JPS61152279A - Cultivation of microorganism - Google Patents

Abstract

PURPOSE:To produce a desired protein in high efficiency, by culturing a transformed microorganism for a specific period in a medium containing inorganic phosphorus of an amount necessary and sufficient to induce the protein- synthesis capability. CONSTITUTION:A microorganism is transformed by a chimera plasmid produced by integrating a gene coding exogenic protein under the control of a plasmid having a promoter originated from alkaline phosphatase, and the obtained transformed microorganism is cultured in a medium containing inorganic phosphorus of an amout necessary to induce the protein-synthesis capability from the later stage of the logarithmic proliferation stage to the former stage of the resting stage in the proliferation of the microorganism.

Description

BACKGROUND OF THE INVENTION Technical Field The present invention relates to a method for culturing microorganisms transformed by genetic engineering techniques.

More specifically, the present invention aims at producing a desired exogenous protein using recombinant DNA technology. The present invention relates to a method for culturing microorganisms under conditions that induce induction.

PRIOR ARTCurrently, methods for producing useful substances through genetic engineering using recombinant DNA and A technology are being established.For specific methods, please refer to various documents, documents, published patent publications, and patent publications. I can do it.

Generally, a method for producing a substance using this method involves transforming a microorganism using a vector created by incorporating a gene encoding the desired substance, culturing the obtained transformant, and then injecting the desired substance. It consists of collecting.

By the way, when non-secreted proteins are produced by the above method, the proteins produced in microbial cells are degraded (Proc, NaN, A
cad, Sci, USA, 79°1830-1833
(1982)], it is difficult to obtain the desired protein stably and in large quantities. In addition, when recovering such non-secreted proteins, the host microorganism is physically destroyed and then the desired protein is isolated and purified. As a result, the yield of the desired protein is low,
There is a problem in that the substances that determine activity may be deactivated during the recovery operation. Therefore, in order to deal with the above problems, we developed a signal sequence [protein/nucleic acid/enzyme, zl] that is involved in the passage of proteins through membranes.

386-394 (1981)), various methods have been proposed for secreting proteins produced within host microbial cells to the outside of the cell or the cytoplasmic membrane (Public Patent Publication No. 1981-19092). , No. 55-45395, No. 56-137896, No. 56-145221,
56-154999, etc.). Recently, co-researchers of the present inventors have proposed a method for producing proteins that skillfully utilizes such a method (Japanese Patent Application No. 1983-1999).
No. 159703) (hereinafter referred to as the prior invention).

As a member of the method of this prior invention, the plasmid pTA1529 is equipped with a promoter and signal sequence derived from alkaline phosphatase, and is capable of ligating a gene encoding a desired foreign protein immediately after the signal sequence. is used. In fact, pTA1529 contains a gene [for example, human epidermal growth factor (hUG/EGFo)] that encodes a foreign protein.
(hereinafter simply referred to as hUG)] into a chimeric plasmid (pTA1522 after incorporating the structural gene of hUG)
We have constructed a plasmid and are transforming microorganisms using this plasmid. Therefore, microorganisms transformed with such plasmids (hereinafter, transformed microorganisms are referred to as 1
When a host cell (sometimes a microorganism is simply referred to as a bacterium) is cultured, a foreign protein is first expressed within the host cell, and then this protein is secreted outside the cell by the action of a signal sequence. be done. Here, "secreted outside the bacterial body" means that when the host bacteria is a Gram-negative bacteria such as Bacillus Otomi, the protein is transferred from the bacterial body to the periplasm (the space between the cell membrane and the outer membrane). In Durham-positive bacteria such as Bacillus subtilis, it means that it migrates outside the cell.

In the prior invention, the signal sequence is hydrolyzed by deenzyme signal peptidase when the protein is secreted outside the bacterial cell, so the protein bound immediately after the signal sequence is accompanied by extra amino acids. It is secreted outside the bacterial cell as a complete mature protein without any oxidation.

