FEMS Yeast Research 2 (2002) 563^573

www.fems-microbiology.org

The dynamics of the Saccharomyces carlsbergensis brewing yeast

transcriptome during a production-scale lager beer fermentation
Kjeld Olesen  , Troels Felding, Claes Gjermansen, Jrgen Hansen
Carlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark
Received 14 March 2002; received in revised form 13 June 2002; accepted 2 August 2002
First published online 25 September 2002

Abstract
The transcriptome of a lager brewing yeast (Saccharomyces carlsbergensis, syn. of S. pastorianus), was analysed at 12 different time
points spanning a production-scale lager beer fermentation. Generally, the average expression rapidly increased and had a maximum value
on day 2, then decreased as the sugar got consumed. Especially genes involved in protein and lipid biosynthesis or glycolysis were highly
expressed during the beginning of the fermentation. Similarities as well as significant differences in expression profiles could be observed
when comparing to a previous transcriptome analysis of a laboratory yeast grown in YPD. The regional distribution of various expression
levels on the chromosomes appeared to be random or near-random and no reduction in expression near telomeres was observed.
5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Expression ; Gene regulation; Chromatin structure; Telomeres; Hierarchical clustering ; Transcriptome analysis ; Saccharomyces cerevisiae ;
Saccharomyces pastorianus

1. Introduction
During the process of beer fermentation, carbohydratecontaining wort is converted into alcohol-containing beer.
From the point of view of the yeast the growth conditions
change radically during this process. Prior to the fermentation the yeast is kept in storage tanks at a very high cell
density and basically without any nutrients. The yeast is
then transferred to fresh wort, which, due to a content of
approximately 100 g l31 of carbohydrates, subjects the
cells to high osmotic stress. The progress of fermentation
gradually causes new forms of stress for the cells in the
form of nutrient depletion and an increasing concentration
of alcohol. When all fermentable carbohydrates have been
utilised, the temperature is lowered from typically 11^14C
to 7^8C to mature the beer and, in some cases, to assist
harvesting of the yeast. Ultimately the yeast is collected
and transferred back to storage tanks. Presumably, the
yeast responds to all these changes in growth conditions

* Corresponding author. Tel. : +45 3327 5309 ; Fax: +45 3327 4764.
E-mail address : kol@crc.dk (K. Olesen).

by regulating the transcription of appropriate genes according to the given condition.

A higher degree of understanding of how the brewing
yeast senses and responds to the changing environment
during beer fermentation might be obtained by studies
of the transcriptome, the proteome and/or the metabolome. Transcriptome analysis has been extensively used
to analyse the transcriptional regulation response of Saccharomyces cerevisiae to various genetic and environmental conditions, such as the diauxic shift [1], the cell cycle
[2,3], sporulation [4], adaptive evolution [5], environmental
changes [6], phosphate accumulation [7], and change to a
non-fermentable carbon source [8]. With respect to transcriptome analysis, the lager type brewing yeast, Saccharomyces carlsbergensis (syn. of S. pastorianus), presents a
special challenge, as it is a hybrid between a S. cerevisiae
and another unknown Saccharomyces species. Thus, for
each gene there is usually a version derived from S. cerevisiae and a version derived from the other parental species. While the genes derived from the S. cerevisiae-like
parent have been found to be nearly 100% identical to
the published sequence of the S. cerevisiae S288C genome
[9^14] the genes derived from the other parental species
have been found to be only about 75^85% identical based
on the few genes that have been cloned and sequenced so
far [9^18]. Thus, it would obviously be desirable to be able

1567-1356 / 02 / $22.00 5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 5 5 - 1

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to distinguish between the expression of the two dierent

types of genes. However, as the sequence of most of the
non-cerevisiae-like genome is unknown, this is not possible
at present. Consequently, no matter which type of methodology one chooses, either oligonucleotide-based microarrays (Aymetrics) or DNA-based arrays (e.g. GeneFilters), mRNA from the non-cerevisiae-like part of the
transcriptome will either be undetectable (in case of a
low degree of identity) or it will be measured as part of
the expression from the S. cerevisiae-derived genes.
In spite of such shortcomings, a transcriptome analysis
of a lager brewing yeast during the time course of beer
fermentation will nevertheless provide valuable new insight into the cellular response to the various changes in
growth conditions. Here, we report such a study of the
transcriptome of a lager brewing yeast, using GeneFilter
arrays, spanning the whole fermentation process from the
yeast storage tank through inoculation and fermentation
of the wort and back to the storage tank.

