Selection of Pof - Saccharomyces eubayanus Variants for the Construction of S. cerevisiae × S. eubayanus Hybrids With Reduced 4-Vinyl Guaiacol Formation.

Saccharomyces pastorianus is an interspecies hybrid between S. cerevisiae and S eubayanus. The identification of the parental species of S. pastonanus enabled the de novo reconstruction of hybrids that could potentially combine a wide array of phenotypic traits. Lager yeasts are characterized by their inability to decarboxylate ferulic acid present in wort, a phenotype also known as Pof(-) (phenolic off-flavor). However, all known S. eubayanus strains characterized so far produce clove-like aroma specific of 4-vinyl guaiacol, a decarboxylated form of ferulic acid. This study explored a non-GMO approach to construct Pof(-) S. eubayanus variants derived from the parental strain S. eubayanus CBS 12357. To rapidly screen a population of UV-mutagenized cells two complementary assays were developed. The first assay was based on the difference of light absorption spectra of ferulic acid and 4-vinyl guaiacol, while the second was based on the difference of sensitivity of Pof(-) and Pof(+) strains to cinnamic acid The S. eubayanus variant HTSE042 was selected and was confirmed not to produce 4 vinyl guaiacol. Whole genome sequencing revealed that this variant lost the subtelomeric region of the CHRXIII right arm that carried the two clustered genes SePAD1-SeFDC1 whose deletion in a naive S. eubayanus strain (CBS 12357/FM1318) resulted in an identical phenotype. Subsequently, the Pof(-) variant was crossed with a Pof(-) S cerevisiae partner. The resulting hybrid was not able to convert ferulic acid demonstrating the undisputable value of the mutagenized variant HTSE042 to eventually construct S. cerevisiae x S. eubayanus hybrids with phenotypic characteristics of S. pastonanus.