ORIGINAL RESEARCH
published: 16 September 2021
doi: 10.3389/ffunb.2021.733655
Frontiers in Fungal Biology | www.frontiersin.org 1September 2021 | Volume 2 | Article 733655
Edited by:
Chris Todd Hittinger,
University of Wisconsin-Madison,
United States
Reviewed by:
Matthias Sipiczki,
University of Debrecen, Hungary
Paula Gonçalves,
New University of Lisbon, Portugal
*Correspondence:
Brian Gibson
†Present address:
Jia-Xing Yue,
State Key Laboratory of Oncology in
South China, Collaborative Innovation
Center for Cancer Medicine, Sun
Yat-sen University Cancer Center,
Guangzhou, China
Specialty section:
This article was submitted to
Fungal Genomics and Evolution,
a section of the journal
Frontiers in Fungal Biology
Received: 30 June 2021
Accepted: 20 August 2021
Published: 16 September 2021
Citation:
Krogerus K, Magalhães F, Castillo S,
Peddinti G, Vidgren V, De Chiara M,
Yue J-X, Liti G and Gibson B (2021)
Lager Yeast Design Through Meiotic
Segregation of a Saccharomyces
cerevisiae ×Saccharomyces
eubayanus Hybrid.
Front. Fungal Biol. 2:733655.
doi: 10.3389/ffunb.2021.733655
Lager Yeast Design Through Meiotic
Segregation of a Saccharomyces
cerevisiae ×Saccharomyces
eubayanus Hybrid
Kristoffer Krogerus1,2, Frederico Magalhães1,2, Sandra Castillo1, Gopal Peddinti1,
Virve Vidgren1, Matteo De Chiara3, Jia-Xing Yue3†, Gianni Liti 3and Brian Gibson 1,4*
1VTT Technical Research Centre of Finland, Espoo, Finland, 2Department of Biotechnology and Chemical Technology,
Aalto University, School of Chemical Technology, Espoo, Finland, 3Institute for Research on Cancer and Ageing of Nice
(IRCAN), CNRS UMR 7284, INSERM U1081, University of Nice Sophia Antipolis, Nice, France, 4Brewing and Beverage
Technology, Technische Universität Berlin, Berlin, Germany
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.
Keywords: lager yeast, S. eubayanus, brewing, hybrid, tetraploid, sporulation
INTRODUCTION
Industrial lager yeast are derived from limited genetic stock. The Saccharomyces pastorianus
yeast strains used for lager beer fermentation are natural interspecies hybrids of S. cerevisiae and
S. eubayanus (Liti et al., 2005; Dunn and Sherlock, 2008; Nakao et al., 2009; Libkind et al., 2011;
Walther et al., 2014; Gallone et al., 2019; Langdon et al., 2019). Exactly when or how the original
Krogerus et al. Lager Yeast Design Through Meiosis
hybridization occurred has been debated but the yeast in use
today have originated from a limited number of strains which
were isolated from lager fermentations in Central Europe in
the late nineteenth century, when the use of pure cultures in
brewing became common (Gibson and Liti, 2015; Gallone et al.,
2019; Gorter De Vries A. R. et al., 2019). Lager strains originally
arose after one or possibly two hybridization events that probably
occurred when a domesticated strain of S. cerevisiae encountered
a contaminant S. eubayanus strain during a traditional ale
fermentation (Dunn and Sherlock, 2008; Walther et al., 2014;
Baker et al., 2015; Monerawela et al., 2015; Okuno et al., 2015;
Gallone et al., 2019; Salazar et al., 2019). A hybrid of the
two species would have benefited by inheriting the superior
fermentation performance of the ale strain, in particular the
ability to use the key wort sugar maltotriose (Gibson et al., 2013),
and the cryotolerance of the S. eubayanus strain (Gibson et al.,
2013; Hebly et al., 2015). No naturally-occurring strains of S.
pastorianus have been (knowingly) isolated since the nineteenth
century and it is unlikely that such strains will be found in
the future. In addition, being interspecies hybrids and mostly
aneuploid, existing strains exhibit low sporulation efficiency and
spore viability. As such, increasing diversity through meiotic
recombination and sexual mating, while possible, remains
challenging (Gjermansen and Sigsgaard, 1981; Sanchez et al.,
2012; Ota et al., 2018; Turgeon et al., 2021), in particular without
the aid of targeted genetic intervention (Ogata et al., 2011; Xu
et al., 2015; Alexander et al., 2016; Xie et al., 2018). Greater
functional diversity amongst lager brewing yeast would be of
advantage to the brewing industry, particularly as there now
exists a demand for more efficient resource utilization and an
increased trend for variety in beer characteristics (Kellershohn
and Russell, 2015).
The discovery of S. eubayanus (Libkind et al., 2011) has,
for the first time, allowed creation of de novo S. cerevisiae ×
S. eubayanus hybrids, and strains thus formed show strong
fermentation performance compared to the parental strains as
well as producing distinct flavor profiles (Hebly et al., 2015;
Krogerus et al., 2015, 2016, 2017; Mertens et al., 2015; Alexander
et al., 2016; Gorter de Vries et al., 2019). However, both
sporulation efficiency and spore viability of de novo interspecies
yeast hybrids are limited (Marinoni et al., 1999; Greig et al.,
2002; Sebastiani et al., 2002; Bozdag et al., 2021) just as they
are in the naturally occurring S. pastorianus strains. Post-zygotic
infertility is a defining feature of allodiploid yeast (Naumov,
1996). However, sterility is not necessarily an obstacle to a
hybrid’s fitness as clonal propagation allows such strains to
survive indefinitely, and potentially to take advantage of the
inherited phenotypes from both parental strains. The lager yeast
S. pastorianus is, in fact, the classic example of this phenomenon
(Kielland-Brandt and Nilsson-Tillgren, 1995). A number of
factors may contribute to hybrid sterility, though recent research
suggest that the inability of diverged chromosomes to undergo
recombination is a key factor (Bozdag et al., 2021). Regardless
of the mechanism involved, a consequence of sterility is that
increased diversity through normal chromosomal recombination
and cross-over during meiosis is not possible. However, there are
mechanisms by which fertility can be recovered. One such route
is endoreplication, whereby a sterile diploid hybrid doubles its
genome content to become an allotetraploid capable of producing
viable diploid spores (Sebastiani et al., 2002). The species barrier
can similarly be overcome by mating diploid parents to generate
an allotetraploid hybrid (Gunge and Nakatomi, 1972; Greig
et al., 2002; Krogerus et al., 2017; Charron et al., 2019; Naseeb
et al., 2021). Meiotic segregants derived from such crosses may
be expected to vary considerably due to the segregation of
orthologous genes from the parental strains and the creation
of unique biochemical pathways and regulatory mechanisms
(Landry et al., 2007), particularly if there exists a high degree of
heterozygosity in the parental strains.