2. Materials and methods

2.1. Strains and growth conditions
A production strain (S. carlsbergensis) was used for this
investigation. The strain will not be available to other
scientists while it is still being used as a production strain.
Yeast cells were harvested from storage or fermentation
tanks during a production fermentation of standard lager
beer. Thus, cells were grown in standard wort (14 Plato)
in 5000-hl cylindroconical fermentation tanks using standard conditions. Cells from 8 l of the ferment were harvested by centrifugation at 2000Ug for 10 min at 0C in
four 2-l centrifuge bottles. The supernatant was stored for
chemical analyses while cells were resuspended in one volume of supernatant and stored in 10-ml aliquots at
380C, after rapid freezing in a dry ice/ethanol bath.
2.2. RNA preparation
Total RNA was prepared from an equal amount of cells
taken each day of the fermentation, using the FastRNA
RED kit from Bio-101 (Qbiogene, Illkirch, France). The
quality of total RNA was ascertained by running aliquots
on 1% agarose gels. mRNA was puried from total RNA
using the PolyATtract mRNA Isolation System IV from
Promega (Madison, WI, USA).
2.3. Probe preparation and GeneFilter hybridisation
Radioactive cDNA probes were prepared from mRNA
puried from equal amounts of total RNA using the Gibco BRL cDNA Synthesis System (Life Technologies, Invitrogen Corp., Carlsbad, CA, USA) and [33 P]dCTP
(NEN, Boston, MA, USA) for labelling. For cells har-

FEMSYR 1511 11-11-02

vested during exponential growth phase the amount of

mRNA should correspond to approximately 250 ng, based
on assumed mRNA recovery and approximately 2% of the
total RNA being mRNA. The labelled cDNA was separated from unincorporated [33 P]dCTP by passage through
a Sephadex G50 NICK1 column (Amersham Pharmacia
Biotech AB, Uppsala, Sweden) and specic activity measured in a scintillation counter. GeneFilters (Cat. No.
GF100, Research Genetics, Huntsville, ON, Canada)
were prehybridised and hybridised at 42C in a Hybaid
Micro 4 hybridisation oven (Hybaid Ltd., Ashford, UK)
with 50% formamide, 6U SSC, 5U Denhardts solution
and 1% SDS, in a total volume of 5 ml. Prehybridisation
was for 2 h, and included 0.5 Wg ml31 poly-dA and 0.2 mg
ml31 denatured salmon sperm DNA as carriers. For each
hybridisation equal amounts of heat-denatured probe (approximately 5U108 cpm) was then added to the prehybridisation mixture, and hybridisation allowed to proceed for
15 h. The GeneFilters were washed in a plastic box with
slow agitation, twice at 50C with 2U SSC and 1% SDS
for 20 min, and once at 20C with 0.5U SSC and 1% SDS
for 15 min. Stripping of lters for rehybridisation was
performed by placing the lters in a 1-l glass beaker and
pouring in a boiling solution of 0.5% SDS. The solution
was allowed to cool slowly to room temperature and remaining radioactivity on the lters was measured regularly. When necessary, the procedure was repeated.
2.4. Data acquisition
GeneFilters were analysed by scanning exposed Storage
Phosphor Screens on a PhosphorImager (both Molecular
Dynamics, Sunnyvale, CA, USA). To enable quantitative
measurements of highly as well as weakly expressed open
reading frames (ORFs), exposures of 1, 6, 42 and 120 h
were obtained. The resulting PhosphorImager .gel les
were exported into 24-bit BMP les, that could subsequently be read by the analysis software. During this export the dynamic range of 10 000 of the PhosphorImager
.gel les is non-linearly converted into a dynamic range
of 256 according to the following formula : Y = X0:602 . The
resulting four 24-bit image les were analysed using a
proprietary software developed specically for this purpose. This software reads the intensity of each spot from
the highest exposure, where the intensity of that spot does
not exceed 90% of the dynamic range. The software also
takes into account the duration of the exposure, the halflife of the radioactive probe, the intensity of neighbouring
spots, local background intensity and the non-linear conversion described above. The output from the software is
an array of entries containing spot positions, an arbitrary
number that is a measure of the intensity of the spot, and
a ag indicating the relative intensity of neighbouring
spots. After generating a database of the intensity of all
spots for all days during the fermentation, this database
was merged with two databases containing spot positions

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K. Olesen et al. / FEMS Yeast Research 2 (2002) 563^573

