Relationship between lipid composition, frequency of ethanol-induced respiratory deficient mutants, and ethanol tolerance in Saccharomyces cerevisiae

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1 Journal of Applied Microbiology 1999, 86, elationship between lipid composition, frequency of ethanol-induced respiratory deficient mutants, and ethanol tolerance in Saccharomyces cerevisiae Z. Chi 1 and N. Arneborg 2 1 The State Key Laboratory of Microbial Technology, Shandong University, China, and 2 Department of Dairy and Food Science, The oyal Veterinary and Agricultural University, Frederiksberg, Denmark 7025/01/99: received 8 January 1999 and accepted 9 March 1999 Z. CHI AND N. ANEBOG The frequency of ethanol-induced respiratory deficient mutants and lipid composition in two Saccharomyces cerevisiae strains showing different degrees of ethanol tolerance were investigated. The more ethanol-tolerant strain exhibited a lower frequency of ethanol-induced respiratory deficient mutants than the less ethanol-tolerant strain. In addition, the more ethanol-tolerant strain contained a higher ergosterol/ phospholipid ratio, a higher proportion of phosphatidylcholine, a lower proportion of phosphatidylethanolamine, a higher incorporation of long-chain fatty acids in total phospholipids, and a slightly higher proportion of unsaturated fatty acids in total phospholipids than the less ethanol-tolerant strain. These results show a clear relationship between the lipid composition, the frequency of ethanol-induced respiratory deficient mutants, and the ethanol tolerance of S. cerevisiae. A possible explanation of this relationship is discussed. INTODUCTION It is well established that different strains of the yeast Saccharomyces cerevisiae differ in their ability to tolerate ethanol (ose 1993) but although this phenomenon has been widely investigated throughout the last five decades, it is still not known why some S. cerevisiae strains are more ethanol tolerant than others. It is well known that ethanol is a powerful inducer of respiratory deficient (D) mutants in S. cerevisiae (Bandas and Zakharov 1980; Jimenez and Benitez 1988; Jimenez et al. 1988; Norton et al. 1995; Ibeas and Jimenez 1997). The mechanisms leading to ethanol-induced respiratory deficiency are, however, still unknown. Taking into consideration the facts that ethanol is known to fluidize S. cerevisiae membranes (Mishra and Prasad 1988; Alexandre et al. 1994) and that ethanol-induced D mutations in S. cerevisiae are thought to be caused by damage to the mitochondrial membrane rather than by DNA damage (Ibeas and Jimenez 1997), it is likely that the membrane fluidizing effect of ethanol may be responsible for the formation of ethanol-induced D mutants in S. cerevisiae. Correspondence to: Dr Nils Arneborg, Department of Dairy and Food Science, The oyal Veterinary and Agricultural University, olighedsvej 30, DK-1958 Frederiksberg C, Denmark ( na@kvl.dk). Membrane lipids are well known modulators of membrane fluidity (ussell 1989), and they are considered to play an essential role in the ethanol tolerance of S. cerevisiae (Mishra and Kaur 1991; Sajbidor 1997). Thus, it may be suggested that the lipid composition of a given S. cerevisiae strain, by affecting the fluidity of its membranes, may influence the formation of ethanol-induced D mutants, and that this, in turn, may affect the ethanol tolerance of the strain. The existence of such a relationship in S. cerevisiae seems, however, not to have been reported. In this study, the existence of a relationship between the lipid composition, the frequency of ethanol-induced D mutants and the ethanol tolerance in two different strains of S. cerevisiae is demonstrated, and a possible explanation of this relationship is discussed. MATEIALS AND METHODS Micro-organism and growth The experiments were carried out with a wild type strain, S. cerevisiae 1200, which is a highly ethanol-producing yeast (Chi and Liu 1993; Ohta et al. 1993), and a commercial strain of lager yeast, S. cerevisiae AJL 2155 (The Collection of Pure 1999 The Society for Applied Microbiology

