Hydrogenase Expression Research Articles

Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production

Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10-15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae.

Identification of Novel Drug Targets in HpB38, HpP12, HpG27, Hpshi470, HpSJM180 Strains of Helicobacter pylori : An In Silico Approach for Therapeutic Intervention

Helicobacter species colonizes the stomach and are associated with the development of gastritis disease. Drugs for treatment of Helicobacter infection relieve pain or gastritis symptoms but they are not targeted specifically to Helicobacter pylori. Therefore, there is dire need for discovery of new drug targets and drugs for the treatment of H. pylori. The main objective of this study is to screen the potential drug targets by in silico analysis for the potent strains of H. pylori which include HpB38, HpP12, HpG27, Hpshi470 and HpSJM180. Genome and metabolic pathways of pathogen H. pylori and the host Homosapien sapiens are compared and genes which were unique to H. pylori were filtered and catalogued. These unique genes were subjected to gene property analysis to identify the potentiality of the drug targets. Among the total number of genes analysed in different strains of H. pylori nearly 558, 569, 539, 569, 567 number of genes in HpB38, HpP12, HpG27, Hpshi470 and HpSJM180 found qualified as unique molecules and among them 17 qualified as potential drug targets. Membrane fusion protein of hefABC efflux system, 50 S ribosomal protein L33, Hydrogenase expression protein/formation of HypD, Cag pathogenecity island protein X, Apolipoprotein N acyl transferase, DNA methyalse, Histone like binding protein, Peptidoglycan-associated lipoprotein OprL were found to be critical drug targets to H. pylori. Three (hefABC efflux system, Hydrogenase expression protein/formation of HypD, Cag pathogenecity island protein X) of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lending credence to our unique approach.

Pentoses, hexoses and glycerin as substrates for biohydrogen production: An approach for Brazilian biofuel integration

Biofuels production in Brazil is a traditional activity, and it has becoming more important each year. Within this context, biohydrogen production could exploit residual streams from first generation ethanol (ethanol 1G), second generation ethanol (ethanol 2G) and biodiesel production. Therefore hexoses, pentoses and glycerin were tested as substrates for hydrogen production. Firstly, the effects of different inoculum pretreatments (acid, alkaline and heat) on bacterial communities' performance were evaluated through the levels of Clostridium hydrogenase expression. The heat pretreated inoculum provided the highest yield of H2 (4.62 mol H2/mol sucrose) and also the highest level of hydrogenase expression, 64 times higher when compared with untreated inoculum after 72 h. Then C5 and C6 sugars and also glycerin were tested for H2 production (35 °C and pH 5.5), which resulted in promising yields of H2: sucrose (4.24 mol H2/mol sucrose), glucose (2.19 mol H2/mol glucose), fructose (2.09 mol H2/mol fructose), xylose (1.88 mol H2/mol xylose) and glycerin (0.80 mol H2/mol glycerin).

De novo transcriptomic analysis of hydrogen production in the green alga Chlamydomonas moewusii through RNA-Seq

BackgroundMicroalgae can make a significant contribution towards meeting global renewable energy needs in both carbon-based and hydrogen (H2) biofuel. The development of energy-related products from algae could be accelerated with improvements in systems biology tools, and recent advances in sequencing technology provide a platform for enhanced transcriptomic analyses. However, these techniques are still heavily reliant upon available genomic sequence data. Chlamydomonas moewusii is a unicellular green alga capable of evolving molecular H2 under both dark and light anaerobic conditions, and has high hydrogenase activity that can be rapidly induced. However, to date, there is no systematic investigation of transcriptomic profiling during induction of H2 photoproduction in this organism.ResultsIn this work, RNA-Seq was applied to investigate transcriptomic profiles during the dark anaerobic induction of H2 photoproduction. 156 million reads generated from 7 samples were then used for de novo assembly after data trimming. BlastX results against NCBI database and Blast2GO results were used to interpret the functions of the assembled 34,136 contigs, which were then used as the reference contigs for RNA-Seq analysis. Our results indicated that more contigs were differentially expressed during the period of early and higher H2 photoproduction, and fewer contigs were differentially expressed when H2-photoproduction rates decreased. In addition, C. moewusii and C. reinhardtii share core functional pathways, and transcripts for H2 photoproduction and anaerobic metabolite production were identified in both organisms. C. moewusii also possesses similar metabolic flexibility as C. reinhardtii, and the difference between C. moewusii and C. reinhardtii on hydrogenase expression and anaerobic fermentative pathways involved in redox balancing may explain their different profiles of hydrogenase activity and secreted anaerobic metabolites.ConclusionsHerein, we have described a workflow using commercial software to analyze RNA-Seq data without reference genome sequence information, which can be applied to other unsequenced microorganisms. This study provided biological insights into the anaerobic fermentation and H2 photoproduction of C. moewusii, and the first transcriptomic RNA-Seq dataset of C. moewusii generated in this study also offer baseline data for further investigation (e.g. regulatory proteins related to fermentative pathway discussed in this study) of this organism as a H2-photoproduction strain.