In the method for producing proteins according to the prior invention, the following J: Una culture method is adopted for transformed microorganisms that have a promoter derived from alkaline phosphatase. That is, this culture method takes advantage of the fact that the synthesis of alkaline phosphatase is suppressed in the presence of high concentrations of inorganic phosphorus and induced in the presence of low concentrations of inorganic phosphorus [Biochem, Biophys, Acta
, 38.460 (1960), Nature ,
, LfL2L, 1529 (1959)). First, host microorganisms E and c transformed with pTA1522 into which the structural gene of hUG has been incorporated.
oli' with 12C600 (pTAL 522) for 20
One medium containing μg/d of ampicillin [1% Bactodrypton, 0.5% yeast extract, 0.5% NaC+, 0
．． 1% glucose) 8 (7).Then, the pre-cultured bacteria was cultured in a synthetic medium containing 0.64 mM potassium dihydrogen phosphate (Biochem, 31ophys).
, A cta , TG+20 medium) 2.4
．． 1) and cultured with shaking overnight at 37°C (hereinafter referred to as the "first stage" for convenience).
The cells were transferred to the above synthetic medium containing μM potassium dihydrogen phosphate and cultured with shaking at 37° C. for 5 hours (hereinafter referred to as "second stage" for convenience). Here TG+
20 medium means glucose 2g/J, NaC+ 4
．． 68g/, 11. KCI 1.49g/, 1).

NH4CI 1.09y/J), Na2SO40,
43y#, M(] C1295, 21mg#.

CaCl22, 20 mg/, 11-F
e Cl 3324, 42 u g/, Q, Z
nCl 2272.56μ'; i/-Q, KH2
It consists of PO487, 1mg/0 and O, 1M Tris-HCl buffer (1) l-17,2). According to this J: eel culture method, it is possible to propagate the transformed microorganism in the first step, and then perform culture accompanied by induction of protein synthesis ability in the second step.

By the way, a transformed microorganism is generally loaded with a plasmid introduced into the microorganism (J
our own of generalylicr
obiology, 118.253-261 (19
80)), the growth rate is slower than that of non-transformed microorganisms, and the introduced plasmid is easily shed, and microorganisms from which the plasmid has been shed are known to easily become the preferred species [Δnnals]. of tl
lo New York Academy of 5
Science, 369.1-14 (1981), Journal of General Micr.
biology).

The problem of high concentration cultivation of transformed microorganisms arising from this fact could be overcome by the cultivation method of the prior invention. However, when this method is applied to the production of industrial substances, it is necessary to culture at a higher concentration in the first step. In addition, in the second step of this method, after inoculating the bacteria, the protein synthesis ability is induced once, but as the culture continues, the inorganic phosphorus necessary for the growth of the bacteria runs out, and the bacterial cells themselves disappear. Therefore, there is a problem that the desired protein cannot be obtained in large quantities.

Therefore, in order to solve the above problems, the present inventors proposed a method for culturing microorganisms, which was filed on the same day as the present invention. In this culture method, a microorganism that has been transformed to produce a desired protein using II'1 transgenic DNA technology is first cultured for microbial growth, and then cultured to induce protein synthesis ability. It consists of two steps.

According to this method, it is possible to culture transformed microorganisms at a high concentration, and furthermore, it has become possible to efficiently produce a desired protein in large quantities. However, since this invention consists of a two-step culture process, it is complicated due to operations such as collecting bacteria and washing the inoculated bacteria, and there is also a high possibility of contamination of the culture medium. Problems remain, such as the operation cost of the culture itself being high due to the time required for culturing, and improvements to these issues were necessary.

DETAILED DESCRIPTION OF THE INVENTION The present invention aims to provide a solution to the above-mentioned points, and aims to provide a solution to the above-mentioned problems.In the growth process of microorganisms, protein synthesis ability is induced (in fact, host bacterial cell This purpose is achieved by culturing microorganisms transformed by recombinant DNA technology in a medium containing a sufficient amount of inorganic phosphorus to achieve this goal. The aim is to achieve this.

Therefore, the method for culturing a microorganism according to the present invention involves transforming a microorganism with a chimeric plasmid constructed by incorporating a gene encoding a foreign protein under the control of a plasmid having a promoter derived from alkaline bosphatase. It is characterized in that it is cultured in a medium containing an amount of inorganic phosphorus necessary to induce protein synthesis ability from the late logarithmic growth phase to the early arrest phase in the growth process of microorganisms.

