JPH0346112B2 - - 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,
It is used for pharmaceutical purposes and as a feed additive, and an inexpensive manufacturing method is desired. <Prior art> As a method for producing L-threonine by a fermentation method using microorganisms belonging to the genus Brevibacterium, α-amino-β-hydroxyvaleric acid (hereinafter referred to as
Mutant strain resistant to AHV
-26708 Publication) Mutant strains that are resistant to AHV and have L-lysine or L-isoleucine auxotrophy (Japanese Patent Publication No. 48-44876), AHV and S-(2-aminoethyl)-L-cysteine ( Artificial mutant strains that are resistant to AEC (hereinafter referred to as AEC) and co-produce L-lysine (US Pat. No. 3,732,144), AHV and
Resistant to AEC, requires L-leucine,
Mutant strain that co-produces L-lysine (Japanese Patent Application Laid-Open No. 50-31093
Methods using AHV mutants resistant to S-methylcysteine sulfoxide (hereinafter referred to as S-MCS) (Japanese Patent Publication No. 56-3038) are known. <Problems to be Solved by the Invention> However, the accumulated concentration of L-threonine or the production yield of L-threonine from raw materials such as sugars by these methods were not fully satisfactory. <Means and effects for solving the problem> As a result of intensive research into a method for producing L-threonine with higher productivity, the present inventors discovered that L-threonine belongs to the genus Brevibacterium and has the ability to produce L-threonine. -We have discovered that the accumulation concentration and production yield of L-threonine can be significantly improved by imparting resistance to aspartate antimetabolites, and have thus arrived at the present invention. That is, the present invention involves culturing a microorganism that belongs to the genus Brevibacterium, is resistant to aspartate antimetabolites, and has the ability to produce L-threonine, and contains L-threonine in the culture solution.
Threonine is produced and accumulated, and L
- A method for producing L-threonine by a fermentation method characterized by collecting threonine. Mutant strains of microorganisms belonging to the genus Brevibacterium that are resistant to aspartate antimetabolites are
It has never been separated. Furthermore, it is not known that mutant strains of microorganisms belonging to the genus Brevibacterium that are resistant to aspartate antimetabolites can produce and accumulate significant amounts of L-threonine. Here, aspartate antimetabolite is a substance that (A) inhibits the growth of microorganisms belonging to the genus Brevibacterium and whose growth inhibition is recovered by the addition of L-aspartic acid, or (B) a substance that inhibits the growth of microorganisms belonging to the genus Brevibacterium. A substance that suppresses or inhibits enzymes involved in biosynthetic systems. Examples of aspartate antimetabolites include aspartate hydroxamate, α-methylaspartate, β-methylaspartate,
cysteine sulfinic acid, difluorosuccinic acid,
Hadashidin, meso-2,3-diaminosuccinic acid, and the like. The microorganism used in the present invention belongs to the genus Brevibacterium (Japanese Society of Agricultural Chemistry, Vol. 36, No. 2,
P141-159 (1962)) is a microorganism resistant to aspartate antimetabolites. As long as they have such properties, those with other requirements and other drug resistance are also included within the scope of the present invention. In addition, auxotrophy for L-isoleucine and auxotrophy for L-leucine, AHV
Resistance to threonine analogs such as S
- Since resistance to MCS effectively affects L-threonine production ability, microorganisms having some or all of these characteristics are more preferably used as parent strains. Auxotrophy here has a broad meaning and includes incomplete deletion types (so-called leaky types). Furthermore, it also includes cases where the requirement is satisfied by the biosynthetic precursor of the required substance. Typical mutant strains used in the present invention include, for example, the following. Brevibacterium lactofamentum AHXR−11 (FERM
P-8774). This mutant strain is Brevibacterium lactofamentum FERM P-4190 (AHV resistant, L-isoleucine auxotrophic, L-leucine auxotrophic, S-
MCS-resistant) was obtained as a parent strain using the usual mutation treatment method, and is a mutant strain that is resistant to aspartate hydroxamate. Mutant strains are induced by irradiating the parent strain with ultraviolet rays or treating it with a mutagenic agent (e.g., N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), and then treating the parent strain with a substance that makes it incapable of growing. A strain that can grow on a solid medium containing a high concentration of aspartate antimetabolite may be collected. An aspartate antimetabolite inhibitor-resistant mutant strain refers to a strain that is more resistant to aspartate antimetabolites than the parent strain, and preferably at a concentration of the aspartate antimetabolite that exhibits a relative growth rate of 30% or less of the parent strain. A mutant strain that exhibits a relative growth rate of 60% or more within the range. Here, the relative growth rate is expressed by measuring the absorbance of the culture solution at 660 nm and expressing the absorbance of the culture solution to which the aspartate antimetabolite of each strain is not added as 100%. A commercially available aspartate antimetabolite was used for testing resistance. Bacterial strain used in the present invention and its parent strain DL-
The results of testing for resistance to aspartate hydroxamate are shown in Example 2. The medium for producing L-threonine in the present invention is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, other organic micronutrients. As the carbon source, carbohydrates such as glucose, sucrose and molasses containing these, starch hydrolysates, organic acids such as citric acid and succinic acid, alcohols such as glycerol, and others can be used. Suitable nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, and urea. As the inorganic ions, potassium ions, phosphate ions, magnesium ions, etc. are added as appropriate. As organic micronutrients,
Required substances such as L-isoleucine and L-leucine are added to the medium, and vitamins and the like are further used as appropriate. Cultivation is preferably carried out under aerobic conditions. The most favorable results can be obtained by adjusting the pH of the medium to 5 to 9 and the temperature to 24 to 37°C during cultivation. L-threonine can be collected from the fermentation liquor by a conventional method, for example, by an ion exchange resin method. <Examples> Hereinafter, the present invention will be specifically explained using examples. Example 1 (Isolation of DL-aspartate hydroxamate resistant strain) Brevibacterium lactofamentum
FERM-P4190 (AHV resistant, L-isoleucine auxotrophic, L-leucine auxotrophic, S-MCS resistant) cells were treated with N-methyl-N'-nitro-N by a conventional method.
-Nitrosoguanidine treatment (300 μg/ml, 37°C
After 10 minutes), the cells were grown on an agar medium (glucose 0.5%, ammonium sulfate 0.15%,
Potassium 0.1%, dibasic potassium phosphate 0.3%, magnesium sulfate heptahydrate 0.01%, calcium chloride 2
Hydrate 0.0001%, Urea 0.15%, Biotin 0.000005
%, thiamine hydrochloride 0.00001%, L-isoleucine 0.01%, L-leucine 0.01%). Next, it was cultured at 37°C for 5 days, and the resulting large colonies were isolated by fishing.
P-8774) was obtained. Example 2 (Resistance of DL-aspartate hydroxamate resistant mutant 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 bacteria were collected. Washed with physiological saline. This bacterial suspension
DL-aspartate hydroxamate 0, 0.1,
Minimal medium (glucose 0.5%, ammonium sulfate 0.15%, potassium monophosphate 0.1%, dipotassium phosphate 0.3%, magnesium sulfate heptahydrate 0.01%, Calcium chloride 2
Hydrate 0.0001%, Urea 0.15%, Biotin 0.000005
%, thiamine/hydrochloride 0.00001%, L-isoleucine 0.01%, L-leucine 0.01%) were inoculated into 5 ml of the microorganism, cultured at 30°C for 48 hours, and the growth rate of each strain was examined. The results are shown in Table 1. Brevibacterium lactofamentum AHXR-11 (FERM
P-8774)), the parent strain Brevibacterium
Compared to Lactofermentum FERM-P4190, growth was not inhibited by high concentrations of DL-aspartate hydroxamate, indicating that it had acquired strong DL-aspartate hydroxamate resistance.

