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.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

Redox state-dependent changes in the crystal structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough

Hydrogenases are enzymes that can potentially be used in bioelectrical devices or for biological hydrogen production, the most studied of which are the [NiFe] type. Most [NiFe] hydrogenases are inactivated by oxygen and the few known O₂-tolerant enzymes are hydrogen-uptake enzymes, unsuitable for hydrogen production, due to strong product inhibition. In contrast, the [NiFeSe] hydrogenases, where a selenocysteine is bound to the nickel, are very attractive alternatives because of their high hydrogen production activity and fast reactivation after O₂ exposure. Here we report five high-resolution crystallographic 3D structures of the soluble form of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough in three different redox states (oxidized as-isolated, H₂-reduced and air re-oxidized), which revealed the structural changes that take place at the active site during enzyme reduction and re-oxidation. The results provide new insights into the pathways of O₂ inactivation in [NiFe], and in particular [NiFeSe], hydrogenases. In addition, they suggest that different enzymes may display different oxidized states upon exposure to O₂, which are probably determined by the protein structure.     

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.     

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.     

A polyene-based polymer functionalized with a model of [FeFe]-hydrogenase and film electrodes assembled from the polymer via spin-coating

A diiron hexacarbonyl complex possessing an alkynyl group as a model complex of the diiron sub-unit of [FeFe]-hydrogenase was polymerized under the catalysis of WCl₆-SnPh₄. The polymer (Poly-{Fe₂}) functionalized with {Fe₂(CO)₆} units which is dominated by cis-form was fully characterised using FTIR, NMR, SEM, TEM, and TGA techniques. Through modifying the monomer, the properties, for example, solubility, of the resultant polymer could be tuned and much larger molecule weight, which was estimated as 7.93 × 10⁵ g mol⁻¹ using static light scattering technique was achieved without compromising its solubility. Spin-coating the functionalized polymer onto the surface of vitreous carbon electrode with or without multi-wall carbon nanotubes (MWCNTs) produced film electrodes which show electrochemical responses. Adding MWCNTs into the film enhances significantly the electrochemical response probably via not only improving the conductivity of the film, but also the increase in its effective surface area after being doped with MWCNTs.     

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.     

Hydrogen producing activity by Escherichia coli hydrogenase 4 (hyf) depends on glucose concentration

Escherichia coli produces molecular hydrogen (H₂) during glucose fermentation. This production of H₂ occurs via multiple and reversible membrane-associated hydrogenases (Hyd). Dependence of H₂ producing rate (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ by Hyd-4 (hyf) on glucose concentration was studied at different pHs. During growth on 0.2% glucose at pH 7.5 in JRG3615 (hyfA-B) and JRG3621 (hyfB-R  ) mutants (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was decreased ∼6.7 and ∼5 fold, respectively, compared to wild type. Only in JRG3621 mutant at pH 6.5 and 5.5 (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was severely decreased ∼7.8 and ∼3.8 fold, respectively. But when cells were grown on 0.8% glucose no difference between wild type and mutants was detected at any of the tested pHs. The results indicate Hyd-4 H₂ producing activity inhibition by high concentration of glucose mainly at pH 7.5. This is of significance to regulate Hyd activity and H₂ production by E. coli during fermentation.     

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.     

Hydrogenases, accessory genes and the regulation of [NiFe] hydrogenase biosynthesis in Thiocapsa roseopersicina

The purple (Sulphur) phototrophic bacterium, Thiocapsa roseopersicina BBS contains several [NiFe] hydrogenases, of which two are membrane bound. Mutant T. roseopersicina cells, carrying deletions in both gene clusters showed hydrogenase activity. This activity was located in the cytoplasm. The structural gene cluster hoxEFUYH was identified and sequenced. In addition, genes homologous to hupUV/hoxBC, the hydrogen sensing hydrogenase have been identified and sequenced.Regulation of hydrogenase biosynthesis was studied in detail for HydSL (renamed HynSL). A random mutagenesis system was optimised for T. roseopersicina. One of the mutations was in a gene similar to that coding for the HypF proteins in other organisms. Inactivation of the hypF gene resulted in a 60-fold increase in hydrogen evolution under nitrogen fixing conditions. In addition to hypF, the following accessory genes were identified: hydD, hupK, hypC1, hypC2, hypDE. Characterisation of the corresponding gene products and search for additional accessory genes are in progress.     

