Molecular genetic study of introgression between Saccharomyces bayanus and S. cerevisiae

The genomic constitution of different S. bayanus strains and natural interspecific Saccharomyces hybrids has been studied by genetic and molecular methods. Unlike S. bayanus var. uvarum, some S. bayanus var. bayanus strains (the type culture CBS 380, CBS 378, CBS 425, CBS 1548) harbour a number of S. cerevisiae subtelomeric sequences: Y', pEL50, SUC, RTM and MAL. The two varieties, having 86-100% nDNA-nDNA reassociation, are partly genetically isolated from one another but completely isolated from S. cerevisiae. Genetic and molecular data support the maintaining of var. bayanus and var. uvarum strains in the species S. bayanus. Using Southern hybridization with species-specific molecular markers, RFLP of the MET2 gene and flow cytometry analysis, we showed that the non-S. cerevisiae parents are different in lager brewing yeasts and in wine hybrid strains. Our results suggest that S. pastorianus is a hybrid between S. cerevisiae and S. bayanus var. bayanus, while S. bayanus var. uvarum contributed to the formation of the wine hybrids S6U and CID1. According to the partial sequence of ACT1 gene and flow cytometry analysis, strain CID1 is a triple hybrid between S. cerevisiae, S. kudriavzevii and S. bayanus var. uvarum.     

The effect of Saccharomyces cerevisiae and Saccharomyces bayanus yeast on colour properties and pigment profiles of a Cabernet Sauvignon red wine

Red wine was made from the same batch of Cabernet Sauvignon grapes but with two different yeasts, Saccharomyces cerevisiae (SC) or Saccharomyces bayanus (SB). The colour properties and pigment profiles of SC and SB wines were compared at 8 days and 387 days after yeast inoculation. Grape anthocyanin concentration was found to be lower in SB wines than in SC wines at day 8 and 387, but SB wine exhibited greater wine colour density, a* and b*. The anthocyanin concentration did not correlate with wine colour density. The levels of pigmented polymers and SO₂ non-bleachable pigments were found to be higher in SB wine at day 387, demonstrating that the formation of stable pyranoanthocyanins and pigmented polymers was enhanced by SB yeast. Electrospray mass spectrometry was used for the compositional analysis of pyranoanthocyanins, and direct and ethyl-bridged linked condensation dimers of malvidin 3- O -glucoside (MvG) with flavanols (catechin or epicatechin). The data demonstrated that the formation of acetaldehyde-mediated pigments was enhanced by the use of the SB yeast. The compositional analysis suggested that the differences in colour properties and pigment profiles of SC and SB wines were largely due to the greater production of acetaldehyde-mediated pigments by the use of SB yeast.     

Co-existence of two types of chromosome in the bottom fermenting yeast,Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.     

Developing methods and strains for genetic studies in the Saccharomyces bayanus var. uvarum species

For years, Saccharomyces cerevisiae has been used as a model organism to gain insight into complex biological processes. The study of closely related yeast species may be critical for understanding the molecular mechanism of evolution. Among those species, S. bayanus var. uvarum could be particularly pertinent because of the availability of its genome sequence. However, to date, in that species genetic studies are problematical due to the lack of standard strains collection and genetic methods. Here, we have developed heterothallic S. bayanus var. uvarum strains and obtained stable haploid strains. We further used UV-induced mutation and gene disruption to create a collection of auxotrophic derivatives. Finally, we have elaborated or improved methods to cultivate cells, obtain zygotes and spores and to transform this species. All these tools can now be used by the scientific community to study the biology of this species.     

Genetic homology between Saccharomyces cerevisiae and its sibling species S. paradoxus and S. bayanus: electrophoretic karyotypes.

Chromosomal DNAs of many monosporic strains of the biological species Saccharomyces cerevisiae, S. paradoxus and S. bayanus were analysed using contour-clamped homogeneous electric field electrophoresis. Southern blot hybridization with eight cloned S. cerevisiae genes (ADC1, CUP1, GAL4, LEU2, rDNA, SUC2, TRP1 and URA3) assigned to different chromosomes was used to study homology and chromosomal location of the genes in the three sibling species. A comparative study of Ty1, Ty2 and telomere-associated Y' sequences having multiple chromosomal location was also done. Chromosome length polymorphism was found in cultured strains of S. cerevisiae. Wild S. cerevisiae and S. paradoxus strains yielded chromosome banding patterns very similar to each other. The karyotype pattern of S. bayanus was readily distinguishable from that of S. cerevisiae and S. paradoxus. Southern blot analysis revealed a low degree of homology between the S. cerevisiae genes studied and the corresponding S. paradoxus and S. bayanus genes. The number of chromosomes appears to be 16 in all three species.     

Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae

The cDNA copies of the two endo-beta-1,4-glucanase genes, egl1 and egl3, from the filamentous fungus Trichoderma reesei were expressed in yeast Saccharomyces cerevisiae under the control of the yeast phosphoglycerate kinase gene promoter. Active EGI and EGIII enzyme was produced and secreted by yeast into the growth medium. The recombinant EGI enzyme was larger and more heterogeneous in size than the native enzyme secreted by Trichoderma, due to differences in the extent of N-glycosylation between these two organisms. The morphology of the yeast cells producing EGI or EGIII was clearly different from control strain.     

Sequence comparison of the ARG4 chromosomal regions from the two related yeasts,Saccharomyces cerevisiae and Saccharomyces douglasii

A 3·6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). The ARG4 and the YSD83 coding regions differ from their S. cerevisiae homologs by 8.1% and 12·5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2·8%) than in Ysc83 (12·4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non-coding regions are less conserved, with small AT-rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double-strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.     

Genomic differences between Candida glabrata and Saccharomyces cerevisiae around the MRPL28 and GCN3 loci

We report the sequences of two genomic regions from the pathogenic yeast Candida glabrata and their comparison to Saccharomyces cerevisiae. A 3 kb region from C. glabrata was sequenced that contains homologues of the S. cerevisiae genes TFB3, MRPL28 and STP1. The equivalent region in S. cerevisiae includes a fourth gene, MFA1, coding for mating factor a. The absence of MFA1 is consistent with C. glabrata's asexual life cycle, although we cannot exclude the possibility that a-factor gene(s) are located somewhere else in its genome. We also report the sequence of a 16 kb region from C. glabrata that contains a five-gene cluster similar to S. cerevisiae chromosome XI (including GCN3) followed by a four-gene cluster similar to chromosome XV (including HIS3). A small-scale rearrangement of gene order has occurred in the chromosome XI-like section.     

贝酵母耐硫相关基因FZF1的克隆与分析

FZF1基因在酿酒酵母中对亚硫酸盐转运蛋白编码基因SSU1具有正向调节作用,但在贝酵母中尚无相关报道,本研究拟通过对该基因进行克隆和生物信息学分析为后续研究做出参考。首先对贝酵母FZF1基因进行克隆,并利用在线分析工具Prot Param、Prot Scale、TMHMM、Predict Protein、Swiss-Model等软件对其编码蛋白质的基本理化性质进行分析,同时预测了该基因所编码蛋白质的二级结构和三级结构。结果表明:该核苷酸序列含有一个长900 bp的开放阅读框,可编码299个氨基酸;编码的蛋白质为在细胞核中行使调控功能的亲水蛋白,含有18个丝氨酸（S）激酶潜在磷酸化位点、一个Coil区和4个锌指结构域,与酿酒酵母FZF1基因所编码的蛋白质结构和性质极为相似。可初步认为贝酵母FZF1基因与细胞的耐硫性相关;而贝酵母FZF1基因所编码的蛋白质中仅有4个锌指,则可能是贝酵母硫耐受能力比酿酒酵母弱的重要原因。      

海藻糖与酿酒酵母乙醇耐受性相关性的研究进展

为了得到具有高富锌能力的酵母菌,该研究以面包来源的酵母菌DLY28为出发菌株,重复驯化后筛选获得一株优良耐锌酵母,并通过单因素试验及响应面试验对其富锌培养基进行优化。结果表明,通过驯化筛选得到一株优良耐锌酵母S7,其富锌的最优培养基组成为蔗糖含量82 g/L、胰蛋白胨含量26 g/L、锌含量404 mg/L。在此最优条件下,富锌酵母S7的锌吸附量为18.79 mg/g,生物量（OD600 nm值）为1.49。该研究为富锌酵母的生产以及食品有机锌的开发应用提供理论依据。     

An improved method for whole protein extraction from yeast Saccharomyces cerevisiae

A new method for protein extraction from yeast Saccharomyces cerevisiae cells is described. This method involves the use of LiAc and NaOH to enhance the permeability of yeast cell wall prior to protein extraction with SDS-PAGE sample buffer. It was safe and efficient compared to other methods reported so far in the literature. The proteins extracted with this new method retained their immunoreactive properties and are suitable for most applications in molecular biology studies.     

