Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae

We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.     

DNA replication in a diploid strain of Saccharomyces cerevisiae homozygous for the rad6-1 mutation

The generation time of a diploid strain homozygous for the rad6-1 mutation was 160 min, and the duration of the S phase was 80 min; in the parental heterozygote, these values were 90 and 40 min, respectively. Analysis of DNA sedimentation in an alkaline sucrose gradient revealed that heterozygote high-molecular-weight DNA appeared after 60 min, and homozygote high-molecular weight DNA only after a 100-min pulse.     

Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae

Deoxyribonucleic acid (DNA) synthesis was examined in asynchronous and synchronous cultures of a number of cdc (cell division cycle) temperature-sensitive mutant strains. The kinetics of DNA synthesis after a shift to the restrictive temperature was compared with that obtained after inhibition of protein synthesis at the permissive temperature, a condition that specifically blocks the initiation of new rounds of DNA replication, but does not block those in progress. Mutations in three genes (cdc 4, 7, and 28) appear to block a precondition for DNA synthesis since cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to restrictive temperature, but can finish rounds that were in progress. These three genes are classified as having roles in the "initiation" of DNA synthesis. Mutations in two genes (cdc 8 and 21) block DNA synthesis, itself, since cells harboring these lesions that had started DNA synthesis at the permissive temperature arrest synthesis abruptly upon a shift to the restrictive temperature. Mutations in 13 other cdc genes do not impair DNA synthesis in the first cell cycle at the restrictive temperature.     

Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains

Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100 mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway.     

Heterozygous screen in Saccharomyces cerevisiae identifies dosage-sensitive genes that affect chromosome stability

Current techniques for identifying mutations that convey a small increased cancer risk or those that modify cancer risk in carriers of highly penetrant mutations are limited by the statistical power of epidemiologic studies, which require screening of large populations and candidate genes. To identify dosage-sensitive genes that mediate genomic stability, we performed a genomewide screen in Saccharomyces cerevisiae for heterozygous mutations that increase chromosome instability in a checkpoint-deficient diploid strain. We used two genome stability assays sensitive enough to detect the impact of heterozygous mutations and identified 172 heterozygous gene disruptions that affected chromosome fragment (CF) loss, 45% of which also conferred modest but statistically significant instability of endogenous chromosomes. Analysis of heterozygous deletion of 65 of these genes demonstrated that the majority increased genomic instability in both checkpoint-deficient and wild-type backgrounds. Strains heterozygous for COMA kinetochore complex genes were particularly unstable. Over 50% of the genes identified in this screen have putative human homologs, including CHEK2, ERCC4, and TOPBP1, which are already associated with inherited cancer susceptibility. These findings encourage the incorporation of this orthologous gene list into cancer epidemiology studies and suggest further analysis of heterozygous phenotypes in yeast as models of human disease resulting from haplo-insufficiency.     

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R² analysis) shows that there are over 500 genes for which >70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.     

Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains

Yeasts used in bread making are exposed to freeze-thaw stress during frozen-dough baking. To clarify the genes required for freeze-thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze-thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze-thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H(+)-ATPase. Next, the cross-sensitivity of the freeze-thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze-thaw stress. The results showed that defects in the functions of vacuolar H(+)-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze-thaw injury: oxidative stress generated during the freeze-thaw process, and defects in cell wall assembly.     

Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae.

