Adaptive Evolution of Wine Yeast Strains

Although the origin of S. cerevisiae is a matter of controversy, its original genome has been subjected to strong selective pressures since its first unconscious use in controlled fermentation processes. Useful phenotypic traits, such as fast growth in sugar-rich media, high alcohol production and tolerance, and good flavor production, which have been selected over billions of generations, have had strong influences on the S. cerevisiae genome (Perez-Ortin et al. 2002). We now will analyze some of the molecular mechanisms that explain this wine yeast adaptation to vinification.

4.2.1 Stress Adaptation

During the alcoholic fermentation, yeast cells are subject to a number of stresses (Attfield 1997), the most important being osmotic and ethanol stresses. Osmotic stress is due to the high sugar content in the must and yeast cells must resist this stress in order to start growing and to carry out the alcoholic fermentation. Ethanol stress is related to the progressive production of this compound throughout vinification. Alcohol is highly toxic to yeast metabolism and growth (Ingram and Buttke 1984) and the cell membrane is the primary target for its action. Also it is important to mention the stress due to glucose starvation, which could take place towards the end of the fermentation stage. Supraoptimal temperatures constitute another kind of stress conditions that can take place during fermentation, although it is not very significant for most vinifications, given the sophisticated temperature control systems currently used in wineries (Fleet and Heard 1992). Similarly, adverse growth conditions, such as high ethanol and acetaldehyde concentrations and oxidative stress due to respiratory metabolism, can be found in the biological ageing of fino sherry wine. Acetaldehyde is a known inhibitor of a wide range of metabolic activities and is more toxic than ethanol (Jones 1990). Response to stress conditions requires the activation of signal transduction pathways, which involves the synthesis of protective molecules, including heat shock proteins. One of these proteins (Hsp104p) has been shown to be responsible for stress tolerance in laboratory yeast strains carrying out respiratory metabolism (Lindquist and Kim 1996), but not for wine yeast strains growing under fermentation conditions in a glucose-rich medium (Carrasco et al. 2001) or for brewery yeast strains (Brosnan et al. 2000). Aranda et al. (2002) analyzed the responses of several S. cerevisiae strains (some of them isolated during the production of fino sherry wine) to several adverse conditions. In strains that are dominant during biological ageing, there was a clear correlation between resistance to ethanol and acetaldehyde, the high induction of HSP genes by these compounds and its presence as the predominant strain in most levels of several "soleras." It is interesting to note that the dominant strains during biological ageing are the less resistant ones and the strains isolated during the alcoholic fermentation are more resistant to the osmotic stress.

4.2.2 Gene Expression Variability in Wine Yeast

The molecular basis of the technological properties of wine yeast strains are still largely unknown. However, the obvious possibility is that the adaptation of these strains to the enological environment is dependent on specific expression profiles of their genomes (for a review see Perez-Ortin et al. 2002). Comparative analyses of gene expression between industrial and nonindustrial strains and between different industrial strains could lead to the identification of genes involved in the fitness of the strains in industrial environments. To date, the study of gene expression during wine fermentation has focused on genes induced in the stationary phase in order to express specific activities at the end of the process (Puig et al. 1996; Puig et al. 2000; Riou et al. 1997).

Due to the different properties of the particular wine strains, it seems of great interest to study real commercial wine strains. Consequently, Puig et al. (1996); Puig and

PĂ©rez-Ortin (2000) determined the levels and expression patterns of several genes during wine microvinifications by using a commercial strain (T73, Lallemand). Genes such as POT1, HSP104, and SSA3, which are expressed late in laboratory culture conditions, are expressed in wine yeast cells only during the first few days in microvinifications. The reason for this could be the very different growth conditions used in microvinification (and therefore in real vinifications) compared to those of laboratory conditions. Vinifications are characterized by very high contents of glucose and fructose (20-25%) compared with the usual laboratory conditions (1-3%), and anaerobic conditions vs highly aerated laboratory conditions (Puig et al. 1996).

The completion of the sequence of the genome of S. cerevisiae allowed the development of tools for the evaluation of the expression of the entire complement of genes encoded in the genome (transcriptome). The DNA microarray hybridization analysis was used to investigate how interesting genes change their expression during a biological process; several attempts have been made with wine yeast (Cavalieri et al. 2000; Backhus et al. 2001; Hauser et al. 2001). The knowledge of genetic features as well as the specific expression profiles wine yeast strains under different growth conditions could help us to understand better the biological process of fermentation at the molecular level and how the gene expression is regulated in relation to changes in the physical and chemical properties of the growth medium.

It is surprising that the genes involved in sulphur (SUL1-2) and ammonia (MEP2) transport (Cavalieri et al. 2000), or that (sulphite efflux, SSU1) involved in sulfite resistance (Hauser et al. 2001), were found to be overexpressed in wine yeast strains. They investigated in great detail the possible mechanisms for regulation of the expression of the SSU1 gene of the T73 wine yeast strain. A rearrangement of the promoter of SSU1 was detected, leading to up-regulation in its expression. This can be interpreted as being fixed through its evolution as a result of the selective pressures imposed by winemaking procedures (Perez-Ortin et al. 2002).

4.2.3 Wine Yeast Genome Evolution

The flexibility of the yeast genome to adapt to externally introduced environmental changes has been demonstrated in several experiments. Thus, it has postulated that an ancient duplication of the entire yeast genome may have been instrumental in its evolutionary adaptation to anaerobic, fermentative growth by providing new and specialized gene functions (Wolfe and Shields 1997). Wine yeast strains have the ability to reorganize their chromosomes during mitotic growth (Longo and Vezinhet 1993). In contrast to laboratory strains, wine yeast strains seem not to remain genetically uniform because of this exacerbated capacity to undergo genomic changes (Pretorius 2000). A reason for this is the ability of many "natural" strains to change their mating type when haploid (homothallism). Using this capability, yeast cells could evolve very quickly by means of three successive steps: sporulation (producing haploid cells thorough meiotic reduction), mating type switching of the daughter cells and conjugation with any of the mothers of the same single-spore colony (Mortimer et al. 1994). This process (called "genome renewal") would produce highly homozygous strains that eliminate deleterious mutations by natural selection. Yeast strains, however, are usually aneuploid (Bakalinsky and Snow 1990; Guijo et al. 1997) and heterozygous for many loci (Barre et al. 1992; Kunkee and Bisson 2000), which is not in agreement with the genome renewal hypothesis (Puig et al. 2000). There are several other ways in which wine strains change over time. Apart from spontaneous mutation, which occurs at comparatively very low rates, Ty-promoted chromosomal translocations (Rachidi et al. 1999), mitotic crossing-over (Seehaus et al. 1985) and gene conversion will provoke faster adaptation to environmental changes (Puig et al. 2000). Extra copies of chromosome XIII, which contains the alcohol dehydrogenase genes ADH2and ADH3, are found in flor wine yeasts, these genes are of special interest to oxidize ethanol (Guijo et al. 1997). Finally, (Perez-Ortin et al. 2002) found that the SSU1-R allele, which confers sulfite resistance to yeast cells, is the product of a reciprocal translocation between chromosomes VIII and XVI due to unequal crossing-over mediated by microhomology between very short sequences on the 5' upstream regions of the SSU1 and ECM34 genes, which put the SSU1 coding region under the control of the ECM34 promoter. They also showed that this translocation is only present in wine yeast strains, suggesting that the use of sulfite as a preservative in wine production over millennia could have favored its selection. This is the first time that a gross chromosomal rearrangement has been shown to be involved in the adaptive evolution of S. cerevisiae.

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