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.

Study of the structure-function relationship of formate dehydrogenase- an important enzyme for Staphylococcus aureus biofilms by rational design.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of this bacterium, especially in the form of biofilms. Understanding its mechanism and structure-function relationship can help to find special inhibitors of this enzyme, which can be used as medicines against staphylococci. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. This enzyme has the highest kcat value among the described FDHs and also has a high temperature stability compared to other enzymes of this group. That is why it can also be considered as a promising catalyst for NAD(P)H regeneration in the processes of chiral synthesis with oxidoreductases. In this work, the principle of rational design was used to improve SauFDH catalytic efficiency. After bioinformatics analysis of the amino acid sequence in combination with visualization of the enzyme structure (PDB 6TTB), 9 probable catalytically significant positions 119, 194, 196, 217-219, 246, 303 and 323 were identified, and 16 new mutant forms of SauFDH were obtained and characterized by kinetic experiments. The introduction of the mentioned substitutions in most cases leads to a decrease in stability at high temperatures and an increase at low temperatures. Substitutions in positions 119 and 194 lead to a decreasing of KMNAD+. A consistent decrease in the Michaelis constant in the Ile-Val-Ala-Gly series at position 119 of SauFDH is shown. KMNAD+ of mutant SauFDH V119G decreased by 27 times compared to the wild-type enzyme. After substitution Phe194Val KMNAD + decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. For this mutant form, an increase in catalytic efficiency was demonstrated through the use of a multicomponent buffer system.

NADH-dependent formate dehydrogenase mutants for efficient carbon dioxide fixation.

Bioconversion of CO2 to high-valuable products is a globally pursued sustainable technology for carbon neutrality. However, low CO2 activation with formate dehydrogenase (FDH) remains a major challenge for further upcycling due to the poor CO2 affinity, reduction activity and stability of currently used FDHs. Here, we present two recombined mutants, ΔFDHPa48 and ΔFDHPa4814, which exhibit high CO2 reduction activity and antioxidative activity. Compared to FDHPa, the reduction activity of ΔFDHPa48 was increased up to 743 % and the yield in the reduction of CO2 to methanol was increased by 3.16-fold. Molecular dynamics identified that increasing the width of the substrate pocket of ΔFDHPa48 could improve the enzyme reduction activity. Meanwhile, the enhanced rigidity of C-terminal residues effectively protected the active center. These results fundamentally advanced our understanding of the CO2 activation process and efficient FDH for enzymatic CO2 activation and conversion.

Functionally redundant formate dehydrogenases enable formate-dependent growth in Methanococcus maripaludis.

Methanogens are essential for the complete remineralization of organic matter in anoxic environments. Most cultured methanogens are hydrogenotrophic, using H2 as an electron donor to reduce CO2 to CH4, but in the absence of H2 many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for the reduction of coenzyme F420 or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis. Furthermore, methanogens that use formate encode at least two isoforms of Fdh in their genomes, but how these different isoforms participate in methanogenesis is unknown. Using Methanococcus maripaludis, we undertook a biochemical characterization of both Fdh isoforms involved in methanogenesis. Both Fdh1 and Fdh2 interacted with Hdr to catalyze the flavin-based electron bifurcating reaction, and both reduced F420 at similar rates. F420 reduction preceded flavin-based electron bifurcation activity for both enzymes. In a Δfdh1 mutant background, a suppressor mutation was required for Fdh2 activity. Genome sequencing revealed that this mutation resulted in the loss of a specific molybdopterin transferase (moeA), allowing for Fdh2-dependent growth, and the metal content of the proteins suggested that isoforms are dependent on either molybdenum or tungsten for activity. These data suggest that both isoforms of Fdh are functionally redundant, but their activities in vivo may be limited by gene regulation or metal availability under different growth conditions. Together these results expand our understanding of formate oxidation and the role of Fdh in methanogenesis.

NADP+ or CO2 reduction by frhAGB-encoded hydrogenase through interaction with formate dehydrogenase 3 in the hyperthermophilic archaeon Thermococcus onnurineus NA1.

IMPORTANCE: The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the frhAGB-encoded hydrogenase of Thermococcus onnurineus NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P)+ is significant in that the enzyme can function to supply reducing equivalents, just as F420-reducing hydrogenases in methanogens use coenzyme F420 as an electron carrier. Additionally, it was identified that T. onnurineus NA1 could produce formate from H2 and CO2 by the concerted action of frhAGB-encoded hydrogenase and formate dehydrogenase Fdh3.

Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD+ to NADP.

