Hybrid Yeast Research Articles

Hybrid adaptation is hampered by Haldane’s sieve

Hybrids between species exhibit plastic genomic architectures that could foster or slow down their adaptation. When challenged to evolve in an environment containing a UV mimetic drug, yeast hybrids have reduced adaptation rates compared to parents. We find that hybrids and their parents converge onto similar molecular mechanisms of adaptation by mutations in pleiotropic transcription factors, but at a different pace. After 100 generations, mutations in these genes tend to be homozygous in the parents but heterozygous in the hybrids. We hypothesize that a lower rate of loss of heterozygosity (LOH) in hybrids could limit fitness gain. Using genome editing, we first demonstrate that mutations display incomplete dominance, requiring homozygosity to show full impact and to entirely circumvent Haldane’s sieve, which favors the fixation of dominant mutations. Second, tracking mutations in earlier generations confirmed a different rate of LOH in hybrids. Together, these findings show that Haldane’s sieve slows down adaptation in hybrids, revealing an intrinsic constraint of hybrid genomic architecture that can limit the role of hybridization in adaptive evolution.

Understanding brewing trait inheritance in de novo Lager yeast hybrids.

Our study investigates the principles of lager yeast hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus. This process gave rise to the lager yeast Saccharomyces pastorianus. By examining how these novel hybrids perform during fermentation and the aromas they produce, we uncover the genetic bases of brewing trait inheritance. We successfully generated polyploid hybrids using diverse strains and lineages from both parent species, predominantly triploids and diploids. Although these hybrids did not show improved fermentation capacity, they exhibited varied traits, especially in utilizing maltotriose, a key sugar in brewing. Remarkably, the aroma profiles of these hybrids were primarily influenced by the S. cerevisiae parent, with Beer lineage hybrids adopting aroma characteristics from their S. eubayanus parent. These insights reveal the complex genetic interactions in hybrid yeasts, opening new possibilities for crafting unique brewing yeasts with desirable traits.

Chromosome-level subgenome-aware de novo assembly provides insight into Saccharomyces bayanus genome divergence after hybridization.

Interspecies hybridization is prevalent in various eukaryotic lineages and plays important roles in phenotypic diversification, adaptation, and speciation. To better understand the changes that occurred in the different subgenomes of a hybrid species and how they facilitate adaptation, we have completed chromosome-level de novo assemblies of all chromosomes for a recently formed hybrid yeast, Saccharomyces bayanus strain CBS380, using Oxford Nanopore Technologies' MinION long-read sequencing. We characterize the S. bayanus genome and compare it with its parent species, Saccharomyces uvarum and Saccharomyces eubayanus, and other S. bayanus genomes to better understand genome evolution after a relatively recent hybridization event. We observe multiple recombination events between the subgenomes in each chromosome, followed by loss of heterozygosity (LOH) in nine chromosome pairs. In addition to maintaining nearly all gene content and synteny from its parental genomes, S. bayanus has acquired many genes from other yeast species, primarily through the introgression of Saccharomyces cerevisiae, such as those involved in the maltose metabolism. Finally, the patterns of recombination and LOH suggest an allotetraploid origin of S. bayanus The gene acquisition and rapid LOH in the hybrid genome probably facilitated its adaptation to maltose brewing environments and mitigated the maladaptive effect of hybridization. This paper describes the first in-depth study using long-read sequencing technology of an S. bayanus hybrid genome, which may serve as an excellent reference for future studies of this important yeast and other yeast strains.

Development of a genome-scale metabolic model for the lager hybrid yeast S. pastorianus to understand the evolution of metabolic pathways in industrial settings.

Genome-scale metabolic models (GSMM) have been successfully applied to predict cellular behavior and design cell factories in several model organisms, but no models to date are currently available for hybrid species due to their more complex genetics and general lack of molecular data. In this study, we generated a bespoke GSMM, iSP_1513, for this industrial aneuploid hybrid Saccharomyces pastorianus, which takes into account the aneuploidy and functional redundancy from orthologous parental alleles. This model will (i) help understand the metabolic capabilities and adaptive potential of S. pastorianus (domestication processes), (ii) aid top-down predictions for strain development (industrial biotechnology), and (iii) allow predictions of evolutionary trajectories of metabolic pathways in aneuploid hybrids (evolutionary genetics).

