Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation
© Teixeira et al.; licensee BioMed Central Ltd. 2012
Received: 24 May 2012
Accepted: 13 July 2012
Published: 27 July 2012
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.
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 3 H-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.
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.
Saccharomyces cerevisiae is extensively used in wine-making and brewing processes, and in bio-ethanol production . The successful performance of these industrial alcoholic fermentations depends on the ability of the used yeast strains to cope with a number of stress factors occurring during the process [2–4]. Stress induced by increasing amounts of ethanol, accumulated to highly inhibitory toxic concentrations during yeast alcoholic fermentation, is the major responsible for reduced ethanol productivity and for stuck and sluggish fermentations . Thus, yeast strains that can endure stress imposed by high ethanol concentrations are highly desired.
A number of studies based on detailed physiological and molecular analyses [2, 6–8] or on genome-wide surveys [9–15] have contributed to increase the understanding of the processes underlying ethanol toxicity and yeast tolerance to stress induced by this metabolite. Among the determinants of ethanol stress resistance identified so far, FPS1, described as an aquaglyceroporin involved in the control of the intracellular level of glycerol [16–21], has been reported to influence the intracellular accumulation of ethanol, upon sudden exposure to this compound . Although the exact mechanism of Fps1 action in this context is not clear, Fps1 may affect ethanol partitioning either directly  or through its effect in plasma membrane ergosterol content . Consistently, increased FPS1 expression was shown to increase the final ethanol concentration achieved by yeast cells, in conditions leading to high ethanol titters .
In the present study, the participation of a number of plasma membrane multidrug resistance (MDR) transporters of the ATP-Binding Cassette (ABC) superfamily (Pdr5, Pdr10, Pdr11, Pdr12, Pdr15, Pdr18, Snq2 and Yor1) and Major Facilitator Superfamily (MFS) (Aqr1, Atr1, Azr1, Dtr1, Flr1, Qdr1, Qdr2, Qdr3, Tpo1, Tpo2, Tpo3 and Tpo4) in ethanol stress resistance was scrutinized. Being implicated in the resistance to a variety of chemical stresses and involved in yeast detoxification from these compounds [23, 24], multidrug resistance transporters are plausible candidates for conferring yeast resistance to ethanol. Based on this screening, PDR18, encoding a plasma membrane  multidrug resistance transporter of the ABC superfamily , was the sole MDR transporter found to confer resistance to toxic concentrations of ethanol. Pdr18 was recently characterized as conferring resistance to chemical stress agents, including the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and barban, the agricultural fungicide mancozeb, and to cadmium, copper, manganese and zinc . Based on a genome-wide screening, PDR18 expression was also found to confer resistance to the anticancer drugs cisplatin and carboplatin and the antifungal drug nocodazole . Pdr18 was found to play a role in plasma membrane sterol incorporation, and this physiological trait proposed to contribute to its action as a multidrug resistance determinant . Indeed, the demonstrated effect that PDR18 expression has in plasma membrane ergosterol concentration and transmembrane potential is likely to affect transport across cell membranes and drug partition between the cell interior and the extracellular medium . The participation of Pdr18 in reducing the intracellular concentration of ethanol in yeast cells was examined in this study and PDR18 expression found to increase yeast ability to achieve higher final concentrations of ethanol during alcoholic fermentation. An engineered S. cerevisiae strain, in which the natural PDR18 promoter was replaced by the stronger PDR5 promoter, was obtained and shown to attain higher ethanol concentrations in a high gravity fermentation-like medium.
The expression of the ABC transporter Pdr18 increases yeast tolerance to ethanol
PDR18 expression decreases intracellular ethanol accumulation and ethanol-induced plasma membrane permeabilization in yeast
Given the Pdr18 role in ergosterol incorporation in the plasma membrane, the effect of Pdr18 expression on ethanol-induced plasma membrane permeabilization was examined. The measurement of the intracellular accumulation of the fluorescent marker propidium iodide was used as an indirect method to assess plasma membrane permeability. Based on this method, the permeability of ethanol-stressed yeast cells was seen to depend deeply on the expression of PDR18 (Figure 3B). Indeed, even in control conditions Δpdr18 deletion mutant cells exhibited a nearly 2-fold increase in fluorescence levels, compared to the parental strain, whereas the PDR18 over-expressing strain displayed a 10-fold decrease in cell fluorescence. Even a mild concentration of ethanol (4 %) was found to be sufficient to increase membrane permeability. In these stress conditions, the expression of PDR18 was found to be required to restrain ethanol-induced plasma membrane permeabilization (Figure 3B).
