KR100724699B1 - Novel Corynebactrium glutamicum which is Used For Industrial Production of L-Valine and Manufacturing Method of L-Valine Using The Microorganism - Google Patents

Abstract

The present invention relates to a novel microorganism capable of producing L-valine and a method for producing L-valine using the same microorganism. More specifically, it has the ability to produce L-valine, and is resistant to norvaline, a metabolite of L-valine, the final product, and at the same time, Corynebacterium glutamicum and its strains, which require specific amino acids for growth. It relates to a method for producing L-valine using.

Corynebacterium glutamicum, L-valine, L-leucine, norvaline

Description

Novel corynebactrium glutamicum which is used for industrial production of L-Valine and manufacturing method of L- using novel Corynebacterium glutamicum and the same strain for industrial production of L-valine Valine Using The Microorganism}             

1 is a flow chart showing the biosynthetic pathway of L-valine, the final product of the present invention.

  The present invention relates to the genus Corynebacterium, new microorganisms and a method for producing L-valine (L-valine) using the same.

L-valine, one of essential amino acids, is a kind of natural amino acid that has an effect of protecting muscle cells and preventing muscle breakdown, and is widely used in medicine, food, and other animal feeds. More specifically, it is used as a medicament for fluids, synthetic amino acid preparations and the like, and for foods, it is used for health supplements and beverages.

L-valine production by known microbial fermentation is a method for producing L-valine directly from a carbon source and a nitrogen source using a microorganism having resistance to D, L-aminobutyric acid (D, L-amninobutyric acid) Japanese Patent Laid-Open No. S63-160592), a method using microorganisms resistant to thiazolealanine and having the requirement of leucine, isoleucine, or threonine (Japanese Patent Publication S52-116), Method using microorganisms resistant to aminoethylcysteine (Japanese Patent Publication S58-2678), method using microorganisms resistant to L-valine in acetic acid addition medium and susceptible to pyruvic acid in glucose addition medium (US Pat. No. 5,521,074, Korean Patent No. 199595-0005133), a method using microorganisms resistant to polyketides (Korean Patent No. 1996-0016871) and the like are known.

Meanwhile, a method for improving L-valine production with valine resistance through mutated genes of Corynebacterium or Brevibacterium and evolutionary species has been proposed (European Patent 0477000), in particular β-fluoropyruvate, β -Evolution of species with Corynebacterium or Brevibacterium that is resistant to various pyruvate analogs, such as chloropyruvate, β-mercaptopyruvate or trimethylpyruvate It was characterized. However, European Patent No. 0477000 is similar to the present invention in terms of using Corynebacterium, but feedback inhibition to L-valine, which is a final product, is expected, and thus it is subject to constraints for industrial mass production.

The present invention has been made to overcome the problems of the prior art described above, an object of the present invention is to have the ability to produce L- valine, and to endurance resistance to norvaline (Norvaline), a metabolite of the final product L-valine Eggplant is to provide Corynebacterium glutamicum (Corynebacterium glutamicum).

Another object of the present invention is Corynebacterium glutamime that has the ability to produce L-valine, is resistant to norvaline, a metabolite of L-valine, the final product, and at the same time requires specific amino acids for growth. To provide a com.

Still another object of the present invention is to provide a method of producing L-valine in large quantities by culturing the Corynebacterium glutamicum.

The present invention has the ability to produce L-valine, and includes Corynebacterium glutamicum, which is resistant to norvaline, a metabolite of L-valine, the final product, and particularly requires the growth of specific amino acids for growth. Corynebacterium glutamicum is provided.

1 is a flowchart showing the biosynthetic pathway of L-valine, in which L-valine is derived from pyruvic acid, acetoactic acid, dihydroxy isovaleric acid, and ketoisovaleric acid. biosynthesis via ketoisovaleric acid). These intermediate metabolites include acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B ( Produced by reaction catalyzed by transaminase B).

