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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
DOI: http://dx.doi.org/10.1111/j.1567-1364.2008.00371.x 574-585 First published online: 1 June 2008

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 cell's 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.

Keywords
  • yeast
  • antioxidants
  • stress
  • brewing
  • propagation
  • 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 et al., 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 et al., 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 dissolved oxygen (DO) used will typically be in the range of 8–12 mg L−1 for a standard gravity (12° 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 L−1 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 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 (O2●−), hydrogen peroxide (H2O2) and the hydroxyl radical (OH), which can cause lipid peroxidation (Girotti et al., 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’ (O'Rourke 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 et al., 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 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 L−1 Zn2+ (added as zinc sulphate heptahydrate) and was oxygenated with a ramped supply of molecular oxygen (5–100 L min−1) to give a consistent dissolved oxygen concentration of 7–8 mg L−1. Temperature of the wort was maintained throughout at 20 °C. Specific gravity of the wort was 17° 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 L−1 zinc and was oxygenated in-line before vessel filling to achieve a DO concentration of 25 mg L−1. An in-line 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 vessel's 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 °C and in under 5 min. Cells and wort were separated by centrifugation at 4 °C. Cell pellets for RNA extraction were flash-frozen in liquid nitrogen and stored at −80 °C 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.

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 protein−1 min−1. Concentrations of reduced glutathione (GSH) were determined using a glutathione assay kit (Calbiochem-Novabiochem, UK) and were expressed as μmol GSH mg protein−1.

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 μL), was injected onto an amino column (250 mm × 4.6 mm ID, 5 μm particle size, Spherisorb NH2, Waters) and the sugars eluted using acetonitrile : water (80 : 20, v/v) at a flow rate of 3 mL min−1. 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].

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. (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, http://www.affymetrix.com). Following scanning, nonscaled RNA signal intensity (CEL) files were generated using the GeneChip® 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 GeneChip® array. The RNA CEL file contains signal intensity values for the 11 perfect match probes and 11 mismatch probes within each probe set; more than 100 000 signal intensity values for the Yeast Genome 2.0 GeneChip® 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 two-step process: (1) genes that were at least 1.5-fold up- or down-regulated were selected, and (2) a Welch's t-test was performed to identify genes that were differentially expressed between time points (P<0.05). 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

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 mL−1 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).

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 (▲), maltotriose (△), 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.

During fermentation the initial cell density increased from 15 × 106 to 37 × 106 mL−1 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 mL−1. 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).

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 μmol mg protein−1 occurred and was followed by an increase to c. 250 μmol mg protein−1 (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 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 down-regulation 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).

View this table:
1

Fold-change in expression of oxidative stress genes in the lager strain CB11 during industrial propagation and fermentation

