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Improvement of isoamyl acetate productivity in sake yeast by isolating mutants resistant to econazole
JOURNAL
OF BIOSCIENCE
AND BIOENGWEERING
Vol. 87, No. 5, 697-699. 1999
Improvement of Isoamyl Acetate Productivity in Sake Yeast by Isolating Mutants Resistant to Econazole TADAO ASANO,* TAKAYUKI
INOUE, NAOTAKA KUROSE, NOBUTSUGU HIRAOKA, SADAO KAWAKITA
AND
Alcoholic BeveragesResearch Laboratories, Takara Shuzo Co. Ltd., 3-4-l Seta, Otsu, Shiga 520-2193, Japan Received 13 May 1998/Accepted 18 February 1999
Sake yeast strains were improved so as to produce larger amounts of isoamyl acetate than the parental strain by isolating econazole-resistant mutants. Econazole, au imidazole antimycotic, directly interacts with unsaturated fatty acids in the yeast cell membrane, where it also inhibits the synthesis of ergosterol and decreasesthe ratio of unsaturated to saturated fatty acids. In contrast, alcohol acetyltransferase (AATase), which catalyzes the synthesisof isoamyl acetate, is inhibited by unsaturated fatty acids. Fifty econazole-resistant mutants were isolated from a sake yeast, Kyokal no. 701, several of which produced approximately 1.4 to 2.4 times more isoamyl acetate and an almost equal amount of isoamyl alcohol compared with the parental strain. The AATase activities of the mutants in koji extract were 1.2 to 1.4 times higher, and the unsaturated to saturated fatty acid ratios were lower, than in the parental strain. [Key words: econazole, sake yeast, isoamyl acetate, alcohol acetyltransferase,
fatty acids in the membrane and decreases the ratio of unsaturated to saturated fatty acids in viva at a concentration inhibitory to ergosterol biosynthesis (10-13). From this, we inferred that genetically stable mutants giving a lower ratio of unsaturated to saturated fatty acids in cells may be obtainable from among those resistant to imidazole antimycotics. We therefore isolated mutants that produced larger amounts of i-AmOAc in sake mash than the parental strain from among those resistant to econazole. A sake yeast, Kyokai no. 701 (K701), was mutagenized with 3% ethyl methanesulfonate according to the method described by Lindegren et al. (14). The survival rate of K701 was 25%. The cells were spread on an SD agar plate (0.67% Difco yeast nitrogen base without amino acids, 2% glucose, and 2% agar) containing 5 pg/ ml econazole, at which concentration K701 was not viable. The frequency of mutants resistant to econazole was about 10e5 and large colonies grown on the selective agar plate were isolated. A koji extract medium (brix: 10%) was prepared by mixing koji (polished and steamed rice on which the mold Aspergillus oryzae has been cultivated) with distilled water and then incubating this mixture at 55°C overnight followed by filtration. Fifty econazole-resistant mutants of K701 were grown in 30ml of the koji extract at 13°C for 12 d. Flavor components in the supernatant of the culture were analyzed by head-space gas chromatography. Figure 1 shows the distribution of the i-AmOAc concentration in koji extract prepared with econazole-resistant mutants. Among the 50 mutants tested, 17 produced higher concentrations of i-AmOAc than the parental strain, K701, with several of them producing 1.4 times more i-AmOAc than K701. Thirty mutants yielded lower i-AmOAc concentrations than the parental strain. Hirohata et al. (9) isolated a mutant that produced more alcohol but gave a lower i-AmOAc concentration in sake mash than its parental strain from among clotrimazole-resistant sake yeast mutants. This mutant has a mutation site that induces the overexpression of PDRS, a gene encoding an ATP-binding cassette
