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Involvement of methionine salvage pathway genes of Saccharomyces cerevisiae in the production of precursor compounds of dimethyl trisulfide (DMTS)
Journal of Bioscience and Bioengineering VOL. 116 No. 4, 475e479, 2013 www.elsevier.com/locate/jbiosc
Involvement of methionine salvage pathway genes of Saccharomyces cerevisiae in the production of precursor compounds of dimethyl trisulfide (DMTS) Kou Wakabayashi,1, 2 Atsuko Isogai,1, 2, * Daisuke Watanabe,1 Akiko Fujita,1 and Shigetoshi Sudo1, 2 National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashihiroshima 739-0046, Japan1 and Graduate School of Advanced Science of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima 739-8530, Japan2 Received 15 January 2013; accepted 12 April 2013 Available online 15 June 2013
Dimethyl trisulfide (DMTS) is one of the components responsible for the unpalatable aroma of stale Japanese sake, called “hineka”. Recently, a precursor compound of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), was identified. It was speculated that the yeast methionine salvage pathway (MTA cycle) might participate in the formation of DMTS-P1, because the chemical structure of DMTS-P1 was similar to one of the intermediate compounds of that pathway. Here, we carried out sake brewing tests using laboratory yeast strains with disrupted MTA cycle genes and found that DMTS-P1 was hardly produced by Dmeu1, Dmri1, and Dmde1 strains. Furthermore, the DMTS producing potential (production of DMTS during storage of sake) decreased in sake made with Dmri1 and Dmde1. We constructed sake yeast strains with a disrupted MRI1 or MDE1 gene and confirmed a decline in the DMTS-P1 content and DMTS producing potential of sake made with these disruptants. The results of sake brewing tests using MTA cycle disruptants suggested that SPE2 is responsible for the production of DMTS precursors other than DMTS-P1: although the DMTS-P1 content was higher in Dspe2 sake than in Dmri1 or Dmde1 sake, the DMTS producing potential of Dspe2 sake was as low as that of Dmri1 or Dmde1 sake. Sake brewing tests using BY4743 Dspe2 Dmri1 double disruptants revealed that the DMTS producing potential was further decreased as compared with the Dspe2 or Dmri1 single disruptant. These results suggest that MRI1, MDE1, and SPE2 are promising targets for breeding yeast to suppress the formation of DMTS during storage of sake. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Dimethyl trisulfide; 1,2-Dihydroxy-5-(methylsulfinyl)pentan-3-one; Methionine salvage pathway; Hineka; Sake; Saccharomyces cerevisiae]
Storing sake causes changes in the color, taste, and aroma. The aroma of stored sake is called “hineka” in Japanese. Hineka consists of several kinds of compounds (1), of which dimethyl trisulfide (DMTS) is one of the main components (2). The odor of DMTS is described as sulfury, cooked onion, or Japanese pickle-like odor. An excess amount of DMTS deteriorates the aroma of sake. Hineka can be reduced in the refining step by adsorbing it onto activated carbon. However, it reforms by the chemical reactions of precursor compounds during storage or distribution in the market. The formation of hineka can be reduced by suppressing these chemical reactions; for example, by storing it in a refrigerator, or reducing the amount of dissolved oxygen in sake (3). However, such methods need expensive equipment. Therefore, another technical approach is required that will facilitate control of the formation of hineka. We recently identified one of the precursor compounds of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), a previously unknown compound (4). Although the origin of DMTS* Corresponding author at: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashihiroshima, 739-0046, Japan. Tel.: þ81 82 420 8077; fax: þ81 82 420 8079. E-mail address: [email protected] (A. Isogai).
P1 is not known, the increment in its concentration during fermentation suggested the participation of yeast in its formation (5). Furthermore, if the genes responsible for the production of DMTS-P1 were clarified, control of the formation of DMTS in sake could be achieved by breeding yeast deficient in the relevant genes. The chemical structure of DMTS-P1 led us to consider the involvement of the methionine salvage pathway, because one of the intermediate compounds in this pathway has a structure similar to DMTS-P1 (4). The methionine salvage pathway, also called the 50 methylthioadenosine (MTA) cycle, recycles sulfur from MTA, which is a by-product of the biosynthesis of polyamines such as spermidine and spermine (6). For the synthesis of these polyamines (Fig. 1), S-adenosylmethionine is converted into S-adenosylmethioninamine by S-adenosylmethionine decarboxylase (encoded by SPE2). Next, an aminopropyl group of S-adenosylmethioninamine is transferred to putrescine by spermidine synthase (encoded by SPE3) and spermidine by spermine synthase (encoded by SPE4) to yield spermidine and spermine, respectively (7), and at the same time MTA is formed as a by-product. Because the assimilation of sulfur is strongly energy-consuming in the form of redox equivalents (8), most organisms have evolved recycling pathways to reuse sulfur from MTA in the regeneration of methionine, i.e., the MTA cycle.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.04.016
476
WAKABAYASHI ET AL.
J. BIOSCI. BIOENG.,
1,2-dihydroxy-5-(methylthio)1-penten-3-one
DMTS-P1 O S O
S
O OH
S
OH
OH
O
ARO8, ARO9, BAT1, BAT2
COOH
ADI1
NH 2
OH
S
O
SAM1, SAM2
NH 2 OPO32-
S O
MDE1 OH
O
S-adenosylmethionine HO (SAM) SPE2 H 2N
MRI1 O
SPE3
OPO 32-
MEU1
S HO
OH
O
S+
HOOC
OPO32-
S OH
COOH
methionine
UTR4
S HO
O
Ade OH
S+ HO
O
Ade OH
Ade OH
putrescine
spermidine SPE4 spermine
5’-methylthioadenosine (MTA) FIG. 1. The structure of DMTS-P1 and the MTA cycle in S. cerevisiae.
The MTA cycle is found in a wide variety of organisms including bacteria (9,10), yeast (11,12), plants (13), and mammals (9), with some differences among organisms. In the case of Saccharomyces cerevisiae, MTA phosphorylase (encoded by MEU1) converts MTA into 5-methylthioribose-1-phosphate, which subsequently undergoes successive enzymatic reactions by 5-methylthioribose1-phosphate isomerase (encoded by MRI1), 5-methylthioribulose1-phosphate dehydratase (encoded by MDE1), and 2,3-dioxomethylthiopentane-1-phosphate enolase/phosphatase (encoded by UTR4) to yield 1,2-dihydroxy-5-(methylthio)-1-penten-3-one, the structure of which resembles DMTS-P1. This compound is then converted into 4-methylthio-2-oxobutyrate by aci-reductone dioxygenase (encoded by ADI1), and finally, transamination of the keto acid catalyzed by four kinds of transaminase (encoded by ARO8, ARO9, BAT1, and BAT2) yields methionine. All of the genes encoding this pathway in S. cerevisiae have recently been identified (12). As stated above, the chemical structure of 1,2-dihydroxy-5(methylthio)-1-penten-3-one is similar to DMTS-P1 except that the sulfide group of the former is oxidized to sulfoxide in the latter, and the enediol structure of the former is replaced by diol in the latter (Fig. 1). We therefore speculated that DMTS-P1 might be derived from MTA cycle. In this study we investigated the potential involvement of MTA cycle genes in the formation of DMTS-P1 by using MTA cycle gene disruptants. We also studied the formation of DMTS during the storage of sake brewed with these disruptants and discussed the possibility for the target of breeding yeast to suppress hineka. MATERIALS AND METHODS Yeast strains The S. cerevisiae strains used in this study are listed in Table S1. The laboratory strain BY4743, BY4743 strains Daro8, Daro9, Dbat2, Dsam1, Dsam2, Dspe2, Dspe3, Dspe4, Dmeu1, Dmri1, Dmde1, Dutr4 and Dadi1, BY4741 strain Dspe2, and BY4742 strain Dspe2 were provided by EUROSCARF. The sake yeast strain Kyokai no. 7 (K7) was provided by the Brewing Society of Japan. BY4743, BY4743derived disruptants, and sake yeast are diploid strains. Disruption of the MRI1 or MDE1 gene in K7 was performed using a PCR-based method. Because K7 is a diploid strain, two copies of each gene were replaced by two kinds of marker cassette containing a kanMX4 or natMX4 gene (14). The marker cassettes were synthesized by fusion PCR (15) using the primers listed in Table S2. In the first step, the regions flanking the target genes were amplified with primers C
