Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.Saccharomyces cerevisiae has been used for centuries in the production of bread and alcoholic beverages. Along with ethanol and carbon dioxide, fermenting cultures of this yeast produce a variety of low-molecular-weight flavor compounds (including alcohols, diacetyl, esters, organic acids, organic sulfides, and carbonyl compounds). The compounds 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-1-propanol, commonly known as fusel alcohols, and their esters make an important contribution to the flavor of alcoholic beverages and bread (1, 14).A metabolic pathway for production of fusel alcohols by yeast was first proposed by Ehrlich (6). The Ehrlich pathway starts with the enzyme-catalyzed decarboxylation of branched-chain 2-oxo acids to the corresponding aldehydes. Subsequently, the aldehyde is reduced to the corresponding fusel alcohol by an alcohol dehydrogenase (11, 16, 24). The branched-chain 2-oxo acid substrates for the Ehrlich pathway can be produced by the deamination of l-leucine, l-isoleucine, or l-valine. Growth of S. cerevisiae with any of these three amino acids as the nitrogen source results in the accumulation of the corresponding fusel alcohol (2, 3, 21). Alternatively, branched-chain 2-oxo acids may be synthesized de novo from carbohydrates as intermediates of branched-chain amino acid synthesis (13).The conversion of branched-chain oxo acids into their respective aldehydes and alcohols via the Ehrlich pathway resembles the fermentative metabolism of pyruvate, which yields ethanol and carbon dioxide. In both cases, the decarboxylation of a 2-oxo acid is followed by the reduction of the resulting aldehyde. Partially purified preparations of yeast pyruvate decarboxylase have been shown to catalyze the decarboxylation of various 2-oxo acids, including the putative intermediates of the Ehrlich pathway (8, 12, 16, 21). However, it has not been conclusively proven that pyruvate decarboxylase is essential for or even involved in fusel alcohol production by S. cerevisiae.Dickinson and Dawes (4) have reported that, at least under some conditions, oxidative decarboxylation by a mitochondrial branched-chain oxo acid dehydrogenase complex (17) is involved in the catabolism of branched-chain 2-oxo acids. Mutants that did not express the lipoamide dehydrogenase subunit of this enzyme complex accumulated branched-chain oxo acids in batch cultures grown on media containing leucine, isoleucine, or valine (4), thus casting some doubt on the exclusive role of pyruvate decarboxylase in the decarboxylation of branched-chain oxo acids.The aim of this study was to reinvestigate the role of pyruvate decarboxylase in the production of fusel alcohols by S. cerevisiae. The S. cerevisiae genome harbors three structural genes (PDC1, PDC5, and PDC6) that can each encode an active pyruvate decarboxylase (9). In wild-type yeast strains, PDC6 expression is either very low or absent (7, 9). However, revertants of pdc1-pdc5 double mutants, in which a recombination event has caused a fusion of the PDC1 promoter and the PDC6 open reading frame, express a functional enzyme (10). Therefore, studies on the physiological effects of pyruvate decarboxylase deficiency are most easily interpreted when they are performed with strains in which all three PDC genes are disrupted.In the present study, the decarboxylation of branched-chain 2-oxo acids was studied in cell extracts of wild-type S. cerevisiae and in extracts of an isogenic pyruvate decarboxylase-negative mutant. Furthermore, conversion of branched-chain amino acids to the corresponding fusel alcohols by intact cells was analyzed in ethanol-grown cultures of a wild-type S. cerevisiae strain and in those of the Pdc⁻ mutant.