Diploid Yeast Research Articles

Functional analysis of a S-adenosylmethionine-insensitive methylenetetrahydrofolate reductase identified in methionine-accumulating yeast mutants.

Essential amino acids (EAAs) are important for the maintenance of brain functions. Therefore, the yeast Saccharomyces cerevisiae that accumulate EAAs would help elderly people ingest appropriate levels of EAAs, which in turn could slow neurodegeneration, extend the healthy lifespan and improve quality of life. Here, we isolated two mutant strains, ETH-80 and ETH-129, that accumulate the EAA methionine. Both strains were derived from a diploid laboratory yeast by conventional mutagenesis and carry a novel mutation in the MET13 gene, which encodes the Ser443Phe variant of methylenetetrahydrofolate reductase. Enzymatic analysis revealed that the Ser443Phe substitution abolished the sensitivity to S-adenosyl methionine (SAM)-mediated inhibition even in the presence of 2mM SAM, while increasing the activity for NADPH-dependent reduction. Furthermore, yeast cells expressing the Ser443Phe variant showed a fourfold increase in intracellular methionine content compared to the wild-type Met13. These findings will be useful for the future development of methionine-accumulating yeast strains.

Unveiling the genotypic diversity of Candida albicans and Candida dubliniensis in the oral cavities of drug abusers in Ahvaz, Iran

Candida albicans is a diploid yeast that, under certain conditions, can cause oral or oropharyngeal infections, particularly in immunocompromised individuals. Recent molecular investigations have classified genotypes A, B, and C for Candida albicans, along with genotype D for Candida dubliniensis. This study aimed to identify the different genotypes of the C. albicans complex in drug abusers in Iran. Oral swabs were collected from drug abusers and cultured on CHROMagar Candida. A 21-plex PCR method was employed for the detection of isolates, and the Candida 25S rDNA gene was amplified using primer pairs CA-INT-L and CA-INT-R for ABC genotyping of C. albicans. Out of the 245 substance abusers screened, 151 individuals (61.63 %) were found to harbor the C. albicans complex. The most common genotype among patients was genotype D (39.1 %), followed by genotype A (31.12 %), genotype B (9.93 %), and genotype C (5.29 %). Additionally, 14.56 % of patients had a mixed genotype. Notably, significant differences in genotype distribution were observed in relation to age, underlying diseases, and marital status (P < 0.05). This study highlights the significance of molecular genotyping in understanding the epidemiology of C. albicans and C. dubliniensis in at-risk groups.

Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid’s precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis.Key points• Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis.• Perillic acid was first synthesized de novo in engineered microorganisms.• The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.

Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.

Multi-signal regulation of the GSK-3β homolog Rim11 controls meiosis entry in budding yeast

Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcriptional activation of early meiotic genes (EMGs) hinges on the master regulator Ime1, its DNA-binding partner Ume6, and GSK-3β kinase Rim11. Phosphorylation of Ume6 by Rim11 is required for EMG activation. We report here that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibition of PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. In addition, Ime1 is an anchor protein enabling Ume6 phosphorylation by Rim11. Subsequently, Ume6-Ime1 coactivator complexes form and induce EMG transcription. Our results demonstrate how various signaling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signaling-regulatory network elucidated here generates robustness in cell-fate control.

Molecular Mechanisms of Genotype-Dependent Lifespan Variation Mediated by Caloric Restriction: Insight from Wild Yeast Isolates.

Caloric restriction (CR) is known to extend lifespan across different species and holds great promise for preventing human age-onset pathologies. However, two major challenges exist. First, despite extensive research, the mechanisms of lifespan extension in response to CR remain elusive. Second, genetic differences causing variations in response to CR and genetic factors contributing to variability of CR response on lifespan are largely unknown. Here, we took advantage of natural genetic variation across 46 diploid wild yeast isolates of Saccharomyces species and the lifespan variation under CR conditions to uncover the molecular factors associated with CR response types. We identified genes and metabolic pathways differentially regulated in CR-responsive versus non-responsive strains. Our analysis revealed that altered mitochondrial function and activation of GCN4-mediated environmental stress response are inevitably linked to lifespan variation in response to CR and a unique mitochondrial metabolite might be utilized as a predictive marker for CR response rate. In sum, our data suggests that the effects of CR on longevity may not be universal, even among the closely related species or strains of a single species. Since mitochondrial-mediated signaling pathways are evolutionarily conserved, the dissection of related genetic pathways will be relevant to understanding the mechanism by which CR elicits its longevity effect.

