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1.
Appl Microbiol Biotechnol ; 105(14-15): 5861-5872, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34331557

ABSTRACT

In times of global climate change and the fear of dwindling resources, we are facing different considerable challenges such as the replacement of fossil fuel-based energy carriers with the coincident maintenance of the increasing energy supply of our growing world population. Therefore, CO2 capturing and H2 storing solutions are urgently needed. In this study, we demonstrate the production of a functional and biotechnological interesting enzyme complex from acetogenic bacteria, the hydrogen-dependent CO2 reductase (HDCR), in the well-known model organism Escherichia coli. We identified the metabolic bottlenecks of the host organisms for the production of the HDCR enzyme complex. Here we show that the recombinant expression of a heterologous enzyme complex transforms E. coli into a whole-cell biocatalyst for hydrogen-driven CO2 reduction to formate without the need of any external co-factors or endogenous enzymes in the reaction process. This shifts the industrial platform organism E. coli more and more into the focus as biocatalyst for CO2-capturing and H2-storage. KEY POINTS: • A functional HDCR enzyme complex was heterologously produced in E. coli. • The metabolic bottlenecks for HDCR production were identified. • HDCR enabled E. coli cell to capture and store H2 and CO2 in the form of formate.


Subject(s)
Acetobacterium , Hydrogen , Carbon Dioxide , Deuterium , Escherichia coli/genetics , Formates
2.
Front Microbiol ; 9: 2911, 2018.
Article in English | MEDLINE | ID: mdl-30564206

ABSTRACT

Hydrogenases are key enzymes of the energy metabolism of many microorganisms. Especially in anoxic habitats where molecular hydrogen (H2) is an important intermediate, these enzymes are used to expel excess reducing power by reducing protons or they are used for the oxidation of H2 as energy and electron source. Despite the fact that hydrogenases catalyze the simplest chemical reaction of reducing two protons with two electrons it turned out that they are often parts of multimeric enzyme complexes catalyzing complex chemical reactions with a multitude of functions in the metabolism. Recent findings revealed multimeric hydrogenases with so far unknown functions particularly in bacteria from the class Clostridia. The discovery of [FeFe] hydrogenases coupled to electron bifurcating subunits solved the enigma of how the otherwise highly endergonic reduction of the electron carrier ferredoxin can be carried out and how H2 production from NADH is possible. Complexes of [FeFe] hydrogenases with formate dehydrogenases revealed a novel enzymatic coupling of the two electron carriers H2 and formate. These novel hydrogenase enzyme complex could also contribute to biotechnological H2 production and H2 storage, both processes essential for an envisaged economy based on H2 as energy carrier.

3.
Biotechnol Biofuels ; 11: 237, 2018.
Article in English | MEDLINE | ID: mdl-30186365

ABSTRACT

BACKGROUND: Replacing fossil fuels as energy carrier requires alternatives that combine sustainable production, high volumetric energy density, easy and fast refueling for mobile applications, and preferably low risk of hazard. Molecular hydrogen (H2) has been considered as promising alternative; however, practical application is struggling because of the low volumetric energy density and the explosion hazard when stored in large amounts. One way to overcome these limitations is the transient conversion of H2 into other chemicals with increased volumetric energy density and lower risk hazard, for example so-called liquid organic hydrogen carriers such as formic acid/formate that is obtained by hydrogenation of CO2. Many homogenous and heterogenous chemical catalysts have been described in the past years, however, often requiring high pressures and temperatures. Recently, the first biocatalyst for this reaction has been described opening the route to a biotechnological alternative for this conversion. RESULTS: The hydrogen-dependent CO2 reductase (HDCR) is a highly active biocatalyst for storing H2 in the form of formic acid/formate by reversibly catalyzing the hydrogenation of CO2. We report the identification, isolation, and characterization of the first thermostable HDCR operating at temperatures up to 70 °C. The enzyme was isolated from the thermophilic acetogenic bacterium Thermoanaerobacter kivui and displays exceptionally high activities in both reaction directions, substantially exceeding known chemical catalysts. CO2 hydrogenation is catalyzed at mild conditions with a turnover frequency of 9,556,000 h-1 (specific activity of 900 µmol formate min-1 mg-1) and the reverse reaction, H2 + CO2 release from formate, is catalyzed with a turnover frequency of 9,892,000 h-1 (930 µmol H2 min-1 mg-1). The HDCR of T. kivui consists of a [FeFe] hydrogenase subunit putatively coupled to a tungsten-dependent CO2 reductase/formate dehydrogenase subunit by an array of iron-sulfur clusters. CONCLUSIONS: The discovery of the first thermostable HDCR provides a promising biological alternative for a chemically challenging reaction and might serve as model for the better understanding of catalysts able to efficiently reduce CO2. The catalytic activity for reversible CO2 hydrogenation of this enzyme is the highest activity known for bio- and chemical catalysts and requiring only ambient temperatures and pressures. The thermostability provides more flexibility regarding the process parameters for a biotechnological application.