versus SGDID numbers (gf100_nal_data_071201, Research Genetics, Invitrogen Corp.) and SGDID numbers
versus ORF name, gene name, ORF length, ORF homologies, genetic function and cellular process [19]. It should
be noted that prior to December 2001 the data le which
Research Genetics was supplying to describe the identity
of spots on the GeneFilters was based on partially incorrect annotations made by MIPS/SGD. Prompted by our
discovery of these errors, Research Genetics has in collaboration with SGD produced an updated version (gf100_nal_data_071201) which now identies spots based on the
unique SGDID number. The updated data le was used
for this investigation. The resulting data have been included in the Gene Expression Omnibus repository at
NCBI [20] with the accession number GSE79.
2.5. Hierarchical clustering
Clustering was performed using the programmes Cluster and Treeview [21] (both available at http://rana.lbl.gov/EisenSoftware.htm). Cluster was used for hierarchical
clustering while Treeview was used for visualisation of the
resulting clustered tree. Parameters used for Cluster are
described in the Fig. 11 legend.
2.6. Northern blotting
Electrophoretic separation of RNA was done on a 1.2%
agarose gel (1U MOPS buer, 6.5% formaldehyde, and
0.05 Wg ml31 ethidium bromide). Running buer was 1U
MOPS. RNA samples were prepared of 40 Wl denaturation
buer (50% formamide, 2.2 M formaldehyde, 1U MOPS),
9 Wl loading buer (5% Ficoll, 0.125% bromphenol blue,
0.125% xylene cyanol, 50% formamide) and 5^10 Wg RNA
in a volume of 10 Wl. Samples were denatured for 10 min
at 65C and chilled on ice until loading. After separation
of the RNA, an image was stored for use in normalisation
of the amounts of RNA loaded. Transfer of the RNA was
performed with conventional capillary blotting (over
night) using nylon membrane (Hybond-XL, Amersham
Biosciences, Sunnyvale, CA, USA) and 10USSC. UVirradiation (UV Stratalinker 1800, Stratagene, La Jolla,
CA, USA) was applied to cross-link the RNA to the membrane.
Probes for Northern blots were prepared using a Random Primed DNA labelling kit (Roche Applied Science,
Rotkreuz, Switzerland). Template for making probe was a
PCR fragment of the desired ORF and its promoter.
Amount of template DNA was approximately 10 Wg.
The membrane was soaked in 2U SSC and placed in a
hybridisation tube (Hybaid, Ashford, UK) containing 10
ml hybridisation buer (ULTRAhyb, Ambion, Huntingdon, UK). Prehybridisation was for at least 30 min at
42C. After hybridisation overnight the membrane was
washed twice (3 and 30 min at 65C) with wash buer-1
(2U SSC, 0.1% SDS), followed by one wash in buer-2

FEMSYR 1511 11-11-02

565

(1U SSC, 0.1% SDS, at 65C) for 30 min. The membrane

was then semidried, wrapped in plastic foil, and placed in
a PhosphorImager cassette (Molecular Dynamics) for exposure for a minimum of 4 h. The blot was visualised on a
PhosphorImager (Molecular Dynamics, Amersham Biosciences) using the dedicated software (ImageQuant, Molecular Dynamics). Expression values were quantied by
integrating the area corresponding to each band.
2.7. Dot blotting
300-bp DNA fragments, corresponding to the 3P-end of
the ORFs studied by Northern analysis, were made by
PCR. These DNA fragments were blotted onto a nylon
membrane (Hybond XL, Amersham Pharmacia Biotech)
using a dot blot device (BioDot0 SF Module, Bio-Rad,
Hercules, CA, USA) and cross-linked using UV light.
First strand cDNA was made from a total RNA preparation from day 2 in the production-scale fermentation
(cDNA Synthesis System, Gibco BRL, Invitrogen Corp.,
Carlsbad, CA, USA). This was labelled with [32 P]dCTP
during the reverse transcriptase reaction. The protocol
supplied with the kit was followed, with the modication
of using three times higher non-radioactive nucleotide
(dGTP+dTTP+dATP) concentration, four times labelled
nucleotide ([32 P]dCTP) concentration, and two times reverse transcriptase concentration. The blot was visualised
as described above for Northern blots. Expression values
were quantied by integrating circular areas of equal sizes
covering each spot.

3. Results
3.1. Data set construction
mRNA purication followed by GeneFilter analysis was
performed for lager brewing yeast harvested from the
yeast storage tank either just prior to or just after the
actual fermentation, as well as from 10 dierent time
points during the fermentation. A set of Research Genetics
GF100 GeneFilters, which were used for the analysis, contained 6144 spots. In some cases the GeneFilters contained
two spots for each ORF (e.g. FLO8) while in a few cases
the production of a particular spot failed (annotated with
an error ag). Also, some ORFs have been dened after
the design of the lters, and hence were not represented.
Thus, merging of the analysed data with data tables (SGD
and Research Genetics) of SGDID, ORF names, gene
names, ORF length, and function resulted in a data set
containing data for 6084 ORFs. Thus, a total number of
73 008 data points of arbitrary magnitude were generated.
It should be noted that an updated and corrected Research Genetics data le (gf100_nal_data_071201) was
used for describing the identities of spots on the GeneFilters (see Section 2.4).

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K. Olesen et al. / FEMS Yeast Research 2 (2002) 563^573

Fig. 3. X^Y plot of expression values determined by GeneFilters versus

Northern blot/dot blot.
Fig. 1. X^Y plot of ORF length (SGD) versus TAG frequency (Velculescu et al., [22]).

