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Effects of elevated expression of the CYP51 (P45014DM) gene on the sterol contents of Saccharomyces cerevisiae
JOURNALOF FERMENTATION AND BIOENGINEERING Vol. 79, No. 1, 64-66. 1995
Effects of Elevated Expression of the CYP.51 (P45014& Gene on the Sterol Contents of Saccharomyces cerevisiae HITOSHI
TAINAKA,‘*
HIDEAKI
HASHIMOTO,‘t
YURI AOYAMA,Zt
AND
YUZO YOSHIDA2
The Nikka Whisky Distilling Co. Ltd., 967Masu0, Kashiwa 277’ and Faculty of Pharmaceutical Science, Mukogawa Women’s University, II-68 Koshien 9-Bancho, Nishinomiya 663,2 Japan Received 6 June 1994IAccepted 30 September 1994
Lanosterol metabolism was elevated in yeast cells cultured both aerobically and semi-anaerobically by increased expression of lanosterol 14a-demethylase P450 (P450 14u~ or CYP51). However, despite increases in the diene-intermediates of ergosterol biosynthesis, the ergosterol content was not affected. These results suggested that the levels of cellular sterols are controlled in at least two stages, and that the concomitant elevation of P45014uMand ergostadienols desaturase (P450 22~~)is required to increase the cellular ergosterol content. [Key words: Saccharomyces cerevisiae, P450, CYPSl. sterol content] The cellular sterol content is considered to be one of the important factors that affects the fermentability and viability of yeasts (1). Certain enzymes involved in yeast sterol biosynthesis control ergosterol levels under various conditions (2). In this study, we investigated the effects of the overexpression of lanosterol 14n-demethylase P450 (P45O,,DM or CYP51) (3, 4) on the levels of cellular sterols. This P450 enzyme catalyses the 14a-demethylation of lanosterol, which is the initial step in the synthesis of ergosterol from lanosterol (3). The yeast Saccharomyces cerevisiae SGl, which lacks this enzyme, does not contain ergosterol and accumulates lCmethylated sterols (5, 6). P450,4nM is essential for the growth of yeast, and the gene-disruption of this enzyme results in an ergosterol auxotroph (7). We isolated a DNA clone which contains the structural gene of P45014nM from a chromosomal library of S. cerevisiae S288C (8) according to the method of Kalb et al. (7). A ketoconazole-sensitive strain, OP3C (MATa, ura3, feu2), was derived from a diploid strain produced by crossing AH22 (MATa, leu2, his4, canl) and KK4 (MATa, leu2-3, leu2-112, ura3, trpl, his, ga180), both strains were kindly supplied by Dr. T. Fukasawa (Keio University School of Medicine, Tokyo). We used OP3C as a host strain throughout this work. A few ketoconazole-resistant (20 ppm) transformants were obtained by screening the library and plasmid DNA was segregated. Two clones contained a BamHI-Hind111 fragment (4.7 kbp, see reference 7) bearing the P45Or4nM StrUCtural gene together with its own promoter. The BamHIHind111 fragment was excised and ligated into a YEptype shuttle vector YEUp (kindly provided by Dr. Fujita, Fermentation Research Institute, Osaka). The resulting plasmid, pBH22, was introduced into OP3C cells using the lithium acetate method (9). Control (YEUp3) and transformed (pBH22) cells were grown on solid media consisting of 2% glucose, 0.67% yeast nitrogen base without amino acids (Difco, MI,
USA) and 2% agar supplemented with leucine (60 pg/ml). Liquid seed cultures (100ml) were maintained in the same medium without agar. Large-scale (5 0 liquid cultures were performed in YEPD medium containing 1% yeast extract, 2% peptone and 2% glucose under semi-anaerobic conditions (4). Semi-anaerobic culture conditions were achieved using a cylindrical vessel filled with liquid medium, which was slowly stirred with a magnetic stirrer (50rpm). Aerobic cultures were done in Erlenmeyer flasks, shaken vigorously using a rotary shaker (100 rpm). All cultures were maintained at 30°C. The pH of the medium was unadjusted. The P450 content was measured spectrophotometrically from the reduced carbon monoxide difference spectra (10) of the cell suspensions. In semi-anaerobically grown S. cerevisiae, P4%&,M accounts for more than 80% of the total cellular P45Os (3, 4). Accordingly, the P450 content