JP2954663B2 - Gene DNA involved in leucine analog resistance and responsible for leucine biosynthesis, and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a DNA having a LEU4 gene region derived from a leucine analog-resistant strain, a plasmid having the LEU4 gene region incorporated in a vector, and a transformant using the plasmid. The present invention relates to a method for producing yeast of the genus Saccharomyces and fermented foods and drinks using the yeast.

[Conventional technology]

In general, aroma is an important factor in determining the quality of alcoholic beverages and foods, and isoamyl acetate, which is a flavor ester, is one of highly preferred flavor components similar to fruit aroma, and is similar to ethyl caproate, for example. It is the main component of the scent of Ginjo sake, and its main ingredient is isoamyl alcohol.

However, in general, the production amount of these flavor components of practical yeasts is low, so when producing fermented foods and drinks rich in aroma, etc., artificial mutation is generated in the yeast to impart a high productivity of higher alcohols or esters. A method has already been known for producing a fragrant alcoholic beverage or the like using a mutant yeast having resistance to a leucine analog (Japanese Patent Laid-Open No. 62-6669).

[Problems to be solved by the invention]

As for the improvement of microorganisms producing fermented foods and beverages with aroma, the above is known.However, in the conventional mutation treatment method, isoamyl alcohol and isoamyl alcohol are selectively amplified by selectively amplifying a specific gene. It is virtually impossible to increase the amount of isoamyl acetate produced. In addition, there is a risk that excellent brewing characteristics are lost by the mutation treatment.

In general, auxotrophs such as amino acids are mainly used as markers for detecting plasmid vectors used in yeast recombinant DNA technology, but practical yeasts do not have auxotrophy and mutation It is difficult to impart auxotrophy with the use of liposomes, and the danger that useful traits are impaired cannot be ignored.

Thus, the use of a resistance gene to a drug such as G418 has been reported as a marker that can be selected without causing a mutation in such a yeast, but sake yeast has a resistance to such a drug. It is difficult to select high and efficient transformants.

[Means for solving the problem]

The present invention has been made for the purpose of solving the above-mentioned disadvantages all at once.

Therefore, the present inventors first focused on the following pathways for leucine biosynthesis in yeast.

In the leucine biosynthesis system, α-isopropylmalate synthase (IPM) which is the rate-limiting enzyme in this pathway
Is usually subject to feedback inhibition by the end product leucine. However, it was confirmed that one of the 5-5-5-trifluoroleucine resistant strains obtained by the present inventors did not undergo feedback inhibition of IPM by leucine, and that three types of IPM structural genes were used. Isozymes (LEU4, LEU7, LEU8) exist,
It was also confirmed that the LEU4 gene was mutated.

Focusing on the findings as described above, the present inventors have repeated studies using gene recombination technology for isoamyl alcohol and / or isoamyl acetate-producing yeast breeding. As a result, the LEU4 gene derived from a leucine analog-resistant strain was identified. When the yeast was transformed with the integrated plasmid, it was found that most of the transformants could be specifically selected as leucine analog resistant strains, and at the same time, these transformant strains were isolated in the medium by isoamyl alcohol and / or Alternatively, they have found that they accumulate more isoamyl acetate, and completed the present invention.

That is, the present invention relates to DN derived from leucine analog-resistant bacteria.
A, a plasmid incorporating the same, a method for transforming a yeast using the plasmid, a method for producing a yeast transformed with the plasmid, and a method for producing fermented foods and drinks using the yeast.

 Hereinafter, the present invention will be described in detail.

The DNA of the present invention is obtained from a microorganism having leucine analog resistance.

The term "leucine analog-resistant strain" used herein refers to a microorganism derived from a yeast of the genus Saccharomyces as a parent strain, and has a genetic property change such that the strain can grow on a medium containing a high concentration of leucine analog so that the parent strain cannot grow. Say microorganisms. Leucine analogs are substances having a structure similar to leucine, for example, 4-aza-D, L-
Leucine, 5-5-5-trifluoro-D, L-leucine and the like. The leucine analog resistance referred to in the present invention may be a resistance to at least one of leucine analogs.

