JP3433807B2 - Method for producing a useful substance using Bacillus brevis incorporating an expression vector carrying a novel amino acid sequence and a DNA encoding the amino acid sequence - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to biotechnology, and more specifically, the present invention comprises a novel amino acid sequence and an expression vector having a DNA encoding the amino acid sequence and a foreign gene linked thereto. The present invention relates to a method for producing a product, which comprises culturing Bacillus brevis in which a plasmid is incorporated, generating and accumulating a foreign gene in the culture, and collecting the foreign gene.

[0002]

Utaka et al. Are Bacillus brevis.
It was found that there are many strains that do not produce protease in Llus brevis, and that strain 1 Bacillus brevis 47 (FERM P-7224: JP-A-60-5)
8074, JP-A-62-201589), the major extracellular proteins [H. Yamagata
Et al., J. Bacteriol. , 169 , 1239 (1
987): Norihiro Tsukakoshi, Journal of Japanese Society of Agricultural Chemistry, 61 , 68.
(1987) and JP-A-62-201583, "outer wall protein an", respectively.
d middle wall protein ", described as" extracellular protein "] gene promoter and MW, which is one of the major extracellular proteins
Protein (middle wall protei
[alpha] -amylase using this strain as a host (Japanese Patent Application Laid-Open No. 62-201583, H. Yamaga).
ta et al. Bacteriol. , 169 , 1239
(1987) and Butapepsinogen (Shigezo Utaka, Proceedings of the 62nd Annual Meeting of the Japan Society for Agricultural Chemistry, p837-83).
8; Norihiro Tsukakoshi, Journal of Japan Society for Agricultural Chemistry, 61 , 68 (198)
7)) has been successfully secreted and produced.

Takagi et al. Also reported that Bacillus brevis HPD31 (bacterus brevis H102 (FERM BP-108) in the present specification is a strain that does not extracellularly produce a protease in Bacillus brevis.
7), which is the same strain as 7), is isolated and used as a host for highly secreted production of thermostable α-amylase (Agric.Bio.
l. Chem. , 58 , 2779-2380 (198).
9)) and highly secreted production of human EGF by Yamagata et al.
oc. Natl. Acad. Sci. USA. 86,3
589-3593 (1989)).

[0004]

The productivity of a foreign gene product using Bacillus brevis as a host bacterium is dramatically improved as compared with a system using Escherichia coli or Bacillus subtilis as a host bacterium. Conventionally, in order to secrete and express a foreign gene product using Bacillus brevis as a host bacterium, a vector capable of replicating with Bacillus brevis is used, and further, a promoter, an SD sequence downstream thereof, and a secretion signal sequence starting from the translation initiation codon ATG The foreign gene is connected after. However, depending on the type of foreign gene, it was also found that the amount of secreted expression of the foreign gene product was very small only by applying such a conventional technique, and further technological development was required for industrial application. It was

[0005]

The present inventors have found that Bacillus
Brevis has conducted extensive studies to enable secretory expression of a foreign gene product, which was difficult to produce by secretory expression in the conventional technique. As a result, it was found that the secretory production of the foreign gene product, which was conventionally difficult to secrete and express, was significantly improved by modifying the basic region, the hydrophobic region and the amino acids near the cleavage point of the signal peptide of the MW protein, We have succeeded in developing a novel signal peptide suitable for Bacillus brevis and completed the present invention.

[0006] That is, the present invention relates to modification of a signal peptide suitable for secretory expression of a foreign gene product which was conventionally difficult to secrete and express in Bacillus brevis, and for efficient secretory expression. Still further, the present invention cultivates Bacillus brevis in which a vector obtained by binding a foreign gene to a DNA encoding a novel amino acid sequence is cultured, and the foreign gene product is produced and accumulated in the culture solution, which is collected. It is a method for producing the characteristic product. The present invention will be described in detail below.

The present invention relates to the novel amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listings shown in Tables 1 and 2 below. The nucleotide sequence of DNA encoding the amino acid sequence is Any base sequence may be used as long as it encodes the above amino acid sequence.

