Methane Monooxygenase Research Articles

Characteristics and activity of SpmoB domain of particulate methane monooxygenase expressed in Escherichia coli BL21 (DE3)

Particulate methane monooxygenase is one of the methane monooxygenases of methanotrophic bacteria. The enzyme can convert methane to methanol in mild conditions. Cupredoxin domain recombinant protein (SpmoB) of particulate methane monooxygenase can oxidize methane to methanol. This protein was expressed in Escherichia coli BL21 (DE3) with Lac operon-based induction. SpmoB protein was isolated and refolded from the E. coli recombinant inclusion body. The pH and temperature dependence of SpmoB activity was also investigated to increase its activity. the SpmoB protein expressed by E. coli BL21 (DE3) is about 39 kDa. The SpmoB inclusion body was solubilized in 8 M urea followed by stepwise dialysis to get the active form SpmoB protein. The specific activity of the refolded protein was 0.46 methanol mg protein-1 min-1, which was higher than that of SpmoB from the previous study. The SpmoB was an acidic protein with the highest methanol production at pH six and a temperature of 30°C, which are higher than full-length pMMO. The SpmoB activity was stable at pH 6 to 8, and the Vmax and Km were 0.380 μM methanol s-1 and 44.27 μM, respectively.

Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals.

MmoD regulates soluble methane monooxygenase and methanobactin production in Methylosinus trichosporium OB3b.

Aerobic methanotrophs play a critical role in the global carbon cycle, particularly in controlling net emissions of methane to the atmosphere. As methane is a much more potent greenhouse gas than carbon dioxide, there is increasing interest in utilizing these microbes to mitigate future climate change by increasing their ability to consume methane. Any such efforts, however, require a detailed understanding of how to manipulate methanotrophic activity. Herein, we show that methanotrophic activity is strongly controlled by MmoD, i.e., MmoD regulates methanotrophy through the post-transcriptional regulation of the soluble methane monooxygenase and controls the ability of methanotrophs to collect copper. Such data are likely to prove quite useful in future strategies to enhance the use of methanotrophs to not only reduce methane emissions but also remove methane from the atmosphere.

Unraveling the Valence State and Reactivity of Copper Centers in Membrane-Bound Particulate Methane Monooxygenase.

Particulate methane monooxygenase (pMMO) plays a critical role in catalyzing the conversion of methane to methanol, constituting the initial step in the C1 metabolic pathway within methanotrophic bacteria. However, the membrane-bound pMMO's structure and catalytic mechanism, notably the copper's valence state and genuine active site for methane oxidation, have remained elusive. Based on the recently characterized structure of membrane-bound pMMO, extensive computational studies were conducted to address these long-standing issues. A comprehensive analysis comparing the quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulated structures with cryo-EM data indicates that both the CuC and CuD sites tend to stay in the Cu(I) valence state within the membrane environment. Additionally, the concurrent presence of Cu(I) at both CuC and CuD sites leads to the significant reduction of the ligand-binding cavity situated between them, making it less likely to accommodate a reductant molecule such as durohydroquinone (DQH2). Subsequent QM/MM calculations reveal that the CuD(I) site is more reactive than the CuC(I) site in oxygen activation, en route to H2O2 formation and the generation of Cu(II)-O•- species. Finally, our simulations demonstrate that the natural reductant ubiquinol (CoQH2) assumes a productive binding conformation at the CuD(I) site but not at the CuC(I) site. This provides evidence that the true active site of membrane-bound pMMOs may be CuD rather than CuC. These findings clarify pMMO's catalytic mechanism and emphasize the membrane environment's pivotal role in modulating the coordination structure and the activity of copper centers within pMMO.

