Yeasts have been largely explored as cell factories to produce substances for food and industrial biotechnological
applications. Among these chemicals, glutathione (GSH) is an important antioxidant molecule involved in
several processes, including the control of redox potential, protection against oxidative stress, detoxification and
transport of organic sulfur. Due to its functional roles, GSH is widely used in the pharmaceutical, food and
cosmetic industries. Recently, GSH has received growing attention also in the winemaking field, to control
oxidative spoilage damage; to limit the amount of browning pigments; to avoid the formation atypical aging
characters; and to exert a protective effect on various aromatic compounds. At present GSH is successfully
produced on an industrial scale through fermentation by high GSH-producing Saccharomyces cerevisiae strains,
and several methodological tools have been reported for increasing efficiency and yield of the bioprocess. In this
study, we have applied an evolution-based strategy that combines the sexual recombination of spores with the
application of molybdate Mo(VI), a sulfate analogue toxic for the cells at high concentration, as specific
selective pressure, to generate evolved S. cerevisiae strains with enhanced GSH production. To achieve this aim
we used the 21T2 wine strain from the Unimore Microbial Culture Collection (UMCC) and we exploited its
resistance to Mo(VI) as a rapid and high-throughput screening method for the selection of the evolved strains
improved in GSH production. By this strategy, we obtained two evolved strains, Mo21T2-5 and Mo21T2-12,
both able to enhance GSH content in wine with an increase of 100% and 36%, respectively, compared with the
parental strain 21T2, and 120% and 50% compared with initial GSH content in the must. Our strategy, unlike the
standard evolutionary approaches, has the advantage of not requiring multiple rounds of screening and extensive
cultivation periods because the evolved strains are recognized through a selectable phenotype. The Mo(VI)
resistance has proved to be effective for the selection of the desired evolved strains, probably by activating the
yeast common metal response that involves sulfur assimilation and GSH biosynthesis
