Brewing properties of sake yeast whose EST2 gene encoding isoamyl acetate-hydrolyzing esterase was disrupted

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 85, No. 1, 101-106. 1998

Brewing Properties of Sake Yeast Whose EST2 Gene Encoding Isoamyl Acetate-Hydrolyzing Esterase Was Disrupted?

KIYOSHI FUKUDA,‘* NAG1 YAMAMOTO,’ YOSHIFUMI KIYOKAWA,’ TOSHIYASU YANAGIUCHI,’ YOSHINORI WAKAI,’ KATSUHIKO KITAMOT0,2 YOSHIHARU INOUE,3 AND AKIRA KIMURA3

Kizakura Sake Brewing Co. Ltd., 223 Shioya-machi, Fushimi-ku, Kyoto 612,l Department of Biotechnology, The University of Tokyo, 1-I-I Yayoi, Bunkyo-ku, Tokyo 113,2 and Research Institute for

Food Science, Kyoto University, Uji, Kyoto 611,3 Japan

Received 24 March 1997/Accepted 17 October 1997

The EST2 gene, encoding an isoamyl acetate-hydrolyzing esterase, was disrupted in a diploid strain of Sac- charomyces cerevisiae UT-1 (MATa/MATa ura3/ura3 trpl/trpl EST2/EST2), which is derived from the industrial sake yeast Kyokal no. 701 (strain K-701), by using two disruption plasmids (pDest2U, est2::URAJ; and pDest2T, est2:: TRPl) sequentially. Genomic Southern blot analysis revealed that both loci of the EST2 gene on the chromosome of strain UT-1 were disrupted. The resultant mutants were named UTUT-1 and UTUT-2 (MATa/MATa ura3/ura3 trpl/trpl est2::lJRAJ/est2:: TRPI). Deficiency in Est2p esterase was also confirmed by activity staining of the gel after native-polyacrylamide gel electrophoresis of cell extracts of the two mutant strains. Small scale sake brewing was carried out using these sake yeasts and the strains they were derived from, and their brewing properties were compared. The fermentation profiles of the four strains (strains K-701, UT-l, UTUT-1, and UTUT-2) were largely similar. The components of the resulting sake were also similar except for Ihe acetate ester concentration, although strains UTUT-1 and UTUT-2 produced approximately 24imes more isoamyl acetate than the wild type K-701. These results strongly suggest that the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in the sake mash. Strains UTUT-1 and UTUT-2, deficient in Est2p esterase, are suitable for sake brewing.

[Key words: isoamyl acetate, esterase, Saccharomyces cerevisiae, sake brewing, sake yeast]

Many compounds are responsible for determining the flavor quality of sake and other alcohol beverages. Esters, such as isoamyl acetate and ethyl caproate, impart some of the most important flavors in sake. Isoamyl acetate is a positive flavor determinant that gives a fruit- like flavor to sake. Isoamyl acetate is thought to be synthesized from isoamyl alcohol and acetyl CoA by alcohol acetyltransferase (AATFase, EC 2.3.1.84) in yeast (l-5), and is hydrolyzed by esterases at the same time in the sake mash (6-11). We have thus speculated that the accumulation of isoamyl acetate is dependent upon the ratio of the activity of AATFase to the activities of esterases toward isoamyl acetate (6, 9, 10). We previously succeeded in isolating mutants from sake and laboratory yeast strains of Saccharomyces cerevisiae having low esterase activity by ethylmethane sulfonate (EMS) treatment followed by a diazo staining method (6, 12). These mutants accumulated a large amount of isoamyl acetate and isobutyl acetate in the sake mash compared with their corresponding parent strains. Mutants derived from sake yeast retained slight isoamyl acetate hydrolyzing activity of Est2p, whereas the mutant derived from laboratory yeast was completely deficient in Est2p activity, although its fermentation ability was lower than that of the sake yeasts. Since the treatment of yeast by a mutagen may damage some other genes that are important for

* Corresponding author. t The Mechanism of Isoamyl Acetate Production by Sake Yeast

(VI. At the suggestion of the Succharomyces Genome Database, Stan-

ford University, the EST2 gene has been renamed ZAHZ. However, since this paper is one of a series of studies, we have retained the formers name for consistency and to avoid confusion.

