JPH0365190A - Recombinant plasmid and microorganism containing the same - Google Patents

Abstract

PURPOSE:To enable efficient mass-production of gene products by introducing an extracellular cellulase gene and a specific gene into the cloning site of the vector plasmid for Escherichia coli. CONSTITUTION:A recombinant plasmid (A) is prepared by introducing the gene promoter and the signal sequence of extracellular cellulase from Bacillus subtilis IFO 3034 strain into the cloning site of a vector plasmid of Bacillus bacteria selected form Bacillus subtilis and its analogues, then a structural gene of penicillinase originating from Escherichia coli coding a useful protein such as lactamase on a terminal site of the sequence to give a recombinant plasmid (A). Then, the plasmid (A) is added to Bacillus subtilis N-24(B) belonging to Bacillus and the strain B is cultured in a desired medium to extracellularly produce the gene products such as lactamase.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a recombinant plasmid and a microorganism containing the plasmid that is capable of accumulating a heterologous gene product in the periplasm or extracellularly.

BACKGROUND ART Attention has been focused on methods for mass-producing the gene products using microorganisms using cloned genes for physiologically active proteins such as enzymes.

As one such technique, an attempt was made to connect the gene of a heterologous protein to a vector plasmid for E. coli and introduce it into E. coli to produce a heterologous protein. Problems still remain in the mass production of gene products, as production inhibition has been observed due to inactivation of proteins due to the formation of clumps and inhibition of growth of the microorganisms themselves.

Many extracellular enzyme genes have been cloned and their nucleotide sequences determined. As a result, it was revealed that most bacterial extracellular enzyme genes have a part called a signal sequence, and that their products are translated and synthesized as a precursor with a signal peptide attached to the amino terminus. The signal peptide consists of a methionine at the amino terminal, followed by a region with many basic amino acids such as lysine and arginine, and a region of hydrophobic amino acids, and has a total length of about 17 to 36 amino acids. This protein plays an important role in in vitro secretion, so we would like to attempt the extracellular production of useful proteins mainly using Bacillus subtilis as a host by incorporating a structural gene derived from a foreign organism to encode a useful protein downstream of the signal sequence. Written by Kunio Yamane, Protein Nucleic Acid Enzymes,
Tomaki 925 (1983), Nakayama et al., J. Bio
Technol, 5.71 (1987)>, the signal sequences used in this case were derived from the α-amylase and protease genes of Bacillus subtilis. However, in the case of Bacillus subtilis, protease is also secreted outside the bacterial cell along with protein, so the produced and secreted foreign protein is degraded by this protease, inhibiting the improvement of productivity.

Problems to be Solved by the Invention Therefore, there is a need for a method for producing useful substances such as proteins using Escherichia coli using a novel recombinant plasmid, which can be efficiently mass-produced without deactivation. Furthermore, in the technology using Bacillus subtilis, it is necessary to increase the expression efficiency of useful proteins and to provide an effective means for preventing the secreted and produced useful proteins from being degraded by proteases. It is requested. Furthermore, it is desired to provide a novel recombinant plasmid that can be introduced into both Escherichia coli and Bacillus subtilis. It is an object of the present invention to meet such demands.

Means for Solving the Problems According to the present invention, the above-mentioned objects of the invention are (1) adding the promoter and signal sequence of the extracellular cellulase gene of Bacillus subtilis IF 0 3034 strain to the cloning site of the vector plasmid for E. coli; A recombinant plasmid incorporating a structural gene derived from a heterologous organism to encode a specific useful substance downstream thereof, and a vector plasmid of a Bacillus selected from Bacillus ubtilis and its related species are inserted into the cloning site of the above-mentioned vector plasmid. It is distantly related to a recombinant plasmid obtained by enzymatically cutting and ligating the promoter, signal sequence, and structural gene portion of the recombinant plasmid described in Section 1 to the replicon portion contained in the plasmid.

The present invention utilizes the secretory production ability of the gene for cellulase, which is one of the extracellular enzymes of Bacillus subtilis, to impart a secretory function to a foreign protein, thereby eliminating the accumulation of the foreign protein produced within the E. coli body. The main idea is to release it into the periplasmic space or culture medium. That is, Bacillus subtilis I F 0
A chimeric gene was created by connecting the promoter and signal sequence of the extracellular cellulase gene of strain 3034 with a structural gene derived from a heterologous organism to encode a useful protein downstream, and this was used as a cloning site for a vector plasmid for E. coli. The recombinant plasmid thus constructed is introduced into Escherichia coli to obtain a transformant.

