JPH02257876A - Mutant of bacillus brevis and use thereof - Google Patents

Abstract

PURPOSE:To produce alpha-amylase of human salivary gland and a specific beta-amylase by using a mutant of Bacillus brevis capable of forming extremely large amount of protein out of the cell as a host microorganism for genetic recombination and culturing the cell. CONSTITUTION:Bacillus brevis 47 (FERM P-7224) is treated with nitrosoguanidine, etc., to obtain a new mutant, Bacillus brevis 47K (FERM BP-2308) capable of secreting extremely large amount of proteins in the cultured product compared with the parent strain. Transformation is carried out by conventional method using a plasmid as a manifestation vector for the manifestation of a gene such as human salivary gland amylase gene obtained from an mRNA extracted from the human salivary gland and using the Bacillus brevis 47K as a host. The transformant is cultured at 15-42% and, after the completion of culture, the culture product is separated into bacterial cells and supernatant by centrifugal separation, etc. The supernatant is purified to obtain the objective substance such as human salivary gland alpha-amylase.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is directed to a novel mutant Bacillus
s) and its transformants or their uses.

More specifically, the present invention relates to a novel mutant Bacillus brevis as a host bacterium in genetic recombination.

In addition, the present invention provides a human saliva α-amylase gene (h
The present invention relates to a mutant Bacillus brevis that retains DNA containing AMY) and a method for producing human salivary gland α-amylase using the mutant Bacillus brevis. Also.

The present invention relates to Clostridium thermosu
β-amylase gene (R
Mutant Bacillus carrying DNA containing AM”/)
brevis and C1ostridiu+ using them
The present invention relates to a method for producing a thermosulfuroenes β-amylase.

(Prior art) Conventionally, much recombinant DNA research has been carried out using Escherichia coli (E, Co1).
t), and many heterologous genes have already been expressed in E. coli. However, in this method, since the expressed gene product accumulates within the bacterial body, it not only takes a great deal of time and effort to extract the target substance from the bacterial body and purify it from the extract; It was not easy to obtain the desired substance in its pure form.

On the other hand, bacteria of the genus Bacillus have long been used industrially as producing bacteria for various extracellular enzymes, and the DNA regions encoding the promoters and signal peptides of the genes of these extracellular enzymes have been cloned. It is thought that by linking the structural gene of the target protein to the downstream thereof and introducing this into a bacterium of the genus Bacillus, it will be possible to secrete the target protein as a gene product to the outside of the bacterium.

In Bacillus bacteria, secreted proteins are synthesized as precursors with a signal peptide consisting of approximately 20 to 30 amino acid residues at the amino terminus, and this portion is then cleaved by signal peptidase to form the mature protein. It is believed that Bacillus amyloliquefaciens (Bacillus amyloliquefacens)
liquefaciens) α-amylase gene (
1, Pa1va et al., Gene, 22.22
9 (1983)), Bacillus licheniformis (Ba
Benicillinase gene (S, Chang et al., Molecular
Cloning and Gene Regulation in Bachirai (Molecular Cloning
and Gene Regulation 1n13a
cilli), Academic Press, 6
59 pages, 1982], Bacillus subtilis (Bacillus
α-amylase gene of S. illus 5ubtilis (Hl Yamazaki et al., Journal of Bacteriol.) 156
．． 327 (1983)) have been cloned, and secretion of heterologous proteins using these signal peptides has been reported.

Bacillus subtilis is mainly used as a host for secretory production of the above-mentioned heterologous proteins, but in order to produce large amounts of extracellular protease, this microorganism can be secreted using recombinant DNA technology. The foreign protein that has been used is degraded, and its accumulated amount is significantly reduced.

In contrast, Udaka et al.
We discovered that there are many strains of Bacillus brevis that produce almost no protease, and one of them, Bacillus brevis 47 [FERM P-7224;
Main extracellular proteins [)1. Yamagat
a et al., J. Bacteriol.

169, 1239 (1987), Norihiro Tsukagoshi, Journal of the Japanese Society of Agricultural Chemistry, 61, 68 (1987) and JP-A-62-2
Publication No. 01583 includes “outer wall
protein and m1ddle wallp
It has been described as "rotein", "major extracellular protein" and "cell surface protein". ] A secretion vector was prepared using the promoter of the gene and a region encoding the signal peptide of the MV protein (middle wall protein), which is a type of major extracellular protein, and α-amylase [JP-A-Sho 62
-201583 publication, H. Yamagata et al., J.
Bacteriol,, 169.1239 (19g
? )) and porcine pepsinogen (Juzo Uru 1 Collection of lecture abstracts for the 1986 meeting of the Japanese Society of Agricultural Chemistry, p. 837-838;
Norihiro Tsukagoshi 1 Journal of the Japanese Society of Agricultural Chemistry, 61.68 (1987)
) has been successfully secreted and produced.

