Comparative Genomics Between Saccharomyces kudriavzevii and S. cerevisiae Applied to Identify Mechanisms Involved in Adaptation

Yeasts belonging to the Saccharomyces genus play an important role in human-driven fermentations. The species S. cerevisiae has been widely studied because it is the dominant yeast in most fermentations and it has been widely used as a model eukaryotic organism. Recently, other species of the Saccharomyces genus are gaining interest to solve the new challenges that the fermentation industry are facing. One of these species is S. kudriavzevii, which exhibits interesting physiological properties compared to S. cerevisiae, such as a better adaptation to grow at low temperatures, a higher glycerol synthesis and lower ethanol production. The aim of this study is to understand the molecular basis behind these phenotypic differences of biotechnological interest by using a species-based comparative genomics approach. In this work, we sequenced, assembled and annotated two new genomes of S. kudriavzevii. We used a combination of different statistical methods to identify functional divergence, signatures of positive selection and acceleration of substitution rates at specific amino acid sites of proteins in S. kudriavzevii when compared to S. cerevisiae, and vice versa. We provide a list of candidate genes in which positive selection could be acting during the evolution of both S. cerevisiae and S. kudriavzevii clades. Some of them could be related to certain important differences in metabolism previously reported by other authors such us DAL3 and ARO4, involved in nitrogen assimilation and amino acid biosynthesis. In addition, three of those genes (FBA1, ZIP1, and RQC2) showed accelerated evolutionary rates in Sk branch. Finally, genes of the riboflavin biosynthesis were also among those genes with a significant higher rate of nucleotide substitution and those proteins have amino acid positions contributing to functional divergence

The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae

[Background] Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci.[Results] In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions’ adaptive nature.[Conclusions] The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.This work has been financially supported from the Spanish Government through MINECO and FEDER funds (AGL2013-47300-C3-3-R and PCIN-2015-143 grants) and from Generalitat Valenciana through PROMETEOII/2014/042 grant, awarded to JMG. This study has been carried out in the context of the European Project ERA-IB “YeastTempTation” EGR thanks the Spanish government for an FPI grant BES-2011-044498 and MM also thanks the Generalitat Valenciana for a VALi+d ACIF/2015/194 grant. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Interspecific hybridization and aneuploidy as adaptive mechanisms in Saccharomyces yeasts

With the explosion of genome sequencing technologies, mechanisms such as aneuploidy, polyploidy, and hybridization are emerging as being more frequent and relevant in genomes evolution than what was considered earlier. An interesting model to study such mechanisms are Saccharomyces yeasts as much data is available on its genome structure and evolution and they are easily manipulated in laboratory. Yeast from this genus have small and compact genomes that make them an interesting model for genomics studies on eukaryotic organisms. Moreover, hybrids between the different species of the genus are known and relevant for industrial processes. In this doctoral thesis, we aimed to investigate different aspects of the adaptive value of aneuploidy and interspecific hybridization in Saccharomyces. In the first chapter, we were interested in studying what genomic differences were underlying the different ethanol tolerance observed in S. cerevisiae strains. The most interesting genomic change we observed was a shared polysomy of chromosome III in the highest ethanol tolerant strains. We could determine that this correlation between ethanol tolerance and chromosome III copy number also appeared in different background and we confirmed it was an adaptive mechanism on ethanol stress. In the second chapter of this work, we asked how S. cerevisiae x S. kudriavzevii hybrids mate and how this mechanism would influence the genomic and adaptability outcome of these hybrids. We found that rare-mating was the most frequent but not the only mechanism use. As most of the hybrid were triploid and had a diploid heterozygous contribution of S. cerevisiae, this mating mechanism was the most probable. However, two hybrids were tetraploid. One had an extreme reduction of the S. kudriavzevii subgenome and heterozygous S. cerevisiae. This structure seemed more compatible with the outcome of artificial crossing in strain improvement programs. The last strain had a diploid homozygous contribution of each subgenome what indicates a spore to spore mating followed by whole-genome duplication. An interesting point of the ploidy outcome is that it influenced the phenotype of the strain and its evolvability. In chapter 3, we studied in detail the genome of the hybrid VIN7 and showed that its genome was unstable. The instability influenced the stress resistance of the strain suggesting that genomic instability, probably triggered by hybridization, is an important factor of phenotypic variability, and therefore to adaptability. We aimed to investigate the factors that influenced polysomy frequency for each chromosome and found that the least interacting chromosomes and the smallest were the most frequent polysomic ones. The fourth chapter of this work deals with short-term evolution of artificial hybrids between S. cerevisiae and S. kudriavzevii. We wanted to know how the genome content changed in conditions in which the species that form the hybrid had different phenotypes: ethanol, were S. cerevisiae is better fit, and cold temperature, where the best species is S. kudriavzevii. We showed that recombination between subgenome was less frequent than between the two copies of the S. cerevisiae subgenome. This suggests that the distance between the genome sequence influences the recombination rate in hybrid cells. We found that ploidy was strongly influencing transcription and the evolution mechanisms available for the hybrids. The hybrids evolved at cold temperature showed an aneuploidy on chromosome XII. At the transcriptomic level, these were the only ones showing a modification of the global transcriptome after the evolution process. We determined also that the selection on transcriptional rewiring during the evolution occurred at process level instead of genes or subgenomes

