Traditional Norwegian Farmhouse ale yeasts, also known as kveik, have captured the attention of the brewing community in recent years. Kveik were recently reported as fast fermenting thermo- and ethanol tolerant yeasts with the capacity to produce a variety of interesting flavor metabolites. They are a genetically distinct group of domesticated beer yeasts of admixed origin with one parent from the “Beer 1” clade and the other unknown. While kveik are known to ferment wort efficiently at warmer temperatures, their range of fermentation temperatures and corresponding fermentation efficiencies, remain uncharacterized. In addition, the characteristics responsible for their increased thermotolerance remain largely unknown. Here we demonstrate variation in kveik strains at a wide range of fermentation temperatures and show not all kveik strains are equal in fermentation performance and stress tolerance. Furthermore, we uncovered an increased capacity of kveik strains to accumulate intracellular trehalose, which likely contributes to their increased thermo- and ethanol tolerances. Taken together our results present a clearer picture of the future opportunities presented by Norwegian kveik yeasts and offer further insight into their applications in brewing.

Background Biofilm formation or flocculation is a major phenotype in wild type budding yeasts but rarely seen in laboratory yeast strains. Here, we analysed flocculation phenotypes and the expression of FLO genes in laboratory strains with various genetic backgrounds. Results We show that mutations in histone chaperones, the helicase RRM3 and the Histone Deacetylase HDA1 de-repress the FLO genes and partially reconstitute flocculation. We demonstrate that the loss of repression correlates to elevated expression of several FLO genes, to increased acetylation of histones at the promoter of FLO1 and to variegated expression of FLO11. We show that these effects are related to the activity of CAF-1 at the replication forks. We also demonstrate that nitrogen starvation or inhibition of histone deacetylases do not produce flocculation in W303 and BY4742 strains but do so in strains compromised for chromatin maintenance. Finally, we correlate the de-repression of FLO genes to the loss of silencing at the subtelomeric and mating type gene loci. Conclusions We conclude that the deregulation of chromatin maintenance and transmission is sufficient to reconstitute flocculation in laboratory yeast strains. Consequently, we propose that a gain in epigenetic silencing is a major contributing factor for the loss of flocculation phenotypes in these strains. We suggest that flocculation in yeasts provides an excellent model for addressing the challenging issue of how epigenetic mechanisms contribute to evolution.

Traditional Norwegian Farmhouse ale yeasts, also known as kveik, have captured the attention of the brewing community in recent years. Kveik were recently reported as fast fermenting thermo- and ethanol tolerant yeasts with the capacity to produce a variety of interesting flavour metabolites. They are a genetically distinct group of domesticated beer yeasts of admixed origin with one parent from the Beer 1 clade and the other unknown. While kveik are known to ferment wort efficiently at warmer temperatures, its range of fermentation temperatures and corresponding flavour metabolites produced, remain uncharacterized. In addition, the characteristics responsible for its increased thermotolerance remain largely unknown. Here we demonstrate variation in kveik strains at a wide range of fermentation temperatures and show not all kveik strains are equal in fermentation performance, flavour metabolite production and stress tolerance. Furthermore, we uncovered an increased capacity of kveik strains to accumulate intracellular trehalose, which likely contributes to its increased thermo- and ethanol tolerances. Taken together our results present a clearer picture of the future opportunities presented by Norwegian kveik yeasts and offer further insight into their applications in brewing.

Chromatin Assembly Factor I (CAF-I) plays a central role in the reassembly of H3/H4 histones during DNA replication. In S. cerevisiae CAF-I is not essential and its loss is associated with reduced gene silencing at telomeres and increased sensitivity to DNA damage. Two kinases, Cyclin Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), are known to phosphorylate the Cac1p subunit of CAF-I, but their role in the regulation of CAF-I activity is not well understood. In this study we systematically mutated the phosphorylation target sites of these kinases. We show that concomitant mutations of the CDK and DDK target sites of Cac1p lead to growth retardation and significant cell cycle defects, altered cell morphology and increased sensitivity to DNA damage. Surprisingly, some mutations also produced flocculation, a phenotype that is lost in most laboratory strains, and displayed elevated expression of FLO genes. None of these effects is observed upon the destruction of CAF-I. In contrast, the mutations that caused flocculation did not affect gene silencing at the mating type and subtelomeric loci. We conclude that dysfunctional CAF-I produces severe phenotypes, which reveal a possible role of CAF-I in the coordination of DNA replication, chromatin reassembly and cell cycle progression. Our study highlights the role of phosphorylation of Cac1p by CDK and a putative role for DDK in the transmission and re-assembly of chromatin during DNA replication.

The impermeability of the capsule to cryoprotectants (CPA) has been blamed for poor cryosurvival of equine embryos. We hypothesised that ethylene glycol, with its lower molecular weight and higher permeability, would be preferred over glycerol for equine embryos. The purpose of this study was to determine concentrations of glycerol and ethylene glycol within equine blastocoele fluid of Day 9 to 11 equine embryos following 30 min of exposure to a 1.5 M solution. Fourteen grade 1 embryos were recovered from May through September, using standard transcervical lavage techniques. Day of ovulation (Day 0) was determined as first appearance of the corpus luteum by daily transrectal ultrasonography, following insemination and administration of an ovulatory induction agent (Chorulon, 2500 IU; Intervet, Canada). Embryos were graded, measured, and washed in holding media (emP3TM Partnar Animal Health, Ontario, Canada), blocked by age, and randomly assigned to either 1.5 M ethylene glycol in holding solution (EG; n = 7) or 1.5 M glycerol in holding solution (Gly; n = 7) each containing 1% dimethyl sulfone as the internal standard, for 30 min at room temperature. Following CPA exposure, embryos were moved to a sterile, dry dish, all fluid was removed with a micropipette, and the dish dried with fine tissue. The embryonic capsule was punctured, and aspirated blastocoele fluid was analysed for CPA concentration by gas chromatography using a CP-3800 gas chromatograph (Varian Canada Inc., Mississauga, Ontario, Canada). The CPA concentration was determined by comparing the internal standard count to the accumulated CPA in millimoles per liter by gas chromatograph. A two-sample Kolmogorov-Smirnov test for equality of distribution was used because data were not normally distributed. Post-hoc 2-way t-test with unequal variances was used to compare mean effective CPA concentration across treatments, with significance set at P = 0.05. Collected embryos ranged from 1470 μm to 9240 μm in diameter, but there was no difference in embryo size between groups (P = 0.59). Mean concentrations of EG from blastocoele fluid were significantly higher than Gly (71.58 ± 18.9 mmol L–1 v. 50.71 ± 11.2 mmol L–1, respectively; P = 0.016). This represents 4.8 and 3.4% of available CPA, respectively. Mean EG concentrations by embryo age were not different (Day 9: 68.3 ± 23.1 mmol L–1; Day 10: 83.0 ± 15.4 mmol L–1; Day 11: 71.0 ± 21.9 mmol L–1; P = 0.75). Mean Gly concentrations by embryo age were not different (Day 9: 47.0 ± 12.7 mmol L–1; Day 10: 46.0 ± 7.1 mmol L–1; Day 11: 56.3 ± 13.6 mmol L–1; P = 0.35). The low concentrations of EG and Gly found in this preliminary study are in agreement with indirect evidence from other studies. Ethylene glycol reached higher concentrations inside the embryo than glycerol because of lower molecular weight and higher permeability. However, permeation appeared to be minimal for both agents despite exposure to solutions of high CPA molarity.