JPH01148194A - Production of l-threonine through fermentation process - Google Patents

Abstract

PURPOSE:To obtain L-threonine of high purity simply in a practical scale, by culturing a specific mutant in a medium. CONSTITUTION:Providencia rettgeri is OTR28-31 strain is subjected to mutagenic treatment such as ultraviolet irradiation to obtain a mutant, Providencia rettgeri SVSS II-29 (FERM P-9349), which is sensitive to a acetohydroxyacid inhibitor such as methyl 2-[[[[(4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl] benzoate. The microorganism strain is inoculated in a medium containing 2-15wt.% of carbon source such as glucose, 0.5-4wt.% of nitrogen source such as urea, 0.001-0.4wt.% of organic trace nutrients such as L-isoleucine, and cultured aerobically at a pH of 5-9, 24-37 deg.C, for 48-120hours to collect the culture mixture (B). Cell bodies are removed, the filtrate is adjusted in its pH to about 2, treated with acidic cationic exchange resin to produce L- threonine.

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, there is a method using a mutant strain resistant to ethionine (Japanese Patent Laid-Open No. 61-2166
98), a method using a mutant strain with L-leucine auxotrophy (Japanese Patent Application Laid-Open No. 61-260891), a method using a mutant strain with reduced pyruvate kinase (Japanese Patent Application Laid-open No. 62-44)
191), a method using a mutant strain resistant to aspartate antimetabolites (Japanese Patent Application Laid-Open No. 62-44192), a method using a mutant strain resistant to isoleucine antimetabolites (Japanese Patent Application Laid-Open No. 62-44193). Publication No.) etc. are known. In addition, Burge's Manual of Systematic Bacteriology Volume 1 (1984) describes a method for producing L-threonine by a fermentation method using microorganisms belonging to the former genus Teus, which has been partially reclassified to the genus Providencia. For example, a method using L-isoleucine auxotrophic strain (Japanese Patent Publication No. 43-4440) and α-amino-
A method using a microorganism that is resistant to β-droxyvaleric acid and has a monoisoleucine requirement (Proceedings of the Japanese Society of Agricultural Chemistry, p. 9 (1970)) is known.

<Problems to be Solved by the Invention> However, all of the conventional methods produce a large amount of alanine and valine as by-products, which reduces the purification yield of L-threonine, and it is difficult to obtain highly pure L-threonine. This method required a complicated purification process, which was disadvantageous for industrial use.

Means and action for solving the problems> As a result of intensive study by the present inventors on a method for maintaining high productivity of L-threonine and at the same time suppressing the by-product of valine,
We have discovered that the by-production of valine can be significantly suppressed by using a mutant strain of a microorganism that belongs to the genus Bidensia and has the ability to produce L-threonine and is sensitive to an acetohydroxy acid synthase inhibitor. Reached.

That is, the present invention involves culturing a mutant strain belonging to the genus Videncia that has the ability to produce L-threonine and is sensitive to an acetohydroxy acid synthase inhibitor, and injects L into the culture solution.
- L- by a fermentation method characterized by producing and accumulating threonine and collecting L-threonine from the culture solution.
This is a method for producing threonine.

Here, the acetohydroxy acid synthase inhibitors include:
For example, (methyl 2- (((((4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]amino)
Examples include sulfonyl]benzoate (hereinafter referred to as sulfometuron methyl), 2-thiazolealanine, L-valine, and L-isoleucine. Among them, sulfometuron methyl is preferably used.

The microorganism used in the present invention is a microorganism that belongs to the genus Providencia and is sensitive to acetohydroxy acid synthase inhibitors (according to Burge's Manual of Systematic Bacteriology, Vol. 1 (1984), pp. 475-496). be. 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 1eaky-type auxotrophy for isoleucine or L-leucine, resistance to threonine analogs such as α-amino-β-hydroxyvaleric acid, and methionine analogs such as ethionine. Since resistance to L-threonine effectively affects the ability to produce L-threonine, microorganisms having some or all of these characteristics are preferably used.

Typical mutant strains used in the present invention include, for example, 10 bidencia retogeri 5VSSI[-29
(FBRM P-9349).

This mutant strain is Providencia letgeri 0TR28-3
1 (α-amino-β-hydroxyvaleric acid resistance, L-isoleucine requirement, L-ethionine resistance, L-leucine requirement, thiaisoleucine resistance, oxythiamine resistance) was obtained by a normal mutation treatment method using the parent strain. This is a mutant strain that is sensitive to sulfometuron methyl.

Mutant strains can be induced by conventional mutation treatment methods. In other words, in order to obtain a mutant strain that is sensitive to acetohydroxy acid synthase inhibitors, either irradiation with ultraviolet rays or
Alternatively, a mutagenic agent (e.g., N-methyl-N゛-
After treatment with nitro-N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), isolate the strain that grows significantly less well than the parent strain on a solid medium containing an acetohydroxy acid synthase inhibitor at a concentration that allows the parent strain to grow. All you have to do is collect it.

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 sulfometurone methyl-sensitive strain, the parent strain is 0.1 mtr/sulfometuron methyl that can be grown
1. For those that can hardly grow on the added medium,
It is called a sulfometuron methyl sensitive strain.

The medium for L-threonine production in the present invention is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and other organic trace components as necessary.

