JPH0523742B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to Bacillus subtilis, which has a low protease productivity and is particularly useful for producing peptides and the like by genetic recombination. [Problems to be solved by conventional technology and the invention] Bacillus subtilis does not produce endotoxins, and it does not parasitize or coexist with humans or animals as a pathogenic microorganism, so it is less effective than E. coli. Highly safe. By using such Bacillus subtilis as a host bacterium for genetically modified organisms, useful products having peptide bonds such as foreign gene products such as peptides and proteins can be secreted and produced outside the bacterium. Bacillus subtilis is
In addition to its safety, its industrial usefulness is attracting attention. However, Bacillus subtilis secretes and produces a large amount of protease outside the bacterial body. Therefore, even if a large amount of useful foreign secreted products derived from foreign genes are produced, foreign secreted products having peptide bonds are degraded by various extracellular proteases secreted by Bacillus subtilis into the medium. Therefore, useful products cannot be recovered in high yield. In order to solve this problem, research has recently been conducted to reduce the protease activity of the host Bacillus subtilis. Bacillus subtilis strain 104HL, which lacks both alkaline protease and neutral protease, which are the main extracellular proteases of Bacillus subtilis , has low protease productivity. Research Communications (Biochem.Biophys.Res.
Commun.), 128; 601-606, 1985] and, as previously proposed by the applicant, Bacillus subtilis.
Bacillus subtilis DY-16 strain in which the pap gene was introduced into the 104HL strain (Japanese Patent Application Laid-Open No. 1983-37284)
is publicly known. However, Bacillus subtilis 104HL
Since protease activity still remains in the DY-16 strain and the DY-16 strain, useful secreted products are degraded in the medium due to the remaining protease activity. Therefore, an object of the present invention is to provide Bacillus subtilis which is highly safe and has low protease productivity, allowing the recovery of useful secreted products having peptide bonds in high yield. [Structure of the Invention] The present inventors focused on the fact that most of the protease production in Bacillus subtilis occurs during the sporulation stage, and as a result of intensive studies to obtain Bacillus subtilis with low protease productivity, By introducing a specific gene into Bacillus subtilis, which lacks the ability to produce the two main proteases, alkaline protease and neutral protease, and whose protease activity is less than 3% of the wild strain, the protease activity is higher than that of the parent strain Bacillus subtilis. It was discovered that Bacillus subtilis with significantly reduced oxidation can be obtained, and the present invention was completed. That is, the present invention lacks the ability to produce alkaline protease and neutral protease, which are the two main proteases of Bacillus subtilis, and moreover, the protease activity is lower than that of the wild type strain.
The spo OAΔ677 gene (Ellis, et al, Recombinant DNA Technical
Bulletin, 4, 1-3 (1981) is introduced, and the Bacillus subtilis has reduced protease productivity. In the present invention, Bacillus subtilis into which the spo OAΔ677 gene is introduced is a Bacillus subtilis strain that has lost the ability to produce alkaline protease and neutral protease, which are the two main proteases of Bacillus subtilis, and whose protease activity has decreased to 3% or less of the wild type. It only needs to be bacteria. Even if the spo OAΔ677 gene is introduced into a wild strain or Bacillus subtilis whose protease activity exceeds 3% of the wild strain,
Protease activity remains and the recovery of secreted products with peptide bonds is reduced. It can be easily inferred that the lower the protease activity of Bacillus subtilis, the lower the resulting protease activity. Examples of such Bacillus subtilis include the above-mentioned Bacillus subtilis strain 104HL, Bacillus subtilis DY-
These include 16 stocks. In addition, Bacillus subtilis DY-16 strain is Bacillus subtilis 104HL.
