JPH0355105B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention involves culturing a host microbial cell containing a plasmid incorporating deoxyribonucleic acid (DNA) carrying specific genetic information involved in the extracellular selective production of polymeric substances, which are metabolites. The purpose of this method is to use this specific genetic information to generate and accumulate a polymeric substance outside the bacterial body, and to collect a significant amount of the produced substance. A method has been proposed to produce relatively low-molecular substances such as amino acids and peptides by introducing a plasmid containing DNA carrying specific genetic information into a host microorganism and culturing this microorganism. Plasmids capable of producing molecular substances have different propagation levels depending on the host microorganism, and it is not possible to achieve a sufficient expression function of the plasmid, and no effective culture method has been established. Conventionally, when a microorganism belonging to another genus is transformed as a host with a plasmid containing DNA carrying the genetic information for producing macromolecular substances, which are metabolites of microorganisms, outside the microbial cell, only a specific product can be produced. However, it has not been possible to produce a significant amount of the microorganisms outside the bacterial cells, and this has not been possible even through plasmid-based transformation using microorganisms of the genus Escherichia that can normally be used as hosts. In response, the present inventor introduced into a microorganism belonging to the genus Escherichia a plasmid incorporating DNA carrying genetic information involved in the extracellular selective production of a polymeric substance, which is a metabolite of a microorganism belonging to the genus Bacillus, and By discovering a method for culturing this microorganism, we succeeded in producing a significant amount of polymeric substances, typified by enzyme proteins, outside the microbial cells, making it possible for the first time to achieve the above-mentioned objective. The method of the present invention will be explained in detail below. The microorganism used in the method of the present invention contains a plasmid (extrachromosomal DNA, or vector DNA) in a format that can proliferate in cultured cells, such as the colicin E ₁ factor known as an extrachromosomal gene (plasmid) of the genus Escherichia. , a microorganism belonging to the genus Escherichia represented by Escherichia coli, containing a plasmid incorporating foreign genetic DNA. Foreign gene incorporated into the vector DNA
DNA (chromosomal DNA fragment) is DNA that carries information related to the extracellular selective production of specific metabolites of microorganisms of the genus Bacillus, and is a DNA that carries information related to the extracellular selective production of specific metabolites of microorganisms of the genus Bacillus.
Examples include DNA that carries information that allows enzyme proteins such as glucanase to be produced outside the bacterial cell. The vector DNA may be extracted from naturally occurring DNA, or may be one in which part of the DNA other than the part essential for proliferation is missing.
Examples include the ColE ₁ strain, the pMB9 strain, the pSC101 strain, the R6K strain, and the lambda phage strain. Further, the foreign gene may be added to the vector DNA.
Any known method can be applied to incorporate DNA. For example, a method is used in which DNA is selected and treated with an appropriate restriction enzyme (Endonuclease) to cleave the DNA, then mixed with similarly treated DNA to be used as a vector, and religated with ligase. When the conjugate of the foreign 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 multiplied until it becomes stable as a genetic trait,
A transformed strain having both the desired genetic traits on the chromosome and the traits of the vector DNA can be obtained. To culture the microorganism thus obtained, a medium suitable for producing a substance produced by specific genetic information and suitable for the growth of the host microorganism of the genus Escherichia can be used. However, in the method of the present invention,
The composition of the medium used is to grow and proliferate in a medium that essentially contains inorganic salts required for growth.
It is necessary to continue culturing in the same medium for a period of time from when the amount of bacterial cells reaches the maximum until the production and accumulation of polymeric substances, which are metabolites, substantially cease in the medium. As an inorganic salt,
Particularly effective is sodium chloride, and potassium chloride can also be used. Examples of media containing inorganic salts include glucose, sucrose, lactose,
In addition to carbon sources such as maltose and glycerol, nitrogen sources such as aqueous ammonia and ammonium salts, and inorganic ions, nutrients such as amino acids and vitamins can be added as necessary, and are usually used as a growth medium for Escherichia coli. LB medium (tryptone, yeast extract, salt), BPB medium (Difco; polypeptone, yeast extract, potassium phosphate), nutrients, agar medium (Difco0001), tryptone, salt medium, etc. are used as basic media. Bye. In the above-mentioned medium that does not contain inorganic salts, more than 80% of the substances are produced within the bacterial cells, so the presence of inorganic salts is essential in order to produce most of the produced substances outside the bacterial cells. Further, the appropriate amount of inorganic salt to be used is 0.5 to 2.0% based on the medium composition. The effect of carbon source is glucose in the LB medium,
Particularly good results were also obtained when glycerol was further added, each at 0.1% relative to the medium composition.
It is desirable to add 0.2%. The culture method can adopt conditions suitable for the growth of microorganisms of the genus Escherichia, such as pH, temperature, enzyme supply amount, etc., but after inoculating the microorganisms into the medium,
