Characterization and improvement of non-conventional Saccharomyces yeasts to solve new challenges in the wine industry: Application of omics technologies

Las levaduras Saccharomyces participan en procesos fermentativos de valor en la industria alimentaria, como la elaboración de vino. La industria del vino utiliza levaduras seleccionadas para realizar la fermentación del vino de forma controlada y para producir un vino homogéneo. En la actualidad, S. cerevisiae es la especie del género Saccharomyces más utilizada en la industria vitivinícola para llevar a cabo el proceso fermentativo, por ser muy resistente al etanol, compuesto tóxico que se produce durante las fermentaciones. Hoy en día, como consecuencia del cambio climático, los vinos finales contienen niveles de alcohol y pH más altos y una acidez total menor a la esperada, características indeseables en el vino. En el género Saccharomyces existen otras especies con interés enológico, como S. kudriavzevii y S. uvarum, y cepas híbridas entre estas cepas y S. cerevisiae. Estas especies de Saccharomyces están ganando popularidad ya que producen vinos más aromáticos, con menor contenido de etanol y mayor glicerol. Sin embargo, son menos tolerantes al etanol que las cepas de S. cerevisiae. Teniendo en cuenta todo lo anterior, en esta tesis se propuso la caracterización y mejora de diferentes cepas de levadura del género Saccharomyces con el objetivo de mejorar su comportamiento durante el proceso de fermentación del vino para obtener un mejor producto: el vino final. Nos hemos centrado en mejorar la tolerancia al etanol, ya que, como hemos mencionado, la presencia de una alta concentración de alcohol durante los procesos de fermentación es un factor de estrés de alto impacto para las levaduras. Además, hemos hecho hincapié en intentar relacionar el diferente comportamiento de las levaduras al etanol con su composición de membrana y con la respuesta del genoma y transcriptoma de las levaduras, mediante el uso de tecnologías ómicas. Las conclusiones obtenidas durante esta tesis doctoral son varias. Por un lado, se encontró que la tolerancia al etanol es variable entre diferentes cepas de S. cerevisiae y que puede correlacionarse con la composición de la membrana y con la respuesta transcriptómica en presencia de etanol de cada cepa. Por otro lado, se determinó que es posible obtener por hibridación una nueva cepa de levadura que mejora dos parentales con diferentes características de interés, en nuestro caso, alta tolerancia al etanol y buena producción de aromas, glicerol y tolerancia a bajas temperaturas. . A su vez, un híbrido obtenido de esta manera se puede adaptar en presencia de alta concentración de sulfito y concentraciones crecientes de etanol, lo que conduce a la selección de diferentes características genómicas que finalmente otorgan una mayor tolerancia a estos factores de estrés. Finalmente, se determinó que la evolución adaptativa de diferentes especies del género Saccharomyces en un medio etanólico provoca diferentes cambios en sus genomas y en la fluidez de sus membranas, revelando así la presencia de una gran variedad de mecanismos evolutivos que pueden actuar en presencia de etanol.Saccharomyces yeasts participate in fermentative processes of value in the food industry, such as the production of wine. The wine industry uses selected yeasts to carry out wine fermentation in a controlled manner and to produce a homogeneous wine. At present, S. cerevisiae is the species within the Saccharomyces genus most widely used in the wine industry as starter, because it is very resistant to ethanol, a toxic compound which is produced during fermentations. Nowadays, as a result of climate change, final wines contain higher alcohol and pH levels and a lower total acidity than expected, which are undesirable characteristics in wine. In the Saccharomyces genus there are other species with enological interest, such as S. kudriavzevii and S. uvarum, and hybrids strains between these strains and S. cerevisiae. These Saccharomyces species are gaining popularity as they produce more aromatic wines, with lower ethanol content and higher glycerol. However, they are less ethanol tolerant than S. cerevisiae strains. Taking into account all of the above, in this thesis we proposed the characterization and improvement of different yeast strains of the Saccharomyces genus with the aim of improving their behavior during the wine fermentation process to obtain a better product: the final wine. We have focused on improving ethanol tolerance, since, as we have mentioned, the presence of a high concentration of alcohol during fermentation processes is a high impact stress factor for yeasts. In addition, we have emphasized on trying to relate the different behavior of yeasts to ethanol with their membrane composition and with the genome and transcriptome response of the yeasts, by using omics technologies. The conclusions obtained during this doctoral thesis are several. On the one hand, it was found that ethanol tolerance is variable among different strains of S. cerevisiae and that it can be correlated with the membrane composition and with the transcriptomic response in the presence of ethanol of each strain. On the other hand, it was determined that it is possible to obtain by hybridization a new yeast strain that improves two parents with different characteristics of interest, in our case, high tolerance to ethanol and good production of aromas, glycerol and tolerance to low temperatures. In turn, a hybrid obtained in this way can be adapted in the presence of high sulfite concentration and increasing ethanol concentrations, which leads to the selection of different genomic characteristics that ultimately provide greater tolerance to these stress factors. Finally, it was determined that the adaptive evolution of different species of the genus Saccharomyces in an ethanol media causes different changes in their genomes and in their membrane fluidity, thus revealing the presence of a great variety of evolutionary mechanisms that can act in the presence of ethanol