A heterologously-expressed thermostable Pyrococcus furiosus cytoplasmic [NiFe]-hydrogenase I used as the catalyst of H2/air biofuel cells

Hydrogenase-catalyzed H₂/air biofuel cells have attracted wide attention. However, how to obtain thermostable and highly active hydrogenase remains challenging. Herein, we developed a thermophilic archaea host system to heterologously express a thermostable Pyrococcus furiosus cytoplasmic [NiFe]-hydrogenase I (PfSHI). The recombinant hydrogenase could be easily isolated and then immobilized onto a carbon nanotube-modified carbon felt electrode for further electrochemical characterization. The bioanode was able to work at a wide range of temperatures from 40 °C to 80 °C and no high potential deactivation was observed until 0.3 V at 50 °C. Cyclic voltammograms at elevated temperatures for reverse H₂ oxidation performance indicated that an inactive state might be formed and then reversed when scanning to negative position. Also, the power density of a whole cell reached 1.08 mW cm^?2 using Pt/C as the cathode with a brilliant open circuit potential reaching 1.2 V. The voltage could retain 90% of its initial value after 33 h discharge at a current of 10 μA. Besides, the bioanode exhibited decent oxygen tolerance both in ambient and elevated temperatures. These results suggest that this recombinant PfSHI can be a good candidate for catalyzing H₂/air biofuel cells.     

Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii

The direct relationship between hydrogenase gene conformation and its function in green alga Chlamydomonas reinhardtii has been investigated. We have analyzed the conformation in the 29 kilobase (kb) chromosome region containing [FeFe]-hydrogenase gene (hydA1) of C. reinhardtii in aerobic and anaerobic conditions using chromosome conformation capture technique (3C). The results showed a loop organization in the [FeFe]-hydrogenase gene region under aerobic conditions when the hydrogenase gene is silenced. In contrast, under anaerobic conditions, when the hydrogenase gene is active, no loop conformation in the gene region is present.     

Expression of different types of [FeFe]-hydrogenase genes in bacteria isolated from a population of a bio-hydrogen pilot-scale plant

[FeFe]-hydrogenases are the enzymes responsible for high yield H₂ production during dark fermentation in bio-hydrogen production plants. The culturable bacterial population present in a pilot-scale plant efficiently producing H₂ from waste materials was isolated, classified and identified by means of 16S rDNA gene analysis. The culturable part of the mixed population consists of nine bacterial species that include non-hydrogen producers (Lactobacillus, Enterococcus and Staphylococcus) and several Clostridium that are directly responsible for H₂ production.     

The proton iron-sulfur cluster environment of the [FeFe]-hydrogenase maturation protein HydF from Thermotoga neapolitana

[FeFe]-hydrogenases contain a complex [4Fe–4S]-2Fe cluster (H-cluster) and are able to efficiently reduce protons to H₂. Due to their potential exploitation for renewable energy production biotechnologies, significant efforts have been put into understanding the mechanisms driving the H-cluster assembly, which involves three conserved proteins. Among them, HydF works as scaffold upon which the H-cluster precursor is synthesized and carrier to deliver it to the hydrogenase, resulting in its activation. A FeS cluster binding sequence (CxHx₄₆-₅₃HCxxC) is conserved in all HydF proteins and should in principle provide four ligands to coordinate the Fe atom. However, we found that alternative metal coordination may exist in different HydF proteins and that only the three cysteines are strictly required, whereas the fourth ligand may vary and is, in any case, readily exchangeable. In this work we analyzed by EPR/HYSCORE the FeS cluster proton environment of HydF from Thermotoga neapolitana to determine the possible role of surrounding residues in the non-cysteinyl iron ligation of the protein.     

