Hydrogenases In Cyanobacteria Research Articles

Novel concepts and engineering strategies for heterologous expression of efficient hydrogenases in photosynthetic microorganisms.

Hydrogen is considered one of the key enablers of the transition towards a sustainable and net-zero carbon economy. When produced from renewable sources, hydrogen can be used as a clean and carbon-free energy carrier, as well as improve the sustainability of a wide range of industrial processes. Photobiological hydrogen production is considered one of the most promising technologies, avoiding the need for renewable electricity and rare earth metal elements, the demands for which are greatly increasing due to the current simultaneous electrification and decarbonization goals. Photobiological hydrogen production employs photosynthetic microorganisms to harvest solar energy and split water into molecular oxygen and hydrogen gas, unlocking the long-pursued target of solar energy storage. However, photobiological hydrogen production has to-date been constrained by several limitations. This review aims to discuss the current state-of-the art regarding hydrogenase-driven photobiological hydrogen production. Emphasis is placed on engineering strategies for the expression of improved, non-native, hydrogenases or photosynthesis re-engineering, as well as their combination as one of the most promising pathways to develop viable large-scale hydrogen green cell factories. Herein we provide an overview of the current knowledge and technological gaps curbing the development of photobiological hydrogenase-driven hydrogen production, as well as summarizing the recent advances and future prospects regarding the expression of non-native hydrogenases in cyanobacteria and green algae with an emphasis on [FeFe] hydrogenases.

Diversity in hydrogen evolution from bidirectional hydrogenases in cyanobacteria from terrestrial, freshwater and marine intertidal environments

We characterized a set of 36 strains of cyanobacteria isolated from terrestrial, freshwater and marine intertidal settings to probe their potential to produce hydrogen from excess reductant, in the hope of finding novel strains with improved traits for biohydrogen production. The set was diverse with respect to origin, morphology, taxonomy and phylogeny. We found that about one half of the strains could produce hydrogen from hydrogenases in standard assays, a trait that corresponded invariably with the presence of homologues of the gene hoxH, coding for subunit H in the bidirectional Ni–Fe hydrogenase. Strains from freshwater and intertidal settings had a high incidence of hydrogen producing, hoxH containing strains, but all terrestrial isolates were negative for both. While specific rates of hydrogen production varied among strains, some novel strains displayed rates several fold higher than those previously reported. We detected two different patterns in hydrogen production. Pattern 1, corresponding to that previously known in Synechocystis PCC 6803, encompassed strains whose hydrogenase system produced hydrogen only temporarily to revert to hydrogen consumption within a short time and after reaching moderate hydrogen concentrations. Cyanobacteria displaying pattern 2, in the genera Lyngbya and Microcoleus, tended to have higher rates, did not reverse the direction of the reaction and reached much higher concentrations of hydrogen at steady state, making them of interest as potential platforms for biohydrogen production.

Metabolic Pathways for Photobiological Hydrogen Production by Nitrogenase- and Hydrogenase-containing Unicellular Cyanobacteria Cyanothece

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.

Heterologous Expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] Hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H2 evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.

The role of the bidirectional hydrogenase in cyanobacteria

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni–Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed.

Occurrence of Hydrogenases in Cyanobacteria and Anoxygenic Photosynthetic Bacteria: Implications for the Phylogenetic Origin of Cyanobacterial and Algal Hydrogenases

Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely.

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.

Uptake hydrogenase in cyanobacteria: novel input from non-heterocystous strains

Most of the research to date on cyanobacterial uptake hydrogenases has been performed on filamentous heterocystous strains. However, recent results on the hup gene cluster organization and its transcriptional regulation in non-heterocystous strains has contributed to the widening of knowledge in this field. In the present study, we outline the recent findings on uptake hydrogenases from non-heterocystous cyanobacteria, comparing it with the presently available data from heterocystous strains, and draw attention to potential areas for future research.

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.

Hydrogen metabolism in organisms with oxygenic photosynthesis: hydrogenases as important regulatory devices for a proper redox poising?

Abstract Three different hydrogenases and the nitrogenase putatively participate in the hydrogen metabolism of micro-organisms carrying out oxygenic photosynthesis. Hydrogenases either produce hydrogen or split hydrogen into protons and electrons depending on their redox partners, whereas the nitrogenase produces hydrogen unidirectionally as a byproduct during the reduction of nitrogen to ammonia. Hydrogenases are well-characterized enzymes on the enzymatic, structural and genetic level, especially in prokaryotic micro-organisms. They can be classified regarding the metal composition of their active site (Fe-only, NiFe or metal-free), their preferential direction of reaction (uptake only or bidirectional/reversible) and their in vivo electron donors or acceptors. The main physiological role of the uptake hydrogenase in cyanobacteria is probably recapturing the hydrogen produced by nitrogenase. The role of the bidirectional hydrogenase in phototrophs is still a matter of debate. Based on recent results which showed it to be of the NAD(P)-reducing type, a model for its physiological function is suggested. This model includes that this type of hydrogenase is linked to complex I of the respiratory electron-transport chain and might be an important electron valve during photosynthesis under rapidly changing light conditions. The existence of an Fe-only hydrogenase as well as an NiFe-hydrogenase in green algae is still enigmatic and is discussed as hydrogenases either participating in the production of hydrogen or during fermentation.