Isoamyl acetate (i-AmOAc) is recognized as an imbanana-like flavor component of Japanese sake. Acetate esters, such as i-AmOAc and ethyl acetate, are synthesized from acetyl-CoA by alcohol acetyltransferase (AATase, EC 2.3.1.84), which is inhibited by unsaturated fatty acids and aerobic conditions, and is thermolabile (1, 2). At present, large amounts of i-AmOAc are accumulated by polishing rice to reduce the content of unsaturated fatty acids and low temperature fermentation, which preserves AATase activity. However, such procedures increase sake-brewing costs. To overcome this problem, attempts have been made to improve the i-AmOAc production of sake yeasts by mutation. Ashida et al. (3) reported that mutants resistant to 5,5,5-trifluoro-DL-leucine, an analogue of Lleucine, produced high concentrations of both i-AmOAc and isoamyl alcohol (i-AmOH) through elimination of feedback inhibition and repression of cu-isopropylmalate synthase by L-leucine accumulation. Yanagiuchi et al. (4) obtained mutants with a low level of esterase activity, an enzyme hydrolyzing i-AmOAc. These mutants accumulated large amounts of i-AmOAc in sake mashes. Another method of obtaining mutants with low esterase activity by isolating those resistant to i-AmOXAc (X= F,CI), which is hydrolyzed by esterase into toxic XAcOH, has been reported (5, 6). Some of these mutants had less than 15% of the esterase activity of their parental strain and accumulated 1.5 to 2.0 times more i-AmOAc and isobutyl acetate in sake mashes. There are some reports on yeast mutants resistant to antimycotics, such as nystatin, ketoconazole and clotrimazole, interacting with the yeast cell membrane (7-9). However, these mutants did not produce larger amounts of acetate esters than the parental strains. Nystatin, the preferred polyene antibiotic, effectively binds ergosterol but not interact with unsaturated fatty acids. Econazole, one of the imidazole antimycotics that has been found to interact with the yeast cell membrane by inhibiting ergosterol synthesis, interacts directly with unsaturated portant
* Corresponding
fatty acids]
author. 697
698
ASANO ET AL.
J. BIOSCI. BIOENG.,
12
z
0.4
r-0
iyl,ylry,
/
0
1.0
4.0
lsoamyl
acetate
concentration
of materials used for sake brewing test Addition Intermediate 60 47 13 85
Total 200 150 41 304 0.23 The additionally used a rice was polished to 77% of its original weight. The rice used to produce the koji was polished to 75% of its original weight. Total rice (g) Additional rice (g) Koji rice (g) Water (ml) Lactic acid (ml)
(B) Analysis of
sake
Initial 33 21 12 51 0.23
Final 107 91 16 168
brewed with parental and mutant strains
K701 Alcohol (%) 19.6 Sake meter -2.5 Acidity (ml) 2.70 Amino acidity (ml) 3.15 Acetaldehyde (ppm) 21 Ethyl acetate (ppm) 115 n-Propyl alcohol (ppm) 172 Isobutyl alcohol (ppm) 119 Isoamyl acetate (ppm) 8.0 Isoamyl alcohol (ppm) 226 Ethyl caproate (ppm) 0.7
l.O0.6ox0.4. 0.2-
Time (d)
(ABC) transporter (15). It may be that our 30 lower iAmOAc-producing mutants included a similar type of mutation, but further investigation is needed to verify why strains with lower i-AmOAc productivity than K701 should occur among econazole-resistant mutants. The second screening for the desired mutants was carried out through a sake brewing test (rice: 2OOg), the details of which are shown in Table 1A. After being fermented at 15°C for 14d, the sake mash was centrifuged at 4000 xg for 20min and the supernatant was filtered using a cellulose acetate membrane with a pore size of 10 pm. The brewed sake was assayed according to methods authorized by the Japanese National Tax Administration (16). Table 1B shows that 4 of the mutants (E18, E27, E37, and E43) accumulated approximately 1.4 to 2.4 times more i-AmOAc in the sake than the parental strain, while the amounts of i-AmOH they produced were very similar to that of the parental strain. 1. (A) Proportions
1.2-
4.6
(x 0.1 ppm)
FIG. 1. Distribution of isoamyl acetate concentration in koji extract medium (30 ml) brewed with econazole-resistant mutants derived from K701. All the mutants were grown at 13°C for 12 d.
TABLE
8 g 'i: e E 8 c 8 al E $j m x 8 E _a
El8 19.5 -3.0 2.85 2.93
Strain E27 18.7 - 10.0 2.95 3.30
E37 19.2 -3.5 2.95 2.98
E43 19.1 -5.5 3.05 3.20
16 134 218 115 11.1 236 0.7
25 183 138 110 19.3 196 0.6
21 135 131 119 11.7 247 0.5
22 171 163 120 18.3 228 0.7
FIG. 2. Changes in isoamyl acetate concentration (solid lines) and AATase activity (broken lines) of strains K701 (0) and E27 (0). The strains were pre-cultured in 50 ml YPD medium (1% yeast extract, 2% Polypepton, 5% glucose) at 30°C for 24 h, inoculated into 300 ml koji extract, and grown at 13°C for 12 d without shaking.
