Diploid Yeast Strain Research Articles

Clinopodium vulgare L. Extract Counteracts DNA Damaging Action of Radiomimetic Zeocin Accelerating DSBS Rejoining in Saccharomyces Cerevisiae

The aim of the present work was to evaluate the potential of Clinopodium vulgare water leaves extract to decrease the DNA damaging action of zeocin and to throw more light on the mode of action.
 Single and combined treatments with 10, 100, and 1000 µg/ml plant extract and 100 µg/ml zeocin were performed on 551 (haploid) and D7ts1 (diploid) yeast strains. The protective potential, the magnitude of the repair capacity, and the role of incubation conditions were studied based on double strand breaks' (DSB) induction and rejoining.
 The results provided new evidence that despite the different ploidy both strains are DSBs repair proficient but differ in their repair capacity although comparable levels of initially induced DSBs were measured. The diploid strain D7ts1 was found to possess better-expressed repair capacity than the haploid strain 551. The combined treatment with Clinopodium vulgare L. extract at concentrations' range of 10—1000 µg/ml and zeocin resulted in a significant decrease in DSBs levels. It could be suggested that priming the cells with various concentrations of the extract results in an acceleration of DSBs rejoining when 30 min recovery time at optimal experimental conditions is given. When incubation is performed on ice the repair processes are impeded but not fully blocked. Such a study confirms that the difference in the repair capacity of the cells could be one of the possible mechanisms, participating in cell protection against different DNA damaging factors. The combined application of Clinopodium vulgare L. extract at concentrations range of 10—1000 µg/ml protects yeast DNA against zeocin-induced DSBs by acceleration of DSB rejoining.

UGDR: a generic pipeline to detect recombined regions in polyploid and complex hybrid yeast genomes

MotivationIn eukaryotes, homologous recombination between the parental genomes frequently occurs during the evolutionary conserved process of meiosis, generating the genetic diversity transmitted by the gametes. The genome-wide determination of the frequency and location of the recombination events can now be efficiently performed by genotyping the offspring’s polymorphic markers. However, genotyping recombination in complex hybrid genomes with existing methods remains challenging because of their strain and ploidy specificity and the degree of diversity and complexity of the parental genomes, especially in >2n polyploids.ResultsWe present UGDR, a pipeline to genotype the polymorphisms of complex hybrid yeast genomes. It is based on optimal mapping strategies of NGS reads, comparative analyses of the allelic ratio variation and read depth coverage. We tested the UGDR pipeline with sequencing reads from recombined hybrid diploid yeast strains and various clinical strains exhibiting different degrees of ploidy. UGDR allows to plot the markers distribution and recombination profile per chromosome.ConclusionUGDR detects and plots recombination events in haploids and polyploid yeasts, which facilitates the discovery and understanding of the yeast genetic recombination map and identify new out-performing recombinants.

Biotechnology: use of available carbon sources on the planet to generate alternatives energy

Biotechnology has been an essential tool in the search for solutions and in the optimization of bioprocesses associated with issues of human, plant, animal, energy and also the balance of ecosystems on planet Earth. The objective of this research was to present an unconventional substrate (cellulose), in abundance on the planet, to be used as a substitute source of carbon and energy for biotechnology processes, with the possibility of increasing industrial production of biomass and energy. As basis for the research, an extensive literature review and quantitative and qualitative analyzes were carried out. Genetic Engineering techniques were used to enable the yeast Saccharomyces cerevisiae for partial cellulose degradation, through the use of genetic transformation methods to insert a plasmid carrying the cellobiohydrolase cDNA. It was found that the recombinant and biologically active cellobiohydrolase protein was expressed and excreted in haploid and diploid laboratory yeast strains. The analyzes allowed the visualization of cellulolysis halos around colonies of recombinant strains grown in solid YPD medium with 1% microgranular cellulose. The recombinant clones derived from the haploid lineage yielded in average of 1.70 mg ART/mL, while recombinant clones derived from the diploid lineage produced in average of 2.05 mg ART/mL.

