Metabolism of Yarrowia lipolytica Grown on Ethanol under Conditions Promoting the Production of α-Ketoglutaric and Citric Acids: A Comparative Study of the Central Metabolism Enzymes

A comparative study of the enzymes of tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing -ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.     

Central metabolism in Acinetobacter sp. grown on ethanol

The ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The proportion between these enzymes was different in the exponential and the stationary growth phases. The addition of the C4-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of the microbial exopolysaccharide ethapolan on C2-substrates.     

Some Characteristics of Central Metabolism in Acinetobacter sp. Grown on Ethanol

Ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The ratio of these enzymes was different in the exponential and the stationary growth phases. The addition of the C₄-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of microbial exopolysaccharide ethapolan on C₂-substrates.     

Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different carbon sources

1. The activities of the enzymes of the citric acid cycle, the glyoxylate by-pass and some other enzymes acting on the substrates of these cycles have been measured at the pH of the yeast cell during the aerobic growth of yeast on different carbon sources and in different growth media. 2. Sugars induced an anaerobic type of metabolism as measured by ethanol production. Glucose was much more effective in inducing the anaerobic pathways than was galactose. The production of ethanol by cells grown on pyruvate was very small. 3. Glucose was also a more effective repressor than was galactose of the citric acid-cycle enzymes but both were equally effective in repressing almost completely the enzymes of the glyoxylate by-pass. 4. Disappearance of the sugars from the growth medium resulted in an increase in the activities of the enzymes of the citric acid cycle and in the appearance of substantial activities of the enzymes of the glyoxylate cycle. By contrast, the activities of purely biosynthetic enzymes (glutamate-oxaloacetate transaminase, NADP(+)-linked glutamate dehydrogenase) and of pyruvate decarboxylase were decreased. 5. The 2-oxoglutarate-oxidase system was found to be the least active enzyme of the citric acid cycle. 6. The regulatory control at the levels of pyruvate and acetaldehyde and the control of the citric acid cycle are discussed.     

Dependence of the structure of malate dehydrogenase on the type of metabolism in fresh water filamentous colorless sulfur bacteria of the Beggiatoa species

Major pathways of carbon metabolism were studied in strains D-402 and D-405 of freshwater colorless sulfur bacteria of the genus Beggiatoa grown organotrophically and mixotrophically. The bacteria were found to possess all the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles. When organotrophic growth changed to mixotrophic one, the activity of the TCA cycle enzymes decreased 2- to 3-fold, but the activity of enzymes of the glyoxylate cycle increased threefold. It follows that, in the oxidation of thiosulfate, organic compounds no longer play the leading part in the energy metabolism, and most of electrons that enter the electron transport chain (ETC) derive from inorganic sulfur compounds. A connection was established between the structure and kinetic characteristics of malate dehydrogenase--an enzyme of the TCA and glyoxylate cycles--and the type of carbon metabolism in the strains studied. Malate dehydrogenase in organotrophically grown cells of strains D-402 and D-405 is dimeric, whereas in strain D-402 grown mixotrophically it is tetrameric.     

Stimulation of the glyoxylate shunt in gamma-irradiated banana

Succinic dehydrogenase was the most susceptible among the TCA cycle enzymes to gamma irradiation in preclimacteric banana. Maximum inhibition occurred at the 3rd day after irradiation. Impairment of this activity did not affect operation of the TCA cycle, assessed from the incorporation pattern of acetate [2-¹⁴C] into the organic acids such as citric, malic and succinic. Nevertheless, incorporation into keto acids like glyoxylate, α-keto-glutarate and oxaloacetate showed a difference. The rate of labelling into α-ketoglutarate and oxaloacetate was reduced on the 3rd day while incorporation into glyoxylate was increased indicating the operation of glyoxylate shunt pathway. Studies on the individual enzymes of this pathway, isocitrate lyase and malate synthetase confirmed its operation. The reduction in oxalo-acetate has been attributed to the increased gluconeogenesis.     

Dependence of Malate Dehydrogenase Structure on the Type of Metabolism in Freshwater Filamentous Colorless Sulfur Bacteria of the Genus Beggiatoa

Major pathways of carbon metabolism were studied in strains D-402 and D-405 of freshwater colorless sulfur bacteria of the genus Beggiatoa grown organotrophically and mixotrophically. The bacteria were found to possess all the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles. When organotrophic growth changed to mixotrophic growth, the activity of the TCA cycle enzymes decreased 2- to 3-fold, but the activity of enzymes of the glyoxylate cycle increased threefold. It follows that, in the oxidation of thiosulfate, organic compounds no longer play the leading part in the energy metabolism, and most of electrons that enter the electron transport chain (ETC) derive from inorganic sulfur compounds. A connection was established between the structure and kinetic characteristics of malate dehydrogenase—an enzyme of the TCA and glyoxylate cycles—and the type of carbon metabolism in the strains studied. Malate dehydrogenase in organotrophically grown cells of strains D-402 and D-405 is dimeric, whereas in strain D-402 grown mixotrophically it is tetrameric.     

Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysosporium exposed to exogenous benzoic acid

Intracellular processes of the white-rot basidiomycete Phanerochaete chrysosporium involved in the metabolism of benzoic acid (BA) were investigated at the proteome and metabolome level. Up-regulation of aryl-alcohol dehydrogenase, arylaldehyde dehydrogenase, and cytochrome P450s was observed upon addition of exogenous BA, suggesting that these enzymes play key roles in its metabolism. Intracellular metabolic shifts from the short-cut TCA/glyoxylate bicycle system to the TCA cycle and an increased flux in the TCA cycle indicated activation of the heme biosynthetic pathway and the production of NAD(P)H. In addition, combined analyses of proteome and metabolome clearly indicated the role of trehalose as a storage disaccharide and that the mannitol cycle plays a role in an alternative energy-producing pathway.     

Citric acid cycle biomimic on a carbon electrode

The citric acid cycle is one of the main metabolic pathways living cells utilize to completely oxidize biofuels to carbon dioxide and water. The overall goal of this research is to mimic the citric acid cycle at the carbon surface of an electrode in order to achieve complete oxidation of ethanol at a bioanode to increase biofuel cell energy density. In order to mimic this process, dehydrogenase enzymes (known to be the electron or energy producing enzymes of the citric acid cycle) are immobilized in cascades at an electrode surface along with non-energy producing enzymes necessary for the cycle to progress. Six enzymatic schemes were investigated each containing an additional dehydrogenase enzyme involved in the complete oxidation of ethanol. An increase in current density is observed along with an increase in power density with each additional dehydrogenase immobilized on an electrode, reflecting increased electron production at the bioanode with deeper oxidation of the ethanol biofuel. By mimicking the complete citric acid cycle on a carbon electrode, power density was increased 8.71-fold compared to a single enzyme (alcohol dehydrogenase)-based ethanol/air biofuel cell.     

Enzymes of the tricarboxylic acid cycle in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate pathway were investigated in adults and infective larvae of Ancylostoma ceylanicum and Nippostrongylus brasiliensis, and their activities were compared with those obtained in rat liver. A complete sequence of enzymes of the TCA cycle, with most of them showing activities quite similar to those in the rat liver homogenate, was detected in adults of both species. All the enzymes except fumarase and malate dehydrogenase were located predominantly in mitochondria where they showed a variable distribution of activities between the soluble and the membranes fractions. Malate dehydrogenase and fumarase were found in both the mitochondria and the 9,000-g supernatant fraction. Succinyl CoA synthetase, which was present in minimum activity, appeared rate limiting. Enzymes of the glyoxylate pathway, particularly isocitrate lyase, seemed to aid the functioning of the Krebs cycle by allowing the formation of succinate from isocitrate. The infective larvae of both species also were found equipped with all the enzymes of the Krebs cycle. Nonetheless, only isocitrate lyase of the glyoxylate pathway could be detected in these parasites.     

Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens.

Oxalic acid plays a pivotal role in the adaptation of the soil microbe Pseudomonas fluorescens to aluminum (Al) stress. Its production via the oxidation of glyoxylate necessitates a major reconfiguration of the enzymatic reactions involved in the tricarboxylic acid (TCA) cycle. The demand for glyoxylate, the precursor of oxalic acid appears to enhance the activity of isocitrate lyase (ICL). The activity of ICL, an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells. However, the activity of isocitrate dehydrogenase, a competitor for the substrate isocitrate, appeared to be diminished in cells exposed to Al compared to the control cells. While the demand for oxalate in Al-stressed cells also negatively influenced the activity of the enzyme alpha-ketoglutarate dehydrogenase complex, no apparent change in the activity of malate synthase was recorded. Thus, it appears that the TCA cycle is tailored in order to generate the necessary precursor for oxalate synthesis as a consequence of Al-stress.     

Directing vanillin production from ferulic acid by increased acetyl-CoA consumption in recombinant Escherichia coli

The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5alpha (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5alpha (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli.     

A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes

 Biochemical roles of the representative enzymes involved in carbon metabolism of glucose were investigated in relation to the fruit-body formation of the basidiomycete Flammulina velutipes. Changes in specific activities of the enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate (GLOX) and gluconeogenesis pathways were measured at different stages of development of the fungus. The enzyme activities of malate synthase (MS) and fructose bisphosphatase (FBP) as the key enzymes for the GLOX-gluconeogenesis pathways increased in mycelia during the fruit-body formation. The activities of isocitrate dehydrogenase (IDH) for the TCA cycle and NADP-linked glutamate dehydrogenase (GLTDH (NADP)) for glutamate synthesis increased more markedly. Moreover, the mycelial mat of the cultures producing fruit bodies yielded greater enzyme activities of isocitrate lyase (ICL), MS, FBP, and IDH than that of the cultures that did not produce fruit bodies. These results suggest that the GLOX-gluconeogenesis pathways as well as the glutamate synthesis have a strong correlation with the fruit-body formation of F. velutipes. Received: January 22, 2002 / Accepted: May 10, 2002     

Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase

1. The effects of glyoxylate on partially purified preparations of aconitate hydratase, isocitrate dehydrogenase and oxoglutarate dehydrogenase were compared with those of oxalomalate and hydroxyoxoglutarate (obtained by condensation of glyoxylate with oxaloacetate and pyruvate respectively). 2. Glyoxylate (1mm) did not affect aconitate hydratase and isocitrate dehydrogenase, whereas oxalomalate (1mm) inhibited the enzyme activities completely. 3. Glyoxylate (0.025mm) inhibited oxoglutarate dehydrogenase irreversibly, whereas the same concentrations of oxalomalate and hydroxyoxoglutarate were ineffective. This inhibitory effect was prevented if oxoglutarate, pyruvate or oxaloacetate was mixed with the enzyme before the glyoxylate. 4. Incubation of oxoglutarate dehydrogenase with radioactive glyoxylate produced radioactive carbon dioxide; radioactivity was also recovered in the portion of the enzyme identified with thiamin pyrophosphate. 5. The behaviour of glyoxylate in producing multiple inhibitions of the citric acid cycle, either by direct interaction with oxoglutarate dehydrogenase, or by means of its condensation compounds which inhibit aconitate hydratase and isocitrate dehydrogenase, is discussed.     

Biochemical characterization of the yeast Yarrowia lipolytica overproducing carboxylic acids from ethanol. Nitrogen metabolism enzymes

A comparative assay of nitrogen metabolism enzymes in the Yarrowia lipolytica mutant N1 grown under conditions promoting the overproduction of either alpha-ketoglutaric acid (KGA) or citric acid showed that the overproduction of KGA correlates with an increase in the activities of the NAD- and NADP-linked glutamate dehydrogenase, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase reactions. These reactions are likely to be responsible for the overproduction of KGA by this mutant. In contrast, the overproduction of citric acid correlated with a decline in the activities of the NAD- and NADP-linked glutamate dehydrogenases and with an increase in the activities of glutamine synthetase and glutamate synthase.     

Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids

The yeast Yarrowia lipolytica is one of the most intensively studied “non-conventional” yeast species. Its ability to secrete various organic acids, like pyruvic (PA), citric, isocitric, and alpha-ketoglutaric (KGA) acid, in large amounts is of interest for biotechnological applications. We have studied the effect of the alpha-ketoglutarate dehydrogenase (KGDH) complex on the production process of KGA. Being well studied in Saccharomyces cerevisiae this enzyme complex consists of three subunits: alpha-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and lipoamide dehydrogenase. Here we report the effect of overexpression of these subunits encoding genes and resulting increase of specific KGDH activity on organic acid production under several conditions of growth limitation and an excess of carbon source in Y. lipolytica. The constructed strain containing multiple copies of all three KGDH genes showed a reduced production of KGA and an elevated production of PA under conditions of KGA production. However, an increased activity of the KGDH complex had no influence on organic acid production under citric acid production conditions.     

Regulation of Enzyme Synthesis of the Glyoxylate,the Citric Acid,and the Methylcitric Acid Cycles in Candida lipolytica

Syntheses of the key enzymes of the glyoxylate cycle, in Candida lipolytica, were highly repressed by glucose. Syntheses of the key enzymes of the methylcitric acid cycle were also slightly repressed by glucose but the degrees of repression in the syntheses of these enzymes were nearly equal to those of repression in the syntheses of several enzymes of the citric acid cycle. All enzyme syntheses repressed by glucose were derepressed during incubation with succinate as well as with n-alkanes: enzyme syntheses of the methylcitric acid cycle did not necessitate the addition of propionate or odd-carbon n-alkanes. The enzymes of the methylcitric acid cycle seem to be constitutive, similarly as those of the citric acid cycle.

In the parent strain, the respective enzyme levels of the cells grown on an odd-numbered n-alkane were similar to those of the cells grown on an even-numbered n-alkane. But in the mutant strain lacking 2-methylisocitrate lyase, the cells grown on the odd-numbered alkane contained aconitate hydratase, NADP-Iinked isocitrate dehydrogenase, isocitrate lyase, 2- methylcitrate synthase and 2-methylaconitate hydratase all at higher levels than the cells grown on the even-numbered alkane. Both the parent cells and the mutant cells grown on the same carbon source contained at individually similar levels of the following six enzymes; citrate synthase, NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, and malate synthase. The pleiotropic changes of enzyme activities in the mutant cells grown on the odd-numbered alkane seem to be ascribable to direct or indirect stimulation caused by threo-d_s-2-methylisocitric acid accumulation.     

Activity of NAD-dependent isocitrate dehydrogenase, isocitrate lyase, and malate dehydrogenase in Mucor circinelloides var. lusitanicus INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of gamma-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of gamma-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

Adaptation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> to Al-Citrate: Involvement of Tricarboxylic Acid and Glyoxylate Cycle Enzymes and the Influence of Phosphate

The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles. While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase. On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity. This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles. These observations were further confirmed by ¹H NMR and ¹³C NMR spectroscopy. No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1). This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.