KR920007396B1 - Method for culture of microorganism - Google Patents

Abstract

A cattle growth hormone is prepd. by fermenting E. coli, LUCK- BST-1E KFCC 10693 (I) in the medium contg. sorbitol or mannitol as carbon source. The strain (I) has Trp, Lac, Tac or PL promotor as expression vector. The acetic acid prodn. during the fermentaion is inhibited by adding adding sorbitol or mannitol as carbon source into the culture medium. The cattle growth hormone is obtd. in high yield by inhibiting acetic acid prodn. in the fermentation.

Description

Inhibition of acetic acid production in microbial culture

1 is a graph showing the effect of acetic acid on the specific growth rate of recombinant E. coli,

Figure 2 shows the effect of acetic acid on the expression of protein by SDS-PAGE,

Figure 3 shows the expression level of the protein according to the carbon source by SDS-PAGE,

4 is a graph showing the growth curve and the amount of acetic acid produced by recombinant E. coli when glucose was used as a carbon source.

5 shows the expression level of protein using SDS-PAGE when glucose is used as a carbon source.

6 is a graph showing the growth curve and the amount of acetic acid produced by recombinant E. coli when sorbitol is used as a carbon source,

Figure 7 shows the expression level of the protein when using sorbitol as a carbon source by SDS-PAGE.

The present invention relates to a method of inhibiting acetic acid production in culturing microorganisms, and more particularly, to inhibit acetic acid production as much as possible in culturing recombinant microorganisms capable of producing useful substances such as enzymes or other bioactive substances. It relates to a cultivation method to be able to produce effectively.

Conventionally, as a method for culturing recombinant microorganisms to produce bioactive substances, such as enzymes, antibiotics, amino acids, hormones, etc., batch culture is usually performed by simply adding a medium, but the productivity is low. There was a downside.

Since then, methods such as fed-batch culture or addition of inducers have been used to increase productivity. However, the fed-batch culture method has a problem that it is difficult to produce the material to be obtained by the production of by-products during the culture is not only suppressed the production of useful substances but also the growth of microorganisms. The fact that the production of by-products in Escherichia coli culture significantly reduces the growth of microorganisms and protein synthesis. Technol., Vol. 66, No. 2,187-191 (1988). Among these by-products, acetic acid has been reported to have a strong inhibitory effect on the intake of L-serine and other amino acids [J. Bacteriol., Vol. 8, 525-530 (1972)], when the concentration of acetic acid is more than 0.1M it is reported that the growth rate of the cells is sharply reduced and the cell growth is stopped at 0.15M [Mikrobiologiya 54, 252 (1985)].

As described above, acetic acid inhibits the production of useful proteins by reducing protein synthesis in the case of recombinant Escherichia coli, and many studies have been conducted to develop a microorganism culturing method that can minimize the production of acetic acid. In particular, the effect of acetic acid is more problematic in high-density cell cultures, such as fed-batch cultures, and in the mass production of enzymes or other bioactive substances than in batch cultures.

To solve this problem, dialysis culture [J. Gen. Microbiol., 103,345 (1977)], Cultured by acetic acid monitoring method [J. Ferm. Tech., Vo1. 66, No. 2, 187-191 (1988)], a culture method for adjusting the dissolved oxygen concentration [Biotech, Letters, Vo1. 11 3,155-160 (1989)], the addition method by the carbon source concentration gradient [Biokphorm, No. 11 41 (1987)] and the like to try to maximize the production of protein and enzyme by minimizing the production of acetic acid as a by-product Came, but no obvious results.

Accordingly, the present inventors have intensively researched to develop a culture method for effectively producing useful substances such as physiologically active substances by maximally inhibiting the production of acetic acid in culturing microorganisms and plant animal cells. In particular, the use of several kinds of sugar alcohols as a carbon source significantly lowers the production of acetic acid even at high concentrations, which is very useful for producing useful proteins such as enzymes and other bioactive substances by culturing microorganisms at high concentrations. The development of the present invention has led to the completion of the present invention.

That is, an object of the present invention is to provide a method for culturing microorganisms which can maximize the production of useful substances such as protein or other bioactive substances by minimizing the production of acetic acid.

Hereinafter, the present invention will be described in detail.

The present invention is to provide a method for minimizing acetic acid production, characterized in that sorbitol or mannitol as a carbon source of the embryo medium in the culture of the microorganism.

Such a present invention is suitable for application to recombinant microorganisms, in particular, recombinant Escherichia coli, and particularly, for application to culture of microorganisms having a Trp, Lac, Tac or pL promoter as an expression vector.

