Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae

Glucoamylase (SGA) was purified approximately 250-fold from sporulating Saccharomyces cerevisiae cells. The partially purified enzyme was active against glycogen, starch, maltotriose and maltose. It exhibited maximum catalytic activity against glycogen at pH 5.5. The enzyme appears to be glycosylated, because it bound to lentil-lectin Sepharose. SGA was expressed in vegetatively growing cells under the control of the GAL1 promoter, and the cellular location of the enzymatic activity determined by fractionation techniques. SGA was preferentially recovered in fractions which were enriched for the vacuolar hydrolases, carboxypeptidase Y and alpha-mannosidase.     

Relating individual behaviour to population dynamics

How do the behavioural interactions between individuals in an ecological system produce the global population dynamics of that system? We present a stochastic individual-based model of the reproductive cycle of the mite Varroa jacobsoni, a parasite of honeybees. The model has the interesting property in that its population level behaviour is approximated extremely accurately by the exponential logistic equation or Ricker map. We demonstrated how this approximation is obtained mathematically and how the parameters of the exponential logistic equation can be written in terms of the parameters of the individual-based model. Our procedure demonstrates, in at least one case, how study of animal ecology at an individual level can be used to derive global models which predict population change over time.     

Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae

To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.     

Role of reserve carbohydrates in the growth dynamics of Saccharomyces cerevisiae

The purpose of this study was to explore the role of glycogen and trehalose in the ability of Saccharomyces cerevisiae to respond to a sudden rise of the carbon flux. To this end, aerobic glucose-limited continuous cultures were challenged with a sudden increase of the dilution rate from 0.05 to 0.15 h(-1). Under this condition, a rapid mobilization of glycogen and trehalose was observed which coincided with a transient burst of budding and a decrease of cell biomass. Experiments carried out with mutants defective in storage carbohydrates indicated a predominant role of glycogen in the adaptation to this perturbation. However, the real importance of trehalose in this response was veiled by the unexpected phenotypes harboured by the tps1 mutant, chosen for its inability to synthesize trehalose. First, the biomass yield of this mutant was 25% lower than that of the isogenic wild-type strain at dilution rate of 0.05 h(-1), and this difference was annulled when cultures were run at a higher dilution rate of 0.15 h(-1). Second, the tps1 mutant was more effective to sustain the dilution rate shift-up, apparently because it had a faster glycolytic rate and an apparent higher capacity to consume glucose with oxidative phosphorylation than the wild type. Consequently, a tps1gsy1gsy2 mutant was able to adapt to the dilution rate shift-up after a long delay, likely because the detrimental effects from the absence of glycogen was compensated for by the tps1 mutation. Third, a glg1Deltaglg2Delta strain, defective in glycogen synthesis because of the lack of the glycogen initiation protein, recovered glycogen accumulation upon further deletion of TPS1. This recovery, however, required glycogen synthase. Finally, we demonstrated that the rapid breakdown of reserve carbohydrates triggered by the shift-up is merely due to changes in the concentrations of hexose-6-phosphate and UDPglucose, which are the main metabolic effectors of the rate-limiting enzymes of glycogen and trehalose pathways.     

Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae

A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme. This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues. Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35%–56%, and most homology with that of Aspergillus niger. The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADH1). Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability.     

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.     

Localization of glucoamylase genes of Saccharomyces cerevisiae by pulsed field gel electrophoresis

The chromosomal locations of four glucoamylase-specifying genes in the yeastSaccharomyces cerevisiae have been determined. Chromosomes were separated by pulsed field gel electrophoresis and blots were probed with radiolabelledSTA2 and marker DNA from specific yeast chromosomes. The three genes encoding extracellular glucoamylases,STA1 (DEX2), STA2 (DEX1) andSTA3 (DEX3) are located on chromosomes IV, II and XIV, respectively.SGA, specifying the sporulation-specific glucoamylase, was positioned on chromosome IX.     

Heterogeneity of stress gene expression and stress resistance among individual cells of Saccharomyces cerevisiae

Knowledge of gene expression and cellular responses in microorganisms is derived from analyses of populations consisting of millions of cells. Analytical techniques that provide data as population averages fail to inform of culture heterogeneity. Flow cytometry and fluorescence techniques were used to provide information on the heterogeneity of stress-responsive gene expression and stress tolerance in individual cells within populations. A sequence of DNA encoding the heat shock and stress response elements of the Saccharomyces cerevisiae HSP104 gene was used to express enhanced green fluorescent protein (EGFP). When integrated into the genome of yeast strain W303-1A, intrinsic expression of EGFP increased about twofold as cells progressed from growth on glucose to ethanol utilization in aerobic batch cultures. Staining of cells with orange/red fluorescent propidium iodide (PI), which only enters cells that have compromised membrane integrity, revealed that the population became more tolerant to 52 degrees C heat stress as it progressed from growth on glucose and through the ethanol utilization phase of aerobic batch culture. Exposure of cultures growing on glucose to a mild heat shock (shift from 25 degrees C to 37 degrees C) resulted in significantly increased expression of EGFP in the population. However, there was heterogeneity in the intensity of fluorescence of individual cells from heat-shocked cultures, indicating variability in the strength of stress response in the clonal population. Detailed analysis of the heterogeneity showed a clear positive trend between intensity of stress response and individual cell resistance, measured in terms of PI exclusion, to heat stress at 52 degrees C. Further experiments indicated that, although the mean gene expression by a population is influenced by the genetic background, the heterogeneity among individual cells in clonal populations is largely physiologically based.     

