Polyphosphates and exopolyphosphatases in cytosol and mitochondria of Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus

Content and chain lengths of inorganic polyphosphates (polyP) as well as exopolyphosphatase activities were compared in cytosol and mitochondria of the yeast Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus. PolyP metabolism in cytosol and mitochondria was substantially dependent upon the carbon source. Acid-soluble polyP accumulated mainly in cytosol using either glucose or ethanol. The level of the accumulation was lower during growth on ethanol compared to that on glucose. Increase in polyP content in mitochondria was observed during growth on glucose, but not on ethanol. In cytosol the activity of exopolyphosphatase PPN1 was increased and the activity of exopolyphosphatase PPX1 was decreased independently of the carbon source under phosphate surplus conditions. Growth on ethanol caused exopolyphosphatase PPN1 to appear in the soluble mitochondrial fraction, while during growth on glucose only exopolyphosphatase PPX1 was present in this fraction.     

A novel phosphatase specific for pyridoxal 5'-phosphate in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Abstract An acid phosphatase highly spcific for pyridoxal 5'-phosphate (PLP) was found and partially purified from the aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114. The enzyme showed a pH optimum at 5.5; its activity was stimulated by magnesium ions. This enzyme also hydrolyzed p -nitrophenyl phosphate (NPP) and flavin mononucleotide (FMN). The enzyme level varied depending on growth conditions. Supplementing the growth medium with glycerol, glucose, xylose or mannitol increased the level of phosphatase activity. An inverse relationship between free phosphate content in the cells and enzyme level was observed.     

Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Isolation of a Regulatory Mutant of Fructose-1, 6-diphosphatase in Saccharomyces carlsbergensis

By selecting for growth on glycerol, but absence of growth on glucose, a mutant of Saccharomyces carlsbergensis was isolated which does not grow on glucose, fructose, mannose, or sucrose, which shows long-term adaptation to maltose, but which can grow normally on galactose, ethanol, or glycerol. In the mutant, fructose diphosphatase is not inactivated after the addition of glucose, fructose or mannose to the medium, resulting in the simultaneous presence of fructose diphosphatase and phosphofructokinase activity. Under these conditions, a cycle is probably catalyzed between fructose-6-phosphate and fructose-1,6-diphosphate, resulting in the net consumption of adenosine triphosphate and an immediate stop of protein synthesis.     

Optimization of biphasic growth of Saccharomyces carlsbergensis in fed-batch culture

The optimal glucose feeding policy for the fed-batch culture of Saccharomyces carlsbergensis is presented. The biphasic nature of growth results in a singular feed rate policy that is unique to this organism. When the operating cost is high, the reduction in operating time forces the cells to utilize both glucose and ethanol toward the end of fermentation time and results in a decreasing rate of glucose addition, unlike the normally observed in creasing feed rate. The optimal feeding policy depends heavily on the initial conditions and is highly sensitive to changes in kinetic parameters. A semiempirical scheme for feedback optimization is suggested for the fed-batch yeast culture.     

在不同碳源培养条件下酿酒酵母的蛋白质组解析

为了分析酿酒酵母在不同培养条件下的代谢调控过程的差异,采用固相pH梯度-SDS聚丙烯酰胺双向凝胶电泳对其利用不同碳源时细胞的总蛋白进行了分离,银染显色,使用2D蛋白质图像分析系统Image Master-2D Elite对双向电泳图谱进行分析,查询SWISS-2D-PAGE蛋白质组数据库,识别了约500个蛋白质点。对与糖酵解途径、磷酸戊糖途径、三羧酸循环和几种回补反应相关的大部分关键的蛋白质进行了差异分析。探讨了酿酒酵母利用不同碳源时及生长的不同阶段代谢机理的变化和在蛋白质水平的调控。     

Subcellular fractionation by differential and zonal centrifugation of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis

1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 10(5)g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH- and NADPH-cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH-cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions.     

Rhizomorph Formation in Fungi III. The Effect of Ethanol on the Synthesis of DNA and RNA and Uptake of Asparagine and Phosphate in Armillaria mellea

Growth and rhizomorph formation in Armillaria mellea (Vahl ex Fr.) Quél. can be stimulated by ethanol when grown on a synthetic glucose medium. The content of DNA (deoxy-ribonucleic acid) and RNA (ribonucleic acid) in cultures of A. mellea have been followed during a growth period in relation to growth (dry weight) and ethanol uptake with 3 different initial ethanol concentrations. The continuous increase in dry weight as well as DNA and RNA contents during growth showed similar exponential rates as long as ethanol was present in the medium. The final amounts were proportional to the initial ethanol concentration. A further supply of ethanol caused a similar proportional increase in dry weight and also in the DNA and RNA contents, which indicates that the growth stimulated by ethanol is caused by cell divisions rather than an accumulation of lipids or polysaccharides. Uptake of asparagine and phosphate were also stimulated by ethanol.     

