Gene dosage influences the functionalattributes of de novo lager yeast hybrids
The 5th International Young Scientists Symposium onMalting, Brewing and DistillingApril 21-23, 2016 – Chico, California, USAKristoffer Krogerus
215/04/2016 2
Background
Pale lager is the most popular beer style worldwide
Cold fermentation and clean flavour profile
Made with lager yeast: Saccharomyces pastorianus
© Joel Olives
315/04/2016 3
Background
Lager yeast, S. pastorianus, is a natural hybrid between S.cerevisiae and S. eubayanus:
Saccharomyces cerevisiae(cold-sensitive, good fermentation)
Saccharomyces eubayanus(cold-tolerant, poor fermentation)
Saccharomyces pastorianus(Good fermentation performance at low temperature, lager brewing)
415/04/2016 4
Background
Poor diversity among the traditional lager yeastsBelong to one of two distinct lineages:
SaazFrohberg
Group 1(Saaz)
Group 2(Frohberg)
Weak fermentationVery cold-tolerantS. eubayanus genomedominates
Strong fermentationCold-tolerantS. cerevisiae genomedominates
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Background
Can we increase the diversity of lager yeast by creatingnew lager yeast hybrids?
Mating S. eubayanus with selected S. cerevisiae parents?
S. cerevisiae S. eubayanus
Lager yeast hybrid
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Our previous research
De novo lager yeast hybrids canoutperform their parent strains during fermentationproduce beer with higher concentrations of aroma compounds
715/04/2016 7
Controlling the properties of the hybrids
How does the contribution of the parental subgenomes affectimportant phenotypic traits?
Fermentation performanceAroma productionStress tolerance
Group 1(Saaz)
Group 2(Frohberg)
Weak fermentationVery cold-tolerantS. eubayanus genomedominatesAllotriploid
Strong fermentationCold-tolerantS. cerevisiae genomedominatesAllotetraploid
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Hybrid A2(allodiploid)
Hybrid B3(allotriploid)
Hybrid C4(allotetraploid)
Controlling the properties of the hybrids
S. cerevisiae A62 S. eubayanus C902
XX XX
XX XXX XXXX
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Aims
Compare the performance of these hybrids in 2-litrefermentations using 15 and 25 °P wort.How does the DNA content of the lager yeast hybrids affect
fermentation performancearoma productionresistance to harsh conditions of high gravity wort
Elucidate the relationship between gene expression andaroma formation in the strains.
© Joel Olives
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Creation
Incubation
Selection
Purification
Spores oryeast cellcultures of
parent strains
Sc A62 ura- Se C902 lys-
Confirmation
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Ploidy estimation
The DNA content of thehybrids were estimated withflow cytometry
Parent strains: diploid
Hybrid A2: diploid
Hybrid B3: triploid
Hybrid C4: tetraploid
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Sequencing
Each hybrid strain was analysed with Illumina pair-endDNA sequencingThese were compared to the parent genomes
Sc A62 sequence was obtained through the hybrid assemblyof Illumina and PacBio sequencing dataSe C902 sequence from previous studies (Baker et al. 2015)
ATGTGACTTACCATGTAGGCATAGCATGGATCATTAGCGCATGGGCATACGA
© Illumina
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Sequencing – allodiploid Hybrid A2
1 copy of each chromosome from both subgenomes
1n
XX
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Sequencing – allotriploid Hybrid B3
2 copies of each chromosome from S. cerevisiaeException in chromosome III (1 copy)
1 copy of each chromosome from S. eubayanus
1n
2n
XXX
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Sequencing – allotetraploid Hybrid C4
2 copies of each chromosome from S. cerevisiaeException in chromosome III (1 copy)
2 copies of each chromosome from S. eubayanusException in chromosome X (1 copy)
1n
2n
XXXX
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Growth at various temperaturesSc
A62Se
C902
Hybrids have a broadertemperature range ofgrowth
The same strain can beused for low and hightemperaturefermentations?