High gravity alcoholic fermentation with an engineered yeast strain exhibiting increased PDR18 expression levels produces a higher final ethanol concentration
This study describes the protective role of the yeast ABC transporter Pdr18 against ethanol stress, and the subsequent development of an engineered yeast strain able to achieve higher final ethanol concentrations in a high gravity fermentation media suitable for bio-ethanol production.
Based on a screening of mutants devoid of MFS or ABC multidrug transporters, PDR18 was found to be a determinant of resistance to inhibitory concentrations of ethanol in Saccharomyces cerevisiae. PDR18 gene expression was shown to decrease the intracellular accumulation of radiolabelled ethanol against a concentration gradient in stressed yeast cells. Although it is surprising to find out that a lipophilic molecule such as ethanol can be accumulated in yeast cells, a previous report had shown that ethanol also accumulates intracellularly in the absence of the Fps1 aquaglyceroporin , or during the first hours of alcoholic fermentation in media containing 20 % glucose . Whether Pdr18 facilitates the direct efflux of ethanol across the yeast plasma membrane, or indirectly affects its intracellular accumulation, remains to be established. Given that apparently a highly lipophylic molecule such as ethanol does not require a transporter since it may easily cross the lipid bilayer by passive diffusion, it is likely that its accumulation in yeast cells may occur at the level of the plasma membrane and depend on its lipid composition. This is a possible explanation for the differential accumulation of ethanol in yeast cells devoid of FPS1 or PDR18 (this work), whose deletion is known to affect at least the plasma membrane ergosterol composition [22, 26].
In this work it was found that Pdr18 expression contributes to increased ethanol tolerance, by controlling ethanol-induced plasma membrane permeabilization. The results obtained herein, correlating the decrease in PDR18 expression with the increase in plasma membrane permeability, are consistent with the effect of Pdr18 in the incorporation of ergosterol in the yeast plasma membrane . Indeed, the sterol plasma membrane composition has been shown to affect the level of plasma membrane permeabilization under stress conditions, particularly those leading to dehydration and changes in cell volume, a described effect of ethanol stress .
The obtained results led us to hypothesize that Pdr18 expression could also increase yeast capability to produce higher ethanol concentrations, being an advantage for industrial processes. Indeed, in conditions that lead to high ethanol production (medium containing 30 % glucose and no other limiting nutrient), the deletion of PDR18 was shown to reduce the concentration of ethanol reached when compared to the wild-type strain, whereas the engineered PDR18-overexpressing strain was shown to reach even higher ethanol concentrations in this industrial-like fermentation medium. Furthermore, the PDR18-overexpressing yeast strain was found to exhibit an increased ethanol production yield, favouring an improved use of the available glucose. This observation correlates with the effect of Pdr18 in restraining plasma membrane permeabilization occurring throughout alcoholic fermentation. It may also be related to the role of Pdr18 in ergosterol incorporation in the yeast plasma membrane, since a recent study shows that increasing ergosterol concentration in model membranes protects the membrane by preventing the ethanol-induced formation of an interdigitated phase, maintaining optimal membrane thickness as ethanol concentration increases during anaerobic fermentations . The over-expression of PDR18 in industrial yeast strains is pointed out as a promising strategy to further increase bio-ethanol production yield in industrial-scale processes. The strategy used in this study might also be a good choice for the manipulation of PDR18 expression in such industrial strains, since the PDR5 promoter is stronger than the natural PDR18 promoter and was shown in this study to lead to higher transcriptional levels during alcoholic fermentation than that induced by the PDR18 promoter. This choice further allows the use of high gravity rich media, similar to those used for industrial bio-ethanol production, in which the selective pressure required for plasmid maintenance cannot be assured or would become too expensive.
Higher ethanol yield is highly desired to reduce production costs associated to bio-ethanol production, in order to increase the sustainability of this process of obtaining energy from renewable sugar-rich substrates. In this study, PDR18 expression was found to increase yeast tolerance to highly inhibitory concentrations of ethanol, possibly through its role in decreasing ethanol-induced plasma membrane permeabilization and reducing the intracellular ethanol concentration. The final ethanol production reached by yeast cells in fermentation medium leading to high ethanol production was found to be higher in yeast cells over-expressing this gene. The over-expression of the PDR18 gene from its chromosomal locus in industrial yeast strains appears to be a promising strategy to increase the capacity for sustainable production of higher ethanol concentrations in industrial processes.
