JPH09135693A - Transformant microorganism and production of heterogene product using the same microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new microorganism, capable of remarkably increasing the production amount of a heterogene product by culturing and useful for production, etc., of an epidermal cell growth factor, etc., by holding the heterogene in a chromosome and further holding an expression vector bonding the heterogene thereto. SOLUTION: This transformant microorganism is a new transformant microorganism, obtained by holding a heterogene (e.g. a gene for expressing an epidermal cell growth factor) in a chromosome and further holding an expression vector having the heterogene bonded thereto such as a microorganism of the genus Bacillus (e.g. Bacillus brevis HPD31-int2) and capable of remarkably increasing the production amount of a heterogene product such as the epidermal growth factor by culturing the microorganism in a culture medium. The transformant microorganism is prepared by integrating a neomycin-resistant gene, a promoter of a cell wall protein gene and the heterogene such as a human epidermal cell growth factor into a plasmid obtained from a microorganism, etc., of the genus Bacillus preparing a recombinant plasmid and transducing the resultant plasmid into the microorganism of the genus Bacillus, etc., according to an elecrtoporation method, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to biotechnology. More specifically, the present invention has a chromosome carrying a heterologous gene, and a transformed microorganism carrying an expression vector to which the heterologous gene is ligated, and the microorganism are cultured to produce and accumulate a heterologous gene product in the culture. The present invention relates to a method for producing a heterologous gene product, which is characterized by collecting this.

[0002]

2. Description of the Related Art Currently, the production of heterologous gene products using recombinants is widely used in the food, pharmaceutical, cosmetics and other industries. Bacteria such as Escherichia coli and Bacillus subtilis, yeast, filamentous fungi, etc. are used as the genetically modified host bacterium. Utaka and his colleagues Bacillus brev
We found that there are many strains that do not produce protease, and one strain of Bacillus brevis 47 (S.Ud
akaand H. Yamagata, Methods in Enzymology, 217 , 23-
33 (1993)) major extracellular protein (H. Yamagata et. Al.,
J. Bacteriol., 169 , 1239 (1987): Norihiro Tsukakoshi, Journal of Japanese Society of Agricultural Chemistry, 61 , 68 (1987) and JP-A-62-20156.
No. 3 gazette describes "outer wall protein and middle
wall protein "," extracellular protein ".) Secretion using a gene promoter and a region encoding a signal peptide of MW protein (middle wall protein), which is one of the major extracellular proteins. A vector was prepared and α-amylase using this strain as a host (Japanese Patent Application Laid-Open No. 6-58242)
2-201583, H. Yamagata et. Al., J. Bacterio.
l., 169 , 1239 (1987)) and Butapepsinogen (Ushige Shigezo, Proceedings of the 62nd Annual Meeting of the Japan Society for Agricultural Chemistry, 1987, p837).
-838; Norihiro Tsukakoshi, Journal of the Japanese Society of Agricultural Chemistry, 61 , 68 (1987)) has been successfully secreted. In addition, Takagi et al. Isolated Bacillus brevis HPD31 (which is the same strain as Bacillus brevis H102 (FERM BP-1087)) which does not extracellularly produce protease in Bacillus brevis. , Using this as a host, highly secretory production of thermostable α-amylase (Agric. Biol. Chem., 53 ,
2279-2280 (1989)), and Yamagata et al.
Highly secreted production of (human epidermal growth factor) (Proc. Nati. A
cad. Sci. USA, 86 , 3589-3593 (1989)). To produce a heterologous gene product by gene recombination using such a microorganism, the heterologous gene is ligated to a vector replicable in the host bacterium, the host bacterium is transformed with the vector, and the heterologous gene is transformed into the host bacterium by the vector. It is carried out by holding and expressing it inside.

Also, SD Ehrlich et al. Linked the chloramphenicol (Cm) resistance gene and the cellulase gene derived from Clostridium thermocellum to homologous recombination into the Bacillus subtilis chromosome to increase the Cm concentration. By doing so, the Cm resistance gene and cellulase gene are amplified, and cellulase production has been successfully increased (BIO / TECHNOLOG
Y, 8 , 559-563 (1990)).

