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.

双乙酰（Diacetyl）是一种感官阈值较低的邻二酮（vicinal diketone）类化合物，是啤酒中主要的不良风味物质，需通过延长后熟过程将其降解为无风味活性的化合物以消除不良影响；在啤酒酿造中，通常使用细菌来源的α-乙酰乳酸脱羧酶（α-acetolactate decarboxylases），通过将其前体α-乙酰乳酸直接转化为乙偶姻来缓解双乙酰的生成。本研究首次报道了真核来源的功能性α-乙酰乳酸脱羧酶的发现与表征，该类酶来源于酵母菌亚门（Saccharomycotina subphylum）中的酵母菌株，通过对真菌基因组数据库开展基于同源性的搜索，我们在仅3个属（Lipomyces属、Dipodascus属和Wickerhamiella属）的18个酵母物种中鉴定出29个候选基因，并将其划分为不同的系统发育类群；系统发育分析显示，这些酶同时具有真菌与潜在细菌起源，提示其存在进化保守性与水平基因转移事件。我们选取7个基因在巴斯德酿酒酵母（Saccharomyces pastorianus）拉格啤酒酿造菌株中进行异源表达，实验室规模隔垫式烧瓶与欧洲酿酒协会（E.B.C.）高型发酵管的发酵试验均表明，酵母来源的α-乙酰乳酸脱羧酶可显著降低双乙酰含量，部分酶的效果甚至优于参比的短短芽孢杆菌（Brevibacillus brevis）来源的酶；这些重组菌株同时表现出正常的发酵动力学特性，所酿造啤酒的双乙酰浓度低于感官阈值，无需再进行延长后熟处理。本研究发现了出芽酵母中此前未被认知的酶活性，并提出酵母来源的α-乙酰乳酸脱羧酶可作为极具潜力的非细菌替代方案，以提升拉格啤酒生产的工艺效率与可持续性。