JP3504955B2 - Novel DNA fragment, plasmid vector carrying the same, recombinant microorganism and method for producing protein using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA fragment capable of promoting gene expression in various host microorganisms, a novel plasmid vector containing the same, a recombinant microorganism containing the plasmid vector, and a protein using the recombinant microorganism. Manufacturing method.

[0002]

2. Description of the Related Art Recently, researches on genetic engineering have been actively conducted, and an example in which a useful protein is produced in a large amount by using a recombinant microorganism has been reported. Vectors used for large-scale production of proteins in genetic engineering include various promoter regions of genes involved in the production of extracellular enzymes such as amylase, protease and levansucrase, and signals related to the extracellular secretion of these proteins. Expression / secretion vectors that apply the peptide coding region have been used.

These expression / secretion vectors are the same as those described above.
Target downstream of the motor and secretory signal peptide regions
Of the large intestine
Fungus (Escherichia coli), Bacillus subtilis (Bacillus subtili
s), Baker's yeast (Saccharomyces cerevisiae) Etc. suitable
A large amount of the target protein was obtained by introducing it into the host bacterium.
It is possible to produce the secretion.

Specifically, Bacillus amyloliquefaci
Ens (Bacillus amyloliquefacience) Α-Amira
Bacillus subtilis lodging with an expression and secretion vector that utilizes a protease gene
Production of human interferon α2 mainly [I. Palva et al.
Gene, 22, 229-235 (1983)], α-amylase of Bacillus subtilis
Escherichia coli β-lactamer in Bacillus subtilis host using gene
Ze production [K. Ohmura et al.J. Biochem., 95. 87-93 (198
4)], the alkali and Bacillus amyloliquefaciens
And Bacillus subtilis host cells using a neutral and neutral protease gene
Tafilococcus (Staphylococcus) Protein A raw
Production [N. Vasantha and L. D. Thompson,J. Bacteriol., 1
65, 837-842 (1986)], Bacillus amyloliquefaciens
Bacillus subtilis host using the alkaline protease gene
Production of E. coli alkaline phosphatase [MS Payn
e and E. N. Jackson,J. Bacteriol., 173, 2278-2282 (1
991)], Escherichia coli using the outer membrane protein gene of Escherichia coli
Production of β-galactosidase in the host [J-H Seo et al.Bio
technol. Bioeng., 32. 725-730 (1988)], Bacillus subtilis
Utilizing the bancheuculase gene
Lostridium (Clostridium) End Glucaner
Production of ZE A [G. Joliff et al.Appl. Environ. Microbio
l., 55, 2739-2744 (1989)] and the like have been reported.

[0005]

However, in the protein production using the vector as described above, the production amount cannot be said to be sufficient, and examples of industrial production of useful proteins by the application of genetic engineering are extremely difficult. The current situation is that there are few. In view of such circumstances, a novel plasmid vector that enables the large-scale production of useful proteins by finding DNA fragments that promote the expression of various structural genes encoding useful proteins in a wide range of host microorganisms Is extremely significant and is a major issue in genetic engineering.

[0006]

Means for Solving the Problems The present inventors have cloned an alkaline cellulase gene derived from a bacterium of the genus Bacillus, and have an effect of the upstream region of the gene, that is, a promoter region or a secretory signal peptide region, on the expression of a downstream gene. Was analyzed. As a result, a structural gene for a heterologous protein is ligated to the downstream of the DNA fragment of maximum 639 bp existing upstream of the gene, and this is inserted into an appropriate vector and introduced into an appropriate host bacterium to obtain the downstream. The inventors have found that the production amount of the bound structural gene product can be significantly increased, and have completed the present invention.

That is, the object of the present invention is to promote the expression of a structural gene linked to a downstream region having a base sequence derived from the promoter region and secretory signal peptide region of an alkaline cellulase-producing gene derived from a bacterium of the genus Bacillus. It is to provide a fragment (hereinafter referred to as "high expression-inducing DNA") and a novel plasmid vector containing the fragment.

Another object of the present invention is to provide a microorganism containing a recombinant plasmid in which a structural gene of a protein is linked downstream of high expression-inducing DNA of this novel plasmid vector.

Still another object of the present invention is to provide a method for producing a protein by culturing the above recombinant microorganism.

The present invention will be described in detail below. The high expression-inducing DNA of the present invention is present in the upstream part of the alkaline cellulase-producing gene of a bacterium belonging to the genus Bacillus capable of producing alkaline cellulase and encodes a promoter region and a secretory signal peptide region.

For example, the high expression-inducing DNA is Bacillus sp. KS which is a Bacillus microorganism.
Alkaline cellulase K- of M-64 (FERM P-10482) strain
It exists in the upstream region of 64 genes and has a nucleotide sequence as shown in FIG.

This Bacillus sp. KSM-64 strain
The high expression-inducing DNA possessed consists of 639 bases and has 10
From the 0th base to the 132nd base and the 208th salt
Reverse repeats in the 236th base region from the base
(IR-1 and IR-2) are present. Also, 206
234th to 234th base region, 365th salt
396th from the base and 515th from the 487th base
Promoter-like sequences (P-1,
P-2 and P-3) [C. P. Moran et al.Mol.Gen. Gene
t., 186, 339-346 (1982)] exists.