determined as above was considered to represent the P450r4nM levels. Although some portions of the plasmids might be lost in non-selective YEPD media, the pBH22 transformants produced more P45O(s) than the control cells (Fig. 1). This indicated that the introduction of pBH22 elevated the expression level of the P450r4nM gene under semi-anaerobic conditions. However, under aerobic conditions, the P450 levels were lowered both in the transformed and control cells, and other cytochromes interfered with the spectrophotometric determination of P450. Therefore, higher production in the transformant was not confirmed by this means. Total lipid was extracted from lyophilized cells with dimethylsulfoxide and saponified (modified from the method in reference 11). Unsaponified substances were extracted with diethylether and analyzed by gas-liquid chromatography (GC) with an OV-1 capillary column. Lanosterol, 4,4-dimethylzymosterol, zymosterol and ergosterol were identified by comparing their mass spectra (MS) with those of authentic samples. In addition, six more peaks were assigned as ergostenols and ergostadienols by GC-MS. Since these sterols were considered as intermediates of the ergosterol biosynthetic they were combined and determined as pathway, “unidentified intermediates. ’ Figure 1 shows the growth and P450 production by the transformant and control cells under semi-anaerobic conditions. The P450 production was significantly higher
* Corresponding author. Present address: Amersham K.K., 28021 Hiratsuka, Shiroi-machi, Inba-gun 270-14, Japan. t Present address: Unkai Distillery Co. Ltd., 1800-5 Minamimata, Aya-machi, Higashimorogata-gun 880-13, Japan. t Present address: Faculty of Engineering, Soka Univ., I-236 Tangi-cho, Hachioji 192, Japan. 64
NOTES
VOL. 79, 1995
TABLE 2.
Sterol contents in aerobically grown transformant control cells at the stationary phase (20 h) Transformant control (nmol/mg dry wt.)
Lanosterol 4,4-Dimethylzymosterol Zymosterol Ergosterol Unidentified Total 4
8
65 and
Transformanti control
0.6 0.4 2.5 7.7 8.1
1.3 0.1 1.7 8.3 4.8
0.46 4.00 1.47 0.93 1 69
19.3
16.2
1.19
12
Time (h)
0
4
8
12
Time (h) FIG. 1. conditions.
Cell growth and P450 content under semi-anaerobic Symbols: 0, control; and 0, transformant.
in the transformant and the maximum level was reached at 8.5 h. At this time, the cells were collected and the sterol content was determined (Table 1). In the transformant cells, the levels of 4,4-dimethylzymosterol and the unidentified intermediates were increased, indicating that the elevated P450 levels accelerated lanosterol metabolism. Although the P450 level was not elevated, as discussed above, an essentially similar alteration in sterol composition was also evident in the transformant and control cells grown under aerobic conditions (Table 2). The lanosterol level was decreased and that of 4,4dimethylzymosterol was increased in the transformed cells. The contents of zymosterol and unidentified intermediates were also increased in the transformant. These findings suggest that the level of P450,,,, was higher in the transformant, which in turn, promoted the metabolism of lanosterol. However, it is noteworthy that no increase was evident in the ergosterol content of the transformant under both aerobic and semi-anaerobic conditions. In cells grown under the latter conditions, the elevated P450i4nM level did not increase the zymosterol content, and lanosterol was not decreased but rather increased slightly in the transformant. Although the reason for the discrepancies between the aerobically and the semi-anaerobically grown cells is TABLE 1. Sterol contents in semi-anaerobically grown transformant and control cells at the late-log phase (8.5 h) Transformant control (nmol/mg dry wt.) Lanosterol 4,4-Dimethylzymosterol Zymosterol Ergosterol Unidentified Total
Transformant/ control
1.9 0.9 1.1 3.2 7.2
1.3 0.3 1.0 4.3 3.3
1.46 3.00 1.10 0.74 2.18
14.3
10.2
1.40