Hence, the i-AmOAc to i-AmOH ratio, an important index in determining the quality of a sake, was increased. These results suggested that the activity of AATase, which catalyzes i-AmOAc formation from acetyl-CoA and isoamyl alcohol, was increased by mutation. To confirm that the AATase activity of the mutants obtained was increased, they were grown in 300ml koji extract. Figure 2 shows the time courses of changes in the i-AmOAc concentration and AATase activity of the mutant strain E27 and the parental strain K701. Strain E27 was selected because it gave the highest i-AmOAc productivity in the sake mash. The two strains were precultured in 50ml YPD (1% yeast extract, 2% Polypepton, 5% glucose) medium at 30°C for 24 h and then inoculated into 300 ml koji extract and grown at 13°C for 12 d without shaking. Their AATase activities were measured by the method of Minetoki et al. (17), using n-amyl alcohol as an internal standard. The protein content of the cell extracts was measured with a Bio-Rad Protein Assay Kit. Both strains accumulated the maximal iAmOAc in the koji extract on the 5th day and exhibited the highest AATase activity on the 3rd day after inoculation. However, strain E27 produced approximately 1.2 times more isoamyl acetate on day 5 and showed 1.4fold higher AATase activity on the day 3 compared with the parental strain. The AATase activities of the other isolated mutants (E18, E37, E43) on day 3 (shown in Table 2) were also higher than that of strain K701. It was surmised from these results that the fatty acid composition in the cells of the four mutants whose AATase activities were increased might have been altered. The expression of the AATase gene (ATFZ) is mainly adjusted at the transcriptional level in the presence of oxygen and unsaturated fatty acids (2, 18). AATase is directly inhibited by unsaturated fatty acids in vitro (17) as well as in vivo (19). That is, the decrease in the ratio of unsaturated to saturated fatty acids in the cells enhances the expression of the ATFl gene and AATase activity. Recently, Omori et al. (20) and Kajiwara et al. (21) enhanced the ATFZ gene expression and i-AmOAc production of brewing yeasts by heat shock treatment. The ratio of unsaturated to saturated fatty acids in heatshocked cells decreased in comparison with that in pretreated cells. To elucidate intracellular fatty acid changes in the cells of the 4 mutants, the fatty acid compositions of
NOTES
VOL. 81, 1999 TABLE
2.
Fatty acid composition in cells and AATase activity K701 36.0 40.5 6.0 17.5
El8 36.9 39.6 6.5 17.1
Strain E27 38.5 39.3 6.2 16.0
E37 36.0 39.8 6.7 17.5
E43 36.7 39.4 7.6 16.3
Total saturated fatty acids (%) 42.0 Total unsaturated fatty acids (%) 58.0
43.4 56.6
44.7 55.3
42.7 57.3
44.3 55.7
AATase activity (ppm/h/mg protein)
14.0
15.2
13.8
15.0
Fatty acid (% of total weight) Pahnitic acid (Cl6 : 0) Palmitoleic acid (Cl6 : 1) Stearic acid (Cl8 : 0) Oleic acid (Cl8 : 1)
11.3
The strains pre-cultured in 50 ml YPD medium (1% yeast extract, 2% Polypepton, 5% glucose) at 30°C for 24 h were inoculated into 300 ml koji extract and grown at 13°C for 3 d without shaking. Yeast cells used for fatty acid analysis were collected by centrifugation and lyophilized after washing with de-ionized water, methanol, and ethyl ether.
