KR0174830B1 - Microorganism producing l-tryptophan - Google Patents

Abstract

The present invention relates to Escherichia coli CJ 18 (KFCC 10902) capable of accumulating L-tryptophan at high concentrations while minimizing the accumulation of acetic acid in a culture medium, wherein the strain is resistant to tryptophan analogues, The parent strain was obtained by mutating the parent strain using E. coli CA 15 (KFCC 10861), which is a tryptophan producing strain susceptible to L-arginine, as a parent strain.

The present invention also relates to a method for producing L-tryptophan by accumulating E. coli CJ 18 (KFCC 10902) in a culture medium containing glucose directly by fermentation.

Description

Microorganisms Producing L-Tryptophan

In the present invention, Escherichia coli KFCC 10861, which is resistant to tryptophan analogues, has nutritional composition to L-tyrosine, which is a kind of aromatic amino acid, and susceptibility to arginine, As a microorganism selected by an artificial mutant method, the present invention relates to a microorganism that produces tryptophan while suppressing accumulation of acetic acid in a culture medium during fermentation. The present invention also relates to a method for producing L-tryptophan in which L-tryptophan is accumulated in a culture medium by culturing in a medium containing glucose in which acetic acid production is inhibited by direct fermentation.

Tryptophan is one of the essential amino acids, and is widely used as an amino acid solution because of its high price compared to its wide range of use as food and feed additives and pharmaceutical raw materials. However, tryptophan, along with methionine and lysine, has proved to be very effective as a feed additive, so the demand for this is rapidly increasing as it has been recently supplied at a low price.

Tryptophan production methods include chemical synthesis, enzyme synthesis, precursor fermentation, and direct fermentation.However, after 1990, the introduction of biotechnology led to the development of high titer strains, which produced production by direct fermentation, which is more competitive than other processes. ought. In the case of chemical synthesis, high-temperature, high-pressure manufacturing conditions are required, and D-type tryptophan is produced as a by-product, and D-type tryptophan has a problem in that it is not easy to be separated in the purification process with L-type tryptophan having biological value. In the case of the enzyme synthesis method, L-tryptophan is produced by reacting indole and serine in the reaction mixture using an enzyme as in the patent of Mitsui Toatsu, Japan (Korean Patent Publication 90-5773), but the prices of substrates indole and serine It is higher than that and increases the production cost due to the stability of the enzyme.

Patents using direct fermentation methods published in Korea have been used to provide resistance to tryptophan analogs or to use mutant strains with nutritional composition (Korean Patent Publication No. 87-1813, 90-8251, 92-7405) and recombinant strains. It can be classified as a case (Korean notification number 90-5772, 91-5627).

As a method of using a mutant strain, a method of manufacturing L-tryptophan using E. coli mutant strain (KCTC 8164P) has been patented (Korea Patent Publication 87-1318), which is a tryptophan analog after inducing a mutant strain by UV treatment. Characterized by selecting a strain resistant to the final tryptophan production concentration was 13.6g / l. In addition, L-tryptophan and L-varin analogues are resistant to L-phenylalanine and L-tyrosine, E. coli mutant strains (KCTC 8373P) using the direct fermentation method to produce L- tryptophan as a result of oil price Tryptophan up to 15.6 g / l was produced by the food culture method (Korean Patent Publication 90-8251). In addition, the present inventors have invented an innovative strain that produces tryptophan with a fermentation concentration of 20.4 g /, which has significantly increased productivity compared to the existing mutants by inventing a strain (E. coli KFCC 10861) that is sensitive to L-arginine, an amino acid. (Korean Patent Application No. 95-18728).

However, when a medium containing glucose is used to produce amino acids such as L-tryptophan by direct fermentation, acetic acid accumulates as a by-product in the fermentation broth unless the dissolved oxygen content in the fermentation broth is sufficiently increased. The biochemical mechanism of acetic acid accumulation is to enter the TCA cycle when glycolysis or excess intracellular pyrubinic acid produced by the PTS (phosphoenolpyruvate: suger phosphotransferase system) cannot normally supply oxygen to cells due to dissolved oxygen restriction in the culture medium. It is known that it is not oxidized and is released into the cell after being converted into acetic acid by the enzymes phosphotransacetylase and acetate kinase via acetyl CoA (J. Gen. Microbiol., 135, 2875). -2883, 1989). The acetic acid accumulated in the culture medium inhibits the growth of microorganisms and consequently decreases the productivity of the target amino acid. In order to prevent the accumulation of acetic acid, fermentation is performed by increasing the stirring speed or increasing the aeration rate during fermentation. Is not a fundamental solution and can be a problem in the process of industrialization.

