JPH0142674B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel plasmid, a method for its preparation, and a novel microorganism containing the plasmid.
The purpose of this invention is to provide a new plasmid incorporating deoxyribonucleic acid (DNA) that carries specific genetic information involved in the extracellular selective production of penicillinase, a metabolite, and a new microorganism transformed with the plasmid. It is. Plasmids are extrachromosomal genes found within the cells of microorganisms, which are circular molecules of DNA.
In recent years, it has been used as a means of genetically modifying microorganisms, and its importance in research in the field of fermentation industry has been increasing. In recent years, as seen in the examples of amino acids and peptides, DNA carries genetic information related to the specific requirements necessary for the growth of microorganisms and the ability to produce metabolites.
Research has been carried out on plasmids containing the plasmids, and some plasmids have been introduced into host microorganisms and transformed strains have been obtained. The present invention has succeeded in preparing a new plasmid that incorporates DNA carrying genetic information involved in the extracellular selective production of penicillinase, a metabolite of a microorganism belonging to the genus Bacillus, and furthermore, the plasmid has been incorporated into a DNA that carries genetic information related to the extracellular selective production of penicillinase, a metabolite of a microorganism belonging to the genus Bacillus. The present invention was completed by successfully obtaining a new and useful transformed strain, Escherichia coli HB101 (pEAP2), by introducing the transformant into Escherichia coli HB101 strain. The present invention will be explained in detail below. The host microorganism used in the present invention is colicin, which is known as a plasmid of the genus Escherichia.
A microorganism belonging to the genus Bacillus as a foreign gene DNA is added to a plasmid (vector DNA) in a format that can proliferate in cultured cells, such as factor _E1 .
It is a microorganism belonging to the genus Escherichia represented by Escherichia coli, which contains a plasmid that incorporates a chromosomal DNA fragment prepared from Bacillus No. 170 (Feikoken Bacteria No. 3221). The chromosomal DNA fragment prepared from Bacillus No. 170 to be incorporated into the vector DNA is
Responsible for the genetic information involved in the extracellular selective production of penicillinase, a metabolite of Bacillus bacteria.
The vector DNA may be extracted from naturally occurring DNA, or may be one in which part of the DNA other than the part necessary for proliferation is missing, such as the ColE ₁ strain, pMB9, etc. lineage of,
Examples include the pSC101 strain, the R6K strain, and the lambda phage strain. Furthermore, any known method can be applied to incorporate the chromosomal DNA fragment into the vector DNA. For example, a method is used in which a suitable restriction enzyme (Endonuclease) is selected and treated to cut DNA at a specific site, and then the DNA is mixed with similarly treated DNA to be used as a vector and religated using ligase. The conjugate of the chromosomal DNA fragment and vector DNA obtained in this way is introduced into the cells of a recipient microorganism of the genus Escherichia by a transformation method, and is grown until it becomes stable as a genetic trait. A transformed strain that has both the genetic traits on the chromosome and the traits of the vector DNA can be obtained. The transformed strain obtained in the Examples described below is
A novel vector obtained by using pMB9 plasmid DNA as vector DNA and incorporating a chromosomal DNA fragment prepared from Bacillus No. 170 into it.
pEAP2 plasmid was added to Escherichia coli
Escherichia coli HB101 (pEAP2) is a new microorganism obtained by introducing the HB101 strain (a hybrid strain of Escherichia coli K-12 and Escherichia coli B strains) into a transformation reaction.
[Escherichia coli HB101 (pEAP2)] (referred to as FERMP-6939).
The restriction enzyme cleavage map of pEAP2 plasmid is
As shown in the figure. As is clear from Figure 4, this plasmid contains the Hind site of the restriction site of pMB9 plasmid DNA.
The site contains a penicillinase DNA fragment that carries the genetic information involved in the extracellular selective production of penicillinase from Bacillus No. 170.
DNA circular molecule, i.e. pMB9 plasmid DNA and approx.