Furthermore, an operator-like sequence relating to suppression of catabolites is located in the nucleotide sequence region from the 399th position to the 414th position [WL Nicholson et al., J. Mol. Biol. , 198, 609-618.
(1987)], the ribosome binding site (SD sequence) [PJ Hager and JCR in the 600th to 606th base region.
abinowitz, The Molecular Biology of the Bacilli, vo
l. II, pp. 1-32. DA Dubnau Edition Academic Press] exists. Then, the structural gene of alkaline cellulase K-64 starts from the 610th position.

This high expression-inducing DNA promotes the expression of the structural gene bound to its downstream region and enables the large-scale production of the structural gene product, as will be clarified in the Examples below. For example, when the Bacillus sp. KSM-64 strain-derived alkaline cellulase K-64 gene is ligated downstream of the high expression-inducing DNA and introduced into a Bacillus subtilis or E. coli host, 4
~ 6 times or more productivity of alkaline cellulase K-64 is recognized (Table 1).

On the other hand, in the case where the acid cellulase K-330 gene derived from Bacillus sp. The production amount of acid cellulase K-330 is 4 times or more in Escherichia coli (Table 4) and 5 times or more in Bacillus subtilis (Table 5).

Furthermore, high expression-inducible DN in Bacillus subtilis host
Alkaline cellulase K-635 gene derived from Bacillus sp. KSM-635 strain or chloramphenicol resistance gene is ligated downstream of A to give alkaline cellulase K-635 (Table 8) or chloramphenicol acetyltransferase. (Table 10) can be produced in large quantities.

The high expression-inducing DNA is isolated from the chromosomal DNA of Bacillus sp. KSM-64 strain and other Bacillus bacterium having alkaline cellulase-producing ability. For example, the method of Marmur [ J.
Mol. Biol. , 3, 208-218 (1961)] and the method of Saito and Miura [ Biochim. Biophys. Acta. , 72, 619-629 (1963)].
Etc., and other similar methods can be used instead.

The isolated chromosomal DNA is cleaved with an appropriate restriction enzyme and then ligated to an appropriate vector DNA linearized to clone the desired high expression-inducing DNA. .

The vector used for cloning the high expression-inducing DNA of the present invention is capable of self-replicating in a host strain, and is stable to the high expression-inducing DNA and a structural gene encoding the target protein linked downstream thereof. Anything can be used as long as it is held in.

Examples of such a vector include Escherichia coli
And a shuttle vector pHY30 capable of replicating in Bacillus subtilis
0PLK [H. Ishiwa and H. Shibahra,Jpn. J. Gene
t., 60,235-243 (1985)], pUB1 capable of replicating in Bacillus subtilis
10 [K. H. Keggins et al.Proc.Natl. Acad. Sci.,US
A., 75, 1423-1427 (1978)], and p that is capable of replication in E. coli.
BR322 [F. Bolivar et al., Gene, 2, 95-113 (1977)]
And pUC19 [J. Messing,Methods Enzymol., 101, 2
0-78 (1983)] and the like.

On the other hand, as the type of host bacterium, any strain may be used as long as it can stably retain and replicate the desired recombinant plasmid. For example, in the case of E. coli host, When the HB101 strain, C600 strain, JM101 strain, etc. are B. subtilis hosts, B
D170 strain, 168 strain, ISW1214 strain and the like.

The method for transforming a host bacterium with a recombinant DNA molecule is not particularly limited. For example, in the case of an E. coli host, the method of Mandel and Higa [ J. Mol. Biol. , 53, 159].
-162 (1970)] and Hanahan's method [ J. Mol. Biol. , 166, 5
57-580 (1983)], and in the case of Bacillus subtilis host, the Competent cell method [S. Contente and D. Da.
bnau, Mol. Gen. Genet. , 167, 251-258 (1979)] and the protoplast method [S. Chang and SN Cohe
n, Mol. Gen. Genet. , 168, 111-115 (1978)] and the like.

As a method for selecting a target clone, a method using an increase in the expression level of an appropriate gene bound on a vector DNA as an index, or a probe DNA prepared based on the nucleotide sequence shown in FIG. 1 is used. It is possible to use the method by hybridization, etc., but it is preferable to carry out cloning by using the productivity of alkaline cellulase by the alkaline cellulase gene existing downstream of the present high expression-inducing DNA as an index, and then obtaining the obtained recombinant A method of subcloning the desired high expression-inducing DNA from the plasmid may be used.

[0024] To obtain a specifically high expression-inducing DNA, for example, by cutting with the restriction enzyme Hin dIII chromosomal DNA of Bacillus sp KSM-64 strain First, high expression induced DNA and alkaline cellulase K-
64 DNA was prepared containing the gene, a plasmid pUC this the same was linearised by restriction enzyme Hin dIII
After mixing with 19, a ligation reaction with T ₄ DNA ligase was carried out, and high expression-inducing DNA and alkaline cellulase K-
A plasmid pUCL64 having 64 genes and capable of self-replication in an E. coli host is prepared (FIG. 2).