4.
Biotechnol Biofuels ; 11: 93, 2018.
Article in English | MEDLINE | ID: mdl-29619089

ABSTRACT

BACKGROUND: Molecular hydrogen (H2) is an attractive future energy carrier to replace fossil fuels. Biologically and sustainably produced H2 could contribute significantly to the future energy mix. However, biological H2 production methods are faced with multiple barriers including substrate cost, low production rates, and low yields. The C1 compound formate is a promising substrate for biological H2 production, as it can be produced itself from various sources including electrochemical reduction of CO2 or from synthesis gas. Many microbes that can produce H2 from formate have been isolated; however, in most cases H2 production rates cannot compete with other H2 production methods. RESULTS: We established a formate-based H2 production method utilizing the acetogenic bacterium Acetobacterium woodii. This organism can use formate as sole energy and carbon source and possesses a novel enzyme complex, the hydrogen-dependent CO2 reductase that catalyzes oxidation of formate to H2 and CO2. Cell suspensions reached specific formate-dependent H2 production rates of 71 mmol gprotein-1 h-1 (30.5 mmol gCDW-1 h-1) and maximum volumetric H2 evolution rates of 79 mmol L-1 h-1. Using growing cells in a two-step closed batch fermentation, specific H2 production rates reached 66 mmol gCDW-1 h-1 with a volumetric H2 evolution rate of 7.9 mmol L-1 h-1. Acetate was the major side product that decreased the H2 yield. We demonstrate that inhibition of the energy metabolism by addition of a sodium ionophore is suitable to completely abolish acetate formation. Under these conditions, yields up to 1 mol H2 per mol formate were achieved. The same ionophore can be used in cultures utilizing formate as specific switch from a growing phase to a H2 production phase. CONCLUSIONS: Acetobacterium woodii reached one of the highest formate-dependent specific H2 productivity rates at ambient temperatures reported so far for an organism without genetic modification and converted the substrate exclusively to H2. This makes this organism a very promising candidate for sustainable H2 production and, because of the reversibility of the A. woodii enzyme, also a candidate for reversible H2 storage.

5.
Appl Environ Microbiol ; 82(14): 4056-4069, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27208103

ABSTRACT

Acetogenic bacteria are a diverse group of strictly anaerobic bacteria that utilize the Wood-Ljungdahl pathway for CO2 fixation and energy conservation. These microorganisms play an important part in the global carbon cycle and are a key component of the anaerobic food web. Their most prominent metabolic feature is autotrophic growth with molecular hydrogen and carbon dioxide as the substrates. However, most members also show an outstanding metabolic flexibility for utilizing a vast variety of different substrates. In contrast to autotrophic growth, which is hardly competitive, metabolic flexibility is seen as a key ability of acetogens to compete in ecosystems and might explain the almost-ubiquitous distribution of acetogenic bacteria in anoxic environments. This review covers the latest findings with respect to the heterotrophic metabolism of acetogenic bacteria, including utilization of carbohydrates, lactate, and different alcohols, especially in the model acetogen Acetobacterium woodii Modularity of metabolism, a key concept of pathway design in synthetic biology, together with electron bifurcation, to overcome energetic barriers, appears to be the basis for the amazing substrate spectrum. At the same time, acetogens depend on only a relatively small number of enzymes to expand the substrate spectrum. We will discuss the energetic advantages of coupling CO2 reduction to fermentations that exploit otherwise-inaccessible substrates and the ecological advantages, as well as the biotechnological applications of the heterotrophic metabolism of acetogens.


