Saccharomyces cerevisiae as cell factory for antioxidant organic seleno-compounds biosynthesis

The unique redox chemistry of selenium (Se) makes this element an essential trace element for humans. Se-dependent antioxidative enzymes protect againstperoxides that increase cell damages due to high oxidative stress level. Not only Se-proteins, but also low molecular weight organic Se-compounds derived from cellular metabolism of Se have been demonstrated to have high cancer prevention potential. In particular, Se-methylselenocysteine (SeMCys), -glutamyl-Se-methylselenocysteine (G-SeMCys) and their derivative methylselenol (MeSeH) are the most effective Se-compounds in trial cancer therapies, when Se is supplied at supra-nutritional doses. SeMCys and G-SeMCys are present in some plants belonging to Astragalus, Brassica and Allium families. These plants are known as Se accumulators because, thanks to the action of the enzyme Se-Methyltransferase (SeMT), they convert Se-Cys in the more stable methylated form SeMCys. However, increase of Se in-take to reach effective anti-cancer doses is not feasible by consuming a Northern European diet with natural Se compounds. The aim of this work is to enhance metabolic fluxes in the yeast Saccharomyces cerevisiae towards SeMCys biosynthesis, in order to exploit S. cerevisiae as a cell factory to produce bioactive LMW Se-compounds. To this aim, optimization of growth conditions to maximize yeast Se up-take and genetic engineering of S. cerevisiae were the two main approaches considered. The foremost factor considered for enhancing Se uptake is the competition between sulphur and selenium for the same yeast transporter. Given that, yeast has been grown in the presence of Se under S limited and non-limited condition, respectively. Se depletion in the extracellular medium and accumulation of Se in the cells during batch fermentations were monitored, showing that, even though S limitation combined with Se presence affects yeast growth rate, the level of Se uptake is drastically increased under S shortage. Furthermore, in order to promote the biosynthesis of SeMCys, S. cerevisiae has been transformed with expression vectors carrying SeMT gene from Brassica oleracea (BoSeMT). The transformant strains expressing BoSeMT at different levels have been grown in batch fermentations under S limited and non-limited conditions in the presence of selenate: Se uptake and intracellular Se-metabolites have been analysed by HPLC-ICP-MS. The ensemble of results we obtained will help in clarify the routes of Se metabolism in S. cerevisiae, so that further metabolic engineering strategy and/or growth condition modifications will be considered towards the production of LMW Se-compounds.