KR100338375B1 - L-threonine producing microorganism and methods for preparing L-threonine using the same - Google Patents

Abstract

The present invention relates to E. coli Ly1053 (KFCC-11132) capable of producing high concentrations and high purity of L-threonine without the addition of an osmotic counterpart, and a method for preparing L-threonine using the same.

Description

L-threonine producing microorganism and methods for preparing L-threonine using the same

The present invention relates to E. coli (Escherichia coli, hereinafter referred to as E. coli) Ly 1053 (KFCC-11132) capable of producing L-threonine, and to a method for producing L-threonine using the same. More specifically, the present invention relates to E. coli Ly 1053 (KFCC-11132) resistant to lysozyme, a cell wall degrading enzyme by mutating from E. coli KFCC-10718 (Korean Patent Publication 92-8365), and L-threonine using the same. It relates to a manufacturing method.

L-threonine is an essential amino acid, which is used as a feed additive because it improves the feed quality by adding it to the feed, and is used as a food additive, a pharmaceutical sap, and a synthetic raw material of medicines. Currently, the method of producing L-threonine is mainly composed of fermentation methods using nutrient-forming strains and metabolic control strains. When L-threonine is produced by fermentation, artificial mutants derived from wild strains of E. coli, coryneform bacteria, Seritia bacteria and Providencia strains are used. Such mutant strains are known to have amino acid analogs and drug-resistant mutants or artificial mutants which have been given diaminopimeric acid, methionine, lysine, and isoleucine nutrient composition (see Japanese Patent Application Laid-Open No. 2-219582, Appl. Microbiol.Biotechnol., 29, 550-553 (1988), Korean Patent Publication No. 92-8365).

However, there are limitations in enhancing fermentation yield, such as delay in fermentation time, when amino acid analogs, drug resistance, amino acid demands, etc., are imparted, and cell walls are weakened. Is generated. If a large amount of these amino acid by-products accumulate, it is difficult to crystallize the L-threonine purification process and the purification yield is deteriorated. In order to solve this problem, Korean Patent Publication No. 96-16873 discloses a method of adding an osmotic counterpart, proline or choline chloride, but this method also requires the addition of expensive substances such as proline and choline chloride. There is a disadvantage of being raised.

Thus, the present inventors studied a method that can strengthen the cell wall to overcome the above problems. As a result, a mutant strain resistant to lysozyme, a cell wall degrading enzyme, improves L-threonine yield, suppresses the production of by-product L-glutamic acid, and improves the sugar consumption rate even without adding an osmotic counterpart. The present invention has been accomplished by discovering that L-threonine can be produced economically.

Accordingly, an object of the present invention is to produce a high concentration and high purity of L-threonine without the addition of an osmotic counterpart, E. coli mutant strain resistant to lysozyme and the production of L-threonine using the same To provide a way.

The present invention relates to an E. coli artificial mutant strain resistant to lysozyme and a method for producing L-threonine using the same. More specifically, the present invention relates to an artificial mutant strain having resistance to lysozyme as a parent strain of E. coli KFCC 10718 strain and a method for producing L-threonine using the same.

The E. coli artificial mutant strain of the present invention is suspended in a buffer using the E. coli KFCC 10718 strain as a parent strain, and then treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce artificial mutations. After treatment by suspending in the lysozyme-added buffer, the treatment solution was cultured and treated with lysozyme again, and then single colonies formed by plating them on the medium were selected. More specific screening methods are described in detail in Example 1.

L-threonine productivity and byproduct L-glutamic acid production and sugar consumption of the resistant strain and known parent strain KFCC 10718 were compared. As a result, the resistant strain against lysozyme inhibited the production of by-products compared to the KFCC 10718 strain, it was confirmed that it can produce high concentration and high purity L-threonine by improving the sugar consumption rate (Experimental Example 2 and Experimental Example 3). Reference).

In the following Examples, the present invention will be described in more detail, but the following Examples do not limit the scope of the present invention.

Example 1. Selection of resistant strain through artificial mutation

Suspended at 10 ⁷ -10 ⁸ cell / ml in 0.1M phosphate buffer (pH 7.0) or 0.1M citrate buffer (pH 5.5) using the E. coli KFCC-10718 strain as parent strain and N-methyl-N'-nitro N-nitrosoguanidine was added to a final concentration of 100-500 mg / L and treated for 20 minutes at room temperature to 33 ° C. to induce artificial mutations.

