Analysing transcriptome data: II the antioxidant response of yeast during brewery propagation and fe

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Analysing transcriptome data II the antioxidant
response of yeast during brewery propagation and
fermentationBrian R. Gibson, Stephen J.
Lawrence, Chris A. Boulton, Wendy G. Box, Robert
S.T. Linforth, Katherine A. Smart
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Yeast handling in the Brewery
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Brewing as a stress continuum
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Oxygen use in the brewery

• Propagation and early fermentation
• UFA and sterol synthesis
• Cell replication
• Potential source of toxic reactive oxygen species

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Reactive oxygen species

• Unstable oxygen-containing molecules
• 1 (or more) unpaired electrons
• Superoxide (O2-)
• Hydrogen peroxide (H2O2)
• Hydroxyl radical (OH)

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Chemical origin of ROS
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Mitochondrial Intermembrane Space
Mitochondrial Matrix
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Cellular targets of ROS (O2-, H2O2, OH)
Lipid
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Yeast antioxidants

• Enzymatic defences
• Catalase
• Superoxide dismutase
• Cytochrome c peroxidase
• Glutathione peroxidase
• Glutathione reductase
• Thioredoxin peroxidase
• Thioredoxin reductase

• Non-enzymatic defences
• Glutathione
• Thioredoxins
• Glutaredoxins
• Ascorbic acids
• Flavohaemoglobin
• Metallothioneins
• Polyamines
• Ubiquinol
• Trehalose
• Ergosterol

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Catalase

• Eliminates cellular H2O2
• H2O2 ? 2H2O O2
• Yeast possesses 2 catalase enzymes
• Catalase A and Catalase T

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Glutathione

• A non-enzymatic polypeptide
• Has general ROS-scavenging ability
• Regulated by glutathione reductase

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Oxidative stress response stimuli

• Excess O2
• Growth phase
• Glucose derepression
• Anaerobic/aerobic transition
• Fatty acid metabolism
• Heat shock
• Gralla and Kosman (1992), Adv Gen 30, 251
  Jamieson (1998), Yeast 14, 1511

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Does oxygen exposure initiate the antioxidant
response in the brewery?

• Yeast cells exposed to oxygen in the brewery
• Yeast cells initiate an oxidative stress response
  when exposed to oxygen
• Does this occur in the brewery?

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Aims I

• Determine antioxidant status of yeast cells
  during industrial brewery handling
• Identify factors influencing the observed changes
  in the antioxidant response

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Propagation

• Two-tank propagation
• 8 hl seed vessel
• 140 hl growth vessel
• 30 h propagation

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Propagation sampling

• 140 hl principal growth vessel
• Triplicate samples
• 0 30 hours
• Cell density and budding index measured
• Antioxidant potential assessed
• Catalase activity
• Glutathione concentration
• Wort retained for sugar analysis
• RNA extracted for transcriptional analysis

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Cell growth during propagation
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Antioxidant activity during propagation
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Sugar profile during propagation
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Fermentation

• 3275 hl FV
• O2 25ppm

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Fermentation sampling

• Triplicate samples
• 0 102 hours
• Cell density and budding index measured
• Antioxidant potential assessed
• Catalase activity
• Glutathione concentration
• Wort retained for sugar analysis
• RNA extracted for transcriptional analysis

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Cell growth during fermentation
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Antioxidant activity during fermentation
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Sugar profile during fermentation
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Summary

• Temporal changes in cellular antioxidant levels
  during brewery handling
• Changes coincide with glucose depletion (glucose
  derepression)
• Changes in antioxidant levels unrelated to oxygen
  status of wort

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Data acquisition

• Extracted RNA reverse transcribed to produce cDNA
• In vitro transcription of cDNA to produce cRNA
• cRNA hybridized to Yeast Genome 2.0 GeneChip
  array (Affymetrix)
• Chip scanned to generate signal intensity (.CEL)
  files

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Data analysis

• GeneSpring analysis software (GeneSpring 7.3
  Silicon Agilent Technologies, USA)
• .CEL files normalized using Robust Multichip
  Average (RMA) pre-normalization algorithm
• Data filtered based on fold change and
  statistical significance (Welchs t-test P0.05)
  

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Aims II

• Elucidate genomic response of yeast during
  brewery handling
• Determine if antioxidant response is regulated at
  the transcriptional level

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Samples chosen for transcriptional analysis
(early propagation)
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Samples chosen for transcriptional analysis
(early propagation)

• 0 hour Stationary cells recently transferred
  from the seed vessel (low sugar continuous
  oxygenation)
• 8 hour Early exponential phase cells exposed to
  high sugar concentrations (including glucose) for
  8 hours continuous oxygenation

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Transcriptional changes during propagation
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Transcriptional changes during early propagation
(P0.05)
2513 genes ??
901 genes ?
1612 genes ?
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Transcriptional changes during early propagation
(P0.05)
2513 genes ??
901 genes ?
1612 genes ?
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Transcriptional changes during early propagation
(P0.05)
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Transcriptional changes during early propagation
(P0.05)
20 genes ??
2 genes ?
18 genes ?
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Samples chosen for transcriptional analysis (late
propagation)
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Samples chosen for microarray analysis (late
propagation)

• 8 hour Early exponential phase cells exposed to
  high sugar concentrations (including glucose) for
  8 hours continuous oxygenation
• 30 hour Early stationary phase, low sugar
  (glucose depleted) continuous oxygenation

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Transcriptional changes during late propagation
(P0.05)
566 genes ??
396 genes ?
170 genes ?
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Transcriptional changes during late propagation
(P0.05)
566 genes ??
396 genes ?
170 genes ?
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Transcriptional changes during late propagation
(P0.05)
genes ??
genes ?
genes ?
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Transcriptional changes during late propagation
(P0.05)
6 genes ??
5 genes ?
1 gene ?
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Transcriptional changes during fermentation
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Samples chosen for transcriptional analysis
(early fermentation)
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Samples chosen for microarray analysis (early
fermentation)

• 8 hour exponential phase cells, high sugar
  (including glucose), oxygen present.
• 30 hour early stationary phase, reduced sugar
  (including glucose), anaerobic.

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Transcriptional changes during early fermentation
(P0.05)
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Transcriptional changes during early fermentation
(P0.05)
40 genes ??
23 genes ?
17 genes ?
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Transcriptional changes during early fermentation
(P0.05)
genes ??
genes ?
genes ?
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Samples chosen for transcriptional analysis (late
fermentation)
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Samples chosen for transcriptional analysis
(late fermentation)

• 30 hour early stationary phase, reduced sugar
  (minimal glucose), anaerobic.
• 102 hour stationary phase, low sugar (glucose
  depleted), anaerobic.

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Transcriptional changes during late fermentation
(P0.05)
1605 genes ??
796 genes ?
809 genes ?
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Transcriptional changes during late fermentation
(P0.05)
1605 genes ??
796 genes ??
809 genes ??
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Transcriptional changes during late fermentation
(P0.05)
genes ??
genes ?
genes ?
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Transcriptional changes during late fermentation
(P0.05)
12 genes ??
10 genes ?
2 genes ?
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Conclusions

• Antioxidant response is regulated at the
  transcriptional level
• Response is initiated by glucose derepression
• Response is initiated irrespective of presence of
  other fermentable carbohydrates
• Response is not influenced by oxygen exposure or
  aerobic/anaerobic transition

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Future genomic analyses