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RESEARCH ARTICLE

The oxidative stress response of a lager brewing yeast strain during


industrial propagation and fermentation
Brian R. Gibson, Stephen J. Lawrence, Chris A. Boulton, Wendy G. Box, Neil S. Graham, Robert S.T.
Linforth & Katherine A. Smart
School of Biosciences, University of Nottingham, Loughborough, Leicestershire, UK

Correspondence: Katherine A. Smart,


School of Biosciences, University of
Nottingham, Sutton Bonington Campus,
Loughborough, Leicestershire LE12 5RD, UK.
Tel.: 144 (0) 1159516214; fax: 144 (0)
1159516162; e-mail:
katherine.smart@nottingham.ac.uk
Received 30 October 2007; revised 17 January
2008; accepted 22 January 2008.
First published online 28 March 2008.
DOI:10.1111/j.1567-1364.2008.00371.x
Editor: Ian Dawes
Keywords
yeast; antioxidants; stress; brewing;
propagation; fermentation.

Abstract
Commercial brewing yeast strains are exposed to a number of potential stresses
including oxidative stress. The aim of this investigation was to measure the
physiological and transcriptional changes of yeast cells during full-scale industrial
brewing processes with a view to determining the environmental factors influencing the cells oxidative stress response. Cellular antioxidant levels and
genome-wide transcriptional changes were monitored throughout an industrial
propagation and fermentation. The greatest increase in cellular antioxidants and
transcription of antioxidant-encoding genes occurred as the rapidly fermentable
sugars glucose and fructose were depleted from the growth medium (wort) and the
cell population entered the stationary phase. The data suggest that, contrary to
expectation, the oxidative stress response is not influenced by changes in the
dissolved oxygen concentration of wort but is initiated as part of a general stress
response to growth-limiting conditions, even in the absence of oxygen. A
mechanism is proposed to explain the changes in antioxidant response observed
in yeast during anaerobic fermentation. The available data suggest that the yeast
cell does not experience oxidative stress during industrial brewery handling. This
information may be taken into consideration when setting parameters for
industrial brewery fermentation.

Introduction
Yeast involved in the industrial fermentation of brewery
wort are exposed to a number of stresses, each with the
potential to cause cellular damage and impair fermentation
performance (Gibson et al., 2007). Despite the enrichment
of wort with oxygen, little is known about the oxidative
stress response of yeast cells during industrial brewery
handling. Oxygen is an essential component of the brewing
process and is required for the synthesis of unsaturated fatty
acid and sterols (Lorenz & Parks, 1991). The presence of
oxygen can, however, lead to the generation of reactive
oxygen species (ROS), normal by-products of cellular metabolism under aerobic conditions (Halliwell & Gutteridge,
1999). The brewing yeast cell is continuously exposed to
oxygen during brewery propagation, a process whereby yeast
cells are grown from stock cultures to a sufficient quantity
for transfer into fermentation vessels. Such exposure may be
via aeration or oxygenation and the final concentration of
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dissolved oxygen (DO) used will typically be in the range of


812 mg L1 for a standard gravity (121 Plato) wort. The
propagated yeast is then pitched (inoculated) into a fermentation vessel containing wort with an initial DO concentration which may be as high as 30 mg L1 depending on wort
type, fermentation temperature and tank pressure (Briggs
et al., 2004).
Oxygen is typically depleted within the first 12 h of
fermentation and wort fermentation continues under anaerobic conditions. Towards the end of the fermentation
process yeast sediments out of suspension, resulting in beer
clarification and, in the case of typical lager fermentation,
will collect at the bottom of a cylindroconical fermentation
vessel (Briggs et al., 2004). The yeast cells can then be
removed (cropped) and reused in another fermentation
where they will be re-exposed to oxygen as before. This
repitching of a yeast slurry can be repeated a number of
times, though in the case of industrial lager fermentations a
batch of yeast will typically be used in no more than ten
FEMS Yeast Res 8 (2008) 574585

575

Oxidative stress response of lager brewing yeast

successive fermentations due to progressive deterioration of yeast quality and fermentation performance (Briggs
et al., 2004).
Common reactive oxygen species (ROS) generated endogenously under aerobic conditions include the superoxide
anion (O
2 ), hydrogen peroxide (H2O2) and the hydroxyl
radical (OH), which can cause lipid peroxidation (Girotti,
1998), protein inactivation (Cabiscol et al., 2000), damage
to nucleic acids within the nucleus (Salmon et al., 2004;
Ribeiro et al., 2006) and mitochondria, with the latter effect
commonly inducing the formation of respiratory-deficient
petites (ORourke et al., 2002; Doudican et al., 2005;
Gibson et al., 2006). As ROS are known to have a direct role
in cellular ageing (Halliwell & Gutteridge, 1999) and replicative lifespan in yeast is known to be related to antioxidant
potential of the cell (Barker et al., 1999; Van Zandycke et al.,
2002), the number of times a batch of yeast can be repitched
might be determined by its exposure to oxygen and its
ability to mitigate the effects of endogenous ROS production. The relative importance of oxidative stress and the
oxidative stress response during industrial brewery handling
has received only limited attention. It has, however, been
shown that cellular antioxidant levels in production strains
of brewing yeast can change rapidly in response to changes
in the oxygen levels of semi-defined wort medium (Clarkson
et al., 1991). Microarray analyses have also demonstrated
changes in the transcription of antioxidant-encoding genes
during small-scale wort fermentation (Higgins et al., 2003;
James et al., 2003). A relative decrease in the transcription of
several oxidative stress response genes during such a fermentation with a lager brewing yeast strain has been
reported (Higgins et al., 2003). A second transcriptomic
study, however, found a relative increase in the transcription
of such genes in the later stages of a small-scale wort
fermentation (James et al., 2003).
The available evidence for an oxidative stress response by
brewing yeast exposed to oxygen during wort fermentation
is somewhat contradictory. Furthermore, previous investigations have been carried out only with small-scale fermentations. The aim of this study was to analyse the
physiological and transcriptional changes which occur during full-scale industrial propagation and fermentation in
order to determine the factors which influence the expression of the oxidative stress response in a lager brewing yeast
strain during brewery handling. Collection of samples from
both the propagation vessel and the fermentation vessel
allowed for the comparison of a fully aerobic system and a
system involving an aerobic/anaerobic transition. Changes
in the transcription of other stress response genes were
analysed to determine if the observed changes in the
transcription of the antioxidant-encoding genes belong to a
general or specific stress response. It was hypothesized that
the oxidative stress response, if evident, would be most
FEMS Yeast Res 8 (2008) 574585

