Membrane transport in an osmotically fragile mutant of Saccharomyces cerevisiae

Transport properties of the osmotically fragile strain VY1160 of Saccharomyces cerevisiae were compared with those of the parent S288c strain. Mediated diffusion of 6-deoxy-D-glucose was practically unaffected; membrane-potential-dependent transport of D-glucosamine was very much depressed in the fragile strain. The H+-driven transport of L-lysine and L-proline, as well as that of the hitherto uninvestigated D-glucose-6-phosphate, were also very depressed. 2-Deoxy-D-glucose transport displayed slightly different kinetic parameters. Primary H+ extrusion by the plasma membrane H-ATPase was not diminished although the ATP-splitting activity was depressed by about 50%. The overall proton-motive force (pmf) of the fragile mutant at pH 5.5 was only 20 mV while in the parent strain it was 108 mV. In parallel with this, spontaneous acidification of the external medium (a CO2-associated event) was only about 2% of that in the parent strain. The defect in this, together with the inability to stimulate transport protein synthesis by glucose, may account for the generally poorer transport performance of the fragile mutant.     

Assembly of phosphofructokinase-1 from Saccharomyces cerevisiae in extracts of single-deletion mutants

Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the β-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits. Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.     

Isolation and characterization of barosensitive mutants of Saccharomyces cerevisiae obtained by UV mutagenesis

Using UV mutagenesis, 2 high-pressure (HP) sensitive (barosensitive) mutants of Saccharomyces cerevisiae were obtained. The HP inactivation of the mutants, as well as their parent strains, followed 1st-order kinetics in the range of 175 to 250 MPa within 600 s. Both mutants showed larger 1st-order inactivation rate constant values or significant loss of viabilities, compared with their parent strains in the pressure range tested. The inactivation rate constant value of one of the mutants was comparable with that of a previously reported highly barosensitive strain, which was generated by deletion of hsp104 in a trehalose deficient strain. The activation volume values of HP inactivation reactions in the 2 mutants were apparently equivalent with those of their parent strains. This suggested that the mutation did not bring drastic volume changes of the key molecules for HP inactivation. Their auxotrophic properties, growth, and ethanol fermentation were identical in mutant and parent strains. The mutants could therefore be useful for fermentations where control by HP processing is desired.     

Polyploid fragile strains of Saccharomyces cerevisiae--a novel source of proteins for nutritional purposes

A series of prototrophic fragile strains of different ploidy (2n, 3n and 4n) has been genetically constructed on the basis of haploid srb1 containing segregants of the fragile Saccharomyces cerevisiae mutant VY 1160. The strains have been characterized by several criteria. In regard to generation time, biomass yield, and nucleic acids content of the cells, the tetraploid srb1 homozygous hybrid is indistinguishable from an industrial strain of S. cerevisiae. However, it is characterized by a higher protein content. Unlike any other laboratory or industrial strains, the original mutant and these hybrids possess an ability for lysis upon suspension in hypotonic solutions. The dependence of the percentage of lysed cells on the growth phase and concentration of osmotic stabilizer in the medium has been investigated. The quantity of proteins in the soluble and insoluble fractions obtained after lysis of these strains by osmotic shock has been determined. These hybrids can be considered as a potential industrial source of proteins for nutritional purposes.     

Increased transformation levels in intact cells of Saccharomyces cerevisiae aculeacin A-resistant mutants

Several Saccharomyces cerevisiae mutants resistant to the cell wall synthesis inhibitor aculeacin A exhibit higher transformation levels than the parental strain. Mutant acr2 has been studied in more detail. It is transformed up to ten-fold more efficiently than the wild-type strain with episomal, centromeric and integrative plasmids, and dimethyl sulfoxide has an additive effect improving transformation efficiency. Transformation with linear DNA molecules is not as much affected in acr2 cells. The observed effects may be caused by the altered plasma membrane composition of the mutants.     

Glucan structure in a fragile mutant of Saccharomyces cerevisiae

The phenotype of VY1160 fragile Saccharomyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased permeability of cells growing in osmotically stabilized media. Two mutations, srb1 and ts1, have been identified in VY1160 cells and previous studies have shown that the increased permeability is due to the ts1 mutation which causes a shortening of mannan side-chains. Here we report that the srb1 mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans provide evidence that srb1 mutation leads to i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain beta(1-6) inter-residue linkages and ii) insufficient filling of the space between the fibrils due to a shortage of the alkali-soluble glucan. Although growing exponentially in osmotically stabilized media, the srb1 cells cannot resist an osmotic shock and, hence, burst immediately.     

Functional analysis reports. Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction

We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.     

Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160

The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent and consistent with a fluid-phase mechanism of endocytosis. The rate of steady-state accumulation of the dye at 37 degrees C is 145 ng/micrograms DNA per h for VY1160 mutant and 23 ng/micrograms DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24 degrees C and shifted at 38 degrees C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38 degrees C. The analysis of ts1 x wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might explain the generally increased permeability of VY1160 mutant cells.     