Next, by cutting the plasmid pUCL64 by restriction enzymes Bgl II and Pst I, the method of Henikoff [Gene, 28, 351-359 ( 1984)] according to, exonuclease, mung bean (mung bean) nuclease and DNA polymerase After digesting the structural gene region of alkaline cellulase by treating with a large fragment (Klenow enzyme), a high expression-inducing DNA and a part of the signal sequence of alkaline cellulase K-64 were subjected to self-cyclization reaction by T ₄ DNA ligase. A plasmid vector pUS64 containing the encoding DNA is obtained (Fig. 2).

Furthermore, this plasmid vector pUS64
Restriction enzymes Eco RI and Hin dIII or Eco
1.8 kb obtained by RI and ScaI treatment
Alternatively, a 679 bp DNA fragment was digested with the restriction enzyme EcoRI.
And smoothing the cut after ends with Hin dIII treated or Hi n dIII, further shuttle vector p that Eco RI treated
By mixing each with HY300PLK and ligating using T ₄ DNA ligase, a plasmid vector pHS64 or pHSP64 containing a high expression-inducible DNA in a limited manner and capable of self-replicating in Bacillus subtilis and Escherichia coli host can be prepared ( (Fig. 2).

By inserting the structural gene of the protein to be produced into the downstream region of the high expression-inducing DNA of the thus obtained plasmid vector, a plasmid for producing the target protein in a large amount can be constructed.

In the downstream region of the high expression-inducing DNA of the obtained plasmid vectors pUS64 and pHSP64 (FIG. 2), Sal I, Xba I, Bam HI, Sma I and Kpn were included.
A multi-cloning site (MCS) having I, Sac I and Eco RI restriction enzyme sites is ligated, and is useful for cloning a foreign protein structural gene.

Further, Bacillus sp. KSM is provided in the downstream region of the high expression inducing DNA of pUS64 and pHSP64.
-64 strain S derived from the alkaline cellulase K-64 gene
D sequence, translation initiation codon and part of the signal region (10
It is also possible to express it as a fusion protein by matching the open reading frame (ORF) of the gene of interest with the ORF of this signal region.

High expression-inducing DNA obtained as described above
It is possible to bind various structural genes to the downstream thereof to produce a protein encoded by the structural gene.

As an example of the recombinant plasmid in which the structural gene is bound to the high expression inducing DNA, Bacillus sp.
PHCL64 (FIG. 3) in which a 4.4 kb fragment containing the high expression-inducing DNA of the SM-64 strain and the alkaline cellulase K-64 gene was inserted into the shuttle vector pHY300PLK, acid cellulase K-330 gene derived from Bacillus sp. KSM-330 strain Recombinant plasmid pUS-KC (Fig. 5) in which a 1.8 kb fragment containing the same was inserted into plasmid vector pUS64, and recombinant plasmid p in which the 1.8 kb fragment was inserted into plasmid vector pHS64.
HS-KC (Fig. 6), Bacillus SP KSM-63
5 (FERM P-8872) strain-derived alkaline cellulase K-6
PHSP-BC (Fig. 7) in which a 1.0 kb fragment encoding a region of Ala228-Leu584 essential for activity expression of No. 35 was inserted into pHSP-64, and a 0.8 kb fragment containing a chloramphenicol resistance gene was used as a plasmid vector. Recombinant plasmid pHSP inserted into pHSP64
-CmSB (FIG. 8) and the like.

Among the above, pHSP-BC and pHS
In the case of P-CmSB, the ORF of the inserted gene coincides with the ORF of the signal region of alkaline cellulase existing downstream of the high expression-inducing DNA, and the protein of interest is expected to be expressed as a fusion protein. It

By transforming a suitable host bacterium with the recombinant plasmid thus obtained and culturing this in a suitable medium under suitable culture conditions, a large amount of the desired protein can be produced. it can.

The type of medium used is not particularly limited as long as it can grow the recombinant microorganism and produce the enzyme. For example,
Recombinant Bacillus subtilis ISW1214 containing high expression inducing DNA
(PHCL64) strain by alkaline cellulase K-6
When producing 4, as shown in Table 2, as a carbon source, monosaccharides such as glucose and fructose, disaccharides such as sucrose, maltose or cellobiose, or polysaccharides such as CMC and soluble starch, etc. As shown in 3, a nitrogen source such as yeast extract, zest 70, polypeptone, meat extract, fish meat extract, cultivator, corn steep liquor (CSL), pharma media, soybean powder, and adipron E ₂ can be used. .

Similarly, in the case of the production of acid cellulase K-330 by the recombinant Bacillus subtilis ISW1214 (pHS-KC) strain containing a high expression-inducing DNA, as shown in Table 5 , yeast extract, fish meat were used as nitrogen sources. Extract, Zest 7
0, cultivator, CSL or the like can be used. These nitrogen source and carbon source may be appropriately combined at appropriate concentrations, and in addition to these, inorganic salts at appropriate concentrations such as sodium chloride, sodium phosphate, sodium sulfate, etc. may be added.

The pH of the medium is not particularly limited as long as it can grow the recombinant microorganism to be used, but it is preferable to adjust the pH to 6.5 to 7.5. Further, regarding the culture conditions, it may be carried out by shaking at 15 ° C. to 50 ° C., preferably at 25 ° C. to 35 ° C. for 1 to 4 days or by aerating and stirring.