and D (for the upstream flanking region) or E and F (for the downstream flanking region), and S. cerevisiae chromosomal DNA as a template. The marker genes were amplified using the plasmid pFA6-kanMX4 (for kanMX4 gene) or pAG25 (for natMX4 gene) as a template, and primers A and B. These primers contained sequences complementary to the primer D or E, in addition to the template marker plasmid sequences. Next, the marker DNA fragments, and upstream and downstream flanking regions were fused by fusion PCR to synthesize the marker cassettes, mri1kanMX4 and mri1-natMX4, or mde1-kanMX4 and mde1-natMX4. These DNA fragments were transformed into K7 by the lithium acetate method to generate K7 Dmri1::kanMX4/Dmri1::natMX4 or K7 Dmde1::kanMX4/Dmde1::natMX4 (referred to as K7 Dmri1 or K7 Dmde1, respectively). The mutations were confirmed by PCR using the primers G and H. The SPE2 and MRI1 double deletion mutant was generated by deletion of MRI1 in the background of BY4741 Dspe2 and BY4742 Dspe2 using a PCR-based method with the primers MRI1-D-f and MRI1-D-r (Table S2) and the plasmid pAG25 as a template, to generate BY4741 Dspe2::kanMX Dmri1::natMX4 and BY4742 Dspe2::kanMX Dmri1::natMX4. Both strains were mated to generate BY4743 Dspe2::kanMX/ Dspe2::kanMX Dmri1::natMX4/Dmri1::natMX4 (referred to as BY4743 Dspe2 Dmri1). One-step sake brewing test of BY4743 deletion strains A small-scale sake brewing with one-step mashing was carried out as described by Kitagaki et al. (16) with some modifications. Although one-step mashing is not a usual method for the industrial sake brewing, we consider it is enough for screening purpose. Yeast cells were precultured in nutrient broth at 30 C for 3 days without shaking and then harvested by centrifugation. About 2.0 109 cells were mixed with 200 ml of ion exchange water, 540 ml of 7.5% lactic acid, 23 g of dried koji (Tokushima seikiku, Awa), and 60 g of dried rice (Tokushima seikiku). After gentle mixing, fermentation was carried out at 15 C. The weight of the mash was measured every day to monitor carbon dioxide evolution as a measure of fermentation progress. After 19 days, the mash was centrifuged and the supernatant was filtered with a membrane filter of 0.45 mm pore size. Three-step sake brewing test of K7 Dmri1 and K7 Dmde1 A small-scale sake brewing with three mashing steps was carried out as described by Nanba et al. 8 (17) using 300 g of total rice. Precultured yeast (8.6 10 cells) was added to a mixture of 84 ml of ion exchange water, 2 ml of 7.5% lactic acid, and 15 g of dried koji, and incubated at 15 C overnight. The next day, 37.5 g of polished rice (Nakateshinsenbon, milling ratio 70%) was steamed and added to it (Soe). Two days later, 115 ml of ion exchange water, 15 g of dried koji, and 82.5 g (the weight as polished rice) of steamed rice were added, and the mash was fermented at 9 C (Naka). The following day, 201 ml of ion exchange water, 30 g of dried koji, and 120 g of steamed rice were added, and the mash was fermented at 7 C (Tome). The fermentation temperature was then increased by 1.0 C/day to a maximum temperature of 15 C, which was maintained for 8 days. Next, the temperature was decreased by 2.0 C/day to 7 C. Twenty-one days after Tome, the mash was centrifuged and filtered with a membrane filter as described above. Measurement of DMTS-P1 The concentration of DMTS-P1 was measured as described previously (5), except that [methyl-d3]-DMTS-P1 was used as an internal standard instead of 1,5-pentanediol (18).
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INVOLVEMENT OF MTA CYCLE IN DMTS PRECURSOR PRODUCTION
Measurement of DMTS producing potential The amount of DMTS produced during the storage of sake for 7 days at 70 C was defined as the DMTS producing potential, which was measured by a previously described method (5). Other chemical analyses Ethanol concentration, sake meter, acidity, and amino acidity were measured according to the reference (19). When the amount of sake was not enough, the ethanol concentration was measured using an ethanol analyzer (Riken Keiki, Shizuoka). Sensory analysis The effect of deletion of MRI1 or MDE1 on the odor of sake after storage was investigated by sensory analysis. Five sake samples brewed with K7, K7 Dmri1 (K7-mri1_1, K7-mri1_2), and K7 Dmde1 (K7-mde1_1, K7-mde1_2) were treated with activated carbon (1 g/L) and filtered. They were then pasteurized at 63 C for 5 min. After cooling, they were stored at 40 C for two months. Sensory evaluation was performed by eight panelists who work at our institute. The five stored sake samples were coded using three-digit random numbers. To avoid bias caused by the color of the samples, they were poured into amber-colored glasses. To avoid an order effect, each panelist evaluated the samples in a different order. Panelists were instructed to smell the samples and to evaluate the intensity of hineka and sulfury odor using the labeled magnitude scales (barely detectable, 1.4; weak, 6.1; moderate, 17.2; strong, 35.4; very strong, 53.3; strongest imaginable, 100) (20,21). Because drinking sake samples made with genetically modified organisms is prohibited in our institute, panelists evaluated the samples only by orthonasal olfaction. Data were expressed as means of the logarithms of the labeled magnitude values and subjected to the two-way (panelists samples) analysis of variance (ANOVA) and TukeyeKramer HSD test to study the statistical differences.
RESULTS
1.2
0.5
1.0
0.4
0.8
0.3
0.6
0.2
0.4
0.1
0.2
0.0
0.0
DMTS producing potential (µg/L)
0.6
WT Δaro8 Δ aro9 Δbat2 Δsam1 Δ sam2 Δ spe2 Δ spe3 Δspe4 Δ meu1 Δ mri1 Δmde1 Δutr4 Δ adi1
DMTS-P1 (mg/L)
Involvement of MTA cycle genes in the formation of DMTSP1 and the DMTS producing potential of sake Using 13 yeast disruptants in which one of the MTA cycle genes was disrupted in the background of BY4743, we carried out small-scale sake brewing tests with one-step mashing. After fermentation was finished, the concentration of DMTS-P1 and the DMTS producing potential were determined. As shown in Fig. 2, a lower DMTS-P1 content was observed for sake brewed with the Daro9, Dspe2, Dmeu1, Dmri1, Dmde1, and Dutr4 strains. In particular, DMTS-P1 was hardly detected in Dmeu1, Dmri1, and Dmde1 sake. The results suggest that the MTA cycle in yeast is responsible for the formation of DMTS-P1, and that the MEU1, MRI1, and MDE1 genes are essential for it. The DMTS producing potential of sake made with Dmri1 or Dmde1 was also greatly decreased as compared with the parent strain. Although the DMTS-P1 content in Dmeu1 sake was as low as that in Dmri1 or Dmde1 sake, the DMTS producing potential of
FIG. 2. The influence of deletion of MTA cycle genes on the formation of DMTS-P1 (black) and the DMTS producing potential (gray) of brewed sake. Using the BY4743 strain and 13 yeast disruptants in which one of the MTA cycle genes was disrupted in the background of BY4743, small-scale sake brewing tests with one-step mashing were carried out. The concentration of DMTS-P1 and the DMTS producing potential of brewed sake were measured. Data represent the mean values and standard deviations of two independent experiments.
