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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
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
575
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.
576
Results
Changes in cell and wort characteristics
577
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.
578
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).
579
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
8 vs. 60 h
1.5
5.1w
1.3
1.0
2.3w
1.3
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
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
1.2
3.0w
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
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
8 vs. 30 h
580
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
1.7w
1.2
2.4w
4.9w
1.8w
1.8w
3.4w
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.
581
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
582
583
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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