Adaptive laboratory evolution of maltose utilization in wild Irish isolates of Saccharomyces eubayanus

The first European isolates of Saccharomyces eubayanus were discovered in Ireland in 2022 and belong to the same clade as the S. eubayanus parent of the hybrid lager yeast Saccharomyces pastorianus. Here, we report the isolation of 10 additional Irish strains and we explore maltose metabolism. Maltose metabolism genes are clustered in subtelomeric MAL loci including maltase (MALS), maltose transporter (MALT), and regulator (MALR). Despite having intact MAL loci, the Irish strains do not grow on maltose. Two Irish S. eubayanus strains (UCD650 and UCD926) were passaged in medium containing maltose as the sole carbon source until they acquired the ability to metabolize maltose. The UCD650-derived lineage acquired an I243N substitution in MalR, and a 30-kb duplication of the MAL locus. The UCD926 parent has a similar MAL locus duplication and its evolved lineage acquired a W307L substitution in MalR. Expression of MALT and MALS was increased in the evolved isolates. Introducing the W307L mutation into UCD926 by CRISPR-Cas9 editing fully recapitulates the maltose utilization phenotype. The I243N variant alone has little effect on expression of maltose genes. We find that maltose utilization is influenced both by the copy number of MAL genes and by gain-of-function mutations in MalR.