffer). The microcapillary substituted for the standard metal probe supplied with this instrument samples very low quantities of total gas making up an inconsequential amount of total gas volume for the duration of the experiment. Hydrogen (100%, Westair) was injected to a final concentration of 10% hydrogen headspace and verified by mass spectrometry. For microaerobic samples, air was injected to create a final 1% oxygen mixture. Anaerobic samples were incubated for 2h at 30°C to ensure quantitative enzyme recovery, followed by injection of 0.1 mL D2O, followed by monitoring of the developing m/z = 3, until χHD > 1000 ppm. Microaerobic samples, prepared in parallel, were then injected with 0.1 mL D2O followed by overnight mass spectrometric monitoring at m/z = 3. Oxygen tolerance was measured by filtering spikes from the progress curve, calculating enzyme rate and normalizing the slope of the microaerobic activity against the slope of the anaerobic activity. This process was subjected to three biological replicates; the geometric mean and geometric standard errors are reported.
Cleared lysate from E. coli strains containing plasmid pIY085 and pIY086 was further purified using Streptactin magnetic bead resin (Qiagen). Cleared lysate was prepared as above but in NP buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl) with 1 mM dithiothreitol and NeutrAvidin (Thermo) at 10 μg mL-1 final concentration. Streptactin resin (100 μl) was combined with cleared lysate (400 μl) and incubated at 4°C for 1 h with end-over-end rotation. The supernatant was removed and the resin was washed three times for five minutes each at room temperature in NP buffer containing 0.05% sodium dodecyl sulfate (SDS). This was followed by two washes in NP buffer supplemented with 0.01% Tween 20 as above. Final elution was performed at room temperature in 100 μl NP buffer supplemented with 0.01% Tween 20 and 10 mM biotin.
Lysates were diluted to 5 mg mL-1 total protein. One lysate from an overexpression of WT hydrogenase was further treated by denaturation at 65°C for 10 minutes and clearing by centrifugation, at 20,000 x g for 10 minutes. 25 μL of 4x NuPAGE SDS-PAGE loading buffer (Invitrogen) was added to 75 μL aliquots, followed by boiling for 10 minutes. These samples were frozen at −80°C and on a subsequent day thawed and 10 μL was separated onto a 10% NuPAGE Bis-Tris gel with the NuPAGE MOPS-SDS running buffer system (Invitrogen). Gel electrophoresis was performed at 150 V for 5 hours at 4°C, and the gel was cut separating the gel into two halves at approximately 75 kDa. The top gel was stained using the Sypro Ruby kit (Invitrogen) as a total protein loading control; the bottom gel was transferred onto nitrocellulose using NuPage transfer buffer (Invitrogen) with 30% MeOH for 10 hours at 10 mA constant current. These samples were then hybridized using rabbit-anti-HynL primary antibody  and goat-anti-rabbit-DyLight secondary antibody (Thermo), and imaged using a Typhoon fluorescence scanning imager (GE).
For gels associated with protein purification, samples were prepared as described above, but for cleared lysate, approximately 5 μg was loaded per lane and for purified proteins, approximately 0.5 μg protein was loaded per lane. Electrophoresis of the gels for western blot analysis of Strep-tagged HynS was only performed for 1 h at 150 V. Monoclonal antibody to the strep-tag antigen (Qiagen) was used with goat-anti-mouse-DyLight secondary antibody (Thermo) and imaged as above.
1In this paper the amino acid sequence numbers used for A. macleodii HynS are relative to the translational start methionine as annotated (NCBI: YP_006975190.1), not the putative starting residue after N-terminal processing by the TAT system, as this site has not been experimentally verified.
We thank Pin-Ching Maness for advice on experimental procedures. We thank Phillip Wright for kindly providing us with the BL21ΔH4 strain. Funding for all aspects of this work was provided by the U.S. Department of Energy, Fuel Cell Technologies Office (DE-FG36-05GO15027).