Effects As described above, the method for culturing microorganisms of the present invention uses recombinant DNA
Microorganisms transformed by this technique are grown in a medium containing a necessary and sufficient amount of inorganic phosphorus to induce protein synthesis ability during the growth process of the microorganisms, from the late logarithmic growth phase to the early arrest phase. Since the above-mentioned culture is performed in a single culture system, the present invention provides the following advantages.

(1) The culture operation is simple, the possibility of medium contamination is low, and the operating costs of the culture system are kept low.
Ki6. E. The "microorganism culture method" proposed by the present inventors as patent m (↓) on the same day as the present invention is a microorganism growth process (first step) as described above.
The culture is divided into two steps: and a culture step (second step) that involves induction of protein synthesis ability. Therefore, the culture operation is complicated because it involves collecting bacteria, cleaning the inoculated bacteria, and constant-rate feeding operation, and there is a high possibility of contamination of the medium.Also, since the two culture systems require the same amount of culture time, it is difficult to operate the culture. The cost was high. On the other hand, the present invention solves the above-mentioned problems because microorganism growth and protein synthesis ability can be induced in a single culture system.

(2) A desired protein can be efficiently produced.

Conventionally, microorganisms have been cultured in two steps as described above in IC. Therefore, since the culture is interrupted and the culture medium is changed when moving from the first step to the second step, the protein synthesis ability of the microorganism may decrease. On the other hand, according to the culture method of the present invention, the objectives of the above two steps are achieved in a single culture system, so that the desired protein can be efficiently produced in a short time without interrupting the culture.

Further, the method of the prior invention proposed by the joint researchers of the present inventors (Japanese Patent Application No. 59-159703)
By combining with No.), the desired protein can be obtained efficiently. The method disclosed in Japanese Patent Application No. 59-159703 is a method for producing proteins by genetic engineering using a plasmid that is equipped with a promoter derived from alkaline phosphatase and also has various signal sequences. In this method, plasmid pTA
1529 is used. pTA1529 is equipped with a promoter and signal sequence derived from alkaline phosphatase, and allows a gene encoding a desired foreign protein to be ligated immediately after the gene encoding the signal sequence.

Therefore, if a gene encoding a desired foreign protein is incorporated into a chimeric plasmid, a microorganism is transformed using this, and the transformant is cultured according to the method of the present invention, the desired protein can be efficiently and efficiently produced. can be obtained in large quantities. That is, by subjecting the transformant to the culture of the present invention, the transformant first grows. During this bacterial growth process, a protein that induces protein synthesis ability is synthesized from the late logarithmic growth phase to the early arrest phase. Roughly speaking, proteins produced within the transformant are secreted by the action of the signal sequence of the plasmid and accumulated in the periplasm (the space between the cytoplasmic membrane and the outer membrane).

The proteins accumulated here can then be extracted using the osmotic shock method (J, Biol, Chem, 240.3
685 (1965)), it is easily released outside the bacterial cells. In addition, when the desired protein released here is transferred to the periplasm by the action of the signal sequence, the signal sequence is cleaved by deenzyme signal peptidase.
Moreover, since the signal sequence and the protein are directly linked, the protein accumulated in the periplasm is a complete mature protein with no extra amino acids attached. Also,
Since the constituent components of the solution from which the protein has been released are known, it is easy to recover the protein from the solution. Therefore, transformants grown at high concentrations can efficiently synthesize proteins, and since the proteins can be easily recovered, desired proteins can be obtained efficiently and in large quantities.

Detailed Description of the Invention Plasmid The plasmid used in the present invention is based on the fact that alkaline phosphatase is affected by the amount of inorganic phosphorus present in the culture method of the present invention [3 iochem above.

B 1ophys, Acta, ), any promoter may be used as long as it has a promoter derived from alkaline phosphatase and can proliferate in the intended host cell. Even more preferred is a plasmid that also utilizes the function of a signal sequence (secretion of protein outside the bacterial body).