477
Dmeu1 sake was higher than that of Dmri1 or Dmde1 sake. By contrast, the DMTS producing potential of Dspe2 sake was as low as that of Dmri1 or Dmde1 sake, although the DMTS-P1 content was higher in Dspe2 sake than in the other two strains. These discrepancies suggest the presence of another DMTS precursor, which is increased by deletion of MEU1 and decreased by deletion of SPE2. The general properties of sake (ethanol content, sake meter, acidity, and amino acidity) made with MTA cycle gene disruptants were almost the same as those of the parent strain (Table 1). Disruption of the MDE1 or MRI1 gene in sake yeast Next, we deleted the MRI1 and MDE1 genes in sake yeast strain K7 to confirm that they are also involved in the formation of DMTS-P1 and the DMTS producing potential in this sake strain as well as in the laboratory strain. Because K7 is diploid, we disrupted the two copies of MRI1 or MDE1 on homologous chromosomes by inserting two different kinds of antibiotic-resistant markers. As a result, seven strains of K7 Dmri1 and six strains of K7 Dmde1 were obtained. Among them, two strains of each gene disruptant (K7mri1_1, K7-mri1_2, K7-mde1_1, and K7-mde1_2) were used for a small-scale sake brewing test. The sake brewing test was carried out with three mashing steps, in the same way as pilot-scale brewing, to confirm the effect of gene disruption in sake yeast under the conditions similar to actual sake brewing. As shown in Fig. 3, the content of DMTS-P1 in sake brewed with K7 Dmri1 or Dmde1 was greatly reduced as compared with the parent strain. The results suggest that the MRI1 and MDE1 genes are involved in the formation of DMTS-P1 in sake yeast K7 as well as in the laboratory yeast BY4743. In addition, deletion of MRI1 or MDE1 caused more than a ten-fold decrease in the DMTS producing potential (Fig. 3), suggesting that these genes are promising as targets for breeding sake yeast in which the formation of DMTS is repressed. The ethanol fermentation process (data not shown) and the general properties of sake (Table 2) brewed with the deletion strains were almost the same as those of the parent strain. Sensory analyses were carried out to investigate the effect of deletion of MRI1 or MDE1 on the odor of stored sake. After storage at 40 C for two months, the sake samples were evaluated for sulfury odor and hineka by olfaction. As shown in Fig. 4, the intensity of sulfury odor in sake made with K7 Dmri1 or Dmde1 seemed to decrease as compared with the parent strain. Although the differences between samples were not statistically significant by oneway ANOVA because of variance among the panelists, two-way ANOVA revealed significant differences between both samples (p ¼ 0.045) and panelists (p < 0.001). TukeyeKramer HSD test indicated a significant difference between K7 and K7-mri1_1. The intensity of hineka of sake made with K7 Dmri1 or Dmde1 also tended to decrease as compared with K7. Statistical differences were observed between samples (p ¼ 0.036) and panelists
TABLE 1. Properties of sake brewed with MTA cycle gene disruptants of BY4743. Strain
Ethanol (%)
Sake meter
Acidity
Amino acidity
WT Daro8 Daro9 Dbat2 Dsam1 Dsam2 Dspe2 Dspe3 Dspe4 Dmeu1 Dmri1 Dmde1 Dutr4 Dadi1
12.5 12.3 12.1 12.1 12.6 12.7 11.8 13.5 12.0 12.9 13.1 12.6 12.9 12.7
16 16 20 18 15 14 23 5 22 13 11 14 14 15
3.6 3.8 3.7 3.8 3.7 3.5 3.6 3.7 3.5 3.6 3.7 3.8 3.6 3.7
1.5 1.4 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Data represent the mean values of two independent experiments.
J. BIOSCI. BIOENG.,
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
FIG. 3. The influence of deletion of MRI1 and MDE1 in the background of sake yeast K7 on the formation of DMTS-P1 (black) and the DMTS producing potential (gray) of brewed sake. Using sake yeast strain K7, K7 Dmri1, and K7 Dmde1, small-scale sake brewing test was carried out with three mashing steps. The concentration of DMTS-P1 and the DMTS producing potential of brewed sake were measured. Data represent the mean values and standard deviations from three independent experiments.
(p < 0.001) by two-way ANOVA. The difference between K7 and K7mri1_1 was significant by TukeyeKramer HSD test. Effect of a Dspe2 Dmri1 double deletion on the DMTS producing potential As mentioned above, the possibility was suggested that SPE2 is involved in the formation of DMTS precursors other than DMTS-P1 (Fig. 2). Thus, we expected that the DMTS producing potential of sake made with a Dspe2 Dmri1 double disruptant would be even lower than that of the single disruptant. We constructed a BY4743 Dspe2 Dmri1 double disruptant and carried out small-scale sake brewing tests with one-step mashing. As a result, the DMTS producing potential of sake made with Dspe2 Dmri1 decreased by about 50% as compared with Dspe2, and 40% as compared with Dmri1 although the difference between the single and double disruptants was not statistically significant (Fig. 5). The content of DMTS-P1 in sake made with Dspe2 Dmri1 was less than that in sake made with Dspe2, and as little as that in sake made with Dmri1, which suggested that the decrease in DMTS-P1 content in Dspe2 Dmri1 sake was caused by the deletion of MRI1. The ethanol content, sake meter, acidity, and amino acidity of sake brewed with Dspe2 Dmri1 were almost the same as those of the parent strain (BY4743), Dspe2 and Dmri1 (data not shown). DISCUSSION DMTS is one of the main components of hineka, the “off-flavor” of stored Japanese sake. During storage of sake, DMTS is produced through the chemical conversion of precursor compounds. We previously identified one of the precursors, DMTS-P1, and have demonstrated here that the MTA cycle of yeast is involved in the formation of this precursor.
Strongest imaginable
2.0 1.6
Very strong Strong
aa ab bb
1.2
ab
ab ab ab ab
0.8
Moderate
Weak
0.4
FIG. 4. Sensory analysis of stored sake made with K7 and K7 deletion strains Dmri1 and Dmde1. Sake samples made with K7, K7 Dmri1, and K7 Dmde1 were stored at 40 C for two months, and sensory analyses were carried out to investigate the intensity of hineka (black) and sulfury odor (gray). Data represent the means and standard errors of mean from the evaluation of eight panelists. Values with different letters are significantly different at p < 0.05 by TukeyeKramer HSD test.
Sake brewing tests using MTA cycle gene disruptants of BY4743 revealed that the MEU1, MDE1, and MRI1 genes are indispensable for the formation of DMTS-P1, and the DMTS producing potential of sake made with the disruptant Dmde1 or Dmri1 strain was greatly reduced (Fig. 2). Deletion of the MDE1 or MRI1 gene in sake yeast K7 also caused a marked decline in the concentration of DMTS-P1 and in the DMTS producing potential of sake (Fig. 3). Even after storage for 1 week at 70 C, the DMTS concentration in sake made with these deletion strains was far below its detection threshold (0.18 mg/l). The effect of the deletion of MDE1 or MRI1 was also observed in the sensory analyses. The intensity of the sulfury odor and hineka tended to decrease as compared with the parent strain (Fig. 4). These results indicate that the formation of hineka can be suppressed by breeding sake yeast that is deficient in the MDE1 or MRI1 gene.
0.25
0.5
a
0.4
0.20 a
0.3
0.15 0.10 0.05 0.00
0.2
b b b c
c
b
c
b
0.1 0.0
DMTS producing potential (µg/L)
0.4
Log perceived intensity of odor
0.5
DMTS-P1 (mg/L)
0.5
DMTS producing potential (µg/L)
WAKABAYASHI ET AL.
DMTS-P1 (mg/L)
478
TABLE 2. Properties of sake brewed with K7 and K7 deletion strains Dmri1 and Dmde1. Strain
Ethanol (%)
K7 K7-mri1_1 K7-mri1_2 K7-mde1_1 K7-mde1_2
17.3 17.3 17.1 17.0 17.1
0.4 0.5 1.0 0.3 0.3
Sake meter 10 10 12 12 13
0.0 2.5 6.4 1.8 0.8
Acidity 2.1 2.3 2.4 2.1 2.2
0.0 0.2 0.1 0.1 0.1
Amino acidity 1.7 1.7 1.7 1.8 1.7
0.1 0.1 0.0 0.1 0.0
Data represent the mean values and standard deviations from three independent experiments.
FIG. 5. The effect of double deletion of SPE2 and MRI1 on the concentration of DMTS-P1 (black) and the DMTS producing potential (gray) of brewed sake. Small-scale sake brewing tests with one-step mashing were carried out using the BY4743, BY4743 strains Dspe2, Dmri1, and Dspe2 Dmri1 double disruptant. The concentration of DMTSP1 and the DMTS producing potential of brewed sake were measured. Data represent the mean values and standard deviations from three independent experiments. Values with different letters are significantly different at p < 0.05 by TukeyeKramer HSD test.
