Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

真菌的多向耐药性ABC转运蛋白

真菌的多向耐药性ABC转运蛋白(ATP-binding cassette transporters)是导致多药耐药性和抗真菌药物治疗效果明显下降的主要原因。文章对酿酒酵母(Saccharomyces cerevisiae)和主要致病真菌白色假丝酵母(Candida albicans)、新型隐球酵母(Cryptococcus neoformans)和烟曲霉(Aspergillus fumigatus)中的多向耐药性ABC转运蛋白的种类、药物外排机制以及基因表达调控网络的研究进展作一综述,为深入了解真菌的多向耐药性机制以及探讨克服多向耐药性的策略和提高药效提供参考。     

Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.     

The metabolic response of Saccharomyces cerevisiae to continuous heat stress

A study has been initiated to integrate molecular and physiological responses of Saccharomyces cerevisiae to heat stress conditions. We focus our research on a quantification of the energetics of the stress response. A series of continuous heat stresses was applied to exponentially growing cells of the strain X2180-1A at 28°C, by increasing the growth temperature to 37, 39, 40, 41, 42, or 43°C. Here, the results on cell growth and viability, as well as on anabolic and catabolic rates are presented. We observed a surprisingly thin line for the cells between growing, surviving, and dying, with regard to growth temperature. The heat stress showed a dual effect on catabolism: immediately after the temperature increase a strong peak was seen, after which a new, steady level was reached. In addition, the yield on glucose decreased with increasing temperature. Our results indicate that life at elevated temperatures is energetically unfavourable and a non-lethal heat stress invokes a redistribution of catabolic and anabolic fluxes.     

Cultivator for NMR studies of suspended cell cultures

When nuclear magnetic resonance (NMR) spectroscopy is employed for physiological experiments with suspended cells, providing for adequate nutrient and oxygen delivery is particularly important, because the inherent insensitivity of NMR requires that concentrated cell suspensions be used. In addition, it is desirable to be able to manipulate the growth rate of cells during a NMR experiment. To address these concerns, a continuous cell cultivator that provides convective oxygen and nutrient transport has been constructed for NMR experiments. The NMR detector coil is located within the cultivator volume. The location is advantageous because the rapid exchange of cells in and out of the coil leads to a small apparent spin lattice relaxation time, thus allowing for rapid pulsing and fast signal averaging. In this article we present the physical principles on which the cultivator's design is based. (31)P spectra showing the response of continuously cultivated Saccharomyces cerevisiae cultures to a phosphate bolus and growth rate shift are then given. (c) 1992 John Wiley & Sons, Inc.     

Absence of Gup1p in Saccharomyces cerevisiae results in defective cell wall composition, assembly, stability and morphology

Saccharomyces cerevisiae Gup1p and its homologue Gup2p, members of the superfamily of membrane-bound O-acyl transferases, were previously associated with glycerol-mediated salt-stress recovery and glycerol symporter activity. Several other phenotypes suggested Gup1p involvement in processes connected with cell structure organization and biogenesis. The gup1Delta mutant is also thermosensitive and exhibits an altered plasma membrane lipid composition. The present work shows that the thermosensitivity is independent of glycerol production and retention. Furthermore, the mutant grows poorly on salt, ethanol and weak carboxylic acids, suggestive of a malfunctioning membrane potential. Additionally, gup1Delta is sensitive to cell wall-perturbing agents, such as Calcofluor white, Zymolyase, lyticase and sodium dodecyl sulphate and exhibits a sedimentation/aggregation phenotype. Quantitative analysis of cell wall components yielded increased contents of chitin and beta-1,3-glucans and lower amounts of mannoproteins. Consistently, scanning electron microscopy showed a strikingly rough surface morphology of the mutant cells. These results suggest that the gup1Delta is affected in cell wall assembly and stability, although the Slt2p/MAP kinase from the PKC pathway was phosphorylated during hypo-osmotic shock to a normal extent. Results emphasize the pleiotropic nature of gup1Delta, and are consistent with a role of Gulp1p in connection with several pathways for cell maintenance and construction/remodelling.     

