WO2000009660A1

WO2000009660A1 - Novel microorganisms and method for producing l-threonine using the same

Info

Publication number: WO2000009660A1
Application number: PCT/KR1998/000340
Authority: WO
Inventors: Jae Chun Han; Young Kil Choi; Sang Tae Park; In Suk Choi; Byung Rak Lim; Bun Sam Lim
Original assignee: Daesang Corporation
Priority date: 1998-08-14
Filing date: 1998-10-28
Publication date: 2000-02-24
Also published as: ZA9810327B; KR20000013853A; KR100270510B1

Abstract

From a strain which is resistant to α-methylserine and diaminosuccinic acid, and susceptible to fluoropyruvate and requires L-methionine and L-isoleucine at once for growth, an Escherichia coli mutant (KCCM-10132) is mutated so as to have resistance to L-threonine and L-glutamic acid additionally. This mutant is culturel in media containing, as a carbon source, sugars, such as glucose, under an aerobic condition, to accumulate a large quantity of L-threonine in the culture.

Description

NOVEL MICROORGANISMS AND METHOD FOR PRODUCING L-THREONINE USING THE SAME

Technical Field

The present invention relates, in general, to a novel microorganism and a method for producing L-threonine using the same and, more particularly, to a Escherichia sp. which is resistant to α-methylserine, diaminosuccinic acid, L-threonine and L-glutamic acid and susceptible to fluoropyruvate and requires L-methionine and L-isoleucine at once for growth and a method for producing a large amount of L-threonine from the bacteria efficiently even in the late state of fermentation.

Background Art

L-threonine, an essential amino acid, is a second limited amino acid of rice. As well known, this amino acid is used as a component for, e.g., amino acid transfusion liquid or general amino acid tablets, and as a nutrient. In recent, there has been greatly increasing a demand for L- threonine because it, together with L-lysine, is used as an additive of feedstuff.

Many methods of producing L-threonine through fermentation were disclosed. For example, Japanese Pat. Publication No. Heisei 5-10076 teaches use of a recombinant DN A which contains the genetic information for asparto kinase, homoserine kinase, homoserine dehydrogenase, and threonine synthase in production of a great quantity of threonine from a L-threonine-producing Serratia sp. Japanese Pat. Publication No. Heisei 1-289493 discloses that a DNA taken from a Providencia sp. resistant to methionine metabolic antagonist is genetically engineered and used to increase the productivity of L-threonine. In order to produce L-threonine, there are used a threonine metabolic antagonist-resistant Escherichia sp. which requires methionine or diaminopimelic acid for growth in Japanese Pat. Publication No. Sho. 56-10037, and a strain which can grow in a medium of L-serine and ethionine in EP 91103569.9.

Disclosure of the Invention While the present inventors have being researched production of L- threonine from Escherichia coli KCCM- 10133, the productivity is significantly lowered in the late stage of culture owing to the accumulation of L-threonine and unknown by-products. The thorough and intensive research repeated by the present inventors, aiming to solve the problem, resulted in the finding that a mutant strain resistant simultaneously to L-threonine and L-glutamic acid can accumulate a larger amount of L-threonine in culture than does the parent strain.

Accordingly, it is an object of the present invention to overcome the above problems encountered in prior arts and to provide a novel strain, which is reinforced in anti-osmosis against L-threonine, unknown by-products and other amino acids, so as to efficiently produce L- threonine even in the late stage of culture.

It is another object of the present invention to provide a method for producing L-threonine using the novel strain.

Best Modes for Carrying Out the Invention

The novel strain of the present invention, which can well grow in the presence of L-threonine and L-glutamic acid, e.g., resistant to L- threonine and L-glutamic acid, is mutated from the strain DSM 9806 (KCCM- 10133) by common mutation techniques. In detail, the strain DSM 9806 (KCCM- 10133) is treated with UV or with chemical mutagens, NTG (N-methyl-N'-nitro-N-nitroso guanidine) and DES (diethylsulfate). The colonies thus obtained are streaked on complete agar plates containing 7% of L-threonine to select 1-threonine-resistant colonies. After being applied with the same mutation, the selected colonies are streaked on the complete agar plates containing 240 mM of L-glutamic acid, an intermediate in the L-threonine biosynthesis pathway, and cultured at 37 °C for 2-3 days. Thereafter, replicas of the colonies grown are made on a minimal agar plate containing 7% of L-threonine and 240 mM of L-glutamic acid and a minimal agar plate devoid of both of them. Of the colonies which survive on the agar plate containing L- threonine and L-glutamic acid, those which are clearly grown are separated, and compared with the parent strain, as to microbiological properties. This mutant strain was named DSM 9807. A complete broth for the selection of the mutant strain has a - composition comprising yeast extract 1.0%, peptone 1.0%, beef broth 0.3%, NaCl 0.5% and glucose 0.5% at pH 7.0 while a complete agar plate comprises agar 2% additionally. As for the composition of the minimal plates on which the microbiological properties of the mutant and parent strains are compared, it comprises glucose 1.0%, ammonium sulfate 0.2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, diaminopimelic acid 100 mg/L, and agar 2% at pH 7.3. L-methionine 200 mg/L and L-isoleucine 200 mg/L are respectively used in order to determine whether they are needed for the growth of the novel strain.