An example of such a DNA donor is Saccharomyces cerevisiae 7-F-13 (FERM P-8236), but is not limited to this strain.

The method for extracting chromosomal DNA may be performed according to a conventional method. After culturing such a DNA donor microorganism, the microbial cells are collected, and this is, for example, the method of Marmur et al. Or the method of Rodriguez et al. What is necessary is just to extract according to literature 10 and 12).

Using the chromosomal DNA obtained in this manner to create a gene library according to a conventional method, plaque or colony hybridization using a probe,
Isolate the desired LEU4 gene.

After cloning in this manner, the DNA containing the LEU4 gene is ligated to a plasmid to form a recombinant DNA.

The yeast is transformed with the obtained plasmid,
Since the transformant according to the present invention has leucine analog resistance, only a target transformant can be specifically and positively selected by using a leucine analog-containing medium.

By using the transformant thus obtained, isoamyl alcohol and / or isoamyl acetate can be produced in a remarkable amount. Production of these aromatic components, and various fermented foods using these aromatic components (Ginjo sake, sake) , Wine, shochu, bread, cider, beer, amazake, etc.). In addition, by using the thus cloned LEU4 gene and / or a DNA fragment containing it as a marker, various target transformants obtained by genetic manipulation can be positively selected, and screening is extremely efficient. Not only oversight, but also very accurate without oversight or mistakes,
Very good.

In order to carry out the present invention having such excellent characteristics, first, after extracting chromosomal DNA, L
The EU4 gene is cloned. The LEU4 gene is cloned, for example, by using the chromosomal DNA described above in Reference 5.
A genomic library prepared according to the method described in Literature 11 is isolated by performing plaque or colony hybridization using a synthetic DNA probe prepared based on the wild-type LEU4 gene sequence reported in Literature 11 by the method described in Literature 5. can do.

As a host used for cloning, Escherichia coli Q359 (Reference 1), P2392 (Reference 2) and the like, and as a vector, a λ phage cloning vector EMBL3 (Reference 3) and λ2001 (Reference 4) can be used. However, other hosts and vectors can also be used as long as they achieve the purpose of the present cloning. Fragmentation of the chromosomal DNA and cleavage of the phage vector can be performed, for example, by a normal enzyme reaction using a restriction enzyme such as Sau3AI or BamHI.

Ligation of a DNA fragment and a phage vector is performed, for example, by T
4 It can be performed by a normal ligation reaction using an enzyme such as DNA ligase. The ligated DNA is introduced into phage particles by the method described in Reference 5, for example.
It can impart infectivity to E. coli. Also,
As a synthetic DNA probe, LEU described in Reference 11
From any part of the DNA sequence of the four genes, a synthetic DNA equivalent to a sequence of any length of 17 bases or more can be prepared and used.

The recombinant phage selected by the synthetic DNA probe was
For example, the phage is grown in large amounts by the method of Reference 5, DNA containing the LEU4 gene is recovered from the phage, and Escherichia coli vectors such as pBR322 (Reference 6) and pUC19 (Reference 7);
7 (Reference 8), YEp13 (Reference 9), or the like, or can be used for yeast transformation using only a chromosome fragment containing the LEU4 gene.

For example, when Sake Yeast Association No. 7 (JCM1818) is used, this yeast is cultured overnight in a YEPD medium and sterilized (121 ° C., 1 ° C.).
atm, 15 min) TE buffer (10 mM Tris-HCl, 1 mM E
DTA, pH 8.0), then with 0.2M lithium acetate at 30 ℃
After adding and mixing 10 μg of plasmid DNA (20 μ) to 100 μl of the bacterial solution incubated for 60 minutes, the incubation is continued for another 30 minutes. Next, an equal volume of 70% PEG-4000 was added and mixed, and the mixture was further incubated for 60 minutes.
Hold for a minute. After washing this bacterial solution three times with sterile water, 100
A colony growing on an SD agar medium (2% glucose, 0.67% yeast nitrogen base, 2% agar) containing μM 5-5-5-trifluoro-D, L-leucine was used to grow the colony. Selection can be easily performed using the leucine analog resistance of the subject as an index.