VOL. 116, 2013
INVOLVEMENT OF MTA CYCLE IN DMTS PRECURSOR PRODUCTION
The decline in the DMTS producing potential by the deletion of MRI1 or MDE1 in K7 seemed more significant than that of BY4743 (70e75% decrease for BY4743, more than 90% decrease for K7, Figs. 2 and 3). Since DMTS-P1 was hardly produced by these disruptants, the residual DMTS producing potential may be attributed to DMTS precursors other than DMTS-P1. It is possible that the formation of these precursors by K7 disruptants was lesser than BY4743 disruptants. At present, however, we cannot decide either genetic background or brewing conditions caused the differences. Recently, Okimori et al. reported that sake brewing conditions, such as death of yeast greatly affect the DMTS producing potential (Okimori, Y., Nishibori, N., Fujii, T., Sasaki, K., Kanai, M., Isogai, A., Kanda, R., Goto, N., and Yamada, O., Abstr. Annu. Meet. J. Soc. Biosci. Biotechnol. Agrochem., p. 688, 2012). The chemical structure of DMTS-P1 most resembles that of 1,2dihydroxy-5-(methylthio)-1-penten-3-one, which is produced by Utr4p (Fig. 1). Among the MTA cycle genes, however, UTR4 was not the most influential in the formation of DMTS-P1 (Fig. 2). The presence of isozymes of Utr4p is unlikely because Pirkov et al. reported that only UTR4 had a role in conversion of MTA to methionine among two candidate genes for 2,3-dioxomethylthiopentane-1-phosphate enolase/phosphatase found by homology BLAST search (12). Another possibility is that the MTA cycle partly proceeds reversely. The slight decrease in the DMTS-P1 content observed in Daro9 sake (Fig. 2) is not inconsistent with this hypothesis. The sake brewing tests using MTA cycle gene disruptants suggest the possibility that SPE2 is responsible for the formation of other precursors of DMTS; the DMTS producing potential of Dspe2 sake was as low as that of Dmde1 or Dmri1 despite its higher DMTSP1 content as compared with them (Fig. 2). We reasoned that deletion of SPE2, in addition to MRI1 or MDE1, could lead to a further decrease in the DMTS producing potential through the decrease in the other DMTS precursors in addition to DMTS-P1. Indeed, the potential of sake made with the Dspe2 Dmri1 double disruptant was decreased as compared with the Dspe2 or Dmri1 single disruptant, although the difference was not significant by TukeyeKramer HSD test at the 5% level (Fig. 5). From these results, it is considered that SPE2, as well as MRI1 and MDE1, is a promising target for breeding yeast to suppress the formation of hineka. We have not identified the other DMTS precursors; however, previous studies about MTA cycle gene disruption lead us to speculate about it. Chattopadhyay et al. reported that Dmeu1 strain accumulated MTA both in culture medium and cells, whereas Dmeu1 Dspe2 double mutant did not (22). As mentioned above, our experiments using MTA cycle gene disruptants suggested that the precursors increase by deletion of MEU1 and decrease by deletion of SPE2. Taken together, MTA is considered one of the candidates of another DMTS precursor. The accumulation of MTA was also reported for Dmri1 and Dmde1 strains, although the levels decreased as the steps proceed along the pathway, dependent of the equilibrium constants of the reactions (12). The residual DMTS producing potential of sake made with Dmri1 or Dmde1 (Figs. 2 and 3) may be partly attributed to the accumulated MTA. However, further study is needed to confirm the contribution of MTA to DMTS formation. In conclusion, we found that the DMTS-P1 content and DMTS producing potential of sake can be reduced by the deletion of yeast MTA cycle genes, especially MRI1, MDE1, or SPE2. However, the yeast strains used in this study were genetically modified, and the application of such strains to industrial sake brewing is difficult because of consumers’ concerns about genetically modified foods. Breeding yeast deficient in these genes by an industrially applicable method is currently under study.
479
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.04.016. ACKNOWLEDGMENTS We wish to thank the panelists for participating in the sensory analysis, and Ms. Ryoko Kanda and Ms. Michiko Endo for technical assistance. References 1. Takahashi, K.: The aroma of aged sake, J. Soc. Brew. Jpn., 75, 463e468 (1980) (in Japanese). 2. Isogai, A., Utsunomiya, H., Kanda, R., Iwata, H., and Nakano, S.: Aroma compounds responsible for “hineka” in commercial sake, J. Soc. Brew. Jpn., 101, 125e131 (2006) (in Japanese). 3. Okamoto, M., Yamauchi, T., Yano, S., Kurose, N., Kawakita, S., Takahashi, K., and Nakamura, T.: Preservation of sake quality by decreasing the dissolved oxygen concentration, J. Soc. Brew. Jpn., 94, 827e832 (1999) (in Japanese). 4. Isogai, A., Kanda, R., Hiraga, Y., Nishimura, T., Iwata, H., and GotoYamamoto, N.: Screening and identification of precursor compounds of dimethyl trisulfide (DMTS) in Japanese sake, J. Agric. Food Chem., 57, 189e195 (2009). 5. Isogai, A., Kanda, R., Hiraga, Y., Iwata, H., and Sudo, S.: Contribution of 1,2dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) to the formation of dimethyl trisulfide (DMTS) during the storage of Japanese sake, J. Agric. Food Chem., 58, 7756e7761 (2010). 6. Albers, E.: Metabolic characteristics and importance of the universal methionine salvage pathway recycling methionine from 50 -methylthioadenosine, IUBMB Life, 61, 1132e1142 (2009). 7. Walters, D. and Cowley, T.: Polyamine metabolism in Saccharomyces cerevisiae exposed to ethanol, Microbiol. Res., 153, 179e184 (1998). 8. Thomas, D. and Surdin-Kerjan, Y.: Metabolism of sulfur amino acids in Saccharomyces cerevisiae, Microbiol. Mol. Biol. Rev., 61, 503e532 (1997). 9. Wray, J. W. and Abeles, R. H.: The methionine salvage pathway in Klebsiella pneumoniae and rat liver: identification and characterization of two novel dioxygenases, J. Biol. Chem., 270, 3147e3153 (1995). 10. Sekowska, A., Denervaud, V., Ashida, H., Michoud, K., Haas, D., Yokota, A., and Danchin, A.: Bacterial variations on the methionine salvage pathway, BMC Microbiol., 4, 9 (2004). 11. Marchitto, K. S. and Ferro, A. J.: The metabolism of 50 -methylthioadenosine and 5-methylthioribose-1-phosphate in Saccharomyces cerevisiae, J. Gen. Microbiol., 131, 2153e2164 (1985). 12. Pirkov, I., Norbeck, J., Gustafsson, L., and Albers, E.: A complete inventory of all enzymes in the eukaryotic methionine salvage pathway, FEBS J., 275, 4111e4120 (2008). 13. Kushad, M. M., Richardson, D. G., and Ferro, A. J.: Intermediates in the recycling of 5-methylthioribose to methionine in fruits, Plant Physiol., 73, 257e261 (1983). 14. Goldstein, A. L. and McCusker, J. H.: Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae, Yeast, 15, 1541e1553 (1999). 15. Amberg, D. C., Botstein, D., and Beasley, E. M.: Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction, Yeast, 11, 1275e1280 (1995). 16. Kitagaki, H. and Shimoi, H.: Mitochondrial dynamics of yeast during sake brewing, J. Biosci. Bioeng., 104, 227e230 (2007). 17. Nanba, K., Obata, T., Kayashima, S., Yamazaki, Y., Murakami, M., and Shimoda, T.: The setting of the small sake brewing, J. Soc. Brew. Jpn., 73, 295e300 (1978) (in Japanese). 18. Isogai, A., Kanda, R., Sudo, S., and Matsumaru, K.: Quantitation of DMTS precursor, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) in alcoholic beverages and fermented solutions by a stable isotope dilution analysis, J. Soc. Brew. Jpn., 108, 605e614 (2013) (in Japanese). 19. Okazaki, N.: Seishu, and Seishu, Gousei, pp. 13e24, in: Seishu, and Seishu, Gousei Official methods of analysis of National Tax Administration Agency, 4th ed. The Brewing Society of Japan, Tokyo (1993) (in Japanese). 20. Green, B. G., Shaffer, G.. S., and Gilmore, M. M.: Derivation and evaluation of a semantic scale of oral sensation magnitude with apparent ratio properties, Chem. Senses, 18, 683e702 (1993). 21. Green, B. G., Dalton, P., Cowart, B., Shaffer, G., Rankin, K., and Higgins, J.: Evaluating the labeled magnitude scale for measuring sensations of taste and smell, Chem. Senses, 21, 323e334 (1996). 22. Chattopadhyay, M. K., Tabor, C. W., and Tabor, H.: Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1D mutant, Biochem. Biophys. Res. Commun., 103, 203e207 (2006).
