Enhanced glutathione production via precision cysteine feeding in whole-cell S. cerevisiae biocatalysis under oxidative stress

A whole‐cell biocatalytic process was developed to transform surplus Saccharomyces cerevisiae biomass—such as brewers’ or distillers’ yeast—into a high‐value, glutathione–rich ingredient. A central composite design (CCD) was used to maximize total glutathione accumulation, evaluating the effects of cysteine concentration and oxidative stress under two dosage strategies: single dosage and continuous feeding. Compared to baseline, resting (non-growing) yeast cells can achieve a four-fold increase in reduced and total glutathione. Cysteine uptake saturates at ~11 mM, but a continuous feed of 13 mM cysteine with 1.2 mM hydrogen peroxide delivers the best performance for total and reduced glutathione (2.47% and 1.59%, respectively). Significant quadratic effects, particularly associated with cysteine and hydrogen peroxide concentrations, were observed, evidencing increased GSH accumulation at the central point of the design. The phenomenological analysis supported by multi-objective optimization demonstrated that it is not possible to simultaneously increase total and reduced glutathione. These findings validate using yeast as a whole-cell biocatalyst, under moderate oxidative conditions and controlled cysteine feeding, as a scalable strategy for producing GSH-enriched biomass. By repurposing waste yeast with minimal additional capital investment, breweries and wineries can unlock a new revenue stream and meet growing demand for natural antioxidant supplements. • Peroxide increases total and oxidized glutathione but decreases reduced glutathione • Continuous cysteine feeding preserves cell viability while maintaining its gradient • Oxidative stress with constant cysteine boost total glutathione accumulation 4-fold • The maximum cysteine yeast uptake was 71 g per 100 g fed • The maximum total glutathione was 2.43 g per 100 g DCW for continuous addition