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. 2025 Oct 16;109(1):227.
doi: 10.1007/s00253-025-13626-8.

Construction of yeast with extremely high 2,3-butanediol tolerance by introducing point and structural mutations and partial elucidation of the mechanism of 2,3-butanediol tolerance

Kaito Nakamura  1 , Ryosuke Yamada  2 , Rumi Sakaguchi  1 , Takuya Matsumoto  1 , Hiroyasu Ogino  1
Affiliations

Construction of yeast with extremely high 2,3-butanediol tolerance by introducing point and structural mutations and partial elucidation of the mechanism of 2,3-butanediol tolerance

Kaito Nakamura et al. Appl Microbiol Biotechnol. .

Abstract

Microbial production of valuable chemicals is a promising and sustainable approach, offering high energy efficiency and minimal waste generation. Production of 2,3-butanediol (2,3-BDO) by the safe industrial yeast Saccharomyces cerevisiae holds potential as a sustainable bioprocess. However, the low tolerance of 2,3-BDO in yeast remains a major challenge. In this study, we aimed to improve 2,3-BDO tolerance in S. cerevisiae by introducing DNA point and structural mutations using techniques developed in previous studies, thereby advancing the sustainable industrial production of 2,3-BDO. Through point and structural mutagenesis, we successfully obtained the mutant strain YPH499/Co58, which exhibited a 122-fold higher OD600 value than the parent strain after 96 h of cultivation in a medium containing 175 g/L 2,3-BDO. Transcriptome analysis of four mutants with particularly high 2,3-BDO tolerance suggested that the upregulation of genes related to the proteasome, peroxisome, TCA cycle, mitochondria, and transcriptional regulation was closely related to 2,3-BDO tolerance. The use of these mutant strains represents a major step toward realizing the sustainable industrial production of 2,3-BDO. Additionally, the insights gained in this study regarding 2,3-BDO tolerance may contribute to improving yeast tolerance to various stresses, including ethanol, heat, and low pH. The mutagenesis technique developed in this study holds promise for the construction of yeast strains with enhanced robustness for various applications. KEY POINTS: DNA point and structural mutations enhanced 2,3-BDO tolerance in yeast. Engineered yeast mutant showed 122-fold higher growth in 175 g/L 2,3-BDO. Transcriptome analysis revealed key factors for 2,3-BDO tolerance.

Keywords: Saccharomyces cerevisiae; 2,3-Butanediol; Mutagenesis; Stress tolerance; Transcriptome analysis; Yeast.

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Conflict of interest statement

Declarations. Ethics approval: This article does not contain any studies with human participants or animals performed by any authors. Competing Interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
ΔOD600 of mutant strains YPH499/PMSM_CoX (X: 1–94) after 48 h of cultivation in YPD medium containing 150 g/L 2,3-BDO. Mutant strains are presented in descending order from left to right. The ΔOD600 value of the parental strain is represented by a horizontal line. Data is presented from a single experiment
Fig. 2
Fig. 2
Growth curves of mutant strains YPH499/CoX (X: 36, 40, 53, 58) and its parent strain YPH499 in YPD medium containing A 0 g/L, B 100 g/L, C 150 g/L, and D 175 g/L of 2,3-BDO. Data are presented as the average of three independent experiments. Error bars represent standard deviation
Fig. 3
Fig. 3
Growth curve of mutant strains YPH499/CoX (X: 36, 40, 53, 58) and the parent strain YPH499 under three conditions: ethanol stress (containing 70 g/L ethanol), heat stress (cultured at 37 °C), and low pH stress (buffered at pH 2.5). Data are presented as the average of three independent experiments. Error bars represent standard deviation
Fig. 4
Fig. 4
Venn diagram showing the number of genes with A increased expression and B decreased expression in selected mutant strains YPH499/CoX (X: 36, 40, 53, 58)
Fig. 5
Fig. 5
Gene ontology enrichment analysis of genes with common expression changes in selected mutants YPH499/CoX (X: 36, 40, 53, 58). A Upregulated genes, B downregulated genes. P-values were adjusted using the Benjamini–Hochberg procedure; the smallest -log10 P-value was 3.5
See this image and copyright information in PMC

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