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Hybrid nature of lager yeast

The physiology of lager yeasts differs fundamentally from the physiology of other brewing yeasts (Barnett, 2000; Pasteur, 1876; Rees, 1870). One of the most peculiar differences is the inability of S. pastorianus to sporulate and form viable spores, a property that is still present in many ale-type (and other S. cerevisiae) yeast strains (Anderson and Martin, 1975; Kodama et al., 2006; Snoek et al., 2015; Steensels et al., 2014). This inability to form viable offspring is a trait typically encountered in interspecific cross-breeding, e.g. mules (horse x donkey) or ligers (lion x tiger). Indeed, early genetic analysis showed that S. pastorianus harboured genetic material of (at least) two different species, and was thus not a clean yeast lineage, but rather the result of a hybridization event between S. cerevisiae and another (non-cerevisiae) Saccharomyces species. The first molecular evidence ofthe hybrid nature oflager yeast was obtained by a technique called kar-medi- ated single chromosome transfer (Nilsson-Tillgren et al., 1981), and revealed that the chromosomes of lager yeasts could be divided into three types: (i) homologous (cerevisiae-like) chromosomes, (ii) homeologous (non-cerevisiae like) chromosomes, and (iii) mosaic chromosomes, i.e. chromosomes composed of both homologous and homeologous segments. Later, these findings were confirmed by several DNA hybridization experiments (e.g. by using southern blot or S. cerevisiae-specific gene arrays) (Dunn and Sherlock, 2008; Paul Casey, 1986; Tamai et al., 1998; Yamagishi and Ogata, 1999).

By comparing the DNA sequence of 11 independent loci, Dunn and Sherlock further suggested that the S. cerevisiae parent of S. pastorianus was closely related to ale-type S. cerevisiae strains, and not to wild isolates or strains used in other fermentation industries (Dunn and Sherlock, 2008). This result was in line with the previous work in which allelic variation in 12 microsatellite loci of 651 diverse S. cerevisiae and 15 S. pastorianus strains was investigated (Legras et al., 2007).

The first research suggesting a potential origin of the non-cerevisiae part of the lager genome was published in 1985 (Vaughan Martini and Kurtzman, 1985). Using DNA-DNA hybridization methods, the authors revealed a similarity of72% between the non-cerevisiae moiety of the lager yeast CBS1513 (at that time classified as S. carlsbergensis) and S. bayanus, a cold tolerant species commonly encountered in wine fermentations. This finding was later confirmed by PCR/RFLP analysis of 48 genes of the same lager yeast strain (Rainieri et al., 2006). Interestingly, this hypothesis was later questioned when it was established that part of the S. pastorianus MET2 gene showed a significant sequence difference with the corresponding S. bayanus sequence, suggesting a closely related Saccharomyces species, rather than S. bayanus, as the non-cerevisiae parent (Hansen and Kielland-Brandt, 1994). In 2009, the analysis of the first whole-genome sequence of a lager strain (Weihenstephan 34/70) confirmed this hypothesis. By aligning annotated open reading frames (ORFs) of the lager brewing strain Weihenstephan 34/70 to annotated ORFs of S. cerevisiae S288c and S. bayanus CBS7001 reference genomes, the cerevisiae-type subgenome showed very high similarity to S. cerevisiae S288c (> 99%) as opposed to the bayanus-type subgenome, exhibiting lower sequence identity with S. bayanus CBS7001 (average of 92.7%) (Nakao et al., 2009). Moreover, the authors identified eight genes in the lager yeast genome that were not present in the genomes of the S. cerevisiae yeast S288c nor in the genome of the S. bayanus yeast CBS7001, further indicating that probably a different, yet closely related species is the second lager yeast parent.