(Problems to be Solved by the Invention) As mentioned above, various attempts have been made to secrete and produce heterologous proteins using Bacillus brevis as a host, and it has been shown that the system is very efficient compared to E. coli, Bacillus subtilis, etc. Understood.

However, the amount of protein produced remains within a certain limit, and in industry there is a need for hosts that can produce even greater amounts of protein.

(Means for Solving the Problems) The present inventors (1) conducted intensive research to create a more efficient secretory production system for heterologous proteins using Bacillus brevis as a host bacterium, and found that Bacillus brevis 47
By treating (FORM P-7224) with a mutagen, an extremely superior bacterial cell was created. The present invention is a novel mutant Bacillus brevis that produces a significant amount of protein outside the bacterial cell. Our goal is to provide the following.

Another object of the present invention is to provide a novel method for producing human salivary gland α-amylase using a novel mutant Bacillus brevis as a host.

Furthermore, the present invention provides Clostridium thermosulfur using a novel mutant Bacillus brevis as a host.
An object of the present invention is to provide a new method for producing β-amylase derived from P. genus.

The present inventors have conducted intensive research in order to provide a host bacterium that can efficiently secrete and produce heterologous proteins.

Bacillus brevis 47 (FERM P-7224)
By treating Bacillus brevis with nitrosoguanidine, we succeeded in creating a new mutant bacterium that has the ability to secrete and produce a significantly higher amount of protein in culture than the parent strain, Bacillus brevis 47, and completed the present invention. did.

That is, the present invention provides (1) a mutant Bacillus brevis that produces a significant amount of protein outside the bacterial body;

(2) A host bacterium for genetic recombination consisting of a mutant Bacillus brevis that produces a significant amount of protein outside the bacterium.

(3) A mutant Bacillus strain that produces a significant amount of protein outside the bacterial body by ligating the DNA encoding human salivary gland α-amylase to the 3' end of the DNA containing the promoter region.
Brevis.

(4) C at the 3' end of the DNA containing the promoter region.
lostridium thermosulfurog
A mutant Bacillus brevis that produces an extremely large amount of protein outside the bacterial body and retains DNA bound to DNA encoding β-amylase derived from B. enes.

(5) Human salivary gland α-amylase, which is characterized by culturing the mutant Bacillus brevis of item 3 in a medium, producing and accumulating human salivary gland α-amylase in the culture, and collecting the same.
Production method of amylase.

(6) Cultivate the mutant Bacillus brevis of Section 4 in a medium, and Clostridium thermosulfur in the culture.
Genes-derived β-amylase is produced and accumulated,
C1ostridium characterized by collecting this
+a β derived from thermosulfurogenes
-A method for producing amylase.

It is.

Any method of mutating Bacillus brevis 47 may be used as long as it induces mutations in microorganisms, such as ultraviolet irradiation, gamma ray irradiation, or treatment with drugs such as nitrosoguanidine. Examples include:

When performing mutation treatment on Bacillus brevis 47, Bacillus brevis 47 may be treated while retaining a plasmid into which a gene encoding a heterologous protein has been inserted.

As a promoter for expressing a gene encoding a foreign protein in mutant Bacillus brevis.

Any promoter that functions in Bacillus brevis may be used, but promoters derived from Bacillus brevis are preferred, such as the promoter of the cell wall protein gene, which is a major extracellular protein of Bacillus brevis 47. One or more types of promoters may be contained.

The DNA containing the promoter region must contain, in addition to the above-mentioned promoter, an SD sequence, a translation initiation codon, etc., and may further contain a part of a major extracellular protein gene.

In the present invention, the heterologous protein may be accumulated either inside or outside the mutant Bacillus brevis cells;
Facilitates separation and purification of heterologous proteins.

Furthermore, in order to increase the production amount, it is preferable to accumulate a heterologous protein outside the bacterial body. In this case, the DNA containing the promoter region has a region encoding a signal peptide at the 3' end of the DNA. included.

Any signal peptide may be used as long as it secretes and expresses a heterologous protein outside the mutant Bacillus brevis cells, such as the signal peptide of the cell wall protein gene of Bacillus brevis 47.

The expression vector used for gene expression may be any vector as long as it functions in mutant Bacillus brevis, and examples include the plasmid obtained in Reference Example.