Furthermore, a new recombinant plasmid was created by ligating the promoter, signal sequence, and structural gene part of the secretion vector described above to the replicon part contained in the plasmid to the vector plasmid for Bacillus subtilis, and this was introduced into Bacillus subtilis. By obtaining transformants, it becomes possible to efficiently produce heterologous proteins in Bacillus subtilis.
ira Nakamura: Eur, J, Bio
chem. It is intended to be used not only for Bacillus subtilis but also for E. coli.

Hereinafter, the present invention will be described as a structural gene derived from a heterologous organism for encoding a useful protein.
Bacillus subtilis cellulase used in the present invention is constitutively produced during the breeding period of the bacterium. In addition, 36 amino acids function as a signal peptide,
It is cleaved when penetrating the bacterial cell membrane and is secreted outside the bacterial cell as a mature protein. In the present invention, the promoter and signal sequence of the extracellular cellulase gene of Bacillus subtilis, I F 0 3034 strain are connected to the amino terminal side of the mature lactamase gene, and this chimeric gene is incorporated into a vector plasmid for E. coli to create a recombinant plasmid. (pBTDl) was introduced into Escherichia coli to produce lactamase, and the above recombinant plasmid from the chimera gene portion to the replicon portion was incorporated into a vector plasmid for Bacillus subtilis to create a recombinant plasmid (pCesec2). This was introduced into Bacillus subtilis or Escherichia coli to produce lactamase.

EXAMPLES The present invention will be explained in more detail and specifically with reference to Examples below.

Construction of niblasmid pBTD1 Plasmid T modified from vector plasmid pBR322 for E. coli
G 2 (Kadonaga J, T, ;J, Bi
ol, chem, 259.2149-2154 (1
984) 1 B and restriction enzyme BstEII to New England Biolabs 162) 10 units to 30
- B s t E 11Mg solution (10mM Tris
) IcI (pH 8,0) 10mM Mgc12
150++M NaCI) and reacted at 37°C for 1 hour. 50-M NTP (triphosulfate mixture, manufactured by Sigma) was added thereto and reacted with 4.5 units of polymerase I large fragment (Takara Shuzo 2140A) at 37°C for 30 minutes to repair the 5' overhanging end and make it blunt. 4366b was determined by agarose electrophoresis.
A DNA fragment of p (fragment a) was recovered. This fragment a, 15 units of restriction enzyme BamHI (Toyobo BAH-105) and 201N1 BamHI&! I! r solution (10mM T
ris HCI (pH7,5) 7mM MgC1□
150+nM KCI) and a digestion reaction was performed at 37°C for 1 hour. Approx. 3 by agarose electrophoresis
．． A 7 Kb DNA fragment (fragment A) was recovered.

The promoter and signal sequence region of the in vitro cellulase gene of Bacillus subtilis I F 0 3034 strain were transferred to the plasmid p.
1 mg of plasmid pBR3igo1 subcloned into the EcoRI, C1aI restriction sites of BR322
New England Biolabs 1 to the restriction enzyme DdeI
75) Add 6 units of NTP to a final concentration of 5mM in 20rIIl of DdeIlli solution and incubate with polymerase Large Fragment (Takara Shuzo 214OA
) 4.5 units were reacted at 37° C. for 30 minutes to repair the 5° protruding end and make it a blunt end.

Approximately 600 bp fragment (fragment b) by agarose electrophoresis
was recovered. This fragment contains 10 units of restriction enzyme EcoRI (Toyobo Eco-103) and 20+*I Ec.

RIII solution (100+*M Tris HCI
(pH 7,5) 7mM 14gcI260mM
NaC1) was mixed and a digestion reaction was performed at 37°C for 1 hour. A 564 bp DNA fragment (fragment B) was recovered by agarose electrophoresis.

Plasmid pBR322 was digested with restriction enzyme EcoRI.
EcoRI in 10 units, BamHI 15 units
m solution (100mM Tris HCI (pH 7,
5) 7sM MgCl2 60 rm
The digestion reaction was carried out in MNaCI) 20nd at 37°C for 1 hour. D of 377 bp was determined by agarose electrophoresis.
The NA fragment (fragment C) was recovered.

Three fragments A, B, and C were treated with T4 ligase (Takara Shuzo 2011
B) Ligase M[r solution (66mM T
ris-HCI (pH 7,6) 6.6mM Mg
CI2MgCl21O66mMATP) 30d for 10 hours at 4°C. The bound DNA was used to transform E. coli RRI using the calcium method (Norgard, M.