[0004]

In the production of a heterologous gene product by gene recombination using a microorganism, the productivity of the heterologous gene product has been dramatically improved by the various technical improvements as described above. However, further technological development was required to supply the industrially necessary amount at low cost.

[0005]

[Means for Solving the Problem] The present inventors have constructed an expression vector in which a gene for expressing epidermal growth factor is inserted into the chromosome of Bacillus brevis and a gene for expressing epidermal growth factor is linked. We succeeded in retaining it, and by culturing it, it was possible to remarkably increase the yield of human epidermal growth factor.

That is, the present inventors have conducted extensive research based on the integration technology and the plasmid technology, and as a result, have integrated a heterologous gene into the chromosome of the host bacterium and, at the same time, expressed an expression vector in which the same heterologous gene is bound to the host bacterium. The present invention has been completed by succeeding in achieving the introduction into S. cerevisiae and increasing the production amount of the target heterologous gene product.

The present invention cultivates a transformed microorganism having a heterologous gene in the chromosome of a host bacterium and an expression vector to which the heterologous gene is bound, and the microorganism,
The present invention relates to an efficient method for producing a heterologous gene product, which comprises collecting and collecting the heterologous gene product in a culture.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, when a desired heterologous gene is integrated into a chromosome of a host bacterium, a gene originally possessed by the host bacterium on the chromosome can be used. That is, if the heterologous gene is linked to a gene originally possessed by the host bacterium on the chromosome, and then integrated into a plasmid and introduced into the host bacterium, the heterologous gene is integrated with high probability into the chromosome of the host bacterium. The gene on the chromosome of the host bacterium used in this case may be any gene or DNA fragment as long as it is derived from the chromosome of the host bacterium, and its chain length is preferably 200 bp or more. For example, a secreted and expressed heterologous gene product. When a gene on the chromosome encoding a protease or peptidase having the function of degrading the enzyme is targeted, the ability of the host bacterium to produce the protease or peptidase is lost, so that the degradation of the expressed heterologous gene product can be suppressed.

[0009] Specifically, when the host bacterium is Bacillus brevis HPD31, the lon gene on the chromosome encoding the protease lon (J. Bacteriol., 174 , 2281-2287).
(1992)) and so on. Also,
As described above, if a drug resistance gene is linked to a gene encoding a heterologous protein linked to a gene originally possessed by the host strain on the chromosome, a transformant in which the heterologous gene is integrated into the chromosome using drug resistance as a marker Can be selected. Furthermore, the gene encoding the heterologous protein of interest can be amplified by increasing the drug concentration and amplifying the incorporated drug resistance gene, and thus the production amount of the heterologous protein of interest can be increased. As the drug resistance gene, a resistance gene for any drug may be used as long as it is a resistance gene originally not possessed by the host bacterium, and for example, if a known resistance gene such as neomycin, erythromycin, ampicillin or chloramphenicol is used. Good.

The DNA fragment in which the heterologous gene of interest, the gene originally possessed by the host bacterium on the chromosome and the drug resistance gene are ligated is introduced into the host bacterium cell by a plasmid or the like and integrated into the host bacterium chromosome. It is necessary to use a plasmid that cannot self-replicate in the host fungus. This is because the confirmation of the integration of the heterologous gene into the chromosome is carried out using the drug resistance gene incorporated at the same time as a marker, as described above. This is because the transformant acquires drug resistance by self-replication, and thus it becomes impossible to confirm the integration of the target heterologous gene into the chromosome. For example, when a bacterium belonging to the genus Bacillus is used as a host bacterium, a heterologous gene can be introduced into cells of the host bacterium by using plasmids such as pUC119 and pBR322 for E. coli.

In the present invention, the production amount of a heterologous gene product can be dramatically increased by introducing a vector carrying the same heterologous gene into a host bacterium in which the heterologous gene is integrated into the chromosome. In this case, a plasmid capable of replicating in the host can be used as the vector for introducing and retaining the heterologous gene in the host bacterium. For example, in a system using Escherichia coli as a host, pUC19, pUC119 and their derivatives can be used. In the system in which the host bacterium is Bacillus subtilis or Bacillus brevis, pUB110, pNU200 (Ushitaka Shigezo, Journal of the Japanese Society of Agricultural Chemistry, 61 , 669 (1987)), pHY700 (Biosci. Biotech.
Biochem., 56 , 812-813 (1992)), pHT110 (JP-A-6-
133382) and plasmids such as these derivatives can be used.

