Bidirectional Hydrogenase Research Articles

Increasing biohydrogen production by metabolic engineering

Two types of enzymes can catalyze the reduction of protons to H 2, namely nitrogenase and hydrogenase. Although much progress has been made in the elucidation of gene expression, structure and regulation of these key enzymes, no practical and economically competitive process for the continuous production of biological H 2 (biohydrogen) has, as yet, been put on the market. One of the difficulties is due to the fact that H 2 output represents an energy loss for the cell and that microbial metabolic network has evolved for rationalization of energy use and optimization of specific growth rate. The study of the physiology of genetically modified photosynthetic microorganisms has shown that electron flux could be redirected to the bidirectional hydrogenase in a ndhB mutant of Synechocystis and that a change in carbon metabolism in mutants of Rhodobacter capsulatus unable to grow photoautotrophically could affect the flow of reducing equivalent from organic substrates to nitrogenase. Increasing the flux through an existing pathway or redirecting enzyme-catalyzed reactions is an approach referred to as metabolic engineering. Various “naturally engineered” organisms are found in Nature. An example is provided by the bacterium Dehalococcoides ethenogenes, which is the only bacterium known to reductively dechlorinate the ground water pollutants tetrachloroethene and trichloethene to ethene. D. ethenogenes exhibits an unusual metabolic specialization; it uses only H 2 as an electron donor and chlorinated compounds as electron acceptors to support growth. In accordance, the sequence of its genome has revealed the presence of five hydrogenase complexes.

Metal dependence and intracellular regulation of the bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803

Investigating the regulation of the cyanobacterial bidirectional hydrogenase on the molecular level is a prerequisite to learn more about the details of its function and to enhance biohydrogen production. Several deletion mutants of histidine kinase genes ( hik) of Synechocystis were analysed concerning their hydrogenase activity. hik7 showed no hydrogenase activity at all. It is supposed that the decreased activity is due to an improper processing of the enzyme. Furthermore, the effect of iron deficiency on the bidirectional hydrogenase was investigated on the level of transcription and enzyme activity. Low iron supply elicited low hydrogenase activity but a high promoter activity. Genetic and phenotypic differences have been repeatedly described for different wild-type (WT) strains of Synechocystis. Here, we report the identification of a substrain that has a severely reduced hydrogenase activity compared to the strain used throughout this study. The hydrogenase activity of the substrain was found to be partly complemented by the addition of nickel. Thus, revealing an inhibited nickel uptake into the cell or insertion into the hydrogenase. It emphasizes the importance of the right choice of WT strain for investigations concerning the hydrogen production capacities.

LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator

The bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 is encoded by five genes (hoxEFUYH) which are transcribed as one unit. The transcription of the hox-operon is regulated by a promoter situated upstream of hoxE. The transcription start point was located at -168 by 5'Race. Several promoter probe vectors carrying different promoter fragments revealed two regions to be essential for the promoter activity. One is situated in the untranslated 5'leader region and the other is found -569 to -690 nucleotides upstream of the ATG. The region further upstream was shown to bind a protein. Even though an imperfect NtcA binding site was identified, NtcA did not bind to this region. The protein binding to the DNA was purified and found to be LexA by MALDI-TOF. The complete LexA and its DNA binding domain were overexpressed in Escherichia coli. Both were able to bind to two sites in the examined region in band-shift-assays. Accordingly, the hydrogenase activity of a LexA-depleted mutant was reduced. This is the first report on LexA acting not as a repressor but as a transcriptional activator. Furthermore, LexA is the first transcription factor identified so far for the expression of bidirectional hydrogenases in cyanobacteria.

LexA, a transcription regulator binding in the promoter region of the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis PCC 6803 contains a single pentameric bidirectional hydrogenase encoded by hoxEFUYH. Transcriptional experiments demonstrated that the five hox genes are part of a single transcript together with three ORFs with unknown functions. The transcription start point was localized by 5′ RACE to 168bp upstream the hoxE ATG start codon. DNA affinity assays demonstrated a specific interaction between the hox regulatory promoter region and a protein which, using mass spectrometry, was identified to be LexA. Overexpressed His-tagged Synechocystis LexA and EMSA showed a specific binding to the promoter region of the hox operon. Increasing concentrations of the purified LexA resulted in two retarded LexA–DNA complexes, in agreement with the presence of two putative LexA binding sites upstream the determined TSP.

