Pressurized cultivation strategies for improved microbial hydrogen production by Thermococcus onnurineus NA1.

While the hydrogen economy is receiving growing attention, research on microbial hydrogen production is also increasing. Microbial water-gas shift reaction is advantageous as it produces hydrogen from by product gas including carbon monoxide (CO). However, CO solubility in water is the bottleneck of this process by low mass transfer. Thermococcus onnurineus NA1 strain can endure a high-pressure environment and can enhance hydrogen production in a pressurized reactor by increasing CO solubility. As CO causes cell toxicity, two important factors, pressure and input gas flow rate, should be considered for process control during cultivation. Hence, we employed different operational strategies for enhancing hydrogen production and obtained 577 mmol/L/h of hydrogen productivity. This is the highest hydrogen productivity reported to date from microbial water-gas shift reaction.

An overview of 25 years of research on Thermococcus kodakarensis, a genetically versatile model organism for archaeal research.

Almost 25 years have passed since the discovery of a planktonic, heterotrophic, hyperthermophilic archaeon named Thermococcus kodakarensis KOD1, previously known as Pyrococcus sp. KOD1, by Imanaka and coworkers. T. kodakarensis is one of the most studied archaeon in terms of metabolic pathways, available genomic resources, established genetic engineering techniques, reporter constructs, in vitro transcription/translation machinery, and gene expression/gene knockout systems. In addition to all these, ease of growth using various carbon sources makes it a facile archaeal model organism. Here, in this review, an attempt is made to reflect what we have learnt from this hyperthermophilic archaeon.

Genes regulated by branched-chain polyamine in the hyperthermophilic archaeon Thermococcus kodakarensis.

Branched-chain polyamine (BCPA) synthase (BpsA), encoded by the bpsA gene, is responsible for the biosynthesis of BCPA in the hyperthermophilic archaeon Thermococcus kodakarensis, which produces N4-bis(aminopropyl)spermidine and spermidine. Here, next-generation DNA sequencing and liquid chromatography-mass spectrometry (LC-MS) were used to perform transcriptomic and proteomic analyses of a T. kodakarensis strain (DBP1) lacking bpsA. Subsequently, the contributions of BCPA to gene transcription (or transcript stabilization) and translation (or protein stabilization) were analyzed. Compared with those in the wild-type strain (KU216) cultivated at 90 °C, the transcript levels of 424 and 21 genes were up- and downregulated in the DBP1 strain, respectively. The expression levels of 12 frequently-used tRNAs were lower in DBP1 cells than KU216 cells, suggesting that BCPA affects translation efficiency in T. kodakarensis. LC-MS analyses of cells grown at 90 °C detected 50 proteins in KU216 cells only, 109 proteins in DBP1 cells only, and 499 proteins in both strains. Notably, the transcript levels of some genes did not correlate with those of the proteins. RNA-seq and RT-qPCR analyses of ten proteins that were detected in KU216 cells only, including three flagellin-related proteins (FlaB2-4) and cytosolic NiFe-hydrogenase subunit alpha (HyhL), revealed that the corresponding transcripts were expressed at higher levels in DBP1 cells than KU216 cells. Electron microscopy analyses showed that flagella formation was disrupted in DBP1 cells at 90 °C, and western blotting confirmed that HyhL expression was eliminated in the DBP1 strain. These results suggest that BCPA plays a regulatory role in gene expression in T. kodakarensis.

Oxygen-mediated growth enhancement of an obligate anaerobic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

A linear pathway for mevalonate production supports growth of Thermococcus kodakarensis.

