KR20020029213A - E.coli variety producing L-threonine and producing method of L-threonine using thereof - Google Patents

Abstract

PURPOSE: An L-threonine producing mutant strain and a method for producing L-threonine using the same strain are provided, thereby producing the L-threonine in higher yield. CONSTITUTION: The L-threonine producing mutant strain is obtained by transduction between E. coli(ATCC 21148) and E. coli(ATCC 21151) to obtain E. coli HT101; treating E. coli HT101 with NTG as a mutagen to select E. coli HT201 having AHV resistance; spreading the selected E. coli HT201 on a medium without isoleucine and DAP to select E. coli MT101 having slow growth rate; culturing E. coli MT101 in a medium containing high concentration of aspartate and homoserine to obtain E. coli MT201(KCTC 18048P). The method for producing L-threonine using E. coli MT201(KCTC 18048P) comprises the steps of: inserting pyruvate carboxylase gene derived from Corynebacterium glutamicum(ATCC 31831) into a plasmid pTrc99A to construct a recombinant plasmid pPC; transforming E. coli MT201(KCTC 18048P) with the plasmid pPC to produce E. coli MT201/pPC; and fed batch fermenting E. coli MT201/pPC in a medium containing glucose and sodium citrate.

Description

L-threonine-producing mutants and L-threonine-producing method using the same

The present invention relates to L-threonine production mutant strain and L-threonine production method using the same. More specifically, the present invention was obtained by obtaining a mutant strain having resistance to amino acid requirements and amino acid analogs from known E. coli and inserting a pyruvate carboxylase structural gene obtained from the test strain into a plasmid. Recombinant plasmids are introduced into the mutant strain to produce high-efficiency L-threonine, which is an essential amino acid. The present invention relates to a fermentation method for producing L-threonine with high efficiency using this strain.

Threonine is a chemical that has two free carbons and four types of stereoisomers. Among them, L-threonine is an essential amino acid that is indispensable to our body. L-threonine is widely used as an additive in animal feed and food, and is used as a medicinal product (eg, amino acid preparations, etc.) as a fluid preparation, a synthesis and a production raw material. However, it is not easy to chemically separate only L-type threonine in nature. The amino acids produced by the fermentation method are all L-type, and since the specific amino acids can be produced and accumulated in a high yield by a simple process from a cheap raw material, and are easily separated, they are widely used as industrial amino acid production methods.

α-amino-β-hydroxyvaleric acid (AHV) is an L-threonine analogue that inhibits the growth of bacteria. AHV is structurally similar to L-threonine, and enzymes that are inhibited by the over-accumulated L-threonine are also inhibited by AHV. As a result, cell growth in the presence of AHV is inhibited through this pathway. E. coli strains resistant to AHV (Escherichia coli),Brevibacterium (Brevibacterium), Corynebacterium (CorynebacteriumIt is isolated from microorganisms such as the genus), and this resistance mutation is characterized by the release of feedback inhibition by the accumulation of L-threonine.

For mass production of L-threonine, a method of expressing a large amount of structural genes of a group of enzymes correlated to L-threonine synthesis using gene recombination methods in general [US Pat. No. 4,278,765. (1981), Japanese Patent Laid-Open No. 61-70983, EP 0,131,171, Appl. Microbiol. biotechnol., 34. 617-622 (1991)] and the like, but these methods have a problem that the stability of the plasmids produced during the culture for the mass production of L-threonine is greatly reduced. In order to improve the stability of the L-threonine operon-containing plasmid to improve the stability of several methods [US Pat. No. 4,278,765. (1981), Gene, 31.275-277. (1984), Republic of Korea Patent No. 107376] is known, In order to maintain stability, methods such as adding antibiotics during large-scale fermentation were also used. However, the plasmids produced by the above-mentioned methods show undesirable aspects in terms of product cost and stability in the fermentation process for mass production of L-threonine.