In an effort to produce diverse strains of S. cerevisiae ×
S. eubayanus for use in the brewing industry, an allotetraploid
hybrid strain was here created through rare mating of an ale
strain and the type strain of S. eubayanus. This hybrid strain was
sporulated and four sibling spores derived from a single ascus
were isolated. The brewing fermentation performance of each
F1 meiotic segregant derived from this strain was characterized
and compared with that of its siblings and the original tetraploid
strain as well as the original diploid S. cerevisiae and S. eubayanus
parents. Two of the F1 meiotic segregants were found to be
tetraploids capable of producing viable spores. The isolation of
F2 ascus siblings from the best-performing strain was carried out
in order to further improve fermentation performance and flavor
production. In an effort to assess the genotypic and phenotypic
stability of the hybrids, one of the F2 spore clones was passaged
10 times in all-malt brewer’s wort and fermentation performance
of this serial repitched yeast slurry and three single cell cultures
derived from this population were assessed. Genome sequences
were analyzed to determine the main genetic changes (SNP,
CNV, structural variation) associated with the observed changes.
It is our contention that this approach is a feasible method
for selectively producing natural, genetically and phenotypically
diverse strains for the lager brewing industry.
MATERIALS AND METHODS
Yeast Strains
The two parental strains were S. cerevisiae VTT-A-81062 (VTT
Culture Collection, Finland), an industrial brewer’s yeast strain,
and the S. eubayanus type strain VTT-C12902 (VTT Culture
Collection, Finland; deposited as CBS12357 at CBS-KNAW
Fungal Biodiversity Centre). The industrial lager strain A-63015
was included to compare performance of de novo hybrids with
that of an industrial strain. An alloaneuploid hybrid (A-81062
×C12902) strain was created in a previous study (Krogerus
et al., 2017) and is deposited in the VTT Culture Collection
as A-15225. Meiotic segregants of this strain derived from an
individual ascus are deposited as A-15226, A-15227, A-15228 and
A-15229. Further meiotic segregants of the tetraploid strain A-
15227 are deposited as A-16232, A-16233, A-16234, A-16235.
Strain A-16235 was further passaged through 10 consecutive
batch fermentations in 15 ◦Plato wort, after which three single
cell isolates were isolated from the yeast slurry. These isolates are
here referred to as A235 G10 1-3. The strains will be referred to
by their “short codes” throughout the manuscript (Table 1).
Frontiers in Fungal Biology | www.frontiersin.org 2September 2021 | Volume 2 | Article 733655
Krogerus et al. Lager Yeast Design Through Meiosis
TABLE 1 | Strains used in this study and their spore viabilities, flocculation potential, and post-fermentation viability.
VTT Code Short Code Strain Spore
viability (%)
Flocculation
potential (%)
Post-
fermentation
viability (%)
A-81062 A62 S. cerevisiae ale strain 8 99 ±0.0 97 ±0.2
A-63015 A15 S. pastorianus lager strain 0 ND 92 ±0.4
C-12902 C902 S. eubayanus type strain 96 3.0 ±3.1 64 ±2.0
A-15225 A225 Hybrid of A-81062 and C-12902 55 92 ±1.3 76 ±2.0
A-15226 A226 Meiotic segregant of A-15225 63 96 ±1.1 71 ±3.4
A-15227 A227 Meiotic segregant of A-15225 95 4.2 ±0.1 76 ±0.5
A-15228 A228 Meiotic segregant of A-15225 0 88 ±0.8 98 ±0.1
A-15229 A229 Meiotic segregant of A-15225 0 2.8 ±4.0 95 ±0.1
A-16232 A232 Meiotic segregant of A-15227 78 0.6 ±0.1 94 ±0.1
A-16233 A233 Meiotic segregant of A-15227 0 1.0 ±4.9 93 ±0.2
A-16234 A234 Meiotic segregant of A-15227 78 0.0 ±3.1 17 ±2.1
A-16235 A235 Meiotic segregant of A-15227 86 6.9 ±4.1 6 ±0.6
NA A235 G10 1 Single cell isolate after 10 consecutive batch
fermentations with A-16235
NA ND 93 ±0.4
NA A235 G10 2 Single cell isolate after 10 consecutive batch
fermentations with A-16235
NA ND 93 ±0.1
NA A235 G10 3 Single cell isolate after 10 consecutive batch
fermentations with A-16235
NA ND 83 ±0.5
Spore viability was assessed by dissecting at least 16 tetrads by micromanipulation and observing colony formation after 4 days (YPM media, 24◦C).
ND, not determined; NA, not available.
Generation of Meiotic Segregants
The meiotic segregants of the tetraploid interspecific hybrid A-
15255 were obtained by first culturing A-15255 in YPM medium
(1% yeast extract, 2% peptone, 4% maltose) at 20◦C overnight.
It was then transferred to pre-sporulation medium (0.8% yeast
extract, 0.3% peptone, 10% glucose) at a starting OD600 of
0.3 and allowed to grow for 20 h at 20◦C. The yeast was then
washed with 1% potassium acetate and a thick suspension was
plated onto sporulation agar (1% potassium acetate and 2%
agar). The yeast was allowed to sporulate for 7 days at 25◦C.
Meiotic segregants were obtained by dissecting tetrad ascospores
treated with Zymolyase 100T (US Biological, USA) on YPD agar
with a micromanipulator. Spore viability was calculated based
on the amount of colonies formed from the dissection of up to
20 tetrads.
DNA Content by Flow Cytometry
Flow cytometry was performed on the yeast strains essentially as
described by Haase and Reed (2002) and Krogerus et al. (2016).
Briefly, the yeast strains were grown overnight in YPD medium
(1% yeast extract, 2% peptone and 2% glucose), after which
cells were fixed in 70% ethanol, treated with RNAse A (0.25 mg
ml−1) and Proteinase K (1 mg ml−1), stained with SYTOX Green
(2 µM; Life Technologies, USA), and their DNA content was
determined using a FACSAria cytometer (Becton Dickinson).
Measurements were performed on duplicate independent yeast
cultures, and 100,000 events were collected per sample during
flow cytometry.
