Yeast Candida Utilis Research Articles

Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation.

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-1 h-1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in qO2 to 13 mmol g-1 h-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 to 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 extracellular 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 of S. cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1 h-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 and Hansenula 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 mu(max) 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and characterization of an inducible L-lysine: 2-oxoglutarate 6-aminotransferase from Candida utilis.

L-Lysine:2-oxoglutarate 6-aminotransferase (EC 2.1.6.36) was purified 202-fold from the yeast Candida utilis. The subunit Mr estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 40 kDa. The Mr of the native enzyme was estimated to be 83 kDa by gel filtration, suggesting a dimeric structure. The enzyme exhibits absorption maxima at 280, 340 and 420 nm, and binds 2 mol pyridoxal-5-phosphate/mol of the native enzyme. The aminotransferase has a maximum activity at pH 8.5 and at 4 degrees C. 2-Oxoglutarate is the best amino acceptor with L-lysine as amino donor. Lower activity is observed with oxaloacetate (38%), pyruvate (19%) and 2-oxoadipate (7%) as acceptor or with L-thialysine (13%) as donor. The Km values are 2.5 mM for L-lysine, 3.8 mM for 2-oxoglutarate and 0.015 mM for pyridoxal-5-phosphate. The enzyme activity is induced in cells grown in the presence of L-lysine.

Fluorescence measurements under aerobic and anaerobic conditions on Pichia stipitis, Pachysolen tannophilus and Candida utilis grown on D-xylose

Batch growth of the yeasts Candida utilis, Pachysolen tannophilus and Pichia stipitis on 1% D-xylose was monitored using a commercial fluorosensor with an excitation wavelength of 340 nm and a detection wavelength of 460 nm. Step changes in oxygen concentration were made and in the presence of 0.3 g/l of xylose, step changes from aerobic to anaerobic conditions resulted in an increase of the fluorescence level by about 40% for the non-fermentative yeast C. utilis. However, the increases of the fluorescence levels for P. tannophilus and P. stipitis stayed below 10%. These measurements indicate better control of (or better redox balance for) intracellular NADH concentration in P. tannophilus and P. stipitis than in C. utilis.

Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid and simple method for DNA preparation fromCandida utilis

A method for the extraction of genomic DNA from the industrial yeast Candida utilis is described. The method is rapid, simple and produces DNA that is sufficiently pure for restriction analysis and should be suitable for Southern blotting and the construction of gene libraries.

Formaldehyde as a carbon and energy source in yeast growth

Summary Formaldehyde can simultaneously be utilized by yeasts during growth on another heterotrophic substrate (e.g. glucose, xylose, methanol). From transient state growth experiments it became evident with Hansenula polymorpha that up to a molar mixing ratio of formaldehyde to glucose of 3 the former seems to play the role of an energy donor for glucose carbon incorporation. This also holds for the non-methylotrophic yeast Candida utilis. At higher mixing ratios formaldehyde is used as a carbon and energy source besides glucose in the case of H. polymorpha. Formaldehyde utilization in the mixture is limited by the capacity of formate dehydrogenase. In contract, with C. utilis formaldehyde dehydrogenase activity limits consumption of this substrate in the mixture. The results with this yeast suggest that formaldehyde is not used significantly as a carbon source.

Temperature adaptation in yeasts: the role of fatty acids

Studies on the yeasts Candida oleophila, Candida utilis, Lipomyces starkeyi, Rhodosporidium toruloides and Saccharomyces cerevisiae revealed the existence of three different temperature adaptation responses involving changes in fatty acid composition. These conclusions were drawn by determining the growth rates, total cellular fatty acid content, fatty acid composition, degree of unsaturation, and the mean chain length of fatty acids over a range of growth temperatures. Within temperatures permitting growth, there were no changes in the major fatty acids of any of the yeasts, but the absolute amounts and relative compositions of the fatty acids did alter. In S. cerevisiae there were temperature-induced changes in the mean fatty acid chain length, whereas in R. toruloides there were changes in the degree of unsaturation. C. oleophila, C. utilis and L. starkeyi showed both responses, depending on whether the growth temperature was above or below 20-26 degrees C. Below 20-26 degrees C temperature-dependent changes were observed in the mean chain length whereas above 20-26 degrees C there were changes in the degree of unsaturation.

A new procedure of a two‐step cultivation of microorganisms. Part II. Application to the procedure of SCP‐production on the basis of sucrose

AbstractThe two‐step cultivation of microoganisms described in part I of this series will be explained by referring to its application to the production of SCP on the basis of sucrose/molasses as carbon sources. The inactivation of the yeast Candida utilis is carried out thermally, and the specific death rate was determined to be between 119 × 10−4 s−1, and 324 × 10−4 s−1, respectively. With this new procedure the apparent productivity could be increased by 96.6%, the yield coefficient of molasses increased by 5.8% and of sucrose by 12.9%. The crude protein content of the product in the case of sucrose rose by 104%. The variability of process design is demonstrated.

Production of ethanol by the yeastCandida utilis under autoanaerobic conditions

Candida utilis CCY 29-38-65 converts glucose to ethanol under autoanaerobic conditions. On aeration switch-on the produced ethanol is utilized as carbon source and the specific rate of biomass production increases.

Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts ( Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50–150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.

Control of Escherichia coli–Candida utilis continuous, competitive mixed‐culture system using the dynamic matrix control algorithm

The control of the continuous, competitive mixed culture of the yeast Candida utilis (ATCC 8205) and the bacterium Escherichia coli B/r (ATCC 12407) was examined. A modified version of the dynamic matrix control (DMC) algorithm was tested in simulation and then was successfully implemented. This represents the first report of the location and stabilization of a competitive mixed culture at a metastable equilibrium point using automatic control.

Growth of yeast colonies on solid media.

Colonies on nutrient agar of the aerobic yeast Candida utilis show linear increases in diameter and height with time throughout most of the growth cycle. The concentration of glucose in the agar has a negligible effect on radial growth rate although an increase in the glucose concentration prolongs the linear radial growth phase. The rate of increase in height of the colony is proportional to the square root of the initial glucose concentration. A new model that considers both glucose diffusion and oxygen diffusion in the colony is consistent with the observed colony profiles.

Adaptation of the Kinetics of Glucose Transport to Environmental Conditions in the Yeast Candida utilis CBS 621: a Continuous-culture Study

SUMMARY: The relation between the kinetic parameters of glucose transport and the physiology of Candida utilis CBS 621 was studied in chemostat cultures. In glucose-limited cultures the transport parameters were dependent on the growth rate of the yeast. Three different transport systems were found which differed by an order of magnitude in their affinity constants, namely a high-affinity (K m 25 μM), a medium-affinity (K m 190 μM), and a low-affinity uptake system (K m 2000 μM). Cells growing at a dilution rate of 0.45 h-1 or less had the high- and medium-affinity uptake systems. At a dilution rate of 0.52 h-1 the high-affinity system was absent and both the medium-and low-affinity systems were present. At a dilution rate close to μmax (0.57 h-1) only the low-affinity system was detected. The in situ contribution of each of the transport systems to glucose consumption in glucose-limited cultures was estimated on the basis of their kinetic parameters (K m and V max) and the residual glucose concentration in these cultures. The sum of the calculated rates of transport corresponded to the in situ rate of glucose consumption by the cultures as determined from the yield constant and the dilution rate. The dependence of the transport parameters on the growth rate and hence on the environmental sugar concentration was also evident in cells grown under nitrogen limitation. In contrast to carbon-limited cells, nitrogen-limited cultures growing at D = 0.15 h-1 did not exhibit the high-affinity glucose uptake system, whereas the medium- and low-affinity systems were present.

Inhibition of germination of sporangia of Peronospora hyoscyami by cation deprivation: the effects of substrate and chelating agents

Germination of sporangia of Peronospora hyoscyami de Bary (tobacco blue mould) was affected by the germination substrate. Washed sporangia germinated freely in water on glass slides, but failed to germinate on agarose. Germination was reduced on detached tobacco leaves, being lower on cv. Hicks Q46 than on cv. ZZ100. Inhibition of germination by agarose was reversed by the addition to the inoculum of an extract obtained by centrifuging a suspension of non‐living, powdered torula yeast Candida utilis (Hennenberg) Lodder & van Rij. Yeast extract also improved germination in vivo. The siderophore rhodotorulic acid, the chelating agents ferric and sodium citrate, and riboflavin combined with calcium and magnesium salts, also stimulated germination of sporangia on agarose. The chelating agent ethylenediaminetetraacetic acid (EDTA) inhibited germination on glass slides. Inhibition by EDTA was partially reversed by the addition of ferric salts. The inhibition of germination by agarose and stimulation by additives may have been due to effects on the availability of cations to sporangia ofP. hyoscyami. The reduction in germination on tobacco leaves may have involved a similar mechanism.

Morphological basis for multiple interactions of ethidium bromide (EB) with yeast Candida utilis

Exposure of yeast cells to EB produced multiple effects on the cellular organelles; a) changes in the plasma membrane characterized by 75 to 110 nm deep pits; b) polymorphisms of the mitochondria ranging from cup-shaped, ring-shaped, ribbon-shaped, dumbbell-shaped structures to finally the formation of very elongated mitochondria (up to 4.5 μm in length); c) an increase in the length and number of endoplasmic reticulum; d) an increase in the number of cytoplasmic vesicles whose diameter varied between 25 to 45 nm. Furthermore, EB inhibited cytochrome c oxidase and cytochrome b biosynthesis, stimulated cytochrome c biosynthesis and uncoupled oxidative phosphorylation.

Evaluation of Escherichia coli and Candida utilis as a model continuous, competitive mixed culture system

The yeast Candida utilis and the bacterium Escherichia coli B/r were evaluated as a candidate experimental, continous, competitive mixed culture system under ammonia-nitrogen limited conditions at 30 degrees C. High dilution rates favored yeast dominance, while low dilution rates favored bacterial dominance. The hydrogen ion concentration was also demonstrated to be an effective manipulative variable for control of the yeast-bacterial mixed culture. Through trial-and-error manipulation of the pH for the mixed culture operating at constant dilution rate, it was possible to locate a metastable equilibrium point and to operate in the vicinity of that point for more than 24h. The reproducible emergence of a variant E. coli was also noted in this study.

Restoration of respiratory competence in the yeastCandida utilis after somatic fusion withSaccharomyces cerevisiae

After somatic fusion between a mitochondrial mutant ofCandida utilis andSaccharomyces cerevisiae respiratory competent strain, the complex III of the respiratory chain seems to be restored. Fusion products, FP, synthesizing normal apocytochromeb were recovered and showed normal-shaped mitochondria along the cytoplasm as in theCandida utilis original, respiratory-competent strain.

Complete amino-acid sequence of the natural ATPase inhibitor from the mitochondria of the yeast Candida utilis.

The complete alignment of the 63 residues of the mitochondrial ATPase inhibitor from the yeast Candida utilis has been determined. The sequence study was carried out mainly by automatic (liquid and solid-phase) methods. Peptides were obtained by enzymatic digestion with clostripain and purified by reverse-phase high-performance liquid chromatography. The ATPase inhibitor contains three sets of repetitive sequences and eight clusters of charged residues, as also found in the inhibitor of Saccharomyces cerevisiae, with which it shares 58.7% homology of conserved residues. When the two yeast ATPase inhibitor sequences were compared to that of beef heart, 20 residues remained common to the three alignments, although the latter protein contained a long histidine-rich insertion, only found in this inhibitor. Most of the homologous residues were clustered near the center of the protein, which by partial proteolytic digestion of the beef heart ATPase inhibitor [Dianoux, A.C. et al. (1982) FEBS Lett. 140, 223-228] has already been shown to be involved in the biological function.

Growth ofCandida utils on single- and multicomponent-sugar substrates and on waste banana pulp liquors for single-cell protein production

Large quantities of cull bananas are available world-wide, and often in regions lacking indigenous sources of high-quality protein animal feedstuffs. One possible method of utilizing these wastes is to upgrade them to microbial protein by means of bioconversion of the fermentable sugars of the ripe or over-ripe pulp by the yeastCandida utilis, which is nutritionally well-accepted. Studies of ripeMusa sapientum var. ‘Gros Michel’ bananas were conducted in order to obtain both the microbial and the engineering data necessary for the development of such an aerobic fermentation process. For banana pulp fermentation liquors in the concentration range 10 to 35% (w/v) fresh weight (FW) banana, the corresponding dry weight banana concentrations and the fraction of insoluble solids before and after sterilization (121°C, 30 min) have been measured and are reported. In addition to fermentation studies of banana liquor prepared from 10% (w/v) fresh whole banana, comparative kinetic studies of the batch growth and substrate assimilation pattern were performed on media containing as the sole soluble carbohydrate source either one of the major banana sugars (glucose, fructose, sucrose) or a mixture of the three. Wherever feasible, the maximum specific growth rate (μmax) was evaluated by four different methods: dry weight biomass, optical density, crude protein content of broth insoluble solids and dissolved oxygen history. In addition to the comparative μmax results, values of the biomass yield coefficient and the specific respiration rates for each case are presented and discussed. A preliminary process design of the fermentation section of a moderate-scale microbial protein production plant based on 20,000 kg FW banana/day feedstock is presented. The results of this study indicate that such a plant would yield about 2,900 kg/day of protein-enriched product containing 5 wt% moisture and 44 wt% (c. 1,400 kg/day) crude protein.

A 31P NMR study of the internal pH of yeast peroxisomes.

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by 31P nuclear magnetic resonance (NMR) spectroscopy. 31P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent Pi-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic Pi at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected Pi at a considerably lower pH of approximately 5.8-6.0. Pi at a considerably lower pH of approximately 5.8-6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two Pi-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the Pi-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to Pi in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated Pi was confirmed cytochemically.