Involvement of methionine salvage pathway genes of Saccharomyces cerevisiae in the production of precursor compounds of dimethyl trisulfide (DMTS) Kou Wakabayashi,1, 2 Atsuko Isogai,1, 2, * Daisuke Watanabe,1 Akiko Fujita,1 and Shigetoshi Sudo1, 2 National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashihiroshima 739-0046, Japan1 and Graduate School of Advanced Science of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima 739-8530, Japan2 Received 15 January 2013; accepted 12 April 2013 Available online 15 June 2013
Dimethyl trisulfide (DMTS) is one of the components responsible for the unpalatable aroma of stale Japanese sake, called “hineka”. Recently, a precursor compound of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), was identified. It was speculated that the yeast methionine salvage pathway (MTA cycle) might participate in the formation of DMTS-P1, because the chemical structure of DMTS-P1 was similar to one of the intermediate compounds of that pathway. Here, we carried out sake brewing tests using laboratory yeast strains with disrupted MTA cycle genes and found that DMTS-P1 was hardly produced by Dmeu1, Dmri1, and Dmde1 strains. Furthermore, the DMTS producing potential (production of DMTS during storage of sake) decreased in sake made with Dmri1 and Dmde1. We constructed sake yeast strains with a disrupted MRI1 or MDE1 gene and confirmed a decline in the DMTS-P1 content and DMTS producing potential of sake made with these disruptants. The results of sake brewing tests using MTA cycle disruptants suggested that SPE2 is responsible for the production of DMTS precursors other than DMTS-P1: although the DMTS-P1 content was higher in Dspe2 sake than in Dmri1 or Dmde1 sake, the DMTS producing potential of Dspe2 sake was as low as that of Dmri1 or Dmde1 sake. Sake brewing tests using BY4743 Dspe2 Dmri1 double disruptants revealed that the DMTS producing potential was further decreased as compared with the Dspe2 or Dmri1 single disruptant. These results suggest that MRI1, MDE1, and SPE2 are promising targets for breeding yeast to suppress the formation of DMTS during storage of sake. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Dimethyl trisulfide; 1,2-Dihydroxy-5-(methylsulfinyl)pentan-3-one; Methionine salvage pathway; Hineka; Sake; Saccharomyces cerevisiae]
Storing sake causes changes in the color, taste, and aroma. The aroma of stored sake is called “hineka” in Japanese. Hineka consists of several kinds of compounds (1), of which dimethyl trisulfide (DMTS) is one of the main components (2). The odor of DMTS is described as sulfury, cooked onion, or Japanese pickle-like odor. An excess amount of DMTS deteriorates the aroma of sake. Hineka can be reduced in the refining step by adsorbing it onto activated carbon. However, it reforms by the chemical reactions of precursor compounds during storage or distribution in the market. The formation of hineka can be reduced by suppressing these chemical reactions; for example, by storing it in a refrigerator, or reducing the amount of dissolved oxygen in sake (3). However, such methods need expensive equipment. Therefore, another technical approach is required that will facilitate control of the formation of hineka. We recently identified one of the precursor compounds of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), a previously unknown compound (4). Although the origin of DMTS* Corresponding author at: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashihiroshima, 739-0046, Japan. Tel.: þ81 82 420 8077; fax: þ81 82 420 8079. E-mail address: [email protected] (A. Isogai).
P1 is not known, the increment in its concentration during fermentation suggested the participation of yeast in its formation (5). Furthermore, if the genes responsible for the production of DMTS-P1 were clarified, control of the formation of DMTS in sake could be achieved by breeding yeast deficient in the relevant genes. The chemical structure of DMTS-P1 led us to consider the involvement of the methionine salvage pathway, because one of the intermediate compounds in this pathway has a structure similar to DMTS-P1 (4). The methionine salvage pathway, also called the 50 methylthioadenosine (MTA) cycle, recycles sulfur from MTA, which is a by-product of the biosynthesis of polyamines such as spermidine and spermine (6). For the synthesis of these polyamines (Fig. 1), S-adenosylmethionine is converted into S-adenosylmethioninamine by S-adenosylmethionine decarboxylase (encoded by SPE2). Next, an aminopropyl group of S-adenosylmethioninamine is transferred to putrescine by spermidine synthase (encoded by SPE3) and spermidine by spermine synthase (encoded by SPE4) to yield spermidine and spermine, respectively (7), and at the same time MTA is formed as a by-product. Because the assimilation of sulfur is strongly energy-consuming in the form of redox equivalents (8), most organisms have evolved recycling pathways to reuse sulfur from MTA in the regeneration of methionine, i.e., the MTA cycle.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.04.016
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WAKABAYASHI ET AL.
J. BIOSCI. BIOENG.,
1,2-dihydroxy-5-(methylthio)1-penten-3-one
DMTS-P1 O S O
S
O OH
S
OH
OH
O
ARO8, ARO9, BAT1, BAT2
COOH
ADI1
NH 2
OH
S
O
SAM1, SAM2
NH 2 OPO32-
S O
MDE1 OH
O
S-adenosylmethionine HO (SAM) SPE2 H 2N
MRI1 O
SPE3
OPO 32-
MEU1
S HO
OH
O
S+
HOOC
OPO32-
S OH
COOH
methionine
UTR4
S HO
O
Ade OH
S+ HO
O
Ade OH
Ade OH
putrescine
spermidine SPE4 spermine
5’-methylthioadenosine (MTA) FIG. 1. The structure of DMTS-P1 and the MTA cycle in S. cerevisiae.
The MTA cycle is found in a wide variety of organisms including bacteria (9,10), yeast (11,12), plants (13), and mammals (9), with some differences among organisms. In the case of Saccharomyces cerevisiae, MTA phosphorylase (encoded by MEU1) converts MTA into 5-methylthioribose-1-phosphate, which subsequently undergoes successive enzymatic reactions by 5-methylthioribose1-phosphate isomerase (encoded by MRI1), 5-methylthioribulose1-phosphate dehydratase (encoded by MDE1), and 2,3-dioxomethylthiopentane-1-phosphate enolase/phosphatase (encoded by UTR4) to yield 1,2-dihydroxy-5-(methylthio)-1-penten-3-one, the structure of which resembles DMTS-P1. This compound is then converted into 4-methylthio-2-oxobutyrate by aci-reductone dioxygenase (encoded by ADI1), and finally, transamination of the keto acid catalyzed by four kinds of transaminase (encoded by ARO8, ARO9, BAT1, and BAT2) yields methionine. All of the genes encoding this pathway in S. cerevisiae have recently been identified (12). As stated above, the chemical structure of 1,2-dihydroxy-5(methylthio)-1-penten-3-one is similar to DMTS-P1 except that the sulfide group of the former is oxidized to sulfoxide in the latter, and the enediol structure of the former is replaced by diol in the latter (Fig. 1). We therefore speculated that DMTS-P1 might be derived from MTA cycle. In this study we investigated the potential involvement of MTA cycle genes in the formation of DMTS-P1 by using MTA cycle gene disruptants. We also studied the formation of DMTS during the storage of sake brewed with these disruptants and discussed the possibility for the target of breeding yeast to suppress hineka. MATERIALS AND METHODS Yeast strains The S. cerevisiae strains used in this study are listed in Table S1. The laboratory strain BY4743, BY4743 strains Daro8, Daro9, Dbat2, Dsam1, Dsam2, Dspe2, Dspe3, Dspe4, Dmeu1, Dmri1, Dmde1, Dutr4 and Dadi1, BY4741 strain Dspe2, and BY4742 strain Dspe2 were provided by EUROSCARF. The sake yeast strain Kyokai no. 7 (K7) was provided by the Brewing Society of Japan. BY4743, BY4743derived disruptants, and sake yeast are diploid strains. Disruption of the MRI1 or MDE1 gene in K7 was performed using a PCR-based method. Because K7 is a diploid strain, two copies of each gene were replaced by two kinds of marker cassette containing a kanMX4 or natMX4 gene (14). The marker cassettes were synthesized by fusion PCR (15) using the primers listed in Table S2. In the first step, the regions flanking the target genes were amplified with primers C
