Formate Dehydrogenase Research Articles

Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

A new strategy of carbon-energy coupling transfer enhancement by formate on phototrophic green microalga Chlamydomonas reinhardtii

Low efficient CO2 and solar energy transfer in aquatic microalgae cultivation are always challenges and have consumed huge efforts during the last two decades. With the development of solar fuels technologies, formate can come from the reduction of CO2 with green H2. As a water-soluble solar fuel, formate is a good carbon-energy coupling enhancement carrier for biosynthesis. However, formate is also an inhibitor to the electron transfer chain (ETC) in the cell. Based on the chloroplast engineering, a series of Chlamydomonas reinhardtii mutants with chloroplast transit peptide chimeric NADPH-dependent formate dehydrogenase (FDH) were constructed, and these mutants were named as PsFDHs. Results showed that exogenous FDH alleviated the inhibition of formate on the photosynthetic system and promoted the growth, and a win–win result obtained between the natural and artificial photosynthesis: 40 % more carbon from formate was fixed than heterotrophic only, and 62 % increase in dry weight (DW), while no obvious formate consumption was detected in the wide type (WT). The maximum overall biomass productivity was 15.4 mg·L−1·h−1, which was 36 % higher than that of WT. It’s postulated that formate enhanced the carbon and energy transportation close to carbon fixation reaction center, and photosynthesis made up for energy shortages in the conversion of formate to biomass. The results showed the potential of formate in achieving a higher-than-nature artificial-natural hybrid photosynthesis by microalgae, to meet requirements of both the carbon neutralization and protein supply for human nutrients.

Biotransformation of free cyanide to formic acid by a cyanide hydratase−formamidase cascade reaction

Free cyanide (fCN) consisting of HCN and CN- is highly hazardous. Today, removal of cyanide from industrial (mining, plating, coke-plant) wastewaters largely relies on physicochemical processes followed by microbial degradation. Enzymatic processes are gaining ground but are still at a low technological stage. The cyanide-converting enzymes of interest are primarily cyanide dihydratases (CynDs; EC 3.5.5.1.), which hydrolyze HCN to formic acid and ammonia, and cyanide hydratases (CynHs; EC 4.2.1.66), which hydrate HCN to formamide. CynHs usually have much higher specific activities and a broader pH profile especially in the alkaline region compared to CynDs. However, the product of CynH, formamide, although much less toxic than fCN, still poses a significant health risk. Therefore, it is attractive to combine the CynH with an amidase that converts formamide to formic acid and ammonia. Here we demonstrate on a laboratory scale a two-step “one-pot” detoxification of fCN (5 mM) to formic acid using recombinantly produced purified enzymes − CynH from Exidia glandulosa and formamidase (EC 3.5.1.49) from Bacillus cereus. The reaction proceeds at pH 9.0–10.0, which reduces the risk of HCN escape. We also hypothesize that the cascade can be used for fCN determination after coupling an NAD-dependent formate dehydrogenase.

Enzymatic conversion of CO2 to formate: The potential of tungsten-containing formate dehydrogenase in flow reactor system

This study introduces a novel approach for CO2 reduction to formate using the recombinant formate dehydrogenase 1 (MeFDH1) from Methylorubrum extorquens AM1 as biocatalyst, addressing challenges in activity, productivity, and long-term stability of enzyme. We demonstrate that immobilized MeFDH1 supported by electrochemical reaction system enhances formate production and stability, achieving over 1.7 M concentration with an initial rate of 20 mM/h and near-unity Faradaic efficiency for over 200 hours. In further, the reusability of immobilized MeFDH1 was obtained without significant declination of productivity and selectivity. The electrochemical study of MeFDH1 found the product inhibition in continuous CO2 conversion. To overcome this challenge and build efficient process, the integration of a flow reactor system and in-situ separation unit further improved the system's performance and scalability. This advancement in enzymatic CO2 conversion suggests the potential of biocatalysis towards addressing global warming through sustainable chemical synthesis.

Isolation of an H2-dependent electron-bifurcating CO2-reducing megacomplex with MvhB polyferredoxin from Methanothermobacter marburgensis.