A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination

BackgroundBudding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern.ResultsWe developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 “leaky bowl”-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging.ConclusionsThe DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells.Graphical

Computational method for untargeted determination of cycling yeast metabolites using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

A computational method for the untargeted determination of cycling yeast metabolites using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) dataset is presented. The yeast metabolomic cycle for the diploid yeast strain CEN.PK with a 5 h cycle period relative to the O2 concentration level is comprehensively examined to determine the metabolites that exhibit cycling. Samples were collected over only two cycles (10 h with a total of 24 time-point sampling intervals at 25 min each) as an experimental constraint. Due to the limited number of cycles expressed in the dataset, a computational method was devised to determine with statistical significance whether or not a given metabolite exhibited a temporal signal pattern that constituted cycling in the context of the 5 h cycle period. The computational method we report compares the experimentally obtained 24 time-point metabolite signal sequences to randomly generated signal sequences coupled with statistically based confidence level LOF metrics to determine whether or not a given metabolite expresses cycling, and if so, what is the phase of the cycling. Initially the GC×GC-TOFMS dataset was analyzed using tile-based Fisher ratio (F-ratio) analysis. Since there were 24 time-point intervals, this constituted 24 sample classes in the F-ratio calculation which produced 672 metabolite hits. Next, application of the computational method determined that there were 210 of the 672 metabolites exhibiting cycling: 55 identified metabolites and 155 unknown metabolites. Furthermore, the 210 cycling metabolites were categorized into four groups, and where applicable, a phase determined: 1 cycle/5 h period (106 metabolites), 2 cycles/5 h period (13 metabolites), spiky pattern (12 metabolites), or multimodal pattern (79 metabolites).

The interplay of additivity, dominance, and epistasis on fitness in a diploid yeast cross

In diploid species, genetic loci can show additive, dominance, and epistatic effects. To characterize the contributions of these different types of genetic effects to heritable traits, we use a double barcoding system to generate and phenotype a panel of ~200,000 diploid yeast strains that can be partitioned into hundreds of interrelated families. This experiment enables the detection of thousands of epistatic loci, many whose effects vary across families. Here, we show traits are largely specified by a small number of hub loci with major additive and dominance effects, and pervasive epistasis. Genetic background commonly influences both the additive and dominance effects of loci, with multiple modifiers typically involved. The most prominent dominance modifier in our data is the mating locus, which has no effect on its own. Our findings show that the interplay between additivity, dominance, and epistasis underlies a complex genotype-to-phenotype map in diploids.

CYP1B1 converts procarcinogens into genotoxins in Saccharomyces cerevisiae

CYP1B1 activates many chemical carcinogens into potent genotoxins, and allelic variants are risk factors in lung, breast, and prostate cancer. However, few eukaryotic genetic instability endpoints have been directly measured for CYP1B1-activated metabolites. In this study, we expressed human CYP1B1 in yeast strains that measure DNA damage-associated toxicity and frequencies of chromosomal translocations. DNA damage-associated toxicity was measured in a rad4 rad51 strain, defective in both DNA excision and recombinational repair. Frequencies of chromosomal translocations were measured in diploid yeast strains containing two his3 fragments. These strains were exposed to benzo[a]pyrene-7,8-dihydrodiol (BaP-DHD), aflatoxin B1 (AFB1), and the heterocyclic aromatic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). We observed that AFB1, BaP-DHD, IQ, and MeIQx conferred toxicity in the DNA repair mutant expressing CYP1B1. Translocation frequencies increased eight-fold and three-fold after exposure to 50 μM AFB1 and 33 μM BaP-DHD respectively. A DNA damage response was observed after AFB1 exposure, as measured by the induction of the small subunit of ribonucleotide reductase, Rnr3. While CYP1B1-mediated activation of BaP-DHD and heterocyclic aromatic amines was expected, activation of AFB1 to become a potent recombinagen was not expected. These studies demonstrate that chromosomal rearrangement is a useful genotoxic endpoint for CYP1B1-mediated carcinogen activation.

