JPH0346114B2 - - 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. <Prior art> As a method for producing L-threonine by a fermentation method using microorganisms belonging to the genus Providencia, L-
A method using a mutant strain that is resistant to ethionine (Japanese Unexamined Patent Publication No. 61-216698), a method that uses a mutant strain that requires L-leucine (Japanese Unexamined Patent Publication No. 61-260891), a mutant strain with decreased pyruvate kinase. (Japanese Unexamined Patent Publication No. 62-44191), a method using a mutant strain resistant to aspartate antimetabolites (Japanese Patent Application Laid-Open No. 62-44192), and a method using a mutant strain resistant to isoleucine antimetabolites. A method using the method (Japanese Patent Application Laid-Open No. 62-44193) is known. In addition, Bergey's Manual of Systematic Bacteriology Volume 1 (1984) describes a method for producing L-threonine by fermentation using microorganisms belonging to the former genus Proteus, which has been partially reclassified to the genus Providencia. , a method using an L-isoleucine auxotrophic strain (Japanese Patent Publication No. 43-4440) and a method using a strain resistant to α-amino-β-hydroxyvaleric acid and L-isoleucine.
- A method using a microorganism that requires isoleucine (Japan Society of Agricultural Chemistry Proceedings, p. 9 (1970)) is known. <Problems to be Solved by the Invention> However, in all conventional methods, a large amount of alanine and valine are produced as by-products, which reduces the purification yield of L-threonine, and it is difficult to obtain highly pure L-threonine. This required a complicated purification process, which was disadvantageous for industrial use. <Means and effects for solving the problems> As a result of intensive studies by the present inventors on a method for maintaining high productivity of L-threonine and at the same time suppressing the side effects of valine, the present inventors found that L-threonine, which belongs to the genus Providencia, The present inventors have discovered that the by-production of valine can be significantly suppressed by using a mutant strain of microorganisms capable of producing threonine that is sensitive to acetohydroxy acid synthase inhibitors, thereby achieving the present invention. That is, it belongs to the genus Providencia of the present invention,
Cultivating a mutant strain that has the ability to produce L-threonine and is sensitive to an acetohydroxy acid synthase inhibitor, producing and accumulating L-threonine in the culture solution, and collecting L-threonine from the culture solution. This is a method for producing L-threonine using fermentation ability, which is characterized by: Here, as the acetohydroxy acid synthase inhibitor, for example, (methyl 2-[[[[(4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl]benzoate (hereinafter referred to as
sulfomethyuron methyl), 2-thiazolealanine, L-valine, L-isoleucine, and the like. Among them, sulfomethyuron methyl is preferably used. The microorganisms used in the present invention belong to the genus Providencia (Manual of Systematic Bacteriology, Vol. 1 (1984), Vol.
(according to pages 475-496) are microorganisms sensitive to acetohydroxy acid synthase inhibitors. As long as it has such properties, it falls within the scope of the present invention, regardless of requirements and drug resistance.
In particular, in addition to acetohydroxy acid synthase inhibitors, auxotrophy or leaky auxotrophy for L-isoleucine or L-leucine, resistance to threonine analogs such as α-amino-β-hydroxyvaleric acid, and resistance to methionine analogs such as ethionine. has an effective effect on the ability to produce L-threonine, so microorganisms having some or all of these characteristics are preferably used. Typical mutant strains used in the present invention include, for example, Providencia retogeri
SVSS-29 (FERM P-9349) is mentioned. This mutant strain is Providencia letgeri
OTR28-31 (α-amino-β-hydroxyvaleric acid resistant, L-isoleucine auxotrophic, L-ethionine resistant, L-leucine auxotrophic, thiaisoleucine resistant, oxythiamine resistant) was used as the parent strain and was subjected to normal mutation treatment. It is a mutant strain that is sensitive to sulfomethyuron methyl. Mutant strains can be induced by conventional mutation treatment methods. In other words, to obtain a mutant strain that is sensitive to acetohydroxy acid synthase inhibitors,
After irradiation with ultraviolet light or treatment with mutagenic agents (e.g., N-methyl-N-nitro-N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), acetohydroxy acid is synthesized at a concentration that allows the parent strain to grow. It is sufficient to collect a strain that grows significantly less well than the parent strain on a solid medium containing an enzyme inhibitor. In the present invention, the acetohydroxy acid synthase inhibitor-sensitive strain refers to a strain that is more sensitive to acetohydroxy acid synthase inhibitors than the parent strain; for example, in the case of a sulfomethyuron methyl-sensitive strain, the parent strain is A strain that can grow but can hardly grow in a medium supplemented with 0.1 mg of sulfomethyluron methyl is called a sulfomethyluron methyl sensitive strain. 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,
Starch and cellulose hydrolysates, sugars such as molasses, organic acids such as fumaric acid, citric acid, succinic acid, etc., alcohols such as glycerol, etc. 2-15%, organic ammonium salts such as ammonium acetate as a nitrogen source. , ammonium sulfate, ammonium chloride, ammonium phosphate,
inorganic ammonium salts such as ammonium nitrate;
Ammonia gas, ammonia water, urea, etc. 0.5
~4.0%, organic micronutrients include 0.001~0.4% of required substances such as L-isoleucine, or as necessary, carbonated liquor, peptone,
A medium containing an appropriate amount of yeast extract or the like from 0 to 4% is preferably used. In addition to these, potassium phosphate, magnesium sulfate, ferrous sulfate heptahydrate, manganese sulfate 4-6 hydrate, and the like are added in small amounts as trace components. Cultivation is performed under aerobic conditions. During cultivation, the medium
Adjust the pH to 5 to 9, the temperature to 24 to 37℃, and the temperature to 48 to 9.
Favorable results can be obtained by culturing 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, then the solution is passed through an acidic cation exchange resin, the adsorbed components are eluted with aqueous ammonia, and the adsorbed components are removed after removal of 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 sulfomethyuron methyl sensitive strain Providencia letgeli OTR28-31 (α-
Amino-β-hydroxyvaleric acid resistant, L-isoleucine auxotrophic, L-ethionine resistant, L-leucine auxotrophic, thiaisoleucine resistant, oxythiamine resistant) bacterial cells were treated with N-methyl-N-nitro-N by a conventional method. - After nitrosoguanidine treatment (300 μg/ml, 10 minutes at 30°C), the cells were placed on an agar medium supplemented with 0.1 mg of sulfomethyuron methyl (glucose 0.5%, ammonium sulfate 0.1%,
Potassium phosphate 0.3%, potassium phosphate 0.7%, magnesium sulfate heptahydrate 0.01%,
L-isoleucine 0.001%, L-leucine 0.005
%, minimal medium containing 0.02% L-valine). Next, the cells were cultured at 30°C for 6 to 8 days, and small colonies among the resulting colonies were isolated. Among the isolated strains, we selected strains that grew on an agar plate medium without the addition of sulfomethyuron methyl and had significantly poor growth on an agar plate medium supplemented with 0.1 mg of sulfomethyuron methyl. obtained. B. Assay of sulfomethyuron methyl-susceptible mutant strains The strains listed in Table 1 below were tested on a broth agar medium.
Cultivate for 24 hours, scrape off a very small amount of the bacterial cells,
Apply a thin layer to a synthetic agar plate medium with the following composition without the addition of sulfomethyuron methyl and with the addition of 0.1 mg of sulfomethyuron methyl, and incubate at 30°C for 30 minutes.
The cells were cultured for days and the presence or absence of growth was observed. Synthetic agar medium Glucose 0.5% (NH ₄ ) ₂ SO ₄ 0.1% MgSO ₄・7H ₂ O 0.01% KH ₂ PO ₄ 0.3% K ₂ HPO ₄ 0.7% L-isoleucine 0.001% L-leucine 0.005% L-valine 0.02% Agar 2.0% The results are shown in Table 1, and the Providencia retogeri SVSS used in the method of the present invention
-29 is the parent plant Providencia letgeri
Comparison with OTR28-31 clearly shows that sulfomethyuron methyl sensitivity has been acquired.

