KR20020061133A - Novel Corynebacterium glutamicum producing L-lysine which has high assimilation of gluconic acid and process for producing L-lysine using the same - Google Patents

Abstract

PURPOSE: Provided are novel Corynebacterium glutamicum which produces L-lysine and has high assimilation of gluconic acid and a process for producing L-lysine using the same. CONSTITUTION: The novel Corynebacterium glutamicum CJG-18(KFCC-11245) is prepared by treating Corynebacterium glutamicum producing L-lysine with mutagens, such as NTG or nitrous acid. L-lysine is manufactured by culturing Corynebacterium glutamicum CJG-18(KFCC-11245) in a medium, accumulating L-lysine therein and separating L-lysine therefrom.

Description

Novel corynebacterium glutamicum producing L-lysine which has high assimilation of gluconic acid and process for L-lysine with increased magnetization to gluconic acid producing L-lysine using the same}

The present invention novel glues for the chemical conversion of the acid character producing L- lysine, characterized by increased Corynebacterium Com (Corynebacterium glutamicum) and by him relates to a method of producing L- lysine, and more particularly Preferably it is derived from Corynebacterium glutamicum KFCC 11043 according to Korean Patent Application No. 98-39305 (filed September 22, 1998), L- lysine using a strain having a high magnetizing properties for gluconic acid Relates to a method of increasing productivity.

L-lysine, along with methionine, threonine, and tryptophan, is an essential amino acid that is used as feed additives, food additives, and pharmaceutical ingredients in livestock. In particular, L-lysine can improve the feed efficiency by adding to the grain feed of livestock with low content, and the demand is increasing according to the recent movement to regulate nitrogen and phosphorus in livestock manure. The market size of L-lysine as a feed additive amounted to about 500,000 tons in 1998, and is expected to increase demand by 12-15% per year on average. Therefore, the improvement of productivity of L-lysine by the development of L-lysine producing strain or improvement of fermentation process can bring a great economic effect.

To date, the production of L-lysine is metabolized by adding biosynthetic precursors (e.g., α-aminoadipic acid and α-ketoadipic acid), and the two-stage method using mutant strains (diaminopimelic acid). Production method)), enzyme production method (conversion of DL-α-aminocaprolactam) and direct fermentation method using improved microorganisms through classical mutation methods, among which direct use of improved microorganisms is known. Fermentation law is the main driver.

Patents for microorganisms producing L-lysine are described in Korean Patent Publication Nos. 79-1782, 81-1746 and 92-7402, Japanese Patent Publication No. Hei 10-165180, Hei 5-111386, Hei 4-166092, etc. The main strain consists of strains that have given amino acid requirements, resistance to amino acid analogs, and antibiotics by means of artificial mutation. However, in the case of the classical mutation method described above, there is a problem in that fermentation productivity is decreased due to the lengthy incubation time due to the continuous mutation, and the possibility of the mutation occurring in other than the specific metabolic part desired by the researcher cannot be excluded.

Therefore, in recent years, with the rapid development of genetic engineering techniques such as cell fusion and genetic recombination, it is possible for researchers to amplify or eliminate specific genes in production strains, thus modifying metabolic pathways to efficiently produce the desired product. Many methods are used for strain improvement, and actual amino acids such as tryptophan are produced using recombinant microbial strains.

In the case of Corynebacteria, these gene recombination techniques have been used in many aspects to develop lysine producing strains. To increase the productivity of lysine, several researchers have performed amplification of enzymes involved in lysine biosynthesis and amplification of enzymes involved in producing precursors for lysine biosynthesis. Representative of these studies is the study of phosphoenolpyruvate carboxylase and pyruvate carboxylase involved in synthesizing oxaloacetate (OAA) from phosphoenolpyruvate (PEP) and pyruvate. Can be mentioned. However, phosphoenolpyruvate carboxylase has been thought to be a major source of OAA but has not been shown to be important for lysine production (FEMS Microbiol Lett (1993) 112: 269-274). In addition, several attempts have been made to increase carbon flow from PEP to OAA, but have little effect on lysine production (Appl Environ Microbiol (1991) 57: 1746-1752, Appl Environ Microbiol (1994) 60: 2494-2500, Biotecnol Prog (1994) 10: 320-326). From these facts, the assumption that the metabolic pathway directly linked to the production of a particular product is the bottleneck is incorrect.

