JPH02207790A - Dna sequence derived from chromosome of bacillus subtilis related to suppression of protease formation - Google Patents

Abstract

PURPOSE:To reduce activity level of extracellular protease by synthesizing a DNA sequence to suppress formation of extracellular enzyme such as alpha-amylase, protease or alkali phosphatase. CONSTITUTION:A DNA sequence derived from chromosome of Bacillus subtilis, having functions of suppressing formation level of at least extracellular enzyme such as alpha-amylase, protease or alkali phosphatase and intracellular protease is partially digested with a restriction enzyme. Then the DNA sequence is inserted and bonded to a vector plasmid replicable with Bucillus subtilis and Bacillus subtilis is transformed by using the prepared recombinant plasmid to give strain MT-121 (FERM-BP-2229) having reduced productivity of protease.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a DNA sequence that suppresses the production of extracellular enzymes α-amylase, protease, alkaline phosphatase and levansucrase.

This DNA sequence suppresses the production of intracellular proteases in addition to the enzymes mentioned above. Therefore, focusing on protease, the present invention can be said to relate to a DNA sequence that suppresses protease production inside and outside of Bacillus subtilis.

[Prior art and problems to be solved by the invention] In recent years, recombinant DNA technology has made it possible to express heterologous genes using microorganisms as hosts and produce desired heterologous gene products. Bacteria such as Escherichia coli and Bacillus subtilis, or yeast are commonly used as hosts.

Recently, attempts to secrete heterologous gene products into culture supernatants have been widely studied from the viewpoint of substance production, which avoids contamination with other proteins due to cell destruction and complicated purification processes. As the host in this case, it is thought that bacteria of the genus Bacillus, which originally have the property of secreting large amounts of amylase, protease, etc. outside the bacterial body, are desirable. In particular, among Bacillus bacteria, Bacillus subtilis has attracted attention because it has a lot of genetic, biochemical, molecular biological, and applied microbiological knowledge and is highly safe. Efforts are being made to create host-vector systems for extracellular secretion of gene products.

However, it is known that foreign gene products secreted and accumulated in the culture solution are often degraded by the extracellular protease activity of Bacillus subtilis itself. Therefore, in order to avoid such degradation, protease activity can be suppressed by using a protease-deficient strain as a host or by adding a protease inhibitor to the culture supernatant of Bacillus subtilis, thereby producing a heterologous gene product that does not undergo degradation. Efforts are being made to produce it, but it has not yet reached the stage of practical use (References 1.2).

As mentioned above, it is extremely important to suppress host protease production in order to prevent the degradation of the target gene product.

[Means to solve the problem]

In view of this, the present inventors have conducted intensive studies, and have searched for chromosomal DNA fragments that suppress the production of proteases and other extracellular enzymes inside and outside Bacillus subtilis cells, and have finally found such an effect. The present invention was completed by successfully isolating a chromosomal DNA fragment containing the chromosomal DNA fragment and elucidating its DNA sequence. Hereinafter, this DNA sequence will be referred to as a protease production suppressor gene for convenience, because we focused on the suppression of protease production, which is the most important property from the viewpoint of substance production among the several properties of this DNA sequence.

The present invention will be explained in detail below.

It is generally known that the production of protease in Bacillus subtilis is regulated by genes that perform complex positive and negative control. Among those that positively regulate production, 5
acQ (Reference 3), prtR (Reference 4), se
n (ref. 5) and 5acU (ref. 6.7)
has been reported. Examples of those that suppress production by negative 11 nodes include 5in (Reference 8) and hpr
(Reference document 9) has been reported.

From this, it is considered possible to reduce the extracellular protease activity of Bacillus subtilis by utilizing this negatively regulated gene, for example, by increasing its expression level or modifying its structure.

