AU699075B2 - Transformed microorganism and process for producing foreign gene product using the transformed microorganism - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: HIGETA SHOYU CO., LTD.

Actual Inventor(s): Shogo EBISU; and Hiroaki TAKAGI Address for Service: CALLINAN LAWRIE, 278 High Street, Kew, 3101, Victoria, Australia Invention Title: "TRANSFORMED MICROORGANISM AND PROCESS FOR PRODUCING FOREIGN GENE PRODUCT USING THE TRANSFORMED MICROORGANISM" The following statement is a full description of this invention, including the best method of performing it known to me:- It -~1c -1- V Ii* 14

A-

TRANSFORMED MICROORGANISM AND PROCESS FOR PRODUCING FOREIGN GENE PRODUCT USING THE TRANSFORMED

MICROORGANISM

EIETAILED DESCRIPTION OF THE INVENTION Field of the Invention: The present invention relates to biotechnology. More specifically, the present invention relates to a transformed microorganism obtained by integrating a foreign gene into the chromosome of a microorganism and introducing an expression orr) *vector into which the same foreign gene has been bound into ~o the microorganism, and a process for producing a foreign gene o| e *microorganism, forming and accumulating the foreign gene product in a medium, and collecting the foreign gene product.

Prior Art: At present, the production of foreign gene products a.c* using recombinants has found wide acceptance in industries of food, medicaments, toiletries and the like. As host microorganisms for gene recombination, bacteria such as Escherichia coli and Bacillus subtilis, yeasts, filamentous fungi and the like are used.

Udaka et al. discovered that many strains belonging to species of Bacillus brevis do not produce protease. They successfully constructed a secretory vector by using the promoter of the gene of the main extracellular protein 2 [which is referred to as "outer wall protein and middle wall protein" or "extracellular protein" in H. Yamagata et al., J. Bacteriol., 169, 1239 (1987), N. Tsukagoshi, Nippon Kaishi, 61, 68 (1987)] of Bacillus brevis 47 ES. Udaka and H. Yamagata, Methods in Enzymology, 217, 23-33 (1993)] which is one of these strans, and the region encoding the signal peptide of the middle wall protein (MW protein) which is one type of the above-mentioned main extracellular protein to secrete and produce C-amylase 1JP-A 62-201,583 o*

I

(1987), and H. Yamagata et al., J. Bacteriol., 169, 1239 *too 1 9 8 7 )J or swine pepsinogen Udaka, Preprint of 1987 Meeting of Agricultural Chemical Society of Japan, pp. 837- 838, and N. Tsukagoshi, Nippon Nogeikagaku Kaishi, 61, 68 S (1987) 3 by using the above-mentioned strain as a host.

.i Further, Takagi et al. successfully isolated Bacillus brevis HPD31 [which is the same strain as Bacillus brevis H102 (FERM BP- :g a 1087)] that does not produce any detectable extracellular a. proteases, and secreted and produced a heat-resistant Q-amylase at high level by using the above-mentioned strain as a host [Agric. Biol. Chem., 53, 2279-2280 (1989)] Yamagata et al.

succeeded in secretive production of human epidermal growth factor (human EGF) [Proc. Nati. Acad. Sci., USA, 86, 3589-3593 (1989)] at high level by using the same strain as a host.

Such a production of a foreign gene product using a Smicroorganism is conducted by inserting a foreign gene to a i i "I I A

I

-I c~ 4a 4 cOa 4 C

C

4. E

'C~

*t 4 *t t 4 a Ci 3 vector capable of being replicated within a host microorganism, transforming the host microorganism with the resulting plasmid to express the foreign gene therein.

S. D. Ehrlich et al. succeeded in integrating a chloramphenicol (Cm)-resistant gene and a cellulase gene derived from Clostridium thermocellum into the chromosome of Bacillus subtilis, and increasing the amount of the cellulase produced by amplification of the Cm-registant gene and the cellulase gene which is induced by increased Cm amount in cultivation(s) of the resulting Bacillus subtilis [Biotechnology, 8, 559-563 (1990)] Problems to be solved by the Invention: In the production of a foreign gene product through the genetic recombination using a microorganism, the productivity of a foreign gene product has been rapidly improved by various technological improvements. However, further technological development has been demanded to supply a foreign gene product in an industrially necessary amount at low costs.

