Studies on the Respiratory Physiology of Euglena gracilis Cultured on Acetate or Glucose

SYNOPSIS. The glyoxylate cycle operates at a high level in Euglena gracilis when acetate is the only carbon source, and at a low level when glucose is the only carbon source, as indicated by activities of malate synthase. Altho glucose causes a moderate repression of some of the enzymes of the glyoxylate cycle, it neither represses nor inhibits malate synthase. The specific activity of the malic enzyme was about 5-fold greater in acetate-grown Euglena than in glucose-grown cells, but the absolute rate of CO₂ fixation was about twice as great in cells grown on glucose. The respiratory quotient was unity regardless of substrate.     

Characteristics of TCA Cycle of Cycloheximide-resistant Mutant of Zygosaccharomyces soja

Glucose catabolism in Zygosaccharomyces soja was carried out. through fermentation, whereas riboflavin producing mutant obtained by cycloheximicie treatment was found to utilize oxidative mechanisms. For instance, growth of mutant was sustained in minimal medium containing TCA cycle intermediates as the sole carbon source, whereas the mother strain could not grow on such culture media. Furthermore, malonate inhibited the oxidation of succinate with resting cells of mutant. As these results support the existence of TCA cycle in mutant, various enzyme activities relating to TCA cycle were investigated by the use of cell-free extracts of both strains. Aconitase, α-ketoglutaric decarboxylase, succinic dehydrogenase, fumarase, isocitritase and glyoxylic reductase were not detected in mother strain. These data indicated that both the TCA cycle and glyoxylate cycle did not operate in mother strain. On the other hand, all enzyme activities relating to TCA cycle and glyoxylate cycle were verified in mutant. This finding indicates that both the TCA cycle and glyoxylate cycle performed a main role in carbohydrate catabolism in mutant. The evidence that alteration of the mode of glucose catabolism from fermentation in case of mother strain to respiration of mutant elicited by the action of cycloheximicie was thus explained.     

Ethanol catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.     

Pathways of glucose catabolism in Mycobacterium smegmatis.

Glucose metabolism in Mycobacterium smegmatis was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Glucose is oxidized in this organism mainly through the Embden-Meyerhof-Parnas pathway, irrespective of the carbon source used for growth. The pentose phosphate pathway plays only a minor role and its extent depends on the carbon source used for growth. Enzymes of glycolytic and oxidative pathways were detected in cells grown on glucose, glycerol, or pyruvate but enzymes of the Entner-Duodroff pathway could be detected only in glucose-grown cells. Labeled acetate is utilized by cells cultured on glucose, glycerol, and pyruvate. In all cases more of C1 of acetate was converted to CO2 while incorporation into cellular constituents was maximum from C2 of acetate.     

Proteomic and metabolomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous using different carbon sources

Background

Astaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable).

Results

A total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis.Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions.

Conclusions

The use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1484-6) contains supplementary material, which is available to authorized users.     

Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates.

Alanine: glyoxylate aminotransferase (EC 2.6.1.44), which is involved in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates in Saccharomyces cerevisiae, was highly purified and characterized. The enzyme had Mr about 80 000, with two identical subunits. It was highly specific for L-alanine and glyoxylate and contained pyridoxal 5'-phosphate as cofactor. The apparent Km values were 2.1 mM and 0.7 mM for L-alanine and glyoxylate respectively. The activity was low (10 nmol/min per mg of protein) with glucose as sole carbon source, but was remarkably high with ethanol or acetate as carbon source (930 and 430 nmol/min per mg respectively). The transamination of glyoxylate is mainly catalysed by this enzyme in ethanol-grown cells. When glucose-grown cells were incubated in medium containing ethanol as sole carbon source, the activity markedly increased, and the increase was completely blocked by cycloheximide, suggesting that the enzyme is synthesized de novo during the incubation period. Similarity in the amino acid composition was observed, but immunological cross-reactivity was not observed among alanine: glyoxylate aminotransferases from yeast and vertebrate liver.     

Constitutive uptake and degradation of fatty acids by Yersinia pestis.

Yersinia pestis was found to utilize palmitic acid as a primary carbon and energy source. No inhibition of growth by palmitic acid was observed. Comparison of palmitic acid uptake by cells pregrown either with or without palmitic acid demonstrated that fatty acid uptake was constitutive. High basal levels of two enzymes of beta-oxidation, beta-hydroxyacyl-coenzyme A dehydrogenase and thiolase, and the two enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, were found in cells grown in defined medium with glucose. Elevated levels of all four enzymes were found when cells were grown with acetate as a primary carbon and energy source, and even higher levels were observed when palmitic acid was provided as a primary carbon and energy source. High-pressure liquid chromatography was used to demonstrate that, in the presence of glucose, uniformly labeled [14C]palmitic acid was converted to intermediates of the tricarboxylic acid cycle and glyoxylate shunt. Pregrowth with palmitic acid was not required for this conversion. Strains lacking the 6- or the 47-megadalton plasmid did not take up [3H]palmitic acid but did possess levels of enzyme activity comparable to those observed in the wild-type strain.     

Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Amperometric detection of the bacterial metabolic regulation with a microbial array chip

A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose.     