Identification of a glucose-insensitive variant of Gal2 from Saccharomyces cerevisiae exhibiting a high pentose transport capacity

As abundant carbohydrates in renewable feedstocks, such as pectin-rich and lignocellulosic hydrolysates, the pentoses arabinose and xylose are regarded as important substrates for production of biofuels and chemicals by engineered microbial hosts. Their efficient transport across the cellular membrane is a prerequisite for economically viable fermentation processes. Thus, there is a need for transporter variants exhibiting a high transport rate of pentoses, especially in the presence of glucose, another major constituent of biomass-based feedstocks. Here, we describe a variant of the galactose permease Gal2 from Saccharomyces cerevisiae (Gal2N376Y/M435I), which is fully insensitive to competitive inhibition by glucose, but, at the same time, exhibits an improved transport capacity for xylose compared to the wildtype protein. Due to this unique property, it significantly reduces the fermentation time of a diploid industrial yeast strain engineered for efficient xylose consumption in mixed glucose/xylose media. When the N376Y/M435I mutations are introduced into a Gal2 variant resistant to glucose-induced degradation, the time necessary for the complete consumption of xylose is reduced by approximately 40%. Moreover, Gal2N376Y/M435I confers improved growth of engineered yeast on arabinose. Therefore, it is a valuable addition to the toolbox necessary for valorization of complex carbohydrate mixtures.

Saccharomyces cerevisiae quiescent cells: cadmium resistance and adaptive response

The budding yeast Saccharomyces cerevisiae is a widely used model organism to investigate the changes occurring in the eukaryotic cell and to predict its possible ‘reaction’ to different environmental factors. Recently it was also shown that these microorganisms possess another advantageous ability: to ‘enter’ into quiescent state (G0). Yeast G0 cells have similar physiological characteristics to those of higher eukaryotes making them a better model for toxicology studies. As cadmium could affect severely human health, the main aim of the present study was to use Saccharomyces cerevisiae quiescent cells to investigate the resistance potential and corresponding adaptive response of eukaryotic cells to elevated cadmium concentrations. Both diploid and haploid yeast strains in logarithmic, quiescent and non-quiescent state were exposed to different concentrations of Cd(NO3)2. The half-maximal inhibitory concentration (IC50) for all tested cell types was 100 µmol/L Cd(NO3)2. The deleterious effects of cadmium on intracellular macromolecule structures were analyzed through evaluating the levels of accumulated reactive oxygen species (ROS) and carbonylated proteins. The highest ROS concentration was measured in logarithmic cells: up to 50% versus about 8% in G0 cells. Significant damage in protein molecules was observed in haploid cells where the protein carbonylation reached levels of 25 µmol/mg. Studying the adaptive response to elevated Cd2+ concentrations revealed that quiescent and non-quiescent cells respond with increased expression of key elements from the antioxidant defense system: reduced glutathione, superoxide dismutase (SOD) and catalase. Furthermore, an additional SOD izoenzyme was detected when diploid and haploid cell populations were exposed to Cd2+.

Effects of a novel variant of the yeast \u03b3-glutamyl kinase Pro1 on its enzymatic activity and sake brewing

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

Improved cellulase expression in diploid yeast strains enhanced consolidated bioprocessing of pretreated corn residues

In an effort to find a suitable genetic background for efficient cellulolytic secretion, genetically diverse strains were transformed to produce core fungal cellulases namely, β-glucosidase (BGLI), endoglucanase (EGII) and cellobiohydrolase (CBHI) in various combinations and expression configurations. The secreted enzyme activity levels, gene copy number, substrate specificities, as well as hydrolysis and fermentation yields of the transformants were analysed. The effectiveness of the partially cellulolytic yeast transformants to convert two different pre-treated corn residues, namely corn cob and corn husk was then explored. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants produced up to 1.34 fold higher glucose concentrations (g/L) than a control composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted ratio of 1:15 (cellulase activity unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments and yielded 4.05 g/L ethanol in fermentations. We demonstrate that the choice of optimal genetic background and cellulase activity secretion ratio can improve cellulosic ethanol production by consolidated bioprocessing yeast strains.

Increased Acetate Ester Production of Polyploid Industrial Brewer's Yeast Strains via Precise and Seamless "Self-cloning" Integration Strategy.