These enzymes are also involved in L-isoleucine biosynthesis starting from ketobutyric acid and pyruvic acid, and 2-isopropylmalic acid and 3-isopropyl from intermediate metabolites ketoisovaleric acid. L-Leucine is biosynthesized via 3-acid isopropylmalic acid and ketoisocaproic acid. L-valine, L-isoleucine, and L-leucine, which are thus biosynthesized, are called branched-chain amino acids, and because the same enzyme is used in the biosynthesis process, one branched amino acid is industrially fermented. It is known that there is a difficulty in manufacturing.

Meanwhile, Corynebacterium glutamicum NV-44 according to the present invention (depository institution: Biotechnology Research Institute Gene Bank, Accession No .: KCTC 10707BP) has the production capacity of L-valine, preferably the Corynebacterium gluten Tamicom NV-44 (KCTC 10707BP) is a L-isoleucine requester and a parent strain of MS-03178 (KFCC-10011) in Corynebacterium that produces L-leucine. (N-methyl-N'-nitro-N-nitrosoguanidine) and the like were selected step by step using a conventional mutation treatment method. Firstly, L-leucine is required to grow L-leucine from Corynebacterium dongae MS-03178 (KFCC-10011), which has the ability to produce L-leucine, and has L-valine production capacity. After -27 was selected, NV-44 (KCTC 10707BP) resistant to novalin, a metabolic antagonist of L-valine, was isolated using DL-27 as a parent.

Although it is not clear about the mechanism by which the selected strains accumulate large amounts of L-valine in the medium, the activity of acetohydroxy acid synthase, a major rate-regulating enzyme of L-valine biosynthesis, was enhanced compared to the parent strain. By imparting resistance to metabolic antagonists of the final product, L-valine, it is believed that the enzyme is not subject to feedback inhibition or inhibition by L-valine.

In addition, the present invention to solve the problem of oxygen deficiency caused by the overgrowth of cells from the strain resistant to the metabolic antagonist of the L-valine, the final product, by using the L- leucine requirement of the desired degree during the fermentation process Corynebacterium glutamicum is mutated to maintain cell mass.

Hereinafter, the isolation process of the strain having resistance to norvaline, which is a metabolite of L-valine, a final product, based on the Corynebacterium sinus MS-03178 (KFCC-10011) strain, will be described in more detail. Shall be.

The following cells were incubated at 30 ° C. for 24 hours in a complete plate medium having the composition of Table 1 and incubated at 30 ° C. for 24 hours. After incubation, the cells were centrifuged and washed twice with 0.05 M TM buffer (pH 6.2). After diluting the cells with the same buffer so as to have a concentration of 10 ^{6-10 8} / ml, NTG was added at 250 µg / ml and treated at 30 ° C for 30 minutes. The treated cells were washed twice with physiological saline, then plated to form a single colony in a complete flat medium, and the formed single colonies were then sequentially placed in the minimum flat medium of Table 2 and the minimum flat medium to which the corresponding amino acid was added. As a result of the replication, strains which were impossible to grow in the minimum medium but were grown in the medium to which specific amino acids were added were selected.

In addition, isolates of mutant strains resistant to specific components were treated with NTG, washed twice with physiological saline, then plated on a minimal flat medium supplemented with a suitable agent and incubated at 30 ° C. for 3-5 days. The grown colonies were separated.

Table 1 Full Reputation Badges

ingredient pH glucose Juicy Yeast extract peptone saline Agar 1.0% 0.5% 0.5% 1.0% 0.25% 1.5-2.0% 7.2

Table 2 Minimum Reputation Badges

Ingredients and pH glucose 2.0% Ammonium Sulfate 0.5% Element 0.15% Potassium phosphate monobasic 0.1% Dibasic Potassium Phosphate 0.1% Magnesium sulfate 0.05% Isoleucine 50 mg / l Biotin 200 μg / ℓ Thiamine Hydrochloride 100 μg / ℓ Trace elements 1 μg / ℓ, 1.5-2.0% Agar 1.5-2.0% pH 7.2

※ Trace elements: Copper sulfate (CuSO ₄ · 5H ₂ O) 270 mg / l, Zinc sulfate (ZnSO ₄ · 7H ₂ O) 10 mg / l, Iron sulfate (FeSO ₄ 7H ₂ O) 870 mg / l, Manganese sulfate ( MnSO ₄ H ₂ O) 7.2 mg / l

In the above manner, DL-27, an L-leucine-producing strain and L-valine, was isolated from parent strain Corynebacterium Dong-a MS-03178 (KFCC-10011). The result of confirming the leucine requirement of the separated leucine requirement DL-27 in the liquid minimum medium is shown in Table 3 below.

