Chiara Nadai

The impact of genomic variability on gene expression in environmental Saccharomyces cerevisiae strains.

Environmental Saccharomyces cerevisiae strains are crucially important, as they represent the large pool from which domesticated industrial yeasts have been selected, and vineyard strains can be considered the genetic reservoir from which industrial wine strains with strong fermentative behaviour are selected. Four vineyard strains with different fermentation performances were chosen from a large collection of strains isolated from Italian vineyards. Their genomes were sequenced to identify how genetic variations influence gene expression during fermentation and to clarify the evolutionary relationship between vineyard isolates and industrial wine strains. RNA sequencing was performed on the four vineyard strains, as well as on the industrial wine yeast strain EC1118 and on the laboratory strain S288c, at two stages of fermentation. We showed that there was a large gene cluster with variable promoter regions modifying gene expression in the strains. Our results indicate that it is the evolvability of the yeast promoter regions, rather than structural variations or strain-specific genes, that is the main cause of the differences in gene expression. This promoter variability, determined by variable tandem repeats and a high number of single-nucleotide polymorphisms together with 49 differentially expressed transcription factors, explained the strong phenotypic differences in the strains.

Oxidative stress response and nitrogen utilization are strongly variable in Saccharomyces cerevisiae wine strains with different fermentation performances

We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization.

Different mechanisms of resistance modulate sulfite tolerance in wine yeasts

From a technological point of view, yeast resistance to sulfite is of great interest and represents an important technological character for winemaking. Several mechanisms are involved, and strain-dependent strategies to obtain SO2 resistance can deeply influence wine quality, although this choice is less relevant in determining the technological performance of the strain during fermentation. In this study, to better understand the strain-specific mechanisms of resistance, 11 Saccharomyces cerevisiae strains, whose genomes have been previously sequenced, were selected. Their attitude towards sulfites, in terms of resistance and production, was evaluated, and RNA-sequencing of four selected strains was performed during fermentation process in synthetic grape must in the presence of SO2. Results demonstrated that at molecular level, the physical effect of SO2 triggered multiple stress responses in the cell and high tolerance to general enological stressing condition increased SO2 resistance. Adaptation mechanism due to high basal gene expression level rather than specific gene induction in the presence of sulfite seemed to be responsible in modulating strain resistance. This mechanism involved higher basal gene expression level of specific cell wall proteins, enzymes for lipid biosynthesis, and enzymes directly involved in SO2 assimilation pathway and efflux.

Biocontrol Ability and Action Mechanism of Starmerella bacillaris (Synonym Candida zemplinina) Isolated from Wine Musts against Gray Mold Disease Agent Botrytis cinerea on Grape and Their Effects on Alcoholic Fermentation.

Gray mold is one of the most important diseases of grapevine in temperate climates. This plant pathogen affects plant growth and reduces wine quality. The use of yeasts as biocontrol agents to apply in the vineyard have been investigated in recent years as an alternative to agrochemicals. In this work, fermenting musts obtained from overripe grape berries, therefore more susceptible to infection by fungal pathogens such as Botrytis cinerea, were considered for the selection of yeasts carrying antifungal activity. Thirty-six isolates were identified as Starmerella bacillaris, a species recently proven to be of enological interest. Among them 14 different strains were studied and antifungal activity against B. cinerea was demonstrated, for the first time, to be present in S. bacillaris species. The production of volatile organic compounds (VOCs), tested in vitro, was found to be the main responsible of S. bacillaris antifungal effects. All the strains were able to reduce B. cinerea decay on wounded grape berries artificially inoculated with gray mold. The colonization level of wound was very high reaching, after 5 days, a concentration of 106 cells per ml of grape juice obtained after berry crushing. At this cell concentration S. bacillaris strains were used to ferment synthetic and natural musts. The sequential yeast inoculation, performed by adding S. cerevisiae 48 h after S. bacillaris, was needed to complete sugar consumption and determined a significant increase in glicerol content and a reduction of ethanol and acetic acid concentrations. The high wound colonization ability, found in this work, together with the propensity to colonize grape berry and the interesting enological traits possessed by the selected S. bacillaris strains allow the use of this yeast as biocontrol agent on vine and grape berries with possible positive effects on must fermentation, although the presence of S. cerevisiae is needed to complete the fermentation process. This work introduces new possibilities in wine yeast selection programs in order to identify innovative wine yeasts that are simultaneously antifungal agents in vineyards and alternative wine starters for grape must fermentation and open new perspective to a more integrated strategy for increasing wine quality.

Selection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in the presence of sulfite.

Sulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions. In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite. Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must.