and D (for the upstream flanking region) or E and F (for the downstream flanking region), and S. cerevisiae chromosomal DNA as a template. The marker genes were amplified using the plasmid pFA6-kanMX4 (for kanMX4 gene) or pAG25 (for natMX4 gene) as a template, and primers A and B. These primers contained sequences complementary to the primer D or E, in addition to the template marker plasmid sequences. Next, the marker DNA fragments, and upstream and downstream flanking regions were fused by fusion PCR to synthesize the marker cassettes, mri1kanMX4 and mri1-natMX4, or mde1-kanMX4 and mde1-natMX4. These DNA fragments were transformed into K7 by the lithium acetate method to generate K7 Dmri1::kanMX4/Dmri1::natMX4 or K7 Dmde1::kanMX4/Dmde1::natMX4 (referred to as K7 Dmri1 or K7 Dmde1, respectively). The mutations were confirmed by PCR using the primers G and H. The SPE2 and MRI1 double deletion mutant was generated by deletion of MRI1 in the background of BY4741 Dspe2 and BY4742 Dspe2 using a PCR-based method with the primers MRI1-D-f and MRI1-D-r (Table S2) and the plasmid pAG25 as a template, to generate BY4741 Dspe2::kanMX Dmri1::natMX4 and BY4742 Dspe2::kanMX Dmri1::natMX4. Both strains were mated to generate BY4743 Dspe2::kanMX/ Dspe2::kanMX Dmri1::natMX4/Dmri1::natMX4 (referred to as BY4743 Dspe2 Dmri1). One-step sake brewing test of BY4743 deletion strains A small-scale sake brewing with one-step mashing was carried out as described by Kitagaki et al. (16) with some modifications. Although one-step mashing is not a usual method for the industrial sake brewing, we consider it is enough for screening purpose. Yeast cells were precultured in nutrient broth at 30 C for 3 days without shaking and then harvested by centrifugation. About 2.0 109 cells were mixed with 200 ml of ion exchange water, 540 ml of 7.5% lactic acid, 23 g of dried koji (Tokushima seikiku, Awa), and 60 g of dried rice (Tokushima seikiku). After gentle mixing, fermentation was carried out at 15 C. The weight of the mash was measured every day to monitor carbon dioxide evolution as a measure of fermentation progress. After 19 days, the mash was centrifuged and the supernatant was filtered with a membrane filter of 0.45 mm pore size. Three-step sake brewing test of K7 Dmri1 and K7 Dmde1 A small-scale sake brewing with three mashing steps was carried out as described by Nanba et al. 8 (17) using 300 g of total rice. Precultured yeast (8.6 10 cells) was added to a mixture of 84 ml of ion exchange water, 2 ml of 7.5% lactic acid, and 15 g of dried koji, and incubated at 15 C overnight. The next day, 37.5 g of polished rice (Nakateshinsenbon, milling ratio 70%) was steamed and added to it (Soe). Two days later, 115 ml of ion exchange water, 15 g of dried koji, and 82.5 g (the weight as polished rice) of steamed rice were added, and the mash was fermented at 9 C (Naka). The following day, 201 ml of ion exchange water, 30 g of dried koji, and 120 g of steamed rice were added, and the mash was fermented at 7 C (Tome). The fermentation temperature was then increased by 1.0 C/day to a maximum temperature of 15 C, which was maintained for 8 days. Next, the temperature was decreased by 2.0 C/day to 7 C. Twenty-one days after Tome, the mash was centrifuged and filtered with a membrane filter as described above. Measurement of DMTS-P1 The concentration of DMTS-P1 was measured as described previously (5), except that [methyl-d3]-DMTS-P1 was used as an internal standard instead of 1,5-pentanediol (18).
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INVOLVEMENT OF MTA CYCLE IN DMTS PRECURSOR PRODUCTION
Measurement of DMTS producing potential The amount of DMTS produced during the storage of sake for 7 days at 70 C was defined as the DMTS producing potential, which was measured by a previously described method (5). Other chemical analyses Ethanol concentration, sake meter, acidity, and amino acidity were measured according to the reference (19). When the amount of sake was not enough, the ethanol concentration was measured using an ethanol analyzer (Riken Keiki, Shizuoka). Sensory analysis The effect of deletion of MRI1 or MDE1 on the odor of sake after storage was investigated by sensory analysis. Five sake samples brewed with K7, K7 Dmri1 (K7-mri1_1, K7-mri1_2), and K7 Dmde1 (K7-mde1_1, K7-mde1_2) were treated with activated carbon (1 g/L) and filtered. They were then pasteurized at 63 C for 5 min. After cooling, they were stored at 40 C for two months. Sensory evaluation was performed by eight panelists who work at our institute. The five stored sake samples were coded using three-digit random numbers. To avoid bias caused by the color of the samples, they were poured into amber-colored glasses. To avoid an order effect, each panelist evaluated the samples in a different order. Panelists were instructed to smell the samples and to evaluate the intensity of hineka and sulfury odor using the labeled magnitude scales (barely detectable, 1.4; weak, 6.1; moderate, 17.2; strong, 35.4; very strong, 53.3; strongest imaginable, 100) (20,21). Because drinking sake samples made with genetically modified organisms is prohibited in our institute, panelists evaluated the samples only by orthonasal olfaction. Data were expressed as means of the logarithms of the labeled magnitude values and subjected to the two-way (panelists samples) analysis of variance (ANOVA) and TukeyeKramer HSD test to study the statistical differences.
RESULTS
1.2
0.5
1.0
0.4
0.8
0.3
0.6
0.2
0.4
0.1
0.2
0.0
0.0
DMTS producing potential (µg/L)
0.6
WT Δaro8 Δ aro9 Δbat2 Δsam1 Δ sam2 Δ spe2 Δ spe3 Δspe4 Δ meu1 Δ mri1 Δmde1 Δutr4 Δ adi1
DMTS-P1 (mg/L)
Involvement of MTA cycle genes in the formation of DMTSP1 and the DMTS producing potential of sake Using 13 yeast disruptants in which one of the MTA cycle genes was disrupted in the background of BY4743, we carried out small-scale sake brewing tests with one-step mashing. After fermentation was finished, the concentration of DMTS-P1 and the DMTS producing potential were determined. As shown in Fig. 2, a lower DMTS-P1 content was observed for sake brewed with the Daro9, Dspe2, Dmeu1, Dmri1, Dmde1, and Dutr4 strains. In particular, DMTS-P1 was hardly detected in Dmeu1, Dmri1, and Dmde1 sake. The results suggest that the MTA cycle in yeast is responsible for the formation of DMTS-P1, and that the MEU1, MRI1, and MDE1 genes are essential for it. The DMTS producing potential of sake made with Dmri1 or Dmde1 was also greatly decreased as compared with the parent strain. Although the DMTS-P1 content in Dmeu1 sake was as low as that in Dmri1 or Dmde1 sake, the DMTS producing potential of
FIG. 2. The influence of deletion of MTA cycle genes on the formation of DMTS-P1 (black) and the DMTS producing potential (gray) of brewed sake. Using the BY4743 strain and 13 yeast disruptants in which one of the MTA cycle genes was disrupted in the background of BY4743, small-scale sake brewing tests with one-step mashing were carried out. The concentration of DMTS-P1 and the DMTS producing potential of brewed sake were measured. Data represent the mean values and standard deviations of two independent experiments.