[0008]

[Table 1]

[0009]

[Table 2]

The novel amino acid sequence of the present invention and the amino acid sequence
Preparation of DNA encoding a sequence and inserted into a vector is known
DNA synthesis method (Itakura, K., JJR)
ossi, and R.S. B. Wallace. Ann
u. Rev. Biochem. ，53, 323 (198
4)) is used, but Bacillus Brevis 47 MW
It was prepared using the DNA sequence of the protein signal peptide.
It can also be manufactured. For example, due to site-specific mutation, M
Salts such as lysine and arginine in the basic region of the WP signal
Add 0 to 3 basic amino acids, or add to the hydrophobic region
Hydrophobic amino acids such as syn, isoleucine, valine, and alanine
2 to 10 amino acids inserted, or other amino acids near the cleavage point
It can be prepared by substituting the amino acid of. Ami of the present invention
As a vector carrying DNA encoding the no acid sequence
Can be duplicated in Bacillus brevis
Although it may be offset, for example, pNU200 (Shigezo Utaka, Sun
This Journal of Agricultural Chemistry,61, 669 (1987))
PNU210 (BIOINDUSTRY,9
(2), (1992)) or its derivatives
It To be stably retained in the host, use antibiotics, etc.
Against the resistance gene (for example, erythromycin resistance)
It can also be held.

Any gene can be used as the gene encoding the promoter and SD sequence as long as it functions in the host. The promoter and SD sequence derived from Bacillus brevis 47 or Bacillus brevis H102 (FERM BP-1087) can be used. It is preferred to use the encoding gene.

For example, the DNA encoding the amino acid sequence of the present invention is ligated downstream of the Bacillus brevis 47-derived MWP promoter and the gene encoding the SD sequence to more effectively enhance the secretory expression of the foreign gene product. You can

The foreign gene linked downstream of the DNA encoding the novel amino acid sequence of the thus constructed expression vector may be any gene so long as it can be expressed in the host bacterium, and examples thereof include human interleukin 2 and human. Amylases of salivary glands, fish growth hormones such as Thailand, salmon, trout, yellowtail, flounder, etc. (JP-A-60-214798, JP-A-63-152985, JP-A-2-23884)
Is mentioned.

As a method for constructing a plasmid, a conventional method is appropriately used, for example, a molecular clone.
ing, A Laboratory Manual, C
old Spring Harbour Laborat
Examples include the method described in ory (1982).

If a foreign gene of interest is inserted in the downstream side of the promoter of the expression vector and the subsequent DNA sequence encoding the amino acid sequence of the present invention according to a conventional method and Bacillus brevis is transformed, secretory expression is difficult in the past. It is possible to obtain a transformant capable of secretory expression of the foreign gene product.

The Bacillus brevis transformed with a vector incorporating a foreign gene may be any Bacillus brevis capable of expressing a vector or a promoter, for example, Bacillus brevis 47 (FERM).
P-7224), Bacillus brevis H102 (FE
RM BP-1087) and the like.

The method for transforming Bacillus brevis may be a known method, for example, the method of Takahashi et al. (J. Bacteriol., 156 , 1130).
(1983)) or the method of Takagi et al. (Agri)
c. Biol. Chem. , 53 , 3099-3100
(1989)) and the like.

The medium used for culturing the obtained transformant may be any medium as long as the transformant can grow and produce the desired foreign gene product.

The carbon source contained in the medium may be, for example, glucose, glycerol, starch, dextrin, molasses, urea, organic acid or the like. The nitrogen source contained in the medium includes casein, polypeptone, meat extract,
Organic nitrogen sources such as yeast extract, casamino acid, glycine,
Inorganic nitrogen sources such as ammonium sulfate and ammonium nitrate are used. Alternatively, the culture may be performed using a synthetic medium mainly containing sugar and an inorganic nitrogen source. For auxotrophic bacteria, nutrient substances necessary for their growth may be added to the medium. Examples of the nutritional substance include amino acids, vitamins, nucleic acids, salts and the like.