2 1048 Z. CHI AND N. ANEBOG Cultures of Brewing Yeasts, Alfred Jorgensen Laboratory Ltd, Copenhagen, Denmark). The yeast strains were maintained at 4 C on YPD agar (containing l 1 : 20 g glucose, 20 g peptone, 10 g yeast extract and 20 g agar). The yeast cells were grown for 20 h to early stationary growth phase at 30 C in 100 ml defined medium with 20 g l 1 glucose (DMD) (Arneborg et al. 1995) in a rotary shaker (200 rev min 1 ). The growth experiments for each yeast strain were carried out in duplicate. Ethanol shock treatment At each early stationary growth phase, one cell sample was harvested and washed twice with sterile water. The cell pellet was resuspended in 10 ml defined medium without glucose (DM) and with 18% (v/v) ethanol. In addition, an unstressed control, i.e. cells suspended in DM without ethanol, was carried out for each cell harvest. Both the ethanol-stressed cells and the unstressed controls were incubated at 30 C in a rotary shaker (100 rev min 1 ). disrupted cells were mixed with 80 ml chloroform/methanol (2 : 1), transferred to a 500 ml flask and stirred with a magnetic bar for 2 h on ice. The mixture was filtered and washed with another 40 ml chloroform/methanol (2 : 1). The filtrate was mixed with 20 ml 0 034% MgCl 2 and the mixture was left to separate overnight at 4 C. The lower organic phase was mixed with 3 ml benzene/ethanol (3 : 2) and taken to dryness on a rotary evaporator at 40 C. The residue was immediately dissolved in 2 ml chloroform/methanol (2 : 1). The lipid sample was stored at 20 C. The separation of phospholipids from neutral lipids, and the analysis of ergosterol, were performed according to Arneborg et al. (1995). Phospholipids were separated by twodimensional thin-layer chromatography (TLC) using silica gel 60 plates (Merck) (Wagner and Paltauf 1994). The separated phospholipids were scraped off the TLC plate and analysed according to the method of ouser et al. (1970). Fatty acids of total phospholipids were analysed according to Arneborg et al. (1995). Determination of cell survival Samples were taken periodically from the cell suspension, and appropriate dilutions were plated on YPD agar in triplicate. The plates were incubated for 72 h at 30 C before counting. The survival of cells was taken as the percentage of survivors after exposure to ethanol compared with the unstressed control. Determination of frequency of D mutants espiratory deficient mutants were identified by overlaying the yeast colonies on the above mentioned plates with a 2,3,5-triphenyltetrazoliumchloride (TTC) staining medium (containing l 1 : 0 5 g TTC, 5 g glucose and 6 25 g agar). After incubation at 30 C for 1 h, D mutants were identified as white colonies that were unable to reduce the tetrazolium salt from colourless to red. The frequency of D mutants was taken as the percentage of white colonies compared with the total number of cells on a plate. The respiratory deficiency was confirmed by an inability of the white colonies to grow on YPG agar (containing l 1 : 30 ml glycerol, 20 g peptone, 10 g yeast extract and 20 g agar) and, at the same time, an ability to grow on YPD agar (Ernandes et al. 1993). Lipid analyses At each early stationary growth phase, one cell sample was harvested and washed, and the yeast cells were disrupted with glass beads (diameter mm) in a MM2 mixer mill (K. etsch GmbH, Haan, Germany) for 30 min at 4 C and a frequency of 1800 rev min 1 (Arneborg et al. 1995). The Miscellaneous analytical procedures The O.D. 525 was measured using a UVIKON 942 spectrophotometer (Kontron Instruments, Neufahrn, Germany). Cell dry weight (CDW) was determined by filtering 3 ml of the growth medium using dry Micron Separations Inc. filters (pore diameter 0 45 mm; MSI, Westboro, MA, USA) and vacuum suction. The filters with cell deposits were washed twice with 3 ml water, dried in a microwave oven for 30 min and desiccated for 30 min in a desiccator before weighing. Statistical analysis Statistical analysis of data was carried out by one-way analysis of variance (ANOVA). In the ethanol tolerance and D mutant experiments, probabilities lower than or equal to 0 1 were considered significant. In the lipid composition experiments, probabilities lower than or equal to 0 05 were considered significant. ESULTS Ethanol tolerance The survival of S. cerevisiae 1200 cells when exposed to 18% (v/v) ethanol for 2 h was significantly (P ³ 0 1) higher than the survival of S. cerevisiae AJL 2155 cells (Fig. 1), showing that S. cerevisiae 1200 cells were more ethanol tolerant, as measured by viability, than S. cerevisiae AJL 2155 cells.