GenePropagationFermentationDescription of gene product
0 vs. 8 h8 vs. 30 h8 vs. 30 h8 vs. 60 h
Superoxide dismutase
SOD1−10.81.91.55.1Cytosolic superoxide dismutase
Cytochrome c peroxidase and associated molecule
CCP1−2.5−1.11.32.3Mitochondrial cytochrome-c peroxidase
SCO11.5−1.11.01.3Mitochondrial membrane protein
Catalases
CTA1−3.21.01.77.5Peroxisomal catalase A
CTT1−1.81.21.11.9Cytosolic catalase T
Glutathione synthesis and regulation
GSH11.51.21.22.3γ-Glutamylcysteine synthetase
Glutaredoxins
GRX1−18.01.61.12.4GSH-dependent disulfide oxidoreductase
GRX2−7.81.51.32.4GSH-dependent disulfide oxidoreductase,
GRX3−6.5−1.51.01.2GSH-dependent oxidoreductase
GRX41.01.21.52.9GSH-dependent oxidoreductase
GRX5−2.31.01.01.9Mitochondrial oxidoreductase
Thioredoxins and associated molecules
TRX1−3.4−1.21.12.1Cytoplasmic thioredoxin
TRX2−4.81.41.33.0Cytoplasmic thioredoxin
TRX3−2.02.11.55.5Mitochondrial thioredoxin
TRR1−3.6−1.51.11.4Cytoplasmic thioredoxin reductase
TRR21.21.1−1.12.1Mitochondrial thioredoxin reductase
PRX1−3.71.61.22.5Mitochondrial peroxiredoxin
TSA1−5.31.01.22.7Ubiquitous thioredoxin peroxidase
Glutathione peroxidase
GPX1−1.41.01.73.1Phospholipid hydroperoxide glutathione peroxidase
HYR1−3.71.61.23.4Thiol peroxidase
Heat shock protein
HSP12−2.02.11.23.0Plasma membrane protein
Transcription factors and associated molecules
AFT21.01.01.52.7Iron-regulated transcriptional activator
AHP1−3.3−1.11.12.1Thiol-specific peroxiredoxin
SCH91.11.91.32.9Protein kinase
SKN70.81.51.02.2Nuclear response regulator/transcription factor
yap1−2.71.21.13.8Basic leucine zipper transcription factor
YBP11.81.21.02.0Protein required for oxidation of cysteine residues of yap1p
UBA4−2.31.51.03.6Activates Urm1p, a target is Ahp1p
Other genes involved in the oxidative stress response
ASK10−2.0−1.10.81.0Component of RNA polymerase II
ATX11.81.41.04.0Cytosolic copper metallochaperone
CYR1−3.31.21.21.8Adenylate cyclase
DOT5−5.01.21.11.7Nuclear thiol peroxidase
EOS11.71.11.01.2N-Glyosylation protein
ERV1−2.5−1.11.01.8Sulfhydryl oxidase
FAP7−10.0−1.10.91.6NTPase
GAD1−1.11.21.32.5Glutamate decarboxylase
GND1−5.01.11.00.86-Phosphogluconate dehydrogenase
LOT63.0−1.11.01.6NAD(P)H:quinone reductase
LTV1−3.31.31.22.7Component of the GSE complex
MCR1−2.01.11.22.5Mitochondrial NADH-cytochrome b5 reductase
MXR1−1.81.31.55.2Peptide methionine sulfoxide reductase
NCE103−5.01.21.13.0Carbonic anyhdrase
OCA14.21.11.31.7Putative tyrosine phosphatase
OXR1−2.31.2−1.11.4Protein of unknown function
POS51.61.21.11.7Mitochondrial NADH kinase
SLN1−1.71.71.01.4Histidine kinase osmosensor
SVF1−10.01.20.91.7Protein required for diauxic growth shift
TMA19−5.5−1.71.41.2Ribosome-associated protein
UGA21.31.01.22.4Succinate semialdehyde dehydrogenase
URM1−3.31.31.14.9Ubiquitin-like protein
YAR1−5.01.20.91.8Protein of unknown function
YBL055C1.41.60.81.83′→5′ exonuclease and endonuclease
YCL033C−7.01.21.23.4Protein-methionine-R-oxide reductase
  • * Descriptions following those of the Saccharomyces Genome Database (Hong et al., 2007).

  • Values showing a statistically significant change in expression compared with the reference sample, as determined by Welch's 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<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.

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 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 peroxiredoxin-encoding 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 up-regulated), 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<0.05) in transcription (Table 1).

Differential transcription of stress response genes during brewery handling

During early propagation (0–8 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 up-regulated (from a total of 901 up-regulated genes). This 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.

2

Numbers of genes from various stress categories up- or down-regulated 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<0.05) with at least a 1.5-fold change between the two time points.

Between the 8 and 30 h sample times a relative up-regulation 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 8–30 h period, yeast cells changed 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.

3

Numbers of genes from various stress categories up- or down-regulated 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<0.05) with at least a 1.5-fold change between the two time points.

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 et al., 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. (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. (2003) have shown increased transcription of a number of stress-related genes coincident with entry into stationary phase of a wine yeast strain during microvinification of natural and synthetic wine must (Puig & Pérez-Ortín et al., 2000; Rossignol et al., 2003). Similar results have been reported for small-scale wort fermentations. Brosnan. (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. (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 et al., 1972; Petko & Lindquist et al., 1986; Bissinger et al., 1989; Borkovich et al., 1989; Hwang et al., 1989; Werner-Washburne et al., 1989; Praekelt & Meacock et al., 1990; Francois et al., 1992; Jamieson et al., 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). Boy-Marcotte. (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.

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. (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 et al., 1999; Estruch et al., 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 required for the activation of CTT1 in response to oxidative stress (Martinez-Pastor et al., 1996; Schmitt & McEntee et al., 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 STRE-regulated 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 et al., 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 et al., 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 et al., 1996). CUP1 transcription is also known to be activated by Hsf1p in response to glucose limitation (Tamai et al., 1994; Hahn & Thiele et al., 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 antioxidant-encoding 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 et al., 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 the function of certain antioxidants such as glutathione (Wu & Cederbaum et al., 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 Boy-Marcotte. (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 environmental 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.

Footnotes

  • Editor: Ian Dawes

References

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