The inventors have attempted mass production of L-threonine to improve this. In order to produce L-threonine, the glucose metabolism was altered to achieve an efficient mass production system of L-threonine. First of all, the present invention induced an artificial mutant having resistance to amino acid nutritional components and amino acid analogs for mass production of L-threonine. In addition, in order to mass-produce L-threonine, an oversynthesis of oxaloacetate, a precursor, is required (FIG. 1). For this, Corynebacterium glutamicum (Corynebacterium glutamicum) The pyruvate carboxylase gene obtained from ATCC31831 was inserted into plasmid pTrc99A where the pyruvate carboxylase structural gene is expressed. E. coli recombinant plasmid pPC expressing the structural gene of pyruvate carboxylase L-threonine high-efficiency production strain was obtained by transforming and introducing into MT201 strain. In order to improve the productivity of L-threonine using a recombinant strain, growth of the strain is essential, and thus, the productivity of L-threonine was examined while adding glucose and citrate at a constant ratio for sufficient supply of citrate.

Accordingly, it is an object of the present invention to provide an L-threonine producing E. coli MT201 resistant to amino acid nutritional components and amino acid analogs. Another object of the present invention by using the recombinant E. coli MT201 / pPC improved L-threonine production capacity and strain growth produced by introducing the recombinant vector pPC constructed by inserting the pyruvate carboxylase gene into pTrc99A It is to provide a fermentation culture method for producing a recombinant L-threonine with high efficiency.

The object of the present invention is to use E. coli ATCC 21148, E. coli ATCC 21151, etc. to obtain a mutant strain for improving the production of L-threonine Met^-, Ile^leaky, DAP^leaky, Thr analogue^r, Asp^r, Hom^rAfter obtaining the present invention mutant Escherichia coli MT201 having the characteristics of, Corynebacterium glutamicum (Corynebacterium glutamicum) After obtaining a pyruvate carboxylase gene derived from ATCC 31831 and inserting the gene into the plasmid pTrc99A to construct a recombinant vector pPC, the recombinant vector pPC was introduced into the above-mentioned artificial strain E. coli MT201 to obtain L-threonine. Production Recombinant Escherichia coli MT201 / pPC was prepared and fermented by adding glucose and sodium citrate, which are carbon sources, to the medium component of the strain, thereby achieving L-threonine with high efficiency.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a TCA cycle (cycle) showing the production process of the threonine of the present invention.

Figure 2 is a known strain Corynebacterium glutamicum (Corynebacterium glutamicum) The cleavage map of the recombinant plasmid pPC constructed by inserting the pyruvate carboxylase structural gene recovered and purified from ATCC 31831 into plasmid pTrc99A is shown.

The present invention is directed to E. coli ATCC 21148 (Met) to obtain a mutant strain that is resistant to amino acid nutritional requirements and amino acid analogs to improve L-threonine production^-, DAP^-) And E. coli ATCC 21151 (Ile^-, DAP^-) Strains were crossed by intercellular transduction to yield Met^-, Ile^-, DAP^-E. coli HT101 strains exhibiting characteristics were obtained, and E. coli HT201 (Met), an L-threonine-producing strain resistant to AHV, was obtained by treating Mutant NTG with the E. coli HT101 strain.^-, Ile^-, DAP^-, Thr analogue^r), And then the selected E. coli HT201 strain was diluted and smeared in a medium without addition of isoleucine and DAP.^-, Ile^leaky, DAP^leaky, Thr analogue^r) And E. coli MT101 (E. coli) MT201 (Met.^-, Ile^leaky, DAP^leaky, Thr analogue^r, Asp^r, Hom^rSelecting); The pyruvate carboxylase gene, which produces oxaloacetate directly from pyruvate of the corresponding system, was identified as Corynebacterium glutamicum (Corynebacterium glutamicumObtaining from ATCC 31831; Digesting plasmid pTrc99A with NcoI and HindIII restriction enzymes and then ligating the pyruvate carboxylase gene to construct a recombinant plasmid pPC for pyruvate carboxylase expression; Colied strain MT201 (Met of the present invention)^-, Ile^leaky, DAP^leaky, Thr analogue^r, Asp^r, Hom^r) To introduce the recombinant plasmid pPC into the strain by electric field transformation method to prepare L-threonine high efficiency E. coli MT201 / pPC; In order to compare the production of L-threonine and the growth of strains E. coli MT201 and recombinant E. coli MT201 / pPC, the culture of fermenter added with glucose, a single carbon source, and then measuring the production and quantitative analysis of L-threonine; In order to determine the effect of citrate compounds on L-threonine production and strain growth of the strain E. coli MT201 and recombinant strain MT201 / pPC, the production of L-threonine was measured and quantitatively analyzed after fermentation of citrate and glucose-containing flasks. Performing; Determination of L-threonine production and strain growth changes of the strain E. coli MT201 and recombinant strain MT201 / pPC according to the addition ratio of glucose and sodium citrate, and L-threonine production in the milk culture of the recombinant strain MT201 / pPC and Investigate strain viability.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Acquisition of Aspartate Metabolic Mutant MT201

In order to obtain aspartate-based metabolic mutant strains to improve the production of L-threonine, Escherichia coli was mutated in the following order to obtain Escherichia coli MT201 mutant strains. E. coli was cultured at 37 ° C. using LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose).