If necessary during the culture, antibiotics such as erythromycin and neomycin are added to the medium. Further, if necessary, an antifoaming agent such as soybean oil,
Lard oil Various surfactants may be added to the medium. The initial pH of the medium is 5.0 to 9.0, more preferably 6.5.
Is about 7.5. The culture temperature is usually 15 ° C to 42 ° C, more preferably 24 ° C to 37 ° C, and the culture time is usually 16
~ 166 hours, more preferably 24-96 hours.

According to the present invention, by culturing the transformant as described above, a significant amount of a foreign gene product such as human interleukin 2 is efficiently produced in the culture, that is, the cells and the culture solution. , It will be accumulated, so after culturing,
A microbial cell and a supernatant can be separated by a method known per se, such as centrifugation and filtration, and an exogenous gene product can be separated and purified from each by a conventional method.

The Bacillus brevis to be transformed may be any Bacillus brevis, but Bacillus brevis 47 or Bacillus brevis H102 is preferably used. These are Tris-
Not only can it be easily transformed by the PEG method or the electric pulse method, but it also has the excellent property of producing the desired product outside the cells, and thus the culture obtained as described above. The foreign gene product contained in the supernatant can be easily obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[0024]

Example 1 Preparation of vector in which MWP signal sequence is replaced and converted

(1) Construction of plasmid M13MWP pNU210 was digested with restriction enzymes EcoRI and HindIII (manufactured by Takara Shuzo Co., Ltd.), and the resulting DNA fragment of about 330 bp was transformed into plasmid M13mp18 using EcoRI and Hin.
Inserted into the digested with dIII, plasmid M13MW
P was obtained. The base sequence from the MWP promoter to the signal sequence is shown in FIG.

(2) Construction of plasmid M13R2 Site-directed mutagenesis was performed using the M13MWP single-stranded DNA obtained above as a template and synthetic primer R2 (FIG. 2). The site-specific mutation is performed by the Amersham kit (Olig
onlinetide-directed in v
intro mutagenesis system, A
mersham). Next, the nucleotide sequence of the DNA was confirmed, and the obtained target plasmid was designated as M13R2.

(3) Construction of plasmid M13L4 Using the plasmid M13MWP single-stranded DNA as a template and the synthetic primer L4 (FIG. 2), a plasmid M13L4 was obtained in the same manner as in Example 1- (2).

(4) Construction of plasmid M13L4PA Using the plasmid M13L4 single-stranded DNA obtained above as a template and the synthetic primer PA (FIG. 2) in the same manner as in Example 1- (2), the plasmid M13L4PA was prepared.
Got This signal sequence (L4PA) is shown in FIG.

(5) Construction of plasmid M13L4MN Using the plasmid M13L4 single-stranded DNA as a template and the synthetic primer MN (FIG. 2), a plasmid M13L4MN was obtained in the same manner as in Example 1- (2).

(6) Construction of Plasmid M13L5 Using the plasmid M13L4 single-stranded DNA as a template and the synthetic primer L5 (FIG. 2), a plasmid M13L5 was obtained in the same manner as in Example 1- (2).

(7) Construction of plasmid M13L6 Using the plasmid M13L4 single-stranded DNA as a template and the synthetic primer L6 (FIG. 2), a plasmid M13L6 was obtained in the same manner as in Example 1- (2).

(8) Construction of plasmid pNUR2L4MN M13R2 obtained in (2) was digested with EcoRI and HpaI, and the obtained 228 bp DNA fragment and M13L4MN obtained in (5) were digested with HpaI and HindIII. DNA fragment of 108 bp and pNU210
Inserted into digested with coRI and HindIII, pNU
R2L4MN was obtained. This signal sequence (R2L4MN)
Is shown in FIG.

(9) Construction of plasmid pNUL4PA M13L4PA obtained in (4) was EcoRI, HindII.
The 330 bp DNA fragment digested with I was digested with pNU2
10 was inserted into a digested product with EcoRI and HindIII to obtain a plasmid pNUL4PA.