Therefore, as part of host construction, for the purpose of cloning negatively regulated genes, Bacillus subtilis chromosomal DNA was partially digested with the restriction enzyme 5au3AI, and then inserted and ligated into a vector plasmid that can be replicated in Bacillus subtilis. Bacillus subtilis was transformed using the recombinant plasmid. In a strain in which such a recombinant plasmid is retained within the bacterial body, the antibiotic resistance gene present in the vector plasmid is expressed and becomes resistant to the antibiotic. Therefore, such antibiotic-resistant strains were transplanted onto a casein agar medium and their halo-forming ability, which is an indicator of protease activity, was assayed. As a result, a strain, MT-121 (FERM-BP-2229), whose halo-forming ability was significantly suppressed, was found, and the plasmid carried by this strain was recovered and analyzed. As a result, this plasmid has approximately 2.5 kb of chromosome D.
It turned out to be a recombinant plasmid containing an NA fragment. Therefore, the plasmid was named pPA1121, and the base sequence of the chromosomal DNA fragment contained therein was determined. In addition, the transformed strain carrying this pPA1121 was cultured, and the levels of amylase activity, protease activity, alkaline phosphatase activity, levanshephrase activity, and intracellular protease activity in the culture supernatant were examined. As a result, they found that the activity levels of all of these enzymes were reduced. Furthermore, this p
PA1121 is a strain with high protease production, Hayasa 111.
32 strains of sacU (H) (Ohio University Bacillus Stosic Center: Address: 8411est 12th^ven
ue Columbus 0haio 43210
US + preserved strain IA95 strain), the protease activity level is reduced, and the double-deficient neutral and alkaline extracellular protease activity strain MT
-400 (FERM-BP-1078), it was found that the level of protease activity remaining in the protease-deficient strain was reduced.
The effectiveness of PA1121 was demonstrated.

As mentioned above, pPA1121 was found to have the property of reducing the levels of extracellular amylase activity, protease activity, and alkaline phosphatase activity, as well as reducing the level of intracellular protease activity. Therefore, in order to specify the DNA sequence of the part that has this property, we created a deletion or insertion in a 2.5 kb inserted DNA fragment and conducted a deletion test to determine whether the suppression of protease production was lost. . As a result, pPAl
The region involved in suppressing protease production contained in the inserted DNA fragment of No. 121 was determined as shown in the Examples. According to the results of DNA sequence analysis, this region contains the 3° end (3' UCUUUCC) of the 16s RNA of Bacillus subtilis.
A DNA sequence considered to be a ribosome binding region (5' GA
There was an open reading frame (ORF, Figure 1) consisting of 209 amino acids having AGGGTGG3') at the 51st end, and this was determined to be the main body of the gene involved in suppressing protease production.

It was also found that when 5DS-polyacrylamide electrophoresis was performed using a cell lysate of a Bacillus subtilis transformed strain having this pP/1121, a large amount of protein having a size of around 20 kD was accumulated.

This indicates that the above-mentioned ORF actually encodes a protein, and this was also supported by the fact that the 20 kD protein disappeared by creating a deletion in this ORF.

As a known protease production suppressor gene,
5in (Reference 8) and hpr (Reference 9), but the DNA sequence of the present invention has a completely different structure from these, and it has been revealed that it is a novel gene.

The DNA sequence encoding the ORF (Fig. 1) can be obtained from Bacillus subtilis chromosomal DNA, as shown in the Examples, but it can also be obtained by chemical synthesis.

The protease activity suppressing gene of the present invention is thought to normally exist in one copy in the Bacillus subtilis chromosome, but one way to amplify its effects is to have it exist in a multicopy state in the Bacillus subtilis host. . In order to make the protease production suppressing gene exist in multiple copies, the above protease production suppressing gene is inserted into a plasmid that exists in multiple copies, or,
This can be done by having multiple copies present in chromosomal DNA. Furthermore, as another method for amplifying the effect of the protease production suppressing gene of the present invention, there is also a method of efficiently expressing the protease production suppressing gene. To this end, a possible method is to increase the expression level by modifying the region that controls the expression of the ORF itself, and then incorporate it into the chromosomal DNA.

As a method of applying the present protease production suppressing gene to the production of a heterologous gene product by Bacillus subtilis to increase productivity, it is possible to coexist the present protease production suppressing gene in a recombinant plasmid for producing a heterologous gene product. By introducing such a recombinant plasmid into Bacillus subtilis, it is possible to simultaneously produce a target heterologous gene product and reduce the protease activity level in Bacillus subtilis.
A strain containing this plasmid has already been used as MT-2.
08 (FERM-P-10028). A new recombinant plasmid pPAIH20B was constructed using this PNPH208 and a DNA fragment containing the protease production suppressor gene coexisting in pPA1121 (
(Fig. 2), Bacillus subtilis was transformed using pPAIH20B and pNPH208, and the secreted and accumulated amount of hirudin was compared and examined. The result (Figure 3) PSLI
(PNPH208), the secreted and accumulated amount of hirudin decreased from 25 hours after the start of culture, whereas PSLI
(pPAIH208), the secreted and accumulated amount of hirudin increased with culture time, confirming the effect of this protease production suppressing gene.

