JPH0832240B2 - Novel gene, vector, transformant using the same, and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to Saccharomyces cerevisiae (Saccharo).
The present invention relates to a killer KHR gene located on chromosome 9 obtained from myces cerevisiae), a vector containing the killer KHR gene, a transformant obtained by transferring the vector into cells 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 (KHR) was discovered.

This killer toxin, unlike the conventional toxin genetically dominated by the double-stranded ribonucleic acid plasmid of Saccharomyces cerevisiae with respect to optimum pH and temperature stability, shows a lethal effect on a wide range of yeasts when concentrated. , Is also novel in protein chemistry.

(Problems to be Solved by the Invention) The present invention has been made for the purpose of elucidating a gene that controls a killer toxin (KHR) controlled by chromosomal DNA and relatively stable at high temperature and high pH as described above. Is.

(Means for Solving Problems) Therefore, in order to achieve the above-mentioned object, the present inventors have conducted various studies on the isolation of the gene that controls this toxin,
After succeeding in isolating the gene and examining its structure, it was found that this gene has an excellent expression / secretion function in Saccharomyces cerevisiae and also has a dominant marker of killer property. Was confirmed and 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 15.8 kbp (kilobase pair), which contains a KHR killer structural gene (about 0.9 kbp) inside. A kb DNA containing a novel gene having a promoter for expressing this killer toxin in the yeast of the genus Saccharomyces in the upstream region of the structural gene and having a secretory mechanism for secreting the killer toxin. It is also possible to use the KHR gene as a dominant marker or as a vector capable of confirming insertion of a foreign gene in yeast by utilizing a restriction enzyme site in the KHR gene.

 Hereinafter, the present invention will be described in detail.

The KHR 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 KHR gene on the chromosome is prepared. Suitable strains include, for example, Saccharomyces cerevisiae.
No. 11 strain or No. 18 ρ ^- part. Total DNA is prepared from these strains, partially digested with a restriction enzyme, and a fragment of about 5 to 10 kb is electrophoretically recovered.

Several pieces of the recovered DNA are ligated to the E. coli-yeast shuttle vector. As the shuttle vector, one known in the art is appropriately used, and examples thereof include YEp13 and YCpG11. 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 is recovered from the above gene bank and introduced into a recipient bacterium such as S. cerevisiae DBY746 strain 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, for example, Candida glabrata, 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 KHR 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 KHR gene on its chromosome, and specifically, for example, No. 11 strain.

To extract genomic DNA from this bacterium, for example, Cryer et al.
Methods in Cell Biology, Vol. 12, 39-44 (1973), the method for extracting chromosomal DNA of Saccharomyces cerevisiae is performed.

Next, the obtained genomic DNA was treated with a restriction enzyme Sau 3A and partially digested, and then a 5-10 kbp nucleic acid fragment was fractionated by agarose gel electrophoresis to obtain a vector as shown in FIG. Connected. This YCpG11 vector is Agric.Biol.C.
hem., Vol.50, 1177-1182 (1986), ARS1 capable of replicating in yeast, pBR322 ori capable of replicating in Escherichia coli, yeast TRP1 as a marker gene, and Ba as an E. coli cloning site.
It is composed of a tetracycline resistance gene containing an mHI site, an ampicillin resistance gene, and the cen4 gene which limits the number of vectors in yeast to 1-2. The insertion site for heterologous DNA is the BamHI site. Restriction enzyme for YCpG11 vector
After BamHI digestion, add fragmented genomic DNA, mix, and
4 Ligase-ligated and introduced into E. coli JA221, selected ampicillin-resistant Escherichia coli that has lost tetracycline resistance, selected as recombinant vector containing yeast gene, and further amplified vector containing yeast-derived nucleic acid Let

The vector obtained above was recovered, and the yeast Saccharomyces cerevisiae DBY746 strain (Botstein D. et al.,: Gen
e8,17-24 (1979)), a transformant strain is selected by complementation of a tryptophan auxotrophic marker on the vector, and a KHR killer detection medium at pH 6 from which KHR killer expression can be confirmed Proliferated above, sprayed with Candida glabrata IFO 0622 strain as an indicator bacterium, selected strains showing growth inhibition of Candida glabrata around DBY746 strain, and showed KHR (property as shown in Table 1). C) The strain was acquired. This transformant (S. cerevisiae KHR (C))
FERM P-10927 has been deposited with the Institute of Microtechnology.