101

sake brewing, and sake yeasts are polyploid, it is quite difficult to specifically disrupt the EST2 gene in sake yeasts. However, the gene disruption technique is common by employed in making knockout mutants of S. cerevisiae. Recently, we succeeded in cloning the EST2 gene from S. cerevisiae (12).

In the work reported here, we constructed an est2- deficient diploid sake yeast by a one-step gene disruption method using two different disruption plasmids sequentially, and studied the sake brewing profiles of the resulting mutants.

MATERIALS AND METHODS

Strains, media, and plasmids The strains of S. cerevisae used in this study are listed and summarized in Table 1. Escherichia coli JMl09 was used as a host for plasmid amplification. YPD medium (1% yeast extract, 2% Polypepton, 2% glucose; pH 5.5) and SD minimal medium (2% glucose, 0.67% yeast nitrogen base without amino acids; pH 5.5) were used to culture the yeast strains. Appropriate amounts of amino acids and nucleo- bases were added to SD minimal medium according to the auxotrophy of the strain used. Plasmids YRp7 (14) and YEp24 (15) and bacteriophage Ml3mp18 (16) were used for construction of the disruption plasmids.

Disruption of EST2 gene Two plasmids, pDest2U (est2:: URA3) and pDest2T (est2::TRPI), were constructed to disrupt the EST2 gene of S. cerevisiae. A plasmid carrying the EST2 gene (pIAHll8) (12) was digested with Hind111 and PstI, and electrophoresed on a low- melting agarose gel. A DNA fragment (approximately 2.3 kb) carrying an open reading frame (ORF) of the EST2 gene was purified and recloned into the HindIII-PstI site

102 FUKUDA ET AL. J. FERMENT. BIOENG..

TABLE 1. Yeast strains used in this study

Strain

IF0 10506 YPU 1 YPTl K-701 UT-l UTU-1 UTUT-I UTUT-2

Genotype

MATtr trp-05 his3-KVO 1~~2-801 let&U ade2-101 ura3-52 MATa trp436 his34200 lys2-801 leu2-AI ade2-101 ura3-52 est2:: URA3 MATtr trp436 his3-A200 lys2-801 leu2-hl ade2-101 ura3-52 est2:: TRPI MATa/MATa non-forming sake yeast derived from Kyokai no. 7 MATa/MATa ura3/ura3 trpl/trpl ESTZ/ESTZ MATa/MATa ura3/ura3 trplkrpl est2::URA3/ESTZ MA Ta/MATn ura3/ura3 trpl/trpl est2:: URA3/est2:: TRPI MA Ta/MATa ura3/ura3 trpl/trpl est2:: URA3/est2:: TRPI

Source or reference ----- IF@ This study This study Brewing Society of Japan (13) This study This study This study

a IFO, Institute for Fermentation, Osaka, Japan.

of M13mp18 (16). The resultant RF form of phage DNA was named pHPmp18; it was digested by AccIII, a unique site in pHPmp18 located in the ORF of the EST2 gene, and treated by Klenow fragment. To construct pDest2U, the URA3 gene from YEp24 (15), was inserted into the AccIII site of pHPmp18. Similarly, the TRPI gene from YRp7 (14) was inserted into the AccIII site of pHPmp18 to construct pDest2T. To obtain the estt::URA3 fragment, pDest2U was digested by SphI, and a 2.0-kb fragment was purified by low-melting agarose gel electrophoresis; to obtain the est2::TRPl fragment, pDest2T was digested by DraI and SphI, and a 2.2-kb fragment was purified in the same manner (Fig. 1A).

