JPS6058074A - Plasmid and transformation of bacillus brevis using it - Google Patents

Abstract

NEW MATERIAL:A plasmid having at least a drive unit range of plasmid pUB 110, and contained in the cell of Bacillus brevis. USE:A transforming agent for Bacillus brevis. Since Bacillus brevis has low protease activity, formed protein is not decomposed. PREPARATION:For example, plasmid PHC79 and plasmid pUB110 are linked at EcoR I site, a gene having erythromycin resistance derived from plasmid pHW1 obtained by scissoring at Hind III and Cla I is inserted into it, to give shuttle vector pEB3. BamH I fragment of 0.9kb is removed from this plasmid pEB3, to give plasmid pHT-1.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a plasmid and a method for transforming Bacillus plevis using the same.

Bacillus brevig
) accumulates large amounts of surface proteins outside the cells and has little protease activity.

Based on these characteristics, we considered this bacterium to be suitable as a recipient bacterium for recombinant DNA.

However, no plasmid with a high copy number has been found in Bacillus plevis so far. Due to these circumstances, the outstanding properties of Bacillus brejvis as a DNA recipient bacterium have not been utilized.Therefore, there is a strong demand for the development of plasmid vectors that can proliferate well within Bacillus brevis cells.

The present inventors developed pUB 110 (
Gryawan, T. J., 8.0 onten.
te tmdD.

Dubnan (19ys) J, Acteriol
, 154.318-529), and when this plasmid vector pUB i 10 was used to transform Bacillus plevis, pUl 110 was transformed into Bacillus. It was found that the plasmid pUB 110 proliferated extremely well in Bacillus plevis cells, and that the information of this foreign gene was efficiently expressed when the plasmid pUB 110 was transformed.

The present invention firstly provides a plasmid that has at least the drive unit region of plasmid pUB j 10 and is contained in Bacillus plevis cells, and secondly, it provides a plasmid that is contained in Bacillus plevis cells. The present invention provides a method for transforming Bacillus plevis, which is characterized by introducing a plasmid having at least the drive unit region of plasmid pUB110.

Here, the drive unit region is the same as the ori region, and refers to a DNA region that controls the function of plasmid propagation within a host cell. Plasmid of the invention pUB1
As a plasmid having at least 10 drive unit regions, in addition to the drive unit region, when introduced into a host cell such as drug resistance, it transforms the host and pressures the host so that it can be distinguished from a host in which the plasmid has not been introduced. It is desirable to use one that contains a so-called marker gene. Furthermore, a promoter sequence may be inserted, and in addition, a gene encoding a protein to be expressed, IIA, etc. necessary for secreting the protein to the outside of the cell may be inserted. That is, these marker genes, promoter sequences, foreign genes and/or DNA for protein secretion.
Any plasmid into which is inserted is included in the plasmids of the present invention.

Specifically, the new plasmids derived from pUB110 of the present invention are as follows. That is, the restriction enzyme cleavage map is shown in Figure 1. Plasmid plB-2 is pUB
110 and TIBB! +22 is combined with Ashi, Escherichia coli (E, coli) and Bacillus plebis (Ea).
ail18brevis), and is characterized by a high copy number in all bacteria. In the figure, Amp' is ampicillin resistance;
Tar indicates tetracycline resistance, and Nmr indicates neomycin resistance. FIG. 2 shows the restriction enzyme cleavage maps of pKB-5 and pET-1, which are new plasmids of the present invention, and the progress of their construction. pHT-1 is pUBll
It is a plasmid with a large copy number in Bacillus plebis, similar to O, and contains an erythromycin resistance gene (m-') in addition to the neomycin resistance gene (Nmr). Furthermore, in addition to the restriction enzyme cleavage site present in pUB110 DNA, it has one site that is cleaved by H1n4. Furthermore, the restriction enzyme cleavage map of plasmid pBAM-102 obtained by ligating pUBllo with α-amylase is shown in FIG.

E. coli and No.
All of the Plebis strains have been deposited with the Institute of Fine Technology, and the strain names and deposit numbers are as follows.

11! echeriahia coli HB101/
pH1B-2FICRMP-7227HB 101/Momi B-5FIl+RM P-7228B&cillus
brevis 47/pHT-1a] IinM P-7
226147/pBAM-102'I! HRM P-7
'225 etc. Furthermore, examples of plasmid vectors include the four types of plasmids described above.