3 ETHANOL TOLEANCE IN S. CEEVISIAE Survival (%) Frequency of D mutants (%) Time (h) Fig. 1 Percentage survival after exposure to 18% (v/v) ethanol at 30 C of aerobic, early stationary phase Saccharomyces cerevisiae AJL 2155 (Ž) and 1200 () cells. Values for each yeast strain are means of two independent growth experiments (see Materials and Methods). Vertical bars represent standard deviations Time (h) Fig. 2 Frequency of respiratory-deficient mutants among the survivors after exposure to 18% (v/v) ethanol at 30 C of aerobic, early stationary phase Saccharomyces cerevisiae AJL 2155 (Ž) and 1200 () cells. Values for each yeast strain are means of two independent growth experiments (see Materials and Methods). Vertical bars represent standard deviations espiratory deficient mutants The frequency of D mutants among the survivors of the S. cerevisiae 1200 cells after 2 h of ethanol shock treatment was significantly (P ³ 0 1) lower than that of the S. cerevisiae AJL 2155 cells (Fig. 2). For both yeast strains, no D mutants were observed in the unstressed controls (data not shown). These results indicate that the mitochondria in S. cerevisiae 1200 were more resistant to the ethanol shock treatment than the mitochondria in S. cerevisiae AJL Lipid composition Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constituted the major part of the phospholipids (approximately 70%) in both yeast strains (Table 1). Saccharomyces cerevisiae 1200 had a significantly (P ³ 0 05) higher proportion of PC and a significantly (P ³ 0 05) lower proportion of PE than S. cerevisiae AJL 2155 (Table 1). The proportions of the other phospholipids, including phosphatidylinositol (PI), were not significantly (P ³ 0 05) different in the two yeast strains (Table 1). In both yeast strains, the total phospholipids contained mainly C 16:0,C 16:1,C 18:0 and C 18:1 fatty acids (Table 2). The fatty acid compositions of the total phospholipids were characterized by a low amount of C 18:0 and a high amount of C 16:1 (Table 2). The S. cerevisiae 1200 cells contained a significantly (P ³ 0 05) higher proportion of unsaturated fatty acids (DUFA) in total phospholipids than the S. cer- evisiae AJL 2155 cells, mainly due to a significantly (P ³ 0 05) higher amount of C 18:1 and a significantly (P ³ 0 05) lower amount of C 16:0 in the phospholipids (Table 2). Moreover, the S. cerevisiae 1200 cells incorporated significantly (P ³ 0 05) more long-chain fatty acids, i.e. C 18:0 and C 18:1, into total phospholipids at the expense of shorter-chain fatty acids, i.e. C 16:0 and C 16:1, compared with the S. cerevisiae AJL 2155 cells (Table 2). The ergosterol/phospholipid ratio was significantly (P ³ 0 05) higher in the S. cerevisiae 1200 cells than in the S. cerevisiae AJL 2155 cells, due to a significantly (P ³ 0 05) higher ergosterol content in the S. cerevisiae 1200 cells (Table 3). DISCUSSION The two S. cerevisiae strains used in this study have significantly different ethanol tolerances as determined by viability, i.e. S. cerevisiae 1200 is more ethanol tolerant than S. cerevisiae AJL 2155 (Fig. 1). Furthermore, the results show that the formation of ethanol-induced D mutants in the more ethanol-tolerant strain is significantly lower than in the less ethanol-tolerant strain (Fig. 2). These results are in accordance with results described in the literature (Jimenez and Benitez 1988; Jimenez et al. 1988), indicating that the ethanol tolerance of S. cerevisiae is dependent on the maintenance of functional mitochondria during ethanol stress.

