Hyperthermophilic Archaeon Pyrococcus Furiosus Research Articles

Heterologous Expression of Chaperones from Hyperthermophilic Archaea Inhibits Aminoglycoside-Induced Protein Misfolding in Escherichia coli.

Aminoglycoside antibiotics affect protein translation fidelity and lead to protein aggregation and an increase in intracellular oxidative stress level as well. The overexpression of the chaperonin GroEL/GroES system promotes short-term tolerance to aminoglycosides in Escherichia coli. Here, we demonstrated that the coexpression of prefoldin or Hsp60 originating from the hyperthermophilic archaeon Pyrococcus furiosus in E. coli cells can rescue cell growth and inhibit protein aggregation induced by streptomycin exposure. The results of our study show that hyperthermophilic chaperones endow E. coli with a higher tolerance to streptomycin than the GroEL/GroES system, and that they exert better effects on the reduction of intracellular protein misfolding, indicating that these chaperones have unique features and functions.

Fixation of bioactive compounds to the cuticle of Artemia

Artemia is extensively used in aquaculture to feed early stages of cultured marine species. A problem associated with this practice is that Artemia fails to supply some essential nutrients. As a possible solution, we have devised a procedure to make Artemia a vehicle for exogenous nutrients and other bioactive compounds. It consists of the construction of chimeric proteins composed of a chitin-binding domain, which binds to the cuticle of Artemia, and a carrier domain that conveys a functional property. As confirmatory examples, we describe the successful fixation to Artemia's metanauplii of two hybrid proteins: a β-galactosidase from the thermophilic bacterium Thermotoga maritima and the jellyfish green fluorescent protein (GFP), both linked to the CBM2 chitin-binding domain from the hyperthermophilic archaeon Pyrococcus furiosus. Positive results of experiments carried out ex vivo and in vivo show the validity of this approach. The methodology used could become a general procedure for the attachment of different kinds of bioactive compounds, such as enzymes, hormones, antibiotics, etc., to the cuticle of Artemia as well as other arthropods. Statement of relevanceOur results overcome shortcomings of Artemia as a feedstock.

Reverse gyrase is essential for microbial growth at 95 °C.

Reverse gyrase is an enzyme that induces positive supercoiling in closed circular DNA in vitro. It is unique to thermophilic organisms and found without exception in all microorganisms defined as hyperthermophiles, that is, those having optimal growth temperatures of 80 °C and above. Although its in vivo role has not been clearly defined, it has been implicated in stabilizing DNA at high temperatures. Whether or not it is absolutely required for growth at these high temperatures has yet to be fully determined. In a previous study with an organism that has an optimal growth temperature of 85 °C, it was shown that the enzyme is not a prerequisite for life at extreme temperatures as disruption of its gene did not result in a lethal phenotype at the supraoptimal growth temperature of 90 °C. Herein we show that the enzyme is absolutely required for microbial growth at 95 °C, which in this case is a suboptimal growth temperature. Deletion of the gene encoding the reverse gyrase of the model hyperthermophilic archaeon Pyrococcus furiosus, which has an optimal growth temperature of 100 °C, revealed that the gene is required for growth at 95 °C, as well as at 100 °C. The results suggest that a temperature threshold above 90 °C exists, wherein the activity of reverse gyrase is absolutely necessary to maintain a correct DNA twist for any organism growing at such temperature extremes.

Ethanol production by the hyperthermophilic archaeon Pyrococcus furiosus by expression of bacterial bifunctional alcohol dehydrogenases.

