Activation of two different resistance mechanisms in Saccharomyces cerevisiae upon exposure to octanoic and decanoic acids

Abstract

Medium-chain fatty acids (octanoic and decanoic acids) are well known as fermentation inhibitors. During must fermentation, the toxicity of these fatty acids is enhanced by ethanol and low pH, which favors their entrance in the cell, resulting in a decrease of internal pH. We present here the characterization of the mechanisms involved in the establishment of the resistance to these fatty acids. The analysis of the transcriptome response to the exposure to octanoic and decanoic acids revealed that two partially overlapping mechanisms are activated; both responses share many genes with an oxidative stress response, but some key genes were activated differentially. The transcriptome response to octanoic acid stress can be described mainly as a weak acid response, and it involves Pdr12p as the main transporter. The phenotypic analysis of knocked-out strains confirmed the role of the Pdr12p transporter under the control of WAR1 but also revealed the involvement of the Tpo1p major facilitator superfamily proteins (MFS) transporter in octanoic acid expulsion. In contrast, the resistance to decanoic acid is composite. It also involves the transporter Tpo1p and includes the activation of several genes of the beta-oxidation pathway and ethyl ester synthesis. Indeed, the induction of FAA1 and EEB1, coding for a long-chain fatty acyl coenzyme A synthetase and an alcohol acyltransferase, respectively, suggests a detoxification pathway through the production of decanoate ethyl ester. These results are confirmed by the sensitivity of strains bearing deletions for the transcription factors encoded by PDR1, STB5, OAF1, and PIP2 genes.

FIG. 1.

Variability of Saccharomyces cerevisiae wine strain resistance to octanoic acid (x axis) and decanoic acid (y axis) as revealed by drop test (resistance levels are given in Table 1). The dimension of the spots is related to the number of strains in each category indicated in the spots. The arrow indicates the group containing U13.

FIG. 2.

Correlation between the two responses. Triangles, expression ratio of genes whose expression level varied significantly compared to that of the control for one modality; spheres, expression ratio of genes whose expression level varied significantly for C8 compared to that of C10; these points were excluded for the estimation of the correlation between the two responses.

FIG. 3.

Venn diagram presenting the genes differentially expressed in these three conditions. C8, 20-min exposure to octanoic acid at 50 mM; C10, 20-min exposure to octanoic acid at 50 mM; C8/C10, comparison of exposure to C10 to exposure to C8. Repressed genes are followed by a minus sign; induced genes are followed by a plus sign.

FIG. 4.

Factorial component analysis comparing the involvement of each transcription factor in the different stress responses. The proportion of each stress response explained by one transcription factor has been calculated from the Yeastract website. Codes for the different stresses: OleicAc, oleic acid; POELE, polyoxyethylene-9-laurylether; SorbicAc, sorbic acid; 2-4D, 2,4-dichlorophenoxyacetic acid; DCP, 2,4-dichlorophenol; C10whole, decanoic acid; C8whole, octanoic acid. The octanoic and decanoic shared response (C8-C10 shared) is analyzed as a supplementary individual and given in italic. Transcription factors with a cos² lower than 0.1 are not drawn. Main transcription factors involved in the stress response are in boldface.

FIG. 5.

Drop test presenting the sensitivity provoked by the deletion of different transporters on YPD (pH 4.5) medium containing 0.6 mM octanoic acid (C8) or 0.25 mM decanoic acid (C10) compared to that of the control (T). Cells used to prepare the spots were grown on liquid YPD (pH 4.5) medium until a standardized OD₆₆₀ of 0.8 (a) and diluted to an OD₆₆₀ of 0.08 (b). The growth observed for the nondiluted spot of the wild-type By laboratory strain corresponds to a rank of three in the first figure.

FIG. 6.

Drop test presenting the sensitivity of diploid strains deleted from one or two copies of PDR12 and TPO1 transporter genes on YPD (pH 4.5) medium containing 0.6 mM octanoic acid (C8) or 0.25 mM decanoic acid (C10) compared to that of the control (T). Cells used to prepare the spots were grown on liquid YPD (pH 4.5) medium until a standardized OD₆₆₀ of 0.8 (a) and diluted to an OD₆₆₀ of 0.08 (b).

FIG. 7.

Drop test presenting the sensitivity of strains deleted for different transcription factors on YPD (pH 4.5) medium containing 0.6 mM octanoic acid (C8) or 0.25 mM decanoic acid (C10) compared to that of the control (T). Cells used to prepare the spots were grown on liquid YPD (pH 4.5) medium until a standardized OD₆₆₀ of 0.8 (a) and diluted to an OD₆₆₀ of 0.08 (b).