Gene 3.279 (1978)) and transformed with tetracycline-containing agar medium (7 μgird
Colonies selected and grown on an ampicillin-containing agar medium (50 μg/rlfi) were subjected to two-step selection, and a plasmid pBTD1 of approximately 4.5 Kb was obtained from both resistant strains.

'1 Construction of JULL niblasmid pCesec2 11 mg of plasmid pBTD was treated with restriction enzyme Ec.

RI (Toyobo Eco-103>10 units, P v u
II (Boehringer Mannheim 643,703) 9
EcoRIII solution (1001M Tris-H
CI (pH7,5)? +aMMgCh, 60mM
Digested for 1 hour at 37°C in NaCl) 20°C, and analyzed by agarose electrophoresis. The OKb DNA fragment (fragment α) is recovered.

Next, plasmid PU B 110 (Grycza
n T, J, Proc.

Natl, Acad, Sci, 745463 (1
979)) 1 mg was converted into EcoR using 10 units of restriction enzymes EcoRI (Toyobo Eco-103) and 9 units of PvulI (Boehringer Mannheim 643.703).
II! solution (100mM Tris-HCI (pH 7)
,5) 7i+M MgCh 60s+14 NaCl
20-20 at 37° C. for 1 hour, and a DNA fragment of approximately 3.5 Kb (fragment β) was recovered by agarose electrophoresis. DNA fragment α and fragment β were treated with 350 units of T4 ligase (Takara Shuzo 2011B') in ligase buffer (66
szM Tris-HCI (p) 17.6) 6.6 g
eMMgCIalOdDTT 66mMATP) 3
The reaction was carried out in Od for 10 hours at 4°C. The bound DNA was used to transform E. coli RRI using the calcium method (Norgard,
M, V, Gene 3, 279 (1978)'') and ampicillin-containing agar medium (50 μg/d).
An ampicillin-prone strain was obtained. A plasmid pCesec2 of approximately 6.6 Kb was obtained from this transformant.

Bacillus subtilis B, 5ubtilis RM141 and B, 5u using two plasmids pCesec2
Transformation of bt is N-24 and acquisition of recombinant strain Bacillus subtilis B, 5ubtilis RM 141 strain was mixed with L-broth (10 g of bactodryptone, 5 g of bacto yeast extract, 3 g of NaCl, 1 g of glucose).
, 1 ffl of water) was cultured overnight at 37°C in a test tube, and then the culture was mixed with 51111 Antibiotic Medium 3 (Bact Beef Extract 1.
5. Bacto Yeast Extract, Bactodorypton 5g, Vatdextrose 1g, NaCt 3.
5g, NaHPO43,68g, KHPO41,
32g, water 1ffl) was inoculated with 100N1 at 37°C.
The cells were cultured for 2 hours. After that, the cells were left standing in ice water for 5 minutes, centrifuged (7000 rpm, 5 in) to collect bacteria, and the culture supernatant was removed. 50ONM SMMP (shake 0.5M, MAL/inic acid 0.02M, MgCl2
0.02M, PH 6,5) dissolved in double concentration anti-biotic medium, reacted at 37°C for 90 minutes, and centrifuged (7000 rpm, 5 win).
, ) were collected, suspended in SMMP, washed, and centrifuged again.
of SMMP was added to make a protoplast suspension. S M M
After adding the DNA solution dissolved in P30Fd to the protoplast suspension, immediately add 40% polyethylene glycol 60005MM solution (0.5M shoecloth 0.02M
Maleic acid, 0.02M Mgc12, PH6゜5)1
．． 5- was added and left standing at room temperature for 2 minutes. 5- SMMP
was further added, centrifuged, and the collected bacteria was suspended in SMMPI- and then slowly cultured under shaking for 90 minutes. Finally, DM3 agar medium (0.8% agar, 0.5% casamino acids, 0.5%
% Bakto Yeast Extract, 0.35%, HP
O,,0,15%KH2PO,,0,5% glucose,
0.02M Mgc12゜0.01% Bobyle Serum Albumin, 0.5M Sodium Succinate, 300μg
A 200-ml protoplast suspension was spread on the plate (Kanamycin) and grown at 37°C for 2 days to observe colony formation. When this colony was transferred to an agar medium containing ampicillin, an ampicillin-resistant strain was obtained.