Genome Sequencing and Analysis
Genome assemblies of both parent strains, S. cerevisiae A-
81062 and S. eubayanus C-12902, were first obtained in order
to create a reference genome to which sequencing reads from
the hybrid strains could be aligned. A long-read assembly of
S. eubayanus C-12902 was obtained from Brickwedde et al.
(2018).S. cerevisiae A-81062 has been sequenced previously by
our group using an Oxford Nanopore Technologies MinION
(Krogerus et al., 2019) and with Illumina technology (Krogerus
et al., 2016). Reads were accessed from SRR9129759 and
SRR2911435, respectively. Here, the long reads were de novo
assembled using the LRSDAY (version 1.4) pipeline (Yue and Liti,
2018). The initial assemblies were produced with smartdenovo
(available from https://github.com/ruanjue/smartdenovo) using
default settings. The assembly was first polished with medaka
(1.2.0; available from https://github.com/nanoporetech/medaka),
followed by two rounds of short-read polishing with Pilon
(version 1.23; Walker et al., 2014). Alignment of long reads
for medaka was performed with minimap2 (version 2.17-r941;
Li, 2018). The contigs in the polished assemblies were then
scaffolded with Ragout (version 2.3; Kolmogorov et al., 2014)
to S. cerevisiae S288C (R64-2-1). Because of the relatively high
heterozygosity of S. cerevisiae A-81062, two haplotypes were
further produced through phasing in WhatsHap (version 1.0;
Martin et al., 2016). Short reads were first mapped to above
scaffolds, and variants were called with FreeBayes (version 1.32;
Garrison and Marth, 2012). Long reads were also mapped to
the above scaffolds with minimap2, and the resulting VCF and
long-read BAM files were then passed to WhatsHap. The two
haplotypes of S. cerevisiae A-81062 were then extracted from
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Krogerus et al. Lager Yeast Design Through Meiosis
the resulting phased VCF. Assembly statistics are available in
Table S1 and Figure S1, while the A-81062 assembly is available
as Supplementary Data 1. A reference genome for the analysis of
the hybrid strains was produced by concatenating S. cerevisiae A-
81062 haplotype 1 with the obtained assembly of S. eubayanus
C-12902. The genomes of both parent strains were also
separately annotated using MAKER2 (Holt and Yandell, 2011) as
implemented in the LRSDAY pipeline. A horizontal gene transfer
event from Torulaspora microellipsoides in the S. cerevisiae A-
81062 genome was identified by mapping chromosome XV to
scaffold FYBL01000004.1 of T. microellipsoides CLIB830 (NCBI
GCA_900186055.1; Galeote et al., 2018) using minimap2 (with “-
x asm20” parameter). Alignments were visualized with the “pafr”
-package for R (https://github.com/dwinter/pafr).
The tetraploid hybrid A-15225 and all derived spore clones
and G10 isolates were sequenced by Biomedicum Genomics
(Helsinki, Finland). The sequencing of A-15225 has been
described previously in Krogerus et al. (2017) and reads
are available from NCBI-SRA SRR5141258 (referred to as
“Hybrid H1”). In brief, an Illumina KAPA paired-end 150
bp library was prepared for each strain and sequencing
was carried out with a NextSeq 500 instrument. The newly
described Illumina sequencing reads have been submitted to
NCBI-SRA under BioProject number PRJNA357993. Paired-
end reads from the NextSeq 500 sequencing were trimmed and
filtered with fastp using default settings (version 0.20.1; Chen
et al., 2018). Trimmed reads were aligned to the concatenated
reference genome described above using BWA-MEM (Li and
Durbin, 2009), and alignments were sorted and duplicates were
marked with sambamba (version 0.7.1; Tarasov et al., 2015).
Variants were jointly called in the 12 hybrid strains using
FreeBayes (version 1.3.2; Garrison and Marth, 2012). Variant
calling used the following settings: —min-base-quality 30—min-
mapping-quality 30—min-alternate-fraction 0.25—min-repeat-
entropy 0.5—use-best-n-alleles 70—p 2. The resulting VCF file
was filtered to remove variants with a quality score <1,000
and with a sequencing depth below 10 per sample using
BCFtools (Li, 2011). The haplotype blocks in the meiotic
segregants were obtained from the filtered VCF file by clustering
consecutive reference (haplotype 1) or alternative (haplotype 2)
allele calls using the vcf_process.pl script from https://github.
com/wl13/BioScripts. Variants were annotated with SnpEff
(version 4.5covid19; Cingolani et al., 2012). Visualizations were
performed in R using the ‘karyoploter’ package (Gel and Serra,
2017). Chromosome copy numbers were estimated based on
the median coverage in 10 kb windows across each contig in
the reference genome as calculated with mosdepth (version
0.2.6; Pedersen and Quinlan, 2018). Alignment of reads to the
right arm of S. cerevisiae chromosome XV was visualized with
samplot (https://github.com/ryanlayer/samplot).
Structural variations in the S. cerevisiae A-81062 parent strain
were identified using long sequencing reads. Long reads were
first aligned to the de novo assembly produced above using
NGMLR (version 0.2.7; Sedlazeck et al., 2018), after which
structural variations were called from the alignment using Sniffles
(version 1.0.12; Sedlazeck et al., 2018). Variants were annotated
with SnpEff (Cingolani et al., 2012). Gene ontology enrichment
analysis on the set of genes affected by heterozygous structural
variants was carried out with YeastMine (Balakrishnan et al.,
2012). Structural variations in the hybrid strains were estimated
from split and discordant Illumina reads using LUMPY (Layer
et al., 2014) and genotyped with svtyper (Chiang et al., 2015)
as implemented in smoove (version 0.2.6; https://github.com/
brentp/smoove). Variations in all 12 hybrid strains were jointly
called, and the resulting VCF was filtered to remove sites with an
imprecise breakpoint or a quality score <100 using BCFtools (Li,
2011).
Fermentations
Yeast performance was determined in fermentations carried out
at 15◦C in a 15 ◦Plato all-malt wort. Yeast was propagated
essentially as described previously (Krogerus et al., 2015) with
the use of a ‘Generation 0’ fermentation prior to the actual
experimental fermentations. The experimental fermentations
were carried out in duplicate, in 2-L cylindroconical stainless
steel fermenting vessels, containing 1.5 L of wort medium. The 15
◦Plato wort was produced at the VTT Pilot Brewery from barley
malt and was oxygenated to 15 mg L−1prior to pitching. Yeast
was inoculated at a rate of 5 g L−1to the wort. Wort samples were
drawn regularly from the fermentation vessels aseptically, and
placed directly on ice, after which the yeast was separated from
the fermenting wort by centrifugation (9000 ×g, 10 min, 1◦C).
Samples for yeast-derived flavor compounds and fermentable
sugars were taken from the beer.
Wort and Beer Analysis
The specific gravity, alcohol level (% v/v) and pH of samples
was determined from the centrifuged and degassed fermentation
samples using an Anton Paar Density Meter DMA 5000 M
(Anton Paar GmbH, Austria) with Alcolyzer Beer ME and pH
ME modules (Anton Paar GmbH, Austria). Concentrations of
fermentable sugars (glucose, fructose, maltose and maltotriose)
were measured by HPLC using a Waters 2695 Separation Module
and Waters System Interphase Module liquid chromatograph
coupled with a Waters 2414 differential refractometer (Waters
Co., Milford, MA, USA). An Aminex HPX-87H Organic Acid
Analysis Column (300 ×7.8 mm, Bio-Rad) was equilibrated with
5 mM H2SO4(Titrisol, Merck, Germany) in water at 55◦C and
samples were eluted with 5 mM H2SO4in water at a 0.3 ml/min
flow rate. Maltose and maltotriose consumption was calculated
by comparing the beer maltose and maltotriose concentrations
with the initial concentrations in the unfermented wort.