Means taken for Solving the Problems: The present inventors have successfully integrated the gene for expression of an epidermal growth factor into the chromosome of Bacillus brevis and introduced an expression vector having the same gene into the treated Bacillus brevis, and have enabled producing the epidermal growth factor in a remarkably high yield by culturing the obtained recombinant.

The present inventors have studied using integration t-

S;

I 1

I

i *449 44, .4 4 '.44 4* o Ct

'I

:I

L:

4 technology and plasmid technology in combination. They have integrated a foreign gene into the chromosome of a host microorganism, and introduced an expression vector having the same foreign gene into the host microorganism, and resultantly succeeded in increasing the production amount of the desired foreign gene product by using the obtained transformant. Thus, they have completed the present invention.

The present invention relates to a transformant obtained by integrating a foreign gene into the chromosome of a microorganism and introducing an expression vector having the same foreign gene into said microorganism, and a process for producing efficiently a foreign gene product, which comprises culturing the above-mentioned transformant, forming and accumulating the foreign gene product in a medium and/or in the cells, and collecting the foreign gene product therefrom.

Brief Description of Drawings: Fig. 1 shows the construction of pUC119-lon'.

Fig. 2 shows the construction of pUC119-lon'-Nm.

Fig. 3 shows the construction of pUCll9-lon'-Nm-EGF.

Fig. 4 shows the arrangement of genes of the integrated portion.

Fig. 5 shows the gene for expression of hEGF.

Embodiments of the Invention: When the desired foreign gene is integrated into the chromosome of the host microorganism in the present invention, i i i i i i: i' ii /r

I

i ;ii Ull_~i IYr 1LI_*1III .Llli Li;Cj 5 a gene that the host microorganism inherently contains on the chromosome can be used. That is, if the foreign gene is bound to the gene that the host microorganism inherently contains on the chromosome and inserted into a plasmid and then introduced into the host microorganism, the foreign gene is integrated into the chromosome of the host microorganism at a high frequency. At this time, any gene or DNA fragment present on the chromosome of the host microorganism can be used and the chain length of such a gene or DNA fragment is preferably 200 bp or more. For example, when a gene on the chromosome that encodes a protease or a peptidase degrading a foreign gene product expressed and secreted is chosen as a target for integrating, the ability to produce the protease or peptidase of the host microorganism is lost and therefore the degradation of the foreign gene product expressed is suppressed.

Specifically, when the host microorganism is Bacillus brevis HPD31, the lon gene on the chromosome encoding protease La Bacteriol., 174, 2281-2287 (1992)] or the like may be targeted.

When an antibiotic-resistant gene connected to a gene encoding a foreign protein is integrated into a gene that the host microorganism inherently contains on the chromosome as mentioned above, it is possible to select a transformant, in which the foreign gene has been integrated into the chromosome, by utilizing the antibiotic resistance as a marker. Further, the antibiotic-resistant gene can be amplified by increasing the concentration of antibiotics in cultivation(s) and accordingly the desired foreign protein- 'i i d ii- fi it 6 encoding gene connected to said gene can be also amplified.

Thus, the production amount of the desired foreign proten can be increased. Any antibiotic-resistant genes that the host microorganism does not contain inherently may be used.

For example, genes which are resistant to known antibiotics such as neomycin, erythromycin, amplicillin and chloramphenicol may be used.