It is a 7.7Kb DNA circular molecule consisting of 2000 base pairs of DNA. Next, the said Escherichia coli HB101
The mycological properties of (pEAP2) are the same as those of the DNA recipient strain Escherichia coli HB101, except for penicillin resistance [Molecular
Cloning A Laboratory Manual, p.504 (1982)
Reference, genetic traits: F ^- , hsd S 20 (r _B , m _B ),
rec A13, ara−14, proA2, lacY1, galK2,
rpsL20(Sm′), xyl-5, mtl-1, supE44λ ^- ],
Other properties include the properties of the pEAP2 plasmid;
In other words, it carries the genetic information for penicillinase production ability.
Its unique feature is that it has the added property of selectively producing penicillinase outside the bacterial body using the pEAP2 plasmid. As shown in the examples below, the extracellular selective production of penicillinase by Escherichia coli HB101 (pEAP2) reaches more than 80% of the total enzyme production, including intracellular production, and the production amount remains constant for a long time. sustained. In contrast, in penicillinase production by the well-known Escherichia coli HB101 (pBR322), more than 80% of the total enzyme production is intracellular production;
In addition, the known extracellular production of penicillinase by Bacillus No. 170 lacks long-term sustainability, and these points are in complete contrast. Therefore, the said Escherichia coli HB101
(pEAP2) is novel and useful in that it creates a microorganism that has the ability to selectively produce an enzyme protein extracellularly, which was previously absent, and provides a microorganism that can produce penicillinase extremely advantageously. It is something. In addition, the produced substances are not only a single target polymer substance, but also a plurality of enzyme proteins, each of which is produced (secreted) in large amounts outside the bacterial cell, and can be collected together. i.e. Escherichia coli
By culturing the HB101 strain, alkaline phosphatase, β-galactosidase, and a protein group consisting of about 10 proteins, which had been detected only inside the bacterial cells, were released outside the bacterial cells, as described in the Examples below. It was revealed that it was secreted in large amounts. This means that the approximately 2000 base pairs of DNA contained in the pEAP2 plasmid obtained by the present invention confers on the host the ability to selectively produce (secrete) metabolites outside the cell. be. Below, the method for preparing the plasmid of the present invention, the method for preparing the transformed strain containing the plasmid, Escherichia coli HB101 (pEAP2), and the effects of the transformed strain, regarding extracellular selective production of penicillinase, etc. This will be explained using an example. Example 1 (1) Preparation of chromosomal DNA with genetic information for penicillinase production ability Alkaliphilic Bacillus No. 170 that has the ability to produce and accumulate penicillinase outside the bacterial body (Feikoken Bacillus No. 3221) Medium [(g/): glycerol 2.0, yeast extract 5.0, polypeptone
5.0, _{K2HPO4 1.0} , _MgSO4・_7H2O0.2
pH adjusted to 9.0 with NaHCO ₃ 10], 30℃
After culturing with shaking for 19 hours and collecting bacterial cells in the late logarithmic growth stage, chromosomal DNA was extracted and purified using the phenol DNA extraction method.
5 mg of DNA was obtained. (2) Insertion of chromosomal DNA fragment into vector Take 10μg of chromosomal DNA obtained in (1), add restriction endonuclease Hind, and incubate at 37℃ for 5 minutes.
Partial cleavage was performed after reacting for 10, 20, 30, and 60 minutes. On the other hand, the tetracycline-resistant (Tet ^r ) pMB9 plasmid used as a vector
DNA (manufactured by Bethesda Research Laboratories (USA)) was completely cut with Hind and incubated at 65°C.
After 5 minutes of heat treatment, the mixture was mixed with the former and incubated at 10°C for 24 hours using _T4 phage-derived DNA ligase.
A ligation reaction of the DNA strands was carried out, and after heat treatment at 65°C for 5 minutes, twice the volume of ethanol was added to the reaction solution to precipitate and collect plasmid DNA incorporating chromosomal DNA. (3) Transformation with a plasmid carrying a gene for extracellular selective production of penicillinase Escherichia coli HB101 strain, a hybrid strain of Escherichia coli K-12 and Escherichia coli B [Molecular Cloning A
See Laboratory Manual p.504 (1982)]
(Genetic trait: F ^- , hsd S 20 (r _B , m _B ), rec
A13, ara−14, pro A2, lac Y1, gal K2,
rps L20 (Sm′), xyl-5, mtl-1, sup
E44λ ^- ) in 10 ml of LB medium (10 g of tryptone (Difco), 5 g of yeast extract, 1 g of glucose, and 10 g of _NaCl per pure water adjusted to pH 7.0)
The cells were inoculated, cultured with shaking at 37°C, grown to late logarithmic growth, and then harvested. This was sequentially suspended in a solution of 0.03M CaCl ₂ at a final concentration under ice cooling to obtain competent cells. The plasmid DNA solution obtained in (2) was added to this cell suspension, reacted for 60 minutes on ice, and then subjected to a heat shock at 42°C for 1 to 2 minutes to extract the plasmid DNA.
