JPH0455671B2 - - Google Patents

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 gene of interest obtained from animals, plants, microorganisms, etc. has been introduced, and producing a gene product of interest in large quantities. [Background of the Invention] It is known that genes of certain complex plasmids are expressed by adding an inducer during culture. For example, "Nature" vol.291, 503
See page 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 Escherichia 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. [Object 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 involves culturing a microorganism that maintains in its cells a complex plasmid consisting of a target gene, a vector, and a promoter, and has the ability to express the target gene.
When expressing the target gene and collecting its products, the change in dissolved oxygen (DO) concentration during culture is used as an indicator, and an inducer and a nutrient are added at the same time. In the present invention, as a specific example showing the culture control method,
The trp promoter is used as a promoter, and β-
A culture control method using E. coli carrying a complex plasmid in which gal (β-galactosidase) gene is linked is shown. Figure 1 shows the structure of the composite plasmid pTREZ1 in which the trp promoter and β-gal gene are linked. trp
The promoter part is the promoter of the E. coli trp operon, and includes a portion of the tip of trpL (soda peptide) and trpE (anthranilate synthase).
It is a DNA fragment of 500bp (base pairs),
This was inserted into the EcoRI site of the pBR322 plasmid. On the other hand, the β-gal gene is expressed in pMC1403 (J.Ba-
cteriol.vol.143, pp. 971-980, 1980), with a size of 6.2 kb, which contains the trp promoter.
It was inserted between the EcoRI site and the SalI site of pBR322. The composite plasmid pTREZ1 with the structure described above was transferred to E. coli M182, a strain lacking the β-gal gene on the chromosome.
A genetically modified bacterium that produces β-gal was created by introducing it into a strain (Biko Kenkyuken No. 7650). β-gal production is under the control of the trp promoter, and the function of the promoter was effectively used to improve β-gal production. It is known that the gene of a complex plasmid with a trp promoter is expressed by adding IA (3-β-idole acrylic acid) during culture (Nature, vol. 291, pp. 503-506, 1981 ).
This is because the repressor that suppresses gene transcription is IA.
This is because mRNA synthesis by RNA polymerase is initiated. In order to efficiently produce β-gal using a genetically modified bacterium that produces β-gal, the present inventors
After various studies on culture control methods, the inventors succeeded in producing large amounts of β-gal and 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, there are a method of adding IA at the first hour of culture as described in the above-mentioned literature, and a method of using the bacterial cell concentration as an index (Japanese Patent Application Laid-open No. 141796/1983). The present inventors have discovered a method of adding IA using changes in DO concentration as an indicator, in order to enable addition of IA suitable for the physiologically active state of bacterial cells in contrast to these methods. As the bacteria grow while keeping the stirring speed and aeration constant, the DO concentration decreases, and the substrate glucose concentration also decreases due to consumption by the bacteria. Next, when the substrate is consumed, the DO
rises. At this point, by simultaneously adding nutrients that serve as synthesis materials for IA and β-gal, β-gal can be synthesized.
-gal is produced in large quantities. In other words, by adding IA and nutrients when bacterial cell growth due to substrate consumption has stopped, β-gal production can be started at the maximum bacterial cell concentration. The optimal amount of IA added in the present invention was 15 μg/ml as shown in FIG. From now on, 15μg/
It is considered that it is sufficient to add about ml of IA during culture. In addition, nutritional substances added at the same time as IA should be added first.
As shown in the table, casamino acids were effective, and glucose decreased the production of β-gal. Since casamino acids are a mixture of amino acids, 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.

[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 M182 strain harboring complex plasmid pTREZ1. Medium: M9-casamino acid medium, composition is NH ₄ Cl1
g, _Na2HPO46g , _KH2PO43g , _{NaCl5g ,}
MgSO ₄・7H ₂ O 0.1 g, CaCl ₂・2H ₂ O 15 mg, glucose 5 g, casamino acid 2.5 g, distilled water 1, PH
It is 7.0. Note that ampicillin (Ap) was added at 50 μg/ml to this medium in order to proliferate only E. coli carrying the complex plasmid. Culture conditions: E. coli carrying complex plasmid
The mixture was inoculated into 20 500 ml shaking flasks containing 100 ml of M9-casamino acid medium, and cultured overnight at 37°C using a shaking incubator at an amplitude of 7 cm and shaking 115 times/min. The cultured cells were collected by centrifugation and suspended in 50 ml of M9-casamino acid medium. This was used as a seed and inoculated into a 5-jar fermenter containing 2-9-casamino acid medium to start culturing. At this time, two drops of Leocon 1705W (manufactured by Lion) were added as an antifoaming agent. Culture at 37℃ and PH
7.2, the rotation speed of the stirrer was 800 rpm, and the aeration rate was 2/min for 6 hours. Results: As shown in FIG. 4, the DO concentration rapidly increased after 1.5 hours of culture, so IA and casamino acids were added at 15 μg/ml and 2.5 mg/ml, respectively.
β-gal production when IA and casamino acids are added is
The concentration was 16.6U/ml, but at the 6th hour of culture, the concentration was 1.3 times higher.
It improved to 21.8U/ml. After the addition of IA and casamino acids, the bacterial cell concentration hardly increased, and it is thought that most of the added casamino acids were consumed for β-gal production. The above results revealed that β-gal production can be significantly improved by adding IA and casamino acids using DO concentration as an indicator. That is, in the embodiment of the present invention shown in FIG. 4, a sudden increase in DO can be easily detected because DO is rising and is above a predetermined value, or because the amount of change in increase in DO is above a predetermined value. It can be detected. Example 2 Strain: Same as Example 1. Medium: Same as Example 1. Culture conditions: 2.5 casamino acids when DO concentration increases
Same as Example 1 except that mg/ml was added. Results: As shown in Figure 5, the DO concentration rapidly increased at 1.5 hours of culture, so casamino acids were added.
2.5 mg/ml was added. In this case, the β-gal production amount and bacterial cell concentration hardly increased after the addition of casamino acids. Therefore, IA is necessary to improve β-gal production.
It was found that the addition of [Effects 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 the drawing]

Figure 1 is a structural diagram of the composite plasmid pTREZ1.
Figure 2 is a characteristic diagram showing the relationship between the amount of IA added and the amount of β-gal produced, Figure 3 is a schematic diagram of an example of the culture device of the present invention, and Figure 4 is a diagram showing the relationship between IA and casamino acids using DO concentration as an index. FIG. 5 is a characteristic diagram showing the addition experiment of casamino acid using DO concentration as an index. 1... Culture tank, 2... Stirrer, 3... Control electronic computer, 4... Substrate tank, 5... Addition tank, 6, 7
... Metering pump, 8, 8 ... Conduit, 10 ... Dissolved oxygen meter, 11 ... Dissolved oxygen sensor, 12, 13 ...
…conduit.

Claims (1)

[Scope of Claims] 1. A method for 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 a change in dissolved oxygen concentration of a culture solution during culture is used as an indicator, and when the dissolved oxygen concentration increases rapidly, an inducer and a nutrient are added almost simultaneously. 2. The method for controlling the culture of genetically modified bacteria according to claim 1, wherein the promoter is a trp (tryptophan) promoter. 3. A method for controlling the culture of genetically modified bacteria according to claim 1, wherein the inducer is IA (3-β-indoleacrylic acid). 4. The method for controlling the culture of genetically modified bacteria according to claim 1, wherein the nutritional substance is casamino acid. 5. The method for controlling the culture of a genetically modified bacterium according to claim 1, wherein the microorganism carrying the complex plasmid is Escherichia coli.