[Table] (Note) +: Growth -: No growth Example 2 (Culture of L-threonine producing bacteria and production of L-threonine) Each strain shown in Table 2 was grown in a liquid broth medium at 30°C for 16 hours. After shaking and pre-culturing, 800 ml of the main fermentation medium having the following composition, which had been previously steam sterilized at 115°C for 10 minutes, was inoculated into a small glass jar fermenter, and cultured with aeration at 30°C, 800 rpm, and aeration rate of 1 vvm. Fermentation medium Glucose (sterilized separately) 4% (NH ₄ ) ₂ SO ₄ 0.5% KH ₂ PO ₄ 0.1% MgSO ₄・7H ₂ O 0.04% Fe ⁺⁺ 2ppm Mn ⁺⁺ 2ppm L-isoleucine 0.0025% L-leucine 0.06 % PH 7.0 (neutralized with KOH) PH adjustment and nitrogen source supply were done with 25% aqueous ammonia and the PH was maintained between 6.5 and 8.0. The cells were cultured for 69 hours while adding glucose, KH ₂ PO ₄ , MgSO ₄ .7H ₂ O, L-leucine and L-isoleucine as appropriate. After culturing, the concentrations of L-threonine, alanine, and valine in the solution from which bacterial cells and calcium carbonate had been removed were determined using an automatic amino acid analyzer (JLC300 manufactured by JEOL Ltd.), and the results are shown in Table 2. I got it.

[Table] As is clear from Table 2, according to the examples of the present invention, there was no significant difference in the accumulated concentration of L-threonine compared to the comparative examples, and the amount of valine by-product was small. After culturing, remove the bacterial cells from each culture solution and add 100ml of it.
Strong cation exchange resin “Diaion” SKIB
(H type) column. After washing each column with water,
Elute the adsorbed components of the column with 2N ammonia water,
After decolorization, it was concentrated under reduced pressure and crystallized. As a result of collecting and drying the crystals, Provitencia retogeri
6.3 g of L-threonine crystals with a purity of 92% from the culture solution of OTR28-31 strain, Providencia retogeri
5.8 g of L-threonine crystals with a purity of 98% were obtained from each strain of SVSS-29. <Effects of the Invention> The present invention has made it possible to suppress the by-product of valine and produce highly pure L-threonine. Since there is no valine by-product, a complicated purification process is unnecessary, which is advantageous for industrial practical application.

Claims (1)

[Claims] 1 Belongs to the genus Providencia,
Cultivating a mutant strain that has the ability to produce L-threonine and is sensitive to an acetohydroxy acid synthase inhibitor, producing and accumulating L-threonine in the culture solution, and collecting L-threonine from the culture solution. A method for producing L-threonine using a characteristic fermentation method. 2 The acetohydroxy acid synthase inhibitor is (methyl 2-[[[[(4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl]
A method for producing L-threonine by the fermentation method according to claim 1, which is benzoate.