JPH0832241B2 - Novel gene, vector, transformant using the same, and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Field of the Invention The present invention relates to Saccharomyces cerevisiae ( Saccharo
The present invention relates to a killer KHS gene located on chromosome 5 obtained from myces cerevisiae ), a vector containing the killer KHS gene, a transformant obtained by transferring the vector into a bacterium such as Saccharomyces cerevisiae, and the use thereof.

(Prior Art) Most killer of Saccharomyces yeast is genetically controlled by a cytoplasm-dependent double-stranded ribonucleic acid (ds RNA) plasmid.

However, apart from such killer,
A DNA-controlled killer factor (KHS) was discovered.

This killer toxin is the same as the conventional toxin genetically dominated by the double-stranded ribonucleic acid plasmid of Saccharomyces cerevisiae in terms of optimum pH and temperature stability,
The killer property is different even under the condition of plasmid removal due to drug treatment or high temperature culture.

When concentrated, this toxin has a lethal effect on a wide range of yeasts, and is novel in protein chemistry.

(Problems to be Solved by the Invention) The present invention has been made for the purpose of elucidating a gene controlling a killer toxin (KHS) controlled by a chromosomal DNA as described above, and a novel method utilizing this gene This is a pioneering breeding method.

(Means for Solving Problems) Therefore, in order to achieve the above-mentioned object, the present inventors have conducted various studies on the isolation of a gene controlling this toxin, and as a result, have succeeded in isolating the gene. As a result of further examination of the structure, it was confirmed that this gene has an excellent expression / secretion function in Saccharomyces cerevisiae and also has a dominant marker of killer property at the same time, and thus the present invention was completed.

That is, the present invention is a novel plasmid characterized by the restriction enzyme cleavage map of FIG. 2 having a molecular weight of about 16.0 kbp (kilobase pair), which contains 5.3 Kbp killer structural gene (about 2 kbp) inside. , Which has a promoter for expressing this killer toxin in the yeast of the genus Saccharomyces in the upstream region of the structural gene, and contains a novel gene having a secretory mechanism for secreting the killer toxin. It is also possible to use the KHS gene as a dominant marker or as a vector capable of confirming the insertion of a foreign gene in yeast using the restriction enzyme site in the KHS gene. Furthermore, yeast with the KHS gene is enriched in K
Survival and growth are possible even in the presence of HS enzyme, and yeast transformants into which this gene has been introduced also acquired immunity to KHS toxin, allowing selective enrichment of transformants in the presence of concentrated KHS toxin. Is also available.

 Hereinafter, the present invention will be described in detail.

The KHS gene according to the present invention is as outlined above, and its cloning is performed by the method described below.

First, a strain containing the KHS gene on its chromosome is prepared. Suitable strains include, for example, Saccharomyces cerevisiae.
No.115 strain or No.18ρ ^- strain. Total DNA was prepared from these strains, partially digested with restriction enzymes,
A fragment of about 4-10 kbp is collected by electrophoresis.

Several pieces of the recovered DNA are ligated to the E. coli-yeast shuttle vector. As the shuttle vector, those known in the art are appropriately used, and examples include YEp13 (Broach J.et
al.,: Gene 8, 121-133 (1979), YCpG11 and the like. The ligated body of the recovered DNA fragment and the shuttle vector is introduced into Escherichia coli such as JA221 strain to prepare a gene bank.

A vector was recovered from the above gene bank and used as a recipient strain such as Saccharomyces cerevisiae DBY746 (Bots
tein D. et al.,: Gene 8, 17-24 (1979) for transformation. For these transformants, auxotrophic primary selection was performed, and then secondary selection was performed on a killer activity detection medium depending on the presence or absence of killer property against an indicator bacterium, such as Candida glabrate, to obtain the target transformant strain. To screen.

From the transformant thus obtained, the total DNA is recovered by the same method as described above, introduced into Escherichia coli to recover the plasmid, and the desired KHS gene is recovered. .