〔Example〕

The present invention will be explained below using specific examples, but the present invention is not limited to these examples in any way.

Example 1 (Isolation of protease production suppressor gene and assay of effect) Chromosomal DNA of Bacillus subtilis Marburg strain was digested with restriction enzyme 5a.
Partially digested with u3A+, 2.5-4.5 kb DN
A fragment of BamH1 of vector plasmid pUB110
Bacillus subtilis 207-25 was transformed by the protoplast method using the recombinant plasmid inserted into the site. When the obtained transformed strains exhibiting kanamycin resistance were transplanted onto a casein agar medium, one strain out of 3000 strains was found to have suppressed halo formation. A plasmid was prepared from this strain and the size of the inserted DNA fragment was examined.
It was found that this plasmid had a donor DNA fragment of approximately 2.5 kb inserted, and this plasmid was transformed into pP.
It was named A1121. The restriction enzyme map of pPA1121 is shown in FIG.

In order to analyze the characteristics of pPA1121, this plasmid was introduced into the PSL 1 strain (Ohio University Bacillus Stock Center stock, storage number 1A310), which was used as a strain MT-1.
121 (FERM-BP-2229) was constructed. MT
-121 and psL1 strains were cultured in 5heafer of sporulation medium (Reference 11), and the activity levels of protease activity, amylase activity, and alkaline phosphatase activity in the culture supernatant were examined. Measurement of amylase activity and alkaline phosphatase activity is performed using Bioc
hemica Information (Reference 1
The method described in 2) was followed. As a result, pPA
The introduction of 1121 reduced alkaline protease to 2.0%, neutral protease to 14.2%, amylase to 48.1%, and alkaline phosphatase to below 2.0% levels compared to the control PSL 1. It became clear that. Furthermore, as a result of examining the protease activity in the cells of the same culture, it was found that pPA1121 lowered the intracellular protease activity level to a level of 30% compared to the control.

Furthermore, MT-121 strain and PSLI strain were cultured in LB medium containing 2% sucrose, and levansucrase activity was measured using Dedonder's method (Reference 13). As a result, it was found that introduction of pPA1121 also reduced levansucrase activity to a level below 30% compared to the control. 6 Next, we investigated 5acU (H
pPA1121 was introduced into strain 32 (Ohio University Bacillus Stock Center stock, storage number IA95), and its effect was examined by halo formation on a casein agar medium. As a result, it was found that pPA1121 also suppresses the protease production ability of high protease producing strains. moreover,
In order to investigate the effect of the protease-deficient strain on suppressing residual proteases, pPA1121 was used as the MT400 strain (F
ERM-BP-1078) and assayed for halo-forming ability on a casein agar medium. As a result, it was surprisingly found that pPA1121 can also suppress the production of residual protease.

Example 2 (Determination of region involved in inhibition of protease production) pPA
In order to determine the region involved in suppressing protease production among the 1121 inserted DNA fragments, the inserted DNA fragments were miniaturized.

That is, as a result of constructing various plasmids in which deletions were made in the inserted DNA fragment using the restriction enzyme cleavage site of pPA1121 DNA shown in FIG. “It was revealed that the deletion plasmid contains the terminal Accll and HindI [sites].An example of a deletion plasmid is described below.
After cleaving pPA1121 with indll and collecting the larger DNA fragment, it was ligated with T4 ligase to construct a recombinant plasmid pPAI427 (Figure 5).
This pPAI427 was used to transform the PSLI strain,
PSLI (pPAI427) strain was constructed. This transformed strain, PSLI (pPA1121) strain and PSL
As a result of culturing I strain in 2XLB medium at 30'C for 18 hours and measuring extracellular protease activity using casein degrading activity as an index, pPAI427 no longer had the ability to suppress protease production (Table 1). Due to this, among the inserted DNA fragments of pPAl121, Hind
The region containing the I[[ site was found to be a region involved in the ability to suppress protease production.