Table 3 Comparison of growth with addition of L-leucine

L-Leucine Concentration (mg / L) Growth ¹⁾ KFCC-10011 DL-27 0 0.482 0.018 10 0.475 0.045 50 0.489 0.247 100 0.480 0.396 500 0.401 0.412

1) Measure the absorbance at 610 nm by diluting the culture medium shaken for 24 hours in the minimum liquid medium 10-fold.

To increase valine production from leucine demand, NV44 (KCTC 10707BP), which is resistant to norvaline, known as a metabolic antagonist of valine, was derived from DL-27. The resistance of each strain to norvaline is shown in Table 4 below. Parents, KFCC-10011 and DL-27, had growth of less than about 20% compared to the no addition group when 2,000 mg / l of norvalin was added, whereas NV-44 (KCTC 10707BP) showed more than 40% of growth.

Table 4 Comparison of resistance to norvaline

Norvalin concentration (mg / l) Growth rate ²⁾ KFCC-10011 DL-27 NV-44 0 0.504 0.492 0.485 100 0.346 0.423 0.438 500 0.197 0.316 0.341 1,000 0.143 0.171 0.246 2,000 0.074 0.067 0.208 5,000 0.027 0.020 0.115

2) Measure the absorbance at 610 nm by diluting the culture medium shaken for 24 hours in a minimum liquid medium 10 times

The present invention is to cultivate Corynebacterium glutamicum having a production capacity of L- valine, and resistant to norvaline, a metabolite of L-valine, the final product, and L-valine from the culture. It includes a method of producing.

In the method for producing L-valine using the microorganism of the present invention, L-valine accumulated in the medium was recovered by culturing in a medium containing a common carbon source, nitrogen source, minerals and other nutrients.

As the main carbon source used in the present invention, sugars such as glucose, sugar, starch hydrolyzate and waste molasses can be used alone or in combination. As nitrogen sources, ammonium sulfate, ammonium chloride, urea, ammonia gas, peptone, gravy, yeast Extracts, defatted soybean meal hydrolysates and the like can be used. Potassium phosphate, magnesium sulfate, salt, iron sulfate, manganese sulfate and the like could be used as the inorganic substance. In the case of amino acid requirements, the appropriate amino acid was added to the medium and cultured.

The culture is carried out under normal aerobic conditions, preferably incubated for 2 to 4 days in the culture temperature of 25 ~ 35 ℃, pH 5.5 ~ 7.5 range. PH control during the culturing is possible with ammonia gas or ammonia water, calcium carbonate and the like.

Analysis of L-valine in culture was analyzed using a Hitachi Amino Acid Analyzer (L-8500A), and growth was analyzed at 610 nm using a Beckman spectrophotometer (DU-70) by diluting the culture 100-fold. However, when the pH was adjusted using calcium carbonate, absorbance was measured after 5 N HCl was added to decompose the calcium carbonate.

Hereinafter, the content of the present invention will be described in more detail with reference to preferred embodiments.

<Example 1>

Used strain: KFCC-10011, DL-27, NV-44

Species medium: glucose 5%, yeast extract 1.0%, urea 0.3%, corn steep liquor (CSL) 0.5%, peptone 1.0%, salt 0.25%, biotin 200 ㎍ / ℓ, thiamine hydrochloride 100 ㎍ / ℓ, (pH 7.2 5N caustic soda solution