cells grown in 300 ml koji extract for 3 d were measured as following manner. Cells (5 x lOlo) were saponified by 50% KOH in a 50% aqueous methanol solution at 70°C for 30min. After acidification with cont. HCl, fatty acids were extracted with chloroform : methanol (2 : 1) and dried with Na2S0+ Samples containing the fatty acids were prepared as methyl esters with boron trifluoride in methanol and were analyzed by a gas chromatograph equipped with a flame ionization detector and a DEGS column ($2.6 mm x 3.1 m). The fatty acid compositions of the 4 mutants differed from that of the parental strain (Table 2) in that the proportions of total unsaturated fatty acids in the mutants were reduced. The Cr&Cr6.0 and Cr~.r/Cr~.~ ratios of the mutants were all lower in comparison with those of K701. The AATase activities of the mutants increased in accordance with the reduction in total unsaturated fatty acids. The proportion of total unsaturated fatty acids in strain E27, which accumulated most iAmOAc in the sake mash and had the highest AATase activity, decreased 2.7% in comparison with that of K701. The C16.1/C16.0ratios were 1.02 and 1.13, and the C18,1/C18.0ratios were 2.58 and 2.92 in cells of strains E27 and K701, respectively. These results suggest that a reduction in intracellular unsaturated fatty acids caused an increase in the AATase activity of the yeast and, therefore, the econazoleresistant mutants of K701 produced larger amounts of i-AmOAc in the sake mashes than the parental strain. Further genetic studies are required to determine whether the unsaturated fatty acid reduction in the mutant strains mainly enhances the ATFI gene expression or decreases direct repression of the AATase enzyme. The 4 mutants obtained in this study were grown on an SD agar plate containing 7.5 pg/ml clotrimazole and 5 pg/ml miconazole, at which concentrations K701 was not viable. That is, the econazole-resistant mutants were also resistant to other imidazole antimycotics. Thus, the selection of imidazole antimycotic-resistant mutants with reduced intracellular unsaturated fatty acids appears to be a useful method for the isolation of brewing yeasts that will produce larger amounts of i-AmOAc in sake mashes. REFERENCES 1. Yosbioka, K. and Hasbimoto, N.: Ester formation by alcohol acetyltransferase from brewers’ yeast. Agric. Biol. Chem., 45,
699
2183-2190 (1981). 2. Malcorps, P., Cbevai, J. M., JamIl, S., and Dufour, J. P.: A new model for the regulation of ester synthesis by alcohol acetyltransferase in Succharomyces cerevisiae during fermentation. J. Am. Sot. Brew. Chem., 49, 47-53 (1991). 3. Asbida, S., Ichikawa, E., Snginami, K., and Imayasu, S.: Isolation and application of mumnts producing sufficient isoamyl acetate, a sake flavor component. Anric. Biol. Chem.. 51, 2061-2065 (1987). 4. Yanagiucbi, T., Kiyokawa, Y., and Wakai, Y.: Isolation of sake-yeast strains accumulating large amounts of isoamyl acetate. Hakkokogaku, 67, 159-165 (1989). 5. Watanabe, M., Tanaka, N., Misbima, H., and Takemura, S.: Isolation of sake yeast mutants resistant to isoamyl monofluoroacetate to improve isoamyl acetate uroductivity. J. Ferment. Bioeng., 76, 229-231 (1993). 6. Watanabe, M., Nagai, H., and Kondo, K.: Properties of sake yeast mutants resistant to isoamyl monochloroacetate. J. Ferment. Bioeng., 80, 291-293 (1995). 7. Parks, L. W:,-Bottema, C. D. K.; Rodriguez, R. J., and Lewis, T. A.: Yeast sterols: yeast mutants as tools for the studv of sterol metabolism. Methods Enzymol., 111, 333-346 (1985): 8. Kalb, V. F., Loper, J. C., Dey, C. R., Woods, C. W., and Sutter, T. R.: Isolation of a cytochrome P-450 structural gene from Saccharomyces cerevisiae. Gene, 45, 237-245 (1986). 9. Hbobata, S., Watanabe, M., Nisbimura, A., and kondo, K.: Brewing properties of clotrimazole-resistant mutants isolated from sake yeast. Seibutsu-kogaku, 72,283-289 (1994). 10. Yamagucbi, I-I.: Antagonistic action of lipid components of membranes from Candida albicans and various other liuids on two imidazole antimycotics, clotrimazole and miconazole. Antimicrob. Agents Chemother., 12, 16-25 (1977). 