Appl Microbiol Biotechnol (1989) 30:294--298 Applied Microbiology

Biotechnology © Springer-Verlag 1989

Relationship between ethanol tolerance and fatty acyl composition of Saccharomyces cerevisiae

Prashant Mishra and Rajendra Prasad

School of Life Sciences, Jawaharlal Nehru University, New Delhi-l10 067, India

Summary. The effect of ethanol on exponential phase cultures of S. cerevisiae has been examined using L-alanine uptake and proton effiux as in- dices of ethanol tolerance. Preincubation with 2 M ethanol inhibited L-alanine uptake, proton efflux and fermentation rates. However, the effect of ethanol varied in yeast cells enriched with dif- ferent fatty acyl residues. It was observed that cells enriched with polyunsaturated fatty acids ac- quired greater tolerance to ethanol as compared to monounsaturated fatty acids. By varying the degree of unsaturation of supplemented fatty acid, a sequential insertion of double bonds in yeast membrane lipid was achieved. Results de- monstrated that S. cerevisiae became more resist- ant to ethanol with an increase in the degree of unsaturation and that membrane fluidity could be an important determinant of ethanol tolerance.

Introduction

Saccharomyces cerevisiae is by far the most alco- hol resistant eucaryotic organism. It is known that ethanol producing organisms, including yeast, cease to grow beyond a certain threshold level of its concentration. The toxicity of ethanol pro- duced by yeast acts as a limiting factor in achiev- ing high yields of alcohol. There is increasing evi- dence that toxicity is exerted at the level of cyto- plasmic membrane. Thus, an understanding of the relationship between plasma membrane composi- tion and ethanol tolerance could be of great po- tential in increasing the yield of ethanol produc- tion. Investigations so far have revealed that etha- nol in S. cerevisiae inhibits cell growth (Rose 1980), viability (Thomas et al. 1978; Beavan et al.

Offprint requests to: R. Prasad

1982), accumulation of various nutrients (Thomas and Rose 1979; Loureiro-Dias and Peinado 1982; Leao and Van Uden 1982; 1983; 1984a), H + fluxes (Leao and Van Uden 1984b; Cartwright et al. 1986; Juroszek et al. 1986) and fermentation rate (Casey and Ingledew 1986).

Earlier studies based on a correlation between ethanol potency and lipid solubility led to the conclusion that hydrophobic sites of plasma membrane are most likely to be the prime target of ethanol action (Ingrain and Buttke 1984). It ap- pears that amongst various membrane compo- nents, lipids are the main site of ethanol toxicity (Thomas et al. 1978; Thomas and Rose 1979; In- gram and Buttke 1984). Thus fluctuations in lipid contents are likely to affect sensitivity of cells to- wards ethanol. Observed changes in phospho- lipids and fatty acyl residues in presence of etha- nol have been attributed to an adaptive response of the organism (Ingram 1986).

Rose and his co-workers have shown that var- iations in fatty acid and sterol composition affect ethanol tolerance of S. eerevisiae (Thomas et al. 1978; Thomas and Rose 1979) but a correlation between the degree of unsaturation and ethanol sensitivity has yet not emerged. The availability of an unsaturated fatty acid auxotroph of S. cerevi- siae, KD115, provided us with an opportunity to predictably modify the unsaturation index and fluidity of plasma membrane. In the present study the physical and physiological response of yeast with varying degrees of fatty acyl unsaturation has been studied to gain an insight into the mech- anism underlying ethanol action.

Materials and methods

Strain and culture conditions. S. cerevisiae KDl l5 , an auxo- troph of unsaturated fatty acid, was obtained from Yeast Ge-

P. Mishra and R. Prasad: Ethanol tolerance in S. eerevisiae 295

netic Centre, Berkeley and maintained on YEPD slants and enriched with Tween 80. The cells were specifically enriched with different fatty acyl residues as described earlier (Mishra and Prasad 1987), harvested and washed thrice with citrate buffer (10 mM, pH 4.5) before use for further studies.

Fatty acid analysis. Fatty acids were extracted and methyl es- ters were prepared and analysed as described earlier (Mishra and Prasad 1987) using GLC (Shimadzu GC 9-A) equipped with flame ionization detector and chromatopac CR-2A auto- matic integrator.