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.     

Escherichia coli wild type and hydrogenase mutant cells growth and hydrogen production upon xylose and glycerol co-fermentation in media with different buffer capacities

In this study, the new strategy for long term bio-hydrogen (H₂) production using different substrates and waste materials is presented. Growth characteristics and H₂ production were investigated upon consumption of 0.4% xylose and 1% glycerol alone (which were optimal) or their mixture by Escherichia coli BW25113 wild type parental strain (PS) and ΔhyaB, ΔhybC, ΔhycE, ΔhyfG mutants with genes deletions for key subunits of hydrogenase (Hyd)-1 to Hyd-4, respectively, in high and low buffer capacity peptone (HPM, LPM) mediums, pH 5.5 and 7.5. Overall, pH 5.5 negatively affected bacterial growth and H₂ production. At pH 7.5, apart from Hyd-3 and Hyd-4 mutants, upon growth of PS, Hyd-1 and Hyd-2 mutants drop of Pt redox electrode readings from positive (～+150 mV) to negative (of ?400 to ?550 mV) values was detected during log growth phase mentioning H₂ formation. Xylose and glycerol co-utilization did not affect PS and Hyd-1 and Hyd-2 mutant's biomass and H₂ formation during log growth phase in LPM, but ～1.5 fold stimulated these parameters, especially in HPM, pH 7.5, during prolonged 96 h bacterial growth. Roles of Hyd-3 and Hyd-4 in H₂ production; and Hyd-1 and Hyd-2 in H₂ oxidation during bacterial log growth phase were stated under xylose and glycerol co-fermenting conditions. The results obtained might be valuable for industrial long term H₂ production by bacteria using mixture of carbon sources and combining various organic waste materials.     

Specificity and selectivity of HypC chaperonins and endopeptidases in the molecular assembly machinery of [NiFe] hydrogenases of Thiocapsa roseopersicina

The purple photosynthetic bacterium, Thiocapsa roseopersicina harbours at least three functional [NiFe] hydrogenases. Two of them are attached to the periplasmic membrane (HynSL, HupSL), while the third one is apparently localized in the cytoplasm (HoxEFUYH). Two hypC-type genes, coding for putative small maturation proteins, were found and their roles were studied by activity measurements performed with hypC mutants. Protein–protein interaction experiments confirmed that each HypC-type protein participates in the maturation of at least two [NiFe] hydrogenase large subunits via direct interaction. Endopeptidases perform the last step of the complex [NiFe] hydrogenase maturation process. A separate endopeptidase (HynD, HupD, HoxW) cleaves off the C-terminus of each large subunit and they are strictly specific for their corresponding hydrogenases. The results demonstrate a sophisticated assembly of these functionally active redox metalloenzymes through specific and selective protein–protein interactions and imply some diversity in the hydrogenase assembly machinery among the various microbes.     

Ferredoxin has a pivotal role in the biosynthesis of the hydrogen-oxidizing hydrogenases in Escherichia coli

Iron–sulphur clusters (FeS) are essential cofactors in the small, electron-transfer subunits of [NiFe]-hydrogenases (Hyd). In this study we analyzed the in vivo role of ferredoxin in the biosynthesis of three of the Hyd in Escherichia coli. Our results reveal that a fdx mutant, which is unable to synthesize ferredoxin, lacks the activity of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2. In the case of Hyd-2 this was due to the absence of the FeS cluster-containing small subunit. In the case of Hyd-1, stability of the catalytic subunit was also impaired. Partial activity of the hydrogen-evolving Hyd-3 enzyme, as well as that of both respiratory formate dehydrogenases was retained in the fdx mutant. Analysis of lacZ fusions demonstrated that the fdx mutation had a limited effect on expression of the operon encoding Hyd-1. Rather, these data suggest that ferredoxin has a role in the maturation or assembly of the hydrogen-oxidizing [NiFe]-hydrogenases.