477
Dmeu1 sake was higher than that of Dmri1 or Dmde1 sake. By contrast, the DMTS producing potential of Dspe2 sake was as low as that of Dmri1 or Dmde1 sake, although the DMTS-P1 content was higher in Dspe2 sake than in the other two strains. These discrepancies suggest the presence of another DMTS precursor, which is increased by deletion of MEU1 and decreased by deletion of SPE2. The general properties of sake (ethanol content, sake meter, acidity, and amino acidity) made with MTA cycle gene disruptants were almost the same as those of the parent strain (Table 1). Disruption of the MDE1 or MRI1 gene in sake yeast Next, we deleted the MRI1 and MDE1 genes in sake yeast strain K7 to confirm that they are also involved in the formation of DMTS-P1 and the DMTS producing potential in this sake strain as well as in the laboratory strain. Because K7 is diploid, we disrupted the two copies of MRI1 or MDE1 on homologous chromosomes by inserting two different kinds of antibiotic-resistant markers. As a result, seven strains of K7 Dmri1 and six strains of K7 Dmde1 were obtained. Among them, two strains of each gene disruptant (K7mri1_1, K7-mri1_2, K7-mde1_1, and K7-mde1_2) were used for a small-scale sake brewing test. The sake brewing test was carried out with three mashing steps, in the same way as pilot-scale brewing, to confirm the effect of gene disruption in sake yeast under the conditions similar to actual sake brewing. As shown in Fig. 3, the content of DMTS-P1 in sake brewed with K7 Dmri1 or Dmde1 was greatly reduced as compared with the parent strain. The results suggest that the MRI1 and MDE1 genes are involved in the formation of DMTS-P1 in sake yeast K7 as well as in the laboratory yeast BY4743. In addition, deletion of MRI1 or MDE1 caused more than a ten-fold decrease in the DMTS producing potential (Fig. 3), suggesting that these genes are promising as targets for breeding sake yeast in which the formation of DMTS is repressed. The ethanol fermentation process (data not shown) and the general properties of sake (Table 2) brewed with the deletion strains were almost the same as those of the parent strain. Sensory analyses were carried out to investigate the effect of deletion of MRI1 or MDE1 on the odor of stored sake. After storage at 40 C for two months, the sake samples were evaluated for sulfury odor and hineka by olfaction. As shown in Fig. 4, the intensity of sulfury odor in sake made with K7 Dmri1 or Dmde1 seemed to decrease as compared with the parent strain. Although the differences between samples were not statistically significant by oneway ANOVA because of variance among the panelists, two-way ANOVA revealed significant differences between both samples (p ¼ 0.045) and panelists (p < 0.001). TukeyeKramer HSD test indicated a significant difference between K7 and K7-mri1_1. The intensity of hineka of sake made with K7 Dmri1 or Dmde1 also tended to decrease as compared with K7. Statistical differences were observed between samples (p ¼ 0.036) and panelists
TABLE 1. Properties of sake brewed with MTA cycle gene disruptants of BY4743. Strain
Ethanol (%)
Sake meter
Acidity
Amino acidity
WT Daro8 Daro9 Dbat2 Dsam1 Dsam2 Dspe2 Dspe3 Dspe4 Dmeu1 Dmri1 Dmde1 Dutr4 Dadi1
12.5 12.3 12.1 12.1 12.6 12.7 11.8 13.5 12.0 12.9 13.1 12.6 12.9 12.7
16 16 20 18 15 14 23 5 22 13 11 14 14 15
3.6 3.8 3.7 3.8 3.7 3.5 3.6 3.7 3.5 3.6 3.7 3.8 3.6 3.7
1.5 1.4 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Data represent the mean values of two independent experiments.
J. BIOSCI. BIOENG.,
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
FIG. 3. The influence of deletion of MRI1 and MDE1 in the background of sake yeast K7 on the formation of DMTS-P1 (black) and the DMTS producing potential (gray) of brewed sake. Using sake yeast strain K7, K7 Dmri1, and K7 Dmde1, small-scale sake brewing test was carried out with three mashing steps. The concentration of DMTS-P1 and the DMTS producing potential of brewed sake were measured. Data represent the mean values and standard deviations from three independent experiments.
(p < 0.001) by two-way ANOVA. The difference between K7 and K7mri1_1 was significant by TukeyeKramer HSD test. Effect of a Dspe2 Dmri1 double deletion on the DMTS producing potential As mentioned above, the possibility was suggested that SPE2 is involved in the formation of DMTS precursors other than DMTS-P1 (Fig. 2). Thus, we expected that the DMTS producing potential of sake made with a Dspe2 Dmri1 double disruptant would be even lower than that of the single disruptant. We constructed a BY4743 Dspe2 Dmri1 double disruptant and carried out small-scale sake brewing tests with one-step mashing. As a result, the DMTS producing potential of sake made with Dspe2 Dmri1 decreased by about 50% as compared with Dspe2, and 40% as compared with Dmri1 although the difference between the single and double disruptants was not statistically significant (Fig. 5). The content of DMTS-P1 in sake made with Dspe2 Dmri1 was less than that in sake made with Dspe2, and as little as that in sake made with Dmri1, which suggested that the decrease in DMTS-P1 content in Dspe2 Dmri1 sake was caused by the deletion of MRI1. The ethanol content, sake meter, acidity, and amino acidity of sake brewed with Dspe2 Dmri1 were almost the same as those of the parent strain (BY4743), Dspe2 and Dmri1 (data not shown). DISCUSSION DMTS is one of the main components of hineka, the “off-flavor” of stored Japanese sake. During storage of sake, DMTS is produced through the chemical conversion of precursor compounds. We previously identified one of the precursors, DMTS-P1, and have demonstrated here that the MTA cycle of yeast is involved in the formation of this precursor.
Strongest imaginable
2.0 1.6
Very strong Strong
aa ab bb
1.2
ab
ab ab ab ab
0.8
Moderate
Weak
0.4
FIG. 4. Sensory analysis of stored sake made with K7 and K7 deletion strains Dmri1 and Dmde1. Sake samples made with K7, K7 Dmri1, and K7 Dmde1 were stored at 40 C for two months, and sensory analyses were carried out to investigate the intensity of hineka (black) and sulfury odor (gray). Data represent the means and standard errors of mean from the evaluation of eight panelists. Values with different letters are significantly different at p < 0.05 by TukeyeKramer HSD test.
Sake brewing tests using MTA cycle gene disruptants of BY4743 revealed that the MEU1, MDE1, and MRI1 genes are indispensable for the formation of DMTS-P1, and the DMTS producing potential of sake made with the disruptant Dmde1 or Dmri1 strain was greatly reduced (Fig. 2). Deletion of the MDE1 or MRI1 gene in sake yeast K7 also caused a marked decline in the concentration of DMTS-P1 and in the DMTS producing potential of sake (Fig. 3). Even after storage for 1 week at 70 C, the DMTS concentration in sake made with these deletion strains was far below its detection threshold (0.18 mg/l). The effect of the deletion of MDE1 or MRI1 was also observed in the sensory analyses. The intensity of the sulfury odor and hineka tended to decrease as compared with the parent strain (Fig. 4). These results indicate that the formation of hineka can be suppressed by breeding sake yeast that is deficient in the MDE1 or MRI1 gene.
0.25
0.5
a
0.4
0.20 a
0.3
0.15 0.10 0.05 0.00
0.2
b b b c
c
b
c
b
0.1 0.0
DMTS producing potential (µg/L)
0.4
Log perceived intensity of odor
0.5
DMTS-P1 (mg/L)
0.5
DMTS producing potential (µg/L)
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DMTS-P1 (mg/L)
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TABLE 2. Properties of sake brewed with K7 and K7 deletion strains Dmri1 and Dmde1. Strain
Ethanol (%)
K7 K7-mri1_1 K7-mri1_2 K7-mde1_1 K7-mde1_2
17.3 17.3 17.1 17.0 17.1
0.4 0.5 1.0 0.3 0.3
Sake meter 10 10 12 12 13
0.0 2.5 6.4 1.8 0.8
Acidity 2.1 2.3 2.4 2.1 2.2
0.0 0.2 0.1 0.1 0.1
Amino acidity 1.7 1.7 1.7 1.8 1.7
0.1 0.1 0.0 0.1 0.0
Data represent the mean values and standard deviations from three independent experiments.
FIG. 5. The effect of double deletion of SPE2 and MRI1 on the concentration of DMTS-P1 (black) and the DMTS producing potential (gray) of brewed sake. Small-scale sake brewing tests with one-step mashing were carried out using the BY4743, BY4743 strains Dspe2, Dmri1, and Dspe2 Dmri1 double disruptant. The concentration of DMTSP1 and the DMTS producing potential of brewed sake were measured. Data represent the mean values and standard deviations from three independent experiments. Values with different letters are significantly different at p < 0.05 by TukeyeKramer HSD test.
