Craft beer mon amour: an exploration of Italian craft consumers

Appl Microbiol Biotechnol

Fletcher²,

Rettberg

et al. 2021

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase β-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high β-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for β-lyase activity. Selected phenolic off-flavour positive (POF +) hybrids were further sporulated to generate meiotic segregants with high β-lyase activity, efficient wort fermentation, and lack of POF, all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast. Key points • CRISPR/Cas9-based mating-type switching was applied to industrial yeast strains. • Transformed strains could be readily mated to form intraspecific hybrids. • Hybrids exhibited heterosis for a number of brewing-relevant traits.

Section: Introductionmentioning

confidence: 99%

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

Appl Microbiol Biotechnol

Fletcher²,

Rettberg

et al. 2021

“…Interest towards beer with novel and diverse flavours is increasing (Aquilani et al, 2015;Carbone and Quici, 2020;Gonzalez Viejo and Fuentes, 2020), and the results here suggest that hybridization and subsequent sporulation can give rise to lager yeast strains with both enhanced and diverse production of aroma-active compounds. 3-methylbutyl acetate, with its banana-and pear-like aroma, is one of the most important yeast-derived flavor compounds in beer (Pires et al, 2014).…”

Section: Aroma Diversitymentioning

confidence: 73%

“…Creating new flavour profiles, e.g. in response to the increased consumer demand for higher product quality and beer with novel and diverse flavours (Aquilani et al, 2015; Carbone and Quici, 2020; Gonzalez Viejo and Fuentes, 2020), is hampered by the low level of diversity amongst commercial brewing yeast strains. Previous research has shown that interspecific hybridization is an effective way of introducing new aromatic diversity among lager yeasts (Krogerus et al, 2015; Mertens et al, 2015; Nikulin et al, 2018; Turgeon et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Lager yeast design through meiotic segregation of a fertile Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid

Magalhães

Castillo

et al. 2021

Preprint

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae x Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15 C, 15 Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

“…Interest toward beer with novel and diverse flavors is increasing (Aquilani et al, 2015;Carbone and Quici, 2020;Gonzalez Viejo and Fuentes, 2020), and the results here suggest that hybridization and subsequent sporulation can give rise to lager yeast strains with both enhanced and diverse production of aroma-active compounds. 3-methylbutyl acetate, with its banana-and pear-like aroma, is one of the most important yeastderived flavor compounds in beer (Pires et al, 2014).…”

Section: Aroma Diversitymentioning

confidence: 74%

Lager Yeast Design Through Meiotic Segregation of a Saccharomyces cerevisiae × Saccharomyces eubayanus Hybrid

Magalhães

Castillo

et al. 2021

Front. Fungal Biol.

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae × Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15°C, 15 °Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.