Preparation of cell-free extracts Cells were grown in a 500-ml flask containg 300ml YPD medium without shaking at 20°C for 2 d. Cells were collected by centrifu- gation at 5,OOOxg for 10min at 4”C, washed once with distilled water, and suspended in 5 ml ice-chilled 0.2 M sodium phosphate buffer (pH 7.0). All the procedures described hereafter were done at 0-4”C. The cell suspension was transferred into a 15-ml test tube and an equal amount of glass beads (0.25-0.5 mm) was added. The mixture was vigorously agitated using a vortex mixer for 10min (1 min vortexing followed by 1 min interval), and then centrifuged at 20,000 X g for 15 min. The resultant supernatant was used as an enzyme source. The protein concentration was measured by the method of Lowry et al. (17).

Diazo staining Native-polyacrylamide gel electrophoresis (PAGE) was done with 7.5% polyacrylamide gel at 4°C using the buffer system of Laemmli (18) without SDS. The amount of the sample applied on each lane was adjusted to 300 pg of protein. After electrophoresis, the gel was stained for esterase activity by incubating it in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.025% 1-naphtyl acetate and 0.04% Fast Blue B salt at room temperature for 20 min.

Enzyme assay Esterase activity was assayed in a reaction mixture (1 .O ml) containing 0.1 mM p-nitrophenyl acetate, 100 mM sodium phosphate buffer (pH 7.0), and the enzyme at 30°C for 15min (8). To stop the reaction, 300,ul of 10% trichloroacetic acid was added to the mixture and it was kept on ice for 10min. The mixture was then centrifuged in a microfuge tube at 14,000 rpm, 4°C for 5 min. The absorbance at 330 nm of the resultant surpernatant was measured. One unit of the activity was defined as the amount of enzyme forming 1 .O pmol of p-nitrophenol in 1 h at 30°C.

Isoamyl acetate-hydrolyzing activity was measured by monitoring the production of acetate or isoamyl alcohol from isoamyl acetate. A reaction mixture (1.5 ml) containing 1OOppm isoamyl acetate, 50mM sodium phosphate buffer (pH 7.0), 7 mM MgC12, and the enzyme in a

25-ml vial sealed with a silicone rubber septum was incu- bated at 25°C for 1 h. Solid NaCl (l.Og) was added to the reaction mixture to stop the reaction, and supernatant was withdrawn to measure the acetate using an F- kit (Boehringer), or the reaction was stopped by the addi- tion of NaCl and the mixture was directly applied to head space gas chromatography as described by Yoshioka and Hashimoto (1). One unit of isoamyl acetate- hydrolyzing activity was defined as the amount of enzyme forming 1 ppm of acetate or isoamyl alcohol in 1 h at 25°C.

AATFase activity was measured by the method of Minetoki et al. (2). One unit of AATFase activity was defined as the amount of enzyme forming 1.0 ppm of isoamyl acetate in 1 h at 25’C.

DNA manipulation Transformation of S. cerevisiae was carried out according to the method of Ito et al. (19). Other DNA manipulations were carried out according to Sambrook et al. (20).

Southern blot analysis Approximately 10 /lg of chromosomal DNA from S. cerevisiae was digested by the restriction endonuclease SphI, separated by 0.8% agarose gel electrophoresis, and transferred onto a nylon membrane (Hybond-N+; Amersham, UK) using 0.4N NaOH solution as a transfer buffer. The band was hybri- dized and detected using an ECL kit (Amersham) according to the vendor’s specifications. The SphI (0.8 kb) fragment from pIAH (12) containing the open reading frame of the EST2 gene was used as a probe.

Sake brewing by est2 disruptant Laboratory scale sake brewing using the est2 disruptants were carried out as described by Namba et al. (21). Sake mash containing 170g of rice was fermented for 18 d at 15°C. Esters formed in the sake mash were dertermined by head space gas chromatography as described in (21).

Chemicals Isoamyl acetate, 1-naphtyl acetate, and p-nitrophenyl acetate were purchased from Nacalai Tesque, Kyoto. Restriction endonucleases, T4 DNA ligase, and the Klenow fragment were obtained from Takara Shuzo, Kyoto. Fast Blue B salt was purchased from Merck, Darmstadt, Germany. An ECL directed nucleic acid labeling and detection kit was obtained from Amersham, UK and a combination acetate kit (F- kit) from Boehringer Mannheim, Mannheim, Germany. All other reagents were of analytical grade.

