Development of Freeze-Thaw Tolerant Yeast Strains via a Hybrid Fusion Evolutionary Strategy.

The freeze-thaw cycle presents a significant challenge to Saccharomyces cerevisiae in industrial applications, impacting its resilience and viability. Traditional genetic modifications in industrial yeast strains have achieved limited success in enhancing freeze-thaw tolerance. To address this, we explored the untapped genetic diversity of wild diploid strains and developed a hybrid fusion evolution (HFE) strategy. This approach involved isolating haploids, implementing targeted modifications to increase glycerol accumulation and aquaporin expression, and subjecting these modified strains to cycles of fusion and selection. The HFE method enabled the development of robust yeast strains with significantly improved freeze-thaw tolerance, from an average of no more than 12% for the initial parents to an average of 67% for the third-generation strains, alongside enhanced resilience to other stresses such as high ethanol and moderate heat. Our findings demonstrate the potential of combining microbial breeding and gene regulation to exploit natural genetic diversity, providing a promising solution to improve industrial yeast strains for sustainable food production under environmental stress conditions.