3.2. Data validation ^ inuence of ORF length

Before endeavouring into the analysis of what the data
show, we carried out a validation analysis of the raw data.
Firstly, it was investigated if the ORF length aected the
apparent expression value in any way. The hybridisation
of the GeneFilters was done using 33 P-labelled cDNA.
Thus, it was possible that relatively longer ORFs would
result in relatively higher intensities read from the lters,
since longer cDNAs should incorporate more label than
shorter cDNAs.
To determine rst if there actually was a correlation
between ORF length and expression level, we examined
the expression values as determined by SAGE analysis,
reported by Velculescu et al., [22]. Due to the nature of
SAGE analysis ORF length should not aect the results.
By plotting ORF length versus mRNA abundance from
this data set we concluded that there was no general correlation between ORF length and expression level (Fig. 1).
Similarly, plotting the spot intensities from the GeneFilter
analysis versus the ORF lengths in an X^Y plot (Fig. 2)

Fig. 2. X^Y plot of ORF length (SGD) versus GeneFilter spot intensities.

FEMSYR 1511 11-11-02

also did not reveal any correlation between ORF length

and spot intensity. This indicated that the average cDNA
length in the experiment was much shorter than full-length
mRNAs. Thus, the spot intensities read from the GeneFilters could be used as a direct measure of relative expression levels without any correction for ORF length.
3.3. Data validation ^ correlation to Northern analysis
However, analysis of the GeneFilter exposures still
presents certain possibilities of errors. Erroneously high
intensity values may result in cases where the spots to be
analysed have one or more neighbouring spots of much
higher intensity. Although the analysis software has been
designed to prevent this, the eect cannot be completely
eliminated.
Thus, to verify the validity of the obtained data, we
analysed the expression of a small subset of ORFs by
high stringency Northern analysis. The absolute expression values for dierent genes obtained by the Northern

Fig. 4. Expression prole of TPI1 (YDR050C) as determined by GeneFilters (a) and Northern blot (b).

Cyaan Magenta Geel Zwart

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567

Fig. 5. Average expression value (a) of all ORFs and sugar content of
maltose (E), maltotriose (F), and glucose (b) plotted against time.

blots were normalised to each other by measuring the relative expression values on day 2 on dot blots containing
equal amounts of DNA of the genes being analysed (data
not shown). Based on the correlation of all corresponding
expression values we estimated that the obtained expression values were accurate within a factor of Y 3 (Fig. 3). It
was found that data for highly expressed ORFs correlated
well between the GeneFilter analysis and the Northern
analysis, especially with respect to the expression proles
(Fig. 4), while relative expression strengths correlated less
well. However, based on the Northern analysis it was also
evident that the expression data derived from GeneFilter

Table 1A
The 25 ORFs with the lowest average expression values
ORF

Gene

Average

YLR235C
YLR041W
YLR315W
YGL024W
YKL131W
YDR457W
YLR233C
YAL001C

TOM1
EST1
TFC3

656
721
756
848
902
917
935
943

YLR124W
YML092C

PRE8

953
968

YPL221W
YKR027W
YLR042C
YKL139W
YKL123W
YIR020C
YKL055C
YFR019W
YPR130C
YLR318W
YDR431W
YAL058C-A
YLR423C

BOP1

CTK1

OAR1
FAB1
EST2
KRE20
APG17

974
976
988
990
1000
1015
1082
1082
1091
1103
1112
1118
1166

SGD annotated process

biological process unknown
biological process unknown
biological process unknown
biological process unknown
biological process unknown
polyubiquitylation
transcription initiation from Pol III
promoter
biological process unknown
ubiquitin-dependent protein
degradation
biological process unknown
biological process unknown
biological process unknown
protein phosphorylation
biological process unknown
biological process unknown
respiration
vacuole organisation and biogenesis
biological process unknown
biological process unknown
biological process unknown
autophagy

FEMSYR 1511 11-11-02

Fig. 6. Plot of distribution of minimum, average and maximum expression values throughout the fermentation.

