A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH

The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change.

A shuttle-vector system allows heterologous gene expression in the thermophilic methanogen Methanothermobacter thermautotrophicus ΔH

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis), because of their short doubling times and robust growth with high growth yields. Yet, a genetic system for these model microbes was missing despite intense work for four decades. Here, we report the establishment of tools for genetic modification of M. thermautotrophicus. We developed the modular Methanothermobacter vector system, which provided shuttle-vector plasmids (pMVS) with exchangeable selectable markers and replicons for both Escherichia coli and M. thermautotrophicus. For M. thermautotrophicus, a thermostable neomycin-resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The pMVS-plasmid DNA was transferred from E. coli into M. thermautotrophicus via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assays, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus on formate as the sole growth substrate, while this was not possible for the empty vector control. These genetic tools provide the basis to investigate hypotheses from four decades of research on the physiology and biochemistry of Methanothermobacter spp. on a genetic level.Significance StatementThe world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production, but also for production of high-value platform chemicals in power-to-x processes.

Draft Genome Sequence of Methanothermobacter thermautotrophicus WHS, a Thermophilic Hydrogenotrophic Methanogen from Washburn Hot Springs in Yellowstone National Park, USA

ABSTRACT A thermophilic methanogen was enriched in coculture from Washburn Hot Springs (Yellowstone National Park, USA), grown on carbon dioxide and hydrogen, and subsequently sequenced. The reconstructed 1.65-Mb genome sequence for Methanothermobacter thermautotrophicus WHS contributes to our understanding of hydrogenotrophic, CO2-reducing methanogenesis in geothermal ecosystems.

Zhaonella formicivorans gen. nov., sp. nov., an anaerobic formate-utilizing bacterium isolated from Shengli oilfield, and proposal of four novel families and Moorellales ord. nov. in the phylum Firmicutes

A novel obligately anaerobic, thermophilic and formate-utilizing bacterium K32T was isolated from Shengli oilfield of China. Cells were straight rods (0.4–0.8 µm × 2.5–8.0 µm), Gram-stain-positive, non-spore-forming and slightly motile. Optimum growth occurred with pH of 7 and 0.5 g l–1 NaCl under temperature of 55–60 °C. Nitrate could be reduced into nitrite, syntrophic formate oxidation to methane and carbon dioxide occurred when co-culturing strain K32T and Methanothermobacter thermautotrophicus ΔH. The main cellular fatty acids were iso-C15 : 0 (24.0 %), anteiso-C15 : 0 (21.7 %), C16 : 0 (12.7 %) and C14 : 0 (10.8 %), and the main polar lipid was phosphatidylglycerol. The G+C content of the genomic DNA was 46.3 mol%. The 16S rRNA gene sequence of K32T shared ≤90.4 % of sequence similarity to closest type strains of Desulfitibacter alkalitolerans , Calderihabitans maritimus and members of the genus Moorella . Based on the phenotypic, biochemical and genotypic characterization, Zhaonella formicivorans gen. nov., sp. nov. is proposed with K32T (=CCAM 584T =DSM 107278T=CGMCC1.5297T) as the type strain, which is the first representative of Zhaonellaceae fam. nov. In addition, the order Thermoanaerobacterales and family Peptococcaceae were reclassified, and three novel families in the novel order of Moorellales ord. nov. were also proposed.

Functional Group Distribution of the Carrier Surface Influences Adhesion of Methanothermobacter thermautotrophicus

Various support carriers are used for high-density retention of methanogenic archaea in anaerobic wastewater treatment systems. Although the physicochemical properties of carrier materials and microorganisms influence the adhesion of methanogenic archaea, details about the underlying mechanism remain poorly characterized. We applied seven types of chemical surface modifications to carbon felts to clarify the adhesion properties of Methanothermobacter thermautotrophicus, a representative thermophilic hydrogenotrophic methanogen. The relationship between carrier surface properties and methanogen adhesion was evaluated. M. thermautotrophicus adhesion was significantly increased up to 2.6 times in comparison with control on carbon felts treated with NaOH, HCl, H2SO4, or Na2HPO4. Treated carbon felts showed a lower water contact angle, but no correlation between the carrier surface contact angle and methanogen adhesion was observed. On the other hand, at the surface of the carrier that showed improved adhesion of methanogens, the ratio of -COOH : -OH was 1 : 0.65. Such a ratio was not observed with treated carriers for which methanogen adhesion was not improved. Therefore, in the adhesion of M. thermautotrophicus, the functional group abundance was important as well as physical surface properties such as the hydrophobicity. Hydrogenotrophic methanogens are involved in active methanation during the startup of anaerobic digestion. Additionally, these methanogenic archaea function as methanogenic cathode catalysts. Therefore, anaerobic digestion performance will greatly improve by controlling the adhesion of hydrogenotrophic methanogens such as M. thermautotrophicus.

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.