Specific examples of such preferable plasmids include plasmids pTA529 (Japanese Patent Application No. 140748/1983), pTA529 (Japanese Patent Application No. 58-140748), which was previously proposed by the present inventors'co-researchers;
1529 (Japanese Patent Application No. 59159703 @), etc. All of these plasmids are equipped with a promoter and a signal sequence derived from alkaline phosphatase, and a gene encoding a foreign protein can be ligated directly to the gene encoding the signal sequence. Here, pTA1529 is pTA529
(pYK283 (E, coli K12C600
Deposited as (DYK283) at Micro-Lab
56)] based on this] Patent Application 1987-J407
4B) created according to the method described in Mt.
)I-181 (this plasmid is pBR322(E,
coli K12C600 (pBR322) deposited with FIKEN (FeIKEN Article No. 235)] was subjected to restriction enzyme EC.
This Eco
RI-1-1indl [with the [ part replaced with the following synthetic linker (see JP-A-59-71692)]
It was created from. For details of the procedure for constructing this plasmid, please refer to the specification of Japanese Patent Application No. 159703/1983.

EcoRI l-1paI SmaI
pTA' 2539 is also used in the member of the present invention, but this has the ampicillin resistance gene of the above pTA1529 removed, a kanamycin resistance gene introduced in its place, and an alkaline phosphatase derived gene. The signal sequence was changed to one derived from β-lactamase.

Transformant microorganisms can be transformed according to conventional methods known in the field of recombinant DNA technology.

For example, if the above plasmid contains a desired foreign protein M3
11 genes to create a chimeric plasmid, and using this chimeric plasmid, microorganisms can be cultured using known methods (for example, the Kushner method (Qene).
ttc Engineering, 1978.17
(1978)')) and then obtain the desired transformant (usually by using a plasmid marker).

= 15 - The microorganism that can be transformed in this way may be any microorganism as long as the above-mentioned plasmid can proliferate within its bacterial body, and specific examples include Escherichia coli, yeast, and Bacillus subtilis. Among them, 12 strains are relatively commonly used in E. coli, and in the examples of the present invention, mutant strains E, Co11 K12YK537,
(Miken Bacteria No. 7941) is used.

In addition, the structural genes of the desired foreign proteins to be incorporated into the above-mentioned plasmids may include hormones, immune-related substances, neuropeptide enzymes, and the like. Methods for preparing these structural genes include methods for obtaining them from natural chromosomal DNA and methods for artificially synthesizing them.For actual methods for preparing structural genes, please refer to various books and literature. (the above-mentioned prior invention, etc.).

This J: eel transformant used in the present invention was obtained by artificially synthesizing h
Chimeric plasmid pTA incorporating the tJG structural gene
'1522, and then used this to create E, col
iK12YK537 was transformed using the Kushchi method (E.

coli K12YK537 (pTA1522))
.

For details of the procedure for creating transformants, please refer to the reference examples below. Similarly, the hUG structural gene was inserted into DTA2539 to create pTA2532, and this was used to transform transformants E and coli.
K12YK537 (pTA2532) was obtained and cultured according to the method of the present invention (see reference side below for details).

Cultivation of Microorganisms The cultivation according to the present invention targets microorganisms transformed with a plasmid having a promoter derived from alkaline phosphatase, and the characteristics of the culture method of the present invention are that The properties of the plasmid (the protein synthesis ability may or may not be induced depending on the amount of inorganic phosphorus [see above [3ioche]
m, 31ophys.

Acta, NatUre') can be skillfully used to grow microorganisms and subsequently induce protein production by the microorganisms in a single culture system.

Details of such a culture method are as follows.

1) Cultivation method The method for culturing microorganisms according to the present invention involves culturing transformed microorganisms in single-cell pure isolation, and then subjecting them to preculture (the above is a well-known conventional method for culturing microorganisms; You can refer to literature and collections. For example, the collection "Microbiology Experimental Methods" (published by Kodansha), "Microbiology Experimental Methods"
(Published by Kyoritsu Shuppan) and ``Bacteriology Practice Summary'' (Published by Maruzen). ), which consists of culturing in a single culture system.

This single culture system belongs to the category of aerated agitation culture, and specifically, after inoculating bacteria in the culture system, sterile air (with pure oxygen mixed in as necessary) is added to the culture medium.
The microorganisms are introduced into the microorganism, and the microorganisms are physically stirred while culturing is carried out while adjusting pl-1, temperature, dissolved oxygen concentration, etc. under suitable conditions to suit the growth conditions of the microorganism to be cultured.