RESULTS

Disruption of the EST2 gene S. cerevisiae UT-l is derived from a non-forming sake yeast, strain K-701 (13). Since strain UT-l is a diploid (MATa/MATu ura3/ura3 trpl/trpl EST2/EST2), two plasmids were constructed to disrupt both of the EST2 loci on the chro-

VOL. 85, 1998 ISOAMYL ACETATE PRODUCTION BY ESTZ-DEFICIENT SAKE YEAST 103

mosome. 1 2345678 First, the est2::URA3 fragment from pDest2U (Fig.

1A) was introduced into strain UT-l, and Ura+ cells were selected on an SD minimal agar plate containing 20 pg/ml L-Trp. Several Ura+ cells were randomly selected, and genomic Southern blot analysis was done to confirm the disruption of one of the EST2 loci. As a con- trol experiment, the same fragment was also introduced into a haploid strain, IF0 10506. Chromosomal DNAs were prepared from each Ura+ cell, digested by SphI and analyzed by Southern blotting using a 0.8-kb (SphI- SphI) fragment (Fig. 1A) as a probe. As shown in Fig. lB, the wild type strains of the laboratory yeast (lane 1, IF0 10506) and of the sake yeasts (lane 4, K-701; lane 5, UT-l) gave a single band 0.8-kb long. On the other hand, the Ura+ cell (lane 2, YPUl) from IF0 10506 gave a 2.0-kb band using the same probe. This was due to the insertion of the URA3 gene in the AccIII site of the EST2 gene (Fig. 1A). On the other hand, the SphI- digest of chromosomal DNA from UTU-1 gave two bands of 0.8 and 2.0 kb. The 0.8-kb band corresponded to the EST2 gene itself, and the 2.0-kb band was est2::URA3, indicating that one of the EST2 loci on the genome was disrupted. Data in Fig. IB are representa- tive of each clone.

Est2p -_)

FIG. 2. Activity staining of est2 disruptants. Lanes: 1, IF010506; 2, YPUl; 3, YPTI; 4, K-701; 5, UT-I; 6, UTU-1; 7, UTUT-1; 8, UTUT-2.

We also used the TRPI marker to disrupt the EST2 gene. The est2::TRPI fragment was introduced into IF0 10506, and Trp+ cells were selected on an SD minimal agar plate without L-Trp. Chromosomal DNA of Trp+

cells (YPTl) was prepared, digested by SphI, and separated by the agarose gel electrophoresis. As shown in Fig. lB, the strain YPTl (lane 3) gave a 2.2-kb band. This was due to the insertion of the TRPI gene in the AccIII site of the EST2 gene.

(Ai Pmhe

pDest2U

(B)

1.2 kh,

7.0 kh -

0.8 kh-

Y -2.2 kh Y

12345678

FIG. 1. Construction of EST2 disruption plasmids (A) and genomic Southern blot analysis (B). The Sphl fragment (0.8 kb) containing the EST2 gene was used as a probe. Lanes: 1, IF010506; 2, YPUl; 3, YPTl; 4, K-701; 5, UT-l; 6, UTU-1; 7, UTUT-1; 8, UTUT-2.

We then disrupted another locus of the EST2 gene of strain UTU-1 by the TRPZ marker as described above. Chromosomal DNAs were prepared from two arbitrary Trp+ transformants (strains UTUT-1 and UTUT-2) obtained from UTU-1, digested by SphI and analyzed by Southern blotting as described above. As shown in Fig. lB, both of the strains UTUT-1 (lane 7) and UTUT-2 (lane 8) gave the 2.0-kb band (est2::URA3) and the 2.2- kb band (est2::TRPI). Therefore, both of the EST2 loci of strain UT-l were disrupted by the URA3 and TRPI marker genes.

