Membrane-bound Hydrogenase Research Articles

Mechanism of hydrogen evolution in the membrane-bound hydrogenase

Mechanism of hydrogen evolution in the membrane-bound hydrogenase

Mechanistic Principles of Hydrogen Evolution in the Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+ exchange across the membrane. Despite recent structural advances, the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L toward the [NiFe] site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the [NiFe] active site of Mbh to drive PCET reactions and ion transport.

Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus.

A genome-scale metabolic model, encompassing a total of 623 genes, 727 reactions, and 865 metabolites, was developed for Pyrococcus furiosus, an archaeon that grows optimally at 100°C by carbohydrate and peptide fermentation. The model uses subsystem-based genome annotation, along with extensive manual curation of 237 gene-reaction associations including those involved in central carbon metabolism, amino acid metabolism, and energy metabolism. The redox and energy balance of P. furiosus was investigated through random sampling of flux distributions in the model during growth on disaccharides. The core energy balance of the model was shown to depend on high acetate production and the coupling of a sodium-dependent ATP synthase and membrane-bound hydrogenase, which generates a sodium gradient in a ferredoxin-dependent manner, aligning with existing understanding of P. furiosus metabolism. The model was utilized to inform genetic engineering designs that favor the production of ethanol over acetate by implementing an NADPH and CO-dependent energy economy. The P. furiosus model is a powerful tool for understanding the relationship between generation of end products and redox/energy balance at a systems-level that will aid in the design of optimal engineering strategies for production of bio-based chemicals and fuels. IMPORTANCE The bio-based production of organic chemicals provides a sustainable alternative to fossil-based production in the face of today's climate challenges. In this work, we present a genome-scale metabolic reconstruction of Pyrococcus furiosus, a well-established platform organism that has been engineered to produce a variety of chemicals and fuels. The metabolic model was used to design optimal engineering strategies to produce ethanol. The redox and energy balance of P. furiosus was examined in detail, which provided useful insights that will guide future engineering designs.

Stepwise assembly of the active site of [NiFe]-hydrogenase.

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2e- and 2H+ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN- molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

Conformational and mechanical stability of the isolated large subunit of membrane-bound [NiFe]-hydrogenase from Cupriavidus necator.

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel-iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator (CnMBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes.

An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A–E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Molecular mechanism of hydrogen production in membrane-bound hydrogenase

Molecular mechanism of hydrogen production in membrane-bound hydrogenase

Molecular insight of the mechanism of an ancient membrane-bound hydrogenase

Molecular insight of the mechanism of an ancient membrane-bound hydrogenase

A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1.

The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H2 production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP+ as electron acceptors, respectively. While most FDHs exhibited NAD+-dependent formate oxidation activity, the Fdh3 of T. onnurineus NA1 showed a strong preference for NADP+ over NAD+ as a cofactor. The catalytic efficiency (kcat/Km) of Fdh3 for NADP+ was measured to be 5,281 mM−1 s−1, which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg204 and Arg205 of Fdh3B may contribute to the stabilization of the 2′-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO2 reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin.

Functional Dynamics of an Ancient Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) is a redox-driven Na+/H+ transporter that employs the energy from hydrogen gas (H2) production to catalyze proton pumping and Na+/H+ exchange across cytoplasmic membranes of archaea. Despite a recently resolved structure of this ancient energy-transducing enzyme [Yu et al. Cell2018, 173, 1636–1649], the molecular principles of its redox-driven ion-transport mechanism remain puzzling and of major interest for understanding bioenergetic principles of early cells. Here we use atomistic molecular dynamics (MD) simulations in combination with data clustering methods and quantum chemical calculations to probe principles underlying proton reduction as well as proton and sodium transport in Mbh from the hyperthermophilic archaeon Pyrococcus furiosus. We identify putative Na+ binding sites and proton pathways leading across the membrane and to the NiFe-active center as well as conformational changes that regulate ion uptake. We suggest that Na+ binding and protonation changes at a putative ion-binding site couple to proton transfer across the antiporter-like MbhH subunit by modulating the conformational state of a conserved ion pair at the subunit interface. Our findings illustrate conserved coupling principles within the complex I superfamily and provide functional insight into archaeal energy transduction mechanisms.

