Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii

International Microbiology - Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major...     

Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.     

Isolation of carbon monoxide dehydrogenase from Acetobacterium woodii and comparison of its properties with those of the Clostridium thermoaceticum enzyme.

An oxygen-labile carbon monoxide dehydrogenase was purified to at least 98% homogeneity from fructose-grown cells of Acetobacterium woodii. Gel filtration and electrophoresis experiments gave molecular weights of 480,000 and 153,000, respectively, of the active enzyme. The molecular weights for the subunits are 80,000 and 68,000; the subunits occur in equal proportion. The small subunit of the A. woodii enzyme differs in size from that of the Clostridium thermoaceticum enzyme; however, the large subunits are similar. The specific activity of the A. woodii enzyme, measured at 30 degrees C and pH 7.6, is 500 mumol of CO oxidized min-1 mg-1 with 20 mM methyl viologen as the electron acceptor. Analysis revealed (number per dimer) iron (9), acid-labile sulfide (12), nickel (1.4), and magnesium or zinc (1). This metal content is quite similar to that of the C. thermoaceticum enzyme (Ragsdale et al., J. Biol. Chem. 258:2364-2369, 1983). The nickel as well as the iron-sulfur clusters are redox-active, as was found for the C. thermoaceticum enzyme (Ragsdale et al., Biochem. Biophys. Res. Commun. 108:658-663, 1982). CO can reduce and CO2 can oxidize the iron-sulfur clusters. The enzyme is inhibited by cyanide, but CO2 in the presence of reduced methyl viologen or CO alone can reverse or prevent this inhibition. Several ferredoxins, flavodoxin, and rubredoxin and some artificial electron carriers were tested for their relative rates of reaction with the CO dehydrogenases from A. woodii, C. thermoaceticum, and Clostridium formicoaceticum. Rubredoxin was by far the most reactive acceptor and is proposed to be the primary natural electron carrier for the acetogenic CO dehydrogenases.     

Single-carbon catabolism in acetogens: analysis of carbon flow in Acetobacterium woodii and Butyribacterium methylotrophicum by fermentation and 13C nuclear magnetic resonance measurement.

The catabolism of methanol, formate, or carbon monoxide to acetate or butyrate or both was examined in two acetogenic bacteria. Butyribacterium methylotrophicum simultaneously transformed methanol and formate mainly to butyrate with concomitant H2 and CO2 production and consumption. In contrast, methanol plus CO was primarily converted to acetate, and only slight amounts of CO2 were produced. In vivo 13C nuclear magnetic resonance analysis of [13C]methanol transformation by B. methylotrophicum indicated that methanol was predominantly incorporated into the methyl of acetate. 13CO2 was produced and then consumed, and butyrate was formed from the condensation of two acetate precursors. The analysis of the position of acetate labeled by a given 13C single-carbon substrate when B. methylotrophicum or Acetobacterium woodii was grown in the presence of a second one-carbon substrate indicated two trends: when methanol was consumed, CO, CO2, or formate predominantly labeled the acetate carboxyl; when CO was consumed, CO2 and formate were principally funneled into the acetate methyl group, and CO remained a better carboxyl precursor. These data suggest a model of acetate synthesis via the combined operation of two readily reversible single-carbon pathways which are linked by CO2.     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii.

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.     

Catechol production by O-demethylation of 2-methoxyphenol using the obligate anaerobe, Acetobacterium woodii

Acetobacterium woodii produced catechol (up to 7.84 mM) by demethylating 2-methoxyphenol during growth in the presence or absence of fructose. The highest product concentrations were obtained when 2-methoxyphenol was the sole energy source but the highest substrate conversion (97%) was obtained in fructose-limited chemostat culture. Growing cells were the most suitable form of the biocatalyst since the catalytic activity was 5-fold higher than in harvested cells.     

Enhanced biotransformation of carbon tetrachloride by Acetobacterium woodii upon addition of hydroxocobalamin and fructose.

The objective of this study was to evaluate the effect of hydroxocobalamin (OH-Cbl) on transformation of high concentrations of carbon tetrachloride (CT) by Acetobacterium woodii (ATCC 29683). Complete transformation of 470 microM (72 mg/liter [aqueous]) CT was achieved by A. woodii within 2.5 days, when 10 microM OH-Cbl was added along with 25.2 mM fructose. This was approximately 30 times faster than A. woodii cultures (live or autoclaved) and medium that did not receive OH-Cbl and 5 times faster than those controls that did receive OH-Cbl, but either live A. woodii or fructose was missing. CT transformation in treatments with only OH-Cbl was indicative of the important contribution of nonenzymatic reactions. Besides increasing the rate of CT transformation, addition of fructose and OH-Cbl to live cultures increased the percentage of [(14)C]CT transformed to (14)CO(2) (up to 31%) and (14)C-labeled soluble materials (principally L-lactate and acetate), while decreasing the percentage of CT reduced to chloroform and abiotically transformed to carbon disulfide. (14)CS(2) represented more than 35% of the [(14)C]CT in the presence of reduced medium and OH-Cbl. Conversion of CT to CO was a predominant pathway in formation of CO(2) in the presence of live cells and added fructose and OH-Cbl. These results indicate that the rate and distribution of products during cometabolic transformation of CT by A. woodii can be improved by the addition of fructose and OH-Cbl.     

