Institut für Lebensmittelwissenschaft und Biotechnologie, Fg. Gärungstechnologie (150f), Universität Hohenheim, Garbenstr. 23, D-70599 Stuttgart, Germany.
Appl Environ Microbiol. 2011 Feb;77(3):727-31. doi: 10.1128/AEM.01579-10. Epub 2010 Dec 3.
Diacetyl (2,3-butanedione) imparts an unpleasant "butterscotch-like" flavor to alcoholic beverages such as beer, and therefore its concentration needs to be reduced below the sensory threshold before packaging. We examined the mechanisms that lead to highly elevated diacetyl formation in petite mutants of Saccharomyces cerevisiae during beer fermentations. We present evidence that elevated diacetyl formation is tightly connected to the mitochondrial import of acetohydroxyacid synthase (Ilv2), the key enzyme in the production of diacetyl. Our data suggest that accumulation of the matrix-targeted Ilv2 preprotein in the cytosol is responsible for the observed high diacetyl levels. We could show that the Ilv2 preprotein accumulates in the cytosol of petite yeasts. Furthermore, expression of an Ilv2 variant that lacks the N-terminal mitochondrial targeting sequence and thus cannot be imported into mitochondria led to highly elevated diacetyl levels comparable to a petite strain. We further show that expression of a mutant allele of the γ-subunit of the F(1)-ATPase (ATP3-5) could be an attractive way to reduce diacetyl formation by petite strains.
双乙酰（2,3-丁二酮）为啤酒等酒精饮料赋予一种令人不快的“奶油硬糖味”，因此在包装前需要将其浓度降低到感官阈值以下。我们研究了在啤酒发酵过程中酿酒酵母 petite 突变体中双乙酰高度升高的形成机制。我们提供的证据表明，双乙酰的形成与乙酰羟酸合酶（Ilv2）的线粒体导入紧密相关，Ilv2 是双乙酰生产的关键酶。我们的数据表明，靶向基质的 Ilv2 前体蛋白在细胞质中的积累是导致观察到的高双乙酰水平的原因。我们可以证明 Ilv2 前体蛋白在 petite 酵母的细胞质中积累。此外，表达一种缺乏 N 端线粒体靶向序列且因此不能导入线粒体的 Ilv2 变体导致双乙酰水平显著升高，与 petite 菌株相当。我们进一步表明，表达 F（1）-ATP 酶（ATP3-5）γ亚基的突变等位基因可能是降低 petite 菌株双乙酰形成的一种有吸引力的方法。