In addition, the results of separate analysis of the inserted DNA fragment of pPA1121 revealed that this Hindl11 site exists at the 3° terminal side of the ORF consisting of about 200 amino acids. This ORF has a 5″ end lined with three ATGs and consists of 209 amino acids at its longest (Figure 1), and upstream of this 5″ end is Bacillus subtilis 16s rRN.
This O
It was considered that there is a high possibility that RF encodes a protein. Therefore, MT-121 strain and PSLI (pUB
llo) strain was cultured at 30° C. for 18 hours using 2XLB medium, and the appearance of proteins in the bacterial cells was examined by 5DS-polyacrylamide gel electrophoresis. As a result, it was found that the introduction of pPAI121 caused a significant accumulation of proteins having a size of around 2OkD. A protein with this molecular size corresponds to about 200 amino acids when calculated assuming that the molecular size of one amino acid is 0.1 kD. This strongly suggests that the above-mentioned ORF encodes a 20kD protein. Furthermore, in the bacterial cells of the PSLI (pPAI427) strain, this protein having a size of 20 kD was not observed, but the presence of a protein having a size of 15 kD was observed. This revealed that the above-mentioned ORF (Fig. 1) is involved in suppressing protease production.

From the above results, it is considered that the ORF encodes a protein that has the ability to suppress protease production.

Table 1 Transformed strains Relative protease activity PSLI (pP
A1121) 12.0PSLI (G
IP^1427) 77.0PSLI
(pUBloo) 100.0PSLI
The casein degrading activity of (pUBllo) was set as 100.

Example 3 (Secretory production of hirudin using protease production suppressor gene) pPAIH20B was constructed using pNPH20B and PPA1121 according to the strategy shown in FIG. First, pPA+121 (1 μg) was added to the restriction enzyme Pstl (
The sticky ends generated after digestion with T4 polymerase (0.5 units) were made into blunt ends using EcoR.
Bacillus subtilis PSL 1 was transformed by inserting a synthetic NA linker (lpg) (manufactured by Takara Shuzo) having one site. As a result of recovering and examining the plasmid from the obtained transformant, it was found that this plasmid contained E at the Pst1 site of pPAI121.
It turned out to be a recombinant plasmid with a coRI linker inserted therein, and this was named pPA I 307. This pPAI307 DNA was digested with restriction enzyme Eco.
After digestion with RI, a DNA fragment of approximately 20 Kb was recovered and the DNA
Next, a type D in which the 3° end of the hirudin gene was linked immediately after the 5° end of the region encoding the promoter and signal sequence of the neutral protease gene.
pNPH20B (1 μg) containing the NA fragment was digested with restriction enzyme EcoRI, treated with alkaline phosphatase,
Phenol treatment and ether treatment were performed sequentially. This material (5 μg) and DNA fragment A (5 μg) were mixed with T4 ligase (
1 unit) at 16°C for 16 hours, Bacillus subtilis PSL 1 was transformed. As a result of preparing and examining a plasmid from the obtained transformed strain, it was found that this plasmid was pNP.
It turned out to be a recombinant in which DNA fragment A was inserted into the EcoR1 site of H20B, and this was transformed into pPAI.
It was named H20B. pNPH208, pPA1121
are Bacillus subtilis MT-208 (FERM-P-1), respectively.
0028), MT-121 (FERM-BP-2229
). Next, PSLI (pPAIH20B
) was used to investigate the extracellular production of hirudin. PSLI (PNPI (20B) strain was used as a control. Culture was performed using 2XLB medium with shaking at 30"C, sampled over time, and measured antithrombin activity in the culture supernatant. Antithrombin activity was determined by measuring the degree of inhibition of the ice-melting activity of the synthetic substrate H-D-phenylalanyl-L-bipecolyl-monoarginyl-p-nitroanilide against thrombin (Reference Reference 14), as a result, the accumulated amount of hirudin secreted in the culture supernatant of the PSLI (pNPH20B) strain decreased from 20 hours after the start of culture, whereas the accumulated secreted amount of hirudin by PSLI (pPAIH208) decreased over time. As a result of investigating the molecular size of secreted and accumulated hirudin by Western blotting using the same culture supernatant (Fig. 3),
It has become clear that the cause of the decrease in the amount of hirudin secretion accumulated in PSLI (PNPH20B) is miniaturization due to the decomposition of hirudin.

In addition, electrophoresis, enzyme reaction in Example 1.2.3,
All buffers used for DNA extraction are provided in the instructions and
It uses a general composition described in many references (for example, Reference 15) or manuals.