- Muellerlanger F, Tzimas E, Kaltschmitt M, Peteves S: Techno-economic assessment of hydrogen production processes for the hydrogen economy for the short and medium term. International Journal of Hydrogen Energy 2007, 32: 3797-3810. 10.1016/j.ijhydene.2007.05.027View Article
- Beer LL, Boyd ES, Peters JW, Posewitz MC: Engineering algae for biohydrogen and biofuel production. Current opinion in biotechnology 2009, 20: 264-271. 10.1016/j.copbio.2009.06.002View Article
- Angermayr SA, Hellingwerf KJ, Lindblad P, De Mattos MJT: Energy biotechnology with cyanobacteria. Current opinion in biotechnology 2009, 20: 257-263. 10.1016/j.copbio.2009.05.011View Article
- Ghirardi ML, Dubini A, Yu J, Maness P-C: Photobiological hydrogen-producing systems. Chemical Society reviews 2009, 38: 52-61. 10.1039/b718939gView Article
- Vignais PM, Billoud B: Occurrence, classification, and biological function of hydrogenases: an overview. Chemical reviews 2007, 107: 4206-4272. 10.1021/cr050196rView Article
- Tamagnini P, Leitão E, Oliveira P, Ferreira D, Pinto F, Harris DJ, Heidorn T, Lindblad P: Cyanobacterial hydrogenases: diversity, regulation and applications. FEMS microbiology reviews 2007, 31: 692-720. 10.1111/j.1574-6976.2007.00085.xView Article
- Houchins JP, Burris RH: Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120. J. Bacteriol 1981, 146: 209-214.
- Cournac L, Guedeney G, Peltier G, Vignais PM, De BL: Sustained Photoevolution of Molecular Hydrogen in a Mutant of Synechocystis sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex. Journal of Bacteriology 2004, 186: 1737-1746. 10.1128/JB.186.6.1737-1746.2003View Article
- Ghirardi ML, Posewitz MC, Maness P-C, Dubini A, Yu J, Seibert M: Hydrogenases and hydrogen photoproduction in oxygenic photosynthetic organisms. Annual review of plant biology 2007, 58: 71-91. 10.1146/annurev.arplant.58.032806.103848View Article
- Parkin A, Sargent F: The hows and whys of aerobic H2 metabolism. Current opinion in chemical biology 2012, 16: 26-34. 10.1016/j.cbpa.2012.01.012View Article
- Fritsch J, Lenz O, Friedrich B: Structure, function and biosynthesis of O(2)-tolerant hydrogenases. Nature reviews. Microbiology 2013, 11: 106-114. 10.1038/nrmicro2940View Article
- McIntosh CL, Germer F, Schulz R, Appel J, Jones AK: The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production. Journal of the American Chemical Society 2011, 133: 11308-11319. 10.1021/ja203376yView Article
- Maróti G, Tong Y, Yooseph S, Baden-Tillson H, Smith HO, Kovács KL, Frazier M, Venter JC, Xu Q: Discovery of [NiFe] hydrogenase genes in metagenomic DNA: cloning and heterologous expression in Thiocapsa roseopersicina. Applied and environmental microbiology 2009, 75: 5821-5830. 10.1128/AEM.00580-09View Article
- Vargas WA, Weyman PD, Tong Y, Smith HO, Xu Q: [NiFe] hydrogenase from Alteromonas macleodii with unusual stability in the presence of oxygen and high temperature. Applied and environmental microbiology 2011, 77: 1990-1998. 10.1128/AEM.01559-10View Article
- Weyman PD, Vargas WA, Chuang R-Y, Chang Y, Smith HO, Xu Q: Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli. Microbiology (Reading, England) 2011, 157: 74-1363.View Article
- Weyman PD, Vargas WA, Tong Y, Yu J, Maness P-C, Smith HO, Xu Q: Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus. PloS one 2011, 6: e20126. 10.1371/journal.pone.0020126View Article
- Bock A, King P, Blokesch M, Posewitz M: Maturation of Hydrogenases. Advances in Microbial Physiology 2006, 51: 1-225.View Article
- Wells MA, Mercer J, Mott RA, Pereira-Medrano AG, Burja AM, Radianingtyas H, Wright PC: Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase. Metabolic engineering 2011, 13: 445-453. 10.1016/j.ymben.2011.01.004View Article
- Sun J, Hopkins RC, Jenney FE, McTernan PM, Adams MWW: Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production. PloS one 2010, 5: e10526. 10.1371/journal.pone.0010526View Article
- Kim JYH, Jo BH, Cha HJ: Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli. Journal of biotechnology 2011, 155: 312-319. 10.1016/j.jbiotec.2011.07.007View Article
- Lukey MJ, Parkin A, Roessler MM, Murphy BJ, Harmer J, Palmer T, Sargent F, Armstrong FA: How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. The Journal of biological chemistry 2010, 285: 3928-3938. 10.1074/jbc.M109.067751View Article
- Abou Hamdan A, Dementin S, Liebgott P-P, Gutierrez-Sanz O, Richaud P, De Lacey AL, Rousset M, Bertrand P, Cournac L, Léger C: Understanding and tuning the catalytic bias of hydrogenase. Journal of the American Chemical Society 2012, 134: 8368-8371. 10.1021/ja301802rView Article