A DNA fragment, which is a link of the desired foreign gene, a gene or DNA fragment inherently present on the *0Ow chromosome of the host microorganism and the antibiotic- *04* resistant gene, is introduced into the host microorganism cell by using a plasmid and is integrated into the chromosome of the host m.croorganism. However, at this time, it is necessary to use a plasmid that cannot replicate in the host microorganism. The reason is as follows. When using a plasmid which can replicate in the host microorganism, the obtained transform;nt comes to have antibiotic resistance, P so that the integration of the desired foreign gene into the Schromosome cannot be confirmed. To avoid this, the plasmid which cannot replicate in the host microorganism has to be used. For instance, when a bacterium of the genus Bacillus is used as the host microorganism, the foreign gene can be introduced into the cell of the host bacterium by using pUCll9, pBR322 or the like which can be maintained only in Escherichia coli.

i j i j i ij+t l i m ii iij rTT~ ir~i-r l r 7 a 6 case "09 0 O. .1 O4.

**uo 0 6 do «o 06*0 C 60 o i 0 Oo a 69 In the present invention, the production amount of the foreign gene product can be greatly increased by introducing a vector containing the foreign gene into the host microorganism in which the same foreign gene has been integrated into the chromosome. At this time, as the vector for introducing the foreign gene into the host microorganism and maintaining it, a plasmid which is replicable in the host must be used. For example, in a system in which Escherichia coli is used as the host, pUC19, pUC119 or derivatives thereof may be used as the plasmid. In a system in which Bacillus subtilis or Bacillus brevis is used as the host, pUBll0, pNU200 IS. Udaka, Nippon Nbgeikagaku Kaishi, 61, 669 (1987)] pHY700 [Biosci. Biotech. Biochem., 56, 812-813 (1992)] pHTll0 [JP-A 6-133782 (1994)] or derivatives thereof may be used as the plasmid.

These plasmids are constructed by a known method. For example, the method described in Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, 1989 is taken up.

The foreign gene to be introduced into the host microorganism in the present invention may be any gene and can be used to produce gene products (enzymes, hormones, interferons, immunoglobulins, other physiological peptides, antigen proteins and the like) derived from human, animal, fowl, fish, microorganism, virus and other organism.

8- For example, it is possible to produce gene products such as an epidermal growth factor (EGF) of human, mouse, sheep and the others.

Examples of a microorganism which is used as the host microorganism in the present invention include bacteria and yeasts. When bacteria are used as the host microorganism, Escherichia coli or bacteria of the genus Bacillus may be used. Preferable examples of the bacteria of the genus Bacillus include Bacillus brevis and Bacillus choshinensis.

The host microorganism may be transformed by a known method. Examples of the known method include the method of Chang and Cohen LS. Chang and S. N. Cohen, Molec. gen.

Genet., 168, 111-115 (1979)1 and the method of Takahashi et al. Bacteriol., 158, 1130 (1983)] and the method of Takagi et al. [Agric. Biol. Chem., 53, 3099-3100 (1989)] concerning Bacillus brevis.

Any culture media in which the resulting transformant can grow and produce the desired foreign gene product may be used *i« to culture the transformant.

Examples of a carbon source to be contained in the 44 culture medium include glucose, sucrose, glycerol, starch, Stc dextrin, molasses, urea and organic acids. Examples of a nitrogen source include organic nitrogen sources such as casein, peptone, meat extract, yeast extract, casamino acid and glycine, and inorganic nitrogen sources such as ammonium ;i

A

I 1 ~g~p;i-i"i 9 sulfate Further, inorganic salts such as potassium chloride, potassium phosphate monobasic, potassium phosphate dibasic, sodium chloride and magnesium sulfate may be added to the culture medium as required. Then using an auxotrophic microorganism, nutrient necessary for the growth of it may be added to the culture medium. Examples of the nutrient include amino acids, vitamins and nucleic acid bases.

Antibiotics such as penicillin, erythromycin, chloramphenicol, ba'-itracin, D-cycloserine, ampicillin and neomycin may be added to the culture medium in the cultivation as required. Further, an antifoamer such as a soybean oil, a lard oil and a surfactant may be added to the culture medium as required.