Activity staining Activity staining (Fig. 2) was done to confirm the phenotype of the est2 disruptants. An Est2p esterase band was detected in the cell extracts of the wild type strains (lane 1, IF0 10506; lane 4, K- 701; lane 5, UT-l). Est2p esterase was the major esterase in both the laboratory and sake yeasts. In the est2 knockout mutants of the haploid strain (lane 2, YPUl; lane 3, YPTl), the Est2p band disappeared, but it is still ap- peared in the cell extract of UTU-1 (lane 6). This esterase was from another locus of the EST2 gene of UTU-1, because, as shown in lanes 7 and 8, no Est2p esterase band

TABLE 2. Esterase and AATFase activities of the EST2 disruptants

Strain

lsoamyl acetate-hydrolyzing Esterase activity AATFase activity

activity (units/mg-protein) (units/ma-protein)

IF0 10506 YPUl YPT 1 K-701 UT- 1 UTU-1 UTUT- 1 UTUT-2

(units/mg protein)

0.757*0.114 0.011 iO.018 0.007~0.011 0.505f0.062 0.666kO.078 0.282 t 0.056 0.017iO.008 0.018i0.018

0.518kO.095 0.394iO.060 0.307f0.024 0.281 kO.094 0.394iO.200 0.248 20.063 0.243kO.092 0.234-+0.060

0.955kO.467 0.924kO.439 1.454t0.821 3.90522.502 3.88812.282 3.58Ok2.135 3.827+-2.071 3.96Ok2.117

The values given are the meansFS.D. of six different tests.

104 FUKUDA ET AL. J. FERMENT. BIOENG..

TABLE 3. Analysis of alcohols and esters in fermented YPD medium

Strain

IF0 10506 YPUl YPTl K-701 UT-1 UTU-1 UTUT-1 UTUT-2

Ethanol (X)

0.8710.26 0.56kO.27 1.26kO.30 1.9810.76 1.68-tO.90 2.0020.79 2.01 kO.81 2.02kO.81

Ethyl acetate (ppm)

0.81 t-O.31 0.62iO.11 1.47kO.56 3.6lk2.17 5.16k3.47 5.45t3.36 5.x1+3.03 5.93t3.29

lsobutyl acetate (ppm)

ND ND ND

0.06?0.03 0.09*0.08 0.12?0.08 0.14io.07 0.13t0.08

lsobutyl alcohol (ppm)

10.8t4.9 11.2t3.2 6.6k2.6

23.1 i7.6 17.7i8.1 21.717.5 22.2-tl.2 22.4k7.7

Isoamyl acetate (ppm)

O.OSiO.06 0.07io.05 0.23iO.09 0.7650.42 0.88-tO.53 1.29kO.72 1.40+-0.70 1.42i0.65

Isoamyl alcohol (ppm)

25.8* 7.3 17.6t 6.6 33.5111.1 60.3k20.3 48.Oi 18.6 67.4i23.8 66.6k22.0 65.8k23.6

Ethyl caproate (ppm)

-- 0.03 i 0.05 ND

0.15 j-O.06 0.42 20.24 0.45 to.29 0.41~0.29 0.54rto.35 0.58 +0.36

The values given are the means-+S.D. of six different tests.

is seen in the cell extracts of strains UTUT-1 and UTUT- 2. These results confirmed that the EST2 genes were specifically destroyed in strains UTUT-1 and UTUT-2.

