Adaptive Laboratory Evolution of Baker’s Yeast for Improved Trehalose Accumulation and Freeze–Thaw Tolerance

Cas9-based genome engineering is a powerful tool for yeast strain development, but its use in the food industry is limited due to GMO concerns. As a non-GMO alternative, adaptive laboratory evolution (ALE) was applied to enhance trehalose accumulation, a stress protectant, in <i>Saccharomyces cerevisiae</i>. To avoid ethanol-centric metabolism and promote storage carbohydrate production, ALE was conducted under nitrogen limitation with ethanol as the sole carbon source. The evolved strain 65EV showed a 2.3-fold increase in trehalose (11.51% vs 5.25%), resulting in enhanced cell viability (77.7% vs 5.11%) and bread loaf volume (107.2 mL vs 61.5 mL) after freeze/thaw stress. Amino acid profiling revealed distinct metabolic shifts, including elevated intracellular proline and extracellular glutamate. Whole-genome sequencing and reverse engineering identified unique mutations, <i>TSL1</i> (V887A) and <i>SSA2</i> (F105L), associated with trehalose regulation. These findings demonstrate potential of ALE as a non-GMO strategy for improving yeast performance in food applications.