Such cultivation is usually carried out using a Schille fermenter, and it goes without saying that appropriate scale-up is possible [Book ``Biochemical Engineering'' (Part 1), (
(see below), University of Tokyo Press).

2) Medium The medium composition of the present invention uses LB medium (trypone, yeast extract, NaC+) as a basic medium, and other components (e.g. MgSO4.7H2o, antibiotics, etc.) are added as necessary. Furthermore, it is composed of a substance containing inorganic phosphorus in an amount necessary and sufficient to induce protein synthesis ability from the late logarithmic growth phase to the early arrest phase of the growth process of the transformed microorganism. In addition, in the specific example of the present invention, E, colt K1 are added as necessary antibiotics.
When culturing 2YK537 (pTA1522), ampicillin is added, and E. coli K12
When culturing YK537 (pTA2532), kanamycin is added. In these specific examples of the present invention, the plasmid retained by each transformant is
Each of them is equipped with ampicillin-resistant and kanamycin-resistant genes, and the transformants are ampicillin-resistant or kanamycin-resistant, so bacteria whose respective plasmids have fallen off during culture (not resistant) may be affected by ampicillin or kanamycin in the medium. Since growth is inhibited by kanamycin, only host bacteria that carry the plasmid will grow. Thus, the addition of antibiotics to the culture medium
This is a means for facilitating the growth of host bacteria.

3) Culture conditions (1) Culture temperature The culture temperature may be any temperature at which the microorganisms used can multiply or grow and the product is stable. In the case of commonly used E. coli and E. coli used in the present invention, 3
7°C is preferred.

(2) Culture time The culture time of the present invention is preferably the time at which the production amount of the desired substance is maximized. One embodiment of the invention employs approximately 10-11 hours. Such examination of culture time can be performed using as indicators the determination of glucose and inorganic phosphorus in the medium, the measurement of 0D66o, the measurement of the number of viable bacteria, and the determination of produced substances. Glucose was determined using the glucose oxidase method using the euglucostat "Fujisawa" Kira]. ), the number of viable bacteria is E, coli K 12YK
537 (pTA1522), ampicillin (E, coli K12YK537 (pTA2
In the case of 532), inorganic phosphorus was determined by measuring the number of colonies growing on agar of L-medium (described above) containing kanamycin] according to the molybdenum blue method (industrial wastewater test method: JIS K 0102). Also,
The product hUG can be quantified using the osmotic shock method [J, Biol, supra.

After treatment by the Radioreceptor Assay (R, RA) method (J.

Biol, Chem, 257, 3053 (198
2)] (for details of hUG quantification, see Reference Examples below and the specification of Japanese Patent Application No. 159703/1983).

(3) Pl-1 of the culture medium Pl-1 may be any medium that allows microorganisms to grow, and a preferable pl-1 may be appropriately set depending on the microorganism used. When using E. coli, the viable pl-1 of E. coli is usually 4.6 to 8.8, of which 1
) Particularly preferred is H7.0 to 8.0.

(/I) Antifoaming agent If there is significant foaming during culture, use an antifoaming agent (higher alcohol,
It is a common practice to add vegetable oil, etc.). Since the antifoaming agent used in the present invention controls the protein synthesis ability of microorganisms by the presence of inorganic phosphorus, it is important that it does not contain inorganic phosphorus. A specific example of such an antifoaming agent is Andihome's Mulsion (Hangetsu Kagaku).

(5) Dissolved Oxygen (Do) Dissolved oxygen refers to molecular oxygen dissolved in the liquid phase.

It is generally known that in aerated agitation culture, if Do is excessive, the growth of microorganisms is inhibited, while if Do is less than 1 ppm, growth is similarly inhibited. Therefore, it is preferable to control DOffl so that it does not become an inhibitory factor for microbial growth. In the case of one embodiment of the present invention, a DO controller 1-roll device [Oriental Electric ■F
C-4 type] to maintain DOM at 4 ppm.

Experimental Example Reference Example 1 According to the method below, transformant strain E, coli K1
2YK537 (pTAL 522) was constructed.

pTA1529 (details of construction are in patent application No. 59-15970)
5 μq (see specification of No. 3) was added to 50 μm buffer solution (10
mMl-Tris-HCl M solution (hereinafter referred to as Tris-HCI) (
1) l-17,5), 10mM MgCl, 50mM
4 units of restriction enzyme 1-l ind in NaCl)
m (Takara) (Hereinafter, hydrolyzed using 1-1indI[I> at 37°C for 1 hour. Ethanol precipitation was then performed, and the resulting precipitate was added to 30 μρ of the reaction solution (67
mM Tris-FICl.