In the hydrogenotrophic methanogenic pathway, formylmethanofuran dehydrogenase (Fmd) catalyzes the formation of formylmethanofuran through reducing CO2. Heterodisulfide reductase (Hdr) provides two low potential electrons for the Fmd reaction using a flavin-based electron-bifurcating mechanism. [NiFe]-hydrogenase (Mvh) or formate dehydrogenase (Fdh) complexes with Hdr and provides electrons to Hdr from H2 and formate, or the reduced form of F420, respectively. Recently, an Fdh-Hdr complex was purified as a 3-MDa megacomplex that contained Fmd, and its three-dimensional structure was elucidated by cryo-electron microscopy. In contrast, the Mvh-Hdr complex has been characterized only as a complex without Fmd. Here, we report the isolation and characterization of a 1-MDa Mvh-Hdr-Fmd megacomplex from Methanothermobacter marburgensis. After anion-exchange and hydrophobic chromatography was performed, the proteins with Hdr activity eluted in the 1- and 0.5-MDa fractions during size exclusion chromatography. Considering the apparent molecular mass and the protein profile in the fractions, the 1-MDa megacomplex was determined to be a dimeric Mvh-Hdr-Fmd complex. The megacomplex fraction contained a polyferredoxin subunit MvhB, which contains 12 [4Fe-4S]-clusters. MvhB polyferredoxin has never been identified in the previously purified Mvh-Hdr and Fmd preparations, suggesting that MvhB polyferredoxin is stabilized by the binding between Mvh-Hdr and Fmd in the Mvh-Hdr-Fmd complex. The purified Mvh-Hdr-Fmd megacomplex catalyzed electron-bifurcating reduction of [13C]-CO2 to form [13C]-formylmethanofuran in the absence of extrinsic ferredoxin. These results demonstrated that the subunits in the Mvh-Hdr-Fmd megacomplex are electronically connected for the reduction of CO2, which likely involves MvhB polyferredoxin as an electron relay.

A native CO2-reducing bacterium: Discovery, implementation and interests

Stenotrophomonas maltophilia has been demonstrated herein to reduce CO2 without any cofactor, photon or hydrogen (H2) addition during reaction. S. maltophilia reduces 13CO2 into 13C-labeled formate in batch mode. Two intracellular enzymes are curently being considered for their ability to catalyze the CO2 reduction reaction: a Fe-nitrogenase and a formate dehydrogenase (FeS-FDH). The reaction was intensified by implementing the bacteria in an electrolysis cell continuously fed with CO2. In this configuration, CO2 removal reached up to 25% v/v at 30°C and atmospheric pressure.

One-pot Twostep Chemobiocatalytic Synthesis of a Furan Amino Acid from 5-Hydroxymethylfurfural.

Recently, catalytic valorization of biomass-derived furans has received growing interest. 5-Aminomethyl-2-furancarboxylic acid (AMFC), a furan amino acid, holds great promise in the aeras of polymer and pharmaceutical, but its synthesis remains limited. In this work, we report a chemobiocatalytic route toward AMFC by combining laccase-TEMPO system and recombinant Escherichia coli (named E. coli_TAF) harboring ω-transaminase (TA), L-alanine dehydrogenase (L-AlaDH) and formate dehydrogenase (FDH), starting from 5-hydroxymethylfurfural (HMF). In the cascade, HMF is oxidized into 5-formyl-2-furancarboxylic acid (FFCA) by laccase-TEMPO system, and then the resulting intermediate is converted into AMFC by E. coli_TAF via transamination with cheap ammonium formate instead of costly organic amine donors, theoretically generating H2O and CO2 as by-products. The tandem process was run in a one-pot twostep manner, affording AMFC with approximately 81 % yield, together with 10 % 2,5-furandicarboxylic acid (FDCA) as by-product. In addition, the scale-up production of AMFC was demonstrated, with 0.41 g/L h productivity and 8.6 g/L titer. This work may pave the way for green manufacturing of the furan-containing amino acid.

