L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

Crystal structure of GTP-dependent dephospho-coenzyme A kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases. Here, we report the crystal structure of DPCK and its complex with GTP and a magnesium ion from the archaeal hyperthermophile Thermococcus kodakarensis. The crystal structure demonstrates why GTP is the preferred substrate of this kinase. We also report the activity analyses of site-directed mutants of crucial residues determined based on sequence conservation and the crystal structure. From these results, the key residues involved in the reaction of phosphoryl transfer and the possible dephospho-CoA binding site are inferred.

Thermococcus thermotolerans sp. nov., a hyperthermophilic archaeon isolated from a chimney in the Southwest Indian Ocean.

An anaerobic hyperthermophilic archaeon was isolated from a black smoker chimney with a snail attachment at a water depth of 2 739 m in the Southwest Indian Ocean. The sample was taken from the chimney exterior wall. The enrichment was conducted under a continuous culture with temperature fluctuation of 80-130 °C over 24 h for 42 days at 28 MPa. The isolation was performed at 90 °C at 0.1 MPa. Cells of the isolated strain 813A4T were irregular cocci. Strain 813A4T grew at 60-94 °C (optimal growth at 85 °C) at 0.1 MPa, and growth was detected at up to 99 °C at 28 MPa. At 85 °C, the strain was able to grow at pressures ranging from 0.1 to 110 MPa (optimal pressure, 0.1-40 MPa). At 85 °C, the cells of 813A4T grew at pH 5.5-9 (optimal, pH 7.0) and a NaCl concentration of 1.0-4.0 % (w/v; optimum concentration, 2.5 % NaCl). Strain 813A4T utilized yeast extract, tryptone and peptone as single carbon sources for growth. Elemental sulphur stimulated its growth. The G+C content of the complete genome was 53.48 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 813A4T belonged to the genus Thermococcus, with the highest sequence similarity to Thermococcus barossii SHCK-94T (99.73 %). The average nucleotide identity between strains 813A4T and SHCK-94T was 82.56 %. All these data indicated that strain 813A4T should be classified as representing a novel species of the genus Thermococcus, for which Thermococcus thermotolerans sp. nov. is proposed. The type strain is 813A4T (=JCM 39367T=MCCC M28628T).

Synthesis and degradation of the cyclic dinucleotide messenger c-di-AMP in the hyperthermophilic archaeon Pyrococcus yayanosii.

Cyclic di-adenosine monophosphate (c-di-AMP) is a newly identified prokaryotic cyclic dinucleotide second messenger well elucidated in bacteria, while less studied in archaea. Here, we describe the enzymes involved in c-di-AMP metabolism in the hyperthermophilic archaeon Pyrococcus yayanosii. Our results demonstrate that c-di-AMP is synthesized from two molecules of ATP by diadenylate cyclase (DAC) and degraded into pApA and then to AMP by a DHH family phosphodiesterase (PDE). DAC can be activated by a wider variety of ions, using two conserved residues, D188 and E244, to coordinate divalent metal ions, which is different from bacterial CdaA and DisA. PDE possesses a broad substrate spectrum like bacterial DHH family PDEs but shows a stricter base selection between A and G in cyclic dinucleotides hydrolysis. PDE shows differences in substrate binding patches from bacterial counterparts. C-di-AMP was confirmed to exist in Thermococcus kodakarensis cells, and the deletion of the dac or pde gene supports that the synthesis and degradation of c-di-AMP are catalyzed by DAC and PDE, respectively. Our results provide a further understanding of the metabolism of c-di-AMP in archaea.

NADP+ or CO2 reduction by frhAGB-encoded hydrogenase through interaction with formate dehydrogenase 3 in the hyperthermophilic archaeon Thermococcus onnurineus NA1.

IMPORTANCE: The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the frhAGB-encoded hydrogenase of Thermococcus onnurineus NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P)+ is significant in that the enzyme can function to supply reducing equivalents, just as F420-reducing hydrogenases in methanogens use coenzyme F420 as an electron carrier. Additionally, it was identified that T. onnurineus NA1 could produce formate from H2 and CO2 by the concerted action of frhAGB-encoded hydrogenase and formate dehydrogenase Fdh3.