Escherichia coli ATCC 21148 (Met^-, DAP^-) And E. coli ATCC 21151 (Ile^-, DAP^-) The strain was crossed by intracellular transduction to achieve Met^-, Ile^-, DAP^-Among the strains exhibiting characteristics, E. coli HT101 strain was obtained. Treatment of the E. coli HT101 strain with mutant NTG, among the 350 strains resistant to threonine analogue (AloV) AHV HT201 (Met^-, Ile^-, DAP^-, Thr analogue^r) Strains were selected. The selected HT201 strain was repeatedly diluted and smeared in a medium without adding isoleucine and DAP, and thus, a slow growing strain MT101 (Met^-, Ile^leaky, DAP^leaky, Thr analogue^r) Was selected. In addition, the mutant strain MT101 was grown in a medium containing high concentrations of aspartate and homoserine to obtain a mutant strain of the present invention having resistance to high concentrations of aspartate and homoserine. This is Escherichia coli MT201 (Met^-, Ile^leaky, DAP^leaky, Thr analogue^r, Asp^r, Hom^r). The properties of the strains used for L-threonine production are shown in Table 1.

Characteristics of the strains used in the present invention Strain characteristic Production rate (g / L) Escherichia coli ACTT21148 Met ^-, DAP ^- 12.3 Escherichia coli ACTT21151 Ile ^-, DAP ^- 10.2 Escherichia coli HT101 Met ^-, Ile ^-, DAP ^- 13.4 Escherichia coli HT201 ^{Met -, Ile -, DAP -} , Thr analogue r E. coli MT101 ^{Met -, Ile leaky, DAP leaky} , Thr analogue r 19.0 E. coli MT201 Met ^-, Ile ^leaky, DAP ^leaky, Thr analogue ^{r, r} Asp Hom ^r 24.0 Escherichia coli JM109 rec A1 sup E44 end A1 hsd R17 gyr A96 rel A1 thi △ ( lac - pro AB)

The mutation-induced aspartate-based metabolic mutant strain MT201 was deposited in the gene bank of the Biotechnology Research Institute under accession number KCTC 18048P as of October 6, 2000.

Example 2 Preparation of plasmid and preparation of transformant for preparation of L-threonine high efficiency production strain

Mass production of L-threonine requires the overproduction of the precursor oxaloacetate (Figure 1). To this end, an expression plasmid was prepared for the excessive production of pyruvate carboxylase, which functions to produce oxaloacetate directly from pyruva-te of the corresponding system.

First step. Isolation and Purification of the pyruvate carboxylase gene

The pyruvate carboxylase structural gene is known as Corynebacterium glutamicum (C).orynebacterium glutamicum) It was obtained by PCR using primers from ATCC 31831. Pyruvate carboxylase genepycFor further information, see Koffas et al., Peters-Wendisch et al. Microbiol. Biotechnol., 50 (3), 346-352, (1998), Microbiology, 144 (Pt 4), 915-927, (1998).HindEach primer having a III restriction enzyme site was prepared. Amplification of the pyruvate carboxylase gene was performed by using the Delta Cycler II system (Ericomp, USA) to obtain a fragment of about 3.4 kb containing the pyruvate carboxylase gene. These DNA fragments are restriction enzymesHindTreated with III toHindFragmentation of the pyruvate carboxylase structural gene was achieved by fragmentation with DNA having a III cleavage site.

Second step. Construction of Recombinant Vector pPC Inserted with the Pyruvate Carboxylase Gene

After the plasmid pTrc99A having a molecular weight of 4.2 kb of pyruvate carboxylic expressing decarboxylase Structural genes in Escherichia coli First, after smoothed by cutting with restriction enzymes NcoI by attaching Hind III linker, cleaved with finally a restriction enzyme Hind III is part the pyruvate A 3.4 kb DNA fragment containing the pyruvate carboxylase gene was linked to construct a recombinant plasmid pPC for 7.5 kb expression and a cleavage map is shown in FIG. 2. The properties of the plasmids are shown in Table 2.

