Diacetyl is one of the main components of beer off-flavor. The acetolactate decarboxylase (ALDC) gene was introduced into brewing yeast in order to reduce the production of total diacetyl (tatal DA; vicinal diketones and their precursors, acetohydroxy acids) during beer fermentation. The fermentation performance and other characteristics of the ALDC-producing yeasts were identical to the parent yeast except total DA production, even though ALDC decarboxylates intermediates in the valine-sioleucine-biosynthetic pathway. The ALDC produced by the ALDC-producing yeast was located in the cytoplasm, but the valine-isoleucine biosynthetic enzymes are known to be associated with the mitochondria. It was suggested that ALDC only decarboxylates the acetohydroxy acids leaked from mitochondria, so the ALDC-producing yeast can grow and ferment normally. The ALDC-producing yeast reduced total DA production under normal conditions of brewing and conditions known to produce high levels of total DA. If the ALDC gene is introduced into other fermentation microorganisms where DA formation is a problem, these transformants would be expected to be able to reduce total DA production successfully.