A 14CO2 ratios method for detecting pyruvate carboxylation

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state 14CO2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14CO2 production from two isotope pairs, [2-14C]pyruvate:[3-14C]pyruvate and [1-14C]acetate:[2-14C]acetate. These ratios are defined as the "pyruvate 14CO2 ratio" and the "acetate 14CO2 ratio," respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14CO2 ratio will be less than the acetate 14CO2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14CO2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.     

Proton Magnetic Resonance Spectroscopy of Klebsiella Capsular Polysaccharides

The presence of acetate and pyruvate groups in Klebsiella capsular polysaccharides may be demonstrated and estimated quantitatively by running the proton magnetic resonance spectrum of the polysaccharide (as sodium salt) in deuterium oxide at 95 C. Such spectra also permit an assessment to be made of the number of alpha- and beta-linkages in the repeat unit of the polysaccharide structure.     

Composition of extracellular polysaccharides ofRhizobium trifolii

Analysis of the extracellular polysaccharides of some related strains ofRhizobium trifolii indicated the presence of glucose, galactose, glucuronic acid, pyruvate and acetate. Small differences in composition between strains were not related to the ability to nodulate or to the capacity of the symbiotic organisms to fix nitrogen. I wish to thank R. D. Woods for sedimentation analyses, R. J. Avery and E. Bird for nitrogen and ash determinations and Miss P. Watson for technical assistance.     

Roles of acetate and pyruvate in the metabolism of Streptococcus diacetilactis

Streptococcus diacetilactis required acetate, contained acetate kinase and phosphotransacetylase, and incorporated both radioactive exogenous acetate and acetate from citrate into cell lipids. dl-alpha-Lipoic acid replaced acetate and was required for the oxidation of pyruvate. Stimulation of S. diacetilactis by citrate was found to depend on pyruvate oxidation. Resting cells of the organism produced acetate from 73% of the pyruvate they utilized. However, molar growth yields from glucose were not greater under aerobic compared to anaerobic conditions or when lipoic acid or citrate plus lipoic acid was used in the medium in place of acetate. Data indicate that the growth of S. diacetilactis is limited by the rate of acetyl-coenzyme A synthesis, that the rate of synthesis from pyruvate is higher than the rate from acetate, and that lack of acetyl-coenzyme A not required for growth limits the production of diacetyl and precludes the formation of adenosine triphosphate from acetyl-coenzyme A.     

The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides cells

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate pathway, is studied. It is found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate pathway. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.     

The effect of acetate on the regulation of the branched chain α-keto acid and the pyruvate dehydrogenase complexes in the perfused rat liver

The regulatory consequences of acetate infusion on the pyruvate and the branched chain α-keto acid dehydrogenase reactions in the isolated, perfused rat liver were investigated. Metabolic flux through these two decarboxylation reactions was monitored by measuring the rate of ¹⁴CO₂ production from infused 1-¹⁴C-labeled substrates. When acetate was presented to the liver as the sole substrate the rate of ketogenesis which resulted was maximal at concentrations of acetate in excess of 10 mm. The increase in hepatic ketogenesis during acetate infusion was not accompanied by an alteration of the mitochondrial oxidation-reduction state as measured by the ratio of β-hydroxybutyrate/ acetoacetate in the effluent perfusate. While acetate infusion did not affect the rate of α-keto[1-¹⁴C]isocaproate decarboxylation, the rate of α-keto[1-¹⁴C]isovalerate decarboxylation was stimulated appreciably upon acetate addition. No change was observed in the amount of extractable branched chain α-keto acid dehydrogenase during acetate infusion. The rate of [1-¹⁴C]pyruvate decarboxylation was stimulated in the presence of acetate at low (<1 mm) but not at high (>1 mm) perfusate pyruvate concentrations. The stimulation of the metabolic flux through the pyruvate dehydrogenase reaction upon acetate infusion was accompanied by an increase in the activation state of the pyruvate dehydrogenase complex from 25.7 to 35.6% in the active form. In a liver perfused in the presence of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, at a low concentration of pyruvate (0.05 mm) the infusion of acetate did not affect the rate of pyruvate decarboxylation. As the rate of mitochondrial acetoacetate efflux is increased during acetate infusion the stimulation of pyruvate and α-ketoisovalerate decarboxylation is attributed to an accelerated rate of exchange of mitochondrial acetoacetate for cytosolic pyruvate or α-ketoisovalerate on the monocarboxylate transporter.     

Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80～100 units for mixotrophic cultures grown on acetate and 200～230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.     

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

Fed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO₂ production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.     

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.     

Acetoin production in Leuconostoc mesenteroides NCDO 518

Abstract Cell suspensions of Leuconostoc mesenteroides NCDO 518 converted pyruvate to acetoin and a small amount of lactate and acetate. Acetoin was not produced from mixtures of pyruvate and glucose unless the ratio of pyruvate to glucose was greater than 2:1. In the presence of glucose, external pyruvate was first used as an electron acceptor, being reduced to lactate, and was converted to acetoin only after the exhaustion of glucose. Use of added pyruvate as an electron acceptor suppressed ethanol formation and the products of glucose fermentation were then lactate and acetate; 2 mol of pyruvate per mol of glucose were required to completely suppress ethanol formation. It is suggested that acetoin is produced by heterofermentative organisms when available pyruvate is in excess of that required for reoxidation of all NADH produced during glucose fermentation.     

Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli

Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly ( approximately 10-fold). The acetyl-CoA levels also increased ( approximately 5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.     

Pyruvate metabolism and the phosphorylation state of isocitrate dehydrogenase in Escherichia coli

During growth of Escherichia coli on acetate, isocitrate dehydrogenase (ICDH) is partially inactivated by phosphorylation and is thus rendered rate-limiting in the Krebs cycle so that the intracellular concentration of isocitrate rises which, in turn, permits an increased flux of carbon through the anaplerotic sequence of the glyoxylate bypass. A large number of metabolites stimulate ICDH phosphatase and inhibit ICDH kinase in the wild-type (E. coli ML308) and thus regulate the utilization of isocitrate by the two competing enzymes, ICDH and isocitrate lyase. Addition of pyruvate to acetate grown cultures triggers a rapid dephosphorylation and threefold activation of ICDH, both in the wild-type (ML308) and in mutants lacking pyruvate dehydrogenase (ML308/Pdh-), PEP synthase (ML308/Pps-) or both enzymes (ML308/Pdh-Pps-). Pyruvate stimulates the growth on acetate of those strains with an active PEP synthase but inhibits the growth of those strains that lack this enzyme. When pyruvate is exhausted, ICDH is again inactivated and the growth rate reverts to that characteristic of growth on acetate. Because pyruvate stimulates dephosphorylation of ICDH in strains with differing capabilities for pyruvate metabolism, it seems likely that pyruvate itself is a sufficient signal to activate the dephosphorylation mechanism, but this does not discount the importance of other signals under other circumstances.     

Physiological Role of Pyruvate Carboxylase in a Thermophilic Bacillus

A prototrophic, thermophilic bacillus is in a state of biotin insufficiency when grown in medium consisting of inorganic salts and a carbon source. The effect of this biotin deficiency on the growth rate is severe only if the functioning of pyruvate carboxylase is essential for the utilization of the particular growth substrate. A mutant, PC2, of the thermophile devoid of active pyruvate carboxylase has been isolated. The properties of this mutant confirm the anaplerotic role of this enzyme in the utilization for growth of compounds like glucose and lactate which are catabolized via pyruvate. This conclusion is supported by the finding that revertants isolated from strain PC2 have regained simultaneously the ability to synthesize active pyruvate carboxylase and the ability to utilize glucose or lactate for growth. The growth of mutant PC2 on acetate, unlike that of the parent wild type, is inhibited when glucose or lactate is added to the medium. Secondary mutants obtained from PC2, which are resistant to such inhibition, still carry the original pyruvate carboxylase lesion but are derepressed for isocitrate lyase. This suggests that the inhibition of the growth of mutant PC2 is due to a block in the functioning of the glyoxylate cycle, produced by the glucose or lactate supplement.     

Pathway of Succinate and Propionate Formation in Bacteroides fragilis

Cell suspensions of Bacteroides fragilis were allowed to ferment glucose and lactate labeled with (14)C in different positions. The fermentation products, propionate and acetate, were isolated, and the distribution of radioactivity was determined. An analysis of key enzymes of possible pathways was also made. The results of the labeling experiments showed that: (i) B. fragilis ferments glucose via the Embden-Meyerhof pathway; and (ii) there was a randomization of carbons 1, 2, and 6 of glucose during conversion to propionate, which is in accordance with propionate formation via fumarate and succinate. The enzymes 6-phosphofrucktokinase (pyrophosphate-dependent), fructose-1,6-diphosphate aldolase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, and methylmalonyl-coenzyme A mutase could be demonstrated in cell extracts. Their presence supported the labeling results and suggested that propionate is formed from succinate via succinyl-, methylmalonyl-, and propionyl-coenzyme A. From the results it also is clear that CO(2) is necessary for growth because it is needed for the formation of C4 acids. There was also a randomization of carbons 1, 2, and 6 of glucose during conversion to acetate, which indicated that pyruvate kinase played a minor role in pyruvate formation from phosphoenolpyruvate. Phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase, and malic enzyme (nicotinamide adenine dinucleotide phosphate-dependent) were present in cell extracts of B. fragilis, and the results of the labeling experiments agreed with pyruvate synthesis via oxaloacetate and malate if these acids are in equilibrium with fumarate. The conversion of [2-(14)C]- and [3-(14)C]lactate to acetate was not associated with a randomization of radioactivity.     

13C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance 13C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The L-alanine level increases further upon incubation with the non-permissive substrate D-glucose. L-Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with D-glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on D-fructose and L-glutamate, alanine also accumulates within 3 h upon transfer to D-glucose.     