Properties of Plasmids Used in the Invention Plasmid characteristic pTrc99A Ptrc, Amp ^r , 4.2kb pPC pyruvate carboxylase gene, Ptrc, Amp ^r , 7.5kb

Third step. Preparation of the transformant MT201 / pPC incorporating the recombinant plasmid pPC

In order to increase the production of L-threonine, the recombinant plasmid pPC containing the pyruvate carboxylase gene of the second step is introduced into the present invention mutant Escherichia coli MT201 by electric field transformation (electroporation) to transform recombinant E. coli MT201 / pPC. Was prepared. Electric field transformation is as follows.

First, the mutant strain E. coli MT201 of the present invention was precultured in an LB medium overnight, and then inoculated in 0.01 mL in 10 mL of LB medium and incubated until the absorbance (OD _{600 nm} ) was about 0.8. The culture solution was centrifuged, only the cells were collected, suspended in 5 ml of ice-cold sterilized glycerolol solution (10% greserol, 90% ultrapure water), and centrifuged again. After repeating the above operation, 0.1 ml of ice-cold sterilized solution of glycerol was added to the precipitated cells, and then suspended, and only 0.04 ml was taken and used for transformation. 0.1 μg of the recombinant plasmid pPC was added to the obtained cell suspension and mixed well, and then subjected to a single electric shock under a voltage of 1,500 V, a current of 25 mA, and a resistance of 150 Ω using Electroporator II (Invitrogen, USA). Immediately, these cell solutions were mixed in 1 ml of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 20 mM glucose) and incubated for 1 hour at 37 ^° C. . Appropriate amount of this culture was plated on LB plate medium to which ampicillin was added, and then the transformants grown were selected to obtain a recombinant recombinant E. coli MT201 / pPc.

Example 3 Measurement of L-Threonine Production of L-Threonine Production Mutant Escherichia Coli MT201 and Transgenic Recombinant E. Coli MT201 / pPC

The ninhydrin test method was used as a method for quantifying L-threonine. Take 1.75 ml of ninhydrin solution, 0.25 ml of sodium citrate-HCl buffer (pH 3.0), and 0.3 ml of centrifuged culture supernatant, dilute 100 times with distilled water, and react for 8 minutes at 70 ℃. _It was measured at OD ₅₇₆ nm. It was measured simultaneously with an amino acid analyzer as needed.

First step. Comparison of L-Threonine Production of MT201 and MT201 / pPC in a Single Carbon Source in Fermentor Cultures

L-threonine production of the present invention L-threonine mutant Escherichia coli MT201 and transformed recombinant E. coli MT201 / pPC was measured and compared under a single carbon source of glucose addition medium.

As a seed medium for fermenter culture, 10 g of glucose per liter, 10 g of tryptone, 7 g of yeast extract, 1 g of (NH ₄ ) ₂ SO ₄ , ₂ g of NaCl ₁₀ g, and KH ₂ PO ₄ were added thereto, and the reaction was performed at 30 ° C. at 500-700 rpm. .

In a fed-batch culture using a strain of E. coli MT201, L-threonine producing strain, in a 5L fermenter containing only glucose as a carbon source, the maximum yield of L-threonine was 81 g / L (Table 3). The above-mentioned recombinant recombinant prepared by introducing the recombinant plasmid pPC of Example 2 containing pyruvate carboxylase gene in E. coli MT201 which is a variant strain of the present invention for the purpose of increasing the production of L-threonine Escherichia coli MT201 / pPC was cultured in the medium containing only glucose as a carbon source, and as a result, L-threonine production of 48.9 g / L was obtained (Table 3). Therefore, it can be seen that E. coli MT201 / pPC reduced both growth and L-threonine production than E. coli MT210 of the present invention under glucose as a single carbon source.