Comparative Genomics Between Saccharomyces kudriavzevii and S. cerevisiae Applied to Identify Mechanisms Involved in Adaptation

Yeasts belonging to the Saccharomyces genus play an important role in human-driven fermentations. The species S. cerevisiae has been widely studied because it is the dominant yeast in most fermentations and it has been widely used as a model eukaryotic organism. Recently, other species of the Saccharomyces genus are gaining interest to solve the new challenges that the fermentation industry are facing. One of these species is S. kudriavzevii, which exhibits interesting physiological properties compared to S. cerevisiae, such as a better adaptation to grow at low temperatures, a higher glycerol synthesis and lower ethanol production. The aim of this study is to understand the molecular basis behind these phenotypic differences of biotechnological interest by using a species-based comparative genomics approach. In this work, we sequenced, assembled and annotated two new genomes of S. kudriavzevii. We used a combination of different statistical methods to identify functional divergence, signatures of positive selection and acceleration of substitution rates at specific amino acid sites of proteins in S. kudriavzevii when compared to S. cerevisiae, and vice versa. We provide a list of candidate genes in which positive selection could be acting during the evolution of both S. cerevisiae and S. kudriavzevii clades. Some of them could be related to certain important differences in metabolism previously reported by other authors such us DAL3 and ARO4, involved in nitrogen assimilation and amino acid biosynthesis. In addition, three of those genes (FBA1, ZIP1, and RQC2) showed accelerated evolutionary rates in Sk branch. Finally, genes of the riboflavin biosynthesis were also among those genes with a significant higher rate of nucleotide substitution and those proteins have amino acid positions contributing to functional divergence.This work was funded by grant AGL2015-67504-C3-3-R from the Spanish Government and European Union ERDF-FEDER to EB and by ERA CoBioTech MeMBrane Project PCI2018-093190. LM was supported by the aforementioned grant associated to EB. MM was supported by a Ph.D. student contract ACIF/2015/194 from the Regional Government of Valencia. CT acknowledged a “Juan de la Cierva” postdoctoral contract JCI-2012-14056 from the Spanish Government.Peer reviewe

Genomic instability in an interspecific hybrid of the genus Saccharomyces: a matter of adaptability

Ancient events of polyploidy have been linked to huge evolutionary leaps in the tree of life, while increasing evidence shows that newly established polyploids have adaptive advantages in certain stress conditions compared to their relatives with a lower ploidy. The genus Saccharomyces is a good model for studying such events, as it contains an ancient whole-genome duplication event and many sequenced Saccharomyces cerevisiae are, evolutionary speaking, newly formed polyploids. Many polyploids have unstable genomes and go through large genome erosions; however, it is still unknown what mechanisms govern this reduction. Here, we sequenced and studied the natural S. cerevisiae × Saccharomyces kudriavzevii hybrid strain, VIN7, which was selected for its commercial use in the wine industry. The most singular observation is that its nuclear genome is highly unstable and drastic genomic alterations were observed in only a few generations, leading to a widening of its phenotypic landscape. To better understand what leads to the loss of certain chromosomes in the VIN7 cell population, we looked for genetic features of the genes, such as physical interactions, complex formation, epistatic interactions and stress responding genes, which could have beneficial or detrimental effects on the cell if their dosage is altered by a chromosomal copy number variation. The three chromosomes lost in our VIN7 population showed different patterns, indicating that multiple factors could explain the mechanisms behind the chromosomal loss. However, one common feature for two out of the three chromosomes is that they are among the smallest ones. We hypothesize that small chromosomes alter their copy numbers more frequently as a low number of genes is affected, meaning that it is a by-product of genome instability, which might be the chief driving force of the adaptability and genome architecture of this hybrid.This work was supported by grants RTI2018-093744-B-C31 and -C32 from the Spanish Government and European Union ERDF-FEDER to A.Q. and E.B., respectively. M.M. was supported by a Ph.D. student contract (ACIF/2015/194) from the Regional Government of Valencia. C.I. acknowledges the Spanish Government for its Ministerio de Ciencia e Innovación studentship (FPI). C.T. was supported by an EMBO long-term fellowship (ALTF 730–2011) and a ‘Juan de la Cierva’ postdoctoral contract (JCI-2012–14056) from the Spanish Ministerio de Economía y Competitividad.Peer reviewe

Genome structure reveals the diversity of mating mechanisms in Saccharomyces cerevisiae x Saccharomyces kudriavzevii hybrids, and the genomic instability that promotes phenotypic diversity