Biodiversity of Hydrogenases in Frankia

Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.

Cyanobacterial-type, heteropentameric, NAD+-reducing NiFe hydrogenase in the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina.

Structural genes coding for two membrane-associated NiFe hydrogenases in the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (hupSL and hynSL) have recently been isolated and characterized. Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction. The enzyme responsible for this activity was partially purified, and the gene cluster coding for a cytoplasmic, NAD+-reducing NiFe hydrogenase was identified and sequenced. The deduced gene products exhibited the highest similarity to the corresponding subunits of the cyanobacterial bidirectional soluble hydrogenases (HoxEFUYH). The five genes were localized on a single transcript according to reverse transcription-PCR experiments. A sigma54-type promoter preceded the gene cluster, suggesting that there was inducible expression of the operon. The Hox hydrogenase was proven to function as a truly bidirectional hydrogenase; it produced H2 under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N2. In-frame deletion of the hoxE gene eliminated hydrogen evolution derived from the Hox enzyme in vivo, although it had no effect on the hydrogenase activity in vitro. This suggests that HoxE has a hydrogenase-related role; it likely participates in the electron transfer processes. This is the first example of the presence of a cyanobacterial-type, NAD+-reducing hydrogenase in a phototrophic bacterium that is not a cyanobacterium. The potential physiological implications are discussed.

Accumulation of O 2-tolerant phenotypes in H 2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidly inactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H 2-evolving activities of up to 10 times the O 2-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H 2-metabolism clones following exposure to increasing amounts of O 2, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H 2 following exposure to O 2 concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H 2-production activity once O 2 is removed. These results suggest that O 2-tolerance can be increased by successive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by genetic means. Other results show that the hydrogenase is less sensitive to O 2 when it is actively catalyzing H 2 evolution.

Photoproduction of H 2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: from laboratory experiments to outdoor culture

We have analyzed the filamentous cyanobacterium Anabaena PCC 7120 (wildtype) containing one nitrogenase, one uptake hydrogenase and one bidirectional hydrogenase and its hydrogen uptake deficient mutant AMC 414 for their H 2 production capacities. Anabaena PCC 7120 and AMC 414 had similar growth rates in turbidostat mode with increased growth rates at higher light intensity. Rates of C 2H 2 reduction were similar for both strains. In contrast to the wildtype, AMC 414 produced H 2 in a PhotoBioReactor (PhBR) using air as the lifting gas. The rate of H 2 production increased with light intensity and was not even saturated at 456 μE m −2 s −1 . H 2 production increased significantly when replacing the air with argon. The maximal H 2 production during outdoor conditions was recorded using AMC 414 with a peak at 14.9 ml H 2 h −1 l −1 . Despite the relatively high production, maximal efficiency of solar energy to H 2 conversion was only 0.042%. A molecular method was developed to analyze the relative abundancies of weight and mutant in competition experiments in the PhBR.

Hydrogenases and photobiological hydrogen production utilizing nitrogenase system in cyanobacteria

We constructed three hydrogenase mutants from Anabaena sp. PCC 7120: Δ hupL (deficient in uptake hydrogenase), Δ hoxH (deficient in bidirectional hydrogenase), and Δ hupL/Δ hoxH (deficient in both genes), and showed that the Δ hup and Δ hupL/Δ hoxH produced H 2 at a rate 4–7 times that of wild-type under optimal conditions (Appl. Microbiol. Biotechnol. 58 (2002) 618). We have studied H 2 producing activity of Δ hup in more detail. H 2 producing activity of Δ hup cells was moderately improved in older cultures when 1% CO 2 was added to the bubbling air. The efficiency of light energy conversion to H 2 by the Δ hupL mutant at its highest H 2 production stage was 1.0–1.6% at an actinic visible light intensity of lower than 50 W/ m 2 under argon atmosphere, and the activity lasted for at least 35 min . At 250 W/ m 2 , H 2 producing activity gradually decreased with illumination time.

A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133

The hupL gene, encoding the uptake hydrogenase large subunit, in Nostoc sp. strain ATCC 29133, a strain lacking a bidirectional hydrogenase, was inactivated by insertional mutagenesis. Recombinant strains were isolated and analysed, and one hupL − strain, NHM5, was selected for further study. Cultures of NHM5 were grown under nitrogen-fixing conditions and H 2 evolution under air was observed using an H 2 electrode.

