The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53

Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner_; however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.     

The influence of carbon substrate on the activity of the periplasmic nitrate reductase in aerobically grown Thiosphaera pantotropha

The periplasmic nitrate reductase was assayed in intact cells of Thiosphaera pantotropha, after aerobic growth with either malate, succinate, acetate, butyrate or caproate present as sole carbon source. The level of enzyme activity was largely dependent upon carbon source and was lowest on malate and succinate, intermediate on acetate and highest on butyrate and caproate. The presence or absence of nitrate did not effect enzyme activity. The results indicate that, during aerobic growth, activity of the periplasmic nitrate reductase increases with the extent of reduction of the carbon substrate.Abbreviation MV⁺ reduced methylviologen     

典型煤层水微生物产甲烷潜力及其群落结构研究

内蒙古自治区二连盆地、海拉尔盆地是我国重要的煤层气产区,其中生物成因煤层气是煤层气的重要来源,但复杂物质转化产甲烷相关微生物群落结构及功能尚不清楚。【目的】研究煤层水中的微生物代谢挥发性脂肪酸产甲烷的生理特征及群落特征。【方法】以内蒙古自治区二连盆地和海拉尔盆地的四口煤层气井水作为接种物,分别添加乙酸钠、丙酸钠和丁酸钠厌氧培养;定期监测挥发性脂肪酸降解过程中甲烷和底物的变化趋势,应用高通量测序技术,分析原始煤层气井水及稳定期产甲烷菌液的微生物群落结构。【结果】除海拉尔盆地H303煤层气井微生物不能代谢丙酸外,其他样品均具备代谢乙酸、丙酸和丁酸产生甲烷的能力,其生理生态参数存在显著差异,产甲烷延滞期依次是乙酸<丁酸<丙酸;最大比产甲烷速率和底物转化效率依次是丙酸<乙酸<丁酸。富集培养后,古菌群落结构与煤层气井水的来源显著相关,二连盆地优势古菌为氢营养型产甲烷古菌Methanocalculus (相对丰度13.5%–63.4%)和复合营养型产甲烷古菌Methanosarcina (7.9%–51.3%),海拉尔盆地的优势古菌为氢营养型产甲烷古菌Methanobact...     

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.     

Synthesis of geraniol esters in a solvent-free system catalysed by Candida antarctica lipase

Summary Candida antarctica lipase was investigated for the synthesis of short chain fatty acid esters of geraniol in a solvent-free system. Maximal activity occurred at 60°C. High yields (about 100%) were obtained with propionate and butyrate, while acetate showed much lower reactivity. The enzyme was used in four consecutive batch reactions with only a 10% loss of activity.     

Isolation from a methanogenic ferulate degrading consortium of an anaerobe that converts methoxyl groups of aromatic acids to volatile fatty acids

An anaerobic, non-motile, rod shaped bacterium is described which cleaves the phenylether bonds of methoxylated aromatic substrates to give the corresponding hydroxy aromatic derivatives and mixed volatile fatty acids, chain length, C₁, C₂ and C₄. The bacterium was isolated from an anaerobic digestor fed with contents from a wood fiber to alcohol fermentation plant, using anaerobic rolltube medium with ferulate as the carbon and energy source. Moles fatty acid produced per 100 mole of methoxyl group of aromatic substrate fermented were approximately: acetate, 14; butyrate, 18; and formate, 15. For the fermentation of equimolar amounts of methoxylated aromatic compounds, growth yields were proportional to the number of methoxylated groups per molecule, and the amount of cells per methoxyl group did not alter when phenylacrylate derivatives were used as substrates. The organism was unable to reduce the side-chain double bond of phenylacrylate derivatives. Coculture of the bacterium on ferulate with Methanospirillum hungatei, or Desulfovibrio in the presence of SO ₄ ⁼ resulted in no nett production of formate, and small quantities of methane and sulfide were produced respectively. The isolate utilized glucose, fructose, and lactate, but not methanol or H₂–CO₂ as growth substrates. Lactate, butyrate, acetate, formate and small quantities of H₂ were produced from glucose fermentation. No reduction of SO ₄ ⁼ or NO ₃ ^- occurred during fermentation of glucose or methoxylated aromatics and no growth occurred in the presence of oxygen.     

A new irreversible enzyme-aided esterification method in organic solvents

A new irreversible esterification method for carboxylic acids catalyzed by a lipase from Candida antarctica (Novozyme 435) in organic solvents has been developed. The water produced during the process is chemically destroyed by a corresponding ester of acetoacetate, which acts as a sacrificial substrate in this reaction. The flavour esters isobutyl acetate, methyl butyrate, ethyl butyrate and benzyl butyrate were synthesized either in small scale (0.05 mol) or large scale (1 mol). The yields range from 82 to 92% within 24 h at 52°C. Optimal molar ratios of reactants were 1:1:1 (carboxylic acid:alcohol:acetoacetate).     

Propionate and butyrate dependent bacterial sulfate reduction at extremely haloalkaline conditions and description of Desulfobotulus alkaliphilus sp. nov.

