Jae Kyu Lim

Formate-driven growth coupled with H2 production

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H2 (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol−1) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H2 consumption by the methanogens, which lowers the H2 partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H2. Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H2-producing growth. The actual ΔG values for the formate metabolism are calculated to range between −8 and −20 kJ mol−1 under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.

The complete genome sequence of Thermococcus onnurineus NA1 reveals a mixed heterotrophic and carboxydotrophic metabolism.

Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO(2), thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.

CO-Dependent H2 Production by Genetically Engineered Thermococcus onnurineus NA1

ABSTRACT Hydrogenogenic CO oxidation (CO + H2O → CO2 + H2) has the potential for H2 production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H2, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H2, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na+/H+ antiporter and a 1.8-fold-higher specific activity for CO-dependent H2 production than did the wild-type strain. The H2 production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H2 production rate of the engineered strain was severalfold higher than those of any other CO-dependent H2-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H2 production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.

Identification of a Novel Class of Membrane-Bound [NiFe]-Hydrogenases in Thermococcus onnurineus NA1 by In Silico Analysis

ABSTRACT In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Thermococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFe]-hydrogenases-based on sequence motifs.

One-carbon substrate-based biohydrogen production: Microbes, mechanism, and productivity

Among four basic mechanisms for biological hydrogen (H2) production, dark fermentation has been considered to show the highest hydrogen evolution rate (HER). H2 production from one-carbon (C1) compounds such as formate and carbon monoxide (CO) is promising because formate is an efficient H2 carrier, and the utilization of CO-containing syngas or industrial waste gas may render the industrial biohydrogen production process cost-effective. A variety of microbes with the formate hydrogen lyase (FHL) system have been identified from phylogenetically diverse groups of archaea and bacteria, and numerous efforts have been undertaken to improve the HER for formate through strain optimization and bioprocess development. CO-dependent H2 production has been investigated to enhance the H2 productivity of various carboxydotrophs via an increase in CO gas-liquid mass transfer rates and the construction of genetically modified strains. Hydrogenogenic CO-conversion has been applied to syngas and by-product gas of the steel-mill process, and this low-cost feedstock has shown to be promising in the production of biomass and H2. Here, we focus on recent advances in the isolation of novel phylogenetic groups utilizing formate or CO, the remarkable genetic engineering that enhances H2 productivity, and the practical implementation of H2 production from C1 substrates.

Thermodynamics of formate-oxidizing metabolism and implications for H2 production.

ABSTRACT Formate-dependent proton reduction to H2 (HCOO− + H2O → HCO3 − + H2) has been reported for hyperthermophilic Thermococcus strains. In this study, a hyperthermophilic archaeon, Thermococcus onnurineus strain NA1, yielded H2 accumulation to a partial pressure of 1 × 105 to 7 × 105 Pa until the values of Gibbs free energy change (ΔG) reached near thermodynamic equilibrium (−1 to −3 kJ mol−1). The bioenergetic requirement for the metabolism to conserve energy was demonstrated by ΔG values as small as −5 kJ mol−1, which are less than the biological minimum energy quantum, −20 kJ mol−1, as calculated by Schink (B. Schink, Microbiol. Mol. Biol. Rev. 61:262-280, 1997). Considering formate as a possible H2 storage material, the H2 production potential of the strain was assessed. The volumetric H2 production rate increased linearly with increasing cell density, leading to 2,820 mmol liter−1 h−1 at an optical density at 600 nm (OD600) of 18.6, and resulted in the high specific H2 production rates of 404 ± 6 mmol g−1 h−1. The H2 productivity indicates the great potential of T. onnurineus strain NA1 for practical application in comparison with H2-producing microbes. Our result demonstrates that T. onnurineus strain NA1 has a highly efficient metabolic system to thrive on formate in hydrothermal systems.

Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1

ABSTRACT Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(Nε-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km, 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 min at 80°C. This study represents the first characterization of a hyperthermophilic archaeon APP.

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

ABSTRACT 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.

Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic Archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS–PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70–80 °C (k cat⁄K m=8.3 mM−1 s−1). In comparison with that of P. furiosus CP (k cat⁄K m=667 mM−1 s−1), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.

Characterization of the frhAGB-encoding hydrogenase from a non-methanogenic hyperthermophilic archaeon

The F420-reducing hydrogenase has been known as a key enzyme in methanogenesis. Its homologs have been identified in non-methanogenic hyperthermophilic archaea, including Thermococcus onnurineus NA1, but neither physiological function nor biochemical properties have been reported to date. The enzyme of T. onnurineus NA1 was distinguished from those of other methanogens and the members of the family Desulfurobacteriaceae with respect to the phylogenetic distribution of the α and β subunits, organization of frhAGB genes and conservation of F420-coordinating residues. RT-qPCR and Western blot analyses revealed frhA gene is not silent but is expressed in T. onnurineus NA1 grown in the presence of sulfur, carbon monoxide, or formate. The trimeric enzyme complex was purified to homogeneity via affinity chromatography from T. onnurineus NA1 and exhibited catalytic activity toward the electron acceptors such as viologens and flavins but not the deazaflavin coenzyme F420. This is the first biochemical study on the function of the frhAGB-encoding enzyme from a non-methanogenic archaea.