11. Yamaguebi, H. and Iwata, K.: Effect of fatty acyl group and sterol composition on sensitivity of lecithin liposomes to imidazole antimycotics. Antimicrob. Agents Chemother., 15, 706711 (1979). 12. Bossibe, H. V. D., WiIIemsens, G., Cools, W., Lanwers, W. F. J., and Le Jeuoe, L.: Biochemical effects of miconazole on fungi. II. Inhibition of ergosterol biosynthesis in Cundida albicans. Chem. Biol. Interactions, 21, 59-78 (1978). 13. Georgopapadakon, N. H., Dii, B.A., Smith, S. A., Freudenberger, J., and Funke, P. T.: Effect of antifungal agents on lipid biosvnthesis and membrane inter&v in Candida albicans. &m&b. Agents Chemother., 31, ?6-51 (1987). 14. Lmdegren, G., Hwang, Y. L., Oshima, Y., and Liodegreo, C. C.: Genetical mutants induced by ethyl methanesulfonate in Saccharomyces. Can. J. Genet. Cytol., 7, 491-499 (1965). 1.5. MIzogucbI, H., Watanabe, M., Nisbimura, A., and Kondo, K.: Improvement of fermentative activity of sake yeast by giving resistance to clotrimazole. Seibutsu-kogaku, 76, 194-199 (1998). _ 16. The Brewing Society of Japan: Annotation of the Official Analytical Methods of the National Tax Administration Agency of Japan (4th ed.), p. 13-24. The Brewing Society of Japan, Tokyo (1993). (in Japanese) 17. Minetoki, T., Bogaki, T., Iwamatsu, A., Fujii, T., and Hamacbi, M.: The purification, properties and internal peptide sequences of alcohol acetyltransferase isolated from Saccharomyces cerevisiae Kyokai no. 7. Biosci. Biotech. Biochem., 57, 20962098 (1993). 18. Fujii, T., Kobayasbi, O., Yosbimoto, H., Furukawa, S., and Tamai, Y.: Effect of aeration and unsaturated fatty acids on expression of the Saccharomyces cerevisiae alcohol acetyltransferase gene. Appl. Environ. Microbial., 63, 910-915 (1997). 19. Yosbioka, K. and Hasbimoto, N.: Cellular fatty acid and ester formation by brewers’ yeast. Agric. Biol. Chem., 47, 22872294 (1983). 20. Omoh, T.; Umemoto, Y., Ogawa, K., Kajiwara, Y., Sbimoda, M., and Wada. H.: A novel method for screenine high glycerol- and ester-producing brewing yeasts (Saccharimyces cerevisiae) by heat shock treatment. J. Ferment. Bioeng., 83, 64-69 (1997). 21. Kajiwara, Y., Ogawa, K., Takashita, H., Omori, T., Sbimoda, M., and Wada, H.: Intracellular fatty acid formation and alcohol acetyl transferase gene expression in brewing yeast (Saccharomyces cerevisiae) treated with heat shock. J. Ferment. Bioeng., 84, 594-598 (1997).
OF BIOSCIENCE
AND BIOENGWEERING
Vol. 87, No. 5, 697-699. 1999
Improvement of Isoamyl Acetate Productivity in Sake Yeast by Isolating Mutants Resistant to Econazole TADAO ASANO,* TAKAYUKI
INOUE, NAOTAKA KUROSE, NOBUTSUGU HIRAOKA, SADAO KAWAKITA
AND
Alcoholic BeveragesResearch Laboratories, Takara Shuzo Co. Ltd., 3-4-l Seta, Otsu, Shiga 520-2193, Japan Received 13 May 1998/Accepted 18 February 1999
Sake yeast strains were improved so as to produce larger amounts of isoamyl acetate than the parental strain by isolating econazole-resistant mutants. Econazole, au imidazole antimycotic, directly interacts with unsaturated fatty acids in the yeast cell membrane, where it also inhibits the synthesis of ergosterol and decreasesthe ratio of unsaturated to saturated fatty acids. In contrast, alcohol acetyltransferase (AATase), which catalyzes the synthesisof isoamyl acetate, is inhibited by unsaturated fatty acids. Fifty econazole-resistant mutants were isolated from a sake yeast, Kyokal no. 701, several of which produced approximately 1.4 to 2.4 times more isoamyl acetate and an almost equal amount of isoamyl alcohol compared with the parental strain. The AATase activities of the mutants in koji extract were 1.2 to 1.4 times higher, and the unsaturated to saturated fatty acid ratios were lower, than in the parental strain. [Key words: econazole, sake yeast, isoamyl acetate, alcohol acetyltransferase,
