YEAST, YEAST EXTRACT CONTAINING GAMMA-GLU-ABU, AND A METHOD FOR PRODUCING THE SAME
A yeast extract containing 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract is produced by culturing a yeast, such as Saccharomyces cervisiae or Candida utilis, in a medium containing a compound selected from Abu (L-2-aminobutyric acid) and γ-Glu-Abu (L-γ-glutamyl-L-2-aminobutyric acid), and preparing a yeast extract from the obtained cells.
The present invention relates to a yeast and yeast extract containing γ-Glu-Abu (L-γ-glutamyl-L-2-aminobutyric acid), as well as a method for producing the same. The yeast extract of the present invention is useful in the field of foodstuffs such as seasonings and health foods.
BACKGROUND ARTYeast extracts have a function of imparting atsumi (thickness), umami, etc. to foodstuffs, and have been widely used as seasonings in the field of foodstuffs. Especially, glutathione (henceforth also referred to as “GSH”), which is a tripeptide consisting of glutamic acid, cysteine and glycine, is known to impart kokumi to foodstuffs (Non-patent documents 1 and 2), and seasonings containing GSH have been developed.
Meanwhile, although the calcium sensing receptor (CaSR), which is a G-protein classified into the class C, has been reported to respond to GSH (Non-patent document 3), the physiological significance thereof has not been clarified. Moreover, this CaSR is present also in the lingual cells, and it was considered to show a certain taste response (Non-patent document 4). Then, it has recently been clarified that this CaSR participates in recognition of kokumi in humans (Non-patent document 5). This reference reported that not only GSH that has been recognized as a kokumi substance, but also several γ-glutamyl compounds similarly respond to CaSR. Furthermore, it has been reported that peptides represented by the general formula γ-Glu-X or γ-Glu-X-Gly (X can represent an amino acid or amino acid derivative other than Cys), for example, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val-Gly, etc. have a kokumi-imparting effect (Patent document 1). Moreover, the group of esters including S- or O-carboxyalkylated γ-glutamyl or β-asparagyl peptides, and so forth are also reported as kokumi substances (Patent document 2). Although these peptides impart kokumi to foodstuffs like GSH, they do not have a reduced SH group unlike GSH. It is known that a substance having the reduced SH group such as GSH is generally unstable, and titer thereof is reduced with formation of disulfide bond (Patent document 2). However, γ-Glu-X, γ-Glu-X-Gly etc. are considered useful from the viewpoint that the kokumi-imparting peptides not having the reduced SH group are stable.
Tastes sensed after eating change with time, and the tastes are called initial taste, middle taste, and aftertaste in the order from the taste sensed immediately after eating. Although tastes imparted by various substances change with time in various patterns, concerning especially kokumi, a kokumi-imparting substance showing a taste-imparting pattern that imparts strong initial taste is highly desired. It is known that the γ-glutamyl compound, γ-Glu-Abu-Gly, has a kokumi-imparting action that mainly imparts initial-middle taste (Patent documents 3 and 4).
It is known that the synthesis and decomposition of glutathione, which is one of the γ-glutamyl compounds, is catalyzed by several enzymes which make up the γ-glutamyl cycle. In particular, γ-glutamyl transpeptidase is known to transfer the glutamate of GSH at the γ-position to another compound having an amino group, resulting in decomposition of GSH to cysteinylglycine (Non-patent document 6). It is considered that, if the compound having an amino group in this reaction is an amino acid, a dipeptide of γ-Glu-X can be generated as a by-product. However, research about producing these compounds effectively using microorganisms has not been positively performed to date, partially because they are by-products.
An analysis of the fermentation broth of Micrococcus glutamicus can be noted as findings about the dipeptide γ-Glu-X (Non-patent document 7). This reference reported that the fermentation broth was loaded onto various columns to separate peptides and the like, resulting in the isolation of γ-Glu-Glu, γ-Glu-Val, and γ-Glu-Leu. However, these were found as a result of separation with various columns, and the amounts of these peptides contained in the broth were not determined. In addition, there was not reported in all the above examples that γ-Glu-Abu was contained.
GSH is biosynthesized by two kinds of enzymes called γ-glutamylcysteine synthetase, which binds Glu and Cys to generate γ-Glu-Cys, and glutathione synthetase, which binds the generated γ-Glu-Cys and Gly to generate GSH. The substrate specificities of the enzymes were investigated in in vitro enzymatic reactions, and it was reported that γ-Glu-Abu was generated from Glu and Abu as the substrates (Non-patent document 8). However, this report concerns an example using a bacterium, Proteus mirabilis, and does not concern investigation using yeast. Furthermore, although Abu can be used as a substrate in an in vitro enzymatic reaction, any Abu synthetic pathway is not known for yeasts.
Yeast extracts produced from yeast cells as a raw material are seasonings that have been widely used in the field of foodstuffs, and are highly accepted by consumers. Therefore, a yeast extract is more preferred as a carrier of taste substances. Yeast strains containing minerals can be exemplified as the investigation concerning the use of yeast as a carrier of taste substances. It is known that if a metal is added to a medium, yeasts uptake the metal into the cells (Non-patent document 9). In particular, if trace elements such as zinc, iron, copper, manganese, selenium, molybdenum and chromium are added to the medium, yeasts can be used as a supply source of the desired enriched elements as foodstuffs (Patent document 5). From this point of view, methods for producing mineral-containing yeast have been developed (Patent documents 6 to 8).
Furthermore, yeast incorporating such minerals may also enjoy a merit concerning on taste. For example, there can be mentioned the yeast containing a large amount of magnesium (Patent document 9). This reference describes that although magnesium-enriched foodstuffs containing inorganic magnesium salt were also marketed, a strong bitterness and astringency was noted due to the mineral salt. As a result, it was quite more difficult to routinely eat the magnesium-enriched foodstuffs containing inorganic magnesium salt as compared to foodstuffs containing naturally occurring magnesium. In that context, Patent document 9 discloses a method to produce a foodstuff containing magnesium in natural form by letting yeast uptake magnesium. As for nutritional merit of yeast that uptakes minerals, the technique disclosed in Patent document 10 can be exemplified. According to this reference, although zinc contributes to improvement of taste disorder and generative function, etc., zinc is still not taken in sufficient amounts. If zinc is added during the yeast cultivation process, yeast uptakes zinc into cells. In this case zinc is not accumulated in the cells as water-soluble form, but zinc is highly accumulated in the cells as amorphous zinc form which binds with a protein or an amino acid. When the amorphous zinc is taken into the human body, the amorphous zinc is more efficiently absorbed into the body compared with crystalline zinc. As a result, absorption of zinc into the body can be improved by taking zinc-containing yeast, as compared to simply taking zinc itself.
As described above, a method comprised by making yeast uptake a target substance and adding either the yeast or a yeast extract to foodstuffs can enjoy various advantages as compared to simply adding the target substance itself to foodstuffs. However, unlike minerals, which are essential nutrients, the ability of yeast to uptake an amino acid or a peptide is strictly controlled, and simply applying the technique for incorporating minerals into yeast to the techniques for uptake of amino acid or peptides was considered to be difficult.
As described above, although yeast cells or yeast extracts are preferred as a carrier of a γ-glutamyl compound such as γ-Glu-X as a kokumi-imparting agent, there have been substantially no reports about yeast cells containing such a γ-glutamyl compound, and a method for producing an extract prepared from the cells.
PRIOR ART REFERENCES Patent Documents
- Patent document 1: WO2007/055393
- Patent document 2: WO2007/042288
- Patent document 3: WO2008/139945
- Patent document 4: WO2008/139946
- Patent document 5: Japanese Patent Laid-open (Kokai) No. 2004-298014
- Patent document 6: Japanese Patent Laid-open (Kokai) No. 54-157890
- Patent document 7: Japanese Patent Laid-open (Kokai) No. 60-75279
- Patent document 8: Japanese Patent Publication (Kokoku) No. 6-16702
- Patent document 9: Japanese Patent Laid-open (Kokai) No. 8-332081
- Patent document 10: Japanese Patent Laid-open (Kokai) No. 2008-099578
- Non-patent document 1: Ueda et al., Agric. Biol. Chem., 54, 163-169 (1990)
- Non-patent document 2: Ueda et al., Biosci. Biotechnolo. Biochem., 61, 1977-1980 (1997)
- Non-patent document 3: Wang et al., Journal of Biological Chemistry, 281, 8864-8870 (2006)
- Non-patent document 4: Gabriel et al., Biochemical and Biophysical Research Communications, 378, 414-418 (2009)
- Non-patent document 5: Ohsu et al., Journal of Biological Chemistry, 285, 1016-1022 (2010)
- Non-patent document 6: Protein Nucleic acid Enzyme, 1988-7, VOL. 33, NO. 9, ISSN 003909450, Special Issue “Epoch of glutathione research”, pp. 1432-1433
- Non-patent document 7: Ronald et al., Journal of Biological Chemistry, 240, p 2508-2511 (1965)
- Non-patent document 8: Nakayama et al., Agric. Biol. Chem., 45(12), 2839-2845 (1981)
- Non-patent document 9: B. Volesky, H. A., Appl. Microbiol. Biotechnol., 42; 797-806 (1995)
An object of the present invention is to provide a yeast extract having a kokumi-imparting effect of initial taste-imparting type, and a method for producing it.
Means for Achieving the ObjectThe inventors of the present invention previously found that γ-Glu-Abu (L-γ-glutamyl-L-γ-aminobutyric acid) had high CaSR agonist activity and extremely excellent kokumi-imparting effect, and in particular, it had a taste-imparting pattern of initial taste-imparting type. Then, they found that yeasts took up Abu (α-aminobutyric acid) or γ-Glu-Abu into cells thereof, and by preparing a yeast extract from yeast cultured in a medium containing Abu or γ-Glu-Abu, a yeast extract containing γ-Glu-Abu could be produced. Moreover, they also found that if the aminotransferase activity or/and the α-ketobutyric acid synthetase activity were increased, intracellular Abu production advanced. Further, they also found that by allowing γ-glutamyltransferase to act on a yeast extract raw material to which Abu had been added, a yeast extract containing γ-Glu-Abu could be produced. The present invention was accomplished on the basis of these findings.
The present invention thus relates to the followings.
(1) A yeast extract containing 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract.
(2) A yeast extract containing 0.5% or more of γ-Glu-Abu based on dry weight of the yeast extract.
(3) A yeast extract containing 1.0% or more of γ-Glu-Abu based on dry weight of the yeast extract.
(4) The yeast extract as mentioned above, wherein the yeast belongs to the genus Saccharomyces or Candida.
(5) The yeast extract as mentioned above, which is obtained from Saccharomyces cervisiae.
(6) The yeast extract as mentioned above, which is obtained from Candida utilis.
(7) A method for producing a yeast extract containing γ-Glu-Abu, which comprises culturing a yeast in a medium to which a compound selected from Abu and γ-Glu-Abu is added, and preparing a yeast extract from the obtained cells.
(8) The method as mentioned above, wherein the compound is added to the medium in an amount of 10 ppm or more in the case of Abu, or 1 ppm or more in the case of γ-Glu-Abu, and the yeast extract contains 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract.
(9) The method as mentioned above, wherein the yeast belongs to the genus Saccharomyces or Candida.
(10) The method as mentioned above, wherein the yeast is Saccharomyces cervisiae.
(11) The method as mentioned above, wherein the yeast is Candida utilis.
(12) The method as mentioned above, wherein the yeast has one or both of the following characteristics:
(a) γ-glutamylcysteine synthetase activity is enhanced,
(b) glutathione synthetase activity is attenuated.
(13) A yeast having an increased γ-Glu-Abu content, which has been modified so that
activity of aminotransferase or/and activity of α-ketobutyric acid synthetase are enhanced, and,
activity of γ-glutamylcysteine synthetase is enhanced, or/and activity of glutathione synthetase is attenuated.
(14) The yeast as mentioned above, wherein the aminotransferase is an enzyme encoded by the BAT1 gene.
(15) The yeast as mentioned above, wherein the aminotransferase is an enzyme encoded by the UGA1 gene.
(16) The yeast as mentioned above, wherein the α-ketobutyric acid synthetase is an enzyme encoded by the CHA1 gene.
(17) The yeast as mentioned above, wherein a peptidase activity is further attenuated.
(18) A yeast extract produced from the yeast as mentioned above.
(19) A method for producing a yeast extract containing γ-Glu-Abu, which comprises allowing a γ-glutamyltransferase to act on a yeast extract raw material to which Abu has beeb added.
(20) The method as mentioned above, wherein Abu is added in an amount of 0.1% or more based on dry weight of the yeast extract raw material, and the yeast extract contains 0.2% or more of Abu based on dry weight of the yeast extract.
Hereafter, the present invention will be explained in detail.
The yeast extract of the present invention is a yeast extract containing 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract.
The yeast extract of the present invention contains γ-Glu-Abu in an amount of 0.2% or more, preferably 0.5% or more, more preferably 1.0% or more, particularly preferably 2.0% or more, based on dry weight of the yeast extract.
The yeast used as the raw material of the yeast extract of the present invention is the same as the yeast used for the method of the present invention described later.
