Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

The H₂-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum.     

Maturation and processing of the recombinant [FeFe] hydrogenase from Desulfovibrio vulgaris Hildenborough (DvH) in Escherichia coli

The need of an efficient and well-characterized heterologous expression system of [FeFe]-hydrogenase for the production of O₂-resistant mutants prompted us to explore the use of Escherichia coli as a possible expression system. O₂-resistant hydrogenase mutants could be instrumental when coupling oxygenic photosynthesis with hydrogen bio-production. In general, expression of Desulfovibrio vulgaris Hildenborough active enzyme in E. coli was very modest indicating that the co-expression of the HydE, HydF and HydG maturases with hydrogenase structural genes in this bacterium is not optimal. A 28-fold increase in activity was obtained when these proteins were co-expressed with the Iron–Sulfur Cluster operon, indicating that one of the problems with over-expression is the correct insertion of FeS clusters. However, the measured activity is still about 4000-fold lower than the one measured in the native hydrogenase indicating that additional, so far unidentified factors may be necessary for optimal heterologous expression of [FeFe]-hydrogenase.     

Expression of the [FeFe] hydrogenase in the chloroplast of Chlamydomonas reinhardtii

Biological hydrogen generation from phototrophic organisms is a promising source of renewable fuel. The nuclear-expressed [FeFe] hydrogenase from Chlamydomonas reinhardtii has an extremely high turnover rate, and so has been a target of intense research. Here, we demonstrate that a codon-optimized native hydrogenase can be successfully expressed in the chloroplast. We also demonstrate a curiously strong negative selective pressure resulting from unregulated hydrogenase expression in this location, and discuss management of its expression with a vitamin-controlled gene repression system. To the best of our knowledge, this represents the first example of a nuclear-expressed, chloroplast-localized metalloprotein being synthesized in situ. Control of this process opens up several bioengineering possibilities for the production of biohydrogen.     

[FeFe]-hydrogenase gene quantification and melting curve analysis from hydrogen-fermenting bioreactor samples

In this study, quantitative PCR (qPCR) was used to quantify [FeFe]-hydrogenases and subsequently melting curves were analyzed from hydrogen-fermenting, mixed-culture bioreactor samples. Denaturing gradient gel electrophoresis (DGGE) analysis was also performed to the reactor samples revealing a clostridial dominance in the reactor. Primers targeting [FeFe]-hydrogenases were designed based on known clostridial [FeFe]-hydrogenase gene sequences and tested with several clostridial strains. The results show that amplification efficiencies of four different clostridia are highly similar and melting curves of the clostridial strains were within 1 °C of each other. We compared the melting curves to the hydrogen percentage and observed a correlation between the results. The closer the melting curves were to those of clostridia, the better the hydrogen production. Based on these results, the primers and melting curve analysis of [FeFe]-hydrogenase amplicons can be used for analysing hydrogenase genes from bioreactor samples.     

Characterization and overexpression of a [FeFe]-hydrogenase gene of a novel hydrogen-producing bacterium Ethanoligenens harbinense

Ethanoligenens, a novel ethanologenic and hydrogen-producing genus, has capability of hydrogen production at low pH. A [FeFe]-hydrogenase gene with [4Fe-4S] and [2Fe-2S] clusters from Ethanoligenens harbinense YUAN-3 was cloned and overexpressed in a non-hydrogen-producing Escherichia coli BL-21. This hydA gene consisted of an open reading frame of 1743 bp encoding 580 amino acids with an estimated molecular weight of 63 188.1 Da. Six characteristic sequence signatures were present within the H-cluster domain of [FeFe]-H₂ases, and three of them were described previously. The overexpressed and purified hydrogenases from recombinant cells showed catalytic activity in vitro and in vivo.     

Homologous overexpression of [FeFe] hydrogenase in Enterobacter cloacae IIT-BT 08 to enhance hydrogen gas production from cheese whey

The present study investigated the influence of increase in intracellular [FeFe] hydrogenase levels, in Enterobacter cloacae IIT-BT 08, on the formation of molecular hydrogen. The hydA gene from E. cloacae IIT-BT 08 was successfully amplified and cloned downstream of a tac promoter in an Escherichiacoli-Enterobacter reconstructed pGEX-Kan shuttle vector and introduced into E. cloacae. Finally E. cloacae strain carrying multiple copies of pGEX-Kan-hydA vector was developed. Homologous overexpression of the [FeFe] hydrogenase gene increased the hydrogenase activity by1.3-fold as compared to the wild type. SDS-PAGE confirmed the successful expression of the GST-tagged hydA protein. The hydrogen yield and rate of production in recombinant strain were found to be 1.2-fold and 1.6-fold higher, respectively, compared to the wild type strain. This was found to be concomitant with the shift in the metabolic pathway. In addition, feasibility of using cheese whey as a substrate for biohydrogen production and the effect of its supplementation with yeast extract as nitrogen source was studied for both the wild type and the recombinant strain. It was found that supplementation with 0.3% (w/v) yeast extract enhanced hydrogen production from whey. Further, the yield and rate of hydrogen production from the recombinant was found to be more promising as compared to the wild type.     

Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases oxygen consumption and promotes hydrogen production

In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii can produce hydrogen (H₂) using hydrogenase. The activity of hydrogenase is inhibited at the presence of molecular oxygen, forming a major barrier for large scale production of hydrogen in autotrophic organisms. In this study, we engineered a novel pathway to consume oxygen and correspondingly promote hydrogen production in Chlamydomonas reinhardtii. The pyruvate oxidase from Escherichia coli and catalase from Synechococcus elongatus PCC 7942 were cloned and integrated into the chloroplast of Chlamydomonas reinhardtii. These two foreign genes are driven by a HSP70A/RBCS2 promoter, a heat shock inducing promoter. After continuous heat shock treatments, the foreign genes showed high expression levels, while the growth rate of transgenic algal cells was slightly inhibited compared to the wild type. Under low light, transgenic algal cells consumed more oxygen than wild type. This resulted in lower oxygen content in sealed culture conditions, especially under low light condition, and dramatically increased hydrogen production. These results demonstrate that pyruvate oxidase expressed in Chlamydomonas reinhardtii increases oxygen consumption and has potential for improving photosynthetic hydrogen production in Chlamydomonas reinhardtii.     

Engineered clostridial [FeFe]-hydrogenase shows improved O2 tolerance in Chlamydomonas reinhardtii

Hydrogenase intolerance to oxygen remains a critical hurdle on the road to photosynthetic hydrogen production for sustainable energy demands. Although the engineering of the intrinsic oxygen tolerance mechanism of hydrogenase using mutagenesis is an ambitious approach, recent in-vitro studies reported a novel and improved synthetic [FeFe]-Hydrogenase variants. To corroborate these findings in-vivo, we expressed either an engineered variant or its cognate wild type enzyme in the chloroplast genome of Chlamydomonas reinhardtii. We characterized their activity using a customized photosynthetic hydrogen production in-vivo assay to test whether the improved variant could maintain a greater fraction of its activity following oxygen exposure. We found that the mutated variant exhibited a superior oxygen tolerance while persevering its photosynthetic performance in terms of hydrogen production yield. Importantly, we show for the first time that this approach can potentially address the inherent O₂ sensitivity of [FeFe]-Hydrogenases for photosynthetic hydrogen production.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

Comparison between heterofermentation and autofermentation in hydrogen production from Arthrospira (Spirulina) platensis wet biomass

Hydrogen production from Arthrospira (Spirulina) platensis wet biomass through heterofermentation by the [FeFe] hydrogenase of hydrogenogens (hydrogen-producing bacteria) and autofermentation by the [NiFe] hydrogenase of Arthrospira platensis was discussed under dark anaerobic conditions. In heterofermentation, wet cyanobacterial biomass without pretreatment was hardly utilized by hydrogenogens for hydrogen production. But the carbohydrates in cyanobacterial cells released after cell wall disruption were effectively utilized by hydrogenogens for hydrogen production. Wet cyanobacterial biomass was pretreated with boiling and bead milling, ultrasonication, and ultrasonication and enzymatic hydrolysis. Wet cyanobacterial biomass pretreated with ultrasonication and enzymatic hydrolysis achieved the maximum reducing sugar yield of 0.407 g/g-DW (83.0% of the theoretical reducing sugar yield). Different concentrations (10 g/l to 40 g/l) of pretreated wet cyanobacterial biomass were used as substrate to produce fermentative hydrogen by hydrogenogens, which were domesticated with the pretreated wet cyanobacterial biomass as carbon source. The maximum hydrogen yield of 92.0 ml H₂/g-DW was obtained at 20 g/l of wet cyanobacterial biomass. The main soluble metabolite products (SMPs) in the residual solutions from heterofermentation were acetate and butyrate. In autofermentation, hydrogen yield decreased from 51.4 ml H₂/g-DW to 11.0 ml H₂/g-DW with increasing substrate concentration from 1 g/l to 20 g/l. The main SMPs in the residual solutions from autofermentation were acetate and ethanol. The hydrogen production peak rate and hydrogen yield at 20 g/l of wet cyanobacterial biomass in heterofermentation showed 110- and 8.4-fold increases, respectively, relative to those in autofementation.     

Iron–iron hydrogenase active subunit covalently linking to organic chromophore for light-driven hydrogen evolution

The first photocatalytic [FeFe]-hydrogenase ([FeFe]-H₂ase) mimic 3 with noble-metal-free benzothiazole as donating photosensitizer had been successfully constructed via an easily accessible approach, and fully characterized by various spectroscopic and X-ray crystallographic techniques. Steady-state spectroscopy and electrochemistry revealed the evidences indicating that the photo-induced electron transfer occurred in 3. The reduced [Fe^IFe⁰] species was further confirmed by laser flash photolysis and considered to be responsible for the light-driven H₂ evolution. As a result, the photocatalytic system consisting of the photocatalyst 3 and the sacrificial electron donor in the presence of proton source indeed produced H₂ with a turnover number (TON) of 24.2 under light irradiation. The TON indicated a remarkably photocatalytic efficiency for an [FeFe]-H₂ase mimic assembled by the covalent combination of a photosensitizer to the catalytic center. The results demonstrated the tremendous potential of present synthetic strategy for the construction of compact, inexpensive, easily accessible [FeFe]-H₂ase model complexes as photocatalysts.     