Up- and Down-Regulation of Enzyme Activity in Aggregates with Gold-Covered Magnetic Nanoparticles Triggered by Low-Frequency Magnetic Field.

The modern global trend toward sustainable processes that meet the requirements of "green chemistry" provides new opportunities for the broad application of highly active, selective, and specific enzymatic reactions. However, the effective application of enzymes in industrial processes requires the development of systems for the remote regulation of their activity triggered by external physical stimuli, one of which is a low-frequency magnetic field (LFMF). Magnetic nanoparticles (MNPs) transform the energy of an LFMF into mechanical forces and deformations applied to enzyme molecules on the surfaces of MNPs. Here, we demonstrate the up- and down-regulation of two biotechnologically important enzymes, yeast alcohol dehydrogenase (YADH) and soybean formate dehydrogenase (FDH), in aggregates with gold-covered magnetic nanoparticles (GCMNPs) triggered by an LFMF. Two types of aggregates, "dimeric" (with the enzyme attached to several GCMNPs simultaneously), with YADH or FDH, and "monomeric" (the enzyme attached to only one GCMNP), with FDH, were synthesized. Depending on the aggregate type ("dimeric" or "monomeric"), LFMF treatment led to a decrease (down-regulation) or an increase (up-regulation) in enzyme activity. For "dimeric" aggregates, we observed 67 ± 9% and 47 ± 7% decreases in enzyme activity under LFMF exposure for YADH and FDH, respectively. Moreover, in the case of YADH, varying the enzyme or the cross-linking agent concentration led to different magnitudes of the LFMF effect, which was more significant at lower enzyme and higher cross-linking agent concentrations. Different responses to LFMF exposure depending on cofactor presence were also demonstrated. This effect might result from a varying cofactor binding efficiency to enzymes. For the "monomeric" aggregates with FDH, the LFMF treatment caused a significant increase in enzyme activity; the magnitude of this effect depended on the cofactor type: we observed up to 40% enzyme up-regulation in the case of NADP+, while almost no effect was observed in the case of NAD+.

Autotrophic growth of Escherichia coli is achieved by a small number of genetic changes

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO2. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for Escherichia coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis (pgi), central-carbon regulation (crp), and RNA transcription (rpoB). The pgi mutation reduces the enzyme’s activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (rpiB) and the energy module (fdoGH), as well as an increased ratio of NADH/NAD+ - the cycle’s electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.

Autotrophic growth of Escherichia coli is achieved by a small number of genetic changes.

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO2. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for Escherichia coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis (pgi), central-carbon regulation (crp), and RNA transcription (rpoB). The pgi mutation reduces the enzyme's activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (rpiB) and the energy module (fdoGH), as well as an increased ratio of NADH/NAD+ - the cycle's electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.

Enhanced synthesis of chloramphenicol intermediate L-threo-p-nitrophenylserine using engineered L-threonine transaldolase and by-product elimination

L-threo-p-nitrophenylserine (component 2) is an important intermediate during synthesis of chloramphenicol. However, its biosynthesis is limited by enzyme activity and stereoselectivity. In this study, we achieved a breakthrough in the high-efficiency production of 2 by employing engineered Chitiniphilus shinanonensis L-threonine transaldolase (ChLTTA) in conjunction with a by-product elimination system within a one-pot reaction. Notably, a novel visual stepwise high-throughput screening method was developed for the directed evolution of ChLTTA, leveraging its characteristic color. The engineered mutant F70D/F59A (Mu6 variant) emerged as a star performer, exhibiting a remarkable 2.6-fold increase in catalytic efficiency over the wild-type ChLTTA, coupled with an outstanding 91.5 % diastereoisomer excess (de). Molecular dynamics (MD) simulations unraveled the mechanism responsible for the enhanced catalytic performance observed in the Mu6 variant. Meanwhile, the Mu6 variant was coupled with Saccharomyces cerevisiae ethanol dehydrogenase (ScADH) and Candida boidinii formate dehydrogenase (CbFDH) to create a high-efficiency cascade system (E.coli/pRSF-Mu6-ScADH-CbFDH). Under optimized conditions, this cascade system demonstrated unparalleled performance, yielding 201.5 mM of 2 with an impressive conversion of 95.9 % and a de value of 94.5 %. This achievement represents the highest reported yield to date. This study offers a novel insight into the sustainable and efficient production of chloramphenicol intermediate.

Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1)

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Titanium carbide-based nanocomposite: A promising reinforcing material for enzymatic CO2 conversion

Enzymatic conversion of CO2 to formate is a highly significant pathway for CO2 utilization, but current level of conversion efficiency remains unsatisfactory due to low solubility of CO2 under mild conditions. CO2-philic materials can significantly augment the reaction by raising local concentration of substrate. Herein, the intercalated Ti3C2Tx, nano-silica (SiO2) and carbon nitride (CN) with abundant CO2 adsorption sites were introduced to fabricate Ti3C2Tx based nanocomposites, Ti3C2Tx/SiO2/CN (TSCN), for the first time. As the controls, binary composites including Ti3C2Tx/SiO2 (TS), Ti3C2Tx/CN (TCN) and SiO2/CN (SCN) were also studied. For each kind of material, the composition, structure, morphology, and application performance in the enzymatic CO2 conversion were characterized and influencing factors were investigated. Among these composites, TSCN exhibited the best application performance and the enzyme reaction was accelerated up to 23.9 times that of the blank system. The value could reach as high as 35.3 after polyethyleneimine (PEI) modification. Following that, PEI modified composites were used as carrier of formate dehydrogenase (FDH) and apparent specific activity was up to 2.8 times that of free enzyme, which could retain 88.2% of the initial value after 10 cycles associated with the structural integrity. This work provides new impetus to CO2 capture and conversion in a clean and mild way that shows bright application prospects.

Unlocking Catalytic Diversity of a Formate Dehydrogenase: Formamide Activity for NADPH Regeneration and Amine Supply for Asymmetric Reductive Amination

Unlocking Catalytic Diversity of a Formate Dehydrogenase: Formamide Activity for NADPH Regeneration and Amine Supply for Asymmetric Reductive Amination

Redox potentials elucidate the electron transfer pathway of NAD+-dependent formate dehydrogenases

Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.

Methanobrevibacter massiliense and Pyramidobacter piscolens Co-Culture Illustrates Transkingdom Symbiosis.

Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1-2-µm-long and 0.6-0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.

Foam fractionation Tags (F-Tags) enabling surfactant-free, activity-preserving recovery of enzymes

Enzymes have become important tools in many industries. However, the full exploitation of their potential is currently limited by a lack of efficient and cost-effective methods for enzyme purification from microbial production. One technology that could solve this problem is foam fractionation. In this study, we show that diverse natural foam-stabilizing proteins fused as F-Tags to β-lactamase, penicillin G acylase, and formate dehydrogenase, respectively, are able to mediate foaming and recovery of the enzymes by foam fractionation. The catalytic activity of all three candidates is largely preserved. Under appropriate fractionation conditions, especially when a wash buffer is used, some F-Tags also allow nearly complete separation of the target enzyme from a contaminating protein. We found that a larger distance between the F-Tag and the target enzyme has a positive effect on the maintenance of catalytic activity. However, we did not identify any particular sequence motifs or physical parameters that influenced performance as an F-tag. The best results were obtained with a short helical F-Tag, which was originally intended to serve only as a linker sequence. The findings of the study suggest that the development of molecular tags that enable the establishment of surfactant-free foam fractionation for enzyme workup is a promising method.Key points• Foam-stabilizing proteins mediate activity-preserving foam fractionation of enzymes• Performance as an F-Tag is not restricted to particular structural motifs• Separation from untagged protein benefits from low foam stability and foam washingsGraphical

Preparation of amino-functionalized polyethylene-silica composite membrane and FDH immobilization

