Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway

Aims: An evolution-based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically molybdate and chromate resistance was used as selectable phenotype to select sulfate permease deficient variants unable to produce sulfites and hydrogen sulfide (H2S).Methods and Results: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the gametes conjugation. Then the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty-four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfites production (SO2 <10 mg l-1) and unable to produce H2S in grape must without added sulfites, were selected.Conclusions: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly resistance to toxic analogues of sulfate allowed to detect strains unable to assimilate sulfates.Significance and Impact of the Study: This strategy, that combines spores sexual recombination and application of a specific selective pressure, provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H2S.