Pascal Ribéreau-Gayon - Handbook of Enology - Volume 1

TABLE 1.4 Physiological Races of S. cerevisiae Grouped Under a Single SpeciesS. cerevisiae by Yarrow and Nakase (1975)

Ga, D‐galactose; Su, sucrose; Ma, maltose; Ra, raffinose; Me, melibiose; St, soluble starch.

TABLE 1.5 DNA/DNA Reassociation Percentages Between the Four SpeciesBelonging to the Saccharomyces Genus in the Strict Sense (Vaughan Martini and Martini, 1987)

Saccharomyces pastorianus replaces the former name Saccharomyces carlsbergensis , given to brewery yeast strains, used for low‐temperature fermentations (lager) and until then included in the cerevisiae species. The placement of S. bayanus, S. uvarum , and S. pastorianus within the Saccharomyces genus was the subject of controversies among yeast scientists for years. The latest works, based on DNA sequencing and the recent discovery of the S. eubayanus species, have definitively determined the taxonomic positions of S. bayanus and S. pastorianus ( Tables 1.4and 1.5; Nguyen and Gaillardin, 2005; Libkind et al ., 2011; Nguyen and Boekhout, 2017). It has been clearly established that S. bayanus and S. pastorianus refer to hybrid individuals, composed of S. eubayanus , S. uvarum , and S. cerevisiae genomes. On the other hand, S. uvarum and S. eubayanus are considered as being of genetically pure lineage.

Thus, S. bayanus is now considered a distinct hybrid species of S. cerevisiae and S. uvarum according to taxonomists. Nevertheless, enologists and winemakers use the name of bayanus (ex oviformis) to designate a physiological race of S. cerevisiae that does not ferment galactose. It possesses a greater resistance to ethanol than S. cerevisiae . The implementation of molecular biology methods, based on DNA analysis, has helped establish the position of the winemaking yeasts formerly designated as var. bayanus and var. uvarum in agreement with the current taxonomy.

For some 30 years, fragment amplification of the genome by the polymerase chain reaction (PCR) has provided an excellent discrimination tool for winemaking yeast species.

Since its discovery by Saiki et al . (1985), PCR has often been used to identify different plant and bacteria species. This technique consists in enzymatically amplifying one or several gene fragments in vitro . The reaction is based on the hybridization of two oligonucleotides that frame a target region on a double strand of DNA or template. These oligonucleotides have different sequences and are complementary to the DNA sequences that frame the strand being amplified. Figure 1.21illustrates the various stages of the amplification process. The DNA is first denatured at a high temperature (95°C). The reaction mixture is then cooled to a temperature between 37 and 55°C, enabling the hybridization of these oligonucleotides on the denatured strands. The strands serve as primers from which a DNA polymerase enables the step‐by‐step addition of deoxyribonucleotide units in the 5′–3′ direction. The DNA polymerase ( Figure 1.22) requires four deoxyribonucleoside‐5Π‐triphosphates (dATP, dGTP, dTTP, and dCTP). A phosphodiester bond is formed between the 3Π–OH end of the primer and the innermost phosphorus of the activated deoxyribonucleoside. Pyrophosphate is thus released. The newly synthesized strand is elongated on the template. A heat‐resistant enzyme, TAQ DNA polymerase, comes from the heat‐resistant bacteria Thermus aquaticus . It is used to conduct a large number of amplification cycles (25–40) in vitro without having to add DNA polymerase after each denaturation. In this manner, the DNA fragment amplified during the first cycle serves as the template for the following cycles. In consequence, each successive cycle doubles the target DNA fragment, which is thus amplified by a factor of 10 5to 10 6during 25–30 amplification cycles.

FIGURE 1.21 Principle of the polymerization chain reaction (PCR).

Hansen and Kielland‐Brandt (1994) proposed MET 2 gene PCR amplification to differentiate between S. cerevisiae and S. uvarum (formerly S. bayanus ), when working on strain types of these two species. This gene, which codes for the synthesis of homoserine acetyltransferase, has different sequences in the two species. Part of the gene is initially amplified by using two complementary oligonucleotides of the sequences bordering the fragment to be amplified. The fragment obtained (about 600 bp) is the same size for typical strains of the S. cerevisiae and S. uvarum species. Different restriction endonucleases, which recognize certain specific DNA sequences, then digest the amplified fragment. Figure 1.23gives an example of the mode of action of the Eco R1 restriction endonuclease. This enzyme recognizes the base sequence GAATTC and cuts at the location indicated by the arrows. Electrophoresis is used to separate the obtained fragments. As a result, restriction fragment length polymorphism (RFLP) can be assessed. The restriction profiles obtained differ between S. cerevisiae and S. uvarum .