key: cord-0281208-mk5tidzl authors: Krogerus, Kristoffer; Magalhães, Frederico; Castillo, Sandra; Peddinti, Gopal; Vidgren, Virve; De Chiara, Matteo; Yue, Jia-Xing; Liti, Gianni; Gibson, Brian title: Lager yeast design through meiotic segregation of a fertile Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid date: 2021-07-01 journal: bioRxiv DOI: 10.1101/2021.07.01.450509 sha: 505e3918d9c53f4b7f4347a0eaba3d492deb3c44 doc_id: 281208 cord_uid: mk5tidzl 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. Contribution to the field Lager beer fermentations have traditionally been carried out with natural S. cerevisiae × S. eubayanus hybrids. These strains possess both the ability to tolerate low temperatures and the ability to utilize efficiently wort sugars. However, being closely related, strains within the group exhibit limited phenotypic variability. Since the recent discovery of wild strains of S. eubayanus, it has been possible to generate lager yeast hybrids artificially, thereby increasing the genetic and phenotypic diversity of lager brewing strains. Here, to demonstrate the potential for further increased diversity, a constructed tetraploid hybrid was sporulated and spore clones derived from a single ascus were evaluated with respect to fermentation performance (sugar utilization, stress tolerance and volatile aroma synthesis). Meiosis introduced variability in a number of key parameters. One fertile spore clone from this F1 generation was sporulated to introduce further diversity and to demonstrate the potential of clone selection in steering phenotypes in a desirable direction. Genome instability of hybrids was observed, but this can be exploited to further increase diversity. This was demonstrated by assessing performance of variants isolated after ten consecutive rounds of fermentation. The approach allows for the introduction of phenotypic diversity without the need for targeted genetic modification. Genome sequencing and analysis 154 Genome assemblies of both parent strains, S. cerevisiae A-81062 and S. eubayanus C-12902, were 155 first obtained in order to create a reference genome to which sequencing reads from the hybrid strains Hybrid generation and genomic analysis 263 The set of 12 de novo hybrid strains used in this study were generated according to Figure 1 . The 264 tetraploid interspecies hybrid A225, from a cross between the S. cerevisiae A62 ale strain and the S. The assembly was polished once with long reads in Medaka, and twice with Illumina reads in Pilon. The resulting assembly consisted of 21 scaffolds (including the 16 chromosomes and mitochondrial The four F1 hybrid spores were found to include two tetraploid strains (A226 and A227) and two Of the four F2 segregants derived from A227, two were again diploid (A232 and A233) and two were compounds. Extensive phenotypic variation was observed between the strains (Figure 4 ). Both were also seen in F2 hybrids, though in this case only for the two tetraploid strains. Fermentation performance In addition to greater aroma diversity, brewers also demand strains with efficient fermentation. As Both the domesticated strains studied here had a low level of sporulation and spore viability. In the 444 A15 lager strain, sporulation was not observed and in the S. cerevisiae A62 ale strain, it was only 445 observed at a low level (21%) and of these only 8% were found to be viable. In contrast, the 446 sporulation efficiency of the S. eubayanus strain was high and spores were generally viable (Table 447 1). Sporulation in the A225 tetraploid strain was intermediate between the parents with spore viability 448 measured as 55%. In the F1 and F2 generation, sporulation and spore viability was largely influenced 449 by ploidy with spore viability ranging from 0% to 95%. Diploid strains were found to have low 450 sporulation efficiency and to be sterile. An exception was the diploid F2 spore clone A232, which 451 had a spore viability of 78% (Table 1) . Phenotypic stability of an F2 spore clone 453 The phenotypic stability of the three G10 isolates of the F2 segregant A235, isolated after 10 isolates were similar or slightly lower than A235 ( Figure 5C ). Futhermore, while A235 was able 465 to sporulate, none of the three single cell isolates produced ascospores when inoculated onto 466 potassium acetate agar (Table 1) . combined, but aroma formation is often improved compared to either of the parents from heterosis. Here, we show that sporulation of fertile allotetraploid hybrids could be exploited to further improve 513 aroma production, as beer concentrations of two important aroma-active esters 3-methylbutyl acetate 514 and ethyl hexanoate were up to 2.5-fold higher in the F2 spore clones compared to the best parent. The variation between spore clones can also be exploited to tailor the de novo hybrid towards specific 516 desired traits. It must, however, be emphasised, that much of the phenotypic variation observed here 517 was likely due to segregation and loss-of-heterozygosity in the heterozygous S. cerevisiae sub-518 genome. Phenotypic stability is an essential trait in any industrial yeast and this is particularly relevant for 520 interspecies hybrids where genomes are known to be inherently unstable. Here, the stability of the F2 521 spore clone A235 was assessed after consecutive wort fermentations. The results showed clearly 522 differences in performance between A235 and the G10 population but also between the single-cell suggested that these changes may be an early adaptive response to ethanol, which would be followed 533 by more refined changes with additional exposure. It may be that the G10 yeast in this study are 534 similarly showing signs of early adaptation to ethanol, which reached up to and over 7% in these 535 fermentations. The higher cell viability of G10 populations is consistent with an improved tolerance, 536 though the exact relationship between these specific CNVs and phenotype has yet to be resolved. 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