Isolation and characterization of haploid heterothallic beer yeasts

Abstract

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.

Keywords: HO gene; Breeding; Domestication; Fermentation; Lager yeast; Ploidy.

Fig. 1

Life cycle of Saccharomyces cerevisiae. A Haploid a and α cells may proliferate by budding or interact via pheromone signalling to mate and form a/α diploid cells. Diploid cells proliferate or under adverse conditions develop into asci, enter meiotic divisions and form four ascospores per ascus, two with a and α mating type each. An a cell is determined by MATa and an α cell by MATα, while diploids carry both mating type loci and, hence, are MATa/α. B In haploid mother cells, the Ho endonuclease initiates a mating type switch by introducing a double-strand break (DSB) at the MAT locus in the G₁ phase of the cell cycle. This form of homothallism allows rapid diploidization of a colony derived from a single spore in the wildtype (Haber 2012)

Fig. 2

Halo assay for mating type assessment. A Use of pheromone-sensitive MATa bar1 or MATα sst2 tester strains in halo assays with query strains. MATα query strains will induce larger halos in tester strains due to their more diffusible α-factor. Halos in MATa tester strains tend to be very small. B A selection of lager yeast spore clones is shown (numbers correspond to GYBC strain collection numbers) that induced halos when used against the MATα tester strain. C The mating type configuration of these strains was tested in Mating type PCR/gel electrophoresis and confirmed to be MATa in all cases. Spore clones of three lager yeast strains, W164, W195 and W34/70, were tested. MATa and MATα bands (right lanes) were used as controls; size marker in the left lane

Fig. 3

Isolation of heterothallic Kveik spore clones. A–D Four halo assay plates inoculated with 10–11 query strains each are shown. Query strains that induced halos are encircled. Strain assignments were as follows: 1, Odin; 2, Thor; 3, Freya; and 4, Vör. Isolate numbers correspond to samples in E. E Gel image of mating type PCR reactions assigning either MATa or MATα to the spore clones (also indicated by their GYBC number, see Table 1). MATa and MATα bands (right lanes) were used as controls; size marker in the left lane. Note: halo assay results and MAT-PCR diagnostics confirm genotype-matched phenotype in all strains

Fig. 4

Analysis of HO gene sequences of heterothallic strains. A The S288C HO allele is shown with its mutations resulting in amino acid changes. The G223S amino acid exchange, due to a G > A transition, abolishes the Ho function in this strain. B Sequence analyses of the HO genes of the indicated heterothallic Kveik yeasts. Chromatograms are shown covering the region 216–223 of the Ho protein. Mutations resulting in amino acid changes are highlighted. C Alignment of Ho protein sequences of S. carlsbergensis (CBS 1513), Odin, Freya (identical to Thor and Vör) and S288C. Matching residues are shaded in black. Residues differing from Freya are shaded with solid deep magenta. Note: Odin’s Ho sequence matches that of lager yeast. The alignment was generated with DNASTAR MegAlign V12.1.0. Ho proteins of S288C and CBS 1513 were obtained from accession numbers NM_001180287 and AZCJ01000000, respectively

Fig. 5

Budding patterns in heterothallic strains. A Schematic drawing of haploid and diploid S. cerevisiae cells highlighting the position of chitin rings/bud scars as remnants of previous rounds of cell division. Budding in haploid cells is restricted to one pole (the proximal pole with which the cell was connected to its mother), while bud site selection in diploid cells can utilize both poles. B–F Representative fluorescence microscopy images of calcofluor-stained cells of the indicated strains clearly distinguish D–F axial and B, C bipolar budding patterns

Fig. 6

Breeding of heterothallic strains with haploid tester strains. A, B Freya and Vör were selected as MATα and MATa strains and mated with compatible BY-strains using marker-assisted breeding and selection of zygotes. C, D Zygotes (Freya × B006 for dissection plate C; Vör × B013 for panel D) were sporulated and tetrads were dissected via micromanipulation. Four tetrads are numbered. E Mating type PCR diagnostics of zygotes and spore clones from complete tetrads. While zygotes showed both mating types as expected, tetrads of the crosses yielded a 2:2 segregation of MATa and MATα. MATa and MATα bands (right lanes in each panel) were used as controls; size marker in the left lanes of each panel. Spore clones are also indicated with their GYBC collection number. F Halo assays of spore clones of one tetrad each derived from the Freya × B006 and Vör × B013 crosses. Strains that elicited a halo in the tester strain background are encircled, and strain identifiers are listed

Fig. 7

Fermentation analyses of heterothallic strains. A–D Fermentation curves of the indicated heterothallic Kveik strains (solid lines) compared to their parental strains (dashed lines). Curves are based on the cumulative loss of CO₂ measured in daily intervals. Fermentations were carried out in triplicate. Bars indicate a standard deviation of the mean