010: Deletion of Nth1 and Fks3 Increases the Environmental Stress Tolerance of Brewers’ Yeast

Introduction

Improving the tolerance of Saccharomyces cerevisiae to environmental stress can improve fermentation and slow down beer deterioration caused by yeast autolysis. This study aims to delete the nth1gene (coding for trehalose-degrading enzyme) and fks3gene (coding for 1,3-β-glucan synthase) in the S. cerevisiae WLP029 strain (Kölschbrewing yeast) to improve its tolerance toward environmental stress.

Methods

Gene deletion was achieved using CRISPR/Cas9 gene editing technology to construct three gene knockout strains: S. cerevisiae WLP029Δnth1, S. cerevisiae WLP029Δfks3, and S. cerevisiae WLP029Δfks3Δnth1. The stress tolerance of these strains was assessed through several tests, including cell survival after freeze-drying, and survival rate and the degree of autolysis during beer fermentation and refrigerated storage. Additionally, RNA-seq analysis was conducted to evaluate the impact of nth1 and fks3 deletions on genome expression.

Results

The results showed that the survival rate and activity of the three gene knockout strains after freeze-drying were significantly higher than those of the wild-type strain. During fermentation and beer storage, WLP029Δnth1Δfks3 exhibited the highest survival rate and the lowest degree of autolysis. RNA-seq analysis revealed that deleting nth1, or fks3, or both nth1 and fks3 simultaneously could increase the expression of antioxidant and stress response-related proteins, suggesting that these deletions might enhance the environmental tolerance of the strains.

Significance

The three gene knockout S. cerevisiae WLP029 strains constructed in this study are suitable for beer brewing, as they may enhance the degree of beer fermentation and delay beer deterioration.

Authors: Chih-Yuan Tan , Zong-Tang Chen, Huan-Yu Lin, Yun-Chin Chung