Thermococcus argininiproducens sp. nov., an arginine biosynthesis archaeal species isolated from the Central Indian Ocean ridge.

A strictly anaerobic hyperthermophilic archaeon, designated strain IOH2T, was isolated from a deep-sea hydrothermal vent (Onnuri vent field) area on the Central Indian Ocean Ridge. Strain IOH2T showed high 16S rRNA gene sequence similarity to Thermococcus sibiricus MM 739T (99.42 %), Thermococcus alcaliphilus DSM 10322T (99.28 %), Thermococcus aegaeus P5T (99.21 %), Thermococcus litoralis DSM 5473T (99.13 %), 'Thermococcus bergensis' T7324T (99.13 %), Thermococcus aggregans TYT (98.92 %) and Thermococcus prieurii Bio-pl-0405IT2T (98.01 %), with all other strains showing lower than 98 % similarity. The average nucleotide identity and in silico DNA-DNA hybridization values were highest between strain IOH2T and T. sibiricus MM 739T (79.33 and 15.00 %, respectively); these values are much lower than the species delineation cut-offs. Cells of strain IOH2T were coccoid, 1.0-1.2 µm in diameter and had no flagella. Growth ranges were 60-85 °C (optimum at 80 °C), pH 4.5-8.5 (optimum at pH 6.3) and 2.0-6.0 % (optimum at 4.0 %) NaCl. Growth of strain IOH2T was enhanced by starch, glucose, maltodextrin and pyruvate as a carbon source, and elemental sulphur as an electron acceptor. Through genome analysis of strain IOH2T, arginine biosynthesis related genes were predicted, and growth of strain IOH2T without arginine was confirmed. The genome of strain IOH2T was assembled as a circular chromosome of 1 946 249 bp and predicted 2096 genes. The DNA G+C content was 39.44 mol%. Based on the results of physiological and phylogenetic analyses, Thermococcus argininiproducens sp. nov. is proposed with type strain IOH2T (=MCCC 4K00089T=KCTC 25190T).

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo.

In spite of the development of genome-editing tools using CRISPR-Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5'-phosphorylated (5'-P) or 5'- hydroxylated (5'-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn2+ or Mg2+ and displayed no obvious preference for the 5'-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.

Thermophilic Inorganic Pyrophosphatase Ton1914 from Thermococcus onnurineus NA1 Removes the Inhibitory Effect of Pyrophosphate.

Pyrophosphate (PPi) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PPi can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg2+ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32-41%, while that of short-chain DNA was only improved by 9.5-15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.

Transformation Techniques for the Anaerobic Hyperthermophile Thermococcus kodakarensis.

Genetic manipulation is an essential tool to investigate complex microbiological phenomena. In this chapter we describe the techniques required to transform the model hyperthermophilic, anaerobic archaeon Thermococcus kodakarensis. T. kodakarensis can support two modes of genetic manipulation, dependent either on homologous recombination into the genome or through retention of autonomously replicating plasmids. The robust genetic system developed in T. kodakarensis offers a variety of selectable and counterselectable markers for complex, accurate and iterative genetic manipulations offering greater flexibility to probe gene function in vivo.

The Exploration of the Thermococcus barophilus Lipidome Reveals the Widest Variety of Phosphoglycolipids in Thermococcales.

One of the most distinctive characteristics of archaea is their unique lipids. While the general nature of archaeal lipids has been linked to their tolerance to extreme conditions, little is known about the diversity of lipidic structures archaea are able to synthesize, which hinders the elucidation of the physicochemical properties of their cell membrane. In an effort to widen the known lipid repertoire of the piezophilic and hyperthermophilic model archaeon Thermococcus barophilus, we comprehensively characterized its intact polar lipid (IPL), core lipid (CL), and polar head group compositions using a combination of cutting-edge liquid chromatography and mass spectrometric ionization systems. We tentatively identified 82 different IPLs based on five distinct CLs and 10 polar head group derivatives of phosphatidylhexoses, including compounds reported here for the first time, e.g., di-N-acetylhexosamine phosphatidylhexose-bearing lipids. Despite having extended the knowledge on the lipidome, our results also indicate that the majority of T. barophilus lipids remain inaccessible to current analytical procedures and that improvements in lipid extraction and analysis are still required. This expanded yet incomplete lipidome nonetheless opens new avenues for understanding the physiology, physicochemical properties, and organization of the membrane in this archaeon as well as other archaea.