SummaryEthanol is an important target for the renewable production of liquid transportation fuels. It can be produced biologically from pyruvate, via pyruvate decarboxylase, or from acetyl‐CoA, by alcohol dehydrogenase E (AdhE). Thermophilic bacteria utilize AdhE, which is a bifunctional enzyme that contains both acetaldehyde dehydrogenase and alcohol dehydrogenase activities. Many of these organisms also contain a separate alcohol dehydrogenase (AdhA) that generates ethanol from acetaldehyde, although the role of AdhA in ethanol production is typically not clear. As acetyl‐CoA is a key central metabolite that can be generated from a wide range of substrates, AdhE can serve as a single gene fuel module to produce ethanol through primary metabolic pathways. The focus here is on the hyperthermophilic archaeon Pyrococcus furiosus, which grows by fermenting sugar to acetate, CO 2 and H2. Previously, by the heterologous expression of adhA from a thermophilic bacterium, P. furiosus was shown to produce ethanol by a novel mechanism from acetate, mediated by AdhA and the native enzyme aldehyde oxidoreductase (AOR). In this study, the AOR gene was deleted from P. furiosus to evaluate ethanol production directly from acetyl‐CoA by heterologous expression of the adhE gene from eight thermophilic bacteria. Only AdhEs from two Thermoanaerobacter strains showed significant activity in cell‐free extracts of recombinant P. furiosus and supported ethanol production in vivo. In the AOR deletion background, the highest amount of ethanol (estimated 61% theoretical yield) was produced when adhE and adhA from Thermoanaerobacter were co‐expressed.

Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.

Characterization and Low-Resolution Structure of an Extremely Thermostable Esterase of Potential Biotechnological Interest from Pyrococcus furiosus.

Enzymes isolated from extremophiles often exhibit superior performance and potential industrial applications. There are several advantages performing biocatalysis at elevated temperatures, including enhanced reaction rates, increased substrate solubility and decreased risks of contamination. Furthermore, thermophilic enzymes usually exhibit high resistance against many organic solvents and detergents, and are also more resistant to proteolytic attack. In this study, we subcloned and characterized an esterase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Est) that exhibits optimal activity around 80°C using naphthol-derived substrates and p-nitrophenyl palmitate (pNPP). According to the circular dichroism spectra, the secondary structure of P. furiosus esterase, which is predominantly formed by a β-sheet structure, is very stable, even after incubation at 120°C. We performed SAXS to determine the low-resolution structure of Pf_Est, which is monomeric in solution at 80°C and has a molecular weight of 28kDa. The Km and V max values for this esterase acting on pNPP were 0.53mmol/L and 6.5×10-3U, respectively. Pf_Est was most active in the immiscible solvents and retained more than 50% in miscible solvents. Moreover, Pf_Est possesses transesterification capacity, presenting better results when isobutanol was used as an acyl acceptor (2.69±0.14×10-2μmol/minmg) and the highest hydrolytic activity toward olive oil among different types of oils testes in this study. Collectively, these biophysical and catalytic properties are of interest for several biotechnological applications that require harsh conditions, including high temperature and the presence of organic solvents.

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus.

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO3- and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO2 into 3HP production in metabolically engineered P. furiosus, and hint at the important role that CA and BPL likely play in the native 3HP/4HB pathway in M. sedula. Biotechnol. Bioeng. 2016;113: 2652-2660. © 2016 Wiley Periodicals, Inc.

Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea.

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5′-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria.

Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria.

Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C5 and C6 sugars primarily to hydrogen gas, lactate, acetate, and CO2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.

Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I.

Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination. Recently, we identified a novel single-strand specific 3′-5′ exonuclease, PfuExo I, from the hyperthermophilic archaeon Pyrococcus furiosus, which may be involved in the Thermococcales-specific DNA repair system. PfuExo I forms a trimer and cleaves single-stranded DNA at every two nucleotides. Here, we report the structural basis for the cleavage mechanism of this novel exonuclease family. A structural analysis of PhoExo I, the homologous enzyme from P. horikoshii OT3, showed that PhoExo I utilizes an RNase H-like active site and possesses a 3′-OH recognition site ∼9 Å away from the active site, which enables cleavage at every two nucleotides. Analyses of the heterotrimeric and monomeric PhoExo I activities showed that trimerization is indispensable for its processive cleavage mechanism, but only one active site of the trimer is required.

Biochemical characterization of a thermostable adenosylmethionine synthetase from the archaeon Pyrococcus furiosus with high catalytic power.

