JPH0313875B2 - - Google Patents

Description

[Detailed description of the invention]

<Industrial Application Field> The present invention relates to a method for producing L-threonine by a fermentation method. L-threonine is one of the essential amino acids and is important as a pharmaceutical and feed additive, and an inexpensive method for producing it is desired. <Prior Art> There is no known method for producing L-threonine by fermentation using microorganisms belonging to the genus Providencia. However, L-threonine can be produced by fermentation using microorganisms belonging to the former Proteus genus, which was partially reclassified to the Providencia genus in Virgies Manual of Systematic Bacteriology Volume 1 (1984).
Method using isoleucine auxotrophic strain (Special Publication No. 43-
4440) and a method using microorganisms that are resistant to α-amino-β-hydroxyvaleric acid and require isoleucine (Japanese Society of Agricultural Chemistry Abstracts, p. 9 (1970)) are known. <Problems to be Solved by the Present Invention> However, the production and accumulation concentration of L-threonine by these methods, or the concentration of L-threonine from raw materials such as sugar,
The threonine production yield was not fully satisfactory. <Means and effects for solving the problems> As a result of intensive research into a method for producing L-threonine with even higher productivity, the present inventors found that a microorganism belonging to the genus Providencia and having the ability to produce L-threonine. The inventors have discovered that by imparting resistance to L-isoleucine antimetabolites, the accumulated concentration and production yield of L-threonine can be significantly improved, and the present invention has been achieved. It is not yet known that the production amount and yield of L-threonine can be significantly improved by using microorganisms imparted with resistance to isoleucine antimetabolites as in the present invention. That is, the present invention involves culturing a microorganism that is resistant to isoleucine antimetabolites among microorganisms belonging to the genus Providencia and having the ability to produce L-threonine, producing and accumulating L-threonine in the culture solution, and This is a method for producing L-threonine by a fermentation method, which is characterized by collecting L-threonine from a liquid. Next, the present invention will be explained in detail. The microorganisms used in the present invention belong to the genus Providencia (Versey's Manual of Systematic Bacteriology, Vol. 1, Nos. 495-496).
(1984)) and is a microorganism resistant to isoleucine antimetabolites. Here, isoleucine antimetabolite inhibits (1) the growth of microorganisms belonging to the genus Providencia, and the growth inhibition is L-
substances that are recovered by the addition of isoleucine, or
(2) A substance that exhibits suppressive and inhibitory effects on the L-isoleucine biosynthesis system. Here, examples of isoleucine antimetabolites include isoleucine hydroxamate, thiaisoleucine, o-
Examples include methylthreonine and β-methylnorleucine. As long as it has such properties, it also falls within the scope of the present invention, even if it has properties such as other requirements and drug resistance. In addition to the above-mentioned resistance, the microorganism used in the present invention preferably has L-isoleucine or auxotrophy or leaky auxotrophy for L-isoleucine, and the threonine biosynthesis system controls the feedback of threonine. It is resistant to Typical microorganisms used in the present invention include, for example, Providencia letgelii TP6-28
(FERM P-8348). This mutant strain is a Providencia retogeri
Providencia retogeri TP3- with α-amino-β-halodroxyvaleric acid resistance, L-ethionine resistance, L-isoleucine requirement or leaky type requirement, and L-leucine requirement obtained from ATCC21118 (L-isoleucine requirement) 105, and is a mutant strain that is resistant to thiaisoleucine, an isoleucine antimetabolite. Mutant strains can be induced relatively easily by conventional mutation treatment methods. That is, to obtain a mutant strain resistant to isoleucine antimetabolites, the parent strain must be irradiated with ultraviolet rays, or a mutagenic agent (for example, N-methyl-N'-nitro-N
- After treatment with nitrosoguanidine, ethyl methanesulfonic acid, etc.), a strain that grows significantly better than the parent strain can be obtained on a solid medium containing an isoleucine antimetabolite in an amount that does not allow the parent strain to grow sufficiently. . In the present invention, the isoleucine antimetabolite-resistant strain refers to a strain that has stronger resistance than its parent strain, and preferably the isoleucine antimetabolite concentration is such that the relative growth after 24 hours of the parent strain is 40% or less. The relative growth rate is 50% or more when cultured in a medium containing For example, in the case of a thiaisoleucine-resistant strain, a strain whose growth rate after 24 hours when cultured in a medium supplemented with 5mM thiaisoleucine is 50% or more of that without the addition of thiaisoleucine is called a thiaisoleucine-resistant strain. Here, the degree of growth is measured by measuring the absorbance of the culture solution at 660 nm, and the absorbance of the culture solution without the addition of isoleucine antimetabolite of each strain is 100%.
It shall be expressed as relative absorbance when expressed as . The medium for producing L-threonine in the present invention is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and other organic trace components as necessary. Carbon sources include glucose, fructose,
Hydrolyzed products of starch and cellulose, sugars such as molasses, organic acids such as fumaric acid, citric acid, succinic acid, etc., alcohols such as glycerol, etc.
~15%, organic ammonium salts such as ammonium acetate, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonia gas, aqueous ammonia, urea, etc. as nitrogen sources, 0.5 to 4.0% as organic micronutrients. contains 0.001 to 0.4% of required substances such as L-leucine, or 0 to 4% of corn staple liquor, peptone, yeast extract, etc. as required.
A medium containing appropriate amounts of each is used. In addition to these, potassium phosphate, magnesium sulfate,
Trace components such as ferrous sulfate heptahydrate and manganese sulfate 4-6 hydrate are added in small amounts. Cultivation is performed under aerobic conditions. During cultivation, the pH of the medium was adjusted to 5 to 9, the temperature was adjusted to 24 to 37°C, and the temperature was adjusted to 48°C.
Favorable results are obtained with shaking or aeration for ~120 hours. To collect L-threonine from a culture solution, for example, the culture solution from which bacterial cells have been removed is adjusted to pH 2 with hydrochloric acid, and then the solution is passed through a strongly acidic cation exchange resin, and the adsorbed components are eluted with dilute ammonia water. After ammonia, concentrate. L-threonine can be obtained by adding alcohol to this and collecting the generated crystals under refrigerated storage. <Example> Hereinafter, the present invention will be specifically explained with reference to Examples. Example 1 A (Isolation of thiaisoleucine-resistant strain) Providencia retogeri TP3-105 (α-amino-β-haloidroxyvaleric acid resistant, L-isoleucine auxotrophy or leaky auxotrophy, L
N-methyl-N'-nitro-
N-nitrosoguanidine treatment (300μg/ml,
After 20 minutes at 30°C, the cells were transformed into D,L-
Thiaisoleucine 1.5g/, L-leucine 2
g/, L-valine 2g/agar medium (completely synthetic medium containing glucose 0.5%, ammonium sulfate 0.1%, monopotassium phosphate 0.3%, dipotassium phosphate 0.7%, magnesium sulfate heptahydrate 0.01%) ) was applied. Next, the culture was carried out at 30° C. for 5 to 7 days, and the resulting large colonies were isolated to obtain a thiaisoleucine-resistant strain (Providencia retogeri TP6-28). B (Resistance level of thiaisoleucine-resistant strains) Each strain shown in Table 1 below was cultured with shaking at 30°C for 16 hours using a liquid broth medium, and the grown cells were collected and thoroughly washed with physiological saline. . This bacterial cell suspension was mixed with thiaisoleucine 0, 2.5,
Minimal medium (medium composition: glucose 0.5%, ammonium sulfate 0.1%, phosphoric acid
Potassium 0.3%, dibasic potassium phosphate 0.7%, magnesium sulfate heptahydrate 0.01%, L-isoleucine 0.001%, L-leucine 0.05%, L-valine 0.05%)) was inoculated into 5 ml and kept at 30℃. After culturing for 24 hours, the growth rate of each strain was examined. The result is
As shown in Table 1. However, a commercially available thiaisoleucine (manufactured by Sigma) was used. In the thiaisoleucine-resistant strain (TP6-28) used in the method of the present invention, growth is not inhibited by thiaisoleucine and it has acquired resistance to thiaisoleucine, compared to the parent strain Providencia retogeri TP3-105. That is clear.