Synergistic action of Acinetobacter baumannii and Talaromyces sp.: Function of enzymes in crude oil degradation

The purpose of this study was to explore the role of intracellular and extracellular enzymes of Acinetobacter baumannii and Talaromyces sp. in the synergistic degradation of crude oil. The extracellular enzymes of Talaromyces sp. contributed more to the degradation of n-alkanes. The degradation rate of n-hexadecane by extracellular enzymes of Talaromyces sp. was 71.83% after 5 days. The concentration of n-hexadecane decreased from 7700 mg/L to 2169 mg/L. This is related to the high extracellular expression of methane monooxygenase (particulate) and aldehyde dehydrogenase (NAD+) in Talaromyces sp. The extracellular enzymes of Acinetobacter baumannii had a better effect on the degradation of aromatic hydrocarbons. The degradation rate of xylene by extracellular enzymes of Acinetobacter baumannii was 35.65% after 5 days. The concentration of xylene decreased from 8600 mg/L to 5514 mg/L. Catechol 1,2-dioxygenase plays a key role in this process. Meanwhile, the degradation enzyme systems of Acinetobacter baumannii (long-chain alkane monooxygenase, alkane 1-monooxygenase, catechol 1,2-dioxygenase, salicylate hydroxylase, and benzoate 1,2-dioxygenase) and Talaromyces sp. (methane monooxygenase (particulate), benzaldehyde dehydrogenase (NAD+), catechol 2,3-dioxygenase, and gentisate 1,2-dioxygenase) complement each other in the degradation process of n-alkanes and aromatic hydrocarbons, thus improving the degradation ability. These results indicated that bacteria and fungi rely on the difference of degrading enzymes to achieve synergistic degradation.

DFT mechanistic study of biomimetic diiron complex catalyzed dehydrogenation: Unexpected Fe(III)Fe(III)-1,1-μ-hydroperoxy active species for hydride abstraction

The diiron active site is pivotal in catalyzing transformations in both biological and chemical systems. Recently, a range of biomimetic diiron catalysts have been synthesized, drawing inspiration from the active architecture of soluble methane monooxygenase (sMMO). These catalysts have been successfully deployed for the dehydrogenation of indolines, marking a significant advancement in the field. Using density functional theory (DFT) calculations, we have identified a novel mechanistic pathway that governs the dehydrogenation of indolines catalyzed by a biomimetic diiron complex. Specifically, this reaction is facilitated by the transfer of a hybrid atom from the C1 position of the substrate to the distal oxygen atom of the Fe(III)Fe(III)-1,1-μ-hydroperoxy active species. This transfer serves as the rate-limiting step for the heterolytic cleavage of the OO bond, ultimately generating the substrate cation. The mechanism we propose aligns well with mechanistic investigations incorporating both kinetic isotope effect (KIE) measurements and evaluations of stereochemical selectivity. This research contributes to the broader scientific understanding of catalysis involving biomimetic diiron complexes and offers valuable insights into the catalytic behaviors of non-heme diiron metalloenzymes.

Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

Product analog binding identifies the copper active site of particulate methane monooxygenase.

Nature's primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyzes the oxidation of methane to methanol. pMMO activity requires copper, and decades of structural and spectroscopic studies have sought to identify the active site among three candidates: the CuB, CuC, and CuD sites. Challenges associated with the isolation of active pMMO have hindered progress toward locating its catalytic center. However, reconstituting pMMO into native lipid nanodiscs stabilizes its structure and recovers its activity. Here, these active samples were incubated with 2,2,2,-trifluoroethanol (TFE), a product analog that serves as a readily visualized active-site probe. Interactions of TFE with the CuD site were observed by both pulsed ENDOR spectroscopy and cryoEM, implicating CuD and the surrounding hydrophobic pocket as the likely site of methane oxidation. Use of these orthogonal techniques on parallel samples is a powerful approach that can circumvent difficulties in interpreting metalloenzyme cryoEM maps.