The form of the yeast extract of the present invention is not particularly limited, and it may be in the form of powder or solution. The yeast extract can have the same uses as that of conventional yeast extracts, for example, seasonings, food additives, health foods, and so forth. The yeast extract of the present invention is excellent in its kokumi-imparting effect. Since the kokumi-imparting effect is more strongly reinforced in the presence of umami or salty taste, an umami substance such as sodium L-glutamate and tasty nucleotides, and/or a salty substance such as sodium chloride may be added to the yeast extract. Further, since the yeast extract of the present invention has especially a superior effect of imparting kokumi as the initial taste, it may be mixed with a kokumi-imparting substance showing a different kokumi-imparting pattern, such as GSH or γ-Glu-Val-Gly, or a yeast extract containing such a substance. Moreover, an umami substance and/or a salty substance may be added to seasonings, food additives or health foods together with the yeast extract of the present invention. The tastes are classified into initial taste, middle taste, and aftertaste. Although these are relative concepts, they are usually defined as tastes sensed in the periods of 0 to 2 seconds, 2 to 5 seconds, and after 5 seconds after eating, respectively. The “initial-middle taste” mentioned above is a taste sensed in the period of 0 to 5 seconds after eating, and the “middle-after taste” mentioned later is a taste sensed in the period of 2 second to about 30 seconds after eating.
The yeast extract of the present invention can be produced by, for example, the method of the present invention described below.
The first method of the present invention is a method for producing a yeast extract containing γ-Glu-Abu, which includes the steps of culturing a yeast in a medium to which a compound selected from Abu and γ-Glu-Abu is added, and preparing a yeast extract from the obtained cells.
Any yeast from any wild-type strains, or various mutant strains or recombinant strains can be used for this invention, so long as the chosen yeast can intracellularly uptake Abu and/or γ-Glu-Abu and accumulate γ-Glu-Abu in the cells. Examples of the mutant strains or recombinant strains include a strain with enhanced activity of γ-glutamylcysteine synthetase (GSH1), a strain with attenuated activity of glutathione synthetase (GSH2), and a strain having two of the foregoing characteristics. Examples of the mutant strains or recombinant strains also include a strain with attenuated activity of a peptidase that decompose intracellular peptides, for example, an enzyme encoded by the DUG1 gene, DUG2 gene, DUGS gene, or ECM38 gene. Further, in addition to the enhanced activity of GSH1 and/or attenuated activity of GSH2, a peptidase activity of the yeast may be attenuated. The nucleotide sequences of the aforementioned genes are disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/).
The yeast is not particularly limited, so long as the chosen yeast can accumulate γ-Glu-Abu in the cells thereof. Examples include yeasts belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, those belonging to the genus Candida such as Candida utilis, those belonging to the genus Pichia such as Pichia pastoris, and those belonging to the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Among these, Saccharomyces cerevisiae and Candida utilis are preferred, which are frequently used for production of yeast extracts. The yeast may be a monoploid, or may have diploidy or a further higher polyploidy.
Methods for enhancing activity of an enzyme or protein such as GSH1 include a method of enhancing expression of a gene coding for it. Methods for enhancing expression of a gene include a method of replacing the promoter of the gene on the chromosome with a stronger promoter, a method of inserting the target gene into the chromosome to increase copy number thereof, a method of incorporating a plasmid containing the target gene into the yeast, a method of activating a transcription factor of the gene coding for the target enzyme, and so forth.
As the promoter, a highly active conventional promoter may be obtained by using various reporter genes, or a known high expression promoter such as ADH1, PGK1, PDC1, TDH3, TEF1 and HXT7 may be used. Alternatively, for increasing copy number of a target gene, for example, a plasmid having the replication origin of CEN4, or a multi-copy plasmid having the replication origin of 2 μm DNA, to which a target gene is inserted, may be used. Furthermore, a transposon may be used in order to introduce a target gene into an arbitrary region of the chromosome, or the target gene may be introduced by using rDNA sequences as a target, which is present in a copy number of 150 in the cell.
Methods for attenuating activity of an enzyme or protein such as GSH2 and peptidase include a method of replacing the promoter of the gene coding for any of these on the chromosome with a weaker promoter to attenuate the expression, a method of introducing a mutation into the target enzyme to reduce the activity thereof, a method of deleting a part of or the entire gene of the target enzyme from the chromosome, a method of inactivating the target enzyme gene by inserting another sequence into the gene, and so forth.
The attenuated enzyme activity include the activity lower than that of a wild-type strain and complete deficiency of the enzyme activity.
Enhancement of the activity of γ-glutamylcysteine synthetase is disclosed in, for example, U.S. Pat. No. 7,553,638; Otake Y. et al., Bioscience and Industry, volume 50, No. 10, pp. 989-994, 1992, and so forth. Although disruption of the glutathione synthetase gene is disclosed in U.S. Pat. No. 7,553,638, the glutathione synthetase activity can also be reduced by inactivating YAP1, which is a transcription factor of the gene coding for γ-glutamylcysteine synthetase.
The nucleotide sequences of the genes coding for GSH1, GSH2, and YAP1 of Saccharomyces cerevisiae are disclosed in Saccharomyces Genome Database (http://www.yeastgenome.org/). The nucleotide sequences of the genes coding for GSH1 and GSH2 of Candida utilis are disclosed in U.S. Pat. No. 7,553,638. The nucleotide sequence of the gene coding for YAP1 of Candida utilis is disclosed in Japanese Patent Laid-open (Kokai) No. 2006-75122.
Such a yeast as mentioned above may be a yeast obtained by screening from nature, breeding based on mutagenesis, or breeding based on genetic engineering. For the mutagenesis, various agents such as EMS, DAPA, and NTG can be used, and an objective mutant strain can be isolated by spreading yeast cells subjected to a mutation treatment on an optimal medium, and choosing a strain of which GSH1 activity is enhanced or a strain of which GSH2 activity is attenuated from the grown strains.
The method for breeding by genetic engineering is not particularly limited, and conventional methods can be used. In particular, genetic engineering methods for Saccharomyces cervisiae are specifically described in many books. Moreover, various kinds of methods have been reported also for Candida utilis in recent years, and they may be used. Specific methods are described in such prior references as, for example, Norihiko Misawa, Chemical Engineering, June, 1999, pp. 23-28; Luis Rodriguez et al., FEMS Microbiology Letters, 165, 335-340 (1998); WO98/07873; Japanese Patent Laid-open (Kokai) No. 8-173170; WO95/32289; Keiji Kondo et al., Journal of Bacteriology, December 1995, Vol. 177, No. 24, pp. 7171-7177; WO98/14600; Japanese Patent Laid-open (Kokai) Nos. 2006-75122, 2006-75123, 2007-089441, and 2006-101867, and these can be appropriately referred to.
The method for producing the yeast extract is explained below.
First, a yeast is cultured in a medium. The medium is not particularly limited, so long as a medium in which the yeast can proliferate is chosen, and is not limited to the SD medium described in the examples. A medium usually used for industrial purposes can be used. Examples of the medium include, for example, media containing glucose, sucrose, molasses, ethanol, acetic acid, spent sulfite liquor, or the like as a carbon source, urea, ammonia, ammonium sulfate, ammonium chloride, nitrate, or the like, or corn steep liquor, casein, yeast extract, peptone, soybean protein decomposition product, or the like as a nitrogen source, phosphoric acid, potassium phosphate, ammonium phosphate, superphosphate of lime, potassium chloride, potassium hydroxide, magnesium sulfate, magnesium chloride, and so forth as phosphoric acid, potassium, and magnesium sources, and an appropriate combination of mineral salts of copper, manganese, zinc, iron ion, etc. as trace elements.
When the yeast is cultured, Abu, γ-Glu-Abu, or both of these is/are added to the aforementioned medium. Abu represents α-aminobutyric acid, and Glu represents glutamic acid. Abu and Glu are L-forms. These compounds may be present in the medium from the start of the culture, or may be added to the medium at an arbitrary time during the culture. When the compounds are added to the medium during the culture, they can be preferably added at 0 to 50 hours before the end of the culture (0 hour means that the culture is terminated immediately after the addition), more preferably 0.1 to 24 hours before the end of the culture, particularly preferably 0.5 to 6 hours before the end of the culture. Furthermore, when the peptides are added during the culture, they may be continuously added.
Abu and/or γ-Glu-Abu to be added to the medium may be a purified product (pure substance), or may be a composition containing such a compound or compounds, so long as the composition contains required amounts of these compounds.
Prior to the culture in the medium containing the compound(s), a preculture may be performed. The medium used for the preculture may or may not contain the compound(s).
When the compound is added to the medium at the start of the culture, Abu is added in an amount of usually 10 ppm or more, preferably 25 ppm or more, more preferably 50 ppm or more, further preferably 100 ppm, and γ-Glu-Abu is added in an amount of usually 1 ppm or more, preferably 5 ppm or more, more preferably 10 ppm or more, in terms of the final concentration in the culture broth at the time of the addition. When both Abu and γ-Glu-Abu are added, concentrations thereof can be determined to be within the aforementioned ranges. Although the maximum amount of the compound is not particularly limited, it is, for example, 100,000 ppm or less from an aspect of production cost, and it is usually 10,000 ppm or less, preferably 1,000 ppm or less, more preferably 500 ppm or less. When the compound is added during an arbitrary period in the middle of the culture, or it is continuously added, it may be added in a total amount equivalent to an amount that can provide the final concentration mentioned above when the compound is added in such an amount at the time of the start of the culture.
As the culture conditions, the same conditions as those used for usual production of yeast extracts can be used, and they may be suitably changed according to the chosen yeast. Arbitrary methods such as batch culture, fed-batch culture, and continuous culture may be used. When the yeast is Saccharomyces cerevisiae or Candida utilis, it is preferably aerobically cultured by shaking or the like at 25 to 35° C., more preferably 27 to 33° C., still more preferably 28 to 32° C.
If the yeast is cultured as described above, γ-Glu-Abu accumulates in the cells of the yeast. When Abu is added to the medium, Abu accumulates in the cells, and in addition, γ-Glu-Abu also accumulates. This is because Abu taken up into the cells is converted into γ-Glu-Abu by the action of intracellular γ-glutamylcysteine synthetase as shown in the example section mentioned later. As shown in the example section, the content of γ-Glu-Abu or γ-Glu-Abu-Gly in the yeast did not correlate with the content of GSH in the cells, and therefore it is considered that yeast extracts produced by the conventional methods do not contain γ-Glu-Abu at a high concentration, even if they are produced from a yeast containing GSH at a high concentration. It is estimated that this is because generation of Abu is limited in the cells as described in the example section mentioned later. According to a preferred embodiment, the yeast cultured as mentioned above contains γ-Glu-Abu in an amount of 0.04% or more, preferably 0.1% or more, more preferably 0.15% or more, still more preferably 0.2% or more, particularly preferably 0.4% or more, based on dry weight of the cells.
The yeast extract can be prepared from the obtained yeast in the same manner as that used for conventional production of yeast extracts. The yeast extract may be obtained by subjecting the yeast cells to hot water extraction and processing the extract, or by digesting the yeast cells by self-digestion or with an enzyme and processing the digestion product. Furthermore, the obtained yeast extract may be concentrated, may be in the form of paste, or may be dried and thereby made into powdered form, as required.
In such a manner as described above, a yeast extract containing an increased amount of γ-Glu-Abu can be obtained. According to a preferred embodiment, the yeast extract contains γ-Glu-Abu in an amount of 0.2% or more, more preferably 0.5% or more, still more preferably 1.0% or more, particularly preferably 2.0% or more, based on dry weight of the yeast extract.
The second method of the present invention will be explained. The second method relates to a yeast of which Abu synthesis ability is enhanced within the yeast cell. Although Abu synthetic pathway in a yeast cell had not conventionally been known, it was found that it was produced from α-ketobutyric acid with aminotransferase in yeast as shown in the example section. Therefore, if Abu synthesis ability is enhanced in a yeast cell, the γ-Glu-Abu accumulation ability is improved. The Abu synthesis ability can be enhanced by enhancing the aminotransferase activity or the α-ketobutyric acid synthetase activity. The activity of aminotransferase or α-ketobutyric acid synthetase can be enhanced in the same manner as that used for GSH1 and so forth.
One embodiment of the yeast of the present invention is a yeast of which aminotransferase activity is enhanced. Another embodiment of the yeast of the present invention is a yeast of which α-ketobutyric acid synthetase activity is enhanced. Still another embodiment of the yeast of the present invention is a yeast both of which aminotransferase activity and α-ketobutyric acid synthetase activity are enhanced. In such a yeast of which aminotransferase activity or/and α-ketobutyric acid synthetase activity are enhanced, the activity of γ-glutamylcysteine synthetase (GSH1) may be enhanced, or the activity of glutathione synthetase (GSH2) may be attenuated as in the first method of the present invention. Alternatively, the yeast may have two of these characteristics. Furthermore, a peptidase that decomposes intracellular peptides, for example, an enzyme encoded by the DUG1 gene, DUG2 gene, DUG3 gene, or ECM38 gene, may be attenuated.
A yeast of which Abu synthesis ability is enhanced, for example, a yeast modified so that the γ-glutamylcysteine synthetase activity and the aminotransferase activity, or/and the α-ketobutyric acid synthetase activity are enhanced accumulates a marked amount of γ-Glu-Abu, even when it is cultured in a medium to which Abu and γ-Glu-Abu are not added. The yeast of a preferred embodiment, for example, such a yeast of which activity of aminotransferase encoded by the BAT1 mentioned below, and the GSH1 activity are enhanced, and of which GSH2 activity is attenuated, preferably contains γ-Glu-Abu in an amount of 0.04% or more, more preferably 0.1% or more, still more preferably 0.15% or more, further preferably 0.2% or more, particularly preferably 0.4% or more, based on dry weight of the cells. A yeast extract prepared from such a yeast contains 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract.
