Abstract <p>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 <i>Saccharomyces cerevisiae</i> 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 <i>S. cerevisiae</i> 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&#xa0;h of cultivation in a medium containing 175&#xa0;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.</p> Key points <p><UnorderedList Mark="Bullet"> <ItemContent> <p><i>DNA point and structural mutations enhanced 2,3-BDO tolerance in yeast.</i></p> </ItemContent> <ItemContent> <p><i>Engineered yeast mutant showed 122-fold higher growth in 175&#xa0;g/L 2,3-BDO.</i></p> </ItemContent> <ItemContent> <p><i>Transcriptome analysis revealed key factors for 2,3-BDO tolerance.</i></p> </ItemContent> </UnorderedList></p>