[0037]

Industrial Applicability According to the present invention described above, it is possible to mass-produce a target protein. For example, ISW1214
(PHCL64) strain 32 per 1 liter of medium,
Alkaline cellulase K-64 ( Bacillus 400 U or more)
sp. KSM-64 strain) can be obtained,
In the ISW1214 (pHS-KC) strain, 20,500 U or more of acid cellulase K-330 per liter of the medium was used.
(From Bacillus sp. KSM-330 strain) can be obtained.

Further, ISW1214 (pHSP-BC)
51,400 U per liter of medium when using strains
Alkaline cellulase K-635 ( Bacillus sp.
KSM-635 strain derived, ISW1214 (pHSP-Cm
In strain SB), it is possible to obtain 68,600 U or more of chloramphenicol acetyltransferase per liter of medium.

[0039]

The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. Although the above description was made in accordance with the specific Bacillus sp. KSM-64 strain and the nucleotide sequence of the strain, a similar microorganism or a nucleotide sequence having identity with the above sequence, that is, one of the nucleotide sequences. Needless to say, those having no deletion, substitution, insertion, etc. of a base in the part that does not impair the function of the original base sequence can be used as well.

Furthermore, cellulase (CMCase) activity was measured as follows. That is, 0.4% of 2.5% carboxymethyl cellulose (CMC; Sunrose AO1MC manufactured by Sanyo Kokusaku Pulp Co., Ltd.), 0.5M glycine buffer (pH 9.0; in the case of alkaline cellulase) or citrate buffer (pH 5.2; For acid cellulase) 0.
0.1 ml of enzyme solution was added to a substrate solution consisting of 2 ml and deionized water 0.3 ml, and after reacting at 40 ° C. for 20 minutes,
The reducing sugar thus produced was subjected to the 3,5-dinitrosalicylic acid method [GL Miller et al . , Anal. Biochem. , 2, 127-132].
(1960)]. The enzyme titer was defined as 1 unit of the amount of enzyme that produced a reducing sugar corresponding to 1 μmol of glucose per minute under the above conditions.

Chloramphenicol acetyltransferase activity was measured as follows [WV Shaw,
Methods Enzymol. , 43, 737-755 (1975)]. That is, 0.4 mg / ml 5,5-dithiobis-2-nitrobenzoic acid, 0.1 mM acetyl coenzyme A and 100
1m solution consisting of mM Tris-HCl buffer (pH 7.8)
10 μl of the crude enzyme solution and 20 μl of 0.1 mM chloramphenicol were added to 1 and the change in absorbance at 412 nm was measured at 37 ° C. The enzyme titer was defined as 1 unit of the amount of enzyme that changes the absorbance at 412 nm corresponding to the acetylation of 1 μmol of chloramphenicol per minute under the above conditions.

The amount of protein was measured using a Bio-Rad protein assay kit (manufactured by Bio-Rad),
Bovine plasma albumin was calculated as a standard protein.

Example 1 Bacillus, a producer of alkaline cellulase K-64.
SP KSM-64 with 0.5% polypeptone (JP
This pharmaceutical company), 0.5% yeast extract (manufactured by Difco),
0.1% KH₂PO_Four, 0.02% MgSO_Four・ 7H₂O
And 0.5% Na₂ CO₃100 ml of medium consisting of
The cells were cultured at 30 ° C. for 12 hours with shaking. After culturing,
Saito and Miura's method from bacterial cells collected by heart separation [Bi
ochim. Biophys.Acta, 72, 619-629 (1963)] by
10 μg of the obtained chromosomal DNA was prepared by a conventional method such as [T. Mani
atis et al., Molecular Cloning, Cold Spring Harbar Labo
ratry, (1982)]HindIII (Baby
It was cut with Ringer Mannheim). Then this
Same asHinPlasmid pUC1 cut with dIII
9 (manufactured by Takara Shuzo) mixed with 1 μg, and_FourDNA liger
The binding reaction with ZE (Boehringer Mannheim)
I did.

Escherichia coli HB101 strain produced by this ligation reaction product
Competent cells (manufactured by Takara Shuzo)
The bacterial suspension after treatment is treated with 50 μg / ml amp.
LB agar plate medium containing picillin (manufactured by Sigma)
[1.0% tryptone (manufactured by Difco), 0.5% yeast
Extract (Difco), 1.0% NaCl, 1.5%
Bact agar (manufactured by Difco)] and apply at 37 ° C for 12
Incubated for hours. 0.5 on the colonies of the transformants that appeared.
% CMC, 1 mg / ml lysozyme (manufactured by Sigma) and
And 50 mM glycine buffer (pH 9) 1.0
After overlaying with% agar, it was left for about 6 hours. After this, the village
Strains that decomposed the surrounding CMC were treated with the Congo Red method [R.
 M. Teather and P. J. Wood,Appl. Environ. Microbio
l., 43,777-780 (1982)], and the target group
The exchanged E. coli was isolated.

Example 2 The recombinant plasmid harbored by the recombinant Escherichia coli obtained in Example 1 was prepared by a conventional method [eg, T. Maniatis et al., Molecular Clonin].
g, Cold Spring Harbar Laboratry, (1982)] and analyzed by agarose gel electrophoresis [JA Meyers et al., J. Bacteriol. , 127, 1529-15].
37 (1976)]. As a result, next apparent that Alkaline Cellulase K-64 gene is present in the Hin dIII fragment of about 4.4 kb, E. coli HB101 strain containing pUCL64, pUCL64 plasmid (7.1 kb) containing this HB101 (PUCL64 ) Strain.