4 1050 Z. CHI AND N. ANEBOG Table 1 Phospholipid composition of aerobic, early stationary phase Saccharomyces cerevisiae AJL 2155 and 1200 cells in % of total phospholipids Percentage of total phospholipids (2 S.D.) Strain PC PI PE PS CL PA AJL (2 0 5) b 19 5 (2 1 4) a 38 1 (2 2 2) a 3 4 (2 1 1) a 2 2 (2 0 4) a 1 5 (2 0 4) a (2 0 4) a 17 6 (2 1 8) a 29 4 (2 0 8) b 5 1 (2 1 2) a 3 0 (2 0 1) a 1 4 (2 0.05) a Values for each yeast strain are means 2 S.D. of two independent growth experiments (see Materials and Methods). Values in the same column with different letters as superscript are significantly different (P ³ 0 05). PC, phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PS, phosphatidylserine; CL, cardiolipin; PA, phosphatidate. Table 2 Fatty acid composition of total phospholipids in aerobic, early stationary phase Saccharomyces cerevisiae AJL 2155 and 1200 cells Percentage of fatty acids (2 S.D.) (C 18:0 +C 18:1 )/ DUFA (C 16:0 +C 16:1 ) Strain C 16:0 C 16:1 C 18:0 C 18:1 Others (2 S.D.) (2S.D.) AJL (2 0 5) a 47 7 (2 0 4) a 5 7 (2 0 01) a 28 1 (2 1 0) b 1 1 (2 0 1) a 0 76 ( ) b 0 52 (2 0 02) b (2 1 6) b 42 9 (2 0 6) b 6 2 (2 0 2) a 39 1 (2 1 3) a 1 1 (2 0 1) a 0 82 (2 0 02) a 0 85 (2 0 04) a Values for each yeast strain are means 2 S.D. of two independent growth experiments (see Materials and Methods). Values in the same column with different letters as superscript are significantly different (P ³ 0 05). DUFA, proportion of unsaturated fatty acids. Table 3 Phospholipid and ergosterol content in aerobic, early stationary phase Saccharomyces cerevisiae AJL 2155 and 1200 cells Phospholipid: Ergosterol: Ergosterol: CDW (2 S.D.) CDW (2 S.D.) phospholipid Strain (mg g 1 )* (mg g 1 ) (2S.D.) (mg mg 1 ) AJL (2 2 8) a 6 9 (2 0 04) b 0 23 (2 0 02) b (2 0 04) a 8 9 (2 0 3) a 0 37 (2 0 01) a *In the calculations, a mean molecular weight of 760 g mol 1 was used for the phospholipids. Values for each yeast strain are means 2 S.D. of two independent growth experiments (see Materials and Methods). Values in the same column with different letters as superscript are significantly different (P ³ 0 05). CDW, cell dry weight. In addition, the two S. cerevisiae strains exhibit significantly distinct lipid compositions. The more ethanol-tolerant strain contains a higher proportion of PC and a lower proportion of PE than the less ethanol-tolerant strain (Table 1). Very little attention has been given to the role of phospholipids in etha- nol tolerance, although these lipids are essential for membrane structure and function (ussell 1989). Mishra and Prasad (1988) found that phosphatidylserine (PS)-enriched cells of S. cerevisiae were more ethanol tolerant than PC- and PEenriched cells, and that this difference in ethanol tolerance was attributed to an altered anion/zwitterion ratio of the phospholipids. The present results, however, do not sustain these findings. In contrast, supplementation of PC to the growth medium has been found to enhance the ethanol tolerance of S. sake cells (Hayashida and Ohta 1980; Shin et al. 1995). These findings are strongly supported by our results. The mechanisms behind the role of PC in the ethanol tolerance of S. cerevisiae, however, remain to be clarified. Although the degree of fatty acid unsaturation is considered to play a major role in the ethanol tolerance of S. cerevisiae (Mishra and Kaur 1991), conflicting results on this issue exist in the literature. Most authors find that the ethanol tolerance of S. cerevisiae correlates with an increased degree of fatty acid unsaturation (Mishra and Kaur 1991), whereas others find either no correlation (Aguilera and Benitez 1986; Swan and Watson 1997) or even that it correlates with a decreased degree of fatty acid unsaturation (del Castillo Agudo 1992). In this study, it was found that the more