In addition, the present invention can be particularly preferably applied to high concentration culture such as fed-batch culture of recombinant microorganisms.

In addition, the method for minimizing acetic acid production of the present invention may or may not use an inducer.

Referring to the culture method of the microorganism according to the method for minimizing acetic acid production of the present invention in more detail as follows.

[I. microbe]

The method for culturing microorganisms according to the present invention can be widely applied to the cultivation of various microorganisms, plants and animal cells for the purpose of producing useful proteins such as enzymes or other physiologically active substances. It is preferred for application to microorganisms having Trp, Lac, Tac or pL promoters.

In the following experiment to prove the present invention, E. coli LUCK-BST-1E deposited by Korean Applicant No. 10693 dated May 25, 1990 to the Korean spawn association, which was incorporated by the applicant.

Escherichia coli LUCK-BST-1E [KFCC-10693] has a hybrid plasmid (ptrp hs BGH) in which a Trp promoter and a calcined enterophore gene are combined. This hybrid plasmid (ptrp hs BGH) was treated with pSOD, the final E. coli expression carrier, using the restriction enzymes NcoI and SalI to isolate the genes of the entire plastic field hormone conjugated with the artificial synthesis linker, which was then transferred to the E. coli expression carrier (ptrp 322H SGH). Recloned vehicle (ptrp hs BGH 1-13). Here, pSOD, the final E. coli expression vector carrier, was prepared by cloning a SalI-SalI fragment of a plastic field hormone of about 526 base pairs in a carrier pSOD treated with NcoI and SalI together with an artificial synthetic linker.

[II. Effect of Acetic Acid on Growth and Expression of Microorganisms]

In order to investigate the effect of acetic acid on the growth and expression of microorganisms, it was realized as follows.

In 1 liter of distilled water, 1 g of chlorinated chloride, 3 g of calcium phosphate, 7 g of potassium diphosphate, 0.5 g of sodium chloride, 0.1 g of magnesium sulfide, 0.015 g of calcium chloride, 4 g of casamino acid, and 10 g of sorbitol were dissolved and the pH was adjusted to 7.0 to 7.2. Amino acid medium was prepared. Ampicillin was added to the M-9 casamino acid medium at a concentration of 50 μg / ml for selection.

50 ml of the medium was added to a 250 ml shaker incubator, and subjected to E. coli LUCK-BST-1E [KFCC-10693], followed by incubation at 37 ° C. under a rotation speed of 120 rpm and a rotation radius of 7 cm.

To determine the effect of acetic acid on the specific growth rate of E. coli, the specific growth rate was measured by adding acetic acid to the culture medium after 2 hours of inoculation of E. coli. The results are shown in FIG. As shown in FIG. 1, after adding acetic acid for each concentration, the specific growth rate was measured. As the concentration of acetic acid increased, the non-proliferation rate rapidly decreased, and as the concentration of acetic acid became more than 1%, It was reduced to about 1/4 or less compared with when acetic acid was not added.

On the other hand, in order to determine the effect of acetic acid on the expression of foreign proteins, E. coli LUCK-BST-1E [KFCC-10693] was incubated overnight in the same manner as above and then added acetic acid to the culture medium by concentration, calcined hormone The expression level of was confirmed by SDS-PAGE (sodium dodecyl sulfite-electrophoresis). The results are shown in FIG. In FIG. 2, the first lane is a control group without adding acetic acid, and the result of adding acetic acid at concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0% from lanes 2 to 8, respectively. As shown in FIG. 2, when the concentration of acetic acid reached 1%, the synthesis of calcined hormone was sharply decreased compared to the control, and the synthesis of calcined hormone was completely inhibited at 1.5% or more.

As can be seen from FIG. 1 and FIG. 2, the concentration at which the growth of recombinant E. coli cells is inhibited and the concentration at which the expression of foreign proteins are suppressed are almost the same.

As a result of analyzing the E. coli culture by gas chromatography to assay a substance that inhibits cell growth and protein synthesis, it was found that the inhibitor was acetic acid.

From the above results, it can be seen that acetic acid has a significant effect on inhibiting protein synthesis as well as the specific growth rate of recombinant E. coli.

[III. Selection of Carbon Sources in Culture Medium]

In order to select a carbon source in the microbial culture medium was tested as follows.

Escherichia coli LUCK-BST-1E [KFCC-10693] was incubated in the same manner as above, except that the type of carbon source was different. As the carbon source, grape net, glycerol, mannose, fructose, sorbitol and the like were used.