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Fermentation performance (15 °C, 15 °P)
0
1
2
3
4
5
6
7
0 48 96 144 192 240 288 336
Alco
hol(
%)
Fermentation time (hours)
A81062 (Sc)C12902 (Se)Hybrid C4Hybrid B3Hybrid A2
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Fermentation performance (15 °C, 25 °P)
0123456789
1011
0 48 96 144 192 240 288 336 384 432 480 528
Alco
hol(
%)
Fermentation time (hours)
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Sugar use (15 °P)
0 48 96 144 192 240 288 336Fermentation time (hours)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 48 96 144 192 240 288 336Fermentation time (hours)
A81062 (Sc)C12902 (Se)Hybrid C4Hybrid B3Hybrid A2
Maltose Maltotriose
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Flocculation & ethanol tolerance
0
10
20
30
40
50
60
70
80
90
100
Sc A62 Se C902 Hybrid C4 Hybrid B3 Hybrid A2
Flocculation potential (%)
Loss of flocculation ability in diploid Hybrid A2
0%
20%
40%
60%
80%
100%
5% EtOH 10% EtOH
Relative growth
A81062 (Sc)C12902 (Se)Hybrid C4Hybrid B3Hybrid A2
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Diacetyl
Highest diacetyl peaks were observed for diploid Hybrid A2and tetraploid Hybrid C4
No clear pattern between hybrid strains and the parent strainsMore rapid diacetyl removal in diploid hybrid A2
aa
aa bb
b
c
cc
d
d
d
d
d
0.0
0.2
0.4
0.6
0.8
1.0
1.2
33% 60% Beer
mg/
L
15 °P A81062 (Sc)C12902 (Se)Hybrid C4Hybrid B3Hybrid A2
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Aroma compounds (15 °P) – acetate esters
© Mary Siebert
© bitsorf
a
b b
cd
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Beer
mg/
L
3-methylbutyl acetate
a
b
c
a a
0.0
0.5
1.0
1.5
2.0
Beer
mg/
L
2-phenylethyl acetate
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Aroma compounds (15 °P) – ethyl esters
© arsheffield
© Première Moisson
a
b
c aa
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Beer
mg/
L
Ethyl hexanoate
a,b
a,c
b
a,c
c
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Beer
mg/
LEthyl octanoate
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Formation of aroma compounds
Condensationreaction betweenan alcohol andacyl-CoA
Acetate estersATF1ATF2
Ethyl estersEEB1EHT1 Pires et al. 2014
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Gene copy numbers
Chromosome
Genes located on
chromosomeHybrid A2 Hybrid B3 Hybrid C4
Scer Seub Scer Seub Scer Seub Scer Seub
II Sc-EHT1 1 1 2 1 2 2
IV Se-EHT1 1 1 2 1 2 2
VII Sc-ATF2 Se-ATF2 1 1 2 1 2 2
VIII Sc-BAT1 Se-ATF1 1 1 2 1 2 2
XV Sc-ATF1 Se-BAT1 1 1 2 1 2 2
XVI Sc-EEB1 Se-EEB1 1 1 2 1 2 2
Estimated from the sequence coverageSupported by qPCR analysis
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Transcriptional analysis
Gene expression: information from a gene is used in thesynthesis of a functional gene product.
Transcription is the first step of gene expression, in which aparticular segment of DNA is copied into mRNA
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Transcriptional analysis (TRAC)
Transcriptional analysis with the aid of affinity capture
Target mRNA
Magnet
Streptavidin-coated
magneticbeads
Fluorescent probes for detectionBiotinylated Poly-dTfor capture
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Transcriptional analysis (TRAC)
0
500
1000
1500
2000
2500
3000
3500
4000
24 hr 33 % 60 %
FLU
Uni
ts
Sc-ATF1 A81062 (Sc)C12902 (Se)Hybrid C4Hybrid B3Hybrid A2
0
1000
2000
3000
4000
5000
6000
24 hr 33 % 60 %
FLU
Uni
ts
Se-ATF1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Beer
mg/
L
3-methylbutyl acetate
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Transcriptional analysis (TRAC)
02000400060008000
100001200014000160001800020000
24 hr 33 % 60 %
FLU
Uni
ts
Sc-EEB1
0
5000
10000
15000
20000
25000
24 hr 33 % 60 %
FLU
Uni
ts
Se-EEB1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Beer
mg/
L
Ethyl hexanoate
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Correlation – Transcription vs Gene Copies
Positive correlation between gene copy numbers andtranscription levels
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
0 1 2
Nor
mal
ized
tran
scrip
tion
leve
l
Gene copy number
Pearson correlation: r = 0.88
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Correlation – Transcription vs Aroma
Multiple linear regression between maximum transcriptionlevels and aroma concentrations
Positive correlation observed for several genes and esters
0.5
1
1.5
2
2.5
3
0.5 1.5 2.5
Pred
icte
d
Measured
3-Methylbutyl acetate (mg/L)
Gene 3-methylbutylacetate
2-phenylethylacetate Gene Ethyl hexanoate
Sc-ATF1 NS NS Sc-EHT1 NSSe-ATF1 4.8 · 10-4 3.1 · 10-4 Se-EHT1 -3.8 · 10-5
Sc-ATF2 NS NS Sc-EEB1 1.6 · 10-5
Se-ATF2 5.8 · 10-4 3.7 · 10-4 Se-EEB1 NS
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Conclusions
Physiological properties of hybrid strains can be controlledto some extent with their ploidy and subgenome inheritanceWe observed an increased fermentation performance andstress tolerance with increased ploidy levelEster formation linked to genome contribution from parentstrains
Higher transcription levels of orthologous genes linked toaroma synthesis when present in higher copy numbers.
© Joel Olives
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Take-home message
Yeast hybrids often perform better than the parentsCreate strains with increased flavour diversityEndless possibilties for creating new lager yeastsNo genetic engineering involved
© Joel Olives
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Acknowledgements
VTT:Brian GibsonMikko ArvasSirpa JylhäFrederico MagalhãesMerja OjaVirve VidgrenAnnika Wilhelmson
Aila SiltalaArvi WilpolaEero Mattila
IRCAN:Gianni LitiMatteo De ChiaraJia-Xing Yue
Funding:Alfred Kordelin FoundationSvenska KulturfondenPBL Brewing LaboratorySABMillerAcademy of FinlandFP7 Marie-Curie ITNYEASTCELL © Jaryl Cabuco
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