This recombinant plasmid, pUCL64, was controlled.
Restriction enzymeHinAgarose gel after cutting with dIII
Electrophoresis is performed by the method of McDonnell et al. [J. Mol. Bio
l., 110, 119-146 (1977)], about 4.4 kbH
inThe dIII fragment was isolated. About 4.1 kb of thisXh
oI-HinThe danger region was modified by the Sanger method [F. Sanger et al.Pro
c. Natl. Acad. Sci.,USA, 74, 5463-5467 (1977)]
Alkaline cellular consisting of 2466bp
The structural gene for zeK-64 was found.

Upstream of this structural gene, as shown in FIG. 1, promoter-like sequences (P-1, P-2, P-
3), inverted repeat sequences (IR-1, IR-2), operator-like sequences for catabolite repression (==
=) And a ribosome binding site (SD sequence, ΔΔΔ), etc. were observed.

[0048] The Hin dIII fragment of about 4.4kb obtained in implementation example 3 Example 2, according to the strategy shown in Figure 3, was inserted into a shuttle vector pHY300PLK, to prepare a recombinant plasmid PHCL64. In addition, according to the strategy shown in FIG. 3, from the obtained recombinant plasmid pHCL64, a Sma I- Sca I 1.1 kb fragment or Sma I
-Plasmid pH deficient in the Hpa I 1.5 kb fragment
CL64a and pHCL64b were prepared and recombinant E. coli HB101 containing these recombinant plasmids (pHC
L64) strain, HB101 (pHCL64a) strain and H
B101 (pHCL64b) strain was acquired.

Then, the same procedure as in Example 2 was carried out from both recombinant Escherichia coli.
Using each purified recombinant plasmid extracted in this way,
Herb fungus ISW1214 strain (leuA8,metB5,hsrM1) Trait
The conversion is based on the protoplast method [S. Chang and S. N. Choen,Mo
l. Gen. Genet., 168, 111-115 (1978)]
Protoplus containing 5 μg / ml tetracycline
DM3 medium for regeneration [0.5% sodium succinate (p
H7.3), 0.5% casamino acid, 0.5% yeast extract
Su, 0.35% K₂HPO_Four, 0.15% KH₂PO_Four ,
0.5% glucose, 20 mM MgCl₂, 0.01%
 Transformation using bovine serum albumin (Sigma)
I chose my body.

As a result, recombinant Bacillus subtilis ISW1214 (pHCL64) strain containing these recombinant plasmids, IS
W1214 (pHCL64a) strain, ISW1214 (p
HCL64b) strain was acquired.

Example 4 Each recombinant Bacillus subtilis strain or E. coli strain obtained in Example 3 was treated with PY medium containing 15 μg / ml tetracycline (2%
Polypeptone, 0.1% yeast extract, 0.1% KH ₂
PO ₄ , 0.5% NaCl) or 50 μg / ml ampicillin in LB medium at 30 ° C. or 37 ° C. with shaking, and the alkaline cellulase activity in the culture medium or cell-free extract was measured. A plasmid containing DNA, namely pHCL64 or pHCL64a
It is clear that the productivity of alkaline cellulase K-64 by the recombinant Bacillus subtilis ISW1214 strain or Escherichia coli HB101 strain that retains is about 4 to 6 times that of the recombinant strain that retains pHCL64b that does not contain high expression induction DNA. , And the effect of promoting enzyme production by high expression-inducing DNA was shown (Table 1).

[0052]

Recombinant Bacillus subtilis ISW1214 (pHCL64) strain having high expression-inducing DNA was used in various carbon sources of 0.2% (however, CMC and soluble starch were 1.0%).
And PY medium containing 15 μg / ml tetracycline and various nitrogen sources (0.2% of total nitrogen, ie 2.6
% Yeast Extract, 2.9% Zest 70, 1.8% Polypeptone, 2.0% Meat Extract, 3.4% Fish Meat Extract, 9.
N medium containing 6% cultivator, 8.0% CSL, 2.8% Pharmamedia, 3.6% soybean flour or 2.6% adipron E ₂ ) and 15 μg / ml tetracycline [0.5% fish meat extract ( NE40), 0.05% yeast extract (B-2), 0.1% KH 2 PO 4, 0.02% M
gSO ₄ .7H ₂ O, 1.0% maltose]. As a result, the recombinant Bacillus subtilis ISW1214 (pHCL64) strain produced alkaline cellulase K-64 with higher productivity.
Was confirmed to be obtained (Tables 2 and 3).

[0054]

[0055]

[0056] The recombinant plasmid pUCL64 obtained in implementation example 5 Example 2 was cleaved by restriction enzymes Bgl II and Pst I, using a de-translation kit (Takara Shuzo), subjected to digestion of alkaline cellulase structural gene After this, a self-cyclization reaction was carried out using T ₄ DNA ligase. Then, E. coli was transformed with the ligation product in the same manner as in Example 1, and the recombinant plasmid of the transformant that appeared was analyzed in the same manner as in Example 2 to give the alkaline cellulase K-64 structural gene. Recombinant Escherichia coli H carrying the plasmid vector pUS64 (FIG. 2) deleted in
B101 (pUS64) was selected.