(10) Construction of plasmid pNUR2L5 M13R2 obtained in (2) was digested with EcoRI and HpaI, and the obtained 228 bp DNA fragment was obtained in (6).
1 obtained by digesting 13L5 with HpaI and HindIII
11 bp DNA fragment and pNU210 to EcoR
The plasmid p was inserted into the digested product with I and HindIII.
NUR2L5 was obtained. This signal sequence (R2L5) is shown in FIG.

(11) Construction of plasmid pNUR2L6 M13R2 obtained in (2) was digested with EcoRI and HpaI, and the obtained 228 bp DNA fragment was obtained in (7).
1 obtained by digesting 13L6 with HpaI and HindIII
14 bp DNA fragment with pNU210 with EcoRI,
Plasmid pNU inserted into HindIII digested product
R2L6 was obtained. This signal sequence (R2L6) is shown in FIG.

[0036]

Example 2 Construction of hIL2 Secretion Vector and Secretion Production of hIL2 by Transformant Plasmid pIL-50A having hIL2 (human interleukin 2) gene
(Taniguchi, T. et al. NATURE
302 , 305-310 (1983)) with restriction enzyme H
It was digested with giAI and DraI to obtain a DNA fragment carrying the hIL2 gene. Next, the plasmid pNU210 obtained in Example 1- (1) and Examples 1- (8), 1- (9),
Plasmid pNUR obtained in 1- (10) and 1- (11)
2L4MN, pNUL4PA, pNUR2L5, pNU
R2L6 as PstI, HindIII (fill in)
Each of them was digested with, and the DNA fragment containing the hIL2 gene obtained above was inserted into the plasmid pNU210-IL2.
And pNUR2L4MN-IL2, pNUL4PA-I
L2, pNUR2L5-IL2, pNUR2L6-IL
Got 2.

Each of the resulting plasmids was transformed into Bacillus
Bacillus brevis H102 / pNU210-IL2 obtained by transformation into Brevis H102 and Bacillus
Brevis H102 / pNUR2L4MN-IL2, Bacillus brevis H102 / pNUL4PA-IL2, Bacillus brevis H102 / pNUR2L5-IL2,
Bacillus brevis H102 / pNUR2L6-IL2
Was cultured in PY medium at 30 ° C. for 2 days with shaking. The amount of hIL2 produced in the supernatant was scarcely produced when the native MWP signal peptide was used, whereas that produced by modifying the MWP signal peptide was 40 m.
It was g / l or more, and hIL2 could be secreted and produced (Table 3 below).

[0038]

[Table 3]

[0039]

INDUSTRIAL APPLICABILITY A novel signal peptide has been developed according to the present invention. This novel signal peptide is particularly suitable for Bacillus brevis, and enables the secretory expression or increase of the amount of a foreign gene that was difficult to secrete and produce by the conventional technique. It is possible to do. Therefore, according to the present invention, Bacillus
It is possible to further improve the efficiency of various bioactive substance production systems using brevis and further increase the usefulness.

[Brief description of drawings]

FIG. 1 is a diagrammatic representation of this signal sequence.

FIG. 2 illustrates synthetic primers used for site-directed mutagenesis.

Continuation of front page (56) Reference JP-A-2-31682 (JP, A) J Biol Chem, March 5 1992, 267 (7), p. 4882-4888 J Biol Chem, February 15 1990, 265 (5), p. 2873-2880 J Biol Chem, March 15 1990, 265 (8), p. 4358-4363 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 SwissProt / PIR / GeneSeq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A novel peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (shown in the following chemical formula 1) in the sequence listing. [Chemical Formula 1] Met Lys Lys (Arg) _n Val Val Asn Ser Val Leu (Leu) _m Ala Ser 1 5 10 Ala Leu Ala Leu Thr Val Ala X ₁ X ₂ Ala Phe Ala 15 20 25 where n and m are respectively 0 to 2 Arg and 3 to Leu
It indicates that 5 cells are arranged in succession. X ₁ is Pro or Al
a and X ₂ are Met or Asn. 2. A foreign gene product is produced and accumulated in the culture by culturing Bacillus brevis carrying a plasmid having the foreign gene inserted immediately after the gene encoding the amino acid sequence of claim 1. A method for producing the product, which comprises collecting the product.