Dynamic aspects of radiorespirometry during the cell cycle of Candida utilis: Some observations in phased culture under carbon-, nitrogen-, and phosphorus-limited growth

Many changes that occur in a cell during the cell cycle can be demonstrated in synchronous cultures and can reveal dimensions of cell metabolism not attainable by the study of balanced growth of asynchronous populations in batch cultures or the steady state in chemostat cultures. The release of ¹⁴CO₂ from specifically labeled glucose by phased (continuously synchronized) cultures follows a characteristic pattern (profile) that depends upon the stage in the cell cycle and the period of labeling used. Successive profiles throughout a cycle showed differences that were altered under different nutrient-limiting growth conditions. Profiles obtained with glucose-1-¹⁴C, glucose-2-¹⁴C, glucose-3,4-¹⁴C, and glucose-6-¹⁴C and phased cells of Candida utilis under N-, P-, and C-limited growth demonstrated the variable character of the metabolic activity that occurred in the cells while contour changes within the profiles across the cycle indicated possible correlations with activities of the hexose monophosphate, Embden-Meyerhof-Parnas, and tricarboxylic acid cycle pathways during the cell cycle. The basis of these changes and their use as elementary parameters for study of problems of physiological changes in vivo are considered.     

Effect of aeration and mixing in various types of cultivation devices on the cell cycle of budding yeasts

When investigating the effect of aeration capacityK _L a of a cultivation device on the cell cycle of daughter cells ofCandida utilis it was found that the length of a phase (S + G₂) of the cell cycle is influenced by the rate of oxygen transfer. An increase ofK _L a, of a cultivation device achieved by increasing the specific output of mechanical energy for air dispersion and mixing may lead to cell damage and to changes in the cell cycle. The effect of high intensity of aeration and mixing is thus invalidated.     

Changes in acid soluble nucleotide components of candida utilis during the cell cycle

Acid soluble extracts obtained at 30 min intervals from cells of C. utilis growing in synchrony in a phased culture (cycle time 51/2 hr) were fractionated on a Dowex-1-formate column. The series of fractionation profiles showed changes in number and amounts of components over the cell cycle. Transient accumulations of numerous components over the complex pool were observed. The significance of the changes are discussed in relation to practical applications and cell metabolism.     

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on pectic materials

Hydrolysis of pectin by Saccharomycopsis fibuliger and cell growth on the products of hydrolysis by Candida utilis require different incubation conditions. A three-stage sequential culture is described in which S. fibuliger was first grown under aerobic conditions to generate cell mass. The concentration of dissolved oxygen was then reduced to promote pectolytic activity and reduce the number of viable cells in the culture. Finally culture conditions were adjusted to promote the growth of C. utilis in mixed culture with S. fibuliger. The presence of C. utilis increased the rate of pectolytic activity by S. fibuliger. A yeast product, containing 98% C. utilis cells, was obtained from the mixed culture grown on 10 g l⁻¹ pectin. Cell yields using starch or an equal mixture of starch and pectin were similar to those reported in the Symba process, although lower cell yields were recorded using pectin alone.     

Growth of yeast Candida utilis in crotonaldehyde containing media

Summary The inhibitory influence of the higher concentration of 20butenal, crotonaldehyde was followed during the batch and long-term continuous fermentation of Candida utilis growing on synthetic ethanol. Most crotonaldehyde is removed from the medium by biotransformation. Crotonaldehyde inhibits the growth, lengthens the lag phase and decreases the biomass yield and the content of crude proteins in the biomass. The yeast C. utilis is capable of growing on media containing very high concentrations of inhibitor in the in-flow during continuous cultivation. Uncharacteristic transport oscillations of the content of crotonaldehyde were observed for which acidic groups on the cell membrane are probably responsible. A sensitive method which is suitable for measuring very low concentration of crotonaldehyde in aqueous solutions is described. Crotonaldehyde acts as an uncompetitive inhibitor with slight mixed type of inhibition. An equation describing the kinetics of inhibition was derived.     

Age-related distribution of cells in cell cycle phases in dependence on specific rate of growth in chemostat cultures ofCandida utilis

Individual cells in chemostat cultures ofCandida utilis (D=0.05, 0.1, 0.2, 0.25, 0.3 and 35/h) were analyzed according to the number of bud scars and according to their position in the cell cycle. The distribution in the cell cycle phases depends both on the genealogical age and the specific rate of growth.     

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.     

Comparison of various ways of extraction of nucleic acids and of preparation of yeast extract from saccharomyces cerevisiae and candida utilis

Six different variations of the extraction procedure applied to yeast cells of Saccharomyces cerevisiae and Candida utilis to optimize the production of yeast extract and isolation of nucleic acids were compared. The autolysis of C. utilis at 50 to 52°C without adding chemical agents was found to be the best for the production of yeast extract. The most suitable procedures used for the extraction of nucleic acids were those which were carried out from C. utilis at pH 7.5 (92°C) and the other with 0.4 M NH₄OH (40°C). Both these modifications yielded the highest amounts of polymer nucleic acids. Applying all procedures compared to S. cerevisiae an increased content of sterols (including Δ^5.7-sterols, predominantly ergosterol) was detected.     

