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Yeast dealing with stress in fermentative environments
Revisiting physiological models in light of molecular biology
Cecília Leão
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Growth /Fermentation
NutrientsConsumption
Cell Death andAging
Yeast in fermentative environments
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Physiology
1980s
1990s
2000s
Biology/Molecular Genetics
Yeast as a model
1990s
2000s
I. Yeast performance in fermentative environments: physiological advances of the 80-90s
II. Molecular mechanisms
Ethanol toxicity: temperature profile and nutrient membrane transport
Negative effects of weak carboxylic acids: the example of acetic acid
Molecular players of cell death induced by : -acetic acid -ammonium
Yeast dealing with stress in fermentative environmen tsRevisiting physiological models in light of molecular biology
III. Integrating the molecular biology with physiological models: from S. cerevisiae to Z. bailii
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Grape Must Ethanol
Temperature
Alcoholic fermentation
Ethanol Toxicity: a central question in the decade of the 80s
Nutrients: carbon and nitrogen
sources
Cell growth and metabolism
Wine production
Alcoholic beverages
Bio-ethanol industries
Mechanisms underlying the ethanol toxicity in S. cerevisiae?
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Ethanol Toxicity: a central question in the decade of the 80s
van Uden’s Lab
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Effects of ethanol on the yeast temperature profile
S. cerevisiae
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C Cabeça-Silva, A Madeira Lopes, N van Uden 1982. FEMS Microbiology Letters, 15, 149-151
6 % ethanol
Inner mitochondrial membrane as target
6% Ethanol
Control
Ethanol enhances “petite”
mutation in S. cerevisiae and at
any given ethanol concentration
the temperature profile of
“petite” mutation is located
between the T op and the T max
that are established at the same
ethanol concentration.
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bioethanol and red
wine in warm countries
“heat sticking” of fermentations
Ethanol
high alcohol beer,
champagne
Increased yeast ethanol-sensitive at intermediate temperatures
High temperature
fermentations
Low temperature
fermentations
Effects of ethanol on the yeast temperature profile
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Grape Must Ethanol
Temperature
Alcoholic fermentation
Ethanol Toxicity: a central question in the decade of the 80s
Nutrients: carbon and nitrogen
sources
Cell growth and metabolism
Wine production
Alcoholic beverages
Bio-ethanol industries
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Nutrient’s transport
Leão C, Van Uden N. Biotechnol Bioeng. 1983.
Inhibition by ethanol of growth andfermentation at permissive temperatures
k i MIC
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Sequential inactivation of ammonium and glucose transport during fermentation
Glucose + ammonium
Glucose + ammonium + Ethanol (10%)
Glucose + ammonium + Ethanol (12%)
Glucose + ammonium + Ethanol (14%)
1012
14
5
Ethanol (%)
Inactivation of ammonium transportGlycineAlaninePhenylanineTyrosineTryptophan
Cardoso H, Leão C. FEMS Microbiol Lett. 1992.
Cycloheximide
Cycloheximide
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inner mitochondrial membrane
Plasma membrane
plateau of maximum tolerance
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Grape Must Ethanol
Temperature
Alcoholic fermentationNutrients: carbon
and nitrogen sources
Cell growth and metabolism
Wine production
Alcoholic Beverages
Bio-ethanol industries
By-products
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● MEMBRANE TRANSPORT SYSTEMS AND THEIR REGULATION
● EFFECTS OF ACETIC ACID AND OTHER MONOCARBOXYLIC ACIDS
ON CELL DEATH
● SYNERGISTIC EFFECTS WITH ETHANOL
● IMPLICATIONS ON THE SURVIVAL OF THE YEAST IN ACIDIC
ENVIRONMENTS
Effects of weak carboxylic acids on yeast performance
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Debaryomyces hanseniiGlucose and xylose
Dekkera anomalaAcetic acid
Zygosaccharomyces bailiiAcetic acid
Kluyveromyces marxianusMalic acid
Candida utilisMalic, citric, lactic, succinic, fumaric, oxaloacetic and alpha-ketoglutaric acids
Saccharomyces cerevisiaeLactic and acetic acids, ammonium and glucose
Torulaspora delbrueckiiAcetic and lactic acids
Schizosaccharomyces pombeMalic acid and glucose
Hansenula anomalaFumaric, alpha-ketoglutaric, oxaloacetic and malic acids
Characterized transport systems
Department of Biology, University of Minho
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Wine and food spoilage yeasts
Yeasts resistant to extreme environments
Zygosaccharomyces bailii…….
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Acid and glucose media
Zb - Zygosaccharomyces bailiiSc - Saccharomyces cerevisiae
International patent nº PCT/PT00/00004
Wine and food spoilage yeasts
Schuller D, Corte-Real, Leão C. J Food Prot, 2000.