The novel strain DSM 9807 of the present invention was deposited in Korean Culture Center of Microorganisms on July 16, 1998 (Deposition No. KCCM-10132). As shown in Tables 1 and 2, below, the novel strain DSM 9807 (KCCM-10132) maintains the characteristics of the parent strain, including requirement of L-methionine and L-isoleucine for growth and resistance to α-methylserine and diaminosuccinic acid and susceptibility to fluoropyruvate.

TABLE 1

Resistance to L-Thr. and L-Glu. of KCCM- 10133 and KCCM-10132

Concentration Strains

L-Thr.(%) L-Glu.(m M) KCCM-10133 KCCM-10132

0 0 1.722 1.892

100 0.501 1.883

3.0 210 0.210 1.897 240 0.100 1.901

100 0.410 1.821

5.0 210 0.181 1.834 240 0.092 1.799 -A-

100 0.286 1.767

7.0 210 0.127 1.742

240 0.054 1.705 note : As for growth of the strains, it was measured by absorbance at 610 nm after culturing them in minimal broths containing L-thr. and L-glu.

TABLE 2

The comparison of characteristics of KCCM- 10133 and KCCM- 10132

Concentration (mg/L) Strains^*

L-Met L-Ile α-Me F-pyr. Di- DSM 9806 DSM 9807

Ser aminosuc. (KCCM- 10133) (KCCM-10133)

200 • • • • - _

• 200 .

200 200 40mM +++ +++

200 200 40mM 40mM + +

200 200 40mM 40mM 2.5g/L + +

: growth state after being cultured on the minimal agar plates for 24 hours. - no growth, + growth, +++ good growth

Concerning the growth and the productivity of L-threonine, the novel strain DSM 9807 (KCCM-10132) and the parent strain DSM 9806 (KCCM-10133) were compared according to L-glutamic acid concentrations, and the results are given as shown in Table 3, below. The data demonstrate that the novel strain of the present invention is superior in productivity of L-threonine to the parent strain.

TABLE 3

Growth and Productivity of KCCM- 10133 and KCCM- 10132

Strains

L-Glu Cone.³ KCCM-10133 KCCM-10132

Growth¹ 0.837 0.861

O mM

L-Thr.² 16.57 19.60

Growth 0.711 0.877

100 mM

L-Thr. 16.26 19.67

Growth 0.430 0.851

210 mM

L-Thr. 15.98 19.98

Growth 0.355 0.836

L-Thr. 16.04 20.65

Growth 0.311 0.527

270 mM

L-Thr. 12.25 12.23 note 50-fold diluted solutions of the cultures incubated for 72 hours from the production media (Example I)were measured by absorbance at 610 nm (Beckman DU-70).

² L-Thr accumulated in cultures as measured by an amino acid automatic analyzer (Hitachi L-8500A). ³ dissolved in distilled water and separately added after sterilization.

Why the novel strain of the present invention accumulates much more L-threonine in culture compared with the parent strain, is not clearly verified, but is, to our knowledge, attributed to the following facts: the improved resistance to L-threonine and L-glutamic acid allows the novel strain to surmount the feedback inhibition of the L-threonine accumulated in the culture; and L-glutamic acid plays as a osmosis protective against the object fermentation product, L-threonine, and other by-products. In fermenting the novel strain of the present invention, sucrose, glucose, raw sugar catabolites, etc are available as carbon sources while ammonia gas, ammonia water, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, etc available as nitrogen sources. Also, the culture media for the fermentation may comprise other natural nutrient sources and inorganic salts.

Under the following conditions, the novel strain of the present invention is cultured in a fermenting bath. Culturing is carried out at about 30 °C for 3-4 days with aeration at 0.8-1.5 wm and stirring at 600- 700 rpm. With ammonia water or liquified ammonia, the pH of the culture is adjusted to 6.5-7.0. After the completion of the fermentation, the L-threonine contained in the culture media can be isolated by adsorption to ion exchange resins. The elute from the ion exchange resins is treated with ethanol to give crude L-threonine crystals.

A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.

EXAMPLE 1

° Strain used : DSM 9807 (KCCM-10132) ° Pre-culture medium composition : Glucose 0.5%, Yeast Extract 1.0%, Peptone 1.0%, NaCl 0.5%, Beef Broth 0.3%, pH 7.0.