Enhanced H2 production by deletion of the Tfx family DNA-binding protein in the hyperthermophilic archaeon Thermococcus onnurineus NA1

A gene encoding an archaeal transcription regulator (TON_1525) homologous to the Tfx family DNA-binding protein has been identified to affect gene expression of carbon monoxide metabolism in the hyperthermophilic archaeon Thermococcus onnurineus NA1. To broadly understand gene regulation by the TON_1525 gene, a gene deletion mutant (Δ1525) was constructed and transcriptome changes were investigated. A total of 351 genes were found to be differentially expressed in the Δ1525 mutant. The expression levels of the hydrogenase genes were significantly changed; mbh (membrane-bound hydrogenase) and mch (membrane-bound carbon monoxide-dependent hydrogenase) genes were increased, and mfh2 (membrane-bound formate-dependent hydrogenase) and soluble hydrogenase genes were decreased. The Δ1525 mutant was cultured in a medium supplemented with maltodextrin or carbon monoxide to investigate whether changes in gene expression lead to physiological changes. The maximum H2 production rates of the Δ1525 mutant were significantly enhanced on both substrates compared to the wild-type strain. These results indicate that the TON_1525 gene can affect the expression of various genes including mbh and mch, consequently regulating the metabolism of T. onnurineus NA1. This study expands our understanding of the functional role of the Tfx family DNA-binding proteins.

PH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus.

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

Genome-Scale Metabolic Model of Caldicellulosiruptor bescii Reveals Optimal Metabolic Engineering Strategies for Bio-based Chemical Production.

ABSTRACTMetabolic modeling was used to examine potential bottlenecks that could be encountered for metabolic engineering of the cellulolytic extreme thermophile Caldicellulosiruptor bescii to produce bio-based chemicals from plant biomass. The model utilizes subsystems-based genome annotation, targeted reconstruction of carbohydrate utilization pathways, and biochemical and physiological experimental validations. Specifically, carbohydrate transport and utilization pathways involving 160 genes and their corresponding functions were incorporated, representing the utilization of C5/C6 monosaccharides, disaccharides, and polysaccharides such as cellulose and xylan. To illustrate its utility, the model predicted that optimal production from biomass-based sugars of the model product, ethanol, was driven by ATP production, redox balancing, and proton translocation, mediated through the interplay of an ATP synthase, a membrane-bound hydrogenase, a bifurcating hydrogenase, and a bifurcating NAD- and NADP-dependent oxidoreductase. These mechanistic insights guided the design and optimization of new engineering strategies for product optimization, which were subsequently tested in the C. bescii model, showing a nearly 2-fold increase in ethanol yields. The C. bescii model provides a useful platform for investigating the potential redox controls that mediate the carbon and energy flows in metabolism and sets the stage for future design of engineering strategies aiming at optimizing the production of ethanol and other bio-based chemicals.IMPORTANCE The extremely thermophilic cellulolytic bacterium, Caldicellulosiruptor bescii, degrades plant biomass at high temperatures without any pretreatments and can serve as a strategic platform for industrial applications. The metabolic engineering of C. bescii, however, faces potential bottlenecks in bio-based chemical productions. By simulating the optimal ethanol production, a complex interplay between redox balancing and the carbon and energy flow was revealed using a C. bescii genome-scale metabolic model. New engineering strategies were designed based on an improved mechanistic understanding of the C. bescii metabolism, and the new designs were modeled under different genetic backgrounds to identify optimal strategies. The C. bescii model provided useful insights into the metabolic controls of this organism thereby opening up prospects for optimizing production of a wide range of bio-based chemicals.

Engineering the Calvin\u2013Benson\u2013Bassham cycle and hydrogen utilization pathway of Ralstonia eutropha for improved autotrophic growth and polyhydroxybutyrate production

BackgroundCO2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin–Benson–Bassham (CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO2 fixation efficiency of cells is an important subject of biological research with significant application potential.ResultsIn this work, the autotrophic microbe Ralstonia eutropha (Cupriavidus necator) was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha, we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were functional during autotrophic growth of R. eutropha. SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system(A quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins) and RbcX(A chaperone in the folding of Rubisco), the culture OD600 of engineered strain increased 89.2% after 72 h of autotrophic growth, although the difference was decreased at 96 h, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and lead to improved growth in comparison to the host specific enzyme. Meanwhile, expression of hydrogenases was optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD600 to 93.4% at 72 h. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.7%.ConclusionsTo our best knowledge, this is the first report of successfully engineering the CBB pathway and hydrogenases of R. eutropha for improved activity, and is one of only a few cases where the efficiency of CO2 assimilation pathway was improved. Our work demonstrates that R. eutropha is a useful platform for studying and engineering the CBB for applications.