Identification of hox genes and analysis of their transcription in the unicellular cyanobacterium Gloeocapsa alpicola CALU 743 growing under nitrate-limiting conditions

The unicellular non-N 2-fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectional hydrogenase. Parts of all structural genes, encoding the hydrogenase, were identified, cloned and sequenced. When comparing the sequences with analogous sequences from other cyanobacteria the highest similarity was observed with hox genes from Synechocystis sp. PCC 6803. The hydrogenase activity increased considerably when the cells were grown aerobically in a medium with limiting concentrations of nitrate. However, the relative abundances of hoxH and hoxY transcripts, detected by RT-PCR, did not change significantly, demonstrating that the increase in the activity of G. alpicola hydrogenase was not a result of the increase of the transcription. In contrast, in Anabaena variabilis the induction of a bidirectional hydrogenase activity correlated with the relative level of hoxH and hoxY transcripts.

Identification of hox genes and analysis of their transcription in the unicellular cyanobacterium Gloeocapsa alpicola CALU 743 growing under nitrate-limiting conditions.

The unicellular non-N(2)-fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectional hydrogenase. Parts of all structural genes, encoding the hydrogenase, were identified, cloned and sequenced. When comparing the sequences with analogous sequences from other cyanobacteria the highest similarity was observed with hox genes from Synechocystis sp. PCC 6803. The hydrogenase activity increased considerably when the cells were grown aerobically in a medium with limiting concentrations of nitrate. However, the relative abundances of hoxH and hoxY transcripts, detected by RT-PCR, did not change significantly, demonstrating that the increase in the activity of G. alpicola hydrogenase was not a result of the increase of the transcription. In contrast, in Anabaena variabilis the induction of a bidirectional hydrogenase activity correlated with the relative level of hoxH and hoxY transcripts.

HoxE—a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria

NAD(P) +-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 μmol H 2 evolved (min mg protein) −1 and 15 μmol H 2 evolved (min mg protein) −1, respectively. H 2-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 °C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH) 2.

Hydrogenases and hydrogen metabolism of cyanobacteria.

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect--the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.

Disruption of the uptake hydrogenase gene, but not of the bidirectional hydrogenase gene, leads to enhanced photobiological hydrogen production by the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120

In order to determine the effects of the deletion of hydrogenase genes on nitrogenase-based photobiological H(2) productivity by heterocystous N(2)-fixing cyanobacteria, we have constructed three hydrogenase mutants from Anabaena sp. PCC 7120: hupL(-) (deficient in the uptake hydrogenase), hoxH(-) (deficient in the bidirectional hydrogenase), and hupL(-)/ hoxH(-) (deficient in both genes). The hupL(-) mutant produced H(2) at a rate four to seven times that of the wild-type under optimal conditions. The hoxH(-) mutant produced significantly lower amounts of H(2) and had slightly lower nitrogenase activity than wild-type. H(2) production by the hupL(-)/ hoxH(-) mutant was slightly lower than, but almost equal to, that of the hupL(-) mutant. The efficiency of light energy conversion to H(2) by the hupL(-) mutant at its highest H(2) production stage was 1.2% at an actinic visible light intensity of 10 W/m(2) (PAR) under argon atmosphere. These results indicate that deletion of the hupL gene could be employed as a source for further improvement of H(2) production in a nitrogenase-based photobiological H(2) production system.

Quantitative analysis of expression of two circadian clock-controlled gene clusters coding for the bidirectional hydrogenase in the cyanobacterium Synechococcus sp. PCC7942.

Hydrogen metabolism is of central interest in cyanobacterial research because of its potential applications. The gene expression and physiological role of the cyanobacterial bidirectional NAD(P)+-reducing hydrogenase are poorly understood. Transcription rates of hoxEF and hoxUYH encoding this enzyme have been studied in Synechococcus sp. PCC7942. PhoxU activity was about three times higher than that of PhoxE. Circadian phasing of both promoters was found to be synchronous and influenced expression levels by at least one order of magnitude. This is the first demonstration of circadian control of gene expression for any hydrogenase. For the majority of PhoxU-driven messages, transcription presumably terminates between hoxU and hoxH. Being part of a polycistronic hoxUYHW... operon, hoxW, encoding a protease involved in C-terminal processing of the hydrogenase large-subunit HoxH, is mainly expressed by its own promoter, PhoxW. The complex transcript formation may be a key feature for controlling bidirectional hydrogenase expression in vivo.

