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  • 1
    ISSN: 0749-503X
    Keywords: Hexose monophosphate pathway ; NADPH ; radiorespirometry ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A comparative radiorespirometric study of glucos emetabolism in glucose-limited chemostat cultures of Saccharomyces cerevisiae, Candida utilis and Rhodosporidium toruloides was performed in an attempt to estimate the contribution of the hexose monophosphate (HMP) pathway to glucose metabolism. Radioactively labelled glucose was administered directly to the cultures in a constant substrate feed, without disturbance of the steady state. The 14CO2 yields from [1-14C]- and [6-14C]-glucose demonstrated that the HMP pathway activities for the three yeasts were very similar. Furthermore, a quantitative analysis of results indicated that the HMP pathway activities were close to the theoretical minimum needed to cover the NADPH requirement for biomass formation.
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  • 2
    ISSN: 0749-503X
    Keywords: Yeasts ; fermentation ; ethanol ; Durham tube test ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: All type strains of ‘non-fermentative’ yeasts, available in the culture collection of the Centraalbureau voor Schimmelcultures, were reinvestigated for their capacity to ferment glucose in the classical Durham tube test. Although visible gas production was absent, nearly all strains produced significant amounts of ethanol under the test conditions. Under conditions of oxygen-limited growth, even strong alcoholic fermentation may occur in a number of yeasts hitherto considered as non-fermentative. Thus, shake-flask cultures of Hansenula nonfermentans and Candida silvae fermented more than half of the available sugar to ethanol. It is concluded that the taxonomic test for fermentation capacity, which relies on detection of gas formation in Durham tubes, is not reliable for a physiological classification of yeasts as fermentative and non-fermentative species.
    Additional Material: 4 Ill.
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  • 3
    ISSN: 0749-503X
    Keywords: Dihydroxyacetone reductase ; 2,3-butanediol dehydrogenase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Candida utilis CBS 621 contained four different enzymes capable of reducing carbonyl compounds such as dihydroxyacetone, acetoin, diacetyl, acetol, methylglyoxal and acetone, namely alcohol dehydrogenase, acetone reductase, dihydroxyacetone reductase and 2,3-butanediol dehydrogenase. The dihydroxyacetone reductase of C. utilis did not oxidize glycerol, thus providing evidence that this enzyme cannot function as a glycerol-2-dehydrogenase during growth of the yeast on glycerol. This enzyme may, however, play a role in the assimilation of 2,3-butanediol by C. utilis. The organism also contained a separate 2,3-butanediol dehydrogenase which was unable to reduce dihydroxyacetone. Both dihydroxyacetone reductase and 2,3-butanediol dehydrogenase were present at very high activities during growth of C. utilis on a variety of substrates, including 2,3-butanediol.
    Additional Material: 2 Ill.
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  • 4
    ISSN: 0749-503X
    Keywords: Crabtree effect ; sugar transport ; growth kinetics ; yeast ; chemostat ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The glucose transport capacity of Saccharomyces cerevisiae CBS 8066 was studied in aerobic glucose-limited chemostat cultures. Two different transport systems were encountered with affinity constants of 1 and 20 mM, respectively. The capacity of these carriers (Vmax) was dependent on the dilution rate and the residual glucose concentration in the culture. From the residual glucose concentration in the fermenter and the kinetic constants of glucose transport, their in situ contribution to glucose consumption was determined. The sum of these calculated in situ transport rates correlated well with the observed rate of glucose consumption of the culture.The growth kinetics of S. cerevisiae CBS 8066 in glucose-limited cultures were rather perculiar. At low dilution rates, at which glucose was completely respired, the glucose concentration in the fermenter was constant at 110 μM, independent of the glucose concentration in the reservoir. At high dilution rates, characterized by the occurrence of both respiration and alcoholic fermentation, the residual substrate concentration followed Monod kinetics. In this case, however, the overall affinity constant was dependent on the reservoir glucose concentration.
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  • 5
    ISSN: 0749-503X
    Keywords: benzoic acid: Yeasts ; Crabtree effect ; respiration ; fermentation ; mitochondria ; metabolic flux ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Addition of benzoate to the medium reservoir of glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h-1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g-1h-1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in (qO2) to 13 mmol g-1h-1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash-out.As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest of study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D=0.10 h-1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the entracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate.Growth ofS. cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1h-1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume.Also with the Crabtree-negative yeasts Candida utilis, Kluyveromyces marxianus andHansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S.Cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h-1 in the presence of benzoate was equal to, or lower than the qO2 attainable at μmax without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K.Marxianus when the glucose flux was increased by the presence of benzoate.The maximal aerobic fermentation rate at D = 0.10 h-1 of the Crabtree-negative yeasts at high benzoate concentrations was considerably lower than for S. cerevisiae. This is probably due to the fact that under aerobic conditions these yeasts are unable to raise the low basal pyruvate decarboxylase level: cultivation without benzoate under oxygen-limited conditions resulted in rates of alcoholic fermentation and levels of pyruvate decarboxylase comparable to those of S. cerevisiae.