Examples of carbon sources include glucose, fructose, starch and cellulose hydrolysates, sugars such as molasses, organic acids such as fumaric acid, citric acid, succinic acid, alcohols such as glycerol, etc., and nitrogen sources. As, 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.
4.0%, and as organic micronutrients, required substances such as isoleucine are o, ooi ~ 0.4%, or cornstarch liquor, peptone, yeast extract, etc. are 0 to 4% as necessary. A medium containing an appropriate amount is preferably used. In addition to these, small amounts of potassium phosphate, magnesium sulfate, sulfuric acid 1#C7 hydrate, manganese sulfate 4-6 hydrate, etc. are added as trace components.

Cultivation is carried out under aerobic conditions.During cultivation, the pH of the medium is 5.
to 9, the temperature was adjusted to 24-37℃, and the temperature was adjusted to 48-120℃.
Favorable results can be obtained by culturing with shaking or aeration for 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
After passing the solution through an acidic cation exchange resin, the adsorbed components are eluted with aqueous ammonia, deammoniated, and then concentrated. 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 sulfometuron methyl-susceptible strain 10 Bidentsia letgeri 0TR28-31 (α-amino-β-hydroxyvaleric acid resistant, L-isoleucine auxotrophic, L-ethionine resistant, L-leucine auxotrophic, Thiisoleucine resistant, oxythiamine resistant) bacterial cells were treated with N-methyl-N-12 and N-nitrosoguanidine (3
00μg/ml for 10 minutes at 30°C), the cells were grown on an agar medium (glucose 0.5%, ammonium sulfate 0.1%, Potassium 0.3%, potassium phosphate 0.7
%, magnesium sulfate heptahydrate 0.01%, L-isoleucine o,ooi%, L-leucine 0°005%, L-
0x3 applied to minimal medium containing 0.02% valine)
After culturing at 0°C for 6 to 8 days, small colonies were isolated from the resulting colonies. Among the isolated strains, they grew on an agar plate medium without sulfometuron methyl. A strain that grew significantly poorly on an agar plate medium supplemented with 1/i was selected, and a mutant strain sensitive to sulfometuron methyl was obtained.

B. Assay of sulfometuron methyl-susceptible mutant strains The strains shown in Table 1 below were cultured on a bouillon agar medium for 24 hours, and a very small amount of the bacterial cells were scraped off. .1■/
The cells were spread on a synthetic agar plate medium having the composition shown below and cultured at 30° C. for 3 days, and the presence or absence of growth was observed.

Synthetic agar medium glucose 0.5% (NH+)2
SO40,1% M g SO4・7H200,01%KH2PO40
, 3% to 2HP0. 0.7% L-isoleucine 0.001% L-leucine 0
．． 005% L-valine 0.02% agar 2.0% The results are shown in Table 1. Providencia retogeri 5vSSH-29 used in the method of the present invention was compared with the parent strain Providencia retogeri 0TR28-31. It has clearly acquired sulfometuron methyl sensitivity.

Table 1 (Note) +: Growth -: No growth Example 2 (Culture of L-threonine producing bacteria and production of L-threonine) Each strain shown in Table 2 was incubated in liquid broth medium for 30 min.
After pre-incubation at 11°C for 16 hours with shaking,
Main fermentation medium 80 with the following composition steam sterilized at 5°C for 10 minutes
0 ml was inoculated into a glass-type small jar fermenter, and cultured with aeration at 30°C, 800 rpm, and aeration volume of 1 vvm.

Fermentation medium Glucose (separately sterilized) 4% (NH4)2 SO40.5% KH2PO40.1% Mg5O+ ・7M20 0.04%Fe”
21) rlM n ” ”
21) 11 Iw isoleucine
0.0025% Leucine 0.06
%p)I 7.0 (neutralized with OH)
pH adjustment and nitrogen source supply were performed with 25% ammonia water, and the pH was maintained at 6.5-8.0. The cells were cultured for 69 hours while adding glucose, KH2PO4, Mg5O+.7H20, L-leucine, and isoleucine as appropriate. Cultivate My Hi°.

After the cultivation, L- in the furnace liquid from which calcium was removed was removed.
The concentrations of threonine, alanine, and valine were determined using an automatic amino acid analyzer (JLC300 manufactured by JEOL Ltd.), and the results shown in Table 2 were obtained.

Table 2 *Trace: Indicates a trace amount of less than lr/J.

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, the bacterial cells were removed from each culture solution, and 100 ml thereof was passed through a column of strong cation exchange resin "1 Diaion + 13 KIB (H type)". After washing each column with water, the absorption components of the column were eluted with 2N ammonia water, decolorized, and concentrated under reduced pressure.
crystallized.

As a result of collecting and drying the crystals, 6.3 g of L-threonine crystals with a purity of 92% and Providencia retgeri 5VS were obtained from the culture solution of Providencia retgeri 0TR28-31 strain.
5.8 g of L-threonine crystals with a purity of 98% were each obtained from the SII-29 strain.

<Effects of the Invention> The present invention makes 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 (2)

[Claims] (1) A mutant strain that belongs to the genus Providencia and has L-threonine producing ability and is sensitive to an acetohydroxy acid synthase inhibitor is cultured, and L-threonine is produced and accumulated in the culture solution. A method for producing L-threonine by a fermentation method, which comprises collecting L-threonine from a culture solution. (2) Acetohydroxy acid synthase inhibitor (methyl 2
-[[[[(4,6-dimethyl-2-pyrimidinyl)-
L- by the fermentation method according to claim 1, which is amino]carbonyl]amino]sulfonyl]benzoate.
Method for producing threonine.