The sole unifying feature of Archaea is the use of isoprenoid-based glycerol lipid ethers to compose cellular membranes. The branched hydrocarbon tails of archaeal lipids are synthesized via the polymerization of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but many questions still surround the pathway(s) that result in production of IPP and DMAPP in archaeal species. Isotopic-labeling strategies argue for multiple biological routes for production of mevalonate, but biochemical and bioinformatic studies support only a linear pathway for mevalonate production. Here, we use a combination of genetic and biochemical assays to detail the production of mevalonate in the model archaeon Thermococcus kodakarensis. We demonstrate that a single, linear pathway to mevalonate biosynthesis is essential and that alternative routes of mevalonate production, if present, are not biologically sufficient to support growth in the absence of the classical mevalonate pathway resulting in IPP production from acetyl-CoA. Archaeal species provide an ideal platform for production of high-value isoprenoids in large quantities, and the results obtained provide avenues to further increase the production of mevalonate to drive isoprenoid production in archaeal hosts.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway.IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+ from nicotinamide (Nm) in the hyperthermophilic archaeon Thermococcus kodakarensis The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+ itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.

An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis.

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.

The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis.

The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells.

Structure of histone-based chromatin in Archaea.

Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

Adaptive evolution of a hyperthermophilic archaeon pinpoints a formate transporter as a critical factor for the growth enhancement on formate.

Previously, we reported that the hyperthermophilic archaeon Thermococcus onnurineus NA1 could grow on formate and produce H2. Formate conversion to hydrogen was mediated by a formate-hydrogen lyase complex and was indeed a part of chemiosmotic coupling to ATP generation. In this study, we employed an adaptation approach to enhance the cell growth on formate and investigated molecular changes. As serial transfer continued on formate-containing medium at the serum vial, cell growth, H2 production and formate consumption increased remarkably. The 156 times transferred-strain, WTF-156T, was demonstrated to enhance H2 production using formate in a bioreactor. The whole-genome sequencing of the WTF-156T strain revealed eleven mutations. While no mutation was found among the genes encoding formate hydrogen lyase, a point mutation (G154A) was identified in a formate transporter (TON_1573). The TON_1573 (A52T) mutation, when introduced into the parent strain, conferred increase in formate consumption and H2 production. Another adaptive passage, carried out by culturing repeatedly in a bioreactor, resulted in a strain, which has a mutation in TON_1573 (C155A) causing amino acid change, A52E. These results implicate that substitution of A52 residue of a formate transporter might be a critical factor to ensure the increase in formate uptake and cell growth.

Metabolism Dealing with Thermal Degradation of NAD+ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

NAD+ is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD+ is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD+ concentrations and possibly remove and/or reuse undesirable degradation products of NAD+ Here we confirmed that at 85°C, thermal degradation of NAD+ results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD+ in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD+ breakdown.IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD+ is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal degradation to ADP-ribose and nicotinamide. Here we show that an ADP-ribose pyrophosphatase homolog from the hyperthermophilic archaeon Thermococcus kodakarensis converts the detrimental ADP-ribose to ribose 5-phosphate and AMP, compounds that can be directed to central carbon metabolism. This physiological role for ADP-ribose pyrophosphatases might be universal in hyperthermophiles, as their homologs are widely distributed among both hyperthermophilic bacteria and archaea.

Colonization of Black Smokers by Hyperthermophilic Microorganisms.

Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but they house hyperthermophiles in substantial amounts in later stages. No hard data exist on the mechanisms by which hyperthermophiles colonize newly erupted black smokers. Here I propose a scenario - based on various experimental data - for how hyperthermophiles colonize black smokers. Hyperthermophiles which are present in cold sea water in minute amounts are transferred by chance to the outside of black smokers and react within seconds to the high temperature by very fast movements. After reaching an optimal temperature region they scan the surface via a zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms.

Thermococcus piezophilus sp. nov., a novel hyperthermophilic and piezophilic archaeon with a broad pressure range for growth, isolated from a deepest hydrothermal vent at the Mid-Cayman Rise.

A novel strictly anaerobic, hyperthermophilic archaeon, designated strain CDGST, was isolated from a deep-sea hydrothermal vent in the Cayman Trough at 4964m water depth. The novel isolate is obligate anaerobe and grows chemoorganoheterotrophically with stimulation of growth by sulphur containing compounds. Its growth is optimal at 75°C, pH 6.0 and under a pressure of 50MPa. It possesses the broadest hydrostatic pressure range for growth that has ever been described for a microorganism. Its genomic DNA G+C content is 51.11mol%. The novel isolate belongs to the genus Thermococcus. Phylogenetic analyses indicated that it is most closely related to Thermococcus barossii DSM17882T based on its 16S rRNA gene sequence, and to 'Thermococcus onnurineus' NA1 based on its whole genome sequence. The average nucleotide identity scores with these strains are 77.66% for T. barossii and 84.84% for 'T. onnurineus', respectively. Based on the draft whole genome sequence and phenotypic characteristics, strain CDGST is suggested to be separated into a novel species within the genus Thermococcus, with proposed name Thermococcus piezophilus (type strain CDGST=ATCC TSD-33T=UBOCC 3296T).