<i>Methylomonas Montana </i>sp. nov., the First Unpigmented Methanotroph of the Genus <i>Methylomonas</i>, Isolated from Mountain River Sediments

Abstract—Aerobic methanotrophic bacteria of the genus Methylomonas inhabit a wide spectrum of habitats including freshwater bodies, river sediments, wetlands, rice paddies, landfill cover soils, and hydromorphic soils. Majority of the currently described species of this genus are represented by neutrophilic, motile, growing on methane rod-shaped bacteria, whose pigmentation varies from yellow to pink and red. This study reports characterization of a novel, unpigmented isolate of these bacteria, strain MW1T, which was obtained from sediments of the mountain river Khosta, Krasnodar region, Russia. Strain MW1T grew on methane and methanol within a temperature range of 8–37°C (optimum at 25–30°C) and at рН 5.5–7.5 (optimum at 6.3–7.0). The 16S rRNA gene sequence of strain MW1T displayed 95.48–98.47% similarity to those in earlier described Methylomonas. The closest taxonomically characterized phylogenetic relative of strain MW1T was M. fluvii EbBT, isolated from the river Elbe sediments. Complete genome sequence of strain MW1T was 4.6 Mb in size and contained three rRNA operons and about 4200 protein-encoding genes, including the gene cluster pmoCAB coding for membrane methane monooxygenase. Soluble methane monooxygenase was not encoded in the genome. The G+C DNA content was 52.4%. The average nucleotide identity of the genome of strain MW1T with those in earlier described representatives of the genus Methylomonas was 79.4–82.1%. We propose to classify this isolate as representing a novel species of the genus Methylomonas, M. montana sp. nov. Strain MW1T (=VKM 3737T = UQM 41536T) is the type strain of the newly proposed species.

Transcriptional and metabolomic responses of Methylococcus capsulatus Bath to nitrogen source and temperature downshift

Methanotrophs play a significant role in methane oxidation, because they are the only biological methane sink present in nature. The methane monooxygenase enzyme oxidizes methane or ammonia into methanol or hydroxylamine, respectively. While much is known about central carbon metabolism in methanotrophs, far less is known about nitrogen metabolism. In this study, we investigated how Methylococcus capsulatus Bath, a methane-oxidizing bacterium, responds to nitrogen source and temperature. Batch culture experiments were conducted using nitrate or ammonium as nitrogen sources at both 37°C and 42°C. While growth rates with nitrate and ammonium were comparable at 42°C, a significant growth advantage was observed with ammonium at 37°C. Utilization of nitrate was higher at 42°C than at 37°C, especially in the first 24 h. Use of ammonium remained constant between 42°C and 37°C; however, nitrite buildup and conversion to ammonia were found to be temperature-dependent processes. We performed RNA-seq to understand the underlying molecular mechanisms, and the results revealed complex transcriptional changes in response to varying conditions. Different gene expression patterns connected to respiration, nitrate and ammonia metabolism, methane oxidation, and amino acid biosynthesis were identified using gene ontology analysis. Notably, key pathways with variable expression profiles included oxidative phosphorylation and methane and methanol oxidation. Additionally, there were transcription levels that varied for genes related to nitrogen metabolism, particularly for ammonia oxidation, nitrate reduction, and transporters. Quantitative PCR was used to validate these transcriptional changes. Analyses of intracellular metabolites revealed changes in fatty acids, amino acids, central carbon intermediates, and nitrogen bases in response to various nitrogen sources and temperatures. Overall, our results offer improved understanding of the intricate interactions between nitrogen availability, temperature, and gene expression in M. capsulatus Bath. This study enhances our understanding of microbial adaptation strategies, offering potential applications in biotechnological and environmental contexts.

Methylococcus mesophilus sp. nov., the first non-thermotolerant methanotroph of the genus Methylococcus, from a rice field.