Interspecific hybridization has played an important role in the evolution of eukaryotic organisms by favouring genetic interchange between divergent lineages to generate new phenotypic diversity involved in the adaptation to new environments. This way, hybridization between Saccharomyces species, involving the fusion between their metabolic capabilities, is a recurrent adaptive strategy in industrial environments. In the present study, whole-genome sequences of natural hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii were obtained to unveil the mechanisms involved in the origin and evolution of hybrids, as well as the ecological and geographic contexts in which spontaneous hybridization and hybrid persistence take place. Although Saccharomyces species can mate using different mechanisms, we concluded that rare-mating is the most commonly used, but other mechanisms were also observed in specific hybrids. The preponderance of rare-mating was confirmed by performing artificial hybridization experiments. The mechanism used to mate determines the genomic structure of the hybrid and its final evolutionary outcome. The evolution and adaptability of the hybrids are triggered by genomic instability, resulting in a wide diversity of genomic rearrangements. Some of these rearrangements could be adaptive under the stressful conditions of the industrial environment.This work was supported by grants RTI2018-093744-B-C31 and -C32 from the Spanish Government and European Union ERDF-FEDER to A.Q. and E.B., respectively. M.M. was supported by a Ph.D. student contract ACIF/2015/194 from the Regional Government of Valencia. GOT was supported by doctoral scholarship 176060 from CONACYT, Mexican Government. C.T. acknowledges a ‘Juan de la Cierva’ postdoctoral contract JCI-2012–14056 from the Spanish Government.Peer reviewe

Aneuploidy and ethanol tolerance in Saccharomyces cerevisiae

Response to environmental stresses is a key factor for microbial organism growth. One of the major stresses for yeasts in fermentative environments is ethanol. Saccharomyces cerevisiae is the most tolerant species in its genus, but intraspecific ethanol-tolerance variation exists. Although, much effort has been done in the last years to discover evolutionary paths to improve ethanol tolerance, this phenotype is still hardly understood. Here, we selected five strains with different ethanol tolerances, and used comparative genomics to determine the main factors that can explain these phenotypic differences. Surprisingly, the main genomic feature, shared only by the highest ethanol-tolerant strains, was a polysomic chromosome III. Transcriptomic data point out that chromosome III is important for the ethanol stress response, and this aneuploidy can be an advantage to respond rapidly to ethanol stress. We found that chromosome III copy numbers also explain differences in other strains. We show that removing the extra chromosome III copy in an ethanol-tolerant strain, returning to euploidy, strongly compromises its tolerance. Chromosome III aneuploidy appears frequently in ethanol-tolerance evolution experiments, and here, we show that aneuploidy is also used by natural strains to enhance their ethanol tolerance.This work was funded by grant AGL2015-67504-C3-R from the Spanish Government and European Union ERDF-FEDER to EB. MM was supported by a Ph.D. student contract ACIF/2015/194 from the Regional Government of Valencia. ML-P acknowledges a Ph.D. student FPU contract FPU15/01775 from the Spanish Government. LM and RP-T are supported by the aforementioned grant AGL2015-67504-C3-R. CT acknowledges a “Juan de la Cierva” postdoctoral contract JCI-2012-14056 from the Spanish Government.Peer reviewe

Additional file 6: Table S2. of The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae

List of genes used in the RH analysis with the BY4741 strain that are present in the subtelomeric regions and are not essential. (XLSX 13 kb

Comparative genomics of infective Saccharomyces cerevisiae strains reveals their food origin

Fungal infections are less studied than viral or bacterial infections and often more difficult to treat. Saccharomyces cerevisiae is usually identified as an innocuous human-friendly yeast; however, this yeast can be responsible for infections mainly in immunosuppressed individuals. S. cerevisiae is a relevant organism widely used in the food industry. Therefore, the study of food yeasts as the source of clinical infection is becoming a pivotal question for food safety. In this study, we demonstrate that S. cerevisiae strains cause infections to spread mostly from food environments. Phylogenetic analysis, genome structure analysis, and phenotypic characterization showed that the key sources of the infective strains are food products, such as bread and probiotic supplements. We observed that the adaptation to host infection can drive important phenotypic and genomic changes in these strains that could be good markers to determine the source of infection. These conclusions add pivotal evidence to reinforce the need for surveillance of food-related S. cerevisiae strains as potential opportunistic pathogens.This work was supported by the Fundación Areces awarded to AQ. CP is supported by a CSIC Fellowship (JAEIntro). Thanks to the Spanish government MCIN/AEI to the Center of Excellence Accreditation Severo Ochoa CEX2021-001189-S.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S)Peer reviewe

Additional file 3: Figure S2. of The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae

Workflow of populations’ selection and sequencing. Cells were grown in complete media (YPD) and synthetic must (SM), and were incubated at either optimum temperature (28 °C) or low temperature (15 °C) until the stationary phase was reached. At this time, the volume required to inoculate at an OD of 0.2 was re-inoculated into 60 mL of fresh medium. The experiment was carried out 8 times after which the selected populations were analyzed and sequenced. (PDF 43 kb