WO2000009661A1

WO2000009661A1 - Novel microorganisms and method for producing l-threonine using the same

Info

Publication number: WO2000009661A1
Application number: PCT/KR1998/000341
Authority: WO
Inventors: In Suk Choi; Hyo Hun Yi; Jae Chun Han; Byung Rak Lim; Bun Sam Lim
Original assignee: Daesang Corp
Current assignee: Daesang Corp
Priority date: 1998-08-14
Filing date: 1998-10-28
Publication date: 2000-02-24
Anticipated expiration: 2001-02-14
Also published as: KR100270509B1; ZA9810331B; KR20000013852A

Abstract

An Escherichia coli mutant (KCCM-10133), which is resistant to α-methylserine and diaminosuccinic acid and susceptible to fluoropyruvate and requires L-methionine and L-isoleucine at once for growth, is cultured 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 and diaminosuccinic 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 even in a poor aerobic condition.

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 have been disclosed. For example, Japanese Pat. Publication No. Heisei 5- 10076 teaches use of a recombinant DNA 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. In order to efficiently obtain L-threonine, Escherichia sp. microorganisms are usually cultured under aeration with stirring. As the preexisting Escherichia sp. Microorganisms are proliferated, they need more oxygen. However, the dissolved oxygen in the culture is not fully replenished by current aeration techniques, so exhaustion of the dissolved oxygen occurs, causing anaerobic fermentation. Where the bacteria is grown in an anaerobic condition, acetic acid is accumulated in the culture, lowering the productivity of L-threonine.

Disclosure of the Invention

Through thorough and intensive research, the present inventors found that a large quantity of L-threonine is accumulated in the culture of a mutant of DSM 454, a. Escherichia sp., susceptible to fluoropyruvate and resistant to diaminosuccinic acid and α-methylserine, even under poor aeration.

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 able to efficiently produce L-threonine even under an anaerobic condition.

It is another object of the present invention to provide a method for producing L-threonine using the novel strain. The novel strain of the present invention, a mutant of DSM 454, can overcome the problem attributable to the physical factor upon culturing and improve the productivity of L-threonine.

Why the novel strain of the present invention accumulates much more L-threonine in culture compared with the mother strain, is not clearly verified, but is, to our knowledge, attributed to the following facts: the resistance to -methylserine which is an analogue of L-threonine allows the novel strain to surmount the inhibition or suppression for L-threonine in

L-threonine biosynthesis pathway; the citric acid cycle, through which microorganisms generally obtain energy from the oxidation of sugars, is less activated in the novel strain owing to the susceptibility to fluoropyruvate than in the parent strain, so less oxygen is required for growth; and thus, the reduced number of the cycle allows phosphoenolpyruvate carboxylase to supply more L-threonine precursors.

In addition, the resistance of the novel strain to aminosuccinic acid overcomes the inhibition or suppression of the enzymes involved in the L- threonine biosynthesis, contributing accumulation of a large quantity of L- threonine in culture. Best Modes for Carrying Out the Invention

The novel strain of the present invention, which is susceptible to fluoropyruvate and resistant to diaminosuccinic acid and α-methylserine, is mutated from Escherichia coli ATCC 21272, which requires L- methionine and L-isoleucine at once for growth. For this, the E. coli ATCC 21272 is first treated with chemical mutagens, NTG (N-methyl-N -nitro-N-nitroso guanidine) and DES (diethylsulfate). After the chemical treatment, replicas of the colonies thus obtained are made on a minimal agar plate and a minimal agar plate containing 40 mM of α-methylserine and, then, incubated at 37 for 2-3 days. The colonies which grow on the agar plates supplemented with α-methylserine are isolated and compared in microbiological properties with the parent strain which grows in a minimal broth. This mutant strain which is resistant to α-methylserine was named DSM 454. The strain DSM 454 is subjected to mutation, again, after which its replicas are made on a minimal agar plate and a minimal agar plate containing 40 mM of fluoropyruvate. After incubation at 37 °C for 2-3 days, selection is made for the colonies which grow on the plate devoid of fluoropyruvate, but not on the plate supplemented with fluoropyruvate. The microbiological properties of this selected strain are compared with those of its parent strain DSM 454. The same mutation procedure as above is repeated for the selected strain, followed by culturing it on an agar plate which contains 2.5 g/L of diaminosuccinic acid. The colonies which grow on the agar plate are isolated and named DSM 9806.

A complete broth for the selection of the mutant strain has a composition comprising yeast extract 1%, 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 described above, as much as 40 mM of each of α-methylserine and fluoropyruvate and 2.5 g/L of diaminosuccinic acid are preferably used for screening the colonies which are resistant to them. As for the composition of the minimal plates on which the microbiological properties of mutant and parent strains are compared, it comprises fructose 5.0%, ammonium sulfate 1.4%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1%, 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 9806 of the present invention was deposited in Korean Culture Center of Microorganisms on July 16, 1998 (Deposition No. KCCM-10133). As shown in Table 1, below, the novel strain DSM 9806 (KCCM-10133) requires L-methionine and L-isoleucine for growth, like the strain ATCC 21272, and resistant to α-methylserine like the parent strain DSM 454. However, the novel strain is different from the comparative strains in that it is susceptible to fluoropyruvate as well as resistant to diaminosuccinic acid.