Strain 16-5T, a mesophilic methanotroph of the genus Methylococcus, was isolated from rice field soil sampled in Chungcheong Province, Republic of Korea. Strain 16-5T had both particulate and soluble methane monooxygenases and could only grow on methane and methanol as electron donors. Strain 16-5 T cells are Gram-negative, white to light tan in color, non-motile, non-flagellated, diplococcoid to cocci, and have the typical type I intracytoplasmic membrane system. Strain 16-5T grew at 18-38 °C (optimum, 27 °C) and at pH 5.0-8.0 (optimum, pH 6.5-7.0). C16 : 1 ω7c (38.8%), C16 : 1 ω5c (18.8%), C16 : 1 ω6c (16.8%) and C16 : 0 (16.9%) were the major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phospholipid were the major polar lipids. The main respiratory quinone was methylene-ubiquinone-8. Strain 16-5T displayed the highest 16S rRNA gene sequence similarities to other taxonomically recognized members of the genus Methylococcus, i.e. Methylococcus capsulatus TexasT (98.62%) and Methylococcus geothermalis IM1T (98.49 %), which were its closest relatives. It did, however, differ from all other taxonomically described Methylococcus species due to some phenotypic differences, most notably its inability to grow at temperatures above 38 °C, where other Methylococcus species thrive. Its 4.34 Mbp-sized genome has a DNA G+C content of 62.47 mol%, and multiple genome-based properties such as average nucleotide identity and digital DNA-DNA hybridization value distanced it from its closest relatives. Based on the data presented above, this strain represents the first non-thermotolerant species of the genus Methylococcus. The name Methylococcus mesophilus sp. nov. is proposed, and 16-5T (=JCM 35359T=KCTC 82050T) is the type strain.

Resilience and robustness of alphaproteobacterial methanotrophs upon methane feast-famine scenarios.

Most of methane (CH4) emissions contain low CH4 concentrations and typically occur at irregular intervals, which hinders the implementation and performance of methane abatement processes. This study aimed at understanding the metabolic mechanisms that allow methane oxidizing bacteria (MOB) to survive for long periods of time under methane starvation. To this aim, we used an omics-approach and studied the diversity and metabolism of MOB and non-MOB in bioreactors exposed to low CH4 concentrations under feast-famine cycles of 5 days and supplied with nutrient-rich broth. The 16S rRNA and the pmoA transcripts revealed that the most abundant and active MOB during feast and famine conditions belonged to the alphaproteobacterial genus Methylocystis (91–65%). The closest Methylocystis species were M. parvus and M. echinoides. Nitrifiers and denitrifiers were the most representative non-MOB communities, which likely acted as detoxifiers of the system. During starvation periods, the induced activity of CH4 oxidation was not lost, with the particulate methane monooxygenase of alphaproteobacterial MOB playing a key role in energy production. The polyhydroxyalkanoate and nitrification metabolisms of MOB had also an important role during feast-famine cycles, maintaining cell viability when CH4 concentrations were negligible. This research shows that there is an emergence and resilience of conventional alphaproteobacterial MOB, being the genus Methylocystis a centrepiece in environments exposed to dilute and intermittent methane emissions. This knowledge can be applied to the operation of bioreactors subjected to the treatment of dilute and discontinuous emissions via controlled bioaugmentation.

Biosynthesis of chiral diols from alkenes using metabolically engineered type II methanotroph

Methanotrophs are environmentally friendly microorganisms capable of converting gas to liquid using methane monooxygenases (MMOs). In addition to methane-to-methanol conversion, MMOs catalyze the conversion of alkanes to alcohols and alkenes to epoxides. Herein, the efficacy of epoxidation by type I and II methanotrophs was investigated, and type II methanotrophs were observed to be more efficient in converting alkenes to epoxides. Subsequently, three (Epoxide hydrolase) EHs of different origins were overexpressed in the type II methanotroph Methylosinus trichosporium OB3b to produce 1,2-diols from epoxide. Methylosinus trichosporium OB3b expressing Caulobacter crescentus EH produced the highest amount of (R)-1,2-propanediol (251.5 mg/L) from 1-propene. These results demonstrate the possibility of using methanotrophs as a microbial platform for diol production and the development of a continuous bioreactor for industrial applications.

Methylacidiphilum caldifontis gen. nov., sp. nov., a thermoacidophilic methane-oxidizing bacterium from an acidic geothermal environment, and descriptions of the family Methylacidiphilaceae fam. nov. and order Methylacidiphilales ord. nov.