According to another embodiment, for example, the aforementioned yeast of which activity of serine (threonine) deaminase encoded by CHA1 is further enhanced preferably contains γ-Glu-Abu in an amount of 0.1% or more, more preferably 0.15% or more, still more preferably 0.2% or more, further preferably 0.4% or more, particularly preferably 0.5% or more, based on dry weight of the cells. When a yeast of which Abu synthesis ability is enhanced is cultured, Abu and/or γ-Glu-Abu may be added to the medium.
The yeast is not particularly limited, so long as the chosen yeast can accumulate γ-Glu-Abu in the cells thereof. Examples include yeasts belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, those belonging to the genus Candida such as Candida utilis, those belonging to the genus Pichia such as Pichia pastoris, and those belonging to the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Among these, Saccharomyces cerevisiae and Candida utilis are preferred, which are frequently used for production of yeast extracts. The yeast may be a monoploid, or may have diploidy or a further higher polyploidy.
Examples of aminotransferase of yeast include alanine: glyoxylate aminotransferase, branched-chain amino acid transaminase, aspartate aminotransferase, γ-aminobutyrate transaminase, and so forth. The genes coding for these enzymes in Saccharomyces cervisiae have already been specified, and they are encoded by AGX1 (systematic name: YFL030W), BAT1 (systematic name: YHR208W), BAT2 (systematic name: YJR148W), AAT1 (systematic name: YKL106W), AAT2 (systematic name: YLR027C), and UGA1 (systematic name: YGR019W), respectively. Moreover, various genes have been also reported for Candida utilis in recent years, and homologue genes thereof can also be easily specified by identifying the total genome sequence using recent advanced sequencers, and used. Among these, BAT1 and UGA1 are preferred, and BAT1 is particularly preferred, since it shows a marked effect as described in the example section described later. Although activity of one kind of aminotransferase may be enhanced, activities of arbitrary two or more kinds of aminotransferases may also be enhanced.
Examples of the α-ketobutyric acid synthetase of yeast include the serine (threonine) deaminase encoded by the CHA1 gene (systematic name: YCL064C), and the threonine deaminase encoded by the ILV1 gene (systematic name: YER086W). Although activity of one kind of α-ketobutyric acid synthetase may be enhanced, activities of arbitrary two or more kinds of α-ketobutyric acid synthetases may also be enhanced.
The activities of aminotransferase and α-ketobutyric acid synthetase can be enhanced by enhancing expression of genes coding for the enzymes as in the case of enhancement of the GSH1 activity.
Production of a yeast extract using a yeast having enhanced activity of aminotransferase or/and enhanced activity of α-ketobutyric acid synthetase, and containing γ-Glu-Abu can be performed in the same manner as that explained for the first method.
The third method of the present invention is a method for producing a yeast extract containing γ-Glu-Abu, which includes the step of allowing a γ-glutamyltransferase to act on a yeast extract raw material to which Abu has been added.
If a γ-glutamyltransferase is allowed to act on Abu, γ-Glu-Abu is generated. Therefore, a yeast extract containing γ-Glu-Abu can also be obtained by allowing a γ-glutamyltransferase to act on a yeast extract containing Abu. The yeast extract containing Abu may be prepared from a yeast cultured in a medium containing Abu, or may be obtained by adding Abu to a yeast extract raw material.
As the yeast extract raw material, a yeast extract obtained by a conventional method can be used.
Abu is usually added to the yeast extract in an amount of 0.1% or more, preferably 1% or more, more preferably 5% or more, still more preferably 10% or more, based on dry weight of the yeast extract raw material.
The reaction catalyzed by the γ-glutamyltransferase is performed in an aqueous solvent such as water or buffers. Specifically, for example, the yeast extract raw material is dissolved in the aqueous solvent, and the γ-glutamyltransferase is added. The reaction conditions can be suitably determined according to the γ-glutamyltransferase to be used. The reaction is usually allowed at pH 3 to 9 and 15 to 70° C. for 1 to 300 minutes, preferably at pH 5 to 8 and 30 to 70° C. for 5 to 150 minutes.
Concentration of the yeast extract raw material in the aqueous solvent may be determined in view of ease of handling. The concentration is usually 0.1 to 50%, preferably 0.5 to 20%, in terms of dry weight of the yeast extract raw material.
Examples of the γ-glutamyltransferase include glutaminase, γ-glutamyl transpeptidase (γ-GTP), and so forth. As for the amount of the enzyme, in the case of γ-GTP, it is usually 0.001 to 1000 units/ml, preferably 0.005 to 100 units/ml, more preferably 0.01 to 25 units/ml, most preferably 0.05 to 10 units/ml, wherein 1 unit is defined to be the activity of liberating 1.0 μmole of p-nitroaniline from γ-glutamyl-p-nitroanilide per 1 minute in a solution at pH 8.5 and 25° C. (definition described in Sigma General Catalogue, 2008-2009 Edition, p. 917). The amount of glutaminase can also be determined in a manner similar to that for γ-GTP.
After the enzymatic reaction, a treatment for inactivating the γ-glutamyltransferase, for example, a heat treatment at 80 to 100° C., may be performed, or may not be performed.
As a substrate of the γ-glutamyltransferase, a γ-glutamyl compound, for example, GSH, may be added to the reaction mixture. GSH contained in the yeast extract may also be used as a substrate. In this case, a yeast extract prepared from a yeast in which the content of GSH is increased, for example, a yeast in which activities or activity of GSH1 and/or GSH2 is enhanced, can be used. Although a greater GSH content in the yeast extract is more preferred, it is usually 1 to 50%, preferably 1 to 30%, more preferably 5 to 20%, based on dry weight of the yeast extract. Alternatively, glutamine can also be used like GSH.
In such a manner as described above, a yeast extract in which the amount of γ-Glu-Abu is increased is obtained. According to a preferred embodiment, the yeast extract contains γ-Glu-Abu in an amount of 0.02% or more, more preferably 0.5% or more, still more preferably 1.0% or more, particularly preferably 2.0% or more, based on dry weight of the yeast extract.
The obtained yeast extract may be concentrated, or may be in the form of paste, or may be dried and thereby made into powdered form, as required.
Another kokumi substance may be added to a yeast extract obtained by any of the aforementioned first to third methods of the present invention. Examples of such a kokumi substance include, for example, a peptide such as γ-Glu-X and γ-Glu-X-Gly (X represents an amino acid or an amino acid derivative), specifically GSH and γ-Glu-Val-Gly, and a yeast extract containing these. As described above, a yeast extract that has kokumi for a broad range from the initial taste to aftertaste can be produced. In the case of GSH, in particular, in view of the balance of kokumi of the initial taste and the aftertaste, the ratio of GSH with respect to γ-Glu-Abu is preferably 0.3 or more, more preferably 0.5 or more, still more preferably 1.0 or more, particularly preferably 3.0 or more.
EXAMPLESHereafter, the present invention will be more specifically explained with reference to examples.
However, the present invention is no way limited by the following examples. Unless specially mentioned, the amino acids and amino acid derivatives mentioned in the examples are L-forms.
Reference Example 1 Evaluation of Kokumi-Imparting Activity of γ-Glu-AbuDegree of kokumi-imparting activity of γ-Glu-Abu was investigated through a quantitative organoleptic evaluation test.
The quantitative organoleptic evaluation test was carried out as follows. Strength of the kokumi-imparting activity of a test compound was measured by using a mixture containing the test compound at 0.001 to 0.5 g/dl in distilled water containing sodium glutamate (0.05 g/dl), inosine monophosphate (0.05 g/dl), and sodium chloride (0.5 g/dl). A sample of the distilled water containing sodium glutamate, inosine monophosphate, and sodium chloride to which any test compound was not added was used as a no addition control. Samples that became acidic relative to the no addition control after dissolution of the test compound were used after pH of them was adjusted with NaOH to be within the range of ±0.2 from that of the no addition control.
The test was performed with organoleptic scores of 0 for the control, 3 for strong effect, and 5 for extremely strong effect, and n=4. Moreover, in order to make the criteria more definite, the scores of 0.001 g/dl of γ-Glu-Val-Gly for the initial taste and the middle-after taste were defined to be 3.0. The “middle-after taste” is a taste sensed in the periods for the middle taste and the aftertaste. Specifically, the initial taste, middle taste, and aftertaste are tastes sensed in the periods of 0 to 2 seconds, 2 to 5 seconds, and after 5 seconds, after eating, respectively, and the “middle-after taste” is a taste sensed in the period from 2 seconds to around 30 seconds. For the scoring, the linear scale method was used, in which determined scores were noted on a straight line on which positions of scores of from −5 to 5 were indicated. The panelists were consisted of persons who had experience of development of seasonings for foodstuffs for one year or longer in total, and could judge the difference of the titers of γ-Glu-Cys-Gly and γ-Glu-Val-Gly added to the tasty and salty solution to be about 10 times (this ability was periodically confirmed). Although γ-Glu-Abu showed kokumi-imparting activity in a broad range within the aforementioned addition concentration range, the result for a typical concentration is shown in Table 1.
The result of similar evaluation for γ-Glu-Ala is also shown in Table 2. The both are initial taste-imparting type substances giving high scores for the initial taste, but it was found that γ-Glu-Abu was a dipeptide showing an extremely high titer.
Kokumi-imparting activities of γ-Glu-Cys and other dipeptides were determined by the same quantitative organoleptic evaluation test as mentioned above. The results are shown in Table 2.
It was found that γ-Glu-Abu had superior kokumi-imparting activity, and showed marked rise of the initial taste in the taste pattern. This rise of the initial taste is one of the extremely advantageous characteristics of γ-Glu-Abu compared with γ-Glu-Cys. Moreover, γ-Glu-Abu shows superior storage stability, and this is also one of the advantageous characteristics compared with γ-Glu-Cys. Further, since the number of residues contained in γ-Glu-Abu is as small as two, it can be more easily produced at lower cost compared with tripeptides containing three amino acid residues, and this is industrially extremely advantageous.
Example 1 Detection of Abu, γ-Glu-Abu and γ-Glu-Abu-Gly in Various Yeast ExtractsAbu, γ-Glu-Abu and γ-Glu-Abu-Gly contents in various yeast extracts were measured by fluorescence derivatization of the compounds with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), and detection by LC-MS/MS according to the method described below. Specifically, to 2.5 μL of a sample diluted to an appropriate concentration, or 2.5 μL each of standard solutions containing 1 μM Abu, γ-Glu-Abu or γ-Glu-Abu-Gly, 2.5 μL of Milli-Q water, 5 μL of a 5 μM internal standard substance solution (3-methyl-His-d2 (Sigma) or Gly-d2 (Sigma), both are labeled with stable isotope), and 30 μL of a borate buffer (attached to AccQ-Fluor (registered trademark) Reagent Kit, Nihon Waters) were added. To each mixture, 10 μL of an AQC reagent solution (prepared by dissolving the reagent powder of the aforementioned reagent kit in 1 mL of acetonitrile) was added. This mixture was heated at 55° C. for 10 minutes, and then 100 μL of 0.1% formic acid aqueous solution was added to the mixture to prepare a sample for analysis.
Then, the sample for analysis prepared as described above was subjected to separation by the reverse phase liquid chromatography described below, and then introduced into a mass spectrometer. The separation conditions were as follows.
(1) HPLC: Agilent 1200 Series(2) Separation column: Unison UK-Phenyl; internal diameter, 2.0 mm; length, 100 mm; particle size, 3 μm (Imtakt)
(3) Column oven temperature: 40° C.
(4) Mobile phase A: 25 mM Ammonium formate (pH 6.0, adjusted with aqueous ammonia)
(5) Mobile phase B: methanol
(6) Flow rate: 0.25 mL/min
(7) Elution conditions: Elution was performed by using mixtures of the mobile phase A and the mobile phase B. The ratios of the mobile phase B to the mixtures are as follows: 0 minute (5%), 0 to 17 minutes (5 to 40%), 17 to 17.1 minutes (40 to 80%), 17.1 to 19 minutes (80%), 19 to 19.1 minutes (80 to 5%), 19.1 to 27 minutes (5%).
Then, derivatized compounds of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly eluted under the aforementioned separation conditions were introduced into a mass analyzer, and quantified by mass chromatography. The analysis conditions were as follows.
(1) Mass analyzer: AB Sciex API3200 QTRAP
(2) Detection mode: Selected Ion Monitoring (positive ion mode)
The derivatized compounds of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly were quantified by using analysis software, Analyst ver. 1.4.2 (AB Sciex). As the internal standard substance for performing the quantification, a derivatized compound of 3-methyl-His-d2 was used in the case of the derivatized compound of Abu, and a derivatized compound of Gly-d2 was used in the case of the derivatized compounds of γ-Glu-Abu and γ-Glu-Abu-Gly. The analysis results (mass chromatograms) of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly as well as the derivatized internal standard amino acid are shown in
At the time of the quantification of γ-Glu-Abu, a contaminated peak was very rarely observed, and in such a case, the quantification was performed by using selected ion of 145.2 or 104.1 for the second mass analyzer.
