JPH0722509B2 - Temperature-sensitive repressor and gene expression method using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a temperature sensitive repressor and a gene expression method using the temperature sensitive repressor. Using this method, the production of useful gene products can be regulated by temperature.

2. Description of the Related Art In recent years, it has become possible to transform a microorganism with a plasmid containing a DNA carrying useful genetic information to allow the microorganism to produce a useful gene product. To date, it has been reported that not only various proteins derived from microorganisms but also peptides produced by organisms of extremely different types such as mammals, such as growth hormone, insulin, lymphokines, etc. can be produced. In the production of this useful gene product, it is extremely convenient for the host to be in charge of a system that regulates and controls the production. It is known that in the absence of such a regulatory system, production of a useful gene product construct often damages the growth rate of the host, or even survival itself. Such a regulatory system uses the promoter / operator of the lac operon in Escherichia coli, which is a gram-negative bacterium (Itakura, K., et al., Science, 198, 1056.197).
7) and, those that use the P _L promoter / operator of lambda phage (Derynck., R., et al ., Nature, 287, 19
3, 1980) and many others have been reported. However, in the Bacillus bacterium, which is a Gram-positive bacterium, which is considered to be an industrially important microorganism due to its safety, high protein production capacity, and the possibility of secretory production into the medium, the trp promoter / operator is used. Used (Shimotsu, H.,
et al., Proc. Natl. Acad. Sci. USA 81, 503 , 1984) and only a few examples have been reported. In addition, all of them require the addition of an inducing reagent such as indole acrylic acid for expression regulation, and a regulation method that is simpler and allows easier purification of the product has been desired.

Problems to be Solved by the Present Invention The present invention uses industrially important Bacillus bacterium as a host,
It is intended to provide a system in which the production of useful gene products can be controlled by the temperature.

The Bacillus licheniformis penP gene is a gene encoding the secretory enzyme penicillinase, and many peptide secretion vectors have been constructed using its signal sequence (Himeno, T., et al., FEMS Microbiol. Lett., 35, 1
7, 1986). The expression of penP is present in close contact with pen I
It is regulated by the repressor protein that is the product of the gene. The repressor protein binds to the penP operator site and terminates promoter function.

The present inventors mutated the wild-type pen I gene to induce pen I (pen Its), which became to encode a temperature-sensitive repressor. The temperature-sensitive repressor is a protein whose higher-order structure is changed by the change of temperature and loses its function. The gene that carries the genetic information of this protein, pen Its, is inserted into a plasmid in which the penP promoter / operator is inserted, or is inserted into a plasmid that is not incompatible with the plasmid in which the promoter / operator is inserted, and coexists in the same Bacillus host. Thus, it is possible to construct a phenotype expression system capable of changing the temperature-sensitive repressor protein activity by changing the culture temperature and regulating the function of the promoter.

The method of inducing pen Its will be described in detail below. B. lich
Both the penP and Pen I genes of eniformis have a 4.2 kb Eco
Coded on RI fragment (Imanaka, T., et
al., J. Bacteriol., 147, 776 , 1981). The region containing both genes can be cut out with a restriction enzyme and inserted into a plasmid vector. The restriction enzyme used may be any enzyme as long as it does not cleave the inside of both genes, and the plasmid vector may be any plasmid capable of growing in the genus Bacillus. Transformation of a Bacillus genus plasmid containing this region was performed by the protoplast method or the competent method (Imanaka T., et al., J. Bacter.
iol., 146, 1091,1981) such can be implemented in various ways.

A method for mutating the cloned Pen I gene is to mutate the DNA itself in vitro, for example, a method using hydroxyamine or the like to transform a plasmid carrying a plasmid into N-methyl-N. ′ -Nitro-
Either a method of treating with a mutant agent such as N-nitrosoguanidine (NTG) and then removing the plasmid and screening the mutant plasmid may be used.

For example, when B. subtilis was transformed with the mutated plasmid and a plate test of penicillinase was performed, the transformant strain carrying the plasmid containing the desired Pen Its gene secreted a large amount of penicillinase at high temperatures. It forms halos, but only small halos can be formed at low temperatures. The low temperature may be 30 ° C or lower and the high temperature may be 35 ° C or higher.

The plasmid containing the Pen Its gene is, for example, Lovett
Et al. (Levett, PSet al., Methods in Enzymology,
68, can be mass adjusted by 342,1979). In order to obtain the DNA fragment inserted from the recombinant plasmid, it may be cut with a restriction enzyme and used for preparative agarose gel electrophoresis or preparative acrylamide gel electrophoresis.

The DNA fragment thus obtained is, for example, the method of Maxam and Gilbert (AWMaxam et al., Met.
The nucleotide sequence can be determined by hod in Enzymology, 65 , 499, (1980)).

When the function of the penP promoter is regulated by the temperature-sensitive repressor protein, as in the case of the screening of Pen Its described above, the method in which the Pen Its and the penP promoter are present in the same plasmid, or the incompatibility as described above, is used. There is a method of dividing and presenting in a heterogeneous plasmid having no sex. In either case, if a gene carrying useful genetic information is inserted downstream of the penP promoter, Baci
It is useful for applications because useful peptides can be produced in llus bacteria while controlling the expression by changing temperature.

Example 1 Mutation induction and nucleotide sequence of PenIts 4.2 kb EcoR encoding both wild-type penP and Pen I genes
The plasmid pTTE 21 (T.Im
anaka et al., J. Bacterial, 147, 776 , 1981) was excised from the above EcoR I fragment of 4.2 kb and cut with EcoR I to cut low copy number plasmid pTB522 (Imanaka, T. et al., J. Ge.
n.Microbiol., 131, 1753, 1985) with T ₄ DNA ligase. With this reaction mixture, B. subtilis MI 113 (arg-
15, trp C2, r ^- _M , m ^- _M ) was transformed with L agar medium (1% bactotryptone, containing 25 μg / ml tetracycline).
Transformed strains were selected with 0.5% yeast extract, 0.5% NaCl, 1.5% agar, pH 7.0).
The one with the incorporated fragment was selected. The resulting plasmid is designated as pPTB 60 (Fig. 1). B. subtilis MI113 carrying pPTB 60 is treated with 100 μg / ml NTG for 30 minutes and then plated on L agar medium containing 25 μg / ml tetracycline. A replica of the emerged colony was used to test the penicillinase plate at 48 ° C (T.
Imanaka, et al., J. Bacteriol., 147, 776 , 1981). Of the approximately 150,000 colonies, 200 colonies had pP at 48 ° C.
It formed a larger halo than B. subtilis, which carried TB60. When these colonies were further used for plate test at 30 ° C., two colonies formed smaller halos than B. subtilis carrying pPTB50 having an inactive repressor gene. Each of these two candidate strains
We named them D13 and E24 and extracted the plasmid.
lis MI 113 was transformed. After selecting the transformants on L agar medium containing 25 μg / ml tetracycline, the same plate test was repeated to confirm the phenotype of Pen Its. These plasmids were designated pPTB 60 D13 and pPTB, respectively.
It was named 60 E24 (Fig. 1).

B.subtilis MI 113 / pPTB 60 D13 and B.subtilis MI 113 / p
PTB 60 E24 is FERM P-8963 (AJ12304), FERM
P-8964 (AJ12305) has been deposited with the Institute of Mechanical Engineering.

Example 2 Expression of Penicillinase Gene in B. subtilis Carrying the Pen Its Plasmid In order to investigate whether the Pen Its induced in Example 1 functions as expected, penicillinase in a liquid medium is performed as follows. I investigated the production of.

Plasmid pPTB 60 D13, pPTB 60 E24, pPTB 50 or p
B. subtilis MI 113 carrying PTB 60 was cultured in L medium overnight at 37 ° C., and 1% of each was inoculated into L medium containing 25 μg / ml of tetracycline. 30, 37, 42, and 48 each
℃ were cultured to 0.D ₆₆₀ 1, the measured penicillinase activity (T.Imanaka et al., J.Bacteriol. , 147, 776,198
1). The results are shown in Table 1.

Plasmids pPTB 60 and pPTB 50 were used as controls for complete repression and complete depletion.

The results in Table 1 show that the repressor encoded by Pen Its can be used to control the production of penicillinase, and more specifically the activity of the penP promoter, by temperature.

[Brief description of drawings]

Figure 1 shows the plasmids pPTB 50, pPTB 60, pPTB 60 D13 and pPT.
The restriction map of B60E24 is shown. The symbol in the figure is E; Ec
The cleavage sites of oR I, H; Hind III, P; Pst I are shown respectively. FIG. 2 shows the nucleotide sequences and amino acid sequences of the wild-type Pen I gene and its temperature-sensitive mutant gene. The first base in the encoded region of the protein was set to +1. The amino acid sequence of the encoded region is shown below. pPTB 60 D13
The base substitutions in (D13) and pPTB 60 E24 (E24) are marked on the upper side of the base sequence of the wild-type gene. In addition, the amino acid substitution corresponding to this was marked at the bottom of the amino acid sequence of the wild-type gene product.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12P 21/02 C 9282-4B (C12N 15/09 C12R 1:07) (C12N 1/21 C12R 1: 125) (C12P 21/02 C12R 1: 125) C12R 1:07)

Claims (3)

[Claims] 1. A DNA encoding a repressor having an amino acid sequence represented by the following formula. However, X represents an amino acid other than Ala and Y represents an amino acid other than Pro. 2. A vector containing a DNA encoding a repressor having an amino acid sequence represented by the following formula. However, X represents an amino acid other than Ala and Y represents an amino acid other than Pro. 3. A bacterium belonging to the genus Bacillus carrying a vector containing a DNA encoding a repressor having an amino acid sequence represented by the following formula. However, X represents an amino acid other than Ala and Y represents an amino acid other than Pro.