Optimal secretion of thermostable Beta-glucosidase in Bacillus subtilis by signal peptide optimization.

Commercial applications of ß-glucosidase (BGL) demands its purity and availability on a large scale. In the present study, we aim to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus (PfuBGL) in B. subtilis strain RIK1285. Initial studies with base strain BV002 harboring aprE signal peptide (aprESP) showed PfuBGL yield of 0.743 ± 0.19 pNP U/ml only. A library of 173 different homologous SPs from B. subtilis 168 genome was fused with target PfuBGL gene (PF0073) in pBE-S vector and extracellularly expressed in RIK1285 strain to identify optimal SP for PfuBGL secretion. High-throughput screening of the resulting SP library for BGL activity with a synthetic substrate followed by systematic scaling of the clones yielded a gene construct with CitHSP reporting a sixteen fold enhancement of PfuBGL secretion in comparison to base strain. Batch fermentation (7.5 L scale) PfuBGL yield of the BV003 strain with CitHSP-PF0073 fusion was observed to be 12.08 ± 0.21 pNP U/ml with specific activity of 35.52 ± 0.53 U/mg. Thus, the study represents report on the secretory expression of thermostable PfuBGL using B. subtilis as a host organism and demonstrating its high potential for industrial production of any protein/enzyme.

N-terminal domain replacement changes an archaeal monoacylglycerol lipase into a triacylglycerol lipase.

BACKGROUND: Lipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C2-C12) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from Archaeglobus fulgidus. Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil-water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon Thermococcus onnurineus (strain NA1) into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase-Thermomyces lanuginosus (TLIP). RESULTS: TON-LPL and rc-TGL were cloned and overexpressed in E. coli, and the proteins were purified by Ni-NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 °C in its optimum temperature (Topt). The far UV-CD spectrums were consistent with a well-folded α/ß-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates. CONCLUSIONS: Here, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals.

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase.

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

SGNH hydrolase-type esterase domain containing Cbes-AcXE2: a novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium, is rich in hydrolytic and accessory enzymes that can degrade untreated biomass, but the precise role of many these enzymes is unknown. One of such enzymes is a predicted GDSL lipase or esterase encoded by the locus Athe_0553. In this study, this probable esterase named as Cbes-AcXE2 was overexpressed in Escherichia coli. The Ni-NTA affinity purified enzyme exhibited an optimum pH of 7.5 at an optimum temperature of 70 °C. Cbes-AcXE2 hydrolyzed p-nitrophenyl (pNP) acetate, pNP-butyrate, and phenyl acetate with approximately equal efficiency. The specific activity and K M for the most preferred substrate, phenyl acetate, were 142 U/mg and 0.85 mM, respectively. Cbes-AcXE2 removed the acetyl group of xylobiose hexaacetate and glucose pentaacetate like an acetyl xylan esterase (AcXE). Bioinformatics analyses suggested that Cbes-AcXE2, which carries an SGNH hydrolase-type esterase domain, is a member of an unclassified carbohydrate esterase (CE) family. Moreover, Cbes-AcXE2 is evolutionarily and biochemically similar to an unclassified AcXE, Axe2, of Geobacillus stearothermophilus. Thus, we proposed a novel family of carbohydrate esterase for both Cbes-AcXE2 and Axe2.

Heterologous expression and biochemical studies of a thermostable glucose tolerant ß-glucosidase from Methylococcus capsulatus (bath strain).

Glucose inhibition of ß-glucosidase (BG) is a bottleneck in biomass hydrolysis. In this study, a glucose resistant GH1 ß-glucosidase gene- Mbgl from Methylococcus capsulatus (bath strain) was cloned and overexpressed in E.coli. The Ni-NTA affinity purified Mbgl displayed an optimum temperature of 70°C and optimum pH was 6.0. The calculated KM of the enzyme was 48.6mM and 0.12mM for cellobiose and 4-Nitrophenyl ß-d-glucopyranoside (PNPG) respectively. PNPG hydrolysis in presence of various glucose concentrations showed that the enzyme was stimulated by ∼2.2 fold at 50mM glucose and was not inhibited up to 450-500mM glucose. Homology modeling and structural comparisons of Mbgl with a glucose tolerant ß-glucosidase of Humicola insolens (HiBG) revealed that the Mbgl has a much broader active site unlike to a deep and narrow active site pocket of HiBG. The difference in active site shape reflects on an alternative mechanism of glucose tolerance in Mbgl. Supplementing a commercial cellulase enzyme mixture CTec with Mbgl in the hydrolysis of the pretreated rice straw enhanced the glucose yield by 10-15%. In addition, Mbgl was also stable in organic solvents, detergents and oxidative conditions which would be advantageous for biotechnological applications.