Effect of glucose on potassium contracture tension in rat heart

The effect of substrates on potassium contracture tension of the isolated rat ventricle strip was investigated. The contracture tension magnitude of ventricle strips exposed to potassium rich medium was markedly greater with medium containing pyruvate or acetate than with glucose as the substrate. The effect of substrates on contracture tension was not related to their ability to maintain the ATP stores of the heart, for there was not a significant difference in the ATP levels in ventricle strips incubated in medium containing pyruvate, pyruvate + glucose, or glucose. Glucose reduced the K-contracture tension magnitude of heart strips suspended in medium containing pyruvate or acetate as substrate; 3-0-methylglucose and 2-deoxy-D-glucose did not have this action on K-contracture tension. The reduction of K-contracture tension by glucose was inhibited by iodoacetate and fluoride. Under anaerobic conditions, 50 mM glucose significantly reduced the K-contracture tension of ventricle strips suspended in pyruvate medium. The findings of this study suggest that glucose metabolism has an action on K-contracture tension in cardiac muscle that is not shared by acetate or pyruvate.     

Pyruvate as a substrate for growth and methanogenesis forMethanosarcina barkeri

Methanosarcina barkeri strains (227, MS, and UBS) were tested for their ability to utilize pyruvate for growth and methanogenesis. All three strains grown on methanol required 4–5 weeks of adaptation for growth on pyruvate, whereas they required only 2–3 weeks of adaptation for growth on acetate. The adapted cells had a lag of 3–4 days for growth on acetate and 5–10 days for growth on pyruvate. Equimolar amounts of methane were produced from acetate, whereas 0.6–0.7 mol of methane was produced per mol of pyruvate. The optimal concentration of pyruvate for growth and methanogenesis for all three strains was 100 mM, and doubling times were in the range of 135–170 h.     

Growth of Methanosarcina barkeri (Fusaro) under nonmethanogenic conditions by the fermentation of pyruvate to acetate: ATP synthesis via the mechanism of substrate level phosphorylation.

A mutant of Methanosarcina barkeri (Fusaro) is able to grow on pyruvate as the sole carbon and energy source. During growth, pyruvate is converted to CH4 and CO2, and about 1.5 mol of ATP per mol of CH4 is formed (A.-K. Bock, A. Prieger-Kraft, and P. Schönheit, Arch. Microbiol. 161:33-46, 1994). The pyruvate-utilizing mutant of M. barkeri could also grow on pyruvate when methanogenesis was completely inhibited by bromoethanesulfonate (BES). The mutant grew on pyruvate (80 mM) in the presence of 2 mM BES with a doubling time of about 30 h up to cell densities of about 400 mg (dry weight) of cells per liter. During growth on pyruvate, the major fermentation products were acetate and CO2 (about 0.9 mol each per mol of pyruvate). Small amounts of acetoin, acetolactate, alanine, leucine, isoleucine, and valine were also detected. CH4 was not formed. The molar growth yield (Yacetate) was about 9 g of cells (dry weight) per mol of acetate, indicating an ATP yield of about 1 mol/mol of acetate formed. Growth on pyruvate in the presence of BES was limited; after six to eight generations, the doubling times increased and the final cell densities decreased. After 9 to 11 generations, growth stopped completely. In the presence of BES, suspensions of pyruvate-grown cells fermented pyruvate to acetate, CO2, and H2. CH4 was not formed. Conversion of pyruvate to acetate, in the complete absence of methanogenesis, was coupled to ATP synthesis. Dicyclohexylcarbodiimide, an inhibitor of H(+)-translocating ATP synthase, did not inhibit ATP formation. In the presence of dicyclohexylcarbodiimide, stoichiometries of up to 0.9 mol of ATP per mol of acetate were observed. The uncoupler arsenate completely inhibited ATP synthesis, while the rates of acetate, CO2, and H2 formation were stimulated up to fourfold. Cell extracts of M. barkeri grown on pyruvate under nonmethenogenic conditions contained pyruvate: ferredoxin oxidoreductase (0.5 U/mg), phosphate acetyltransferase (12 U/mg), and acetate kinase (12 U/mg). From these data it is concluded that ATP was synthesized by substrate level phosphorylation during growth of the M. barkeri mutant on pyruvate in the absence of methanogenesis. This is the first report of growth of a methanogen under nonmethanogenic conditions at the expense of a fermentative energy metabolism.     

Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroides and role of pyruvate dehydrogenase in anaerobic fermentation

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate --> 1 lactate + 1 acetate + 1 CO(2)). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.     

Metabolism of pyruvate and citrate in lactobacilli

Lactobacillus acidophilus, L. bulgaricus, L. casei, L. delbrueckii , L. lactis and L. plantarum contained a pyruvate oxidase for the oxidation of pyruvate to acetyl phosphate and acetate. The presence of an acetate kinase converted the acetyl phosphate to acetate. L. casei and L. plantarum produced lactate and acetoin, in addition to acetate, under the conditions used while L. casei also produced diacetyl. L. casei and L. plantarum were the only species to utilize citrate. L. helveticus and L. helveticus subsp. jugurti did not utilize pyruvate under the conditions used.