5 ETHANOL TOLEANCE IN S. CEEVISIAE 1051 ethanol-tolerant strain contains a slightly higher DUFA than the less ethanol-tolerant strain (Table 2), thus supporting the majority of the findings reported in the literature. Moreover, it was found that the more ethanol-tolerant strain incorporates more long-chain fatty acids, i.e. C 18:0 and C 18:1, into the membrane phospholipids at the expense of shorter-chain fatty acids, i.e. C 16:0 and C 16:1, compared with the less ethanol-tolerant strain (Table 2). This finding does not correlate with the results of del Castillo Agudo (1992) who showed that the phospholipids of more ethanol-tolerant strains contain a higher amount of short-chain fatty acids (mainly C 12 ) compared with less ethanol-tolerant strains. This discrepancy may be due to differences in the yeast strains and analytical procedures used. Our results indicate that longchain fatty acids may play an important role in the ethanol tolerance of S. cerevisiae. The results in Table 3 show that the more ethanol-tolerant strain contains a higher ergosterol/phospholipid ratio than the less ethanol-tolerant strain. These results correspond to those reported in the literature (del Castillo Agudo 1992), indicating that ergosterol also plays an important role in the ethanol tolerance of S. cerevisiae. It is, as yet, unknown how long-chain fatty acids and ergosterol affect the ethanol tolerance of different S. cerevisiae strains. Our results, however, show a clear relationship between a high incorporation of long-chain fatty acids in the phospholipids (Table 2), a high ergosterol/phospholipid ratio (Table 3), and a low susceptibility to the formation of ethanolinduced D mutants (Fig. 2) in the more ethanol-tolerant S. cerevisiae strain. It has recently been suggested that ethanolinduced D mutations in S. cerevisiae are due to mitochondrial membrane damage rather than DNA damage (Ibeas and Jimenez 1997). In fact, ethanol is known to fluidize S. cerevisiae membranes (Mishra and Prasad 1988; Alexandre et al. 1994) and this membrane-fluidizing effect of ethanol may therefore be considered as an explanation for the formation of ethanol-induced D mutants in S. cerevisiae. It is well known that long-chain fatty acids decrease the fluidity of cellular membranes (ussell 1989). In addition, a high ergosterol/phospholipid ratio in S. cerevisiae is typically associated with a low fluidity of the membranes (Zinser et al. 1991; Alexandre et al. 1994). Thus, the difference in ethanol tolerances observed between the two S. cerevisiae strains in this study may be explained by the ability of the more ethanoltolerant strain to obtain a lower fluidity of its membranes, thereby resulting in a better counteraction against the membrane-fluidizing effect of ethanol and thus, a better maintenance of functional mitochondria during ethanol stress, than the less ethanol-tolerant strain. In conclusion, the two S. cerevisiae strains used in this study exhibited significantly different ethanol tolerances, significantly different susceptibilities to the formation of ethanol-induced D mutants, and significantly different lipid compositions. The results indicate that the lipid composition of a given S. cerevisiae strain may play an important role in the maintenance of functional mitochondria during ethanol stress, e.g. by affecting the membrane fluidity, which, in turn, may affect the ethanol tolerance of the strain. ACKNOWLEDGEMENTS This work was supported by the Sino-Danish Committee on Scientific and Technological Co-operation. EFEENCES Aguilera, A. and Benitez, T. (1986) Ethanol-sensitive mutants of Saccharomyces cerevisiae. Archives of Microbiology 143, Alexandre, H., ousseaux, I. and Charpentier, C. (1994) elationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata. FEMS Microbiology Letters 124, Arneborg, N., Hoy, C.E. and