Unusual gene arrangement of the bidirectional hydrogenase and functional analysis of its diaphorase subunit HoxU in respiration of the unicellular cyanobacterium anacystis nidulans

The bidirectional, NAD+-dependent hydrogenase from cyanobacteria is encoded by the structural genes hoxFUYH, which have been found to be clustered, though interspersed with different open reading frames (ORFs), in the heterocystous, N2-fixing Anabaena variabilis and in the unicellular Synechocystis PCC 6803. In another unicellular, non N2-fixing cyanobacterium, Anacystis nidulans, hoxF has now been identified as being separated by at least 16 kb from the residual structural genes hoxUYH. An ORF (termed hoxE gene) is located immediately upstream of hoxF in A. nidulans and in Synechocystis. Its deduced amino acid sequence shows similarities to the NuoE subunit of NADH dehydrogenase I of E. coli, to the homologous subunit of respiratory complex I in mitochondria, and also to the first 104 amino acids of HoxF in A. nidulans and Synechocystis. The diversity in the arrangement of hydrogenase genes in cyanobacteria is puzzling. The subunits HoxE, HoxF, and HoxU of the diaphorase part of the bidirectional hydrogenase have been discussed to be shared both by respiratory complex I and bidirectional hydrogenase in cyanobacteria. Different hoxU mutants were obtained by inserting a lacZKmR cassette into the gene both in A. nidulans and Anacystis PCC 7942. Such mutants showed reduced H2-evolution activities catalyzed by the bidirectional hydrogenase, but had nonimpaired respiratory O2-uptake. A common link between respiratory complex I and the diaphorase part of the bidirectional hydrogenase in cyanobacteria may still exist, but this hypothesis could not be verified in the present study by analyzing defined mutants impaired in one of the diaphorase genes.

Molecular biological analysis of a bidirectional hydrogenase from cyanobacteria.

An 8.9-kb segment with hydrogenase genes from the cyanobacterium Anabaena variabilis has been cloned and sequenced. The sequences show homology to the methyl-viologen-reducing hydrogenases from archaebacteria and, even more striking, to the NAD(+)-reducing enzymes from Alcaligenes eutrophus and Nocardia opaca as well as to the NADP(+)-dependent protein from Desulfovibrio fructosovorans. The cluster from A. variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxY) and for the diaphorase moiety (hoxU and hoxF) described for the A. eutrophus enzyme. In A. variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxY, respectively), and a probably non-coding 0.9-kb segment in an unusual way. The hoxH partial sequence from Anabaena 7119 and Anacystis nidulans was amplified by PCR. Using the labeled segment from A. 7119 as probe, Southern analysis revealed homologous gene segments in the cyanobacteria A. 7119, Anabaena cylindrica, Anacystis nidulans and A. variabilis. The bidirectional hydrogenase from A. nidulans was purified and digests were sequenced. The amino acid sequences obtained showed partial identities to the amino acid sequences deduced from the DNA data of the 8.9-kb segment from A. variabilis. Therefore the 8.9-kb segment contains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria. Crude extracts from A. nidulans perform NAD(P)H-dependent H2 evolution corroborating the molecular biological demonstration of the NAD(P)(+)-dependent hydrogenase in cyanobacteria.

Localization of the Reversible Hydrogenase in Cyanobacteria

Abstract The reversible hydrogenase of the cyanobacterium Anacystis nidulans was purified 250-fold by classical methods. Activity staining on gels obtained by native PAGE allowed to identify two bands. A ntibodies were raised against the electrophoretically homogeneous protein. The molecu­lar weight of the hydrogenase was determined by SD S-PAGE follow ed by Western blot analysis with these antibodies. Crude extracts from the unicellular Anacystis and from heterocysts and vegetative cells of Anabaena variabilis showed precipitation bands of 56 and 17 kd. The reversible hydrogenase appeared to be com posed o f a larger and a smaller subunit as the NiFeS-hydrogen-ases from other bacteria. The hydrogenase subunits, particularly the smaller on e, were subject to proteolysis. By application of the protein A-immuno-gold labeling technique the reversible hydro­ genase was shown to associate with the cytoplasmic membrane in Anacystis. When A nacystis was grown microaerobically, the increase in the specific activity of the reversible hydrogenase paral­leled the raise in the gold-labeling at the cell periphery as measured from the electron micro­ graphs. It is concluded that Anacystis has two different hydrogenases: the reversible or bidirec­tional hydrogenase which is located exclusively at the cytoplasmic membrane of the cells, and a thylakoid-bound enzyme which catalyzes only the uptake of H2.