A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferredoxin in the stroma. In this work, by analyzing wild type and mutants affected in H2 production, we show that maximal PSII photosynthetic electron transfer during the first seconds of illumination after a prolonged dark-anaerobiosis period is linearly related to hydrogenase capacity. Based on the specific chlorophyll fluorescence induction kinetics typical of hydrogenase-deficient mutants, we set up an in vivo fluorescence imaging screening protocol allowing to isolate mutants impaired in hydrogenase expression or activity, as well as mutants altered in related metabolic pathways required for energy production in anaerobiosis. Compared to previously described screens for mutants impaired in H2 production, our screening method is remarkably fast, sensitive and non-invasive. Out of 3000 clones from a small-sized insertional mutant library, five mutants were isolated and the most affected one was analyzed and shown to be defective for the hydrogenase HydG assembly factor.

Genomic and Physiological Characterization of the Chromate-Reducing, Aquifer-Derived Firmicute Pelosinus sp. Strain HCF1

Pelosinus spp. are fermentative firmicutes that were recently reported to be prominent members of microbial communities at contaminated subsurface sites in multiple locations. Here we report metabolic characteristics and their putative genetic basis in Pelosinus sp. strain HCF1, an isolate that predominated anaerobic, Cr(VI)-reducing columns constructed with aquifer sediment. Strain HCF1 ferments lactate to propionate and acetate (the methylmalonyl-coenzyme A [CoA] pathway was identified in the genome), and its genome encodes two [NiFe]- and four [FeFe]-hydrogenases for H(2) cycling. The reduction of Cr(VI) and Fe(III) may be catalyzed by a flavoprotein with 42 to 51% sequence identity to both ChrR and FerB. This bacterium has unexpected capabilities and gene content associated with reduction of nitrogen oxides, including dissimilatory reduction of nitrate to ammonium (two copies of NrfH and NrfA were identified along with NarGHI) and a nitric oxide reductase (NorCB). In this strain, either H(2) or lactate can act as a sole electron donor for nitrate, Cr(VI), and Fe(III) reduction. Transcriptional studies demonstrated differential expression of hydrogenases and nitrate and nitrite reductases. Overall, the unexpected metabolic capabilities and gene content reported here broaden our perspective on what biogeochemical and ecological roles this species might play as a prominent member of microbial communities in subsurface environments.

Improvement of bacterial hydrogen production by ATP in mixed organic compounds extracted from Rhodobacter sphaeroides aerobically cultured under dark conditions

The aim of this study was to increase the hydrogen production of recombinant Escherichia coli harboring HupSL hydrogenase by supplementing physiologically activating compounds extracted from Rhodobacter sphaeroides cultured under anaerobic dark condition after treating them with dimethyl sulfoxide, and the 0.5% extracts contained 4×10−8M ATP, which was 100-fold higher than that in the extracts from E. coli. In addition, it was found that the hydrogen production from recombinant E. coli harboring HupSL hydrogenase isolated from R. sphaeroides was doubled under anaerobic conditions when it was supplemented by the extracts from R. sphaeroides cultured aerobically in dark conditions, and this also showed consistent pattern with the increased level of HupSL hydrogenase expression. Therefore, we conclude that the mixed organic compounds extracted from R. sphaeroides have an ATP which enhances the hydrogen production by increasing the amount of HupSL hydrogenase.

Production of hydrogenases as biocatalysts

Hydrogen is emerging as one of the most promising energy carriers for the future. Most of the methodologies used for hydrogen production employ use of hydrogenases, known to play a critical role in biological hydrogen production as well as its utilization. These hydrogenases display fascinating redox-chemical properties with tremendous efficiency as a biocatalyst for hydrogen fuel. However, Due to the complex structure and maturation process of hydrogenases, their heterologous production has been a challenging task. This results in the limitation of the viable application of this metalloprotein for hydrogen fuel. This review briefly discusses various hydrogenase expression systems and their perspectives.

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38°C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40°C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.

Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

BackgroundSolar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions.ResultsRecombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%.ConclusionHere, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

BackgroundFeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2.ResultsWe have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.ConclusionsRational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts.

High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

BackgroundThe realization of hydrogenase-based technologies for renewable H2 production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.Principal FindingsIn this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H2 evolution with rates comparable to those of enzymes isolated from their respective native organisms.SignificanceThe production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H2-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

BackgroundHydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H2 production involves consumption of 2H+, hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms.Methods and Principal FindingsWe analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H2 to 2H+. Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H2 production and consumption was tested using a H2-specific Clark-type electrode. Hyd-3-dependent H2 production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H2 consumption was maximal at alkaline pH. H2 production, was unaffected by a shift in external or internal pH. H2 production was associated with hycE expression levels as a function of external pH.ConclusionsAnaerobic growing cultures of E. coli generate H2 via Hyd-3 at low external pH, and consume H2 via Hyd-2 at high external pH. Hyd-3 proton conversion to H2 is required for acid resistance in anaerobic cultures of E. coli.