[Table] The absorbance of the culture solution of each strain without the addition of thiaisoleucine is expressed as 100%.
Example 2 Each strain shown in Table 2 was pre-cultured in a liquid bouillon medium at 30°C by shaking for 16 hours, and then 500ml containing 50ml of the main fermentation medium with the following composition, which had been steam sterilized at 115°C for 10 minutes, was prepared. Inoculate into a shake flask and incubate under the conditions of 30℃, 110rpm, and 5cm amplitude.
Cultured for 90 hours. Fermentation medium Glucose (separately sterilized) 8% (NH ₄ ) ₂ SO ₄ 2.5% KH ₂ PO ₄ 0.1% MgSO ₄・7H ₂ O 0.04% Fe 2ppm Mn 2ppm L-isoleucine 0.005% L-leucine 0.08% CaCO ₃ ( Separately sterilized) 4% PH 7.0 (neutralized with KOH) After culturing, the L-threonine concentration in the solution from which bacterial cells and calcium carbonate were removed was determined using an automatic amino acid analyzer (JEOL JLC200A). Table 2 shows the results. The results shown are obtained.

[Table] The threonine production yield was expressed as the weight yield of the produced threonine relative to the consumed glucose. Providencia retogeri TP6-28 according to the present invention
In this case, both the accumulated concentration and production yield of L-threonine were significantly improved. Example 3 Each strain shown in Table 3 was grown in a liquid broth medium.
After culturing with shaking at 30°C for 16 hours, 800 ml of the fermentation medium of Example 2 containing 0.5% (NH ₄ ) ₂ SO ₄ and 4.0% glucose was dispensed into a small glass jar fermenter. were inoculated at a concentration of 10%. Aerated agitation culture was started at 30°C, 800 rpm, and aeration rate of 1 vvm. PH adjustment and nitrogen source supply are performed with 25% ammonia water, and the PH is 6.5 ~
I kept it at 8.0. Glucose, KH ₂ PO ₄ ,
When cultured for 70 hours while appropriately adding MgSO ₄ .7H ₂ O, L-leucine and L-isoleucine, the results shown in Table 3 were obtained.

[Table] Bacterial cells were removed from the culture solution of Providencia retogeri TP6-28, and 500 ml of it was passed through a column of strong cation exchange resin Diaion SK.1B (H type).
After washing the column with water, the adsorbed components of the column were eluted with 2N aqueous ammonia, decolorized, and concentrated under reduced pressure. Ethanol was added thereto, the mixture was cooled, and the resulting crystals were collected and dried to obtain 32.3 g of L-threonine crystals with a purity of 96% or higher. <Effects of the Invention> The method of the present invention makes it possible to produce L-threonine with high yield and high accumulated concentration, and makes it possible to produce L-threonine at a lower cost.

Claims (1)

[Claims] 1 Belongs to the genus Providencia,
Resistant to isoleucine antimetabolites and L
- Cultivating a microorganism capable of producing threonine,
A method for producing L-threonine by a fermentation method, which comprises producing and accumulating L-threonine in a culture solution, and collecting L-threonine from the culture solution. 2. The method for producing L-threonine by the fermentation method according to claim 1, wherein the isoleucine antimetabolite is thiaisoleucine.