Measurement of rate of amino acid uptake. Transport assay procedure was similar to that described earlier (Mishra and Prasad 1987; Kaur et al. 1988). Inhibition constant Ki(EtOH) values for L-alanine uptake were derived from reciprocal plots of velocities of amino acid uptake against increasing concen- trations of ethanol (0.5 to 2.0 M) at two fixed substrate con- centrations (Dixon 1953).

Measurement of rates of proton flux. Glucose-stimulated pro- ton efflux was measured by the addition of glucose (100 mM) to a suspension (100 mg wet wt • m1-1) of cells enriched with different fatty acyl residues. A constant temperature of 30 ° C was maintained by circulating water through a jacketed cham- ber. The cell suspension in the chamber was constantly stirred. Changes in pH values of suspension were recorded using a REC 80 Servograph with REA 105 pH mV unit (Radiometer, Copenhagen). The effect of ethanol on glucose induced H + efflux was measured by supplementing the cell suspension with ethanol after adjusting its pH value to 4.0 prior to the addition of glucose (100 raM). Proton flux was then followed over a 5 min period (Cartwright et al. 1986). The pH range was fixed in order to avoid denaturation of the cellular compo- nents of energized organisms (Juroszek et al. 1986).

Fluorescence measurements. Two mM 1,6-diphenyl-l,3,5-hexa- triene (DPH) was prepared in tetrahydrofuran and 100 ~1 of it was added to 50 ml rapidly stirring phosphate buffer (10 mM, pH 6.8). Excess of tetrahydrofuran was removed by flushing with nitrogen. Spheroplasts of normal as well as fatty acyl enriched cells were prepared as described earlier (Jayakumar et al. 1981). These spheroplasts were washed with phosphate buffer (20 mM, pH 6.0) containing 10 mM MgSO4 and 0.6 M sorbitol and incubated with 2 p~M DPH for 60 rain at 30°C. Fluorescence polarization was measured and anisotropy was calculated according to Haggerty et al. (1978).

Measurement of fermentation rates. The ability of S. cerevisiae (altered in fatty acyl composition) to ferment glucose was de- termined with a Gilson single-valve differential respirometer. Cells (100 mg wet wt- m1-1) were suspended in citrate buffer (50 mM, pH 4.5). Glucose (300 raM) placed in the side arm of the Warburg flasks was mixed with the cell suspension after incubating for 10 min at 30 ° C. Prior to the addition of glucose flasks were shaken (95 oscillations min-1) and the system was continuously flushed with nitrogen gas passed through a trap containing pyrogallol solution. The supply of nitrogen gas was cut off before the addition of glucose to buffered cell suspen- sion to start fermentation. Evolution of COz was then followed over a period of 5 min (Cartwright et al. 1986).

Chemicals. Palmitoleic acid, oleic acid, linoleic acid, linolenic acid, DPH, tetrahydrofuran, L-alanine and standard fatty acids were purchased from Sigma Chemical Co., St. Louis, USA. 14C L-alanine was purchased from Bhaba Atomic Re- search Centre, India. All other chemicals were of analytical grade.

Table 1. Unsaturation index of K D l l 5 enriched in different fatty acyl residues

Fatty acid Fatty acyl Percentage Unsaturation supplemented residue of index* (40 ~M) enriched enrichment (Amole- 1)

Palmitoleate Palmitoleyl 64 0.80 Oleate Oleyl 78 0.83 Linoleate Linoleyl 64 1.45 Linolenate Linolenyl 70 2.22

* Unsaturation index (Amole- i ) was calculated as:

Amole- 1 = I x (% monoene) q 2 x (% diene) + 3 x (% triene) 100 100 100

Results and discussion

Modification of fatty acyl groups in S. cerevisiae

The auxotrophic strain of S. cerevisiae, KD115, is defective in A 9 desaturase (Keith et al. 1969) and thus requires unsaturated fatty acid (UFA) sup- plementation for its continued growth (Resnick and Mortimer 1966). Since the requirement of UFA is not very specific, this auxotroph serves as an excellent tool for dialling the desired fatty acyl composition (Walenga and Lands 1975; Mishra and Prasad 1987). It was observed that the growth of KDll5 cells in presence of different UFA re- suited in an enrichment of the corresponding sup- plemented fatty acid between 64% and 78% (Table 1). It was apparent that increase in the number of double bonds of supplemented UFA increased the unsaturation index (Table 1).