This is a strain in which the pap gene has been introduced, and it has been deposited as Microtechnical Research Institute No. 9488. As detailed in the Examples, these strains have significantly lower protease activity than the wild strain Bacillus subtilis strain 207-25. In other words, Bacillus subtilis strain 104HL is approximately 2.8% of the wild type 207-25 strain,
The DY-16 strain produces only about 1% of the protease of the wild type 207-25 strain. By the way, it is known that most of the protease production in Bacillus subtilis occurs during the sporulation period. Therefore, the present inventors have discovered that by introducing the spo OAΔ677 gene into the Bacillus subtilis host, which has low protease activity, the protease activity remaining in the host can be further reduced. The spoOAΔ677 mutation is a deletion mutation that blocks sporulation at the earliest stages of sporulation. That is, spores are produced by Bacillus subtilis when exposed to heat, ultraviolet light,
Although it is normally a form that can survive in nature even when exposed to chemicals and desiccation, the mutant gene destroys the spore-forming ability in Bacillus subtilis. Therefore, Bacillus subtilis into which the spo OAΔ677 gene has been introduced does not have spore-forming ability. The spo OAΔ677 gene used in the present invention is not particularly limited, and strains having the spo OAΔ677 gene, such as Bacillus subtilis
ATCC35148 strain, ATCC39090 strain, ATCC39091 strain,
The spo OAΔ677 gene such as ATCC39092 strain, ATCC39094 strain, ATCC39096 strain can be used,
These strains are part of the American Type Culture Collection.
Correction). Bacillus subtilis ATCC39090 strain is also known as Bacillus subtilis BGSC1S53.
ATCC39096 strain is Bacillus subtilis
Bacillus genetics as BCSCW1S58
Stock Center (Bacilus Genetic Stock
It is also available from the Center. The spo OAΔ677 gene can be introduced into the host Bacillus subtilis using conventional methods, such as (1) the competent cell transformation method, in which all the chromosomal DNA of the spo OAΔ677 gene donor bacterium is introduced into the host Bacillus subtilis; 2) Cut the chromosomal DNA gene of the spo OAΔ677 gene donor bacterium with a restriction enzyme, integrate the DNA fragment containing the spo OAΔ677 gene into a plasmid,
Examples include a method of transforming the host Bacillus subtilis, and (3) a method of transforming the host Bacillus subtilis using the spo OAΔ677 gene incorporated into phage particles in the same manner as the above plasmid. Regarding the transformation methods mentioned in (1), (2), and (3) above, please refer to "Microbial genetic experiment method,"
You can refer to Volume 3, p.96-116, Kyoritsu Shuppan. Bacillus subtilis lacking spore-forming ability is selected from among the transformed strains in which the spo OAΔ677 gene is introduced into the Bacillus subtilis host in this way. A strain deficient in spore-forming ability can be identified by (1) inoculating a transformed strain into a sporulation medium, and the colony loses the ability to produce melanin pigment; (2) it is resistant to heat treatment at 80°C for 10 minutes. and (3) the absence of sporulation by microscopic examination. Furthermore, by selecting among Bacillus subtilis that does not have spore-forming ability, the bacteria whose protease productivity is lower than that of the host Bacillus subtilis can be used to obtain the Bacillus subtilis of the present invention whose protease productivity is lower than that of the host Bacillus subtilis. Strains with reduced protease productivity are spo
Bacillus subtilis, in which the OAΔ677 gene has been introduced and has become a sporulation-deficient strain, is cultured using a plate containing casein, and the clear zone (Halo) formed by the protease secreted by the bacteria is smaller than that of the host. Obtained by selecting bacterial strains.
Further, as a method for investigating protease productivity in more detail, there is a method using casein and the synthetic substrates azocol and FITC-casein. With this method, protease activity in the culture supernatant can be examined by measuring the degradation of these substrates using a spectrophotometer or fluorophotometer, so strains with low protease productivity can be reliably selected. . Among the Bacillus subtilis strains of the present invention with reduced protease productivity, a particularly preferred strain is Bacillus subtilis strain SP011 (Feikoken Bacterial Serial No. 10987) obtained by introducing the spo OAΔ677 gene into Bacillus subtilis strain 104HL. and Bacillus subtilis DY
-16 strain, Bacillus subtilis SPL14 strain obtained by introducing the spo OAΔ677 gene
No. 10988). The Bacillus subtilis mutant strain with low protease productivity of the present invention retains the general properties of Bacillus subtilis, except that it does not have spore-forming ability. To explain the preferred strain more specifically, Bacillus
Bacillus subtilis strain 104HL, which is the parent strain of Bacillus subtilis strain SP011, has a genetic marker of his
It has leu npr R2 npr E4Δ apr A3. On the other hand, the mutant strain Bacillus subtilis
The SP011 strain has the genetic marker his npr R2 npr E4Δ apr
A3 has spo OAΔ677, the parent strain is deficient in leu, and spo OAΔ677 has been introduced into the parent strain. In addition, Bacillus subtilis strain DY-16, which is the parent strain of Bacillus subtilis strain SPL14, has his leu npr R2 npr E4Δ apr A3 cyc −r as a genetic marker.