Overexpression of OsNAR2.1 by OsNAR2.1 promoter increases drought resistance by increasing the expression of OsPLDα1 in rice

BackgroundpOsNAR2.1:OsNAR2.1 expression could significantly increase nitrogen uptake efficiency and grain yield of rice.ResultThis study reported the effects of overexpression of OsNAR2.1 by OsNAR2.1 promoter on physiological and agronomic traits associated with drought tolerance. In comparison to the wild-type (WT), the pOsNAR2.1:OsNAR2.1 transgenic lines exhibited a significant improvement in survival rate when subjected to drought stress and then irrigation. Under limited water supply conditions, compared with WT, the photosynthesis and water use efficiency (WUE) of transgenic lines were increased by 39.2% and 28.8%, respectively. Finally, the transgenic lines had 25.5% and 66.4% higher grain yield than the WT under full watering and limited water supply conditions, respectively. Compared with the WT, the agronomic nitrogen use efficiency (NUE) of transgenic lines increased by 25.5% and 66.4% under full watering and limited water supply conditions, and the N recovery efficiency of transgenic lines increased by 29.3% and 50.2%, respectively. The interaction between OsNAR2.1 protein and OsPLDα1 protein was verified by yeast hybrids. After drought treatment, PLDα activity on the plasma membrane of the transgenic line increased 85.0% compared with WT.ConclusionThese results indicated that pOsNAR2.1:OsNAR2.1 expression could improve the drought resistance of rice by increasing nitrogen uptake and regulating the expression of OsPLDα1.

Development of freezing-resistant hybrid yeast from Saccharomyces cerevisiae for French bread dough

This study aimed to develop non-GMO freezing-resistant hybrid yeast for frozen French bread dough. The hybrids were generated by direct mating, using Saccharomyces cerevisiae strains from the ethanol industry (PE-2) and a baker's yeast (FLE). Four viable hybrids were generated and named F5P1, F5P33, F5P40, and F5P42. According to the 96-well microplate analyses, the growth curves of the hybrids after freezing and subsequent thawing presented similar behavior to those without freezing. In a performance test after the freezing step, F5P1 and F5P40 achieved a higher development index, with values of (147.1 ± 2.5) % and (102.1 ± 5.6) %, respectively. The peak reached by F5P1, after 50 min, was similar (p > 0.05) to that of FLE, which was at 110 min. The difference between the time required for F5P1 to achieve the growth peak with and without freezing was 10 min, whereas for FLE, this difference was three times higher, which highlights the improving freezing resistance of the hybrid. Therefore, the hybrid F5P1 showed a leavening capacity similar to FLE (p > 0.05) and improved freezing resistance, which indicates its potential for application to frozen French bread dough.

The genomic landscape of transposable elements in yeast hybrids is shaped by structural variation and genotype-specific modulation of transposition rate

Transposable elements (TEs) are major contributors to structural genomic variation by creating interspersed duplications of themselves. In return, structural variants (SVs) can affect the genomic distribution of TE copies and shape their load. One long-standing hypothesis states that hybridization could trigger TE mobilization and thus increase TE load in hybrids. We previously tested this hypothesis (Hénault et al., 2020) by performing a large-scale evolution experiment by mutation accumulation (MA) on multiple hybrid genotypes within and between wild populations of the yeastsSaccharomyces paradoxusandSaccharomyces cerevisiae. Using aggregate measures of TE load with short-read sequencing, we found no evidence for TE load increase in hybrid MA lines. Here, we resolve the genomes of the hybrid MA lines with long-read phasing and assembly to precisely characterize the role of SVs in shaping the TE landscape. Highly contiguous phased assemblies of 127 MA lines revealed that SV types like polyploidy, aneuploidy, and loss of heterozygosity have large impacts on the TE load. We characterized 18 de novo TE insertions, indicating that transposition only has a minor role in shaping the TE landscape in MA lines. Because the scarcity of TE mobilization in MA lines provided insufficient resolution to confidently dissect transposition rate variation in hybrids, we adapted an in vivo assay to measure transposition rates in variousS. paradoxushybrid backgrounds. We found that transposition rates are not increased by hybridization, but are modulated by many genotype-specific factors including initial TE load, TE sequence variants, and mitochondrial DNA inheritance. Our results show the multiple scales at which TE load is shaped in hybrid genomes, being highly impacted by SV dynamics and finely modulated by genotype-specific variation in transposition rates.

The genomic landscape of transposable elements in yeast hybrids is shaped by structural variation and genotype-specific modulation of transposition rate.