The expression plasmid prepared using the above DNA may be any plasmid as long as it functions in Bacillus brevis. pH
Examples include 5^5.

Methods for constructing these plasmids include, for example, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory
(Molecular Cloning, ALabo
ratoryManual, Co1d Spring
(1982)
On the other hand, hosts used for plasmid construction include Escherichia coli (E. coli) and Bacillus subtilis.
), Bacillus brevis
Any microorganism belonging to is) may be used, such as Escherichia coli HBIOI, Escherichia coli, Bacillus subtilis R.
M141 (J, Bacteriol, [5g,
1054 (1984)), Bacillus brevis 47 (
FEBN P-7224), Bacillus brevis 47K
(FERM BP-2308) and the like.

Specifically, Bacillus brevis 47K (FERM BP-2308) is preferably used as a host for gene expression.

The method for transforming Bacillus brevis may be any known method. For example, the method of Takahashi et al. [J.

Bacteriol, 156.1130 (1983
)) or the method of Chang and Cohen [Molecular and General Genetics (Mo1.
Gen, Genet, ), 168°111 (19
79)).

Any medium may be used for culturing the resulting transformant, as long as the transformant can grow and produce the desired protein.

Examples of the carbon source contained in the medium include glucose, sucrose, glycerol, starch, dextrin, molasses, urea, and organic acids. Nitrogen sources contained in the medium include casein, polypeptone, meat extract, and yeast extract.

Amino acids such as casamino acids and glycine, organic nitrogen sources such as NZ-amine, and inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, and ammonium nitrate are used. In addition, inorganic salts such as potassium chloride, potassium phosphate, dipotassium phosphate, sodium chloride, and magnesium sulfate are added to the medium as necessary. Alternatively, the culture may be performed using a synthetic medium containing mainly sugar and an inorganic nitrogen source. When using a strain that exhibits auxotrophy, nutritional substances necessary for its growth may be added to the medium. Examples of the nutritional substances include amino acids, vitamins, nucleobases, and the like.

Also, if necessary during culture, add antibiotics (
Examples, penicillin, erythromycin, chloramphenicol, bacitracin, D-cycloserine, etc.) are added. Further, if necessary, an antifoaming agent (eg, soybean oil, lard oil, etc.) may be added to the medium.

The initial pH of the medium is 5.0 to 9.0, more preferably 6.5 to 7.5.

The culture temperature is usually 15°C to 42°C, more preferably 24°C.
℃ to 37℃, and the culture time is usually 16 to 166 hours.

More preferably, it is 24 to 96 hours.

After completion of the culture, the bacterial cells and the supernatant are separated by a method known per se, such as centrifugation or filtration.

Heterologous proteins in the culture supernatant are purified using conventional protein purification methods, such as salting out, isoelectric precipitation, gel filtration, ion exchange chromatography, hydroxyapatite, and high performance liquid chromatography (HPLC, FPLC, etc.). , the desired heterologous protein can be obtained.

The novel Bacillus brevis 47 of the present invention is capable of efficiently secreting the product of genetic recombination to the outside of the bacterial cell, making it an extremely excellent host bacterium for genetic recombination.

Next, manufacturing examples and examples of the present invention will be described.

Production example Plasmid PHA containing human salivary gland amylase cDNA
Construction of MY5 Y, NaKa+mura et al.
Gang, 28 (1984) 263-270, from RNA extracted from human salivary glands.
DNA was created and human salivary gland α-amylase (hAM
Y) Plasmids PHS^7 and pH5A15 were constructed in which the entire cDNA region was inserted into plasmid pBR322. Furthermore, a plasmid pBR5X was constructed in which the region other than the hAMY gene region was removed as much as possible. The Ban n site present at the signal peptide cleavage site of hAMY cDNA on this plasmid was cleaved with Ban1l, and T
After treatment with 4-DNA polymerase, a BamHI linker was added (, )lAMY2).

pHAMY2 DNA was cut with XhoI and hAMY c
The fragment containing the DNA was transferred to the plasmid p)111 (S,
Horinouchi.

B, Veisblum, J, Bacteriol
, 150804 (1982)) was inserted into the 5aQ1 site of plasmid pRU101, in which a 5aQI linker was added to the Pvun site, and then inserted into the 5aQ1 site of plasmid pRU101, which had a 5aQI linker added to the Pvun site of Bacterio1. ．． 156.1130 (1983)
) was introduced into B. brevis47-5 (ρF
IAMY3).

Next, add B, brev to the BamllI site of pHAMY3.
A 600 bp AlluI fragment containing one promoter site of the is 47 MlilP gene [■.