The Stephanopoulos et al. (Biotechnol Bioeng (1992) 39: 565-574) under the high-lysine-producing speed from having to increase emissions of CO ₂ in the production of lysine, and a bar indicate that the path is important HMP (hexose monophosphate), ¹³ Through C-NMR analysis, several reports have shown that lysine production is superior when carbon flow in HMP pathway is higher than glycolysis (Agric Biol Chem (1986) 50: 2453-2459, J Gen Appl Microbiol ( 1991) 37: 157-165, Biotechnol Bioeng 49: 111-129). In addition, the report shows that the production efficiency of lysine was increased by 20% when glucose and gluconic acid were simultaneously administered as a carbon source (Appl Microbiol Biotechnol (1998) 49: 9-15). . However, a method for producing L-lysine using microorganisms artificially strengthening the HMP pathway has not been developed yet.

Therefore, the present inventors focused on the fact that the gluconic acid is introduced into the HMP pathway without passing through the corresponding pathway in the cell, so that the microorganism that can use the gluconic acid with higher efficiency than the parent strain would have enhanced the HMP pathway. Mutant strains characterized by increased magnetization of gluconic acid with the mother strain of Corynebacterium glutamicum were developed. As a result, the strain strain developed as a novel strain was previously unknown. It was confirmed that to produce a high efficiency and came to complete the present invention.

Accordingly, it is an object of the present invention to provide a novel Corynebacterium glutamicum strain that produces L-lysine, characterized by increased magnetization to gluconic acid in order to enhance the HMP pathway.

The new strain according to the present invention can be usefully used for lysine production in the following points. First, since gluconic acid is introduced into cells by permease and not through phosphotransferase system (PTS), the consumption of PEP, an intermediate of lysine biosynthesis, is less than that of parent strains. The production of NADPH, which is used as an energy source in biosynthesis reaction, can be increased. Third, if the magnetization to gluconic acid is increased, gluconic acid can be used as a carbon source in addition to raw sugar, glucose, etc. There is an advantage to it.

Further, another object of the present invention by culturing the new Corynebacterium Com (Corynebacterium glutamicum) state, characterized by a chemical conversion character to the gluconic acid is increased to produce L- lysine at a high efficiency from the culture To provide a way.

In order to achieve the object of the present invention, the present invention is derived from a microorganism belonging to Corynebacterium glutamicum species and having L-lysine productivity as a parent strain, and the parent strain in a minimal medium containing gluconic acid. It provides a L- lysine high-productivity Corynebacterium glutamicum strain characterized by increased magnetization to the gluconic acid obtained by selecting a strain having a relatively high growth.

More specifically, the present invention is Corynebacterium characterized in that the magnetization of gluconic acid is increased by using the parent strain of Corynebacterium glutamicum KFCC 11043 as disclosed in Korean Patent Application No. 98-39305 Corybacterium glutamicum CJG-18 (Accession No. KFCC-11245), which is a novel strain that has not been known at all, L- lysine production efficiency is significantly superior to the parent strain This can be confirmed (see Table 2).

In order to achieve another object of the present invention, the present invention is characterized by induced Corynebacterium glutamicum having a L- lysine productivity as a parent strain and increased magnetization to gluconic acid A method of producing L-lysine, comprising culturing Corynebacterium glutamicum strain in a medium, accumulating L-lysine in the culture, and collecting L-lysine from the culture. To provide.

More specifically, the present invention, Corynebacterium glutamicum CJG-18 (Accession No. KFCC-11245) to produce L- lysine, characterized in that the magnetization to gluconic acid is increased The present invention relates to a method for producing L-lysine from the culture by direct fermentation. According to the present invention, the fermentation concentration and yield of L-lysine may be improved, and thus may be usefully used for the production of L-lysine.

Hereinafter, the present invention will be described in more detail.