It has pap-S. On the other hand, the mutant strain Bacillus subtilis SPL14 is his
leu npr R2 npr E4Δ apr A3 cyc −r pap −S spo
OAΔ677 lin 2, and the parent strain has spo OAΔ677 and lin
2 genetic traits have been introduced. As is clear from the above genetic marker, the Bacillus subtilis of the present invention usually has at least a histidine auxotrophy, and therefore is useful as a host for genetic recombination experiments. In addition, strains containing the spo OAΔ677 gene, for example,
Bacillus subtilis strain ATCC39096, the donor of the spoOAΔ677 gene, has thy
It has A1 thy B1 pyr D1 aro 10 lin 2 amy 3 spo OAΔ677. The meanings of the genetic marker symbols described herein are as follows. leu : leucine auxotrophic mutation his : histidine auxotrophic mutation npr R2: neutral protease regulatory gene mutation npr E4: neutral protease gene deletion mutation Δ apr A3: alkaline protease gene deletion mutation cyc -r: cycloserine resistance mutation pap - S: Genetic mutation affecting α-amylase and protease productivity spo OAΔ677: Deletion mutation lin causing a block in the earliest stages of sporulation 2: Lincomycin resistance mutation thy A1, thy B1: Thymine and thymidine Pyr D1 confers auxotrophy for pyrimidine and confers trimethoprim resistance Aro 10: Mutation confers auxotrophy for the aromatic amino acids phenylalanine, tyrosine, and tryptophan [Effects of the Invention] As described above, the Bacillus subtilis with low protease productivity of the present invention has the spo OAΔ677 gene introduced into Bacillus subtilis with low protease activity, so it is highly safe and has a useful peptide bond. Secreted products can be recovered in high yield. Therefore, the Bacillus subtilis of the present invention is useful as a host when producing peptides and the like using genetic recombination technology. [Examples] The present invention will be described in more detail below based on Examples. Note that the present invention is not limited to these examples. Example 1 Bacillus subtilis ATCC39096 strain, which is a Bacillus subtilis having the spo OAΔ677 gene and the lincomycin resistance gene (hereinafter referred to as Lin ^r ), was grown at a temperature of 37°C to the logarithmic growth phase using the LB medium shown in Table 1. Cultured with shaking. Table 1 1.0% by weight Tryptone 0.5% by weight Yeast extract 0.5% by weight NaC PH 7.5 The bacterial cells contained in 50ml of culture solution were collected and subjected to the method of Saito and Miura (H.Saito, K.Miura, Biochim.
Chromosomes according to Biophis. Acta, 72, 619 (1963))
DNA was extracted and purified. Using the obtained chromosomal DNA, Bacillus subtilis strain 104HL was transformed by the copytent cell transformation method. The transformed Bacillus subtilis was spread on a minimal agar medium containing 50 mg/histidine and cultured at a temperature of 37°C for 48 hours. Strains that met the following indicators 1 to 3 were selected as spore-forming ability-deficient strains. 1. The transformed strain was inoculated into a sporulation medium and the colony lost the ability to produce melanin pigment. 2. It did not show resistance to heat treatment at 80°C for 10 minutes, and 3. No sporulation was observed by microscopic examination. thing. Among the obtained 173 strains lacking spore-forming ability, the strain with the lowest protease activity was selected, and the protease activity was measured by the following method. That is, a transformed strain lacking sporulation ability and the parent strain Bacillus subtilis strain 104HL were separately inoculated onto an LB agar plate medium containing 1% by weight of casein, and cultured at a temperature of 37°C for 24 hours. .