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INVOLVEMENT OF MTA CYCLE IN DMTS PRECURSOR PRODUCTION
The decline in the DMTS producing potential by the deletion of MRI1 or MDE1 in K7 seemed more significant than that of BY4743 (70e75% decrease for BY4743, more than 90% decrease for K7, Figs. 2 and 3). Since DMTS-P1 was hardly produced by these disruptants, the residual DMTS producing potential may be attributed to DMTS precursors other than DMTS-P1. It is possible that the formation of these precursors by K7 disruptants was lesser than BY4743 disruptants. At present, however, we cannot decide either genetic background or brewing conditions caused the differences. Recently, Okimori et al. reported that sake brewing conditions, such as death of yeast greatly affect the DMTS producing potential (Okimori, Y., Nishibori, N., Fujii, T., Sasaki, K., Kanai, M., Isogai, A., Kanda, R., Goto, N., and Yamada, O., Abstr. Annu. Meet. J. Soc. Biosci. Biotechnol. Agrochem., p. 688, 2012). The chemical structure of DMTS-P1 most resembles that of 1,2dihydroxy-5-(methylthio)-1-penten-3-one, which is produced by Utr4p (Fig. 1). Among the MTA cycle genes, however, UTR4 was not the most influential in the formation of DMTS-P1 (Fig. 2). The presence of isozymes of Utr4p is unlikely because Pirkov et al. reported that only UTR4 had a role in conversion of MTA to methionine among two candidate genes for 2,3-dioxomethylthiopentane-1-phosphate enolase/phosphatase found by homology BLAST search (12). Another possibility is that the MTA cycle partly proceeds reversely. The slight decrease in the DMTS-P1 content observed in Daro9 sake (Fig. 2) is not inconsistent with this hypothesis. The sake brewing tests using MTA cycle gene disruptants suggest the possibility that SPE2 is responsible for the formation of other precursors of DMTS; the DMTS producing potential of Dspe2 sake was as low as that of Dmde1 or Dmri1 despite its higher DMTSP1 content as compared with them (Fig. 2). We reasoned that deletion of SPE2, in addition to MRI1 or MDE1, could lead to a further decrease in the DMTS producing potential through the decrease in the other DMTS precursors in addition to DMTS-P1. Indeed, the potential of sake made with the Dspe2 Dmri1 double disruptant was decreased as compared with the Dspe2 or Dmri1 single disruptant, although the difference was not significant by TukeyeKramer HSD test at the 5% level (Fig. 5). From these results, it is considered that SPE2, as well as MRI1 and MDE1, is a promising target for breeding yeast to suppress the formation of hineka. We have not identified the other DMTS precursors; however, previous studies about MTA cycle gene disruption lead us to speculate about it. Chattopadhyay et al. reported that Dmeu1 strain accumulated MTA both in culture medium and cells, whereas Dmeu1 Dspe2 double mutant did not (22). As mentioned above, our experiments using MTA cycle gene disruptants suggested that the precursors increase by deletion of MEU1 and decrease by deletion of SPE2. Taken together, MTA is considered one of the candidates of another DMTS precursor. The accumulation of MTA was also reported for Dmri1 and Dmde1 strains, although the levels decreased as the steps proceed along the pathway, dependent of the equilibrium constants of the reactions (12). The residual DMTS producing potential of sake made with Dmri1 or Dmde1 (Figs. 2 and 3) may be partly attributed to the accumulated MTA. However, further study is needed to confirm the contribution of MTA to DMTS formation. In conclusion, we found that the DMTS-P1 content and DMTS producing potential of sake can be reduced by the deletion of yeast MTA cycle genes, especially MRI1, MDE1, or SPE2. However, the yeast strains used in this study were genetically modified, and the application of such strains to industrial sake brewing is difficult because of consumers’ concerns about genetically modified foods. Breeding yeast deficient in these genes by an industrially applicable method is currently under study.
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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.04.016. ACKNOWLEDGMENTS We wish to thank the panelists for participating in the sensory analysis, and Ms. Ryoko Kanda and Ms. Michiko Endo for technical assistance. References 1. Takahashi, K.: The aroma of aged sake, J. Soc. Brew. Jpn., 75, 463e468 (1980) (in Japanese). 2. Isogai, A., Utsunomiya, H., Kanda, R., Iwata, H., and Nakano, S.: Aroma compounds responsible for “hineka” in commercial sake, J. Soc. Brew. Jpn., 101, 125e131 (2006) (in Japanese). 3. Okamoto, M., Yamauchi, T., Yano, S., Kurose, N., Kawakita, S., Takahashi, K., and Nakamura, T.: Preservation of sake quality by decreasing the dissolved oxygen concentration, J. Soc. Brew. Jpn., 94, 827e832 (1999) (in Japanese). 4. Isogai, A., Kanda, R., Hiraga, Y., Nishimura, T., Iwata, H., and GotoYamamoto, N.: Screening and identification of precursor compounds of dimethyl trisulfide (DMTS) in Japanese sake, J. Agric. Food Chem., 57, 189e195 (2009). 5. Isogai, A., Kanda, R., Hiraga, Y., Iwata, H., and Sudo, S.: Contribution of 1,2dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) to the formation of dimethyl trisulfide (DMTS) during the storage of Japanese sake, J. Agric. Food Chem., 58, 7756e7761 (2010). 6. Albers, E.: Metabolic characteristics and importance of the universal methionine salvage pathway recycling methionine from 50 -methylthioadenosine, IUBMB Life, 61, 1132e1142 (2009). 7. Walters, D. and Cowley, T.: Polyamine metabolism in Saccharomyces cerevisiae exposed to ethanol, Microbiol. Res., 153, 179e184 (1998). 8. Thomas, D. and Surdin-Kerjan, Y.: Metabolism of sulfur amino acids in Saccharomyces cerevisiae, Microbiol. Mol. Biol. Rev., 61, 503e532 (1997). 9. Wray, J. W. and Abeles, R. H.: The methionine salvage pathway in Klebsiella pneumoniae and rat liver: identification and characterization of two novel dioxygenases, J. Biol. Chem., 270, 3147e3153 (1995). 10. Sekowska, A., Denervaud, V., Ashida, H., Michoud, K., Haas, D., Yokota, A., and Danchin, A.: Bacterial variations on the methionine salvage pathway, BMC Microbiol., 4, 9 (2004). 11. Marchitto, K. S. and Ferro, A. J.: The metabolism of 50 -methylthioadenosine and 5-methylthioribose-1-phosphate in Saccharomyces cerevisiae, J. Gen. Microbiol., 131, 2153e2164 (1985). 12. Pirkov, I., Norbeck, J., Gustafsson, L., and Albers, E.: A complete inventory of all enzymes in the eukaryotic methionine salvage pathway, FEBS J., 275, 4111e4120 (2008). 13. Kushad, M. M., Richardson, D. G., and Ferro, A. J.: Intermediates in the recycling of 5-methylthioribose to methionine in fruits, Plant Physiol., 73, 257e261 (1983). 14. Goldstein, A. L. and McCusker, J. H.: Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae, Yeast, 15, 1541e1553 (1999). 15. Amberg, D. C., Botstein, D., and Beasley, E. M.: Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction, Yeast, 11, 1275e1280 (1995). 16. Kitagaki, H. and Shimoi, H.: Mitochondrial dynamics of yeast during sake brewing, J. Biosci. Bioeng., 104, 227e230 (2007). 17. Nanba, K., Obata, T., Kayashima, S., Yamazaki, Y., Murakami, M., and Shimoda, T.: The setting of the small sake brewing, J. Soc. Brew. Jpn., 73, 295e300 (1978) (in Japanese). 18. Isogai, A., Kanda, R., Sudo, S., and Matsumaru, K.: Quantitation of DMTS precursor, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) in alcoholic beverages and fermented solutions by a stable isotope dilution analysis, J. Soc. Brew. Jpn., 108, 605e614 (2013) (in Japanese). 19. Okazaki, N.: Seishu, and Seishu, Gousei, pp. 13e24, in: Seishu, and Seishu, Gousei Official methods of analysis of National Tax Administration Agency, 4th ed. The Brewing Society of Japan, Tokyo (1993) (in Japanese). 20. Green, B. G., Shaffer, G.. S., and Gilmore, M. M.: Derivation and evaluation of a semantic scale of oral sensation magnitude with apparent ratio properties, Chem. Senses, 18, 683e702 (1993). 21. Green, B. G., Dalton, P., Cowart, B., Shaffer, G., Rankin, K., and Higgins, J.: Evaluating the labeled magnitude scale for measuring sensations of taste and smell, Chem. Senses, 21, 323e334 (1996). 22. Chattopadhyay, M. K., Tabor, C. W., and Tabor, H.: Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1D mutant, Biochem. Biophys. Res. Commun., 103, 203e207 (2006).