yet to be determined, elevation of the cellular P450i4nM level evidently accelerated the metabolism of lanosterol to ergostadiene intermediates under both aerobic and semianaerobic growth conditions. The accumulation of these intermediates m P450L4nM overexpressed cells suggests that the cellular level of ergosterol is regulated at the final step of the biosynthetic pathway (the conversion of ergostadienol to ergosterol), which is catalyzed by another P450 enzyme (P4502& (12). This study revealed that the elevated P450,4DM level accumulated intermediates of ergosterol biosynthesis under aerobic and anaerobic conditions, although the cellular ergosterol content is lower in cells cultured semi-anaerobically than in those grown aerobically (Tables 1 and 2). The growth environment for fermenting yeasts in the brewing industry is considered semi-anaerobic, the supply of oxygen being maintained only by absorption from the surface of liquid media. Thus, yeast strains used in commercial fermentation processes are considered to experience a shortage of oxygen for sterol biosynthesis (13, 14). Under these conditions, the cellular level of is raised. We have shown here that cellular p450,4DM ergosterol intermediates were increased by elevating the P450i4nM level. These facts suggest that an elevated P450i4nM level improves the cellular sterol metabolism, thus increasing the sterol contents under conditions of a low oxygen supply. However, elevation of the P450,,nM level alone did not increase the content of ergosterol but resulted in an accumulation of ergostadienol intermediates. Since another regulatory P450 enzyme (P450ZZDS) that converts ergostadienol to ergosterol is involved in ergosterol synthesis, the concomitant elevation of both P45Os is necessary to improve the ergosterol content in fermenting yeast. REFERENCES
1. Quain, D. E.: Studies on yeast physiology-impact on fermentation performance and product quality. J. Inst. Brew., 95, 31% 323 (1988). 2. Ratledge, C. and Evans, C. T.: Lipids and their metabolism, p. 428. In Rose, A. H. and Harrison, .I. S. (ed.), The yeasts, vol. 3. Academic Press, London (1989). 3. Aoyama, Y., Yoshida, Y., and Sato, R.: Yeast cytochrome P450 catalyzing lanosterol 14a-demethylation. II. Lanosterol metabolism by purified P-450,.,nM and by intact microsomes. J. Biol. Chem., 259, 1661-1666 (1984). 4. Yoshida, Y. and Aoyama, Y.: Yeast cytochrome P-450 catalyzing lanosterol 14a-demethylation. I. Purification and spectral properties. J. Biol. Chem., 259, 1655-1660 (1984). 5. Aoyama, Y., Yoshida, Y., Nishino, T., Katsuki, H., Maitra, U. S., Mohan, V. P., and Sprinson, D. B.: Isolation and characterization of an altered cytochrome P-450 from mutant defective in lanosterol 14w-demethylation. J. Biol. Chem., 262, 14260-14264 (1987).
66
J. FERMENT.BIOENG.,
TAINAKA ET AL.
6. Trocha,
7.
8.
9.
10.
P. J., Jasne, S. T., and Sprinson, D. B.: Yeast mutants blocked in removing the methyl group of lanosterol at C14. Separation of sterols by high-pressure liquid chromatography. Biochemistry, 16, 4721-4726 (1977). Kalb, V. F., Woods, C. W., Turi, T. G., Dey, C. R., Sutter, T. R., and Loper, J. C.: Primary structure of the P-450 lanosterol demethylase gene from Succharomyces cerevisiue. DNA, 6, 529-537 (1987). Carlson, M. and Botstein, D.: Two differentially regulated mRNAs with different 5’ ends encode secreted and intracellular forms of yeast invertase. Cell, 28, 145-154 (1982). Ito, H., Fukuda, Y., Murata, K., and Kimura, A.: Transformation of intact yeast cells treated with alkali cations. J. Bacteriol., 153, 163-168 (1983). Omura, T. and Sato, R.: The carbon monoxide-binding
11.
12.
13.
14.
pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239, 2370-2378 (1964). Taylor. F. R. and Parks, L. W.: Metabolic interconversion of free stkrols and steryl esters in Saccharomyces cerevisiae. J. Bacterial., 136, 531-537 (1978). Hata, S., Nishino, T., Katsuki, H., Aoyama, Y., and Yoshida, Y.: Characterization of A22-desaturation in ergosterol biosynthesis of yeast. Agric. Biol. Chem., 51, 1349-1354 (1987). Aries, V. and Kirsop, B.: Sterol biosynthesis by strains of Saccharomyces cerevisiae in the presence and absence of dissolved oxygen. J. Inst. Brew., 84, 118-122 (1978). Ahvenainen, J.: Lipid composition of aerobically and anaerobically propagated brewer’s bottom yeast. J. Inst. Brew., 88, 367-370 (1982).
