Tuning FLO1 Expression via Promoter Engineering Modulates Flocculation Degree and Acetic Acid Stress Tolerance in Saccharomyces cerevisiae

Robust yeast tolerance to inhibitors is essential for lignocellulosic biorefinery. Although cell flocculation is known to enhance acetic acid stress tolerance, the impact of its intensity remains unclear. In this study, engineered <i>S. cerevisiae</i> strains with distinct floc sizes were constructed through promoter engineering. The native <i>FLO1</i> promoter in the non-flocculating laboratory strain BY4741 was replaced with either the constitutive strong promoter <i>PGK1p</i> or the ethanol-inducible promoter <i>TPS1p</i> using CRISPR-Cas9-mediated genome editing, resulting in strongly and moderately flocculating strains BY4741 <i>PGK1p-FLO1</i> and BY4741 <i>TPS1p-FLO1</i>, respectively. It was revealed that the BY4741 <i>PGK1p-FLO1</i> showed a survival advantage in the late-stage fermentation and severe stress condition in the presence of 7.5 g/L acetic acid, while BY4741 <i>TPS1p-FLO1</i> exhibited superior growth and fermentation performance under 5.0 g/L acetic acid stress. Further studies suggested that the enhanced acetic acid tolerance in flocculating cells was associated with their ability to maintain significantly higher intracellular ATP levels under stress. Our work highlights the importance of optimizing flocculation properties for robust industrial fermentation, and also provides a strategic basis for engineering stress-tolerant yeast strains for efficient fermentation in inhibitor-rich cellulosic hydrolysates.