    Additional Material: 10 Ill.
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  • 6
    ISSN: 0749-503X
    Keywords: Cytochrome c peroxidase ; hydrogen peroxide ; energetics ; yeast ; anaerobic respiration ; chemostat ; mitochondria ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Chemostat cultures of a catalase-negative mutant of Hansenula polymorpha CBS 4732 were able to decompose hydrogen peroxide at a high rate. This was apparent from experiments in which yeast was grown under carbon limitation in chemostat culture on mixtures of glucose and H2O2. The enzyme responsible for H2O2 degradation is probably the mitochondrial enzyme cytochrome c peroxidase (CCP), which was present at very high activities. This enzyme was partially purified and shown to be specific for reduced cytochrome c as an electron donor; no reaction was observed with NAD(P)H. Thus, reducing equivalents for H2O2 degradation by CCP must be provided by the respiratory chain.That H2O2 can act as an electron acceptor for reducing equivalents could be confirmed with experiments in which cells were incubated with ethanol and H2O2 in the absence of oxygen. This resulted in oxidation of ethanol to equimolar amounts of acetate.Energetic aspects of mitochondrial H2O2 decomposition via CCP and the physiological function of CCP in yeasts are discussed.
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  • 7
    ISSN: 0749-503X
    Keywords: yeast ; Candida utilis ; alcoholic fermentation ; Kluyver effect ; oxygen limitation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar-limited growth to oxygen-limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose-grown cultures to oxygen limitation was very similar to that of glucose-grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D-lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose- and glucose-grown cultures diverged: ethanol disappeared from the maltose-grown cultures, whereas fermentation continued in steady-state, oxygen-limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen-limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.
    Additional Material: 6 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 12 (1996), S. 1607-1633 
    ISSN: 0749-503X
    Keywords: Yeast ; glycolysis ; TCA cycle ; sugar metabolism ; metabolic engineering ; pyruvate decarboxylase ; pyruvate carboxylase ; pyruvate dehydrogenase complex ; alcoholic fermentation ; Crabtree effect ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation. This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase. Special attention is devoted to physiological studies on S. cerevisiae strains in which structural genes encoding these key enzymes have been inactivated by gene disruption.
    Additional Material: 7 Ill.
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  • 9
    ISSN: 1572-9699
    Keywords: Saccharomyces cerevisiae ; pyruvate carboxylase ; anaplerotic reactions ; sugar metabolism ; yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.
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  • 10
    ISSN: 0006-3592
    Keywords: glutamine limitation ; mammalian cells ; chemostat ; specific metabolic rates ; hybridoma ; medium optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Glutamine is a major source of energy, carbon, and nitrogen for mammalian cells. The amount of glutamine present in commercial mammalian cell media is, however, not necessarily balanced with cell requirements. Therefore, the effects of glutamine limitation on the physiology of two mammalian cell lines were studied in steady-state chemostat cultures fed with IMDM medium with 5% serum. The cell lines used were MN12, a mouse-mouse hybridoma, and SP2/0-Ag14, a mouse myeloma often used in hybridoma fusions. Cultures, grown at a fixed dilution rate of 0.03 h-1, were fed with media containing glutamine concentrations ranging from 0.5 to 4 mmol L-1. Biomass dry weight and cell number were linearly proportional to the glutamine concentrations fed, between 0.5 and 2 mmol L-1, and glutamine was completely consumed by both cell lines. From this it was concluded that glutamine was the growth-limiting substrate in this concentration range and that the standard formulation of IMDM medium contains a twofold excess of glutamine. In glutamine-limited cultures, the specific rates of ammonia and alanine production were low compared to glutamine-excess cultures containing 4 mmol L-1 glutamine in the feed medium. The specific consumption rates of nearly all amino acids decreased with increasing glutamine feed, indicating that, in their metabolic function, they may partially be replaced by glutamine. Both cell lines reacted similarly to differences in glutamine feeding in all aspects investigated, except for glucose metabolism, In SP2/0-Ag14 glutamine feed concentrations did not affect the specific glucose consumption, whereas in MN12 this parameter increased with increasing amounts of glutamine fed. This systematic study using controlled culture conditions together with a detailed analysis of culture data shows that, although cells may react similarly in many aspects, cell-line-specific characteristics may be encountered even with respect to fundamental physiological responses like the interaction of the glutamine and glucose metabolism. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 272-286, 1997.
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