Exploiting Ethanol to Improve Production of Glutathione by Saccharomyces cerevisiae

Glutathione (GSH) is a crucial antioxidant tripeptide with extensive applications in pharmaceuticals, food, and cosmetics. Nevertheless, its microbial production is constrained by low yields, intracellular accumulation, and feedback inhibition. While Saccharomyces cerevisiae presents a promising host for GSH synthesis, the efficient secretion of GSH into the culture medium poses a significant challenge. In this study, we implemented a multi-stage bioengineering strategy aimed at maximizing extracellular GSH production by Saccharomyces cerevisiae. Statistical optimization of fermentation parameters and medium composition enhanced the extracellular glutathione (GSH) yield in shake-flask culture from 18.56 mg/L to 63.60 mg/L, representing a 3.43-fold increase. Furthermore, scaling up the fermentation process to a 30-L high-density fed-batch bioreactor resulted in a significant increase in GSH production, elevating the extracellular GSH concentration from 63.660 mg/L in the optimized shake-flask culture to 1033.33 mg/L. Moreover, the addition of 1% (v/v) ethanol during the later stages of cultivation enhanced both the biosynthesis and secretion of GSH by increasing membrane permeability. This treatment resulted in a 35.27% increase in total GSH content, reaching 94.80 mg/g, and redirected 76.76% of the total GSH into the extracellular fraction. Collectively, these strategies effectively addressed the cell-membrane transport barrier and significantly improved secretion efficiency. The integrated process offers a robust and scalable platform for industrial-scale extracellular GSH production.