In 2011, Argentinean researchers sampling for cryotolerant Saccharomyces yeasts in the Patagonian forest stumbled upon a new Saccharomyces species (Libkind et al., 2011). The draft genome obtained through WGS of this species (dubbed Saccharomyces eubayanus), showed a remarkable high degree of similarity (99.56%) to the non-cerevisiae portion of the lager yeast genome, indicating that this species is very likely the missing link in the S. pastorianus origin. The authors further suggest a possible scenario where the initial hybridization event between a diploid S. cerevisiae cell and a diploid S. eubayanus cell gave rise to an allotetraploid hybrid (the original S. pastorianus strain), which was subsequently subjected to extensive genome rearrangement and mitotic recombination, resulting in loss of heterozygosity and recombinant chimeric chromosomes. Since these adaptations occurred in the highly selective and man-made environment of (lager) beer fermentation, they considered this the ‘domestication' of lager yeasts (Fig. 6.2). However, several questions still remain unanswered. While S. eubayanus was originally discovered in Argentina, it is rather unlikely that lager yeasts originated in South America. Initially, Libkind and coworkers hypothesized that the South American S. eubayanus strain was introduced in Europe via the transatlantic travel between Europe and America (Libkind et al., 2011; Peris et al., 2014). However, while transatlantic travel was only established after Columbus' first voyage to the new world and the first reports of the bottom-fermenting phenotype predate Columbus' travels, lager brewing yeasts most likely originated earlier (probably in the early 1400s in Bavaria) (Bond, 2009; Gibson and Liti, 2014; Hornsey, 2003; Kodama et al., 2006; Smart, 2007). More recent discoveries of genetically distinct lineages of S. eubayanus in other parts of the world (North America and China) suggest that S. eubayanus is not unique to South America (Bing et al., 2014; Peris et al., 2014). Moreover, genetic evidence suggests that the non-cerevisiae moiety of the Weihenstephan 34/70 is more closely related to a S. eubayanus lineage isolated in Tibet (sequencing of 12 loci indicated a 99.82% similarity to the non-cerevisiae moiety of lager yeast of the Asian S. eubayanus isolate, compared to the 99.56% similarity obtained by WGS with the Argentinean isolate described by Libkind et al., 2011), suggesting that this lineage is more likely the direct ancestor of this lager yeast (Bing et al., 2014). Therefore, it is now hypothesized that S. eubayanus made his way to Europe via the 2000-year-old Silk Road. Nevertheless, S. eubayanus is up till now not yet discovered in Europe and it could be that this yeast species occupies a highly specific niche in Europe and still awaits discovery (Gibson and Liti, 2014).

However, the question of when and how S.

The origin of lager yeast

Figure 6.2 The origin of lager yeast. Current hypothesis about the origin of lager yeast involves (1) a contamination of ancient Bavarian fermentation (suggested to be originally conducted by ale yeast S. cerevisiae) by a wild S. eubayanus yeast contaminant; (2) a rare interspecific hybridization event between both yeast species; (3) selection of the interspecific hybrid due to its likely ability to combine the fermentation capacity and ethanol tolerance of its S. cerevisiae parent with the cold tolerance of its S. eubayanus parent; (4-5) followed by genome stabilization and diversification of the ancient S. pastorianus yeast to its current conformation. This whole process happened at least twice in history, giving rise to the two groups of lager yeasts (Saaz and Frohberg types).

eubayanus got into contact with the lager-brewing environment remains open. It is hypothesized that this event took place 500-600 years ago, triggered by a law enforcing brewing at cold temperatures in Bavaria. The non-cerevisiae parent (S. eubayanus) is thought to occur as a wild yeast contaminant around the brewing environment, and being better equipped to withstand the fermentation conditions in cold temperatures compared to the native ale yeasts. However, phenotypic analysis of the first two S. eubayanus strains isolated showed an inferior fermentation profile of S. eubayanus compared to S. cerevisiae (e.g. it is unable to ferment maltotriose, shows a lower ethanol tolerance, and produces an inferior aroma profile), prohibiting its use as starter cultures for lager beer fermentation. An interspecific hybridization event (between the S. eubayanus contaminant and the ale-type S. cerevisiae), that probably happened within the brewing tank, resolved the shortcomings of both species, and resulted in a hybrid species (S. pastorianus) that possessed the combined advantage of cold tolerance and fermentation capacity. This species was therefore able to outcompete its parental strains in lager beer fermentation, and was in this way (unintentionally) selected by the brewers, as traditionally part of the fermented beer was used to inoculate the next batch (Gibson and Liti, 2014).

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