An object of the present invention is to significantly increase the productivity of tryptophan by accumulating high concentrations of L-tryptophan in a culture medium of direct fermentation while inhibiting acetic acid accumulation using a medium containing glucose.

That is, the present invention can not significantly metabolize acetic acid can significantly reduce the accumulation of acetic acid in the culture medium during fermentation, as a result provides a new strain to significantly increase the tryptophan fermentation concentration and productivity, and also to the culture medium using such a strain Provided are methods for accumulating high concentrations of L-tryptophan.

The present invention is described in detail as follows.

A strain which cannot metabolize acetic acid according to the present invention is obtained by the following method.

Tryptophan-producing strains (E. coli KFCC 10861) that are resistant to tryptophan analogs and nutritional to tyrosine and susceptible to arginine (E. coli KFCC 10861) are referred to as N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as NTG). After treatment, the cells are cultured in a minimal medium containing glucose. When cultured cells are transferred to a minimal medium containing acetic acid as the only carbon source, and then treated with ampicillin, ampicillin-treated metabolites are lysed by the action of the antibiotic ampicillin to obtain a strain that cannot metabolize acetic acid. . The strain thus obtained is incubated for 2-3 days by tooth picking on a minimal plate medium containing glucose and acetic acid as the only carbon source, respectively. Strains that grow well on glucose-containing minimal plate media will not grow on minimal plate media containing acetic acid as the only carbon source.

The composition of the minimum medium was as follows. Tyrosine, a nutrient-constituting amino acid, was added with 100 mg / l.

The strains obtained by the above mutation treatment were subjected to aerobic conditions (shaking culture 200-300 rpm in the case of flasks, agitation speed 400-1000 rpm and aeration 0.5-1.5 vvm in the case of fermenters) and incubation temperature 30-37 ℃, pH 6.0-8.0 (In the case of fermentation tank, L-tryptophan was accumulated in the culture medium by incubation at pH 6.9 to 7.1 with ammonia water). In the case of flask culture, L-tryptophan was accumulated in the culture solution for 48 to 60 hours. In addition, L-tryptophan was produced by an organic fermentation method of supplying additional sugars several times when cultured in a fermentation tank. In the present invention, L-tryptophan production capacity of the strain was measured by an organic fermentation method.

Minimum medium and composition of LB medium

The cell growth of the culture was measured by optical density at 562 nm, and the sugar analysis was performed by Bertrand method. Meanwhile, quantification of L-tryptophan and acetic acid was analyzed by HPLC (High Performance Liquid Chromatography).

The following examples illustrate the present invention in detail, and these examples merely illustrate the present invention, but the scope of the present invention is not limited thereto.

Example 1

E. coli CA15 (KFCC 10861) with tryptophan production capacity was cultured in a 5 L fermenter in a fed-batch mode with a culture temperature of 30 to 32 ° C., a culture pH of 6.9 to 7.1, and an aeration rate of 1.0 vvm. Tryptophan and organic acid accumulated in the culture medium and the cell growth was measured. Here, to compare the accumulation of acetic acid with dissolved oxygen and the productivity of tryptophan, fermentation was carried out by fixing the stirring speed at 400 to 1000 rpm and the effects thereof were examined and the results are summarized in Table 3.

Here, as the stirring speed was lowered, it was confirmed that the dissolved oxygen in the culture was limited. In fact, the dissolved oxygen was maintained at 15% or more during the entire fermentation process while maintaining the stirring speed of 1,000rpm, while the dissolved oxygen in the culture medium was limited from a few minutes to several tens of minutes each time the medium containing glucose was added during fermentation. . On the other hand, when maintaining the stirring speed of 400rpm dissolved oxygen was limited throughout the entire fermentation process from 10 hours from the start of fermentation, if the fermentation proceeds below 600rpm normal fermentation proceeds without metabolizing glucose in the medium. As can be seen from the results of Table 3, it can be seen that tryptophan productivity is affected by the concentration of acetic acid accumulated, and it can be seen that the excess accumulation of acetic acid has a fatal effect on cell growth.

From these results, selection of strains that do not accumulate acetic acid in the culture medium can increase cell growth, and also predict the possibility of improving tryptophan productivity.

