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

Abstract

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.

Fruits of their labour: biotransformation reactions of yeasts during brewery fermentation

“…Selection of ure2 mutants has been successfully demonstrated through growth on agar plates containing the toxic ammonium analogue methylamine and proline as sole nitrogen sources (Salmon and Barre 1998 ). A number of brewing strains carrying heterozygous nonsense mutations in URE2 have also been identified among the ‘United Kingdom’ sub-group of the ‘Ale beer’/’Beer 1’ clade (Krogerus et al 2021 ). Expression of IRC7 in S. cerevisiae is also affected by its location in the sub-telomeric region of the right arm of chromosome VI (Holt et al 2019 ).…”

“…The most well reported of these is a 38-bp deletion ( IRC7 S ), which results in the formation of a truncated 360 amino acid protein with considerably lower activity than the full-length 400 amino acid Irc7p (Roncoroni et al 2011 ). This 38-bp deletion is surprisingly widespread among wine strains and found in heterozygous form among many ‘Mosaic beer’/ ‘Beer 2’ brewing strains, but it is not so common in ‘Ale beer’/’Beer 1’ brewing or other domesticated strains (Cordente et al 2019 ; Ruiz et al 2021 ; Krogerus et al 2021 ). The S. cerevisiae S288C reference genome also contains the 38-bp deletion in IRC7 .…”

Evolutionary Optimisation of Beer Organoleptic Properties: A Simulation Framework

“…In related research by Rodman et al [15], the aim has been to gain insight into the brewing process and provide an investigation into the influence of byproduct (diacetyl and ethyl acetate) threshold levels on obtainable fermentation performance by computing optimal operating temperature profiles for a range of constraint levels on byproduct concentrations in the final product. Some recent research has also been exploring faults detection in beers using artificial intelligence methods, as well as using strain development methodology to breed industrial brewing yeast [16,17].…”

Unlocking the functional potential of polyploid yeasts

“…This suggests that indeed commercial strains are not yet optimized, leaving room for improvement despite the long domestication period 10 – 12 . However, attempts to cross industrial yeasts through selective breeding or induce genomic recombination through meiosis are often unsuccessful due to the inherent sterility of the strains, which is a hallmark of yeast domestication 6 , 13 and breeding them relies on a CRISPR/Cas9 engineering 14 . The domestication syndrome might have derived from the lack of selection on sexual reproduction, random genetic drift or adaptation to specific fermentation niches, leading to the accumulation of punctuated deleterious loss-of-functions (LOF) alleles that inactivate genes involved in the gametogenesis (sporulation in yeast).…”