Towards cryogenic neutron crystallography on the reduced form of [NiFe]-hydrogenase.

A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni-Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni-Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni-Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions

The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) in C. necator H16, while the growth of which is restricted. Under autotrophic culture using carbon dioxide, hydrogen, and oxygen as substrates, the oxygen concentration below 6.9% (v/v) in the mixture is considered as a safe condition. It also expected to achieve cell rapid growth and large accumulation of PHB simultaneously under the oxygen-limiting condition in C. necator H16. In this study, a metabolically engineered strain capable of both rapid growth and large accumulation of PHB under oxygen-limiting conditions was constructed based on the transcriptomic analysis. In the comparative transcriptomic analysis, the genes related to energy-generating of C. necator H16 at autotrophic culture were downregulated under oxygen-limiting conditions (3%, v/v). Besides, the genes related to the key intermediates (pyruvate and acetyl-CoA) metabolism in PHB biosynthetic pathway were analyzed. Most of which were downregulated, except the genes ldh, iclA, and ackA2 respectively encoding L-lactate dehydrogenase, isocitrate lyase, and acetate kinase were upregulated under oxygen-limiting conditions (3%, v/v). The Vitreoscilla hemoglobin (VHb) has the ability to promote aerobic metabolism and energy generation. To promote the bacterium growth and improve the energy generation in C. necator H16 under oxygen-limiting conditions, the VHb gene was introduced into C. necator H16 with the optimized promoter PphaC1-j5. Moreover, VHb was localized to the periplasmic space of the bacterium by the traction of membrane-bound hydrogenase (MBH) signal peptide. By optimizing the knockout of different genes, it was found that knockout of ldh can improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression of vgb and ldh knockout in C. necator H16. Compared with the control (Reh (p2)) under oxygen-limiting conditions (3%, v/v), the dry cell weight (DCW), PHB content, and PHB production of Reh01 (p2M-pj-v) increased by 31.0%, 30.9%, and 71.5%, respectively. From the perspectives of transcriptome and metabolic engineering, the work provides new ideas to achieve rapid cell growth and large PHB accumulation in C. necator under oxygen-limiting and autotrophic conditions.

Metabolic Adaptation to Sulfur of Hyperthermophilic Palaeococcus pacificus DY20341T from Deep-Sea Hydrothermal Sediments.

The hyperthermo-piezophilic archaeon Palaeococcus pacificus DY20341T, isolated from East Pacific hydrothermal sediments, can utilize elemental sulfur as a terminal acceptor to simulate growth. To gain insight into sulfur metabolism, we performed a genomic and transcriptional analysis of Pa. pacificus DY20341T with/without elemental sulfur as an electron acceptor. In the 2001 protein-coding sequences of the genome, transcriptomic analysis showed that 108 genes increased (by up to 75.1 fold) and 336 genes decreased (by up to 13.9 fold) in the presence of elemental sulfur. Palaeococcus pacificus cultured with elemental sulfur promoted the following: the induction of membrane-bound hydrogenase (MBX), NADH:polysulfide oxidoreductase (NPSOR), NAD(P)H sulfur oxidoreductase (Nsr), sulfide dehydrogenase (SuDH), connected to the sulfur-reducing process, the upregulation of iron and nickel/cobalt transfer, iron–sulfur cluster-carrying proteins (NBP35), and some iron–sulfur cluster-containing proteins (SipA, SAM, CobQ, etc.). The accumulation of metal ions might further impact on regulators, e.g., SurR and TrmB. For growth in proteinous media without elemental sulfur, cells promoted flagelin, peptide/amino acids transporters, and maltose/sugar transporters to upregulate protein and starch/sugar utilization processes and riboflavin and thiamin biosynthesis. This indicates how strain DY20341T can adapt to different living conditions with/without elemental sulfur in the hydrothermal fields.

