Thermococcus Onnurineus NA1 Research Articles

An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Development of natural seawater-based continuous biohydrogen production process using the hyperthermophilic archaeon Thermococcus onnurineus NA1

Previously, we reported that the hyperthermophilic archaeon, Thermococcus onnurineus NA1 could produce hydrogen from various resources including carbon monoxide (CO) with high productivity. In this study, a high-performance and cost-effective fermentation process was developed as a prerequisite for the industrial implementation of H2 production from CO using the strain. Firstly, an economical medium was formulated on the basis of natural seawater. Cysteine as a reducing agent was found to be an important factor to consider. Secondly, three variables, initial concentration of yeast extract, CO supply rate and agitation rate, were statistically optimized in batch culture by applying a response surface methodology. Notably, biomass productivity of 0.39 g/L/h could be observed and cell density reached 14 optical units at 600 nm, which is exceptionally high among members of the order Thermococcales. Finally, continuous cultures were tested at various dilution rates with new medium formulation and optimized culture conditions. As a result, the steady state of H2 production and cell growth were stably maintained, yielding H2 production rate of 471.5 mmol/L/h at a dilution rate of 0.1 h−1, which is one of the highest values at ambient CO pressure to date. Our study shows that T. onnurineus NA1 has outstanding potential as an economical and high performance industrial strain suitable for long-term processes for CO-dependent H2 production.

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

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

Chitosan/oleamide nanofluid as a significant medium for enhancing gas utilization efficiency in C1-gas microbial biotransformation

Microbial biotransformation of C1 gas feedstocks such as CH4 and CO is a notable technique for sustainable, carbon–neutral chemical and fuel production. However, low C1-gas utilization efficiency in biological processes has hampered the efficient production of value-added materials, despite efficient, nonnative strains having been recently developed. Here, we constructed a nanofluid material mainly composed of chitosan and oleamide (CS/OA), which was stably suspended with a particle size of 120.7 ± 39.0 nm in aqueous culture media below pH 7.5 and attached to the cell surface, leading to a change in hydrodynamic properties mainly due to the decrease of surface tension and a decrease in mass-transfer resistance on the cell surface. The nanofluid was applied to three C1-gas-utilizing strains, Methylomonas sp. DH-1 (a type I methanotroph), Methylosinus trichosporium OB3b (a type II methanotroph), and Thermococcus onnurineus NA1 156 T (a hyperthermophilic CO-utilizing archaeon). Changes in culture media and cell membranes from the applied CS/OA nanoparticles increased specific cell growth rates (μmax). Remarkably, the strains adapted from the seed culture using the CS/OA nanofluid media also had increased μmax in a subsequent subculture and the main culture using conventional culture media, resulting in higher C1 gas consumption, cell growth, and metabolite production, such as succinate production. These results showed that the CS/OA nanofluid could be an effective medium to increase gas utilization efficiency and cell growth, and it is expected that the nanofluid can be widely used for increasing the production of value-added metabolites in C1-gas microbial biotransformation.

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

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

Long-term Operation of Continuous Culture of the Hyperthermophilic archaeon, Thermococcus onnurineus for Carbon Monoxide-dependent Hydrogen Production

In this study, we performed a kinetic analysis of CO-dependent H2 production by metabolically engineered Thermococcus onnurineus NA1, MC01 in terms of the cell activity as well as mass transfer rate. We conducted continuous cultures with varying dilution rate, CO flow rate, or agitation speed. Despite oscillations in cell density, the cultures reached steady states at all operating conditions. As the dilution rate increased from 0.1 to 0.3 h−1, specific activity of H2 production (SAHP) and volumetric cell production rate were linearly increased. Also, the SAHP remained almost constant at the fixed dilution rate of 0.3 h−1 even though the CO transfer rate was changed by adjusting the CO flow rate or the agitation speed. This relationship could be expressed as a typical Luedeking-Piret model, implying that high cell density culture with a sufficient growth rate is essential to obtain higher H2 productivity. On the other hand, more elevated CO transfer at the same dilution rate improved H2 production rate (HPR) by the increase of the cell density, not in the rise of SAHP. Through the continuous culture, 108 mmol/g-cell/h and 121 mmol/L/h of SAHP and HPR, respectively, could be achieved at a dilution rate of 0.3 h−1 with CO supply rate of 0.07 vvm and agitation speed of 700 rpm. Considering high H2 production activity and long-term stability of the strain over 1,000 h, MC01 is confirmed to have an outstanding potential for CO-dependent H2 production.