Functional analysis of feedback inhibition-insensitive aspartate kinase identified in a threonine-accumulating mutant of Saccharomyces cerevisiae.

Humans and mammals need to ingest essential amino acids (EAAs) for protein synthesis. In addition to their importance as nutrients, EAAs are involved in brain homeostasis. However, elderly people are unable to efficiently consume EAAs from their daily diet due to reduced appetite and variations in the contents of EAAs in foods. On the other hand, strains of the yeast Saccharomyces cerevisiae that accumulate EAAs would enable elderly people to intakegest adequate amounts of EAAs and thus might slow down the neurodegenerative process, contributing to the extension of their healthy lifespan. In this study, we isolated a mutant (strain HNV-5) that accumulates threonine, an EAA, derived from a diploid laboratory yeast by conventional mutagenesis. Strain HNV-5 carries a novel mutation in the HOM3 gene encoding the Ala462Thr variant of aspartate kinase (AK). Enzymatic analysis revealed that the Ala462Thr substitution significantly decreased the sensitivity of AK activity to threonine feedback inhibition even in the presence of 50 mM threonine. Interestingly, Ala462Thr substitution did not affect the catalytic ability of Hom3, in contrast to previously reported amino acid substitutions that resulted in reduced sensitivity to threonine feedback inhibition. Furthermore, yeast cells expressing the Ala462Thr variant showed an approximately threefold increase in intracellular threonine content compared to that of the wild-type Hom3. These findings will be useful for the development of threonine-accumulating yeast strains that may improve the quality of life in elderly people.IMPORTANCEFor humans and mammals, essential amino acids (EAAs) play an important role in maintaining brain function. Therefore, increasing the intake of EAAs by using strains of the yeast Saccharomyces cerevisiae that accumulate EAAs may inhibit neurodegeneration in elderly people and thus contribute to extending healthy lifespan and improving their quality of life. Threonine, an EAA, is synthesized from aspartate. Aspartate kinase (AK) catalyzes the first step in threonine biosynthesis and is subject to allosteric regulation by threonine. Here, we isolated a threonine-accumulating mutant of S. cerevisiae by conventional mutagenesis and identified a mutant gene encoding a novel variant of AK. In contrast to previously isolated variants, the Hom3 variant exhibited AK activity that was insensitive to feedback inhibition by threonine but retained its catalytic ability. This resulted in increased production of threonine in yeast. These findings open up the possibility for the rational design of AK to increase threonine productivity in yeast.

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.&#x0D; 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.&#x0D; 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.

Molecular mechanisms of genotype-dependent lifespan variation mediated by caloric restriction: insight from wild yeast isolates.

Caloric restriction (CR) is known to extend lifespan across different species and holds great promise for preventing human age-onset pathologies. However, two major challenges exist. First, despite extensive research, the mechanisms of lifespan extension in response to CR remain elusive. Second, genetic differences causing variations in response to CR and genetic factors contributing to variability of CR response on lifespan are largely unknown. Here, we took advantage of natural genetic variation across 46 diploid wild yeast isolates of Saccharomyces species and the lifespan variation under CR conditions to uncover the molecular factors associated with CR response types. We identified genes and metabolic pathways differentially regulated in CR-responsive versus non-responsive strains. Our analysis revealed that altered mitochondrial function and activation of GCN4-mediated environmental stress response are inevitably linked to lifespan variation in response to CR and a unique mitochondrial metabolite might be utilized as a predictive marker for CR response rate. In sum, our data suggests that the effects of CR on longevity may not be universal, even among the closely related species or strains of a single species. Since mitochondrial-mediated signaling pathways are evolutionarily conserved, the dissection of related genetic pathways will be relevant to understanding the mechanism by which CR elicits its longevity effect.

The impact of the GLOSSY2 and GLOSSY2-LIKE BAHD-proteins in affecting the product profile of the maize fatty acid elongase.

The maize glossy2 and glossy2-like genes are homologs, which encode proteins that belong to the BAHD family of acyltransferases. In planta genetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploid Saccharomyces cerevisiae strains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.

Homozygous gene disruption in diploid yeast through a single transformation

As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5'- and 3'-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5'- and 3'-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3/ura3 lys5/lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2/leu2 and leu2/leu2 his3/his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae.