Yamagata at al., J. Bacte
riol, 169.1239 (1987))
600b cut with BamHI after adding mHI linker
The P fragment was inserted in the same direction as the hAMY gene to obtain PIIAMY4.

On the other hand, pBR5X was cut with Ran II and T4 DN
1 (paI-Fnu4) isolated from the signal peptide-encoding part of the MVP gene after treatment with A polymerase.
HI fragment (Klanov fragment treatment) was inserted to obtain plasmid PHKI, and pHYl on MVP
The base sequence of the connecting part of the gene and hAMY gene is pHK
The XhoI-Bgfl II 330bp fragment was isolated from 1 and subcloned into the PυC118BamHI-3aQI site. The base sequence of the connecting portion is shown below.

5' ・-・-・ATG AAG CTCTTT TG
G TTG CTT TTCACCATT GGG. Amylase was measured by Saito's method (Arch.

Bioch, Biophy, 155.290-29
8 (1973).

TATπ°C... PHAMY4 was cleaved with ApaLI and BdIt, the small fragment was removed, and pH5A15 was obtained by replacing it with the ApaLI-BgQ II small fragment of pHK1.

Example I B, B in which p)lAMY5 (plasmid containing the hAMY (human salivary gland amylase) gene) obtained in the production example of Brevis 47 was introduced
, -brevis 47 (in log phase) at 50mM
The cells were treated with nitrosoguanidine in phosphate buffer (pH 7) (survival rate approximately 10%), cultured for several hours, and then spread on an agar medium containing soluble starch. The resulting colonies were stained with KI-I, and those with a significantly large transparent area (halo) around them were separated.

This was designated as mutant strain 47. As shown in Table 1, the hAMY production amount of this mutant strain is more than 5 times higher than that of the parent strain.The characteristic of this mutant strain is that the amount of hAMY in the medium gradually increases even during long-term culture (7 to 8 days). The point is to keep doing it. In addition, although the medium pH in the late stage of the culture was relatively high, it was possible to suppress P)I to 7.5 to 8 by adding glucose (3%) 24 hours after the start of the culture. The growth of this mutant strain is somewhat temperature sensitive (growth is suppressed at 42°C).

To remove the plasmid from the mutant strain, treat the mutant strain with nitrosoguanidine and use the replica method to remove the erythromycin-sensitive strain that does not produce hAMY (B).

brevis 47K) could be separated.

This strain was designated as Bacillus previs 47 and sent to FER.
It has been deposited as M BP-2308.

The morphology and physiological properties of B. brevis 47 are shown in Table 2.

Table 2 B, Morphological and physiological properties of Brevis 47 Example 2 B, Secretion production of human salivary gland amylase by Brevis 47 Journal of the Chemical Society, 61,
669-676 (1987)) and pHK1
m, ApaLI, and the latter fragment containing the hAMY gene was inserted between the former Hindll and ApaLI sites to obtain PH5A5.

B, brevis by the method of Takahashi
47K, was introduced into FERM BP-2308.

2% polypeptone, 0.5% meat extract, 0.4% yeast extract, 1% glucose, 2mM CaCQ, 1
Brevis47K (pH 3A5) was cultured with shaking at 37° C. in a medium containing 0 μg/cod erythromycin pH 7.0, and after 24 hours, 3% glucose was added and the culture was continued. The amylase activity of the culture filtrate after 5 days of culture was 2,950 LI/vsQ, and the specific activity showed 22 tag.
/Q human salivary gland amylase was produced. When the parent strain B, brevis 47, carrying the same plasmid was cultured under the same culture conditions, the amylase activity of the culture filtrate after 5 days of culture produced salivary gland amylase with an amylase activity of 2500/SR (approximately 2 sg/1). From this, mutant strain B,
Brevis 47 produced approximately 11 times more salivary gland amylase than the parent strain. On the other hand, an attempt was made to secrete and produce salivary gland amylase by introducing the same <PH5A5 using yeast and Bacillus subtilis as host bacteria, but almost no amylase activity was detected in the culture solution. .

Example 3 Clostridi by B, brevis 47K
β of umther + mosulfuro enesis
- Production method of amylase C1ostridium the
The rmosulfuro enesis β-amylase structural gene was directly ligated after the MVP promoter and signal sequence to obtain plasmid PMM2.

C1, β of thermosulfur enesis
-Amylase cloning method, nucleotide sequence, and plasmid pNK1 containing the gene are available from Kitamoto
et al.