As a method for constructing these plasmids, known methods are appropriately used.
Cloning, A Laboratory Manual 2nd
Edition, Cold Spring Harbor Laboratory (Molecular Cloning, A Laboratory Manual 2nd ed,
The method described in Cold Spring Harbor Laboratory, 1982) is exemplified. The heterologous gene to be introduced into the host bacterium in the present invention may be a gene derived from any organism, such as human, animal,
Gene products derived from birds, fish, microorganisms, viruses and other organisms (enzymes, hormones, interferons, immunoglobulins, other bioactive peptides, antigen proteins, etc.)
Applicable to production of. For example, gene products such as human, mouse, sheep, and other epidermal growth factor (EGF) can be produced.

In the present invention, bacteria, yeasts, filamentous fungi and the like can be used as the microorganisms as the host fungus. When bacteria are used, Escherichia coli and Bacillus bacteria can be used. In the case of Bacillus bacterium, Bacillus subtilis, Bacillus brevis, Bacillus choshinensis and the like can be preferably used. The method for transforming the host bacterium may be a known method. For example, for Bacillus subtilis, the method of Chang and Cohen (SC
hang and SNCohen, Molec. gen. Genet., 168 , 111-1
15 (1979)), in Bacillus Brevis, the method of Takahashi et al.
(J. Bacteriol., 158 , 1130 (1983)) or the method of Takagi et al. (Agric. Biol. Chem., 53 , 3099-3100 (1989)) and the like.

The medium used for culturing the obtained transformant may be any medium as long as the transformant can grow and produce the desired heterologous gene product. Examples of the carbon source contained in the medium include glucose, sucrose, glycerol, starch, dextrin, molasses, urea, organic acids and the like. As the nitrogen source, casein, polypeptone, meat extract, yeast extract,
Organic nitrogen sources such as casamino acid and glycine, inorganic nitrogen sources such as ammonium sulfate, etc. are used. In addition, inorganic salts such as potassium chloride, monopotassium phosphate, dipotassium phosphate, sodium chloride, and magnesium sulfate are added to the medium as needed. For auxotrophic bacteria, nutrient substances necessary for their growth may be added to the medium. Examples of the nutritional substance include amino acids, vitamins, nucleic acids and the like. In addition, antibiotics such as penicillin, erythromycin, chloramphenicol, bacitracin, D-cycloserine, ampicillin, neomycin and the like are added to the medium if necessary during the culture. If necessary, an antifoaming agent such as soybean oil, lard oil, various surfactants and the like may be added to the medium.

The initial pH of the medium is 5.0 to 9.0, and more preferably 6.5 to 7.5. Culture temperature is usually 15
℃ ~ 42 ℃, more preferably 24 ℃ ~ 37 ℃,
Cultivation time is usually 16 to 166 hours, more preferably 2
4 to 96 hours. In the present invention, the heterologous gene product is produced and accumulated in the culture by culturing the transformant under the above conditions. The heterologous gene product thus obtained can be purified by a known method, for example, by membrane treatment, ammonium sulfate fractionation method, chromatography and the like (basic experimental method for protein / nucleic acid, Nankodo, (1985)).
In the present invention, various heterologous gene products can be stably produced at a high level by retaining the heterologous gene in the chromosome of the host bacterium and in the expression vector. Hereinafter, the present invention will be described in more detail by way of examples, which are merely illustrative and the present invention is not limited thereto.