Example 6 Recombinant plasmid pUS64 was extracted and purified in the same manner as in Example 2, and the restriction plasmids Eco RI and Hin dIII were used for extraction.
Cleavage was carried out and a DNA fragment of 1.8 kb in the upstream region of the alkaline cellulase K-64 gene was isolated. Were combined using a shuttle vector pHY300PLK and T ₄ DNA ligase was cleaved with restriction enzymes Eco RI and Hin dIII to this.

On the other hand, the recombinant plasmid pUS was similarly prepared.
After cutting 64 with restriction enzymes Eco RI and Sca I, 679 bp in the upstream region of alkaline cellulase K-64
Was isolated. And this DNA fragment was cleaved with the restriction enzyme Hin dIII, the ends blunted with DNA Blunting Kit (Takara Shuzo), further restriction enzyme
Shuttle vector pHY300P cut with Eco RI
It was mixed with LK and ligated with T ₄ DNA ligase.

The transformation treatment of Escherichia coli with both ligation products was carried out according to Example 1, and the mixture was smeared on an LB agar plate medium containing 15 μg / ml of tetracycline (manufactured by Sigma) and cultured at 37 ° C. for 12 hours. The emerged transformant colony was inoculated into 5 ml of LB medium containing tetracycline, the restriction enzyme cleavage point of the recombinant plasmid obtained in the same manner as in Example 2 was analyzed, and the plasmid vector pHS64 is retained. Recombinant E. coli HB101
(PHS64) strain and plasmid vector pHSP6
Escherichia coli HB101 (pHSP64) strain carrying 4 was selected (FIG. 2).

Example 7 About 1.8 kb Hin dI containing the acid cellulase K-330 gene derived from Bacillus sp. KSM-330 strain
II- HpaI fragment [K. Ozaki et al., J. Gen. Microbiol. , 1
37, 2299-2305 (1991)] was blunted with a DNA blunting kit (Takara Shuzo). A linear vector pUC1 cleaved with the restriction enzyme Sma I
9, plasmid vector pUS64, plasmid vector pHS64 and shuttle vector pHY300PL
K was mixed separately and ligated with T ₄ DNA ligase.

Transformation of Escherichia coli with the ligation reaction product was carried out in the same manner as in Example 1, the restriction enzyme cleavage points of the obtained plasmid were analyzed, and the DNA fragment was inserted into the Sma I site of each plasmid. Recombinant plasmids, namely pUKC331 and pUKC331R
(FIG. 4), pUS-KC and pUS-KCR (FIG. 5),
pHS-KC and pHS-KCR (FIG. 6), and pH
Recombinant E. coli HB101 strains containing KC331 and pHKC331R (FIG. 4) were obtained.

In the same manner as in Example 3, pHY30
Recombinant Bacillus subtilis ISW1214 strain was transformed with each recombinant plasmid derived from 0PLK.
1214 (pHKC331) strain, ISW1214 (pH
KC331R) strain, ISW1214 (pHS-KC) strain and ISW1214 (pHS-KCR) strain were acquired.

Example 8 Each of the recombinant Escherichia coli and Bacillus subtilis strains obtained in Example 7 was inoculated into LB medium containing ampicillin or PY medium containing tetracycline, and shake culture was carried out at 37 ° C. or 30 ° C. As a result, a recombinant Bacillus subtilis ISW1214 (pHS-KC) carrying a plasmid in which the high expression-inducing DNA is present upstream of the acid cellulase K-330 structural gene
Productivity of acidic cellulases strains, recombinant Bacillus subtilis ISW1214 (pHKC 331 without the high expression induced DNA
R ) strain and a recombinant Bacillus subtilis ISW1214 (pHS-KC) present downstream of the acid cellulase K-330 structural gene.
It was revealed that it was about 5 to 6 times higher than that of the R) strain (Table 4), and the efficacy of the high expression induction DNA was shown here. Also, a recombinant Bacillus subtilis IS containing a high expression-inducing DNA
W1214 (pHS-KC) strain contains N containing various nitrogen sources.
It showed good production of acid cellulase K-330 in the medium (Table 5).

[0064] * See FIG. 4, ** See FIG. 6 + Shows the direction of the secretory signal peptide present in the highly expressed inducible DNA with respect to the transcription direction.

[0065]

Similarly, in recombinant Escherichia coli, a recombinant E. coli (pU) carrying a plasmid in which high expression-inducing DNA is present upstream of acid cellulase K-330 structural gene
High acid cellulase activity was observed in the cell-free extract of (S-KC) (Table 6).

[0067]

Example 9 Bam of about 1.0 kb containing the alkaline cellulase K-635 gene derived from Bacillus sp. KSM-635 strain.
HI- Bam HI fragment was cleaved with a restriction enzyme Bam HI, vector pHY300PLK, and about 1.0 kb Bam containing alkaline cellulase K-635 structural gene.
HI- a Sal I fragment was mixed respectively with restriction enzymes Bam HI- Sal I at the cleaved linear vector pHSP64, bound with T ₄ DNA ligase.