Since casein is degraded by protease secreted by the bacterial cells, the protease production ability was lower than that of the parent strain, Bacillus subtilis strain 104HL, as measured by the size of the halo around the bacterial cells formed by the decomposition of casein. 24 bacterial strains were isolated. Furthermore, these strains were liquid cultured in LB medium, and the activity of protease present in the culture supernatant was measured using Azocol (manufactured by Sigma) as a substrate. i.e. Bacillus subtilis 104HL
strain and transformed strain 24 with reduced protease activity.
After culturing each strain overnight in 10 ml of LB medium, 1 ml of the culture solution was centrifuged, and the supernatant was used as a protease enzyme solution. In addition, as a substrate solution, 0.48g of Azocol was added.
Suspend in 20ml of a mixture of 50mM Tris-HCl (PH7.5) and 1mM calcium chloride, and add 750μ
was mixed with 50μ of the enzyme solution and incubated at a temperature of 37°C for 30 minutes. After adding 800 μl of 0.5M trichloroacetic acid as a reaction stop solution to the reaction solution, the mixture was centrifuged, and the absorbance of the supernatant was measured at a wavelength of 520 nm. In addition, protease activity (PU) is determined by Kaken Pharmaceutical Co., Ltd.
It was calculated in terms of the activity of actinase E manufactured by Co., Ltd. At that time, actinase E uses casein as a substrate at a temperature of 37°C and a pH of 7.5, at a rate of 1 μmol per minute.
The activity of releasing trichloroacetic acid-soluble peptide corresponding to tyrosine was defined as one unit. That is, in place of the supernatant protease enzyme solution, the same reaction as above is carried out using the actinase E, the absorbance of the supernatant is measured, a calibration curve is created, and the protease activity is determined based on this calibration curve. Calculated. Then, four strains with significantly lower protease activity than the parent strain Bacillus subtilis 104HL were obtained, and these strains were designated as Bacillus subtilis spo6 strain, spo7 strain, spo9 strain, and spo11 strain, respectively. The protease activities of these four strains were measured in more detail. i.e. Bacillus subtilis
The 104HL strain and 4 transformed strains with reduced protease activity were cultured in 10 ml of LB medium for 18 hours, and then cultured in 50 ml of Medium A medium (Journal of
Bacteriology, 165, 796-804, 1986) and cultured for 24 and 48 hours, and then the protease activity in the culture supernatant was measured in the same manner as above. The results are shown in Table 2. [Table] From Table 2, among the obtained sporulation-defective strains,
It was confirmed that the SPO7 and SPO11 strains have extremely low protease activity compared to the parent strain. especially
The SPO11 strain has a higher protease activity than the parent strain.
Since it is about 1% of the 104HL strain, it is extremely useful as a host for secretory production of peptides, etc. In addition, the SPO11 strain has the above-mentioned genetic marker. All strains also have a requirement for histidine. Therefore, Bacillus subtilis spo6 strain, spo9 strain, especially spo7 strain,
Among them, Bacillus subtilis strain SPO11 is safe and useful as a host that can efficiently produce peptides. Example 2 Chromosome of Bacillus subtilis ATCC39096 strain
Using the DNA, Bacillus subtilis strain DY-16 was transformed by a competent cell transformation method. Next, the obtained transformed strain was added at 5 μg/ml.
The cells were cultured using an LB agar plate medium containing lincomycin, and strains in which Lin ^r was introduced were selected. As a result, about 800 lincomycin-resistant transformants were obtained. In addition, in the same manner as in Example 1, 51 strains lacking sporulation ability were isolated from these lincomycin-resistant transformants. Among the obtained sporulation ability-deficient strains, the strain with the smallest protease activity was treated in the same manner as in Example 1,
and the protease activity was measured. That is, the sporulation-deficient transformant and the parent strain Bacillus subtilis DY-16 were separately inoculated onto an LB agar plate medium containing 1% by weight of casein, and cultured at a temperature of 37°C for 24 hours. As in Example 1, 40 strains with protease production ability lower than that of Bacillus subtilis strain DY-16 were isolated using the size of the halo around the bacterial cells as an index. These strains were cultured in liquid culture in LB medium, and the protease activity present in the culture supernatant was measured using FITC-
Measurement was carried out using casein (manufactured by Sigma) as a substrate.