BackgroundEnhancing the industrial yeast strains ethyl acetate yield through a precise and seamless genetic manipulation strategy without any extraneous DNA sequences is an essential requisite and significant demand.ObjectivesFor increasing the ethyl acetate yield of industrial brewer’s yeast strain, all the ATF1 alleles were overexpressed through “self-cloning” integration strategy.Material and MethodsEscherichia coli strain DH5α was utilized for plasmid construction. ATF1 alleles were overexpressed through a precise and seamless insertion of the PGK1 promoter in industrial brewer’s yeast strain S6. In addition, growth rates, ATF1 mRNA levels, AATase activity, the fermentation performance of the engineered strains, and gas chromatography (GC) analysis was conducted.ResultsThe two engineered strains (S6-P-12 and S6-P-30) overexpressed all ATF1 alleles but unaffected normal growth. The ATF1 mRNA levels of the S6-P-12 and S6-P-30 were all 4-fold higher than that of S6. The AATase (Alcohol acetyl transferases, encoded by ATF1 gene) activity of the two engineered strains was all 3-fold higher than that of the parent strain. In the beer fermentation at 10 ℃, the concentrations of ethyl acetate produced by the engineered strains S6-P-12 and S6-P-30 was increased to 23.98 and 24.00 mg L-1, respectively, about 20.44% and 20.54% higher than that of S6.ConclusionsThese results verify that the ethyl acetate yield could be enhanced by the overexpressed of ATF1 in the polyploid industrial brewer’s yeast strains via “self-cloning” integration strategy. The present study provides a reference for target gene modification in the diploid or polyploid industrial yeast strains.

Gene overexpression screen for chromosome instability in yeast primarily identifies cell cycle progression genes

Loss of heterozygosity (LOH) in a vegetatively growing diploid cell signals irregularity of mitosis. Therefore, assays of LOH serve to discover pathways critical for proper replication and segregation of chromosomes. We screened for enhanced LOH in a whole-genome collection of diploid yeast strains in which a single gene was strongly overexpressed. We found 39 overexpression strains with substantially increased LOH caused either by recombination or by chromosome instability. Most of them, 32 in total, belonged to the category of “cell division”, a broadly defined biological process. Of those, only one, TOP3, coded for an enzyme that uses DNA as a substrate. The rest related to establishment and maintenance of cell polarity, chromosome segregation, and cell cycle checkpoints. Former studies, in which gene deletions were used, showed that an absence of a protein participating in the DNA processing machinery is a potent stimulator of genome instability. As our results suggest, overexpression of such proteins is not comparably damaging as the absence of them. It may mean that the harmful effect of overexpression is more likely to occur in more complex and multistage processes, such as chromosome segregation. We also report a side finding, resulting from the fact that we worked with the yeast strains bearing a 2-micron plasmid. We noted that intense transcription from such a plasmid led to an enhanced rate of an entire chromosome loss (as opposed to LOH produced by recombination). This observation may support models linking segregation of 2-micron plasmids to segregation of chromosomes.

Recombinant Diploid Saccharomyces cerevisiae Strain Development for Rapid Glucose and Xylose Co-Fermentation

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L−1 glucose and 95 g L−1 xylose generated 120.6 g L−1 ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g−1, and ethanol productivity of 5.26 g L−1·h−1.

Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.

PacBio sequencing, de novo assembly and haplotype phasing of diploid yeast strains

PacBio sequencing, de novo assembly and haplotype phasing of diploid yeast strains

Construction of low-ethanol\u2013wine yeasts through partial deletion of the Saccharomyces cerevisiae PDC2 gene

We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduction in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Compared to the control, the diploid BY4743pdc2Δ519 mutant strain reduced up to 7.4% the total ethanol content in lab scale-vinifications. The residual sugar and volatile acidity was not significantly affected by this ethanol reduction. Remarkably, we got a much higher ethanol reduction of 10 and 15% when the pdc2Δ519 mutation was tested in a native and a commercial wine yeast strain against their respective controls. Our results demonstrate that the insertion of the pdc2Δ519 mutation in wine yeast strains can reduce the ethanol concentration up to 1.89% (v/v) without affecting the fermentation performance. In contrast to non-GMO based strategies, our approach permits the insertion of the pdc2Δ519 mutation in any locally selected wine strain, making possible to produce quality wines with regional characteristics and lower alcohol content. Thus, we consider our work a valuable contribution to the problem of high ethanol concentration in wine.