转新疆雪莲去饱和酶基因sikSAD重组酵母低温和酒精耐受性分析

根据GenBank收录的sikSAD基因序列, 采用反转录PCR技术从新疆雪莲(Sasussured involucrata Kar. et Kir)中克隆了sikSAD基因, 并构建了pYES2-sikSAD大肠杆菌/酵母穿梭表达载体, 通过电击法转化酿酒酵母288C菌株, 并利用PCR和SDS-PAGE对转化酿酒酵母进行鉴定, 最后通过低温胁迫和酒精胁迫进行抗性初步分析及方差分析。结果表明: 低温胁迫实验中, 转sikSAD基因酿酒酵母在低温条件下仍能存活, 并且在温度恢复到28 °C时能迅速生长, 生长状态良好, 不饱和脂肪酸油酸的含量有明显的变化。酒精胁迫实验中, 其能耐受一定浓度的酒精, 并且耐受能力比非转基因酿酒酵母提高了十几个百分点。可见, 在低温胁迫和高浓度酒精条件下, 转新疆雪莲sikSAD基因酿酒酵母表现出了优良的活性和生长优势, 显示出较强的抗性特征, 用分子手段改造酿酒酵母, 为工业生产提供高质量的酿酒酵母奠定实验基础。     

Immobilization of UDP-Galactose 4-Epimerase from Escherichia coli on the Yeast Cell Surface

UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, the Gal E gene from E. coli, coding UDP-galactose 4-epimerase, was cloned into pYD1 plasmid and then transformed into Saccharomyces cerevisiae EBY100 for expression of Gal E on the cell surface. Enzyme activity analyses with EBY100 cells showed that the enzyme displayed on the yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It took about 3 min to reach equilibrium from UDP-galactose to UDP-glucose.     

酿酒酵母产油脂条件的优化

微生物细胞通常仅含2%3%油脂,但少数微生物含油脂率却可达70%以上,所以高含油脂量使微生物油脂实际开发成为可能。目前用于生产多不饱和脂肪酸的微生物主要为藻类和真菌。尽管微生物油脂是当前的研究热点,已经引起广大研究者的重视,但目前国内外研究大都集中在含油脂量在干重20%以上的微生物,如浅白色隐性酵母、粘红酵母等,而对于酿酒酵母来说,则很少见到研究其产油脂的相关报道。     

Reaction kinetics of d-glucose fermentation by Saccharomyces cerevisiae

Data obtained on the conversion of d-glucose to alcohol using Saccharomyces cerevisiae in batch culture has been analysed kinetically. The effects of different kinetic parameters, e.g. rates of ethanol and biomass formation, rate of d-glucose utilization and variation of pH have been studied. Analysis of data was made on the basis of Michaelis-Menten, Leudeking-Piret and simple kinetics. Unsteady rate behaviour in the lag phase was observed and explained.     

酵母菌属间原生质体融合构建高温酵母菌株

酿酒酵母(Saccharomyces cerevisiae) A001和克鲁维酵母(Kluyveromyces sp.) Y034属间原生质体融合构建高温酵母菌株。对制备高再生活性原生质体及融合子细胞形态、生理化特征、同工酶性质、遗传稳定性和高温发酵等方面进行了研究。结果表明,融合子AY023和AY680遗传性能稳定,表达了双亲优良性状,获得了在45℃培养条件下产酒率7.4%的属间融合菌株,是目前已见文献报道的产酒率最高的高温(45℃)酵母菌株。     

Genetic evidence of a new flocculation suppressor gene in Saccharomyces cerevisiae

Abstract The flocculation character in strain IM1-8b of Saccharomyces cerevisiae is controlled by a single and dominant gene shown to be allelic to FLO1 . Such a gene has been both mitotically and meiotically mapped on the right arm of chromosome I at 4.7 cM from PHO11 . The phenotype was suppressed by a single gene of wide distribution among non-flocculent strains (proposed as fsu3 ) that, however, was unable to suppress other FLO1 genes in other flocculent strains.     

Production of Extracellular lipases by Saccharomyces cerevisiae

Seven strains of Saccharomyces cerevisiae all produced lipase when grown in shake flask culture. The best strain, DSM 1848, produced 4.0U of lipase in the medium containing olive oil and yeast extract. Production of the lipase was growth-associated.