KR20170106685A - Mutant of Corynebacterium glutamicum Producing L-Histidine and Method for Producing L-Histidine Using the Same - Google Patents

Abstract

The present invention relates to a mutant strain of Corynebacterium glutamicum producing L-histidine, and a method for producing L-histidine using the same. According to the present invention, the mutant strain of Corynebacterium glutamicum (Access number: KCCM11795P) shows resistance to a derivative of L-histidine.

Description

The present invention relates to a mutant of Corynebacterium glutamicum producing L-histidine and a method for producing L-histidine using the mutant of Corynebacterium glutamicum Producing L-Histidine and Method for Producing L-

This application relates to a novel mutant producing L-histidine and a method for producing L-histidine using the same.

L-Histidine is one of the 20 standard amino acids and is classified as an essential amino acid for growing children. L-histidine is involved in important physiological processes such as antioxidation and immune regulation, and is used in the medical industry such as gastro-intestinal ulcer treatment, circulating agent and amino acid solution.

L-histidine is often found in hemoglobin, and is produced mainly by protein hydrolysis extraction using blood as a raw material. However, it has drawbacks such as low efficiency and environmental pollution. On the other hand, L-histidine can be produced through microbial fermentation, but large-scale industrialization has not yet been achieved. This is because biosynthesis of L-histidine competes with PRPP, a nucleotide synthesis precursor, and has a complicated biosynthesis process and regulatory mechanism requiring high energy.

L-histidine production ability of the microorganism used in the fermentation method can be improved by a method of controlling the metabolism of the strain through gene modification as well as a method of mutagenesis and mutant selection.

In order to produce L-histidine by a fermentation method using microorganisms, development of a strain and L-histidine production method capable of producing L-histidine at a stable and high concentration has been demanded.

Chinese Patent Publication No. 104845923

One aspect of the present application is to provide a Corynebacterium glutamicum mutant strain KCCM11795P that produces L-histidine.

Another aspect of the present application is a method for producing Corynebacterium glutamicum , comprising culturing Corynebacterium glutamicum mutant strain KCCM11795P in a medium; And recovering L-histidine from the medium or mutant strain.

Another aspect of the present application is to provide a composition for producing L-histidine comprising Corynebacterium glutamicum mutant strain KCCM11795P.

One aspect of the present application provides Corynebacterium glutamicum mutant strain KCCM11795P which produces L-histidine.

The term " L-histidine " as used in the present application means an amino acid having an imidazole as a side chain and a basicity of the formula C ₆ H ₉ N ₃ O ₂ . L-histidine is an amino acid that promotes the production of hemoglobin and leukocyte in the body, is used as an essential component of histamine synthesis, and plays a role in the regeneration and growth of tissues.

On the other hand, in microorganisms, L-histidine starts with the condensation of 5-phosphoribosyl-1-pyrophosphate (PRPP) with ATP, and through various steps, nitrogen from glutamine L-histidinol is produced and finally can be biosynthesized by histidinol dehydronase. The biosynthetic pathway is complicated as compared with other amino acids, and there is a problem in mass production of L-histidine at a high concentration due to inhibition of feedback against L-histidine or its derivatives. Therefore, a microorganism capable of producing L-histidine at a high yield is obtained by inducing a mutation having resistance to the L-histidine derivative so that the inhibition of feedback against L-histidine or its derivative is released so that it can grow also at a high concentration of L-histidine . Thus, the mutants of the present application are resistant to L-histidine derivatives.

The term " derivative of L-histidine " as used in the present application is a generic term for the binding of L-histidine to another amino acid, substituent or functional group. In the present application, it is meant to include a substance capable of causing feedback inhibition in relation to the biosynthesis of L-histidine, including 1,2,4-triazole-3-alanine But is not limited thereto. The derivative of L-histidine can be used in the process of selecting a mutant strain that produces L-histidine to confirm whether the mutant strain has released feedback inhibition to L-histidine or a derivative thereof.

In the present application, "mutant strain producing L-histidine" means a microorganism mutated to produce L-histidine at a higher concentration than the parent strain. At this time, the mutagenesis of the microorganism can be carried out by various means widely known in the field, and one of the physical or chemical mutagenesis methods can be used. For example, chemical mutagenesis agents suitable for the present application include N-methyl-N'-nitro-Nitrosoguanidine (NTG), diepoxybutane, ethyl Ethylmethane sulfonate, mustard compounds, hydrazine, and nitrous acid may be used but are not limited to these compounds. In addition, the physical mutagenic factors may include, but are not limited to, ultraviolet and gamma radiation. When mutagenized, parental strains are affected by mutagenic factors at concentrations that leave a certain size of viable populations. The size varies depending on the type of mutagenic factors, and the mutation factor depends on the amount of mutation induced within the survival population at a constant rate of killing. For example, in the case of NTG, the killing rate can leave approximately 10% to 50% of the starting population. Mutagenesis by nitrous acid may leave approximately 0.01% to 0.1% of the starting population, and ultraviolet mutagenesis may leave approximately 1.0%, but is not limited thereto.