During the period from when the microorganism grows and reaches its maximum cell mass, that is, from the late stage of logarithmic growth until production and accumulation of polymeric substances in the medium substantially cease,
It is necessary to continue using the same medium. The time from the time when the amount of microorganisms of the genus Escherichia reaches the maximum until the production and accumulation of polymeric substances in the medium substantially stops is in the range of about 16 to 48 hours. Note that the pH condition is not particularly affected, but a pH of 6 to 8 is suitable. In this way, a large amount of polymeric substances are produced outside the cells of the microorganism and can be advantageously collected without further adding inorganic salts and other components required for growth to the medium during culture. By the method of the present invention, not only a single target polymeric substance but also multiple enzyme proteins are produced (secreted) in large amounts outside the bacterial cells, and can be collected together. . That is, Esierihia
By culturing the E. coli HB101 strain, alkaline phosphatase and β, which had previously been detected only within the bacterial body, were detected.
- It has been revealed that galactosidase and a protein group consisting of about 10 proteins are each secreted in significant amounts outside the bacterial cells, as described in the Examples below. This is because the approximately 2000 base pair DNA contained in the plasmid (pEAP2), which will be described later, obtained by the present invention is used for selective extracellular production (secretion) of metabolites.
This means that the host is endowed with this ability. The culture method of the present invention enables the production of enzyme proteins, antibiotics, polysaccharides, and other polymeric fermentation products in large quantities, and furthermore, it is possible to produce a large amount of fermented polymer products such as enzyme proteins, antibiotics, polysaccharides, and other polymer fermentation products. Responsible for genetic information involved in extracellular selective production of microorganisms belonging to
By using plasmids with DNA, it can be applied to mass production of hormone peptides such as insulin and physiologically active proteins such as interferon antibodies, contributing to the industrial fermentation production of polymeric substances. It is extremely large. The case of penicillinase, an enzyme protein, will be explained below as an example of the method of the present invention. First, we will exemplify the preparation of a transformed strain of Escherichia coli using a plasmid incorporating DNA carrying the genetic information for in vitro selection production. (1) Preparation of chromosomal DNA with genetic information for penicillinase production ability In a culture 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 phase of logarithmic growth, chromosomal DNA was extracted and purified using the phenol DNA extraction method to obtain 5 mg of chromosomal DNA. (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 an extracellular selective production gene for penicillidase Escherichia coli HB101 strain, which is a hybrid strain of Escherichia coli K-12 and Escherichia coli B [Molecular Cloning A
See Laboratory Manual p.504 (1982)]
(genetic traits: ^F- , hsds20(r ^- _B , m ^- _B ), rec
A13, ara ^- 14, pro A2, lac Y1, gal K2,
rps L20 (Sm ¹ ), 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 adjusted to pH 7.0 per 1 pure water)
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).
(FERMP-6939). The plasmid (pEAP2) of this transformed strain is a DNA circular molecule containing a penicillinase DNA fragment that carries genetic information involved in the extracellular selective production (secretion) of penicillinase, that is, pMB9 plasmid DNA and approximately 2000 base pairs. consists of the DNA of
It is a 7.7kb DNA circular molecule, and its restriction enzyme cleavage map is as shown in Figure 4. The method of the present invention will be explained below using examples. Example 1 The transformed strain obtained in (3) above, Escherichia
Coli HB101 (pEAP2) (Feikoken Bibori No. 6939)
(EFRMP-6939) in 500 ml containing 100 ml of LB medium (10 g of tryptone, 5 g of yeast extract, 1 g of glucose, 2 g of glycerol, 10 g of NaCl).
The cells were cultured with shaking at 37°C in a large flask. Cell growth (measuring the amount of bacterial cells) is determined by measuring the absorbance (OD) at a wavelength of 660 nm, and the activity of penicillinase produced outside and inside the cells is measured using Sargent.
Variation of [Sawai et al; Antimicrob.Agents
Chemother. 13 , 910 (1978)], 30
The amount of enzyme that hydrolyzes 1 micromole (μmole) of benzylpenylicin per minute at ℃ 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, even if the culture is continued for up to 48 hours, the production amount does not decrease, and the total enzyme activity decreases.
It reached over 80%. In contrast, intracellular penicillinase was observed in the early stage of culture (8 to 20 hours after inoculation), but its production accounts for 10% of the total enzyme activity.
The maximum activity was only 8 U/ml, and its 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 bacteria (Feikoken Bacteria No. 3221) and a known DNA receptor strain Escherichia coli HB101 (pBR322) (carrying the genetic information for penicillinase production ability).
strain containing the pBR322 plasmid) [Boyer et
al; Gene, vol. 2, 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,
_{K2HPO4 1.0} , _MgSO4.7H2O , _{0.2NaHCO3 10}
[adjusted to pH 9.0]] was inoculated into a 500 ml flask containing 100 ml, 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. Example 2 The influence of each culture medium on extracellular penicillinase production of Escherichia coli HB101 (pEAP2) (FERMP-6939) was compared using each medium. Note that the culture conditions were the same as in Example 1.