The total nucleic acid was extracted from this yeast strain 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 cells and the restriction enzyme cleavage map was prepared with various restriction enzymes. The results are shown in FIG. The Escherichia coli transformant strain (E. coli KHR-YCP) has been deposited with RIETI as FERM P-10926.

Detailed restriction enzyme cleavage sites of the inserted yeast nucleic acid-derived portion are shown in FIG.

Using this restriction enzyme cleavage site, the nucleic acid
The result of recombining the fragment containing the yeast-derived nucleic acid with a vector that increases the copy number in yeast is also shown at the same time. The vector used is Gene Vo from Botstein et al.
YEp24 described in l.8,17-24 (1979). This vector is composed of 2 micron DNA capable of replicating in yeast, pBR322 ori capable of replicating in E. coli, yeast URA3 as a marker gene, Escherichia coli tetracycline resistance gene and ampicillin gene. It has BamH I and Sal I sites, which are the only restriction enzyme cleavage sites in this tetracycline gene.

This vector is double-digested with BamHI and SalI, a BamHI-SalI fragment containing the KHR gene is added thereto, and ligated with T4 DNA ligase to obtain the vector KHR-YEp. Then this KH
R-YEp was transferred to Escherichia coli JA221 and purified in a large amount by a conventional method. This transformant (E. coli KHR-YEp) is FERMP-10929.
Has been deposited with the Institute of Micro Engineering.

Subsequently, the purified vector KHR-YEp was added to J. Bac.
-168 (1983) according to the method described in yeast host DBY746
(Α trp1−289 his 3Δ1 leu-2-3−112ura3−52)
And the uracil auxotrophy was complemented to obtain a transformant capable of growing in a uracil-free medium. Similarly, after cleavage with an appropriate restriction enzyme, the KHR killer expression was observed from the vector into which the DNA fragment containing the KHR gene was inserted, and the results of examination of the portion required for KHR killer gene expression are shown in Table 2. . Required for killer activity expression
It was discovered that the DNA resides at 1.7 kb which is cleaved with the restriction enzymes Xho I-EcoR I.

Among the transformants shown in Table 2, those having killer activity show almost the same strength, but the strain having the strongest killer property (hereinafter referred to as KHR (E) strain) will be described.
This strain (S. cerevisiae KHR (E)) has been deposited with MICRO as FERM P-10928. As shown in Table 3, KHR
The killer activity of the strain (E) is secreted into the culture supernatant of the strain having the KHR gene on the chromosome (No. 11 strain) or the strain having the KHR gene on the single copy vector (for example, KHR (C) strain). About 3 to 4 times stronger killer activity was observed.

A transformant having a plurality of KHR genes, such as the KHR (E) strain, was extremely inhibited in growth under the conditions where killer properties were most strongly expressed (pH 6.0, 20 ° C., stationary culture). Therefore, in order to grow yeast cells, citrate-phosphate buffer (pH 4.0) was added to the medium from which uracil had been removed to a concentration of 0.1 M, and the growth inhibitory effect was removed by shaking culture at 30 ° C and recovered. . For the preparation of killer toxin, the recovered cells were cultured in a medium containing 10 volumes of 0.1 M citrate buffer (pH 6.0) without uracil at 20 ° C.
The stationary culture was carried out for 5 days, and the culture supernatant was used.

Killer toxin produced and secreted from this strain was concentrated by freeze-drying and gel filtration (TSK-gel G3000SW (TOSOH)).
And hydrophobic chromatography (SynChropak propyl (SynCh
rom)) can be used for partial purification. The properties of the toxin are shown in Table 4, and the isoelectric point p I5.2-5.
6. It was found to be a protein with an optimum pH 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 level are DN, in terms of pH, temperature stability, optimum pH and filterability by ultrafiltration.
The results are the same as those of the A-supplied strain (No. 11 strain), and it is shown that the same killer toxin is produced.