was taken into cells. Next, this cell suspension was separately inoculated into the LB medium and incubated at 37°C for 3
After carrying out a transformation reaction by culturing with shaking for ~5 hours, the bacteria were harvested and washed to obtain the transformed strain, Escherichia coli HB101 (pEAP2) (Feikoken Bacteria Collection No. 6939).
No.) was obtained. Example 2 The transformed strain obtained in Example 1 (3), Escherichia coli HB101 (pEAP2)
(FERM P-6939) was mixed with LB medium (10 g of tryptone, 5 g of yeast extract, 1 g of glucose per plate).
g, glycerol 2g, NaCl 10g) 100ml
Shaking culture was carried out at 37°C in a 500 ml flask. Cell growth (measurement of bacterial mass) was determined by measuring the absorbance (OD) at a wavelength of 660 nm, and the activity of penicillinase produced outside and inside the bacterial cells was measured using a modified Sargent method [Sawai et al; Antimicrob.
Agents Chemother. 13 , 910 (1978)], 1 micromole per minute at 30°C.
The amount of enzyme that hydrolyzes benzylpenicillin was defined as 1 unit (U) of penicillinase. The amount of bacterial cells of the transformed strain reached its maximum 16 hours after inoculation, and as shown in Figure 1, the activity of extracellular penicillinase began to increase approximately 20 hours later.
The maximum was reached after 28 hours (≈20 U/ml). The produced penicillinase is very stable, and as shown in Figure 1a, the production amount does not decrease even if the culture is continued for up to 48 hours, reaching more than 80% of the total enzyme activity. did. On the other hand, intracellular penicillinase is produced during the initial stage of culture (8 to 20 days after inoculation).
However, its production was only about 10% of the total enzyme activity, and the maximum was only 8 U/ml.
Furthermore, the activity decreased rapidly and was not observed at all after 48 hours. FIG. 1b shows the ratio of extracellular penicillinase and intracellular penicillinase production to the total enzyme activity. For comparison, the DNA-donor Bacillus No. 170 (Feikoken Bacteria No. 3221) and the DNA-recipient strain Escherichia coli HB101 (pBR322) (pBR322 plasmid carrying the genetic information for penicillinase production ability) were used for comparison. strain) [Boyer et al; Gene, vol2,
p.95-113 (1977)] were cultured, and the penicillinase activity was measured. In the case of Bacillus No. 170, the medium used [(g/): glucose (or glycerol)
2.0, yeast extract 5.0, polypeptone 5.0,
K ₂ HPO ₄ 1.0, MgSO ₄ 7H ₂ O 0.2 in NaHCO ₃ 10
[pH adjusted to 9.0]] was inoculated into a 500 ml flask and cultured with shaking at 37°C. The extracellular penicillinase activity of the culture solution was measured every 4 hours,
The activity reached its maximum (19 U/ml) 12 hours after inoculation, and then rapidly decreased until it was no longer observed at 40 hours (see Figure 2). On the other hand, E. coli HB101 (pBR322)
In the case of Escherichia coli HB101
(pEAP2) strain 100ml of LB medium used to culture
It was placed in a 500 ml flask, inoculated, and cultured with shaking at 37°C. As shown in Figure 3a, after inoculation,
At 20 hours, more than 80% of the total activity was observed as intracellular penicillinase (maximum 35 U/ml), but it rapidly decreased thereafter, and only 10% or less of extracellular penicillinase activity was observed. FIG. 3b shows the ratio of production of extracellular penicillinase and intracellular penicillinase to the total enzyme activity. The culture solution of the transformed strain of Example 1 was ultracentrifuged at 10,000 xg for 10 minutes to make an 80% saturated solution with ammonium sulfate. This precipitate was dissolved in water and dialyzed overnight against 0.05M phosphate buffer (PH7.0) containing 0.1M NaCl. This dialysate was equilibrated with Sephadex G-75 (Sephadex G-75), which was equilibrated with the same buffer solution.