Acetic acid concentration in each of the resulting cultures was measured, and the results are shown in Table 1 below. The degree of expression of the plastic window homologue was shown in FIG. 3 by SDS-PAGE. In FIG. 3, the first lane is glucose, the second lane is fructose, the third lane is sorbitol, the fourth lane is glycerol, and the fifth lane is mannose.

According to Tables 1 and 3, the production of acetic acid was much higher in glucose than in other carbon sources, and the expression of calcined hormone was much lower than in other carbon sources, whereas in the case of fructose, sorbitol, mannose, and glycerol, Acetic acid production was lower and protein expression was relatively higher. Particularly, among these carbon sources, sorbitol hardly accumulated acetic acid and the expression rate of calcined hormone was relatively higher.

Table 1. Comparison of Acetic Acid Production by Carbon Sources

In the above experiment, sorbitol was described mainly, but mannitol having a similar structure and properties to sorbitol is also suitable for use as a carbon source of culture medium according to the present invention.

[IV. Feeding Cultivation and Addition of Inducers]

When applying fed-batch culture and culturing with the addition of inducers, the following experiments were performed to compare the production of acetic acid, cell growth, and protein expression using glucose and sorbitol as carbon sources.

200 ml of LB (Luria broth) medium was added to a 500 ml shake incubator, inoculated with E. coli LUCK-BST-1E [KFCC-10693], and the temperature was 37 ° C, the rotation speed was 120 times / min, and the rotation radius was 7 cm. Incubated overnight under the conditions of. The cultured LB medium was inoculated in a 5 L incubator containing M-P casamino acid medium (containing 50 µg / ml ampicillin) containing glucose as a carbon source. At this time, the inoculated culture solution was 2ℓ, and the organic culture medium (glucose 200g, cassanoic acid 100g, distilled water 1L) was added intermittently under the culture conditions of culture temperature 37 ℃, pH 7.0, air injection speed 2ℓ / min, rotation speed 700rpm The culture was carried out while the absorbance of the culture solution was 40 at 660 nm, and IAA (2-β-indolyl acrylic acid) was added as an inducer.

The concentration of acetic acid produced in the culture was measured by gas chromatography (HEWLETT 5890A), and the results are shown in FIG. As shown in FIG. 4, when glucose is used as the carbon source of the culture medium, the production of acetic acid may increase rapidly with cell growth throughout the whole process. Due to the production of acetic acid, the growth rate is slowed down. As a result, the concentration of acetic acid became 1% or more at the time of induction, and thus no protein expression was achieved.

In addition, in order to determine the expression level of the protein in the culture, SDS-PAG after a certain time after induction, and the results are shown in FIG. In FIG. 5, the first, second and third lanes are the results when 1, 2 and 3 hours have elapsed after the induction, respectively. As shown in FIG. 5, when glucose was used as the carbon source of the culture medium, expression was hardly achieved.

On the other hand, Escherichia coli LUCK-BST-1E [KFCC-10693] was cultured in the same manner as described above except for using sorbitol instead of glucose as the carbon source of M-9 casamino acid medium and organic culture medium.

The concentration of acetic acid produced in the culture was measured by gas chromatography (HEWLETT 5890A), and the results are shown in FIG. In addition, SDS-PAGE is shown in Figure 7 after a certain time after the induction to determine the expression level of the protein. In FIG. 7, the first, second and third lanes are the results when 1 hour, 2 hours and 3 hours have elapsed after induction, respectively.

As shown in FIGS. 6 and 7, sorbitol hardly produced acetic acid even at high concentrations of cells, and at 660 nm, acetic acid began to produce acetic acid above 30, but much higher than glucose. The concentration was low, and the expression rate of phytopathogenic hormone was very high.

The method for culturing microorganisms according to the present invention is not only induced with IAA when the promoter of the microorganism is Trp, but also with IPTG (isopropyl β-D-thiolactopyranoside) for Tac or Lac promoters. It can also be applied to induce or increase the temperature as in the case of the pL promoter. Furthermore, it can be applied to other microorganisms, plants and animal cells which are affected by acetic acid production and production of bioactive substances.

Claims (6)

In culturing a recombinant microorganism to produce a bioactive substance, sorbitol or mannitol as a carbon source of the culture medium, characterized in that to minimize the production of acetic acid in the culture medium. The method of claim 1, wherein the recombinant microorganism is recombinant E. coli. The method of claim 2, wherein the recombinant E. coli is characterized in that it has a Trp, Lac, Tac or pL promoter as an expression vector. The method of claim 1, wherein the culture is a fed-batch culture. The method of claim 1, wherein the incubation is added to the culture medium or cultured without addition of the inducer. The method according to claim 1, wherein the bioactive substance is calcined hormone.

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