Data underlying the study on 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 (<em>Lipomyces</em>, <em>Dipodascus</em> and <em>Wickerhamiella</em>) 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 <em>Saccharomyces pastorianus</em> 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 <em>Brevibacillus brevis</em> 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.