Cytosolic Localization of Acetohydroxyacid Synthase Ilv2 and Its Impact on Diacetyl Formation during Beer Fermentation

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 gamma-subunit of the F-1-ATPase (ATP3-5) could be an attractive way to reduce diacetyl formation by petite strains.