Intracellular metabolite profiling of Fusarium oxysporum converting glucose to ethanol

The filamentous fungus Fusarium oxysporum is known for its ability to produce ethanol by simultaneous saccharification and fermentation (SSF) of cellulose. However, the conversion rate is low and significant amounts of acetic acid are produced as a by-product. In this study, the growth characteristics of F. oxysporum were evaluated in a minimal medium using glucose as the sole carbon source in aerobic, anaerobic and oxygen-limited batch cultivations. Under aerobic conditions the maximum specific growth rate was found to be 0.043 h(-1), and the highest ethanol yield (1.66 mol/mol) was found under anaerobic conditions. During the different phases of the cultivations, the intracellular profiles were determined under aerobic and anaerobic conditions. The profiles of the phosphorylated intermediates indicated that there was a high glycolytic flux at anaerobic growth conditions, characterized by high efflux of glyceraldehyde-3-phosphate (G3P) and fructose-6-phosphate (F6P) from the pentose phosphate pathway (PPP) to the Embden-Meyerhof-Parnas (EMP) pathway, resulting in the highest ethanol production under these conditions. The amino acid profile clearly suggests that the TCA cycle was primarily active under aerobic cultivation. On the other hand, the presence of high levels of gamma-amino-n-butyric acid (GABA) under anaerobic conditions suggests a functional GABA bypass and a possible block in the TCA cycle at these conditions.     

Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (K_m 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO₄³⁻-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F⁻, PO₄³⁻) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.     

The proteomic response of Saccharomyces cerevisiae in very high glucose conditions with amino acid supplementation

Ethanol yield by Saccharomyces cerevisiae in very high glucose (VHG) media with an amino acid supplement was investigated. Amino acid supplementation led to positive cell responses, including reduced lag time and increased cell viability in VHG media. A quantitative shotgun proteomic analysis was used to understand how amino acid supplemented S. cerevisiae responds to high osmotic conditions. iTRAQ data revealed that most proteins involved in glycolysis and pentose phosphate pathways were up-regulated under high glucose shock. Reactivation of amino acid metabolism was also observed at the end of the lag phase. The relative abundance of most identified proteins, including aminoacyl-tRNA biosynthesis proteins, and heat-shock proteins, remained unchanged in the hours immediately following application of glucose shock. However, the expression of these proteins increased significantly at the end of the lag phase. Furthermore, the up-regulation of trehalose and glycogen biosynthesis proteins, first maintaining then latterly increasing glycolysis pathway activity was also observed. This was verified by enhanced ethanol yields at 10 and 12 h (0.43 and 0.45 g ethanol/g glucose) compared to 2 h (0.32 g ethanol/g glucose). These data combined with relevant metabolite measurements demonstrates that enhanced ethanol fermentation under VHG conditions can be achieved with the aid of amino acid supplementation.     

Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.     

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.     

Ethanol production from hexoses, pentoses, and dilute-acid hydrolyzate by Mucor indicus

Consumption of hexoses and pentoses and production of ethanol by Mucor indicus were investigated in both synthetic media and dilute-acid hydrolyzates. The fungus was able to grow in a poor medium containing only carbon, nitrogen, phosphate, potassium, and magnesium sources. However, the cultivation took more than a week and the ethanol yield was only 0.2 gg(-1). Enrichment of the medium by addition of trace metals, particularly zinc and yeast extract, improved the growth rate and yield, such that the cultivation was completed in less than 24 h and the ethanol and biomass yields were increased to 0.40 and 0.20 gg(-1), respectively. The fungus was able to assimilate glucose, galactose, mannose, and xylose, and produced ethanol with yields of 0.40, 0.34, 0.39, and 0.18 gg(-1), respectively. However, arabinose was poorly consumed and no formation of ethanol was detected. Glycerol was the major by-product in the cultivation on the hexoses, while formation of glycerol and xylitol were detected in the cultivation of the fungus on xylose. The fungus was able to take up the sugars present in dilute-acid hydrolyzate as well as the inhibitors, acetic acid, furfural, and hydroxymethyl furfural. M. indicus was able to grow under anaerobic conditions when glucose was the sole carbon source, but not on xylose or the hydrolyzate. The yield of ethanol in anaerobic cultivation on glucose was 0.46 g g(-1).     

Saccharomyces carlsbergensis fdp mutant and futile cycling of fructose 6-phosphate.

