Aerobic and anaerobic metabolism in a facultative anaerobe Ep01 lacking cytochrome, quinone and catalase

Abstract A facultative anaerobe, strain Ep01 produced a mixture of pyruvate, formate, acetate and ethanol from glucose anaerobically, and acetate and pyruvate aerobically. Cell extract of anaerobic-grown cells contained active pyruvate formatelyase, aldehyde dehydrogenase and alcohol dehydrogenase, while cell extract of aerobic grown cells contained an active pyruvate dehydrogenase system, NaDH oxidase and NADH peroxidase. Levels of acetate kinase and phosphate acetyltransferase activities were not significantly different in cells grown under either condition. Based on the metabolic products and the emzyme activities, we propose the presence of two metabolic pathways in strain Ep01, namely, a pathway to form formate, acetate and ethanol under anaerobic conditions, and a pathway to form under aerobic conditions. This explains why strain Ep01 can grow well both under strictly anaerobic conditions and well-aerated conditions.     

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91-96% of the pyruvate towards alpha-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD+ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.     

Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.     

Effect of mycoplasma infection on pyruvate dehydrogenase complex activity of normal and pyruvate dehydrogenase complex-deficient fibroblasts

The fermentative mycoplasmas A. laidlawii JS, M. hyorhinis DBS-50, M. hyorhinis GDL and M. pneumoniae FH have very high apparent activities of pyruvate dehydrogenase (PDH) (EC 1.2.4.1) and pyruvate dehydrogenase complex (PDHC). Infection of normal and PDHC-deficient fibroblasts with these mycoplasma species resulted in a marked increase of the specific activity of these two enzymes, and under certain conditions could conceal the enzymatic defect. The non-fermentative mycoplasmas M. salivarium VV and M. arthritidis PG-6 have very low apparent activities of these two enzymes. Normal fibroblasts infected with non-fermentative mycoplasmas could appear as deficient in these two enzymes. The degree of interference depends on the number of mycoplasmas associated with the harvested cells. Besides the mycoplasma species, this depends (1) on the duration of infection which determines mycoplasmal titers and also can have a killing effect on both host cells and/or mycoplasmas; (2) harvest of the cells by scraping or trypsinization; (3) centrifugal force used in the collection of the cells; (4) washing and the inherent mechanical treatment; and (5) other possibilities.     

Comparative Studies of Glucose Catabolism by Escherichia coli Grown in a Complex Medium under Aerobic and Anaerobic Conditions

Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two-dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end-product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end-product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di-and triphosphates, UDPG, and NAD(H). Two-dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically in complex medium.     

Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775.

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.     

Studies of regulatory metabolism in Moniezia expansa: general considerations.

The activities of selected enzymes in the branched metabolic pathway to succinate or lactate were determined in cytosol and mitochondrial fractions. The enzymes of lowest activity in the cytosol, and thus possibly regulatory, are phosphofructokinase and pyruvate kinase. Malic enzyme activity could scarcely be detected in either compartment; phosphoenolpyruvate carboxykinase and malate dehydrogenase occur in both. The end products of metabolism are succinate and lactate; under anaerobic conditions lactate production increases whereas succinate production shows a small decrease. The presence of glucose in the medium does not influence the change, but causes an increase in total endproduct accumulation. Levels of metabolic intermediates in worms incubated aerobically and anaerobically are presented, and ‘cross-over’ plots and calculations of apparent equilibrium constants identify hexokinase, phosphofructokinase and pyruvate kinase as regulatory. Under aerobic conditions a large increase in the size of the malate pool is observed suggesting that the depression of lactate production is produced by its inhibitory effect on pyruvate kinase. Adenine nucleotide levels are maintained whether or not the worm is incubated under anaerobic conditions.     

Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ ratios.

NADH/NAD+ ratios and internal pyruvate concentrations were determined during switches between aerobic and anaerobic steady-state conditions of glucose-limited chemostat cultures of Enterococcus faecalis. During the switch experiments, changes in catabolic fluxes were observed: transition from anaerobic to aerobic conditions resulted in a complete and instantaneous conversion of glucose into acetate and CO2 via the pyruvate dehydrogenase complex, while during a switch from aerobic to anaerobic conditions the culture became homolactic. A similar switch to a homolactic fermentation was observed upon release of the limitation by addition of a glucose pulse to the culture. In sharp contrast to this, a pyruvate pulse resulted in an increase of both pyruvate formate-lyase and pyruvate dehydrogenase complex activity. Furthermore, acetoin was formed during a pyruvate pulse, probably due to a dramatic increase in internal pyruvate concentration. Regulation of the catabolic fluxes over the various pyruvate-catabolizing enzymes is discussed in view of the observed changes in internal pyruvate concentrations and NADH/NAD+ ratios.     