Effects of Elevated Expression of the CYP.51 (P45014& Gene on the Sterol Contents of Saccharomyces cerevisiae HITOSHI
TAINAKA,‘*
HIDEAKI
HASHIMOTO,‘t
YURI AOYAMA,Zt
AND
YUZO YOSHIDA2
The Nikka Whisky Distilling Co. Ltd., 967Masu0, Kashiwa 277’ and Faculty of Pharmaceutical Science, Mukogawa Women’s University, II-68 Koshien 9-Bancho, Nishinomiya 663,2 Japan Received 6 June 1994IAccepted 30 September 1994
Lanosterol metabolism was elevated in yeast cells cultured both aerobically and semi-anaerobically by increased expression of lanosterol 14a-demethylase P450 (P450 14u~ or CYP51). However, despite increases in the diene-intermediates of ergosterol biosynthesis, the ergosterol content was not affected. These results suggested that the levels of cellular sterols are controlled in at least two stages, and that the concomitant elevation of P45014uMand ergostadienols desaturase (P450 22~~)is required to increase the cellular ergosterol content. [Key words: Saccharomyces cerevisiae, P450, CYPSl. sterol content] The cellular sterol content is considered to be one of the important factors that affects the fermentability and viability of yeasts (1). Certain enzymes involved in yeast sterol biosynthesis control ergosterol levels under various conditions (2). In this study, we investigated the effects of the overexpression of lanosterol 14n-demethylase P450 (P45O,,DM or CYP51) (3, 4) on the levels of cellular sterols. This P450 enzyme catalyses the 14a-demethylation of lanosterol, which is the initial step in the synthesis of ergosterol from lanosterol (3). The yeast Saccharomyces cerevisiae SGl, which lacks this enzyme, does not contain ergosterol and accumulates lCmethylated sterols (5, 6). P450,4nM is essential for the growth of yeast, and the gene-disruption of this enzyme results in an ergosterol auxotroph (7). We isolated a DNA clone which contains the structural gene of P45014nM from a chromosomal library of S. cerevisiae S288C (8) according to the method of Kalb et al. (7). A ketoconazole-sensitive strain, OP3C (MATa, ura3, feu2), was derived from a diploid strain produced by crossing AH22 (MATa, leu2, his4, canl) and KK4 (MATa, leu2-3, leu2-112, ura3, trpl, his, ga180), both strains were kindly supplied by Dr. T. Fukasawa (Keio University School of Medicine, Tokyo). We used OP3C as a host strain throughout this work. A few ketoconazole-resistant (20 ppm) transformants were obtained by screening the library and plasmid DNA was segregated. Two clones contained a BamHI-Hind111 fragment (4.7 kbp, see reference 7) bearing the P45Or4nM StrUCtural gene together with its own promoter. The BamHIHind111 fragment was excised and ligated into a YEptype shuttle vector YEUp (kindly provided by Dr. Fujita, Fermentation Research Institute, Osaka). The resulting plasmid, pBH22, was introduced into OP3C cells using the lithium acetate method (9). Control (YEUp3) and transformed (pBH22) cells were grown on solid media consisting of 2% glucose, 0.67% yeast nitrogen base without amino acids (Difco, MI,
USA) and 2% agar supplemented with leucine (60 pg/ml). Liquid seed cultures (100ml) were maintained in the same medium without agar. Large-scale (5 0 liquid cultures were performed in YEPD medium containing 1% yeast extract, 2% peptone and 2% glucose under semi-anaerobic conditions (4). Semi-anaerobic culture conditions were achieved using a cylindrical vessel filled with liquid medium, which was slowly stirred with a magnetic stirrer (50rpm). Aerobic cultures were done in Erlenmeyer flasks, shaken vigorously using a rotary shaker (100 rpm). All cultures were maintained at 30°C. The pH of the medium was unadjusted. The P450 content was measured spectrophotometrically from the reduced carbon monoxide difference spectra (10) of the cell suspensions. In semi-anaerobically grown S. cerevisiae, P4%&,M accounts for more than 80% of the total cellular P45Os (3, 4). Accordingly, the P450 content determined as above was considered to represent the P450r4nM levels. Although some portions of the plasmids might be lost in non-selective YEPD media, the