HU207536B - Process for producing 1-threonine with fermentation - Google Patents

Abstract

PURPOSE:To inexpensively produce L-threonine in good yield by culturing a microorganism belonging to the genus Escherichia and having a resistance against valine analog and simultaneously having L-threonine-producing ability in a prescribed medium. CONSTITUTION:A microorganism belonging to the genus Escherichia such as Escherichia coli H-7538, H-7600, etc., having a resistance against valine analog and simultaneously L-threonine-producing ability is cultured in an ordinary synthetic medium or that of natural source. The culture is preferably carried out in aerobic conditions of shaking incubation, submerged stirring incubation, etc., at about 25-35 deg.C and at neutral pH. Precipitate of bacterial cell, etc., is removed from the culture mixture after finishing culture and ion exchange treatment method, concentration method, salting method, etc., are jointly used to recover L-threonine from the cultured mixture.

Description

The present invention relates to a process for the production of L-threonine by fermentation using a microorganism of the genus Escherichia which is resistant to valine analogue and capable of producing L-threonine.

L-threonine can be used not only as an active ingredient in pharmaceutical amino acid formulations, but also as an additive in animal feed.

To date, various processes for the preparation of L-threonine by fermentation have become known. Examples include a process using an Escherichia genus and a microorganism susceptible to borrelidine (Japanese Patent Application Publication 6572/76), a microorganism belonging to the genus Escherichia and needing diamine-pimelic acid and methionine to grow, threonine feedback inhibition (Japanese Patent Application Laid-Open No. 10037/81), a method of the genus Serratia that is deficient in threonine dehydrogenase and is resistant to a threonine-metric antagonist (Japanese Patent Laid-Open Publication No. 48 197/77), Method using a microorganism of the genus Corynebacterium resistant to α-amino-β-hydroxy-valeric acid and S- (2-aminoethyl) -L-cysteine and requiring methionine (U.S. Pat. No. 19087/72) Japanese Patent Application), a process using a microorganism of the genus Brevibacterium which is resistant to α-amino-β-hydroxyvaleric acid and S- (2-aminoethyl) -L-cysteine and which requires L-leucine (Publication No. 31,093/75). Japanese Patent Application), a method of using a microorganism belonging to the genus Brevibacterium, resistant to α-amino-β-hydroxyvaleric acid and S- (2-aminoethyl) -L-cysteine and requiring L-isoleucine and L-lysine (224684 / Japanese Patent Application Publication No. 83), a process using a microorganism resistant to at least one member of the family Escherichia of the group consisting of rifampicin, lysine, methionine, aspartic acid and homoserine, which has a reduced ability to degrade L-threonine (Publication No. 33693/87) Japanese Patent Application and European Patent Application Publication No. 0237 819) and an Esch a method using a microorganism resistant to rifampicin, lysine, methionine, aspartic acid and homoserine of the genus Erichia (Japanese Patent Application Publication No. 37295/89 and European Patent Application Publication No. 0301 527).

On the other hand, microorganisms resistant to the valine analog are known from the genus Corynebacterium or Brevibacterium capable of producing 2-thiazolylalanine and capable of producing L-valine [Agr. Biol. Chem., 39 (6), 1319-1322 (1975)], a microorganism of the species Serratia marcescens resistant to aminobutyric acid and capable of producing L-valine (Journal of Bacteriology 106 (2), 493-499 (1971)). , a microorganism of the genus Brevibacterium capable of producing α-aminobutyric acid and L-valine (Japanese Patent Application Publication No. 276,286/88) and a microorganism capable of producing valine resistant and isoleucine of the Escherichia coli species [ Microbiol. Rev., 43, 42 (1979)] and the like.

There is a need for a process for the low cost production of L-threonine on an industrial scale.

The inventors have undertaken extensive research to develop a process for the production of Ltreonine by fermentation and have discovered that L-threonine production capacity can be increased by introducing resistance to a valine analogue into an L-threonine-producing microorganism belonging to the genus Escherichia.

The present invention provides a process for the production of L-threonine by fermentation comprising culturing a microorganism of the genus Escherichia that is resistant to valine analogue and capable of producing L-threonine in a culture medium until the accumulation of L-threonine and recovering L-threonine.