Yeast-derived flavor compounds were determined by
headspace gas chromatography with flame ionization detector
(HS-GC-FID) analysis. 4 ml samples were filtered (0.45 µm),
incubated at 60◦C for 30 mins and then 1 ml of gas phase was
injected (split mode; 225◦C; split flow of 30 ml min−1) into a
gas chromatograph equipped with a FID detector and headspace
autosampler (Agilent 7890 Series; Palo Alto, CA, USA). Analytes
were separated on a HP-5 capillary column (50 m ×320 µm
×1.05 µm column, Agilent, USA). The carrier gas was helium
(constant flow of 1.4 ml min−1). The temperature program
involved 50◦C for 3 min, 10◦C min−1to 100◦C, 5◦C min−1to
140◦C, 15◦C min−1to 260◦C and then isothermal for 1 min.
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Krogerus et al. Lager Yeast Design Through Meiosis
Compounds were identified by comparison with authentic
standards and were quantified using standard curves. 1-Butanol
was used as internal standard.
Yeast Analysis
The yeast dry mass content of the samples (i.e. yeast in
suspension) was determined by washing the yeast pellets
gained from centrifugation with 25 ml deionized H2O and then
suspending the washed yeast in a total of 6 ml deionized H2O.
The suspension was then transferred to a pre-weighed porcelain
crucible, and was dried overnight at 105◦C and allowed to cool
in a desiccator before the change of mass was measured. Yeast
viability was measured from the yeast that was collected at the
end of the fermentations using a Nucleocounter R
YC-100TM
(ChemoMetec). Flocculation of the yeast strains was evaluated
using a modified Helm’s assay (D’Hautcourt and Smart, 1999).
The zero-trans maltose and maltotriose uptake rates were
assayed using [U-14C]-maltose and [U-14C]-maltotriose as
described by Lucero et al. (1997). Yeast strains were grown in YP-
Maltose at 20◦C to an OD600 between 4 and 8 prior to uptake
measurement. Yeast was harvested by centrifugation, washed
with ice-cold water and then with ice-cold 0.1 M tartrate-Tris (pH
4.2), and finally resuspended in the same buffer at a concentration
of 200 mg of fresh yeast ml−1. The uptake rate was determined
at 20◦C using 5 mM of [U-14C]-maltose (ARC 488, American
Radiolabeled Chemicals Inc., St. Louis, MO, USA) or [U-14C]-
maltotriose (ARC 627, American Radiolabeled Chemicals Inc.,
St. Louis, MO, USA) in 0.1 M tartrate-Tris (pH 4.2) with 1 min
incubation time. [U-14C]-maltotriose was repurified before use
as described by Dietvorst et al. (2005).
Data and Statistical Analysis
Data and statistical analysis on the fermentation and yeast
data was performed with R (http://www.r-project.org/). One-
way ANOVA and Tukey’s post hoc test was performed using the
“agricolae” package (De Mendiburu and Simon, 2015). Values
were considered significantly different at p<0.05. Heatmaps
were drawn with the “pheatmap” package (Kolde, 2015).
RESULTS
Hybrid Generation and Genomic Analysis
The set of 12 de novo hybrid strains used in this study were
generated according to Figure 1. The alloaneuploid interspecies
hybrid A225, from a cross between the S. cerevisiae A62 ale
strain and the S. eubayanus C902 type strain, was obtained with
‘rare mating’ in a previous study (Krogerus et al., 2017). The
hybrid is nearly allotetraploid, as its genome has a complete
autodiploid S. eubayanus sub-genome and an autoaneuploid S.
cerevisiae sub-genome (lacking one copy of chromosome III).
This interspecies hybrid sporulated efficiently and spores showed
a viability of 55%. A set of four F1 segregants (A226-A229), all
derived from the same ascus, were isolated. F1 segregant A227
also sporulated efficiently, and a set of four F2 segregants (A232–
A235) were derived from this strain. F2 segregant A235 was
further subjected to ten consecutive batch fermentations in 15
◦P wort (corresponding to ∼30–40 cells doublings), and three
single cell isolates (A235 G10 1-3) were randomly selected from
the resulting yeast population.
For the genomic analysis of the hybrid strains, a new de
novo assembly of parent strain S. cerevisiae A62 was produced
for use as reference genome. The genome of A62 has been
assembled previously using a hybrid strategy (assembly from
150 bp Illumina reads, and scaffolding with PacBio reads)
(Krogerus et al., 2016). Here, a long-read assembly was instead
produced with smart de novo using reads generated with the
Oxford Nanopore MinION from our previous study (Krogerus
et al., 2019). 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 DNA) and spanned
a genome size of 12.68 Mbp (assembly statistics available in
Table S1 and Figure S1). A total of 29,517 heterozygous single
nucleotide polymorphisms were detected, corresponding to a
heterozygosity of around 0.23%. The heterozygous SNPs were
phased in whatshap using the long sequencing reads, and the
two haplotypes were extracted. 90% of the heterozygous SNPs
(26,569) were phased into a total of 29 blocks (1.45 per scaffold).
The first haplotype was selected to be used as reference for the
S. cerevisiae A62 parent strain. The reference genome for the S.
eubayanus C902 parent strain was obtained from Brickwedde
et al. (2018). The genomes were separately annotated using the
MAKER-based pipeline in LRSDAY, and a total of 5945 and 5430
protein-coding genes were detected, respectively. For analysis
of the hybrid strains produced in this study, a concatenated
reference genome of S. cerevisiae A62 and S. eubayanus C902
was used.
Chromosome Copy Number Variation
Chromosome copy numbers of the F1 hybrid and derived
spore clones were estimated based on median coverage
of the sequencing reads and flow cytometry with SYTOX
Green-staining (fluorescence histograms available in Figure S2).
Diversity in both ploidy and individual chromosome copy
numbers were observed (Figure 2). The two parent strains have
been previously shown to be diploid, both carrying two copies of
all 16 chromosomes (Krogerus et al., 2016). The genome of the F1
hybrid A225 consisted of two copies of each chromosome from S.
cerevisiae and S. eubayanus. An exception was the S. cerevisiae
chromosome III with only one copy, likely related to the rare
mating. The mitochondrial genome in A225 and derived strains
was inherited from S. eubayanus.
The four F1 hybrid spores were found to include two
alloaneuploid (genome size approximately tetraploid) strains
(A226 and A227) and two allodiploid strains (A228 and A229).
The allodiploid strains contained one copy of each chromosome
from both S. cerevisiae and S. eubayanus (Figure 2). The
alloaneuploid F1 strains contained two copies of nearly all
chromosomes. Exceptions included chromosome I (three copies
from S. eubayanus in strain A227), chromosome III (no copy
from S. cerevisiae in A226 and A227, and an additional copy from
S. eubayanus in A227), chromosome IV (with an additional copy
from S. eubayanus in A227) and chromosome XII (four and three
copies of the S. eubayanus form in A226 and A227, respectively).
