KR100317901B1 - A microorganism producing glutamic acid and a method for producing glutamic acid using said microorganism - Google Patents

Abstract

The present invention is directed to mutant causing agents such as UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG), as a parent strain of Corynebacterium glutamicum , which produces glutamic acid, as a parent strain. The present invention relates to a mutant strain having a change in the metabolic pathway of the strain by modifying the strain of the parent strain to have monofluoroacetate resistance by treatment according to a conventional method, thereby improving the glutamic acid production capacity as compared with the parent strain. The present invention also relates to a method for producing glutamic acid using microorganisms, wherein the mutant strain is cultured in a medium containing waste molasses, glucose, starch hydrolyzate, raw sugar, and the like to extract glutamic acid from the culture.

Description

A microorganism producing glutamic acid and a method for producing glutamic acid using said microorganism}

The present invention relates to a microorganism that produces glutamic acid and a method for producing glutamic acid using the same, and more particularly, has a monofluoroacetate resistance as a mutant of Corynebacterium glutamicum KFCC-10656. The present invention relates to a microorganism having improved production capacity of glutamic acid, and a method for producing glutamic acid using the same, by the metabolic pathway.

In the glutamic acid production metabolic pathway, the citric acid cycle is known to play an important role as a source of metabolic precursors. That is, the supply of α-ketoglutaric acid, a precursor of glutamic acid, is made by the circulation of the citric acid cycle, and thus is advantageous for the production of glutamic acid when the smooth circulation of the citric acid cycle occurs. The citric acid cycle starts with the production of citric acid by the reaction of acetyl-CoA with oxaloacetic acid. Acetyl-CoA is easily produced from acetic acid by biochemical reactions, thus obtaining a high acetic acid magnetizable strain would be advantageous for glutamic acid production in terms of enhancing the citric acid cycle.

However, since a mechanism called metabolite inhibition exists as a mechanism for inhibiting the production of excess metabolites in the metabolic process of microorganisms, it is not easy to allow the formation or accumulation of excess intermediate metabolites. That is, products produced at each metabolic step inhibit the activity of enzymes involved in producing the metabolites, thereby preventing the metabolites from being produced and accumulated more than necessary.

As an existing method of artificially releasing such a regulatory mechanism, a method of obtaining a microbial strain resistant to an analog of a specific metabolite has been commonly used.

The present inventors completed the present invention by selecting strains resistant to acetic acid analogs and resistant to acetic acid analog monofluoroacetate in order to obtain a strain having high acetic acid magnetization.

Microorganism TS1 of the present invention is a parent strain using Corynebacterium glutamicum KFCC 10656 as a parent strain and according to a conventional method as a mutation inducing agent such as UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG), etc. After treatment, the medium to which monofluoroacetate was added [concentration 10g / l glucose, 1g / l potassium phosphate, 0.5g / l potassium diphosphate, 0.5g / l ammonium sulfate, 1g / urea] l, 20 mg / l iron sulfate, 0.5 g / l magnesium sulfate, 20 mg / l manganese sulfate, 200 μg / l thiamine hydrochloride, 200 μg l biotin, 5 g / l molasses, pH 7.0 Medium from which 30-150 g / l of fluoroacetate was added]. At this time, the monofluoroacetate concentration in the medium used for the experiment was up to 150 g / l, and the resistant mutants which were successfully separated were found to be able to grow at 70 g / l monofluoroacetate concentration.

Flask experiments and fermenter culture experiments were performed on the mutant strains obtained therein, and it was demonstrated that glutamic acid productivity was improved by about 11% compared to the existing strain.The strain was named TS1 and was assigned accession number KFCC-11113 on November 2, 1999. It was deposited with the Korean spawn association.

The nutrient medium used for the strain of the present invention is composed of peptone 1, gravy 1, sodium chloride 0.25, yeast extract 1, agar 2, pH 7.2.

