Spontaneous Attenuation of Alcoholic Fermentation via the Dysfunction of Cyc8p in Saccharomyces cerevisiae

Abstract

A cell population characterized by the release of glucose repression and known as [GAR⁺] emerges spontaneously in the yeast Saccharomyces cerevisiae. This study revealed that the [GAR⁺] variants exhibit retarded alcoholic fermentation when glucose is the sole carbon source. To identify the key to the altered glucose response, the gene expression profile of [GAR⁺] cells was examined. Based on RNA-seq data, the [GAR⁺] status was linked to impaired function of the Cyc8p-Tup1p complex. Loss of Cyc8p led to a decrease in the initial rate of alcoholic fermentation under glucose-rich conditions via the inactivation of pyruvate decarboxylase, an enzyme unique to alcoholic fermentation. These results suggest that Cyc8p can become inactive to attenuate alcoholic fermentation. These findings may contribute to the elucidation of the mechanism of non-genetic heterogeneity in yeast alcoholic fermentation.

Keywords: Cyc8p–Tup1p complex; Saccharomyces cerevisiae; [GAR+]; alcoholic fermentation; glucose repression; non-genetic heterogeneity; pyruvate decarboxylase.

Figure 1

[GAR⁺] retards alcoholic fermentation when glucose is the sole carbon source. Carbon dioxide emissions of [gar⁻] (gray) and [GAR⁺] (orange) strains in the K701 background were monitored in YP + 20% glucose (left) or YP + 10% glucose + 10% sucrose (right) medium for 2 d. Data represent mean values ± standard deviations from three independent experiments. Percentages in the graphs indicate how much emissions were affected by [GAR⁺]. Asterisks indicate that emissions significantly decreased by [GAR⁺] (t test, p < 0.05). YP: yeast extract–peptone medium; Glc: glucose; Suc: sucrose.

Figure 2

[GAR⁺] impairs transcriptomic responses to a nonmetabolizable glucose analog. Bar graphs represent the relative FPKM values for each gene normalized by the data of the [gar⁻] strain without addition of glucosamine (GlcN). Blue graphs show data without GlcN addition and red graphs show data with GlcN addition. Values in the graphs indicate fold changes in comparison to the data of each strain without addition of GlcN. Representative data of glucose-repressed genes (A), glucose-inducible hexose transporter genes (B), and glucose-inducible Hog1p target genes (C) are shown. FPKM: fragments per kilobase of exon per million fragments mapped.

Figure 3

Deletion of the CYC8 gene retards alcoholic fermentation when glucose is the sole carbon source. Carbon dioxide emissions of the wild-type (WT; gray) and cyc8Δ (orange) strains in the BY4741 background were monitored in YP + 20% glucose (left) or YP + 10% glucose + 10% sucrose (right) medium for 2 d. Data represent mean values ± standard deviations from three independent experiments. The percentages in the graph indicate how much emissions were affected by cyc8Δ. The asterisks indicate that emissions significantly decreased by cyc8Δ (t test, p < 0.05).

Figure 4

Deletion of the CYC8 gene inactivates pyruvate decarboxylase. Bar graphs in (A) represent fold increases in each metabolite level per cell compared to that in WT. G6P; glucose 6-phosphate. F6P; fructose 6-phosphate. FBP; fructose 1,6-bisphosphate. DHAP; dihydroxyacetone phosphate. GAP; glyceraldehyde 3-phosphate. 1,3-BPG; 1,3-bisphosphoglycerate. 3-PG; 3-phosphoglycerate. 2-PG; 2-phosphoglycerate. PEP; phosphoenolpyruvate. Pyr; pyruvate. AA; acetaldehyde. EtOH; ethanol. G1P; glucose 1-phosphate; UDPG; UDP-glucose. ND; not detected. The schematic diagram in (B) indicates glucose metabolism in S. cerevisiae and the putative point of action of cyc8Δ. 1,3-BG; 1,3-β-glucan.