Biological process for coproduction of hydrogen and thermophilic enzymes during CO fermentation

To develop a thermophilic cell factory system that uses CO gas, we attempted to engineer a hyperthermophilic carboxydotrophic hydrogenic archaeon Thermococcus onnurineus NA1 to be capable of producing thermophilic enzymes along with hydrogen (H2). The mutant strains 156T-AM and 156T-POL were constructed to have another copy of a gene encoding α-amylase or DNA polymerase, respectively, and exhibited growth rates and H2 production rates distinct from those of the parental strain, 156T, in gas fermentation using 100% CO or coal-gasified syngas. Purified α-amylase displayed starch-hydrolyzing activity, and whole-cell extracts of 156T-AM showed saccharifying activity for potato peel waste. PCR amplification was used to demonstrate that purified DNA polymerase was free from bacterial DNA contamination, in contrast to commercial bacteria-made enzymes. This study demonstrated that this archaeal strain could coproduce enzymes and H2 using CO-containing gas, providing a basis for cell factories to upcycle industrial waste gas.

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 577mmol/L/h of hydrogen productivity. This is the highest hydrogen productivity reported to date from microbial water-gas shift reaction.

Formulation of a Low-cost Medium for Improved Cost-effectiveness of Hydrogen Production by Thermococcus onnurineus NA1

Hydrogen has been receiving considerable attention as a clean energy carrier. Thermococcus onnurineus NA1, a hyperthermophilic archaeon, is capable of producing hydrogen using carbon monoxide (CO) in a growth-associated manner, providing a promising platform to upcycle industrial waste gases. In order to improve cost-effectiveness of the biological hydrogen production from carbon monoxide (CO), we developed formulation of a low-cost medium for T. onnurineus 156T by substituting fishmeal and sea salt for yeast extract (YE) and reagentgrade salts, respectively. In serum bottle cultures, hydrogen production by the 156T strain was less than 30% in fishmeal-containing media when compared to that of the YE-containing MM1 medium. However, hydrogen production was increased substantially by adaptive evolution in the fishmeal-sea salt (FMSS) medium. In bioreactor experiments, the resulting strain 156T-FA showed shorter lag phase and 70% higher maximum hydrogen production rate in FMSS medium than the parent strain 156T. Our results showed that the H2 production cost was reduced by ca. 95%, when YE and chemical salts were replaced by fishmeal and sea salt, respectively.

Fuzzy-inference-based failure mode and effects analysis of the hydrogen production process using Thermococcus onnurineus NA1

Abstract Hydrogen energy can be effectively converted from various energy sources, such as renewable energy or hydrocarbon fuel, and therefore, it is a promising energy source. Various hydrogen production processes have been proposed worldwide because conventional energy conversion systems, after minor design modifications, can be used for such conversions. However, hydrogen gas can be more dangerous than other flammable gases if released into the atmosphere, where it can rapidly reach its explosion limit because of its high diffusion velocity. Therefore, a failure mode and effects analysis (FMEA) for hydrogen production processes is required. In this study, FMEA is performed for hydrogen produced from coal syngas using Thermococcus onnurineus NA1, which was discovered in the Indonesian deep sea. A fuzzy inference methodology is introduced for a quantitative analysis of the linguistic ambiguity of risk priority number estimated from a conventional FMEA. To estimate the objective severity ranking, we introduced the potential asset loss combined with the proportionate cost of each piece of equipment under total capital investment and a weight value influenced by environmental and mankind. Moreover, we proposed a fuzzified risk matrix to effectively represent the fuzzy risk priority number (f-RPN) under the risk matrix; variation with and without fuzzification of risk priority number is then expressed to ascertain why this variation has occurred through a vector diagram. Based on the f-RPN vector diagram, we have performed a design revision of the hydrogen production process using Thermococcus onnurineus NA1 to adjust the risk priority number downward from a broadly unacceptable region.