Effect of temperature on the prevalence of Saccharomyces non-cerevisiae species against a S. cerevisiae wine strain in wine fermentation: competition, physiological fitness, and influence in final wine composition

Saccharomyces cerevisiae is the main microorganism responsible for the fermentation of wine. Nevertheless, in the last years wineries are facing new challenges due to current market demands and climate change effects on the wine quality. New yeast starters formed by non-conventional Saccharomyces species (such as S. uvarum or S. kudriavzevii) or their hybrids (S. cerevisiae x S. uvarum and S. cerevisiae x S. kudriavzevii) can contribute to solve some of these challenges. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S. cerevisiae can competitively displace other yeast species from wine fermentations, therefore the use of these new starters requires an analysis of their behavior during competition with S. cerevisiae during wine fermentation. In the present study we analyzed the survival capacity of non-cerevisiae strains in competition with S. cerevisiae during fermentation of synthetic wine must at different temperatures. First, we developed a new method, based on QPCR, to quantify the proportion of different Saccharomyces yeasts in mixed cultures. This method was used to assess the effect of competition on the growth fitness. In addition, fermentation kinetics parameters and final wine compositions were also analyzed. We observed that some cryotolerant Saccharomyces yeasts, particularly S. uvarum, seriously compromised S. cerevisiae fitness during competences at lower temperatures, which explains why S. uvarum can replace S. cerevisiae during wine fermentations in European regions with oceanic and continental climates. From an enological point of view, mixed co-cultures between S. cerevisiae and S. paradoxus or S. eubayanus, deteriorated fermentation parameters and the final product composition compared to single S. cerevisiae inoculation. However, in co-inoculated synthetic must in which S. kudriavzevii or S. uvarum coexisted with S. cerevisiae, there were fermentation performance improvements and the final wines contained less ethanol and higher amounts of glycerol. Finally, it is interesting to note that in co-inoculated fermentations, wine strains of S. cerevisiae and S. uvarum performed better than non-wine strains of the same species

Metabolic differences between a wild and a wine strain of Saccharomyces cerevisiae during fermentation unveiled by multi‐omic analysis

Saccharomyces cerevisiae, a widespread yeast present both in the wild and in fermentative processes, like winemaking. During the colonization of these human‐associated fermentative environments, certain strains of S. cerevisiae acquired differential adaptive traits that enhanced their physiological properties to cope with the challenges imposed by these new ecological niches. The advent of omics technologies allowed unveiling some details of the molecular bases responsible for the peculiar traits of S. cerevisiae wine strains. However, the metabolic diversity within yeasts remained poorly explored, in particular that existing between wine and wild strains of S. cerevisiae. For this purpose, we performed a dual transcriptomic and metabolomic comparative analysis between a wild and a wine S. cerevisiae strains during wine fermentations performed at high and low temperatures. By using this approach, we could correlate the differential expression of genes involved in metabolic pathways, such as sulfur, arginine, and thiamine metabolisms, with differences in the amounts of key metabolites that can explain some important differences in the fermentation performance between the wine and wild strains.RM was supported by an FPI grant from the Ministerio de Economía y Competitividad (ref. BES-2016-078202). ML-P was supported by an FPU contract from Ministerio de Ciencia, Innovación y Universidades (ref. FPU15/01775). This work was supported by the Spanish government and FEDER projects RTI2018-093744-B-C31 awarded to AQ, and RTI2018-093744-B-C32 to EB.Peer reviewe

Differential Contribution of the Parental Genomes to a S. cerevisiae × S. uvarum Hybrid, Inferred by Phenomic, Genomic, and Transcriptomic Analyses, at Different Industrial Stress Conditions