pronounced during propagation, which involves the continuous oxygenation of the yeast culture.

Materials and methods


Yeast sampling
Triplicate samples of the lager yeast strain CB11 were
sampled from a 140 hL (14 000 L) propagation vessel immediately after inoculation and at intervals throughout a
30-h propagation period. The yeast batch had been incubated aerobically in an 8 hL propagation vessel before
incubation in the principal (140 hL) vessel. Propagation
wort (85% malt and 15% sugar adjunct) contained
0.5 mg L1 Zn21 (added as zinc sulphate heptahydrate) and
was oxygenated with a ramped supply of molecular oxygen
(5100 L min1) to give a consistent dissolved oxygen concentration of 78 mg L1. Temperature of the wort was
maintained throughout at 20 1C. Specific gravity of the wort
was 171 Plato. Triplicate samples of the same yeast strain
were also collected from a fourth-generation 3275 hL wort
fermentation, i.e. the yeast batch had been used in four
fermentations previously. The cylindroconical fermentation
vessel was filled in stages, with six successive batches of
oxygenated wort (each between 520 and 570 hL) added. The
wort used for fermentation was the same as that used for the
propagation (85% malt and 15% sugar adjunct), contained
0.2 mg L1 zinc and was oxygenated in-line before vessel
filling to achieve a DO concentration of 25 mg L1. An inline oxygen meter (Orbisphere), upstream of yeast pitching,
was used to measure DO. Yeast was pitched with the 2nd and
4th batches of wort. The first sample for analysis was taken
8 h after all of the yeast had been pitched (and 5 h after the
last batch of oxygenated wort had been added to the
fermentation vessel). The fermentation vessels sample point
was located on the vertical side of the vessel, 1 m above the
cone. The sample valves were hygienic needle-type valves.
Samples were taken at intervals for up to 102 h after pitching
and were transported between the process vessels and the
laboratory at 4 1C and in under 5 min. Cells and wort were
separated by centrifugation at 4 1C. Cell pellets for RNA
extraction were flash-frozen in liquid nitrogen and stored at
80 1C until required.

Cell density and budding index


Cell suspensions were diluted to an appropriate volume and
density was measured using an Improved Neubauer counting chamber (Weber Scientific International Ltd, Middlesex,
UK) and standard light microscope (Zeiss, Oberkocken,
Germany) at  200 magnification. To determine budding
index, a minimum of 500 cells were scored microscopically
and the number of budded cells was calculated as a
percentage of the total.
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576

Cellular catalase activity and reduced


glutathione assays
Catalase activity of fresh samples was determined using the
method of Aebi (1984) and specific activity was expressed as
catalase units mg protein1 min1. Concentrations of reduced glutathione (GSH) were determined using a glutathione assay kit (Calbiochem-Novabiochem, UK) and
were expressed as mmol GSH mg protein1.

HPLC analysis of fermentable sugars in wort


samples
Wort samples (2 mL) were passed through C18 Plus solid
phase extraction cartridges (Waters, Milford) previously
conditioned with 5 mL methanol and equilibrated with
5 mL water. The first 1 mL of sample to pass through the
cartridge was discarded; the second 1 mL of sample was
collected for analysis. An aliquot of the sample (20 mL), was
injected onto an amino column (250 mm  4.6 mm ID,
5 mm particle size, Spherisorb NH2, Waters) and the sugars
eluted using acetonitrile : water (80 : 20, v/v) at a flow rate of
3 mL min1. Detection was achieved using a Wyatt Refractive
Index (RI) Detector (Optilab 903, Wyatt Technology Corporation, Santa Barbara). Peak heights were recorded for
each compound and concentrations determined by reference
to standards of known concentration. The retention times [s]
of the sugars were as follows; fructose [185]; glucose [214];
sucrose [338]; maltose [414] and maltotriose [820].

B.R. Gibson et al.

match probes and 11 mismatch probes within each probe


set; more than 100 000 signal intensity values for the Yeast
Genome 2.0 GeneChips array.

Microarray data analysis


The nonscaled RNA CEL files, representing replicates from
each time point, were loaded into GENESPRING analysis software (GeneSpring 7.3; Agilent Technologies) using the
Robust Multichip Average (RMA) prenormalization algorithm (Irizarry et al., 2003). To establish the optimal
hybridization threshold for interpreting transcriptional
data, the nonscaled RNA CEL files were prenormalized as a
single experimental group.
Per-gene normalizations were applied to the probe-set
signal values as follows. For each replicate, probe-set signal
values were standardized to the median probe-set signal
value for all arrays in the experiment. Probe-sets with
differential hybridization intensities between a given time
point and the reference sample were identified using a twostep process: (1) genes that were at least 1.5-fold up- or
down-regulated were selected, and (2) a Welchs t-test was
performed to identify genes that were differentially expressed between time points (Po0.05). Array data is available from the GEO database (http://www.ncbi.nlm.nih.gov/
geo/; accession series: GSE9423).