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example The genomic DNA used in the present invention is derived from Saccharomyces cerevisiae having the KHS gene on its chromosome, and is specifically No. 115 or No18ρ ⁻ .

To extract genomic DNA from this bacterium, for example, Cryer
Et al Methods in Cell Biology, Vol.12, 39-44 (1973)
Chromosomal DN of Saccharomyces cerevisiae described in
It is carried out according to the method for extracting A.

Next, the obtained genomic DNA was treated with a restriction enzyme Sau 3A and partially digested, and then a nucleic acid fragment of 4-10 kbp was fractionated by agarose electrophoresis, and a vector as shown in FIG. 2 was obtained. Connected. This YEp13 vector Gene 8, 1
21 (1979) is described in, replicable 2μmDNA origin of replication in yeast, capable of replication in E. coli pBR322 ori, yeast LEU2 as a labeling gene, tetracycline resistance gene containing the BamH I site as E. coli cloning site, is composed of the ampicillin resistance gene There is. The insertion site for heterologous DNA is Bam
It is the HI site. After restriction enzyme BamH I degrade YEp13 vector, fragmented genomic DNA added, mixed and ligated with T4 ligase, introduced into E. coli E. coli JA221, selecting the ampicillin resistance, and have lost the tetracycline resistance ability coli Then, a recombinant vector containing a yeast gene was prepared, and a vector containing a yeast-derived nucleic acid was further amplified.

The vector obtained above was recovered and used in J. Bac., 15 of Ito et al.
3, 163 -168 yeast Saccharomyces according to the method of (1983) cerevisiae DBY746 (α trpl-289 his3Δ1 leu
(2-3 ieu2-112 ura3-52) strain, a transformant strain is selected by complementation of the leucine-requiring marker on the vector, and a KHS killer expression medium of pH 4.5 from which KHS killer expression can be confirmed Candida glabrata I grown as an indicator strain
Candida glabrat around the DBY746 strain by spraying with FO 0622 strain
A strain showing growth inhibition of a was selected, and a KHS (E) strain having the properties as shown in Table 1 was obtained. This transformant strain ( S. cerevisiae KHS (E)) has been deposited with MICRO as FERM P-11562.

Total nucleic acid was extracted from this yeast based on the method of Cryer et al., Described above, and the nucleic acid was introduced into Escherichia coli to recover ampicillin-resistant clones. The vector was recovered from the E. coli and the restriction enzyme cleavage map was prepared with various restriction enzymes. The results are shown in FIG. This E. coli transformant ( E.
Escherichia coli KHS-YEp) has been deposited with RIETI as FERM P-11561.

Detailed restriction enzyme cleavage sites of the inserted yeast nucleic acid-derived portion are shown in FIG. ORF consisting of 2,124 bp (Open
Reading Frame) and 9 Asn-X-Thr or Se
There were r sequences (sequences to which sugar chains can attach) and the like. The amino acid sequence (APCQVV) corresponding to the N-terminal sequence shown by the purified toxin was Ala at the 37th position from the beginning of ORF.
Existed from. The estimated protein is 672 amino acids,
The molecular weight was 75,590 da, which was almost in agreement with the molecular weight of about 75 kDa estimated by SDS-PAGE.

After digestion with an appropriate restriction enzyme using this restriction enzyme cleavage site, KH
Table 2 shows the results of examining the portion required for the expression of the KHS killer gene by looking at the expression of the S-killer property. The DNA required for killer activity expression is the restriction enzyme HindIII - HindIII - SalI
It was discovered that it exists at 3.7 kbp which is cleaved by.

The transformants having the killer among the transformants shown in Table 2 showed almost the same strength.