As an example of the transformant thus obtained, Saccharomyces cerevisiae X-7 (FERM P-11) which is a transformant of Saccharomyces cerevisiae K-7 (JCM1818) is used.
424).

The method for producing fermented foods and drinks using the Saccharomyces cerevisiae strain X-4 described above can be carried out in accordance with a conventionally and generally employed production method, and the present invention is singular in this respect.

In describing the present invention, the following documents are cited or referred to in the present specification.

References: 1. T. Maniatis, EfFritsch and J. Sambrook, Molecular
Cloning Cold Spring Habor (1982), 504-506 2.J. Gough and N. Murray, J. Mol. Biol., 166, 1-19. (198
3) 3. Frischauf AMet.al., J. Mol. Biol., 170, 827 (1983) 4. J. Karn, H. Matthes, M. Gait and S. Benner, Gene, 32, 217
224224 (1984) 5. Masanori Muramatsu ed .: “Lab-manual genetic engineering” P73-106 (1
988) 6. Keith WCPeden, Gene, 22, 277 (1983) 7. Yanisch-Perron, C, Vieria, J. and Messing, J., Gene, 3
3,103-119 (1985) 8.Struhl, K., DTStinchcomb, S.Scherer and RWDavi
s, Proc.Natl.Aced.Sci.US, 76,1035, (1979) 9.JRBroach, JNStrathern and JBHick, Gene, 8,121
~ 133 (1979) 10.Cryer, DR, R.Eccleshall and J.Marmur, Methods in
Cell Biology XII (ed. Prescott, DM) 39, Academic P
ress (1975) 11.James P. Beltzer et.al., J. Biol, Chem., 261, 5160-5
167 (1986) 12.Rodoriguez and Tait, Recombinant DNA techniques,
Addisson-Wesley Pub. Co. (1983) [Example] Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 (1) Preparation of chromosomal DNA Saccharomyces cerevisiae 7-F-13 (FERM P) having 5-5-5-trifluoro-D, L-leucine resistance
-8236) into 500 ml of YEPD medium (2% glucose, 2% polypeptone, 1% yeast extract), incubate at 30 ° C overnight, and use the method of MARMUR et al. (Reference 10) from the obtained cells. To prepare chromosomal DNA.

(2) Blotting of DNA digest on nylon filter The DNA obtained in (1) was digested with various restriction enzymes, and agarose electrophoresis was performed based on the molecular weight of lambda-phage DNA (manufactured by Toyobo) Hind III digest. Electrophoresis was performed. Next, the DNA was transferred to the nylon filter from the gel that had been electrophoresed according to the method of Reference 5, and fixed by irradiating with ultraviolet light for 5 minutes.

(3) Detection of chromosome fragment containing LEU4 According to the method of Reference 5, a nylon filter was used in a hybridization solution (5% Ficoll, 5% polyvinylpyrrolidone, 5% bovine serum albumin).
[P] -γ-ATP at the 5 ′ end with T4 DNA kinase at 32 [P]
Hybridization was performed overnight at 60 ° C. using the labeled synthetic DNA (FIG. 1). Next, the nylon filter membrane was washed twice with 2 × SSC (0.3NaCl, 0.03 M sodium citrate, pH 7.0) at 60 ° C. for 15 minutes, and further 0.1 ×
SSC (0.015M NaCl, 0.0015M sodium citrate, 0.1%
(SDS, pH 7.0) at 60 ° C. for 5 minutes. Next, this film was covered with Saran Wrap (Asahi Kasei: registered trademark), brought into close contact with an X-ray film, and exposed to light at -70 ° C overnight.