Example 2 Measurement of γ-Glu-Abu Content in Various Commercially Available Yeast Extractsγ-Glu-Abu contents in various commercially available yeast extracts (based on dry weight of the yeast extracts) were measured by using the method of Example 1. GSH contents were also measured in a conventional manner. The results are shown in Table 4.
As shown in Table 4, the γ-Glu-Abu contents in various yeast extracts were in the range of 15 to 920 ppm. Further, the γ-Glu-Abu/GSH ratio was not constant, and in particular, there was observed a tendency that the ratio was more decreased in a yeast extract having a higher GSH content. It is considered that this is because GSH1 and GSH2 responsible for the GSH biosynthetic pathway can recognize Abu and γ-Glu-Abu as a substrate, but first of all, the intracellular generation amount of Abu is limited, and even if the generation pathway of GSH is enhanced, a large amount of γ-Glu-Abu cannot be accumulated due to lack of Abu, as shown in the following examples. This suggests that the γ-Glu-Abu content in the known high GSH content yeasts is not so high.
Example 3 Effect of Addition of Abu to Saccharomyces cervisiae Type Strain, S288C StrainThen, γ-Glu-Abu content in the S288C strain, which is a type strain of Saccharomyces cervisiae, was measured. Moreover, effect of addition of the precursor, Abu, to the medium was also investigated. The S288C strain is deposited at the independent administrative agency, National Institute of Technology and Evaluation, Biological Resource Center (NBRC, NITE Biological Resource Center, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) with the number of NBRC 1136, and can be provided therefrom. This strain is also deposited at the American Type Culture Collection (12301 Parklawn Drive, Rockville, Md. 20852, United States of America) with the number of ATCC 26108, and can be provided therefrom.
One loop of the S288C strain was inoculated into the SD medium (50 ml in 500 ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hours with shaking at a velocity of 120 rpm.
Composition of SD Medium:
(Nitrogen Base of 10-fold concentration was obtained by dissolving a mixture of 1.7 g of Bacto Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco) and 5 g of ammonium sulfate in 100 ml of sterilized water, adjusting the solution to about pH 5.2, and sterilizing the solution by filter filtration.)
Absorbance of the obtained culture broth was measured, the culture broth was inoculated into the SD medium or the SD medium containing 10, 50 or 100 ppm of Abu as the final concentration (400 ml in a 2 L-volume conical flask with baffle fins), so that OD660 was 0.01 at the start of the culture (absorbance was measured by using DU640 SPECTROPHTOMETER, BECKMAN COULTER), and the yeast cells were cultivated at 30° C. for 19 hours with shaking by rotation at a velocity of 120 rpm. From the obtained culture broth, 400 OD units of the cells (1 OD unit is defined as cells contained in 1 ml of culture broth of which OD660 is 1) were collected by centrifugal separation. The supernatant was removed as much as possible, and the residual cells were suspended in 45 ml of Milli-Q water. The cells were collected again by centrifugal separation, and resuspended in 45 ml of Milli-Q water. By repeating this operation 3 times in total, the medium was completely removed from the cells. The washed cells were suspended in about 1.5 ml of Milli-Q water, and the suspension was heated at 70° C. for 10 minutes. By this step, the extractable components contained in the cells were extracted. Then, the extract and the cell residue were separated by centrifugation.
Cell debris were removed from the extract using a centrifugal filtration membrane of 10 kDa cutoff (Amicon Ultra—0.5 mL 10K, MILLIPORE, Catalogue Number UFC501096)), the obtained fraction was derivatized with the AQC reagent in the same manner as that used in Example 1, and Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly were measured by LC-MS/MS. Furthermore, dry cell weight was measured after the washed cells were dried at 104° C. for 4 hours. From the amounts of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly contained in a certain volume of culture broth and dry cell weight measured as described above, the contents of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly based on weight of dried cells were calculated. The results are shown in Table 5.
As shown in Table 5, it was found that, by adding Abu to the medium, the γ-Glu-Abu content in the cells was increased. Moreover, since not only γ-Glu-Abu but also γ-Glu-Abu-Gly accumulated, it was found that a part of γ-Glu-Abu was converted into γ-Glu-Abu-Gly by a certain enzymatic reaction using γ-Glu-Abu as a substrate in the yeast cells.
In addition, Abu in the washing solution obtained in the cell washing step was measured, but Abu was not contained in the final washing solution. The cell washing operation was performed further once again, and therefore it was confirmed that Abu in the medium was fully removed by the four-step separation in the washing step, and was not carried over into the cell extract. Further, γ-Glu-Abu was not detected in the final washing solution also in the γ-Glu-Abu addition experiments described in the examples mentioned below.
Example 4 Effect of Addition of γ-Glu-Abu to S288C Strain (1)Then, effect of addition of γ-Glu-Abu to the medium at the time of culturing the S288C strain was investigated. One loop of the S288C strain was inoculated into the SD medium (50 ml in a 500 ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hours with shaking at a velocity of 120 rpm. Absorbance of the obtained culture broth was measured (absorbance was measured by using DU640 SPECTROPHTOMETER, BECKMAN COULTER), the culture broth was inoculated into the SD medium or the SD medium containing 10 or 100 ppm of γ-Glu-Abu as the final concentration (400 ml in a 2 L-volume conical flask with baffle fins), so that OD660 was 0.01 at the start of the culture, and the yeast cells were cultivated at 30° C. for 19 hours with shaking by rotation at a velocity of 120 rpm. Extract was obtained from the obtained culture broth in the same manner as that of Example 3, and contents of the compounds in the cells were measured.
As shown in Table 6, it was found that when the S288C strain was cultured in a medium containing γ-Glu-Abu, the S288C strain took up γ-Glu-Abu in the medium and accumulated it in the cells. Moreover, not only γ-Glu-Abu, but also γ-Glu-Abu-Gly was accumulated, as in Example 3.
Then, effect of delayed addition of γ-Glu-Abu to the medium at the time of culturing the S288C strain was investigated. One loop of the S288C strain was inoculated into the SD medium (50 ml in a 500 ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hours with shaking at a velocity of 120 rpm. Absorbance of the obtained culture broth was measured (absorbance was measured by using DU640 SPECTROPHTOMETER, BECKMAN COULTER), the culture broth was inoculated into the SD medium (400 ml in a 2 L-volume conical flask with baffle fins), so that OD660 was 0.01 at the start of the culture, and the yeast cells were cultivated at 30° C. for 18 hours with shaking by rotation at a velocity of 120 rpm. Then, γ-Glu-Abu was added at 10 ppm or 100 ppm as the final concentration for the experiment utilizing delayed addition of γ-Glu-Abu to the medium, or no substance was added for the control, and the culture was continued for further 1 hour (total culture time was 19 hours). Extract was obtained from the obtained culture broth in the same manner as that of Example 3, and contents of the compounds in the cells were measured.
As shown in Table 7, it was found that γ-Glu-Abu in the medium could be uptaken into the cells and accumulated in the cells as in Example 4, even when γ-Glu-Abu was added afterward. Moreover, not only γ-Glu-Abu, but also γ-Glu-Abu-Gly accumulated, as in Examples 3 and 4.
Solid contents of the extracts obtained in Examples 3 to 5 were measured, and γ-Glu-Abu contents based on the solid contents of the extracts were calculated from the γ-Glu-Abu contents based on dry cell weight. As a result, it was found that the γ-Glu-Abu content markedly increased compared with commercially available yeast extracts.
Then, effect of addition of Abu to the Candida utilis type strains, NBRC 10707 strain and NBRC 0988 strain, was investigated. These strains are deposited at the independent administrative agency, National Institute of Technology and Evaluation, Biological Resource Center (NBRC, NITE Biological Resource Center, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) with the number of NBRC 10707 and NBRC 0988, and can be provided therefrom.
One loop each of the NBRC 10707 strain and NBRC 0988 strain was inoculated into the SD medium (50 ml in a 500 ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hours with shaking at a velocity of 120 rpm. Absorbance of the obtained culture broth was measured (absorbance was measured by using DU640 SPECTROPHTOMETER, BECKMAN COULTER), the culture broth was inoculated into the SDP medium (400 ml in a 2 L-volume conical flask with baffle fins, corresponding to the SD medium described in Example 3, provided that 5 g of ammonium sulfate was replaced with 1 g of proline at the time of preparing Yeast Nitrogen Base of 10-fold concentration), so that OD660 was 0.01 at the start of the culture, and the yeast cells were cultivated at 30° C. with shaking by rotation at a velocity of 120 rpm. The NBRC 10707 strain was cultivated for 46.5 hours, and the NBRC 0988 strain was cultivated for 22.5 hours to attain the logarithmic phase for each strain. Then, Abu was added at a final concentration of 100 ppm, and the culture was continued for further 1 hour. As a control group, cultivation was continued for 1 hour without adding the compound. The absorbance of the culture broth to which Abu was added afterward was about 4.5 after the culture, and residual saccharides were also detected. An extract was obtained from the obtained culture broth in the same manner as that of Example 3, and contents of the compounds in the cells were measured. The results are shown in Tables 9 and 10. As a result, without the addition of Abu, γ-Glu-Abu was not detected in the cells, but with addition of Abu, γ-Glu-Abu was detected in the cells. Also from these results, it was found that supply of Abu was important for accumulation of γ-Glu-Abu.
Since it was found that γ-Glu-Abu was produced by a certain enzymatic reaction using Abu as a substrate in the yeast cells on the basis of the results of Example 3, possibility of side reaction with γ-glutamylcysteine synthetase was investigated.
1) Construction of Plasmid pET-GSH1 for Expression of Yeast-Derived γ-Glutamylcysteine Synthetase Gene (GSH1)
A plasmid pET-GSH1 for expression of the GSH1 gene coding for γ-glutamylcysteine synthetase of the Candida utilis ATCC 22023 strain was constructed by the following procedures, and introduced into Escherichia coli. This strain is deposited at the American Type Culture Collection (12301 Parklawn Drive, Rockville, Md. 20852, United States of America) with the number of ATCC 22023, and can be provided therefrom.
(1-1) Construction of Plasmid pAUR-GSH1 for Expression of Yeast GSH1
First, a plasmid pAUR-GSH1 for expression of yeast GSH1 was constructed by the following procedure. The construction was entrusted to Takara Bio.
By PCR using Primer G (SEQ ID NO: 3) and Primer H (SEQ ID NO: 4), which were produced on the basis of the nucleotide sequence (SEQ ID NO: 1) of the GSH1 gene of the Candida utilis ATCC 22023 strain, as well as the chromosomal DNA of the ATCC 22023 strain as the template, the sequence containing the GSH1 gene was amplified. Primer G consisted of a region containing the start codon of the GSH1 gene in the chromosomal DNA of the ATCC 22023 strain, to which the KpnI recognition sequence and a partial sequence of the yeast expression plasmid pAUR123 (Takara Bio) were added at the 5′ end. Primer H consisted of a nucleotide sequence complementary to the C-terminal nucleotide sequence of the GSH1 gene, to which a nucleotide sequence complementary to the sequence coding for the His-tag, a nucleotide sequence complementary to the stop codon (TAA), the XbaI recognition sequence, and a partial sequence of pAUR123 were added. PCR was performed by using PrimeSTAR Max DNA Polymerase (Takara Bio) according to the protocol described in the attached manual. The amplified fragment was introduced into pAUR123 (Takara Bio) at the KpnI-XbaI site by using In-Fusion Advantage PCR Cloning Kit (Takara Bio) to construct the plasmid pAUR-GSH1 for expression of yeast GSH1.
(1-2) Construction of Plasmid pET-GSH1 for Expression of GSH1 of Escherichia coli
Then, a GSH1 expression plasmid pET-GSH1 for Escherichia coli was constructed by the following procedure.
Primer I (SEQ ID NO: 5) and Primer J (SEQ ID NO: 6) were purchased from Japan Bio Service, which were prepared on the basis of the nucleotide sequence of the GSH1 gene of the Candida utilis ATCC 22023 strain (SEQ ID NO: 1). Primer I consisted of a region containing the start codon of the GSH1 gene in the chromosomal DNA of the Candida utilis ATCC 22023 strain, to which a nucleotide sequence containing the SpeI recognition sequence was added at the 5′ end. Primer J consisted of a nucleotide sequence complementary to the nucleotide sequence outside from the stop codon of the GSH1 gene in pAUR-GSH1 mentioned above, to which a nucleotide sequence containing the XhoI recognition sequence was added at the 5′ end.
By PCR using Primer I and Primer J, as well as the aforementioned pAUR-GSH1 as the template, the sequence containing the GSH1 gene was amplified. PCR was performed by preparing 50 μl of a reaction mixture containing the plasmid DNA, 0.2 μmol/L each of the primers, 1.25 units of PrimeSTAR HS DNA Polymerase (Takara Bio), 10 μL of the 5× PrimeSTAR buffer (Takara Bio), and 2.5 mmol/L each of dNTPs (dATP, dGTP, dCTP, and dTTP), and subjecting the reaction mixture to warming at 98° C. for 10 seconds, then 30 cycles of 98° C. for 10 seconds, 56° C. for 5 seconds and 72° C. for 2 minutes, and further warming at 72° C. for 1 minute.
