Nobuhiko Mukai

PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae

The volatile phenols, to which Saccharomyces cerevisiae converts from phenylacrylic acids including ferulic acid, p-coumaric acid, and cinnamic acid, generate off-flavors in alcoholic beverages such as beer and wine. Using gene disruptants, transformants and cell-free extracts of these strains, we have verified that the adjacent PAD1 (phenylacrylic acid decarboxylase, YDR538W) and FDC1 (ferulic acid decarboxylase, YDR539W) genes are essential for the decarboxylation of phenylacrylic acids in S. cerevisiae. Pad1p and Fdc1p are homologous with UbiX and UbiD, respectively, in the ubiquinone synthetic pathway of Escherichia coli. However, ubiquinone was detected quantitatively in all of the yeast single-deletion mutants, Delta pad1, Delta fdc1, and double-deletion mutant, Delta pad1 Delta fdc1.

Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production.

Among industrial yeasts used for alcoholic beverage production, most wine and weizen beer yeasts decarboxylate ferulic acid to 4-vinylguaiacol, which has a smoke-like flavor, whereas sake, shochu, top-fermenting, and bottom-fermenting yeast strains lack this ability. However, the factors underlying this difference among industrial yeasts are not clear. We previously confirmed that both PAD1 (phenylacrylic acid decarboxylase gene, YDR538W) and FDC1 (ferulic acid decarboxylase gene, YDR539W) are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. In the present study, single nucleotide polymorphisms (SNPs) of PAD1 and FDC1 in sake, shochu, wine, weizen, top-fermenting, bottom-fermenting, and laboratory yeast strains were examined to clarify the differences in ferulic acid decarboxylation ability between these types of yeast. For PAD1, a nonsense mutation was observed in the gene sequence of standard top-fermenting yeast. Gene sequence analysis of FDC1 revealed that sake, shochu, and standard top-fermenting yeasts contained a nonsense mutation, whereas a frameshift mutation was identified in the FDC1 gene of bottom-fermenting yeast. No nonsense or frameshift mutations were detected in laboratory, wine, or weizen beer yeast strains. When FDC1 was introduced into sake and shochu yeast strains, the transformants exhibited ferulic acid decarboxylation activity. Our findings indicate that a positive relationship exists between SNPs in PAD1 and FDC1 genes and the ferulic acid decarboxylation ability of industrial yeast strains.

Effect of Soy Peptide on Brewing Beer

Here, we examined the effect of soy peptides (SPs) on the fermentation and growth of Yeast Bank Weihenstephan 34/70 (W34/70), a bottom-fermenting yeast. We compared fermentation for SP with that for a free amino acid (FAA) mixture having the same amino acid composition as SP, as a nitrogen source. Maltose syrup was used as a carbon source, and the medium contained excess amounts of essential minerals and vitamins. We observed that SP was better than FAA mixture at promoting fermentation and growth and that much more beta-phenylethyl alcohol was produced during fermentation with SP than with FAA mixture. Subsequently, we compared fermentations with the FAA mixture and selected mixtures containing various dipeptides of Phe as a nitrogen source. We found that the rates of Phe metabolism and beta-phenylethyl alcohol generation were much higher when Phe was presented as a dipeptide (Phe-Asp, Phe-Leu, or Phe-Phe) than when presented as FAA. These results show that amino acids such as Phe are absorbed more rapidly when presented as a peptide than as FAA, resulting in a more rapid production of beta-phenylethyl alcohol.

Beer brewing using a fusant between a sake yeast and a brewer's yeast.

Beer brewing using a fusant between a sake yeast (a lysine auxotrophic mutant of sake yeast K-14) and a brewers yeast (a respiratory-deficient mutant of the top fermentation yeast NCYC1333) was performed to take advantage of the beneficial characteristics of sake yeasts, i.e., the high productivity of esters, high tolerance to ethanol, and high osmotolerance. The fusant (F-32) obtained was different from the parental yeasts regarding, for example, the assimilation of carbon sources and tolerance to ethanol. A brewing trial with the fusant was carried out using a 100-l pilot-scale plant. The fusant fermented wort more rapidly than the parental brewers yeast. However, the sedimentation capacity of the fusant was relatively low. The beer brewed using the fusant contained more ethanol and esters compared to that brewed using the parental brewers yeast. The fusant also obtained osmotolerance in the fermentation of maltose and fermented high-gravity wort well.