A computer vision and residual neural network (ResNet) combined method for automated and accurate yeast replicative aging analysis of high-throughput microfluidic single-cell images

With the rapid development of microfluidic platforms in high-throughput single-cell culturing, laborious operation to manipulate massive budding yeast cells (Saccharomyces cerevisiae) in replicative aging studies has been greatly simplified and automated. As a result, large datasets of microscopy images bring challenges to fast and accurately determine yeast replicative lifespan (RLS), which is the most important parameter to study cell aging. Based on our microfluidic diploid yeast long-term culturing (DYLC) chip that features 1100 traps to immobilize single cells and record their proliferation and aging via time-lapse imaging, herein, a dedicated algorithm combined with computer vision and residual neural network (ResNet) was presented to efficiently process tremendous micrographs in a high-throughput and automated manner. The image-processing algorithm includes following pivotal steps: (i) segmenting multi-trap micrographs into time-lapse single-trap sub-images, (ii) labeling 8 yeast budding features and training the 18-layer ResNet, (iii) converting the ResNet predictions in analog values into digital signals, (iv) recognizing cell dynamic events, and (v) determining yeast RLS and budding time interval (BTI) ultimately. The ResNet algorithm achieved high F1 scores (over 92%) demonstrating the effectiveness and accuracy in the recognition of yeast budding events, such as bud appearance, daughter dissection and cell death. Therefore, the results conduct that similar deep learning algorithms could be tailored to analyze high-throughput microscopy images and extract multiple cell behaviors in microfluidic single-cell analysis.

Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments.

Adaptation is driven by the selection for beneficial mutations that provide a fitness advantage in the specific environment in which a population is evolving. However, environments are rarely constant or predictable. When an organism well adapted to one environment finds itself in another, pleiotropic effects of mutations that made it well adapted to its former environment will affect its success. To better understand such pleiotropic effects, we evolved both haploid and diploid barcoded budding yeast populations in multiple environments, isolated adaptive clones, and then determined the fitness effects of adaptive mutations in 'non-home' environments in which they were not selected. We find that pleiotropy is common, with most adaptive evolved lineages showing fitness effects in non-home environments. Consistent with other studies, we find that these pleiotropic effects are unpredictable: they are beneficial in some environments and deleterious in others. However, we do find that lineages with adaptive mutations in the same genes tend to show similar pleiotropic effects. We also find that ploidy influences the observed adaptive mutational spectra in a condition-specific fashion. In some conditions, haploids and diploids are selected with adaptive mutations in identical genes, while in others they accumulate mutations in almost completely disjoint sets of genes.

Live while the DNA lasts. The role of autophagy in DNA loss and survival of diploid yeast cells during chronological aging.

Aging is inevitable and affects all cell types, thus yeast cells are often used as a model in aging studies. There are two approaches to studying aging in yeast: replicative aging, which describes the proliferative potential of cells, and chronological aging, which is used for studying post-mitotic cells. While analyzing the chronological lifespan (CLS) of diploid Saccharomyces cerevisiae cells, we discovered a remarkable phenomenon: ploidy reduction during aging progression. To uncover the mechanism behind this unusual process we used yeast strains undergoing a CLS assay, looking for various aging parameters. Cell mortality, regrowth ability, autophagy induction and cellular DNA content measurements indicated that during the CLS assay, dying cells lost their DNA, and only diploids survived. We demonstrated that autophagy was responsible for the gradual loss of DNA. The nucleophagy marker activation at the start of the CLS experiment correlated with the significant drop in cell viability. The activation of piecemeal microautophagy of nucleus (PMN) markers appeared to accompany the chronological aging process until the end. Our findings emphasize the significance of maintaining at least one intact copy of the genome for the survival of post-mitotic diploid cells. During chronological aging, cellular components, including DNA, are exposed to increasing stress, leading to DNA damage and fragmentation in aging cells. We propose that PMN-dependent clearance of damaged DNA from the nucleus helps prevent genome rearrangements. However, as long as one copy of the genome can be rebuilt, cells can still survive.