The reaction mixture after PCR (3 μl) was subjected to agarose gel electrophoresis to confirm that a DNA fragment of about 2.0 kb corresponding to the GSH1 gene fragment was amplified, and then the DNA fragment was purified from the remaining reaction mixture by using Ethachinmate (NIPPON GENE), and dissolved in 25 μl of dH2O. Then, the DNA fragment in the whole volume of the obtained DNA solution was digested with the restriction enzymes SpeI and XhoI, then purified by using MinElute Reaction Cleanup Kit (QIAGEN), and dissolved in 15 μl of Buffer EB (10 mM Tris-HCl, pH 8.5, QIAGEN).
The expression plasmid pET-21a(+) (1 μg, Novagen) was digested with the restriction enzymes NheI and XhoI, then purified by using MinElute Reaction Cleanup Kit, and dissolved in 15 μl of Buffer EB. Then, the DNA fragment in the whole volume of the obtained DNA solution was dephosphorylated with an alkaline phosphatase (calf intestine alkaline phosphatase, CIAP), then purified by using MinElute Reaction Cleanup Kit, and dissolved in 10 μl of Buffer EB.
The DNA fragment of about 2.0 kb containing the GSH1 gene obtained above, and the DNA fragment of about 5.4 kb of the expression plasmid pET-21a(+) (Novagen) obtained above were reacted at 16° C. for 30 minutes by using TaKaRa Ligation Kit Ver. 2.1 (Takara Bio), and thereby ligated. Competent cells of the Escherichia coli DH5α strain (Takara Bio) were transformed by the heat shock method using the above reaction mixture, and the transformants were applied on the LB [10 g/L of Bacto tryptone (Difco), 5 g/L of yeast extract (Difco), and 5 g/L of sodium chloride (Wako)] agar medium containing 100 μg/ml of ampicillin, and cultured overnight at 37° C.
From the grown colonies of the transformants, a plasmid was extracted by a known method, and the nucleotide sequence thereof was determined by a known method. The obtained plasmid was a plasmid consisting of the GSH1 gene derived from the Candida utilis ATCC 22023 strain having the sequence coding for the His-tag at the 3′ end, which was ligated to T7 promoter on the downstream side, and this plasmid was designated pET-GSH1. The nucleotide sequence of the GSH1 gene derived from the Candida utilis ATCC 22023 strain and the amino acid sequence encoded thereby are shown in SEQ ID NOS: 1 and 2, respectively.
Then, competent cells of the Escherichia coli Rosetta2(DE3)pLysS strain (Novagen) were transformed with pET-GSH1 by the heat shock method, and the transformants were applied on the LB agar medium containing 100 μg/ml of ampicillin and 30 μg/ml of chloramphenicol, and cultured overnight at 37° C. Plasmids were extracted from the grown colonies of the transformants in a known manner, and the structures thereof were analyzed by using restriction enzymes to confirm that the transformants harbored pET-GSH1. The Escherichia coli Rosetta2(DE3)pLysS strain harboring pET-GSH1 was designated Escherichia coli Rosetta2(DE3)pLysS/pET-GSH1.
2) Purification of C-Terminal His-Tag-Added Recombinant Gsh1Escherichia coli Rosetta2(DE3)pLysS/pET-GSH1 obtained as described above was inoculated into 3 mL of the LB medium containing 100 μg/ml of ampicillin and 30 μg/ml of chloramphenicol contained in a test tube, and cultured at 37° C. for 16 hours with shaking. The obtained culture broth (2 ml) was inoculated into 100 ml of the LB medium contained in a Sakaguchi flask. Culture was performed at 37° C. for 2 hours with shaking, then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mmol/L, and culture was further continued at 30° C. for 4 hours. The culture broth was centrifuged to obtain wet cells.
The wet cells were suspended in 10 ml of a 100 mmol/L Tris-hydrochloric acid buffer (pH 8.0) containing 300 mM sodium chloride, the cells were disrupted by ultrasonication, and the suspension was centrifuged. From the obtained supernatant, His-tag added recombinant Gsh1 was purified by using a His-tag-added protein purification kit, Ni Sepharose 6 Fast Flow (GE Healthcare), according to the protocol described in the attached manual, and then desalted by using PD-10 column (GE Healthcare) according to the protocol described in the attached manual. This purified and desalted Gsh1 was used for the following experiments as purified Gsh1.
3) Analysis of Substrate Specificity of GSH1A reaction mixture (pH 8.0, 200 μl) containing the purified recombinant GSH1 obtained above (24.6 μg), 100 mmol/L of Tris-HCl (pH 8.0), 12.5 mmol/L of Abu, 12.5 mmol/L of glutamic acid, 12.5 mmol/L of adenosine triphosphate (ATP), 12.5 mmol/L of magnesium sulfate, and 2 mmol/L of dithiothreitol (DTT) was prepared, and the reaction was performed at 37° C. for 16 hours.
After completion of the reaction, the reaction product was analyzed by HPLC. The analysis conditions were as follows.
(1) HPLC: HITACHI L-2000 Series(2) Isolation column: Synergi 4μ Hydro-RP 80A; internal diameter, 4.6 mm; length, 250 mm; particle size, 4 μm (Phenomenex)
(3) Column temperature: 40° C.
(4) Mobile phase A: 50 mM phosphate buffer (pH 2.5)
(5) Mobile phase B: acetonitrile
(6) Flow rate: 1.0 ml/minute
(7) Elution conditions: Elution was performed by using a mixture of the mobile phase A and the mobile phase B. The ratio of the mobile phase B in the mixture was as follows: 0 minute (0%), 0 to 5 minutes (0 to 2.5%), 5 to 15 minutes (2.5%), 15 to 30 minutes (2.5 to 40%), 30 to 30.1 minutes (40 to 0%), and 30.1 to 50 minutes (0%).
As a result of the aforementioned measurement, the retention time of the peak of the reaction product agreed with that of a γ-Glu-Abu sample, and it was judged that the product was γ-Glu-Abu. As a result of quantification, γ-Glu-Abu concentration was found to be 10.6 mM.
These results revealed that GSH1 of yeast recognized Abu as a substrate.
Example 9 Effect of Addition of Abu to Saccharomyces cervisiae GSH1 Expression-Enhanced StrainSince it was revealed by the investigation performed in Example 8 mentioned above based on in vitro enzymatic reaction that GSH1 was responsible for an enzymatic reaction using Abu and Glu as substrates, it was then investigated whether this reaction would actually occur in the yeast cells.
1) Acquisition of Uracil Auxotrophic Strain (Ura3 Mutant)A uracil auxotrophic strain was obtained by introducing an URA3-neighboring DNA except for the URA3 gene into a Saccharomyces cervisiae wild-type strain monoploid (Matα type), and disrupting the URA3 gene, as shown below.
First, a 500-bp upstream region of URA3 was amplified by PCR using the primers of SEQ ID NO: 7 (gataaggaga atccatacaa) and SEQ ID NO: 8 (gtgagtttag tatacatgca tttacttata atacagtttt gatttatctt cgtttcctgc), and the chromosomal DNA of the aforementioned wild-type strain as the template. Furthermore, a 500-bp downstream region of URA3 was also amplified using the primers of SEQ ID NO: 9 (aaaactgtat tataagtaaa) and SEQ ID NO: 10 (cacttatttg cgatacagaa). As for PCR conditions, a cycle consisting of thermal denaturation at 94° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extension at 72° C. for 1 minute was repeated 25 times. Then, overlap PCR was performed by using the above two kinds of DNA fragments purified by ethanol precipitation as templates and the primers of SEQ ID NO: 11 (gataaggaga atccatacaa) and SEQ ID NO: 12 (cacttatttg cgatacagaa) to obtain a 1-kb DNA fragment consisting of the 500-bp upstream region and 500-bp downstream region of the URA3 gene ligated together. The wild-type strain was transformed with this DNA fragment, and then cultured overnight in the SD medium to which uracil was added, and the cells were applied to 5-FOA plate medium. The ura3Δ0 strain was obtained from the resulting transformants. This strain was given a private number AJ14956, and was deposited at the independent administrative agency, Agency of Industrial Science and Technology, International Patent Organism Depository (Tukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Aug. 18, 2010, and assigned an accession number of FERM P-22000. Then, the deposit was converted to an international deposit under the provisions of the Budapest Treaty, and assigned an accession number of FERM BP-11299.
2) Preparation of Template Plasmid for Promoter SubstitutionFirst, the URA3 locus was amplified by PCR using primers of SEQ ID NO: 13 (atagcatgct cataaaattg ataaggaga) and SEQ ID NO: 14 (atagaattca ggacgtcatt agtggcgaa) and the chromosomal DNA of a Saccharomyces cerevisiae wild-type strain as the template (thermal denaturation: 94° C. for 10 seconds, annealing: 50° C. for 10 seconds, extension: 72° C. for 1 minute, 25 cycles). The resulting DNA fragment was purified by ethanol precipitation, and then digested with SphI and EcoRI, and the product was inserted into the plasmid pUC19 at the SphI-EcoRI sites to obtain pUC19-URA3. Then, the ADH1 promoter region was amplified from the chromosomal DNA of the Saccharomyces cerevisiae wild-type strain using the primers of SEQ ID NO: 15 (atactgcaga taatcgatta attttttttt ctttc) and SEQ ID NO: 16 (atactgcaga agtagataat tacttcctt). This DNA fragment was digested with PstI, and inserted into pUC19-URA3 digested with PstI and treated with CIAP at the PstI site to obtain pUC19-ADH1p-URA3. It was confirmed that ADH1p was correctly inserted in the forward direction with respect to the URA3 gene by sequencing the neighboring region thereof. In a similar manner, the ADH1 promoter amplified by using the primers of SEQ ID NO: 17 (atagacgtct aatttttttt tctttc) and SEQ ID NO: 18 (atagacgtct gttttatatt tgttgtaaa) was digested with AatII, and inserted into pUC19-ADH1p-URA digested with AatII and treated with CIAP at the AatII site to obtain pUC19-ADH1p-URA3-ADH1p. It was confirmed that ADH1p was correctly inserted in the forward direction with respect to the URA3 gene by sequencing the neighboring region thereof.
3) Introduction of ADH1 Promoter into GSH1 Gene on Chromosome
PCR was performed by using the primer of SEQ ID NO: 19 (TATTGCCCCAGTGTTCCCTCAACAACCTTGGTAGTTGGAGCGCAATTAGCGTATCCT GTACCATACTAATTCTCTTCTGCTCTTAACCCAACTGCACAGA), which has a GSH1 upstream sequence at the 5′ end, the primer of SEQ ID NO: 20 (ATACCTTCATCCCTTATGTGTTCATTGTACGTCCTAGACTCAAACCACTGCAAAGGC GTGCCCAAAGCTAAGAGTCCCATTGTATATGAGATAGTTGATT), which has a part of a sequence in ORF starting from the start codon of the GSH1 gene, and pUC19-ADH1p-URA3-ADH1p as the template (thermal denaturation: 94° C. for 10 seconds, annealing: 60° C. for 10 seconds, extension: 72° C. for 4 minutes) to prepare a DNA fragment having URA3 between ADH1p promoters. The ura3Δ0 strain was transformed with this DNA fragment, and plated on an SD plate medium to obtain transformants, and a strain in which the GSH1 promoter was replaced with the ADH1 promoter-URA3-ADH1 promoter was obtained from the transformants.
4) Elimination of URA3 Selective Marker and Substitution of Promoter for GSH1 GeneThe strain in which the ADH1p promoter-URA3-ADH1 substitutes for the GSH1 promoter was cultured overnight in a uracil-supplemented SD medium, and an appropriate volume of the culture was applied to 5-FOA plate medium. From the grown colonies, a strain in which URA3 was removed, and the GSH1 promoter was replaced with the ADH1 promoter by homologous recombination between the introduced ADH1 promoters, AG1-ura3Δ0 strain, was obtained. Furthermore, by introducing a DNA amplified by using a wild-type genome as the template and the primers of SEQ ID NO: 21 (AGTTACAGCAATGAAAGAGCAGAGCGAGAG) and SEQ ID NO: 22 (ATTACTGCTGCTGTTCCAGCCCATATCCAA) into the above strain, a strain in which URA3 was returned to wild-type, and the GSH1 promoter was replaced with the ADH1 promoter, was obtained. This strain was designated AG1 strain. In a similar manner, by introducing a DNA amplified by using a wild-type genome as the template and the primers of SEQ ID NO: 23 (AGTTACAGCAATGAAAGAGCAGAGCGAGAG) and SEQ ID NO: 24 (ATTACTGCTGCTGTTCCAGCCCATATCCAA) into the ura3Δ0 strain, AJ14956, a strain in which URA3 was returned to wild-type was obtained. This strain was designated Control strain.
