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Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Holomycin is a member of the dithiolopyrrolone class of secondary metabolites. It contains a cromophore formed by two heterocycles, one of them carrying a disulfide bridge. The holomycin chromophore is also present in thioluthin, aureothricin, the xenorhabdins and the complex thiomarinols. Streptomyces clavuligerus ATCC 27064 contains the holomycin gene cluster (hlm). This antibiotic is formed by a cysteine-activating non-ribosomal peptide synthetase followed by four reduction steps by a set of four different flavoproteins; the intermediate is cyclized by a thiol oxidase and modified by acylation. Holomycin is a broad spectrum antibiotic, reported as antitumoral, acting in vivo on RNA synthesis. It is modified intracellularly by the producer strains by methylation and formation of heterodimers as a way of self-protection. Holomycin might be a lead molecule for the production of new hybrid compounds with higher activity and lower toxicity.