JPS6170982A - Cultivation control of microorganism with recombinant gene - Google Patents

Abstract

PURPOSE:The change in concentration of oxygen dissolved in the culture medium is used as an index, the induction substance and nutrients are simultaneously added to effect high-efficiency cultivation of a recombined microorganism. CONSTITUTION:The culture medium and the seed are charged in the cultivation tank 1 and the culture mixture is stirred with the air fed into the mixture from the pipe 12. D0 sensor 11 and D0 meter 10 are used to measure the D0 concentration in the culture medium and a signal is sent to the metering pumps 6 and 7, when the D0 concentration steeply rises, so that Casamino acid is sent from the substrate tank 4 and the inducer IA from the tank 5 to the cultivation tank 1 simultaneously in certain amounts, respectively. Then, cultivation is continued for a certain time, cell bodies are recovered from the medium and the useful substance such as beta-galactonedase produced in the cell bodies is isolated and purified.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to a method for efficiently culturing microorganisms into which a target gene obtained from animals, plants, microorganisms, etc. has been introduced, and producing a target gene product in large quantities. .

[Background of the invention]

It is known that genes of complex plasmids can be expressed by adding an inducer during culture. For example, Ding NatureJ vot, 291 +503
See p. 506, 11 June 1981.

Recently, genetic recombination technology has been developed that uses a host microorganism that carries a complex plasmid in which a gene having production information for a useful substance has been integrated into the vector plasmid of the host microorganism, and allows the microorganism to mass-produce the above-mentioned useful substances. Human interferon, insulin, etc. are already being produced using this technology, and E. coli is being used as a host microorganism.

However, a method for industrially producing a desired product in large quantities using genetically modified bacteria carrying the target gene has not yet been developed, and there is an urgent need to develop efficient cultivation methods for genetically modified bacteria. There is.

[Purpose of the invention]

An object of the present invention is to provide an efficient method for culturing genetically modified bacteria.

[Summary of the invention]

The present invention maintains a complex plasmid consisting of a target gene, a vector, and a promoter in cells, and cultivates a microorganism capable of expressing the target gene, and when expressing the target gene and collecting the product, Dissolved oxygen (Do
) The trick is to use the change in concentration as an indicator and add the inducer and nutrients at the same time.

In the present invention, as a specific example showing the culture control method, TRP pro Tanabata is used as a promoter, and β-gat (β-
A method for controlling culture using E. coli carrying a complex plasmid in which the galactosidase (galactosidase) gene is linked is shown.

The structure of the composite plasmid p'rEZ1 in which the trp glomotor and the β-gat gene are linked is shown in FIG.

The trp promoter part is the promoter of the E. coli trp operon, trpL (soda peptide) and trpE (
Approximately 5 including part of the tip of anthranilate synthase)
Q Q b p (base pairs), a DNA fragment of the pBR322 plasmid E
It was inserted into the aoRI site.

On the other hand, the β-gat gene was expressed in pMC1403 (J, Ba
-cteriol, vot, 143+ 971~9
80, 1980)] with a size of 6.2 kb and was inserted between the BcoRI site of the trp promoter and the 5atI site of pBR322.

The composite plasmid pTREZl having the structure described above was introduced into Escherichia coli M2B5 strain, which is a chromosomal β-gat gene-deficient strain, to create a genetically modified bacterium that produces β-gat (Feikoken No. 7650). . β-gat production is tr
It is under the control of the p promoter and effectively utilizes the function of the promoter to improve β-gal production.

The complex plasmid containing the trp promoter is
It is known that genes are expressed by adding A (3-β-indole acrylic acid) (Natural
re, vot, 291, pp. 503-506, 19
81). This suppresses the transcription of the gene or pretuna I
This is because since it is inactivated by A, synthesis of mRNA by RNA polymerase is started.

In order to efficiently produce β-gat using a genetically modified bacterium that produces β-gaL, the present inventors investigated various culture control methods and succeeded in producing large amounts of β-gat. , we have completed the present invention.

The trp promoter starts working when the inducer IA is added, and determining the timing of its addition is extremely important for producing the target gene product. As for the timing of IA addition, the method of adding IA at the first hour of culture as in the above-mentioned literature, or the method of using the bacterial cell concentration as an index (Japanese Patent Application Laid-open No. 58-1417
Publication No. 96). The present inventors have discovered a method of adding IA as a DOa ratio change indicator in order to enable the addition of IA suitable for the live kite activity state of the bacterial cells to these methods. After culturing for 10 days with the stirrer rotation speed and aeration rate constant, the DOTIk degree decreases as the bacterial cells proliferate, and the glucose concentration of the substrate also decreases due to consumption by the bacteria. Next, when one substrate is consumed, DO suddenly
rises. At this point, by simultaneously adding IA and nutritional substances that are the synthesis materials for β-gat,
is produced in large quantities. By adding IA and nutrients at the 9th point after the bacterial cell growth due to consumption of the substrate has stopped, β-gat production can be started at the maximum bacterial cell concentration.

The optimum IA addition amount in the present invention is 1 as shown in Figure 2.
5μg/- is a good idea. From now on, about 15μg/- IA
It is considered that it is sufficient to add it during culture. Furthermore, as shown in Table 1, casamino acids were effective as nutritional substances added at the same time as IA, and glucose decreased the production of β-gat. Casamino acids are a mixture of amino acids, so
Similar results can be obtained by adding amino acids other than tryptophan or mixtures thereof. The larger the amount of casamino acid added, the higher the production amount, but the culture time needs to be longer.

Table 1 In the present invention, the case of a complex plasmid having a trp promoter has been explained, but the inducer is IPTG (isopr
opyl-β-]) -thiogalactosid
The same can be applied to the case of adding e) to the taC promoter.

