Turnover numbers ( k cat ) describe intrinsic catalytic capacities of enzymes. Although they have been characterized in vitro for many model organisms, such data is scarce for anaerobic Archaea like the methanogen Methanococcus maripaludis. Moreover, the apparent in vivo catalytic rates of enzymes operating in C1 utilization have neither been quantified experimentally nor predicted computationally. Here, we determined the in vivo catalytic rates of 99 M. maripaludis enzymes during growth on formate as the sole carbon source and electron donor. The determination was performed using previously published proteomics data and genome-scale metabolic model simulations constrained with experimental data across multiple growth rates. The obtained in vivo catalytic rates ( k app ) were compared to sequence-derived maximum turnover numbers ( k cat ) previously predicted by a machine-learning model trained on in vitro data. Sequence-derived turnover numbers and maximum in vivo catalytic rates did not correlate. The quantitative insight into methanogenic C1 metabolism and the in vivo catalytic rates can be used to guide metabolic engineering strategies. Engineered methanogens can serve as hosts in disruptive solutions of future biotechnological chemical production from one-carbon compounds.

Methanosarcina acetivorans is a model methanogen because of its metabolic versatility and genetic tractability. This microbe is not known to natively utilize hydrogen as a catabolic electron donor, despite its genome encoding hydrogenases, and the fact that this microbe can be used to heterologously express functional hydrogenases. Its native hydrogenases are expressed at a very low level and are suggested to have a role in recycling hydrogen that is produced as a byproduct of nitrogen fixation. To explore whether hydrogen can act as a catabolic electron donor, we utilized a previously constructed strain (str. JB-MF) that has the operon encoding for the methyl-H4MPT:CoM methyltransferase (Mtr) disrupted which makes growth dependent on oxidation of electron donors other than methanol. We showed that this M. acetivorans strain can grow by methanol reduction to methane with hydrogen as the sole electron donor. The strain was then used to test the putative electron donors for methyl-reducing methanogenesis: hydrogen, serine, and ethanol. Hydrogen and serine were identified to act as catabolic electron donors for methyl-reducing methanogenesis, which demonstrates the expanded metabolic versatility of M. acetivorans JB-MF. The methanophenazine-reducing hydrogenase (Vht) and the F420-reducing hydrogenase (Frh) were determined to be involved in hydrogen metabolism, because deletion of either made M. acetivorans JB-MF incapable of growing using H2-dependent methyl-reduction. We demonstrate that strain JB-MF is a suitable chassis to screen alternative electron donors for methanogenesis and that this strain may be used for targeted directed evolution of oxidoreductases.

Tetrahydromethanopterin (H4MPT) is the main carbon carrier in methane metabolism, which allows the interconversion of a formyl group into a methyl group. Although typically described as a single carbon (C1) carrier, we identified and characterized N 5-ethyltetrahydromethanopterin (N 5-ethyl-H4MPT) and N 5,N 10-(1,1-ethylene)tetrahydromethanopterin (N 5,N 10-ethylene-H4MPT) in the model methanogen Methanothermobacter marburgensis. The chemical competence of H4MPT to accept C2 moieties was assayed through the study of the spontaneous formation of N 5,N 10-ethylene-H4MPT by the condensation of acetaldehyde with H4MPT under standard conditions. The rate constant for this reaction was determined to be 1.53 +/- 0.05 M-1 s-1, and the equilibrium constant was determined to be (8.8 +/- 0.5) & times; 103 M-1, which is ca. 35 times higher compared to the analogous reaction with the more common carbon carrier tetrahydrofolate. Biochemical assays with M. marburgensis cell lysate suggest that the observed formation of N 5-ethyl-H4MPT relies on the enzymatic reduction of N 5,N 10-ethylene-H4MPT. These findings illustrate the chemical competency of H4MPT to promote biocatalysis with two-carbon moieties and highlight that such reactions might be compatible with established H4MPT-mediated microbial pathways.

Methanogenic archaea (methanogens) are key drivers of global carbon cycling and biomethane production, thriving in diverse anaerobic environments through specialized metabolic and transport systems. While methanogenesis is well understood, transport mechanisms underlying nutrient uptake, ion homeostasis, and macromolecule translocation remain poorly understood. This review compiles current knowledge on transporter proteins and substrate uptake in methanogens. It also highlights fundamental knowledge gaps and outlines experimental procedures for identifying new transporters experimentally and bioinformatic strategies to identify transporter-encoding genes. These approaches enable the investigation of cultured and uncultured methanogens, providing a broader view of transporter diversity and global distribution. Advancing transporter research will enhance insights into archaeal physiology and supports further developing biotechnological applications of methanogens for biofuels and chemical production, and sustainable energy systems.