双亲灭活原生质体融合选育高性能酒精酵母菌的研究

为了选育高性能的酒精酵母菌,利用双亲灭活和PEG诱导,对酿酒酵母GGFS16和GJ2008单倍体细胞进行了原生质体融合。从42株融合子中筛选得到了2株融合菌株RH3和RH5,分别适合于木薯粉和甘蔗汁酒精发酵,且较好的保持了亲本的特性。其中RH3在耐酸能力上,RH5在酒精耐受能力上要优于亲本,RH3的遗传稳定性要优于RH5。     

Effect of low temperature upon vitality of Saccharomyces cerevisiae phospholipid mutants

The phospholipid metabolism of Saccharomyces cerevisiae plays a central role in its adaptation to low temperatures. In order to detect the key genes in this adaptation, various phospholipid mutants from the EUROSCARF collection of Saccharomyces cerevisiae BY4742 were tested to ascertain whether the suppression of some genes could improve the fermentation vitality of the cells at low temperature. The cell vitality and phospholipid composition of these mutants were analysed. Some knockouts improved (hmn1Δ) or impaired (cho2Δ and psd1Δ) their vitality at low temperature (13 °C) but were not affected at optimum temperature (25 °C). A common trait of the mutants that had some defect in vitality was a lower concentration of phosphatidylcholine and/or phosphatidylethanolamine. The supplementation with choline allowed them to recover viability, probably by synthesis through the Kennedy pathway. Hmn1Δ showed a lower concentration of phosphatidylcholine, which explains the dominant role of the de novo pathway in cellular phosphatidylethanolamine and phosphatidylcholine vs the Kennedy pathway. The absence of such genes as CRD1 or OPI3 produced important changes in phospholipid composition. Cardiolipin was not detected in crd1Δ but phosphatidylglycerol circumvents most of the functions assigned to CL. The considerable reduction in PC diminished the cell vitality of opi3Δ at both temperatures, although the decrease at 13 °C was more marked. Copyright © 2012 John Wiley & Sons, Ltd.     

A Simplified Method for the Isolation and Estimation of Cell Wall Bound Glycogen in Saccharomyces cerevisiae

Glycogen is an important storage reserve in yeast. In Saccharomyces cerevisiae glycogen is present in two pools, an intracellular soluble pool and a cell wall bound, insoluble extra‐cellular pool. The present method uses a 20% KOH treatment to separate the two pools, which are then estimated using amyloglucosidase. The amount of soluble glycogen was found to be 6.5 mg/g of wet weight of yeast while that of cell wall bound glycogen was found to be almost three times that of the soluble, viz., 18 mg/g of wet weight of yeast. The data is compared with two earlier commonly used methods of yeast carbohydrate fractionation, which reported glycogen in totality. Reviewing these methods in the light of finding two pools of glycogen revealed that both the methods can be demonstrated to yield soluble glycogen in the range of 6–9 mg/g of yeast and 18–21 mg/g of wet weight of yeast of cell wall bound glycogen.     

Isolation and characterization of Schizosaccharomyces pombe fragile mutants

Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall.     

双歧杆菌和酿酒酵母原生质体融合子筛选方法的探讨

目的:探讨双歧杆菌(Bifidobacterium)和酿酒酵母(Saccharomycescerevisiae)原生质体融合子的筛选方法。方法:将双歧杆菌和酿酒酵母原生质体进行原生质体融合,采用10%乳糖中性红培养基进行融合子的初筛,并分析融合子传代稳定性、生物学特性,采用双歧杆菌属特异性寡核苷酸探针对亲本菌和融合子进行了荧光原位杂交检测比较。结果:结果表明,筛选得到的融合子具有双歧杆菌的特异性序列和亲本菌的生物学特性。结论:双歧杆菌和酿酒酵母原生质体融合,实现了厌氧菌和酵母的跨界融合,为双歧杆菌生物学功能的开发提供新思路、新途径。     

Intracellular and extracellular levels of cyclic AMP during the cell cycle of Saccharomyces cerevisiae

Using the technique of centrifugal elutriation it was demonstrated that during the cell division cycle of the budding yeast Saccharomyces cerevisiae there are stage-specific fluctuations in the intracellular concentration of adenosine 3',5'-cyclic monophosphate (cAMP). Results shown here indicate that the intracellular concentration of cAMP is at its highest during the division cycle, and at its lowest immediately prior to and just after cell separation. Results also show the extrusion of extracellular cAMP into the medium by Saccharomyces cerevisiae, extracellular cAMP levels being ten to one hundred times higher than intracellular levels. During the cell cycle of Saccharomyces cerevisiae the extracellular level of cAMP does not fluctuate.     

