screening and breeding of yeasts leads to a whole new ... · •s. eubayanus most probably entered...
TRANSCRIPT
Screening and breeding of yeasts
leads to a whole new spectrum of
beers with diverse flavour profiles
Gino Baart EBC Brewing Science Group 10th Technical Meeting 2014
Vienna, Austria
Introduction
The Leuven Institute for Beer Research (LIBR)
is embedded in the University of Leuven and
unites four specialized KU Leuven laboratories
with a proven track record of expertise in beer-
and beverage research and development in one
unique research center.
2
3
Laboratory of Enzyme, Fermentation
and Brewing Technology (EFBT)
Prof. Guido Aerts
Prof. Luc De Cooman
Laboratory for Genetics and Genomics /
VIB laboratory for Systems Biology
Prof. Kevin Verstrepen
Malt-Beer Science Laboratory
Prof. Guy Derdelinckx
Laboratory for Process Microbial Ecology
and Bioinspirational Management
Prof. Bart Lievens
Prof. Kris Willems
Expertise
4
• Malting & Brewing Technology
• Enzyme & Fermentation Technology
• Basic & applied yeast genetics (incl. breeding)
• Bioinformatics & Biostatistics
• Microbial Ecology (meta-genomics)
• Molecular Diagnostics
• (Bio)chemical Analysis
• Applied Beer Science (Gushing, Flavour stability)
Mission
• Facilitate the transfer of innovative technologies,
knowledge and knowhow to the brewing industry
• Provide the gateway to participation in research
programs at national and international (EU) level
• Provide access to a multidisciplinary research
team of experts and state-of-the-art facilities
• Carry out cutting-edge fundamental and applied
research projects
5
6
Yeast research
• Fundamental
Why does yeast produce flavours?
• Applied
Development of industrial yeasts with superior
charachteristics (e.g. flavour production)
7
How to obtain superior yeast strains?
8
Steensels et al., FEMS Microbiol Rev, 2014
Exploring natural diversity
9
100
49
2
88
14
149 7
68
10 16
221
22
Cocoa
Lager
Dietetic supplements
Wine
Sake
Ale
Biofuel
Feral
Bakery
Reference & Lab strains
Non-Sacchs
Spirits
Total of 746 yeast strains
Collection of yeast biodiversity
10
Intensive phenotypic screening
Aroma production
Fermentation kinetics
Osmotolerance
Halotolerance
Temperature tolerance
…
Ethanol tolerance
Inhibitor tolerance (HMF)
Metal tolerance
Carbon sources (#15)
Drug tolerance (cyclohex.)
…
11
Intensive phenotypic screening
12
Control
Strains
Control 30°C - glucose
Fructose 30°C Fructose 39°C
Data analysis:
Example: Growth on fructose
13
Differentiate between yeasts on a genetic (DNA) level
“Phylogenetic” tree of yeast strains
Interdelta analysis (QiaXcel)
10
0
95
90
85
80
75
70
65
60
55
50
45
40
Interdelta analysis (QiaXcel)
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Ale
Ale
Ale
Ale
Ale
Ale
Ale
Ale
Ale
Ale
Ale
.
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.
Y128
Y130
Y127
Y131
Y133
Y129
Y134
Y3
Y2
Y4
Y132
DNA fingerprinting
14
Interdelta analysis (QiaXcel)
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95908580757065605550454035302520151050
Interdelta analysis (QiaXcel) Aroma Jan trial 3
Ace
tald
ehyd
e
Eth
yl a
ceta
te
Eth
yl p
ropi
onat
e
Isob
utyl
ace
tate
Eth
yl b
utyr
ate
Pro
pano
l
Isob
utan
ol
Isoa
myl
ace
tate
But
anol
Isoa
myl
alc
ohol
Eth
yl h
exan
oate
Eth
yl o
ctan
oate
Eth
yl d
ecan
oate
Phe
nyl e
thyl
ace
tate
Phe
nyl e
than
ol
Vaskar 04032014
Glu
cose
40%
Glu
cose
46%
Glu
cose
48%
Glu
cose
50%
Fruc
tose
40%
Fruc
tose
46%
Fruc
tose
48%
Fruc
tose
50%
Sor
bito
l 40%
Sor
bito
l 44%
Sor
bito
l 46%
Sor
bito
l 48%
Sor
bito
l 50%
NaC
l 500
mM
NaC
l 100
0mM
NaC
l 150
0mM
KC
l 100
0mM
KC
l 150
0mM
KC
l 200
0mM
LiC
l 10m
M
LiC
l 50m
M
LiC
l 100
mM
LiC
l 200
mM
Eth
OH
5%
Eth
OH
7%
Eth
OH
10%
Eth
OH
11%
Eth
OH
12%
Eth
OH
13%
24°C
37°C
39°C
HM
F 2g
/L
HM
F 3g
/L
HM
F 4g
/L
HM
F 5g
/L
Zn 1
mM
Zn 2
mM
Zn 3
.5m
M
Zn 5
mM
Zn 6
mM
Cd
0.25
mM
Cd
0.5m
M
Cu
0.1m
M
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Genotype
Flavor profile Stress tolerance
Phenotype
Database allows selection of specific strains
15
Ale
Lager
Wine
Sake
Spirits
Bioethanol
Wild
Bakery
31
x range
8x ran
ge
Iso
amyl
ace
tate
(p
pm
) Et
hyl
ace
tate
(p
pm
) Isoamyl acetate
Ethyl acetate
Acetate ester production is a variable trait
16
Phenotypic screening reveals correlations
Steensels et al., AEM, 2014
Production of compounds of the same group of molecules is correlated.