A Structurally Novel Chitinase from the Chitin-Degrading Hyperthermophilic Archaeon Thermococcus chitonophagus.

UNLABELLED: A structurally novel chitinase, Tc-ChiD, was identified from the hyperthermophilic archaeon Thermococcus chitonophagus, which can grow on chitin as the sole organic carbon source. The gene encoding Tc-ChiD contains regions corresponding to a signal sequence, two chitin-binding domains, and a putative catalytic domain. This catalytic domain shows no similarity with previously characterized chitinases but resembles an uncharacterized protein found in the mesophilic anaerobic bacterium Clostridium botulinum Two recombinant Tc-ChiD proteins were produced in Escherichia coli, one without the signal sequence [Tc-ChiD(ΔS)] and the other corresponding only to the putative catalytic domain [Tc-ChiD(ΔBD)]. Enzyme assays using N-acetylglucosamine (GlcNAc) oligomers indicated that both proteins hydrolyze GlcNAc oligomers longer than (GlcNAc)4 Chitinase assays using colloidal chitin suggested that Tc-ChiD is an exo-type chitinase that releases (GlcNAc)2 or (GlcNAc)3 Analysis with GlcNAc oligomers modified with p-nitrophenol suggested that Tc-ChiD recognizes the reducing end of chitin chains. While Tc-ChiD(ΔBD) displayed a higher initial velocity than that of Tc-ChiD(ΔS), we found that the presence of the two chitin-binding domains significantly enhanced the thermostability of the catalytic domain. In T. chitonophagus, another chitinase ortholog that is similar to the Thermococcus kodakarensis chitinase ChiA is present and can degrade chitin from the nonreducing ends. Therefore, the presence of multiple chitinases in T. chitonophagus with different modes of cleavage may contribute to its unique ability to efficiently degrade chitin. IMPORTANCE: A structurally novel chitinase, Tc-ChiD, was identified from Thermococcus chitonophagus, a hyperthermophilic archaeon. The protein contains a signal peptide for secretion, two chitin-binding domains, and a catalytic domain that shows no similarity with previously characterized chitinases. Tc-ChiD thus represents a new family of chitinases. Tc-ChiD is an exo-type chitinase that recognizes the reducing end of chitin chains and releases (GlcNAc)2 or (GlcNAc)3 As a thermostable chitinase that recognizes the reducing end of chitin chains was not previously known, Tc-ChiD may be useful in a wide range of enzyme-based technologies to degrade and utilize chitin.

Entrapment of anaerobic thermophilic and hyperthermophilic marine micro-organisms in a gellan/xanthan matrix.

AIMS: The aims of this study were (i) to develop a protocol for the entrapment of anaerobic (hyper)thermophilic marine micro-organisms; (ii) to test the use of the chosen polymers in a range of physical and chemical conditions and (iii) to validate the method with batch cultures. METHODS AND RESULTS: The best conditions for immobilization were obtained at 80°C with gellan and xanthan gums. After 5-week incubation, beads showed a good resistance to all tested conditions except those simultaneously including high temperature (100°C), low NaCl (<0∙5 mol l(-1) ) and extreme pH (4/8). To confirm the method efficiency, batch cultures with immobilized Thermosipho sp. strain AT1272 and Thermococcus kodakarensis strain KOD1 showed an absence of detrimental effect on cell viability and a good growth within and outside the beads. CONCLUSION: This suggests that entrapment in a gellan-xanthan matrix could be employed for the culture of anaerobic (hyper)thermophilic marine micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: (Hyper)thermophilic marine micro-organisms possess a high biotechnological potential. Generally microbial cells are grown as free-cell cultures. The use of immobilized cells may offer several advantages such as protection against phage attack, high cell biomass and better production rate of desired metabolites.

Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle.

A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.

Intrinsic kinetic parameters of Thermococcus onnurineus NA1 strains and prediction of optimum carbon monoxide level for ideal bioreactor operation.

This study determines and compares the intrinsic kinetic parameters (Ks and Ki) of selected Thermococcus onnurineus NA1 strains (wild-type (WT), and mutants MC01, MC02, and WTC156T) using the substrate inhibition model. Ks and Ki values were used to find the optimum dissolved CO (CL) conditions inside the reactor. The results showed that in terms of the maximum specific CO consumption rates (qCO(max)) of WT, MC01, MC02, and WTC156T the optimum activities can be achieved by maintaining the CL levels at 0.56mM, 0.52mM, 0.58mM, and 0.75mM, respectively. The qCO(max) value of WTC156T at 0.75mM was found to be 1.5-fold higher than for the WT strain, confirming its superiority. Kinetic modeling was then used to predict the conditions required to maintain the optimum CL levels and high cell concentrations in the reactor, based on the kinetic parameters of the WTC156T strain.

Genome expression of Thermococcus barophilus and Thermococcus kodakarensis in response to different hydrostatic pressure conditions.

Transcriptomes were analyzed for two related hyperthermophilic archaeal species, the piezophilic Thermococcus barophilus strain MP and piezosensitive Thermococcus kodakarensis strain KOD1 subjected to high hydrostatic pressures. A total of 378 genes were differentially expressed in T. barophilus cells grown at 0.1, 40 and 70 MPa, whereas 141 genes were differentially regulated in T. kodakarensis cells grown at 0.1 and 25 MPa. In T. barophilus cells grown under stress conditions (0.1 and 70 MPa), 178 upregulated genes were distributed among three clusters of orthologous groups (COG): energy production and conversion (C), inorganic ion transport and metabolism (P) and carbohydrate transport and metabolism (G), whereas 156 downregulated genes were distributed among: amino acid transport and metabolism (E), replication, recombination and repair (L) and nucleotide transport and metabolism (F). The expression of 141 genes was regulated in T. kodakarensis cells grown under stress conditions (25 MPa); 71 downregulated genes belong to three COG: energy production and conversion (C), amino acid transport and metabolism (E) and transcription (K), whereas 70 upregulated genes are associated with replication, recombination and repair (L), coenzyme transport (H) and defense mechanisms (V).

High hydrostatic pressure increases amino acid requirements in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

We have established a defined growth medium for the piezophilic hyperthermophilic archaeon Thermococcus barophilus, which allows growth yields of ca. 10(8) cells/ml under both atmospheric and high hydrostatic pressure. Our results demonstrate a major impact of hydrostatic pressure on amino acid metabolism, with increases from 3 amino acids required at atmospheric pressure to 17 at 40 MPa. We observe in T. barophilus and other Thermococcales a similar discrepancy between the presence/absence of amino acid synthesis pathways and amino acid requirements, which supports the existence of alternate, but yet unknown, amino acid synthesis pathways, and may explain the low number of essential amino acids observed in T. barophilus and other Thermococcales. T. barophilus displays a strong metabolic preference for organic polymers such as polypeptides and chitin, which may constitute a more readily available resource of carbon and energy in situ in deep-sea hydrothermal vents. We hypothesize that the low energy yields of fermentation of organic polymers, together with energetic constraints imposed by high hydrostatic pressure, may render de novo synthesis of amino acids ecologically unfavorable. Induction of this metabolic switch to amino acid recycling can explain the requirement for non-essential amino acids by Thermococcales for efficient growth in defined medium.

A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain.

Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and "Candidatus Korarchaeum cryptofilum" OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR(↑)) compared with those in the wild-type strain. In addition, the ΔCorR/corQR(↑) strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter(-1) h(-1)) and the specific H2 production rate (249.6 mmol g(-1) h(-1)) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production.