(4) Estimation of the size of the chromosome fragment containing LEU4 The size of the band that appeared on the X-ray film was determined by comparing it with the reference DNA for electrophoresis, and the LEU4 gene was contained in the 6.5 kb EcoRI fragment. Was.

(5) Digestion and fractionation of chromosomal DNA by restriction enzyme The DNA obtained in (1) at 0.2 μg / μ was digested with 0.05 nuit / μ restriction enzyme Sau3A I in high buffer at 37 ° C for 15 minutes. The product (200 μg in DNA amount) was centrifuged in 9 ml of a 10 to 40% sucrose density gradient, and 0.5 ml fractions were collected. Centrifugation was performed at 20 ° C., 25,000 rpm for 24 hours using a Hitachi RP-65T rotor. Measure the length of each fraction by electrophoresis, and
Fractions were obtained.

(6) Ligation of DNA and infection of E. coli 4 μg of the DNA fragment obtained in (5) was phage vector EMBL3
2 μg of BamHI arm (manufactured by Amersham) together with T4 DNA ligase in 100 μm of 6 mM MgCl ₂ , 6 mM β-mercaptoethanol, 0.5 mM ATP, 6 mM Tris · HCl (pH 7.6)
After incubating overnight at 20 ° C, 20 µl of this reaction solution is used in an in vitro packaging kit (Amersham).
In vitro packaged and ligated DNA
Was introduced into the phage particles.

Next, 1 µ of the phage solution was mixed with 200 µ of a bacterial solution obtained by culturing the host E. coli P2392 according to the method of Reference 5, incubated at 37 ° C for 15 minutes to infect the host, 10 ml of soft agarose was added and mixed, and 15 cm of agar was added. Plated on a plate. This plate was cultured at 37 ° C. for 4 to 10 hours to form phage plaques on the agar medium. Restriction enzyme and T4DNA ligase were manufactured by Takara Shuzo Co., Ltd., and the activity unit was defined according to the attached manual.

(7) Primary screening According to the method of Reference 5, about 20 phage plaques obtained in (6)
000 pieces were transferred to a nylon filter membrane (Hibond N: manufactured by Amersham), immobilized with ultraviolet light, and then subjected to hybridization and detection using an X-ray film according to the method described in (3), and were placed in the same position as the X-ray film. One phage plaque was isolated as a phage containing LEU4.

(8) Secondary screening Using the phage obtained in (7), a phage lysate was prepared according to the method of Reference 5, and DNA was prepared therefrom. This DNA was cut with a restriction enzyme EcoRI and subjected to agarose electrophoresis. The electrophoresed DNA was subjected to Southern analysis by the method described in (2) and (3), and had a 6.5 kb EcoRI fragment and LEU4
A phage λ-26 having a cleavage map similar to the gene map was selected. FIG. 2 shows a cleavage map of phage λ-26.

(9) Ligation to plasmid Plasmid pL4 was prepared by inserting the 6.5 kb EcoRI fragment of phage λ-26 into pUC19 (manufactured by Takara Shuzo Co., Ltd.), and the autonomous replication origin and TRP1 gene were further connected to pL4. The plasmid pL4T was prepared (FIG. 3).

(10) Transformation of yeast using plasmid pL4 Plasmid p obtained previously from Practical Yeast Association No. 7 (JCM1818)
When transformed with L4, 5-10 transformed cells are obtained per 4 μg of pL, and all these transformed cells are
100 μM of 5-5-5-trifluoro-D, L-leucine resistance was obtained.

Next, from the obtained colonies, 10 strains (X-1 to X-
-10) To confirm that these strains were transformants, the presence or absence of the plasmid was confirmed by the Southern method using pUC19 as a probe.
pL4 has been inserted and 5-5-5-trifluoro-D, L
-It was revealed that transformants can be specifically selected using leucine resistance.