13C Metabolic Flux Analysis of acetate conversion to lipids by Yarrowia lipolytica

Volatile fatty acids (VFAs) are an inexpensive and renewable carbon source that can be generated from gas fermentation and anaerobic digestion of fermentable wastes. The oleaginous yeast Yarrowia lipolytica is a promising biocatalyst that can utilize VFAs and convert them into triacylglycerides (TAGs). However, currently there is limited knowledge on the metabolism of Y. lipolytica when cultured on VFAs. To develop a better understanding, we used acetate as the sole carbon source to culture two strains, a control strain and a previously engineered strain for lipid overaccumulation. For both strains, metabolism during the growth phase and lipid production phase were investigated by metabolic flux analysis using two parallel sodium acetate tracers. The resolved flux distributions demonstrate that the glyoxylate shunt pathway is constantly active and the flux through gluconeogenesis varies depending on strain and phase. In particular, by regulating the activities of malate transport and pyruvate kinase, the cells divert only a portion of the glyoxylate shunt flux required to satisfy the needs for anaplerotic reactions and NADPH production through gluconeogenesis and the oxidative pentose phosphate pathway (PPP). Excess flux flows back to the tricarboxylic acid (TCA) cycle for energy production. As with the case of glucose as the substrate, the primary source for lipogenic NADPH is derived from the oxidative PPP.     

Effect of zwf gene knockout on the metabolism of Escherichia coli grown on glucose or acetate

The mutant deficient in glucose-6-phosphate dehydrogenase (G6PDH) was constructed by disrupting zwf gene by one-step inactivation protocol using polymerase chain reaction primers. The knockout of zwf gene was shown to have different influence on the metabolism of Escherichia coli grown on glucose or acetate. The decreased rates of substrate uptake and CO(2) production were found for the mutant grown on acetate, whereas these two rates were increased during the growth on glucose. The metabolic flux analysis based on (13)C-labeling experiments indicates that the metabolism of the mutant grown on glucose is related to the higher flux via tricorboxylic acid (TCA) cycle to generate anabolic reducing equivalents normally provided by the oxidative pentose phosphate pathway. However, the metabolism of the mutant grown on acetate shows a lower flux towards the TCA cycle as compared with the parent strain. The decreased flux through TCA cycle is associated with an increased flux via the glyoxylate shunt, by which the carbon source can bypass the two decarboxylative steps of TCA cycle in which CO(2) is released, thus conserving more carbon for biosynthesis in response to the decreased uptake rate of the carbon source.     

Glyoxylate cycle in Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was grown in media containing acetate, glutamate, and peptone as carbon sources. The component enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, were present under these conditions throughout the growth cycles. Highest specific activities for each enzyme were found in media with acetate as the carbon source. In an enriched peptone medium containing glucose, neither activity was detected until glucose was exhausted from the medium. Treatment of acetate-grown cells with glucose resulted in a rapid decline in the specific activities of both enzymes. The importance of this cycle in acetate-grown cells was indicated by the ability of itaconic acid (100 mM) to inhibit the growth of M. racemosus in acetate but not glutamate media. Itaconate was also shown to be a potent inhibitor of isocitrate lyase activity in vitro.     

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.     

Metabolism of C2 compounds inAcetobacter aceti

The presence of isocitrate lyase and malate synthase was detected in cell-free extracts ofAcetobacter aceti, grown in a mineral medium with acetate as sole carbon source. The presence of these enzymes explains the ability of this strain to grow with ethanol or acetate as sole carbon source, which is an important characteristic in Frateur's classification system forAcetobacter. In addition to isocitrate lyase and malate synthase, these cell-free extracts were found to contain glyoxylate carboligase, tartronicsemialdehyde reductase and glycerate kinase. The induction of these enzymes during growth on acetate is thought to be caused by the very high activity of isocitrate lyase, which may lead to an accumulation of glyoxylate. The importance of this pathway in cells growing with acetate as sole carbon source for the synthesis of their carbohydrate components is discussed. The presence of the enzymes from the pathway from glyoxylate to 3-phosphoglycerate explains the ability of this strain to grow with ethyleneglycol and glycollate as sole carbon source.     

Changes in the intracellular concentrations of adenosine phosphates and nicotinamide nucleotides during the aerobic growth cycle of yeast on different carbon sources

1. Methods for the quantitative extraction of adenosine phosphates and nicotinamide nucleotides from yeast cells are described. 2. The intracellular concentrations of adenosine phosphates and nicotinamide nucleotides were measured during the aerobic growth cycle of yeast on glucose and galactose. 3. When sugars were still present in the media the intracellular concentrations of NADH and AMP were in general higher in glucose- than in galactose-grown cells, whereas ADP concentration was always lower in glucose-grown cells. 4. The adenylate-kinase reaction was found to be far from equilibrium in the glucose-grown cells and when glucose was still present in the growth medium. 5. The significance of the changes in the intracellular concentrations of adenosine phosphates and nicotinamide nucleotides observed during growth on either sugar is discussed in relation to the metabolism and growth of the cells. 6. The differences observed in the concentrations of these cofactors in glucose- and galactose-grown cells are also discussed in relation to the type of metabolism of these cells. Control of glycolysis at the level of phosphofructokinase in galactose-grown cells and at the level of phosphoglycerate kinase in glucose-grown cells is suggested. 7. ADP is suggested to be the inducer of formation of respiratory enzymes.     

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.