pBH22 transformants produced more P45O(s) than the control cells (Fig. 1). This indicated that the introduction of pBH22 elevated the expression level of the P450r4nM gene under semi-anaerobic conditions. However, under aerobic conditions, the P450 levels were lowered both in the transformed and control cells, and other cytochromes interfered with the spectrophotometric determination of P450. Therefore, higher production in the transformant was not confirmed by this means. Total lipid was extracted from lyophilized cells with dimethylsulfoxide and saponified (modified from the method in reference 11). Unsaponified substances were extracted with diethylether and analyzed by gas-liquid chromatography (GC) with an OV-1 capillary column. Lanosterol, 4,4-dimethylzymosterol, zymosterol and ergosterol were identified by comparing their mass spectra (MS) with those of authentic samples. In addition, six more peaks were assigned as ergostenols and ergostadienols by GC-MS. Since these sterols were considered as intermediates of the ergosterol biosynthetic they were combined and determined as pathway, “unidentified intermediates. ’ Figure 1 shows the growth and P450 production by the transformant and control cells under semi-anaerobic conditions. The P450 production was significantly higher
* Corresponding author. Present address: Amersham K.K., 28021 Hiratsuka, Shiroi-machi, Inba-gun 270-14, Japan. t Present address: Unkai Distillery Co. Ltd., 1800-5 Minamimata, Aya-machi, Higashimorogata-gun 880-13, Japan. t Present address: Faculty of Engineering, Soka Univ., I-236 Tangi-cho, Hachioji 192, Japan. 64
NOTES
VOL. 79, 1995
TABLE 2.
Sterol contents in aerobically grown transformant control cells at the stationary phase (20 h) Transformant control (nmol/mg dry wt.)
Lanosterol 4,4-Dimethylzymosterol Zymosterol Ergosterol Unidentified Total 4
8
65 and
Transformanti control
0.6 0.4 2.5 7.7 8.1
1.3 0.1 1.7 8.3 4.8
0.46 4.00 1.47 0.93 1 69
19.3
16.2
1.19
12
Time (h)
0
4
8
12
Time (h) FIG. 1. conditions.
Cell growth and P450 content under semi-anaerobic Symbols: 0, control; and 0, transformant.
in the transformant and the maximum level was reached at 8.5 h. At this time, the cells were collected and the sterol content was determined (Table 1). In the transformant cells, the levels of 4,4-dimethylzymosterol and the unidentified intermediates were increased, indicating that the elevated P450 levels accelerated lanosterol metabolism. Although the P450 level was not elevated, as discussed above, an essentially similar alteration in sterol composition was also evident in the transformant and control cells grown under aerobic conditions (Table 2). The lanosterol level was decreased and that of 4,4dimethylzymosterol was increased in the transformed cells. The contents of zymosterol and unidentified intermediates were also increased in the transformant. These findings suggest that the level of P450,,,, was higher in the transformant, which in turn, promoted the metabolism of lanosterol. However, it is noteworthy that no increase was evident in the ergosterol content of the transformant under both aerobic and semi-anaerobic conditions. In cells grown under the latter conditions, the elevated P450i4nM level did not increase the zymosterol content, and lanosterol was not decreased but rather increased slightly in the transformant. Although the reason for the discrepancies between the aerobically and the semi-anaerobically grown cells is TABLE 1. Sterol contents in semi-anaerobically grown transformant and control cells at the late-log phase (8.5 h) Transformant control (nmol/mg dry wt.) Lanosterol 4,4-Dimethylzymosterol Zymosterol Ergosterol Unidentified Total
Transformant/ control
1.9 0.9 1.1 3.2 7.2
1.3 0.3 1.0 4.3 3.3
1.46 3.00 1.10 0.74 2.18
14.3
10.2
1.40