Next, in order to check the amount of isoamyl alcohol produced by these transformants, the parent strain Saccharomyces cerevisiae (JCM1818) was used as a control and cultured overnight in an SD5 medium (5% glucose, 0.67% yeast nitrogen base). When the higher alcohol contained in the liquid was measured by head space gas chromatography,
All the strains have acquired high productivity of isoamyl alcohol, and produced 5 to 50 times the isoamyl alcohol of the parent strain. Table 1 shows the results.

Example 2 Transformation of yeast using pL4T Using 10 µg of the plasmid pL4T obtained in Example 1 (9), yeast of Association No. 7 was transformed. All of the obtained transformants had acquired 5-5-5-trifluoro-D, L-leucine resistance. Next, 5 strains (T-1 to T-5) were selected from the obtained colonies, and the presence or absence of the plasmid was determined to confirm that these strains were transformants.
When confirmed by the Southern method using pUC19 as a probe, 4
PL4T was confirmed to be present in the strain,
It was revealed that transformants can be specifically selected using 5-5-5-trifluoro-D, L-leucine resistance.

Example 3 Transformation Using LEU4 DNA Fragment 100 μg of pL4 was digested with a restriction enzyme EcoRI and subjected to agarose electrophoresis based on the molecular weight of lambda-phage DNA (manufactured by Toyobo) HindIII digest. A 6.5 kb DNA fragment was recovered from this agarose according to the method described in Reference 5, and 10 μg of the recovered DNA was used to transform No. 7 yeast, and 10 strains (TF-1 to TF-10) of the transformants were used. When the amount of isoamyl alcohol produced was measured, in about half of the strains, the amount of isoamyl alcohol increased 3 to 6 times that of the parent strain. Table 2 shows the results.

Example 4 Association No. 7 yeast, transformed yeast X-4 (FERM P-1142)
The aroma generation when sake was brewed with the two yeasts in 4) was compared. The preparation was performed in a single-stage preparation, and the yeast was fermented at a constant temperature of 15 ° C. using a yeast suspension (2 × 10 ⁷ per 1 ml of charged water). Table 3 shows the blended ingredients and Table 4 shows the general components of the resulting sake.

As is evident from the above results, isoamyl alcohol and isoamyl acetate were about 4 times and 3 times the parent strain, respectively.
The number increased twice, and the fruity and gorgeous scent was strongly felt in the sensory test.

Usually, in the pathway for producing isoamyl alcohol, α-isopropylmalate synthase (IPM) is subject to feedback inhibition by leucine, and the reaction of changing α-ketoisovaleric acid to α-isopropylmalic acid is performed by leucine. Has been inhibited. However, the transformed yeast clearly shows that the amount of isoamyl alcohol was increased by the introduction of one or more mutant forms of LEU4 to release the inhibition and to increase the enzyme activity due to the increase in the number of genes.

Example 5 Wine yeast Saccharomyces cerevisiae (IAM427)
The aroma generation when wine was brewed with two yeasts, 4) and transformed yeast X-4 (FERM P-11424), was compared.

Koshu grape juice was supplemented with glucose to prepare a sugar content of 24%. To each 1,000 ml of the juice, 2 g of each yeast was added as a wet cell weight and fermented at 18 ° C. for 7 days. Table 5 shows the components of the wine thus obtained.

As is clear from the above results, the wine using X-4 has isoamyl alcohol and isoamyl acetate.
It was confirmed that each of IAM4274 was about 3 times and 2 times as high as IAM4274, and that it also had a refreshing aroma functionally.

Example 6 Shochu Yeast Brewing Association Shochu No. 2 and Transformed Yeast X-4
Using (FERM P-11424), shochu was produced according to the charge formulation shown in Table 6. Add the cultured yeast to the charge formulation shown in Table 6.
2 g was added as a wet cell weight, and one-stage charging was performed. 14 at 15 ° C
After fermentation for days, the mash was distilled under reduced pressure on a rotary evaporator. Table 7 shows the components of the obtained shochu.

