Redox and specific effects of vanadium ions on respiratory enzymes

The effects of vanadium ions on the activities of enzymes of aerobic and anaerobic respiratory chains were investigated in vitro and in situ employing ¹H-, ¹⁴N-, ³¹P- and ⁵¹V- nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and spectrophotometry. Vanadate and vanadyl ions produced either non-specific redox or specific activation or inhibition of respiratory enzymes. The oxidants molybdate and chromate and the reductant dithiothreitol were used to distinguish between non-specific and specific effects of vanadium ions on enzyme activities. The results suggested that components of anaerobic respiratory chains were more susceptible to vanadium ions than those of the aerobic respiratory chain     

Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO₄ ^3- concentration and the growth was completely arrested at a concentration of 20 mM of VO₄ ^3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A mobile and an immobile species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time _r indicated the relative motional freedom at the macromolecular site. A strongly immobilized vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO²⁺ ions with isolated cell walls.     

The effects of vanadate on the yeast Saccharomyces cerevisiae

Growth of Saccharomyces cerevisiae on non-fermentable medium was more sensitive to inhibition by vanadate than growth of fermentable medium. The frequency of petite mutants increased in cultures grown for 18 hours in fermentable medium containing vanadate. However, oxygen uptake markedly increased in yeast cultures grown in the presence of vanadate, a similar effect being produced by phosphate. It was also found that oligomycin toxicity was relieved by vanadate. These results suggest that vanadate may interact with the mitochondria of S. cerevisiae.     

1种用于检测抑制基因功能的酵母株系的建立

酵母被广泛用于分子生物学中基因功能的检测。为扩大酵母株系UCC419在抑制基因活性检测方面的应用,本研究通过向UCC419株系中导入用特殊引物扩增出的包含标记基因TRP1的PCR片段,利用同源重组将UCC419中的筛选标记基因LEU2敲除,并同时插入TRP1,新建立的株系命名为UCC419m（m：modi-fied）。UCC419m为TRP1筛选、leu2突变型菌株,其它基因型均同UCC419。给UCC419m中转入携带LEU2的质粒pDEST32检测是否能恢复其表现型,同时转入不携带LEU2的质粒pDEST22作为阴性对照,将转化子在不含LEU2与URA3的培养基中培养,结果显示,携带LEU2质粒pDEST32的转化子能够在LEU2与URA3缺陷型培养基上正常生长,而不携带LEU2质粒pDEST22的转化子不能生长。本研究结果表明,成功建立了一种适用于基于Invitrogen载体的抑制基因活性检测或从文库中筛选抑制基因的酵母菌株。     

Proteomic analysis of a distilling strain of Saccharomyces cerevisiae during industrial grain fermentation

The fermentation performance of industrial yeast strains is influenced, among other things, by their genetic composition and the nature of the fermentable sugar, availability of nitrogen, and temperature. Therefore, to manipulate the fermentation process, it is important to understand, at a molecular level, the changes occurring in the yeast cell throughout industrial fermentation processes. With this aim in mind, using two-dimensional gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), we have examined the proteome of distillers yeast in an industrial context. Using yeast sampled from a local grain whisky distillery, we have prepared a detailed reference map of the proteome of distillers yeast and have examined in some detail the alterations in protein levels that occur throughout fermentation. In particular, as fermentation progresses, there is a significant increase in the levels of a variety of proteins involved in protecting against stress and nitrogen limitation. These results therefore give an insight into the stresses that yeast are exposed to in industrial fermentations and reveal some of the proteins and enzymes that are either necessary or important for efficient fermentation.     

Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae

The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions. The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth. In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth. Orthovanadate addition to whole cells is known to stimulate various cellular processes. In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with [48V]VO4(3-) and VO2+. ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g. V(V)). Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium. ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium. 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM). These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate. These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms. These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.     

Survival of inoculated Saccharomyces cerevisiae strain on wine grapes during two vintages

AIMS: To investigate the influence of a specific ecological niche, the wine grape, on the survival and development of Saccharomyces cerevisiae. METHODS AND RESULTS: A strain with a rare phenotype was sprayed onto the grape surfaces and monitored through two vintages using a specific indicative medium and analysing the internal transcribed spacer regions in the 5.8S rDNA. During the ripening process, there was a progressive colonization of the surface of the undamaged and damaged grapes by epiphytic yeasts, up to the time of harvest. The damaged wine grapes showed a much greater epiphytic yeast population. However, the inoculated S. cerevisiae strain showed a scarce persistence on both undamaged and damaged wine grapes, and the damaged grapes did not appear to improve the grape surface colonization of this strain. CONCLUSIONS: Results indicated that wine grape is not a favourable ecological niche for the development and colonization of S. cerevisiae species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this work are further evidence that S. cerevisiae is not specifically associated with natural environments such as damaged and undamaged wine grapes.     