In European regions of cold climate, S. uvarum can replace S. cerevisiae in wine fermentations performed at low temperatures. S. uvarum is a cryotolerant yeast that produces more glycerol, less acetic acid and exhibits a better aroma profile. However, this species exhibits a poor ethanol tolerance compared with S. cerevisiae. In the present study, we obtained by rare mating (non-GMO strategy), and a subsequent sporulation, an interspecific S. cerevisiae × S. uvarum spore-derivative hybrid that improves or maintains a combination of parental traits of interest for the wine industry, such as good fermentation performance, increased ethanol tolerance, and high glycerol and aroma productions. Genomic sequencing analysis showed that the artificial spore-derivative hybrid is an allotriploid, which is very common among natural hybrids. Its genome contains one genome copy from the S. uvarum parental genome and two heterozygous copies of the S. cerevisiae parental genome, with the exception of a monosomic S. cerevisiae chromosome III, where the sex-determining MAT locus is located. This genome constitution supports that the original hybrid from which the spore was obtained likely originated by a rare-mating event between a mating-competent S. cerevisiae diploid cell and either a diploid or a haploid S. uvarum cell of the opposite mating type. Moreover, a comparative transcriptomic analysis reveals that each spore-derivative hybrid subgenome is regulating different processes during the fermentation, in which each parental species has demonstrated to be more efficient. Therefore, interactions between the two subgenomes in the spore-derivative hybrid improve those differential species-specific adaptations to the wine fermentation environments, already present in the parental species.ML-P was supported by a FPU contract from Ministerio de Ciencia, Innovación y Universidades (ref. FPU15/01775). This work was supported by projects RTI2018-093744-B-C31 (MCIU/AEI/FEDER, UE) to AQ, RTI2018-093744-B-C32 (MCIU/AEI/FEDER, UE) to EB, and ERACoBioTech MeMBrane project PCI2018-093190 (AEI/FEDER, UE) to AQ.Peer reviewe

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

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

Thermo-adaptive evolution to generate improved Saccharomyces cerevisiae strains for cocoa pulp fermentations

Cocoa pulp fermentation is a consequence of the succession of indigenous yeasts, lactic acid bacteria and acetic acid bacteria that not only produce a diversity of metabolites, but also cause the production of flavour precursors. However, as such spontaneous fermentations are less reproducible and contribute to produce variability, interest in a microbial starter culture is growing that could be used to inoculate cocoa pulp fermentations. This study aimed to generate robust S. cerevisiae strains by thermo-adaptive evolution that could be used in cocoa fermentation. We evolved a cocoa strain in a sugary defined medium at high temperature to improve both fermentation and growth capacity. Moreover, adaptive evolution at high temperature (40 °C) also enabled us to unveil the molecular basis underlying the improved phenotype by analysing the whole genome sequence of the evolved strain. Adaptation to high-temperature conditions occurred at different genomic levels, and promoted aneuploidies, segmental duplication, and SNVs in the evolved strain. The lipid profile analysis of the evolved strain also evidenced changes in the membrane composition that contribute to maintain an appropriate cell membrane state at high temperature. Our work demonstrates that experimental evolution is an effective approach to generate better-adapted yeast strains at high temperature for industrial processes.This work has been financially supported by the Spanish Government through MINECO and FEDER funds (AGL2016-77505-C3-1-R and PCIN-2015-143 grants) awarded to JMG. This study has been carried out as part of the European Project ERA-IB “YeastTempTation”.Peer reviewe

Analysis of lipid composition reveals mechanisms of ethanol tolerance in the model yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyse five representatives with varying ethanol tolerances. The most tolerant strain, AJ4, was dominant in co-culture at 0% and 10% ethanol. Unexpectedly, although it does not have the highest NIC or MIC, MY29 was the dominant strain in co-culture at 6% ethanol, which may be linked to differences in its basal lipidome. Whilst relatively few lipidomic differences were observed between strains, a significantly higher PE concentration was observed in the least tolerant strain, MY26, at 0% and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggests a limited set of membrane compositions that diverse yeast species use to achieve this.This work was supported by projects ERACoBioTech MeMBrane project (UE) to AQ and AG, PCI2018-093190 (AEI/FEDER, UE) to AQ and BBSRC (BB/R02152X/1) to AG.Peer reviewe

Adaptive response to wine selective pressures shapes the genome of a Saccharomyces interspecies hybrid

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII–XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms