JPH0724585B2 - Actinomycetes promoter DNA sequence - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel promoter gene in the gene manipulation technology of actinomycetes.

(Problems to be Solved by Conventional Techniques and Inventions) Promoter genes for expression in gene manipulation techniques using Escherichia coli as a host have already been considerably studied. However, regarding gene manipulation using actinomycetes as a host, research on promoter genes has hardly progressed.

When a useful protein such as interferon is produced in a microorganism by a gene manipulation method, the target product is often harmful to the microorganism. When a foreign gene is expressed using an uncontrollable promoter, the target product continues to be produced during the growth phase of the microorganism, so self-defense cannot be achieved, and the host bacterium is killed, making it difficult to produce the target product in a large amount.

If expression can be artificially controlled by external signals,
It is possible to produce a useful product from the target time, for example, the time when the growth of the microorganism is completed.

As such an example, a study using the promoter (gal P1) of the galactose operon of Streptomyces Iividans has recently been published (Proc.Natl.Acad.Sci.USA, Vol84,2130-2134,1987). ).
However, more useful promoters have been sought in the field of gene manipulation of actinomycetes.

(Means and Actions for Solving the Problems) The present inventors have developed a special promoter gene capable of controlling the expression of thiostrepton, which is widely used for the selection of plasmids in the gene manipulation of actinomycetes, as an inducer of gene expression. I found it.

That is, the present invention relates to a promoter gene whose expression is induced by thiostrepton. More specifically, the seventh
The present invention relates to a part of the gene that substantially functions as a promoter in the DNA sequence of the promoter region of the 19 kd protein induced by thiostrepton contained in the figure.

Furthermore, the present invention also relates to the use of expression of genes encoding various foreign useful substances such as human-derived interferon by incorporating this promoter gene.

The background of the present invention is as follows. Plasmid pIJ in Streptomyces Iividans 66
When 702-introduced strain (FERM BP-739) was cultivated and thiostrepton was added during the growth or stationary phase, a large amount of protein (19 kd) was transferred to Streptomyces.
We found that it was inducibly expressed from the dye pair of Lividance 66 (pIJ702). If a gene encoding this protein is cloned and the expression control site (promoter region) of that gene is used as a tool for expressing a useful gene in actinomycetes, the target gene can be switched by addition of thiostrepton. It can be expressed at a desired time.

Therefore, the gene encoding the above-mentioned 19 kd protein was cloned and its promoter region was determined. Furthermore, it was demonstrated that when this promoter region was used, aminoglycoside phosphatase (aph II) derived from E. coli could be expressed only when thiostrepton was added (Table 1). This indicates that the promoter gene of the present invention can also be used for expression of foreign genes.

Example 1 (1) Purification of 19kd protein Streptomyces lividans 66 into which pIJ702 was introduced 66
(St. Iividans) (FERM BP-739) in YEME medium (80 ml / Sakaguchi flask, 31 ° C, 4 ° C) containing 20 μg / ml thiostrepton.
It was cultured for 8 hours. The 19 kd protein was separated and purified from 3 g of the crude cell extract using FPLC manufactured by Pharmacia. The columns and elution buffer used are as follows.

First step: column MonoQ (Pharmacia), elution buffer, 50 mM Tris-HCl (pH 7.0) NaCl 0 to 1 M gradient Second step: column Superose12 (Pharmacia), elution buffer, 50 mM Tris-HCl (pH 7.0) ) 100 mM NaCl 3rd step: Column ProRPC (Pharmacia), elution buffer, H ₂ O (containing 0.1% trifluoroacetic acid)
→ 80% acetonitrile (containing 0.1% trifluoroacetic acid) The 19 kd protein was detected using Pharmacia Fast Flow System, and the molecular weight was detected by SDS polyacrylamide gel as a marker.

(2) Analysis of N-terminal amino acid sequence of 19kd protein The purified 19kd protein was subjected to Edman degradation using an amino acid sequence analyzer (Applied Biosystems model 470A) to determine 14 kinds of amino acid sequences from the N-terminal. . First
As shown in the figure, it was glycine-isoleucine-aspartic acid-leucine-threonine-proline-glutamic acid-glutamic acid-lysine-phenylalanine-glutamic acid-valine-phenylalanine-glycine.

(3) Preparation of synthetic probe In order to clone the gene encoding the 19kd protein, the DNA sequence corresponding to the 6th amino acid (proline) to the 14th amino acid (glycine) from the N-terminal was determined, and the DNA synthesizer (Applied Biosystems model 3
80 bases) were used to synthesize 27-based oligonucleotides (Fig. 2).

(4) Cloning of DNA encoding the 19kd protein Streptomyces lividans 66 (FERM BP-737) chromosomal DNA was partially digested with the restriction enzyme Sau3AI and preliminarily digested with BamHI λ phage vector EMBL3 (ST
RATA GENE).

This was introduced into Escherichia coli JM101 strain (manufactured by Toyobo) to form plaques. The plaques were transferred to a nitrocellulose membrane, the above 27 base synthetic DNA was labeled with ³² P and used as a probe to obtain 5 hybridizing clones.

(5) Preparation of phage DNA Method such as Grossbergar (Nucleic Aci
DNA was purified from the phage plaque to which the synthetic probe was hybridized according to ds Research Vol 15, No16, 1987). Restriction map of cloned DNA (3rd
(FIG. 3) and at the same time when the 27-base synthetic probe labeled with ³² P was used to determine the hybridizing region by the Southern method, the PstI (C) -SstI ( D) area.

(6) Determination of DNA nucleotide sequence United States Biochemical using cloning method using M13 phage and nucleotide sequence determination method using Dideoxy sequencing method (Proc.Natl.Acad.Sci.USA, 1977,74; 5463-5467) PstI (C)-
When the DNA base sequence of the SstI (D) region was determined, a DNA sequence (Fig. 4) corresponding to 14 N-terminal amino acid sequences of the 19kd protein analyzed above was present.

(7) Determination of mRNA start point by S1 mapping Streptomyces lividans 66 (FERM BP-739) in which pIJ702 was introduced into YEME medium (80 ml / Sakaguchi flask, 31 ° C)
Was inoculated, and after 24 hours, 20 μg / ml thiostrepton was added. After further culturing for 24 hours, the cells are collected and the method such as Hopwood is used (Genetic Manipulation of Strep).
mRNA was isolated according to Tomyces, The John Innes Foundation, England 1985). On the other hand, SmaI (A) -Ss shown in FIG.
27-base synthetic DNA using the single-stranded DNA obtained by cloning the DNA region of tI (D) in M13 phage as a template
Was used as a primer to synthesize DNA using α- ³² P-dATP. Using this DNA and the above mRNA, S1 mapping was carried out according to the method of Hopwood et al., And it was revealed that the start point of the mRNA encoded by the 19 kd protein is at * shown in FIG.

(8) Promoter search test Next, the promoter region was cloned using the promoter search vector pIJ486 (John Innes Culture Collection) that is usually used in actinomycetes. Judging from the results of the previous S1 mapping, SmaI (A) -NaeI shown in FIG.
The region (B) was presumed to have promoter activity, and this region was subcloned into the SmaI side of E. coli vector pUC13 (Pharmacia). This plasmid is then EcoRI
And pIJ4 same as double digest with Hind III
86 was combined with EcoRI and a double digest with Hind III, and introduced into the protoplasts of Streptomyces lividans by a method commonly used in gene manipulation of actinomycetes. Spores of the strain regenerated on R2YE medium were treated with kanamycin 30
Replicating to a nutrient agar (Difco) medium containing μg / ml and thiostrepton 2.5 μg / ml, a growing strain was obtained, from which a plasmid was isolated. As shown in FIG. 6, these strains contain SmaI (A) -NaeI, the promoter region of the gene encoding the 19kd protein.
The region (B) carried the plasmid pAK114 plasmid inserted upstream of the aminoglycoside phosphatase gene.

Streptomyces lividans 66 and pI carrying pAK114
When the expression of kanamycin resistance of Streptomyces lividans 66 carrying J1486 was examined depending on the presence or absence of thiostrepton, as shown in Table 1, Streptomyces lividans 66 carrying pAK114 was found only when thiostrepton was added. It showed kanamycin resistance.

(9) DNA base sequence of SmaI (A) -NaeI (B) fragment From the above results, the promoter region of the 19kd protein was determined to be the SmaI (A) -NaeI (B) region shown in FIG. DNA sequence was determined by cloning with M13 phage and Dideoxy sequencing method. As shown in FIG. 7, it was a region consisting of 149 bases.

(Effects of the Invention) In the field of gene manipulation of actinomycetes, the use of the promoter gene according to the present invention can be useful for the production of useful substances.

[Brief description of drawings]

Figure 1: Shows the N-terminal amino acid sequence of the 19kd protein. Figure 2: Shows the DNA sequence (synthetic probe) corresponding to the N-terminal 6th to 14th amino acids of the 19kd protein. FIG. 3: Restriction enzyme map of phage plaque DNA hybridized with synthetic probe. FIG. 4: DN corresponding to pstI (C) -sstI (D) in FIG.
A base sequence is shown. FIG. 5: Shows a restriction map of the promoter search vector pIJ486. FIG. 6 shows the nucleotide sequence of the promoter site of pAK114. p
SmaI (A) -Nae on the Hind III-EcoRI site of IJ486
(B) The area is inserted. FIG. 7: Shows the DNA base sequence of the SmaI (A) -NaeI (B) region of FIG.

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Claims (2)

[Claims] 1. A promoter gene whose expression is induced by thiostrepton, which comprises the following nucleotide sequence: 2. A method for producing a useful substance by using the promoter gene according to claim 1 when the useful gene is expressed in actinomycetes.