(pl-18,8), 16.6mM ammonium sulfate = 23- [hereinafter referred to as (Nhl) 804), 6.7mM ethylene diamine tetraacetate (hereinafter referred to as EDTA), 0.66mM
dATP, dCTP, dGTP, TTP]
1 unit of T4-DNA polymerase at 37°C.
Processed for 5 minutes. Next, the precipitate obtained by ethanol precipitation was added to a reaction solution (6mM Tris
-1-ICI (1)I-18,0), 6mM
Hydrolysis was carried out at 37°C for 1 hour using 4 units of the restriction enzyme 5alI [Takara] (hereinafter referred to as Sal I) in M (]C1, 150mM NaCl]. After the reaction was completed, it was analyzed by agarose gel electrophoresis. 3900bp D
An NA fragment (■ in Figure 1) was obtained.

Plasmid 1) BR322'-hUG (pBR322
(E, coli K12C600<1) BR'322
) has been deposited as EC0
5 μ3 of a fragment of the artificially synthesized hUG structural gene digested with RI and 3al and then digested with FCORI and Sa1 was added to the reaction mixture of 501 (
100mM-24 = Tris-HCl (pH 7,5), 50m
After hydrolysis at 37°C for 1 hour using 4 units of restriction enzyme EC0RI (Takara) in MNaCl, 50mM M(]Cl2), T4 DNA polymerase treatment was performed in the same manner as above, and further 3al■ treatment was performed. Later, 160bp DNA was extracted by agarose gel electrophoresis.
A fragment (■ in Figure 1) was obtained.

The two DNA fragments prepared above (■ and ■ in Figure 1)
), 30μβ reaction solution (20mM Tris = HCl
(pH 7,5), 10mM MgCl, 10mM
300 units of 74D in DTT, 0.5mM ATP)
The reaction was carried out at 14° C. for 16 hours using NA ligase (Takara).

After the reaction is completed, 12YK537 is transformed into Escherichia coli, and a transformed strain (E, coli K
12YK537 (pTA1522)] was obtained.

Reference Example 2 According to the method below, transformant E, coli K1
2YK537 (+)T△2532) was created.

pTA2539 (■ in Figure 2) 5 μq, 50 μ, 0
Oiling was performed at 37°C for 1 hour using 4 units of restriction enzyme NaeI (N, E, 8 companies) in a buffer solution (6mM Tris-HCl pH 8, 6mM tvloCI, 20mM NaCl). Next, ethanol precipitation was performed, and the resulting precipitate was further heated in the same manner as above.
After reacting with ndI[I4 unit, a DNA fragment of approximately 5oobp was recovered in the same manner as in Reference Example 1 (indicated by ■ in Figure 2).
).

On the other hand, pTA2539 5μ9 was treated with l-
1indI [[(Takara] After digestion, approximately 3800 bp D
It was recovered as an NA fragment (■ in Figure 2).

Roughly 5 μg of pB''R322-, hUG (as described above) (■ in Figure 2) was digested with EC0RI in the same manner as above, treated with T4 DNA polymerase, and then -8a
After the II digestion, approximately 160 bl of DNA fragments were recovered (■ in Figure 2).

Then, ■ was obtained above in the same manner as in Reference Example 1.

■ and ■ were ligated to construct pTA2532, and this was used to transform transformant E, coli K12 YK53.
7 (1) TA2532> was obtained.

Example 1 The microorganism E, coli K12 YK537 (pT
Al 522) was cultured as follows. Single cell pure isolation [:, coli K12YK537 (p
TΔ1522) - Platinum loops were inoculated into a medium 100- consisting of tryptone 10y/41, yeast extract 5Lj/n 1Na C15g/η, ampicillin 20mg/Jl,
Shaking culture was carried out overnight at 37°C in a 00-volume Sakalocolben.