Appearance of a reversible hydrogenase activity in Anabaena cylindrica grown in high light

In vivo H2 evolution by Anabaena cylindrica Lemm. strain PCC 7122 grown in the presence of ammonia at low and high light intensities was studied. We found that after 2 h of anaerobic incubation, H2 evolution [at a rate of 0.5 μmol (mg dry weight)1 h−1] via reversible hydrogenase occurred in high light grown cells, while this kind of activity was not found in low light grown cells. H2 evolution was inhibited by 3‐(3′. 4′‐dichlorophenyl‐1, 1‐dimethylurea (DCMU). Illuminating the cells in the phycocyanin absorption region resulted in a higher rate of H2 evolution than illuminating the cells in the chlorophyll absorption region. The results indicate that reversible hydrogenase receives reducing equivalents from photosynthetic water photolysis and that both photosystem II and photosystem I participate in the H2 production. Hydrogenase activity was found in the soluble fraction after mild sonication in the case of low light grown cells. After this treatment high light grown cells retained 70% of their hydrogenase activity in the particulate fraction, but released it into the soluble fraction in the presence of 2% deoxycholic acid.In vitro H2 evolution did not differ significantly in the low and high light grown cells. Hence, the differences in the in vivo H2 evolution reflect the different availability of endogenous reductants for hydrogenase in the two kinds of cells. On the basis of our results it is suggested that high light grown Anabaena cells eliminate part of the photosynthetically produced excess electrons via an induced reversible hydrogenase activity. This is the first report of H2 evolution associated with water photolysis and catalyzed by hydrogenase in cyanobacteria.

Hydrogenase activities in cyanobacteria

In the unicellular Anacystis nidulans, the expression of both the H2-uptake (with phenazine methosulfate or methylene blue as the electron acceptor) and H2-evolution (with methyl viologen reduced by Na2S2O4) was dependent on Ni in the culture medium. In extracts from Anacystis and Anabaena 7119, H2-evolution and uptake activities were strongly inhibited by Cu2+, p-chloromercuribenzoate and HgCl2 suggesting that at least one functional SH-group is involved in catalysis by hydrogenase. Extracts from the N2-fixing Anabaena 7119 contained two different hydrogenase fractions which could be separated by chromatography on DE-52 cellulose using a linear NaCl concentration gradient. The fraction eluting with 0.13 M NaCl from the column catalyzed only the uptake of H2 with methylene blue as the electron acceptor but virtually not the evolution of H2 ("uptake" hydrogenase fraction). The fraction eluting at a NaCl strength of 0.195 M catalyzed both H2-uptake with methylene blue and H2-evolution with reduced methyl viologen ("reversible" hydrogenase fraction). Growth under anaerobic conditions drastically enhanced the activity levels of the "reversible" but not of the "uptake" hydrogenase fraction. The "uptake" hydrogenase but not the "reversible" protein was activated by reduced thioredoxin. It is suggested that thioredoxin activates the H2-uptake by the membrane-bound "uptake" hydrogenase also in intact cells. The occurrence of the number of hydrogenases in cyanobacteria will be reevaluated.

The Number of Hydrogenases in Cyanobacteria

Cyanobacteria consume H2 by two different pathways: the oxyhydrogen reaction and anaerobic, light-dependent H2 utilization. The two pathways are shown here to be induced differently by incubating cyanobacteria anaerobically under H2.Inthe unicellular Anacystis nidulans and in N2 − and NH+ 4-grown Anabaena cylindrica and Nostoc muscorum,such treatment greatly enhances the activity of the oxyhydrogen reaction in all cell types. In contrast, the light-dependent pathway, determined by the H2-dependent photoreduction of NADP+, is demonstrable with higher activity only in heterocysts. Whereas the activity of the oxyhydrogen reaction isdirectly correlated to the structural integrity of membranes, there is an inverse correlation between membrane integrity and H2 formation catalysed by hydrogenase. These findings, together withphysiological considerations, suggest that a ‘reversible’ soluble hydrogenase does not exist in photoautotrophic cyanobacteria. No definite conclusions about the existence of two membrane-bound uptake hydrogenases are possible at present.