Both R-loop removal and ribonucleotide excision repair activities of RNase H2 contribute substantially to chromosome stability

Cells carrying deletions of genes encoding H-class ribonucleases display elevated rates of chromosome instability. The role of these enzymes is to remove RNA-DNA associations including persistent mRNA-DNA hybrids (R-loops) formed during transcription, and ribonucleotides incorporated into DNA during replication. RNases H1 and H2 can degrade the RNA component of R-loops, but only RNase H2 can initiate accurate ribonucleotide excision repair (RER). In order to examine the specific contributions of these activities to chromosome stability, we measured rates of loss-of-heterozygosity (LOH) in diploid Saccharomyces cerevisiae yeast strains carrying the rnh201-RED separation-of-function allele, encoding a version of RNase H2 that is RER-defective, but partly retains its other activity. The LOH rate in rnh201-RED was intermediate between RNH201 and rnh201Δ. In strains carrying a mutant version of DNA polymerase ε (pol2-M644G) that incorporates more ribonucleotides than normal, the LOH rate in rnh201-RED was as high as the rate measured in rnh201Δ. Topoisomerase 1 cleavage at sites of ribonucleotide incorporation has been recently shown to produce DNA double strand breaks. Accordingly, in both the POL2 and pol2-M644G backgrounds, the LOH elevation in rnh201-RED was suppressed by top1Δ. In contrast, in strains that incorporate fewer ribonucleotides (pol2-M644L) the LOH rate in rnh201-RED was low and independent of topoisomerase 1. These results suggest that both R-loop removal and RER contribute substantially to chromosome stability, and that their relative contributions may be variable across different regions of the genome. In this scenario, a prominent contribution of R-loop removal may be expected at highly transcribed regions, whereas RER may play a greater role at hotspots of ribonucleotide incorporation.

Isolation of baker's yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses

During bread-making processes, yeast cells are exposed to baking-associated stresses such as freeze-thaw, air-drying, and high-sucrose concentrations. Previously, we reported that self-cloning diploid baker's yeast strains that accumulate proline retained higher-level fermentation abilities in both frozen and sweet doughs than the wild-type strain. Although self-cloning yeasts do not have to be treated as genetically modified yeasts, the conventional methods for breeding baker's yeasts are more acceptable to consumers than the use of self-cloning yeasts. In this study, we isolated mutants resistant to the proline analogue azetidine-2-carboxylate (AZC) derived from diploid baker's yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular proline, and among them, 5 mutants showed higher cell viability than that observed in the parent wild-type strain under freezing or high-sucrose stress conditions. Two of them carried novel mutations in the PRO1 gene encoding the Pro247Ser or Glu415Lys variant of γ-glutamyl kinase (GK), which is a key enzyme in proline biosynthesis in S. cerevisiae. Interestingly, we found that these mutations resulted in AZC resistance of yeast cells and desensitization to proline feedback inhibition of GK, leading to intracellular proline accumulation. Moreover, baker's yeast cells expressing the PRO1P247S and PRO1E415K gene were more tolerant to freezing stress than cells expressing the wild-type PRO1 gene. The approach described here could be a practical method for the breeding of proline-accumulating baker's yeasts with higher tolerance to baking-associated stresses.

Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools.

The absolute and relative concentrations of the four dNTPs are key determinants of DNA replication fidelity, yet the consequences of altered dNTP pools on replication fidelity have not previously been investigated on a genome-wide scale. Here, we use deep sequencing to determine the types, rates and locations of uncorrected replication errors that accumulate in the nuclear genome of a mismatch repair-deficient diploid yeast strain with elevated dCTP and dTTP concentrations. These imbalanced dNTP pools promote replication errors in specific DNA sequence motifs suggesting increased misinsertion and increased mismatch extension at the expense of proofreading. Interestingly, substitution rates are similar for leading and lagging strand replication, but are higher in regions replicated late in S phase. Remarkably, the rate of single base deletions is preferentially increased in coding sequences and in short rather than long mononucleotides runs. Based on DNA sequence motifs, we propose two distinct mechanisms for generating single base deletions in vivo. Collectively, the results indicate that elevated dCTP and dTTP pools increase mismatch formation and decrease error correction across the nuclear genome, and most strongly increases mutation rates in coding and late replicating sequences.