Next, take 10 d of this culture solution, glucose 10 g/fJ, tryptone 10 g/fJ, yeast extract 5 y/fJXl.
v1g, so4-7H200, 5g/ft, and ampicillin 20my/fJ.
It was cultured in a mini-jar fermenter with a volume of p. The culture temperature was 37°C, and the ventilation a was 0.5 vvm (i vvm was 1
Volume-volume-minute refers to the volume of culture fluid per minute. This means that 1 p of air is introduced per 1 liter. ), l-1 is 4N
The concentration of dissolved oxygen (Do) was adjusted to 7.2 with NaOH and 4Nl-ICI, and the concentration of dissolved oxygen (Do) was maintained around 4 ppm by changing the stirring speed using a Do control device. Cultivation consists of quantitative determination of glucose and phosphorous, measurement of viable bacterial count, h
By measuring the amount of UG produced over time, hUG
The process was continued until the production amount reached the maximum. Quantification of glucose was carried out according to the glucose oxidase method using Kira 1- from uglucostat "Fujisawa"). The number of viable bacteria was measured using L-medium containing ampicillin 20/j (tryptone 1
0g/, I11 yeast extract'), 59/f: I, Na
This was done by measuring the number of colonies that appeared on an agar plate of C+5g/F). 0D66o was measured as an indicator of the amount of bacterial cells, and inorganic phosphorus was determined by the Motsupu starch method (described above). Quantification of htJG was performed by the following method. That is, 10 Id of the culture solution was taken, centrifuged to collect the bacteria, and the bacteria were mixed with 20% Shunicrose and 0.02M Tris-HCl (pH 8,0
), EDTAo, 001M solution 20#Il! The mixture was suspended in water and left at room temperature for 10 minutes. Then, the cells were collected by centrifugation again, and the cells were suspended in 20 d of cold water (0° C. to 4° C.) and left in water for 10 minutes. This suspension was then centrifuged to obtain a supernatant, which was analyzed by radioreceptor assay (RRA). The RRA method first uses KB cells derived from human nasopharyngeal carcinoma cells (AT CC No.
, CCL 17 ) was cultured in a monolayer in an 800 d flask in Dulbecco's modified Eagle (DME) medium [Japan]. The medium was then removed and the cells were detached using phosphate balanced salt solution (PBS) containing 0.05% EDTA to prepare a cell suspension. Then 20mM H
Cells were washed twice with lunks balanced salt solution (1-IB88) containing Epes (pH 7,4). 3 cells
indin.

5oluNon (DME medium 20mM Hol
leS(1)t717.4)-0.35g/, 1lNa
After FIA, the number of cells was calculated to be 300,000 to 400,000/0.
, 2 m B inding solution, and dispensed into tubes in 0.2 m increments. 0.2 d of sample solution containing various concentrations of hUG and -mEGF (mouse epidermal growth factor (hereinafter referred to as mEGF)) was added to the tube and incubated at 37°C for 1 hour. After washing the cells three times with ice-cold HBSS, they are suspended in 10% trichloroacetic acid (hereinafter referred to as TOA) and fixed using a glass filter. After removing TC△ with acelin, it was counted using a liquid scintillation counter. Then, 8 of hUG was converted to mEGF.

The culture time, OD, number of viable bacteria, amount of inorganic phosphorus,
Table 1 shows the changes over time in the amount of glucose and hUGo).

Table 1 *Number of cells resistant to ampicillin ** Could not be detected.

From this result, it seems that the culture time is preferably about 11 hours.

Example 2゜ Transformants E and coli were grown in the same manner as in Example 1 above except that ampicillin in the medium composition was changed to kanamycin.
K12 YK537(1)TA2532) was cultured. Culture time, number of viable 0D66o1 bacteria, amount of inorganic phosphorus, amount of glucose, and hUG = 31 − * Number of kanamycin-resistant cells ** Undetectable.

From this result, it seems that the culture time is preferably about 10 hours.

