Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.

Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and contribute to the sensory properties of end-products, the yeast S. cerevisiae invariably dominates the final stages of fermentation. The ability of S. cerevisiae to outcompete other microbial species during alcoholic fermentation processes, such as winemaking, has traditionally been ascribed to its high fermentative power and capacity to withstand the harsh environmental conditions, i.e. high levels of ethanol and organic acids, low pH values, scarce oxygen availability and depletion of certain nutrients. However, in recent years, several studies have raised evidence that S. cerevisiae, beyond its remarkable fitness for alcoholic fermentation, also uses defensive strategies mediated by different mechanisms, such as cell-to-cell contact and secretion of antimicrobial peptides, to combat other microorganisms. In this paper, we review the main physiological features underlying the special aptitude of S. cerevisiae for alcoholic fermentation and discuss the role of microbial interactions in its dominance during alcoholic fermentation, as well as its relevance for winemaking.

The early death of two non-Saccharomyces wine strains (H. guilliermondii and H. uvarum) during mixed fermentations with S. cerevisiae was studied under enological growth conditions. Several microvinifications were performed in synthetic grape juice, either with single non-Saccharomyces or with mixed S. cerevisiae/non-Saccharomyces inocula. In all mixed cultures, non-Saccharomyces yeasts grew together with S. cerevisiae during the first 1-3 days (depending on the initial inoculum concentration) and then, suddenly, non-Saccharomyces cells began to die off, regardless of the ethanol concentrations present. Conversely, in both non-Saccharomyces single cultures the number of viable cells remained high (ranging 10(7)-10(8) CFU ml(-1)) even when cultures reached significant ethanol concentrations (up to 60-70 g l(-1)). Thus, at least for these yeast strains, it seems that ethanol is not the main death-inducing factor. Furthermore, mixed cultures performed with different S. cerevisiae/ H. guilliermondii inoculum ratios (3:1; 1:2; 1:10; 1:100) revealed that H. guilliermondii death increases for higher inoculum ratios. In order to investigate if the nature of the yeast-yeast interaction was related or not with a cell-cell contact-mediated mechanism, cell-free supernatants obtained from 3 and 6 day-old mixed cultures were inoculated with H. guilliermondii pure cultures. Under these conditions, cells still died and much higher death rates were found for the 6 days than for the 3 day-old supernatants. This strongly indicates that one or more toxic compounds produced by S. cerevisiae triggers the early death of the H. guilliermondii cells in mixed cultures with S. cerevisiae. Finally, although it has not been yet possible to identify the nature of the toxic compounds involved in this phenomenon we must emphasise that the S. cerevisiae strain used in the present work is killer sensitive with respect to the classical killer toxins, K1, K2 and K28, whereas the H. guilliermondii and H. uvarum strains are killer neutral.

The nature of the toxic compounds produced by Saccharomyces cerevisiae CCMI 885 that induce the early death of Hanseniaspora guilliermondii during mixed fermentations, as well as their ability to inhibit the growth of other non-Saccharomyces wine-related strains, was investigated. The killing effect of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (<10 kDa), which were detected only when death of H. guilliermondii was already established. Death-inducing supernatants were ultrafiltrated by 10 and 2 kDa membranes, respectively, and the inhibitory effect of those permeates were tested in H. guilliermondii cultures. Results indicated that the (2-10) kDa protein fraction of those supernatants seemed to contain antimicrobial peptides active against H. guilliermondii. Thus, the (2-10) kDa protein fraction was concentrated and its inhibitory effect tested against strains of Kluyveromyces marxianus, Kluyveromyces thermotolerans, Torulaspora delbrueckii and H. guilliermondii. Under the growth conditions used for these tests, the (2-10) kDa protein fraction of S. cerevisiae CCMI 885 supernatants exhibited a fungistatic effect against all the strains and a fungicidal effect against K. marxianus.

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.