Enzyme activity When the esterase activities of the wild type and est2 knockout mutants were compared (Table 2), the isoamyl acetate-hydrolyzing esterase activities in strains YPUl and YPTl were found to be approximately only 1% of that of the parent strain IF0 10506. A similar tendency was observed in the case of sake yeasts; isoamyl acetate-hydrolyzing esterase activity in strain UTU-1 (est2::URA3/EST2) was 42x, and in strains UTUT- 1 and UTUT-2 (est2:: URA3/est2:: TRPl) approximately 3% of that of the parent strain UT-l (EST2/EST2). The differences in the isoamyl acetate- hydrolyzing esterase activities of these yeasts is thought to be caused by the copy number of the EST2 gene in each strain. When p-nitrophenyl acetate was used as a substrate, the esterase activities in the est2 disruptants (YPUl and YPTl) were 60-75x compared with that of the wild type strain (IF0 10506). Similary, approximately 60% of the p-nitrophenyl acetate-hydrolyzing esterase activity of UT-l was retained in strains UTU-1, UTUT- 1, and UTUT-2. These results suggest that the Est2p esterase prefers to hydrolyze isoamyl acetate. The specific activities of alcohol acetyltransferase (AATFase) in the wild type and est2 knockout mutants were similar when compared among the haploid (IF0 10506 versus YPUl and YPTl) or diploid (K-701 and UT-l versus UTU-1, UTUT-1, and UTUT-2) strains. Furthermore, a variance analysis of the AATFase activity among the sake yeasts

5 I Cl IS 20 I) 3 10 IS 20

Fermentation time(d) Fermentation time (d)

(K-701, UT-l, UTUT-1, and UTUT-2) showed no significant difference at p<O.O5. However, the activities of the diploid strains were approximately 3.5-times higher than those of the haploid strains, which may be due to the gene dosage effect.

Volatile compounds in YPD medium In order to evaluate the EST2 gene product in ester production, each est2 mutant was cultured at 20°C in YPD medium without shaking, and the concentrations of volatile esters and alcohols in the culture supernatants were then determined (Table 3). A small amount of isoamyl acetate was detected in the cultures of the haploid strains IF0 10506, YPUl, and YPTl. When the isoamyl acetate concentrations in cultures of sake yeasts were compared, the est2 disruptants (UTUT-1 and UTUT-2) were found to produce approximately 1.8-fold more isoamyl acetate than the wild type strain (K-701). Since the AATFase activities in strains K-701, UTUT-1, and UTUT-2 were almost the same, the differences in the isoamyl acetate concentrations in the cultures of these yeasts are thought to be caused by the Est2p esterase activities in each strain.

Sake brewing by est2 disruptauts To investigate the effect of EST2 gene disruption on sake brewing, small scale sake brewing was performed using the est2 knockout mutants derived from the laboratory and sake yeasts. Evolution of CO2 was measured to evaluate the fermentation profiles (Fig. 3). In the case of the laboratory strains (Fig. 3A), fermentation speed was almost the same in the parent strain and the est2 disruptants.

FIG. 3. Sake brewing with the EST2 disruptant. Laboratory scale sake brewing was carried out with (A) laboratory yeasts n , IF0 10506; ??, YPUl; 0, YPTl; and (B) sake yeasts 0, K-701; 0, UT-l; 0, UTU-1; V, UTUT-1; and A, UTUT-2.

VOL. 85, 1998 ISOAMYL ACETATE PRODUCTION BY EST2-DEFICIENT SAKE YEAST 105

TABLE 4. Properties of sake brewing with the EST2 disruptants

Strain Sake meter Ethanol (%)

Acidity (ml)

IF010506 YPUl YPTl K-701 UT-l UTU-1 UTUT- 1 UTUT-2

-62.0 9.8 6.4 -66.0 9.1 5.6 -42.0 11.5 6.3 +3.0 18.9 3.0 f5.0 14.7 7.3

0.0 19.6 3.3 +2.5 19.9 3.0 +4.0 20.5 2.8

Amino acidity aFL.$e Isobutyl W acetate

(ppm) (ppm) 2.2 18.6? 1.9 ND 2.9 30.1* 2.0 0.08+0.00 1.9 27.02 4.3 O.lltO.OO 2.0 122.2+31.5 0.86+-0.26 2.3 62.6f11.8 0.4850.11 2.1 93.71_ 6.2 0.83iO.05 2.1 126.9+ 0.4 1.93&0.03 2.2 153.0& 4.1 2.28kO.03

Isobutyl Isoanlyl Isoamyl Ethyl alcohol acetate alcohol caproate @pm) @pm) @pm) (ppm)