yet to be determined, elevation of the cellular P450i4nM level evidently accelerated the metabolism of lanosterol to ergostadiene intermediates under both aerobic and semianaerobic growth conditions. The accumulation of these intermediates m P450L4nM overexpressed cells suggests that the cellular level of ergosterol is regulated at the final step of the biosynthetic pathway (the conversion of ergostadienol to ergosterol), which is catalyzed by another P450 enzyme (P4502& (12). This study revealed that the elevated P450,4DM level accumulated intermediates of ergosterol biosynthesis under aerobic and anaerobic conditions, although the cellular ergosterol content is lower in cells cultured semi-anaerobically than in those grown aerobically (Tables 1 and 2). The growth environment for fermenting yeasts in the brewing industry is considered semi-anaerobic, the supply of oxygen being maintained only by absorption from the surface of liquid media. Thus, yeast strains used in commercial fermentation processes are considered to experience a shortage of oxygen for sterol biosynthesis (13, 14). Under these conditions, the cellular level of is raised. We have shown here that cellular p450,4DM ergosterol intermediates were increased by elevating the P450i4nM level. These facts suggest that an elevated P450i4nM level improves the cellular sterol metabolism, thus increasing the sterol contents under conditions of a low oxygen supply. However, elevation of the P450,,nM level alone did not increase the content of ergosterol but resulted in an accumulation of ergostadienol intermediates. Since another regulatory P450 enzyme (P450ZZDS) that converts ergostadienol to ergosterol is involved in ergosterol synthesis, the concomitant elevation of both P45Os is necessary to improve the ergosterol content in fermenting yeast. REFERENCES
1. Quain, D. E.: Studies on yeast physiology-impact on fermentation performance and product quality. J. Inst. Brew., 95, 31% 323 (1988). 2. Ratledge, C. and Evans, C. T.: Lipids and their metabolism, p. 428. In Rose, A. H. and Harrison, .I. S. (ed.), The yeasts, vol. 3. Academic Press, London (1989). 3. Aoyama, Y., Yoshida, Y., and Sato, R.: Yeast cytochrome P450 catalyzing lanosterol 14a-demethylation. II. Lanosterol metabolism by purified P-450,.,nM and by intact microsomes. J. Biol. Chem., 259, 1661-1666 (1984). 4. Yoshida, Y. and Aoyama, Y.: Yeast cytochrome P-450 catalyzing lanosterol 14a-demethylation. I. Purification and spectral properties. J. Biol. Chem., 259, 1655-1660 (1984). 5. Aoyama, Y., Yoshida, Y., Nishino, T., Katsuki, H., Maitra, U. S., Mohan, V. P., and Sprinson, D. B.: Isolation and characterization of an altered cytochrome P-450 from mutant defective in lanosterol 14w-demethylation. J. Biol. Chem., 262, 14260-14264 (1987).
66
J. FERMENT.BIOENG.,
TAINAKA ET AL.
6. Trocha,
7.
8.
9.
10.
P. J., Jasne, S. T., and Sprinson, D. B.: Yeast mutants blocked in removing the methyl group of lanosterol at C14. Separation of sterols by high-pressure liquid chromatography. Biochemistry, 16, 4721-4726 (1977). Kalb, V. F., Woods, C. W., Turi, T. G., Dey, C. R., Sutter, T. R., and Loper, J. C.: Primary structure of the P-450 lanosterol demethylase gene from Succharomyces cerevisiue. DNA, 6, 529-537 (1987). Carlson, M. and Botstein, D.: Two differentially regulated mRNAs with different 5’ ends encode secreted and intracellular forms of yeast invertase. Cell, 28, 145-154 (1982). Ito, H., Fukuda, Y., Murata, K., and Kimura, A.: Transformation of intact yeast cells treated with alkali cations. J. Bacteriol., 153, 163-168 (1983). Omura, T. and Sato, R.: The carbon monoxide-binding
11.
12.
13.
14.
pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239, 2370-2378 (1964). Taylor. F. R. and Parks, L. W.: Metabolic interconversion of free stkrols and steryl esters in Saccharomyces cerevisiae. J. Bacterial., 136, 531-537 (1978). Hata, S., Nishino, T., Katsuki, H., Aoyama, Y., and Yoshida, Y.: Characterization of A22-desaturation in ergosterol biosynthesis of yeast. Agric. Biol. Chem., 51, 1349-1354 (1987). Aries, V. and Kirsop, B.: Sterol biosynthesis by strains of Saccharomyces cerevisiae in the presence and absence of dissolved oxygen. J. Inst. Brew., 84, 118-122 (1978). Ahvenainen, J.: Lipid composition of aerobically and anaerobically propagated brewer’s bottom yeast. J. Inst. Brew., 88, 367-370 (1982).