The following methods are available for transforming Bacillus plebis. Protoplasts (naked cells that have lost their cell walls) are produced by the action of lysozyme on cells suspended in a high osmotic solution (also called a hypertonic solution). Using polyethylene glycol to introduce plasmid DNA into cells (cells) that have had surface proteins removed by treatment with alkaline Tris-HCl buffer (cells that have lost only cell wall proteins), as described later, There is a method of introducing plasmid DNA using

By using the plasmid of the present invention, Noctilus
plebis can be easily transformed. This Bacillus plebis has the ability to produce a large amount of protein outside the bacterial body, and since it does not have buptease activity, the produced protein is not degraded. Therefore, the genetic information of the gene is expressed with high efficiency, and the produced protein is secreted outside the bacterial body without being degraded. . In addition,
Bacillus transformed with the plasmid of the present invention
Cultivation of Bacillus plevis may be carried out in the same manner as the usual cultivation of Bacillus plevis.

Next, the present invention will be explained using examples.

Example 1 As shown in FIG. 2, plasmid pH079 (Hoh
n, B, and 0olline, :f, (1
980) Gono 11.291-298) and plasmid pUB 110 (Gryczan, T, J
,.

S, 0ontente and D, Dubnan
(197B) J, Baateriol.

Vself, 31B-129) and II! The one bound at the coPI site was cleaved with ntn(III, Ola H, and Hlndll.

Plasmid pHW 1 (H
orinouQhi.

S and B, Weisblum (1982)
, T. Bacteriol, 150.804-814
Shuttle vector pKE5 was obtained by inserting the erythromycin resistance gene derived from A. ).

Then, from this plasmid pHB5, 0.9 Kb of B
By removing the amHi fragment, the plasmid pHT-
I got 1.

Example 2 Plasmid pBR322 and plasmid pUB110 were ligated at the KcoB 1 site in the same manner as in Example 1 to create the plasmid pFi shown in FIG.
I got B2.

Example 5 (11 Isolation of thermophilic α-amylase gene) Bacillus stearothermophilus (B, 5tearo-the
The α-amylase gene of plasmid pH Engineering 501 containing the thermophilic α-amylase gene of DY-5 was prepared as follows.

Plasmid pH engineering 5o1 (see %FM specification No. 58-50148) was partially hydrolyzed with restriction enzyme] jjaoB I, and then partially hydrolyzed again with Barn Hl,
DNAs of various lengths were prepared and used as the α-amylase gene.

(2) Bacillus spudiris and Bacillus plebis 4
pU13110 was used as a cloning vector plasmid for the thermophilic α-amylase gene into 7.

First, plasmid pUB100 was added with restriction enzyme 'EaoB
A linear plasmid pUB 11 G was prepared by cutting with I and BamHI and removing the 5'-terminal phosphate group by bacterial alkaline phosphatase treatment.

The thermophilic amylase DNA and linear plasmid pUB
110, and both DNAs were ligated using T4DIIA ligase.

The ligated DNA was transformed into Bacillus subtilis (E, 5ubtilis).
Oh=g for IA 289 (α-amylase negative strain)
, S., and 0ohen.

S, ) I, protoplast method (Mo1ea, Qe
n, Gono 7, 168゜111-115 (197
9)), and a large number of transformed strains resistant to kanamycin were obtained. These transformants were treated with Difco A containing 1% soluble starch and 10μν blind kanamycin.
After replicating on an ntibiotic medium 3 plate and culturing overnight, α-amylase producing strains were selected by searching for colonies whose surroundings were colorless by iodostarch reaction. Figure 3 shows plasmid pBAM-102 containing the α-amylase gene.
This is a restriction enzyme cleavage map of .

Birnboim, H. obtained a plasmid from Bacillus subtilis containing a plasmid containing the above α-amylase gene.

0, and Doly, J.'s method (Nucls
ia Ac1ds Ees,,7.

151! 1-1525 (1979)), and the plasmid DNA was further purified by cesium tetralide ethidium bromide ultracentrifugation and used for transformation of Bacillus plebis 47.