Enzymatic CO2 conversion represents a highly efficient, energy-conserving, and environmentally sustainable approach to mitigating the increasing pressure caused by huge carbon emissions and generating valuable products. However, several challenges associate with the process including the instability and recovery difficulty of free enzyme, as well as low solubility of CO2 in mild conditions. Immobilizing enzyme on the amino-functionalized supports presents a viable solution to these issues. Considering the wide application of polymer hollow fiber membranes in the field of CO2 gas–liquid membrane contactor and the characteristics of hydrophilic, biocompatible, and high specific surface area of silica nanoparticles, the amino-functionalized polyethylene (PE)-silica (APS) composite membranes were fabricated via different routes. The morphologies, the loading rates of modified SiO2 and reusability of APS membranes were characterized for a crude screening. Following that, seven kinds of APS membranes were used as the carriers of formate dehydrogenase (FDH) that could catalyze the reduction of CO2 to formate, and their immobilization efficiency and catalytic performance were compared. The results show that 1# and 2# membranes based on carboxyl-modified PE with a loading rate of 5.5 %∼5.6 % and the reusability of above 99 % were more ideal carriers than others. The enzyme activity recovery rate of APS-immobilized FDH (FDH-APS) was separately 367 % and 256 %, proving the intensification action of carriers on the enzymatic reaction. After 10 cycles of use, the relative activity of two kinds of FDH-APS was around 70 % and 78 %, much higher than corresponding particles or membrane attached FDH (25.3 % and 20.4 %). After storage at 4 °C for 20 days, the relative activity of FDH-APS was 82.7 % and 81.0 %, while it was only 61.4 % for free enzyme.

Theoretical hypothesis in a direct electron transfer between non-interacting Fe-S proteins within an artificial fusion.

Reduction of CO2 to formate utilizing formate dehydrogenases (FDHs) has been attempted biologically and electrochemically. However, the conversion efficiency is very low due to the low energy potential of electron donors and/or electron competition with other electron acceptors. To overcome such a low conversion efficiency, I focused on a direct electron transfer between two unrelated redox enzymes for the efficient reduction of CO2 and utilized the quantum mechanical magnetic properties of the [Fe-S] ([iron-sulfur]) cluster to develop a novel electron path. Using this electron path, we connected non-interacting carbon monoxide dehydrogenase and FDH, constructing a synthetic carbon monoxide:formate oxidoreductase as a single functional enzyme complex in the previous study. Here, a theoretical hypothesis that can explain the direct electron transfer phenomenon based on the magnetic properties of the [Fe-S] cluster is proposed.

Facultative endosymbiosis between cellulolytic protists and methanogenic archaea in the gut of the Formosan termite Coptotermes formosanus.

Anaerobic protists frequently harbour methanogenic archaea, which apparently contribute to the hosts' fermentative metabolism by consuming excess H2. However, the ecological properties of endosymbiotic methanogens remain elusive in many cases. Here we investigated the ecology and genome of the endosymbiotic methanogen of the Cononympha protists in the hindgut of the termite Coptotermes formosanus. Microscopic and 16S rRNA amplicon sequencing analyses revealed that a single species, designated here "Candidatus Methanobrevibacter cononymphae", is associated with both Cononympha leidyi and Cononympha koidzumii and that its infection rate in Cononympha cells varied from 0.0% to 99.8% among termite colonies. Fine-scale network analysis indicated that multiple 16S rRNA sequence variants coexisted within a single host cell and that identical variants were present in both Cononympha species and also on the gut wall. Thus, "Ca. Methanobrevibacter cononymphae" is a facultative endosymbiont, transmitted vertically with frequent exchanges with the gut environment. Indeed, transmission electron microscopy showed escape or uptake of methanogens from/by a Cononympha cell. The genome of "Ca. Methanobrevibacter cononymphae" showed features consistent with its facultative lifestyle: i.e., the genome size (2.7 Mbp) comparable to those of free-living relatives; the pseudogenization of the formate dehydrogenase gene fdhA, unnecessary within the non-formate-producing host cell; the dependence on abundant acetate in the host cell as an essential carbon source; and the presence of a catalase gene, required for colonization on the microoxic gut wall. Our study revealed a versatile endosymbiosis between the methanogen and protists, which may be a strategy responding to changing conditions in the termite gut.