In addition, the microorganism which can be mutated in the above may be any mutant producing microorganism or L-histidine without limitation, but may be Escherichia sp., Enterobacter sp. For example, Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp. ( Lactobacillus sp.), Etc. However, the present invention is not limited thereto. Specifically, the microorganism of the present application is a microorganism belonging to the genus Corynebacterium or an Escherichia genus microorganism, more specifically, Corynebacterium glutamicum, but is not limited thereto.

Another aspect of the present application is a method for producing Corynebacterium glutamicum , comprising culturing Corynebacterium glutamicum mutant strain KCCM11795P in a medium; And

And recovering L-histidine from the medium or mutant strain.

The method of producing L-histidine according to one embodiment of the present application is described in detail below.

The term "cultivation" of the present application means that microorganisms are grown under moderately artificially controlled environmental conditions. Methods for culturing the microorganisms of the present application include methods known in the art such as Corynebacterium < RTI ID = 0.0 > ( Corynebacterium < / RTI > glutamicum ). < / RTI > Specifically, examples of the culture method include, but are not limited to, a batch culture, a continuous culture, and a fed-batch culture. These various methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138-176, 1991).

The term " medium " as used in the present application is used for the cultivation of bacteria, particularly Corynebacterium glutamicum strain or mutant strains, and includes a medium containing a carbon source, a nitrogen source and inorganic salts so as to produce L- . The culture medium may include a culture medium prepared for culturing the mutant strain, and various substances in which the microorganism is released into the culture medium during the growth. Specifically, the culture medium may contain L-histidine as a target substance.

The medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacterium strains are known (see, for example, the Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington, D.C., USA, 1981). The carbon source in the medium may be, for example, sugars and carbohydrates such as glucose, sucrose, sodium citrate, fructose, lactose or maltose, soybean oil, sunflower oil, But are not limited to, oils and fats such as castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture.

The nitrogen source in the medium may be, for example, a compound including peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean cake, urea, thiourea, ammonium salt, nitrate and other organic or inorganic nitrogen , But is not limited thereto. The inorganic salts contained in the culture medium may include, but are not limited to, compounds including magnesium, manganese, potassium, calcium, iron, zinc, cobalt and the like.

The number of people that can be used in the culture medium may include, but is not limited to, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or a corresponding sodium-containing salt. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. The compounds may be used individually or as a mixture, but are not limited thereto.

In addition to the components of carbon source, nitrogen source and inorganic salts, amino acids, vitamins, nucleic acids and related compounds may be additionally added to the medium of the present application. The raw materials can be added to the medium in a batch manner or in a continuous manner by an appropriate method.

On the other hand, the pH of the medium can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid in a suitable manner. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (e.g., air) can be injected into the medium to maintain the exhalation state. The temperature of the culture may be usually from 20 캜 to 45 캜, but is not limited thereto. The culture is continued until the desired amount of L-histidine is produced. For this purpose, it is usually, but not limited to, 10 to 160 hours. The produced L-histidine may be released into the culture medium or contained in the microorganism.

The step of recovering L-histidine from the culture medium or the mutant cultured with the mutant may be performed by various methods well known in the art, for example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, .

Another aspect of the present application provides a composition for producing L-histidine comprising Corynebacterium glutamicum mutant strain KCCM11795P.

According to one embodiment, the composition for producing L-histidine may further include a culture of the strain in addition to the Corynebacterium glutamicum mutant strain KCCM11795P.

The term " culture product " in the present application is interpreted as a concept that includes a culture medium obtained by culturing a medium or a strain containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the medium described above for a certain period of time and then removing the strain do. Corynebacterium of the present application, Tommy Com (Corynebacterium glutamicum ) mutant strain KCCM11795P is a strain producing L-histidine at a high concentration. Therefore, L-histidine can be recovered from a composition containing the above strain and culture by various methods well known in the art to produce L-histidine.

Corynebacterium of the present application, Tommy Com (Corynebacterium glutamicum ) mutant strain KCCM11795P is resistant to L-histidine and its derivatives so that feedback inhibition resulting therefrom is canceled, thereby allowing a higher concentration of L-histidine to be stably fermented compared to Corynebacterium glutamicum strains Can be produced.

1 is a chromatogram showing the results of quantitative analysis of L-histidine produced from (a) Corynebacterium glutamicum ATCC 13032 and (b) Corynebacterium glutamicum mutant KCCM11795P.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these embodiments are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these embodiments.

Example  1. L-histidine Production capacity  Screening of mutant strains

In order to obtain a microorganism mutant having the ability to produce L-histidine, a mutation of a microorganism was induced by the following method. Specifically, the glutamic acid production parent strain Corynebacterium Com (Corynebacterium glutamicum ) ATCC 13032 was activated for 16 hours in an activated medium and activated. The activated strain was inoculated on a seed medium sterilized for 15 minutes at 121 ° C, cultured for 14 hours, and 5 ml of the culture broth was recovered. The recovered culture was washed with a citric acid buffer (100 mM), and then N-methyl-N'-nitro-N-nitroguanidine (N-methyl- N-nitrosoguanidine (NTG) was added for 20 min and washed with 100 mM phosphate buffer. The mortality rate of the strain treated with NTG was found to be 85%.

