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Journal of Bacteriology Apr 2013Enterobacteria such as Escherichia coli generate formate, lactate, acetate, and succinate as major acidic fermentation products. Accumulation of these products in the...
Coordination of FocA and pyruvate formate-lyase synthesis in Escherichia coli demonstrates preferential translocation of formate over other mixed-acid fermentation products.
Enterobacteria such as Escherichia coli generate formate, lactate, acetate, and succinate as major acidic fermentation products. Accumulation of these products in the cytoplasm would lead to uncoupling of the membrane potential, and therefore they must be either metabolized rapidly or exported from the cell. E. coli has three membrane-localized formate dehydrogenases (FDHs) that oxidize formate. Two of these have their respective active sites facing the periplasm, and the other is in the cytoplasm. The bidirectional FocA channel translocates formate across the membrane delivering substrate to these FDHs. FocA synthesis is tightly coupled to synthesis of pyruvate formate-lyase (PflB), which generates formate. In this study, we analyze the consequences on the fermentation product spectrum of altering FocA levels, uncoupling FocA from PflB synthesis or blocking formate metabolism. Changing the focA translation initiation codon from GUG to AUG resulted in a 20-fold increase in FocA during fermentation and an ∼3-fold increase in PflB. Nevertheless, the fermentation product spectrum throughout the growth phase remained similar to that of the wild type. Formate, acetate, and succinate were exported, but only formate was reimported by these cells. Lactate accumulated in the growth medium only in mutants lacking FocA, despite retaining active PflB, or when formate could not be metabolized intracellularly. Together, these results indicate that FocA has a strong preference for formate as a substrate in vivo and not other acidic fermentation products. The tight coupling between FocA and PflB synthesis ensures adequate substrate delivery to the appropriate FDH.
Topics: Acetyltransferases; Carboxylic Acids; Escherichia coli; Escherichia coli Proteins; Fermentation; Formates; Gene Expression Regulation, Bacterial; Membrane Transport Proteins
PubMed: 23335413
DOI: 10.1128/JB.02166-12 -
Microbiome Jul 2020Current understanding of the carbon cycle in methanogenic environments involves trophic interactions such as interspecies H transfer between organotrophs and...
BACKGROUND
Current understanding of the carbon cycle in methanogenic environments involves trophic interactions such as interspecies H transfer between organotrophs and methanogens. However, many metabolic processes are thermodynamically sensitive to H accumulation and can be inhibited by H produced from co-occurring metabolisms. Strategies for driving thermodynamically competing metabolisms in methanogenic environments remain unexplored.
RESULTS
To uncover how anaerobes combat this H conflict in situ, we employ metagenomics and metatranscriptomics to revisit a model ecosystem that has inspired many foundational discoveries in anaerobic ecology-methanogenic bioreactors. Through analysis of 17 anaerobic digesters, we recovered 1343 high-quality metagenome-assembled genomes and corresponding gene expression profiles for uncultured lineages spanning 66 phyla and reconstructed their metabolic capacities. We discovered that diverse uncultured populations can drive H-sensitive metabolisms through (i) metabolic coupling with concurrent H-tolerant catabolism, (ii) forgoing H generation in favor of interspecies transfer of formate and electrons (cytochrome- and pili-mediated) to avoid thermodynamic conflict, and (iii) integration of low-concentration O metabolism as an ancillary thermodynamics-enhancing electron sink. Archaeal populations support these processes through unique methanogenic metabolisms-highly favorable H oxidation driven by methyl-reducing methanogenesis and tripartite uptake of formate, electrons, and acetate.
CONCLUSION
Integration of omics and eco-thermodynamics revealed overlooked behavior and interactions of uncultured organisms, including coupling favorable and unfavorable metabolisms, shifting from H to formate transfer, respiring low-concentration O, performing direct interspecies electron transfer, and interacting with high H-affinity methanogenesis. These findings shed light on how microorganisms overcome a critical obstacle in methanogenic carbon cycles we had hitherto disregarded and provide foundational insight into anaerobic microbial ecology. Video Abstract.
Topics: Acetates; Anaerobiosis; Archaea; Chemoautotrophic Growth; Ecosystem; Formates; Methane; Thermodynamics
PubMed: 32709258
DOI: 10.1186/s40168-020-00885-y -
Applied and Environmental Microbiology Aug 1993Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate,...
Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate, propionate, and butyrate as substrates was examined. The propionate and butyrate degradation rates in whole granules were 1.16 and 4.0 mumol/min/g of volatile solids, respectively, and the rates decreased 35 and 25%, respectively, after disintegration of the granules. The effect of adding different hydrogen-oxidizing bacteria (both sulfate reducers and methanogens), some of which used formate in addition to hydrogen, to disintegrated granules was tested. Addition of either Methanobacterium thermoautotrophicum delta H, a hydrogen-utilizing methanogen that does not use formate, or Methanobacterium sp. strain CB12, a hydrogen- and formate-utilizing methanogen, to disintegrated granules increased the degradation rate of both propionate and butyrate. Furthermore, addition of a thermophilic sulfate-reducing bacterium (a Desulfotomaculum sp. isolated in our laboratory) to disintegrated granules improved the degradation of both substrates even more than the addition of methanogens. By monitoring the hydrogen partial pressure in the cultures, a correlation between the hydrogen partial pressure and the degradation rate of propionate and butyrate was observed, showing a decrease in the degradation rate with increased hydrogen partial pressure. No significant differences in the stimulation of the degradation rates were observed when the disintegrated granules were supplied with methanogens that utilized hydrogen only or hydrogen and formate. This indicated that interspecies formate transfer was not important for stimulation of propionate and butyrate degradation.
Topics: Anaerobiosis; Bacillaceae; Biodegradation, Environmental; Butyrates; Butyric Acid; Formates; Hydrogen; Methanobacterium; Propionates; Waste Disposal, Fluid
PubMed: 8368842
DOI: 10.1128/aem.59.8.2546-2551.1993 -
Applied and Environmental Microbiology Oct 2021Escherichia coli is a Gram-negative bacterium that is a workhorse for biotechnology. The organism naturally performs a mixed-acid fermentation under anaerobic conditions...
Escherichia coli is a Gram-negative bacterium that is a workhorse for biotechnology. The organism naturally performs a mixed-acid fermentation under anaerobic conditions where it synthesizes formate hydrogenlyase (FHL-1). The physiological role of the enzyme is the disproportionation of formate into H and CO. However, the enzyme has been observed to catalyze hydrogenation of CO given the correct conditions, and so it has possibilities in bio-based carbon capture and storage if it can be harnessed as a hydrogen-dependent CO reductase (HDCR). In this study, an E. coli host strain was engineered for the continuous production of formic acid from H and CO during bacterial growth in a pressurized batch bioreactor. Incorporation of tungsten, in place of molybdenum, in FHL-1 helped to impose a degree of catalytic bias on the enzyme. This work demonstrates that it is possible to couple cell growth to simultaneous, unidirectional formate production from carbon dioxide and develops a process for growth under pressurized gases. Greenhouse gas emissions, including waste carbon dioxide, are contributing to global climate change. A basket of solutions is needed to steadily reduce emissions, and one approach is bio-based carbon capture and storage. Here, we present our latest work on harnessing a novel biological solution for carbon capture. The Escherichia coli formate hydrogenlyase (FHL-1) was engineered to be constitutively expressed. Anaerobic growth under pressurized H and CO gases was established, and aqueous formic acid was produced as a result. Incorporation of tungsten into the enzyme in place of molybdenum proved useful in poising FHL-1 as a hydrogen-dependent CO reductase (HDCR).
Topics: Bioreactors; Carbon Dioxide; Deuterium; Escherichia coli; Formate Dehydrogenases; Formates; Gases; Molybdenum; Tungsten
PubMed: 34647819
DOI: 10.1128/AEM.00299-21 -
Biochimica Et Biophysica Acta Jan 2001It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited... (Review)
Review
It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.
Topics: Bicarbonates; Electron Spin Resonance Spectroscopy; Electron Transport; Formates; Manganese; Oxidation-Reduction; Oxygen; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Water
PubMed: 11115633
DOI: 10.1016/s0005-2728(00)00222-x -
ACS Synthetic Biology May 2019One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a...
One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.
