The production of glutathione (GSH) or GSH enriched yeast is still in the focus of research driven by a high industrial interest. In this study, an optimal growth rate for GSH production via Saccharomyces cerevisiae Sa-07346 was investigated. To further improve the fermentation process in a way that it is independent of lots, the influence of different WMIX medium compositions on biomass and GSH production was studied. Thereby, the fermentation medium was adjusted based on yeast's elemental composition. The resulting chemically defined fermentation medium led to high cell densities in fed-batches. Therefore, it has the potential to be applied successfully for other high cell density yeast fermentation processes. As cysteine is the key component for GSH production, different cysteine addition strategies were studied and finally, a continuous cysteine feeding was applied in the late stage of fermentation. Thereby, a GSH concentration of 1459 +/- 57 mg/l was reached by continuously feeding cysteine, which meant an increase to 253% compared to the control without cysteine addition (577 mg/l GSH). (C) 2016, The Society for Biotechnology, Japan. All rights reserved.