Aptitude of Saccharomyces yeasts to ferment unripe grapes harvested during cluster thinning for reducing alcohol content of wine.

Among the viticultural techniques developed to obtain wine with reduced alcohol content, the use of unripe grapes with low sugar and high malic acid concentration, harvested at cluster thinning, was recently explored. So far, no studies have evaluated the fermentation performances of Saccharomyces in unripe grape musts, in terms of fermentation ability and reducing malic acid contents, to improve the quality of this low-alcohol beverage. In this work, we evaluated 24 S. cerevisiae strains isolated from Italian and Croatian vineyards with different fermentation aptitudes. Moreover, four S. paradoxus were considered, as previous works demonstrated that strains belonging to this species were able to degrade high malic acid amounts in standard musts. The industrial strain S. cerevisiae 71B was added as reference. Sugar and malic acid contents were modified in synthetic musts in order to understand the effect of their concentrations on alcoholic fermentation and malic acid degradation. S. cerevisiae fermentation performances improved when glucose concentration decreased and malic acid level increased. The conditions that simulate unripe grape must, i.e. low glucose and high malic acid content were found to enhance S. cerevisiae ability to degrade malic acid. On the contrary, S. paradoxus strains were able to degrade high amounts of malic acid only in conditions that resemble ripe grape must, i.e. high glucose and low malic acid concentration. In fermentation trials when low glucose concentrations were used, at high malic acid levels S. cerevisiae strains produced higher glycerol than at low malic acid condition. Malic acid degradation ability, tested on the best performing S. cerevisiae strains, was enhanced in fermentation trials when unripe grape must was used.

The Geographic Distribution of Saccharomyces cerevisiae Isolates within three Italian Neighboring Winemaking Regions Reveals Strong Differences in Yeast Abundance, Genetic Diversity and Industrial Strain Dissemination

In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore, the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over 3 years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota.

New rapid PCR protocol based on high-resolution melting analysis to identify Saccharomyces cerevisiae and other species within its genus

Selection projects aiming at the identification of new Saccharomyces strains are always on going as the use of the suitable yeast can strongly improve fermented food production, particularly winemaking. They are mainly targeted on Saccharomyces cerevisiae, but other species in the Saccharomyces genus are of interest. For this reason, more and more efficient molecular techniques for yeast identification able to accelerate yeast selection process are always needed. Among the Saccharomyces genus, four yeasts are widespread in natural environments: S. cerevisiae; S. uvarum; S. kudriavzevii and S. paradoxus. Therefore, among the Saccharomyces species, their discrimination is of great interest.

Draft Genome Sequence of the Yeast Starmerella bacillaris (syn., Candidazemplinina) FRI751 Isolated from Fermenting Must of Dried Raboso Grapes

ABSTRACT Starmerella bacillaris is an ascomycetous yeast commonly present in enological environments. Here, we report the first draft genome sequence of S. bacillaris FRI751, which will facilitate the study of the characteristics of this interesting enological yeast.

The role of nitrogen uptake on the competition ability of three vineyard Saccharomyces cerevisiae strains

Three vineyard strains of Saccharomyces cerevisiae, P301.4, P304.4 and P254.12, were assayed in comparison with a commercial industrial strain, QA23. The aim was to understand if nitrogen availability could influence strain competition ability during must fermentation. Pairwise-strain fermentations and co-fermentations with the simultaneous presence of the four strains were performed in synthetic musts at two nitrogen levels: control nitrogen condition (CNC) that assured the suitable assimilable nitrogen amount required by the yeast strains to complete the fermentation and low nitrogen condition (LNC) where nitrogen is present at very low level. Results suggested a strong involvement of nitrogen availability, as the frequency in must of the vineyard strains, respect to QA23, in LNC was always higher than that found in CNC. Moreover, in CNC only strain P304.4 reached the same strain frequency as QA23. P304.4 competition ability increased during the fermentation, indicating better performance when nitrogen availability was dropping down. P301.4 was the only strain sensitive to QA23 killer toxin. In CNC, when it was co-inoculated with the industrial strain QA23, P301.4 was never detected. In LNC, P301.4 after 12h accounted for 10% of the total population. This percentage increased after 48h (20%). Single-strain fermentations were also run in both conditions and the nitrogen metabolism further analyzed. Fermentation kinetics, ammonium and amino-acid consumptions and the expression of genes under nitrogen catabolite repression evidenced that vineyard yeasts, and particularly strain P304.4, had higher nitrogen assimilation rate than the commercial control. In conclusion, the high nitrogen assimilation rate seems to be an additional strategy that allowed vineyard yeasts successful competition during the growth in grape musts.