Variation in seed germination between populations of five sub-alpine woody species from eastern Qinghai-Tibet Plateau following dry storage at low temperatures

Effects of cold-dry storage on dormancy break and viability were determined for seeds of the five sub-alpine woody species Philadelphus incanus, Berberis vernae, Berberis dubia, Betula utilis, and Picea purpurea collected along an altitudinal gradient on the eastern part of the Qinghai-Tibet Plateau in China. Germination tests were conducted at 20/5°C for seeds stored dry at ambient room temperature for 4 weeks and then at 3–4°C for 0, 6, 12, and 24 weeks. Dormancy break during dry storage, i.e., afterripening, was indicated by an increase in germination percentages and rates. Duration of cold-dry storage and altitude of seed collection had significant effects on germination. With an increase in duration of storage, germination percentages and rates of P. incanus and B. vernae increased with a decrease in altitude of seed collection, while they increased with an increase in altitude for seeds of B. utilis and P. purpurea. Seeds of B. dubia did not exhibit changes in germination percentages and rates with altitude because a high number of seeds remained dormant during storage. Seed viability after 24 weeks of storage ranged from high (88, 93.3, 92.7%) for B. utilis to low (15% for high altitude) for P. incanus. The potential for dormancy break to occur during cold-dry storage should be considered when studies on basic seed dormancy are conducted or when seeds from various locations are stored prior to propagating plants from them.     

Functional Purification of the Monocarboxylate Transporter of the Yeast Candida utilis

Plasma membranes of the yeast, Candida utilis, were solubilized with octyl-β-d-glucopyranoside and a fraction enriched in the lactate carrier was obtained with DEAE-Sepharose anion-exchange chromatography, after elution with 0.4 M NaCl. The uptake of lactic acid into proteoliposomes, containing the purified protein fraction and cytochrome c oxidase, was dependent on a proton-motive force and the transport specificity was consistent with the one of C. utilis intact cells. Overall, we have obtained a plasma membrane fraction enriched in the lactate carrier of C. utilis in which the transport properties were preserved. Given the similarities between the lactate transport of C. utilis and the one of mammalian cells, this purified system could be further explored to screen for specific lactate inhibitors, with potential therapeutic applications.     

The effective expression of xylanase gene in <Emphasis Type="Italic">Candida utilis</Emphasis> by 18S rDNA targeted homologous recombination in pGLR9K

In order to test whether 18S rDNA can influence positively xylanase gene effective expression in the yeast of Candida utilis, a targeting vector pGLR9K-XA was constructed by adding an interested gene xynA from Streptomyces olivaceoviridis into the vector pGLR9K which is constructed by ourselves. pGLR9K contains the 18S rDNA, GAP promoter and CYH resistance gene sequence, all of which is from C. utilis. Then the vector pGLR9K-XA was transformed into C. utilis. To test the vector and transformed system, PCR, Southern blot and DNS methods were used. The results showed that xylanase gene can be detected in the chromosome DNA of recombinant C. utilis and the enzyme activity of xylanase is up to 60 IU ml⁻¹ in the study. It is suggested that this system can be used to express exogenous genes in C. utilis as a bioreactors. This is the first report that xylanase gene was expressed in C. utilis.     

Novel lignocellulolytic ability of Candida utilis during solid-substrate cultivation on apple pomace

Lignocellulolytic enzymes from conventional and non-conventional yeasts are not commonly studied, and they have never been described for Candida utilis species. After solid-substrate cultivation of C. utilis (CCT 3469) on apple pomace, degradation of cellulose, pectin and lignin fragments was observed. Production of the main lignocellulolytic enzymes by C. utilis was investigated and high activity for pectinase (239 U ml^–1) as well as a significant manganese-dependent peroxidase (19.1 U ml^–1) activity was found. Low cellulase (3.0 U ml^–1) and xylanase (1.2 U ml^–1) activities were also observed suggesting that C. utilis may have lignocellulose degradation ability.     

金佛山方竹对亮叶水青冈幼树种群数量结构的影响

为探索贵州省宽阔水保护区亮叶水青冈(Fagus lucida)种群的更新维持机制,采用样方法研究金佛山方竹(Chimonobambusa utilis)对亮叶水青冈种群数量结构的影响。结果表明,较高的金佛山方竹密度降低了亮叶水青冈种群数量,亮叶水青冈株数随径级和高度级增加都呈现递减趋势;金佛山方竹密度增加有利于亮叶水青冈小径级向中径级个体的转化,而方竹密度减少则有利于中径级向大径级个体的转化;金佛山方竹密度增大降低了亮叶水青冈种群平均胸径,但增加了平均高度。这表明金佛山方竹能够影响亮叶水青冈个体数量和形态建成,亮叶水青冈通过降低胸径和增加高度来适应竹丛环境。     

Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

NADH-linked aldose reductase: the key to anaerobic alcoholic fermentation of xylose by yeasts