To further our studies of protein sorting and biogenesis of the lysosome-like vacuole in yeast, we have isolated spontaneous mutations in 11 new VPL complementation groups, as well as additional alleles of the eight previously described VPL genes. These mutants were identified by selecting for cells that mislocalize vacuolar proteins to the cell surface. Morphological examination of the vpl mutants indicated that most contain vacuoles of normal appearance; however, some of the mutants generally lack a large vacuole, and instead accumulate smaller organelles. Of the 19 VPL complementation groups, 12 were found to be identical to 12 of 33 VPT complementation groups identified in a separate study. Moreover, the end1 mutant and all of the previously reported pep mutants, with the exception of pep4, were found to exhibit a profound vacuolar protein sorting defect, and complementation tests between the PEP, VPL VPT and END1 groups demonstrated that there are extensive overlaps between these groups. Collectively, mutants in these four collections define 49 complementation groups required to deliver or retain soluble vacuolar enzymes, including carboxypeptidase Y (CPY) and proteinase A. We have also isolated 462 new mutants that lack normal levels of vacuolar CPY activity. Among these latter mutants, only pep4 mutants were found to be specifically defective in vacuolar zymogen activation. We conclude that there is a large number of gene products required for sorting or retention of vacuolar proteins in yeast, and only a single gene, PEP4, that is essential for activation of CPY and other vacuolar zymogens.     

A homozygous diploid subset of commercial wine yeast strains

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as ‘genome renewal’.     

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.     

Unpredictable fitness transitions between haploid and diploid strains of the genetically loaded yeast Saccharomyces cerevisiae.

Mutator strains of yeast were used to accumulate random point mutations. Most of the observed changes in fitness were negative and relatively small, although major decreases and increases were also present. The average fitness of haploid strains was lowered by approximately 25% due to the accumulated genetic load. The impact of the load remained basically unchanged when a homozygous diploid was compared with the haploid from which it was derived. In other experiments a heterozygous diploid was compared with the two different loaded haploids from which it was obtained. The fitness of such a loaded diploid was much less reduced and did not correlate with the average fitness of the two haploids. There was a fitness correlation, however, when genetically related heterozygous diploids were compared, indicating that the fitness effects of the new alleles were not entirely lost in the heterozygotes. It is argued here that to explain the observed pattern of fitness transitions it is necessary to invoke nonadditive genetic interactions that go beyond the uniform masking effect of wild-type alleles. Thus, the results gathered with haploids and homozygotes should be extrapolated to heterozygotes with caution when multiple loci contribute to the genetic load.     

Lactose utilization by Saccharomyces cerevisiae strains expressing Kluyveromyces lactis LAC genes

Whey generated in cheese manufacture continues being an industrial problem without a satisfactory solution. Genetic modification of the yeast S. cerevisiae to obtain strains able to utilize lactose, is a prerequisite for the utilization of this yeast to convert cheese whey into useful fermentation products (i.e. biomass, heterologous protein and other recombinant products). Although the construction of S. cerevisiae Lac(+) strains has been achieved by different strategies, most of these strains have unsuitable characteristics, such as genetic instability of the Lac phenotype or diauxic growth. In previous communications we have described the construction of genetically stable strains of S. cerevisiae that assimilate lactose with a high efficiency. These strains carry multiple copies of Kluyveromyces lactis LAC4 and LAC12 genes, which code for a beta-galactosidase and a lactose permease, respectively. In this work we report additional results about the effect of gene dosage, and analyze the performance of a selected strain in the bioconversion of cheese whey. Additionally, we describe the construction of a new strain, which combines the Lac(+) phenotype with additional properties of biotechnological interest: flocculence, and the ability to hydrolyze starch.     

Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.     

Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae

Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype. Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho⁰) or those with deleted mitochondrial DNA (rho^–). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho^– and rho⁰ cells were ~2- to 3-fold higher compared to untreated control and wild-type cells, respectively. Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS). In contrast, inactivation of mitochondrial activity (rho^– and rho⁰) led to decreased intracellular levels of ROS. We also demonstrate that in rho⁰ cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome. However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A. Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.     

A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae

We have constructed a convenient new vector, YEp-DE, for the construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae. The vector contains the yeast LEU2 gene, the 2 mu origin of replication, and a region from pUC18 that includes the ampr gene, the Escherichia coli origin of replication (ori), and the LacZ gene with multiple cloning sites. Five sites (Sac1, Sma1, BamH1, Sal1, Sph1) in this region are unique. This vector has advantages over similar yeast-E. coli shuttle vectors: small size (7291 bp, entirely sequenced), convenient cloning sites, and lacZ selection for detecting recombinant plasmids.