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.

In vitro assembly of thermostable Csm complex in CRISPR-Cas type III/A system.

The CRISPR-Cas system is the prokaryotic immune response that destroys invading foreign nucleic acids. Based on the architecture and distinct mechanism of targeting, the CRISPR-Cas system is classified into six types (I-VI). The Csm complex belongs to the type III system and consists of five subunits (Cas10 and Csm2-5) and a crRNA. The Csm complex targets RNA and RNA-dependent single-strand DNA. Here, we present a protocol for in vitro reconstitution of a Csm complex from a hyperthermophilic archaeon Thermococcus onnurineus NA1 (ToCsm complex). The method consists of coexpression and copurification of the subunits, in vitro synthesis of the crRNA and assembly of the ToCsm complex. Purification with heat treatment and affinity and size exclusion chromatography resulted in homogeneous Cas10/Csm4 and Csm2/Csm5 binary complexes, while in vitro transcription with the T7 promoter enabled synthesis of the crRNA. Addition of each component in the presence of the crRNA with a molar ratio of Cas10/Csm4:Csm3:Csm2/Csm5:crRNA=1:3:2:1 yielded an assembled functional Csm complex. This protocol for reconstitution of the Csm complex is presumably applicable to other thermostable effector complexes, which would allow biochemical, structural, or functional studies of the CRISPR-Cas type III/A system in vitro.

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.

Transcriptomic profiling and its implications for the H2 production of a non-methanogen deficient in the frhAGB-encoding hydrogenase.

The F420-reducing hydrogenase of methanogens functions in methanogenesis by providing reduced coenzyme F420 (F420H2) as an electron donor. In non-methanogens, however, their physiological function has not been identified yet. In this study, we constructed an ΔfrhA mutant, whose frhA gene encoding the hydrogenase α subunit was deleted, in the non-methanogenic Thermococcus onnurineus NA1 as a model organism. There was no significant difference in the formate-dependent growth between the mutant and the wild-type strains. Interestingly, the mutation in the frhA gene affected the expression of genes involved in various cellular functions such as H2 oxidation, chemotactic signal transduction, and carbon monoxide (CO) metabolism. Among these genes, the CO oxidation gene cluster, enabling CO-dependent growth and H2 production, showed a 2.8- to 7.0-fold upregulation by microarray-based whole transcriptome expression profiling. The levels of proteins produced by this gene cluster were also significantly increased not only under the formate condition but also under the CO condition. In a controlled bioreactor, where 100% CO was continuously fed, the ΔfrhA mutant exhibited significant increases in cell growth (2.8-fold) and H2 production (3.4-fold). These findings strongly imply that this hydrogenase is functional in non-methanogens and is related to various cellular metabolic processes through an unidentified mechanism. An understanding of the mechanism by which the frhA gene deletion affected the expression of other genes will provide insights that can be applied to the development of strategies for the enhancement of H2 production using CO as a substrate.

Redox regulation of SurR by protein disulfide oxidoreductase in Thermococcus onnurineus NA1.