Strains, plasmids and growth media
The parental Saccharomyces cerevisiae strain BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) and the derived single deletion mutants Δpdr5, Δpdr10, Δpdr11, Δpdr12, Δpdr15, Δpdr18, Δsnq2, Δyor1, Δaqr1, Δatr1, Δazr1, Δdtr1, Δflr1, Δqdr1, Δqdr2, Δqdr3, Δtpo1, Δtpo2, Δtpo3, Δtpo4, Δyhr048w and Δyor152c (all obtained from the EUROSCARF collection) were used in this study. A plasmid pRS416_PDR18, expressing the PDR18 gene from its natural promoter, and the corresponding cloning vector, pRS416 (both plasmids were obtained from the EUROSCARF collection), were also used for complementation assays. Cells were cultivated in either rich YPD medium, containing 20 g/l glucose (Merck), 10 g/l Yeast Extract (Difco) and 20 g/l Peptone (Difco), or in minimal medium MM4, containing (per liter): 1.7 g yeast nitrogen base without amino acids or NH4+ (Difco), 20 g glucose (Merck), 2.65 g (NH4)2SO4 (Merck), 20 mg methionine (Sigma), 20 mg histidine (Sigma), 60 mg leucine (Sigma) and 20 mg uracil (Sigma). Strains were preserved in YPD medium, supplemented with 30 % glycerol, and stored at −80 °C.
PDR18 over-expression strain construction
To obtain the PDR18 over-expressing strain BY4741_ + PDR18, the natural PDR18 promoter (considered to start around -1000 bp from the start codon) was replaced in S. cerevisiae BY4741 for the stronger promoter of the PDR5 gene through homologous recombination. The replacement cassette was obtained by PCR amplification of the PDR5 promoter region preceded by a kanamycin cassette. This cassette was amplified from the Δyor152c deletion mutant, in which the YOR152c ORF, located at 1500 bp upstream of the PDR5 start codon, was replaced by a kanamycin cassette. DNA amplification was carried out using total DNA extracted from Δyor152c strain as a template and the following primers: 5’-ATGTCAGCGTGCCGCATTGAAAGGTAAAAACTAAAATTAATGAACTTTTCGGCGATTTTTGTGTTTCGTC-3’ and 5’-CTCAATTCCGTATCGCCGTCTTTAACAGTATGGAAATCCATTATGTTTAGGTCGAGTGATAACTAACACAG-3’. These primers include 50 nucleotides homologous to the PDR18 promoter region at the 5’ end and 20 and 21 nucleotides homologous to the flanking region of PDR5 promoter in the Δyor152c mutant (in italic), respectively. The PCR product of around 3000 bp was purified and used to transform S. cerevisiae BY4741. Transformants in which homologous recombination took place were selected on YPD supplemented with geneticin (150 mg/L) and promoter replacement was confirmed by PCR.
Ethanol susceptibility assays
The susceptibility of the parental strain BY4741 and derived mutant strains to toxic concentrations of ethanol was assessed by comparing their growth curves or growth on spot assays in MM4 medium supplemented or not with inhibitory concentrations of ethanol (6 % in liquid medium and 10-12 % in solid medium). Cell suspensions used to prepare the inocula for the growth curves or the spot assays were grown in MM4 medium until mid-exponential phase (OD600nm = 0.5±0.05). Cell growth in liquid media was conducted in 100 ml Erlenmeyer flasks, containing 50 ml of growth medium, at 30 °C, 250 rpm, and was followed by measuring culture OD600nm during batch cultivation. Cell suspension samples were diluted to an OD600nm below 0.4±0.05, prior to OD600nm determination. Cell suspensions used for the spot assays were diluted in sterile water to obtain suspensions with OD 600 nm = 0.05 ± 0.005. These cell suspensions and subsequent dilutions (1:5; 1:25) were applied as 4 μl spots onto the surface of agarized MM4 medium, supplemented with adequate ethanol concentrations. In the assays using wild-type or Δpdr18 cells harbouring the PDR18 expression plasmid of the corresponding empty vector, cells were grown in MM4-uracil selective medium.
