Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

Autoselection systems allow the selection of a genetically engineered population independently of the growth medium composition. The structure of a Saccharomyces cerevisiae population transformed with an autoselection plasmid, in which a carbon-source-dependent modulation of the plasmid copy number occurs, was analysed. By means of flow cytometric procedures we tested the cell viability, dynamics of growth and heterologous protein production at single cell level. Such analyses allow the identification and the tracking of a specific cellular sub-population with a higher plasmid copy number which arises after the carbon source shift. The effects of the cellular plasmid distribution on the dynamics of growth are also discussed.     

Differential chromosome control of ploidy in the yeast Saccharomyces cerevisiae

In Saccharomyces cerevisiae, aneuploidy is well tolerated and stable. We analysed whether the induced loss of a disomic chromosome favours endo-reduplication of the remaining chromosome or the cells prefer to retain the acquired euploidy. Chromosome VIII disomes and trisomes were tagged with GFP (green fluorescent protein), DsRed (red fluorescent protein) and BFP (blue fluorescent protein) integrated at the thr1 locus, using our newly designed STIK (specific targeted integration of kanamycin resistance-associated, non-selectable DNA) plasmid system. A knockout cassette for centromere 8 was constructed with the hygromycin-B marker, which was transformed into the strains. The transformants lost sensitivity to hygromycin, thereby indicating the event of centromere replacement. Quantitative PCR and Southern analysis were performed for chromosome VIII copy number determination by probing the markers located on both the right (ARG4 and THR1) and left (GUT1) arm whereas, for chromosome V, markers such as HIS1, located on right arm, and URA3, on left arm, were used. The loss of an extranumerary chromosome VIII in a disome and trisome leads to stable euploidy. Furthermore, in a wild-type diploid, deletion of a copy of chromosome VIII, leads to monosomy, and restoration of euploidy after 22 generations, by reduplication of chromosome VIII, and consequent loss of heterozygosis (LOH). However, chromosome V knockouts in chromosome VIII trisome, still showed LOH and duplication of chromosome V, with return to the original aneuploid condition. These results suggest that yeast cells could control the integrity of their genetic complement acting at the individual chromosome level.     

酿酒酵母低渗敏感性突变株的选育

为促进酿酒酵母胞内产物的有效释放,以其野生型二倍体(2n)菌株Y_1为出发菌株,经紫外诱变处理,通过测定其胞外FDP浓度及核酸、蛋白质渗透率,对比各突变株在低渗条件下的自溶程度。结果表明:突变株Hs_5~*(n)低渗培养时,条件性自溶程度最高,其胞外FDP浓度达25.82μg/mL,低渗培养6h时,其核酸、蛋白质渗透率高达1.86,2.07。此外突变株Hs_2~*(n)、Hs_1(2n)也具有较好的条件性自溶能力。试验共筛选获得3株低渗敏感性突变株,与野生型菌株相比,条件性自溶的低渗敏感性突变株能有效促进胞内大分子物质的外泌。     

Beta-glucosidase activity in a Saccharomyces cerevisiae wine strain

Beta-glucosidase activity contributes to aroma formation during the winemaking process. This study investigated whether beta-glucosidase activity was expressed by wild Saccharomyces cerevisiae strains and by a laboratory strain. beta-Glucosidase activity was assayed on several culture media and under various growth conditions. The highest activities were obtained in Yeast Extract Peptone medium, but activity was also detected using grape juice as the growth medium, although a 25% drop activity was observed when anaerobic conditions were employed. A number of parameters affecting beta-glucosidase activity were evaluated. Optimal conditions for activity were pH 4 and a temperature of 40-50 degrees C. The results showed beta-glucosidase activity to be present during the process of winemaking, although different from the optimal conditions.     

Air-liquid biofilm formation is dependent on ammonium depletion in a Saccharomyces cerevisiae flor strain

Air–liquid biofilm formation appears to be an adaptive mechanism that promotes foraging of Saccharomyces cerevisiae flor strains in response to nutrient starvation. The FLO11 gene plays a central role in this phenotype as its expression allows yeast cells to rise to the liquid surface. Here, we investigated the role of ammonium depletion in air–liquid biofilm formation and FLO11 expression in a S. cerevisiae flor strain. The data obtained show that increasing ammonium concentrations from 0 to 450 m m reduce air–liquid biofilm in terms of biomass and velum formation and correlate with a reduction of FLO11 expression. Rapamycin inhibition of the TOR pathway and deletion of RAS2 gene significantly reduced biofilm formation and FLO11 expression. Taken together, these data suggest that ammonium depletion is a key factor in the induction of air–liquid biofilm formation and FLO11 expression in S. cerevisiae flor strains. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification and characterization of a drug‐sensitive strain enables puromycin‐based translational assays in Saccharomyces cerevisiae

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C‐terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6?, pdr1? and pdr3? (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin‐based assays in yeast, which will enable new avenues of inquiry into protein production and maturation. Copyright © 2014 John Wiley & Sons, Ltd.     

KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae

Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity.     

Recovery of gene function by gene duplication in Saccharomyces cerevisiae

A prototroph revertant (Rev9) selected from an ATCase^? mutant of the URA2 gene containing three nonsense mutations was shown to contain two ATCase coding sequences. We cloned both ATCase coding areas to show that the duplicated locus (dl9) was the only functional one. Its size corresponded roughly to the second half of the URA2 wild-type gene. Sequence analysis of the 5′ end of dl9 indicated that this duplicated sequence was inserted within the intergenic region close to the MRS3 gene and was transcribed from an unknown promoter divergently from the MRS3 gene. The event leading to the revertant strain Rev9 included a rearrangement that increased the size of chromosome X by about 60 kb. In agreement with such a rearrangement, recombination was undetectable in the vicinity of the locus dl9. Genetic mapping confirms that the MRS3 gene is 2 cM distal to the URA2 gene on the right arm of chromosome X.     

