Yeast Plasma Membrane Fungal Oligopeptide Transporters Display Distinct Substrate Preferences despite Their High Sequence Identity

Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation.

Aromatic Higher Alcohols in Wine: Implication on Aroma and Palate Attributes during Chardonnay Aging

The higher alcohols 2-phenylethanol, tryptophol, and tyrosol are a group of yeast-derived compounds that have been shown to affect the aroma and flavour of fermented beverages. Five variants of the industrial wine strain AWRI796, previously isolated due to their elevated production of the ‘rose-like aroma’ compound 2-phenylethanol, were characterised during pilot-scale fermentation of a Chardonnay juice. We show that these variants not only increase the concentration of 2-phenylethanol but also modulate the formation of the higher alcohols tryptophol, tyrosol, and methionol, as well as other volatile sulfur compounds derived from methionine, highlighting the connections between yeast nitrogen and sulfur metabolism during fermentation. We also investigate the development of these compounds during wine storage, focusing on the sulfonation of tryptophol. Finally, the sensory properties of wines produced using these strains were quantified at two time points, unravelling differences produced by biologically modulating higher alcohols and the dynamic changes in wine flavour over aging.

Heat Shock Improves Random Spore Analysis in Diverse Strains of Saccharomyces cerevisiae

Random spore analysis (RSA) is a classic method in yeast genetics that allows high-throughput purification of recombinant haploid spores following specific crosses. RSA typically involves a number of steps to induce sporulation, purge vegetative cells that fail to sporulate, and disrupt the ascus walls of sporulated cells to release haploid spores. These steps generally require expensive chemicals and/or enzymes that kill diploid cells but have few effects on spores. In the fission yeast Schizosaccharomcyes pombe, heat shock has been reported as an effective addition to RSA protocols, but to our knowledge heat shock has not been used for this purpose in the budding yeast Saccharomyces cerevisiae. Here, we evaluate the effects of heat shock on vegetative and sporulated cultures of four diverse yeast strains: a European wine strain (DBVPG6765), a Japanese sake strain (Y12), a West African palm wine strain (DBVPG6044) and a North American strain isolated from the soil beneath an oak tree (YPS128). We characterize this phenotype under multiple combinations of temperature and incubation time, and find specific conditions that lead to the exclusion of vegetative cells and an enrichment in spores, which differ by strain. We also collected genome sequence data from a recombinant population that experienced multiple rounds of RSA, including one round with a heat shock treatment. These data suggest that when incorporated into an RSA protocol, heat shock leads to increased genetic diversity among the cells that survive and mate. Ultimately, our work provides evidence that short heat treatments can improve existing RSA protocols, though in a strain-specific manner. This result informs applications of high-throughput RSA protocols, such as QTL mapping and experimental evolution research.

Mezcal as a Novel Source of Mixed Yeasts Inocula for Wine Fermentation

Mezcal yeasts were evaluated for their potential as grape-juice fermenters, characterizing their fermentation performance, both in terms of primary and volatile metabolites. Experiments were first carried-out in a semi-synthetic medium and then on grape juice, and population dynamics of the chosen mixed inoculum was assessed in grape juice. Accordingly, we initially tested 24 mezcal yeasts belonging to ten different species, and chose those that were more productive and stress tolerant for the mixed (dual) inoculum, having a final selection of three Saccharomyces cerevisiae strains (plus Fermichamp, a commercial wine strain) and three non-Saccharomyces strains, belonging to Kluyveromyces marxianus, Torulaspora delbrueckii, and Zygosaccharomyces bailii species. For the combination S. cerevisiae/T. delbrueckii (Sc/Td) mixed inoculum, we observed increasing isoamyl alcohol and phenyl ethyl acetate concentrations, as compared with the use of individual Saccharomyces strains, which resulted in a fruitier aroma profile. Alcohol final concentration was in average lower for the Sc/Td inoculum (fermentation power, FP, 13.6) as compared with the individual mezcal Saccharomyces strains (FP 14.3), and it was the highest when Td was co-cultured with the commercial strain (FP 14.6). Overall, our results show the feasibility of using yeasts isolated from mezcal as a novel source of inoculum for wine-type fermentation.