Effect o f ethanol on L-alanine uptake in S. cerev&iae

L-Alanine uptake was selected as an index of membrane function. Earlier studies have con- firmed that its uptake is mediated via the general amino acid permease (GAP) thus reflecting the status of a broad spectrum of amino acids (Cal- derbank et al. 1985). In addition, the uptake of L- alanine is also known to be sensitive to lipid fluc- tuation (Calderbank et al. 1985; Mishra and Prasad 1987). The addition of ethanol reduced the uptake rate of L-alanine and such a reduction in response to ethanol was studied in cells with vary- ing degree of fatty acyl unsaturation. The inhibi- tory effect of ethanol on L-alanine uptake grad- ually decreased with increasing unsaturation in- dex. Thus cells enriched with linolenate exhibited greater resistance to ethanol as compared to those

296

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F o t t y c tcy l r e s i d u e s

Fig. 1. Effect of ethanol on L-alanine uptake in S. cerevisiae enriched with different fatty acyl residues. A = 2 M ethanol

P. Mishra and R. Prasad: Ethanol tolerance in S. cerevisiae

Table 2. Inhibitor constants (Ki(EtOH)) for L-alanine uptake by K D l l 5 enriched in different fatty acyl residues

Fatty acid Fatty acyl Type of Ki(EtOH) supplemented residue inhibition (M x 10-1) (40 lxM) enriched

Palmitoleate Palmitoleyl Non-competitive 2.8 Oleate Oleyl Non-competitive 5.0 Linoleate Linoleyl Non-competitive 7.8 Linolenate Linolenyl Non-competitive 10.0

K i ( E t O H ) values were derived from Dixon plots. Values are a mean of three experiments. Velocity of L-alanine uptake was calculated as described in materials and Methods using linear regression analysis

with lesser degree of unsaturation (Fig. 1). Such an increase in ethanol tolerance in cells contain- ing polyunsaturated fatty acyl residues has been attributed to the fact that increased membrane fluidity compensates for a decreased repulsion between polar head groups (Thomas et al. 1978). Such a condition arises when water molecules surrounding the head groups are replaced by ethanol molecules. Our data based on the effect of ethanol on L-alanine uptake corroborate with earlier reports that ethanol non-competitively in- hibits GAP activity (Leao and Van Uden 1984a; Cartwright et al. 1986). K~(Eton) values of L-ala- nine uptake obtained from Dixon plots (not shown) confirm that the ethanol toxicity de-

creases with an increase in the degree of unsatura- tion (Table 2).

Effect of e t h a n o l on H + efflux of S. cerevisiae

Figure 2 depicts that increasing concentrations of ethanol inhibited the efflux of H ÷ in palmitoleate and oleate enriched cells. It was observed that in linoleate and linolenate enriched cells, however, the net proton effiux was negligible. This was due to the modification of fatty acyl composition of cells. The addition of ethanol to these cells in- duced a backflow (influx) of protons which was concentration and unsaturation dependent.

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Fig. 2. Effect of ethanol on acidifying ability of energized S. cerevisiae. The data represents direct tracing of a typical experiment. The four panels of tracing represent acidification of different cells enriched with either (A) palmitoleyl06:l ), (B) oleylos:a ), (C) linoleyl08:2 ) or (D) linolenyl(18:3) residues

P. Mishra and R. Prasad: Ethanol tolerance in S. cerevisiae 297

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Fig. 3. Effect of ethanol on fermentative acitvity of S. cerevi- siae. Different UFA enriched cells were suspended in citrate buffer (pH 4.5, 50 mM) and fermentation rates were measured as described in Methods in presence and absence of 2 M etha- nol

Effect of ethanol on fermentative activity of S. cerevisiae

Since fermentative ability of an organism is a good indicator of its ethanol producing potential (Casey 1986), it was determined in various UFA enriched cells exposed to 2 M ethanol. It is evi- dent that similar to L-alanine uptake and H + ef- flux, percentage inhibition of fermentative activ- ity was reduced with increasing unsaturation (Fig. 3). As compared to palmitoleate, oleate and lino- leate the inhibitory effect of ethanol on fermenta- tion rate of linolenate supplemented cells was the least (Fig. 3).

