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Phenotypic analysis and transcriptome profiling of Saccharomyces response to medium chain fatty acids
Jean Luc Legras, C. Erny, Y. Adolphe, C. Le Jeune, Marc Lollier, Bruno Blondin, Francis Karst
To cite this version:
Jean Luc Legras, C. Erny, Y. Adolphe, C. Le Jeune, Marc Lollier, et al.. Phenotypic analysis and
transcriptome profiling of Saccharomyces response to medium chain fatty acids. 26. International
Specialised Symposium on Yeasts, Jun 2007, Sorrente, Italy. 1 p., 2007. �hal-02819274�
PDR12 mRNA levels are maximal 15’ after exposure and back to basal level after 1 hour.
Phenotypic analysis and transcriptome profiling of Saccharomyces response to medium chain fatty acids
Legras JL, Erny C, Adolphe Y, Le Jeune C, Lollier M, Delobel P, Blondin B and Karst F
(1) UMR1131 Santé de la Vigne et Qualité du Vin, INRA, Université Louis Pasteur de Strasbourg, Colmar - France
(2) LVBE, Université de Haute Alsace, 33 rue de Herrlisheim, BP 50568, 68008 Colmar Cedex – France. Phone : 33(0)389203136 - [email protected] (3) UMR1083 Sciences pour l’Oenologie, AgroM, INRA, Université Montpellier 1, Montpellier - France
Introduction
Medium chain fatty acids (MCFA) produced by Saccharomyces during fermentation are well known fermentation inhibitors, which toxicity is enhanced by ethanol and low pH encountered in wine making process (1). On the one hand the accumulation of medium chain fatty acids during wine fermentation is considered as a contributor to sluggish fermentation but on the other hand, fatty acid ethyl esters do contribute positively to the sensory aspect of wine. When exposed to these acids, Saccharomyces acquires greater resistance. For example, octanoic acid induction results in the synthesis of a membrane transporter not characterized until now (2).
To investigate the Saccharomyces genes involved in the response to octanoic and decanoic acids we combined both transcriptome profiling and phenotypic analysis.
Results
Transcriptome profiling Phenotypic analysis
The ability of 80 knocked-out strains to grow on YPG media containing octanoic or decanoic acid was tested on solid or liquid YPG (pH 4.5).
1- octanoic and decanoic acids induce different sets of genes partially overlapping
Number of genes whose expression levels differ significantly in two different conditions
5 Method
Results
C10/T 168 gènes C8/T
22 gènes C10/C8
48 gènes
11 20
23 6
139 Alsacian oenological U13 strain
4 independent replicates
Triangular transcriptome comparison
T C8
T C10
C8 C10
Results analysis
- LIMA GUI (R) - FunSpec Cluster Interpreter - Yeastract YPD
28°C
t0
YPD + 0.05 mM C8
YPD + 0.05 mM C10
t20
RNA extraction
YPD
x 7.5 2- the more largely overexpressed genes are different according to inhibitors’
Transporters TPO4, PDR12, (slightly TPO1)
10 other genes, mainly controled by Msn2/Msn4
Energy production and mobilisation C8 :
C10 :
x 4.5 PDR12
Weak acids transporter
Lipid metabolism - EEB1 : AcylCoA Ethanol O-acyltransférase -ELO1 : acyl elongase -FAA1 : acylCoA synthetase
3- Transcription factors potentially involved in MCFA response(Yeastract database) The search for known transcription factors involved in the regulation of the two responses (otanoic or decanoic) revealed the same set of genes. However these transcription factors seemed to be involved in different manners according to the fatty acid :
Response to octanoic acid : mainly under the dependence of MSN2/MSN4 + WAR1 Response to decanoic acid : mainly under the dependence of MSN2/MSN4 + YAP1
YNB YNB + 0.8 mM C8 YNB + 0.2 mM C10
By4741 ΔWar ΔPDR12
ΔPDR16 ΔSef1 ΔTPO1
By4741 ΔWar ΔPDR12
ΔPDR16 ΔSef1 ΔTPO1
By4741 ΔWar ΔPDR12
ΔPDR16 ΔSef1 ΔTPO1
S. cerevisiae response to octanoic acid involves the weak acid transporter PDR12, and to a lower extent, the TPO1 transporter and PDR16.
Response to decanoic acid involves TPO1 but not TPO4 MSF transporter.
Deletion of WAR1 abolishes resistance to octanoic acid, while deletion of either MSN2, MSN4 or YAP1 has little effect on yeast growth.
Conclusion
Octanoic and decanoic acids induce different sets of gene partially overlapping.
Saccharomyces cerevisiae response to octanoic acid share common features with weak acid response (3), essentially with activation of Pdr12 transporter.
Moreover, Tpo1 transporter is also involved in octanoic acid expulsion.
Adaptation to decanoic acid is more complex, including expulsion of C10 by the Tpo1 transporter, and activation of a metabolic pathway able to detoxify decanoic acid and to generate flavour-producing ethyl-esters.
References
(1) : Viegas C. A. and Sa-Correia I., 1997, Int J Food Microbiol 34, 267-277 (2) : Guadalupe Cabral M. et al, 2001, Arch Microbiol 175: 301-307 (3) : Schuller C. et al, 2004, Mol Biol Cell 15, 706-720
The transcriptome of the oenological resistant S. cerevisiae U13 strain was studied after exposure to octanoic or decanoic acid.
0 1 2 3 4 5 6 7 8
relative mRNA level
0 15 30 45 60
time post induction (min) Time course of Pdr12 mRNA level
Analysis of PDR12 mRNA levels
PDR12 mRNA levels were measured by Real Time PCR after exposure to 0.1 mM octanoic acid.
Nucleus C8
C10
Amino acids metabolism
Respiration Sugar
metabolism OH
H H O H
OH CH2OH
O OHOH
HXT6
HXT7
PDR12 PDR16 C8
TPO4
TPO1 C10 ? C10
C10 C10-CoA
FAA1
C12-CoA C14-CoA
ELO1 ELO1
Éthyl-C10 Éthyl-C12
Éthyl-C14
EEB1 EEB1
EEB1
C8
check activation of a new metabolic way to detoxify decanoic acid and produce ethyl-esters …..
ISSY26 – Sorrente –June 2007
