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. 2025 Oct;18(10):e70244.
doi: 10.1111/1751-7915.70244.

Chromosome III Aneuploidy Enhances Ethanol Tolerance in Industrial Saccharomyces cerevisiae by Increasing the TUP1 Copy Number

Sonia Albillos-Arenal  1 , Javier Alonso Del Real  1 , Ana Cristina Adam  1 , Eladio Barrio  1   2 , Amparo Querol  1
Affiliations

Chromosome III Aneuploidy Enhances Ethanol Tolerance in Industrial Saccharomyces cerevisiae by Increasing the TUP1 Copy Number

Sonia Albillos-Arenal et al. Microb Biotechnol. 2025 Oct.

Abstract

Ethanol stress poses a considerable challenge for Saccharomyces cerevisiae during fermentation. Strains carrying an extra copy of chromosome III exhibit enhanced ethanol tolerance. Here, we investigated the underlying mechanisms of this tolerance, focusing on gene dosage effects and differential gene expression under ethanol stress. We compared the gene expression profiles of a strain with three copies of chromosome III and its derivative with two copies, exposed to 6% and 10% ethanol. Our analysis identified TUP1, located on chromosome III, as a key regulator of the ethanol stress response. Deleting one copy of TUP1 in the tolerant strain diminished its ethanol tolerance, suggesting that chromosome III aneuploidy in ethanol-tolerant strains enhances adaptive responses by increasing TUP1 copy number. Our findings offer insights into the genetic basis of ethanol tolerance, with potential applications for optimising industrial fermentation processes and understanding the role of aneuploidy in the domestication of industrial yeasts.

Keywords: Saccharomyces cerevisiae; TUP1; aneuploidy; ethanol tolerance; transcriptomic.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Number of genes with higher expression (orange circles) or lower expression (blue circles) in strain 2‐200‐2 (3 × ChrIII) relative to strain 2‐200‐2‐S4 (2 × ChrIII) during growth with GPY supplemented with 6% or 10% ethanol, measured at 1 or 10 h after inoculation. The figure separately displays the differentially expressed genes (DEGs) located on chromosome III and those located on other chromosomes.
FIGURE 2
FIGURE 2
Cluster analysis of transcription factors regulating genes differentially expressed between strains 200‐2 (3xChrIII) versus 2‐200‐2‐S4 (2xChrIII), performed using the Regulator Cluster tool from RegulatorDB. This tool groups regulators based on the log2 fold change in mRNA expression of their target genes in the corresponding regulator mutants, allowing visualisation of shared regulatory patterns. Regulators identified in this analysis were those significantly affecting the expression of the DEGs, using significance thresholds (p < 0.05; fold change > 1).
FIGURE 3
FIGURE 3
Subset of differentially expressed genes (DEGs) between strain 2–200‐2 (3 × ChrIII) and 2–200‐2‐S4 (2 × ChrIII) that are regulated by TUP1, shown for each fermentation condition. The DESeq2 contrast was specified as ‘2‐200‐2’ versus ‘2‐200‐2‐S4’, so positive log2 fold change values (+, orange circles) indicate higher expression in 3xChrIII strain, whereas negative values (‘–‘, blue circles) indicate higher expression in the evolved strain. Only DEGs with log2 fold change > 1 or < −1 are shown. Genes are colour‐coded by magnitude of expression change: Blue (> 2), green (2–3), orange (3–4) and red (≥ 4). p values were calculated for all genes. The p value was obtained from YEASTRACT's regulation enrichment tool, which uses the hypergeometric test to estimate the probability of observing at least the number of DEGs regulated by TUP1 given the size of the DEG list, compared to the whole YEASTRACT database. This probability is corrected for multiple testing using the Bonferroni method.
FIGURE 4
FIGURE 4
Enriched pathways identified by comparing gene expressions between strains 2‐200‐2 and 2‐200‐2‐S4 exposed to ethanol. The network visually represents the fold enrichment of each pathway, the number of genes associated with it and the false discovery rate (FDR). Node size corresponds to the number of genes involved in the respective pathway.
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