[Table] Table 2 shows the results of looking at the effects of the components of the LB medium.

[Table] The concentrations of tryptone, tryptose, and polypeptone are each 1%, and NaCl is also 1%. Furthermore, the results of the influence of carbon sources in the LB medium are shown in the third section.
Shown in the table.

【table】

[Table] The concentrations of glucose, starch, sucrose, and maltose are each 0.1%, glycerol is 0.2%, and NaCl is 1%. Next, tryptone (1%), glucose (0.1
%), glycerol (0.2%), yeast extract (1%)
Add NaCl to the medium containing and culture for 20 hours.
The influence of NaCl was investigated. The results are shown in Table 4.

[Table] Example 3 Escherichia coli HB101 strain into which the plasmid (pEAP2) is not introduced regarding the extracellularly produced substances of the above Escherichia coli HB101 (pEAP2) (FERM P-6939) (FERM P-6939) as well as
Escherichia coli introduced with pMB9 plasmid
Table 5 shows the results of examining enzyme proteins other than penicillinase in comparison with the substances produced by the HB101 strain. 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.
Reference Example The culture solution of the transformed strain of Example 1 was incubated for 10 minutes.
Ultracentrifuge at 10,000 x g and dilute with ammonium sulfate.
% saturated solution. This precipitate was dissolved in water and overnight in 0.05M phosphate buffer (PH7.0) containing 0.1M NaCl.
Dialyzed with This dialysate was equilibrated with the same buffer solution to Sephadex G-75.
Purified penicillinase was obtained. In addition, the aforementioned Bacillus No. 170 bacterium (Feikoken Bacteria Collection
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°C using buffer solutions of various PH values.
After incubation for 45 minutes, the samples were 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.

[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)

[Claims] 1. Deoxyribonucleic acid (DNA) that carries genetic information involved in the extracellular selective production of penicillinase, a metabolite of Bacillus No. 170, or this DNA and DNA encoding a protein other than penicillinase. A microorganism belonging to the genus Escherichia that contains an integrated plasmid is inoculated into a medium containing an inorganic salt required for growth, and after inoculation, the microorganism is substantially By continuing the culture until the production and accumulation of penicillinase or penicillinase and the protein in the medium stops, penicillinase or penicillinase and the protein are removed outside the microorganism. A method for culturing microorganisms, characterized by producing proteins.