The Yeast Saccharomyces cerevisiae in Molecular and Cellular Biology: A Smaller but not Lower Eucaryote

The baker's yeast Saccharomyces cerevisiae is a single celledeucaryote that is gaining appeal for studies of fundamentalprocesses in cellular and molecular biology. This article describesthree examples of the use of yeast to study problems of importanceto human medicine. Specifically, the enzymes involved in neuropeptideprocessing, the RAS oncogenes, and the regulation of cholesterolbiosynthesis in humans are shown to have remarkable similaritiesto their equivalent processes in yeast. In several cases theyeast and human enzymes are functionally interchangeable, emphasizingthe unity of basic principles in biology.     

Molecular and physiological comparisons between Saccharomyces cerevisiae and Saccharomyces boulardii

In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.     

Molecular insights on DNA delivery into Saccharomyces cerevisiae

Understanding of the molecular system for DNA delivery into eucaryotic cells, a key to human DNA therapy, remains obscure. To understand this system, we undertook a study using the Saccharomyces cerevisiae model into which DNA delivery is easily assessed through competence (transformability) and for which all nonessential gene mutants (about 5000 strains) are available. We analyzed the competence of each of these mutants and identified three low-competence mutants, i.e., sin3Delta, she4Delta, and arc18Delta, and three high-competence mutants, i.e., pde2Delta, spf1Delta, and pmr1Delta. Through further studies using the six mutants, we concluded that the Arp2/3 activation machinery involving the Myo3/5p, Vrp1p, Las17p, Pan1p, and Arp2/3 complex is crucial to delivery (competence), and that high cAMP enhances competence via protein kinase A installing Tpk3p. We also propose that DNA is taken up via an endocytosis-like event, being at least partially different from well-known endocytosis.     

Effects of Fusariotoxin T-2 on Saccharomyces cerevisiae and Saccharomyces carlsbergensis

A Fusarium metabolite, T-2 toxin, inhibits the growth of Saccharomyces carlsbergensis and Saccharomyces cerevisiae. The growth inhibitory concentrations of T-2 toxin were 40 and 100 μg/ml, respectively, for exponentially growing cultures of the two yeasts. S. carlsbergensis was more sensitive to the toxin and exhibited a biphasic dose-response curve. Addition of the toxin at 10 μg/ml of S. carlsbergensis culture resulted in a retardation of growth as measured turbidimetrically, after only 30 to 40 min. This action was reversible upon washing the cells free of the toxin. The sensitivity of the yeasts to the toxin was dependent upon the types and concentrations of carbohydrates used in the growth media. The sensitivity of the cells to the toxin decreased in glucose-repressed cultures. These results suggest that T-2 toxin interferes with mitochondrial functions of these yeasts.     

Molecular genetic diversity of the Saccharomyces yeasts in Taiwan: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii

Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.     

Effects of tellurite on growth of Saccharomyces cerevisiae

The effects of potassium tellurite on growth and survival of rho⁺ and rho⁰ Saccharomyces cerevisiae strains were investigated. Both rho⁺ and rho⁰ strains grew on a fermentable carbon source with up to 1.2 mM K₂TeO₃, while rho⁺ yeast cells grown on a non-fermentable carbon source were inhibited at tellurite levels as low as 50 μM suggesting that this metalloid specifically inhibited mitochondrial functions. Growth of rho⁺ yeast cells in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture, a phenomenon not observed with rho⁰ cultures. Transmission electron microscopy of S. cerevisiae rho⁺ cells grown in the presence of tellurite showed that blackening was likely due to elemental tellurium (Te⁰) that formed large deposits along the cell wall and small precipitates in both the cytoplasm and mitochondria.     

Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae

Industrial biotechnology is a rapidly growing field. With the increasing shift towards a bio-based economy, there is rising demand for developing efficient cell factories that can produce fuels, chemicals, pharmaceuticals, materials, nutraceuticals, and even food ingredients. The yeast Saccharomyces cerevisiae is extremely well suited for this objective. As one of the most intensely studied eukaryotic model organisms, a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance can be capitalized upon to enable a substantial increase in the industrial application of this yeast. Developments in genomics and high-throughput systems biology tools are enhancing one's ability to rapidly characterize cellular behaviour, which is valuable in the field of metabolic engineering where strain characterization is often the bottleneck in strain development programmes. Here, the impact of systems biology on metabolic engineering is reviewed and perspectives on the role of systems biology in the design of cell factories are given.     

Effects of cis-Platin on t-RNA of Saccharomyces cerevisiae

We synthesized three hydantocidin derivatives and evaluated their herbicidal activity in order to elucidate the role of the spirohydantoin system at the anomeric center of hydantocidin. With application to foliage at 1000 ppm, only α-ureidoamide 14 demonstrated activity, the remaining compounds being found to be inactive.     

Molecular specificity of 2-aminopurine in Saccharomyces cerevisiae

The rates of DNA, RNA and protein synthesis were investigated by incorporation of radioactive precursors into the excised root tips of V. faba. 2-h exposure to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate. RNA synthesis was reduced in the range of 20–30%. The same concentration of caffeine did not affect the rate of DNA synthesis even during 12-h incubation, but concentrations higher than 1% caused a significant decrease in [³H]thymidine incorporation.     

Comparative Studies of Tri- and Hexavalent Chromium Cytotoxicity and Their Effects on Oxidative State of Saccharomyces cerevisiae Cells

Chromium is a significant mutagen and carcinogen in environment. We compared the effects of tri- and hexavalent chromium on cytotoxicity and oxidative stress in yeast. Cell growth was inhibited by Cr³⁺ or Cr⁶⁺, and Cr⁶⁺ significantly increased the lethal rate compared with Cr³⁺. Both Cr³⁺ and Cr⁶⁺ can enter into the yeast cells. The percent of propidium iodide permeable cells treated with Cr³⁺ is almost five times as that treated with the same concentration of Cr⁶⁺. Levels of TBARS, O₂ ^?, and carbonyl protein were significantly increased in both Cr⁶⁺- and Cr³⁺-treated cells in a concentration- and time-dependent manner. Moreover, the accumulation of these stress markers in Cr⁶⁺-treated cells was over the Cr³⁺-treated ones. The decreased GSH level and increased activity of GPx were observed after 300 μM Cr⁶⁺-exposure compared with the untreated control, whereas there was no other change of GSH content in cells treated with Cr³⁺ even at very high concentration. Exposure to both Cr³⁺ and Cr⁶⁺ resulted in the decrease of activities of SOD and catalase. Furthermore, the effect of Cr⁶⁺ is stronger than that of Cr³⁺. Null mutation sensitivity assay demonstrated that the gsh1 mutant was sensitive to Cr⁶⁺ other than Cr³⁺, the apn1 mutant is more sensitive to Cr⁶⁺ than Cr³⁺, and the rad1 mutant is sensitive to both Cr⁶⁺ and Cr³⁺. Therefore, Cr³⁺ can be concluded to inhibit cell growth probably due to the damage of plasma membrane integrality in yeast. Although both tri- and hexavalent chromium can induce cytotoxicity and oxidative stress, the action mode of Cr³⁺ is different from that of Cr⁶⁺, and serious membrane damage caused by Cr³⁺ is not the direct consequence of the increase of lipid peroxidation.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k_cat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.     

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 δ-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. δ-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.