Strain IT6T, a thermoacidophilic and facultative methane-oxidizing bacterium, was isolated from a mud-water mixture collected from Pisciarelli hot spring in Pozzuoli, Italy. The novel strain is white when grown in liquid or solid media and forms Gram-negative rod-shaped, non-flagellated, non-motile cells. It conserves energy by aerobically oxidizing methane and hydrogen while deriving carbon from carbon dioxide fixation. Strain IT6T had three complete pmoCAB operons encoding particulate methane monooxygenase and genes encoding group 1d and 3b [NiFe] hydrogenases. Simple carbon-carbon substrates such as ethanol, 2-propanol, acetone, acetol and propane-1,2-diol were used as alternative electron donors and carbon sources. Optimal growth occurred at 50-55°C and between pH 2.0-3.0. The major fatty acids were C18 : 0, C15 : 0 anteiso, C14 : 0 iso, C16 : 0 and C14 : 0, and the main polar lipids were phosphatidylethanolamine, aminophospholipid, phosphatidylglycerol, diphosphatidylglycerol, some unidentified phospholipids and glycolipids, and other unknown polar lipids. Strain IT6T has a genome size of 2.19 Mbp and a G+C content of 40.70 mol%. Relative evolutionary divergence using 120 conserved single-copy marker genes (bac120) and phylogenetic analyses based on bac120 and 16S rRNA gene sequences showed that strain IT6T is affiliated with members of the proposed order 'Methylacidiphilales' of the class Verrucomicrobiia in the phylum Verrucomicrobiota. It shared a 16S rRNA gene sequence identity of >96 % with cultivated isolates in the genus 'Methylacidiphilum' of the family 'Methylacidiphilaceae', which are thermoacidophilic methane-oxidizing bacteria. 'Methylacidiphilum sp.' Phi (100 %), 'Methylacidiphilum infernorum' V4 (99.02 %) and 'Methylacidiphilum sp.' RTK17.1 (99.02 %) were its closest relatives. Its physiological and genomic properties were consistent with those of other isolated 'Methylacidiphilum' species. Based on these results, we propose the name Methylacidiphilum caldifontis gen. nov., sp. nov. to accommodate strain IT6T (=KCTC 92103T=JCM 39288T). We also formally propose that the names Methylacidiphilaceae fam. nov. and Methylacidiphilales ord. nov. to accommodate the genus Methylacidiphilum gen. nov.

Bioinspired Cu(II) Defect Sites in ZIF-8 for Selective Methane Oxidation.

Activating the C-H bonds of alkanes without further oxidation to more thermodynamically stable products, CO and CO2, is a long-sought goal of catalytic chemistry. Inspired by the monocopper active site of methane monooxygenase, we synthesized a Cu-doped ZIF-8 metal-organic framework with 25% Cu and 75% Zn in the nodes and activated it by heating to 200 °C and dosing in a stepwise fashion with O2, methane, and steam. We found that it does oxidize methane to methanol and formaldehyde. The catalysis persists through at least five cycles, and beyond the third cycle, the selectivity improves to the extent that no CO2 can be detected. Experimental characterization and analysis were carried out by PXRD, DRUV-vis, SEM, and XAS (XANES and EXAFS). The reaction is postulated to proceed at open-coordination copper sites generated by defects, and the mechanism of methanol production was explicated by density functional calculations with the revMO6-L exchange-correlation functional. The calculations reveal a catalytic cycle of oxygen-activated CuI involving the conversion of two molecules of CH4 to two molecules of CH3OH by a sequence of hydrogen atom transfer reactions and rebound steps. For most steps in the cycle, the reaction is more favored by singlet species than by triplets.

