JP2810697B2 - Method for producing aromatic amino acids - Google Patents

Description

The present invention relates to a method for producing an aromatic amino acid such as L-tryptophan, L-tyrosine or L-phenylalanine by a fermentation method. L-tryptophan is a useful amino acid in medicine, food or feed, etc., L-tyrosine is particularly useful in medicine, and L-phenylalanine is useful in medicine and food industry.

2. Description of the Related Art As a method for producing L-tryptophan by a fermentation method using a microorganism belonging to the genus Corynebacterium or Brevibacterium, a so-called coryneform glutamate-producing bacterium,
A method using a microorganism belonging to the genus Corynebacterium which requires L-tyrosine and L-phenylalanine and is resistant to one or more of tyrosine analogs or phenylalanine analogs (Japanese Patent Publication No. 51-1903)
7), a method using a microorganism having resistance to a tryptophan analog such as 5-methyltryptophan (Japanese Patent Publication No. 48-78)
18828, JP-B-51-38795, JP-B-53-39517), a method using a histidine-requiring strain (Japanese Patent Publication No. 47-4505), and a method using a microorganism belonging to the genus Brevibacterium having reduced or deleted pyruvate kinase activity. (Japanese Unexamined Patent Publication No. 62-25339
1) is known. In addition, as a method for producing L-tyrosine or L-phenylalanine by fermentation using a coryneform glutamic acid-producing bacterium, auxotrophic mutations of amino acids, resistance mutations to amino acid analogs or reduction or deletion mutations of pyruvate kinase activity, Further, a method using a strain having the properties of these mutations is also known [Journal of the Japanese Society of Agricultural Chemistry, 50 (1), p.R79 (1979),
JP-A-61-128897]. On the other hand, L-
Bacteria producing tyrosine or L-phenylalanine have also been produced.
Recombination containing genes involved in the synthesis of deoxy-D-arabino-heptulosonate-7-phosphate synthase (hereinafter abbreviated as DS), chorismate mutase (hereinafter abbreviated as CM) and prephenate dehydrogenase or pretyrosine aminotransferase L-tyrosine-producing bacteria having body DNA are known (JP-A-60-34197). Examples of L-phenylalanine-producing bacteria include L-phenylalanine-producing bacteria having a recombinant DNA containing a gene involved in the synthesis of DS or CM and prephenate dehydratase (hereinafter abbreviated as PD). (JP-A-60-24192, JP-A-61
-260892, JP-A-61-124375).

Problems to be Solved by the Invention Development of a method for industrially producing L-tryptophan useful as a feed additive or the like, L-tyrosine useful as a pharmaceutical, or L-phenylalanine useful in the pharmaceutical and food industries at a lower cost is needed. It has been demanded.

Means for Solving the Problems The present inventor has previously determined that a coryneform having reduced or deleted phosphoenolpyruvate carboxylase (hereinafter abbreviated as PC) activity and capable of producing L-tryptophan, L-tyrosine or L-phenylalanine. Microorganisms belonging to the type glutamate-producing bacterium are conventional L-tryptophan, L-
It has been found that this amino acid-producing ability is superior to that of tyrosine or L-phenylalanine-producing bacteria (Japanese Patent Application No.
241688). As a result of further studies, it was found that a microorganism in which pyruvate kinase (hereinafter abbreviated as PK) activity was reduced or deleted in addition to PC activity had better amino acid-producing ability, and completed the present invention. Was.

The present invention relates to a coryneform glutamate-producing bacterium, in which a PC activity and a PK activity are reduced or deleted, and a microorganism having an ability to produce L-tryptophan, L-tyrosine or L-phenylalanine is cultured in a culture medium. To produce and accumulate L-tryptophan, L-tyrosine or L-phenylalanine.
-A method for producing L-tryptophan, L-tyrosine or L-phenylalanine, which comprises collecting tyrosine or L-phenylalanine.

 Hereinafter, the present invention will be described in detail.