TABLE 1

The comparison of characteristics of KCCM 10133 and its parent strains

note ^* growth state after being cultured on the minimal agar plates for 24 hours. - not grow, + grow, ++ well grow, +++ prosper

As for growth of the novel strain DSM 9806 (KCCM-10133) and its parent strain DSM 454, it can be measured by absorbance at 610 nm, as usual. For this, after being cultured for 72 hours with agitation, each of them is 50-folds diluted. The novel strain DSM 9806 and its parent strain DSM 454 show the absorbance of 0.603 and 0.907, respectively, as measured by a spectrophotometer, such as Beckman DU-70. Concerning the accumulation concentration of L-threonine, 16.32 mg/ml are measured for the novel strain DSM 9806 while 12.35 mg/ml for the parent strain DSM 454. Accordingly, the strain of the present invention produces more L-threonine than the parent strain even in a poor growth state.

It is certain that the significantly increased productivity of the novel strain DSM 9806 (KCCM-10133) compared with its parent strain DSM 454 is attributed to the fact that the inhibition or suppression of L- threonine in its biosynthesis is overcome by the resistance of the novel strain to α-methylserine, an analogue of L-threonine. However, it is a partial reason. The citric acid cycle, which involves oxidation of a two- carbon acetyl unit to carbon dioxide and water and provides some precursors for amino acids, is less activated in the novel strain of the invention than common microorganisms owing to the susceptibility to fluoropyruvate. So, the novel strain requires less oxygen and the fewer numbers of the citric acid cycle allow phosphoenolpyruvate carboxylate to provide a precursor for L-threonine sufficiently. In addition, the resistance to diaminosuccinic acid is thought to give a contribution to the increased productivity. That is, by virtue of the resistance to diaminosuccinic acid, the novel strain of the present invention can overcome the inhibition or suppression of the enzymes involved in L-threonine biosynthesis.

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. 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 vvm and stirring at 500-700 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

Strains used : DSM 9806 (KCCM-10133) and its parent strain DSM 454. 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 18 xl85mm 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 9806 and its parent strain DSM 454 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 9806 (KCCM-10133) and its parent strain DSM 454 at an amount of 1%. The strains were 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 16.32 mg/ml in the novel strain DSM 9806 (KCCM-10133) culture and 12.35 mg/ml in the parent strain DSM 454 culture.

EXAMPLE II

Strains used : DSM 9806 (KCCM-10133) and its parent strain DSM 454. 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 Primary pre-cultures of DSM 9806 (KCCM-10133) and DSM 454 were obtained in the same manner as that of Example I. They were 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 secondary pre-cultures. 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. After being cooked, the secondary cultures of DSM 9806 (KCCM-10133) and DSM 454 each were inoculated at 2% and incubated at 30 °C for 72 hours with aeration at 0.8-1.5 wm and stirring at 700 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 80.6 mg/ml in the novel strain DSM 9806 (KCCM-10133) culture and 42.6 mg/ml in the parent strain DSM 454 culture. 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 68.51 mg per ml ofthe culture ofDSM 9806 (KCCM-10133) and 36.21 mg per ml ofthe culture of DSM 454.

EXAMPLE III

Inocula of DSM 9806 (KCCM-10133) and DSM 454 were obtained in the same medium conditions and pre-culturing conditions as those of Example II. The production culturing procedure of Example II was repeated, except that the shaking speed was reduced to 500 rpm. After fermentation, L-threonine was found to be accumulated at an amount of 75.41 mg/ml in the novel strain DSM 9806 (KCCM-10133) culture and 37.4 mg/ml in the parent strain DSM 454 culture. 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 64.10 mg per ml ofthe culture of DSM 9806 (KCCM-10133) and 31.79 mg per ml ofthe culture of DSM 454.

COMPARATIVE EXAMPLE I

DSM 454 and ATCC 21272 were cultured under the same conditions as those of Example I except for using, instead of glucose, sucrose in the pre-culture and production culture media for ATCC 21272.

After fermentation, L-threonine was found to be accumulated at an amount of 12.67 mg/ml in the DSM 454 culture and 4.02 mg/ml in the ATCC 21272 culture.

Industrial Applicability

As described hereinbefore, the novel strain ofthe present invention is so specifically altered in L-threonine biosynthesis pathway that it can overcome the conventional problem upon E. coli culturing, that is, the problem that, as the culturing proceeds, accumulation of acetic acid and reduction of L-threonine productivity result from the exhaustion of the dissolved oxygen in the culture because ofthe limit of aerating agitation. Therefore, according to the present invention, a large quantity of L- threonine can be obtained even in a poor aeration state. Different from the conventional techniques in which the limit of aerating agitation tends to be solved by controlling medium amount, the method ofthe invention solves the culturing problems resulting from the inevitable physical factors by altering the microbiological properties.

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 9806 with a deposition No. KCCM-10133 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 α-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 9806 (KCCM-10133).

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