Susceptibility of individual cells of Saccharomyces cerevisiae to the killer toxin K1

The susceptibility of sensitive yeast to killer toxins is known to depend on various factors, such as the selected killer toxin, the exposed yeast strain, its growth phase and the state of culture under given experimental conditions. The aim of this paper was to find whether individual cells from one culture are equally susceptible to the impact of the killer toxin. For this purpose the rhodamine B assay in a modified form was used. In order to observe the fate of individual cell the method of fluorescence video microscopy with a digital picture analysis was applied. Four selected groups of specific cells (with no, small, medium, and large bud, respectively) were investigated. Different sensitivity of Saccharomyces cerevisiae cells to the killer toxin K1 was observed in these cell groups. The most susceptible appeared to be the cells which were in S-phase (cells with the small buds); the least susceptible were the M-phase cells with large buds. The enhanced susceptibility in S-phase results probably from coincidence in higher porosity of the cell wall, accumulation of surface receptors, and enlarged growth activity at the surface cell structures.     

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

High expression and efficient secretion of Rhizopus oryzae glucoamylase in the yeast Saccharomyces cerevisiae

Summary The expression and secretion of Rhizopus oryzae glucoamylase were studied in the yeast Saccharomyces cerevisiae. Rhizopus oryzae glucoamylase was highly expressed and efficiently secreted into a medium to a high level (above 300 mg/l) under control of a yeast promoter and the original signal sequence. Excess expression of the secretable glucoamylase with high copy number plasmid slightly decreased growth of the transformant cells in glucose medium but not in fructose medium.     

Dependency of size of Saccharomyces cerevisiae cells on growth rate.

The mean size and percentage of budded cells of a wild-type haploid strain of Saccharomyces cerevisiae grown in batch culture over a wide range of doubling times (tau) have been measured using microscopic measurements and a particle size analyzer. Mean size increased over a 2.5-fold range with increasing growth rate (from tau = 450 min to tau = 75 min). Mean size is principally a function of growth rate and not of a particular carbon source. The duration of the budded phase increased at slow growth rates according to the empirical equation, budded phase = 0.5 tau + 27 (all in minutes). Using a recent model of the cell cycle in which division is thought to be asymmetric, equations have been derived for mean cell age and mean cell volume. The data are consistent with the notion that initiation of the cell cycle occurs at "start" after attainment of a critical cell size, and this size is dependent on growth rate, being, at slow growth rates, 63% of the volume of fast growth rates. Previous reports are reanalyzed in the light of the unequal division model and associated population equations.     

Maximum glucoamylase production by a temperature-sensitive mutant of Saccharomyces cerevisiae in batch culture

In order to maximize the glucoamylase production by recombinant Saccharomyces cerevisiae in batch culture, first a temperature-controlled expression system for a foreign gene in S. cerevisiae was constructed. A temperature-sensitive pho80 mutant of S. cerevisiae for the PHO regulatory system, YKU131, was used for this purpose. A DNA fragment bearing the promoter of the PHO84 gene, which encodes an inorganic phosphate (P_i) transporter of S. cerevisiae and is derepressed by P_i starvation, was used as promoter. The glucoamylase gene connected with the PHO84 promoter was ligated into a YEp13 vector, designated pKU122. When the temperature-sensitive pho80 ^ts mutant harboring the plasmid pKU122 is cultivated at a lower temperature, the expression of glucoamylase gene is repressed, but at a higher temperature it is expressed. Next the effect of temperature on the specific growth rate, μ, and specific production rate, ρ, was investigated. Maximum values of ρ and ρ at various temperatures were at 30°C and 34°C, respectively. The optimal cultivation temperature strategy for maximum production of glucoamylase by this recombinant strain in batch culture was then determined by the Maximum principle using the relationships of μ and ρ to the cultivation temperature. Finally, the optimal strategy was experimentally realized by changing the cultivation temperature from Tμ (30°C) to Tρ (34°C) at the switching time, t_s. Received 18 September 1997/ Accepted in revised form 07 January 1998     

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.