Approximately 1.7 cleaved by restriction enzymes Xho I and EcoR I, which are the smallest units containing the KHR killer gene shown in Table 2.
Based on a conventional method for determining the nucleic acid sequence centered on the kbp fragment, the deletion mutant vectors were constructed using the sequencing vectors pUC118 and 119, and their sequences were determined by the dideoxy method.

Specifically, a fragment generated by digesting the vector having KHR activity shown in FIG. 2 with restriction enzymes BamHI and SalI was designated as pUC11.
Inserted in 8,119. This vector is Gene, Vol.28,351-3
59 (1984) and Gene, Vol.33, 103-119 (1985), the insert was treated with exonuclease III and mung bean nuclease to shorten the insert, and part of the insert fragment was shed, resulting in a different chain length. Various clones with In the process, a kilo-sequencing deletion kit (Takara Shuzo Co., Ltd.) was used. For the inserts of the various clones obtained, fluorescent primers (Bio-Tec
DN by the dideoxy method (Science, Vol.214,1205-1210 (1981)) using hniques Vol.5,754-765 (1987))
The A was sequenced. A DNA sequencer (Applied Biosystems ABI 370A) was used for this determination. As a result, Saccharomyces cerevisiae KHR
The full-length 1080 bp sequence shown in FIG. 1 was determined for the DNA sequences of the entire coding region of the gene and a part of the adjacent upstream and downstream regions.

In the nucleic acid sequence shown in Fig. 1, about 20 amino acids with highly hydrophobic amino terminus are present and form a secretory sequence.
It was discovered that it encodes a protein composed of amino acid residues of Furthermore, it was discovered that a TATA sequence serving as a promoter and a region containing a large amount of adenine and thymine were present at about 80 bp upstream of the nucleic acid sequence from which amino acids were read out.

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 and a sequence of a promoter for expressing the toxin.

That is, the sequence shown in FIG. 1 contains a restriction enzyme site Pvu II in the secretory sequence, and it is possible to express and secrete it as a protein in yeast by incorporating a structural gene in the downstream region. For example, by ligating the LacZ gene derived from Escherichia coli and expressing it in yeast with 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.

In addition, there are several restriction enzyme sites that can serve as cloning sites in the KHR-YEp vector, and it is possible to utilize two types of uracil-requiring or killer-active markers in yeast Saccharomyces cerevisiae. For example KHR-Y
Restriction enzyme Mlu I, which is uniquely present in the KHR gene of Ep vector
A foreign gene can be efficiently recovered by inserting a foreign gene into the site and growing yeast under optimal conditions for killer activity to recover a strain in which the killer property is lost.

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

As a result, according to the present invention, not only the chromosome-dependent killer protein can be mass-produced, but also the KHR gene can be used 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 KHR gene. 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 KHR gene. The KHR killer gene is present in the portion with the amino acid symbol below. It encodes 296 amino acids starting from methionine of the 139th ATG. Figure 2 is in the tetracycline gene of YCpG11
1 shows a vector having yeast-derived DNA inserted into the BamHI site. The part where killer activity is present, on the EcoR I side
BamHI site is destroyed. The size of the insert fragment containing the KHR gene is about 4.7 kb. FIG. 3 is a cleavage map of the KHR gene in which the BamHI and SalI fragments containing the KHR portion of FIG. 2 were recovered, digested with various restriction enzymes, and the respective cleavage sites were estimated. In these drawings, the abbreviations of the alphabets indicate the following cutting positions by restriction enzymes. B: BamH I, Bg: Bgl II, E: EcoR I Ev: EcoR V, M: Mlu I, N: Nco I, P: Pst I Pv: PVu II, S: Sal I, X: Xho I Sau: Sau3A I

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1: 865) (C12P 21/02 C12R 1: 865) (72) Inventor Yasuhiro Iwatsuki Hiroshima Hiroshima 6-30 Kamihacchobori, Naka-ku, Hiroshima Office of the National Tax Agency Appraisal Office (72) Inventor, Kazuyoshi Kitano 1-5-63, Otemae, Chuo-ku, Osaka City, Osaka Prefecture

Claims (3)

[Claims] 1. A chromosomal DNA-dependent killer KHR as shown in FIG. 1, isolated from Saccharomyces cerevisiae.
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 KHR 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.