The production of specific acetate esters also correlates with the production of the respective higher alcohol that serves as a substrate to produce the ester.
17
It might be difficult to change the production of one
aroma compound without changing other compounds
that show a high positive correlation
Generate artificial diversity: Breeding
18
Steensels et al., FEMS Microbiol Rev, 2014
P1 P2
HYBRIDS
Isoamyl acetate (IA)
Ale
Lager
Wine
Sake
Spirits
Bioethanol
Wild
Bakery
31
x range
Iso
amyl
ace
tate
(p
pm
)
→ Y141 (Sake), Y354 (Ale) and Y397 (Ale) were selected as parental strains
Y141 Y354 Y397
Targeted breeding
→ IA production, fermentation performance, sporulation
19
Three parental strains
77 tetrads dissected
142 spores screened
29 spores selected
46 hybrids developed
(inbreds & outbreds) lab scale beer fermentations (21.5 °P wort, 20 ° C, 7 days)
46 hybrids were developed fermentation performance AND Isoamyl acetate production
20
Outbreeding hybrids 48% more IA production!
5.5 ABV
Steensels et al., AEM, 2014
21
Heterosis (hybrids outperform parents)
What about NEW lager yeasts?
• > 90% of the worldwide beer production
• Lager beers show limited aromatic
complexity and diversity
• 2 main reasons: o Basic/low cost recipe (malt- water- hop- yeast)
o Saccharomyces pastorianus is an interspecies hybrid
22
Liger (lion X tiger)
Zonkey (zebra X donkey)
Wholphin (false killer whale X dolphin)
Saccharomyces pastorianus
(S. cerevisiae X S. eubayanus)
Interspecies hybrids
Peppermint (spear mint X water mint)
Libkind et al., PNAS, 2011
23
• S. eubayanus most probably entered the brewing environment
500 to 600 years ago where it hybridized with S. cerevisiae.
• Only 2 types of industrial S. pastorianus strains can be traced
back to 1 or 2 individual hybridization events:
• SAAZ type S. pastorianus (Czech / Carlsberg group)
• FROHBERG type S. pastorianus (Dutch/Danish group)
• Limited genetic diversity limited phenotypic diversity
Dunn and Sherlock, Genome research, 2008; Walther et al., Genes Genomes Genetics,2014
The origin of lager yeast
24
Libkind et al., PNAS, 2011
25
Interdelta analysis (QiaXcel)
100
98
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Interdelta analysis (QiaXcel)
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.
Interdelta analysis (QiaXcel)
100
50
0
Interdelta analysis (QiaXcel)
X1137
X4037
X1011
X1046
X1031
X1077
X1091
X5501
X1128
X5003
X7065
X7067
Ale
Wine
Ale
Ale
Ale
Ale
Ale
Spirits
Ale
Sake
Feral (eubay.
Feral (eubay.
.
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* Relative to highest value of lager yeasts
Origin Strain
code
[isoamyl
acetate]*
[Ethyl
acetate]*
POF/
flocculation/…
Ale Y470 1.24 1.55 …
Wine Y184 1.83 1.10 …
Ale Y134 1.41 1.35 …
Ale Y245 2.08 1.65 …
Ale Y202 1.56 1.18 …
Ale Y354 1.13 0.88 …
Ale Y397 0.88 1.14 …
Spirits Y377 0.80 1.27 …
Ale Y458 2.58 1.68 …
Sake Y141 1.57 1.29 …
Feral Y565 0.24 0.58 …
Feral Y567 0.35 0.68 …
S. c
ere
vis
iae
S. e
ubayanus
Selecting parental strains for hybridization
# 133 # 139
2n
n
4n
S. eub
S. cer
Hybrid
2n
n
4n
S. eub
S. cer
Hybrid
S. eubayanus (# 2) S. cerevisiae (# 10)
Total: # 272
SAAZ type (nXn) FROHBERG type(2nXn)
26
Developing new interspecies hybrids
31 hybrids (12 °P wort, 16 °C) lab scale fermentations
Hybrids show heterosis for ethanol production
SAAZ-type FROHBERG-type
Interspecific hybrids (Sc X Seub2)
Interspecific hybrids (Sc X Seub1)
Y567 (Seub2)
Y565 (Seub1)
Corresp. S. cerevisiae
Parental strain
Frohberg-type S. pastorianus (ref)
Saaz-type S. pastorianus (ref)
27
Hybrids show a diverse aroma production (A)
SAAZ-type FROHBERG-type
Y567 (Seub2)
Y565 (Seub1)
Corresp. S. cerevisiae
Parental strain
Frohberg-type S. pastorianus (ref)
Saaz-type S. pastorianus (ref)
Interspecific Hybrids
SAAZ type S. pastorianus
FROHBERG type S. pastorianus
Hybrids increase diversity in aroma production
Some hybrids show heterosis for both
ethanol and isoamyl acetate production SAAZ-type FROHBERG-type
Interspecific hybrids (Sc X Seub2)
Interspecific hybrids (Sc X Seub1)
Y567 (Seub2)
Y565 (Seub1)
Corresp. S. cerevisiae
Parental strain
Frohberg-type S. pastorianus (ref)
Saaz-type S. pastorianus (ref)
Interspecific hybrids
(Sc X Seub2) Y567 (Seub2) S. Cerevisiae
parents
Frohberg-type S. pastorianus (ref)
Saaz-type S. pastorianus (ref)
Promising pilot scale results!