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Krogerus et al. Lager Yeast Design Through Meiosis
FIGURE 1 | Overview of the yeast strains generated in this study. The F1 hybrid A225 was generated through rare mating of S. cerevisiae A62 and S. eubayanus
C902. The F1 spore clones A226–A229 were isolated from a single ascospore derived from A225. The F2 spore clones A232–A235 were isolated from a single
ascospore derived from A227. The three stabilized F2 spore clones were isolated from yeast slurry collected after ten consecutive wort fermentations with A235.
As the F1 hybrid carried a total of three copies of chromosome III
(containing the MAT locus), the isolation of two diploid and two
approximately tetraploid spore clones from an ascus, suggests
that all four ascospores were originally diploid, but the two latter
spores were nullisomic for S. cerevisiae chromosome III and thus
hemizygous for mating type. These then self-conjugated to form
the approximately tetraploid spore clones.
The four F2 segregants derived from A227 were all
alloaneuploid. Two had genome sizes approximately diploid
(A232 and A233), while and the other two were approximately
tetraploid (A234 and A235). A232 and A233 contained one copy
of nearly all chromosomes from S. cerevisiae and S. eubayanus,
the exception being chromosomes III and XII for which only the
S. eubayanus was represented in two copies. The F2 segregants
A234 and A235 possessed two copies of nearly all the S. cerevisiae
and S. eubayanus chromosomes with the exception that S.
cerevisiae chromosome III was absent (three and two copies of the
S. eubayanus form were present in A234 and A235 respectively).
In addition, both strains contained four copies of S. eubayanus
chromosomes IV and XII (Figure 2). As with the formation
of the F1 spore clones, A227 carries a total of three copies of
chromosome III (all from S. eubayanus), suggesting two out of
four diploid F2 ascospores were monosomic for chromosome
III and thus able to self-conjugate to form the approximately
tetraploid F2 spore clones A234 and A235.
Further chromosome copy number variation was observed
in the G10 isolates of A235, and interestingly all three single
cell isolates exhibited different profiles (Figure 2). Compared
to A235, all three single cell isolates carried an additional two
copies of S. eubayanus chromosome III. Furthermore, A235 G10
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Krogerus et al. Lager Yeast Design Through Meiosis
FIGURE 2 | Chromosome copy numbers and ploidy of the parent and hybrid strains. Chromosome copy number variations (CNV) in the S. cerevisiae A-81062 (top)
and S. eubayanus C12902 (bottom) sub-genomes of the hybrid strains compared to the parent strains (the numbers inside the cells indicate the estimated absolute
chromosome copy number). A blue color indicates a chromosome loss, while a red color indicates a chromosome duplication compared to the parent strain (e.g., −1
corresponds to one less chromosome in the hybrid compared to the parent strain). NA, not available.
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Krogerus et al. Lager Yeast Design Through Meiosis
1 had lost both copies of S. cerevisiae chromosome XII, while
A235 G10 2 had lost two out of four copies of S. eubayanus
chromosome XII.
Single Nucleotide and Structural Variations
Recombination was observed within the parental sub-genomes
of the F1 spore clones. As the reference genome of S. cerevisiae
A62 was phased, recombination in the S. cerevisiae sub-genome
of the F1 spore clones could be easily observed by presence of
either of the two haplotype blocks (Figure 3). Such visualization
could not be produced for the S. eubayanus sub-genome because
of a considerably lower heterozygosity level (0.002%; Hebly et al.,
2015). Of the 24,726 heterozygous SNPs observed in the A225
F1 hybrid (24,117 and 609 in the S. cerevisiae and S. eubayanus
sub-genomes, respectively), 23,017 segregated in a 2:2 pattern
in the four F1 spore clones. Compared to A225, a total of 132
de novo SNPs were detected in the four F1 spore clones. Of
these, 22 were missense mutations and two conservative in-frame
insertions (Table S2). A 2:2 segregation pattern was observed
for many of these SNPs (i.e., mutation present in two out of
four spore clones), suggesting that the mutation might have been
heterozygous in the F1 hybrid, despite showing a 0/0 genotype
(i.e., only reference allele detected), and therefore not a true de
novo mutation.
A total of 1,726 heterozygous SNPs were observed in the
A227 F1 spore clone which was sporulated to produce the F2
spore clones A232–A235. However, a vast majority of these SNPs
remained heterozygous in all four spore clones (1,337), and
only 38 segregated in a 2:2 pattern. In contrast to A227, only
8de novo SNPs were detected in the four F2 spore clones. Of
these, seven were intergenic and one a silent mutation. Hence,
the four F2 spore clones were almost identical to A227 at a
single nucleotide level, suggesting that any phenotypic differences
between A227 and the four F2 spore clones are a result of
larger-scale genomic variations.
Among the three single cell isolates of A235 that had
undergone 10 consecutive batch fermentations in 15 ◦Plato wort,
a total of 33 de novo SNPs were found. Only three of these SNPs
were shared between all three single cell isolates. Of the 33 SNPs,
three were missense mutations, one was a conservative inframe
deletion, and one a conservative inframe insertion (Table S3).
The affected genes include PYC1 (YGL062W), encoding a
pyruvate carboxylase. Of the remaining, 20 were intergenic and
eight were silent mutations.
Structural variations (SVs) in the S. cerevisiae A62 parent
strain were estimated from the long reads using Sniffles. A total
of 94 heterozygous SVs were identified, including 67 deletions,
27 insertions, 3 inversions, 1 duplication and 1 translocation
(Supplementary Data 2). These SVs affected 18 genes, and
the following cellular component GO terms were significantly
enriched among the list: extracellular region (GO:0005576; p-
value 1.2e−5), anchored component of membrane (GO:0031225;
p-value 6.4e−4), fungal-type cell wall (GO:0009277; p-value
8.2e−4) and cell wall (GO:0005618; p-value 0.001). SVs in the
F1 hybrid and derived spore clones were estimated from split
and discordant Illumina reads using LUMPY through smoove.
A total of 39 SVs were detected across the 12 strains (F1
hybrid, F1 spore clones, F2 spore clones, and G10 isolates),
including 24 deletions, 2 duplications and 13 translocations
(Supplementary Data 3). 12 deletion calls in the S. cerevisiae
sub-genome of the F1 hybrid were supported by the SVs called
for the A62 parent strain using the long reads. Of the 39
SVs in the hybrids, only five were absent from the F1 hybrid,
suggesting few de novo SVs were formed during meiosis and
the 10 consecutive batch fermentations in wort. While there was
evidence of recombination within the S. cerevisiae sub-genome
in the F1 and F2 hybrids, no recombination between the sub-
genomes appears to have taken place, as indicated by the lack
of split reads mapping to chromosomes from both sub-genomes.