Functional genomic analysis of a commercial wine strain of Saccharomyces cerevisiae under differing nitrogen conditions

DNA microarray analysis was used to profile gene expression in a commercial isolate of Saccharomyces cerevisiae grown in a synthetic grape juice medium under conditions mimicking a natural environment for yeast: High-sugar and variable nitrogen conditions. The high nitrogen condition displayed elevated levels of expression of genes involved in biosynthesis of macromolecular precursors across the time course as compared to low-nitrogen. In contrast, expression of genes involved in translation and oxidative carbon metabolism were increased in the low-nitrogen condition, suggesting that respiration is more nitrogen-conserving than fermentation. Several genes under glucose repression control were induced in low-nitrogen in spite of very high (17%) external glucose concentrations, but there was no general relief of glucose repression. Expression of many stress response genes was elevated in stationary phase. Some of these genes were expressed regardless of the nitrogen concentration while others were found at higher levels only under high nitrogen conditions. A few genes, FSP2, RGS2, AQY1, YFL030W, were expressed more strongly with nitrogen limitation as compared to other conditions.     

啤酒酵母菌对无机硒的有机转化

利用啤酒酵母菌对无机硒(亚硒酸钠)进行有机转化。通过在培养基中加入不同浓度的无机硒溶液和不同时间加入无机硒溶液,于28℃、220 r/min摇床条件下培养5 d,离心得菌细胞,测定前样品预处理:破碎菌细胞,显微镜下计数,计算破碎率,破碎后的菌体装入透析袋于蒸馏水中透析除去无机硒。准确测定无机硒,用浓硫酸-高氯酸的消化体系消化样品后,紫外分光光度法于335 nm处测量吸光度,在标准曲线上查出硒含量,计算无机硒的转化率。啤酒酵母菌的最佳加硒时间为24 h,亚硒酸钠浓度大于12μg/mL对啤酒酵母菌转化无机硒有明显抑制作用,啤酒酵母菌对无机硒的摄入率约为62%,转化率约为53%;超生波细胞粉碎仪破碎细胞的破碎率为55%左右。结果表明,啤酒酵母菌可以转化无机硒。     

Multiple Ty-mediated chromosomal translocations lead to karyotype changes in a wine strain of Saccharomyces cerevisiae.

Enological strains of Saccharomyces cerevisiae display a high level of chromosome length polymorphism, but the molecular basis of this phenomenon has not yet been clearly defined. In order to gain further insight into the molecular mechanisms responsible for the karyotypic variability, we examined the chromosomal constitution of a strain known to possess aberrant chromosomes. Our data revealed that the strain carries four rearranged chromosomes resulting from two reciprocal translocations between chromosomes III and I, and chromosomes III and VII. The sizes of the chromosomal fragments exchanged through translocation range from 40 to 150 kb. Characterization of the breakpoints indicated that the translocations involved the RAHS of chromosome III, a transposition hot-spot on the right arm of chromosome I and a region on the left arm of chromosome VII. An analysis of the junctions showed that in all cases Ty elements were present and suggested that the translocations result from recombination between transposable Ty elements. The evidence for multiple translocations mediated by Ty elements in a single strain suggests that spontaneous Ty-driven rearrangement could be quite common and may play a major role in the alteration of karyotypes in natural and industrial yeasts. Received: 18 December 1998 / Accepted: 26 March 1999     

Fermentation of starch to ethanol by a co-culture of Saccharomycopsis fibuligera and Saccharomyces cerevisiae

Static fermentation of starch to ethanol by a co-culture of Saccharomycopsis fibuligera and Saccharomyces cerevisiae without addition of nutritional supplements was investigated with respect to initial starch concentration, pH of the media and initial dry weight ratio of Sps. fibuligera to Sacc. cerevisiae biomass (I _R).Optimal conditions for ethanol production were: starch from 20 to 30 g/l; initial pH values from 5.8 to 6.0; and I _R values of 2.0 or 3.0. The highest attained ethanol concentration, 13.7 g/l, represented 88% of the theoretical yield.K. Pirelová, D. mogroviová and . Balá are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Polytechnic University, Radlinského 9, 81237 Bratislava, Slovak Republic.     

Adaptation of a Saccharomyces cerevisiae strain to high copper concentrations

A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mm). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

Detection of Saccharomyces cerevisiae Atg13 by western blot

The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.     

Detection of Saccharomyces cerevisiae Atg13 by western blot

The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.     

Isolation by genetic and physiological characteristics of a fuel-ethanol fermentative Saccharomyces cerevisiae strain with potential for genetic manipulation

Fuel ethanol fermentation process is a complex environment with an intensive succession of yeast strains. The population stability depends on the use of a well-adapted strain that can fit to a particular industrial plant. This stability helps to keep high level of ethanol yield and it is absolutely required when intending to use recombinant strains. Yeast strains have been previously isolated from different distilleries in Northeast Brazil and clustered in genetic strains by PCR-fingerprinting. In this report we present the isolation and selection of a novel Saccharomyces cerevisiae strain by its high dominance in the yeast population. The new strain, JP1 strain, presented practically the same fermentative capacity and stress tolerance like the most used commercial strains, with advantages of being highly adapted to different industrial units in Northeast Brazil that used sugar cane juice as substrate. Moreover, it presented higher transformation efficiency that pointed out its potential for genetic manipulations. The importance of this strain selection programme for ethanol production is discussed.     

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.