Table 1B
The 25 ORFs with the highest maximum expression values
ORF

Gene

Max

SGD annotated process

YBR118W
YJL052W
YJR009C
YPR080W
YGR192C
YHR007C
YGR254W
YGL055W
YHR174W
YKL060C
YLR167W
YEL034W
YER011W

TEF2
TDH1
TDH2
TEF1
TDH3
ERG11
ENO1
OLE1
ENO2
FBA1
RPS31
HYP2
TIR1

988 290
809 362
765 585
759 467
737 481
622 612
563 517
541 299
521 654
498 716
475 732
462 765
446 434

YDR502C
YIL018W
YGL135W
YJR047C
YMR015C
YGR175C
YBR299W
YER102W
YGL123W
YFR031C-A
YBR181C
YLR044C

SAM2
RPL2B
RPL1B
ANB1
ERG5
ERG1
MAL32
RPS8B
RPS2
RPL2A
RPS6B
PDC1

433 826
395 883
393 986
393 541
389 381
381 017
375 972
369 718
355 866
348 313
347 943
341 951

protein synthesis elongation

glycolysis
glycolysis
protein synthesis elongation
glycolysis
ergosterol biosynthesis
glycolysis
mitochondrion inheritance
glycolysis
glycolysis
protein biosynthesis
protein synthesis initiation
cell wall organisation and
biogenesis
methionine metabolism
protein biosynthesis
protein biosynthesis
protein synthesis initiation
ergosterol biosynthesis
ergosterol biosynthesis

Cyaan Magenta Geel Zwart

protein
protein
protein
protein

biosynthesis
biosynthesis
biosynthesis
biosynthesis

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Table 2
Day-to-day correlation coecients of expression values

Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day

0
1
2
3
4
5
6
7
8
9
11
12

Day 0

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 11

Day 12

1.00

0.22
1.00

0.29
0.84
1.00

0.44
0.69
0.86
1.00

0.49
0.59
0.82
0.94
1.00

0.51
0.48
0.67
0.87
0.87
1.00

0.64
0.38
0.58
0.81
0.86
0.89
1.00

0.67
0.32
0.43
0.71
0.70
0.80
0.92
1.00

0.79
0.33
0.47
0.69
0.71
0.78
0.91
0.91
1.00

0.77
0.30
0.39
0.63
0.65
0.70
0.81
0.87
0.86
1.00

0.65
0.29
0.32
0.49
0.54
0.65
0.60
0.65
0.63
0.79
1.00

0.89
0.24
0.31
0.45
0.54
0.53
0.65
0.65
0.71
0.78
0.73
1.00

observed on day 2 (Fig. 5). A second temporary peak in

average expression coincided with the depletion of available sugars in the wort (Fig. 5). Plotting the distribution of
minimum, average and maximum expression values for all
ORFs during the time course of the experiment revealed
peak values of 400, 3200 and 6400 for minimum, average
and maximum expression, respectively (Fig. 6). Thus, on
average, the expression of genes varied about 16-fold during the fermentation. In comparison, the gene RPS6B
(YBR181C) exhibited a 460-fold expression variation during the fermentation, and 172 genes were found to vary by
more than 100-fold, while 693 genes varied less than 10fold. Amongst the ORFs with a low average expression
value there was, not surprisingly, a large number of genes
with unknown biological function (Table 1A). The genes
with high average expression value were mostly genes involved in protein synthesis, glycolysis, and lipid synthesis
(Table 1B).

spots corresponding to weakly expressed ORFs at least in

some cases could be overestimated due to highly exposed
neighbouring spots.
3.4. Dynamic range and distribution
Since it is dicult to accurately measure the expression
value of weakly expressed ORFs, it also becomes dicult
to distinguish between total lack of expression and low
expression. With this in mind, the lowest expression measured had an arbitrary value of 80 and the highest value
measured was 990 000. Thus, theoretically, the dynamic
range of the analysis is more than 10 000. In comparison,
Velculescu et al. [22] have found mRNA levels to vary
between 0.3 and 200 transcripts per cell by SAGE analysis.
An overall maximum expression level for an individual
gene of 990 000 was observed on day 1 for TEF2. In contrast, the highest average expression level for all genes was

Table 3A
Correlation coecients between DeRisi et al., [1] expression values and GeneFilter expression values

09
11
13
15
17
19
21

h
h
h
h
h
h
h

Day 0

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 11

Day 12

0.16
0.17
0.16
0.18
0.19
0.25
0.27

0.57
0.55
0.52
0.54
0.48
0.33
0.22

0.47
0.46
0.45
0.46
0.42
0.33
0.24

0.38
0.38
0.38
0.39
0.37
0.33
0.26

0.33
0.34
0.33
0.34
0.33
0.31
0.25

0.23
0.23
0.25
0.24
0.22
0.22
0.19

0.20
0.21
0.22
0.21
0.21
0.23
0.22

0.21
0.22
0.22
0.24
0.23
0.30
0.29

0.18
0.19
0.20
0.21
0.20
0.28
0.29

0.19
0.20
0.19
0.21
0.23
0.32
0.32

0.08
0.08
0.10
0.10
0.08
0.12
0.13

0.13
0.14
0.12
0.15
0.16
0.20
0.19

Table 3B
Correlation coecients between DeRisi et al., [1] expression values and GeneFilter expression values