[3 H]-ethanol accumulation assays
Accumulation of 3 H-ethanol was assessed as described before . To estimate the accumulation of ethanol (Intracellular/Extracellular 3 H-ethanol) from yeast cells, the parental strain BY4741 and the mutant strain Δpdr18 were grown in MM4 medium till mid-exponential phase. Cells were washed and resuspended in MM4 medium, to obtain dense cell suspensions (OD600nm = 5.0 ± 0.2, equivalent to approximately 2.2 mg (dry weight) ml-1). After 5 minutes incubation at 30 °C, with agitation (150 rev/min), to thermostat the suspensions, 0.1 μM of 3 H-ethanol (American Radiolabelled Chemicals; 250 μCi/ml) and 6 % (v/v) of unlabelled ethanol were added to the cell suspensions. Incubation proceeded for an additional period of 30 min. During this period of incubation, the intracellular accumulation of labeled ethanol was followed by filtering 200 μl of cell suspension, at adequate time intervals, through pre-wetted glass microfibre filters (Whatman GF/C). The filters were washed with ice-cold TM buffer (0.1 M MES (Sigma), 41 mM Tris (Sigma) adjusted to pH 5.5 with HCl) and the radioactivity measured in a Beckman LS 5000TD scintillation counter. Extracellular 3 H-ethanol was estimated, by radioactivity assessment of 50 μl of the supernatant. Non-specific 3 H-ethanol adsorption to the filters and to the cells (less than 5 % of the total radioactivity) was assessed and taken into consideration. To calculate the intracellular concentration of labeled ethanol, the internal cell volume (Vi) of the exponential cells, grown in the absence of drug and used for accumulation assays, was considered constant and equal to 2.5 μl (mg dry weight)-1 .
Membrane permeability measurement assays
Cell membrane permeability for populations of BY4741, BY4741_Δpdr18 and BY4741_ + PDR18 strains was compared by fluorescence microscopy, using as a fluorescent marker propidium iodide (PI), whose accumulation is considered to be dependent on membrane permeability . Cells were harvested by centrifugation (8,600 x g for 5 minutes at 4 °C) at suitable time points during the fermentation process and after 30 minutes of growth in the absence or presence of ethanol (4 %). Cell pellets were ressuspended in 500 mL fresh medium to an OD600nm = 0,4. PI (30 μM, 750 μL; Sigma) was added to the cell suspensions and incubated in the dark with orbital agitation (15 minutes, 250 rpm). PI-exposed cells were harvested by centrifugation (17,500 x g for 5 minutes) and washed twice and ressuspended in distilled deionized water. Fluorescence was examined with an Axioplan microscope equipped with adequate epifluorescence interface filters (BP450-490 and LP520; Zeiss). Fluorescence emission was collected with a cooled charge-coupled device camera (Cool SNAPFX; Roper Scientific Photometrics), and the images were analyzed with MetaMorph, version 3.5. The fluorescence images were background corrected by using dark-current images. Only the fluorescence of living cells was considered, unviable cells being identified based on whole-cell dispersion of fluorescence emitted upon incorporation of DAPI (4',6-diamidino-2-phenylindole; 50 ng/ml) staining and bright-field analysis of cell morphology. The fluorescence intensity values, considered to be proportional to PI accumulation, were calculated as the average of the fluorescence intensity of a minimum of 50 cells/experiment. The value of fluorescence intensity emitted by each cell was calculated by the software as the average of pixel by pixel intensity in the selected region of interest.
Assessment of ethanol and glucose concentration during alcoholic fermentation
The parental strain BY4741, the derived deletion mutant Δpdr18 and the PDR18 overexpressing strain BY4741_ + PDR18 were grown in YPD medium, supplemented with glucose (Merck), in order to obtain a final concentration of 300 g/l, and with the amino acids for which the strain is auxotrophic (240 mg/L Leucine, 80 mg/L Histidine and 80 mg/L Methionine). Cell suspensions used to prepare the inocula were grown in YPD medium until mid-exponential phase (OD600nm = 0.5±0.05). Cell growth in liquid media was followed by measuring culture OD600nm during batch cultivation. Samples of culture supernatants were harvested by centrifugation and used for the quantification of ethanol and glucose concentrations by HPLC. Cultures supernatants were analysed on an Aminex HPX-87 H Ion Exchange Chromatography column, eluted at room temperature with 0.005 M H2SO4 at a flow-rate of 0.6 mL min-1 during 30 minutes, using a refractive-index detector. Under such experimental conditions glucose had a retention time of 8.3 minutes and ethanol 19.4 minutes. Reproducibility and linearity of the method were tested and concentrations were estimated based on appropriate calibration curves.
Luís R. Raposo is acknowledged for his participation during the early stages of this work. Research described in this article was financially supported by “Fundação para a Ciência e a Tecnologia” (FCT) (Contract ERA-IB/0002/2010 and Post-Doctoral grant to TRC).
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