Accelerated Evolution of Lager Yeast Strains for Improved Flavour Profiles

Abstract

S. pastorianus is an interspecific hybrid resulting from natural hybridization between S. cerevisiae and S. eubayanus. These two species belong to the Saccharomyces genus, a genus that encompasses different species related to fermentative processes. S. pastorianus strains carry out the fermentation of sugars and different nitrogen sources to produce a complex matrix, which is known as Lager beer. These strains can be divided into two groups based on their genomic architecture. Group I strains are allotriploid strains with a reduced S. cerevisiae genomic content while Group II strains are allotetraploid with more S. cerevisiae genomic content. The aim of this work was to obtain evolved strains of S. pastorianus with improved flavour profiles. More specifically, we wanted to obtain strains with an increased flux towards the production of higher alcohols and esters derived from the catabolism of the aromatic amino acid phenylalanine, 2-phenylethanol and 2-phenylethyl acetate. These aromatic compounds are one of the most important in wort as they impart notes of roses and honey-like aromas. To obtain strains with improved aromatic compounds, we decided to follow an accelerated evolution approach. Several methods are widely used, as chemical mutagenesis or UV mutagenesis, or hybridization between species. Chemical and UV mutagenesis normally induce single nucleotide changes in the strains. Hybridization is a potent tool but it implies some difficulty and it is time-consuming. Here, we used two different approaches that target the chaperone Hsp90p. This chaperone folds proteins that are involved in several processes, such as DNA repair. Previous studies have shown the potential of these two approaches, and cells exposed to high temperatures and to Radicicol have shown different chromosomal rearrangements. For this experiment, two different strains were selected and submitted to high temperatures and Radicicol treatment. Then, a characterization of the mutants and small-scale fermentations were carried out. One Group I mutant and one Group II mutant were selected based on their overproduction of 2-phenylethanol and 2-phenylethyl acetate. Results also showed that mutants are overproducing higher alcohols derived from tyrosine and tryptophol, the other two aromatic amino acids. To investigate the changes that high temperatures and Radicicol induced to the cells and to investigate the changes causing this phenotype in the mutant strains, we sequenced the genome of the two mutant strains together with the parental strains. Sequencing showed that mutant strains experienced chromosome loss, chromosome copy loss and chromosome rearrangements. Furthermore, single nucleotide polymorphisms were detected. Two nonsynonymous nucleotide changes were identified in Aro4p, an enzyme that catabolises the first step of the aromatic amino acid biosynthesis. Then, we investigated the effects that these chromosomal changes and point mutations had in the strains in three different conditions: minimal medium without amino acids and small-scale fermentations in wort on Day 2 and 4. We detected an upregulation of ARO9 and ARO10, two genes which products are involved in catabolism of aromatic amino acids. Both mutant strains show differences in transcriptomic regulation compared to their respective parental strains. Also, we reported gene dosage in Group II mutant. We investigated gene dosage in the parental strains and orthologue analysis has been carried out and gene dosage prevail in the cell. Interestingly, we detected a preference for S. eubayanus alleles in Group II parental strain. Analysis between the parental strains showed that despite the different genomic composition, metabolism of the strains goes towards the same direction and differences observed are due to copy chromosome changes.