Example 3 Transformant E, coli K12YK537 (p
TA1522) into 3011 volumes of jar fermenter (
The actual volume was 20ρ). In addition, the medium at that time had the following amounts: glucose 30g/ρ, tryptone 20g/fl, yeast extract 10g/fJ M! ]S.O.
4.7H2'''0 1. Oql, Q, and the inoculation amount of bacteria was 100me (single cell pure isolated culture of E, coli
Kl 2YK537 (1) TAL 522) - platinum looper with trypone 1~10g/ρ, yeast extract 5g/n, Na
Cl 5g/, 11. Ampicillin 20mg/4
Culture was carried out under the same conditions and in the same manner as in Example 1 above, except that the culture medium was inoculated into 100 d of a medium consisting of 100 d of Sakalocolben, and cultured overnight with shaking in 500 d of Sakalocolben. Table 3 shows the changes over time in the culture time, OD, number of viable bacteria, amount of inorganic phosphorus, amount of glucose, and amount of hUG.

Table 3 *Ampicillin resistant; could not be detected at 3.5 times As a result, a culture time of about 11R seems to be preferable.

Mycological properties and accession numbers of related microorganisms The mycological properties and accession numbers of the microorganisms disclosed in the present invention are as follows.

Entrustment date (1) June 9, 1981 (2) April 30, 1981 (3) June 9, 1981 (4) November 14, 1980 *Ministry of International Trade and Industry Industrial Technology Accession number of the National Institute of Microbiology and Technology Mycological Properties (1) F. coliKl 2C600 This bacterium is a Durham-negative bacillus, does not produce spores, and exhibits the general attributes of the genus Escherichia coli, such as being facultatively anaerobic. It does not contain the F factor, lacks the function of the suppressor gene, and has a defect in the recBC gene, which encodes a nuclease involved in genetic recombination. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. Furthermore, taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli. The literature related to Mokukan is as follows.

(A), Genetics, 39,440 (B)
, Nature, 217.1110(2) E. coli
Kl 2C600 (pYK283> This bacterium is a Durham-negative bacillus, does not produce spores, has the general attributes of the genus Bold, such as facultative anaerobic properties, and does not contain the F factor, lacks the function of the suppressor gene E, and has the gene It has a defect in the recBG gene, which encodes a nuclease involved in recombination.

As an auxotroph, it requires threonine and leucine for growth on its minimal medium. The promoter-operator region of the phoAI gene, the replication initiation region from pACYC177 and the bla 3 of pBR322! [It contains the plasmid pYK283, which is composed of the gene, and shows resistance to ampicillin.]

Furthermore, taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli.

In addition, except for the traits derived from plasmid pYK283, the mycological properties of this strain are similar to that of its parent strain E, coli Kl 2C.
It is the same as that of 600.

(3) E, coli Kl 2C600 (pBR322)
This bacterium is a Durum-negative rod, does not produce spores, has the general attributes of the genus Bold, such as being facultatively anaerobic, does not contain the E factor, lacks the function of the suppressor gene E, and has a nuclease involved in genetic recombination. It has a defect in the recBci gene encoding. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. It also contains drug resistance plasmid I) BR322. In addition, regarding plasmid I) BR322, Gene, 2
95 (1977'), NatUre supra for 12C600 in E. coli.

Except for the traits derived from pBR322, E. coli K-3
8 = The mycological properties of 12C600 (pf3R322) are the same as those of the parent strain.

(4) E. coliK 12YK537 Escherichia coli 1
2YK537 is a known strain of Escherichia coli with 12 strains (Hicrobiological Reviews
.

±A, 1-56 (1980)) derivatives of E. coli 12
RR1 (Gene, 2.95 (1977), Bioc
hem, Biophys, Acta, , 655,
243 (1981)), and exhibits the following properties, and is a strain that is no different from that of 12RR1 in terms of its characteristics.

(rec A’1, Pho A8, pro”)

[Brief explanation of drawings]

FIG. 1 is a flow chart for the construction of plasmid pTΔ1522 used to transform [:, coli K12Y537. FIG. 2 is a flow chart of the construction of plasmid pTA25'32 used to transform E. coli K12Y537. Engineering AC

Claims (1)

[Claims] 1. A microorganism transformed with a chimeric plasmid constructed by incorporating a gene encoding a foreign protein under the control of a plasmid containing an alkaline phosphatase-derived promoter is transformed into a microorganism in the late logarithmic growth phase during the growth process of the microorganism. 1. A method for culturing a microorganism, which comprises culturing in a medium containing an amount of inorganic phosphorus necessary for induction of protein synthesis ability from to early arrest phase.