The mutant for producing L-lysine according to the present invention with high efficiency belongs to Corynebacterium glutamicum species, and is characterized in that its magnetization to gluconic acid is increased and L-lysine productivity is increased. Any microorganisms may be used. Such mutant strains belong to Corynebacterium glutamicum species and can be induced by artificial mutations using microorganisms having L-lysine productivity as parent strains.

Strains that can be used as parent strains in the present invention include, for example, Corynebacterium glutamicum KFCC 11043 as disclosed in Korean Patent Application No. 98-39305. The Corynebacterium glutamicum KFCC 11043 strain releases feedback inhibition of L-lysine and L-threonine to aspartokinase, an important metabolic regulatory site in the biosynthetic pathway of L-lysine, and releases homoserine dehydrogenase. It is a strain suitable for L-lysine production because it is resistant to S- (β-aminoethyl) -L-cysteine, a lysine analogue that only sustains inhibition of threonine.

In the present invention, in order to obtain a Corynebacterium glutamicum mutant having a high magnetization to the gluconic acid, artificial mutation of the Corynebacterium glutamicum KFCC 11043 having the L- lysine productivity as a parent strain and glue We tried to obtain mutant strains with faster growth rate than the parent strain in the minimal medium containing only cornic acid as a carbon source.

Examples of artificial mutations include UV irradiation, chemical treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), nitrous acid, and the like. Preferably, in the present invention, N-methyl, which is a kind of alkylating agent, is used. N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was treated at a final concentration of 1,000 μg / ml for 10 minutes at 10 ^{7-10 8} cells / ml. . NTG is degraded in vivo and converted to nitrous acid in acidic conditions and to diazomethane in alkaline conditions, causing mutations by inaccurate replication of DNA fragments.

Then, in order to develop a strain having high magnetism in gluconic acid, mutant strains growing faster than those of the parent strain were selected in the minimal medium containing gluconic acid, and the result of the L-lysine fermentation test through the triangular flask titer test, It was confirmed that the lysine production efficiency was higher than that of the parent strain KFCC 11043.

Therefore, the mutant strain was named Corynebacterium glutamicum CJG-18, and the mutant strain was deposited on December 6, 2000 with the Korean spawn association, and was given accession number KFCC-11245.

Production of L-lysine by the mutant strain according to the present invention can be carried out by a culture method of the microorganisms commonly used. The medium used is a carbon source, a nitrogen source, an inorganic substance, and other microorganisms that contain a small amount of nutrients. The synthetic medium or the natural medium may be used. After the end of the culture, L-lysine can be isolated from the culture solution obtained by removing known cells and using a known purification method such as concentrated crystallization treatment, activated carbon treatment or ion exchange resin treatment.

The process for developing Corynebacterium glutamicum CJG-18 according to the present invention is as follows.

EXAMPLE

In the following experiments, the following agar minimum medium was used as the medium for selection of mutant strains, and the following flask fermentation medium was used as a medium for evaluation of the selected strains. The concentration of L-lysine was measured by HPLC (High Performance Liquid Chromatography), and the equivalent was quantified using Bertrand method if necessary.

Gluconate agar medium

Gluconic acid 10 g, (NH ₄ ) ₂ SO ₄ 2 g, urea 2 g, KH ₂ PO ₄ 1.0 g, K ₂ HPO ₄ 3.0 g, MgSO ₄ 7H ₂ O 0.5 g, FeSO ₄ 7H ₂ O 10 mg, MnSO ₄ 5 H ₂ O 10 mg, Biotin 100 μg, Thiamine-HCl 100 μg, CaCl ₂ H ₂ O 0.1 g, Na ₂ B ₄ O ₇ 10 H ₂ O 80 μg, (NH ₄ ) ₆ MoO ₂₇ 4H ₂ O 40 μg , 10 μg of ZnSO ₄ · 7H ₂ O, 300 μg of CuSO ₄ · 7H ₂ O, 10 μg of MnCl ₂ · 4H ₂ O, 1 mg of FeCl ₃ · 6H ₂ O, 20 g of agar, per liter of distilled water (pH 7.0 (pre-sterilization)) .