Similarly, BamH I to Eco end of the acid (Eco-Sal fragment
The results of recombination of a fragment containing a nucleic acid derived from yeast with a vector (in which a site has been added) into a vector that limits the copy number to 1 in yeast are also shown. The vector used is Agric.Biol.Chem., Vo
YCpGl1 described in I.50, 1177 to 1182 (1986). This vector is capable of replicating ARS1 in yeast and E. coli
It is composed of pBR322 ori, yeast TRP1 as a marker gene, Escherichia coli tetracycline resistance gene, and ampicillin gene. It has BamHI and SalI sites, which are the only restriction enzyme cleavage sites in this tetracycline gene.

This vector was double digested with BamHI and SalI and
A BamHI - SalI fragment containing the KHS gene is added and ligated with T4 DNA ligase to obtain the vector KHS-YCp. Next, this KHS-Y
Cp was transferred into Escherichia coli JA221 and purified in a large amount by a conventional method.

Subsequently, the purified vector KHS-YCp was added to Ito et al. J. Bac.
~ 168 (1983) according to the method described in yeast host DBY746
To obtain a transformant capable of growing in a tryptophan-free medium by complementation of the tryptophan requirement (hereinafter referred to as KHS (C) strain).

As shown in Table 3, the killer activity of the KHS (C) strain is the culture supernatant of the strain having the KHS gene on the chromosome (No. 111) or the strain having the KHS gene on the single copy type vector (eg, KHS (C) strain). Approximately 3 to 4 times the strength of the killer activity secreted therein was observed.

For killer activity, after freeze-drying the medium supernatant, dilute it appropriately in a buffer solution, and put 200 μl of the sample on an agar medium into a penicillin cup with a diameter of 8.5 mm to suppress the growth of the indicator bacteria.
And ND indicates that no activity was detected.

Since it was confirmed that the killer toxin productivity of strains having a plurality of KHS genes such as the KHS (E) strain was improved, the killer toxin was prepared using this bacterium. For preparation, culture medium without leucine containing 0.1 M citrate buffer (pH 4.5) at 20 ° C 4
Static culture was carried out for ~ 5 days, and the culture supernatant was used.

The killer toxin produced and secreted from these cells was concentrated by freeze-drying and subjected to hydrophobic chromatography (SynChropakprop
yl (SynChrom)) and gel filtration (TSK-gel3000SW (TOSO
H)) can be used for partial purification. The properties of the toxin are as shown in Table 4. Isoelectric point p15.3, optimum pH4
It was discovered to be a simple protein of ~ 5.5.

When the isolated killer toxin was concentrated and used, it showed killer activity in many yeast genera as shown in Table 5. These results and the nature of the killer activity at the bacterial cell level are DN, in terms of pH, temperature stability, optimum pH and filterability by ultrafiltration.
The result is the same as that of the A-donating strain (No. 115), which indicates that the same killer toxin is produced. Further, it was discovered that the strain having the killer gene is not affected by the killer toxin, and the gene related to immunity to the killer toxin is also included in this gene.

Approximately 3.7 kbp digested with restriction enzymes HindIII and SalI containing the minimum unit containing the KHS killer gene shown in Table 2.
Based on a standard method for determining the nucleic acid sequence centering on the fragment, the deletion vectors were constructed and the sequence was determined by the dideoxy method using the sequencing vectors pUC118 and 119.

Specifically, a vector having KHS activity shown in FIG. 2 was digested with a restriction enzyme HindIII to generate an approximately 1.4 kb fragment, Hind.
Approximately 2.3 kb fragment generated by digestion with III and SalI and PstI and SalI
Three fragments of about 3.0 kbp generated by decomposition were inserted into pUC118,119. These vectors are described in Gene, Vol. 28, 351-359 (1984).
And Gene, Vol. 33, 103-119 (1985), the insert is treated with exonuclease III and Mung Bean Nuclease to shorten the chain, and a part of the insert fragment is shed, resulting in a different chain length. Various clones with In this step, a kilosequencing deletion kit (Takara Shuzo Co., Ltd.) was used. Regarding the inserts of the various clones obtained, the dideoxy method (Science, Vol. 214, 1205-1212 (198) using a fluorescent primer (Bio-Techniques Vol. 5, 754-765 (1987)) was used.
DNA was sequenced according to 1)). The DNA sequencer (Applied Biosystems Co., Ltd. ABI) was used for this determination.
370A) was used. As a result, with respect to the DNA sequences of the entire coding region of the Saccharomyces cerevisiae KHS gene and a part of the adjacent upstream and downstream regions, a full-length 3668 bp sequence was determined and the sequence containing the KHS gene was shown.