Jorgensen, O.B. (1995) The effect of ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae grown anaerobically in a chemostat. Yeast 11, Bandas, E.L. and Zakharov, I.A. (1980) Induction of rho mutations in yeast Saccharomyces cerevisiae by ethanol. Mutation esearch 71, Chi, Z.M. and Liu, Z.. (1993) High-concentration alcoholic production from hydrolysate of raw ground corn by a tetraploid yeast strain. Biotechnology Letters 15, del Castillo Agudo, L. (1992) Lipid content of Saccharomyces cerevisiae strains with different degrees of ethanol tolerance. Applied Microbiology and Biotechnology 37, Ernandes, J.., Williams, J.W., ussell, I. and Stewart, G.G. (1993) espiratory deficiency in brewing yeast strains effects on fermentation, flocculation, and beer flavor components. American Society of Brewing Chemists Journal 51, Hayashida, S. and Ohta, K. (1980) Effects of phosphatidylcholine or ergosteryl oleate on physiological properties of Saccharomyces sake. Agricultural and Biological Chemistry 44, Ibeas, J.I. and Jimenez, J. (1997) Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts. Applied and Environmental Microbiology 63, Jimenez, J. and Benitez, T. (1988) Yeast cell viability under conditions of high temperature and ethanol concentrations depends on the mitochondrial genome. Current Genetics 13, Jimenez, J., Longo, E. and Benitez, T. (1988) Induction of petite yeast mutants by membrane-active agents. Applied and Environmental Microbiology 54, Mishra, P. and Kaur, S. (1991) Lipids as modulators of ethanol tolerance in yeast. Applied Microbiology and Biotechnology 34, Mishra, P. and Prasad,. (1988) ole of phospholipid head groups in ethanol tolerance of Saccharomyces cerevisiae. Journal of General Microbiology 134, Norton, S., Watson, K. and D Amore, T. (1995) Ethanol tolerance

6 1052 Z. CHI AND N. ANEBOG of immobilized brewers yeast cells. Applied Microbiology and Biotechnology 43, Ohta, K., Hamada, S. and Nakamura, T. (1993) Production of high concentrations of ethanol from inulin by simultaneous saccharification and fermentation using Aspergillus niger and Saccharomyces cerevisiae. Applied and Environmental Microbiology 59, ose, A.H. (1993) Composition of the envelope layers of Saccharomyces cerevisiae in relation to flocculation and ethanol tolerance. Journal of Applied Bacteriology Symposium Supplement 74, 110S 118S. ouser, G., Fleischer, S. and Yamamoto, A. (1970) Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipid by phosphorous analysis of spots. Lipids 5, ussell, N.J. (1989) Functions of lipids: Structural roles and membrane functions. In Microbial Lipids, Vol. 2 ed. atledge, C. and Wilkinson, S.G. pp London: Academic Press. Sajbidor, J. (1997) Effect of some environmental factors on the content and composition of microbial membrane lipids. Critical eviews in Biotechnology 17, Shin, C.S., Song, J.Y., yu, O.H. and Wang, S.S. (1995) Enhancing effect of albumin hydrolysate on ethanol production employing Saccharomyces sake. Biotechnology and Bioengineering 45, Swan, T.M. and Watson, K. (1997) Membrane fatty acid composition and membrane fluidity as parameters of stress tolerance in yeast. Canadian Journal of Microbiology 43, Wagner, S. and Paltauf, F. (1994) Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae. Fatty acid pattern of phospholipid classes and selective acyl turnover at sn-1 and sn-2 positions. Yeast 10, Zinser, E., Sperka-Gottlieb, C.D.M., Fasch, E.-V., Kohlwein, S.D., Paltauf, F. and Daum, G. (1991) Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae. Journal of Bacteriology 173,

EXPERIMENT 13: Isolation and Characterization of Erythrocyte

EXPERIMENT 13: Isolation and Characterization of Erythrocyte Day 1: Isolation of Erythrocyte Steps 1 through 6 of the Switzer & Garrity protocol (pages 220-221) have been performed by the TA. We will be