The biochemical properties of the novel mutant TS1 isolated from the present invention are as described in Table 1 and Table 2. According to these contents, the microorganism of the present invention can be grown even in a medium containing 70 g / l monofluoroacetate, Acetic acid magnetization [acetic acid magnetization experimental medium: 10-100 g / l acetic acid, 1 g / l potassium phosphate, 0.5 g / l dibasic potassium phosphate, 0.5 g / l ammonium sulfate, 1 g of urea / l, iron sulfate 20 mg / l, magnesium sulfate 0.5 g / l, manganese sulfate 20 mg / l, thiamine hydrochloride 200 μg / l, biotin 200 μg / l, molasses 5 g / l, pH 7.0]. Able to know.

The characteristics of the microorganism of the present invention are as described in the following table.

Comparison of resistance to monofluoroacetate Strain Monofluoroacetate (g / l) 0 30 50 70 100 120 150 KFCC-10656 +++ + - - - - - TS1 +++ +++ ++ + - - -

(Note) +; Growth,-; Not grown, incubated for 72 hours at 30 ° C.

Comparison of Acetic Acid Magnetization Strain Acetic acid concentration (g / l) 0 10 20 40 60 80 100 KFCC-10656 +++ +++ +++ ++ + - - TS1 +++ +++ +++ +++ ++ + +

(Note) +; Growth,-; Not grown, incubated for 72 hours at 30 ° C.

The present invention is explained in more detail in the following examples.

Example 1

Use strain: Corynebacterium glutamicum KFCC-10656 and its strain microorganism TS1 (KFCC-11113).

Species medium: peptone 1, yeast extract 0.5, gravy 0.5, glucose 1, sodium chloride 0.25, urea 0.13, pH 7.2.

Fermentation medium: glucose 3, liquid 0.2, biotin 1 μg / l, waste molasses 0.05, ammonium sulfate 0.1, iron sulfate 0.002, manganese sulfate 0.002, magnesium sulfate 0.05, thiamine hydrochloride 200 μg / l, potassium phosphate 0.2, urea 0.95 , pH 7.2.

Fermentation method: 3 ml of the seed medium was dispensed into a test tube with a diameter of 18 mm, inoculated using strain after autoclaving according to a conventional method, and shaken for 18 hours at 30 ° C. to use as a seed culture solution. 40 ml of the fermentation broth was dispensed into a 500 ml shake flask for pressure and sterilized under pressure at 121 ° C. for 10 minutes to inoculate 1 ml of the seed culture solution and incubated for 48 hours. The rotation speed was adjusted to 180 times per minute, 30 ° C., pH 7.2. After incubation, the amount of glutamic acid in the medium was 16.8 g / l for KFCC-10656 and 18.7 g / l for TS1.

As a result of the present Example, the microorganism of the present invention was confirmed that the productivity of glutamic acid improved by about 11 at 3 times the concentration of sugar compared to the existing strain.

Example 2

Strains used: same as Example 1

Primary species: peptone 1, yeast extract 0.7, gravy 0.7, glucose 1, sucrose 1, sodium chloride 0.25, urea 0.3, phenol red 0.001, pH 7.2.

Secondary species: raw sugar 3, glucose 1, fructose 1, magnesium sulfate 0.04, potassium monophosphate 0.1, dipotassium phosphate 0.05, ammonium sulfate 0.8, iron sulfate 0.002, manganese sulfate 0.002, zinc sulfate 0.0002, copper sulfate 0.0002, biotin 500 μg / l, thiamine hydrochloride 2 mg / l, soy protein hydrolyzate 0.2, phenol red 0.001, pH 6.8.

Fermentation medium: Sucrose 6, Glucose 2, Fructose 2, Magnesium sulfate 0.05, Potassium phosphate 0.05, Dipotassium phosphate 0.15, Ammonium sulfate 0.1, Iron sulfate 0.002, Manganese sulfate 0.002, Zinc sulfate 0.0002, Copper sulfate 0.0002, Biotin 30 ㎍ / l, thiamine hydrochloride 1 mg / l, soy protein hydrolyzate 0.2, phenol red 0.001, calcium carbonate 3, pH 6.75.

Primary seed culture: 2.5 ml of the primary seed culture medium was dispensed into an 18 mm test tube, cooled by autoclaving at 121 ° C. for 5 minutes, inoculated with the strain used, and incubated for 12 hours at 30 ° C. at 160 revolutions per minute. .