Fermentation medium: glucose 10%, ammonium sulfate 4.0%, urea 0.3%, potassium monophosphate 0.1%, potassium diphosphate 0.1%, magnesium sulfate 0.1%, corn steep liquor (CSL) 2.0%, L- leucine 200 mg / l, isoleucine 50 mg / l, biotin 200 μg / l, thiamine hydrochloride 100 μg / l, copper sulfate 10 mg / l, zinc sulfate 10 mg / l, iron sulfate 20 mg / l, manganese sulfate 10 mg / l, carbonic acid Calcium (CaCO ₃ ) 5% (pH 7.2 is adjusted using 5N caustic soda solution)

Cultivation method: Dispense 50 ml of the seed medium into a 500 ml shake flask, autoclave sterilize at 121 ° C. for 20 minutes, inoculate the experimental strain, and shake culture at 30 ° C. for 24 hours to obtain a seed culture. The fermentation broth was dispensed with 30 ml in a 500 ml shake flask, autoclaved at 121 ° C. for 20 minutes, and then inoculated with 1 ml of the previously prepared seed culture solution and incubated at 30 ° C. for 40 hours. When KFCC-10011 was excluded, 200 mg / L of L-leucine was added to the fermentation broth, and the results of measuring growth and concentration of L-valine in the culture medium are shown in Table 5 below.

Table 5 Growth and L-valine Production Capacity by Strain

Used strain Growth L-valine concentration (g / l) L-leucine concentration (g / l) KFCC-10011 0.507 0.5 8.1 DL-27 0.523 7.8 - NV-44 0.584 10.3 -

<Example 2>

2 L of the fermentation broth of Example 1, except for calcium carbonate, was placed in a sterile 5 L small fermenter, and 200 mL of the seed culture of the new strain NV-44 (KCTC 10707BP) was inoculated as shown in Example 1, followed by 600 rpm, 1 The culture was carried out at 30 ° C. under vvm conditions. During incubation, the pH was adjusted to 7.0 with ammonia water and 55% glucose solution was added 4 times at 1% residue. The total sugar concentration used for fermentation was 228 g / l. At this time, growth and L-valine productivity according to the concentration of L- leucine added to the fermentation medium are shown in Table 6 below.

TABLE 6 Growth and L-valine Productivity by L-Leucine Concentration of New strain NV-44

 L-Leucine Concentration (mg / L) Growth rate L-valine concentration Fermentation time (hrs) 100 0.436 20.1 73 300 0.842 46.3 56 500 0.913 39.2 48

<Example 3>

In the same manner as in Example 2, 20 l of the fermentation broth added with 300 mg / l L-leucine was sterilized and placed in a 50 l fermenter. vvm, incubated at 30 ° C. under a pressure of 0.5 kg / cm ³ . During incubation, the pH was adjusted to pH 7.0 using ammonia gas, and 55% glucose solution was added four times at 1% residue. The total sugar concentration used for fermentation was 228 g / l. The concentration of L-valine in the fermenter was 43 g / l and the fermentation time was 60 hours.

As described above, the present invention has been described with reference to preferred embodiments and the like, but the content of the present invention is not limited thereto, and various embodiments may be changed within the spirit and scope of the present invention.

When L-valine is prepared using the new strain Corynebacterium glutamicum of the present invention, it is possible to industrially manufacture a large amount without receiving a feedback inhibition to the final product, L-valine. In addition to having resistance to the metabolic antagonist of L-valine, which is the final product, and using L-leucine demand, it is possible to solve the problem of oxygen deficiency due to overgrowth of cells by maintaining the desired cell mass in the fermentation process. have.

Claims (7)

Corynebacterium glutamicum (KCTC 10707BP), which has the ability to produce L-valine and is resistant to norvaline, the metabolite of L-valine, the final product, and requires L-leucine for growth delete delete It is capable of producing L-valine, and is resistant to norvaline, a metabolite of L-valine, the final product, while culturing Corynebacterium glutamicum (KCTC 10707BP), which requires L-leucine for growth. To obtain L-valine from the culture. delete The method of claim 4, wherein Method for producing L-valine is cultured under aerobic conditions in the culture temperature is 25 ~ 35 ℃, pH is 5.5 ~ 7.5 range The method of claim 4, wherein Method for producing L-valine, characterized in that the concentration of the cultured strain is controlled by the addition amount of L- leucine

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