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SUMMARY
Lager yeasts are hybrid organisms that originated by cell-fusion of two species of the genus Saccharomyces, S. cerevisiae and S. eubayanus. Post-hybridization events such as copy number variations (CNV), chromosome loss, reciprocal translocations or inter-chromosomal rearrangements can affect genome size, ploidy or sporulation efficiency. In polyploidy hybrids, the presence of multiple alleles of one gene complicates functional studies that rely on genome manipulation, e.g. gene deletions. Most yeast genetic engineering methods are based on the yeast homologous recombination (HR) machinery, which works excellently in S. cerevisiae but has a low efficiency in lager yeasts.
In this thesis, I developed a strategy to improve the HR efficiency of lager yeast allowing short-flank (sf) PCR-based gene targeting. HR efficiency was successfully improved by overexpressing RAD51, which encodes a key regulator of the HR pathway that performs the search for complementary DNA strands during recombination. The successful improvement of sf PCR-based gene targeting in lager yeast was demonstrated by targeting the S. cerevisiae-derived loci of ADE2 and HSP104 in the allotetraploid lager yeast Weihenstephan 34/70. This technology can now be used to facilitate genetic engineering in lager yeast, e.g. by altering the expression of specific genes involved in wort fermentation and flavor production.
Other mechanisms of genome evolution are by uptake of DNA through horizontal gene transfer or introgression. These were studied in more detail in a cider yeast strain. Genome sequencing revealed that the strain represents a particular Saccharomyces uvarum isolate. Interestingly, in this strain we identified the acquisition of ~100 kb of S. eubayanus DNA harboring 54 gene ORFs at chromosome XIV. An analysis based on DNA polymorphisms suggests that a lager yeast strain is likely parental donor of this DNA rather than a S. eubayanus strain. An additional DNA insertion with high identity to Torulaspora microellipsoides was found at chromosome XII. The introgression of this specific DNA region was previously observed in several wine yeast strains. It harbors 19 wine – related genes such as FOT1/FOT2, encoding the fungal oligopeptide transporter Fot, which confers advantages in nitrogen utilization. Strikingly, the S. uvarum cider isolate outcompetes in standard and cold fermentation regimes, S. cerevisiae EC1118, which is considered a workhorse of the wine industry.
Beer and wine yeasts are characterized by their flocculation properties at the end of fermentation. Flocculation is the ability to produce cell-flocs, a critical trait for industrial yeast, which enables facile separation of cells from the green beer. To implement non-conventional yeast (NCY) in an existing brewing process, these yeasts need to be adaptable to the current brewing technology. Therefore, in this thesis, the flocculation properties of a NCY, Saccharomycopsis fermentans, were characterized. I could show that flocculation of S. fermentans is Ca2+- dependent like in S. cerevisiae. By PacBio genome sequencing, based on the similarity to the FLO1/5/9 genes of S. cerevisiae and the ALS gene family of Candida albicans, 34 flocculation genes were identified in S. fermentans, and termed FAS (for FLO/ALS-like sequences). Moreover, FAS genes are found to be located at sub telomeric and telomeric regions as are the S. cerevisiae flocculation genes. “Telologs” – orthologous or paralogous genes located at telomers – was a novel term introduced to identify genes which share an evolutionary conserved genomic location.