- Dementin S, Belle V, Bertrand P, Guigliarelli B, Adryanczyk-Perrier G, De Lacey AL, Fernandez VM, Rousset M, Léger C: Changing the ligation of the distal [4Fe4S] cluster in NiFe hydrogenase impairs inter- and intramolecular electron transfers. Journal of the American Chemical Society 2006, 128: 5209-5218. 10.1021/ja060233bView Article
- Rousset M, Montet Y, Guigliarelli B, Forget N, Asso M, Bertrand P, Fontecilla-Camps JC, Hatchikian EC: [3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis. Proceedings of the National Academy of Sciences of the United States of America 1998, 95: 11625-11630. 10.1073/pnas.95.20.11625View Article
- Fontecilla-Camps JC, Volbeda A, Cavazza C, Nicolet Y: Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases. Chemical reviews 2007, 107: 4273-4303. 10.1021/cr050195zView Article
- Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, Fontecilla-Camps JC: Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 1995, 373: 580-587. 10.1038/373580a0View Article
- Higuchi Y, Yagi T, Yasuoka N: Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis. Structure 1997, 5: 1671-1680. 10.1016/S0969-2126(97)00313-4View Article
- Kelley LA, Sternberg MJE: Protein structure prediction on the Web: a case study using the Phyre server. Nature protocols 2009, 4: 363-371.View Article
- Parkin A, Goldet G, Cavazza C, Fontecilla-Camps JC, Armstrong FA: The difference a Se makes? Oxygen-tolerant hydrogen production by the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum. Journal of the American Chemical Society 2008, 130: 13410-13416. 10.1021/ja803657dView Article
- Garcin E, Vernede X, Hatchikian E, Volbeda A, Frey M, Fontecilla-Camps J: The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center. Structure 1999, 7: 557-566. 10.1016/S0969-2126(99)80072-0View Article
- Bingemann R, Klein A: Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae. European Journal of Biochemistry 2000, 267: 6612-6618. 10.1046/j.1432-1327.2000.01755.xView Article
- Volbeda A, Darnault C, Parkin A, Sargent F, Armstrong FA, Fontecilla-Camps JC: Crystal Structure of the O(2)-Tolerant Membrane-Bound Hydrogenase 1 from Escherichia coli in Complex with Its Cognate Cytochrome b. Structure 2013, 21: 184-190. 10.1016/j.str.2012.11.010View Article
- Huang H-H, Camsund D, Lindblad P, Heidorn T: Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic acids research 2010, 38: 2577-2593. 10.1093/nar/gkq164View Article
- Bustos SA, Golden SS: Light-regulated expression of the psbD gene family in Synechococcus sp. strain PCC 7942: evidence for the role of duplicated psbD genes in cyanobacteria. Molecular & general genetics 1992, 232: 221-230.
- Theodoratou E, Paschos A, Magalon A, Fritsche E, Huber R, Böck A: Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation. European Journal of Biochemistry 2000, 267: 1995-1999. 10.1046/j.1432-1327.2000.01202.xView Article
- Dementin S, Burlat B, Fourmond V, Leroux F, Liebgott P-P, Abou Hamdan A, Léger C, Rousset M, Guigliarelli B, Bertrand P: Rates of intra- and intermolecular electron transfers in hydrogenase deduced from steady-state activity measurements. Journal of the American Chemical Society 2011, 133: 10211-10221. 10.1021/ja202615aView Article
- Goris T, Wait AF, Saggu M, Fritsch J, Heidary N, Stein M, Zebger I, Lendzian F, Armstrong FA, Friedrich B: others: A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase. Nature Chemical Biology 2011, 1: 1-9.
- Pandelia M-E, Nitschke W, Infossi P, Giudici-Orticoni M-T, Bill E, Lubitz W: Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus. Proceedings of the National Academy of Sciences of the United States of America 2011, 108: 6097-6102. 10.1073/pnas.1100610108View Article
- Shomura Y, Yoon K-S, Nishihara H, Higuchi Y: Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase. Nature 2011, 479: 253-256. 10.1038/nature10504View Article
- Fritsch J, Scheerer P, Frielingsdorf S, Kroschinsky S, Friedrich B, Lenz O, Spahn CMT: The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre. Nature 2011, 479: 249-252. 10.1038/nature10505View Article
- Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods 2009, 6: 343-345. 10.1038/nmeth.1318View Article
- Redwood MD, Mikheenko IP, Sargent F, Macaskie LE: Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production. FEMS microbiology letters 2008, 278: 48-55. 10.1111/j.1574-6968.2007.00966.xView Article
- Studier FW: Protein production by auto-induction in high density shaking cultures. Protein expression and purification 2005, 41: 207-234. 10.1016/j.pep.2005.01.016View Article
- Elhai J, Wolk CP: Conjugal transfer of DNA to cyanobacteria. Methods in enzymology 1988, 167: 747-754.View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.