5) Effect of Addition of Abu to Control Strain and AG1 StrainThen, one loop each of the Control strain and the AG1 strain was inoculated into the SD medium (50 ml in a 500 ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hours with shaking at a velocity of 120 rpm. Absorbance of the obtained culture broth was measured, the culture broth was inoculated into the SD medium containing Abu at various concentrations (50 ml in a 500 ml-volume Sakaguchi flask) so that OD660 was 0.01 at the start of the culture, and the yeast cells were cultivated at 30° C. for 19 hours with shaking at a velocity of 120 rpm. Cells corresponding to 20 OD units were obtained from the resulting culture broth by centrifugation. Thereafter, an extract was obtained, and intracellular contents of the compounds were measured in the same manner as that of Example 3
As a result, as shown in the following tables, in the AG1 strain, the γ-Glu-Abu content markedly increased compared with the Control strain. On the basis of this result, it was revealed that GSH1 recognized Abu as a substrate also in the cells and produced γ-Glu-Abu from Abu. In addition, when the AG1 strain was cultivated in the SD medium not containing Abu, intracellular γ-Glu-Abu content was lower than the detection limit, and therefore it was also found that the intracellular γ-Glu-Abu content was not necessarily increased only by the enhancement of expression of GSH1, and supply of Abu was important.
Effect of addition of Abu to a GSH1 expression-enhanced strain of Candida utilis can also be confirmed in the same manner as that of Example 7. Specifically, by using Candida utilis NBRC 0988 as a parent strain and the known Cre-loxP system, an uracil auxotrophic CUD4F strain in which the URA3 gene on the chromosome is deleted can be obtained (Shigeru Ikushima et al., 2009, Biosci. Biotechnolo. Biochem., 73(4), 879-884). Since information on the gene sequences required for the genetic manipulation is described in WO95/32289, the paper of U. Gueldener et al. (Nucleic Acids Research, 2002, Vol. 30, No. 6, e23), the paper of Gritz L. and Davis J. (Gene, 25, 179-188 (1983)), and so forth, various tools may be prepared on the basis of such sequence information.
A plasmid for expression of GSH1 of Candida utilis can be constructed by a known method as follows. The known plasmid pRI177 (Ryo Iwakiri et al., 2005, Yeast, 22, 1079-1087) can be digested with the restriction enzyme BamHI and purified in a conventional manner to obtain a linear plasmid. In addition, the method for preparing the plasmid YRpGAP, which is equivalent to the plasmid pR177, is also disclosed in Japanese Patent Laid-open (Kokai) No. 2006-75122.
Further, the ORF region of GSH1 is amplified by PCR using the plasmid pAUR-GSH1 containing the sequence of GSH1 of Candida utilis, which was constructed in Example 9, as the template, as well as Primer S (SEQ ID NO: 25, GCAGCCCGGGGGATCATGG-GGCTGCTATCATTAGG, 15 nucleotides of the 5′ end is a sequence homologous to the terminal sequence of the linear plasmid obtained by digestion with the restriction enzyme BamHI) and Primer T (SEQ ID NO: 26, TAGAACTAGTGGATCTTAA-GCCCTTTGGGTTGTTTATC, 15 nucleotides of the 5′ end is a sequence homologous to the terminal sequence of the linear plasmid obtained by digestion with the restriction enzyme BamHI). For this PCR, sequences homologous to the terminal sequences produced by digestion of pR177 with the restriction enzyme BamHI are added to the terminuses of Primers S and T. The PCR product can be purified in a conventional manner, and the purified PCR product and the linear plasmid can be ligated by using In-Fusion Advantage PCR Cloning Kit (Takara Bio). By choosing a plasmid having the objective sequence, autonomously replicable plasmid pCGSH1 containing the GSH1 region of Candida utilis can be constructed. The construction procedure of pCGSH1 is shown in
Further, the URA3 gene as the selection marker can also be introduced into pCGSH1 mentioned above by using In-Fusion Advantage PCR Cloning Kit. Specifically, the full length of pCGSH1 is amplified by PCR using Primer U (SEQ ID NO: 27, TTACGCCAAGCGCGCAATTA) and Primer V (SEQ ID NO: 28, TCATGGTCATAGCTGTTTCC) according to the protocol described in the attached manual. For this PCR, predetermined regions of the primers can be designed according to the protocol described in the attached manual, and a linear plasmid blunt-ended at a desired position can be prepared by using them. The URA3 gene to be introduced can be amplified by PCR using Primer W (SEQ ID NO: 29, GCGCGCTTGGCGTAACAAATAGCTCTCTACTTGCT, 15 nucleotides of the 5′ end constitute a sequence homologous to the terminal sequence of the linear plasmid), and Primer X (SEQ ID NO: 30, CAGCTATGACCATGAGCAATCTACAACTTCGAAA, 15 nucleotides of the 5′ end constitute a sequence homologous to the terminal sequence of the linear plasmid), which can be designed on the basis of the known sequence information (Luis Rodriguez et al., 1998, Yeast, 14, 1399-1406), as well as the genome of the NBRC 0988 strain as the template. In this PCR, by adding terminal sequence of the linear plasmid to the terminuses of the primers, the gene can be ligated with the plasmid by using In-Fusion Advantage PCR Cloning Kit. As described above, an autonomously replicable vector pCGSH1-URA3 for expression of GSH1 containing the URA3 gene as the selection marker can be constructed. Outline of the construction procedures of pCGSH1-URA3 is shown in
Then, pCGSH1-URA3 is introduced into the CUD4F strain by the known electroporation method for Candida utilis (Shigeru Ikushima et al., 2009, Biosci. Biotechnolo. Biochem., 73(4), 879-884). By spreading the obtained transformants on the SD medium and selecting a transformant having the objective plasmid from the grown strains, a GSH1 expression-enhanced strain can be obtained. If this GSH1 expression-enhanced strain is cultivated in the SDP medium containing Abu in the same manner as that of Example 7, γ-Glu-Abu is accumulated in the cells.
Example 11 Analysis of Substrate Specificity of Yeast GSH2Since it was found that a part of accumulated γ-Glu-Abu is metabolized into γ-Glu-Abu-Gly by a certain enzymatic reaction using γ-Glu-Abu as a substrate in yeast cells on the basis of the results of Examples 3, 4, and 5, possibility of side reaction of glutathione synthetase was investigated.
1) Construction of Plasmid pET-GSH2 for Expression of Glutathione Synthetase Gene (GSH2) Derived from Yeast
An expression plasmid pET-GSH2 for the GSH2 gene coding for glutathione synthetase of the Saccharomyces cervisiae S288C strain was constructed by the following procedures, and introduced into Escherichia coli.
(1-1) Construction of Plasmid pAUR-GSH2 for Expression of Yeast GSH2
First, an expression plasmid pAUR-GSH2 for yeast was constructed by the following procedure. The construction was entrusted to Takara Bio.
By PCR using Primer A (SEQ ID NO: 33) and Primer B (SEQ ID NO: 34), which were produced on the basis of the nucleotide sequence (SEQ ID NO: 31) of the GSH2 gene of the Saccharomyces cervisiae S288C strain, as well as the chromosomal DNA of the S288C strain as the template, the sequence containing the GSH2 gene was amplified. Primer A consisted of a region containing the start codon of the GSH2 gene in the chromosomal DNA of the S288C strain, to which the KpnI recognition sequence and a partial sequence of the yeast expression plasmid pAUR123 (Takara Bio) were added at the 5′ end. Primer B consisted of a nucleotide sequence complementary to the C-terminal nucleotide sequence of the GSH2 gene, to which a nucleotide sequence complementary to the sequence coding for the His-tag, a nucleotide sequence complementary to the stop codon (TAA), the XbaI recognition sequence, and a partial sequence of pAUR123 were added. PCR was performed by using PrimeSTAR Max DNA Polymerase (Takara Bio) according to the protocol described in the attached manual. The amplified fragment was introduced into the expression plasmid pAUR123 for yeast (Takara Bio) at the KpnI-XbaI site by using In-Fusion Advantage PCR Cloning Kit (Takara Bio) to construct the expression plasmid pAUR-GSH2 for expression of GSH2 in yeast.
(1-2) Construction of Plasmid pET-GSH2 for Expression of GSH2 in Escherichia coli
Then, a GSH2 expression plasmid pET-GSH2 for Escherichia coli was constructed by the following procedure.
Primer C (SEQ ID NO: 35) and Primer D (SEQ ID NO: 36) were purchased from Japan Bio Service, which were produced on the basis of on the nucleotide sequence of the GSH2 gene of the Saccharomyces cervisiae S288C strain. Primer C consisted of a region containing the start codon of the GSH2 gene in the chromosomal DNA of the Saccharomyces cervisiae S288C strain, to which a nucleotide sequence containing the NdeI recognition sequence was added at the 5′ end. Primer D consisted of a nucleotide sequence complementary to the nucleotide sequence outside from the stop codon of the GSH2 gene in pAUR-GSH2 mentioned above, to which the XhoI recognition sequence was added at the 5′ end.
By PCR using Primer C and Primer D, as well as the aforementioned pAUR-GSH2 as the template, the sequence containing the GSH2 gene was amplified. PCR was performed by preparing 50 μl of a reaction mixture containing the plasmid DNA, 0.2 μmol/L each of the primers, 1.25 units of PrimeSTAR HS DNA Polymerase (Takara Bio), 10 μL of the 5× PrimeSTAR buffer (Takara Bio), and 2.5 mmol/L each of dNTPs (dATP, dGTP, dCTP, and dTTP), and subjecting the reaction mixture to warming at 98° C. for 10 seconds, then 30 cycles of 98° C. for 10 seconds, 56° C. for 5 seconds and 72° C. for 2 minutes, and further warming at 72° C. for 1 minute.
The reaction mixture after PCR (3 μl) was subjected to agarose gel electrophoresis to confirm that a DNA fragment of about 1.5 kb corresponding to the GSH2 gene fragment was amplified, and then the DNA fragment was purified from the remaining reaction mixture by using Ethachinmate (NIPPON GENE), and dissolved in 25 μl of dH2O. Then, the DNA fragment in the whole volume of the obtained DNA solution was digested with the restriction enzymes NdeI and XhoI, then purified by using MinElute Reaction Cleanup Kit (QIAGEN), and dissolved in 15 μl of Buffer EB (10 mM Tris-HCl, pH 8.5, QIAGEN).
The expression plasmid pET-21a(+) (1 μg, Novagen) was digested with the restriction enzymes NdeI and XhoI, then purified by using MinElute Reaction Cleanup Kit, and dissolved in 15 μl of Buffer EB. Then, the DNA fragment in the whole volume of the obtained DNA solution was dephosphorylated with an alkaline phosphatase (calf intestine alkaline phosphatase, CIAP), purified by using MinElute Reaction Cleanup Kit, and dissolved in 10 μl of Buffer EB.
The DNA fragment of about 1.5 kb containing the GSH2 gene obtained above, and the DNA fragment of about 5.4 kb of the expression plasmid pET-21a(+) obtained above were reacted at 16° C. for 30 minutes by using TaKaRa Ligation Kit Ver. 2.1 (Takara Bio), and thereby ligated. Competent cells of the Escherichia coli DH5α strain (Takara Bio) were transformed by the heat shock method using the above reaction mixture, and the transformants were applied on the LB [10 g/L of Bacto tryptone (Difco), 5 g/L of yeast extract (Difco), and 5 g/L of sodium chloride (Wako)] agar medium containing 50 μg/ml of ampicillin, and cultured overnight at 37° C.
From the grown colonies of the transformants, a plasmid was extracted by a known method, and the nucleotide sequence thereof was determined by a known method. The obtained plasmid was a plasmid consisting of the GSH2 gene derived from the Saccharomyces cervisiae S288C strain having the sequence coding for the His-tag at the 3′ end, which was ligated to the T7 promoter on the downstream side, and this plasmid was designated pET-GSH2. The nucleotide sequence of the GSH2 gene derived from the Saccharomyces cervisiae S288C strain and the amino acid sequence encoded thereby are shown in SEQ ID NOS: 31 and 32, respectively.
Then, competent cells of the Escherichia coli BL21(DE3) strain (Novagen) were transformed with pET-GSH2 by the heat shock method, and the transformants were applied on the LB agar medium containing 50 μg/ml of ampicillin, and cultured overnight at 37° C. Plasmids were extracted from grown colonies of the transformants in a known manner, and the structures thereof were analyzed by using restriction enzymes to confirm that the transformants harbored pET-GSH2. The Escherichia coli BL21(DE3) strain harboring pET-GSH2 was designated Escherichia coli BL21(DE3)/pET-GSH2.
2) Purification of C-Terminus His-Tag-Added Recombinant Gsh2Escherichia coli BL21(DE3)/pET-GSH2 obtained as described above was inoculated into 3 mL of the LB medium containing 100 μg/ml of ampicillin in a test tube, and cultured at 37° C. for 16 hours with shaking. The obtained culture broth (2 ml) was inoculated into 100 ml of the LB medium contained in a test tube. Culture was performed at 37° C. for 2 hours with shaking, then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mmol/L, and culture was further continued at 30° C. for 4 hours. The culture broth was centrifuged to obtain wet cells.
The wet cells were suspended in 10 ml of a 100 mmol/L Tris-hydrochloric acid buffer (pH 8.0) containing 300 mM sodium chloride, the cells were disrupted by ultrasonication, and the suspension was centrifuged. From the obtained supernatant, His-tag-added recombinant Gsh2 was purified by using a His-tag-added protein purification kit, Ni Sepharose 6 Fast Flow (GE Healthcare), according to the protocol described in the attached manual, and then desalted by using PD-10 column (GE Healthcare) according to the protocol described in the attached manual. Then, this sample was concentrated by using a centrifugal filtration membrane of 10 kDa cutoff (Amicon Ultra-0.5 mL 10K (catalog number, UFC501096), MILLIPORE) according to the protocol described in the attached manual. This purified, desalted and concentrated Gsh2 was used for the following experiments as purified Gsh2.