Topics: Bacterial Proteins; Biocatalysis; Carbon Dioxide; Formate Dehydrogenases; Formates; Glycine; Metabolic Engineering; Plasmids; Saccharomyces cerevisiae
PubMed: 31002757
DOI: 10.1021/acssynbio.8b00464 -
Journal of Industrial Microbiology &... Mar 2015The review describes efforts toward metabolic engineering of production of organic acids. One aspect of the strategy involves the generation of an appropriate amount and... (Review)
Review
The review describes efforts toward metabolic engineering of production of organic acids. One aspect of the strategy involves the generation of an appropriate amount and type of reduced cofactor needed for the designed pathway. The ability to capture reducing power in the proper form, NADH or NADPH for the biosynthetic reactions leading to the organic acid, requires specific attention in designing the host and also depends on the feedstock used and cell energetic requirements for efficient metabolism during production. Recent work on the formation and commercial uses of a number of small mono- and diacids is discussed with redox differences, major biosynthetic precursors and engineering strategies outlined. Specific attention is given to those acids that are used in balancing cell redox or providing reduction equivalents for the cell, such as formate, which can be used in conjunction with metabolic engineering of other products to improve yields. Since a number of widely studied acids derived from oxaloacetate as an important precursor, several of these acids are covered with the general strategies and particular components summarized, including succinate, fumarate and malate. Since malate and fumarate are less reduced than succinate, the availability of reduction equivalents and level of aerobiosis are important parameters in optimizing production of these compounds in various hosts. Several other more oxidized acids are also discussed as in some cases, they may be desired products or their formation is minimized to afford higher yields of more reduced products. The placement and connections among acids in the typical central metabolic network are presented along with the use of a number of specific non-native enzymes to enhance routes to high production, where available alternative pathways and strategies are discussed. While many organic acids are derived from a few precursors within central metabolism, each organic acid has its own special requirements for high production and best compatibility with host physiology.
Topics: Carbon; Formates; Fumarates; Malates; Metabolic Engineering; Metabolic Networks and Pathways; Oxidation-Reduction; Propionates; Succinic Acid
PubMed: 25502283
DOI: 10.1007/s10295-014-1560-y -
Journal of Bacteriology Jun 2019is a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an...
Dissection of the Hydrogen Metabolism of the Enterobacterium Trabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I.
is a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H-evolving Hyd that is most similar to the uncharacterized formate hydrogenlyase (FHL-2 ) complex. The FHL-2 (FHL-2 ) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2 complex catalyzes the disproportionation of formate to CO and H FHL-2 has activity similar to that of the FHL-1 complex in H evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2 operon in the heterologous host has now enabled us to unambiguously prove FHL-2 activity, and it allowed us to characterize the FHL-2 complex biochemically. Although the formate dehydrogenase (FdhH) gene is not contained in the operon, the FdhH is part of the complex, and FHL-2 activity was dependent on the presence of FdhH. Also, in contrast to , can ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H-oxidizing Hyd-2 and the H-forming FHL-2 under these conditions. Biological H production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H producers, and the prototype FHL-1 complex in is well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2 complex, allow a first biochemical characterization of 's fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.
Topics: Bacterial Proteins; Catalysis; Enterobacteriaceae; Fermentation; Formate Dehydrogenases; Formates; Hydrogen; Oxidation-Reduction; Phylogeny
PubMed: 30962355
DOI: 10.1128/JB.00160-19 -
Biomolecules Jan 2020Current methods for the production of esters, including chemical synthesis and extraction from natural sources, are hindered by low yields and environmental pollution....
Current methods for the production of esters, including chemical synthesis and extraction from natural sources, are hindered by low yields and environmental pollution. The enzymatic synthesis of these compounds could help overcome these problems. In this study, phenethyl formate, a commercially valuable formate ester, was synthesized using commercial immobilized lipases. The effects of specific enzymes, enzyme concentration, formic acid:phenethyl alcohol molar ratio, temperature, and solvent were studied in order to optimize the synthesis conditions, which were identified as 15 g/L of Novozym 435 enzyme, a 1:5 formic acid:phenethyl alcohol molar ratio, a 40 °C reaction temperature, and 1,2-dichloroethane as the solvent. Under these conditions, phenethyl formate was obtained in a conversion yield of 95.92%. In addition, when 1,2-dichloroethane was replaced with toluene as the solvent, the enzyme could be recycled for at least 20 reactions with a steady conversion yield above 92%, testifying to the economic aspects of the process. The enzymatic synthesis of phenethyl formate using the proposed method is more environmentally friendly than methods currently employed in academic and laboratory settings. Moreover, the method has the potential to enhance the value-added properties of formic acid owing to its downstream use in the production of commercially essential esters.
Topics: Biocatalysis; Enzymes, Immobilized; Esterification; Esters; Formates; Formic Acid Esters; Phenylethyl Alcohol; Solvents; Temperature
PubMed: 31906270
DOI: 10.3390/biom10010070 -
Nature Communications Mar 2015The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is...
The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efflux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.
Topics: Animals; Formates; Glycolysis; Lactic Acid; Membrane Transport Proteins; Oocytes; Plasmodium falciparum; Protozoan Proteins; Xenopus laevis
PubMed: 25823844
DOI: 10.1038/ncomms7721