A Genome based approach to characterize genes involved in yeast adaptation to Sherry-like wines’ biological ageing

Wine fermentation and flor ageing are performed by two different lineages of yeast strains, with very different lifestyles. In this thesis we have studied the genome of flor yeast in comparison to wine yeast in order to unravel their specificities. We have first developed a set of haploid flor strains for the molecular evaluation of different targets, and developed as well a synthetic media mimicking wine for that purpose. From the genome sequence of 16 strains (8 wine and 8 flor) from France, Hungary, Italy and Spain we have drawn a phylogeny that showed that flor yeast represent a specific group of yeast different from wine and identified divergent regions. These regions contain genes involved in key functions and several associated with velum growth. Remarkably, many genes involved in FLO11 regulation such as MAP kinase, or Ras/PKA pathways were mutated among flor strains and many variations were encountered in genes involved with metal homeostasis such as zinc and divalent metal transporters. The impact of allelic variation of several genes has been evaluated for: the phosphatidylinositol 4-kinase PIK1 possibly involved in pseudohyphal growth, the high affinity zinc transporter ZRT1, and of the major pyruvate decarboxylase PDC1 in order to assess their role in the flor phenotype

A Set of aploid strains available for genetic studies of <i>Saccharomyces cerevisiae</i> flor yeasts

Flor yeasts of Saccharomyces cerevisiae have been extensively studied for biofilm formation, however the lack of specific haploid model strains has limited the application of genetic approaches such as gene knockout, allelic replacement and Quantitative Trait Locus mapping for the deciphering of the molecular basis of velum formation under biological ageing. The aim of this work was to construct a set of flor isogenic haploid strains easy to manipulate genetically. The analysis of the allelic variations at 12 minisatellite loci of 174 Saccharomyces cerevisiae strains allowed identifying three flor parental strains with different phylogenic positions. These strains were characterized for sporulation efficiency, growth on galactose, adherence to polystyrene, agar invasion, growth on wine and ability to develop a biofilm. Interestingly, the inability to grow on galactose was found associated with a frameshift in GAL4 gene that seems peculiar of flor strains. From these wild flor strains, isogenic haploid strains were constructed by deleting HO gene with a loxP-KanMX-loxP cassette followed by the removal of the kanamycin cassette. Haploid strains obtained were characterized for their phenotypic and genetic properties and compared with the parental strains. Preliminary results showed that the haploid strains represent new tools for genetic studies and breeding programs on biofilm formation