G-75) to obtain purified penicillinase. In addition, the aforementioned Bacillus No. 170 bacterium
3221) was treated in the same manner to obtain purified penicillinase. To see the identity of both penicillinase,
The influence of pH on enzyme activity, thermal stability, molecular weight, etc. were measured. As a result, the identity of the two was confirmed as follows. (a) Enzyme stability was measured at 30% by using buffer solutions with various pH values.
After incubation for 45 minutes at ℃, it was examined. As a result, both enzymes were stable at pH 7 to 10, and the optimum pH was PH 6.0 to 7.0. (b) The thermostability of the enzyme can be determined by dissolving the enzyme in 0.05M phosphate buffer (PH7.0), heat-treating the solution at a specified temperature for 10 minutes, and measuring the residual activity at pH7.0. Examined. As a result, both enzymes
It was stable up to 50℃. (c) The molecular weight of the enzyme was measured by gel filtration method. The molecular weights of both enzymes were estimated to be 27,000 to 22,000. Example 3 Regarding the extracellularly produced substances of Escherichia coli HB101 (pEAP2) (FERM P-6939), Escherichia coli HB101 strain without introducing the plasmid (pEAP2) and
Escherichia coli introduced with pMB9 plasmid
Table 1 shows the results of examining enzyme proteins other than penicillinase in comparison with the substances produced by strain HB101. The culture conditions were the LB medium of Example 1, 37
The enzyme activities of alkaline phosphatase and β-galactosidase were expressed by measuring the absorbance (OD) at a wavelength of 420 nm, and the protein concentration was Expressed as mg per ml of medium, other conditions were the same as in Example 1.

[Table] Unit is mg/ml.

[Brief explanation of drawings]

Figure 1 shows Escherichia
Figure 2 is a graph showing the penicillinase activity by Bacillus No. 170, and Figure 3 is a graph showing the penicillinase activity by the known Escherichia coli HB101 (pBR322). It is. FIG. 4 is a restriction enzyme cleavage map of the plasmid (pEAP2) of Escherichia coli HB101 (pEAP2).

Claims (1)

[Scope of Claims] 1. A plasmid that provides a host with the ability to selectively produce penicillinase in vitro, the restriction site of which
A plasmid that incorporates a deoxyribonucleic acid (DNA) fragment that carries genetic information involved in the extracellular selective production of penicillinase from Bacillus genus No. 170 at the Hind site. 2. The plasmid according to claim 1, wherein the plasmid is a pEAP2 plasmid. 3 The plasmid vector is pMB9 plasmid
The plasmid according to claim 1, which is DNA. 4. The plasmid according to claim 1, wherein the host is a microorganism belonging to the genus Escherichia. 5. The plasmid according to claim 4, wherein the microorganism belonging to the genus Escherichia is Escherichia coli. 6. Preparing, using a restriction enzyme, a chromosomal DNA fragment of Bacillus genus No. 170 that confers the ability to selectively produce penicillinase in vitro;
Extracellular selection of penicillinase for DNA fragments Restricting the vector DNA of the plasmid using a restriction enzyme that does not interfere with the genetic information involved in the production, and inserting the chromosomal DNA into the Hind site of the restriction site of the vector DNA of the restricted plasmid. A method for preparing a plasmid incorporating a DNA fragment carrying genetic information involved in the extracellular selective production of penicillinase, which comprises recombining the fragment and extracting the recombined plasmid. 7. The preparation method according to claim 6, wherein the plasmid is a pEAP2 plasmid. 8. The method for preparing a plasmid according to claim 6, using the DNA of the pMB9 plasmid as the plasmid vector. 9 Escherichia coli HB101 (pEAP2), which has the ability to selectively produce penicillinase in vitro
[Escherichia coli HB101 (pEAP2)].