The initial pH of the culture medium is between 5.0 and preferably between 6.5 and 7.5. The cultivation temperature is usually between 15°C and 42 0 C, preferably between 24 0 C and 37 0 C. The cultivation time is usually between 16 hours and 166 hours, preferably between 24 hours and 96 hours.

In the present invention, the transformant is cultivated under the above-mentioned conditions to form and accumulate the foreign gene product in the culture. The thus-obtained foreign gene product can be purified by a known method such as membrane treatment, ammonium sulfate fractionation, chromatography or the like [Tanpakushitu Kakusan no 1111 10 Kisojikkenhou, Nankodo, (1985)1.

In the present invention, various foreign gene products can be produced stably at high level in such a way that the foreign gene is integrated into the chromosome of the host microorganism and also inserted into the expression vecotr.

The present invention will be illustrated more specifically by referring to the following Examples.

However, the present invention is not limited to these Examples.

Examples I. Cloning of protease lon gene A 2.3-kb HindIII-EcoRI fragment containing most of lon gene from a chromosome of Bacillus brevis HPD31 (FERM BP- 1087) was amplified by a PCR method using two types of primers shown in Sequence Listing No. 1 (lon-5 primer) and No. 2 (lon-3 primer) of the following Table 1. The gene fragment amplified was treated with restriction endonuclease HindIII and EcRI, and then subjected to 0.8% agarose gel electrophoresis. The 2.3-kb HindIII-EcoRI fragment was recovered from an agarose gel by using a Gene Clean (Bio 101 USA). Separately, plasmid pUCll9 (supplied by Takara Shuzo Co., Ltd.) was digested with HindIII and EcoRI, then treated with alkaline phosphatase (BAP), ligated with the aboveobtained 2.3-kb HindIII-EcoRI fragment by using T4 ligase to form plasmid pUC119-lon' as shown in Fig. 1.

.i.

i;r 1 *1 i> I r

K

By their Patent Attorneys: CALLINAN LAWRIE 25/96GS8816.4PP,1 11 Table 1 Sequence Listing Sequence No. 1 Length of sequence: 16 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid synthetic DNA Sequence TAAAAGCTTC TGCTTG Sequence No. 2 r" t .Length of sequence: 16 C" Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid synthetic DNA Sequence TAAGAATTCC GGTCAG .II. Construction of plasmid pUC119-lon'-Nm Plasmid pUB110 derived from Staphylococcus aureus was partially digested with HaeIII, and then subjected to 0.8% agarose gel electrophoresis to recover a 1.0-kb HaeIII fragment containing a neomycin (Nm)-resistant gene. Plasmid pUC119 was digested with Smal, then treated with BAP, and ligated with the above-obtained 1.0-kb HaeIII fragment by using 1; 1 I *w i'

II

ii 1

I

ij r

I

9FOO, 12 ft.,.

ft...

ft...

se T4 ligase to obtain plasmid pUC119-Nm. Said pUC119-Nm was digested with EcoRI and HIndIII, and then subjected to 0.8% agarose gel electrophoresis to obtain a 1.1-kb EcoRI-HindIII fragment containing the Nm-resistant gene. Subsequently, this fragment was treated with a Klenow enzyme to blunt the terminals thereof.

Plasmid pUCll9-lon' obtained in I was digested with SacI. The cut portions were then blunted with T4 polymerase.

The thus-treated plasmid was ligated with the above-obtained 1.1-kb fragment containing the Nm-resistant gene to obtain plasmid pUC119-lon'-Nm as shown in Fig. 2.

III. Construction of plasmid pUC119-lon'-Nm-EGF Plasmid pHT110EGF [JP-A 6-133,782 (1994) prepared from plasmid pHT926 that was derived from Bacillus brevis HP926 (FERM BP-5382) was digested with PstI to obtain a 720-bp PstI fragment in which a promoter of the gene of a cell wall protein (HWP) of Bacillus brevis HPD31 is combined with the structural gene of a human epidermal growth factor (hEGF) as shown in Fig. Plasmid pUC119-lon'-Nm obtained in II was digested with Sse 83871, then treated with BAP, and ligated with the aboveobtained 720-bp PstI fragment by using T4 ligase to obtain plasmid pUC119-lon'-Nm-EGF as shown in Fig. 3.