J. Bacteriol, 170.5848-5
854 (1988) and Japanese Patent Application No. 63-43708.

Plasmid pNK1 was partially cut with MfJII, and the resulting 3.6kb fragment was inserted into the BamHI site of the secretory expression vector PNU200 for B. brevis, the direction of the connection was confirmed, and plasmid pMM1 was created.
The 0th order obtained.

After synthesizing the following four types of DNA, phosphorylate each synthesized DNA, mix the four types, and incubate at 100°C for 3 minutes and at 60°C for 10 minutes.
The DNA was annealed by temperature treatment for 7'' CIO minutes.

An Ava -MflI 1.2 kb fragment encoding the N-terminal side of β-amylase was isolated from plasmid pNK1.

pBR-AN was prepared by inserting the synthetic NA shown below into the NruI-Bai+HI site of PBR322.

Met Ala Phe Ala Ser Ile A
la Pro Asn PheLys Val Ph
a Val Mat Gly3' (Ava II) The DNA sequence obtained by this synthesis corresponds to the C-terminal part of the MVP signal sequence, and by partially changing the base sequence, the restriction enzyme Nco1. A PstI cutting point was introduced. However, the amino acid sequence of the signal peptide translated from this sequence is designed to be exactly the same as the amino acid sequence of the original signal peptide.

Between the NcoI and Baa+)II sites of pBR-AN,
The previously obtained DNA fragment obtained by annealing four types of synthetic NA and the PNKI^vaII-MfflI 1.2 kb fragment were inserted to obtain pBRAN-BN.

ApaLI-3phI fragment (27
0bP) was isolated and inserted between the ApaLI and SphI sites of pMNl to obtain 9MM2.

The nucleotide sequence of the junction between the MVP signal sequence of 9MM2 and the β-amylase gene was determined by the dideoxy method, and as a result, the connection was as designed, as shown below.

ATG AAA AAG GTCGTT AACAGT
GTA TTG GCT AGT GCA CTCM
et Lys Lys Val Val Asn Se
r Val Leu Ala Ser Ala tau
GCA CTr ACT GTr GCT CCCAT
G GCT TrC (ECT AGCATCGCTCC
A AACTTCAAA G'rr TrCGTT A
TG GGT CCA TrA GAA AAA GT
C...Pro Asn Phe Lys Val P
he Val Met Gly Pro Leu Gl
B carrying u Lys ValpMM2, brev
is 47 and its parent strain B, β- of brevis 47
The productivity of amylase was investigated.

2% glucose, 1% polypeptone, 0.5% meat extract, 0.2% yeast extract, 100μg/yaQ uracil,
10 t of medium consisting of 10 μg/IIQ erythromycin
Q was placed in a 100 m Erlenmeyer flask and cultured with shaking at 37°C. The results are shown in FIG. 1, and B, b of the present invention
revis 47K (9MM2) exhibited a β-amylase activity of the culture supernatant of 1.1000/aΩ on the 7th day of culture, indicating a production amount approximately 2.5 times that of the parent strain. Note that β-amylase activity was measured by the DNS method in a 50 mM acetate buffer at 70°C.

[Brief explanation of drawings]

FIG. 1 shows B, brevis in Example 3.
47K (9MM2) and B, brevis 4f (9
MM2) C1ostridium+mthermosu
FIG. 3 is a diagram comparing the secretion amount of β-amylase derived from lfuro enesis. Figure 1

Claims (6)

[Claims] (1) A mutant Bacillus brevis that produces extremely large amounts of protein outside the bacterial body. (2) A host bacterium for genetic recombination consisting of a mutant Bacillus brevis that produces a significant amount of protein outside the bacterium. (3) A mutant Bacillus brevis that produces an extremely large amount of protein outside the bacterial cell, which contains DNA in which DNA encoding human salivary gland α-amylase is bound to the 3' end of the DNA containing the promoter region. (4) At the 3' end of the DNA containing the promoter region
Clostridium￥￥thermosulfu
DN encoding β-amylase derived from S. roenes
A mutant Bacillus brevis that retains A-linked DNA and produces a significant amount of protein outside the bacterial body. (5) Human salivary gland α-amylase, which is characterized by culturing the mutant Bacillus brevis of item 3 in a medium, producing and accumulating human salivary gland α-amylase in the culture, and collecting the same.
Production method of amylase. (6) Cultivate the mutant Bacillus brevis of Section 4 in a medium, and Clostridium thermosulfur in the culture.
Genes-derived β-amylase is produced and accumulated,
¥Clostridi, which is characterized by collecting this
A method for producing β-amylase derived from um\thermosulfuroenes\.