Inhibition of photosystem 2 in starch-enriched Chlamydomonas reinhardtii cells prevents the efficient induction of H2 production in sulfur-depleted cultures

In sulfur-deprived Chlamydomonas reinhardtii cells the activity of photosystem 2 (PSII) has been shown to have a crucial role in the photosynthetic production of H₂, since it allows the synthesis of internal reserves such as starch. In the present investigation, the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was added in starch-enriched and sulfur-depleted C. reinhardtii cultures 1) at the very end of the aerobic phase, and 2) soon after the culture started to evolve H₂. In the former case, production of H₂ on a volumetric basis was completely down-regulated, although starch mobilization was enhanced. In vitro tests showed that the hydrogenase enzyme was active, although its efficiency of utilization in vivo was lowered very soon in the experiment. When an inhibitor of Rubisco such as glycolaldehyde was added under the same conditions, no substantial improvement in H₂ production rates was noted. These findings indicate that, aside starch storage, PSII plays an active role in the induction of the H₂ production process.     

Hydrogen production based on the heterologous expression of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator in different genetic backgrounds of Escherichia coli strains

This study explored the genetic engineering of Escherichia coli for hydrogen (H₂) production. In E. coli W3110, the introduction of NAD⁺-reducing [NiFe]-hydrogenase from Cupriavidus necator, combined with the inactivation of three endogenous [NiFe]-hydrogenases, exhibited not only H₂ production but also H₂ uptake based on exogenous hydrogenase. Although the H₂ production ability was much lower than the H₂ uptake ability, inactivation of the ethanol, lactate, and succinate production pathways resulted in a marked increase in H₂ production, demonstrating the bidirectional hydrogenase function in vivo depending on NADH/NAD⁺. Unexpectedly, H₂ production was completely repressed under conditions for high expression of exogenous hydrogenase. Furthermore, the introduction of the heterologous enzyme markedly repressed the endogenous H₂ production ability of E. coli W3110 but not the HST02. These in vivo behaviors largely correlated with in vitro hydrogenase activity suggested complicated interactions between the native and nonnative functional expression of [NiFe]-hydrogenases.     

Arginine of Chlamydomonas reinhardtii [Fe–Fe] hydrogenase HydA1 plays a crucial role in electron transfer to its catalytic center

[Fe–Fe] hydrogenases, with hydrogen evolution activities outperforming [Ni–Fe] hydrogenases by 3–4 orders of magnitude, are still the most promising enzyme class for hydrogen production purposes. For Chlamydomonas reinhardtii [Fe–Fe] hydrogenase HydA1 the question of catalytic activity and electron transport is of main importance. Here we report the characterization of two mutant forms of C. reinhardtii HydA1. An aspartic acid in place of arginine¹⁷¹ leads to a six-fold increase of the catalytic activity in comparison to the wild type protein during methyl viologen-dependent hydrogen production. Tryptophan in position 171 does not result in any change in methyl viologen-induced activity. At the same time these mutations lead to a strong decrease in ferredoxin-dependent hydrogen production while the catalytic center of mutant forms stays intact. The localization of this amino acid (arginine¹⁷¹) in the environment of CrHydA1 H-cluster indicates that the limitation of the catalytic activity of this hydrogenase is due to the electron transfer step to the catalytic center where the reduction of protons takes place.     

Enhanced aerobic H2 production by engineering an [FeFe] hydrogenase from Clostridium pasteurianum

Biological H₂ production is one of the alternative technologies for producing H₂ in a renewable and sustainable fashion. The technology often relies on the [FeFe] hydrogenase enzyme for catalyzing the H₂ production reaction from protons and electrons as it is highly efficient. The high O₂ sensitivity of the enzyme have been one of the key obstacles for implementing the technology, but recent findings showed the feasibility of improving O₂ tolerance via protein engineering. In this study, we investigated the changes in the O₂ tolerance and aerobic H₂ production activity of an [FeFe] hydrogenase by replacing methionines on the surface with leucines. None of the mutations were detrimental to protein folding, and a few were able to exert either positive or negative influence on the O₂ tolerance of the enzyme. We combined the mutation exhibiting the highest improvement in O₂ tolerance with the rest of the Met – Leu replacements; however, the results indicated non-synergistic impact on the tolerance. Combinations with the other types of mutations that have been reported previously also failed to further improve the O₂ tolerance of the hydrogenase. These results provide new insights on how mutagenesis affects the enzyme's functional properties, as well as the molecular mechanism(s) behind the tolerance.