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Improving ale or lager yeasts by conventional breeding is a non-trivial task. Domestication of lager yeasts, which are hybrids between Saccharomyces cerevisiae and Saccharomyces eubayanus, has led to evolved strains with severely reduced or abolished sexual reproduction capabilities, due to, e.g. postzygotic barriers. On the other hand, S. cerevisiae ale yeasts, particularly Kveik ale yeast strains, were shown to produce abundant viable spores (~ 60%; Dippel et al. Microorganisms 10(10):1922, 2022). This led us to investigate the usefulness of Kveik yeasts for conventional yeast breeding. Surprisingly, we could isolate heterothallic colonies from germinated spores of different Kveik strains. These strains presented stable mating types in confrontation assays with pheromone-sensitive tester strains. Heterothallism was due to inactivating mutations in their HO genes. These led to amino acid exchanges in the Ho protein, revealing a known G223D mutation and also a novel G217R mutation, both of which abolished mating type switching. We generated stable MATa or MATα lines of four different Kveik yeasts, named Odin, Thor, Freya and Vör. Analyses of bud scar positions in these strains revealed both axial and bipolar budding patterns. However, the ability of Freya and Vör to form viable meiotic offspring with haploid tester strains demonstrated that these strains are haploid. Fermentation analyses indicated that all four yeast strains were able to ferment maltose and maltotriose. Odin was found to share not only mutations in the HO gene, but also inactivating mutations in the PAD1 and FDC1 genes with lager yeasts, which makes this strain POF-, i.e. not able to generate phenolic off-flavours, a key feature of lager yeasts. These haploid ale yeast-derived strains may open novel avenues also for generating novel lager yeast strains by breeding or mutation and selection utilizing the power of yeast genetics, thus lifting a block that domestication of lager yeasts has brought about. Key points: • Haploid Kveik ale yeasts with stable MATa and MATα mating types were isolated. • Heterothallic strains bear mutant HO alleles leading to a novel inactivating G217R amino acid change. • One strain was found to be POF- due to inactivating mutations in the PAD1 and FDC1 gene rendering it negative for phenolic off-flavor production. • These strains are highly accessible for beer yeast improvements by conventional breeding, employing yeast genetics and mutation and selection regimes. [ABSTRACT FROM AUTHOR]

Saccharomycopsis yeasts are natural organic sulfur auxotrophs due to lack of genes required for the uptake and assimilation of sulfate/sulfite. Starvation for methionine induces a shift to a predatory, mycoparasitic life strategy that is unique amongst ascomycetous yeasts. Similar to fungal plant pathogens that separated from Saccharomycopsis more than 400 million years ago, a specialized infection structure called penetration peg is used for prey cell invasion. Penetration pegs are highly enriched with chitin. Here we demonstrate that an ancient and conserved MAP kinase signaling pathway regulates penetration peg formation and successful predation in the predator yeast S. schoenii. Deletion of the MAP kinase gene SsKIL1, a homolog of the Saccharomyces cerevisiae ScKSS1/ScFUS3 and the rice blast Magnaporthe oryzae MoPMK1 genes, as well as deletion of the transcription factor SsSTE12 generate non-pathogenic mutants that fail to form penetration pegs. Comparative global transcriptome analyses using RNAseq indicate loss of the SsKil1-SsSte12-dependent predation response in the mutant strains, while a methionine starvation response is still executed. Within the promoter sequences of genes upregulated during predation we identified a cis-regulatory element similar to the ScSte12 pheromone response element. Our results indicate that, re-routing MAP-kinase signaling by re-wiring Ste12 transcriptional control towards predation specific genes contributed to the parallel evolution of this predacious behaviour in predator yeasts. Consequently, we found that SsSTE12 is dispensable for mating. Author summary: Saccharomycopsis yeasts represent a unique yeast genus of predator yeasts that attack and kill fungal prey cells. This necrotrophic mycoparasitism is initiated upon starvation, preferably for organic sulphur/methionine as predator yeasts are methionine auxotrophs. We have characterized morphological and cellular features of predation using time lapse fluorescence microscopy. This indicated prey sensing, chitin enrichment of penetration pegs used in the attack of prey cells and coordination of predation with the cell cycle resulting in cessation of daughter cell growth and a block in mitosis during predation. A predator yeast attack from the onset of penetration peg formation to the disappearance of the nuclear GFP-signal in the Saccharomyces cerevisiae prey took ~25 minutes. We identified the MAP kinase KIL1 and its putative target STE12 as key virulence genes that govern penetration peg formation. Comparative transcriptomics of mutant strains with the wild type identified specific hunger and predation responses and associated genes. Predation genes were found to harbor a conserved DNA element in their promoters. These findings can aid in deepening our understanding of necrotrophic predator-prey interactions. [ABSTRACT FROM AUTHOR]

