Butyrate-Producing Bacteria,Including Mucin Degraders,from the Swine Intestinal Tract

To identify bacteria with potential for influencing gut health, 980 anaerobes were cultured from the swine intestinal tract and analyzed for butyrate production. Fifteen isolates in the order Clostridiales produced butyrate and had butyryl coenzyme A (CoA):acetate CoA transferase activity. Three of the isolates grew on mucin, suggesting an intimate association with host intestinal mucosa.     

Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in Butyrate-Producing Bacteria from the Human Large Intestine

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.     

Acetate utilization and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate-producing bacteria from the human large intestine

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.     

Intermediary Metabolism in Clostridium acetobutylicum: Levels of Enzymes Involved in the Formation of Acetate and Butyrate

The levels of seven intermediary enzymes involved in acetate and butyrate formation from acetyl coenzyme A in the saccharolytic anaerobe Clostridium acetobutylicum were investigated as a function of time in solvent-producing batch fermentations. Phosphate acetyltransferase and acetate kinase, which are known to form acetate from acetyl coenzyme A, both showed a decrease in specific activity when the organism reached the solvent formation stage. The three consecutive enzymes thiolase, β-hydroxybutyrylcoenzyme A dehydrogenase, and crotonase exhibited a coordinate expression and a maximal activity after growth had ceased. Only low levels of butyryl coenzyme A dehydrogenase activity were found. Phosphate butyryltransferase activity rapidly decreased after 20 h from 5 to 11 U/mg of protein to below the detection limit (1 mU/mg). Butyrate no longer can be formed, and the metabolic flux may be diverted to butanol. Butyrate kinase showed a 2.5- to 10-fold increase in specific activity after phosphate butyryltransferase activity no longer could be detected. These results suggest that the uptake of acetate and butyrate during solvent formation can not proceed via a complete reversal of the phosphate transferase and kinase reactions. The activities of all enzymes investigated as a function of time in vitro are much higher than the metabolic fluxes through them in vivo. This indicates that none of the maximal activities of the enzymes assayed is rate limiting in C. acetobutylicum.     

Diversity of human colonic butyrate‐producing bacteria revealed by analysis of the butyryl‐CoA:acetate CoA‐transferase gene

Butyrate‐producing bacteria play an important role in the human colon, supplying energy to the gut epithelium and regulating host cell responses. In order to explore the diversity and culturability of this functional group, we designed degenerate primers to amplify butyryl‐CoA:acetate CoA‐transferase sequences from faecal samples provided by 10 healthy volunteers. Eighty‐eight per cent of amplified sequences showed > 98% DNA sequence identity to CoA‐transferases from cultured butyrate‐producing bacteria, and these fell into 12 operational taxonomic units (OTUs). The four most prevalent OTUs corresponded to Eubacterium rectale, Roseburia faecis, Eubacterium hallii and an unnamed cultured species SS2/1. The remaining 12% of sequences, however, belonged to 20 OTUs that are assumed to come from uncultured butyrate‐producing strains. Samples taken after ingestion of inulin showed significant (P = 0.019) increases in Faecalibacterium prausnitzii. Because several of the dominant butyrate producers differ in their DNA % G+C content, analysis of thermal melt curves obtained for PCR amplicons of the butyryl‐CoA:acetate CoA‐transferase gene provides a convenient and rapid qualitative assessment of the major butyrate producing groups present in a given sample. This type of analysis therefore provides an excellent source of information on functionally important groups within the colonic microbial community.     