That is, Bacillus subtilis DY-16 strain and 40 transformed strains with reduced protease activity were
Each was cultured overnight using 10 ml of LB medium, 1 ml of the culture solution was centrifuged, and the supernatant was used as a protease enzyme solution. 50 μg of enzyme solution and 0.2 μg of FITC-casein,
In a 200μ buffer consisting of 10mM Tris-HCl (PH7.5) and 2mM calcium chloride at a temperature of 37°C.
It reacted for 2 hours. After adding 200μ of 7.5% trichloroacetic acid to the reaction solution and centrifuging, add 500mM Tris-HCl (PH8.5) to 100μ of the supernatant.
900μ was added and the protease activity was measured using a fluorometer. In addition, protease activity (PUD) is measured using casein at a temperature of 37°C and a pH of 7.5 per minute.
The amount of enzyme that releases trichloroacetic acid-soluble peptide corresponding to 1 μmol of tyrosine was calculated as 1 PUD. As a result, the protease activity of the parent strain Bacillus
We obtained four strains that were extremely reduced compared to Bacillus subtilis strain DY-16, and these strains were divided into Bacillus subtilis SPL9 strain, SPL11 strain, SPL14 strain, and Bacillus subtilis strain SPL14, respectively.
39 stocks of SPL were used. Then, in the same manner as in Example 1, Bacillus subtilis strain DY-16 and the transformed strain with reduced protease activity were cultured in 10 ml of LB medium for 18 hours, and then the bacterial cells were transferred to 50 ml of Medium A medium. After culturing for 24 hours and 48 hours, the protease activity in the culture supernatant was measured in the same manner as above. The results are shown in Table 3. [Table] From Table 3, it was confirmed that the four transformed strains had a protease activity that was 1/20 or less lower than that of the parent strain. In particular, it was confirmed that the protease activity of the SPL14 strain was suppressed to about 3% of that of the parent strain DY-16 strain. This SPL4 strain has the above-mentioned genetic marker. Furthermore, all of these strains have auxotrophies for histidine and leucine, and multiple auxotrophies required by the host were confirmed in gene recombination experiments. Therefore,
Bacillus subtilis SPL9 strains, SPL11 strains,
The SPL39 strain, especially the SPL14 strain, is safe and useful as a host that can efficiently produce peptides. The protease activities of Bacillus subtilis 104HL strain and Bacillus subtilis DY-16 strain, which were subjected to transformation in Examples 1 and 2, were compared with Bacillus subtilis 207-25 strain, which is a wild protease strain. , the results shown in Table 4 were obtained. In addition, protease activity is
The bacterial strain was cultured for 24 hours and measured in the same manner as in Example 1. [Table] From Table 4, Bacillus subtilis 104HL strain is
2.8% of wild strain 207-25, Bacillus subtilis
The DY-16 strain produces only about 1% of the protease of the wild type 207-25 strain.

Claims (1)

[Scope of Claims] 1. Subtilis with low protease productivity, in which the spo OAΔ677 mutant gene has been introduced into Bacillus subtilis, which lacks the ability to produce alkaline protease and neutral protease and has protease activity of 3% or less of the wild strain. Bacteria. 2 Bacillus subtilis with low protease productivity is Bacillus subtilis strain 104HL, in which the spo OAΔ677 mutant gene has been introduced.
The Bacillus subtilis according to claim 1, which is strain SP011 (Feikoken Bibori No. 10987). 3. Bacillus subtilis with low protease productivity is Bacillus subtilis DY-16 strain (Feikoken Bacterial Serial No. 9488).
Bacillus subtilis SPL14 strain (Feikoken Bacterium Co., Ltd.) into which the spo OAΔ677 mutant gene has been introduced
10988) according to claim 1.