Strain Growth (OD _660nm ) Production rate (g / L) E. coli MT201 53.5 81.0 Escherichia coli MT201 / pPC 41.6 48.9

Second step. Effect of Citrate Compounds on L-Threonine Production and Growth in Flask Culture

The strain E. coli MT201 and the recombinant strain E. coli MT201 / pPC were confirmed in the first step that the cell growth was reduced when only glucose is supplied as a carbon source (Table 3). It is necessary to supply the carbon skeleton necessary for growth as glucose rotates the TCA circuit through the glycolytic process. Pyruvate carboxylase is active to supply oxaloacetate, but the carbon skeleton necessary for microbial growth is insufficient. (Fig. 1). Therefore, to solve this problem, citric acid, a metabolic product of the TCA cycle, was added in the form of a salt. At this time, the culture medium for the flask with 70g glucose per liter, yeast extract 2g, (NH ₄ ) ₂ SO ₄ 20g, MgSO ₄ 1g, KH ₂ PO ₄ 1.5g, FeSO ₄ 10mg, CaCO ₃ 30g, MnSO ₄ 10mg It was carried out in. The carbon source of the flask medium was either glucose (5%) or glucose (3%) + citrate (2%).

The production of L-threonine was investigated in the case of glucose addition without citrate and in flask culture with glucose and citrate (sodium citrate or ammonium citrate). Inventive mutant strain E. coli MT201 and recombinant E. coli MT201 / pPC were grown overnight in LB and LBAmp plate medium, and then platinum was inoculated into a 500 ml Erlenmeyer flask containing 60 ml of the flask fermentation broth at 30 ° C. at 180-200 rpm. L-threonine was produced by culturing in miracle conditions. Ampicillin was added at a concentration of 50 mg / L if necessary. After fermentation was completed, L-threonine concentration was quantitatively analyzed.

The experimental results are shown in Table 4.

Add glucose process No treatment Ammonium citrate Sodium citrate Strain Growth output Growth output Growth output Coliform MT201 3.0 24.0 0.3 4.0 8.8 28.5 E. coli MT201 / pPC 2.7 14.5 0.5 5.0 8.9 32.7 Growth (OD _660nm ) Production (g / L)

Through this, it can be seen that the medium in which glucose and sodium citrate as a carbon source is added to the transformed recombinant E. coli MT201 / pPC strain in the flask culture has an excellent effect on L-threonine production and growth of the strain.

Third step. Changes in L-Threonine Production by Addition of Glucose and Sodium Citrate

In order to determine the optimal concentration of sodium citrate affecting L-threonine production, the flask medium was adjusted to give a total of 5% glucose and sodium citrate, a single carbon source, in a proportion of 5% for the L- of MT201 and MT201 / pPC. The threonine production was compared to each other. Recombinant Escherichia coli MT201 / pPC was grown overnight in LB and LBAmp plate medium, and then one platinum of the strain was inoculated into a 500 ml Erlenmeyer flask containing 60 ml of the flask fermentation medium of the second step and aerobic conditions of 180-200 rpm at 30 ° C. Incubated to produce L-threonine. Ampicillin was added at a concentration of 50 mg / L if necessary. After fermentation was terminated, L-threonine concentration was quantitatively analyzed.

The experimental results are shown in Table 5.

Strain Escherichia coli MT201 / pPC Glucose: Sodium citrate addition ratio 2.0: 3.0 2.5: 2.5 3.0: 2.0 3.5: 1.5 4.0: 1.0 5.0: 0.0 Production rate (g / L) 30.5 31.0 32.5 43.5 24.8 14.5

According to the addition ratio of glucose and sodium citrate, the production of L-threonine of recombinant E. coli MT201 / pPC was efficiently increased, and the optimal carbon source addition ratio for L-threonine production was glucose: sodium citrate = 3.5: 1.5.

Fourth step. Transgenic Recombinant E. Coli MT201 / pPC L-Threonine Production in Dairy Cultures

Glucose and sodium citrate were added to the medium according to the optimum addition ratio of the carbon source and sodium citrate obtained in the third step (glucose: sodium citrate = 3.5: 1.5), and then inoculated with the recombinant strain E. coli MT201 / pPC and in a 5L fermenter. L-threonine was produced by culturing in a fed-batch culture. The culture was performed using 50 g of glucose per liter, 2.5 g of yeast extract, 10 g of (NH ₄ ) ₂ SO ₄ , 1 g of MgSO ₄ , ₂ g of KH ₂ PO _{4 ,} 10 mg of FeSO ₄ , 5 mg of MnSO ₄ , and glucose, methionine, KH in culture. ₂ PO ₄ was further fed. If necessary, 50-100 mg / L amino acid or ampicillin was added at a concentration of 50 mg / L.