酿酒酵母细胞表面电位与絮凝相关性分析

为探究酿酒酵母细胞表面所带电荷与絮凝的相关性,以两种液态发酵酵母和两种固态发酵酵母为研究对象,并对液态发酵酵母FFC2144和固态发酵酵母L2Y进行连续传代培养,用zeta电位仪测定其zeta电位,以细胞沉降速率的方法考察酵母的絮凝性。结果显示,离子强度为0时,固态发酵酵母zeta电位(绝对值)均小于30 m V,液态发酵酵母均大于30 m V,培养72 h后的固态发酵酵母絮凝率达到了80.2%,液态发酵酵母絮凝率最大仅为14.4%。在发酵过程中不同酿酒酵母电位无明显变化。随着传代次数的增加酿酒酵母FFC2144 zeta电位无明显变化,絮凝性能却逐渐增强,到第20代时絮凝率达到了94.8%。不同酿酒酵母具有不同的zeta电位,其大小客观地反映出酿酒酵母絮凝性的强弱。酿酒酵母在衰老过程中细胞的絮凝性与zeta电位无关。      

Genetic control of chromosome stability in the yeast Saccharomyces cerevisiae

We have identified four new genetic loci: CHL2 (on chromosome XII), CHL3 (on chromosome XII); CHL4 (on chromosome IV), and CHL5 (on chromosome IX), controlling mitotic transmission of yeast chromosomes. The frequency of loss of chromosomes is 10-100-fold higher in chl5, chl2, chl3 and chl4 mutants than observed in wild-type strains. The mutants also show unstable maintenance of artificial circular minichromosomes with various chromosomal replicators (ARS) and one of the centromeric loci (CEN3, CEN4, CEN5 or CEN6). The instability of minichromosomes in the chl5, chl2, and chl4 mutants is due to the loss of minichromosomes in mitosis (1:0 segregation). In the chl3 mutant the instability of artificial minichromosomes is due to nondisjunction (2:0 segregation). The CHL3 gene therefore appears to affect the segregation of chromosomes during cell division.     

Breeding of brewer's yeast by hybridization between a top-fermenting yeast Saccharomyces cerevisiae and a cryophilic yeast Saccharomyces bayanus

To improve the fermentability of a top-fermenting yeast at low-temperature, we performed hybridization trials between four top-fermenting Saccharomyces cerevisiae strains and a cryophilic yeast Saccharomyces bayanus YM84 with good fermentability at low-temperature. The hybrids selected using 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside were checked with pulsed-field gel electrophoresis and their brewing performance at the low-temperature of 10.5 degrees C was observed using small-scale (2 l) fermentation trials.     

Reactive oxygen species may influence the heat shock response and stress tolerance in the yeast Saccharomyces cerevisiae

Moderate levels of reactive oxygen species (ROS) have been implicated as second messengers in a number of biochemical pathways, and in animal cells have been associated with the activation of the heat shock response (HSR). Here, using an intracellular probe, we demonstrate that differential accumulation of ROS in the yeast Saccharomyces cerevisiae is strongly associated with differential induction of an HS reporter gene over a range of heat shock temperatures. There was a good correlation between cellular ROS levels and the levels of HS-induced reporter gene expression between 37 degrees C and 44 degrees C, both reaching maximal values at 41 degrees C. Furthermore, the addition of 150 microM H2O2 to the yeast cells during heat treatment resulted in a 3 degrees C decrease in the temperature required for maximal induction of the HS expression vector--an increased HS sensitivity that corresponded to a concomitant increase in ROS levels at these lower HS temperatures. Conversely, cells treated with 10 mM of the antioxidant ascorbic acid required a temperature that was 2 degrees C above that required in untreated controls for maximal induction of the HS expression vector. This decreased HS sensitivity corresponded to a decrease in ROS levels at these higher HS temperatures. Finally, cell viability assays reveal that intrinsic thermotolerance remains high in control cells despite concomitant decreases in HS-reporter gene expression and ROS accumulation between 41 degrees C and 44 degrees C. We conclude that the sensitivity of the yeast HSR is strongly associated with ROS accumulation, and suggest that ROS-mediated signalling ensures cooperation between the HS and the antioxidant responses.     

Saccharomyces cervisiae在食品发酵工业中的研究进展

从基础理论和应用技术等方面综述了国内外对酿酒酵母(Saccharomyces cervisiae)研究进展及其在食品发酵工业中的最新研究成果。