09
11
13
15
17
19
21

h
h
h
h
h
h
h

Day 0

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 11

Day 12

0.16
0.18
0.15
0.19
0.20
0.27
0.29

0.65
0.63
0.59
0.61
0.54
0.39
0.25

0.54
0.53
0.50
0.51
0.47
0.38
0.26

0.43
0.44
0.42
0.44
0.41
0.38
0.31

0.37
0.38
0.37
0.38
0.36
0.36
0.29

0.26
0.26
0.27
0.27
0.25
0.26
0.22

0.23
0.24
0.24
0.24
0.24
0.29
0.27

0.24
0.26
0.25
0.27
0.26
0.35
0.34

0.23
0.24
0.24
0.27
0.25
0.34
0.34

0.22
0.24
0.21
0.26
0.27
0.40
0.40

0.10
0.10
0.10
0.12
0.11
0.16
0.17

0.13
0.15
0.12
0.15
0.16
0.21
0.20

The GeneFilter data set was ltered to only include ORFs not having neighbouring spots of three times or higher intensity.

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569

3.5. Day-to-day correlation

Expression values obtained for each day were found to
correlate with a correlation coecient above 0.8 with the
values from the previous day as well as the following day
of fermentation (Table 2). The highest correlation coecient of 0.94 was found between days 3 and 4, indicating
that the smallest relative changes in transcription occur
between these two days. A high correlation coecient of
0.89 was also found between yeast harvested from the
yeast storage tanks just before and just after the fermentation. Thus, as judged by the mRNA levels, apparently
the yeast was quite stable during storage at such conditions.
3.6. Comparison to S. cerevisiae
We compared the expression values of our GeneFilter
analysis to the expression values published by DeRisi et al.
[1], perhaps the published experiment where the growth
conditions mostly resemble those presented here. Still,
the two experiments do dier signicantly in several aspects. While our data represent the expression values of
lager brewing yeast grown under standard fermentation
conditions in wort, the DeRisi et al. data represent expression values of a S. cerevisiae laboratory yeast grown at
30C in YPD medium. Thus, not surprisingly, a perfect
correlation was not found between the two data sets. The
highest correlation coecient of 0.57 was observed between our data for day 1 and DeRisi et al. data for
9 h (Table 3A). Filtering the data to only include ORFs
from spots not having any neighbouring spots with more
than three times higher intensities on the GeneFilters, this
correlation coecient increased to 0.65 (Table 3B and Fig.
7). The complementary subset of data, which represents
predominantly weakly expressed genes, correlated with a
correlation coecient of 0.37. This is undoubtedly a re-

Fig. 7. X^Y plot of expression values for time point day 1 versus time
point 9 h from DeRisi et al., [1]. Only data points for spots not having neighbours with more than three times higher intensity are included.
The correlation coecient between the two data sets is 0.65.

FEMSYR 1511 11-11-02

Fig. 8. X^Y plot of expression ratios for day 1/day 9 versus 9 h/21
h from DeRisi et al. [1]. Only data points for spots not having neighbours with more than ve times higher intensity are included. The correlation coecient between the two data sets is 0.36.

ection of the problem of accurately quantifying weakly

expressed ORFs. It was furthermore observed that while
the DeRisi et al. data sets for time points 9 h through
17 h all correlated best with our data from time points
day 1 and day 2, only data for DeRisi-19 h and DeRisi-21
h had highest correlation with time points day 7 to day 9.
This is an indication of the dierence in growth rates at
the two dierent culturing conditions.
Comparing the ratios of expression between day 1/day 9
and DeRisi 9 h/21 h revealed a quite low correlation co-

Fig. 9. Distribution of ratio of expression values between neighbouring

ORFs on day 2 in actual or random order. The random order distribution is based on the average of ve independent random order sortings,
standard deviations being indicated on top of the bars.

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K. Olesen et al. / FEMS Yeast Research 2 (2002) 563^573

Fig. 10. Plot of expression value on day 2 versus distance to closest

telomere.

ecient of 0.33. Filtering data to only include ORFs from

spots not having any neighbouring spots with more that
ve times higher intensities, this correlation coecient increased to only 0.36 (Fig. 8). Thus, not surprisingly, the
quite dierent growth conditions in the two experiments
resulted in dierent expression responses in the two yeasts.
3.7. Does local chromatin structure inuence local gene
expression?
The notion persists that local regions of chromosomes
may exist with either a more open or a more closed chromatin structure, leading to regions or loops with regional
higher or lower than average expression levels. We looked
at our data in various ways, to see if this was reected in
the measured expression values. When plotting either the
expression values of single ORFs, or the average expression values of windows of varying sizes according to either
the base pair position or the ORF number on the chromosome, graphs/images appeared which could mislead to
the interpretation that expression values were non-randomly distributed. However, when deliberately sorting
the expression values randomly, such plots resulted in
graphs/images that depicted similar apparent non-random
distribution.
C
Fig. 11. Cluster display of S. carlsbergensis gene expression during the
beer fermentation. Before clustering of the transcriptome data, adjustments were made to the data set. Genes having a neighbouring spot on
the GeneFilter of more than 10 times higher intensity was ltered out,
resulting in 4215 ORFs being included in the clustering. Day 0 and day
12 (data from the yeast storage tank) were averaged (column A). Days
1, 2 and 3 were used as is (B^D). Days 4^6 and days 7^11 were averaged (E,F, respectively). This data set was mean centred and normalised, using the adjustment functions of Cluster, prior to the hierarchical
clustering analysis. Green represents low expression while red represents
high expression relative to the average expression of each ORF.