142.9f19.3 0.201-0.05 179.9k31.2 0.23kO.05 125.8* 2.3 0.26iO.04 125.7-t 7.6 0.25kO.09 133.7f18.1 0.60*0.10 198.lk34.8 0.24+0.07 191.8k28.0 6.9821.64 304.8t22.8 0.841-0.08 232.1 k24.9 3.02kO.71 335.4k27.0 0.52+0.08 207.2k26.9 6.9420.04 338.Ok26.4 0.45+-0.01 178.4f14.7 10.05+-0.35 285.5k18.4 0.5840.01 187.6i32.4 12.8421.63 304.6k31.4 0.64+-0.00

The values of esters and alcohols expect for ethanol given are the means?S.D. of three different tests.

However, the fermentation speed of the laboratory strains was lower than that of sake yeasts (Fig. 3B).

In the case of the sake yeasts, the CO2 evolution of strain UT-I was delayed compared with that of the original strain K-701. This might have been due to uru3 and/or trpl deficiency. Disruption of the EST2 gene itself did not seem to affect the fermentation profile, because CO2 evolution was almost same in strains UTU-1, UTUT-1, and UTUT-2. Analysis of the general components of the brewed sakes showed that there was no difference between that produced with the wild type strain K-701 and those made with the est2 disruptant strains UTUT-1 and UTUT-2, except for the concentration of acetate esters (Table 4). As shown in the table, the sakes brewed with UTUT-1 and UTUT-2 were found to accumulate 2-fold higher amounts of isoamyl acetate, ethyl acetate, and isobutyl acetate than those brewed with the wild type strain K-701. An isoamyl acetate variance analysis of K-701 versus UTUT-1 and UTUT-2 showed significant differences at p < 0.01. The concentrations of the other flavor alcohols and esters-isobutyl alcohol, isoamyl alcohol and ethyl caproate-were at almost same level as those of the wild type strain K-701.

DISCUSSION

Isoamyl acetate is a major and important determinant of the fruit-like flavor of sake. This ester is synthesized by the action of AATFase and hydrolyzed by esterase(s). In order to produce a large amount of isoamyl acetate in the sake mash, two approaches are possible-over- production of AATFase or disruption of esterase(s) that are active on isoamyl acetate in sake yeast. The gene corresponding to AATFase (ATFI) of S. cerevisiae has been cloned and sequenced (3). A transformant carrying the multicopy plasmid with the ATFI gene produces 27- times more isoamyl acetate compared to that of the con- trol strain in YM medium (3). On the other hand, we have been trying to disrupt the esterase activity in sake yeast to inhibit the hydrolysis of isoamyl acetate in the sake mash. We have cloned and sequenced the EST2 gene whose gene product is thought to be the major esterase that hydrolyzes isoamyl acetate (12).

Small scale sake brewing was carried out using the est2 mutant (IAH-1) derived from laboratory yeast, and the resulting sake was found to have a higher amount of isoamyl acetate than that brewed by the parent strain (12), although the sake meter of the sake produced by these laboratory strains was extremeley low [wild type (IF0 10480), -30.0; mutant (IAH-I), -32.0; versus Kyokai no. 7, f2.01. Although the fermentation profiles of sake yeasts and laboratory strains of S.cerevisiae are

considerably different, sake yeasts are classified as S. cerevisiae because they have been bred for improved fermentation profiles by selection in the sake mash. There- fore, many preferable spontaneous mutations, resulting from gene recombination are expected to occur in the genes corresponding to brewing and flavor ester production. It is of considerable interest to know whether the EST2 gene product has the same function in both sake and laboratory yeasts. Since sake brewing yeasts are polyploids and do not have appropriate auxotrophic markers, it is difficult to disrupt the EST2 gene on the chromosome of brewing yeasts. In the present study, however, we were able to disrupt the EST2 gene using the laboratory strain IF0 10506 and strain UT-I, which is derived from an industrial sake yeast (K-701), and compare the brewing properties of the est2-deficient sake yeasts.