Metabolic Engineering of Wine Strains of Saccharomyces cerevisiae

Modern industrial winemaking is based on the use of starter cultures of specialized wine strains of Saccharomyces cerevisiae yeast. Commercial wine strains have a number of advantages over natural isolates, and it is their use that guarantees the stability and reproducibility of industrial winemaking technologies. For the highly competitive wine market with new demands for improved wine quality, it has become increasingly critical to develop new wine strains and winemaking technologies. Novel opportunities for precise wine strain engineering based on detailed knowledge of the molecular nature of a particular trait or phenotype have recently emerged due to the rapid progress in genomic and “postgenomic” studies with wine yeast strains. The review summarizes the current achievements of the metabolic engineering of wine yeast, the results of recent studies and the prospects for the application of genomic editing technologies for improving wine S. cerevisiae strains.

Sequential Non-Saccharomyces and Saccharomyces cerevisiae Fermentations to Reduce the Alcohol Content in Wine

Over the last decades, the average alcohol content of wine has increased due to climate change and consumer preferences for particular wine styles that resulted in increased grape sugar levels at harvest. Therefore, alcohol reduction is a current challenge in the winemaking industry. Among several strategies under study, the use of non-conventional yeasts in combination with Saccharomyces cerevisiae plays an important role for lowering ethanol production in wines nowadays. In the present work, 33 native non-Saccharomyces strains were assayed in sequential culture with a S. cerevisiae wine strain to determine their potential for reducing the alcohol content in Malvar white wines. Four of the non-Saccharomyces strains (Wickerhamomyces anomalus 21A-5C, Meyerozyma guilliermondii CLI 1217, and two Metschnikowia pulcherrima (CLI 68 and CLI 460)) studied in sequential combination with S. cerevisiae CLI 889 were best able to produce dry wines with decreased alcohol proportion in comparison with one that was inoculated only with S. cerevisiae. These sequential fermentations produced wines with between 0.8% (v/v) and 1.3% (v/v) lower ethanol concentrations in Malvar wines, showing significant differences compared with the control. In addition, these combinations provided favorable oenological characteristics to wines such as high glycerol proportion, volatile higher alcohols, and esters with fruity and sweet character.

Autophagic Proteome in Two Saccharomyces cerevisiae Strains during Second Fermentation for Sparkling Wine Elaboration

A correlation between autophagy and autolysis has been proposed in order to accelerate the acquisition of wine organoleptic properties during sparkling wine elaboration. In this context, a proteomic analysis was carried out in two industrial Saccharomyces cerevisiae strains (P29, conventional sparkling wine strain and G1, implicated in sherry wine elaboration) with the aim of studying the autophagy-related proteome and comparing the effect of CO2 overpressure during sparkling wine elaboration. In general, a detrimental effect of pressure and second fermentation development on autophagy-related proteome was observed in both strains, although it was more pronounced in flor yeast strain G1. Proteins mainly involved in autophagy regulation and autophagosome formation in flor yeast G1, and those required for vesicle nucleation and expansion in P29 strain, highlighted in sealed bottle. Proteins Sec2 and Sec18 were detected 3-fold under pressure conditions in P29 and G1 strains, respectively. Moreover, ‘fingerprinting’ obtained from multivariate data analysis established differences in autophagy-related proteome between strains and conditions. Further research is needed to achieve more solid conclusions and design strategies to promote autophagy for an accelerated autolysis, thus reducing cost and time production, as well as acquisition of good organoleptic properties.

First Proteomic Approach to Identify Cell Death Biomarkers in Wine Yeasts during Sparkling Wine Production

Apoptosis and later autolysis are biological processes which take place in Saccharomyces cerevisiae during industrial fermentation processes, which involve costly and time-consuming aging periods. Therefore, the identification of potential cell death biomarkers can contribute to the creation of a long-term strategy in order to improve and accelerate the winemaking process. Here, we performed a proteomic analysis based on the detection of possible apoptosis and autolysis protein biomarkers in two industrial yeast strains commonly used in post-fermentative processes (sparkling wine secondary fermentation and biological aging) under typical sparkling wine elaboration conditions. Pressure had a negatively effect on viability for flor yeast, whereas the sparkling wine strain seems to be more adapted to these conditions. Flor yeast strain experienced an increase in content of apoptosis-related proteins, glucanases and vacuolar proteases at the first month of aging. Significant correlations between viability and apoptosis proteins were established in both yeast strains. Multivariate analysis based on the proteome of each process allowed to distinguish among samples and strains. The proteomic profile obtained in this study could provide useful information on the selection of wine strains and yeast behavior during sparkling wine elaboration. Additionally, the use of flor yeasts for sparkling wine improvement and elaboration is proposed.