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

Abstract The first European isolates of S. eubayanus were discovered in Ireland in 2022 and belong to the same clade as the S. eubayanus parent of the hybrid lager yeast S. pastorianus. Here, we report the isolation of ten 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 metabolise 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 W307 L substitution in MalR. Expression of MALT and MALS was increased in the evolved isolates. Introducing the W307 L mutation into UCD926 by CRISPR-Cas9 editing fully recapitulates the maltose utilisation phenotype. The I243N variant alone has little effect on expression of maltose genes. We find that maltose utilisation is influenced both by the copy number of MAL genes and by gain-of-function mutations in MalR.