The microorganism of the genus Escherichia, which is resistant to the valine analogue and capable of producing L-threonine, can be used in the process of the present invention.

Valine analogs include valine, valine hydroxamate, N-methylvaline, α-aminobutyric acid and the like.

Strains resistant to the valine analogue are obtained by conferring the above-mentioned resistance property on a L-threonine-producing microorganism of the genus Escherichia by conventional mutation techniques. The strain resistant to the valine analogue includes strains Escherichia coli H-7538, H-7600 and H-8014, each of which is derived from Escherichia coli strain H-4258. Strain H-4258 is deposited with the FRI (Fermentation Research Institute (= Fermentation Research Institute), Agency of Industrial Science and Technology, Japan) under FERM BP-985.

The L-threonine-producing microorganisms of the genus Escherichia have the advantage of producing other amino acids as a by-product.

The following specific examples for the preparation of the above strains are given:

Escherichia coli strain H-7538:

Escherichia coli Η-4258 (requires diaminopimellic acid and methionine. Resistance to α-amino-β-hydroxyvaleric acid and rifampicin) by conventional mutation treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (0, 2 mg / ml, 30 ° C, 30 minutes) and then on a minimal medium containing 1 g / l DL-valine hydroxamal [5 g / l glucose, 2 g / l ammonium chloride, 2 g / l potassium dihydrogen phosphate, 0.1 g / l magnesium sulfate heptahydrate, 20 mg / l ferric sulfate heptahydrate, 50 mg / l diaminopimelic acid, 50 g / l DL-methionine and 20 g / l agar, pH 7.2 ] were plated and cultured at 30 ° C for 2-6 days until mutant colonies resistant to DL-valine hydroxamate were able to grow on this medium. 50 mutant strains were then harvested and subjected to L-threonine production assay

HU 207,536 Β to select the best L-threonine productive strain.

The strain H-7538 so selected has been deposited with the FRI since December 21, 1989 under the accession number FERM BP-2696.

Escherichia coli strains H-7600 and H-8014:

Similarly, an α-amino-butyric acid-resistant or L-valine-resistant mutant strain was obtained from the H-4258 strain using the same mutation technique as described above except that 2 g / l of α-amino-butyric acid was replaced with DL-valine hydroxamate in the medium. and 1 g / liter Lvalin was used. The so-selected α-aminobutyric acid-resistant strain H-7600 and L-valine-resistant strain H-8014, which have the best L-threonine productivity, are deposited under the accession number FRM BP-2698 and FERM BP-3161, respectively. .

The growth rates of the above strain cultured in minimal agar plates containing DL-valine hydroxamate, α-aminobutyric acid or L-valine at 30 ° C for 24 hours at various concentrations are shown in Table 1.

Table 1

Valine-to-analog (G / l). Tribe H-4258 H-7538 H-7600 H-8014 DL-valine hydroxyl roxamát 0 + + + 4- 0.1 - + + 4- 1.0 - + - 4- a-amino-butyric acid 0 + + + + 0.5 - + + + 2.0 - - 4- + L-valine 0 + + 4- + 0.1 - -I- 4- 4- 1.0 - 4- ± 4-

+: adequate growth; ±: moderate growth; no growth

The L-threonine is prepared by culturing the above-mentioned microorganisms in conventional manner on synthetic media containing carbon sources, nitrogen sources, inorganic salts, growth factors and the like until L-threonine accumulates in the culture and k-threonine is collected. there.

Carbon sources include carbohydrates such as glucose, fructose, lactose, molasses, starch hydrolyzate or raw sugar and the like. and organic acids such as pyruvic acid, acetic acid, propionic acid, formic acid, fumaric acid, malic acid and the like. They can be used. Depending on the assimilation capacity of the cultured microorganism, an alcohol such as glycerol and ethanol may also be used as a carbon source.

Nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., amines, other nitrogenous compounds, peptones, meat extracts, maize starch hydrolysates, casein hydrolysates, casein hydrolysates, their products, etc. They can be used.

