Sporulation generates stress-dependent phenotypic variability in the heterothallic industrial Saccharomyces...

The industrial yeast Saccharomyces cerevisiae Ethanol Red is widely used in large-scale bioethanol production and is generally considered phenotypically stable because of its heterothallic diploid background. Here, we investigated whether extensive sporulation of this highly domesticated strain could generate functional diversification without genetic engineering. Thirty monosporal cultures (MSCs) were randomly selected and profiled for growth kinetics, CFU-based cultivability and Fourier-Transform Infrared (FTIR) fingerprints under three industrially relevant stressors, namely furfural (3 g L^- 1), formic acid (3 g L^- 1) and sodium chloride (50 g L^- 1), applied at the half maximal effective concentration (EC₅₀) of the parental strain. Sporulation generated reproducible phenotypic variability among MSCs, with the greatest divergence observed under furfural stress. Sodium chloride caused moderate differences, whereas formic acid was associated with reduced cultivability across the tested MSCs. FTIR analysis highlighted stressor-specific biochemical fingerprints, with the widest dispersion observed under furfural exposure, consistent with the marked phenotypic heterogeneity detected at the physiological level. Furfural exposure generated spectral signatures compatible with envelope perturbation and redox imbalance, whereas formic acid affected protein- and phospholipid-associated regions and sodium chloride elicited membrane-protein adjustments linked to osmotic adaptation. Overall, sporulation revealed hidden adaptive potential, supporting classical non-GMO diversification as a promising strategy for strain improvement in bioethanol and lignocellulosic biorefinery applications.