Diversity of α-acetolactate decarboxylase in the Saccharomycotina yeast subphylum: From discovery to brewing application

Diacetyl, a vicinal diketone with a low sensory threshold, is a prominent off-flavour in beer, necessitating extended lagering to allow its reduction to non-flavour-active compounds. In brewing, bacterial α-acetolactate decarboxylases are commonly used to mitigate diacetyl formation by converting its precursor, α-acetolactate, directly into acetoin. Here, we report the first discovery and characterization of functional α-acetolactate decarboxylases enzymes of eukaryotic origin, specifically from yeasts within the Saccharomycotina subphylum. Using a homology-based search against fungal genomic databases, 29 candidate genes were identified across 18 yeast species from only three genera (Lipomyces, Dipodascus and Wickerhamiella) and classified into distinct phylogenetic groups. Phylogenetic analysis revealed both fungal and possible bacterial origins, suggesting evolutionary conservation and horizontal gene transfer events. Seven genes were heterologously expressed in Saccharomyces pastorianus lager brewing strains. Fermentation trials in both lab-scale septum flasks and E.B.C. tall tubes demonstrated that yeast-derived α-acetolactate decarboxylases significantly reduced diacetyl levels, with some performing comparably or superior to the benchmark Brevibacillus brevis enzyme. These strains also showed normal fermentation kinetics and produced beers with diacetyl concentrations below sensory thresholds, effectively eliminating the need for extended lagering. Our findings uncover a previously unrecognized enzymatic activity in budding yeasts and present yeast α-acetolactate decarboxylases as promising non-bacterial alternatives to improve process efficiency and sustainability in lager beer production.