Secondary seed culture: 30 ml of secondary seed medium is dispensed into a 250 ml shake Erlenmeyer flask, pressurized and sterilized at 121 ° C. for 5 minutes, inoculated with 1 ml of the primary seed culture culture solution, and then inoculated with 150 ml at 31 ° C. Shake culture was performed for 14 hours at rpm.

Fermentation method: The fermentation broth was dispensed 30 ml each in a 250 ml Erlenmeyer flask, 1 ml of 10 urea solution was added and 5 ml of the secondary seed culture solution was inoculated and incubated at 31 ° C. for 150 hours at 150 rpm. In the middle of the culture, 8 ml of 10 urea aqueous solution was added from time to time according to the pH to adjust the pH and supply a nitrogen source. After 2 hours of fermentation, 0.04 of surfactant and 0.3 U / ml of penicillin were added and incubated until all of the sugar was consumed.

After incubation, the concentration of glutamic acid was 45.7 g / l for KFCC-10656 and 51.9 g / l for TS1.

Example 3

Primary species: peptone 0.7, yeast extract 0.7, gravy 0.7, glucose 1, sodium chloride 0.25, urea 0.13, ammonium sulfate 0.1, pH 7.5.

Secondary species: raw sugar 3, molasses 1, magnesium sulfate 0.04, potassium diphosphate 0.1, ammonium sulfate 0.3, iron sulfate 0.001, manganese sulfate 0.001, biotin 500 μg / l, thiamine hydrochloride 2 mg / l, urea 0.1, pH 7.1.

Fermentation medium: raw sugar 1.7, molasses 6.8, phosphoric acid 0.13, magnesium sulfate 0.04, iron sulfate 0.001, manganese sulfate 0.001, fine liquid 0.1, thiamine hydrochloride 200 μg / l, potassium hydroxide 0.18, antifoam 0.005, pH 7.5.

Primary seed culture: 40 ml of the primary seed medium was dispensed into a 500 ml shake Erlenmeyer flask, pressurized and sterilized at 121 ° C. for 10 minutes, inoculated with the strain, cooled, and incubated at 180 revolutions per minute and 20 ° C. for 20 hours.

Secondary seed culture: Dispense the secondary seed medium into 2.5 liters of each fermenter for 5 liters, pressurize and sterilize at 121 ℃ for 10 minutes, cool, inoculate 30 ml of the primary seed culture culture solution, and air Shaking culture was carried out for 18 hours at 900 rpm, 30 ℃ while feeding 0.5 liter.

Fermentation method: Dispense the fermentation broth into a 30 liter test fermentation tank at 9 liters, sterilize for 10 minutes at 121 ° C, cool, inoculate 1.5 liters of secondary culture medium, and 2.5 liters of air per minute. Incubation was performed at 450 rpm and 31 ° C. while feeding.

The culture was carried out under the above conditions, and then penicillin or a surfactant was added between the early and the end of the logarithmic growth period, and the pH of the fermentation broth was continuously adjusted to pH 7.7 with 28 ammonia water. When the concentration of the residual sugar in the culture was 1.5, the sterile pulmonary molasses or a mixture of the waste molasses and the raw sugars was supplied at regular ratios. Further culture was continued until the sum of the sugars added to the initial fermentation medium with molasses or mixed sugar was 22 relative to the amount of fermentation broth. After incubation, the concentration of glutamic acid was 109.6 g / l for KFCC-10656 and 121.1 g / l for TS1.

Corynebacterium glutamicum TS1 according to the present invention can be produced in high yield by culturing in a medium containing glutamate, glucose, starch hydrolyzate and raw sugar as an improved glutamic acid production capacity.

Claims (4)

Microorganism TS1 (KFCC-11113), which produces glutamic acid as a variation of Corynebacterium glutamicum . The microorganism according to claim 1, which is capable of growing at a monofluoroacetate concentration of 70 g / l or more. The microorganism according to claim 1 or 2, wherein the microorganism is capable of growing by using at least 80 g / l of acetic acid as a carbon source in the medium. A method for producing glutamic acid, characterized by culturing Corynebacterium glutamicum TS1 in a medium containing waste molasses, glucose, starch hydrolyzate, raw sugar, and the like.