32
Upcoming conferences
33
Belgian Brewing Conference - Chair J. de Clerck
07- 08 September 2015, Leuven, Belgium
THE BEERS OF TOMORROW
Yeast research
• Fundamental
Why does yeast produce flavours?
• Applied
Development of industrial yeasts with superior
charachteristics (e.g. flavour production)
35
Why are flavours (i.e. esters) produced?
• Specific, dedicated genes are involved Verstrepen et al., AEM, 2003; Saerens et al., J Biol Chem, 2006
• It is costly
Breslow et al., Nature Methods, 2008
• In vitro, no benefit has been discovered
Saerens et al. , Microb Biotech, 2010
Let’s look at nature
• Yeasts are non-motile
• Yeasts are not dispersed by the wind
• Yeasts require a vector for dispersal
• Yeasts are co-isolated with insects
• Acetate esters are attractants/pheromones for various insects
Investigate whether acetate-ester production affects
attraction of fruit flies and yeast dispersal
ATF1-deletion affects Acetate-esters
38
Compound (ppm) p-value
Acetaldehyde 8.98 2.73 9.67 2.89 0.74
Ethyl acetate 14.54 1.53 6.45 0.43 5.20E-05*
Ethyl propionate 0.08 0.01 0.09 0.01 0.19
Ethyl isobutyrate 0.02 0 0.02 0 0.9
Propyl acetate 0.03 0.01 ND ND *
Isobutyl acetate 0.09 0.03 ND ND *
Ethyl Butyrate 20.24 3.26 20.4 2.23 0.94
Isobutanol 13.38 2.57 13.31 1.6 0.96
Isoamyl acetate 0.82 0.2 0.13 0.06 5.68E-04*
Butanol 55.45 2.21 57.67 5.22 0.53
Isoamyl alcohol 37.71 5.69 41.54 3.92 0.31
Ethyl hexanoate 0.13 0.03 0.18 0.01 0.04
Ethyl octanoate 1.08 0.23 1.14 0.13 0.66
Phenyl ethyl acetate 0.1 0.02 ND ND *
Phenyl ethanol 0.13 0.06 0.08 0.03 0.16
Ethanol (% v/v) 6.5 0.05 6.46 0.02 0.14
Acetate (g/l) 24.75 6.25 22.15 3.69 0.32
WT-Y182 atf1 -
Measure through fly-tracking
Cooperation with Neuroelectronics Research Flanders (NERF)
Air Air
Air Air
ΔATF1 Mutant Air
Wild Type Air
Air Air
Air Air
WT-strains are preferred over ΔATF1-mutants
Wild Type
ΔATF1
Complementation restores WT-phenotype
Fly neuronal responses
Measure response using GCamP
Genetically Engineered Protein
Fusion of: GFP
Calmodulin
M13
Allows Monitoring of Action Potentials
Wang et al. J Neuroscience, 2004
Akerboom et al., J Biol Chem, 2009
GCamP-imaging
Neuro responses in fruit flies
Overview
• ΔATF1 alters fly preferences for a yeast species
• Behavioural changes are due to differences in
acetate-ester production
• Does attraction of flies affect yeast dispersal?
• Is this true in nature?
Capture wild fruit flies from nature
Isolation of yeast on wild fruit flies
Wild Isolates are fruity!
Conclusion
Based on our data, we propose that acetate-
ester formation in yeasts has evolved as a
means to stimulate dispersal in nature through
insect vectors like fruit flies
Control 30°C - glucose
Ethanol 12% (v/v) Ethanol 13% (v/v)
Data analysis:
Example: Ethanol tolerance
52
Ethanol production is a variable trait
0
2
4
6
8
10
12
14
16
18
0 50 100 150 200 250 300
Alc
oh
ol
% (
%v
/v)
Strains
Ale Lager Wine Sake Spirits Bio-ethanol Feral Bakery Lab Chocolate
3x ran
ge
53
Origins of genetic variation in yeast
54
Improvement techniques using hybridization
55
Classic breeding yield fabulous results
Applied spore to spore mating technique
57
S. cerevisiae S. eubayanus
Sporulation
Random
Spore
Isolation
Initiate mating with
Micromanipulator
Make
couples Inspect
visually
S.
ce
revis
iae
S.
eu
ba
yanus
=hybrids
Conforming
hybrid nature