Hydrogen production by nitrogenase as a potential crop rotation benefit

Hydrogen production by nitrogenase as a potential crop rotation benefit

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.

The unbearable naivety of legumes in symbiosis

The mechanisms by which legumes choose their rhizobial partners operate independently from their ability to fix nitrogen. As a result of this naivety, symbiotic nitrogen fixation is often suboptimal. The initial recognition of the bacterial partner and the subsequent signal transduction in the host root utilises components that are functionally conserved between legumes and probably actinorhiza host plants. However, the later steps, which largely determine symbiotic performance, are subject to ongoing evolutionary diversification of molecular mechanisms. For example, the impact of bacterial effector proteins, the occurrence of terminal bacteroid differentiation and the expression of bacterial hydrogenase, all depend on the plant genotype. Strategies towards increased nitrogen fixation of legumes in agriculture need to encompass this diversification of mechanisms.

Expression of Ni-Fe hydrogenase structural genes derived from Thiocapsa roseopersicina in Escherichia coli

Obtaining high amounts of pure hydrogenase is a key objective, because this enzyme may be used in fuel cells and H 2 sensors. In this aspect, the HydSL Ni‐Fe hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina , which exhibits a high activity and is resistant to elevated temperatures and oxygen, is of special interest. Unfortunately, since T. roseopersicina is difficult to culture and synthesizes the HydSL Ni‐Fe hydrogenase at a very low level, the production of this enzyme in Escherichia coli would be invaluable. To study this possibility, we investigated the products of expression of structural genes encoding two subunits of the HydSL Ni‐Fe hydrogenase in this heterologous system. The major part of the protein was present in inclusion bodies, and a smaller part was in solution. Electron micrographs revealed different structure and electron density of inclusion bodies corresponding to the products of two different structural genes. We assumed that the difference in the electron density may be due to inclusion of metals in one of the subunits. Hydrogenase (EC 1.12.xx) is an enzyme catalyzing the reaction of molecular oxygen activation. Depending on the metal contained in the active site of the enzyme, all enzymes of this class are divided into Fe-containing hydrogenases, Ni‐Fe-containing hydrogenases, and hydrogenases whose reaction site contains an iron ion that is not incorporated into the protein globule but is present as a cofactor [1]. The interest to hydrogenases is determined by both active studies of the formation of the structure and the mechanism of action of complex metalloenzymes and the possibility of their use in practice in fuel cells and H 2 sensors.

Recombinant and in vitro expression systems for hydrogenases: new frontiers in basic and applied studies for biological and synthetic H2 production

This review focuses on recent progress in developing heterologous and recombinant expression as well as in vitro maturation systems for the biosynthesis of active [FeFe] and [NiFe]-hydrogenases, which catalyze the reversible reaction, H2 <--> 2e- + 2H+. Activities of [FeFe] and [NiFe]-hydrogenases produced from different recombinant and in vitro maturation approaches are compared. Examples of how hydrogenase expression supports basic and applied studies of these enzymes are presented, and barriers to achieving more viable biological and synthetic H2-production systems and catalysts are addressed.

Biochemical kinetics of fermentative hydrogen production by Clostridium butyricum W5

The fermentation process for hydrogen production has been widely reported. However, there is lack of information related to detailed kinetic studies. The aim of this work was to investigate biochemical kinetics of fermentative hydrogen production by a newly isolated strain of Clostridium butyricum W5. The research objectives were to clarify relationships between hydrogen fermentation and biochemical parameters and hydrogenases, and consequently to seek an index for hydrogen production. Time profiles of hydrogen production, cell growth, volatile fatty acid accumulation and [FeFe]hydrogenase expression level were described. The amount of hydrogen produced in a laboratory batch process was 45.45 mmol/L at 10 h and peak production rate was 7.61 mmol/l/h at 9 h. Cell growth rate peaked at 8 h. Lactic acid was a main by-product, followed by butyric acid and acetic acid. Quantification of [FeFe]hydrogenase mRNA was optimized by a real-time reverse transcriptase-PCR. Statistical analysis showed that [FeFe]hydrogenase mRNA levels peak earlier than hydrogen production rate, and cell growth has a linear positive relationship with hydrogen production.

The expression of formate dehydrogenases and hydrogenases in Syntrophobacter fumaroxidans in different growth phases and at different pHs

The expression of formate dehydrogenases and hydrogenases in Syntrophobacter fumaroxidans in different growth phases and at different pHs