Tetrahydromethanopterin (H4MPT) is the main carbon carrier in methane metabolism, which allows the interconversion of a formyl group into a methyl group. Although typically described as a single carbon (C1) carrier, we identified and characterized N5-ethyltetrahydromethanopterin (N5-ethyl-H4MPT) and N5,N10-(1,1-ethylene)tetrahydromethanopterin (N5,N10-ethylene-H4MPT) in the model methanogen Methanothermobacter marburgensis. The chemical competence of H4MPT to accept C2 moieties was assayed through the study of the spontaneous formation of N5,N10-ethylene-H4MPT by the condensation of acetaldehyde with H4MPT under standard conditions. The rate constant for this reaction was determined to be 1.53 ± 0.05 M-1 s-1, and the equilibrium constant was determined to be (8.8 ± 0.5) × 103 M-1, which is ca. 35 times higher compared to the analogous reaction with the more common carbon carrier tetrahydrofolate. Biochemical assays with M. marburgensis cell lysate suggest that the observed formation of N5-ethyl-H4MPT relies on the enzymatic reduction of N5,N10-ethylene-H4MPT. These findings illustrate the chemical competency of H4MPT to promote biocatalysis with two-carbon moieties and highlight that such reactions might be compatible with established H4MPT-mediated microbial pathways.

Methyl-coenzyme M reductase (MCR) is the enzyme responsible for nearly all biologically generated methane 1 . Its active site comprises coenzyme F 430 , a porphyrin-based cofactor with a central nickel ion that is active exclusively in the Ni(I) state 2,3 . How methanogenic archaea perform the reductive activation of F 430 represents a major gap in our understanding of one of the most ancient bioenergetic systems in nature. Here we purified and characterized the MCR activation complex from Methanococcus maripaludis . McrC, a small subunit encoded in the mcr operon, co-purifies with the methanogenic marker proteins Mmp7, Mmp17, Mmp3 and the A2 component. We demonstrated that this complex can activate MCR in vitro in a strictly ATP-dependent manner, enabling the formation of methane. In addition, we determined the cryo-electron microscopy structure of the MCR activation complex exhibiting different functional states with local resolutions reaching 1.8–2.1 Å. Our data revealed three complex iron–sulfur clusters that formed an electron transfer pathway towards F 430 . Topology and electron paramagnetic resonance spectroscopy analyses indicate that these clusters are similar to the [8Fe-9S-C] cluster, a maturation intermediate of the catalytic cofactor in nitrogenase. Altogether, our findings offer insights into the activation mechanism of MCR and prospects on the early evolution of nitrogenase.

Corrinoid-dependent enzymes either catalyze methyltransfer reactions, or they generate substrate radicals using adenosylcobalamin for subsequent rearrangement reactions. The corrinoid-dependent methyltransferases are present in all domains of life and assumed to be exclusive for methyl-groups. In Methanosarcina, however, trace ethane production from ethanol has been shown in vivo, which led to the hypothesis that corrinoid-dependent methanol-specific methyltransferases are promiscuous towards also accepting ethyl-groups. Here, we show that the conversion of ethanol to trace amounts of ethane in Methanosarcina acetivorans involves homologous reactions of the known methanol-to-methane metabolism. The methanol methyltransferase (MtaB) activates ethanol and loads the ethyl-group onto the corrinoid-containing methyl-accepting protein (MtaC). Besides MtaCB, substrate promiscuity in corrinoid:coenzyme M methyltransferase (MtaA) and methyl-coenzyme M reductase (Mcr) are required to grant the microbe the capacity for ethane production. We show that the MtaCB subunits of M. acetivorans can activate ethanol, however, the ethane yields compared to methane are ca. 3 orders of magnitude lower. The ethyl-transfer capability was confirmed for each of the three MtaCB isozyme by quantifying the amount of ethane produced by mtaCB double deletion strains during growth in ethanol-supplemented media and in resting-cell suspensions. Ethane formation requires the cells to be grown on methanol to trigger the expression of the mtaCB genes, and detectable ethane formation starts only after all methanol has been consumed. Demonstrating that corrinoid-dependent methanol-specific methyltransferases process ethyl groups extends the pool of reactions to be considered in metabolic networks and suggests possible routes for biogenic ethane in nature.

