Industrially Applicable De Novo Lager Yeast Hybrids with a Unique Genomic Architecture: Creation and Characterization

Abstract

Lager beer is produced by Saccharomyces pastorianus, which is a natural allopolyploid hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus Lager strains are classified into two major groups based largely on genomic composition: group I and group II. Group I strains are allotriploid, whereas group II strains are allotetraploid. A lack of phenotypic diversity in commercial lager strains has led to substantial interest in the reconstitution of de novo allotetraploid lager strains by hybridization of S. cerevisiae and S. eubayanus strains. Such strategies rely on the hybridization of wild S. eubayanus isolates, which carry unacceptable traits for commercial lager beer such as phenolic off flavors and incomplete utilization of carbohydrates. Using an alternative breeding strategy, we have created de novo lager hybrids containing the domesticated S. eubayanus subgenome from an industrial S. pastorianus strain by hybridizing diploid meiotic segregants of this strain to a variety of S. cerevisiae ale strains. Five de novo hybrids were isolated which had fermentation characteristics similar to those of prototypical commercial lager strains but with unique phenotypic variation due to the contributions of the S. cerevisiae parents. Genomic analysis of these de novo lager hybrids identified novel allotetraploid genomes carrying three copies of the S. cerevisiae genome and one copy of the S. eubayanus genome. Most importantly, these hybrids do not possess the negative traits which result from breeding wild S. eubayanus The de novo lager strains produced using industrial S. pastorianus in this study are immediately suitable for industrial lager beer production.IMPORTANCE All lager beer is produced using two related lager yeast types: group I and group II, which are highly similar, resulting in a lack of strain diversity for lager beer production. To date, approaches for generating new lager yeasts have generated strains possessing undesirable brewing characteristics which render them commercially inviable. We have used an alternative approach that circumvents this issue and created new lager strains that are directly suitable for lager beer production. These novel lager strains also possess a unique genomic architecture, which may lead to a better understanding of industrial yeast hybrids. We propose that strains created using our approach be classified as a third group of lager strains (group III). We anticipate that these novel lager strains will be of great industrial relevance and that this technique will be applicable to the creation of additional novel lager strains that will help broaden the diversity in commercial lager beer strains.

Keywords: Saccharomyces cerevisiae; Saccharomyces eubayanus; Saccharomyces pastorianus; fermentation; hybridization; mating; yeasts.

FIG 1

Temperature tolerance of novel hybrids compared to that of group I (CBS1513), group II (W3470), S. eubayanus (CBS12357), and S. cerevisiae (US-05) controls at 7°C, 25°C, and 37°C. Each spot represents a 10-fold serial dilution (10⁴ to 10¹ cells/spot) on YEG medium.

FIG 2

Phenotypic characterization of lager hybrids during beer fermentations performed isothermally at 13°C in 15°P wort in single replicates. Maltose consumption (top left), maltotriose consumption (bottom left), diacetyl production (top right), and cells in suspension (flocculation) (bottom right), shown over a 14-day time course.

FIG 3

Two-dimensional principal-component analysis (PCA) of volatile compound production by novel lager hybrids and control group I (CBS1513) and group II (W3470) S. pastorianus strains.

FIG 4

Visual representation of the volatile compound production of novel lager hybrids and control group I (CBS1513) and group II (W3470) lager strains. Colors represent the range of compound concentrations normalized for each strain from 0, the lowest concentration (blue), to 1, the highest concentration (red).

FIG 5

Overview of the SNP frequency (red) and scaled depth of sequencing (0 to 1, in blue) along each subgenome (S. cerevisiae on the left and S. eubayanus on the right) of novel lager hybrids. Strains are RB-1141, RB-1186, RB-2215, RB-2251, and RB-2403.

FIG 6

Chromosome-wide parental allele frequency (for S. cerevisiae and S. pastorianus). Vertical lines mark the various ratios, blue as a triploid region mark (at 0.33 and 0.66) and green as diploid/tetraploid region mark (at 0.25, 0.5, and 0.75). Strains are RB-1141, RB-1186, RB-2215, RB-2251, and RB-2403.