Subject(s)
Acetobacterium/metabolism , Energy Metabolism , Heterotrophic Processes , Acetobacterium/growth & development , Alcohols/metabolism , Anaerobiosis , Carbohydrate Metabolism , Carbon Dioxide/metabolism , Hydrogen/metabolism , Lactic Acid/metabolism
6.
FEBS J ; 283(7): 1311-22, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26833643

ABSTRACT

Interconversion of CO2 and formic acid is an important reaction in bacteria. A novel enzyme complex that directly utilizes molecular hydrogen as electron donor for the reversible reduction of CO2 has recently been identified in the Wood-Ljungdahl pathway of an acetogenic bacterium. This pathway is utilized for carbon fixation as well as energy conservation. Here we describe the further characterization of the quaternary structure of this enzyme complex and the unexpected behavior of this enzyme in polymerizing into filamentous structures. Polymerization of metabolic enzymes into similar structures has been observed only in rare cases but the increasing number of examples point towards a more general characteristic of enzyme functioning. Polymerization of the purified enzyme into ordered filaments of more than 0.1 µm in length was only dependent on the presence of divalent cations. Polymerization was a reversible process and connected to the enzymatic activity of the oxygen-sensitive enzyme with the filamentous form being the most active state.


Subject(s)
Bacterial Proteins/metabolism , Carbon Dioxide/metabolism , Formates/metabolism , Hydrogen/metabolism , Oxidoreductases/metabolism , Acetobacterium/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/ultrastructure , Biocatalysis , Enzyme Stability , Kinetics , Magnesium Sulfate/chemistry , Microscopy, Electron , Oxidoreductases/chemistry , Oxidoreductases/ultrastructure , Protein Multimerization
7.
J Bacteriol ; 198(7): 1058-65, 2016 Jan 19.
Article in English | MEDLINE | ID: mdl-26787767

ABSTRACT

UNLABELLED: The acetogenic bacterium Acetobacterium woodii is able to grow by the oxidation of diols, such as 1,2-propanediol, 2,3-butanediol, or ethylene glycol. Recent analyses demonstrated fundamentally different ways for oxidation of 1,2-propanediol and 2,3-butanediol. Here, we analyzed the metabolism of ethylene glycol. Our data demonstrate that ethylene glycol is dehydrated to acetaldehyde, which is then disproportionated to ethanol and acetyl coenzyme A (acetyl-CoA). The latter is further converted to acetate, and this pathway is coupled to ATP formation by substrate-level phosphorylation. Apparently, the product ethanol is in part further oxidized and the reducing equivalents are recycled by reduction of CO2 to acetate in the Wood-Ljungdahl pathway. Biochemical data as well as the results of protein synthesis analysis are consistent with the hypothesis that the propane diol dehydratase (PduCDE) and CoA-dependent propionaldehyde dehydrogenase (PduP) proteins, encoded by the pdu gene cluster, also catalyze ethylene glycol dehydration to acetaldehyde and its CoA-dependent oxidation to acetyl-CoA. Moreover, genes encoding bacterial microcompartments as part of the pdu gene cluster are also expressed during growth on ethylene glycol, arguing for a dual function of the Pdu microcompartment system. IMPORTANCE: Acetogenic bacteria are characterized by their ability to use CO2 as a terminal electron acceptor by a specific pathway, the Wood-Ljungdahl pathway, enabling in most acetogens chemolithoautotrophic growth with H2 and CO2. However, acetogens are very versatile and can use a wide variety of different substrates for growth. Here we report on the elucidation of the pathway for utilization of ethylene glycol by the model acetogen Acetobacterium woodii. This diol is degraded by dehydration to acetaldehyde followed by a disproportionation to acetate and ethanol. We present evidence that this pathway is catalyzed by the same enzyme system recently described for the utilization of 1,2-propanediol. The enzymes for ethylene glycol utilization seem to be encapsulated in protein compartments, known as bacterial microcompartments.