IV. Procurement of integrated-recombinant Bacillus brevis HPD31 (lon: Nm-EGF) Sor ou ,oQ 4 So.

o~ ;lu 'f* ft.

ft S e i a ft ,ft. k« I% K-1

A

I::

C

Ce

C(

C C 13 Bacillus brevis HPD31 was transformed by electroporation [Agric. Biol. Chem., 53, 3099-3100 (1989)] using plasmid pUC119-lon'-Nm-EGF obtained in III to form an Nm-resistant strain. pUC119 is a plasmid for Escherichia coli, and plasmid pUC119-lon'-Nm-EGF is self-replicable in Escherichia coli but not in Bacillus brevis. Accordingly, the above-obtained Nm-resistant strain is a transformant in which the Nm-resistant gene is integrated into the chromosome.

From the thus-obtained Nm-resistant strains, eight EGF production strains were selected by colony immunoassay using an anti-EGF antibody.

Subsequently, each of these eight strains was cultured in 5' PY liquid medium [Agric. Biol. Chem., 53, 2279 (1989) at 30 0 C for 4 days with shaking. The culture supernatant was subjected to SDS-PAGE, and then subjected to the Western blotting using an anti-EGF antibody to select one strain, Bacillus brevis HPD31-intl which produces hEGF in the largest amount.

The culture supernatant of the selected strain was analyzed with HPLC (column: C18-100A, 4 mm (diameter) x 250 mm (length), buffer: 0.1% TFA/H 2 0, 0.1% acetonitrile, linear gradient, detection: UV 276 nm). The amount of hEGF produced was determined by comparing the obtained peak area with the peak area under the same conditions conducted by using commercial hEGF (made by 1- Y;..r

T

fl 14 Funakoshi as a standard product. It was found to be 0.04 g/liter.

In regards to Bacillus brevis HPD31-intl, the presence of the plasmid was examined by the alkali-SDS method [Nucleic Acids Res., 7, 1513 (1979) but it was not detected. Accordingly, a chromosomal DNA was prepared from Bacillus brevis HPD31-Intl by the method of Saito and Miura [Saito, H. and Miura, Biochem.

Biophys. Acta., 72, 639 (1964) digested with HindIII and then subjected to agarose gel electrophoresis.

The agarose gel DNA was transferred onto Hybound (made by Amarsham, UK), and subjected to the Southern blotting using the lon' gene (2.3-kb HindIII-EcoRI) fragment obtained in I as a probe. The 7.5-kb and 5.2-kb fragments were hybridized with the probe, and then the location of the genes at the integrated position was examined. The results that the Nm-EGF genes were integrated at two locations are shown in Fig. 4.

V. Increase in the amount of hEGF produced by gene amplification The Nm-resistant gene of Bacillus brevis HPD31-intl and the hEGF gene were amplified by increasing the Nm concentration. The strain which had been cultured overnight in T2 Nm50 medium (containing 1% peptone, 0.5% meat extract, Bi 0.2% yeast extract, 1% glucose and 50 pg/ml of neomycin, pH spread onto T2 Nm500 agar medium containing

I

11 r ri i g ti A c j 1 500 pg/ml of Nm, and cultured at 30 0 C for 3 days.

From among the plural strains which grew on the T2 Nm500 agar medium, one strain, Bacillus brevis HPD31-int2 was selected. The selected strain, Bacillus brevis HPD31-int2 was cultured in 5' PY liquid medium at 30 0 C for 4 days with shaking. The obtained-culture supernatant was subjected to SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB).

It was found that the amount of hEGF produced was increased.

Further, the amount of hEGF in the culture supernatant was determined with HPLC in the same manner as in IV. It was found to be 0.4 g/l and it was approximately 10 times that of S hEGF produced by using Bacillus brevis HPD31-intl.