Methanosarcinales are versatile methanogens, capable of regulating most types of methanogenic pathways. Despite the versatile metabolic flexibility of Methanosarcinales, no member of this order has been shown to use formate for methanogenesis. In the present study, we identified a cytosolic formate dehydrogenase (FdhAB) present in several Methanosarcinales, likely acquired by independent horizontal gene transfers after an early evolutionary loss, encouraging re-evaluation of our understanding of formate utilization in Methanosarcinales. To explore whether formate-dependent (methyl-reducing or CO2-reducing) methanogenesis can occur in Methanosarcinales, we engineered two different strains of Methanosarcina acetivorans by functionally expressing FdhAB from Methanosarcina barkeri in M. acetivorans. In the first strain, fdhAB was integrated into the N5-methyl- tetrahydrosarcinapterin:coenzyme M methyltransferase (mtr) operon, making it capable of growing by reducing methanol with electrons from formate. In the second strain, fdhAB was integrated into the F420-reducing hydrogenase (frh) operon, instead of the mtr operon, enabling its growth with formate as the only source of carbon and energy after adaptive laboratory evolution. In this strain, one CO2 is reduced to one methane with electrons from oxidizing four formate to four CO2, a metabolism reported only in methanogens without cytochromes. Although methanogens without cytochromes typically utilize flavin-based electron bifurcation to generate the ferredoxins needed for CO2 activation, we hypothesize that, in our engineered strains, reduced ferredoxins are obtained via the Rhodobacter nitrogen fixation complex complex running in reverse. Our work demonstrates formate-dependent methyl-reducing and CO2-reducing methanogenesis in M. acetivorans that is enabled by the flexible nature of the microbe working in tandem with the nurturing provided.

Corrinoid-dependent methyltransferases catalyze methyl-group transfer reactions in all domains of life. These enzymes are generally considered exclusive for C1-substrates (methyl-groups). However, in Methanosarcina trace ethane production from ethanol has been demonstrated in vivo, which led to the hypothesis that corrinoid-dependent methanol specific methyltransferases are promiscuous towards also accepting ethyl-groups. Here we show that the conversion of ethanol to trace amounts of ethane in Methanosarcina acetivorans proceeds via the known methanol-to-methane metabolism, involving the methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB) and a corrinoid-containing methyl-accepting protein (MtaC), but via transfer of ethyl groups instead of methyl groups. We demonstrate that all three isozymes of the methanol specific MtaB subunit and the corrinoid protein MtaC of M. acetivorans are promiscuous towards accepting ethanol, granting the microbe capacity of ethane production via promiscuity downstream in Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercaptoethanesulfonate methyltransferase (MtaA) and methyl-coenzyme M reductase (Mcr). We assessed the ethyl-group transfer efficiency of each of the three isozymes and engineered chimeras that combine 2 different MtaA subunits with the 3 isoforms of MtaCB together to increase the ethane production capability of M. acetivorans. Demonstrating that corrinoid-dependent coenzyme M methyltransferases can catalyze transfer of higher alkyl groups extends the pool of reactions to be considered in metabolic networks.

Methanogenic archaea are crucial in global carbon cycling as around 1 Gt of the potent greenhouse gas, methane, is produced annually. Major contributors belong to the order Methanosarcinales, which contain some of the most versatile methanogens that are capable of acetotrophic, methylotrophic and CO2-reducing methanogenesis. The genetically tractable model methanogen, Methanosarcina acetivorans , by its nature shows versatility in substrate utilization and energy conservation pathways but cannot utilize formate. In this study, we expanded the primary metabolism of M. acetivorans to include formate-dependent methanogenesis. By introducing an exogenous formate dehydrogenase, the two metabolically engineered M. acetivorans strains acquired the capacity for formate-dependent methanogenesis pathways with one capable of formate-dependent methyl-reduction and the other capable of formate-dependent CO2-reduction. Through nurturing the strain capable of CO2-reduction with adaptive laboratory evolution, we were able to enable growth and methanogenesis of M. acetivorans solely on formate, a metabolism only reported in methanogens without cytochromes which are limited by their versatility. M. acetivorans also showed acetogenic potential where the formate-dependent CO2-reducing strain was able to divert ≈ 10% of carbon to acetate instead of methane. Our results show that even though M. acetivorans lacks energy converting hydrogenase and cannot use H2, it has yet-uncharacterized capacity to obtain reduced ferredoxins from oxidizing formate. Our work encourages reevaluation of our understanding of formate utilization in Methanosarcinales. By enabling formate-dependent methanogenesis, we have expanded the substrate spectrum of a versatile model methanogen with cytochromes to include formate as well.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACT Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli . This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H ( Ec FDH-H). Using an Ec FDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active Ec FDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using Ec FDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 Ec FDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes. IMPORTANCE Oxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO 2 and N 2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter-RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the beta-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter-RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter-RBS combinations. Our promoter-RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds. IMPORTANCE Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for M. acetivorans. Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.