Despite the increasing number of publications on non-conventional yeasts (NCYs), many areas in this field remain poorly understood, making the examination of these strains important for determining their potential in wine fermentations. The amino acid metabolic pathways involved, particularly the catabolic Ehrlich pathway but also anabolic pathways such as the leucine biosynthesis pathway, are crucial for producing high-value aroma compounds that contribute to the final flavour of wine. We examined the potential use of Saccharomycopsis fermentans in wine fermentations. We selected mutant strains resistant to the toxic compound trifluoro-leucine (TFL), verified mutations in the SfLEU4 gene, and characterized the ability of the resulting strains to contribute to fermentation bouquets. Resistance to TFL relieves feedback inhibition in the leucine biosynthesis pathway and resulted in increased leucine biosynthesis. Concomitantly, the S. fermentans TFL-resistant mutants generated increased amounts of isoamyl alcohol and isovalerate during wine fermentation. Selection of TFL-resistant strains thus provides a generally applicable strategy for the improvement in NCYs and their utilization in co-fermentation processes for different grape must varieties. [ABSTRACT FROM AUTHOR]

Hybrid formation and introgressions had a profound impact on fermentative yeasts domesticated for beer, wine and cider fermentations. Here we provide a comparative genomic analysis of a British cider yeast isolate (E1) and characterize its fermentation properties. E1 has a Saccharomyces uvarum genome into which ~102 kb of S. eubayanus DNA were introgressed that replaced the endogenous homologous 55 genes of chromosome XIV between YNL182C and YNL239W. Sequence analyses indicated that the DNA donor was either a lager yeast or a yet unidentified S. eubayanus ancestor. Interestingly, a second introgression event added ~66 kb of DNA from Torulaspora microellipsoides to the left telomere of SuCHRX. This region bears high similarity with the previously described region C introgression in the wine yeast EC1118. Within this region FOT1 and FOT2 encode two oligopeptide transporters that promote improved nitrogen uptake from grape must in E1, as was reported for EC1118. Comparative laboratory scale grape must fermentations between the E1 and EC1118 indicated beneficial traits of faster consumption of total sugars and higher glycerol production but low acetic acid and reduced ethanol content. Importantly, the cider yeast strain produced high levels of fruity ester, including phenylethyl and isoamyl acetate. [ABSTRACT FROM AUTHOR]

All laboratories dealing with microbes have to develop a strain maintenance regime. While lyophilization based on freeze-drying may be feasible for large stock centers, laboratories around the world rely on cryopreservation and freezing of stocks at -80 °C. Keeping stocks at these low temperatures requires investments of several thousand kW/h per year. We have kept yeast stocks for several decades at room temperature on agar slants in glass reagent tubes covered with vaspar and sealed with cotton plugs. They were part of the Geisenheim Yeast Breeding Center stock collection that was started in the 19th century, well before -80 °C refrigeration technology was invented. Of these stocks, 60 tubes were analyzed and around one-third of them could be regrown. The strains were typed by sequencing of rDNA PCR fragments. Based on BlastN analyses, twelve of the strains could be assigned to Saccharomyces cerevisiae, two to S. kudriavzevii, and the others to Meyerozyma and Candida. The strains were used in white wine fermentations and compared to standard wine yeasts Uvaferm/GHM (Geisenheim) and Lalvin EC1118. Even with added nitrogen, the strains exhibited diverse fermentation curves. Post-fermentation aroma analyses and the determination of residual sugar and organic acid concentrations indicated that some strains harbor interesting flavor characteristics, surpassing current standard yeast strains. Thus, old strain collections bear treasures for direct use either in wine fermentations or for incorporation in yeast breeding programs aimed at improving modern wine yeasts. Furthermore, this provides evidence that low-cost/long-term culture maintenance at zero-emission levels is feasible. [ABSTRACT FROM AUTHOR]