In Saccharomyces, the addition of glucose to cells grown in media lacking sugars causes irreversible inactivation of fructose bisphosphatase. One function of this process might be to prevent a futile cycle of formation and hydrolysis of fructose 1,6-bisphosphate. We tested such cycling by assessing the labeling of the 1-position of glucose in polysaccharides from [6-14C]glucose (J.P. Chambost and D. G. Fraenkel, J. Biol. Chem. 225:2867-2869, 1980) by using mutants impaired in glucose growth and known not to inactivate the phosphatase normally (i.e., the fdp mutant of Saccharomyces carlsbergensis [van de Poll et al., J. Bacteriol. 117:965-970, 1974] and the similar cif mutant of Saccharomyces cerevisiae [Navon et al., Biochemistry 18:4487-4499, 1979] ), as well as in the wild-type strain tested in the 1-h period before inactivation is complete. There was marginal, if any, cycling in any situation, and we conclude that the phosphatase activity is controlled by means other than inactivation or that the extent of cycling is too low to be significant, or both. For the fdp mutant data are also presented on growth, rate of glucose metabolism, metabolite accumulations, enzyme levels, and glucose transport, but the primary lesion is unknown.     

Regulation of extracellular acid phosphatase biosynthesis by phosphates in proteinase producing fungus Humicola lutea 120-5

The feasibility of using proteinase producing fungus Humicola lutea 120-5 as a source of extracellular acid phosphatase was investigated. To enhance the acid phosphatase yield and significantly reduce the proteolytic activity the composition of casein-glucose medium containing inorganic phosphate (Pi) was modified. The regulation of phosphatase formation was controlled by Pi. The repression influence of Pi on the synthesis of phosphatase was established. A reduction of Pi (KH(2)PO(4)) concentration from 1.0 to 0.01 g/l caused approximately 5-fold increase of the phosphatase (1200 U/I) and 3-fold decrease of the proteinase (10 U/ml). The omission of Pi from the medium in which the casein (phosphoprotein) was the sole phosphatase source resulted in higher phosphatase yield (2000 U/l) and lower proteolytic activity (7.5 U/ml). Different concentrations of glucose and casein were tested to obtain the optimal medium for maximal acid phosphatase production and minimal level of proteinase. The highest acid phosphatase activity of 2500 U/l and the least amount of acid proteinase (5.5 U/ml) were achieved in 72 h shake-flask culture using Pi-free medium containing glucose and casein in concentrations of 20 and 4 g/l, respectively. The ability of the fungus H. lutea 120-5 to dephosphorylate casein providing orthophosphate for cell growth was discussed.     

Identification of nutrient-dependent changes in extracellular pH and acid phosphatase secretion in Aspergillus nidulans

The present study was designed to identify nutrient-dependent changes in extracellular pH and acid phosphatase secretion in the biA1 palC4 mutant strain of Aspergillus nidulans. The palC4 mutant was selected as lacking alkaline phosphatase, but having substantially increased acid phosphatase activity when grown on solid minimal medium under phosphate starvation, pH 6.5. Gene palC was identified as a putative member of a conserved signaling cascade involved in ambient alkaline sensing whose sole function is to promote the proteolytic activation of PacC at alkaline pH. We showed that both poor growth and conidiation of the palC4 mutant strain on solid medium, alkaline pH, were relative to its hypersensitivity to Tris (hydroxymethyl) aminomethane buffer. Also, the secretion of acid phosphatase was repressed when both the wild-type and palC4 mutant strains were grown in low-phosphate yeast extract liquid medium, pH 5.0, indicating that the secretion of this enzyme is not necessary to regenerate inorganic phosphate from the organic phosphate pool present in yeast extract.     

Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose⁻¹) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Expression and low pH increase the activity of an apoplastic acid phosphatase in growing tissues from Zea mays

Auxin‐induced secretion of an acid phosphatase (EC 3.1.3.2) leads to the hypothesis that this enzyme may be involved in plant cell elongation growth (W. Pfeiffer. 1996. Physiol. Plant. 98: 773–779). Elongation growth can be characterized by the effects of pH, phosphate and citrate, and the correlation with a particular region of the root: the elongation region. Therefore, it was investigated whether these parameters may reveal further correlations between acid phosphatase and elongation growth. The following results were obtained. (1) An extracellular acid phosphatase with high substrate affinity was characterized (Michaelis‐Menten constant, 0.03 m M for 4‐methylumbelliferyl phosphate; pH optimum, 3.0). The pH dependence of the enzyme was similar to that of elongation growth of coleoptile segments after pretreatment with phosphate (U. Kutschera and P. Schopfer. 1985. Planta 163: 483–493). (2) Phosphate inhibited both the acid phosphatase and coleoptile growth. Phosphate was a competitive inhibitor of the acid phosphatase (inhibitor constant, 2.5 m M ). (3) Citrate inhibited coleoptile growth and the acid phosphatase in a similar way (inhibitor constant, 21 mM). (4) The elongation region of maize roots contained more apoplastic acid phosphatase than adjacent regions (170%). The pH dependence of the enzyme suggests that the low pH reported for the elongation region would result in an additional increase of the enzymatic activity (pH optimum at 3.0).     

Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol

The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.