fatty acids in the membrane and decreases the ratio of unsaturated to saturated fatty acids in viva at a concentration inhibitory to ergosterol biosynthesis (10-13). From this, we inferred that genetically stable mutants giving a lower ratio of unsaturated to saturated fatty acids in cells may be obtainable from among those resistant to imidazole antimycotics. We therefore isolated mutants that produced larger amounts of i-AmOAc in sake mash than the parental strain from among those resistant to econazole. A sake yeast, Kyokai no. 701 (K701), was mutagenized with 3% ethyl methanesulfonate according to the method described by Lindegren et al. (14). The survival rate of K701 was 25%. The cells were spread on an SD agar plate (0.67% Difco yeast nitrogen base without amino acids, 2% glucose, and 2% agar) containing 5 pg/ ml econazole, at which concentration K701 was not viable. The frequency of mutants resistant to econazole was about 10e5 and large colonies grown on the selective agar plate were isolated. A koji extract medium (brix: 10%) was prepared by mixing koji (polished and steamed rice on which the mold Aspergillus oryzae has been cultivated) with distilled water and then incubating this mixture at 55°C overnight followed by filtration. Fifty econazole-resistant mutants of K701 were grown in 30ml of the koji extract at 13°C for 12 d. Flavor components in the supernatant of the culture were analyzed by head-space gas chromatography. Figure 1 shows the distribution of the i-AmOAc concentration in koji extract prepared with econazole-resistant mutants. Among the 50 mutants tested, 17 produced higher concentrations of i-AmOAc than the parental strain, K701, with several of them producing 1.4 times more i-AmOAc than K701. Thirty mutants yielded lower i-AmOAc concentrations than the parental strain. Hirohata et al. (9) isolated a mutant that produced more alcohol but gave a lower i-AmOAc concentration in sake mash than its parental strain from among clotrimazole-resistant sake yeast mutants. This mutant has a mutation site that induces the overexpression of PDRS, a gene encoding an ATP-binding cassette
Isoamyl acetate (i-AmOAc) is recognized as an imbanana-like flavor component of Japanese sake. Acetate esters, such as i-AmOAc and ethyl acetate, are synthesized from acetyl-CoA by alcohol acetyltransferase (AATase, EC 2.3.1.84), which is inhibited by unsaturated fatty acids and aerobic conditions, and is thermolabile (1, 2). At present, large amounts of i-AmOAc are accumulated by polishing rice to reduce the content of unsaturated fatty acids and low temperature fermentation, which preserves AATase activity. However, such procedures increase sake-brewing costs. To overcome this problem, attempts have been made to improve the i-AmOAc production of sake yeasts by mutation. Ashida et al. (3) reported that mutants resistant to 5,5,5-trifluoro-DL-leucine, an analogue of Lleucine, produced high concentrations of both i-AmOAc and isoamyl alcohol (i-AmOH) through elimination of feedback inhibition and repression of cu-isopropylmalate synthase by L-leucine accumulation. Yanagiuchi et al. (4) obtained mutants with a low level of esterase activity, an enzyme hydrolyzing i-AmOAc. These mutants accumulated large amounts of i-AmOAc in sake mashes. Another method of obtaining mutants with low esterase activity by isolating those resistant to i-AmOXAc (X= F,CI), which is hydrolyzed by esterase into toxic XAcOH, has been reported (5, 6). Some of these mutants had less than 15% of the esterase activity of their parental strain and accumulated 1.5 to 2.0 times more i-AmOAc and isobutyl acetate in sake mashes. There are some reports on yeast mutants resistant to antimycotics, such as nystatin, ketoconazole and clotrimazole, interacting with the yeast cell membrane (7-9). However, these mutants did not produce larger amounts of acetate esters than the parental strains. Nystatin, the preferred polyene antibiotic, effectively binds ergosterol but not interact with unsaturated fatty acids. Econazole, one of the imidazole antimycotics that has been found to interact with the yeast cell membrane by inhibiting ergosterol synthesis, interacts directly with unsaturated portant
* Corresponding
fatty acids]
author. 697
698
ASANO ET AL.