Results
Changes in cell and wort characteristics

RNA processing, hybridization and data


acquisition
Three time points from the propagation cycle (0, 8 and 30 h
after inoculation) and three time points from the fermentation (8, 30 and 60 h after pitching) were chosen for microarray analysis. All analyses were carried out in triplicate
(except for the 30 h sample from the fermentation vessel, for
which only duplicate samples were available for analysis),
with each replicate processed separately. In total, therefore,
17 samples were used in this investigation. RNA extraction
was carried out following the method of Lyne et al. (2003).
RNA yield and purity were determined using an Agilent
2100 Bioanalyser (Agilent Technologies Inc., Santa Clara,
CA). Sample preparation, GeneChip hybridization and
scanning as per manufactures instructions as described in
Affymetrix GeneChip Expression analysis technical manual
(Affymetrix, www.affymetrix.com). Following scanning,
nonscaled RNA signal intensity (CEL) files were generated
using the GeneChips operating system (GCOS version 5.0;
Affymetrix). Nonscaled RNA CEL files contain the raw
signal intensity values for each probe on the array, generated
from the scanned image of the GeneChips array. The RNA
CEL file contains signal intensity values for the 11 perfect
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At the beginning of propagation a lag phase in growth was


observed in the inoculated cells which lasted for at least 4 h.
After this time an exponential increase in cell density to
82  106 cells mL1 was observed (Fig. 1a). No increase in
cell density was observed after 24 h. An increase in budding
index preceded entry into the exponential phase of growth
and peaked at c. 75% between 4 and 8 h. Budding index
decreased to 30% as the cell population left the exponential
growth phase and a further decrease to 16% was observed
during the period of nonproliferation (Fig. 1a).
During fermentation the initial cell density increased
from 15  106 to 37  106 mL1 at the 30 h time point (Fig.
1b). No change in cell density was observed until 102 h, at
which time a reduction in cell density was observed, resulting from the aggregation (flocculation) of cells and the
sedimentation of these aggregates (flocs). As a consequence,
cell density was reduced to 27  106 cells mL1. Within the
first 8 h after pitching the budding index of cells in the
fermentation vessel increased from 2% to 70% (Fig. 1b).
Thereafter, a steady decrease in budding index to c. 10% was
observed until the 60 h sampling time, when cells had ceased
proliferating. This value remained constant until the end of
the sampling period (Fig. 1b).
FEMS Yeast Res 8 (2008) 574585

577

Oxidative stress response of lager brewing yeast

Fig. 1. Changes occurring during an industrial 140 hL propagation (a, c, e) and 3275 hL generation-four wort fermentation (b, d, f) with the lager strain
CB11. Graphs a and b show cell density () and budding index (). Graphs c and d show reduced glutathione (GSH) concentration (&) and catalase
activity (). Graphs e and f show concentrations of maltose (m), maltotriose (n), glucose (), fructose (}) and sucrose (~). Values are means of three
independent measurements. Error bars indicate  SEM. Arrows in figures a and b indicate samples taken for microarray analysis.

Reduced glutathione (GSH) concentration and total


catalase activity of the yeast population were found to vary
during propagation (Fig. 1c). In both cases no change was
observed during the lag phase of growth. A decrease in GSH
concentration was observed between 4 and 8 h as the cells
began to proliferate, followed by a modest increase towards
the end of the sampling period when the cells had entered a
nonproliferative state (Fig. 1c). A similar response was
observed with catalase activity, except that the increase in
activity on entering the nonproliferative stage in the life
cycle was more pronounced, with values rising from 2 to
10.5 catalase units (Fig. 1c). During fermentation, cellular
GSH concentrations were also observed to decrease after an
initial period in which no change was observed (Fig. 1d).
Between 14 and 60 h a reduction in the concentration of
GSH from 215 to 150 mmol mg protein1 occurred and was
FEMS Yeast Res 8 (2008) 574585

followed by an increase to c. 250 mmol mg protein1 (Fig.


1d). Total cellular catalase activity during fermentation
differed to that during propagation in that no decrease in
activity was observed. Rather, the increases in activity
appeared to mirror the changes in growth, with a relatively
low rate of increase in the first 14 h followed by a more rapid
increase until the 60 h time point (Fig. 1d).
A decrease in all wort fermentable sugars was observed
during the propagation (Fig. 1e). A steady reduction in
glucose concentration was observed in the first 8 h and this
sugar was not detected in subsequent samples. Sucrose was
only detected at low concentrations in wort at the beginning
of the propagation and was absent from later wort samples.
Fructose concentration remained constant up to 8 h but was
absent at 24 h. A reduction in both maltose and maltotriose
was observed throughout the propagation. Both of these
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578

sugars were present at 24 h when the other analysed sugars


had been utilized. At 30 h no maltose and only negligible
levels of maltotriose were present in the propagation
wort (Fig. 1e).
Sucrose was the first sugar to be depleted during fermentation and was undetectable at 30 h (Fig. 1f). Fructose and
glucose were present in low concentrations at 30 h and
absent by 60 h. Maltose was only utilized after 14 h and was
still present at the end of the sampling period. Maltotriose
was only utilized after 30 h and was also present at a low
concentration at the end of the sampling period (Fig. 1f).

