Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

Abstract

Background: The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance.

Results: A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased ³H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation.

Conclusions: PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production.

Figure 1

PDR18expression increases yeast tolerance to ethanol. Comparison of the susceptibility to ethanol-induced stress of S. cerevisiae parental strain BY4741 and the derived deletion mutant Δpdr18, through spot assays (A) or cultivation in liquid medium (B). Cells used for the ethanol susceptibility assays were mid-exponential phase cells (OD_600nm = 0.5±0.05) cultivated in minimal growth medium in the absence of stress. A: The susceptibility to inhibitory concentrations of ethanol of BY4741 and derived Δpdr18 strains, harboring a PDR18 expression plasmid or the corresponding cloning vector pRS416 was compared. The cell suspensions used to prepare the spots in lanes b) and c) were 1:5 and 1:25 serial dilutions, respectively, of the suspensions with an OD_600nm = 0.05±0.005 in lane a). B: Wild-type (▵, ▴) and Δpdr18 (□, ■) cell suspensions were also used to inoculate MM4 liquid medium in the absence (▵, □) or presence (▴, ■) of 6 % ethanol, with an initial OD_600nm = 0.05±0.005. Growth curves, followed by the measurement of the OD_600nm and the number of colony forming units (CFU) per ml, are representative of at least three independent experiments.

Figure 2

PDR18expression decreases intracellular ethanol accumulation. Comparison of [³ H]-ethanol accumulation in non-adapted cells of S. cerevisiae parental strain BY4741 (▴) and the derived deletion mutant Δpdr18 (■), during cultivation for 30 min in MM4 liquid medium supplemented with 6 % cold ethanol. The accumulation ([³ H-Eth]intra/[³ H-Eth]extra) values are the means of at least three independent experiments, error bars indicating standard deviation.

Figure 3

PDR18over-expression increases ethanol tolerance and protects from ethanol-induced plasma membrane permeabilization.A: Comparison of the susceptibility to ethanol induced stress of S. cerevisiae parental strain BY4741 and the derived deletion mutant BY4741_Δpdr18, and PDR18 over-expression strain BY4741_ + PDR18 through spot assays. Cells used for the spot assays were mid-exponential phase cells (OD_600nm = 0.5±0.05) cultivated in minimal growth medium in the absence of stress. The cell suspensions used to prepare the spots in lanes b) and c) were 1:5 and 1:25 serial dilutions, respectively, of the suspensions with an OD_600nm = 0.05±0.005 in lane a). B: Comparison of the membrane permeability exhibited by S. cerevisiae yeast strains BY4741, BY4741_Δpdr18 and BY4741_ + PDR18, grown to mid-exponential phase and exposed (white columns) or not (grey columns) for 30’ to mild-stress induced by 4 % ethanol. Cell permeability was assessed based on the fluorescence intensity exhibited by yeast cells upon passive accumulation of propidium iodide. Fluorescence intensity values are the means of at least three independent experiments, error bars indicating standard deviation.

Figure 4

PDR18over-expression leads to higher ethanol production yield in a high gravity-like fermentation medium. Comparison of the PDR18 transcript levels (B), and the extracellular concentrations of ethanol (C) and glucose (D), accumulated/used during cultivation in fermentation medium, containing 30 % glucose, of S. cerevisiae yeast strains BY4741 (◊), BY4741_Δpdr18 (▵) and BY4741_ + PDR18 (□). A: Growth curves, followed by measuring culture OD_600nm, are representative of at least three independent experiments. B: The relative values of PDR18 mRNA/ACT1 mRNA, as obtained through RT-PCR, are the means of at least three independent experiments, error bars indicating standard deviation. The PDR18 mRNA relative value for the parental BY4741 in control conditions was set as 1 and the remaining values were calculated relative to it. C and D: Ethanol and glucose concentration levels, assessed using HPLC, are also mean values of at least three independent experiments, error bars indicating standard deviation.

Figure 5

PDR18over-expression protects from fermentation-induced plasma membrane permeabilization. Comparison of the membrane permeability exhibited by S. cerevisiae yeast strains BY4741, BY4741_Δpdr18 and BY4741_ + PDR18, upon 6 h (white columns; corresponding to exponentially growing cells) or 160 h (grey columns; corresponding to stationary phase cells, upon fermentation arrest) of cultivation in fermentation medium containing 30 % glucose. Cell permeability was assessed based on the fluorescence intensity exhibited by yeast cells upon passive accumulation of propidium iodide. Fluorescence intensity values are the means of at least three independent experiments, error bars indicating standard deviation.