Recombinant expression and subcellular targeting of the particulate methane monooxygenase (pMMO) protein components in plants

Methane is a potent greenhouse gas, which has contributed to approximately a fifth of global warming since pre-industrial times. The agricultural sector produces significant methane emissions, especially from livestock, waste management and rice cultivation. Rice fields alone generate around 9% of total anthropogenic emissions. Methane is produced in waterlogged paddy fields by methanogenic archaea, and transported to the atmosphere through the aerenchyma tissue of rice plants. Thus, bioengineering rice with catalysts to detoxify methane en route could contribute to an efficient emission mitigation strategy. Particulate methane monooxygenase (pMMO) is the predominant methane catalyst found in nature, and is an enzyme complex expressed by methanotrophic bacteria. Recombinant expression of pMMO has been challenging, potentially due to its membrane localization, multimeric structure, and polycistronic operon. Here we show the first steps towards the engineering of plants for methane detoxification with the three pMMO subunits expressed in the model systems tobacco and Arabidopsis. Membrane topology and protein–protein interactions were consistent with correct folding and assembly of the pMMO subunits on the plant ER. Moreover, a synthetic self-cleaving polypeptide resulted in simultaneous expression of all three subunits, although low expression levels precluded more detailed structural investigation. The work presents plant cells as a novel heterologous system for pMMO allowing for protein expression and modification.

Transcriptional regulation of soluble methane monooxygenase via enhancer-binding protein derived from Methylosinus sporium 5.

Methane is a major greenhouse gas, and methanotrophs regulate the methane level in the carbon cycle. Soluble methane monooxygenase (sMMO) is expressed in various methanotroph genera, including Alphaproteobacteria and Gammaproteobacteria, and catalyzes the hydroxylation of methane to methanol. It has been proposed that MmoR regulates the expression of sMMO as an enhancer-binding protein under copper-limited conditions; however, details on this transcriptional regulation remain limited. Herein, we elucidate the transcriptional pathway of sMMO depending on copper ion concentration, which affects the interaction of MmoR and sigma factor. MmoR and sigma-54 (σ54) from Methylosinus sporium 5 were successfully overexpressed in Escherichia coli and purified to investigate sMMO transcription in methanotrophs. The results indicated that σ54 binds to a promoter positioned -24 (GG) and -12 (TGC) upstream between mmoG and mmoX1. The binding affinity and selectivity are lower (Kd = 184.6 ± 6.2 nM) than those of MmoR. MmoR interacts with the upstream activator sequence (UAS) with a strong binding affinity (Kd = 12.5 ± 0.5 nM). Mutational studies demonstrated that MmoR has high selectivity to its binding partner (ACA-xx-TGT). Titration assays have demonstrated that MmoR does not coordinate with copper ions directly; however, its binding affinity to UAS decreases in a low-copper-containing medium. MmoR strongly interacts with adenosine triphosphate (Kd = 62.8 ± 0.5 nM) to generate RNA polymerase complex. This study demonstrated that the binding events of both MmoR and σ54 that regulate transcription in M. sporium 5 depend on the copper ion concentration. IMPORTANCE This study provides biochemical evidence of transcriptional regulation of soluble methane monooxygenase (sMMO) in methanotrophs that control methane levels in ecological systems. Previous studies have proposed transcriptional regulation of MMOs, including sMMO and pMMO, while we provide further evidence to elucidate its mechanism using a purified enhancer-binding protein (MmoR) and transcription factor (σ54). The characterization studies of σ54 and MmoR identified the promoter binding sites and enhancer-binding sequences essential for sMMO expression. Our findings also demonstrate that MmoR functions as a trigger for sMMO expression due to the high specificity and selectivity for enhancer-binding sequences. The UV-visible spectrum of purified MmoR suggested an iron coordination like other GAF domain, and that ATP is essential for the initiation of enhancer elements. Binding assays indicated that these interactions are blocked by the copper ion. These results provide novel insights into gene regulation of methanotrophs.

Methanol on the rocks: green rust transformation promotes the oxidation of methane.