Summary The kinetics and enzymology of d-xylose utilization were studied in aerobic and anaerobic batch cultures of the facultatively fermentative yeasts Candida utilis, Pachysolen tannophilus, and Pichia stipitis. These yeasts did not produce ethanol under aerobic conditions. When shifted to anaerobiosis cultures of C. utilis did not show fermentation of xylose; in Pa. tannophilus a very low rate of ethanol formation was apparent, whereas with Pi. stipitis rapid fermentation of xylose occurred. The different behaviour of these yeasts ist most probably explained by differences in the nature of the initial steps of xylose metabolism: in C. utilis xylose is metabolized via an NADPH-dependent xylose reductase and an NAD⁺-linked xylitol dehydrogenase. As a consequence, conversion of xylose to ethanol by C. utilis leads to an overproduction of NADH which blocks metabolic activity in the absence of oxygen. In Pa. tannophilus and Pi. stipitis, however, apart from an NADPH-linked xylose reductase also an NADH-linked xylose reductase was present. Apparently xylose metabolism via the NADH-dependent reductase circumvents the imbalance of the NAD⁺/NADH redox system, thus allowing fermentation of xylose to ethanol under anaerobic conditions. The finding that the rate of xylose fermentation in Pa. tannophilus and Pi. stipitis corresponds with the activity of the NADH-linked xylose reductase activity is in line with this hypothesis. Furthermore, a comparative study with various xylose-assimilating yeasts showed that significant alcoholic fermentation of xylose only occurred in those organisms which possessed NADH-linked aldose reductase.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

Effects of ribonucleic acid removal methods on proteolytic activity and protein solubility in Candida utilis

Candida utilis was grown under controlled conditions and nucleic acids were removed from whole cells and homogenates by alkaline hydrolysis techniques, en-zymatic methods, and washing with buffer. Homogenization released hydrolytic enzymes and proteolytic activity increased with incubation at elevated temperatures under acidic conditions. Slight proteolysis occurred in all incubated samples and this may contribute to protein insolubilization. Very little protein was lost during incu-bation when compared to similar processes using bakers' yeast. This can be due to lower levels of protease activities in C. utilis. Alkaline hydrolysis methods resulted in hydrolysis of some proteins and irreversible insolubilization of the protein. These methods also destroyed any residual enzymatic activities. Heat denaturation studies suggest that protein insolubilization occurs at neutral pH when heat treatments equivalent to or greater than 85° C for 15 min are used. SDS-PAGE methods were used to characterize and monitor changes in protein. Eighteen proteins and/or sub-units were present at levels of 1% or greater. Results may help to explain changesin functional properties of sample preparations which accompany RNA removal.     

Biological control of oliver scale,Parlatoria oleae (Colvée), in a deciduous fruit Orchard in California

Conclusions Data obtained during the years 1960–1966 show conclusively that the olive scale parasites,Aphytis maculicornis andCoccophagoides utilis, when well-established, are capable of fully effective control of their host on a commerciallty-grown deciduous fruit host.A. maculicornis alone, prior to the establishment ofC. utilis, failed to reduce or maintain control of scale densities. Subsequent to the colonization and establishement ofC. utilis in 1962 and its assumption of a contributing role as an agent of biological control of oliver scale, both species of parasites were able to reduce the host below economic levels within a year. Scale densities have decreased continually since 1963. Continuation of satisfactory biological control is indicated by the low densities of scales now maintained by its parasites. Judicious use of chemical treatments, both in selection and in timing of application, to control other insect and mit problems will ensure the continuing biological control of oliove scale in this deciduous orchard.     

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.     

Significance of yeast peroxisomes in the metabolism of choline and ethanolamine

The yeasts Candida utilis and Hansenula polymorpha were able to grow in media containing choline or ethanolamine as the sole nitrogen source. During growth in the presence of these substrates, large peroxisomes developed in the cells, and extracts of choline-grown C. utilis cells contained increased levels of amine oxidase and catalase. Incubation of whole cells with choline in the presence of the amine oxidase inhibitor aminoacetonitrile led to excretion of dimethylamine and methylamine. Cytochemical experiments in which spheroplasts prepared from choline-grown cells were incubated with CeCl₃ and choline, trimethylamine, dimethylamine or methylamine revealed positively stained peroxisomes, whereas in the presence of 1 mM aminoacetonitrile staining was not observed. This indicated that choline was degraded via methylated amines and that peroxisomes played a role in its metabolism. A similar involvement of peroxisomes in choline degradation was observed in H. polymorpha. Cell-free extracts of ethanolamine-grown C. utilis and H. polymorpha also contained increased levels of amine oxidase and catalase. Ethanolamine was oxidized by cell-free extracts of both organisms after growth in the presence of ethanolamine or choline. Incubation of spheroplasts of ethanolamine-or choline-grown C. utilis with CeCl₃ and ethanolamine resulted in positively stained peroxisomes. In this organism peroxisomes were therefore also involved in ethanolamine degradation.K. B. Zwart was supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).