The above-mentioned culture control method (method of improving gene product production by simultaneously adding an inducer and a nutrient when the M1 degree increases) is not known for culturing genetically modified bacteria or normal microorganisms. First, an example of the apparatus necessary to carry out the method of the present invention, which was discovered for the first time by the present inventors, is shown in Fig. 3.A culture medium and seed bacteria are placed in a culture tank 1, and cultured through a conduit 12. Genetically modified bacteria are cultured while blowing air into tank 1 and stirring the culture solution using stirrer 2. At this time, the dissolved oxygen concentration of the culture solution is measured using the dissolved oxygen sensor 11 and dissolved oxygen meter 10 installed in the culture tank 1. The measured data is sent to the control electronic computer 3, and changes in dissolved oxygen concentration are monitored. When the dissolved oxygen concentration rapidly increases, a signal is sent to the metering pumps 6 and 7, and a constant amount of casamino acid from the substrate tank 4 and IA from the addition tank 5 are simultaneously supplied to the culture tank 1. After that, culture for a certain period of time,
Collect the bacterial cells in the culture solution and collect the β-ga produced within the bacterial cells.
Products are produced by separating and purifying useful substances such as t. Reference numerals 8 and 9 are conduits.

[Embodiments of the invention]

Next, examples of the present invention will be specifically described, but the present invention is not limited thereto.

Example 1 Bacterial cell: Escherichia coli M carrying complex plasmid pTREZ1
2B5 stock.

Medium: M9-casamino acid medium, composition: NH4CLl g
, NazHPO+ 6 g, KHzPO43g +
NaC45g, MgSO4・7H*OO, Ig
, CaCtz 2 Hz 015 mg, glucose 5 g, casamino acid 2.5 g, distilled water 1 t, pH 7.0. In addition, in order to grow only E. coli carrying the complex plasmid, this medium contains 50% ampirin (Ap).
Added μg/at.

Culture conditions: 1 large 'I31 bacterium carrying the complex plasmid
The inoculation was carried out in 20 500-shake flasks containing 00 Go's M9-casamino acid medium, using a shaking culture machine at an amplitude of 7 cm and a number of shakes of 115 times/m.

Cultured overnight at 37C. The cultured cells were collected by centrifugation and suspended in 50 d of M9-casamino acid medium. This was used as a starter and 2 tons of M9-casamino acid medium was added.
The cells were inoculated into J. 7 Armenta and culture was started. At this time, as an antifoaming agent, use 2 times of Rheocon 1705W (manufactured by Lion).
Added dropwise. The culture was carried out at a temperature of 37C, a pH of 7.2, and a stirrer rotation speed of 80 rpm.

The test was carried out for 6 hours at an air flow rate of 2t7.

Results: As shown in Figure 4, DO
Since the concentration increased rapidly, 15 μg/- and Z5 mg/- of EnA and casamino acid were added, respectively. The β-gat production amount when IA and casamino acids were added was 16.6 U/-, but it increased 1.3 times to 21.8 U/Wt after 6 hours of culture. After adding IA and casamino acids, the bacterial cell concentration hardly increased, and most of the added casamino acids were β-gat.
It is thought that it was consumed for production.

From the above results, it was revealed that β-gat production can be significantly improved by adding IA and casamino acid using DO concentration as an indicator.

Example 2 Strain: Same as Example 1.

Medium: Same as Example 1.

Culture conditions: 25 mg/- of casamino acids when DO concentration increases
Same as Example 1 except that it was added.

Results: As shown in Figure 5, DO
Since the concentration increased rapidly, casamino acids were added at Z5mg/d.
Added. In this case, the β-gat production amount and bacterial cell concentration hardly increased after addition of Kazami and noic acids. From this, it was found that addition of IA was necessary to improve β-gal production.

〔Effect of the invention〕

As explained above, according to the culture control method of the present invention, a target gene can be efficiently produced.

[Brief explanation of drawings]

Figure 1 is a structural diagram of the composite plasmid pTREZ 1, Figure 2
The figure is a characteristic diagram showing the relationship between IA addition amount and β-gal production amount, Figure 3 is a schematic diagram of an example of the culture device of the present invention, and Figure 4 is D
FIG. 5 is a characteristic diagram showing the addition experiment of IA and casamino acid using the DOa concentration as an index, and FIG. DESCRIPTION OF SYMBOLS 1...Culture tank, 2...Agitator, 3...Control computer, 4...Substrate tank, 5...Addition tank, 6.7...
- Metering pump, 8.8... Conduit, lO... Dissolved oxygen meter, 11... Dissolved oxygen sensor, 12.13... Conduit.

Claims (1)

[Claims] 1. A method of retaining a complex plasmid consisting of a target gene, a vector, and a promoter in cells, culturing a microorganism capable of expressing the target gene, expressing the target gene, and collecting the product. A method for controlling the culture of genetically modified bacteria, characterized in that an inducer and a nutrient are added at the same time using changes in dissolved oxygen concentration during culture as an indicator. 2. A method for controlling the culture of genetically modified bacteria according to claim 1, wherein the promoter is a trp (tryptophan) promoter. 3. In claim 1, the inducer is I
A (3-β-indole acrylic acid), a method for controlling the culture of a genetically modified bacterium. 4. A method for controlling the culture of genetically modified bacteria according to claim 1, wherein the nutritional substance is casamino acid. 5. In claim 1, the microorganism carrying the complex plasmid is Escherichia coli (Escherichia coli).
A method for controlling the culture of a genetically modified bacterium, characterized in that it is a genetically modified bacterium (coli).