Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H(2) (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 H(2) consumption by the methanogens, which lowers the H(2) 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 H(2). Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H(2)-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.

To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and 25 degrees C, respectively, and rEML1 displayed more than 50% activity at 5 degrees C. The activation energy for the hydrolysis of olive oil was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols acyl-group chains with long chain lengths of > or = 8 carbon atoms and displayed hydrolyzing activities toward various natural oil substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example which developed a new cold-active lipase from a deep-sea sediment metagenome.

bHydrogenogenic CO oxidation (CO ؉ H 2 O ¡ CO 2 ؉ H 2 ) has the potential for H 2 production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H 2 , 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 H 2 , 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 H 2 production than did the wild-type strain. The H 2 production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H 2 production rate of the engineered strain was severalfold higher than those of any other COdependent H 2 -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 H 2 production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe. Carbon monoxide (CO) is highly toxic to most living creatures, but it can be utilized by microorganisms as an energy and carbon source for the production of fuels and chemicals, such as acetate, butyrate, ethanol, butanol, and H 2 . Among those carboxydotrophic microbes, CO-dependent H 2 production has been observed in three distinct groups, i.e., mesophilic bacteria, thermophilic bacteria, and hyperthermophilic archaea (1-3). Generally, growth rates of the mesophilic hydrogenogenic bacteria on CO are low, and high levels of CO are inhibitory. Predominant within this group are nonsulfur purple bacteria, including Rubrivivax gelatinosus and Rhodospirillum rubrum, which require light for optimal cell growth. Although Rhodopseudomonas palustris P4 is capable of hydrogenogenic CO conversion in the dark, it does not grow under this condition (4). Nonphototrophic Citrobacter strain Y19 also converts CO to H 2 , but it only grows slowly under anaerobic conditions and an aerobic growth phase is required to generate sufficient biomass before the anaerobic CO conversion phase (5). The second group includes thermophilic, hydrogenogenic bacteria isolated from freshwater and marine environments with temperatures ranging from 40 to 85°C. Carboxydothermus hydrogenoformans, Carboxydocella thermautotrophica, Thermosinus carboxydivorans, and Caldanaerobacter subterraneus subsp. pacificus are capable of chemolithotrophic growth on high concentrations of CO. The third group includes two hydrogenogenic CO-converting archaea, Thermococcus sp. strain AM4 and Thermococcus onnurineus NA1 (6, 7). Both strains are hyperthermophiles isolated from deep-sea hydrothermal vents and can grow on 100% CO (8).Anaerobic carboxydotroph...

The TON_0002 gene, which is in close proximity to the DNA polymerase locus in Thermococcus onnurineus NA1, has been shown to encode an inorganic pyrophosphatase. Its genomic position and function suggest a role for pyrophosphate hydrolysis during DNA polymerization. This is the first report of an inorganic pyrophosphatase belonging to the haloacid dehalogenase superfamily, in which unique residues in motif I and II have been replaced with Trp and Gly, respectively. The optimum pyrophosphatase activity of the recombinant enzyme occurred at pH 6, and it displayed an absolute dependence on divalent metal ions, among which Ni(2+) was the most efficient. The site-specific mutation of the Gly residue in motif II to Ala or Ser residue exhibited only a slight change in the enzymatic activity and the K(m) value.

Ferredoxin-dependent metabolic engineering of electron transfer circuits has been developed to enhance redox efficiency in the field of synthetic biology, e.g., for hydrogen production and for reduction of flavoproteins or NAD(P)+. Here, we present the bioconversion of carbon monoxide (CO) gas to formate via a synthetic CO:formate oxidoreductase (CFOR), designed as an enzyme complex for direct electron transfer between non-interacting CO dehydrogenase and formate dehydrogenase using an electron-transferring Fe-S fusion protein. The CFOR-introduced Thermococcus onnurineus mutant strains showed CO-dependent formate production in vivo and in vitro. The maximum formate production rate from purified CFOR complex and specific formate productivity from the bioreactor were 2.2 ± 0.2 μmol/mg/min and 73.1 ± 29.0 mmol/g-cells/h, respectively. The CO-dependent CO2 reduction/formate production activity of synthetic CFOR was confirmed, indicating that direct electron transfer between two unrelated dehydrogenases was feasible via mediation of the FeS-FeS fusion protein.