Characterization of a novel thermostable phospholipase C from T. kodakarensis suitable for oil degumming.

The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils' phospholipids. This enzymatic method reduces the gums' volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. KEY POINTS: • A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. • TkPLC was recombinantly produced in Pichia pastoris and successfully purified. • TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.

Temperature dependent autocleavage and applications of recombinant L-asparaginase from Thermococcus kodakarensis for acrylamide mitigation.

This manuscript describes enhancement of soluble production, auto-cleavage analysis and assessment of acrylamide mitigation potential of Tk2246, a plant-type L-asparaginase from Thermococcus kodakarensis. The gene encoding Tk2246 was cloned and expressed in Escherichia coli. Recombinant Tk2246 was produced mainly in insoluble form. Various strategies were utilized to enhance the soluble production, which significantly increased the soluble yield. Interestingly, recombinant Tk2246 was produced even without addition of the inducer, though relatively in a lower amount. To our surprise, Tk2246 was produced in partially cleaved form when the inducer was not added in the culture. When applied for acrylamide mitigation, Tk2246 reduced the acrylamide formation more than 80% in French fries, chapati and yeast-leavened bread. In addition to acrylamide mitigation, Tk2246 exhibited antistaling activity without loss of sensory properties of the food. High activity, thermostability and efficient acrylamide reduction capability make Tk2246 a potential candidate for industrial applications.

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 (k cat /K m) 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.

Genetic and Functional Analyses of Archaeal ATP-Dependent RNA Ligase in C/D Box sRNA Circularization and Ribosomal RNA Processing.

RNA ligases play important roles in repairing and circularizing RNAs post-transcriptionally. In this study, we generated an allelic knockout of ATP-dependent RNA ligase (Rnl) in the hyperthermophilic archaeon Thermococcus kodakarensis to identify its biological targets. A comparative analysis of circular RNA reveals that the Rnl-knockout strain represses circularization of C/D box sRNAs without affecting the circularization of tRNA and rRNA processing intermediates. Recombinant archaeal Rnl could circularize C/D box sRNAs with a mutation in the conserved C/D box sequence element but not when the terminal stem structures were disrupted, suggesting that proximity of the two ends could be critical for intramolecular ligation. Furthermore, T. kodakarensis accumulates aberrant RNA fragments derived from ribosomal RNA in the absence of Rnl. These results suggest that Rnl is responsible for C/D box sRNA circularization and may also play a role in ribosomal RNA processing.

PCNA from Thermococcus gammatolerans: A protein involved in chromosomal DNA metabolism intrinsically resistant at high levels of ionizing radiation.

Proliferating cell nuclear antigen (PCNA) is an essential protein for cell viability in archaea and eukarya, since it is involved in DNA replication and repair. In order to obtain insights regarding the characteristics that confer radioresistance, the structural study of the PCNA from Thermococcus gammatolerans (PCNATg ) in a gradient of ionizing radiation by X-ray crystallography was carried out, together with a bioinformatic analysis of homotrimeric PCNA structures, their sequences, and their molecular interactions. The results obtained from the datasets and the accumulated radiation dose for the last collection from three crystals revealed moderate and localized damage, since even with the loss of resolution, the electron density map corresponding to the last collection allowed to build the whole structure. Attempting to understand this behavior, multiple sequence alignments, and structural superpositions were performed, revealing that PCNA is a protein with a poorly conserved sequence, but with a highly conserved structure. The PCNATg presented the highest percentage of charged residues, mostly negatively charged, with a proportion of glutamate more than double aspartate, lack of cysteines and tryptophan, besides a high number of salt bridges. The structural study by X-ray crystallography reveals that the PCNATg has the intrinsic ability to resist high levels of ionizing radiation, and the bioinformatic analysis suggests that molecular evolution selected a particular composition of amino acid residues, and their consequent network of synergistic interactions for extreme conditions, as a collateral effect, conferring radioresistance to a protein involved in the chromosomal DNA metabolism of a radioresistant microorganism.

