Accelerated Evolution of Lager Yeast Strains for Improved Flavour Profiles
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De La Cerda García-Caro, Roberto, Accelerated Evolution of Lager Yeast Strains for Improved Flavour Profiles, Trinity College Dublin.School of Genetics & Microbiology, 2022Download Item:
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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.
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Author: De La Cerda García-Caro, Roberto
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Bond, UrsulaPublisher:
Trinity College Dublin. School of Genetics & Microbiology. Discipline of MicrobiologyType of material:
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