Cloning and sequencing of the genes encoding the subunits of bidirectional hydrogenase of Anabaena variabilis IAM M58

The enzymes directly involved in hydrogen metabolism in cyanobacteria are hydrogenases and nitrogenase. Functionally, two types of hydrogenase are known: uptake hydrogenase (Hup) and bidirectional hydrogenase (Hox). Distribution of hydrogenases among 15 filamentous cyanobacteria was studied by heterologous Southern hybridizations with hupL and hoxH probes from Anabaena PCC7120 and by direct assay of in vitro hydrogenase activities. All tested 15 strains showed hybridization with the hupL probe. With hoxH probe, hybridization bands are detected in 12 of them, but not in 3 of them. Moreover, the latter have no detectable in vitro Hox activity. The hupL and/or hoxH gene from Anabaena PCC7120 were inactivated. The extracts from the hoxH- strains were completely unable to perform Na2S2O4- and methyl viologen-dependent H2 evolution. H2 uptake with PMS by extracts from N2 fixing cells was undetectable in hupL-. The hupL- and hupL-hoxH- strains produced 4-7 times higher amounts of H2 than the wild-type strains. The nitrogenase activities of these hupL- strains were not reduced compared to the wild-type. Unexpectedly, the hoxH- strains were lower in H2 production activities by 20-50% compared to the wild-type.

Diversity of cyanobacterial hydrogenases, a molecular approach.

In an effort to elucidate the diversity of cyanobacterial hydrogenases, we used a molecular approach. Filamentous strains from a broad range of sources were screened for the presence of hup (uptake hydrogenase), xisC (rearrangement within hupL), and hox (bidirectional hydrogenase) genes. As expected, an uptake hydrogenase seems to be present in all N(2)-fixing cyanobacteria. On the other hand, no evidence was found for the presence of a conventional bidirectional enzyme in several strains. Similarly, the presence of xisC is not a characteristic shared by all the heterocyst-forming cyanobacteria. Although tempting, it is not possible to establish a correlation between the presence/absence of the bidirectional hydrogenase and the occurrence of xisC. The natural molecular variation of hydrogenases in cyanobacteria is certainly a field to explore, both to understand the physiological functions of the respective enzymes and to identify a genetic background to be used when constructing a strain for photobiological H(2) production in a bioreactor.

H 2-Uptake and evolution in the unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758

The unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758 growing photoautotrophically synthesised a hydrogenase which catalysed an in vivo H 2 uptake in the oxyhydrogen reaction at a significant rate and showed only low level of in vitro MV-dependent H 2 evolution. The in vitro hydrogenase activity was not induced under microaerobic or nitrate-limiting conditions. Some correlation observed between the two activities indicated that the same enzyme may be involved in both H 2 uptake and H 2 evolution. Heterologous Southern hybridisations, using cyanobacterial hup and hox DNA fragments as probes, showed the presence of sequences similar to hox (encoding for a bidirectional hydrogenase) in C. thermalis CALU 758 with no indication for the presence of any sequences corresponding to an uptake hydrogenase. Further molecular experiments, using specific primers directed against different conserved regions of the large subunit ( hoxH) of the bidirectional hydrogenase confirmed the presence of corresponding sequences in C. thermalis CALU 758. Low-stringency Southern hybridisations detected only one copy of hoxH within the genome of C. thermalis CALU 758.

The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis.

The activity of the bidirectional hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was found not to be regulated in parallel to respiration but to photosynthesis. A mutant with a deletion in the large hydrogenase subunit gene (hoxH), which contains the active site, was impaired in the oxidation of photosystem I (PSI) when illuminated with light, which excites either PSI alone or both photosystems. The fluorescence of photosystem II (PSII) of this mutant was higher than that of wild-type cells. The transcript level of the photosynthetic genes psbA, psaA and petB was found to be different in the hydrogenase-free mutant cells compared to wild-type cells, which indicates that the hydrogenase has an effect on the regulation of these genes. Collectively, these results suggest that the bidirectional hydrogenase functions as a valve for low-potential electrons generated during the light reaction of photosynthesis, thus preventing a slowing down of electron transport. This conclusion is supported by growth curves demonstrating that the mutant cells need more time to adapt to changing light intensities. Investigations of the wild-type and deltahoxH strains strongly suggest that Synechocystis contains only the bidirectional hydrogenase, which seems to be essentially insensitive to oxygen.