A Differential Medium for the Enumeration of the
Spoilage Yeast Zygosaccharomyces bailii in Wine
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Physiology
1980s
1990s
2000s
Biology/Molecular Genetics
Yeast as a model
1990s
2000s
I. Effects on yeast performance: an overview of the 80-90s
II. Molecular mechanisms
Molecular players of cell death induced by : -acetic acid -ammonium
Yeast dealing with stress in fermentative environmen tsRevisiting physiological models in light of molecular biology
Ethanol and acetic acid: temperature profile and nutrient membrane transport
Negative effects of weak carboxylic acids: the example of acetic acid
III. Integrating the molecular biology with physiological models: from S. cerevisiae to Z. bailii
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Acetic acid induces death of glucose-grown cells of Saccharomyces cerevisiae
Acetic acid is over 30-times more toxic than ethanol at high
process temperatures
Acetic acid at concentrations as may occur during fermentations
induced cell death at intermediate temperatures
Yeast dealing with stress in fermentative environmentsRevisiting physiological models in light of molecular biology
I Pinto, H Cardoso, C Leão, N van Uden 1989. Biotechnology and Bioengineering .
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Detection ofDNA strand
breaks
TUNEL reaction andcolocalization by DNA
staining with PI
Double staining with FITC-Annexin V and PI
Transmission electronmicroscopy analysis
Control Apoptotic40 mM
Necrotic120 mM
ChromatinCondensation
Phosphatidylserineexposure
Ludovico P, Sousa MJ, Silva MT, Leão C, Côrte-Real M. Microbiology. 2001.
Saccharomyces cerevisiae commits to a programmed celldeath process in response to acetic acid
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Respiratory activities
Cytochrome c
Respiratory function is affected
Ludovico P, Rodrigues F, Almeida A, Silva MT, Barrientos A, Côrte-Real M. Mol Biol Cell. 2002
Mitochondria involvement in programmed cell deathinduced by acetic acid in Saccharomyces cerevisiae
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Control Acetic acid
12 Up39 Down
Proteome of total extracts of acetic acid-inducedapoptosing cells
28 Proteins
Almeida B, Ohlmeier S, Almeida AJ, Madeo F, Leão C, Rodrigues F, Ludovico P. Proteomics. 2009.
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Intracellular amino acid pool
Nutrient import
Depletion of the intracellular pool of amino acids in yeast cells upon acetic acid induced apoptosis
Increase of severalenzymes ofaminoacidsbiosynthetic pathways
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Acetic acid Acetic acid
Activation ofMetacaspase
Aif
Cyt c
Glucose
Amino acids/Ammonium
Starvation inGlucose
alteration in respiratory
activity
Production of ROS
Decrease of pH
Acetate + H+Starvation in amino
acids
Fragmentation ofDNA
Celldeath
S. cerevisiae
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Physiology
1980s
1990s
2000s
Biology/Molecular Genetics
Yeast as a model
1990s
2000s
I. Yeast performance in fermentative environments: physiological advances of the 80-90s
II. Molecular mechanisms
Ethanol toxicity: temperature profile and nutrient m embrane transport
Negative effects of weak carboxylic acids: the example of acetic acid
Molecular players of cell death induced by : -acetic acid -ammonium
Yeast dealing with stress in fermentative environmen tsRevisiting physiological models in light of molecular biology
III. Integrating the molecular biology with physiological models: from S. cerevisiae to Z. bailii
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Non-conventional yeasts
Z. bailii ISA 1307
Guerreiro JF, Mira NP, Sá-Correia I. Adaptive
response to acetic acid in the highly resistant
yeast species Z. bailii revealed by quantitative
proteomics. Proteomics. 2012
Rodrigues F, Sousa MJ, Ludovico P, Santos H, Côrte-Real
M, Leão C. The fate of acetic acid during glucose co-
metabolism by the spoilage yeast Z. bailii. PLoS One.
2012;7(12):e52402.
Mira et al. The genome sequence of
the highly acetic acid-tolerant Z.
bailii derived interspecies hybrid
strain ISA1307, isolated from a
sparkling wine plant. Under review
Integrating the molecular biology with physiological models
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S. cerevisiae
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If nowadays the scientific life is difficult at that time it appeared almost impossible.
Van Uden was a visionary and brought to Portugal somany outstanding scientists:
And believe that:
- Overall, we could learn a lot withall the advanced courses at IGCin 80’s spreading knowledgethroughout the entire Portugal.
- His creative thinking and theseminal work he developed werethe inoculum of a growing“yeast scientific community”that fermented and are stillrespiring the knowledgeproduced.
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So for you all, young scientists facing the so difficult days,
believe that with your work, perseverance, intelligence and creativity,
nothing and no one will be able to stop you!
So for you all, young scientists facing the so difficult days,
believe that with your work, perseverance, intelligence and creativity,
nothing and no one will be able to stop you!
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Before Finishing …Before Finishing …Before Finishing …Before Finishing …
A magia de um olhar sobre a
beleza “No fundo das
masseiras”
The magic of a look at the beauty"At the bottom of
the dough troughs“
Yeasts isolated from traditional corn and rye bread doughFrom the Book “Tribute to Prof. Carlos Gancedo, Madrid
Almeida MJ, Pais C. and Leão C. (2007)
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Biology Department, Scienes School (1976-2000)Life and Health Sciences Research Institute, Health Sciences School (since 2000)
Cecília Leão
Yeast dealing with stress in fermentative environments
Revisiting physiological models in light of molecular biology
University of Minho, Braga, Portugal
ComunicaçãMicrobiotec dezembro Vult. 7 dez-2013