The Escherichia coli HB101 strain was transformed with the ligation reaction product in the same manner as in Example 1, and the restriction enzyme cleavage points of the obtained recombinant plasmid were analyzed. As a result, each digested DNA fragment was converted to pHY300PLK B
am HI site or Bam HI at pHSP64, Sal I
PHB, which is each recombinant plasmid inserted between the sites
C115B, pHBC115BR and pHSP-BC
Recombinant Escherichia coli HB101 strains each containing (Fig. 7) were obtained.

The recombinant Bacillus subtilis ISW1214 strain was transformed with each recombinant plasmid extracted in the same manner as in Example 2 as in Example 3.
1214 (pHBC115B) strain, ISW1214 (p
HBC115BR) strain, ISW1214 (pHSP-B
C) The strain was acquired.

Example 10 Each of the recombinant Bacillus subtilis strains obtained in Example 9 was inoculated into a PY medium containing tetracycline, and cultured at 30 ° C. with shaking. As a result, recombinant Bacillus subtilis ISW1214 (pHSP-B carrying a plasmid having high expression-inducing DNA upstream of the alkaline cellulase K-635 structural gene
Strain C) showed high productivity of alkaline cellulase (Table 7). Further, the recombinant Bacillus subtilis ISW1214 (pHSP-BC) strain containing high expression-inducing DNA showed good production of alkaline cellulase K-635 in the N medium containing tetracycline (Table 8).

[0072]

[0073]

Example 11 In the same manner as in Example 7, after blunting the ends of a chloramphenicol resistance gene (Pharmacia) of about 0.8 kb, this was linearized with the restriction enzyme Sma I. The plasmid vector pHSP64 was mixed and ligated with T ₄ DNA ligase. Transformation of E. coli with the ligation reaction was performed according to Example 1. The treated bacterial suspension was smeared on an LB agar plate medium containing 15 μg / ml tetracycline (manufactured by Sigma) and cultured at 37 ° C. for 12 hours.

From the resulting transformants, the restriction enzyme cleavage points of the recombinant plasmid obtained in the same manner as in Example 2 were analyzed, and recombinant Escherichia coli HB101 carrying the recombinant plasmid pHSP-Cm (FIG. 8) was analyzed. (PHSP-Cm) was selected. Further Sal I and Bam this pHSP-Cm
After digestion with HI, digestion with mung bean nuclease and T
_{4 By} recombining with DNA ligase, the secretory signal peptide region in the high expression-inducing DNA and O of chloramphenicol acetyltransferase structural gene
An RF matched pHSP-CmSB was prepared.

The plasmid pHSP-CmSB extracted in the same manner as in Example 2 was used to transform the Bacillus subtilis ISW1214 strain in the same manner as in Example 3, and the recombinant Bacillus subtilis ISW1 carrying the plasmid pHSP-CmSB was transformed.
The 214 (pHSP-CmSB) strain was obtained.

Recombinant Bacillus subtilis ISW1214 (pHSP-
CmSB) strain and ISW1214 (pH
SP64) strain and ISW1214 (pHY300PL
K) strain was inoculated into LB medium containing tetracycline, and 3
When shake culture was performed at 0 ° C., the recombinant Bacillus subtilis harboring the plasmid pHSP-CmSB having high expression-inducing DNA produced about 80 mg / l chloramphenicol acetyltransferase (Table 9). In addition, recombinant Bacillus subtilis ISW1214 (p containing high expression inducing DNA
The HSP-CmSB) strain showed good chloramphenicol acetyltransferase production in N medium containing tetracycline (Table 10).

[0078]

[0079]

[0080]

[Sequence listing] SEQ ID NO: 1 Array length: 639 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA Source: Organism name: Bacillus sp. Share Name: KSM-64 Sequence features: Characteristic symbol: RBS Location: 600..606 How the feature was determined: E Sequence features: Characteristic symbol: peptide Location: 610..639 How the feature was determined: E  Arrangement  AGTACTTACC ATTTTAGAGT CAAAAGATAG AAGCCAAGCA GGATTTGCCG ATGCAACCGG 60  CTTATATTTA GAGGGAATTT CTTTTTAAAT TGAATACGGA ATAAAATCAG GTAAACAGGT 120  CCTGATTTTA TTTTTTTGAA TTTTTTTGAG AACTAAAGAT TGAAATAGAA GTAGAAGACA 180  ACGGACATAA GAAAATTGTA TTAGTTTTAA TTATAGAAAA CGCTTTTCTA TAATTATTTA 240  TACCTAGAAC GAAAATACTG TTTCGAAAGC GGTTTACTAT AAAACCTTAT ATTCCGGCTC 300  TTTTTTTAAA CAGGGGGTGA AAATTCACTC TAGTATTCTA ATTTCAACAT GCTATAATAA 360  ATTTGTAAGA CGCAATATAC ATCTTTTTTT TATGATATTT GTAAGCGGTT AACCTTGTGC 420  TATATGCCGA TTTAGGAAGG GGGTAGATTG AGTCAAGTAG TCATAATTTA GATAACTTAT 480  AAGTTGTTGA GAAGCAGGAG AGAATCTGGG TTACTCACAA GTTTTTTAAA ACATTATCGA 540  AAGCACTTTC GGTTATGCTT ATGAATTTAG CTATTTGATT CAATTACTTT AATAATTTTA 600             1 2 3 4 5 6 7 8 9 10  GGAGGTAAT ATG ATG TTA AGA AAG AAA ACA AAG CAG TTG 639            Met Met Leu Arg Lys Lys Thr Lys Gln Leu

[Brief description of drawings]

FIG. 1 is an upstream region of the alkaline cellulase K-64 gene of Bacillus sp.
It is a figure of the base sequence of A).