Differential transcription of oxidative stress


response genes
An investigation of the transcriptional changes occurring
during brewery handling was carried out to determine if the
continuous oxygenation that occurs during brewery propagation would elicit a specific oxidative stress response and,
likewise, if the anaerobic conditions during fermentation
would lead to a reduction in the oxidative stress response. A
total of 53 genes (of the 68 known to be involved in the
oxidative stress response) were shown to be significantly
differentially transcribed during either propagation or fermentation, or during both processes. This number represents 78% of the genes involved in the oxidative stress
response (Hong et al., 2007). During early propagation
(between 0 and 8 h), a down-regulation of 22 of these genes
occurred alongside an up-regulation of three genes (out of
1612 genes down-regulated and 901 up-regulated during
this period) (Table 1). Genes down-regulated included those
encoding proteins involved in superoxide dismutase activity
(SOD1), catalase activity (CTA1), as well as glutaredoxins
(GRX1, GRX2, GRX5), thioredoxins and thioredoxin-related
proteins (TRX1, TRX2, TRR1, PRX1, TSA1, UBA4), glutathione peroxidase (HYR1), a heat shock protein (HSP12)
and a number of other genes known to be involved in the
oxidative stress response (MCR1, TMA19, GND1, DOT5,
URM1, ASK10, FAP7, OXR1 and YCL033C) (Table 1). Genes
up-regulated included SCO1, encoding a mitochondrial
membrane protein associated with cytochrome c peroxidase
activity; OCA1, a tyrosine phosphatase and LOT6, a
NAD(P)H:quinine reductase (Table 1). The general downregulation in the transcription of genes involved in the
oxidative stress response occurred as the stationary phase
cells transferred to the propagator were exposed to fermentable sugars and began proliferating rapidly. This is shown
by the transcription of the G1 cyclin gene CLN2, whose
transcription increases 14-fold (between 0 and 8 h; data not
shown).
Conversely, at a later stage of the propagation (between 8
and 30 h), two and six genes were down- and up-regulated,
respectively (Table 1) (out of 396 genes up-regulated and
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B.R. Gibson et al.

170 down-regulated between 8 and 30 h). The down-regulated genes included GRX3, which encodes a glutaredoxin
and TMA19, a ribosome-associated protein whose cellular
localization is affected by oxidative stress. The up-regulated
genes included the Cu, Zn superoxide-encoding gene SOD1,
the thioredoxin-encoding gene TRX3, the peroxiredoxinencoding gene PRX1, the heat shock protein-encoding gene
HSP12, SCH9, which encodes a protein kinase and SLN1,
which encodes a histidine kinase osmosensor (Table 1). Of
these genes, the first four (SOD1, TRX3, PRX1 and HSP12)
demonstrated a significant reduction in transcription during early propagation (Table 1). This slight recovery of the
oxidative stress response coincided with the depletion of
rapidly fermentable sugars from the wort and the entry of
the cells into stationary phase. This is shown by the
transcription of CLN2 only increasing by 1.5-fold between
8 and 30 h (data not shown).
In the early stage of the fermentation process (between 8
and 30 h) only a relatively small change in gene transcription
was observed, with just 40 genes showing a differentially
significant change in transcription. No change in the transcription of antioxidant-encoding genes was observed (Table 1). This was despite the fact that the sampling times
encompassed the period in which oxygen was depleted from
the wort and growth rate reduced as cells approached
stationary phase. A comparison of the 8 and 60 h time
points did however show an increase in the transcription of
40 oxidative stress response genes (out of 4384 genes upregulated), with no genes showing a reduced transcription
during this period (out of 346 genes down-regulated). The
cells displaying this relative increase in the transcription of
oxidative stress response genes were those which were no
longer exposed to the monosaccharides glucose and fructose
(Table 1).
The transcriptional changes observed were not confined
to a particular cell compartment, as genes encoding proteins
expressed in the nucleus, mitochondrion, peroxisomes and
cytosol were all affected. Likewise, the changes were not
restricted to genes encoding proteins with a particular
metabolic function, as genes encoding a superoxide dismutase, several peroxidases and general oxidant scavengers
showed significant changes (P o 0.05) in transcription
(Table 1).

Differential transcription of stress response


genes during brewery handling
During early propagation (08 h), a relative decrease in the
transcription of stress response genes was observed (Fig. 2a).
Of the 209 stress-related genes displaying a significant
change in transcription, 155 were down-regulated (from a
total of 1612 down-regulated genes), while only 54 were upregulated (from a total of 901 up-regulated genes). This
FEMS Yeast Res 8 (2008) 574585

579

Oxidative stress response of lager brewing yeast

Table 1. Fold-change in expression of oxidative stress genes in the lager strain CB11 during industrial propagation and fermentation
Propagation
Gene