Shared coordination geometries between metal ions within reactive minerals and enzymatic metal cofactors hints at mechanistic and possibly evolutionary homology between particular abiotic chemical mineralogies and biological metabolism. The octahedral coordination of reactive Fe2+/3+ minerals such as green rusts, endemic to anoxic sediments and the early Earth's oceans, mirrors the di-iron reaction centre of soluble methane monooxygenase (sMMO), responsible for methane oxidation in methanotrophy. We show that methane oxidation occurs in tandem with the oxidation of green rust to lepidocrocite and magnetite, mimicking radical-mediated methane oxidation found in sMMO to yield not only methanol but also halogenated hydrocarbons in the presence of seawater. This naturally occurring geochemical pathway for CH4 oxidation elucidates a previously unidentified carbon cycling mechanism in modern and ancient environments and reveals clues into mineral-mediated reactions in the synthesis of organic compounds necessary for the emergence of life.

Thermophilic methane oxidation is widespread in Aotearoa-New Zealand geothermal fields.

Geothermal areas represent substantial point sources for greenhouse gas emissions such as methane. While it is known that methanotrophic microorganisms act as a biofilter, decreasing the efflux of methane in most soils to the atmosphere, the diversity and the extent to which methane is consumed by thermophilic microorganisms in geothermal ecosystems has not been widely explored. To determine the extent of biologically mediated methane oxidation at elevated temperatures, we set up 57 microcosms using soils from 14 Aotearoa-New Zealand geothermal fields and show that moderately thermophilic (>40°C) and thermophilic (>60°C) methane oxidation is common across the region. Methane oxidation was detected in 54% (n = 31) of the geothermal soil microcosms tested at temperatures up to 75°C (pH 1.5-8.1), with oxidation rates ranging from 0.5 to 17.4 μmol g-1 d-1 wet weight. The abundance of known aerobic methanotrophs (up to 60.7% Methylacidiphilum and 11.2% Methylothermus) and putative anaerobic methanotrophs (up to 76.7% Bathyarchaeota) provides some explanation for the rapid rates of methane oxidation observed in microcosms. However, not all methane oxidation was attributable to known taxa; in some methane-consuming microcosms we detected methanotroph taxa in conditions outside of their known temperature range for growth, and in other examples, we observed methane oxidation in the absence of known methanotrophs through 16S rRNA gene sequencing. Both of these observations suggest unidentified methane oxidizing microorganisms or undescribed methanotrophic syntrophic associations may also be present. Subsequent enrichment cultures from microcosms yielded communities not predicted by the original diversity studies and showed rates inconsistent with microcosms (≤24.5 μmol d-1), highlighting difficulties in culturing representative thermophilic methanotrophs. Finally, to determine the active methane oxidation processes, we attempted to elucidate metabolic pathways from two enrichment cultures actively oxidizing methane using metatranscriptomics. The most highly expressed genes in both enrichments (methane monooxygenases, methanol dehydrogenases and PqqA precursor peptides) were related to methanotrophs from Methylococcaceae, Methylocystaceae and Methylothermaceae. This is the first example of using metatranscriptomics to investigate methanotrophs from geothermal environments and gives insight into the metabolic pathways involved in thermophilic methanotrophy.

Bioinspired microenvironment modulation of metal–organic framework-based catalysts for selective methane oxidation

Inspiration from natural enzymes enabling creationary catalyst design is appealing yet remains extremely challenging for selective methane (CH4) oxidation. This study presents the construction of a biomimetic catalyst platform for CH4 oxidation, which is constructed by incorporating Fe-porphyrin into a robust metal–organic framework, UiO-66, furnished with saturated monocarboxylic fatty acid bearing different long alkyl chains. The catalysts demonstrate the high efficiency in the CH4 to methanol (CH3OH) conversion at 50 °C. Moreover, the selectivity to CH3OH can be effectively regulated and promoted through a fine-tuned microenvironment by hydrophobic modification around the Fe-porphyrin. The long-chain fatty acids anchored on the Zr-oxo cluster of UiO-66 can not only tune the electronic state of the Fe sites to improve CH4 adsorption, but also restrict the amount of H2O2 around the Fe sites to reduce the overoxidation. This behavior resembles the microenvironment regulation in methane monooxygenase, resulting in high CH3OH selectivity.