Transposable elements (TEs) are major contributors to structural genomic variation by creating interspersed duplications of themselves. In return, structural variants (SVs) can affect the genomic distribution of TE copies and shape their load. One long-standing hypothesis states that hybridization could trigger TE mobilization and thus increase TE load in hybrids. We previously tested this hypothesis (Hénault et al., 2020) by performing a large-scale evolution experiment by mutation accumulation (MA) on multiple hybrid genotypes within and between wild populations of the yeasts Saccharomyces paradoxus and Saccharomyces cerevisiae. Using aggregate measures of TE load with short-read sequencing, we found no evidence for TE load increase in hybrid MA lines. Here, we resolve the genomes of the hybrid MA lines with long-read phasing and assembly to precisely characterize the role of SVs in shaping the TE landscape. Highly contiguous phased assemblies of 127 MA lines revealed that SV types like polyploidy, aneuploidy, and loss of heterozygosity have large impacts on the TE load. We characterized 18 de novo TE insertions, indicating that transposition only has a minor role in shaping the TE landscape in MA lines. Because the scarcity of TE mobilization in MA lines provided insufficient resolution to confidently dissect transposition rate variation in hybrids, we adapted an in vivo assay to measure transposition rates in various S. paradoxus hybrid backgrounds. We found that transposition rates are not increased by hybridization, but are modulated by many genotype-specific factors including initial TE load, TE sequence variants, and mitochondrial DNA inheritance. Our results show the multiple scales at which TE load is shaped in hybrid genomes, being highly impacted by SV dynamics and finely modulated by genotype-specific variation in transposition rates.

Screening using loop-mediated isothermal amplification (LAMP) assay and breeding of a Saccharomyces cerevisiae strain isolated from Muramatsu Park, Japan, for sake brewing.

Sake is a Japanese alcoholic beverage produced by fermenting steamed rice and koji (a culture of Aspergillus oryzae on steamed rice) with sake yeast, a strain of Saccharomyces cerevisiae. Sake yeast strains are important for maintaining product quality and process efficiency. In this study, a S. cerevisiae strain from Muramatsu Park, Gosen City, Niigata Prefecture was isolated using a loop-mediated isothermal amplification (LAMP) assay. The yeast strain was cultured using the mass spore-cell/cell-cell mating method with a sake yeast haploid. The resultant hybrid yeast strain, HG-3-F2, exhibited superior efficiency in alcoholic fermentation compared with the HG-3 strain. Our findings support the applicability of these original and mating strains in sake brewing.

Effect of Interspecific Yeast Hybrids for Secondary In-Bottle Alcoholic Fermentation of English Sparkling Wines.

In sparkling winemaking, only a few yeast strains are regularly used for the secondary in-bottle alcoholic fermentation (SiBAF). Recently, advances in yeast development programs have yielded new breeds of interspecific wine yeast hybrids that ferment efficiently while producing novel flavors and aromas. In this work, the chemical and sensorial impacts of the use of interspecific yeast hybrids for SiBAF were studied using three commercial English base wines prepared for SiBAF using two commercial and four novel interspecific hybrids. After 12 months of lees aging, the chemical and macromolecular composition, phenolic profile, foaming, viscosity and sensory properties of the resulting 13 wines were assessed. Chemically, the yeast strains did not result in significant differences in the main wine parameters, while some differences in their macromolecular contents and sensory characteristics were noticeable. The foamability was mostly unaffected by the strain used; however, some effect on the foam stability was noticeable, likely due to the differences in polysaccharides released into the wines by the yeast strains. The wines exhibited different sensory characteristics in terms of aroma and bouquet, balance, finish, overall liking and preference, but these were mostly attributable to the differences in the base wines rather than the strain used for SiBAF. Novel interspecific yeast hybrids can be used for the elaboration of sparkling wines, as they provided wines with chemical characteristics, flavor and aroma attributes similar to those of commonly used commercial Saccharomyces cerevisiae strains.

Evolution of loss of heterozygosity patterns in hybrid genomes of Candida yeast pathogens

BackgroundHybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events.ResultsWe found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids.ConclusionsOur results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events—as expected by mitotic repair of rare chromosomal breaks—but rather of acute episodes involving many LOH events in a short period of time.

Control of meiotic crossing over in plant breeding.

Meiotic crossing over is the main mechanism for constructing a new allelic composition of individual chromosomes and is necessary for the proper distribution of homologous chromosomes between gametes. The parameters of meiotic crossing over that have developed in the course of evolution are determined by natural selection and do not fully suit the tasks of selective breeding research. This review summarizes the results of experimental studies aimed at increasing the frequency of crossovers and redistributing their positions along chromosomes using genetic manipulations at different stages of meiotic recombination. The consequences of inactivation and/or overexpression of the SPO11 genes, the products of which generate meiotic double-strand breaks in DNA, for the redistribution of crossover positions in the genome of various organisms are discussed. The results of studies concerning the effect of inactivation or overexpression of genes encoding RecA-like recombinases on meiotic crossing over, including those in cultivated tomato (Solanum lycopersicum L.) and its interspecific hybrids, are summarized. The consequences of inactivation of key genes of the mismatch repair system are discussed. Their suppression made it possible to significantly increase the frequency of meiotic recombination between homeologues in the interspecific hybrid yeast Saccharomyces cerevisiae × S. paradoxus and between homologues in arabidopsis plants (Arabidopsis thaliana L.). Also discussed are attempts to extrapolate these results to other plant species, in which a decrease in reproductive properties and microsatellite instability in the genome have been noted. The most significant results on the meiotic recombination frequency increase upon inactivation of the FANCM, TOP3α, RECQ4, FIGL1 crossover repressor genes and upon overexpression of the HEI10 crossover enhancer gene are separately described. In some experiments, the increase of meiotic recombination frequency by almost an order of magnitude and partial redistribution of the crossover positions along chromosomes were achieved in arabidopsis while fully preserving fecundity. Similar results have been obtained for some crops.

Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts

Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR-induced double strand breaks is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development.

Novel Saccharomyces cerevisiae × Saccharomyces mikatae Hybrids for Non-alcoholic Beer Production

The popularity of non-alcoholic beers has been increasing over the past few years. Maltose-negative strains of different genera are frequently used to obtain beers of low alcohol content. S. cerevisiae hybrids with other Saccharomyces species offer interesting inherited flavour characteristics; however, their use in non-alcoholic beer production is rare. In this work, we constructed six hybrids of maltose-negative S. cerevisiae parental strains (modified to produce higher amounts of organic acids) and S. mikatae (wild-type). Growth behaviour, osmotolerance and fermentation features of the offspring were compared with parental strains. One hybrid with mitochondrial DNA inherited from both parents was used to produce non-alcoholic beer in which organic metabolites were evaluated by HPLC and HS-SPME-GC-MS. This hybrid produced non-alcoholic beer (≤0.05% (v/v)) with an increased organic acid content, just as its parent S. cerevisiae, but without producing increased amounts of acetic acid. The beer had a neutral aromatic profile with no negative off-flavours, similar to the beer produced by the parent S. mikatae, which was used for the first time to produce non-alcoholic beer. Overall, both parents and hybrid yeast produced non-alcoholic beers with increased amounts of higher alcohols compared with esters.

CRISPR-Cas9 engineering in the hybrid yeast zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.

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.

SCREENING HETEROSIS IN HYBRID YEAST CELLS FOR THEIR LEAVENING PROPERTIES UNDER OSMOTIC STRESS

Bread-making industry occupies an important place in the food sector. This industry is in full process of expansion and automation for obtaining good quality and cost-efficient products. Hybridization in yeast gives rise to offspring carrying an admixture of both parental genotypes which display unique traits that may drive adaptation to changing environments. This study aimed to evaluate the fermentation performance of hybrid yeast cells under sucrose stress, as well as, to elucidate the role of fermentation time in decreasing the weight of fermented bread dough. Two hybrids originated from two hybridizations between three diverged strains of Saccharomyces cerevisiae were used in this study. Hybrids were characterized concerning the parental strains in a wort fermentation of bread wheat containing four sucrose concentrations. Hybrid genotypes have apparent hybrid vigor for reducing the weight of fermented bread dough over time than their parents. This indicated faster fermentation and producing higher quantities of CO2 leading to a decrease in the weight of fermented bread dough under sucrose stress up to 6 g /325 g wheat flour. Results apparent a strong positive correlation in the association between the decrease in fermented bread dough weight and fermentation time. Both variables vary in the same direction because they are dependent on each other. Results suggest that hybrid yeast genotypes are suitable for bread bread-making industry because of their unique properties in fermentation ability and producing high carbon dioxide leading the weight of fermentation bread dough to decrease over time.

High yield co-production of isobutanol and ethanol from switchgrass: experiments, and process synthesis and analysis

Hybrid yeast strain co-produces isobutanol and ethanol at high yields. Reducing hydrolysis enzyme loading and enhancing xylose conversion greatly impact the economic potential of the biorefinery.

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.

Increased volatile thiol release during beer fermentation using constructed interspecies yeast hybrids

Interspecies hybridization has been shown to be a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. Thanks to the popularity of heavily hopped ‘India Pale Ale’-style beers, there is an increased demand from brewers for strains that can boost hop aroma. Here, we explored whether hybridization could be used to construct strains with an enhanced ability to release hop-derived flavours through β-lyase activity, which releases desirable volatile thiols. Wild Saccharomyces strains were shown to possess high β-lyase activity compared to brewing strains, however, they also produced phenolic off-flavours (POF) and showed poor attenuation. To overcome these limitations, interspecies hybrids were constructed by crossing pairs of one of three brewing and one of three wild Saccharomyces strains (S. uvarum and S. eubayanus). Hybrids were screened for fermentation ability and β-lyase activity, and selected hybrids showed improved fermentation and formation of both volatile thiols (4MMP, 3MH and 3MH-acetate) and aroma-active esters compared to the parent strains. Undesirable traits (e.g. POF) could be removed from the hybrid by sporulation. To conclude, it was possible to boost the release of desirable hop-derived thiols in brewing yeast by hybridization with wild yeast. This allows production of beer with boosted hop aroma with less hops (thus improving sustainability issues).