[0016]

【Example】

I. Cloning of protease lon gene Bacillus brevis HPD31 (FERM BP-1
087) and using the two types of primers shown in SEQ ID NO: 1 (lon-5 primer) and SEQ ID NO: 2 (lon-3 primer) in the sequence listing shown in Table 1 below, 2.3 containing most of the lon gene.
The -kb HindIII-EcoRI fragment was amplified by the PCR method. After treating the amplified gene fragment with the restriction enzymes HindIII-EcoRI,
Subjected to 0.8% agarose gel electrophoresis, 2.3-kb HindI
The II-EcoRI fragment was recovered from the agarose gel using Gene Clean (Bio 101 USA). Separately, the plasmid pUC119 (Takara Shuzo) was digested with HindIII-EcoRI and then treated with alkaline phosphatase (BAP) to obtain 2.3-kb HindIII-EcoR obtained earlier.
The I fragment was ligated with T4 ligase to obtain plasmid pUC119-
lon 'was obtained (Fig. 1).

[0017]

[Table 1]

II. Construction of plasmid pUC119-lon'-Nm Staphylococcus aureu
The plasmid pUB110 derived from (s) was partially digested with HaeIII, and
It was subjected to 8% agarose gel electrophoresis, and neomycin (N
m) The 1.0-kb HaeIII fragment containing the resistance gene was recovered.
The plasmid pUC119 was digested with SmaI and then treated with BAP, and the previously obtained 1.0-kb HaeIII fragment was ligated with T4 ligase to obtain a plasmid pUC119-Nm. pUC119-Nm is EcoRI and Hi
After digestion with ndIII, it was subjected to 0.8% agarose gel electrophoresis to obtain a 1.1-kb EcoRI-HindIII fragment containing the Nm resistance gene, and this fragment was treated with klenow enzyme to blunt the ends. The plasmid pUC119-lon 'obtained in I.
After digestion with, the cleavage site was blunted with T4 polymerase, and the 1.1-kb fragment containing the Nm resistance gene obtained above was ligated with T4 ligase to obtain plasmid pUC119-lon'-Nm (Fig. 2).

III. Construction of plasmid pUC119-lon'-Nm-EGF Bacillus brevis HP926 (FERM BP-5
382) possessing the plasmid pHT110EGF (JP-A-6-133782) prepared from the plasmid pHT926, the promoter of the cell wall protein (HWP) gene of Bacillus brevis HPD31 and the structural gene of human epidermal growth factor (hEGF). Concatenated 720-bp Pst (Figure 5)
I fragment was obtained. The plasmid pUC119-lon′-Nm obtained in II.
After digestion with se 8387I, BAP treatment was performed, and then the previously obtained 720-
The bp PstI fragment was ligated with T4 ligase to give the plasmid pUC119-lon'-Nm-EGF (Fig. 3).

IV. Homologous recombinant Bacillus brevis
Acquisition of HPD31 (lon: Nm-EGF) Using the plasmid pUC119-lon'-Nm-EGF obtained from Bacillus brevis HPD31 in III., The electroporation method (Agric. Biol. Chem., 53 , 3099-3100 ( 1989)) to obtain an Nm resistant strain. pUC119 is a plasmid for Escherichia coli, and the plasmid pUC119-lon'-Nm-EGF is capable of self-replication in E. coli but not in Bacillus brevis. Therefore, the Nm-resistant strain obtained here is a transformant in which the Nm-resistant gene is integrated into the chromosome. Eight EGF-producing strains were selected from the obtained Nm-resistant strains by a colony immunoassay method using an anti-EGF antibody. These 8 strains were then added to 5'PY liquid medium (Agri
c. Biol. Chem., 53 , 2279 (1989)) at 30 ° C.
After culturing with shaking for one day, the culture supernatant was subjected to SDS-PAGE and subjected to Western blotting using an anti-EGF antibody, and 1 strain Bacillus brevis HPD31-int1 having the highest EGF production was selected.

The culture supernatant of the selected strain was analyzed by HPLC (column:
C18-100A, diameter 4 mm × length 250 mm, buffer: 0.1% TFA / H ₂ O, 0.1% TFA / 50
% Acetonitrile, linear gradient, detection: UV2
76 nm), and the amount of EGF produced was quantified by comparing it with the peak area when HPLC was performed under the same conditions using commercially available EGF (manufactured by Funakoshi Co., Ltd.) as a standard product.
It was 4 g / l.