不同菌龄酿酒酵母细胞壁蛋白差异性分析

以酿酒酵母为研究对象,比较了完整细胞提取法、稀碱缓冲液提取法及溶菌酶和β-葡聚糖酶复合酶法等三种酵母菌细胞壁蛋白提取方法,分析了不同菌龄酵母细胞壁差异性蛋白。结果表明:溶菌酶和β-葡聚糖酶复合酶液水解纯化好细胞壁提取蛋白的方法具有所得胞壁蛋白条带较多,且纯度较高的优点,确定了此方法为提取酵母细胞壁蛋白的最佳提取方法。同时,通过SDS-PAGE电泳分析发现,不同菌龄酵母细胞壁蛋白存在着较大的差异性,并确定了分子质量在36 ku、17 ku和12 ku为不同酵母代数细胞壁的3个主要差异性蛋白,其中36 ku、17 ku处条带蛋白随着菌龄的增加酵母细胞壁蛋白表达量逐渐减少,而12 ku处条带蛋白随着菌龄的增加酵母细胞壁蛋白表达量逐渐增加。     

重组酿酒酵母合成白藜芦醇的研究

为了获得一种酵母快速合成白藜芦醇的体系,本研究分别从虎杖和烟草中克隆获得白藜芦醇合成途径的关键酶基因:芪合酶基因STS和4-香豆酰辅酶A连接酶基因4CL,引入3个连续重复的GSG作为连接肽,采用Overlap PCR扩增技术构建了融合基因4CL-(GSG)3-STS,进一步获得重组表达载体p ESC-TRP-4CL-(GSG)3-STS后转化至酿酒酵母中,之后利用HPLC分析检测重组酿酒酵母代谢产物,最后对重组菌合成白藜芦醇的诱导时间、底物添加浓度和添加方式进行了优化研究。结果表明:所构建的重组酿酒酵母菌体生长48 h后进行诱导表达,同时添加浓度为6 mg/L底物4-香豆酸,并且每隔2 h添加一次,8 h后白藜芦醇产量即可达7.01 mg/L。利用本体系合成白藜芦醇具有操作简单、生产周期短的特点,为进一步实现微生物大规模生产白藜芦醇提供了基础。     

Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation.

We have isolated two temperature-sensitive Saccharomyces cerevisiae mutants which exhibit a deficiency in mannose outer chain elongation of asparagine-linked oligosaccharide. The size of yeast glycoprotein, secretory form of invertase, of one mutant (och1) was slightly larger than that of the sec18 mutant at the non-permissive temperature, while that of the other mutant (och2) was almost the same as that of the sec18 mutant. Unlike sec mutants, the och mutants were not deficient in secretion of invertase. The och1 mutant showed a 2+:2- cosegregation with regard to the temperature sensitivity and mannose outer chain deficiency, suggesting that a single gene designated as OCH1 is responsible for these two phenotypes. The och1 mutant stopped its growth at the early stage of bud formation and rapidly lost its viability at the non-permissive temperature. The och1 mutation was mapped near the ole1 on the left arm of chromosome VII. The och1 mutant cells accumulated the external invertase containing a large amount of core-like oligosaccharides (Man9-10GlcNAc2) and a small amount of high mannose oligosaccharides (greater than Man50GlcNAc2) at the non-permissive temperature. Production of the active form of human tissue-type plasminogen activator was increased in the och1 mutant compared with the parental strain, suggesting the potential advantage of this mutant for the production of mammalian-type glycoproteins which lack mannose outer chains in yeast.     

A Survey of Industrial Strains of Saccharomyces cerevisiae Reveals Numerous Altered Patterns of Maltose and Sucrose Utilisation

A model fermentation system was used to define the abilities of 25 Saccharomyces cerevisiae strains, representing the brewing, baking, winemaking and distilling industries, to utilise maltose and sucrose in the presence of glucose and fructose. Three categories of sucrose and maltose utilisers were observed; repressible, constitutive and non‐utilisers. In terms of fermentation kinetics, neither high rates of sucrose hydrolysis nor the early onset of maltose utilisation were correlated with reduced fermentation duration in the experimental system used. Instead better positive correlations were found between this parameter and biomass formation (R² = 0.62) and rates of maltose or monosaccharide removal (R² = 0.87 and 0.82, respectively). Additionally, invertase activity of brewing strains was seen to occur in two forms: cell‐associated and non‐cell‐associated. This survey exposed a number of novel phenotypes that could be harnessed as a means of producing strains with rapid and efficient utilisation of fermentable carbohydrates.     

电融合法构建黑曲霉与K氏酵母融合子的研究

对黑曲霉原生质体进行紫外单亲灭活与K氏酵母原生质体在交变电场频率1MHz,交变电场强度700V/cm,脉冲电场强度10kV/cm,脉冲个数10个,脉宽50μS,电极时间5s条件下进行电融合,得到2株遗传性能稳定的融合子AKU-1与AKU-4,其耐酒精度分别为15%、16%(v/v),且2融合子在以料液比为1:3的生料中,35℃静态发酵4d,产酒精量分别为11.2%、12.1%(v/v),残糖量分别为1.07%、1.06%。