Methyl-Coenzyme M reductase (Mcr) plays an important role in the regulation of the global carbon cycle. The enzyme catalyzes the reversible conversion of methyl-coenzyme M and coenzyme B to methane and the corresponding heterodisulfide CoM-S-S-CoB, which constitutes the first step of the anaerobic oxidation of methane. Mcr homologs of varies methanogenic archaea also catalyze the anaerobic oxidation of extended alkanes. Fully active, purified enzyme is exclusively achievable via strictly anaerobic purification from Methanothermobacter marburgensis . This microbe expresses two isoenzymes and most studies were performed with isoenzyme I that exhibits a limited substrate-promiscuity (c.a. 0.5%) towards the homologous substrate ethyl-coenzyme M. Here, cell-free lysates from the different species such as Methanosarcina mazei, Methanococcus maripaludis, Methanothermococcus okinawansis , and Mt. marburgensis were screened for Mcr activity and substrate promiscuity. An assay relying on titanium (III) citrate and cobalamin to regenerate coenzyme B and coenzyme M was used to test the ability of different cell extracts for the catalytic activity towards the C2-substrate ethyl-coenzyme M. Cell extracts from M. mazei showed a ratio of ethane-to methane-production of ca. 8% at 37 °C, and about 14% at 49 °C. The level of substrate-promiscuity towards ethyl-coenzyme M for M. marburgensis cell extracts under our assay conditions were much higher than previously reported for purified isoenzyme I, indicating that isoenzyme II is much more promiscuous for ethane formation than isoenzyme I. Our experiments demonstrate that Mcr activity can be quickly and conveniently studied via cell-free lysates, and that substrate promiscuity towards ethane formation is generally larger than anticipated.### Competing Interest StatementThe authors have declared no competing interest.

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Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans are biochemically characterized and can be genetically engineered using a variety of molecular tools. Methanogens’ anaerobic lifestyle and autofluorescence, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives) which require oxygen for chromophore maturation. Here, we employ the tandem activation and absorption-shifting tag protein 2 (tdFAST2) which is fluorescent when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5DOM) is present. tdFAST2 expression in M. acetivorans and M. maripaludis is not cytotoxic and tdFAST2:HBR-3,5DOM fluorescence can be clearly distinguished from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be clearly distinguished which allows high-throughput investigations of dynamics within single and mixed cultures.Importance Methane-producing archaea play an essential role in the global carbon cycle and have a high potential for biotechnological applications such as biofuel production, carbon dioxide capture, and in electrochemical systems. The oxygen sensitivity and high autofluorescence hinder the use of common fluorescent proteins to study methanogens. By using the tdFAST2:HBR-3,5DOM fluorescence, which is functional also under anaerobic conditions and distinguishable from the autofluorescence, real-time reporter studies and high-throughput investigation of dynamics within (mixed) cultures via flow cytometry are possible. This will accelerate the exploitation of the methanogens’ biotechnological potential.

Methanogenic archaea play an important role in the global carbon cycle and are regarded as promising host organisms for the biotechnological generation of fuels and chemicals from one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to the availability of genetic tools and its versatile substrate range. Although genome editing in M. acetivorans via CRISPR/Cas9 has already been demonstrated, we now describe a user-friendly CRISPR/Cas12a toolbox that recognizes a T-rich (5′-TTTV) PAM sequence. This new system can manage deletions of 3500 bp (i.e., knockout of the entire frhADGB operon) and heterologous gene insertions with 80% efficiency observed in ten Pur R transformants. Our CRISPR/Cas12a system also enables multiplex genome editing at high efficiency, which helps speed up genetic engineering. Deletions of 100 bp generated on two separate sites of the genome yielded 8/8 correctly edited transformants. Simultaneous gene deletion (100 bp) and replacement (100-bp region replaced by the 2400-bp uidA expression cassette) at a separate site was achieved, with 3/6 of transformants being edited correctly. In combination with the Cas9-based system, our CRISPR/Cas12a toolbox enables targeted genome editing at two sites (guanine-rich and thymine-rich, respectively) and, in so doing, hastens the overall genetic engineering of the Methanosarcinales species.

Methanogenic archaea play an important role in the global carbon cycle and may serve as host organisms for the biotechnological production of fuels and chemicals from CO2 and other one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to its large genome, versatile substrate range, and available genetic tools. Genome editing in M. acetivorans via CRISPR/Cas9 has also been demonstrated. Here, we describe a user-friendly CRISPR/Cas12a toolbox that recognizes T-rich (5 ' -TTTV) PAM sequences. The toolbox can manage deletions of 3,500 bp (i.e., knocking out the entire frhADGB operon) and heterologous gene insertions with positive rates of over 80%. Cas12a-mediated multiplex genome editing was used to edit two separate sites on the chromosome in one round of editing. Double deletions of 100 bp were achieved, with 8/8 of transformants being edited correctly. Simultaneous deletion of 100 bp at one site and replacement of 100 bp with the 2,400 bp uidA expression cassette at a separate site yielded 5/6 correctly edited transformants. Our CRISPR/Cas12a toolbox enables reliable genome editing, and it can be used in parallel with the previously reported Cas9-based system for the genetic engineering of the Methanosarcina species.

Methanogenic and methanotrophic archaea play important roles in the global carbon cycle by interconverting CO2 and methane. To conserve energy from these metabolic pathways that happen close to the thermodynamic equilibrium, specific electron carriers have evolved to balance the redox potentials between key steps. Reduced ferredoxins required to activate CO2 are provided by energetical coupling to the reduction of the high-potential heterodisulfide (HDS) of coenzyme M (2-mercaptoethanesulfonate) and coenzyme B (7-mercaptoheptanoylthreonine phosphate). While the standard redox potential of this important HDS has been determined previously to be -143 mV (Tietze et al. 2003 DOI: 10.1002/cbic.200390053), we have measured thiol disulfide exchange kinetics and reassessed this value by equilibrating thiol-disulfide mixtures of coenzyme M, coenzyme B, and mercaptoethanol. We determined the redox potential of the HDS of coenzyme M and coenzyme B to be -16.4±1.7 mV relative to the reference thiol mercaptoethanol (E0 '=-264 mV). The resulting E0 ' values are -281 mV for the HDS, -271 mV for the homodisulfide of coenzyme M, and -270 mV for the homodisulfide of coenzyme B. We discuss the importance of these updated values for the physiology of methanogenic and methanotrophic archaea and their implications in terms of energy conservation.

Metal-dependent formate dehydrogenases (Me-FDHs) are highly active CO2-reducing enzymes operating at low redox potentials and employ either molybdenum or tungsten to reduce the bound substrate. This makes them suitable for electrochemical applications such as fossil-free production of commodity chemicals utilizing renewable energy. Electrocatalytic CO2 reduction by cathode-immobilized Me-FDHs has been recently demonstrated and rational protein engineering can be used to optimize Me-FDHs for various carbon reduction reactions. In the present study, CO2 reduction by soluble monomeric Escherichia coli formate dehydrogenase H (EcFDH-H) was demonstrated and the function of its nucleophilic selenocysteine residue as a transient ligand of a centrally bound molybdenum atom was investigated. Kinetic analysis of the wildtype enzyme revealed maximum CO2 reduction rates of 44 ± 6 s−1 at pH 5.8 that was decreased to 19% and 0% in the case of selenocysteine substitution with the structural homologues cysteine and serine, respectively. Further selenocysteine-to-cysteine substitution effects included an increased acid tolerance as well as stronger inhibition by nitrate and azide indicating a shift of the Mo oxidation state from IV to VI. Conversely, a destabilizing effect on the oxidized Mo(VI) center could be assigned to the native selenocysteine residue that may facilitate the observed efficient CO2 reduction by rapid transition between Mo oxidation states. Taken together, the performed characterization of EcFDH-H as a catalyst for CO2 reduction and the selenocysteine substitution analysis furthers the understanding of the active-site structure of Me-FDHs and thereby supports the development of more efficient biocatalysts for CO2 reduction.

Significance The reductive acetyl-coenzyme A (acetyl-CoA) pathway is the only carbon fixation pathway that can also be used for energy conservation like it is known for acetogenic bacteria. In methanogenic archaea, this pathway is extended with one route toward acetyl-CoA formation for anabolism and another route toward methane formation for catabolism. Which of these traits is ancestral in evolution has not been resolved. By diverging virtually all substrate carbon from methanogenesis to flow through acetyl-CoA, Methanosarcina acetivorans can be converted to an acetogenic organism. Being able to deconstruct methanogenic into the seemingly simpler acetogenic energy metabolism provides compelling evidence that methanogens are not nearly as metabolically limited as previously thought and suggests that methanogenesis might have evolved from the acetyl-CoA pathway. The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide–dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.