After incubating the recombinant Escherichia coli MT201 / pPC overnight in a LBAmp plate medium, one platinum was inoculated into a 500 ml Erlenmeyer flask containing 60 ml of spawn medium and incubated sufficiently at 30 ° C. at 180-200 rpm to incubate 2 L of the culture medium. 5L fermenter containing the inoculation was inoculated in an amount corresponding to 5-10% of the culture medium to 30 ℃, pH 6.8 while supplying ammonia water for adjustment, and maintained at aeration rate of 1vvm. According to the consumption of glucose during the cultivation was incubated by fed-batch fermentation method additionally supplying glucose, methionine, KH ₂ PO ₄ and the final culture was analyzed and expressed as the production of L-threonine. As a result of the above conditions, an excellent recombinant strain producing an excessive amount of L-threonine was produced, and the yield thereof was also able to obtain a breakthrough 105 g / L.

The experimental results are shown in Table 6.

Strain Carbon source Growth (OD _660nm ) Production rate (g / L) Coliform MT201 glucose 53.5 81 E. coli MT201 / pPC glucose 41.6 49 E. coli MT201 / pPC Glucose, sodium citrate 35.0 105

like this, Plasmid pPC in which the pyruvate carboxylase structural gene was inserted into Escherichia coli MT201, which is an L-threonine producing mutant strain prepared by inducing amino acid requirement and inducer resistant mutant strains for mass production of L-threonine The transgenic recombinant E. coli MT201 / pPC prepared by introduction showed that the production of L-threonine and the growth of strains were significantly improved when the medium contained 3.5: 1.5 carbon source glucose: sodium citrate.

In addition, by introducing a recombinant vector inserting genes of various metabolites into E. coli MT201, which is an amino acid requirement and amino acid-resistant substance resistant strain of the present invention, useful strains such as L-threonine high efficiency production strain in Example 2 can be produced. . In the present invention, only the L-threonine producing strain MT201 / pPC has been described by way of example, but the scope of the present invention is not limited thereto.

As is apparent from the above examples, the present invention utilizes E. coli to obtain aspartate-based metabolic mutant strains, such as amino acid requirement and analogue resistant strains such as AHV, that is, L-threonine producing strain E. coli MT201 (Thr). analogue^r, Met^-, Ile^leaky, DAP^leaky, Asp^r, Hom^r) And the published strain Corynebacterium glutamicum (C.glutamicum) A recombinant plasmid pPC was constructed by introducing a pyruvate carboxylase structural gene purified and recovered from ATCC 31831 into plasmid pTrc99A, and transforming the recombinant plasmid pPC into the mutant MT201 for mass production of L-threonine. E. coli, an excellent recombinant strain that produces excess L-threonine by introduction into the process MT201 / pPC was obtained, and the strain was fermented in a medium fed with glucose and sodium citrate, and the result was a recombinant strain MT201 / with improved production and strain growth of L-threonine mutant MT201 and L-threonine. Providing a fermentation production method of L-threonine using pPC has an excellent effect that can be used as animal feed, food additives and medical preparations, so it is a very useful invention in the livestock industry, food processing industry and pharmaceutical manufacturing industry.

Claims (2)

Crossing Escherichia coli ATCC 21148 and Escherichia coli ATCC 21151 by intercellular transduction to obtain Escherichia coli HT101; Selecting Escherichia coli HT201 resistant to AHV by treating Mutant NTG with E. coli HT101; Diluting and smearing the selected E. coli HT201 in a medium without adding isoleucine and DAP to select a slow-growing E. coli mutant strain MT101; And The Escherichia coli MT101 (Esherichia coli MT201) (KCTC 18048P), which is obtained by growing E. coli MT101 in a medium containing high concentrations of aspartate and homoserine. Disclosure strain Corynebacterium glutamicum (Corynebacterium glutamicum) The recombinant plasmid pPC constructed by inserting the pyruvate carboxylase gene derived from ATCC 31831 into the plasmid pTrc99A was introduced into the aspartate-based metabolic mutant Escherichia coli MT201 described in claim 1 to produce L-threonine recombinant E. coli MT201 / pPC. L-threonine fermentation production method characterized in that L-threonine is produced by fermentative fermentation culture in a medium containing glucose and sodium citrate mixed.