No Est2p esterase band was detected in the cell extracts of strains UTUT-1 and UTUT-2 by activity staining (Fig. 2), although trace activity of esterase toward isoamyl acetate was retained in the cell extracts (Table 2). It may be that S. cerevisiae has some esterase(s) active toward isoamyl acetate other than Est2p. However, the Est2p esterase is thought to be the major esterase hydrolyzing isoamyl acetate in the sake mash.

A small scale sake brewing test was performed to evaluate the fermentation properties of the est2-disrupted yeasts. In the case of the laboratory strains (haploid), the sake meter values were within the range -42.0 to -66.0, and the CO* evolution rate was relatively low compared with that of the industrial sake yeast (K-701). These results are consistent with our previous observa- tions using the est2-deficient mutant IAH- (12). However, the isoamyl acetate concentrations in the resultant sake produced by the est2 disruptants (YPUl and YPTl) were 1.3- to 3.0-fold higher than that produced by the parent strain (IF0 10506). In the case of the sake yeasts, the fermentation speed (CO;? evolution) of strain UT-l decreased compared with that of its original strain (K-701), presumably due to the deficiency in ura3 and/or trpl. Kitamoto et al. reported that if w-a3 deficiency was complemented by the introduction of the URA3 gene into the locus, the fermentaion speed was recovered (13). This finding is supported by the data presented in this paper. In the case of the laboratory yeast, the absence of the EST2 gene did not affect the fermentaion speed (Fig. 3A). However, deficiency of the EST2 gene in strain UT-l caused by the introduction of the est2::URA3 fragment, which complements ura3- but disrupts one of the EST2 loci to create the strain UTU-1 (ura3/ura3 trpl/trpl est2:: URA3/EST2), allowed the fermentation speed to be recovered (Fig. 3B). If deficiency

106 FUKUDA ET AL J. FERMENT. BIOENG.,

50 100 150 200 250

Ratio (AATFase/Est?p)

FIG. 4. Relationship between isoamyl acetate production and AATFase/isoamyl acetate-hydrolyzing activity. The concentration of isoamyl acetate in the sake is plotted against the ratio of AATFase/isoamyl acetate-hydrolyzing activity. Enzyme activity and isoamyl acetate concentration values are taken from Tables 2 and 4, respectively.

of the EST2 gene itself did not actually affect the fermentation speed, as was observed in the case of the laboratory strain, recovery of CO2 evolution by strain UTU-1 was presumably caused by the complementation of ur& .

The EST2 gene disruptants derived from laboratory and sake yeasts produced higher amounts of isoamyl acetate in sake than that produced by each parent strain. The band pattern of genomic Southern analysis of the EST2 gene was the same in the laboratory and sake yeasts. These results suggest that the EST2 gene product in both the laboratory and sake yeasts has the same function with regard to the production of isoamyl acetate.

The brewing properties of strains UTUT-1 and UTUT- 2 were similar to those of the industrial sake yeast K-701 except for the productivities of acetate esters-isoamyl acetate, isobutyl acetate, and ethyl acetate. Isoamyl acetate is synthesized by AATFase and hydrolyzed by esterase(s), and we have speculated that accumulation of isoamyl acetate is dependent on the activity ratio of these two enzymes. Thus, we have been trying to change the ratio by destroying the Est2p esterase activity. The concentration of isoamyl acetate in sake was plotted against the ratio of AATFase/isoamyl acetate-hydrolyzing activity (Fig. 4). The amount of isoamyl acetate accumulated in the sake increased in accord with the increasing ratio of AATFase/isoamyl acetate-hydrolyzing activity. Therefore, the accumulation of isoamyl acetate seemed to be related to the balance of these two enzyme activities in the yeast. However, we must, of course, also consider the concentrations of the substrates in the sake mash also, i.e., isoamyl alcohol and acetyl CoA. We are now trying to breed sake yeasts which have various copy numbers of the ATFI and EST2 genes, to verify the relationship between the accumulation of isoamyl acetate and the ratio of these enzyme activities.

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Documents

Brewing properties of sake yeast whose EST2 gene encoding isoamyl acetate-hydrolyzing esterase was disrupted