[Characterization of a family B DNA polymerase from Thermococcus eurythermalis A501 and its application in PCR].

DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl2, 60 mmol/L KCl, 10 mmol/L (NH4)2SO4, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.

Structural basis for peptide recognition by archaeal oligopeptide permease A.

Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.

Deep Conservation of Histone Variants in Thermococcales Archaea.

Histones are ubiquitous in eukaryotes where they assemble into nucleosomes, binding and wrapping DNA to form chromatin. One process to modify chromatin and regulate DNA accessibility is the replacement of histones in the nucleosome with paralogous variants. Histones are also present in archaea but whether and how histone variants contribute to the generation of different physiologically relevant chromatin states in these organisms remains largely unknown. Conservation of paralogs with distinct properties can provide prima facie evidence for defined functional roles. We recently revealed deep conservation of histone paralogs with different properties in the Methanobacteriales, but little is known experimentally about these histones. In contrast, the two histones of the model archaeon Thermococcus kodakarensis, HTkA and HTkB, have been examined in some depth, both in vitro and in vivo. HTkA and HTkB exhibit distinct DNA-binding behaviors and elicit unique transcriptional responses when deleted. Here, we consider the evolution of HTkA/B and their orthologs across the order Thermococcales. We find histones with signature HTkA- and HTkB-like properties to be present in almost all Thermococcales genomes. Phylogenetic analysis indicates the presence of one HTkA- and one HTkB-like histone in the ancestor of Thermococcales and long-term maintenance of these two paralogs throughout Thermococcales diversification. Our results support the notion that archaea and eukaryotes have convergently evolved histone variants that carry out distinct adaptive functions. Intriguingly, we also detect more highly diverged histone-fold proteins, related to those found in some bacteria, in several Thermococcales genomes. The functions of these bacteria-type histones remain unknown, but structural modeling suggests that they can form heterodimers with HTkA/B-like histones.

Genomics and simulated laboratory studies reveal Thermococcus sp. 101C5 as a novel hyperthermophilic archaeon possessing a specialized metabolic arsenal for enhanced oil recovery.

Laboratory evaluation of hyperthermophiles with the potential for Enhanced Oil Recovery (EOR) is often hampered by the difficulties in replicating the in situ growth conditions in the laboratory. In the present investigation, genome analysis was used to gain insights into the metabolic potential of a hyperthermophile to mobilize the residual oil from depleting high-temperature oil reservoirs. Here, we report the 1.9 Mb draft genome sequence of a hyperthermophilic anaerobic archaeon, Thermococcus sp. 101C5, with a GC content of 44%, isolated from a high-temperature oil reservoir of Gujarat, India. 101C5 possessed the genetic arsenal required for adaptation to harsh oil reservoir conditions, such as various heat shock proteins for thermo-adaptation, Trk potassium uptake system proteins for osmo-adaptation, and superoxide reductases against oxidative stress. Microbial Enhanced Oil Recovery (MEOR) potential of the strain was established by ascertaining the presence of genes encoding enzymes involved in the production of the metabolites such as hydrogen, bio-emulsifier, acetate, exopolysaccharide, etc. Production of these metabolites which pressurize the reservoir, emulsify the crude oil, lower the viscosity and reduce the drag, thus facilitating mobilization of the residual oil was experimentally confirmed. Also, the presence of crude oil degradative genes highlighted the ability of the strain to mobilize heavy residual oil, which was confirmed under simulated conditions in sand-pack studies. The obtained results demonstrated additional oil recoveries of 42.1% and 56.5% at 96 °C and 101 °C, respectively, by the strain 101C5, illustrating its potential for application in high-temperature oil reservoirs. To our best knowledge, this is the first report of genome analysis of any microbe assessed for its suitability for MEOR from the high-temperature oil reservoir.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase.

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS-1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS-1 (PPIase KS-1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS-1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS-1. The recombinant E. coli cells expressing the PPIase KS-1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS-1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS-1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.