Kinetic modelling of lactate utilization and butyrate production by key human colonic bacterial species

Butyrate is the preferred energy source for colonocytes and has an important role in gut health; in contrast, accumulation of high concentrations of lactate is detrimental to gut health. The major butyrate-producing bacterial species in the human colon belong to the Firmicutes. Eubacterium hallii and a new species, Anaerostipes coli SS2/1, members of clostridial cluster XIVa, are able to utilize lactate and acetate via the butyryl CoA : acetate CoA transferase route, the main metabolic pathway for butyrate synthesis in the human colon. Here we provide a mathematical model to analyse the production of butyrate by lactate-utilizing bacteria from the human colon. The model is an aggregated representation of the fermentation pathway. The parameters of the model were estimated using total least squares and maximum likelihood, based on in vitro experimental data with E. hallii L2-7 and A. coli SS2/1. The findings of the mathematical model adequately match those from the bacterial batch culture experiments. Such an in silico approach should provide insight into carbohydrate fermentation and short-chain fatty acid cross-feeding by dominant species of the human colonic microbiota.     

Alternative schemes of butyrate production in Butyrivibrio fibrisolvens and their relationship to acetate utilization, lactate production, and phylogeny

Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate, and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production in strain 49 was, however, contradicted by the observations that very low concentrations (< 0.2 mM) of fructose 1,6-bisphosphate gave maximal activity, and continuous cultures did not produce additional lactate when the pH was decreased. The lactate production of strain 49 was clearly inhibited by the presence of acetate in the growth medium. When strain 49 was supplemented with as little as 5 mM acetate, lactate production decreased dramatically, and most of the glucose was converted to butyrate. Strain 49 did not possess butyrate kinase activity, but it had a butyryl-CoA/acetate CoA transferase that converted butyryl-CoA directly to butyrate, using acetate as an acceptor. The transferase had a low affinity for acetate (K _m of 5 mM), and this characteristic explained the acetate stimulation of growth and butyrate formation. Strains D1 and A38 had butyrate kinase but not butyryl-CoA/acetate CoA transferase, and it appeared that this difference could explain the lack of acetate stimulation and lactate production. Based on these results, it is unlikely that B. fibrisolvens would ever contribute significantly to the pool of ruminal lactate. Since relatives of strain 49 (strains Nor37, PI-7, VV1, and OB156, based on 16S rRNA sequence analysis) all had the same method of butyrate production, it appeared that butyryl-CoA/acetate CoA transferase might be a phylogenetic characteristic. We obtained a culture of strain B835 (NCDO 2398) that produced large amounts of lactate and had butyryl-CoA/acetate CoA transferase activity, but this strain had previously been grouped with strains A38 and D1 based on 16S rRNA sequence analysis. Our strain B835 had a 16S rRNA sequence unique from the one currently deposited in GenBank, and had high sequence similarity with strains 49 and Nor37 rather than with strains A38 or D1. Received: 3 December 1998 / Accepted: 18 February 1999     

Uptake and activation of acetate and butyrate in Clostridium acetobutylicum

Summary The pathway for uptake of acids during the solvent formation phase of an acetone-butanol fermentation by Clostridium acetobutylicum ATCC 824 was studied. ¹³C NMR investigations on actively metabolizing cells showed that butyrate can be taken up from the medium and quantitatively converted to butanol without accumulation of intermediates. The activities of acetate phosphotransacetylase, acetate kinase and phosphate butyryltransferase rapidly decreased to very low levels when the organism began to form solvents. This indicates that the uptake of acids does not occur via a reversal of these acid forming enzymes. No short-chain acyl-CoA synthetase activity or butyryl phosphate reducing activity could be detected. Based on our results and a critical analysis of literature data on acetone-butanol fermentations, it is suggested that an acetoacetyl-CoA: acetate (butyrate) CoA-transferase is solely responsible for uptake and activation of acetate and butyrate in C. acetobutylicum. The transferase exhibits a broad carboxylic acid specificity. The key enzyme in the uptake is acetoacetate decarboxylase, which is induced late in the fermentation and pulls the transferase reaction towards formation of acetoacetate. The major implication is that it is not feasible to obtain a batch-wise butanol fermentation without acetone formation and retention of a good yield of butanol.     