Effect of molecular hydrogen and carbon dioxide on chemo-organotrophic growth of Acetobacterium woodii and Clostridium aceticum

During growth of Acetobacterium woodii on fructose, glucose or lactate in a medium containing less than 0.04% bicarbonate, molecular hydrogen was evolved up to 0.1 mol per mol of substrate. Under an H₂-atmosphere growth of A. woodii with organic substrates was completely inhibited whereas under an H₂/CO₂-atmosphere rapid growth occurred. Under these conditions H₂+CO₂ and the organic substrate were utilized simultaneously indicating that A. woodii was able to grow mixotrophically. Clostridium aceticum differed from A. woodii in that H₂ was only evolved in the stationary phase, that the inhibition by H₂ was observed at pH 8.5 but not at pH 7.5, anf that in the presence of fructose and H₂+CO₂ only fructose was utilized.The hydrogenase activity of fructose-grown cells of C. aceticum amounted to only 12% of that of H₂+CO₂-grown cells. With A. woodii a corresponding decrease of the activity of this enzyme was not observed.     

Complexes of horseradish peroxidase with formate, acetate, and carbon monoxide

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the "hydrated" form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP-acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463-468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron-ligand distances of 2.3 and 1.8 A, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 A from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.     

Microbial production of ethanol from carbon monoxide

Production of ethanol from fermentation of CO has received much attention in the last few years with several companies proposing to use CO fermentation in their ethanol production processes. The genomes of two CO fermenters, Clostridium ljungdahlii and Clostridium carboxidivorans, have recently been sequenced. The genetic information obtained from this sequencing is aiding molecular biologists who are enhancing ethanol and butanol production by genetic manipulation. Several studies have optimized media for CO fermentation, which has resulted in enhanced ethanol production. Also, new reactor designs involving the use of hollow fiber membranes have reduced mass transfer barriers that have hampered previous CO fermentation efforts.     

Fermentative production of ethanol from carbon monoxide

'Too much Carbon Monoxide for me to bear…' are the opening lyrics of the CAKE song Carbon Monoxide (from their 2004 album Pressure Chief), and while this may be the case for most living organisms, several species of bacteria both thrive on this otherwise toxic gas, and metabolize it for the production of fuels and chemicals. Indeed CO fermentation offers the opportunity to sustainably produce fuels and chemicals without impacting the availability of food resources or even farm land. Mounting commercial interest in the potential of this process has in turn triggered greater scrutiny of the molecular and genetic basis for CO metabolism, as well as the challenges associated with the implementation and operation of gas fermentation at scale.     

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.     

Tetrahydrofolate enzyme levels in Acetobacterium woodii and their implication in the synthesis of acetate from CO2

Acetate synthesis from CO2 by Acetobacterium woodii may occur as in homoacetate-fermenting clostridia, as indicated by high levels of enzymes of the tetrahydrofolate pathway and by pyruvate-dependent formation of acetate from methyl-B12 and methyltetrahydrofolate.     

Mitochondrial complex I impairment and differential carbon monoxide sensitivity of cytochrome c oxidase in wild type and CMS II mutants of Nicotiana sylvestris

Plant mitochondria unlike their animal counterpart have some unique features with highly branched respiratory chain. The present work was undertaken in order to investigate the effect of loss/dysfunction of plant mitochondrial complex I on the relative flux of electrons through alternative oxidase (AOX) and cytochrome oxidase. Loss of a major subunit of mitochondrial complex I in cytoplasmic male sterile II (CMS II) mutant of Nicotiana sylvestris caused respiratory redox perturbations, as evident from the differential CO sensitivity of cytochrome oxidase. The leaf segments of CMS II mutant when exposed to CO under dark aerobic condition were insensitive to the inhibition of cytochrome oxidase, as against the wild type (WT). The differential CO response of WT and CMS II mutants appeared to be due to differences in the redox state of cytochrome a3 (cyt a3), the terminal electron acceptor during in situ respiration. Cyt a3 appeared to be more in its oxidized form in CMS II and hence unable to form cyt a3-CO complex. Pre-treatment of CMS II leaves with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation increased the CO response. The slight increase in rotenone-insensitive respiration of CMS II could be attributed partly to enhanced flux of electrons through cytochrome pathway to compensate for the loss of phosphorylation site and partly through AOX, which was induced by nitrate.     

Complete degradation of carbohydrate to carbon dioxide and methane by syntrophic cultures of Acetobacterium woodii and Methanosarcina barkeri

Methanosarcina barkeri (strain MS) grew and converted acetate to CO₂ and methane after an adaption period of 20 days. Growth and metabolism were rapid with gas production being comparable to that of cells grown on H₂ and CO₂. After an intermediary growth cycle under a H₂ and CO₂ atmosphere acetateadapted cells were capable of growth on acetate with formation of methane and CO₂. When acetate-adapted Methanosarcina barkeri was co-cultered with Acetobacterium woodii on fructose or glucose as substrate, a complete conversion of the carbohydrate to gases (CO₂ and CH₄) was observed.Abbreviation CMC carboxymethyl cellulose     

Methane formation from fructose by syntrophic associations of Acetobacterium woodii and different strains of methanogens

When Acetobacterium woodii was co-cultured in continuous or in stationary culture with Methanobacterium strain AZ, fructose instead of being converted to 3 mol of acetate was converted to 2 mol of acetate and 1 mol each of carbon dioxide and methane, showing that interspecies hydrogen transfer occurred. In continous culture the organisms formed a close physical association in clumps; the doubling time for each organism was 6h at 33°C. Methane mainly was derived from carbon positions 3 and 4 of the sugar, but other carbons also yielded methane; this was shown to be due to carbon dioxide-acetate exchange reactions by A. woodii in a manner similar to that carried out by Clostridium thermoaceticum. Four other methanogens, Methanobacterium M.o.H. and M.o.H. G, Methanobacterium formicicum, and Methanosarcina barkeri (not acetate-adapted) also produced similar results, when co-cultured with A. woodii.