FIG. 2 is a diagram showing the construction of plasmid vectors pUS64, pHS64 and pHSP64 containing high expression induction DNA.

FIG. 3 is a view showing the construction of a plasmid containing the alkaline cellulase K-64 gene of Bacillus sp. KSM-64 strain.

FIG. 4 is a view showing the construction of a plasmid containing the acid cellulase K-330 gene of Bacillus sp. KSM-330 strain.

FIG. 5 shows the construction of a plasmid containing the acid cellulase K-330 gene of Bacillus sp. KSM-330 strain.

FIG. 6 is a diagram showing the construction of a plasmid containing the acid cellulase K-330 gene of Bacillus sp. KSM-330 strain.

FIG. 7 shows the construction of a plasmid containing the alkaline cellulase K-635 gene of Bacillus sp. KSM-635 strain.

FIG. 8 shows the construction of a plasmid containing a chloramphenicol resistance gene.

[Explanation of symbols]

2 to 8, the bold line indicates the DNA of the inserted gene.
This is a fragment, and the thin line portion is DN derived from the plasmid pUC19 or pHY300PLK shown in each plasmid.
A fragment. On the other hand, the arrow indicates the structural gene region of the introduced various genes, and the hatched portion indicates the region of high expression inducing DNA. Ba Bam HI Bg Bgl II Kp Kpn I Hp Hpa I Hi Hin dIII Ps Pst I Pv Pvu II Sl Sal I Ec Eco RI Sm Sma I Xb Xba I Xh Xho I Sc Sca I Sa Sac I Ap ampicillin resistance gene Tc tetracycline gene AEG Alkaline cellulase gene (multiple cloning site) MCSI Ec-Sa-Kp-Sm-Ba-Xb-
Sl-Ps-Sp-Hi MCSII Ec-Sm-Ba-Sl-Ps-Bg-
Xb-Hi MCSIII Ec-Sa-Kp-Sm-Ba-Xb-
Sl MCSIV Ec-Sa-Kp- (Sm) MCSV Hi-Sp-Ps-Sl-Xb-Ba-
(Sm) MCSVI Sl-Xb-Ba- (Sm) MCSVII Hi-Xb-Bg-Ps-Sl-Ba MCSVIII Sp-Ps-Sl-Xb-Ba-Sm
-Ec MCSIX Hi-Xb-Bg-Ps-Sl-Ba-
Xb-Sl-Ps-Sp MCSX Ba-Sm-Ec MCSXI Ba-Sm-Kp-Sa-Ec

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI C12R 1:19) C12N 15/00 ZNAA (C12N 1/21 C12R 1: 125) (C12N 9/10 C12R 1: 125) (C12N 9/10 C12R 1:19) (C12N 9/42 C12R 1:19) (C12N 9/42 C12R 1: 125) (56) Reference JP-A-4-190793 (JP, A) Biosci. Biotechno l. Biochem. , Vol. 56 (6), p. 872-877 (1992) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09 C12N 1/21 C12N 9/10 C12N 9/42 C12R 1:19 C12R 1: 125 GenBank / EMBL / DDBJ / Gene Seq CA / BIOSIS / MEDLINE / W PIDS (STN)

Claims (6)

(57) [Claims] 1. A D capable of promoting the expression of a structural gene bound to a downstream region having a base sequence derived from the promoter region and secretory signal peptide region of the Bacillus bacterium-derived alkaline cellulase producing gene shown in SEQ ID NO: 1.
NA fragment or one or more of the nucleotide sequences of SEQ ID NO: 1
A base sequence in which several bases have been deleted, replaced or added
A DNA fragment which has the same nucleotide sequence as SEQ ID NO: 1 and can promote the expression of a structural gene bound to the downstream region. 2. A structural gene linked to a downstream region (provided that the alkaline cellulase K-64 gene has a nucleotide sequence derived from the promoter region and secretory signal peptide region of the Bacillus bacterium-derived alkaline cellulase producing gene shown in SEQ ID NO: 1). Vector) or a sequence number containing a DNA fragment for promoting the expression of
One or more bases in the base sequence of No. 1 are deleted or
Includes a DNA fragment having a modified or added base sequence
Only, a DNA fragment having the nucleotide sequence of SEQ ID NO: 1
Structural gene linked to the downstream region in the same manner as the rasmid vector (excluding alkaline cellulase K-64 gene)
A plasmid vector containing a DNA fragment for promoting the expression of 3. An acid cellulase K330 having a structural gene.
The plasmid vector according to claim 2, which is a gene, an alkaline cellulase K-635 gene, or a chloramphenicol resistance gene. 4. A recombinant microorganism containing the plasmid vector according to claim 2 or 3. 5. The recombinant microorganism according to claim 4, wherein the host microorganism is Escherichia coli or Bacillus subtilis. 6. A method for producing a protein, which comprises culturing the recombinant microorganism according to claim 4 or 5, and separating the target protein from the culture.