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

## TL;DR

This study shows that adjusting yeast's ability to clump together improves its survival in harsh fermentation conditions.

## Contribution

The novel contribution is using promoter engineering to control FLO1 expression and demonstrate its impact on acetic acid tolerance and flocculation.

## Key findings

- Strongly flocculating yeast strains showed better survival in late-stage fermentation and severe acetic acid stress.
- Moderately flocculating strains performed better under moderate acetic acid stress with improved growth and fermentation.
- Flocculating cells maintained higher intracellular ATP levels under stress, contributing to enhanced tolerance.

## Abstract

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 S. cerevisiae strains with distinct floc sizes were constructed through promoter engineering. The native FLO1 promoter in the non-flocculating laboratory strain BY4741 was replaced with either the constitutive strong promoter PGK1p or the ethanol-inducible promoter TPS1p using CRISPR-Cas9-mediated genome editing, resulting in strongly and moderately flocculating strains BY4741 PGK1p-FLO1 and BY4741 TPS1p-FLO1, respectively. It was revealed that the BY4741 PGK1p-FLO1 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 TPS1p-FLO1 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.