Dataset of gene cloning and gel filtration chromatography of R-est6.

The data presented in this article are connected to the research article entitled "Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath)" (Soni et al., 2016 [1]). The family 6 carboxylesterases are the smallest and display broad substrate specificity. The 1 kb gene encoding, a family 6 carboxylesterase - R-est6, was amplified from the genome of M. capsulatus (bath strain), and showed in the agarose gel. The corresponding purified protein, after overexpression in Escherichia coli, was biochemically studied in the research article (Soni et al., 2016 [1]). R-est6 has hydrophobic patches on the surface so, it is expected to show oligimeric forms. Here, we have confirmed the presence of oligomers by gel filtration chromatography data and the proteins belonging to the different peaks are shown on a SDS-PAGE.

Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath).

The genome of Methylococcus capsulatus (bath) encodes a protein R-est6 that is annotated as a lipase family 3 protein. The phylogenetic and the sequence analyses linked this protein to the family 6 carboxylesterase. The gene encoding R-est6 was cloned and overexpressed in Escherichia coli and the recombinant 6x-His tagged protein was purified by Ni-NTA affinity chromatography. The buffers used in the purification were modified by adding 1% glycerol instead of the salt to prevent the protein aggregation. Far UV-CD spectrum and gel filtration chromatography of the purified R-est6 confirmed that the protein was well folded like a typical α/ß hydrolase and had the quaternary structure of a tetramer, in addition to a compact monomer. The optimum pH was in the range of 7.0-9.0 and the optimum temperature was at 55 °C for the hydrolysis of pNP-butyrate. As expected, being a member of the family 6 carboxylesterase, R-est6 hydrolyzed triglycerides, pNP esters of the small and the medium fatty acid chain esters and an aryl ester-phenyl acetate. However, R-est6 was also found to hydrolyze the long-chain fatty acid ester which had never been reported for the family 6 carboxylesterase. Additionally, R-est6 was stable and active in the different water-miscible organic solvents. Therefore, the broad substrate range and the structural stability of R-est6 would be advantageous for its application in industrial processes.

The [NiFe]-Hydrogenase of Pyrococcus furiosus Exhibits a New Type of Oxygen Tolerance.

We report the first direct electrochemical characterization of the impact of oxygen on the hydrogen oxidation activity of an oxygen-tolerant, group 3, soluble [NiFe]-hydrogenase: hydrogenase I from Pyrococcus furiosus (PfSHI), which grows optimally near 100 °C. Chronoamperometric experiments were used to probe the sensitivity of PfSHI hydrogen oxidation activity to both brief and prolonged exposure to oxygen. For experiments between 15 and 80 °C, following short (<200 s) exposure to 14 µM O2 under oxidizing conditions, PfSHI always maintains some fraction of its initial hydrogen oxidation activity; i.e., it is oxygen-tolerant. Reactivation experiments show that two inactive states are formed by interaction with oxygen and both can be quickly (<150 s) reactivated. Analogous experiments, in which the interval of oxygen exposure is extended to 900 s, reveal that the response is highly temperature-dependent. At 25 °C, under sustained 1% O2/ 99% H2 exposure, the H2oxidation activity drops nearly to zero. However, at 80 °C, up to 32% of the enzyme's oxidation activity is retained. Reactivation of PfSHI following sustained exposure to oxygen occurs on a much longer time scale (tens of minutes), suggesting that a third inactive species predominates under these conditions. These results stand in contrast to the properties of oxygen-tolerant, group 1 [NiFe]-hydrogenases, which form a single state upon reaction with oxygen, and we propose that this new type of hydrogenase should be referred to as oxygen-resilient. Furthermore, PfSHI, like other group 3 [NiFe]-hydrogenases, does not possess the proximal [4Fe3S] cluster associated with the oxygen tolerance of some group 1 enzymes. Thus, a new mechanism is necessary to explain the observed oxygen tolerance in soluble, group 3 [NiFe]-hydrogenases, and we present a model integrating both electrochemical and spectroscopic results to define the relationships of these inactive states.

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling.