Adenosylmethionine synthetase plays a key role in the biogenesis of the sulfonium compound S-adenosylmethionine, the principal widely used methyl donor in the biological methylations. We report here, for the first time, the characterization of adenosylmethionine synthetase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMAT). The gene PF1866 encoding PfMAT was cloned and expressed, and the recombinant protein was purified to homogeneity. PfMAT shares 51, 63, and 82% sequence identity with the homologous enzymes from Sulfolobus solfataricus, Methanococcus jannaschii, and Thermococcus kodakarensis, respectively. PfMAT is a homodimer of 90 kDa highly thermophilic with an optimum temperature of 90 °C and is characterized by remarkable thermodynamic stability (Tm, 99 °C), kinetic stability, and resistance to guanidine hydrochloride-induced unfolding. The latter process is reversible as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. Limited proteolysis experiments indicated that the proteolytic cleavage site is localized at Lys148 and that the C-terminal peptide is necessary for the integrity of the active site. PfMAT shows kinetic features that make it the most efficient catalyst for S-adenosylmethionine synthesis among the characterized MAT from Bacteria and Archaea. Molecular and structural characterization of PfMAT could be useful to improve MAT enzyme engineering for biotechnological applications.

Improving the thermostability and optimal temperature of a lipase from the hyperthermophilic archaeon Pyrococcus furiosus by covalent immobilization.

A recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL) was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL) and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment) (glyoxyl-DTT PFUL) and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment) (glyoxyl PFUL). The enzyme's properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity), whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity). Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity (E = 22) and enantiomeric excess (ee (%) = 91).

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.

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.

Strategies for reliable and improved large-scale production of Pyrococcus furiosus with integrated purification of hydrogenase I

The hyperthermophilic archaeon Pyrococcus furiosus is an interesting organism for research and application, especially owing to its unique NADPH-dependent hydrogenase I. However, mass production of P. furiosus through fermentation is susceptible to fault because of its sensitivity to oxygen, a short exponential and stationary phase and a rapid cell lysis in typical cultivation process. In this study, significant improvement for pilot plant scale production processes for P. furiosus biomass was made by investigations of the fermentation process with subsequent hydrogenase I enzyme purification. Scale-up in a 300-L stirred tank bioreactor was successfully achieved. A repeated-batch cultivation process with high reproducibility and productivity was realized. Furthermore, the enzyme hydrogenase I was purified, and its activity tested and verified. The improvements in this production process for the production of large amount of P. furiosus biomass and hydrogenase I have been achieved, especially by successfully implementing the following key measures and steps: unsterile cultivation setup, skipping typical intermediate preculture and inoculation steps, accelerating the cultivation process by defining an optimal state of the inoculation, optimal time point of biomass harvesting and finally by choosing a one-step purification procedure for enzyme recovery.

Effects of metal ions on stability and activity of hyperthermophilic pyrolysin and further stabilization of this enzyme by modification of a Ca2+-binding site.

Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na(+), Ca(2+), or Mg(2+) salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca(2+)-binding sites (Ca1 and Ca2) revealed that the binding of Ca(2+) is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca(2+)-binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin.

Characterization of an extremely thermostable but cold-adaptive β-galactosidase from the hyperthermophilic archaeon Pyrococcus furiosus for use as a recombinant aggregation for batch lactose degradation at high temperature

β-Galactosidase (lactase), which catalyzes the hydrolysis of lactose into glucose and galactose, is one of the most important enzymes used in dairy processing. In this study, a gene that encoded an extremely thermostable β-galactosidase from Pyrococcus furiosus (Pflactase) was cloned and expressed in Escherichia coli BL21. The recombinant enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The enzyme displayed optimal activity at 90°C and pH 7.0 in phosphate buffer. The specific activity of the recombinant enzyme on o-nitrophenyl-β-d-galactopyranoside was 10.2U/mg at 0°C and 130.0dU/mg at 90°C. The half-lives of the enzyme were 31423.4, 8168.3, 4017.7, 547.4, 309.6, and 203.5min at 70°C, 80°C, 85°C, 90°C, 95°C, and 100°C, respectively. The recombinant enzyme exhibited both β-galactosidase and β-glucosidase activity. The active inclusion bodies of β-galactosidase were easily isolated by nonionic detergent treatment and directly used for lactose conversion in a repetitive batch mode. More than 54% (90°C) or 88% (10°C) of the original enzyme activity was retained after 10 conversion cycles under optimum conditions. These results suggest that the recombinant thermostable β-galactosidase may be suitable for the hydrolysis of lactose in milk processing.

Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus

The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO2 and acetyl-CoA by the heterologous-expression of three enzymes from the CO2 fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α2β2) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus.

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.