J. BIOSCI. BIOENG.,
12
z
0.4
r-0
iyl,ylry,
/
0
1.0
4.0
lsoamyl
acetate
concentration
of materials used for sake brewing test Addition Intermediate 60 47 13 85
Total 200 150 41 304 0.23 The additionally used a rice was polished to 77% of its original weight. The rice used to produce the koji was polished to 75% of its original weight. Total rice (g) Additional rice (g) Koji rice (g) Water (ml) Lactic acid (ml)
(B) Analysis of
sake
Initial 33 21 12 51 0.23
Final 107 91 16 168
brewed with parental and mutant strains
K701 Alcohol (%) 19.6 Sake meter -2.5 Acidity (ml) 2.70 Amino acidity (ml) 3.15 Acetaldehyde (ppm) 21 Ethyl acetate (ppm) 115 n-Propyl alcohol (ppm) 172 Isobutyl alcohol (ppm) 119 Isoamyl acetate (ppm) 8.0 Isoamyl alcohol (ppm) 226 Ethyl caproate (ppm) 0.7
l.O0.6ox0.4. 0.2-
Time (d)
(ABC) transporter (15). It may be that our 30 lower iAmOAc-producing mutants included a similar type of mutation, but further investigation is needed to verify why strains with lower i-AmOAc productivity than K701 should occur among econazole-resistant mutants. The second screening for the desired mutants was carried out through a sake brewing test (rice: 2OOg), the details of which are shown in Table 1A. After being fermented at 15°C for 14d, the sake mash was centrifuged at 4000 xg for 20min and the supernatant was filtered using a cellulose acetate membrane with a pore size of 10 pm. The brewed sake was assayed according to methods authorized by the Japanese National Tax Administration (16). Table 1B shows that 4 of the mutants (E18, E27, E37, and E43) accumulated approximately 1.4 to 2.4 times more i-AmOAc in the sake than the parental strain, while the amounts of i-AmOH they produced were very similar to that of the parental strain. 1. (A) Proportions
1.2-
4.6
(x 0.1 ppm)
FIG. 1. Distribution of isoamyl acetate concentration in koji extract medium (30 ml) brewed with econazole-resistant mutants derived from K701. All the mutants were grown at 13°C for 12 d.
TABLE
8 g 'i: e E 8 c 8 al E $j m x 8 E _a
El8 19.5 -3.0 2.85 2.93
Strain E27 18.7 - 10.0 2.95 3.30
E37 19.2 -3.5 2.95 2.98
E43 19.1 -5.5 3.05 3.20
16 134 218 115 11.1 236 0.7
25 183 138 110 19.3 196 0.6
21 135 131 119 11.7 247 0.5
22 171 163 120 18.3 228 0.7
FIG. 2. Changes in isoamyl acetate concentration (solid lines) and AATase activity (broken lines) of strains K701 (0) and E27 (0). The strains were pre-cultured in 50 ml YPD medium (1% yeast extract, 2% Polypepton, 5% glucose) at 30°C for 24 h, inoculated into 300 ml koji extract, and grown at 13°C for 12 d without shaking.
Hence, the i-AmOAc to i-AmOH ratio, an important index in determining the quality of a sake, was increased. These results suggested that the activity of AATase, which catalyzes i-AmOAc formation from acetyl-CoA and isoamyl alcohol, was increased by mutation. To confirm that the AATase activity of the mutants obtained was increased, they were grown in 300ml koji extract. Figure 2 shows the time courses of changes in the i-AmOAc concentration and AATase activity of the mutant strain E27 and the parental strain K701. Strain E27 was selected because it gave the highest i-AmOAc productivity in the sake mash. The two strains were precultured in 50ml YPD (1% yeast extract, 2% Polypepton, 5% glucose) medium at 30°C for 24 h and then inoculated into 300 ml koji extract and grown at 13°C for 12 d without shaking. Their AATase activities were measured by the method of Minetoki et al. (17), using n-amyl alcohol as an internal standard. The protein content of the cell extracts was measured with a Bio-Rad Protein Assay Kit. Both strains accumulated the maximal iAmOAc in the koji extract on the 5th day and exhibited the highest AATase activity on the 3rd day after inoculation. However, strain E27 produced approximately 1.2 times more isoamyl acetate on day 5 and showed 1.4fold higher AATase activity on the day 3 compared with the parental strain. The AATase activities of the other isolated mutants (E18, E37, E43) on day 3 (shown in Table 2) were also higher than that of strain K701. It was surmised from these results that the fatty acid composition in the cells of the four mutants whose AATase activities were increased might have been altered. The expression of the AATase gene (ATFZ) is mainly adjusted at the transcriptional level in the presence of oxygen and unsaturated fatty acids (2, 18). AATase is directly inhibited by unsaturated fatty acids in vitro (17) as well as in vivo (19). That is, the decrease in the ratio of unsaturated to saturated fatty acids in the cells enhances the expression of the ATFl gene and AATase activity. Recently, Omori et al. (20) and Kajiwara et al. (21) enhanced the ATFZ gene expression and i-AmOAc production of brewing yeasts by heat shock treatment. The ratio of unsaturated to saturated fatty acids in heatshocked cells decreased in comparison with that in pretreated cells. To elucidate intracellular fatty acid changes in the cells of the 4 mutants, the fatty acid compositions of
NOTES
VOL. 81, 1999 TABLE
2.