The microorganism used in the present invention is a microorganism belonging to the genus Corynebacterium or Brevibacterium known as a coryneform glutamic acid producing bacterium,
Any strain may be used as long as it has the ability to produce L-tryptophan, L-tyrosine or L-phenylalanine, and has reduced or deleted PC activity and PK activity. Typical coryneform glutamate-producing bacteria used as a parent strain for inducing a microorganism having such properties include the following strains.

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoacid film ATCC13870 Corynebacterium herculis ATCC13868 Corynebacterium lilium ATCC15990 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 Brevibacterium dibaricatum ATCC140 Brecobacterium Thiogenitalis ATCC19240 L-glutamic acid-producing bacteria as described above
In order to induce a tryptophan-producing bacterium, the strain may be provided with L-tyrosine and L-phenylalanine auxotrophy and resistance to tryptophan analogs such as 5-methyltryptophan.

In order to obtain an L-tyrosine-producing bacterium from a coryneform glutamic acid-producing bacterium as described above, a strain which requires L-phenylalanine, has resistance to amino acid analogs, or a combination of these properties may be used. In addition, recombinant DN
A method for obtaining the producing bacterium by the A technique is also known, and can be obtained, for example, by introducing a recombinant DNA containing DS, CM, and a gene involved in the synthesis of prephenate dehydrogenase or pretyrosine aminotransferase. (JP-A-60-34197). Furthermore, a recombinant DNA of a vector fragment and a DNA fragment that carries genetic information of enzymes involved in L-tyrosine biosynthesis, such as DS and CM, is introduced into bacteria producing L-tryptophan synthesized via a common biosynthetic pathway. Alternatively, it can be obtained by converting an L-tryptophan-producing bacterium into an L-tyrosine-producing bacterium (JP-A-63-94985).

On the other hand, L-phenylalanine-producing bacteria can be obtained from coryneform glutamate-producing bacteria by inducing L-tyrosine auxotrophy, amino acid analog resistance, or a strain having both of these properties. In addition, an acquisition method using recombinant DNA technology is also known. For example, it can be obtained by introducing a recombinant DNA containing a gene involved in the synthesis of DS or CM and PD (Japanese Patent Application Laid-Open No. 60-24192, JP-A-61-260892, JP-A-61-124
375). Furthermore, L-tryptophan-producing bacteria are transformed with L-tryptophan-producing bacteria into L-phenylalanine-producing bacteria by introducing a recombinant DNA of a vector fragment and a DNA fragment carrying the gene information involved in the synthesis of DS, CM and PD. (Japanese Patent Application Laid-Open No. 63-10568)
8).

L of the present invention having reduced or deleted PC activity and PK activity
-A tryptophan, L-tyrosine or L-phenylalanine-producing bacterium can be obtained by imparting a mutation capable of reducing or deleting PC activity and PK activity to a strain having L-tryptophan, L-tyrosine or L-phenylalanine-producing ability. . Alternatively, by imparting properties such as auxotrophy and analog resistance as described above to a mutant strain having reduced or deleted PC activity and PK activity derived from a wild strain, L-tryptophan, L-tyrosine or L-tyrosine can be obtained. -A phenylalanine-producing strain can be obtained.

The mutant strain having a reduced or deleted PC activity in the present invention is usually used, for example, by ultraviolet irradiation or chemical treatment with N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter abbreviated as NTG), nitrous acid or the like. L-glutamic acid auxotrophs can be obtained from strains mutated by the mutation treatment method. Mutants with reduced PC activity can also be selected as strains that have become sensitive to the affinity labeling reagent at the catalytic center of PC, or as revertants of L-glutamic acid auxotrophic (PC activity deficient) strains. Obtainable. The affinity labeling reagent refers to a compound having specific affinity for the active site of an enzyme and deleting the enzyme, and is an active-site-directed irreversible inhibitor.
eversible inhibitor) [“Biochemical Dictionary”
Tokyo Chemical Dojin (1984)]. Further, a mutant strain having a reduced or deleted PK activity is selected from strains mutated by the above-described mutation treatment method, from those strains which have become sensitive to β-fluoropyruvic acid in a minimal medium containing gluconic acid as a main carbon source. Can be obtained.

