yeast dealing with stress in fermentative environmentsludovico p, rodrigues f, almeida a, silva mt,...

Yeast dealing with stress in fermentative environments

Revisiting physiological models in light of molecular biology

Cecília Leão

Growth /Fermentation

NutrientsConsumption

Cell Death andAging

Yeast in fermentative environments

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

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?


van Uden’s Lab

Effects of ethanol on the yeast temperature profile

S. cerevisiae

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.

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

Grape Must Ethanol

Temperature

Alcoholic fermentation


Nutrients: carbon and nitrogen

sources


Wine production

Alcoholic beverages


Nutrient’s transport

Leão C, Van Uden N. Biotechnol Bioeng. 1983.

Inhibition by ethanol of growth andfermentation at permissive temperatures

k i MIC

Sequential inactivation of ammonium and glucose transport during fermentation

Glucose + ammonium

Glucose + ammonium + Ethanol (10%)



1012

14

5

Ethanol (%)

Inactivation of ammonium transportGlycineAlaninePhenylanineTyrosineTryptophan

Cardoso H, Leão C. FEMS Microbiol Lett. 1992.

Cycloheximide

Cycloheximide

inner mitochondrial membrane

Plasma membrane

plateau of maximum tolerance

Grape Must Ethanol

Temperature

Alcoholic fermentationNutrients: carbon

and nitrogen sources


Wine production

Alcoholic Beverages


By-products

● 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

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

Wine and food spoilage yeasts

Yeasts resistant to extreme environments

Zygosaccharomyces bailii…….

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

Physiology

1980s

1990s

2000s


Yeast as a model

1990s

2000s

I. Effects on yeast performance: an overview of the 80-90s




Ethanol and acetic acid: temperature profile and nutrient membrane transport



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 .

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

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

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.

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

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

Physiology

1980s

1990s

2000s


Yeast as a model

1990s

2000s

I. Yeast performance in fermentative environments: physiological advances of the 80-90s


Ethanol toxicity: temperature profile and nutrient m embrane transport





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

S. cerevisiae

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.

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!

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)

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

yeast dealing with stress in fermentative environmentsludovico p, rodrigues f, almeida a, silva mt,...

Documents