gene dosage influences the functional attributes of de novo lager...

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

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Background

Pale lager is the most popular beer style worldwide

Cold fermentation and clean flavour profile

Made with lager yeast: Saccharomyces pastorianus

© Joel Olives

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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)

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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

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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)

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1

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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%

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100%

0 48 96 144 192 240 288 336Fermentation time (hours)


Maltose Maltotriose

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Flocculation & ethanol tolerance

0

10

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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


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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

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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

15P 25P

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Aroma compounds (15 °P) – acetate esters

© Mary Siebert

© bitsorf

a

b b

cd

0.0

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Beer

mg/

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3-methylbutyl acetate

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b

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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

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Beer

mg/

L

Ethyl hexanoate

a,b

a,c

b

a,c

c

0.0

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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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0

500

1000

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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

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2.0

2.5

3.0

Beer

mg/

L

3-methylbutyl acetate

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02000400060008000

100001200014000160001800020000

24 hr 33 % 60 %

FLU

Uni

ts

Sc-EEB1

0

5000

10000

15000

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25000

24 hr 33 % 60 %

FLU

Uni

ts

Se-EEB1

0.0

0.1

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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

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0.5

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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

gene dosage influences the functional attributes of de novo lager...

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