Inorganic compounds include potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, potassium carbonate and the like. They can be used.

Culturing is carried out under aerobic conditions, in shake culture or under agitation, in immersion culture and the like. The pH of the medium may be in the range of 5-9, and it is preferable to maintain it at a near neutral level. The pH is adjusted with calcium carbonate, inorganic or organic acids, alkaline solutions, ammonia, pH buffer solutions and the like. L-threonine can accumulate in the culture within 2-7 days. After completion of the culture, the precipitates, e.g., cells, by centrifugation, etc., are removed. is removed from the culture and L-threonine can be recovered by the combined application of ion exchange treatment, concentration, salting and the like.

The invention is illustrated by the following example.

Example 1:

L-Threonine production experiments were performed using Escherichia coli strains H-4258, H-7538, H-7600 or H-8014 as inoculum strains.

The inoculum strain was cultivated at 30 ° C for 16 hours with 20 g / l glucose, 10 g / l peptone, 10 g / l yeast extract, 2.5 g / l sodium chloride and 0.5 g / l diaminopimelic acid. containing 7.4 inoculum medium. 100 ml of this inoculum culture is inoculated into a 1 liter small fermenter containing 1 liter of fermentation broth of the following composition and cultured at 30 ° C with 800 rpm stirring and 1 liter / min aeration. hours. In the meantime, 60% by weight glucose solution is added to the fermentation broth and the pH of the broth is adjusted to 6.5 with aqueous ammonium hydroxide solution during culture.

After completion of the culture, the amount of accumulated Ltreonine is determined by high performance liquid chromatography. The results are shown in Table 2 (from single fermentation).

The fermentation broth (pH 7.4) has the following composition:

glucose: 40 g / liter, ammonium sulfate: 12 g / liter, potassium dihydrogen phosphate: 2 g / liter, magnesium sulfate heptahydrate: 1 g / liter, diaminopimelic acid: 0.9 g / liter, DL-methionine: 0.3 g / liter, corn syrup: 5 g / liter. It was obtained by cultivating strain H-7538, L-threonine

Centrifugation of one liter of fermentation broth containing 3000 rpm for 10 minutes removes cells and other contaminants from the solution. The supernatant was obtained with a highly acidic ion-exchange resin, Diaion SK IB (Hydrogen Cycle, Mitsubishi Kasé Corporation,

HU 207 536 Β

(Product of Japan) is passed through a packed column to adsorb L-threonine to the resin. The column was then washed with water and eluted with 0.5 N aqueous ammonium hydroxide; the fractions containing L-threonine were pooled. The combined fractions were concentrated and ethanol was added to the evaporated solution. The mixture was allowed to stand after cooling to give 30.0 g of crystalline L-threonine of at least 98% purity.

Table 2 jq

Tribe L-threonine (g / l iter) H-4258 36.3 H-7538 38.5 H-7600 40.3 H-8034 39.4

The purity of the resulting L-threonine was determined by dissolving 1 g of authentic L-threonine in 100 ml of ammonium hydroxide to prepare a control sample. Also, 1 g of the L-threonine prepared in the example was dissolved in 100 ml of ammonium hydroxide. Both samples were analyzed by high performance liquid chromatography and peaks were measured. Compared to the peak area of the control sample and the test sample, this was 0.98. Because the area under the peaks is proportional to the purity of the material, the L-threonine produced by the process of the present invention is at least 98% pure.

Claims (2)

PATIENT INDIVIDUAL POINTS A method for producing L-threonine by fermentation, characterized in that it is resistant to valine analogue Escherichia coli H-7538 (FERM BP-2696), Escherichia coli H-7600 (FERM BP-2698) or Escherichia coli H8014 (FERM BP-3161). the microorganism is cultured in a suitable medium until L-threonine accumulates in the culture and the L-threonine is recovered. The method according to claim 1, wherein the valine analogue is a Escherichia coli strain resistant to valine hydroxamate, α-aminobutyric acid, valine or N-methylvaline. Published by the National Bureau of Invention, Budapest Responsible for the publication: dr. Gabriella Szvoboda-Dománszky Head of Department ARCANÜM Bt-BUDAPEST