3) Production of γ-Glu-Abu-Gly Using Purified Gsh2By using the purified Gsh2 obtained above, possibility of production of γ-Glu-Abu-Gly using γ-Glu-Abu as the substrate was examined. A reaction mixture having the following composition was prepared, and the enzymatic reaction was performed at 30° C. for 22 hours.
[Composition of Reaction Mixture]
After completion of the reaction, the reaction product was analyzed by HPLC under the same conditions as those used in Example 8. As a result, the retention time of the peak of the reaction product agreed with that of a γ-Glu-Abu-Gly sample, and it was judged that the product was γ-Glu-Abu-Gly. As a result of quantification, γ-Glu-Abu-Gly concentration was found to be about 10 mM.
Example 12 Effect of Addition of Abu to GSH2-Disrupted StrainThe results of Example 11 revealed that γ-Glu-Abu served a substrate of glutathione synthetase of yeast to generate γ-Glu-Abu-Gly. On the other hand, the results of Examples 4 and 5 revealed that a part of intracellularly incorporated γ-Glu-Abu was converted into γ-Glu-Abu-Gly, but γ-Glu-Abu was also accumulated. On the basis of these results, it was considered that all of γ-Glu-Abu accumulated in the cells could not be metabolized into γ-Glu-Abu-Gly with the GSH2 activity of the yeast wild strain, but a lower activity thereof might provide larger accumulation of γ-Glu-Abu. Therefore, effect of addition of Abu to a GSH2-disrupted strain was investigated.
Specifically, Saccharomyces cervisiae S288C gsh2Δ0 strain was obtained by the following procedure. First, a region containing GSH2 including replacement with the kanamycin resistance gene cassette KanMX was amplified by using the primers of SEQ ID NO: 37 (CTAGTGAAAAACAAGAAGTA) and SEQ ID NO: 38 (GCCACATAGAAAAATCGATG) as well as the genome of GSH2-disrupted strain of YEAST KNOCK OUT STRAIN COLLECTION (Funakoshi, YCS1056) as the template. As for PCR conditions, a cycle consisting of thermal denaturation at 94° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extension at 72° C. for 3 minutes was repeated 25 times. Then, the DNA fragment was purified by ethanol precipitation, and then used to transform the S288C strain, and the cells were applied to a YPD plate medium containing G418. From the obtained transformants, the gsh2Δ0 strain was obtained.
Then, effect of addition of Abu to this strain was investigated in the same manner as that used in Example 3. First, one loop of the gsh2Δ0 strain was inoculated into the SD medium (50 ml in 500 ml-volume Sakaguchi flask), and cultured at 30° C. for 48 hours with shaking at a velocity of 120 rpm. Absorbance of the obtained culture broth was measured, the culture broth was inoculated into the SD medium or the SD medium containing 100 ppm of Abu as a final concentration (400 ml in a 2 L-volume conical flask with baffle fins), so that OD660 was 0.01 at the start of the culture (absorbance was measured by using DU640 SPECTROPHTOMETER, BECKMAN COULTER), and culture was performed at 30° C. for 65.75 hours with shaking by rotation at a velocity of 120 rpm. From the obtained culture broth, an extract was obtained, and intracellular content of γ-Glu-Abu was measured in the same manner as that of Example 3.
As a result, when the strain was cultured in the SD medium, the intracellular γ-Glu-Abu content was 128 ppm, but when it was cultured in the medium containing 100 ppm of Abu, the intracellular γ-Glu-Abu content was 10333 ppm. Thus, this result also revealed that γ-Glu-Abu was not accumulated only by deficiency of the GSH2 gene, but supply of Abu was important.
Example 13 Effect of Addition of Abu to GSH2-Disrupted Strain (2)By using a procedure using the cre-loxp system similar to that used for disruption of URA3 of Candida utilis in Example 10 with changing a part of the primer sequences, a GSH2-disrupted Candida utilis CUDF4 strain can be obtained.
Specifically, by using Primer N-59 (SEQ ID NO: 39, AAGTAGCCAATACAACCAGC, sequence of from the −57th to −38th region upstream of ORF of GSH2 of Candida utilis), Primer N60 (SEQ ID NO: 40, CTGCAGCGTACGAAGCTTCAGCTGGCGGGCCACTCACC-CACTCAACATCAC, 31 nucleotide from the 5′ end constitute a direct repeat for overlap PCR), Primer N-295 (SEQ ID NO: 41, GCTGTTTTAGACTCGTTTGC, a region of the 244th to 263rd positions of ORF of GSH2 of Candida utilis), Primer N-296 (SEQ ID NO: 42, CTGCAGCGTACGAAGCTTCAGCTGGCGGCCAGAAGATTCAGACACCGGGA, 29 nucleotides from the 5′ end constitute a direct repeat for overlap PCR), Primer N-61 (SEQ ID NO: 43, ATTAGGTGATATCAGATCCACTAGTGGCCTGGTTTCTTAAGATCTATTCC, 30 nucleotides from the 5′ end constitute a direct repeat for overlap PCR), and Primer N-62 (SEQ ID NO: 44, TAAATGCGGCTCCATCTATTG, region up to the nucleotide of the +18th position downstream of ORF of GSH2 of Candida utilis) instead of the primers IM-59, IM60, IM295, IM296, IM61, and IM-62 used for obtaining the CUDF4 strain described in Shigehito IKUSHIMA et al., Biosci. Biotechnol. Biochem., 73 (4), 879-884, 2009, respectively, a cassette for disruption of GSH2 of Candida utilis can be prepared. By using this cassette for disruption in the same manner as that described in the reference, GSH2 of the CUDF4 strain is disrupted. In addition, if 1 mM GSH and a required amount of uracil are added to the medium in the transformation step and the culture step, the acquisition rate of the strain may be improved. Whether a strain obtained as described above is the objective GSH2-disrupted strain can be confirmed by PCR. Further, by transforming such a GSH2-disrupted strain with pCGSH1-URA3 in the same manner as that of Example 10, a strain of which expression of GSH1 is enhanced, and GSH2 is disrupted can be obtained. If this strain is cultured in a medium containing Abu, a marked amount of γ-Glu-Abu is accumulated in the cells.
Example 14 Search of Intracellular Abu Synthesis EnzymeAlthough the metabolic pathways of Saccharomyces cerevisiae have been well studied, any enzyme that biosynthesizes Abu is not known. However, the inventors of the present invention estimated that an enzyme reported as aminotransferase for another substrate might have the activity for converting AKB (α-ketobutyrate (α-ketobutyric acid)) into Abu, in view of the fact that substrate recognition of aminotransferases was comparatively ambiguous, which had been confirmed in researches using other microorganisms. Therefore, there were bred a strain highly expressing BAT1, reported to be responsible for the transamination reaction of BCAA (branched chain amino acid), and a strain highly expressing UGA1, reported to be responsible for the transamination reaction of GABA (γ-aminobutyric acid).
1) Construction of BAT1 and UGA1 Expression VectorsFirst, in a conventional manner, a constitutive expression promoter of yeast, ADH1p, was introduced into the plasmid pYES2 (Invitrogen), which is a yeast-Escherichia coli shuttle vector. Specifically, by PCR using the genome prepared from a yeast wild strain as the template, as well as primers of SEQ ID NO: 45 (ATAACCGGTGGGTGTACAATATGGACTTC) and SEQ ID NO: 46 (ATAAAGCTTTGTATATGAGATAGTTGATT), the promoter region of ADH1 was amplified (a cycle consisting of thermal denaturation at 94° C. for 10 seconds, annealing at 50° C. for 10 seconds, and extension at 72° C. for 1 minute was repeated 25 times). The obtained DNA fragment was purified by ethanol precipitation, then digested with the restriction enzymes HindIII and AgeI, and inserted into the plasmid pYES2 at the HindIII-AgeI site to obtain pYES2-ADH1p.
Then, in order to insert ORF regions of the genes into this pYES2-ADH1p, amplification products of the genes were each subcloned into the pT7 vector. Specifically, by PCR using the genome prepared from a Saccharomyces cervisiae wild strain as the template, as well as the primers of SEQ ID NO: 47 (GGATCCATGTTGCAGAGACATTCC) and SEQ ID NO: 48 (TCTAGATTAGTTCAAGTCGGC), or the primers of SEQ ID NO: 49 (AAGCTTACAGACAAGAAACCGTC) and SEQ ID NO: 50 (TCTAGAGGCCTCGCTAATATAC), the ORF regions of BAT1 and UGA1 were amplified, respectively. The obtained BAT1 amplification product was digested with the restriction enzymes BamHI and XbaI, and inserted into the pT7 vector at the BamHI-XbaI site to obtain pT7-BAT1. The UGA1 amplification product was digested with the restriction enzymes HindIII and XbaI, and inserted into the pT7 vector at the HindIII-XbaI site to obtain pT7-UGA1.
pT7-BAT1 obtained as described above was treated with the restriction enzymes BamHI and XbaI, and the DNA fragment of BAT1 was purified by separation based on electrophoresis and excision of the objective gene, and introduced into the plasmid pYES2-ADH1p at the BamHI-XbaI site. Further, pT7-UGA1 was treated with the restriction enzymes HindIII and XbaI, and the DNA fragment of UGA1 was purified by separation based on electrophoresis and excision of the objective gene, and introduced into the plasmid pYES2-ADH1p at the HindIII-XbaI site. As described above, a BAT1 high expression vector, pYES2-ADH1p-BAT1, and a UGA1 high expression vector, pYES2-ADH1p-UGA1, were prepared.
The nucleotide sequences of BAT1 and UGA1 are shown in SEQ ID NOS: 51 and 53, respectively. Further, the amino acid sequences encoded by these genes are shown in SEQ ID NOS: 52 and 54, respectively.
2) Breeding of S288Cura3Δ0 strain
In the same manner as that of Example 9, there was bred S288Cura3Δ0 strain corresponding to the S288C strain in which the ORF region of the URA3 gene was deleted.
3) Breeding of Various Expression StrainsBy transforming S288Cura3Δ0 bred in 2) with each of the expression vectors constructed in 1), strains highly expressing each of the genes were bred. Specifically, competent cells of S288Cura3Δ0 were prepared by using Frozen EZ Yeast Transformation II Kit of Zymo Research, and each of the expression vectors was introduced into the cells to obtain S288C/pYES2-ADH1p strain, S288C/pYES2-ADH1p-BAT1 strain, and S288C/pYES2-ADH1p-UGA1 strain.
4) Evaluation of Obtained StrainsThe aforementioned strains were evaluated by culture in the SD medium in the same manner as that of Example 3. The results are shown in Table 13.
As a result, increase of the intracellular Abu content was observed in the BAT1 high expression strain and the UGA1 high expression strain. Further, since Abu synthesized within the cells is used as a substrate for γ-Glu-Abu and γ-Glu-Abu-Gly, the total amounts of these three kinds of compounds were compared in terms of molar concentration. The total amounts were calculated to be 2.46 μmol/g-DCW for the control strain, 7.06 μmol/g-DCW for the BAT1 high expression strain, and 3.91 μmol/g-DCW for the UGA1 high expression strain, and thus it was revealed that the high expression of the various aminotransferases provided increase of amount of intracellular Abu-containing compounds, i.e., increase of Abu biosynthesis ability. Further, in the same manner as that used for obtaining the S288Cgsh2Δ0 strain from the S288Cura3Δ0 strain as a parent strain in Example 12, S288Cura3Δ0gsh2Δ0 strain was obtained. By preparing competent cells of this strain and introducing each of the expression vectors into the cells, S288Cgsh2Δ0/pYES2-ADH1p strain and S288Cgsh2Δ0/pYES2-ADH1p-BAT1 strain were obtained. If the latter strain is cultured in the SD medium, it accumulates γ-Glu-Abu.
Example 15 Effect of High Expression of GSH1 and High Expression of AminotransferaseSince the effect of increasing the intracellular Abu-containing compounds was provided by increase of the activity of aminotransferase, especially increase of the activity of BAT1, in the aforementioned investigation, effect of combination thereof with high expression of GSH1 was examined. The uracil auxotrophic strain obtained in Example 9, AG1-ura3Δ0, was transformed with pYES2-ADH1p-BAT1 prepared in Example 14 to breed a BAT1 and GSH1 high expression strain, AG1/pYES2-ADH1p-BAT1 strain. In the same manner as that of Example 3, this strain was cultured in the SD medium, and the γ-Glu-Abu content in the cells and the γ-Glu-Abu content in solid content of extract were calculated. As a result, the AG1/pYES2-ADH1p-BAT1 strain contained 1813 ppm of γ-Glu-Abu based on the dry cell weight, and the extract obtained from the cells of the strain contained about 4560 ppm of γ-Glu-Abu based on solid content.
Example 16 Yeast Extract Added with Abu and Treated with γ-glutamyltransferaseA 1% aqueous solution of yeast extract containing regent GSH (Wako Pure Chemical Industries) having a GSH content of about 8% based on the solid content was prepared, and adjusted to pH 7.0 with NaCH. Powder Abu was added to this solution at a final concentration of 800 ppm, 1600 ppm, or 8000 ppm in the aqueous solution to prepare test samples. Further, the yeast extract aqueous solution not containing Abu was used as a control. γ-GTP (γ-glutamyltranspeptidase from equine kidney; Sigma; code, G9270-100UN) was added to these test samples at 0.05 mg/ml, and the enzymatic reaction was allowed at 37° C. for 120 minutes. The reaction mixture was immediately cooled on ice after the reaction, and the γ-Glu-Abu content was measured. Further, the solid content was measured by using a part of the reaction mixture, and the content of γ-Glu-Abu produced by the enzymatic reaction based on the solid content was calculated.