Fat metabolism in higher plants: The nonenzymatic acylation of dithiothreitol by acyl coenzyme A

A compound soluble in organic solvents and synthesized from [¹⁴C]acetate by isolated spinach chloroplasts incubated in the dark in the presence of dithiothreitol was shown to be O-acetyl dithiothreitol. The chloroplast system was required for the activation of acetate to acetyl CoA, but the transfer of the acetyl moiety to dithiothreitol was nonenzymatic. The first product of the reaction was shown to be S-acetyl dithiothreitol, but in the presence of an oxidant, simultaneous ring closure and migration of the acetyl group from the thiol to an adjacent hydroxyl group occurred to form an O-acetyl dithiothreitol.The acetyl transfer reaction involving acetyl CoA and dithiothreitol showed a marked pH dependence, being most active at about pH 9 and inoperative below pH 6. All acyl CoAs tested (C₂-C₁₈) rapidly labeled dithiothreitol; acetyl acyl carrier protein, and palmityl acyl carrier protein were much less reactive and free fatty acids were unreactive. The thiol reagents dithioerythritol, glutathione, and cysteine, in addition to dithiothreitol, reacted rapidly with acetyl CoA to form the corresponding acetyl mercaptans. 2-Mercaptoethanol was much less reactive; oxidized dithiothreitol was unreactive. The second-order rate constant for acetyl dithiothreitol synthesis was 12.3 m_?1 min^?1 at pH 8.5 and 30 °C.     

Oxidation of acetate,acetyl CoA and acetylcarnitine by pea mitochondria

Acetylcarnitine was rapidly oxidised by pea mitochondria. (-)-carnitine was an essential addition for the oxidation of acetate or acetyl CoA. When acetate was sole substrate, ATP and Mg²⁺ were also essential additives for maximum oxidation. CoASH additions inhibited the oxidation of acetate, acetyl CoA and acetylcarnitine. It was shown that CoASH was acting as a competitive inhibitor of the carnitine stimulated O₂ uptake. It is suggested that acetylcarnitine and carnitine passed through the mitochondrial membrane barrier with ease but acetyl CoA and CoA did not. Carnitine may also buffer the extra- and intra-mitochondrial pools of CoA. The presence of carnitine acetyltransferase (EC 2.3.1.7) on the pea mitochondria is inferred.     

Enzymology of butyrate formation by Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens is a major butyrate-forming species in the bovine and ovine rumen. The enzymology of butyrate formation from pyruvate was investigated in cell-free extracts of B. fibrisolvens D1. Pyruvate owas oxidized to acetylcoenzyme A (CoA) in the presence of CoA.SH and benzyl viologen or flavin nucleotides. The bacterium uses thiolase, beta-hydroxybutyryl-CoA dehydrogenase, crotonase, and crotonyl-CoA reductase to form butyryl-CoA from acetyl-CoA. Reduction of acetoacetyl-CoA to beta-hydroxybutyryl-CoA was faster with NADH than with NADPH. Crotonyl-CoA was reduced to butyryl-CoA by NADH, but not by NADPH, only in the presence of flavin nucleotides. Reduction of flavin nucleotides by NADH was much slower than the flavin-dependent reduction of crotonyl-CoA. This indicates that flavoproteins rather than free flavin participated in the reduction of crotonyl-CoA. Butyryl-CoA was converted to butyrate by phosphate butyryl transferase and butyrate kinase.     

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g?L^?1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g?L^?1, with a productivity of 0.22 g?L^?1 h^?1. The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of ¹³C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.     

Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes

Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.     

Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii.

Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to beta-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or beta-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in the absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.     

Acetyl coenzyme A biosynthesis in the chloroplast

Acetyl coenzyme A (CoA) biosynthesis in spinach chloroplasts has been investigated by following the incorporation of bicarbonate and acetate into fatty acids under a variety of conditions. Both substrates were readily incorporated into fatty acids in a light-dependent manner by intact photosynthesising chloroplasts, but when the concentrations of these substrates were adjusted to those found in vivo, i.e. 200 M acetate, 10 M bicarbonate, then acetate was found to supply carbon atoms for fatty acids biosynthesis via acetyl CoA at forty times the rate of bicarbonate. It is proposed that extra-chloroplastic free acetate is the pricipal substrate for chloroplasts acetyl CoA biosynthesis in spinach.Abbreviations ACP acyl carrierprotein - CoASH coenzyme A     