Protein disulfide oxidoreductases are redox enzymes that catalyze thiol-disulfide exchange reactions. These enzymes include thioredoxins, glutaredoxins, protein disulfide isomerases, disulfide bond formation A (DsbA) proteins, and Pyrococcus furiosus protein disulfide oxidoreductase (PfPDO) homologues. In the genome of a hyperthermophilic archaeon, Thermococcus onnurineus NA1, the genes encoding one PfPDO homologue (TON_0319, Pdo) and three more thioredoxin- or glutaredoxin-like proteins (TON_0470, TON_0472, TON_0834) were identified. All except TON_0470 were recombinantly expressed and purified. Three purified proteins were reduced by a thioredoxin reductase (TrxR), indicating that each protein can form redox complex with TrxR. SurR, a transcription factor involved in the sulfur response, was tested for a protein target of a TrxR-redoxin system and only Pdo was identified to be capable of catalyzing the reduction of SurR. Electromobility shift assay demonstrated that SurR reduced by the TrxR-Pdo system could bind to the DNA probe with the SurR-binding motif, GTTttgAAC. In this study, we present the TrxR-Pdo couple as a redox-regulator for SurR in T. onnurineus NA1.

Genome-wide primary transcriptome analysis of H2-producing archaeon Thermococcus onnurineus NA1

In spite of their pivotal roles in transcriptional and post-transcriptional processes, the regulatory elements of archaeal genomes are not yet fully understood. Here, we determine the primary transcriptome of the H2-producing archaeon Thermococcus onnurineus NA1. We identified 1,082 purine-rich transcription initiation sites along with well-conserved TATA box, A-rich B recognition element (BRE), and promoter proximal element (PPE) motif in promoter regions, a high pyrimidine nucleotide content (T/C) at the −1 position, and Shine-Dalgarno (SD) motifs (GGDGRD) in 5′ untranslated regions (5′ UTRs). Along with differential transcript levels, 117 leaderless genes and 86 non-coding RNAs (ncRNAs) were identified, representing diverse cellular functions and potential regulatory functions under the different growth conditions. Interestingly, we observed low GC content in ncRNAs for RNA-based regulation via unstructured forms or interaction with other cellular components. Further comparative analysis of T. onnurineus upstream regulatory sequences with those of closely related archaeal genomes demonstrated that transcription of orthologous genes are initiated by highly conserved promoter sequences, however their upstream sequences for transcriptional and translational regulation are largely diverse. These results provide the genetic information of T. onnurineus for its future application in metabolic engineering.

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5′-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1.

해양바이오수소개발 사업의 상업생산을 위한 예비경제성평가

This project sought to conduct an economic feasibility study regarding the commercial production of bio-hydrogen by the marine hyperthermophilic archaeon, Thermococcus onnurineus NA1 using carbon monoxide-containing industrial off-gas. We carried out the economic evaluation of the biohydrogen production process using the raw material of steel mill by-product gas. The process parameter was as follows: H2 production rate was 5.6 L/L/h; the conversion of carbon monoxide was 60.7%. This project established an evaluation criterion for about 10,000 tonne/year. Inflation factors were considered as 3%. The operating costs were recalculated based on prices in 2014. The total investment required for development was covered 30% by capital and 70% by a loan. The operation cost for the 0.5-year test and integration, and the cost for the first three months in the 50% production period were considered as the working capital in the cost estimation. The costs required for the rental of office space, facilities, and other related costs from the construction through to full-scale production periods were considered as continuing expenses. Materials, energy, waste disposal and other charges were considered as the operating cost of the development system. Depreciation, tax, maintenance and repair, insurance, labor, interest rate charges, general and administrative costs, lubrication and miscellaneous expenses were also calculated. The hydrogen price was set at US$ 4.15/kg for the economic evaluation. As a result, the process was considered to be economical with the payback period of 6.3 years, NPV of 18 billion Won and IRR of 26.7%.

Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased. The underlying mechanism for those physiological changes could be explained by using multi-omics approaches including genomic, transcriptomic and epigenomic analyses. A putative transcriptional regulator was newly identified to regulate the expression levels of genes related to CO oxidation. Transcriptome analysis revealed significant changes in the transcript levels of genes belonging to the categories of transcription, translation and energy metabolism. Our study presents the first genome-scale methylation pattern of hyperthermophilic archaea. Adaptive evolution led to highly enhanced H2 productivity at high CO flow rates using synthesis gas produced from coal gasification.

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.