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO2 reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO2-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO2 capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD+ vs. NADP+ reduction. Starting from the NAD+-specific wild-type RcFDH, key residues were exchanged to enable NADP+ binding on the basis of the NAD+-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys157) in the ß-subunit of the enzyme is essential for the binding of NAD+. RcFDH variants that had Glu259 exchanged for either a positively charged or uncharged amino acid had additional activity with NADP+. The FdsBL279R and FdsBK276A variants also showed activity with NADP+. Kinetic parameters for all the variants were determined and tested for activity in CO2 reduction. The variants were able to reduce CO2 using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.

Structural analysis of wild-type and Val120Thr mutant Candida boidinii formate dehydrogenase by X-ray crystallography.

Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source `Turkish DeLight'. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.

The formate dehydrogenase enhances aluminum tolerance of tobacco.

The formate dehydrogenase (FDH) is regarded as a universal stress protein involved in various plant abiotic stress responses. This study aims to ascertain GmFDH function in conferring tolerance to aluminum (Al) stress. The bioinformatics analysis demonstrates that GmFDH from Tamba black soybean (TBS) encodes FDH. Quantitative reverse transcription-PCR (qRT-PCR) showed that GmFDH expression was induced by Al stress with a concentration-time-specific pattern. Moreover, Al stress promotes formate content and activates FDH activity. Further studies revealed that GmFDH overexpression alleviated root growth of tobacco under Al stress inhibition and reduced Al and ROS accumulation in roots. In addition, transgenic tobacco had much more root citrate exudation and much higher activity of antioxidant enzymes than wild type. Moreover, under Al stress, NtMATE and NtALS3 expression showed no changes in wild type and overexpression lines, suggesting that here the known Al-resistant mechanisms are not involved. However citrate synthase activity is higher in transgenic tobaccos than that of wild type, which might be the reason for citrate secretion increase. Thus, the increased Al tolerance of GmFDH overexpression lines is likely attributable to enhanced activities of antioxidant enzymes and promoting citrate secretion. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms and suggest a possible new route towards the improvement of plant growth under Al stress.

Engineering of formate dehydrogenase for improving conversion potential of carbon dioxide to formate.

Formate dehydrogenase (FDH) is a D-2-hydroxy acid dehydrogenase, which can reversibly reduce CO2 to formate and thus act as non-photosynthetic CO2 reductase. In order to increase catalytic efficiency of formate dehydrogenase for CO2 reduction, two mutants V328I/F285W and V354G/F285W were obtained of which reduction activity was about two times more than the parent CbFDHM2, and the formate production from CO2 catalyzed by mutants were 2.9 and 2.7-fold higher than that of the parent CbFDHM2. The mutants had greater potential in CO2 reduction. The optimal temperature for V328I/F285W and V354G/F285W was 55 °C, and they showed increasement of relative activity under 45 °C to 55 °C compared with parent. The optimal pH for the mutants was 9.0, and they showed excellent stability in pH 4.0-11.5. The kcat/Km values of mutants were 1.75 times higher than that of the parent. Then the molecular basis for its improvement of biochemical characteristics were preliminarily elucidated by computer-aided methods. All of these results further established a solid foundation for molecular modification of formate dehydrogenase and CO2 reduction.

Structure and function relationship of formate dehydrogenases: an overview of recent progress.

Formate dehydrogenases (FDHs) catalyze the two-electron oxidation of formate to carbon dioxide. FDHs can be divided into several groups depending on their subunit composition and active-site metal ions. Metal-dependent (Mo- or W-containing) FDHs from prokaryotic organisms belong to the superfamily of molybdenum enzymes and are members of the dimethylsulfoxide reductase family. In this short review, recent progress in the structural analysis of FDHs together with their potential biotechnological applications are summarized.

Engineering Candida boidinii formate dehydrogenase for activity with the non-canonical cofactor 3'-NADP(H).

Oxidoreductases catalyze essential redox reactions, and many require a diffusible cofactor for electron transport, such as NAD(H). Non-canonical cofactor analogs have been explored as a means to create enzymatic reactions that operate orthogonally to existing metabolism. Here, we aimed to engineer the formate dehydrogenase from Candid boidinii (CbFDH) for activity with the non-canonical cofactor nicotinamide adenine dinucleotide 3'-phosphate (3'-NADP(H)). We used PyRosetta, the Cofactor Specificity Reversal Structural Analysis and Library Design (CSR-SALAD), and structure-guided saturation mutagenesis to identify mutations that enable CbFDH to use 3'-NADP+. Two single mutants, D195A and D195G, had the highest activities with 3'-NADP+, while the double mutant D195G/Y196S exhibited the highest cofactor selectivity reversal behavior. Steady state kinetic analyses were performed; the D195A mutant exhibited the highest KTS value with 3'-NADP+. This work compares the utility of computational approaches for cofactor specificity engineering while demonstrating the engineering of an important enzyme for novel non-canonical cofactor selectivity.

Engineering a Formate Dehydrogenase for NADPH Regeneration.

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.

Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella.

Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD+ ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (FdhSNO ) from the soil bacterium Starkeya novella strictly specific for NAD+ . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of FdhSNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+ ) with better catalytic efficiency than NAD+ . The single mutation D221Q enabled the reduction of NADP+ with a catalytic efficiency kCAT /KM of 0.4 s-1 ·mm-1 at 200 mm formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a fivefold increase in catalytic efficiency for NADP+ compared with the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP+ . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of FdhSNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds.

Reduction, evolutionary pattern and positive selection of genes encoding formate dehydrogenase in Wood-Ljungdahl pathway of gastrointestinal acetogens suggests their adaptation to formate-rich habitats.

Acetogens are anaerobes using Wood-Ljungdahl pathway (WLP) as the terminal electron acceptor for both assimilation and dissimilation of CO2 and widely distributed in diverse habitats. However, their habitat adaptation is often unclear. Given that bacterial genome evolution is often the result of environmental selective pressure, hereby we analysed gene copy number, phylogeny and selective pressure of genes involved in WLP within known genomes of 43 species to study the habitat adaption of gastrointestinal acetogens. The gene copy number of formate dehydrogenase (FDH) in gastrointestinal acetogens was much lower than that of non-gastrointestinal acetogens, and in five cases, no FDH genes were found in the genomes of five gastrointestinal acetogens, but that of the other WLP genes showed no difference. The evolutionary pattern of FDH genes was significantly different from that of the other enzymes. Additionally, seven positively selected sites were only identified in the fdhF genes, which means fdhF mutations favoured their adaptation. Collectively, reduction or loss of FDH genes and their evolutionary pattern as well as positive selection in gastrointestinal acetogens indicated their adaptation to formate-rich habitats, implying that FDH genes catalysing CO2 reduction to formate as the first step of methyl branch of WLP may have evolved independently.

Interspecies Formate Exchange Drives Syntrophic Growth of Syntrophotalea carbinolica and Methanococcus maripaludis.

The complete remineralization of organic matter in anoxic environments relies on communities of microorganisms that ferment organic acids and alcohols to CH4. This is accomplished through syntrophic association of H2 or formate producing bacteria and methanogenic archaea, where exchange of these intermediates enables growth of both organisms. While these communities are essential to Earth's carbon cycle, our understanding of the dynamics of H2 or formate exchanged is limited. Here, we establish a model partnership between Syntrophotalea carbinolica and Methanococcus maripaludis. Through sequencing a transposon mutant library of M. maripaludis grown with ethanol oxidizing S. carbinolica, we found that genes encoding the F420-dependent formate dehydrogenase (Fdh) and F420-dependent methylene-tetrahydromethanopterin dehydrogenase (Mtd) are important for growth. Competitive growth of M. maripaludis mutants defective in either H2 or formate metabolism verified that, across multiple substrates, interspecies formate exchange was dominant in these communities. Agitation of these cultures to facilitate diffusive loss of H2 to the culture headspace resulted in an even greater competitive advantage for M. maripaludis strains capable of oxidizing formate. Finally, we verified that an M. maripaludis Δmtd mutant had a defect during syntrophic growth. Together, these results highlight the importance of formate exchange for the growth of methanogens under syntrophic conditions. IMPORTANCE In the environment, methane is typically generated by fermentative bacteria and methanogenic archaea working together in a process called syntrophy. Efficient exchange of small molecules like H2 or formate is essential for growth of both organisms. However, difficulties in determining the relative contribution of these intermediates to methanogenesis often hamper efforts to understand syntrophic interactions. Here, we establish a model syntrophic coculture composed of S. carbinolica and the genetically tractable methanogen M. maripaludis. Using mutant strains of M. maripaludis that are defective for either H2 or formate metabolism, we determined that interspecies formate exchange drives syntrophic growth of these organisms. Together, these results advance our understanding of the degradation of organic matter in anoxic environments.

FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism.

During enterobacterial mixed-acid fermentation, formate is generated from pyruvate by the glycyl-radical enzyme pyruvate formate-lyase (PflB). In Escherichia coli, especially at low pH, formate is then disproportionated to CO2 and H2 by the cytoplasmically oriented, membrane-associated formate hydrogenlyase (FHL) complex. If electron acceptors are available, however, formate is oxidized by periplasmically oriented, respiratory formate dehydrogenases. Formate translocation across the cytoplasmic membrane is controlled by the formate channel, FocA, a member of the formate-nitrite transporter (FNT) family of homopentameric anion channels. This review highlights recent advances in our understanding of how FocA helps to maintain intracellular formate and pH homeostasis during fermentation. Efflux and influx of formate/formic acid are distinct processes performed by FocA and both are controlled through protein interaction between FocA's N-terminal domain with PflB. Formic acid efflux by FocA helps to maintain cytoplasmic pH balance during exponential-phase growth. Uptake of formate against the electrochemical gradient (inside negative) is energetically and mechanistically challenging for a fermenting bacterium unless coupled with proton/cation symport. Translocation of formate/formic acid into the cytoplasm necessitates an active FHL complex, whose synthesis also depends on formate. Thus, FocA, FHL and PflB function together to govern formate homeostasis. We explain how FocA achieves efflux of formic acid and propose mechanisms for pH-dependent uptake of formate both with and without proton symport. We propose that FocA displays both channel- and transporter-like behaviour. Whether this translocation behaviour is shared by other members of the FNT family is also discussed.