Bacillus brevis HPD31-int
Alkaline-SDS method (Nucleic Acids Res.,
7 , 1513, (1979)) examined the presence of the plasmid but did not detect it. So from B.brevis HPD31-int1 method of Saito and Miura (Saito, H., AND Miura, K., Biochem.
Biophys. Acta., 72 , 639 (1964))
Was prepared, digested with HindIII, and subjected to agarose gel electrophoresis. Add agarose gel DNA to Hybond N (+) (Amarsham,
Lon 'gene (2.3-kb HindII obtained in I.
Southern blotting was performed using the (I-EcoRI) fragment as a probe to examine the arrangement of various genes in the reacted integrated portions of 7.5 and 5.2-kb. The results are shown in FIG. 4, and it was revealed that the Nm-EGF gene was integrated at two sites.

V. Improvement of EGF Production by Gene Amplification Next, an attempt was made to amplify Nm resistance gene of Bacillus brevis HPD31-int1 and EGF gene by increasing Nm concentration. T2 Nm50 medium (peptone 1
%, Meat extract 0.5%, yeast extract 0.2%, glucose 1%, neomycin 50 μg / ml, pH 7.0) overnight culture of T2 containing Nm 500 μg / ml
It was applied to Nm500 agar medium and cultured at 30 ° C. for 3 days.

Bacillus brevis H, one of a plurality of strains grown on the T2 Nm500 agar medium obtained here.
PD31-int2 was selected. The selected strain Bacillus brevis HPD31-int2 was used in 5'PY liquid medium for 3
When shake culture was carried out at 0 ° C. for 4 days, and the culture supernatant was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB), it was found that the EGF production amount was clearly improved. Furthermore, the amount of EGF in the culture supernatant was adjusted to HPL as in IV.
When quantified by C, it reached 0.4 g / l. This production is about 10 times that of Bacillus brevis HPD31-int1. Bacillus brevis HPD31-in
Chromosomal DNA was prepared by the method of Saito and Miura from t2, digested with HindIII, and subjected to agarose gel electrophoresis. IV. When Southern blotting was carried out in the same manner as in, it was revealed that the Nm resistance gene and the EGF gene were amplified 10 times or more.

VI. Improvement of EGF production by introducing EGF secretion production plasmid pHT110EGF into Bacillus brevis HPD31-int2 hEGF high secretion production plasmid pHT110EGF obtained by V. homologous recombination gene amplification strain Bacillus brevis HPD31-i
It was introduced into nt2 by the electroporation method to obtain 8 strains growing on T2 agar medium containing 10 μg / ml of erythromycin and 500 μg / ml of Nm. These 8 strains are 5'P
Shaking culture was carried out using Y medium at 30 ° C. for 4 days. When the amount of hEGF produced in the culture supernatant was quantified, all the strains showed high production of hEGF of 1.6 to 1.8 g / l. This production amount was equivalent to the sum of 0.4 g / l of Bacillus brevis HPD31-int2 and 1.2 g / l of Bacillus brevis HPD31 carrying pHT110EGF (Table 2).

[0026]

[Table 2]

[0027]

INDUSTRIAL APPLICABILITY In a transformed microorganism, the production amount of a heterologous gene product can be remarkably increased by allowing a chromosome to carry a heterologous gene and carrying an expression vector to which the heterologous gene is bound.

[Brief description of the drawings]

FIG. 1 shows the construction of puC119-lon ′.

FIG. 2 is a diagram showing the construction of puC119-lon′-Nm.

FIG. 3 shows the construction of puC119-lon′-Nm-EGF.

FIG. 4 shows the arrangement of various genes in the homologous recombination part.

FIG. 5 is a diagram showing genes for hEGF expression.

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Claims (5)

[Claims] 1. A transformed microorganism having a chromosome carrying a heterologous gene and an expression vector having the heterologous gene bound thereto. 2. The transformed microorganism according to claim 1, wherein the transformed microorganism is a bacterium of the genus Bacillus. 3. The transformed microorganism is Bacillus brevis.
Or the transformed microorganism according to 2. 4. The method according to claim 1, wherein the heterologous gene is a gene for expressing epidermal growth factor.
Alternatively, the transformed microorganism of claim 3. 5. A heterologous gene product characterized by producing and accumulating a heterologous gene product in a culture by culturing the transformed microorganism according to claim 1, and collecting the heterologous gene product. Manufacturing method.