Evidence on the utilization of simple fatty acids by sulfate-reducing bacteria (SRB) at extremely haloalkaline conditions are practically absent, except for a single case of syntrophy by Desulfonatronum on acetate. Our experiments with sediments from soda lakes of Kulunda Steppe (Altai, Russia) showed sulfide production with sulfate as electron acceptor and propionate and butyrate (but not acetate) as an electron donor at a pH 10–10.5 and a salinity 70–180 g l^?1. With propionate as substrate, a highly enriched sulfidogenic culture was obtained in which the main component was identified as a novel representative of the family Syntrophobacteraceae. With butyrate as substrate, a pure SRB culture was isolated which oxidized butyrate and some higher fatty acids incompletely to acetate. The strain represents the first haloalkaliphilic representative of the family Desulfobacteraceae and is described as Desulfobotulus alkaliphilus sp. nov.     

Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and medium-chain fatty acids and xenobiotic carboxylic acids.

Two distinct forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) were isolated from human liver mitochondria. They were referred to as HXM-A and HXM-B based on their order of elution from a DEAE-cellulose column. Activity of the two ligases was determined toward 15 different carboxylic acids. HXM-A represented 60-80% of the benzoate activity in the lysate, and kinetic analysis revealed that benzoate was the best substrate (highest V(max)/K(m)). The enzyme also had medium-chain fatty acid:CoA ligase activity. HXM-B had the majority of the hexanoate activity and hexanoate was its best substrate. It was, however, also active toward many xenobiotic carboxylic acids. Comparison of these two human XM-ligases with the previously characterized bovine XM-ligases indicated that they were kinetically distinct. When assayed with benzoic acid as substrate, both HXM-A and HXM-B had an absolute dependence on either Mg(2+) or Mn(2+) for activity. Further, addition of monovalent cation (K(+), Rb(+), or NH(4)(+)) stimulated HXM-A activity by >30-fold and HXM-B activity by 4-fold. For both forms, activity toward straight-chain fatty acids was stimulated less by K(+) than was activity toward benzoate or phenylacetate. A 60 kDa short-chain fatty acid:CoA ligase was also isolated. It had activity toward propionate and butyrate, but not acetate, hexanoate or benzoate. The K(m)(app) values were high but similar for propionate and butyrate (285 microM and 250 microM, respectively) but the V(max)(app) was nearly 6-fold greater with propionate as substrate. While the K(m) values are somewhat high, the enzyme is still more efficient with these substrates than either of the XM-ligases.     

Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum

The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l(-1) with a yield of 0.38-0.59 g g(-1) xylose and a reactor productivity up to 3.19 g l(-1)h(-1). However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.     

Short-chain fatty acid metabolism in temperate marine herbivorous fish

Short-chain fatty acids (SCFAs) were identified and estimated in the gut of three herbivorous fish containing gut endosymbionts, the herring cale Odax cyanomelas (Richardson, 1850) (Family Odacidae), the butterfish O. pullus (Bloch and Schneider, 1801) (Family Odacidae) and the sea carp Crinodus lophodon (Günther, 1859) (Family Aplodactylidae). The highest concentrations of short-chain fatty acids were in the posterior region of the intestine in all species. In O. cyanomelas 85% of the total short-chain fatty acids were found in this region. There was a positive correlation between the distribution of short-chain fatty acids and the microorganisms, suggesting that the short-chain fatty acids were end products of microbial anaerobic metabolism. The major short-chain fatty acid in all three species was acetate, the concentration of which ranged from 20 to 29 mmol·1^-1 in the posterior intestine. Lower concentrations of propionate and butyrate were also found. Additionally, valerate was found in the odacids. The ratio of acetate: propionate:butyrate:valerate in the gut section containing the highest concentration of short-chain fatty acids was 83:8:9:1 in O. cyanomelas, 64:21:14:1 in O. pullus and 74:17:9:0 in C. lophodon. Acetate was present in the blood of O. cyanomelas and C. lophodon at concentrations of 1.74±0.17 and 1.79±0.20 mmol·l^-1, respectively. The presence of the enzyme necessary to activate acetate, acetyl CoA synthetase, in the major tissues of both O. cyanomelas and C. lophodon indicates that these fishes are able to utilise acetate produced in the gut. The highest activity of acetyl CoA synthetase, 3.55±0.51 and 6.48±3.18 nmol·s^-1·g tissue^-1 in O. cyanomelas and C. lophodon, respectively, was found in the kidney. Acetyl CoA hydrolase activity was detected in the liver, heart, muscle, gut and kidney of O. cyanomelas and C. lophodon. The highest activity was in the liver of both species, 91.22±9.03 and 57.35±7.15 nmol·s^-1·g tissue^-1 in O. cyanomelas and C. lophodon, respectively. The presence of acetyl CoA hydrolase in tissues of O. cyanomelas and C. lophodon raises the possibility that some of the acetate in the blood could arise from hydrolysis of endogenously produced acetyl CoA. The results strongly support the hypothesis that short-chain fatty acids produced by endosymbionts in the posterior intestine are used as a blood fuel either for energy purposes or for lipid synthesis by the host fish.Abbreviations DTNB 5,5-dithiobis [2-nitrobenzoic acid] - SCUBA self contained underwater breathing apparatus - SCFA short-chain fatty acid - TCA trichloroacetic acid - TRIS TRIS (hydroxymethyl) amino-methane     

A role for thiamine in the regulation of fatty acid and cholesterol biosynthesis in cultured cells of neural origin.