A chromosomal DNA was prepared from Bacillus brevis HPD31int2 by the method of Saito and Miura, digested with HindIII, and then subjected to agarose gel electrophoresis. The i Southern blotting was conducted in the same manner as in IV.

Consequently, it was found that the Nm-resistant gene and the hEGF gene were amplified to 10 times or more.

t VI. Increase in the amount of hEGF by introduction of plasmid pHT110EGF for secretory production of hEGF into Bacillus brevis HPD31-int2 Plasmid pHT11OEGF for secretory production of hEGF at high level was introduced into the integrated gene amplification strain, Bacillus brevis HPD31-int2 obtained in i.

I a 4 16 V by electroporation, and eight strains which grew on T2 agar medium containing 10 pg/ml of erythromycin and 500 pg/ml of Nm were obtained. These eight strains were cultured in PY liquid medium at 30 0 C for 4 days with shaking. The amounts of hEGF produced in the obtained-culture supernatant were determined. Consequently, all of these eight strains produced hEGF in high amounts of from 1.6 to 1.8 g/liter which corresponded to the sum of hEGF 0.4g/liter by Bacillus brevis HPD31-int2 and hEGF 1.2 g/liter by Bacillus brevis HPD31 containing pHT11OEGF as shown in Table 2.

Table 2 Amount of hEGF in various transformants Strain Amount of hEGF produced* (g/liter) r ect eL 1

I

I't B. brevis HPD31 0 B. brevis HPD31-intl 0.04 B. brevis HPD31-int2 0.40 B. brevis HPD31/pHT110EGF 1.2 B. brevis HPD31int2/pHT110EGF 1.6 1.8 Amount of hEGF produced by shake culture in 5' PY liquid medium at 30 0 C for 4 days.

1 -17 Effect of the Invention: An amount of the foreign gene product can be remarkably increased by using the transformed microorganism formed by integrating the foreign gene into the chromosome of the microorganism and introducing the expression vector having bound thereto the same foreign gene into the microorganism.

Microorganism Deposits The microorganism Bacillus brevis H102 was deposited on 24 June 1986 with the Fermentation Research Institute, Agency of Industrial Science and Technology and assigned accession number FERM BP-1087.

The microorganism Bacillus brevis HP 926 was deposited on 18 December 1991 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry and assigned accession number FERM BP-5382.

This authority accepted petition of transfer to deposit under the Budapest Treaty on 8 February 1996 (transferred custody from Deposit No FERM P-12664 deposited on 18 December 1991.

1 1

'J

Claims (4)

1. A transformed microorganism formed by integrating a foreign gene into the chromosome of a microorganism and introducing an, expression vector having bound thereto the same foreign gene into the microorganism.

2. The transformed microorganism of claim 1, wherein the microorganism is one of the genus Bacillus.

3. The transformed microorganism of claim 1 or 2, wherein the microorganism is Bacillus brevis.

4. The transformed microorganism of claim 1, 2 or 3, wherein the foreign gene is a gene for the expression of an epidermal growth factor. A process for producing a foreign gene product, which comprises culturing the transformed microorganism of any one of claims 1 to 4 to produce and accumulate the foreign gene product in a medium and/or the cells, and collecting the foreign gene product. DATED this 25th day of July 1996 HIGETA SHOYU CO., LTD. By their Patent Trade Mark Attorneys CALLINAN LAWRIE f t i 4* 4C t 44 4 4r 4 (r- amplified by increasing the concentration of antibiotics in cultivation(s) and accordingly the desired foreign protein- 'iL I ,ac I: 19 ABSTRACT The present invention relates to a process for producing a foreign gene product at high level, which comprises culturing a transformed microorganism formed by integrating a foreign gene into the chromosome of a microorganism and introducing an expression vector having bound thereto the same foreign gene into the microorganism to produce and accumulate the foreign gene product in a medium, and collecting the foreign gene product. t ft C- -r~c 1 I