0 vs. 8 h

Fermentation
8 vs. 30 h

Superoxide dismutase
SOD1
10.8w
1.9w
Cytochrome c peroxidase and associated molecule
CCP1
2.5
1.1
SCO1
1.5w
1.1
Catalases
CTA1
3.2w
1.0
CTT1
1.8
1.2
Glutathione synthesis and regulation
GSH1
1.5
1.2
Glutaredoxins
GRX1
18.0w
1.6
GRX2
7.8w
1.5
GRX3
6.5
1.5w
GRX4
1.0
1.2
1.0
GRX5
2.3w
Thioredoxins and associated molecules
TRX1
3.4w
1.2
1.4
TRX2
4.8w
TRX3
2.0
2.1w
w
1.5
TRR1
3.6
TRR2
1.2
1.1
PRX1
3.7w
1.6w
w
TSA1
5.3
1.0
Glutathione peroxidase
GPX1
1.4
1.0
1.6
HYR1
3.7w
Heat shock protein
HSP12
2.0w
2.1w
Transcription factors and associated molecules
AFT2
1.0
1.0
AHP1
3.3
1.1
SCH9
1.1
1.9w
SKN7
0.8
1.5
YAP1
2.7
1.2
YBP1
1.8
1.2
UBA4
2.3w
1.5
Other genes involved in the oxidative stress response
ASK10
2.0w
1.1
ATX1
1.8
1.4
CYR1
3.3
1.2
DOT5
5.0w
1.2
EOS1
1.7
1.1
ERV1
2.5
1.1
1.1
FAP7
10.0w
GAD1
1.1
1.2
GND1
5.0w
1.1
LOT6
3.0w
1.1
LTV1
3.3
1.3
MCR1
2.0w
1.1
MXR1
1.8
1.3
NCE103
5.0
1.2
OCA1
4.2w
1.1
1.2
OXR1
2.3w
POS5
1.6
1.2
SLN1
1.7
1.7w

FEMS Yeast Res 8 (2008) 574585

8 vs. 60 h

Description of gene product

1.5

5.1w

Cytosolic superoxide dismutase

1.3
1.0

2.3w
1.3

Mitochondrial cytochrome-c peroxidase


Mitochondrial membrane protein

1.7
1.1

7.5w
1.9w

Peroxisomal catalase A
Cytosolic catalase T

1.2

2.3w

g-Glutamylcysteine synthetase

1.1
1.3
1.0
1.5
1.0

2.4w
2.4w
1.2
2.9w
1.9w

GSH-dependent disulfide oxidoreductase


GSH-dependent disulfide oxidoreductase,
GSH-dependent oxidoreductase
GSH-dependent oxidoreductase
Mitochondrial oxidoreductase

1.1
1.3
1.5
1.1
1.1
1.2
1.2

2.1w
3.0w
5.5w
1.4
2.1w
2.5w
2.7w

Cytoplasmic thioredoxin
Cytoplasmic thioredoxin
Mitochondrial thioredoxin
Cytoplasmic thioredoxin reductase
Mitochondrial thioredoxin reductase
Mitochondrial peroxiredoxin
Ubiquitous thioredoxin peroxidase

1.7
1.2

3.1w
3.4w

Phospholipid hydroperoxide glutathione peroxidase


Thiol peroxidase

1.2

3.0w

Plasma membrane protein

1.5
1.1
1.3
1.0
1.1
1.0
1.0

2.7w
2.1w
2.9
2.2w
3.8w
2.0w
3.6w

Iron-regulated transcriptional activator


Thiol-specific peroxiredoxin
Protein kinase
Nuclear response regulator/transcription factor
Basic leucine zipper transcription factor
Protein required for oxidation of cysteine residues of Yap1p
Activates Urm1p, a target is Ahp1p

0.8
1.0
1.2
1.1
1.0
1.0
0.9
1.3
1.0
1.0
1.2
1.2
1.5
1.1
1.3
1.1
1.1
1.0

1.0
4.0w
1.8w
1.7
1.2
1.8w
1.6w
2.5w
0.8
1.6w
2.7w
2.5w
5.2w
3.0w
1.7w
1.4
1.7w
1.4

Component of RNA polymerase II


Cytosolic copper metallochaperone
Adenylate cyclase
Nuclear thiol peroxidase
N-Glyosylation protein
Sulfhydryl oxidase
NTPase
Glutamate decarboxylase
6-Phosphogluconate dehydrogenase
NAD(P)H:quinone reductase
Component of the GSE complex
Mitochondrial NADH-cytochrome b5 reductase
Peptide methionine sulfoxide reductase
Carbonic anyhdrase
Putative tyrosine phosphatase
Protein of unknown function
Mitochondrial NADH kinase
Histidine kinase osmosensor

8 vs. 30 h

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580

B.R. Gibson et al.

Table 1. Continued.
Propagation
Gene
SVF1
TMA19
UGA2
URM1
YAR1
YBL055C
YCL033C

Fermentation

0 vs. 8 h

8 vs. 30 h

10.0
5.5w
1.3
3.3w
5.0
1.4
7.0w

1.2
1.7w
1.0
1.3
1.2
1.6
1.2

8 vs. 30 h
0.9
1.4
1.2
1.1
0.9
0.8
1.2

8 vs. 60 h

Description of gene product

1.7w
1.2
2.4w
4.9w
1.8w
1.8w
3.4w

Protein required for diauxic growth shift


Ribosome-associated protein
Succinate semialdehyde dehydrogenase
Ubiquitin-like protein
Protein of unknown function
3 0 ! 5 0 exonuclease and endonuclease
Protein-methionine-R-oxide reductase

Descriptions following those of the Saccharomyces Genome Database (Hong et al., 2007).
w

Values showing a statistically significant change in expression compared with the reference sample, as determined by Welchs t-test.
The 53 genes presented belong to a group of 68 genes whose GO process terms are response to oxidative stress (GO:0006979), response to reactive
oxygen species (GO: 0000302) or age-dependent response to oxidative stress (GO: 0001324). All genes presented show a statistically significant
(P o 0.05) change in expression in either or both brewery-handling processes. Genes with the same GO process terms but showing no statistically
significant change in expression have been omitted.

Fig. 2. Numbers of genes from various stress categories up- or downregulated at different stages of an industrial 140 hL propagation of the
lager yeast CB11. The figure shows the changes in gene expression
between the 0 and 8 h time points (a) and between the 8 and 30 h time
points (b). Empty and filled bars represent up- and down-regulated
genes, respectively. Genes included are those which showed a significant
change in expression (P o 0.05) with at least a 1.5-fold change between
the two time points.