This phenomenon is common in allotetraploid hybrids and is
known as autodiploidisation (Sipiczki, 2018).
In addition to the above mentioned SVs in the S. cerevisiae
A62 parent strain, a heterozygous horizontal gene transfer
event was observed on the right arm of chromosome XV,
which contained an ∼155 kbp region derived from Torulaspora
microellipsoides (Figure S3). This region includes the shorter
65 kb HGT region C that was originally described in S. cerevisiae
EC1118 (Novo et al., 2009; Marsit et al., 2015) and is similar in
size to the one later observed in S. cerevisiae CFC (a brewing
strain) as a likely ancestral event (Peter et al., 2018). Because
of heterozygosity, only two of the F1 spore clones (A226 and
A229) carry this HGT region (Figure S4). The presence of
the HGT region C in wine yeast has been shown to improve
oligopeptide utilization during wine fermentations (Marsit et al.,
2015), yielding an advantage in nitrogen-limited media, but its
effect in wort fermentations remains unclear.
Phenotypic Variation in the Strain Breeding
Panel
A range of brewing-relevant industrial phenotypes were assessed
in the 12 de novo hybrids and the parent strains. These 22
phenotypes included consumption and uptake of maltose
and maltotriose, fermentation rate, flocculation, viability,
growth at 4 and 37◦C, and formation of 11 aroma-active
compounds. Extensive phenotypic variation was observed
between the strains (Figure 4). Both hierarchical clustering based
on Euclidean distance (Figure 4A) and principal component
analysis (Figures 4B,C) grouped the F1 hybrid in between the
parent strains, while F1 and F2 spore clones grouped around the
strain they were derived from (A225 and A227, respectively). As
has been observed in previous studies on de novo brewing yeast
hybrids (Mertens et al., 2015; Krogerus et al., 2016, 2018b), both
mid-parent and best-parent heterosis was observed among the
different hybrid strains and the various phenotypes.
Aroma Diversity
Interest toward beer with novel and diverse flavors is increasing
(Aquilani et al., 2015; Carbone and Quici, 2020; Gonzalez
Viejo and Fuentes, 2020), and the results here suggest that
hybridization and subsequent sporulation can give rise to
lager yeast strains with both enhanced and diverse production
of aroma-active compounds. 3-methylbutyl acetate, with its
banana- and pear-like aroma, is one of the most important yeast-
derived flavor compounds in beer (Pires et al., 2014). Here, we
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Krogerus et al. Lager Yeast Design Through Meiosis
FIGURE 3 | Haplotype blocks (red and blue) in the S. cerevisiae sub-genome of the F1 hybrid and the four F1 spore clones.
measured higher concentrations of this ester in the beer produced
with the F1 hybrid A225 compared to either of the parent strains
(Figure 4D). Of the four F1 spore clones, one (A227) produced
higher levels of 3-methylbutyl acetate than the F1 hybrid. The F1
strain A227 was chosen for further sporulation and spore clone
screening due to its high production of 3-methylbutyl acetate.
Two out of four F2 spore clones produced the highest levels
of 3-methylbutyl acetate among all tested strains, reaching 2.5-
fold higher levels than the most productive parent strain (S.
eubayanus C902). This ester was produced only at very low levels
by the S. cerevisiae A62 parent strain.
Similarly to 3-methylbutyl acetate, considerable variation
was observed for ethyl hexanoate formation. Ethyl hexanoate,
with its apple- and aniseed-like aroma, is another important
yeast-derived flavor compound in beer (Pires et al., 2014).
Again, the F1 hybrid produced higher concentrations of
this ester compared to either parent strain (Figure 4E).
Of the F1 spore clones, A227 again produced the highest
levels of ethyl hexanoate, while the highest levels among
all tested strains was observed in the four F2 spore clones
derived from A227. Two-fold higher ethyl hexanoate
levels were observed in the beers made from these strains
compared to the better parent strain (S. cerevisiae A62).
Low concentrations of this ester were produced by the S.
eubayanus C902 parent strain and the industrial control S.
pastorianus A15.
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Krogerus et al. Lager Yeast Design Through Meiosis
FIGURE 4 | Phenotypic variation in the parent strains and hybrids. (A) Heatmap depicting the variation of the 22 phenotypic traits in the parent strains, F1 hybrid, F1
spore clones and F2 spore clones. (B,C) Principal component analysis of the 22 phenotypic traits. (D) 3-methylbutyl acetate and (E) ethyl hexanoate concentrations in
the beers produced with the above 11 strains and a commercial lager yeast control. (F) The flocculation potential of the above 11 strains as measured by Helm’s test.
(G) The maximum fermentation rate observed among the above 11 strains and a commercial lager yeast control (S. pastorianus A15) during the wort fermentations.
(D-G) Values are means from two independent fermentations and error bars where visible represent the standard deviation. Values with different letters (a–j) above the
bars differ significantly (p<0.05) as determined by one-way ANOVA and Tukey’s test. ABV, alcohol by volume (%), M2, maltose, M3, maltotriose.
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Krogerus et al. Lager Yeast Design Through Meiosis
As 3-methylbutyl acetate and ethyl hexanoate formation was
strongly associated with the two parent strains, S. eubayanus
C902 and S. cerevisiae A62, respectively, hybridization yielded
a strain producing high levels of both. Interestingly, a strain
producing several-fold higher levels of both these esters could be
derived by selecting meiotic segregants. Highest concentrations
of ethyl hexanoate were seen with the four F2 hybrids. In the
case of 3-methylbutyl acetate, the highest concentrations 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 expected based on previous
studies with similar hybrids (Krogerus et al., 2015, 2016, 2017),
the alloaneuploid strain A225 fermented wort more rapidly and
completely than the parental strains (Figures 4A,G). Alcohol
level at the end of the hybrid fermentation was 6.7% (v/v)
compared to 5.7 and 4.9% for the ale and S. eubayanus
strain respectively. A direct comparison of the fermentation
performance of the F1 hybrid and four F1 sibling strains revealed
clear differences that were associated with ploidy. The maximum
fermentation rate of the approximately tetraploid F1 siblings
was slightly higher than that of the parental hybrid (Figure 4G).
Alcohol level was higher relative to the parent (∼6.5% compared
to 6.2%). Fermentation rates of the diploid strains were similar to
that of the parental hybrid in the early stage of the fermentation
(up to 72 h), but were lower thereafter. Final yields of alcohol
in the strains A228 and A229 were 4.2 and 4.4%, respectively.
Similarly to the F1 spore clones, the fermentation performance of
the F2 spore clones appeared to be associated with ploidy. While
little difference was seen in the maximum fermentation rates
(Figure 4G), due to similar performance early in fermentation,
the approximately tetraploid strains A234 and A235 finished
at higher alcohol levels (7.0 and 6.9%, respectively) compared
to the approximately diploid strains A232 and A233 (6.0 and
5.7%, respectively). Of the de novo hybrid strains, A225–A227 all
outperformed the industrial lager yeast A15 that was included as
a reference with respect to maximum fermentation rate.