First, the surface proteins of Bacillus plevis were removed by treatment with an alkaline Tris-HCl solution, and plasmid D
Introduced NA. That is, the bacterial suspension of Bacillus plebis precultured in T medium was diluted to 1/100 in fresh T medium 5-, cultured with shaking at 37°C, and late logarithmic growth (0, D, 660 nm = 1.9), the bacterial cells were collected by centrifugation (5000f, 5 minutes, room temperature) and diluted with 5m7 of 50mM Tris-HCl buffer (pH 7.5).
After washing, 50mM Tris-HCl buffer (pi
e, 5) was suspended in V and shaken at 57°C. After 60 minutes, the bacterial cells were collected by centrifugation (5000G, s min, room temperature) and placed in 0.5 m/ml of TP medium (0,955% KH, PO,
, 0,426% Na2HPO4, 0,5% meat extract, 1
% Polypeptoso, 0.2% F? Mother extract, 1% glucose) K was suspended. Add plasmid DNA solution (10m
M) Liss hydrochloric acid solution, pH 7.5, 1mM IeDT
100 μt of a 1:1 mixture of A) and TP medium was added and mixed. Then, immediately add 40% polyethylene glycol solution (0,953% KH, PO, 0,426% NaH)
PO4, 40% (w/v) polyethylene glycol p
Add 1.5 m of xa6ooo), stir, and heat at 37°C for 10
I went up for a minute. After that, centrifugation (5000ji',
5 minutes at room temperature), the bacterial cells were collected and suspended in T medium supplemented with 20 mM Mgo4. After incubation at 37°C for 150 min, agar medium for transformant selection (60 μF!
-/I medium plate containing neomycin (polypeptone 1%, meat extract 0.5%, yeast extract 0.
2%, glucose 1%, uracil 0.01% + pH 7
) )) was sprayed at a rate of 0.1 m/each. A neomycin-resistant strain was obtained by culturing at 37°C for 30 hours. These transformants were replicated onto a T medium plate containing 1% soluble starch and 60 pf/-/meml neomycin, and after overnight culture, the colonies around them became colorless due to the iodostarch reaction. α-amylase producing strain (Bacillus plevis 47/pBAM-102 (712
11M P-7225)) was selected.

(3) Production and pressure of α-amylase Bacillus subtilis and Bacillus plebis 47 bacteria carrying the plasmids shown in Figure 3 were inoculated into a T2 liquid medium containing 60μ￥/ml neomycin or 10μfI/v+l kanamycin, and incubated at 37°C. Culture filtrate for 22 hours at C [7. Culture filtrate was prepared by centrifugation and α-amylase activity in the culture filtrate was determined using a futuristic method (J, Eioch θm, Dan.

5B! S-605 (1954)) at 40°C.

The results are shown in Table 1.

Table 1 Nosakin Fu1/White 47 Bacteria 550 5600 All α-amylases produced by Bacillus subtilis and Bacillus plevis 47 were secreted outside the cells and showed heat resistance. As shown in Table 1, when Bacillus plevis 47 is used as a host due to the presence of the same plasmid containing the α-amylase gene, approximately 10 times as much α-amylase is produced as in Bacillus subtilis. produced.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme cleavage map of plasmid plB-2 of the present invention, and FIG. 2 shows a restriction enzyme cleavage map of plasmids pK13-3 and pHT-1 of the present invention and the construction progress of these plasmids. FIG. 6 is a restriction enzyme cleavage map of plasmid pBAM-102 of the present invention. Patent Applicant: U Taka Shige 3 Figure 1 Procedural Amendment (Voluntary) October 6, 1980 Commissioner of the Japan Patent Office Kazuo Wakasugi 1. Indication of Case Patent Application 165066/1982 2. Name of the Invention Plasmid and its Use Method for transforming Bacillus plebis 5 Relationship with the case of the person making the amendment Patent applicant Shigezo Udaka 4, Agent 1-1-10-5 Kyobashi, Chuo-ku, Tokyo 104 Invention of the specification subject to the amendment Detailed explanation column and contents of drawings & amendments (1) 6th line from the bottom of page 2 of the specification (' Dubnan
Correct J to "Dub part U". (2) Page 7, line 11 [D, Dubnan J
[D. Corrected by Dubnau J ◎ (3) r 0.9 K? on the second line from the bottom of page 7. )
” is corrected to [6,9Wb J. (4) Revise Figures 1 to 5 as shown in the attached sheet.
Above) Figure 1

Claims (1)

[Claims] 1. Drive unit 9 of plasmid pUB 110
A plasmid that has at least the A region and is contained in Bacillus plevis cells. 2 Plasmid pUB in Bacillus plevis cells
A method for transforming Bacillus 0. plebis, which comprises introducing a plasmid having at least a 110 drive unit region.