In the nucleic acid sequence shown in Fig. 1, about 20 amino acids with highly hydrophobic amino terminus are present, which forms a secretory sequence.
It was discovered that it encodes a protein composed of the amino acid residues of Furthermore, it was discovered that a TATA sequence and a region containing adenine and thymine, which are related to the promoter, are present at about 80,140,260 bp upstream of the nucleic acid sequence that is considered to read out amino acids.

Thus, the nucleic acid sequence of the present invention is a novel secretory sequence,
It was revealed to contain a sequence of a toxin acting on yeast, a sequence relating to immunity to this toxin, and a sequence of a promoter for expressing this toxin.

That is, according to the sequence shown in FIG. 1, there is a recognition site for the restriction enzyme PstI etc. behind the secretory sequence, and by incorporating a structural gene in this downstream region, it is possible to express and secrete as a protein in yeast. . For example, the LacZ gene derived from Escherichia coli was ligated and expression in yeast was controlled by X-Gal.
(5-Bromo-4-chloro-3-indolyl-β-D-gala
Expression can be confirmed by appearance of blue colonies on agar medium containing ctopyranoside) and measurement of galactosidase activity in liquid medium.

Also, there are several restriction enzyme sites that can serve as cloning sites in the KHS-YEp vector, and two types of leucine-requiring or killer-active markers can be used in yeast Saccharomyces cerevisiae. For example, LEU2
A foreign gene can be inserted into the XhoI site that is uniquely present in the gene, and in the presence of KHS killer toxin, the immunity within the KHS gene can be used to selectively recover only the transformant.

(Effects of the Invention) The present invention succeeded in isolating the gene controlling the killer factor KHS for the first time, and determined its structure.

As a result, according to the present invention, not only can the chromosome-dependent killer protein be mass-produced, but the KHS gene can also be used as a dominant marker or as a vector capable of confirming the insertion of a foreign gene using a restriction enzyme site in the vector. The present invention plays an important role in the technical field of genetic engineering.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence around the KHS gene. The KHS killer gene is present in the portion with the amino acid symbol below. From the HindIII site of the cloning site, 708 amino acids starting from methionine of ATG at 454 are encoded. Fig. 2 B in the tetracycline gene of YEp13
1 shows a vector having a yeast-derived DNA inserted at the amHI site. BamHI on both sides of the killer activity
The site was destroyed during insertion. The size of the insert containing the KHS gene is about 5.3 kb. FIG. 3 is a cleavage map of the KHS gene in which the insert fragment containing the KHS portion of FIG. 2 was digested with various restriction enzymes and the respective cleavage sites were deduced.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 865) (C12P 21/02 C12R 1: 865) (72) Inventor Kazuyoshi Kitano Osaka Prefecture 1-563 Otemae, Chuo-ku, Osaka City, Office of Appraisal Office, Osaka National Tax Bureau (72) Inventor: Hiroshi Fukuda 2-11-1, East, Hakata Station, Hakata-ku, Fukuoka, Fukuoka

Claims (3)

[Claims] 1. A chromosomal DNA-dependent killer KHS, which is isolated from Saccharomyces cerevisiae as shown in FIG.
A DNA fragment containing the protein coding region of a gene. 2. A vector derived from Saccharomyces cerevisiae, having a DNA fragment containing the protein coding region of the chromosomal DNA-dependent killer KHS gene shown in FIG. 1 and characterized by the restriction enzyme cleavage map shown below. 3. A transformant having killer activity, which is obtained by transferring the vector according to claim 2 into a bacterium.