Flocculation
The S. cerevisiae A62 parent showed strong flocculation, while
flocculation potential was low in the S. eubayanus C902 parent
strain. The F1 hybrid also showed comparably strong flocculation
relative to the parent strain, and interestingly two out of the
four F1 siblings showed strong flocculation, while the others
showed weak flocculation (Figure 4F). Flocculation potential was
not linked to the ploidy of the spore clones, suggesting that
the heterozygous genotype of the S. cerevisiae A62 parent may
be responsible. Indeed, a number of heterozygous SVs linked
with extracellular region and cell wall were identified, including
a 135 bp deletion in FLO5 and a 65 bp deletion in TIR2
(Supplementary Data 2), which could potentially explain this
loss of flocculation in half the spore clones. A227 and the F2 spore
clones and derived G10 isolates all exhibited weak flocculation.
The TIR2 deletion was identified from the short-read data, and
was present in spore clones A226 (strong flocculation) and A227
(weak flocculation), however the FLO5 deletion was not detected.
Spore Viability
Both the domesticated strains studied here had a low level
of sporulation and spore viability. In the A15 lager strain,
sporulation was not observed and in the S. cerevisiae A62 ale
strain, it was only observed at a low level (21%) and of these
only 8% were found to be viable. In contrast, the sporulation
efficiency of the S. eubayanus strain was high and spores were
generally viable (Table 1). Sporulation in the A225 alloaneuploid
strain was intermediate between the parents with spore viability
measured as 55%. In the F1 and F2 generation, sporulation
and spore viability was largely influenced by ploidy with spore
viability ranging from 0 to 95%. Diploid strains were found to
have low sporulation efficiency and to be sterile. An exception
was the diploid F2 spore clone A232, which had a spore viability
of 78% (Table 1). These results are consistent with previous work
on allotetraploid Saccharomyces hybrids, showing Saccharomyces
species are reproductively isolated by a double sterility barrier
(Pfliegler et al., 2012; Karanyicz et al., 2017; Sipiczki, 2018).
Phenotypic Stability of an F2 Spore Clone
The phenotypic stability of the three G10 isolates of the F2
segregant A235, isolated after 10 consecutive fermentations
in industry-strength all-malt wort, was assessed by comparing
the isolates and the G10 mixed population to A235. In wort
fermentations, the G10 mixed population did not perform as well
as the original A235 strain, despite a relatively rapid fermentation
rate in the first 72 h (Figure 5A). The final alcohol yield was 6.9%,
compared to 7.1% for the original strain. It was however clear that
the G10 population was phenotypically heterogenous in nature.
The three single cell isolates derived from the G10 population
showed clearly different capacities to ferment the wort. Weakest
performance was observed with isolate 2, best performance
with isolate 3 and an intermediate performance with isolate
1. Aroma formation was also affected by the repeated wort
fermentations. Significantly lower amounts of 3-methylbutyl
acetate were formed by the G10 population and single cell
isolates compared to A235 (Figure 5B), while ethyl hexanoate
levels in the G10 isolates were similar or slightly lower than
A235 (Figure 5C). Futhermore, while A235 was able to sporulate,
none of the three single cell isolates produced ascospores when
inoculated onto potassium acetate agar (Table 1).
DISCUSSION
Limited phenotypic and genetic diversity exists between
industrial lager yeasts (Okuno et al., 2015; Gallone et al., 2019;
Langdon et al., 2019). In this study, we sought to explore how
sporulation of a newly created tetraploid S. cerevisiae ×S.
eubayanus interspecies hybrid could be exploited to expand the
phenotypic diversity of this group. Rare mating was used to
produce a polyploid hybrid. This can occur, e.g., by inactivation
of one MAT locus or through spontaneous gene conversion to
produce parental strains that are homozygous for mating type
(MATa/MATa or MATα/MATα) (Gunge and Nakatomi, 1972;
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Krogerus et al. Lager Yeast Design Through Meiosis
FIGURE 5 | Fermentation performance of the G10 isolates and the mixed population. (A) The alcohol content (% volume) of the 15 ◦P wort fermented with the F2
spore clone A235, the tenth generation mixed population derived from it, and the three single cell isolates from the tenth generation population. (B) The 3-methylbutyl
acetate and (C) ethyl hexanoate concentrations in the beers produced with the above strains. Values are means from two independent fermentations and error bars
where visible represent the standard deviation. Values with different letters (a,b) above the bars differ significantly (p<0.05) as determined by one-way ANOVA and
Tukey’s test.
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Krogerus et al. Lager Yeast Design Through Meiosis
Greig et al., 2002; Sipiczki, 2018). In the current study, rare
mating appears to have been facilitated through the former
mechanism. Sequencing of the F1 hybrid suggests that one MAT
locus in the diploid parental S. cerevisiae cell was lost through
whole-chromosome deletion of chromosome III, effectively
producing a cell that was hemizygous for mating type. Similar
losses of the same chromosome have also recently been observed
in artificial S. cerevisiae ×S. kudriavzevii and Saccharomyces
kudriavzevii ×Saccharomyces uvarum hybrids (Karanyicz et al.,
2017; Morard et al., 2020). What induced the parental S.
eubayanus cell to engage in rare mating remains unclear. Loss
of one copy of S. cerevisiae chromosome III has previously
been observed in allotriploid and allotetraploid hybrids derived
from the A62 ale strain (Krogerus et al., 2016). The strain,
therefore, appears susceptible to this change and, as a result, is
particularly suitable for natural allopolyploid hybridization. To
what extent chromosome III loss is responsible for hybridization
in interspecies hybrids requires further investigation.
As observed in previous studies on allotetraploid yeast (Greig
et al., 2002; Sebastiani et al., 2002; Antunovics et al., 2005;
Pfliegler et al., 2012; Karanyicz et al., 2017; Szabó et al., 2020;
Naseeb et al., 2021) there appeared to be no post-zygotic barrier
to reproduction with the F1 hybrid investigated here. The ability
to produce viable spores among the F1 spore clones was also
limited to tetraploid strains (via endomitosis (Sebastiani et al.,
2002) or, as is most likely the case here, self-fertilization of
homo- or hemizygous diploid spores). The inability of allodiploid
F1 spore clones from allotetraploid hybrids to form viable
spores has been attributed to a second sterility barrier caused
by heterozygosity at the MAT locus (Pfliegler et al., 2012).
Interestingly, among the F2 spore clones, both diploid and
tetraploid strains produced viable spores. Antunovics et al.
