Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation

Abstract

The behavior of yeast cells during industrial processes such as the production of beer, wine, and bioethanol has been extensively studied. In contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese, or cocoa fermentation, remains limited. We investigated changes in the transcriptomes of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all three strains exhibit similar changes in expression patterns. At the onset of fermentation, expression of glucose-regulated genes changes dramatically, and the osmotic stress response is activated. The middle fermentation phase is characterized by the induction of genes involved in amino acid metabolism. Finally, at the latest time point, cells suffer from nutrient depletion and activate pathways associated with starvation and stress responses. Further analysis shows that genes regulated by the high-osmolarity glycerol (HOG) pathway, the major pathway involved in the response to osmotic stress and glycerol homeostasis, are among the most differentially expressed genes at the onset of fermentation. More importantly, deletion of HOG1 and other genes of this pathway significantly reduces the fermentation capacity. Together, our results demonstrate that cells embedded in a solid matrix such as bread dough suffer severe osmotic stress and that a proper induction of the HOG pathway is critical for optimal fermentation.

Fig 1

Three genetically distinct strains from different fermentation industries (wine, bread, and bioethanol) with an acceptable dough fermentation capacity were selected. (A) The genetic relatedness of 24 strains from different fermentation industries was determined by an interdelta genetic fingerprinting assay. (B) CO₂ production during dough fermentation indicates that all three strains can ferment dough at an acceptable rate.

Fig 2

(A) A large number of genes are differentially expressed at different time points during bread dough fermentation compared to the 0-min sample. The commercial baker's yeast (“Bread”) strain induces fewer changes in the transcriptome in response to the new environment. Note that differentially expressed genes include both protein- and RNA-coding genes. (B) The transcriptome of cells at the latest fermentation time point shows the highest level of similarity to cells in stationary phase (0-min sample). Shown are pairwise scatter plots and correlation coefficients for all mRNA quantities (as estimated by RPKM values) of the nonfermenting sample (0 min) and the three fermentation time points (30, 60, and 180 min) for the three different yeast strains (bioethanol, wine, and bread yeast). Note that the correlation between biological replicates was very high (R² of between 0.959 and 0.997) (data not shown). (C) Clustering of expression profiles shows that the different yeast strains display largely similar (but not identical) gene expression patterns during bread dough fermentation. The graph shows hierarchical clustering of all expression data (including biological replicates for each strain and each time point). Every horizontal line contains the expression data (RPKM values) for a given strain and sampling time (i.e., one sample). The horizontal lines are ordered based on their overall similarity, and the clustering tree on the right shows the similarity between the samples (see Materials and Methods).

Fig 3

The majority of differentially expressed genes display a transient response. Shown are the normalized expression levels for each gene across the fermentation process (black lines). The genes are grouped per yeast strain (commercial baker's yeast, wine yeast, or bioethanol yeast) based on similarities in expression patterns (i.e., the trend in gene expression from one time point to another). Note that number of genes in different expression categories for all 3 strains includes both protein-coding and RNA-coding genes.

Fig 4

(A) Deletion of key genes involved in the osmotic response results in impaired dough fermentation. Shown is CO₂ production during dough fermentation, which serves as a proxy for the fermentation efficiency. Note that none of these mutants showed differences in activity compared to the wild type during fermentation with standard rich medium (yeast extract-peptone-dextrose) (see Fig. S1 in the supplemental material). (B) The HOG pathway triggers changes in the transcriptome through chromatin modification. Shown is a subnetwork selected by PheNetic for the 100 most differentially expressed genes for each strain at the first time point. Orange edges indicate protein-DNA interactions, and gray edges indicate protein-protein/phosphorylation interactions. The differential expression per node is visualized in a line chart in which the blue lines indicate expression for the bioethanol strain, the green lines indicate expression for the wine strain, and the orange lines indicate expression for the bread strain. Genes involved in chromatin modification are shown in blue.