Example 2

E. coli KFCC 10861 with tryptophan production was shaken overnight in a flask containing LB medium and then washed twice with sterile saline solution and diluted to a final OD of 1.0 with citrate buffer (pH 5.5, 0.1M). The final volume was then adjusted so that the NTG concentration was 500 μg / ml. The solution was treated for 30 minutes in a 37 ° C. incubator, washed three times with pH 7.0 and 0.1 M phosphate buffer, and incubated in a minimal medium containing glucose for one day. The culture solution thus obtained was centrifuged, washed with sterile saline, and incubated for 5 hours in a minimal medium containing primary as the only carbon source. Ampicillin was added to 20 mg / l and the cells were incubated for 2 hours to completely destroy the cells. Ampicillin was removed by centrifugation at 5000 rpm for 20 minutes and then washed several times with sterile saline. Inoculate the washed ampicillin treated strain with an OD ₅₆₂ of 0.2 in a flask containing 3 g / l of pyruvic acid as a carbon source and a minimum medium containing 15 mM of monofluoroacetate, an analogue of acetic acid. , Shaking culture overnight at 37 ℃. The culture solution was diluted with sterile saline, and then plated in a minimal flat medium containing pyruvic acid as a carbon source and containing 15 mM monofluoroacetate, followed by incubation for several days at 37 ° C. Independent colonies obtained in this manner were subjected to tooth picking in succession to a minimal flat medium containing glucose and a minimum flat medium containing acetic acid as the only carbon source, and then incubated for several days at 37 ° C. Then, strains were selected that grew in the minimal plate containing glucose and did not sit in the minimal plate medium containing acetic acid.

As a result of examining the tryptophan production capacity by carrying out flask culturing of a strain of Escherichia coli KFCC 10861, which does not use acetic acid as a carbon source, it was able to obtain a superior strain compared to the existing strain. Among the strains with the highest productivity, tryptophan fermentation concentrations are listed in Table 4.

Example 3

In order to confirm whether it is possible to metabolize acetic acid as the only carbon source for the strain selected in Example 2, the test strain and wild strain W3110 of Example 2 were shaken overnight in glucose-containing minimal medium. This test strain was washed several times with sterile saline to remove glucose, and then inoculated with an appropriate amount of test strain to about 0.3 at OD ₅₆₂ in a test tube containing a minimal medium containing acetic acid as the only carbon source and shaking culture at 37 ° C for 18 hours. After absorbance was measured to measure the growth (OD) of the test bacteria.

As shown in Table 5, wild strains W3110 and KFCC 10861 showed growth in minimal medium containing glucose and acetic acid as the sole carbon source, whereas CJ 15, CJ 18, and CJ 27 selected by artificial variation contained glucose. The minimal media showed no significant difference from the parent strain KFCC 10861, but the cell growth was significantly reduced in the minimal media with acetic acid as the only carbon source.

In the above example, the L-tryptophan concentration in the fermentation broth was improved compared to the parent strain Escherichia coli KFCC 10861, and E. coli CJ 18, which cannot use acetic acid as the only carbon source, was finally selected. Deposited (KFCC 10902).

Example 4

E. coli CJ 18 and the parent strain KFCC 10861, which are not able to metabolize acetic acid as a carbon source, were cultured in 30-L fermenter in a fed-batch, followed by analyzing the accumulation of tryptophan and acetic acid in the final culture solution and measuring the cell growth. It is shown in Table 6. The fermentation conditions were the culture temperature of 32 ℃, pH 7.0 (pH control with ammonia water), aeration rate 1.0vvm, and the stirring speed was 400 ~ 600rpm.

As can be seen from Table 6, E. coli CJ 18 significantly reduced the amount of acetic acid accumulated in the culture medium during fermentation, thereby increasing the cell growth of the final culture medium by about 10%.

On the other hand, the concentration of L-tryptophan in the final culture medium was 25.1g / ℓ compared to the parent strain E. coli KFCC 10861 it was confirmed that 20% and 15% increase in the fermentation concentration and hourly productivity, respectively.

In addition, E. coli CJ 18 fermented about 50% more L-tryptophan than E. coli mutant strains (Korean Patent Publication 90-8251, tryptophan accumulation 15.6 g / l), which produce the largest amount of L-tryptophan by direct fermentation. It was found that the accumulation in the culture solution.

As described above, the present invention is an L-tryptophan production mutant strain that can effectively block accumulation of acetic acid that is produced as a byproduct in the process of producing L-tryptophan by direct fermentation and inhibits cell growth and L-tryptophan production. It can be seen that E. coli CJ 18 (KFCC 10902) is cultured in a medium containing glucose by direct fermentation, which is a useful means for accumulating L-tryptophan in culture medium.

Claims (3)

E. coli mutant CJ 18 (KFCC 10902), which produces L-tryptophan, which cannot use acetic acid as a carbon source. E. coli mutant CJ 18 (CFC 18) according to claim 1, characterized in that the parent strain is E. coli CA 15 (KFCC 10861), which is resistant to tryptophan analogs, shows nutritional requirements for tyrosine, and is susceptible to L-arginine. KFCC 10902). A method for producing L-tryptophan by direct fermentation method using glucose as a carbon source, wherein the E. coli mutant strain CJ 18 (KFCC 10902) which cannot use acetic acid as a carbon source is used.