Kveik Brewing Yeasts Demonstrate Wide Flexibility in Beer Fermentation Temperature Tolerance and Exhibit Enhanced Trehalose Accumulation.

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.

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

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 ß-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 ß-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for ß-lyase activity. Selected phenolic off-flavour positive (POF +) hybrids were further sporulated to generate meiotic segregants with high ß-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.

A Unique Saccharomyces cerevisiae × Saccharomyces uvarum Hybrid Isolated From Norwegian Farmhouse Beer: Characterization and Reconstruction.

An unknown interspecies Saccharomyces hybrid, "Muri," was recently isolated from a "kveik" culture, a traditional Norwegian farmhouse brewing yeast culture (Preiss et al., 2018). Here we used whole genome sequencing to reveal the strain as an allodiploid Saccharomyces cerevisiae × Saccharomyces uvarum hybrid. Phylogenetic analysis of its sub-genomes revealed that the S. cerevisiae and S. uvarum parent strains of Muri appear to be most closely related to English ale and Central European cider and wine strains, respectively. We then performed phenotypic analysis on a number of brewing-relevant traits in a range of S. cerevisiae, S. uvarum and hybrid strains closely related to the Muri hybrid. The Muri strain possesses a range of industrially desirable phenotypic properties, including broad temperature tolerance, good ethanol tolerance, and efficient carbohydrate use, therefore making it an interesting candidate for not only brewing applications, but potentially various other industrial fermentations, such as biofuel production and distilling. We identified the two S. cerevisiae and S. uvarum strains that were genetically and phenotypically most similar to the Muri hybrid, and then attempted to reconstruct the Muri hybrid by generating de novo interspecific hybrids between these two strains. The de novo hybrids were compared with the original Muri hybrid, and many appeared phenotypically more similar to Muri than either of the parent strains. This study introduces a novel approach to studying hybrid strains and strain development by combining genomic and phenotypic analysis to identify closely related parent strains for construction of de novo hybrids.

Traditional Norwegian Kveik Are a Genetically Distinct Group of Domesticated Saccharomyces cerevisiae Brewing Yeasts.

The widespread production of fermented food and beverages has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a farmhouse brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavor negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally, kveik yeasts are reportedly high-temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>28°C) wort. Here, we characterize kveik yeasts from 9 different Norwegian sources via PCR fingerprinting, whole genome sequencing of selected strains, phenotypic screens, and lab-scale fermentations. Phylogenetic analysis suggests that kveik yeasts form a distinct group among beer yeasts. Additionally, we identify a novel POF- loss-of-function mutation, as well as SNPs and CNVs potentially relevant to the thermotolerance, high ethanol tolerance, and high fermentation rate phenotypes of kveik strains. We also identify domestication markers related to flocculation in kveik. Taken together, the results suggest that Norwegian kveik yeasts are a genetically distinct group of domesticated beer yeasts with properties highly relevant to the brewing sector.

Autophagy gene overexpression in Saccharomyces cerevisiae perturbs subcellular organellar function and accumulates ROS to accelerate cell death with relevance to sparkling wine production.

Traditional sparkling wines are produced by the refermentation of a base wine with yeast in the bottle followed by a critical period of aging. During the often lengthy aging process, yeast undergoes cell death and autolysis to release cellular compounds that over time ultimately contribute to the flavor and appearance of the product. While accelerating yeast autolysis for sparkling wine production has been the focus of several studies, employing overexpressed native yeast alleles for this purpose remains poorly explored. Here, we show that the overexpression of native yeast genes, specifically selected autophagic genes, results in accelerated cell death in nitrogen starvation and base wine refermentation. We show ATG3 or ATG4 overexpression has pleiotropic intracellular ramifications including reduced turnover of autophagic cargo, vacuolar fragmentation, abnormal accumulation of lipids, and accelerated accumulation of reactive oxygen species (ROS), all of which precede accelerated cell death. Our findings suggest that the increased expression of autophagy-related genes, such as ATG3 and ATG4, in industrial wine yeast can serve as a suitable marker or breeding strategy to accelerate the cell death and autolysis of wine yeast during sparkling wine production.

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7.

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.