Subject(s)
Acetobacterium/metabolism , Ethylene Glycol/metabolism , Acetic Acid/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Ethanol/metabolism , Gene Expression Regulation, Bacterial/physiology
8.
J Bacteriol ; 197(2): 382-91, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25384483

ABSTRACT

Acetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO2 in the Wood-Ljungdahl pathway. Here, we show that growth of the acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD. NADH is reoxidized by reducing propionaldehyde to propanol. The potential gene cluster coding for the responsible enzymes includes genes coding for shell proteins of bacterial microcompartments. Electron microscopy revealed the presence of microcompartments as well as storage granules in cells grown on 1,2-PD. Gene clusters coding for the 1,2-PD pathway can be found in other acetogens as well, but the distribution shows no relation to the phylogeny of the organisms.


Subject(s)
Acetobacterium/growth & development , Acetobacterium/metabolism , Propylene Glycol/metabolism , Acetobacterium/ultrastructure
9.
Nat Rev Microbiol ; 12(12): 809-21, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25383604

ABSTRACT

Life on earth evolved in the absence of oxygen with inorganic gases as potential sources of carbon and energy. Among the alternative mechanisms for carbon dioxide (CO2) fixation in the living world, only the reduction of CO2 by the Wood-Ljungdahl pathway, which is used by acetogenic bacteria, complies with the two requirements to sustain life: conservation of energy and production of biomass. However, how energy is conserved in acetogenic bacteria has been an enigma since their discovery. In this Review, we discuss the latest progress on the biochemistry and genetics of the energy metabolism of model acetogens, elucidating how these bacteria couple CO2 fixation to energy conservation.


Subject(s)
Acetates/metabolism , Autotrophic Processes , Bacteria, Anaerobic/metabolism , Carbon Dioxide/metabolism , Energy Metabolism , Bacteria, Anaerobic/genetics , Bacteria, Anaerobic/growth & development , Biomass , Carbon Cycle , Metabolic Networks and Pathways , Models, Biological , Oxidation-Reduction , Thermodynamics
10.
J Biol Chem ; 288(44): 31496-502, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-24045950

ABSTRACT

The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na(+)-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD(+) with the generation of a primary electrochemical Na(+) potential across its cytoplasmic membrane. In previous assays in which Ti(3+) was used to reduce ferredoxin, Na(+) transport was observed, but not a Na(+) dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD(+) reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na(+) dependence of this electron transfer reaction. The Km was 0.2 mm. Na(+) could be partly substituted by Li(+). Na(+) dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na(+) as a coupling ion. Electron transport from reduced ferredoxin to NAD(+) was coupled to electrogenic Na(+) transport, indicating the generation of ΔµNa(+). Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of ΔµNa(+), and was accompanied by Na(+) efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na(+) gradient. The physiological significance of this finding is discussed.


Subject(s)
Acetobacterium/enzymology , Bacterial Proteins/metabolism , Ferredoxin-NADP Reductase/metabolism , Membrane Potentials/physiology , Sodium/metabolism , Carbon Monoxide/metabolism , Hydrogen-Ion Concentration , Lithium/metabolism , Oxidation-Reduction
11.
J Biol Chem ; 287(37): 31165-71, 2012 Sep 07.
Article in English | MEDLINE | ID: mdl-22810230

ABSTRACT

The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.


Subject(s)
Acetobacterium/enzymology , Adenosine Triphosphate , Bacterial Proteins , Hydrogenase , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Catalytic Domain , Ferredoxins/chemistry , Ferredoxins/metabolism , Hydrogen/chemistry , Hydrogen/metabolism , Hydrogenase/chemistry , Hydrogenase/isolation & purification , Hydrogenase/metabolism , Oxidation-Reduction
12.
PLoS One ; 7(3): e33439, 2012.
Article in English | MEDLINE | ID: mdl-22479398

ABSTRACT

Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.


Subject(s)
Acetobacterium/metabolism , Adenosine Triphosphate/biosynthesis , Biosynthetic Pathways , Carbon Dioxide/metabolism , Sodium/metabolism , Acetobacterium/genetics , Acetyl Coenzyme A/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon Cycle , Cell Membrane/metabolism , Energy Metabolism , Ferredoxins/metabolism , Genome, Bacterial/genetics , Models, Biological , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism
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