° Production medium composition : Glucose 10%, Corn liquid 3%,

Potassium dihydrogen phosphate 0.1%, Ferrous sulfate 2 mg/L,

Manganese sulfate 2 mg/L, Ammonium sulfate 0.5%, L-Methionine 200 mg/L, L-Isoleucine 200 mg/L, and Calcium carbonate 5% (separately sterilized), pH 7.0.

° Pre-Culturing

The pre-culture medium was aliquoted to 18Φxl 85mm test tubes by 5 ml and autoclaved at 121 °C for 15 min under pressure. After being cooled, the aliquots were inoculated with the novel strain DSM 9807 by use of a sterilized metal loop. They were incubated at 30 °C for 20 hours with shaking at 120 cycles per min.

° Production Culturing

The threonine production media were aliquoted to 500 ml Sakaguchi flasks by 70 ml and autoclaved at 121 °C for 15 min under pressure. After being cooled, the aliquots of the autoclaved threonine were inoculated with the pre-cultures of the novel strain DSM 9807 (KCCM- 10132)at an amount of 1%. The strain was incubated at 30 °C for 72 hours with shaking at 120 cycles per min. After fermentation, L- threonine was found to be accumulated at an amount of 19.60 mg/ml in the novel strain DSM 9806 (KCCM- 10133) culture. The same procedure as in the above was repeated for the parent DSM 454 (KCCM-10133) and L-threonine was found to be accumulated at an amount of 16.57 mg/ml.

EXAMPLE II

° Strain used : DSM 9807 (KCCM-10132) o Primary pre-culture medium composition : Same as the Pre-

Culture medium composition of Example I

° Secondary Pre-culture medium composition : Glucose 2%, Corn liquid 3%, Potassium dihydrogen phosphate 0.1%, Ferrous sulfate 2 ml/L, Manganese sulfate 2 ml/L, Ammonium sulfate 0.05%, Urea 0.6%, L- methionine 200 mg/L, L-isoleucine 200 mg/L, pH 7.0.

° Production medium composition : Glucose 10%, Corn liquid 3%, Potassium dihydrogen phosphate 0.1%, Ferrous sulfate 2 ml/L, Manganese sulfate 2 ml/L, Ammonium sulfate 0.5%, L-Methionine 200 mg/L, and L-Isoleucine 200 mg/L, pH 7.0. ° Pre-Culturing

A primary pre-culture of DSM 9807 (KCCM-10132) was obtained in the same manner as that of Example I. It was inoculated at 1% in 50 ml aliquots of the secondary pre-culture media in Sakaguchi flasks, which had been autoclaved at 121 °C for 15 min. Incubation was carried out at 30 °C for 24 hours with shaking at 120 cycles per min, to give a secondary pre-culture.

⁰ Production Culturing

2L of the production media were bottled in a 5L fermentation bath and then, autoclaved at 121 °C for 15 min under pressure. The secondary culture of DSM 9807 (KCCM-10132) was inoculated at 2% and incubated at 30 °C for 72 hours with aeration at 0.8-1.5 vvm and stirring at 550 rpm. Sugars were added so as to maintain the sugar concentration of the media at 1-3%. The media were adjusted into pH 6.5-7.0 with ammonia water. After fermentation, L-threonine was found to be accumulated at an amount of 95.60 mg/ml in the novel strain DSM 9807 (KCCM-10132) culture. In the same manner as the above, L-threonine was produced from the parent strain DSM 9806 (KCCM-10133) and, measured to be 74.14 mg/ml. 1 L of each of the cultures was centrifuged to harvest the bacteria which were, then, isolated through adsorption into ion-exchange resins and purified to yield L-threonine crystals at an amount of 78.46 mg per ml of the culture of DSM 9807 (KCCM-10132) and 63.02 mg per ml of the culture of DSM 9806 (KCCM-10133).

Industrial Applicability

In contrast to the strain DSM 9806 (KCCM-10133) whose ability to produce L-threonine is lowered in the late stage of fermentation owing to the feedback inhibition of the products, including organic acids and other amino acids as well as L-threonine, the strain DSM 9807 (KCCM- 10132) maintains its ability to produce L-threonine, in the late stage of the fermentation by virtue of the increased resistance to L-threonine as well as L-glutamic acid, known as an osmosis protective.

The present invention has been described in an illustrative manner, and it is to be understood the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims

1. A novel microorganism Escherichia coli

DSM 9807 with a deposition No. KCCM-10132 from Korean Culture Center of Microorganisms on July 16, 1998, which can produce L- threonine.

2. A method for producing L-threonine, in which an Escherichia sp. microorganism requiring L-methionine and L-isoleucine at once for growth, is mutated so as to be resistant to L-threonine and L-glutamic acid as well as ╬▒-methylserine and diaminosuccinic acid, and susceptible to fluoropyruvate and subjected to fermentation.

3. The method as set forth in claim 2, wherein said microorganism is Escherichia coli DSM 9807 (KCCM-10132).