The role of Tyr102 residue in the functioning of bacterial NAD+-dependent formate dehydrogenase of Pseudomonas sp. 101.

Once you have missed the first button …, you'll never manage to button up Johann Wolfgang von Goethe Formate oxidation is a final step of methanol oxidation in methylotrophic prokaryotes and is important for detoxification of formate in other organisms. The structural mechanism of the formate dehydrogenase (FDH) of Pseudomonas sp. 101 has been studied for about 30 years. In the active center of FDH, the oxidation of formic acid into carbon dioxide in a NAD+-dependent way takes place. Residues that form the active center of that enzyme, as well as those that form the so-called substrate channel, are engaged in the catalytic cycle. Our study allowed to characterize a new residue, Tyr102, involved in the work of the enzyme. This residue is located in the outer neck of the substrate channel (at the beginning of the path of the substrate to the active center) and acts as a "button" which connects two enzyme domains into an active, "buttoned up" conformation. Our study of the kinetic parameters of mutant enzymes has shown that Tyr102Phe substitution leads to an approximately 80-fold increase of the Michaelis constant relative to the native enzyme, unlike Phe311Trp and Phe311Tyr substitution of neighboring residue Phe311. Our analysis of the Tyr102Phe mutant in the open conformation by X-ray crystallography has shown that its overall fold remains almost the same as that of the native enzyme. Molecular dynamics simulations of the ternary complexes of the native FDH enzyme and its Tyr102Phe mutant showed that Tyr102Phe substitution results in the loss of an interdomain hydrogen bond between the Tyr102 and Gln313 residues, which, in turn, destabilizes the closed conformation and affects the isolation of the FDH active site from water molecules. Our structural investigations have shown that Tyr102Phe replacement also leads to the destruction of interdomain contacts of Phe102 with Phe311, Pro312 residues, and decreases the stability of the Leu103-Val127 beta bridge. Phylogenetic analysis also confirmed the importance of the Tyr102 residue for enzymes from the FDH family, in which it is absolutely conserved.

Bioconversion of CO to formate by artificially designed carbon monoxide:formate oxidoreductase in hyperthermophilic archaea.

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.

Functional role of formate dehydrogenase 1 (FDH1) for host and nonhost disease resistance against bacterial pathogens.

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In the present study, the metabolic enzyme formate dehydrogenase 1 (FDH1) was identified to associate with nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. The Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that both SA and JA are involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 mainly localized to mitochondria, and the expression level of FDH1 was drastically increased upon infection with host or nonhost pathogens. Furthermore, we identified the potential co-localization of mitochondria expressing FDH1 with chloroplasts after the infection with nonhost pathogens in Arabidopsis. This finding suggests the possible role of FDH1 in mitochondria and chloroplasts during defense responses against bacterial pathogens in plants.

A functional Wood-Ljungdahl pathway devoid of a formate dehydrogenase in the gut acetogens Blautia wexlerae, Blautia luti and beyond.

Species of the genus Blautia are typical inhabitants of the human gut and considered as beneficial gut microbes. However, their role in the gut microbiome and their metabolic features are poorly understood. Blautia schinkii was described as an acetogenic bacterium, characterized by a functional Wood-Ljungdahl pathway (WLP) of acetogenesis from H2  + CO2 . Here we report that two relatives, Blautia luti and Blautia wexlerae do not grow on H2  + CO2 . Inspection of the genome sequence revealed all genes of the WLP except genes encoding a formate dehydrogenase and an electron-bifurcating hydrogenase. Enzyme assays confirmed this prediction. Accordingly, resting cells neither converted H2  + CO2 nor H2  + HCOOH + CO2 to acetate. Carbon monoxide is an intermediate of the WLP and substrate for many acetogens. Blautia luti and B. wexlerae had an active CO dehydrogenase and resting cells performed acetogenesis from HCOOH + CO2  + CO, demonstrating a functional WLP. Bioinformatic analyses revealed that many Blautia strains as well as other gut acetogens lack formate dehydrogenases and hydrogenases. Thus, the use of formate instead of H2  + CO2 as an interspecies hydrogen and electron carrier seems to be more common in the gut microbiome.