For selection of resistant strains against lysozyme, the cells were suspended in 25 mM Tris buffer (pH 8.0) containing 2 g / L of lysozyme and treated for 24 hours. The treated solution was incubated for 14 hours in a minimal medium (Table 1) containing methionine (100 mg / L) and isoleucine (100 mg / L) and treated with lysozyme again. Single colonies were selected by plating on LB agar medium (Table 2).

Minimum medium composition Composition Concentration (g / L) glucose 5.0 Disodium Phosphate 6.0 Potassium phosphate 3.0 Sodium chloride 0.5 Ammonium chloride 1.0 Magnesium sulfate 0.5 Calcium chloride 0.01 L-methionine 0.1 L-isorucin 0.1 pH 7.0

LB Badge Composition Composition Concentration (g / L) Trypton 10 Yeast Extract 5 Sodium chloride 5 Agar 18 pH 7.0

Experimental Example 1 Measurement of Lysozyme Tolerance

The strains selected in the above example and the parent strain E. coli KFCC-10718 were suspended in Tris buffer solution (pH 8.0) containing 2 g / L of lysozyme, and the mortality rate was measured for 24 hours to compare resistance. The results are shown in Table 3.

Comparison of% lysozyme-resistant strain and parental strain E. coli KFCC-10718 Strain % Mortality 0 hours 8 hours 16 hours 24 hours Lysozyme resistant strains 0 30 65 80 E. coli KFCC-10718 0 60 90 98

As can be seen from the table, it can be seen that the lysozyme resistant strain of the present invention survives longer than the parent strain E. coli KFCC-10718. This means that the lysozyme resistant strain of the present invention is a cell wall enhanced strain.

Experimental Example 2 Comparison of By-Product Production

Lysozyme-resistant and parental E. coli KFCC-10718 were incubated overnight in LB plate medium, and then inoculated into a 250 ml Erlenmeyer flask containing 20 ml of threonine production medium (Table 4) for 48-72 hours at 33 ° C. Conditions were shaken to obtain L-threonine, and the amount of L-glutamic acid as a byproduct was compared. The results are shown in Table 5.

Composition of threonine production medium Composition Concentration (per liter) glucose 100 g Potassium phosphate 1.0 g Ammonium Sulfate 28 g Magnesium sulfate 0.5g Manganese sulfate 5 mg Iron sulfate 5 mg Yeast Extract 2.0 g L-methionine 0.15 g Calcium carbonate 30 g pH 7.0

L-Threonine Yield (%) and L-Glutamic Acid Production (g / L) Comparison Strain L-Threonine Yield (%) L-glutamic acid production amount (g / L) Lysozyme resistant strains 28.0 0.9 E. coli KFCC-10718 24.5 5.3

As can be seen from the table, the lysozyme resistant strain of the present invention can be seen that the production amount of by-product L- glutamic acid is very small compared to the parent strain E. coli KFCC-10718.

The resistant strain for lysozyme was named Ly 1053, and was deposited with the Korean spawn association on January 29, 2000 and received Accession No. KFCC-11132.

Experimental Example 3 Sugar Consumption Comparison

Ly 1053 (KFCC-11132) and lysozyme resistant strain Ly. Coli KFCC-10718 were incubated overnight in LB plate medium, and then inoculated in a 250 ml Erlenmeyer flask containing 20 ml of threonine production medium (Table 4) at 33 ° C. The amount of sugar consumption and L-threonine production were compared by shaking culture in aerobic conditions for 60 hours. The results are shown in Table 6.

Comparison of sugar consumption and L-threonine production Strain E. coli Ly 1053 (KFCC-11132) E. coli KFCC-10718 Sugar concentration 0 hours 100 100 14 hours 87 95 24 hours 60 72 48 hours 5 14 60 hours 0 5 L-threonine concentration 0 hours 0 0 14 hours 2.5 1.2 24 hours 11.0 4.0 48 hours 27.0 18.2 60 hours 28.0 24.0

As can be seen from the table, it can be seen that the sugar consumption rate and L-threonine production of the Ly 1053 (KFCC-11132) strain of the present invention was significantly improved compared to the parent strain E. coli KFCC-10718.

As described above, the strain Ly 1053 (KFCC-11132) of the present invention is a strain capable of producing high concentration and high purity L-threonine without adding an osmotic counterpart. It can efficiently produce L-threonine.

Claims (2)

E. coli Ly 1053 (KFCC-11132), which produces L-threonine. A method of producing L-threonine from the culture by culturing the strain according to claim 1.