FEMSYR 1511 11-11-02

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K. Olesen et al. / FEMS Yeast Research 2 (2002) 563^573

As a mathematical approach, assuming local regions of

higher or lower than average expression values, the ratio
of expression values between neighbouring ORFs should
show a distribution enriched for low ratios as compared to
the distribution observed using the same values randomly
sorted. Although this actually turned out to be the case,
the enrichment was too small to substantially support the
existence of such loops with higher or lower than average
expression (Fig. 9). Similarly, Velculescu et al. [22] have
not found any support for such regional expression deviation.
Another notion is that expression of genes near telomeres is lower than average. However, our data did not
show any correlation between expression values and the
distance of each ORF to the nearest telomere (Fig. 10).
The data published by DeRisi et al. [1] and Velculescu et
al. [22] exhibit the same lack of correlation.
3.8. Patterns in gene expression during fermentation
As an approach to try to describe patterns of gene expression during the main fermentation, the transcriptome
data were subjected to hierarchical clustering analysis using the software programmes Cluster and Treeview [19]
(http://rana.lbl.gov/EisenSoftware.htm). Fig. 11 presents

571

the clustering analysis. As can be seen, the majority of

genes were most highly transcribed at day 1 or 2 or
both (clusters 2^6), and a subset of these also had high
expression in the middle part of the fermentation (clusters
4 and 5). The rather small cluster 1 contains genes that
were most highly expressed at day 3, cluster 7 contains
genes that were more or less constitutively expressed
from days 1 to 6, and, nally, the rather large cluster 8
represents genes that were mostly expressed in the middle
part of the beer fermentation.
Table 4 shows the distribution of the most common
gene functions within each cluster. Protein biosynthesis,
chaperone and protein folding genes were best represented
in clusters 2 and 3. This makes sense, as the days of highest expression in these two clusters were days 1 and 2, the
time period where the yeast was most actively dividing and
thus biosynthesising. Genes involved in protein degradation, on the other hand, were best represented in clusters 6
and 7, both with a rather later expression prole. Stress
response genes were pretty much contained in all clusters,
but seemed to be relatively more abundant in clusters 1, 2
and 4. A notable observation was that cluster 1 basically
consisted of genes with unknown function, which is rather
striking, as expression seemed to be almost conned to
day 3. In contrast to this, cluster 2, with its day 1^2 ex-

Table 4
Distribution of common genetic functions within clusters
Cluster

Total

% of cluster

all

Biosynthesis
1
Cell cycle
0
Chaperone
1
Glycolysis
0
Kinase
0
Metabolism
2
mRNA
0
Protein biosyn0
thesis
Protein degrada1
tion
Protein folding
0
RNA
0
rRNA
0
Stress response
4
Structural protein
0
of ribosome
Transcription
2
Transcription fac- 0
tor
Transport
6
tRNA
0
Ubiquitin
2
Unknown
46
Sum
65
TOTAL entries
73
in cluster

