Reduction of off-flavour formation and improved α-oligoglucoside utilization enable use of Saccharomyces eubayanus as a novel lager brewing yeast

Lager beer, made by fermentation of barley malt using the hybrid yeast Saccharomyces pastorianus, is extremely popular worldwide. The low genetic variation of S. pastorianus strains limits the scope of development for novel lager beer types. The wild ancestor Saccharomyces eubayanus has potential for lager brewing, but its application is constrained by formation of phenolic off-flavours (POF+ phenotype) and limited uptake of the wort α-oligoglucosides maltose and maltotriose. Here, we addressed both limitations, using screening and evolutionary engineering of the Himalayan S. eubayanus strain ABFM5L.1, one of the closest known relatives of S. eubayanus sub-genome of modern hybrid lager yeasts. Prior to improving α-oligoglucoside utilisation, 4-vinyl guaiacol formation was abolished by mutagenesis and screening for POF⁻ mutants. Subsequently, evolution for improved sugar utilisation and reverse engineering identified SeMALR1 (the S. eubayanus ortholog of S. cerevisiae MAL63) as a key regulatory control point. Single-nucleotide substitutions in this maltose regulon activator gene enabled rapid maltose uptake and partial maltotriose utilisation in a previously maltose-negative background. The evolved non-genetically modified POF⁻ strains showed near-complete maltose consumption (>90-95%) and partial maltotriose consumption (approximately 25-32%) in industrial wort. These results reveal substantial regulatory flexibility in Holarctic S. eubayanus and support its further development as a novel lager brewing yeast.