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，是啤酒中主要的不良风味物质，通常需要延长后熟工艺才能将其还原为无风味活性的化合物。在啤酒酿造中，细菌源α-乙酰乳酸脱羧酶（α-acetolactate decarboxylases）常被用于缓解双乙酰的生成：该酶可将其前体α-乙酰乳酸（α-acetolactate）直接转化为乙偶姻（acetoin）。
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，这些酶来自酵母亚门（Saccharomycotina subphylum）内的酵母菌株。通过针对真菌基因组数据库的同源性搜索，我们在仅3个属——*油脂酵母属（Lipomyces）*、*双足囊菌属（Dipodascus）*和*威克汉姆酵母属（Wickerhamiella）*——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，这些酶同时存在真菌及潜在细菌起源，提示其既存在进化保守性（evolutionary conservation），也发生过水平基因转移（horizontal gene transfer）事件。
我们将其中7个基因在拉格（lager）啤酒酿造菌株巴氏酵母（Saccharomyces pastorianus）中进行了异源表达。实验室规模带隔垫烧瓶与欧洲酿酒会议（European Brewery Convention，E.B.C.）高管发酵试验均表明，酵母源α-乙酰乳酸脱羧酶可显著降低双乙酰含量，部分酶的效果与参比的短短芽孢杆菌（Brevibacillus brevis）源酶相当甚至更优。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工艺。
本研究揭示了出芽酵母中此前未被发现的酶促活性，并提出酵母源α-乙酰乳酸脱羧酶可作为极具潜力的非细菌替代方案，用于提升拉格啤酒生产的工艺效率与可持续性。