Secretory expression of human growth hormone inSaccharomyces cerevisiae using three different leader sequences

A recombinant human growth hormone (hGH) was expressed as a secretory product in the yeastSaccharomyces cerevisiae. Three different leader sequences derived from the mating factor α1 (MFα1), inulinase and invertase were used to direct the secretion of hGH into the extracellular medium. Among three leader sequences tested, the inulinase leader sequence was found to be the most efficient in the secretory expression of hGH. In contrast, no hGH was detected in the extracellular medium with the invertase leader sequence. After 48 h shake-flask culture, the yields of hGH secreted into the medium by the invertase, MFα1, inulinase and invertase leader sequences were approximately 0, 0.3 and 0.9 mg/L, respectively. The secretion efficiencies were also found to be 0, 3.8 and 13% for the invertase, MFα1 and inulinase leader sequences, respectively.     

Rapid purification of recombinant human lipocortin-I secreted fromSaccharomyces cerevisiae

Human lipocortin-I was expressed as a secretory product bySaccharomyces cerevisiae harboring an expression system consisting ofGAL10 promoter, inulinase signal sequence and lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys²⁶.     

Flow cytometric analysis of nocodazole-induced cell-cycle arrest in the pennate diatom Phaeodactylum tricornutum Bohlin

The microtubule inhibitor, nocodazole (2.5 mg L^-1), can arrest the cell cycle of the pennate diatom Phaeodactylum tricornutum Bohlin at G₂ + M phase. Flow cytometric analysis of cells treated with nocodazole suggest that the proportion of cells at G₂ + M phase can accumulate to over 95%. Even after a 48-h treatment with nocodazole (2.5 mg L^-1), the cells can still exit mitosis, suggesting that the cell-cycle arrest is reversible. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic analysis of inducible sexual agglutination ability in the yeast Saccharomyces cerevisiae

Genetic regulation of the inducibility of sexual agglutination ability in the yeast Saccharomyces cerevisiae was studied. Detailed analysis of the degree of sexual agglutination was carried out; it showed that a greater number of genes are involved in the regulation of inducible sexual agglutination in strain H1-0 than previously assumed. Although dominancy of inducible phenotype over constitutive was confirmed, the effectiveness of one gene changing the constitutive phenotype to the inducible seemed to be somewhat low. Quantity per cell of agglutination substances responsible for sexual agglutination increased as the agglutination ability became greater.     

Scale-up of recombinant hirudin production fromSaccharomyces cerevisiae

Scale-up of hirudin production fromSaccharomyces cerevisiae from bench-scale to pilot-scale was carried out based on constant volumetric oxygen transfer coefficient (K _L a). Fed-batch mode of cultivation using step-wise feeding strategy of galactose was employed for the production of hirudin in a 30-L and a 300-L pilot-scale fermentor. The final hirudin concentrations were achieved 390 mg/L and 286.1 mg/L, and the volumetric productivities were 80.4% and 90.7% with the 30-L and 300-L fermentors, respectively, compared to the productivity of the 5-L bench-scale fermentor.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Acid excreting mutants of yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants acidifying glucose medium containing bromocresol purple were shown to excrete protons when placed in unbuffered water in the absence of any external carbon source. The mutants belong to 16 different complementation groups. Most of them do not grow on glycerol and the excreted protons are associated to particular sets of organic anions such as citrate, aconitate, succinate, fumarate or malate. These novel types of respiratory mutations seem to be located in genes operating in the Krebs or glyoxylate cycle.     

A possible application of ale brewery strains ofSaccharomyces cerevisiae in lager beer production

The physiological characteristics of two strains of brewery ale yeasts,Saccharomyces cerevisiae, with sedimentation abilities, were investigated to see if the strains were suitable for lager beer production. Compared with typical industrial ale strains ofS. cerevisiae and lager strains ofS. uvarum (nowS. cerevisiae), the investigated strains differ in fermentation dynamics, as well as in biological properties. The differences, however, particularly between the two strains and the lager brewing yeasts, were not significant.     

A comparison of fluorescent DNA binding dyes for flow cytometric analysis of sporulating Saccharomyces cerevisiae

Flow cytometry is important tool for investigating DNA replication in sporulating Saccharomyces cerevisiae. However, flow cytometry data from maturing spores is often difficult to interpret due to extensive broadening of the fluorescence peaks. This problem is markedly improved by treatment of the spores with potassium hydroxide prior to staining.     