In order to find resistance mutants to 1,2,4-triazole-3-alanine, a derivative of L-histidine, NTG-treated strains were treated with 1,2,4 -Triazole-3-alanine were respectively applied to the minimum medium of 20 mM, 30 mM, 40 mM and 50 mM, respectively, and cultured at 30 DEG C for 5 days. Among the mutants found at the above four concentrations, three kinds of 1,2,4-triazole-3-alanine-resistant mutants were obtained.

Example  2. Corynebacterium Glutamicum HCJ -86 sorting and L-histidine Production capacity  Confirm

The ability of L-histidine to produce three types of high-density 1,2,4-triazole-3-alanine-resistant mutants obtained in Example 1 was confirmed by the following method.

To a 250 ml Corner-Bubble flask containing 25 ml of the seed culture was added the parent strain Corynebacterium < RTI ID = 0.0 > glutamicum ATCC 13032), and the three mutants were inoculated respectively, followed by shake culture at 200 rpm at 30 DEG C for 20 hours to obtain seed culture. Then, a 250 ml Corner-Bubble flask containing 24 ml of the production medium was inoculated with 1 ml of the seed culture medium and cultured at 30 ° C for 72 hours at 200 rpm to produce L-histidine. After the completion of the culture, the production amount of L-histidine was measured using liquid high-speed chromatography. Detailed analysis methods and concentrations are shown in Table 1 and Table 2 below.

Analysis item Histidine
STD concentration STD 1: 0.011 g / L
STD 2: 0.033 g / L
STD 3: 0.100 g / L
Mobile phase A: 25 mM-KH ₂ PO ₄ + 12 mM-octane sulfonic acid sodium salt pH 2.5 (by H ₃ PO ₄ )
B: 25 mM-KH ₂ PO ₄ + 12 mM-octane sulfonic acid sodium salt: Acetonitrile
= 50: 50 pH 2.5 (by H ₃ PO ₄ )

A: B = 80: 20
analysis
Condition system HPLC Sample injection amount 5 μl Flow rate 1.0 ml / min column CAP CELL PAK C18 (ACR) 3 탆 4.6 x 150 mm Column temperature 40 ℃ Detector UV 210 nm Run time 12 min

L-histidine concentration (g / L) Corynebacterium glutamicum ATCC 13032 0 Mutation 1 1.2 Mutation 2 1.0 Mutant 3 (HCJ-86) 1.6

As a result, it was confirmed that wild type Corynebacterium glutamicum ATCC 13032 did not produce L-histidine, but Mutant 3 produced L-histidine at a concentration of 1.6 g / L (see Table 2 and FIG. 1) . The mutant strain 3 was named Corynebacterium glutamicum HCJ-86 ( Corynebacterium glutamicum HCJ-86) and deposited with the Korean Society for Microbiological Conservation, International Depository under the Budapest Treaty on December 22, 2015, granting accession number KCCM11795P received. These results suggest that Corynebacterium glutamicum HCJ-86, which is resistant to L-histidine and 1,2,4-triazol-3-alanine, consequently inhibits feedback to histidine or its derivatives against wild-type strains Relative to L-histidine, indicating that L-histidine can be produced with high efficiency and high yield.

The composition of the medium used in Examples 1 and 2 is shown in Table 3 below.

Kinds Composition and pH of medium Activation badge Juice 1%, polypeptone 1%, sodium chloride 0.5%, yeast extract 1%, agar 2%, pH 7.2 Bell badge Glucose 5%, bactopeptone 1%, sodium chloride 0.25%, yeast extract 1%, urea 0.4%, pH 7.2 Minimum media 1.0% of glucose, 0.4% of ammonium sulfate, 0.04% of magnesium sulfate, 0.1% of potassium phosphate, 0.1% of urea, 0.001% of thiamine, 200μg of biotin, 2% of agar, pH 7.0 Production badge (5% of glucose, 2% of ammonium sulfate, 0.1% of potassium phosphate monobasic, 7% of magnesium sulfate hepta 0.05%, CSL (corn steep liquor) 2.0%, biotin 200 / / L, calcium carbonate 30g,

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and equivalents thereof are included in the scope of the present invention. .

Korea Microorganism Conservation Center (overseas) KCCM11795P 20151222

Claims (4)

Corynebacterium glutamicum mutant strain KCCM11795P which is resistant to L-histidine derivatives and produces L-histidine.
The method according to claim 1, wherein the L-histidine derivative is 1,2,4-triazole-3-alanine, mutant KCCM11795P.
Culturing the mutant of claim 1 or 2 in a medium; And
And recovering L-histidine from the mutant strain or medium.
A composition for producing L-histidine comprising the mutant of claim 1 or 2.

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