A specific and sensitive enzymatic-isotopic microassay of coenzyme A in pineal gland

The relationship between the concentration of CoASH and the activity of serotonin N-acetyltransferase (NAT) was studied in rat pineal glands in culture. A technique for microdetermination of CoASH was developed by utilizing acetyl CoA synthetase and partially purified rat liver NAT. Initially CoASH was acetylated with [1–³H] acetate using acetyl CoA synthetase. Subsequently, the labelled acetyl group was transferred from [1–³H] acetyl CoA to tryptamine forming [1–³H acetyl-tryptamine which was then extracted into chloroform and measured by scintillation spectrometry. A direct relationship appeared to exist between the concentrations of CoASH and [1–³H] acetyltryptamine. This method is sensitive and specific since it can detect as low as 10–15 pmoles of CoASH but not structurally related substances such as acetyl CoA, ADP, cysteamine, or D-pantothenic acid. After treating the rat pineal glands in culture with 10 μM norepinephrine for six hours, the concentration of CoASH was found to decrease significantly from 31.96 ± 0.68 to 24.44 ± 0.37 pmoles/gland, while the activity of NAT increased 68 fold. This inverse relationship indicates that CoASH does not play a direct role in NAT induction although it does protect darktime NAT activity in pineal homogenates against thermal inactivation. The sensitivity and the adaptability of this method can be utilized to measure CoASH in discrete regions of rat brain and in experimental conditions where the micromeasurement of CoASH may be required.     

Effect of Acetate on Molecular and Physiological Aspects of Clostridium beijerinckii NCIMB 8052 Solvent Production and Strain Degeneration

The addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production and also increase glucose utilization by Clostridium beijerinckii NCIMB 8052. RNA and enzyme analyses indicated that coenzyme A (CoA) transferase was highly expressed and has higher activity in C. beijerinckii NCIMB 8052 grown in MP2 medium containing added sodium acetate than in the microorganism grown without sodium acetate. RNA analysis suggested the existence of a sol operon and confirmed the presence of a ptb-buk operon in C. beijerinckii NCIMB 8052. In addition to CoA transferase, C. beijerinckii NCIMB 8052 grown in MP2 medium containing added acetate demonstrated higher acetate kinase- and butyrate kinase-specific activity than when the culture was grown in MP2 medium containing no added acetate. Southern blot analysis with chromosomal DNA isolated from solventogenic and degenerated C. beijerinckii NCIMB 8052 indicated that C. beijerinckii NCIMB 8052 strain degeneration does not involve loss of the CoA transferase genes. The addition of acetate to MP2 medium may induce the expression of the sol operon, which ensures solvent production and prevents strain degeneration in C. beijerinckii NCIMB 8052.     

Detection of acetyl coenzyme A as an early CO2 assimilation intermediate in Methanobacterium

The pivotal role of acetyl coenzyme A in CO₂ assimilation by autotrophic methanogenic bacteria has been demonstrated by pulse-labelling of growing Methanobacterium thermoautotrophicum with ¹⁴CO₂. After very short incubation with ¹⁴CO₂ (1.5 s) approximately 1% of label incorporated into the soluble cell fraction was contained in acetyl coenzyme A. The percentage distribution of ¹⁴C within acetyl CoA markedly decreased with time, which is indicative for acetyl CoA being an immediate ¹⁴CO₂ fixation product. Label in the acetate molecule first appeared in the carboxyl carbon, but the methyl carbon became equally labelled within only 10 s. The acetyl CoA was compared with authentic material by various criterions and its cellular concentration was determined to be 52 M. This small cellular pool size of acetyl CoA as compared to e.g. alanine (6.4 mM) provides an explanation for the observed labelling kinetics. The data are fully consistent with autotrophic carbon assimilation via a total synthesis of acetyl coenzyme A from 2 CO₂.Dedicated to Professor Dr. Gerhart Drews on occasion of his 60th birthday