98
2
4
3
9
1
9
94

36
1
7
2
6
5
3
33

14
3
2
1
8
13
1
4

23
8
2
1
20
15
8
13

61
12
6
15
30
34
25
24

12
4
5
1
10
7
8
7

12
7
2
0
9
8
15
6

sum
257
37
29
23
92
85
69
181

1
0
1
0
0
3
0
0

30
1
1
1
3
0
3
29

17
0
3
1
3
2
1
16

4
1
1
0
2
3
0
1

3
1
0
0
3
2
1
2

4
1
0
1
2
2
2
2

3
1
1
0
2
2
2
2

2
1
0
0
1
1
2
1

6
1
1
1
2
2
2
4

26

10

48

4
24
6
5
93

5
16
2
3
31

2
12
1
7
4

0
34
6
4
7

6
90
9
9
17

4
32
1
3
6

1
37
1
8
4

22
245
26
43
162

0
0
0
5
0

1
7
2
2
29

2
8
1
1
15

1
3
0
2
1

0
5
1
1
1

0
7
1
1
1

1
7
0
1
1

0
5
0
1
1

1
6
1
1
4

8
3

8
6

16
9

34
16

63
24

20
11

25
12

176
81

3
0

2
1

4
3

4
2

5
2

5
2

4
2

4
2

4
2

12
2
3
94
476
326

12
7
0
74
257
208

14
1
5
223
341
394

21
3
10
410
640
718

51
15
34
690
1241
1362

29
8
12
233
423
457

22
7
5
439
623
685

167
43
71
2209
4066
4215

8
0
3
63
89

4
1
1
29
146

6
3
0
36
124

4
0
1
57
87

3
0
1
57
89

4
1
2
51
91

6
2
3
51
93

3
1
1
64
91

4
1
2
52
96

Numbers represent the number of ORFs in each cluster that contains the query word in either the SGD-annotated Process or Function. Thus, e.g. the
query unknown represents all ORFs with either unknown process or unknown function. Some query words, e.g. metabolism, will return any process or
functions in which metabolism is a substring. Several ORFs will be returned by more than one query.

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K. Olesen et al. / FEMS Yeast Research 2 (2002) 563^573

pression maximum, had a relatively low percentage of

genes with unknown function, only 29% (as opposed to
this type of genes comprising 52% of all ORFs). All in all,
the vast majority of genes were upregulated during the
rst 24^48 h of the beer fermentation. For most of these,
expression gradually decreased until the end of fermentation, while a subset appeared to have a second expression
peak in the middle period (days 4^6, clusters 4 and 5). It is
evident from the cluster analysis that gene expression was
generally very low during yeast storage (days 1 and 12).
This is not surprising, as the yeast is supposed to be in G0
or stationary phase at this point. However, a few genes
were preferentially expressed during storage, e.g. the
ORFs YCL033C, YLR154C and YLR162W (Fig. 12).
Due to the relatively few data points taken, the cluster
analysis can not be used to describe the details of gene
regulation during beer fermentation. However, when scrutinising the data set for expression of genes in specic
functional families, patterns do emerge. One evident example, expression of glycolytic vs. gluconeogenic genes, is
presented in Fig. 13. Whereas transcription of the glycolytic genes peaked at 24 h of fermentation, gluconeogenic
genes only reached their highest level of expression after
100 h.

4. Discussion
To our knowledge, this work represents the rst example of a genome-wide transcription analysis of a lager
brewing yeast during actual production fermentation conditions. Although the transcriptome data obtained as a
result of this investigation was associated with some sources of inaccuracy, such as the ability to distinguish between expression from the two dierent subgenomes,
they nevertheless provide signicant new insight into the
genetics and physiology of an important industrial microorganism.

Fig. 12. Expression proles of genes that are preferentially expressed in

the yeast storage tanks. YLR162W uses the secondary Y-axis. None of
these genes have known biological function.

FEMSYR 1511 11-11-02

Fig. 13. Expression proles of genes involved in glycolysis (red, primary

Y-axis), gluconeogenesis (blue, secondary Y-axis), the glyoxylate cycle
(cyan, secondary Y-axis), and genes involved in glycolysis as well as gluconeogenesis (green, primary Y-axis).

Based on the accumulated literature on genome-wide

transcription analysis of yeast, we might have expected a
more clearly discernible progression of the transcriptional
regulation during the beer fermentation, which would lead
to grouping of related genes into distinct clusters. However, the fact that this was not the case is perhaps the most
important observation of this investigation. It underlines
the fact that in comparison to controlled laboratory experiments, where one growth parameter can be altered at a
time and the response compared to a reference, the progress of beer fermentation presents the yeast with a multitude of gradually altering growth conditions. This in turn
leads to a transcriptional response so complicated that it
becomes dicult at best to fully discern.
Yet, although the overall picture might be somewhat
unclear, when it comes to bioengineering of lager yeast
the strength of the obtained data is apparent. For pathway
engineering, the ability to know the expression proles of
all genes involved as well as the ability to select promoters
to control the expression of selected genes in a desirable
manner is invaluable. In this respect it is also important to
point to another signicant observation. While quite some
similarities were found to the expressional response of
S. cerevisiae grown in YPD [1], many more dierences
existed. Thus, qualied design decisions with respect to
bioengineering of lager brewing yeast should not be based
on the transcriptional regulation of another yeast, grown
under dierent growth conditions.
As a whole, these data do provide a multitude of novel
information about the lager brewing yeast: with respect to
the general regulation of expression in response to the
changing environment during beer fermentation; with respect to relative and absolute expression values; and also
to the expression proles of individual genes and groups

Cyaan Magenta Geel Zwart

K. Olesen et al. / FEMS Yeast Research 2 (2002) 563^573

of genes. If fully understood and exploited, such data may

be especially valuable for the yeast geneticist developing
new yeast strains, but possibly also for the engineer designing new brewing facilities and for the brewer attempting to control the fermentation process and the productivity of the yeast.
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