Electron microscopy of germinating ascospores ofSaccharomyces cerevisiae

The wall of mature ascospores ofSaccharomyces cerevisiae showed in sections under the electron microscope a dark outer layer and a lighter inner layer. The latter was composed of a greyish inner part and a light outer part. During germination, the spore grew out at one side and the dark outer layer was broken. Of the light inner layer, the inner greyish part became the wall of the vegetative cell, but the extented part of the cell had a new wall.     

Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated.     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Heterologous expression and secretion of sweet potato peroxidase isoenzyme A1 in recombinant Saccharomyces cerevisiae

A vector system has been developed to express isoenzyme A1 of sweet potato peroxidase (POD) and was introduced into Saccharomyces cerevisiae. The system contains the signal sequence of Aspergillus oryzae -amylase to facilitate the extracellular secretion of peroxidase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. In a batch culture using YNBDCA medium (yeast nitrogen base without amino acids 6.7 g l^–1, Casamino acids 5 g l^–1 and glucose 20 g l^–1), the recombinant strain expressed the swpa1 gene giving a secretion yield of POD activity of ca. 90% of total expressed peroxidase. Supplementation with PMSF (0.05 mM) and Casamino acids (5 g/50 ml) increased extracellular POD activity to nearly 10 kU ml^–1, equivalent to 1.5 kU g^–1 cell dry wt. This is 9 fold higher than that obtained in medium without PMSF. From SDS-PAGE and native-PAGE analyses POD has an M_r of 53 kDa.     

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare.The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus.

Subjects and methods

Vaginal samples were collected from a total of262 (asymptomaticandsymptomatic) women with vaginitis attending the centre of family planning of General hospital ofPiraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae.

Results

A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker’s yeast.

Conclusions

Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy.     

Metabolic flux analysis of the sterol pathway in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model system for examining the biosynthesis of sterols in eukaryotic cells. To investigate underlying regulation mechanisms, a flux analysis of the ergosterol pathway was performed. A stoichiometric model was derived based on well known biochemistry of the pathway. The model was integrated in the Software COMPFlux which uses a global optimization algorithm for the estimation of intracellular fluxes. Sterol concentration patterns were determined by gas chromatography in aerobic and anaerobic batch cultivations, when the sterol metabolism was suppressed due to the absence of oxygen. In addition, the sterol concentrations were observed in a cultivation which was shifted from anaerobic to aerobic growth conditions causing the sterol pools in the cell to be filled. From time-dependent flux patterns, possible limitations in the pathway could be localized and the esterification of sterols was identified as an integral part of regulation in ergosterol biosynthesis.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Quantification of Saccharomyces cerevisiae viability using BacLight

Yeast viability can be accurately quantified using BacLight, a kit which so far has been used only for bacterial analysis. Upon staining, viable cells can be differentiated from non-viable ones by either confocal laser scanning microscopy (CLSM), epifluorescence microscopy, or flow cytometry. Using Saccharomyces cerevisiae as a model, viabilities quantified by CLSM deviated an average of 1.7% from the actual data, and those determined by flow-cytometry by 1.4%.     

Mutation at the CK2 phosphorylation site on Cdc28 affects kinase activity and cell size in Saccharomyces cerevisiae

We have recently reported that protein kinase CK2 phosphortylates both in vivo and in vitro residue serine-46 of the cell cycle regulating protein Cdc28 of budding yeast Saccharomyces cerevisiae, confirming a previous observation that the same site is phosphorylated in Cdc2/Cdk1, the human homolog of Cdc28. In addition, S. cerevisiae in which serine-46 of Cdc28 has been mutated to alanine show a decrease of 33% in both cell volume and protein content, providing the genetic evidence that CK2 is involved in the regulation of budding yeast cell division cycle, and suggesting that this regulation may be brought about in G1 phase of the mammalian cell cycle. Here, we extended this observation reporting that the mutation of serine-46 of Cdc28 to glutamic acid doubles, at least in vitro, the H1-kinase activity of the Cdc28/cyclin A complex. Since this mutation has only little effects on the cell size of the cells, we hypothesize multiple roles of yeast CK2 in regulating the G1 transition in budding yeast.     

Efficient secretion of human lysozyme from the yeast, <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis, was achieved by using more stable vectors in the order of S11 replication origin-containing episomal vector < full-length K. lactis plasmid pKD1-containing vector < centromeric vector < chromosome-integrated vectors. Cells containing a PGK (phosphoglycerate kinase) promoter-driven integration vector grown in non-selective rich medium achieved the highest level of secretion, 100 g lysozyme secretion ml ^–1 culture: this level was 10-fold higher than that achieved by episomal vectors. An additional copy of the protein disulfide isomerase gene further facilitated the secretion.