Lineage-Specific Adaptive Strategies Shape the Response of de Novo Lager Yeast Hybrids to High-Gravity Fermentation

Lager beer is the most widely consumed fermented beverage worldwide and is produced by the yeast Saccharomyces pastorianus, which originated from hybridization between S. cerevisiae (Sc) and S. eubayanus (Se). Although the genetic factors underlying fermentation performance in de novo hybrids are well understood, how chimeric genomes evolve in relation to the parental background and stabilize under industrial stresses remains unclear. Here, we subjected representative novel hybrids derived from distinct parental lineages to long-term experimental evolution for 250 generations under conditions mimicking high-gravity brewing. We observed a clear pattern of the "rule of declining adaptability", driven by diminishing-returns epistasis, whereby hybrids with lower initial fitness exhibited the greatest evolutionary gains. Overall, 38 of 69 evolved lines produced more CO₂ than their ancestral strains. Adaptive trajectories were strongly influenced by the Sc genetic background, while the Se contribution appeared comparatively limited, highlighting the dominant role of the Sc subgenome in determining brewing performance and evolvability. Genomic analysis of representative hybrids revealed different evolutionary strategies. The evolved HB3-I line (beer Sc parent) showed a large-scale chromosomal rearrangement with few coding-sequence changes, consistent with fine-tuning of an already adapted background. In contrast, HB44-II (bioethanol Sc parent) accumulated multiple gene-disruptive mutations affecting multiple pathways, indicating broader regulatory rewiring. These genomic changes were accompanied by transcriptional shifts in carbon metabolism, nutrient-responsive pathways and membrane-related functions. Together, our results provide a controlled framework for replaying yeast domestication, demonstrating that the adaptive potential of synthetic lager hybrids depends on their lineage, which is mainly shaped by the S. cerevisiae parental background.