As is clear from the above results, the shochu using X-4 was high in the concentration of isoamyl alcohol and isoamyl acetate, and was a new type of shochu in terms of functionality.

Example 7 Beer yeast Saccharomyces uvalam (IFO0565)
The aroma generation when beer was brewed with two types of yeasts, and transformed yeast X-4 (FERM P-11424), was compared.

Water 1 was added to 160 g of malt, saccharified by heating, and then filtered to obtain wort. 2 g of hops were added thereto, and after boiling, hops were removed, and after cooling, water was added to bring the total amount to 1. This wort 1
2 g of each yeast as wet cell weight at 10 ° C at 15 ° C.
Fermented for days. Table 8 shows the components of the beer obtained here.

As is clear from the above results, the beer using X-4 was high in the concentration of isoamyl alcohol and isoamyl acetate, and was a new type of beer in terms of functionality.

Example 8 Commercial baker's yeast and transformed yeast X-4 (FERM P-11
424) was used to produce bread with the composition shown in Table 9. In the composition shown in Table 9, 2 g of the cultured yeast was added as a wet cell weight.
In addition, after fermenting at 30 degrees for 4 hours,
Baking was carried out for 25 minutes in an open at 0 ° C. to produce bread. Table 10 shows the measurement results of the flavor components contained in the bread.

As is clear from the above results, the bread using X-4 had a concentration of isoamyl alcohol about 10 times higher than that of baker's yeast, and was a new type of bread from a sensory viewpoint.

〔The invention's effect〕

According to the present invention, in yeast transformation, 5-5-5
-Only transformants can be efficiently selected using trifluoro-D, L-leucine resistance as an index, and can be used as a marker when introducing a heterologous gene. Further, by introducing only a recombinant plasmid of the resistance gene LEU4 or a gene fragment containing LEU4 into yeast, the amount of isoamyl alcohol and isoamyl acetate produced by yeast can be increased. In addition, by using the transformed yeast, various types of fermented foods and drinks of a new type that is rich in aroma can be produced.

[Brief description of the drawings]

FIG. 1 shows the sequence of a synthetic DNA used for detecting the LEU4 gene. FIG. 2 shows the restriction enzyme recognition site of a 6.5 kb EcoRI fragment containing the LEU4 gene cloned into phage λ-26. FIG. 3 shows a map of a plasmid incorporating the LEU4 gene.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 1/19 C12N 1/19 C12Q 1/68 C12Q 1/68 A // (C12N 15/09 C12R 1: 865) (C12N 1 / 19 C12R 1:85) (C12N 1/19 C12R 1: 865) (72) Inventor Katsuhiro Yasui 2-1, Shimo-Tobasugamacho, Fushimi-ku, Kyoto-shi, Kyoto (56) References JP-A-4-117293 ( JP, A) (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/52

Claims (8)

(57) [Claims] 1. A chromosomal DNA of Saccharomyces genus yeast having resistance to 5,5,5-trifluoro-D, L-leucine and high productivity of isoamyl alcohol and / or isoamyl acetate. About having a cutting point
A 6.5 kb DNA having two Pvu II sites, two BamH I sites, one Sph I site and one Xho I site in the EcoR I fragment. 2. A plasmid into which the DNA according to claim 1 has been incorporated. 3. The plasmid according to claim 2, wherein the plasmid is the plasmid map pL4 or pL4T shown in FIG. 4. A method for detecting a transformant, comprising using the plasmid according to claim 3. 5. The method according to claim 4, wherein the transformant is positively selected. [6] A yeast transformant obtained by transforming yeast using the plasmid according to [3]. 7. The transformed strain according to claim 6, wherein the yeast is a Saccharomyces yeast. 8. The plasmid or LEU4 according to claim 3, wherein the LEU4 gene obtained from chromosomal DNA of Saccharomyces yeast having leucine analog resistance is integrated.
A method for producing fermented foods and drinks, which comprises using a Saccharomyces yeast transformed with the chromosomal DNA according to claim 1 containing a gene.