The use of hydrogen (H2) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H2 and CO2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H2 with a yield of two H2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H2 productivity was increased 3-fold to 32 mmol H2⋅L(-1)⋅h(-1). The productivity was further enhanced to 54 mmol H2⋅L(-1)⋅h(-1) by increasing reaction temperature, substrate, and enzyme concentrations--an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology.

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a catalytically-active nickel-free intermediate form.

The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.

Engineering the respiratory membrane-bound hydrogenase of the hyperthermophilic archaeon Pyrococcus furiosus and characterization of the catalytically active cytoplasmic subcomplex.

The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, carbon dioxide and hydrogen gas (H2), obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is classified as a Group 4 hydrogenase and is encoded by a 14-gene operon that contains hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged 4-subunit cytoplasmic subcomplex of MBH (C-MBH) was engineered and expressed in P. furiosus by differential transcription of the MBH operon. It was purified under anaerobic conditions by affinity chromatography without detergent. Purified C-MBH had a Fe : Ni ratio of 14 : 1, similar to the predicted value of 13 : 1. The O2 sensitivities of C-MBH and the 14-subunit membrane-bound version were similar (half-lives of ∼15 h in air), but C-MBH was more thermolabile (half-lives at 90°C of 8 and 25 h, respectively). C-MBH evolved H2 with the physiological electron donor, reduced ferredoxin, optimally at 60°C. This is the first report of the engineering and characterization of a soluble catalytically active subcomplex of a membrane-bound respiratory hydrogenase.

Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.

In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose.

Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to 15 enzymes under mild reaction conditions (i.e. 37°C and atm). In a batch reaction, the hydrogen yield was 23.2mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74mmol of H2/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.

Mannosylglycerate and di-myo-inositol phosphate have interchangeable roles during adaptation of Pyrococcus furiosus to heat stress.

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.

Hyperthermophile protein behavior: partially-structured conformations of Pyrococcus furiosus rubredoxin monomers generated through forced cold-denaturation and refolding.

Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as 'Trishanku' unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native 'Trishanku' refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.

High-yield production of dihydrogen from xylose by using a synthetic enzyme cascade in a cell-free system.

Let enzymes work: H2 was produced from xylose and water in one reactor containing 13 enzymes (red). By using a novel polyphosphate xylulokinase (XK), xylose was converted into H2 and CO2 with approaching 100 % of the theoretical yield. The findings suggest that cell-free biosystems could produce H2 from biomass xylose at low cost. Xu5P = xylulose 5-phosphate, G6P = glucose 6-phosphate.

Introduction of a thermophile-sourced ion pair network in the fourth beta/alpha unit of a psychophile-derived triosephosphate isomerase from Methanococcoides burtonii significantly increases its kinetic thermal stability.

Hyperthermophile proteins commonly have higher numbers of surface ionic interactions than homologous proteins from other domains of life. PfuTIM, a triosephosphate isomerase (TIM) from the hyperthermophile archaeon, Pyrococcus furiosus, contains an intricate network of 4 ion pairs in its 4th beta/alpha unit, (ß/α)4, whereas MbuTIM, a triosephosphate isomerase from a psychrophile archaeon, Methanococcoides burtonii, lacks this network. Notably, (ß/α)4 is the first element of the structure formed during folding of certain TIM-type (beta/alpha)8 barrel proteins. Previously, we have shown that elimination of PfuTIM's ion pair network in PfuTIM significantly decreases its kinetic structural stability. Here, we describe the reciprocal experiment in which this ion pair network is introduced into MbuTIM, to produce MutMbuTIM. Recombinant MbuTIM displays multi-state unfolding with apparent Tm values of autonomous structural elements approaching, or above, 70°C, when a temperature scanning rate of 90°C/h is used. The protein displays significant intrinsic kinetic stability, i.e., there is a marked temperature scan rate-dependence of the Tm values associated with unfolding transitions. The Tm values drop by as much as ~10°C when the temperature scanning rate is lowered to 5°C/h. MutMbuTIM, incorporating PfuTIM's ion pair network, shows significantly higher apparent Tm values (raised by 4-6°C over those displayed by MbuTIM). MutMbuTIM also displays significantly higher kinetic thermal stability. Thus, it appears that the thermal stability of triosephosphate isomerase can be increased, or decreased, by either enhancing, or reducing, the strength of ion pair interactions stabilizing (ß/α)4, presumably through reduced cooperativity (and increased autonomy) in unfolding transitions.

Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Homologous expression of a subcomplex of Pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase.

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.