DNA Repair Does Not Trigger Genetic Instability of Yeast Cells

The dependence of the delayed formation of colonies on the dose of ionizing radiation (gamma rays of 60Co, dose rate 10 Gy/min) was obtained for six strains of wild-type haploid and diploid yeast, capable of recovering from radiation damage and characterized by sigmoidal survival curves as well as for six haploid and diploid radiosensitive mutants defective in reparation and characterized by exponential survival curves. The delay in the formation of colonies by survival cells after irradiation is considered as a genetic instability display. It was shown that for all diploid strains, genetic instability reached 100% with an increase in the dose of ionizing radiation, regardless of the shape of survival curves and the cell ability to recover from radiation damage. Conversely, for all haploid strains, genetic instability was only close to 20%. In contrast to traditional concepts, these data indicate that the late formation of colonies by surviving yeast cells after irradiation is determined mainly by cell ploidy and does not depend on the shape of the dose-response curves and cell radiosensitivity. This means that DNA repair does not trigger genetic instability in yeast cells.

Structural Variations and Adaptations of Synthetic Chromosome Ends Driven by SCRaMbLE in Haploid and Diploid Yeasts.

Variations and adaptations of chromosome ends play an important role in eukaryotic karyotype evolution. Traditional experimental studies of the adaptations of chromosome ends mainly rely on the strategy of introducing defects; thus, the adaptation methods of survivors may vary depending on the initial defects. Here, using the SCRaMbLE strategy, we obtained a library of haploid and diploid synthetic strains with variations in chromosome ends. Analysis of the SCRaMbLEd survivors revealed four routes of adaptation: homologous recombination between nonhomologous chromosome arms (haploids) or homologous chromosome arms (diploids), site-specific recombination between intra- or interchromosomal ends, circularization of chromosomes, and loss of whole chromosomes (diploids). We also found that circularization of synthetic chromosomes can be generated by SCRaMbLE. Our study of various adaptation routes of chromosome ends provides insight into eukaryotic karyotype evolution from the viewpoint of synthetic genomics.

Apparent diameter and cell density of yeast strains with different ploidy

Optical density at 600 nm (OD600) measurements are routinely and quickly taken to estimate cell density in cultivation and to track cell growth. The yeast Saccharomyces cerevisiae is one of the microorganisms most used in industry, and the OD600 values are frequently adopted as the indicator of yeast cell density, according to the Beer–Lambert law. Because the OD600 value is based on turbidity measurement, the Beer–Lambert law can be applied only for microbial cultivation with low cell densities. The proportionality constants strongly depend on several parameters such as cell size. Typically, yeast strains are categorized into haploids and diploids. It is well known that cell size of diploid yeasts is larger than haploid cells. Additionally, polyploid (especially triploid and tetraploid) yeast cells are also employed in several human-activities such as bread-making and lager-brewing. As a matter of fact, there is almost no attention paid to the difference in the proportionality constants depending on the yeast ploidy. This study presents information for cell size of haploid, diploid, triploid, and tetraploid yeasts with isogenic background, and describes their proportionality constants (k) corresponding to the molar extinction coefficient (ε) in the Beer–Lambert law. Importantly, it was found that the constants are inversely proportional to apparent cell diameters estimated by flow cytometric analysis. Although each cell property highly depends on genetic and environmental factors, a set of results obtained from yeast strains with different ploidy in the current study would serve as a major reference source for researchers and technical experts.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

Engineering a fermenting yeast able to produce the fragrant β-ionone apocarotenoid for enhanced aroma properties in wine.

Wine is composed of multitudinous flavour components and volatile organic compounds that provide this beverage with its attractive properties of taste and aroma. The perceived quality of a wine can be attributed to the absolute and relative concentrations of favourable aroma compounds; hence, increasing the detectable levels of an attractive aroma, such as β-ionone with its violet and berry notes, can improve the organoleptic qualities of given wine styles. We here describe the generation of a new grape-must fermenting strain of Saccharomyces cerevisiae that is capable of releasing β-ionone through the heterologous expression of both the enzyme carotenoid cleavage dioxygenase 1 (CCD1) and its substrate, β-carotene. Haploid laboratory strains of S. cerevisiae were constructed with and without integrated carotenogenic genes and transformed with a plasmid containing the genes of CCD1. These strains were then mated with a sporulated diploid wine industry yeast, VIN13, and four resultant crosses-designated MQ01-MQ04-which were capable of fermenting the must to dryness were compared for their ability to release β-ionone. Analyses of their fermentation products showed that the MQ01 strain produced a high level of β-ionone and offers a fermenting hybrid yeast with the potential to enhance the organoleptic qualities of wine.

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.