Sake yeast is defective for Msn2/4p-dependent gene expression. (A) Expression levels of the known Msn2/4p target genes during sake brewing revealed by DNA microarray analysis. Expression profiles of the sake yeast K701 (closed circles) and the laboratory yeast X2180 (open squares) are shown. (B) qRT-PCR experiments with X2180-1A, X2180-1A Δmsn2 Δmsn4, and K701 cells isolated from sake mash on day 5. The relative expression levels are given as fold differences compared to the induction levels obtained for X2180-1A, using ACT1 as a reference gene. The error bars indicate standard deviations.

The sake yeast is defective for STRE-dependent gene expression, specifically in sake mash. (A) Expression levels of STRE-pCYC1-lacZ fusion during sake brewing. The fusion protein was expressed in K701 (closed circles) or X2180 (open squares) in sake mash. (B) Acute ethanol stress response of the STRE-pCYC1-lacZ reporter fusion in X2180 and K701. Expression levels of the fusion without stress (black) or under acute ethanol stress (8% ethanol, 2 h) (hatched) are shown. The error bars indicate standard deviations.

Modern sake yeast-specific loss-of-function mutations in the MSN4 gene. (A) Point mutation sites in the MSN2 and MSN4 genes of K7. The hatched boxes show the sites of zinc finger motifs. The white circles indicate nonsynonymous polymorphisms in the MSN2 and MSN4 genes of K7. The arrows indicate two point mutation sites, T2C and C1540T, which are responsible for truncation of K7Msn4p at the N and C termini. aa, amino acids. (B) K7Msn4p loses its molecular functions as a stress-responsive transcription factor. Expression levels of STRE-pCYC1-lacZ fusion without stress (black) or under heat shock (39°C; 1 h) (hatched) are shown. WT, wild type. The error bars indicate standard deviations. (C) Distribution of the loss-of-function mutations in MSN4 among S. cerevisiae and Saccharomyces sensu stricto strains. The black and white boxes indicate X2180- and K7-type nucleotide polymorphisms, respectively.

Expression of functional MSN4 delays ethanol fermentation in K7. (A and B) Generation of carbon dioxide gas during small-scale sake-brewing tests of K7 + vector and K7 + ScMSN4. The total amounts of carbon dioxide evolved (A) and the amounts of carbon dioxide each day (B) are shown. *, significantly lower than the control strain; **, significantly higher than the control strain (t test; P < 0.05). The error bars indicate standard deviations. (C and D) Generation of carbon dioxide gas during fermentation tests in 20% glucose-containing YPD medium using K7 + vector and K7 + ScMSN4. The total amounts of carbon dioxide evolved (C) and the amounts of carbon dioxide every 15 min (D) are shown. Representative data from three independent experiments are shown.

Depletion of MSN2 and/or MSN4 increases the fermentation rate in laboratory strains. (A to D) Generation of carbon dioxide gas during small-scale sake-brewing tests of the wild-type (BY4743) (A), Δmsn2 (B), Δmsn4 (C), and Δmsn2 Δmsn4 (D) strains. The amounts of carbon dioxide generated each day are shown. *, significantly higher than the wild type; **, significantly lower than the wild type (t test; P < 0.05). The peak level in the wild type is indicated by the red lines. The error bars indicate standard deviations. (E to H) Generation of carbon dioxide gas during fermentation tests in 20% glucose-containing YPD medium using the wild-type (green), Δmsn2 (orange), Δmsn4 (yellow), and Δmsn2 Δmsn4 (red) strains in the X2180-1A background (E and G) or in the BY4743 background (F and H). The total amounts of carbon dioxide evolved (E and F) and the amounts of carbon dioxide evolved every 15 min (G and H) are shown. Representative data from three independent experiments are shown.