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Eubacterium limosum ZL-II was described to convert secoisolariciresinol (SECO) to its demethylating product 4,4′-dihydroxyenterodiol (DHEND) under anoxic conditions. However, the reaction cascade remains unclear. Here, the O -demethylase being responsible for the conversion was identified and characterized. Nine genes encoding two methyltransferase-Is (MT-I), two corrinoid proteins (CP), two methyltransferase-IIs (MT-II), and three activating enzymes (AE) were screened, cloned, and expressed in Escherichia coli . Four of the nine predicted enzymes, including ELI_2003 (MT-I), ELI_2004 (CP), ELI_2005 (MT-II), and ELI_0370 (AE), were confirmed to constitute the O -demethylase in E. limosum ZL-II. The complete O -demethylase (combining the four components) reaction system was reconstructed in vitro. As expected, the demethylating products 3-demethyl-SECO and DHEND were both produced. During the reaction process, ELI_2003 (MT-I) initially catalyzed the transfer of methyl group from SECO to the corrinoid of ELI_2004 ([Co I ]-CP), yielding demethylating products and [CH 3 -Co III ]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH 3 -Co III ]-CP to tetrahydrofolate, forming methyltetrahydrofolate and [Co I ]-CP. Due to the low redox potential of [Co II ]/[Co I ], [Co I ]-CP was oxidized to [Co II ]-CP immediately in vitro, and ELI_0370 (AE) was responsible for catalyzing the reduction of [Co II ]-CP to its active form [Co I ]-CP. The active-site residues in ELI_2003, ELI_2005, and ELI_0370 were subsequently determined using molecular modeling combined with site-directed mutagenesis. To our knowledge, this is the first study on the identification and characterization of a four-component O -demethylase from E. limosum ZL-II, which will facilitate the development of method to artificial synthesis of related bioactive chemicals.

Sustainable production of carbon-based products is urgently needed.A novel directed flow-through microbial electrosynthesis (MES) reactor was designed and characterized for carbon dioxide (CO2) conversion to C2–C6 carboxylates.Three-times denser biofilm, volumetric current density, and productivity were achieved compared with the state of the art.Biomass-specific production rates were maintained over more than 200 days, yet still an order of magnitude lower than that achieved by acetogens in syngas fermentation.Volumetric productivity in MES was comparable with that from syngas fermentation.Clostridium luticellarii and Eubacterium limosum were the dominant species. Carbon-based products are essential to society, yet producing them from fossil fuels is unsustainable. Microorganisms have the ability to take up electrons from solid electrodes and convert carbon dioxide (CO2) to valuable carbon-based chemicals. However, higher productivities and energy efficiencies are needed to reach a viability that can make the technology transformative. Here, we show how a biofilm-based microbial porous cathode in a directed flow-through electrochemical system can continuously reduce CO2 to even-chain C2–C6 carboxylic acids over 248 days. We demonstrate a threefold higher biofilm concentration, volumetric current density, and productivity compared with the state of the art. Most notably, the volumetric productivity (VP) resembles those achieved in laboratory-scale and industrial syngas (CO-H2-CO2) fermentation and chain elongation fermentation. This work highlights key design parameters for efficient electricity-driven microbial CO2 reduction. There is need and room to improve the rates of electrode colonization and microbe-specific kinetics to scale up the technology. Graphical abstract [Display omitted] Microbial electrosynthesis (MES) of carboxylic acids from CO2 and electricity has been validated for over a decade, now reaching Technology Readiness Levels 3/4 in laboratory settings. However, process optimization is needed before demonstrating an industrial prototype. Key challenges for full-scale implementation include ensuring production stability. Critical areas to investigate and demonstrate are: (i) the impact of CO2 feed stream composition and properties; (ii) the short- and long-term effects of renewable electricity supply intermittency; and (iii) the flexibility of MES operations and the integrated process, including up- and downstream processes. Moreover, a comprehensive market analysis is required for each target product. For instance, hexanoic acid, which serves as a precursor for nylon, plasticizers, lubricants, pharmaceuticals, fragrances, fuels, and animal feed, necessitates the development of business models that consider complete supply chains and systems. Carbon-based products are crucial to society. However, their production from fossil-based carbon is unsustainable. We developed a scalable microbial electrosynthesis process that produces medium-chain carboxylic acids from CO2 and renewable electricity, using microorganisms as a catalyst. This represents a promising avenue for generating low CO2-footprint precursors for the chemical, fuel, feed, and food industries.

Abstract Eubacterium limosum is an acetogenic bacterium of potential industrial relevance for its ability to efficiently metabolize a range of single carbon compounds. However, extracellular polymeric substance (EPS) produced by the type strain ATCC 8486 is a serious impediment to bioprocessing and genetic engineering. To remove these barriers, here we bioinformatically identified genes involved in EPS biosynthesis, and targeted several of the most promising candidates for inactivation, using a homologous recombination-based approach. Deletion of a single genomic region encoding homologues for epsABC, ptkA, and tmkA resulted in a strain incapable of producing EPS. This strain is significantly easier to handle by pipetting and centrifugation, and retains important wild-type phenotypes including the ability to grow on methanol and carbon dioxide and limited oxygen tolerance. Additionally, this strain is also more genetically tractable with a 2-fold increase in transformation efficiency compared to the highest previous reports. This work advances a simple, rapid protocol for gene knockouts in E. limosum using only the native homologous recombination machinery. These results will hasten the development of this organism as a workhorse for valorization of single carbon substrates, as well as facilitate exploration of its role in the human gut microbiota. We developed a rapid, simple protocol for gene deletion in the gas-fermenting microbe Eubacterium limosum, and used this to abolish biofilm formation to improve handling and genetic engineering.