〔Effect of the invention〕

To summarize the effects of the present invention shown in Examples, the chromosomal DNA of Bacillus subtilis is partially digested with restriction enzymes, and then inserted and ligated into a vector plasmid that can be replicated in Bacillus subtilis, resulting in a recombinant plasmid. Strain MT-121 (with reduced protease productivity) was obtained by transforming Bacillus subtilis with
FERM-BP-2229) was obtained. As a result of recovering and examining a plasmid from this strain, this plasmid was found to be 2.5 kb.
It was found that this was a recombinant plasmid in which a chromosomal DNA fragment was inserted into a vector plasmid. This recombinant plasmid consists of 1. A substance that suppresses protease productivity even when introduced into a high protease producing strain, and also suppresses residual protease activity when introduced into a strain that is double deficient in neutral and alkaline extracellular protease activities. It was revealed that this was the case, and its effectiveness was confirmed. We created a Bacillus subtilis strain with reduced extracellular protease activity that carries multiple copies of the protease production suppressor gene, and succeeded in suppressing the extracellular protease activity level to about 12% or less of the parent strain. Furthermore, a recombinant plasmid (111PAIH208) was constructed by inserting this protease production suppressing gene into the separately constructed hirudin secretion plasmid pNP820B. The hirudin activity secreted into the culture medium of the transformed strain of the hirudin-secreting plasmid pNP820B, which does not have the protease production suppressor gene, decreased from 25 hours after the start of culture, but the culture of the transformed strain carrying this pPAIH208 decreased. It was confirmed that hirudin activity in the liquid increased with culture time, demonstrating the effectiveness of the novel gene of the present invention.

From the above results, it is clear that the host-plasmid system obtained by combining a recombinant plasmid that secretes a foreign gene product using a protease production suppressing gene does not degrade the foreign gene product (it secretes and accumulates it in the culture medium). However, the effectiveness of the novel protease production suppressor gene of the present invention was demonstrated.

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[Brief explanation of the drawing]

FIG. 1 is a diagram showing a DNA sequence encoding an ORF involved in suppressing protease production. FIG. 2 is a diagram showing the construction method of pPAIH20B. Figure 3 shows PSLI (pNPH208) strain (mu) and P
It is a figure showing the state of hirudin secretion of SLI (pPAI8208) strain (.). FIG. 4 is a diagram showing the structure of pPA1121. The blacked out area indicates an inserted DNA fragment (approximately 2.5 kb) that has the ability to suppress protease production. FIG. 5 is a diagram showing the construction method of pPAI427. In addition, in FIG. 1, A represents adenine, C represents cytosine, T represents thymine, G represents guanine, and Ala
is alanine, Val is valine, Ile is isoleucine, Met is methionine, Trp is tryptophan, Phe is phenylalanine, Pro is proline, cry is glycine, Set is serine, Thr is threonine, Cys is cysteine, Tyr is tyrosine, Asp is aspartic acid, Glu is glutamic acid, Lys is lysine, His is histidine,
Arg is arginine, Asn is asparagine, G
ln represents glutamine. In FIGS. 2 and 5, LigaLion shows the binding between DNA fragments using T4 ligase, and
In the figure, T4po+ responds to T4 polymerase treatment.
BAP indicates bacterial alkaline phosphatase treatment, EcoRlllinker indicates synthetic NA linker with EcoR1 site, and npr-hir
u refers to a region encoding a fusion protein in which the 5-terminus of the hirudin gene is joined immediately after the 3'-terminus of the region encoding the secretion signal of the neutral protease gene, and frag
mentA refers to a DNA fragment of approximately 2° Okb generated by treating pPAI307 with EcoRI. In addition, in FIG.
This refers to the largest NA fragment produced by treating 121 with Hind.

Claims (5)

[Claims] (1) A DNA sequence derived from the chromosomal DNA of Bacillus subtilis and having the function of suppressing the production level of at least extracellular enzymes such as α-amylase, protease, alkaline phosphatase, and intracellular proteases. (2) The DNA sequence according to claim 1, wherein one strand of the DNA sequence is as follows. [There is a gene sequence] (3) A recombinant plasmid, characterized in that the DNA sequence according to claim 1 is linked to a vector plasmid. (4) A recombinant plasmid, characterized in that the DNA sequence according to claim 2 is linked to a vector plasmid. (5) The recombinant plasmid according to claim 4, wherein the vector plasmid is pUB110.