Tatsushi Mento: Cell fractionation and measurement of β-lactamase activity E. coli RRI (pBTDl>, Bacillus subtilis RM 141 (
pCesec2), Bacillus subtilis N-24 (pCesec2)
In order to confirm the secretion of β-lactamase, the location of β-lactamase activity over time of culture was investigated. For comparison, a similar experiment was also conducted on E. coli RRI (pTG2>).

L-Gloss (ampicillin 50μg/rI11) 3rI
RRl (pTG2), RRI (pBTDl) at t1
, RM141 (pCesec2), N-24 (pC
esec2) were incubated at 7°C for 10 hours, and each was incubated with 2XYT (16 g of Bactodryptone, 10 g of Bacto Yeast Extract, 15 g of NaC, IJ water).
) After 1% inoculation at 37°C in 50-, aliquots of 10- were collected after 3 and 6 hours while measuring the absorbance (650 nm) every hour. The lactamase activity was measured in three fractions of the culture supernatant, and in the case of Bacillus subtilis, the culture supernatant and two intracellular fractions.

For cell fractionation, centrifuge the 10-cell culture solution (15,000 rpm).
(5 minutes) to collect bacteria, save the supernatant, and wash the bacterial cells with washing solution (0
, 85% NaCl, 10mTris-HCI, PH8
,0) twice to remove hypertonic &! Suspend in 20% sucrose, 30mM Tris-HCI, PH8.0). Add 0.25M EDTA to 400-10
After standing at room temperature for a minute, collect the bacteria by centrifugation, and rapidly add distilled water cooled in ice water while shaking to destroy the outer membrane. 1. Remove the insoluble fraction by centrifugation (15,000 rpm, 5 minutes). Separate and use the supernatant as periplasm.

The insoluble fraction was treated with 10mM Tris-HCI [City (PH
7, 5) was added and suspended, the inner membrane was destroyed by sonication, and the insoluble material was precipitated by centrifugation (15,000 rpm, 5 minutes). The supernatant was saved and used as the cytoplasmic fraction.Bacillus subtilis RM14
1 (pCesec2), N-24 (pCesec2
), the supernatant was separated from the culture medium by centrifugation (15,000 rpm, 5 minutes) and diluted with 10 mM Tris-HCIM.
After suspension in street fluid, ultrasonication was performed to obtain a cytoplasmic fraction.

Activity measurement was performed using a substrate solution (ampicillin 14+ag/M
l.

After heating 5- for 5 minutes at 30°C, the enzyme solution of 1- (culture supernatant diluted 10 times, periplasm fraction diluted 50 times, cytoplasm fraction 50
2-fold dilution solution) was added, and the mixture was allowed to react for 30 minutes. Iodine solution (
100g KI, 20.3g I 2 / 1) 2
After the reaction was stopped by adding M acetate buffer pH 4.0, a starch solution (2% cornstarch') 500- was added to develop a blue color.

Hypo solution (3,97g sodium thiosulfate, 10011
Titration was performed with g sodium carbonate and water 11) until the blue color disappeared, and the amount of ampicillin decomposed was calculated from the amount of hypo liquid.

However, a sample in which enzyme was added after iodine was used as a control.

The enzyme activity in the stock solution of the enzyme is expressed by the following formula.

[(Control titration amount ml) - (Reaction titration 1 ml) ] X
8/F
;'r is the reaction time; D is the dilution rate of the enzyme solution added to the reaction solution Table 1 shows the enzyme activity in both fractions converted to culture solution 1-.

Claims (6)

[Claims] (1) The promoter and signal sequence of the extracellular cellulase gene of Bacillus subtilis IFO3034 strain and a structural gene derived from a heterologous organism for encoding a useful protein are incorporated into the cloning site of a vector plasmid for E. coli. Recombinant plasmid. (2) A microorganism that belongs to the Escherichia coli species and is capable of accumulating a gene product in the periplasm, containing the recombinant plasmid according to claim 1. (3) The promoter, signal sequence, and structural gene portion of the recombinant plasmid according to claim 1 are contained in the cloning site of the vector-plasmid of a Bacillus bacterium selected from Bacillus subtilis and its related species. A recombinant plasmid made by enzymatically cutting and ligating the replicon part. (4) A microorganism belonging to the species Escherichia coli and containing the recombinant plasmid according to claim 3, which is capable of accumulating a gene product in the periplasm or secreting it outside the bacterial body. (5) A microorganism belonging to the species Bacillus and containing the recombinant plasmid according to claim 3, which is capable of secreting a gene product to the outside of the cell. (6) The microorganism belonging to the Bacillus species is Bacillus subtilis N-
The microorganism according to claim 5, which is No. 24.