Seung Seob Bae

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.

Description of Croceitalea gen. nov. in the family Flavobacteriaceae with two species, Croceitalea eckloniae sp. nov. and Croceitalea dokdonensis sp. nov., isolated from the rhizosphere of the marine alga Ecklonia kurome

Two novel bacterial strains, designated DOKDO 025(T) and DOKDO 023(T), were isolated on Dokdo Island, Korea, from the rhizosphere of the brown alga Ecklonia kurome. The strains were subjected to a polyphasic taxonomy study and were found to be Gram-negative, aerobic, rod-shaped, non-motile and orange-coloured. The isolates shared 96.3 % 16S rRNA gene sequence similarity. They showed 93.8-95.6 % 16S rRNA gene sequence similarity with respect to members of the genus Muricauda in the family Flavobacteriaceae, but formed a distinct phyletic line. Moreover, the cellular appendages reported for all Muricauda species were absent from strains DOKDO 025(T) and DOKDO 023(T). The predominant cellular fatty acids of strain DOKDO 025(T) were iso-C(15 : 0), iso-C(15 : 1) and one with an equivalent chain-length of 13.565 and those of strain DOKDO 023(T) were iso-C(15 : 0), iso-C(15 : 1) and iso-C(17 : 0) 3-OH. The DNA G+C content of strains DOKDO 025(T) and DOKDO 023(T) were 59.5 and 66.5 mol%, respectively, higher than any values found in recognized members of the family Flavobacteriaceae. The major respiratory quinone was MK-6. On the basis of evidence from the polyphasic study, strains DOKDO 025(T) and DOKDO 023(T) represent two novel species in a new genus, Croceitalea gen. nov., for which the names Croceitalea eckloniae sp. nov. (the type species) and Croceitalea dokdonensis sp. nov. are proposed. The type strain of Croceitalea eckloniae sp. nov. is DOKDO 025(T) (=KCCM 42309(T) =JCM 13827(T)) and that of Croceitalea dokdonensis sp. nov. is DOKDO 023(T) (=KCCM 42308(T) =JCM 13826(T)).

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.

Complete Genome Sequence of Hyperthermophilic Pyrococcus sp. Strain NA2, Isolated from a Deep-Sea Hydrothermal Vent Area

Pyrococcus sp. strain NA2, isolated from a deep-sea hydrothermal vent sample, is a novel marine hyperthermophilic archaeon that grows optimally at 93 °C. The complete genome sequence of the strain contains all the genes for the tricarboxylic acid cycle except for succinate dehydrogenase/fumarate reductase, but the genome does not encode proteins involved in polysaccharide utilization.

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.

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.

Monitoring nutrient impact on bacterial community composition during bioremediation of anoxic PAH-contaminated sediment

Marine harbor sediments are frequently polluted with significant amount of polycyclic aromatic hydrocarbons (PAHs) some of which are naturally toxic, recalcitrant, mutagenic, and carcinogenic. To stimulate biodegradation of PAHs in PAH-contaminated sediments collected from near Gwangyang Bay, Korea, lactate was chosen as a supplementary carbonaceous substrate. Sediment packed into 600 ml air-tight jar was either under no treatment condition or lactate amended condition (1%, w/v). Microbial community composition was monitored by bacteria-specific and archaea-specific PCR-terminal restriction fragment length polymorphism (T-RFLP), in addition to measuring the residual PAH concentration. Results showed that lactate amendment enhanced biodegradation rate of PAHs in the sediment by 4 to 8 times, and caused a significant shift in archaebacterial community in terms of structure and diversity with time. Phylogenetic analysis of 23 archaeal clones with distinctive RFLP patterns among 288 archaeal clones indicated that majority of the archaeal members were closest to unculturable environmental rDNA clones from hydrocarbon-contaminated and/or methanogenesis-bearing sediments. Lactate amendment led to the enrichment of some clones that were most closely related to PAH-degrading Methanosarcina species. These results suggest a possible contribution of methanogenic community to PAH degradation and give us more insights on how to effectively remediate PAH-contaminated sediments.

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.