Fig. 3. Numbers of genes from various stress categories up- or downregulated at different stages of an industrial 3275 hL wort fermentation
with the lager yeast CB11. The figure shows changes in gene expression
between 8 and 30 h after pitching (a) and between 8 and 60 h after
pitching (b). Empty and filled bars represent up- and down-regulated
genes, respectively. Genes included are those which showed a significant
change in expression (P o 0.05) with at least a 1.5-fold change between
the two time points.

pattern of transcription was observed across a number of


stress gene categories. This change in transcription coincided with the transition from the lag to the exponential
phase of growth and the exposure of cells to the rapidly
fermentable carbohydrates glucose and fructose.
Between the 8 and 30 h sample times a relative upregulation of stress-related genes was observed (Fig. 2b). Of
the 52 genes significantly expressed, 43 were up-regulated
during this period. Though the number of genes showing a
significant change in transcription during this period is
lower than during early propagation, the general increase in
transcription still occurred in all of the stress categories. No

down-regulation of genes related to protection against


osmotic stress, starvation or temperature shock was observed (Fig. 2b). These changes in gene transcription coincided with the transition from exponential cell growth to
entry into stationary phase and the depletion of glucose and
fructose from the wort.
A comparison of stress-related gene transcription in the
early stages of an industrial fermentation (8 vs. 30 h)
revealed that the change in gene transcription during this
period was negligible, with only two stress genes showing a
significant change (Fig. 3a), out of 40 genes differentially
expressed. During the 830 h period, yeast cells changed

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FEMS Yeast Res 8 (2008) 574585

581

Oxidative stress response of lager brewing yeast

from a highly proliferative, mid-exponential phase of


growth to a less proliferative, late exponential/early stationary phase of growth. During this period the concentrations
of glucose and fructose were reduced but both were still
detectable in the wort (Fig. 1d). Stress response gene
transcription was found to be greater after depletion of
glucose and fructose (8 vs. 60 h) (Fig. 3b). Of the 193 stress
response genes showing a significant change in transcription, only 11 were down-regulated. The changes represent
the differences in transcription profile between cells growing
exponentially in a glucose-replete medium and cells in the
stationary phase of growth exposed only to nonfermentable
carbon sources and the less readily metabolizable carbohydrates maltose and maltotriose. This reduction in growth is
shown by the transcription of CLN2, reducing 1.3 fold
between 8 and 60 h.

Discussion
Results from this investigation suggest that the increased
transcription of antioxidant-encoding genes is part of a
concerted increase in stress resistance in stationary phase
cells rather than a response to changes in the cells oxygen
environment during industrial scale brewery handling.
Microarray analysis has previously revealed a general increase in the transcription of antioxidant-encoding genes
during small-scale fermentation of wort by an industrial
lager yeast strain (James et al., 2003). Likewise, an increase
in the transcription of several antioxidant-encoding genes
has also been observed during small-scale sake fermentation
with Saccharomyces cerevisiae (Wu et al., 2006). In both of
these cases the increases occurred despite the anaerobic
conditions prevalent in the fermentation media.
A number of other investigations have monitored transcriptional changes in stress responsive genes of industrially
important yeast strains. These studies have monitored
changes during vinification (Riou et al., 1997; Puig &
Perez-Ortin, 2000; Rossignol et al., 2003), sake fermentation
(Wu et al., 2006) and fermentation of very high gravity
media for the production of biofuel (Devantier et al., 2005),
as well as small-scale wort fermentation (Brosnan et al.,
2000; Higgins et al., 2003; James et al., 2003). While each of
these studies involved different experimental conditions and
yeast strains, the majority reported relatively lower transcription of stress response genes in proliferative cells
compared with cells in a nonproliferative state. Riou et al.
(1997), in a study involving microvinification with industrial wine yeast have, for example, reported increased
transcription of genes encoding heat shock proteins as
cultures entered stationary phase (Riou et al., 1997). Likewise, both Puig & Perez-Ortin (2000) and Rossignol et al.
(2003) have shown increased transcription of a number of
stress-related genes coincident with entry into stationary
FEMS Yeast Res 8 (2008) 574585

phase of a wine yeast strain during microvinification of


natural and synthetic wine must (Puig & Pe rez-Ortn, 2000;
Rossignol et al., 2003). Similar results have been reported for
small-scale wort fermentations. Brosnan et al. (2000)
showed an increase in both the basal level of HSP104 gene
transcription and the basal level of Hsp104p in the later
stages of a 50 L wort fermentation by industrial yeast strains
(Brosnan et al., 2000). That study also revealed that cells
sampled from the early stages of the fermentation were
much more responsive to stress, despite the low basal level of
gene and protein expression (Brosnan et al., 2000). Increased transcription of HSP genes in S. cerevisiae has also
been observed during sake fermentation (Wu et al., 2006).
Entry into stationary phase can be induced by a number
of factors including stress and starvation (Werner-Washburne et al., 1993; Gray et al., 2004). The general stress
response observed in this study coincided with the depletion
of the readily-fermentable carbohydrates glucose and fructose and it is therefore possible that entry into stationary
phase and the stress response were both initiated by limitation of fermentable carbohydrate. In an investigation of the
transcriptional changes that occur during diauxic shift,
DeRisi et al. (1997) found that a complete remodelling of
the yeast transcription profile occurs when glucose is
depleted and the cell adapts to respirative, rather than
fermentative, metabolism (DeRisi et al., 1997). This retooling of the yeast transcriptome includes an increase in the
transcription of stress responsive genes (DeRisi et al., 1997).
In fact, a number of studies have demonstrated that the
transcription of such genes is increased when fermentable
sugars such as glucose are exhausted or removed from the
growth medium (Schenberg-Frascino, 1972; Petko & Lindquist, 1986; Bissinger et al., 1989; Borkovich et al., 1989;
Hwang et al., 1989; Werner-Washburne et al., 1989; Praekelt
& Meacock, 1990; Francois et al., 1992; Jamieson, 1992;
DeRisi et al., 1997; Kuhn et al., 2001). Proteomic analysis has
also revealed an increase in the levels of several stress-related
proteins in a wild-type wine yeast strain after depletion of
glucose from YPD media (Trabalzini et al., 2003). BoyMarcotte et al. (1998) have also reported an increase in levels
of proteins encoded by stress response element (STRE)regulated genes (Boy-Marcotte et al., 1998). Evidence from
the current study suggests that a general stress response may
be initiated even in the absence of the oxygen required for
respiration and is therefore not necessarily a result
of the diauxic shift per se. It has also been demonstrated
that the increased tolerance to oxidative stress coincident
with a change from fermentable to nonfermentable carbon
source occurs even in the absence of functional mitochondria (Maris et al., 2001). In other words, the reduced
sensitivity to oxidative stress occurs in response to the loss
of fermentable carbohydrate rather than the initiation of
respiration.
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582