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.     

GIS1基因对酿酒酵母耐受性的研究

该研究主要通过过表达GIS1基因来提高酿酒酵母的耐受性。在GIS1基因的N端加入强启动子PGK1p来实现GIS1基因的过表达,然后通过稀释点板实验来对比其对热激、乙醇、渗透压以及乙酸的耐受性,同时通过高温生长曲线和高温浓醪发酵测定其高温耐受性。结果表明,在相同的稀释倍数下,55 ℃热击4 min之后菌株AY12a-gis1的生长能力明显优于出发菌株,在含有5%（V/V）乙酸的平板上改造菌和出发菌耐受性相差不大,同时通过38 ℃浓醪发酵实验发现菌株AY12a-gis1的酒精度提高了3.79%,残糖略有下降,且发酵时间与亲本菌株相一致。因此通过过表达GIS1基因得到菌株AY12a-gis1,是对工业乙醇发酵有一定应用价值的优良耐逆性菌株。     

Surfome analysis of a wild-type wine Saccharomyces cerevisiae strain

The yeast Saccharomyces cerevisiae, besides being an eukaryotic cell model, plays a fundamental role in the production of fermented foods. In the winemaking industry, yeast cell walls may be involved in numerous processes and contribute substantially to the final chemical and sensorial profiles of wines. Nonetheless, apart from mannoproteins, little is known on the protein components of the yeast cell wall and their changes during the fermentation of must into wine. In this work, we performed a dynamic analysis of the cell surface proteome (surfome) of an autochthonous wine yeast strain (previously selected as a wine fermentation starter) by shaving intact cells with trypsin and identifying tryptic peptides by means of nLC-ESI-LIT-MS/MS. Out of the 42 identified proteins, 16 and 14 were found to be specifically expressed in wine yeast surfome at the beginning and at the end of fermentation, respectively. The molecular functions of these specifically expressed proteins might help in explaining their roles in the cell wall as a response to the alcoholic fermentation-related stresses. Additionally, we provided the identification of 20 new potential cell wall related proteins. Globally, our results might provide new useful data for the selection and characterization of yeast strains to be used in the winemaking industry.     

多优良性状工业化酿酒酵母的选育及其特性研究

通过双亲灭活的原生质体融合技术,将高效的酿酒酵母gxas02和絮凝酿酒酵母A3融合育种,得到的融合子R2-4能在短时间内产生高浓度酒精(发酵液中酒精含量为16.20%vol),发酵时间至少缩短8h,残糖0.076g/L,糖的利用接近理论值,耐酒精能力提高,有一定的工业应用潜力。     

酿酒酵母产泛酸工程菌株的构建与诱变

泛酸具有重要的生理功能,参与糖、脂肪、蛋白质及能量的代谢,广泛用作微生物药物、食品添加剂和饲料添加剂.通过强化酿酒酵母泛酸生物合成途径的酮泛解酸羟甲基转移酶、酮泛解酸还原酶、泛酸合成酶和多胺氧化酶的表达水平,提高酵母泛酸合成量.在不合泛酸的培养基中培养102 h后,泛酸质量浓度达到290 μg/L.进一步对菌株BYPAN01进行微波辐照诱变,筛选到的突变株BYPAN02泛酸质量浓度较菌株BYPAN01和BY4741分别提高了43.3％和203.2％,达到415.7μg/L,为实现泛酸的环保和低成本合成提供了新的思路.     

Mapping of gene controlling thiamine transport in Saccharomyces cerevisiae

A recessive mutation leading to complete loss of thiamine uptake in Saccharomyces cerevisiae was mapped on the left arm of chromosome VII, approximately 56cM centromere-distal to trp5. As the analysed locus is relatively distant from its centromere and from the markers used, its attachment to chromosome VII was confirmed by chromosome loss methods.     

PHA聚合酶基因phaC的克隆及其在酿酒酵母中的表达

对聚羟基脂肪酸酯(PHA)聚合酶基因phaC进行克隆,并将其与穿梭质粒pYES2连接.构建了酿酒酵母表达质粒pYES2-phaC.测序验证后用LiAc/SSDNA/PEG方法将质粒转化至Saccharomyces cerevisiae INVSCI中,经SC-URA培养基筛选得到阳性转化子.由2%半乳糖培养基诱导表达后,提取细胞粗蛋白测定PHA聚合酶酶活力,结果表明,表达phaC基因的重组菌酶活力为2.45 U/mg,证明phaC基因在在cerevisiae INVSCI中得到表达.     

一株耐酸性酿酒酵母的筛选鉴定及特性

基于浓香型白酒回糟酸度高、残糖含量高的生产现状,通过平板划线纯化、WL培养基形态观察及显微镜观察对中高温大曲中酵母菌进行初步鉴定,再通过TTC培养基染色筛选、差异酸度培养基筛选适用于回糟发酵耐酸性产酒精能力强的酵母菌,并进行分子生物学鉴定、生物学特性及回糟发酵小试研究.结果表明:中高温大曲中筛选得到的菌株dq1在pH ...