Anaerobic respiration of superoxide dismutase-deficientSaccharomyces cerevisiae under oxidative stress

The ethanol productivity of superoxide dismutase (SOD)-deficient mutants ofSaccharomyces cerevisiae was examined under the oxidative stress by Paraquat. It was observed that MnSOD-deficient mutant ofS. cerevisiae had higher ethanol productivity than wild type or CuZnSOD-deficient yeast both in aerobic and in anaerobic culture condition. Pyruvate dehydrogenase activity decreased by 35% and alcohol dehydrogenase activity increased by 32% were observed in MnSOD-deficient yeast grown aerobically. When generating oxygen radicals by Paraquat, the ethanol productivity was increased by 40% in CuZnSOD-deficient or wild strain, resulting from increased activity of alcohol dehydrogenase and decreased activity of pyruvate dehydrogenase. However, the addition of ascorbic acid with Paraquat returned the enzyme activities at the level of control. These results imply that SOD-deficiency in yeast strains may cause the metabolic flux to shift into anaerobic ethanol fermentation in order to avoid their oxidative damages by Paraquat.     

Strain variation in Hymenolepis diminuta: enzyme profiles

In comparative study of respiratory metabolism, it was established that the relative proportions of respiratory end-products (succinic, acetic and lactic acids) differed consistently in two strains of Hymenolepis diminuta (Toronto and ANU). The ANU strain produced more lactic acid and less succinic acid under aerobic and anaerobic conditions. In the shift from aerobic to anaerobic conditions both strains compensated by increasing their outputs of succinic acid. The ANU strain possessed significantly higher activities of hexokinase, pyruvate kinase, lactate dehydrogenase, cytosolic and mitochondrial malic enzyme and cytosolic α-glycerophosphate dehy drogenase. The Toronto strain had significantly higher activities of fumarase, succinate dehydrogenase, and fumarate reductase. There were no significant differences in the activities of phosphoenolpyruvate carboxykinase and malic dehydrogenase between strains. The fumarase activity in the Toronto strain was 16 times that of the ANU strain, its K_m (malate) was 0.8mM, as opposed to 2.5 mM, and it was less sensitive to inhibition by NAD or ATP. These observations are consistent with the patterns of end-product formation in the two strains. Ratios of end-products and calculations of approximate redox balance suggest that the Toronto strain may have a greater capacity for aerobic metabolism.     

A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens.

Cell extracts were used to determine the enzymes involved in the intermediary carbon metabolism of several strains of Shewanella putrefaciens. Enzymes of the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) were detected, but those of the Embden-Meyerhof-Parnas pathway were not. While several tricarboxylic acid cycle enzymes were present under both aerobic and anaerobic conditions, two key enzymes (2-oxoglutarate dehydrogenase and pyruvate dehydrogenase) were greatly diminished under anaerobic conditions. Extracts of cell grown anaerobically on formate as the sole source of carbon and energy were positive for hydroxypyruvate reductase, the key enzyme of the serine pathway in other methylotrophs, while no hexulose synthase activity was seen.     

Construction of an Escherichia coli K-12 mutant for homoethanologenic fermentation of glucose or xylose without foreign genes

Conversion of lignocellulosic feedstocks to ethanol requires microorganisms that effectively ferment both hexose and pentose sugars. Towards this goal, recombinant organisms have been developed in which heterologous genes were added to platform organisms such as Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli. Using a novel approach that relies only on native enzymes, we have developed a homoethanologenic alternative, Escherichia coli strain SE2378. This mutant ferments glucose or xylose to ethanol with a yield of 82% under anaerobic conditions. An essential mutation in this mutant was mapped within the pdh operon (pdhR aceEF lpd), which encodes components of the pyruvate dehydrogenase complex. Anaerobic ethanol production by this mutant is apparently the result of a novel pathway that combines the activities of pyruvate dehydrogenase (typically active during aerobic, oxidative metabolism) with the fermentative alcohol dehydrogenase.     

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.     

Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions

In Euglena gracilis, pyruvate:NADP⁺ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP⁺ oxidoreductase. In contrast, pyruvate:NADP⁺ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis.

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

土壤紧实胁迫对黄瓜根系呼吸代谢的影响

用容重分别为1.20和1.55 g·cm^-3的土壤进行盆栽试验,研究了土壤紧实胁迫对‘津春4号’黄瓜根系呼吸代谢的影响.结果表明: 土壤紧实胁迫条件下,黄瓜根系中丙酮酸脱羧酶、乙醇脱氢酶和乳酸脱氢酶活性显著提高;无氧呼吸主要产物(乙醇、乙醛和乳酸)含量显著升高;参与有氧呼吸的苹果酸脱氢酶、琥珀酸脱氢酶和异柠檬酸脱氢酶活性显著下降,丙酮酸和琥珀酸含量显著提高,苹果酸含量显著下降.说明在土壤紧实胁迫条件下,黄瓜根系的有氧呼吸受到显著抑制,无氧呼吸过程加强.     

Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate

Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h(-1). More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, alpha-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.     

Effect of aeration on the activity of gluconeogenetic enzymes in Saccharomyces cerevisiae growing under glucose limitation

1. The effect of aeration on the key enzymes of gluconeogenesis was studied in baker's yeast (Saccharomyces cerevisiae) and in a nonrespiratory variant of S. cerevisiae grown under glucose limitation. 2. In baker's yeast phosphoenolpyruvate carboxykinase, hexosediphophatase and isocitrate lyase were completely repressed under anaerobic conditions. Their repression could be partially reversed by using intense aeration. 3. In the nonrespiratory variant these enzymes were absent independently of aeration. 4. Pyruvate carboxylase of baker's yeast showed maximal activity under anaerobic conditions. In the nonrespiratory variant pyruvate carboxylase had low activity under both anaerobic and aerobic conditions.