Fatty acid composition in cells and AATase activity K701 36.0 40.5 6.0 17.5
El8 36.9 39.6 6.5 17.1
Strain E27 38.5 39.3 6.2 16.0
E37 36.0 39.8 6.7 17.5
E43 36.7 39.4 7.6 16.3
Total saturated fatty acids (%) 42.0 Total unsaturated fatty acids (%) 58.0
43.4 56.6
44.7 55.3
42.7 57.3
44.3 55.7
AATase activity (ppm/h/mg protein)
14.0
15.2
13.8
15.0
Fatty acid (% of total weight) Pahnitic acid (Cl6 : 0) Palmitoleic acid (Cl6 : 1) Stearic acid (Cl8 : 0) Oleic acid (Cl8 : 1)
11.3
The strains pre-cultured in 50 ml YPD medium (1% yeast extract, 2% Polypepton, 5% glucose) at 30°C for 24 h were inoculated into 300 ml koji extract and grown at 13°C for 3 d without shaking. Yeast cells used for fatty acid analysis were collected by centrifugation and lyophilized after washing with de-ionized water, methanol, and ethyl ether.
cells grown in 300 ml koji extract for 3 d were measured as following manner. Cells (5 x lOlo) were saponified by 50% KOH in a 50% aqueous methanol solution at 70°C for 30min. After acidification with cont. HCl, fatty acids were extracted with chloroform : methanol (2 : 1) and dried with Na2S0+ Samples containing the fatty acids were prepared as methyl esters with boron trifluoride in methanol and were analyzed by a gas chromatograph equipped with a flame ionization detector and a DEGS column ($2.6 mm x 3.1 m). The fatty acid compositions of the 4 mutants differed from that of the parental strain (Table 2) in that the proportions of total unsaturated fatty acids in the mutants were reduced. The Cr&Cr6.0 and Cr~.r/Cr~.~ ratios of the mutants were all lower in comparison with those of K701. The AATase activities of the mutants increased in accordance with the reduction in total unsaturated fatty acids. The proportion of total unsaturated fatty acids in strain E27, which accumulated most iAmOAc in the sake mash and had the highest AATase activity, decreased 2.7% in comparison with that of K701. The C16.1/C16.0ratios were 1.02 and 1.13, and the C18,1/C18.0ratios were 2.58 and 2.92 in cells of strains E27 and K701, respectively. These results suggest that a reduction in intracellular unsaturated fatty acids caused an increase in the AATase activity of the yeast and, therefore, the econazoleresistant mutants of K701 produced larger amounts of i-AmOAc in the sake mashes than the parental strain. Further genetic studies are required to determine whether the unsaturated fatty acid reduction in the mutant strains mainly enhances the ATFI gene expression or decreases direct repression of the AATase enzyme. The 4 mutants obtained in this study were grown on an SD agar plate containing 7.5 pg/ml clotrimazole and 5 pg/ml miconazole, at which concentrations K701 was not viable. That is, the econazole-resistant mutants were also resistant to other imidazole antimycotics. Thus, the selection of imidazole antimycotic-resistant mutants with reduced intracellular unsaturated fatty acids appears to be a useful method for the isolation of brewing yeasts that will produce larger amounts of i-AmOAc in sake mashes. REFERENCES 1. Yosbioka, K. and Hasbimoto, N.: Ester formation by alcohol acetyltransferase from brewers’ yeast. Agric. Biol. Chem., 45,
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