[Table] Example 3 (Culture of L-threonine-producing bacteria and L-threonine production) Each strain shown in Table 2 was precultured in a liquid broth medium by shaking at 30°C for 16 hours.
Fermentation medium with the following composition steam sterilized at 120℃ for 10 minutes
5 ml of each culture solution was inoculated into 1-volume Erlenmeyer flasks containing 50 ml, and cultured for 5 days at 30°C, 150 revolutions/minute, and an amplitude of 3 cm. Fermentation medium composition Glucose 10% Ammonium sulfate 4.5% Potassium phosphate 0.1% Magnesium sulfate, heptahydrate 0.1% Ferric sulfate, heptahydrate 0.001% Manganese sulfate, tetrahydrate 0.001% Thiamine, hydrochloric acid Salt 0.00003% Biotin 0.00001% Soybean protein hydrolyzate 1.5% Calcium carbonate (separately sterilized) 5% PH 7.2 (neutralized with KOH) After culturing, remove bacterial cells and calcium carbonate,
When the L-threonine concentration in the culture solution was determined using an automatic amino acid analyzer (JLC-200A manufactured by JEOL Ltd.), the results shown in Table 2 were obtained.

[Table] The threonine production yield was expressed as the weight yield of the produced threonine relative to the consumed glucose. Brevibacterium lactofamentum AHXR−
Bacterial cells were removed from the culture solution No. 11, and 200 ml thereof 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 to this, cooled, and the resulting crystals were collected and dried, resulting in L with a purity of over 96%.
- 2.4 g of threonine crystals were obtained. <Effects of the Invention> According to the present invention, L-threonine can be produced in high yield, and L-threonine can be produced at a lower cost.

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Brevibacterium, resistant to aspartate antimetabolites, and capable of producing L-threonine is cultured, and L-threonine is produced in the culture solution. A method for producing L-threonine by a fermentation method, which comprises producing and accumulating L-threonine and collecting L-threonine from the culture solution. 2. Claim 1, wherein the aspartate antimetabolite is aspartate hydroxamate.
A method for producing L-threonine by the fermentation method described in Section 1.