Flask fermentation medium

Molasses (as reduced sugar) 100 g, yeast extract 4 g, (NH ₄ ) ₂ SO ₄ 40 g, urea 4 g, KH ₂ PO ₄ 1 g, NaCl 2.5 g, MgSO ₄ 7H ₂ O 0.5 g, FeSO ₄ 7H ₂ O 10 mg, 10 mg MnSO ₄ 5H ₂ O, 100 μg biotin, 200 μg thiamine-HCl, 40 g CaCO ₄ , per liter of process water (pH 7.0 (after sterilization)).

Example 1

In order to test the growth of the parental strain KFCC 11043 for the gluconic acid, the time required for colony production was measured after plating on a minimal medium containing 10 g / l of gluconic acid. As a result, it was confirmed that colonies were generated after at least 5 days (Table 1).

Growth Test for Gluconic Acid of Corynebacterium glutamicum KFCC11043 Days of Culture One 2 3 4 5 6 7 Growth - - - - + ++ +++

-: Cell growth does not occur, +: Cell growth occurs

Example 2

In order to develop a bacterium having high magnetism to gluconic acid, Corynebacterium glutamicum KFCC 11043, grown from Luria Bertani liquid medium to the middle of logarithmic phase, was suspended in citric acid buffer (pH 5.5) at 10 ⁷ to 10 ⁸ cells / ml. After the addition, NTG was added to a final concentration of 1,000 μg / ml and treated for 5 minutes on a 30 ° C. shaker. Subsequently, the cells were washed three times with potassium phosphate buffer (pH 7.0), plated in a minimal medium containing 10 g / l of gluconic acid, and colonies generated within 5 days were selected while culturing in a thermostat at 30 ° C. As a result, 20 gluconic acid highly magnetizable strains could be obtained.

The strains obtained above were measured for productivity of L-lysine under conditions of incubation temperature of 30 ° C., agitation speed of 230 rpm (revolution per minute), and incubation time of 72 hours. As a result, three strains with a 3% increase in fermentation concentration were obtained compared to the parent strain KFCC 11043 (Table 2).

Triangular Flask Test Results of Corynebacterium glutamicum KFCC 11043 and Gluconic Acid Highly Magnetizable Strain Strain Fermentation Concentration (g / ℓ) OD562 KFCC 11043 52.1 20 CJG-7 53.5 22 CJG-14 53.5 23 CJG-18 53.8 21

As shown in Table 3, the strains having high magnetization in the gluconic acid of the present invention increased the fermentation concentration by more than 3% compared to the parent strain KFCC 11043, especially in the case of CJG-18 fermentation concentration is significantly higher than the parent strain It was found that the improvement. In addition, it can be seen that the strains having high magnetization to gluconic acid of the present invention have a higher absorbance (OD562) value in proportion to cell proliferation compared to the parent strain KFCC 11043.

In addition, as a result of smearing the gluconic acid-containing medium to reconfirm the growth rate in the gluconic acid of the three strains obtained above, the mother strain produced colonies in 5 days (see Table 1) compared to 3 days in the CJG-18 strain. It was confirmed that colonies were formed and the other two strains were faster than the parent strain, but showed a slow growth rate compared to CJG-18.

According to the present invention, by mutating the mother strain Corynebacterium glutamicum (L-lysine productivity) (Coynebacterium glutamicum) by obtaining a mutant strain (CJG-18, etc.) to increase the magnetization to the gluconic acid, It can be usefully used to improve the productivity of L- lysine by improving the fermentation capacity compared to the strain.

Claims (2)

Corynebacterium having the L- lysine productivity Com (Corynebacterium glutamicum) to it by artificial mutation in the parent strain four gluconic acid productivity and L- lysine, characterized in that a greater chemical conversion character of the Corey tumefaciens glue Tommy Corynebacterium glutamicum CJG-18 strain (Accession No. KFCC-11245). Derived by the Corynebacterium Com (Corynebacterium glutamicum) with L- lysine productivity as the parent strain and the Corynebacterium Com (Corynebacterium glutamicum) strain, characterized in that a greater chemical conversion character of the gluconate A method for producing L-lysine, which is cultured in a medium, L-lysine is accumulated in the culture, and L-lysine is collected from the culture.