Growth of the facultative chemolithoautotroph Ralstonia eutropha on organic waste materials: growth characteristics, redox regulation and hydrogenase activity

BackgroundThe chemolithoautotrophic β-proteobacterium Ralstonia eutropha H16 (Cupriavidus necator) is one of the most studied model organisms for growth on H2 and CO2. R. eutropha H16 is also a biologically significant bacterium capable of synthesizing O2-tolerant [NiFe]-hydrogenases (Hyds), which can be used as anode biocatalysts in enzyme fuel cells. For heterotrophic growth of R. eutropha, various sources of organic carbon and energy can be used.ResultsGrowth, bioenergetic properties, and oxidation–reduction potential (ORP) kinetics were investigated during cultivation of R. eutropha H16 on fructose and glycerol or lignocellulose-containing brewery spent grain hydrolysate (BSGH). BSGH was used as carbon and energy source by R. eutropha H16, and the activities of the membrane-bound hydrogenase (MBH) and cytoplasmic, soluble hydrogenase (SH) were measured in different growth phases. Growth of R. eutropha H16 on optimized BSGH medium yielded ~ 0.7 g cell dry weight L−1 with 3.50 ± 0.02 (SH) and 2.3 ± 0.03 (MBH) U (mg protein)−1 activities. Upon growth on fructose and glycerol, a pH drop from 7.0 to 6.7 and a concomitant decrease of ORP was observed. During growth on BSGH, in contrast, the pH and ORP stayed constant. The growth rate was slightly stimulated through addition of 1 mM K3[Fe(CN)6], whereas temporarily reduced growth was observed upon addition of 3 mM dithiothreitol. The overall and N,N′-dicyclohexylcarbodiimide-sensitive ATPase activities of membrane vesicles were ~ 4- and ~ 2.5-fold lower, respectively, upon growth on fructose and glycerol (FGN) compared with only fructose utilization (FN). Compared to FN, ORP was lower upon bacterial growth on FGN, GFN, and BSGH.ConclusionsOur results suggest that reductive conditions and low ATPase activity might be signals for energy depletion, which, in turn, leads to increased hydrogenase biosynthesis to overcome this unfavorable situation. Addition of fructose or microelements have no, or a negative, influence on hydrogenase activity. Organic wastes (glycerol, BSGH) are promising carbon and energy sources for the formation of biomass harboring significant amounts of the biotechnologically relevant hydrogenases MBH and SH. The results are valuable for using microbial cells as producers of hydrogenase enzymes as catalysts in enzymatic fuel cells.

(Invited) Exoelectrogenicity of Genetically Engineered Hyperthermophiles

A hyperthermophilic archaeon called Pyrococcus furiosus (Pf), is being explored as potential microbial catalyst for use microbial fuel cells (MFCs) [Sekar et al. (2017)]. The two main enzymes membrane bound hydrogenase (MBH) and cytoplasmic soluble hydrogenases (SH) are involved in its oxidative fermentative pathways. In P. furiosus the membrane bound hydrogenase (MBH) uses reducing equivalents from the oxidative fermentative pathway to produce hydrogen, which is believed to be used by soluble hydrogenases (SHI and SHII) for nicotinamide cofactor generation. However, in anaerobic respiratory systems, the oxidative fermentative pathways and the activity of MBH and SH enzyme complexes are not fully understood. In this study, we hypothesize that genetically manipulating the expressions of SH and MBH could influence extracellular electron transfer, which could be characterized by an enhancement in organism’s ability to reduce a metal ion (e.g. soluble ferric citrate or and insoluble ferric oxide). Herein, genetic traceable Pf strain was genetically engineered to produce strains with over-expressed SHI (OESHI), over-expressed MbhJ-N (OEMbhJ-N), knocked-out SHI/SHII (ΔSHI/II) or knocked-out MbhL (ΔMbhL). Soluble ferric citrate and insoluble ferric oxide were used to study the ability of engineered Pf strains to reduce the extracellular oxidants. Experiments revealed that the ΔMbhL strain, produced the highest exo-electrogenic ability among these Pf strains. To the best of our knowledge this is the first report that compares the exoelectrogenic ability of different strains of genetically engineered Pf.

Inactivation of the uptake hydrogenase in the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS enables a biological water-gas shift platform for H2 production.

Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water-gas shift reaction to produce H2 from CO.