The elicitation of a general stress response may explain


why an apparently redundant up-regulation of certain stress
response genes occurred as glucose was depleted and the
yeast cultures entered the stationary phase during both
propagation and fermentation. As well as an increase in the
transcription of antioxidant-encoding genes under anaerobic conditions, there also occurred, for example, an increase
in transcription of osmotic stress genes, despite the reduced
wort gravity during propagation and fermentation. A general increase in the transcription of stress response genes
after glucose depletion would explain why a previous study
has reported an increase in transcription of oxidative stress
response genes during the anaerobic phase of a small-scale
wort fermentation (James et al., 2003). An increase in the
transcription of antioxidant-encoding genes has, however,
been observed during sake fermentation, in which glucose
levels were reduced but not depleted (Wu et al., 2006),
suggesting that glucose derepression was not the primary
regulating factor in that case. While the majority of studies
investigating transcriptional changes during fermentation
have noted an increase in the transcription of oxidative
stress response genes, one particular study (Higgins et al.,
2003) noted a decrease in the transcription of these genes
when comparing samples 1 and 23 h after pitching; a result
which was attributed to a relatively high transcription of
oxidative stress genes in cells exposed to aerobic conditions
at the beginning of the fermentation. It is possible that in
this case the results may have been influenced by the
comparison being made between a stationary phase culture
sample (1 h after inoculation) and an actively proliferating
and glucose-repressed sample (23 h after inoculation). As
details of the sugar concentration of the wort or cell
characteristics were not presented, this alternative explanation is difficult to establish with certainty. The oxidative
stress response observed by Higgins et al. (2003) at the
beginning of the fermentation may have been due to the low
level of cellular ergosterol, which was shown to have a role in
protection against ROS (Higgins et al., 2003). Further
investigation is clearly required to determine the protective
role of ergosterol during full-scale brewery fermentation.
Detection of glucose leads to a transient increase in the
production of cellular cAMP, which ultimately results in
stimulation of growth, mobilization of trehalose, stimulation of glycolysis and repression of STRE-controlled genes
(Thevelein & de Winde, 1999; Estruch, 2000). Low cAMP
levels, which may occur after glucose exhaustion, result in
the transport of the Msn2/Msn4p transcription factors from
the cytoplasm to the nucleus (Gorner et al., 1998), and the
increased transcription of genes containing STRE in their
promoter regions. Many genes involved in the oxidative
stress response are controlled by Msn2p and Msn4p and
yeast strains with high cAMP levels are known to be sensitive
to oxidative stress (Hasan et al., 2002). Msn2p is also
2008 Federation of European Microbiological Societies
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B.R. Gibson et al.

required for the activation of CTT1 in response to oxidative


stress (Martinez-Pastor et al., 1996; Schmitt & McEntee,
1996). It is unlikely that Msn2/4p are regulated directly by
oxidative stress. Rather, the Msn2/4p-mediated response is
regulated by the changes in cAMP levels after exposure to
stress, including oxidative stress. Twenty-seven of the genes
which are related to oxidative stress are known to be STREregulated and, of these, 20 demonstrated a significant
change in transcription during brewery handling. Because
the changes were not related to increases in oxygen exposure, it is possible that the change in transcription of these
genes was a consequence of changes in cellular cAMP levels
as a result of changes in sugar composition of wort.
However, as many of the oxidative stress response genes
listed are not STRE-regulated it is likely that other regulatory pathways are involved in the changes observed.
While the Msn2/4p transcription factors may increase the
transcription of many oxidative stress response genes after
exposure to a number of different stresses, other, more
specific transcription factors, such as Yap1p, exist (Toone &
Jones, 1999). Green fluorescent protein-tagged Yap1p was
observed to accumulate in the nucleus when cells were
transferred from a glucose-rich medium to one containing
either glycerol or no carbon source (Wiatrowski & Carlson,
2003), suggesting a link between glucose availability and
transcriptional activation. Yap1p is directly involved in the
activation of genes encoding the superoxide dismutase and
catalase enzymes. Another transcription factor, Hsf1p is
required for the activation of CUP1, a gene coding for a
metallothionein with a role in protection against oxidative
stress (Liu & Thiele, 1996). CUP1 transcription is also
known to be activated by Hsf1p in response to glucose
limitation (Tamai et al., 1994; Hahn & Thiele, 2004).
There also exists the possibility that a general stress
response is initiated in response to ethanol toxicity. The
increase in ethanol concentration throughout fermentation
may stimulate the expression of a number of genes, including those involved in protection against osmotic and oxidative stress. An increase in the expression of the antioxidantencoding gene CTT1 is, for example, known to be increased
12-fold when cells are exposed to 7% ethanol for 30 min
(Alexandre et al., 2001). In addition, ethanol tolerance of S.
cerevisiae cells has been associated with the activity of the
antioxidant enzyme manganese superoxide dismutase
(MnSOD) (Costa et al., 1993, 1997). The importance of
MnSOD may be related to its location within mitochondria,
the principal producers of ROS during respiration (Halliwell
& Gutteridge, 1999). While the exact cause of increased ROS
production by yeast cells during ethanol exposure is not
known, there are a number of possibilities including interaction of acetaldehyde with proteins and lipids, direct
mitochondrial damage, increased bioavailability of metals
(facilitating reduction of O2 and ROS) and impairment of
FEMS Yeast Res 8 (2008) 574585