(2005) showed persistent fertility of a presumed alloploid hybrid
over several generations, though in that case the fertility was
restricted to allotetraploid cells. The mechanisms that facilitate
this phenomenon are not yet known but appear to be unrelated
to chromosome pairing as fertility was not directly influenced
by ploidy (Greig et al., 2002). Further investigation is necessary
to elucidate the processes involved, and may even help to
clarify those processes that contribute to speciation. Marcet-
Houben and Gabaldón (2015) have, for example, suggested
that an ancient interspecies hybridization may have led to the
creation of the ancestral S. cerevisiae lineage. Regardless of
the mechanisms involved, generation of allotetraploid hybrids
appears to be potentially useful for generating diversity through
meiotic recombination (Bozdag et al., 2021; Naseeb et al., 2021).
Here, no evidence of recombination between the two parental
sub-genomes of the hybrid was observed, rather only within the
parental sub-genomes.
Industrial lager beer fermentation is currently dominated
by Frohberg-type S. pastorianus strains, and there exists little
diversity within the group (Gallone et al., 2019; Langdon et al.,
2019). Creating new flavor profiles, e.g. in response to the
increased consumer demand for higher product quality and beer
with novel and diverse flavors (Aquilani et al., 2015; Carbone
and Quici, 2020; Gonzalez Viejo and Fuentes, 2020), is hampered
by the low level of diversity amongst commercial brewing
yeast strains. Previous research has shown that interspecific
hybridization is an effective way of introducing new aromatic
diversity among lager yeasts (Krogerus et al., 2015; Mertens et al.,
2015; Nikulin et al., 2018; Turgeon et al., 2021). Not only can
distinct aroma profiles of different parent strains be combined,
but aroma formation is often improved compared to either of
the parents from heterosis. Here, we show that sporulation of
allotetraploid hybrids could be exploited to further improve
aroma production, as beer concentrations of two important
aroma-active esters 3-methylbutyl acetate 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 toward specific
desired traits. It must, however, be emphasized, that much of the
phenotypic variation observed here was likely due to segregation
and loss-of-heterozygosity in the heterozygous S. cerevisiae sub-
genome.
Phenotypic stability is an essential trait in any industrial
yeast and this is particularly relevant for interspecies hybrids
where genomes are known to be inherently unstable. Here, the
stability of the F2 spore clone A235 was assessed after consecutive
wort fermentations. The results showed clearly differences in
performance between A235 and the G10 population but also
between the single-cell cultures. Differences were evident mainly
for fermentation capacity, but G10 strains also showed altered
formation of aroma compounds. These differences were not
due to structural variation as no such changes were apparent.
There were however several CNV changes with respect to
chromosomes. The single-cell cultures all gained two extra copies
of S. eubayanus chromosome III. Isolate 1 lost both copies of the
S. cerevisiae chromosome XII, while Isolate 2 lost two copies of S.
eubayanus chromosome XII. Morard et al. (2019) also observed
that copy number gains of chromosome III resulted in increased
ethanol tolerance, possibly from increased expression levels of
stress-related genes located on it. Voordeckers et al. (2015) in a
study of ethanol adaptation also noted changes in the number of
these same chromosomes. In response to high ethanol, several
strains independently gained copies of one or both of these
chromosomes. The authors suggested that these changes may be
an early adaptive response to ethanol, which would be followed
by more refined changes with additional exposure. It may be that
the G10 yeast in this study are similarly showing signs of early
adaptation to ethanol, which reached up to and over 7% alcohol
by volume in these fermentations. This concentration of ethanol
almost completely inhibits the growth of Saaz-type lager yeasts
strains (Gallone et al., 2019). The higher cell viability of G10
populations is consistent with an improved tolerance, though the
exact relationship between these specific CNVs and phenotype
has yet to be resolved.
Genomic stability of brewing yeast is vital from an industrial
point-of-view. This is because, in contrast to other beverage
fermentations, brewing yeast is reused for multiple consecutive
fermentations. The instability that was demonstrated here for
the tetraploid F2 segregant A235, highlights the importance
of stabilizing de novo yeast hybrids before they are suitable
for industrial use. While instability is not a desirable trait for
industrial yeast, rapid genome resolution in interspecies hybrids,
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Krogerus et al. Lager Yeast Design Through Meiosis
such as that seen in this and other studies (Piotrowski et al.,
2012; Dunn et al., 2013; Lopandic et al., 2016; Peris et al., 2017;
Smukowski Heil et al., 2017), suggests that stable genomes may
evolve within a short time and, furthermore, that de novo hybrid
genomes may be amenable to directed evolution to improve their
industrial potential (Krogerus et al., 2018a; Gorter de Vries et al.,
2019). This opens up the possibility of further improving and
developing the strains in a targeted manner.
A key feature of the modern brewing market is a demand
for diversity in beer character. Until now brewers have satisfied
this demand through the creative use of malts and hops.
This study, and related investigations, have shown that there
is also significant potential to direct or fine-tune the flavor
profile of beers through the creation of novel brewing yeast
strains or modification of existing brewing yeast strains. Here, a
number of development steps were undertaken (hybridization,
sporulation, adaptation) to introduce diversity. It is clear
however that further improvement may be achieved through
the addition of even more developmental steps, e.g., further
rounds of sporulation, or evolutionary engineering. Importantly,
all stages in the strain development included here could feasibly
occur in nature. Strains thus produced are therefore suitable
for immediate application in brewing, with the proviso that
genome stabilization has occurred prior to application. Further
investigation is required to determine the dynamics of genome
stabilization following hybridization.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories
and accession number(s) can be found in the
article/Supplementary Material.
AUTHOR CONTRIBUTIONS
BG: conceived the study. KK and BG: designed experiments.
KK, FM, VV, and BG: performed experiments. KK: analysis of
experimental data. KK, SC, GP, MD, J-XY, and GL: analysis of
genome data. KK and BG: wrote the manuscript. All authors
contributed to the article and approved the submitted version.
FUNDING
Research at VTT was supported by the Alfred Kordelin
Foundation, Svenska Kulturfonden—The Swedish Cultural
Foundation in Finland, PBL Brewing Laboratory, the Academy
of Finland (Academy Project 276480). Research in GL lab was
supported by ATIP-Avenir (CNRS/INSERM), ARC (Grant
Number n◦PJA 20151203273), FP7-PEOPLE-2012-CIG (Grant
Number 322035), the French National Research Agency
(Grant Numbers ANR-13-BSV6-0006-01 and 11-LABX-0028-
01), Cancéropôle PACA (AAP émergence 2015) and DuPont
Young Professor Award. JXY was supported by a post-doctoral
fellowship from ARC (PDF20150602803).
ACKNOWLEDGMENTS
Eero Mattila and Niklas Fred are thanked for assistance
in the VTT Pilot Brewery, and Aila Siltala for skilled
technical assistance.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/ffunb.
2021.733655/full#supplementary-material
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Conflict of Interest: KK, FM, SC, GP, VV, and BG affiliated with VTT Technical
Research Centre of Finland Ltd were employed by them.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
The handling editor CTH declared a past co-authorship with the authors
MD and GL.
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