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Fig. 4. Effect of ethanol on fluorescence anisotropic values. Spheroplasts were prepared from cells enriched with (A) pal- mitoleyl06:l), (B) oleylo8:l), (C) linoleylo8:2 ) and (D) linole- nylo8:3 ) residues. Fluorescence measurements were done as de- scribed in Materials and methods

(in presence of 2 M ethanol) versus the degree of unsaturation, a very interesting correlation emerges (Fig. 5). The pattern of inhibition of all the studied parameters in presence of ethanol was the same. Furthermore, it is evident that an in- crease in unsaturation offers protection from the toxic effect of ethanol. It is tempting to speculate that the study of any of these parameters can be taken as a good indicator for the evaluation of

Effect of ethanol on membrane fluidity of S. cerevisiae 100

c 0

The presence of ethanol results in an increase in ~ 80 the ratio of unsaturated to saturated fatty acyl re- Z sidues (Ingram 1976; Beavan et al. 1982). Such a -~

o f f 0 shift in fatty acyl composition (i.e. increased fluidity) is attributed to an adaptive response of 2

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various organisms to ethanol (Ingram 1976). Fluo- ~ 40 x..

rescence studies using DPH as a probe revealed ~_ that increasing concentrations of ethanol decrease 20 the anisotropic values (i. e. increase fluidity) (Fig. 4). Our observation agrees well with an earlier re- port of Curtain et al. 1984, where it was shown that ethanol led to an increase in the fluidity of S. cerevisiae plasma membrane.

On comparing the percentage inhibition of L- alanine uptake, H + effiux and fermentative rate

"o

16:1 18:1 18:2 18:3 Fatty acyl residues

Fig. 5. Effect of ethanol on L-alanine uptake ( A - A), proton efflux ( I - - I ) , fermentative rate ( 0 - - 0 ) and fluorescence anisotropy (O . . . . O). Data compiled from Figs. 1-4

298 P. Mishra and R. Prasad: Ethanol tolerance in S. cerevisiae

e t h a n o l t o l e r a n c e o f yeas t . R e s u l t s a l so c o n f i r m e a r l i e r f i n d i n g s o f R o s e a n d his c o - w o r k e r s t h a t f a t t y a c y l u n s a t u r a t i o n is r e l e v a n t to e t h a n o l t ox - i c i ty ( T h o m a s et al. 1978; T h o m a s a n d R o s e 1979; B e a v a n et al . 1982). I n a d d i t i o n to c h a n g e in m e m b r a n e f lu id i ty , t h e c h a r g e ( a n i o n / z w i t t e r i o n r a t i o ) o f m e m b r a n e p h o s p h o l i p i d o r h e a d g r o u p r a t i o h a s a l so b e e n s h o w n to p l a y a s i g n i f i c a n t r o l e in e t h a n o l t o x i c i t y o f y e a s t ( u n p u b l i s h e d o b - s e rva t i ons ) . I t is p o s s i b l e t h a t m e m b r a n e f l u i d i t y as we l l as c o m p o s i t i o n o f o t h e r m e m b r a n e l i p i d s c o u l d c o n t r i b u t e t o w a r d s t h e s e n s i t i v i t y o f a n or - g a n i s m to e t h a n o l .

Acknowledgements. P. Mishra gratefully acknowledges SRF award from CSIR, New Delhi. This work was supported in part by a grant from Department of Science & Technology (No. 22 (7P/22) 84 STP II) and Indian Council of Medical Research (5/11(1)/87-BMS-II(8700080)), New Delhi, India. The special assistance from University Grant Commission un- der COSIST is also acknowledged.

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Received June 6, 1988/Accepted October 18, 1988