583

Oxidative stress response of lager brewing yeast

the function of certain antioxidants such as glutathione (Wu


& Cederbaum, 2003). The increased expression of many
stress-related genes is likely to be related to the initiation of
the general stress response via STRE, either in response to a
stress such as ethanol toxicity or a reduction in cellular
cAMP. However, not all stress response genes contain STRE
elements in their promoter regions and the induction of
several genes could be related to cellular cAMP levels but
independent of STRE-regulation, as suggested by BoyMarcotte et al. (1998).
The results of this study may have implications for many
aspects of yeast handling and the brewing process. It is clear
that stress resistance is subject to temporal change during
brewery handling but that the increase in particular cellular
defences may be a result of a general stress response rather
than a specific response to one particular stress. This is
exemplified in the case of the oxidative stress defences,
which were observed to decrease in the early stages of
propagation and increase in the later stages of fermentation.
Such changes may suggest that the search for specific stress
bio-markers during brewery handling may be confounded
by the apparent over-riding effect of catabolite repression/
derepression. As stress resistance and fermentation performance are related (Ivorra et al., 1999), and stress resistance is
associated with catabolite repression during brewery handling, it may be beneficial to minimize exposure of yeast to
sugars such as sucrose or glucose (Verstrepen et al., 2004).
While this may not be possible during standard brewery
fermentation due to the risk of changes to the organoleptic
profile of the product, it may be possible to reduce glucose
use by the use of maltose-rich syrup instead of sucrose-rich
syrup during high gravity brewing (Verstrepen et al., 2004).
It may also be speculated that the atypical fermentation
profiles observed with generation-one yeast slurries may be
related to the repressed nature of recently propagated yeast
cultures. It may therefore be advisable to retain propagated
yeast in spent (growth limiting) wort for a number of hours
before pitching in order to maximise the stress resistance of
the cells. The increased resistance to oxidative stress, as well
as other stresses, that develops towards the end of the
fermentation may represent a preconditioning step; allowing the yeast cell to survive the nutrient-poor conditions
during storage and the environmental perturbations associated with pitching. The stressful conditions that occur at
the end of the fermentation therefore obviate the requirement for preconditioning, which is required, for example,
during the production of dried yeast for the brewing
industry (Hottiger et al., 1987; Eleutherio et al., 1997; Crowe
et al., 2001).
In conclusion, it has been observed that the oxidative
stress response of a production lager brewing strain during
full-scale industrial fermentation is influenced by cell
growth phase, which is, itself, influenced by the environFEMS Yeast Res 8 (2008) 574585

mental milieu in which the cells reside. An increase in the


transcription of antioxidant-encoding genes and the accumulation of antioxidants occurs when growth-limiting conditions exist. Carbon catabolite repression, due to the
availability of glucose (and possibly fructose), influences
the general stress response of an industrial lager yeast during
full-scale propagation and fermentation. A number of
transcription factors with a role in initiating a response to
oxidative stress are regulated by glucose (and probably other
sugars). Owing to the fact that changes in cellular levels of
antioxidants and the transcription of antioxidant genes did
not correspond to changes in oxygen concentration of wort
during brewery handling, it is likely that the oxidative stress
response was initiated by other factors such as catabolite
repression or the influence of other stresses such as ethanol
toxicity. The fact that similar responses were observed within a number of other stress response categories and the
prevalence of STRE sequences within the promoter regions
of the antioxidant genes suggests that the general (or global)
stress response has a significant role in determining the
transcription of antioxidant-encoding genes. While yeast
cells are capable of responding to specific stresses without
initiation of the general stress response, it is likely that such
changes will be obscured by the repression and derepression
of stress responses by sugars such as glucose. The initiation
of the stress response coincides with cessation of growth and
entry into stationary phase and is likely to be a mechanism
which allows brewing yeast cells to survive the sub-optimal
conditions to which they are routinely exposed during
brewery handling.

Acknowledgements
K.A.S. is the SABMiller Professor in Brewing Science and
gratefully acknowledges the support provided by SABMiller.
K.A.S. also gratefully acknowledges the support of the Royal
Society, BBSRC and EPSRC for the award of Royal Society
Industrial Fellow. The authors thank the J & J Morison
Trust, The Institute of Brewing and Distilling Grant Fund
and the University of Nottingham for their support. The
authors are grateful to Coors Brewers, UK for permission to
use the CB11 strain and for assistance in sample collection.
The authors are also grateful for the technical support of
Henrik Townsend, Zoe Emmerson and Beatrice Schildknecht at the Nottingham Arabidopsis Stock Centre.

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2008 Federation of European Microbiological Societies


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