Understanding brewing trait inheritance in de novo Lager yeast hybrids

Abstract

Hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus resulted in the emergence of S. pastorianus, a crucial yeast for lager fermentation. However, our understanding of hybridization success and hybrid vigor between these two species remains limited due to the scarcity of S. eubayanus parental strains. Here, we explore hybridization success and the impact of hybridization on fermentation performance and volatile compound profiles in newly formed lager hybrids. By selecting parental candidates spanning a diverse array of lineages from both species, we reveal that the Beer and PB-2 lineages exhibit high rates of hybridization success in S. cerevisiae and S. eubayanus, respectively. Polyploid hybrids were generated through a spontaneous diploid hybridization technique (rare-mating), revealing a prevalence of triploids and diploids over tetraploids. Despite the absence of heterosis in fermentative capacity, hybrids displayed phenotypic variability, notably influenced by maltotriose consumption. Interestingly, ploidy levels did not significantly correlate with fermentative capacity, although triploids exhibited greater phenotypic variability. The S. cerevisiae parental lineages primarily influenced volatile compound profiles, with significant differences in aroma production. Interestingly, hybrids emerging from the Beer S. cerevisiae parental lineages exhibited a volatile compound profile resembling the corresponding S. eubayanus parent. This pattern may result from the dominant inheritance of the S. eubayanus aroma profile, as suggested by the over-expression of genes related to alcohol metabolism and acetate synthesis in hybrids including the Beer S. cerevisiae lineage. Our findings suggest complex interactions between parental lineages and hybridization outcomes, highlighting the potential for creating yeasts with distinct brewing traits through hybridization strategies.

Importance: Our study investigates the principles of lager yeast hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus. This process gave rise to the lager yeast Saccharomyces pastorianus. By examining how these novel hybrids perform during fermentation and the aromas they produce, we uncover the genetic bases of brewing trait inheritance. We successfully generated polyploid hybrids using diverse strains and lineages from both parent species, predominantly triploids and diploids. Although these hybrids did not show improved fermentation capacity, they exhibited varied traits, especially in utilizing maltotriose, a key sugar in brewing. Remarkably, the aroma profiles of these hybrids were primarily influenced by the S. cerevisiae parent, with Beer lineage hybrids adopting aroma characteristics from their S. eubayanus parent. These insights reveal the complex genetic interactions in hybrid yeasts, opening new possibilities for crafting unique brewing yeasts with desirable traits.

Keywords: RNA-seq; beer; hybridization; lager; volatile compounds; yeast.

Fig 1

Hybridization between S. cerevisiae and S. eubayanus strains. (A) Total CO₂ production (g/L) in (A) S. eubayanus and (B) S. cerevisiae strains under 12 °P wort from a broad range of lineages in both species. Black dots depict mean values between the three replicates. (C) Hybridization strategy to generate de novo polyploid lager hybrids using a rare mating approach. Tryptophan (tyr−) and lysine (lys−) auxotrophs were generated in S. eubayanus and S. cerevisiae under 5-fluoroanthranilic acid (5-FAA) and α-aminoadipic acid (α-AA), respectively. Subsequently, rare mating at 12°C was performed, and hybrids were selected using minimal media. (D) Hybridization success rate between S. cerevisiae and S. eubayanus strains. Different letters (from a to g) in panels A and B depict statistically significant differences between strains with a P-value < 0.05, one-way ANOVA with Tukey’s HSD test post hoc.

Fig 2

Fermentative capacity of S. cerevisiae × S. eubayanus hybrids. (A) Average CO₂ production levels in S. eubayanus (blue), Hybrids (black), and S. cerevisiae (red) strains under 12 °P wort. CO₂ production levels in de novo lager hybrids depending on the (B) De novo hybrids grouped by S. cerevisiae parental lineage: Beer (green), Sake (blue), Bioethanol (orange), and Wine (red) lineages and (C) de novo hybrids grouped by S. eubayanus lineages: PB-1 (purple), PB-2 (light blue), PB-3 (light green), and admixed (gold). (D) Maltotriose consumption levels (%) in de novo lager hybrids depending on the S. cerevisiae parental lineage: Beer (green), Sake (blue), Bioethanol (orange), and Wine (red) lineages. (E) Ploidy levels determined by FACS in de novo lager hybrids after rare mating. (F) CO₂ production levels depend on the ploidy level. Mean values are depicted by diamonds. Different letters (a to b) reflect statistically significant differences between strains with a P-value < 0.05, Mann–Whitney–Wilcoxon test. n.s. denotes non-significant differences.

Fig 3

Phenotypic variability across de novo lager hybrids. (A). Heat map depicting the phenotypic diversity in de novo lager hybrids obtained from ten assessed conditions. Strains are grouped by hierarchical clustering from AUC data, and names and colors highlight groups of hybrids with similar phenotypes. The heat maps were elaborated based on a 0–1 normalization within each phenotype, with 0 and 1 representing the lowest and highest growth values, respectively. (B) AUC levels for 12% ethanol tolerance in different hybrids depending on the S. cerevisiae lineage. Black dots depict mean values. Different letters (a to b) reflect statistically significant differences between strains with a P-value < 0.05, Mann–Whitney–Wilcoxon (C) PCA depicting the overall association of hybrids and S. cerevisiae or S. eubayanus parental lineages (D) Plate spotting assay using 10-fold serial dilution of the HB12, HB31 and HB44 hybrids and its corresponding parental strains grown at different temperatures (4°C–20°C and 37°C).

Fig 4

Volatile compound production profile of de novo lager hybrids. Beer wort fermentations were carried out for 14 days at 12°C, and volatile compounds were analyzed by GC-FID at the fermentation endpoint. (A) We used a 0–1 normalization within each phenotype, with 0 and 1 representing the lowest and highest VCs values, respectively. Clusters containing strains with similar aroma profiles are highlighted with colored heatmap sidebars. Hybrids‘ aroma profiles were classified into four distinctive clusters. (B) 4-VG production of hybrids (gray) from Beer and Bioethanol ancestry are represented along with their S. eubayanus (blue) and S. cerevisiae (red) parental strains and the commercial strain W34/70. Diamonds depict mean values. No significant differences were detected among hybrids and their parental species (P-value > 0.05, ANOVA with Tukey’s HSD test post hoc.). Commercial strain W34/70 (light blue) did not show 4-VG production.

Fig 5

Best parent heterosis profile based on volatile compound production in de novo lager hybrids. Normalized positive (red) and negative (blue) BPH values are depicted on a scale from −1 to 1 relative to the highest absolute BPH value. Hybrids’ BPH profiles were classified into four distinctive clusters.

Fig 6

Transcriptome analysis between HB6 and HB41 hybrid strains. (A) Volcano plot depicting differentially expressed genes (DEGs) and upregulated genes in HB41 (orange) and HB6 (green) hybrid strains. (B) Enriched KEEG pathways in HB6 and HB41 hybrids. Colors depict the number of genes in each category. (C) Metabolic relationship between highly produced volatile compounds and differentially expressed genes. Volatile compounds highly produced in HB6 are displayed on the right panel, whereas those by HB41 are displayed on the left. Red color depicts genes and VCs’ greater levels.