Tungsten metabolism was found to be prevalent in the human gut microbiome, which is involved in the detoxification of food and antimicrobial aldehydes, as well as in the production of beneficial SCFAs. In this study, we characterized a protein in the human gut microbe, Eubacterium limosum , that stores tungstate in preparation for its use in enzymes involved in SCFA generation. This revealed several families of tungstate binding proteins that are also involved in tungstate transport and tungstate-dependent regulation and are widely distributed in the human gut microbiome. Elucidating how tungsten is stored and transported in the human gut microbes contributes to our understanding of the human gut microbiome and its impact on human health.

Age-related physiological changes in the gastrointestinal tract, as well as modifications in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota, resulting in a greater susceptibility to infections. By using the Human Intestinal Tract Chip (HITChip) and quantitative PCR of 16S rRNA genes of Bacteria and Archaea, we explored the age-related differences in the gut microbiota composition among young adults, elderly, and centenarians, i.e subjects who reached the extreme limits of the human lifespan, living for over 100 years. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment in facultative anaerobes, notably pathobionts. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammatory status, also known as inflammageing, as determined by a range of peripheral blood inflammatory markers. This may be explained by a remodelling of the centenarians' microbiota, with a marked decrease in Faecalibacterium prauznitzii and relatives, symbiotic species with reported anti-inflammatory properties. As signature bacteria of the long life we identified specifically Eubacterium limosum and relatives that were more than ten-fold increased in the centenarians. We provide evidence for the fact that the ageing process deeply affects the structure of the human gut microbiota, as well as its homeostasis with the host's immune system. Because of its crucial role in the host physiology and health status, age-related differences in the gut microbiota composition may be related to the progression of diseases and frailty in the elderly population.

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in E. limosum is poorly understood, and a system-level characterisation in continuous cultures is yet to be reported. Here, we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280 ± 56 mmol/gDCW/d; gDCW = gramme dry cell weight); however, most carbon went to CO2 (150 ± 11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of phosphotransacetylase (Pta) appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of pyruvate formate ligase (Pfl) to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth.Key pointsFirst Eubacterium limosum steady-state formatotrophic growth omics datasetHigh formate specific uptake rate, however carbon dioxide was the major productFormate may be the cause of intracellular stress and biofilm formation

Background The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. Results In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. Conclusions The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

Background Acetogenic bacteria constitute promising biocatalysts for the conversion of CO 2 /H 2 or synthesis gas (H 2 /CO/CO 2 ) into biofuels and value-added biochemicals. These microorganisms are naturally capable of autotrophic growth via unique acetogenesis metabolism. Despite their biosynthetic potential for commercial applications, a systemic understanding of the transcriptional and translational regulation of the acetogenesis metabolism remains unclear. Results By integrating genome-scale transcriptomic and translatomic data, we explored the regulatory logic of the acetogenesis to convert CO 2 into biomass and metabolites in Eubacterium limosum . The results indicate that majority of genes associated with autotrophic growth including the Wood-Ljungdahl pathway, the reduction of electron carriers, the energy conservation system, and gluconeogenesis were transcriptionally upregulated. The translation efficiency of genes in cellular respiration and electron bifurcation was also highly enhanced. In contrast, the transcriptionally abundant genes involved in the carbonyl branch of the Wood-Ljungdahl pathway, as well as the ion-translocating complex and ATP synthase complex in the energy conservation system, showed decreased translation efficiency. The translation efficiencies of genes were regulated by 5′UTR secondary structure under the autotrophic growth condition. Conclusions The results illustrated that the acetogenic bacteria reallocate protein synthesis, focusing more on the translation of genes for the generation of reduced electron carriers via electron bifurcation, rather than on those for carbon metabolism under autotrophic growth.

One of the bottlenecks of the hydrogen production by dark fermentation is the low yields obtained because of the homoacetogenesis persistence, a metabolic pathway where H2 and CO2 are consumed to produce acetate. The central reactions of H2 production and homoacetogenesis are catalyzed by enzyme hydrogenase and the formyltetrahydrofolate synthetase, respectively. In this work, genes encoding for the formyltetrahydrofolate synthetase (fthfs) and hydrogenase (hydA) were used to investigate the diversity of homoacetogens as well as their phylogenetic relationships through quantitative PCR (qPCR) and next-generation amplicon sequencing. A total of 70 samples from 19 different H2-producing bioreactors with different configurations and operating conditions were analyzed. Quantification through qPCR showed that the abundance of fthfs and hydA was strongly associated with the type of substrate, organic loading rate, and H2 production performance. In particular, fthfs sequencing revealed that homoacetogens diversity was low with one or two dominant homoacetogens in each sample. Clostridium carboxivorans was detected in the reactors fed with agave hydrolisates; Acetobacterium woodii dominated in systems fed with glucose; Blautia coccoides and unclassified Sporoanaerobacter species were present in reactors fed with cheese whey; finally, Eubacterium limosum and Selenomonas sp. were co-dominant in reactors fed with glycerol. Altogether, quantification and sequencing analysis revealed that the occurrence of homoacetogenesis could take place due to (1) metabolic changes of H2-producing bacteria towards homoacetogenesis or (2) the displacement of H2-producing bacteria by homoacetogens. Overall, it was demonstrated that the fthfs gene was a suitable marker to investigate homoacetogens in H2-producing reactors.Key points• qPCR and sequencing analysis revealed two homoacetogenesis phenomena.• fthfs gene was a suitable marker to investigate homoacetogens in H2 reactors.Graphic Abstract

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.