As a result, as shown in
First, samples for organoleptic evaluation were prepared by the following procedure. In the same manner as that used in Example 4, one loop of the S288C strain was inoculated into the SD medium (50 ml in 500 ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hours with shaking at a velocity of 120 rpm. Absorbance of the obtained culture broth was measured, the culture broth was inoculated into the SD medium (400 ml in 2 L-volume conical flask with baffle fins, 4 flasks) or the SD medium containing 200 ppm of γ-Glu-Abu as a final concentration (400 ml in 2 L-volume conical flask with baffle fins, 4 flasks), so that OD660 was 0.01 at the start of the culture (absorbance was measured by using DU640 SPECTROPHTOMETER, BECKMAN COULTER), and the yeast cells were cultivated at 30° C. for 19 hours with shaking by rotation at a velocity of 120 rpm. In the same manner as that used in Example 4, an extract was obtained from the obtained cells, and the γ-Glu-Abu concentration in the extract, and the solid content of the extract were obtained. As a result, the γ-Glu-Abu concentration in the extract prepared in the γ-Glu-Abu addition experiment was about 1,000 ppm, and the solid content thereof was about 0.59% (Extract 1). On the other hand, the solid content of the extract prepared in the no addition experiment was about 1.00% (Extract 2).
Then, kokumi of the following samples was evaluated by six special panelists according to the following method.
Control sample: Aqueous solution containing 0.2% of MSG, and 0.5% of NaCl
Sample 1: Control sample to which Extract 1 is added so that γ-Glu-Abu content is about 40 ppm
Sample 2: Control sample to which Extract 2 is added so that solid content is the same as that of Extract 1 added to Sample 1
As a result, all the panelists evaluated that stronger kokumi of the initial taste was obtained with Sample 1. On the basis of this result, it was confirmed that γ-Glu-Abu exhibited the characteristic thereof, initial taste, even in yeast extract.
Example 18 Organoleptic Evaluation of Yeast Extract Containing γ-Glu-Abu (2)One loop of the Saccharomyces cervisiae AJ14892 strain (Japanese Patent Laid-open (Kokai) No. 2008-61525) that accumulates γ-glutamylcysteine (γ-GC) was inoculated into the SD medium (50 ml in 500 ml-volume Sakaguchi flask, 4 flasks), and cultivated at 30° C. for 48 hours with shaking at a velocity of 120 rpm. Absorbance of the obtained culture broth was measured, the culture broth was inoculated into the SD medium (400 ml in a 2 L-volume conical flask with baffle fins, 4 flasks), so that OD660 was 0.01 at the start of the culture, and the yeast cells were cultivated at 30° C. with shaking by rotation at a velocity of 120 rpm. As for the culture time, residual sugar and absorbance were periodically measured, and the culture was performed for about 42 hours so that the absorbance became about 1.8, which is the absorbance obtained when the S288C strain was cultured in the SD medium for 19 hours. In the same manner as that used in Example 4, an extract was obtained from the cells, and the solid content of the extract were obtained. As a result, the solid content was found to be about 0.71% (Extract 3). The γ-GC content in Extract 3 was about 390 ppm. As described above, Extract 3 having a γ-GC content of about 5.5% based on the solid content was obtained.
Then, kokumi of the following samples was evaluated by six special panelists according to the following method.
Control sample: aqueous solution containing 0.2% of MSG, and 0.5% of NaCl
Sample 3: Control sample to which Extract 3 is added so that solid content is the same as that of Extract 1 added to Sample 1
Sample 4: Control sample to which marketed GSH-rich yeast extract (Aromild UG8, Kohjin Co., Ltd.) was added so that solid content is the same as that of Extract 1 added to Sample 1
For the evaluation, kokumi titer of the control sample was defined as 0.0, and kokumi titer of Sample 4 was defined as 3.0. As a result, as shown in the following table, it was found that the γ-Glu-Abu-containing yeast extract (Sample 1) showed an organoleptic profile different from those obtained with the sample similarly containing a dipeptide, the γ-GC-rich yeast extract (Sample 3), and the sample containing a tripeptide, the GSH-rich yeast extract (Sample 4), and gave a high kokumi titer for the initial taste.
Then, effect of enhancement of α-ketobutyric acid-producing ability was examined. Although it was not known so far that α-ketobutyric acid might serve as a precursor of Abu within yeast cells, whether enhancement of α-ketobutyric acid-producing ability could increase the Abu-producing ability in yeast cells was examined, since activation of aminotransferase increased the Abu-producing ability in yeast cells.
First, in order to insert the ORF region of CHA1 coding for serine (threonine) deaminase into pYES2-ADH1p prepared in Example 13, an amplification product of CHA1 was subcloned into the pT7 vector. Specifically, by PCR using the genome prepared from a Saccharomyces cervisiae wild strain as the template, and the primers of SEQ ID NO: 55 (ATAAAGCTTAACCAGCGAGATGTCG) and SEQ ID NO: 56 (CTCTCTAGAGGGCAAATTGATGCTTC), the ORF region of CHA1 was amplified. The obtained CHA1 amplification product was digested with the restriction enzymes HindIII and XbaI, and inserted into the pT7 vector at the HindIII-XbaI site to obtain pT7-CHA1.
pT7-CHA1 obtained as described above was treated with the restriction enzymes HindIII and XbaI, and the DNA fragment of CHA1 was purified by separation based on electrophoresis and excision of the objective gene, and introduced into the plasmid pYES2-ADH1p at the HindIII-XbaI site. A CHA1 high expression vector pYES2-ADH1p-CHA1 was prepared as described above.
The nucleotide sequence of CHA1 is shown in SEQ ID NO: 57. Further, the amino acid sequence encoded by this gene is shown in SEQ ID NO: 58.
Then, the promoter region of BAT1 in the uracil auxotrophic strain bred in Example 9, AG1-ura3Δ0, was replaced with the promoter region of PGK1 according to the method of Sofyanovich et al. (Olga A. Sofyanovich et al., A New Method for Repeated “Self-Cloning” Promoter Replacement in Saccharomyces cerevisiae, Mol. Biotechnol., 48, 218-227 (2011)). The DNA fragment for the replacement of the promoter was prepared by amplification based on PCR using the pPUP plasmid described in the reference and the primers for replacement of BAT1, SEQ ID NO: 59 (GCCAGGCGGTTGATACTTTGTGCAGATTTCATACCGGCTGTCGCTATTATTACTGAT GAATTGGCTCTCTTTTTGTTTAATCTTAACCCAACTGCACAGA) and SEQ ID NO: 60 (TTGGATGCATCTAATGGGGCACCAGTAGCGAGTGTTCTGATGGAGAATTTCCCCAAC TTCAAGGAATGTCTCTGCAACATTGTTTTATATTTGTTGTAAA). The GSH1- and BAT1-enhanced strain of the uracil auxotrophic strain constructed as described above, AGB-ura3Δ0 strain, was transformed with the CHA1 high expression vector to breed a strain highly expressing GSH1, BAT1 and CHA1. Specifically, in the same manner as that used in Example 14, competent cells of the AGB-ura3Δ0 strain were prepared by using Frozen EZ Yeast Transformation II Kit of Zymo Research, and pYES2-ADH1p-CHA1 was introduced into the cells to obtain AGB-ura3Δ0/pYES2-ADH1p-CHA1 strain.
The aforementioned strain was evaluated by culture in the SD medium in the same manner as that of Example 14. As a result, this strain contained 2024 ppm of γ-Glu-Abu based on the dry cell weight.
Example 20 Effect of Disruption of PeptidaseThen, effect of disruption of an enzyme gene DUG2, reported to be involved in the decomposition of GSH, was investigated. The nucleotide sequence of DUG2 is shown in SEQ ID NO: 61, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 62.
First, by using the primer of SEQ ID NO: 63 having 80 nucleotides upstream from the start codon of DUG2 (TTAAGTGAAAAACTATTTCGAGAAACCGAACAACCCTGTAAGGAAAAGTGAAAAACG AGGGCAGAAGTAATTGTGAAATCGTTCATCATCTCATGGATCT), and the primer of SEQ ID NO: 64 having 80 nucleotides downstream from the stop codon of DUG2 (ACTAATTATCATTAGGTAGAGGCCTACATATGCAAATTGGGTATATATTAAGCACTT TAAAATCAATTGTTTGTAGTTGTAGATTCCCGGGTAATAACTG), the URA3 gene of a wild type strain was amplified. As for PCR conditions, a cycle consisting of thermal denaturation at 94° C. for 10 seconds, annealing at 50° C. for 10 seconds, and extension at 72° C. for 2 minutes was repeated 25 times. The AG1-ura3Δ0 strain was transformed with the obtained DNA fragment, and applied to the SD medium not containing uracil. A dug2Δ strain of the AG1-ura3Δ0 (henceforth referred to as AG1-dug2Δ0 strain) was obtained from the grown transformants. The AG1 strain and the AG1-dug2Δ0 strain were cultured in the SD medium containing 100 ppm of Abu in the same manner as that of Example 9. As a result, it was found that the AG1-dug2Δ0 strain contained a larger amount of γ-Glu-Abu. It was suggested that disruption of the enzyme that decomposed GSH was useful for accumulation of γ-Glu-Abu.
According to the present invention, a yeast containing γ-Glu-Abu and a yeast extract containing γ-Glu-Abu can be produced. The yeast extract containing the peptide has a superior effect of imparting kokumi, especially initial taste type kokumi.
Claims
1. A yeast extract containing 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract.
2. A yeast extract according to claim 1, which contains 0.5% or more of γ-Glu-Abu based on dry weight of the yeast extract.
3. A yeast extract according to claim 1, which contains 1.0% or more of γ-Glu-Abu based on dry weight of the yeast extract.
4. The yeast extract according to claim 1, wherein the yeast belongs to the genus Saccharomyces or Candida.
5. The yeast extract according to claim 2, wherein the yeast belongs to the genus Saccharomyces or Candida.
6. The yeast extract according to claim 3, wherein the yeast belongs to the genus Saccharomyces or Candida.
7. The yeast extract according to claim 4, which is obtained from Saccharomyces cervisiae or Candida utilis.
8. The yeast extract according to claim 5, which is obtained from Saccharomyces cervisiae or Candida utilis.
9. The yeast extract according to claim 6, which is obtained from Saccharomyces cervisiae or Candida utilis.
10. A method for producing a yeast extract containing γ-Glu-Abu, which comprises culturing a yeast in a medium to which a compound selected from Abu and γ-Glu-Abu is added, and preparing a yeast extract from the obtained cells.
11. The method according to claim 10, wherein the compound is added to the medium in an amount of 10 ppm or more in the case of Abu, or 1 ppm or more in the case of γ-Glu-Abu, and the yeast extract contains 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract.
12. The method according to claim 10, wherein the yeast belongs to the genus Saccharomyces or Candida.
13. The method according to claim 11, wherein the yeast belongs to the genus Saccharomyces or Candida.
14. The method according to claim 12, wherein the yeast is Saccharomyces cervisiae or Candida utilis.
15. The method according to claim 13, wherein the yeast is Saccharomyces cervisiae or Candida utilis.
16. The method according to claim 10, wherein the yeast has one or both of the following characteristics:
- (a) γ-glutamylcysteine synthetase activity is enhanced,
- (b) glutathione synthetase activity is attenuated.
17. A yeast having an increased γ-Glu-Abu content, which has been modified so that
- activity of aminotransferase or/and activity of α-ketobutyric acid synthetase are enhanced, and,
- activity of γ-glutamylcysteine synthetase is enhanced, or/and activity of glutathione synthetase is attenuated.
18. The yeast according to claim 17, wherein the aminotransferase is an enzyme encoded by the BAT1 gene.
19. The yeast according to claim 17, wherein the aminotransferase is an enzyme encoded by the UGA1 gene.
20. The yeast according to claim 17, wherein the α-ketobutyric acid synthetase is an enzyme encoded by the CHA1 gene.
21. The yeast according to claim 17, wherein a peptidase activity is further attenuated.
22. A yeast extract produced from the yeast according to claim 18.
23. A method for producing a yeast extract containing γ-Glu-Abu, which comprises allowing a γ-glutamyltransferase to act on a yeast extract raw material to which Abu has been added.
24. The method according to claim 23, wherein Abu is added in an amount of 0.1% or more based on dry weight of the yeast extract raw material, and the yeast extract contains 0.2% or more of Abu based on dry weight of the yeast extract.
Type: Application
Filed: Mar 15, 2013
Publication Date: Oct 24, 2013
Inventors: Hiroaki Nishiuchi (Kawasaki-shi), Misato Morita (Kawasaki-shi), Wataru Hoshino (Kawasaki-shi), Junko Yamazaki (Kawasaki-shi), Takayuki Ito (Kawasaki-shi), Kazuo Yamagishi (Kawasaki-shi)
Application Number: 13/838,065
International Classification: A23L 1/28 (20060101); C12P 13/14 (20060101);