Abstract— Cultured glial (C-6) and neuronal (neuroblastoma) cells were utilized to define the role of thiamine in the regulation of fatty acid and cholesterol biosynthesis. Glial cells subjected to thiamine deficiency exhibited rates of fatty acid synthesis that were only 13% of the rates in thiamine-supple-mented cells. The decrease in fatty acid synthetic rate was accompanied by a comparable decrease in the activities of fatty acid synthetase and acetyl-CoA carboxylase, the two critical enzymes in the pathway. Immunochemical techniques demonstrated that the decrease in activity of fatty acid synthetase reflected a decrease in enzyme content and that this change in content was caused by a decrease in enzyme synthesis. The disturbance of fatty acid synthesis was exquisitely sensitive to thiamine–i.e. marked improvement was evident within hours of replenishment with only 0.01 μ/ml of thiamine. Total recovery occurred in 1–2 days. Thiamine-deficient glia also exhibited reduced rates of cholesterol biosynthesis, i.e. 60% of the rates in thiamine-supplemented cells. This effect was accompanied by a comparable reduction in activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the rate-limiting step in cholesterol biosynthesis. Unlike the glial cells, the neuronal cells exhibited either no or only a slight reduction in lipid synthesis under similar conditions of thiamine deficiency. The data have important implications for the genesis of the neuropathology in states of altered thiamine homeostasis and for the mechanisms of regulation of lipid synthesis.     

Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO₂ is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by ¹⁴N/¹⁵N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in β–oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.     

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.     

Anaerobic degradation of sorbic acid by sulfate-reducing and fermenting bacteria: pentanone-2 and isopentanone-2 as byproducts

Strictly anaerobic bacteria were enriched and isolated from freshwater sediment sources in the presence and absence of sulfate with sorbic acid as sole source of carbon and energy. Strain WoSo1, a Gram-negative vibrioid sulfate-reducing bacterium which was assigned to the species Desulfoarculus (formerly Desulfovibrio) baarsii oxidized sorbic acid completely to CO₂ with concomitant stoichiometric reduction of sulfate to sulfide. This strain also oxidized a wide variety of fatty acids and other organic compounds. A Gram-negative rod-shaped fermenting bacterium, strain AmSo1, fermented sorbic acid stoichiometrically to about equal amounts of acetate and butyrate. At concentrations higher than 10 mM, sorbic acid fermentation led to the production of pentanone-2 and isopentanone-2 (3-methyl-2-butanone) as byproducts. Strain AmSo1 fermented also crotonate and 3-hydroxybutyrate to acetate and butyrate, and hexoses to acetate, ethanol, hydrogen, and formate. The guanine-plus-cytosine content of the DNA was 41.8±1.0 mol%. Sorbic acid at concentrations higher than 5 mM inhibited growth of this strain while strain WoSo1 tolerated sorbic acid up to 10 mM concentration.     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon     

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity

ABSTRACT

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity.     

Effects of Organic Acid Anions on the Growth and Metabolism of Syntrophomonas wolfei in Pure Culture and in Defined Consortia

The effects of organic acid anions on the growth of Syntrophomonas wolfei was determined by varying the initial concentration of the acid anion in the medium. The addition of 15 mM acetate decreased the growth rate of a butyrate-catabolizing coculture containing Methanospirillum hungatei from 0.0085 to 0.0029 per hour. Higher initial acetate concentrations decreased the butyrate degradation rate and the yield of cells of S. wolfei per butyrate degraded. Inhibition was not due to the counter ion or the effect of acetate on the methanogen. Initial acetate concentrations above 25 mM inhibited crotonate-using pure cultures and cocultures of S. wolfei. Benzoate and lactate inhibited the growth of S. wolfei on crotonate in pure culture and coculture. Lactate was an effective inhibitor of S. wolfei cultures at concentrations greater than 10 mM. High concentrations of acetate and lactate altered the electron flow in crotonate-catabolizing cocultures, resulting in the formation of less methane and more butyrate and caproate. The inclusion of the acetate-using methanogen, Methanosarcina barkeri, in a methanogenic butyrate-catabolizing coculture increased both the yield of S. wolfei cells per butyrate degraded and the efficacy of butyrate degradation. Butyrate degradation by acetate-inhibited cocultures occurred only after the addition of Methanosarcina barkeri. These results showed that the metabolism of S. wolfei was inhibited by high levels of organic acid anions. The activity of acetate-using methanogens is important for the syntrophic degradation of fatty acids when high levels of acetate are present.     

1-Butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid β-oxidation

Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid β-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions of the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde dehydrogenase AdhE from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of reversed action of fatty acid β-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.