Production of L-tryptophan, L-tyrosine or L-phenylalanine by the microorganism of the present invention can be carried out by a usual culture method. As the medium to be used, either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and a trace amount of nutrients required by the used strain.

As a carbon source, carbohydrates such as glycol, glycerol, fructose, sucrose, maltose, mannose, starch, starch hydrolysate, molasses, and various organic acids such as polyalcohol, pyruvic acid, fumaric acid, lactic acid, and acetic acid can be used. Furthermore, due to the assimilation of microorganisms,
Hydrocarbons and alcohols are also used. In particular, molasses is preferably used.

Ammonia or ammonium chloride as the nitrogen source,
Various inorganic and organic ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium acetate or urea and other nitrogen-containing substances and peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or Various substances such as nitrogen-containing organic substances such as digests thereof can be used.

Further, as the inorganic substance, potassium hydrogen phosphate, potassium hydrogen phosphate, ammonium sulfate, ammonium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, and the like are used.

Cultivation is performed under aerobic conditions such as shaking culture or aeration and stirring culture. Generally, the culture temperature is preferably from 20 to 40 ° C. It is desirable to maintain the pH of the medium around neutral. The culturing period is usually 1 to 5 days.

The method for collecting L-tryptophan, L-tyrosine or L-phenylalanine from a culture solution is as follows:
It is performed by a known method such as a method of removing cells and performing concentration crystallization, an activated carbon treatment or an ion exchange resin treatment.

 Hereinafter, the present invention will be described specifically with reference to practical examples.

Example 1. Acquisition of mutant strains with reduced PC and PK activity (1) L-tryptophan-producing bacteria L-protein having reduced PC activity disclosed in Japanese Patent Application No. 63-241688
The tryptophan-producing bacterium Corynebacterium glutamicum BPS13 strain (FERM BP-1777) was used as a parent strain. Collect the cells cultured at 30 ° C for 16 hours in a complete medium (medium adjusted to pH 7.2 containing 20 g of powdered broth and 5 g of yeast extract in water 1) and wash with 0.05 M phosphate buffer (pH 7.2) Thereafter, the cells were suspended in the same buffer so that the concentration of the cells was about 10 ⁹ cells / ml.
To this, NTG was added to a final concentration of 500 μg / ml, and 30
Mutation treatment was carried out at 20 ° C. for 20 minutes. After washing the mutated cells with the same buffer, the cells are applied to an agar plate medium containing 1 μg / ml of β-fluoropyruvic acid (hereinafter abbreviated as βFP) in a minimal medium having the composition shown in Table 1. did.

Table 1 Minimal medium composition Sodium gluconate _{10g / (NH 4) H 2} PO 4 1g / KCl 0.2g / MgSO 4 · 7H 2 O 0.2g / FeSO 4 · 7H 2 O 10mg / MnSO 4 · 4~6H 2 O _{0.2mg / ZnSO 4 · 7H 2 O} 0.9mg / CuSO 4 · 5H 2 O 0.4mg / Na 2 B 4 O 7 · 10H 2 O 0.09mg / (NH 4) 6 Mo 7 O 24 · 4H 2 O 0.04mg / Biotin 50μg / p-aminobenzoic acid 2.5mg / thiamine hydrochloride 1mg / L-tyrosine 50mg / L-phenylalanine 50mg / agar 16g / (pH 7.2) Cultured at 30 ° C for 5-10 days and grown on the plate medium Small colonies were picked out of the colonies. Among them, a strain that became more sensitive to βFP than the parent strain was isolated, and among such mutant strains, a strain having reduced PK activity, Corynebacterium glutamicum K81, was obtained.

This strain has been deposited as FERM BP-2409 on May 1, 1989 with the Research Institute of Microbial Industry (MIC) of the Agency of Industrial Science and Technology.

Table 2 shows the βFP sensitivity of the parent strain BPS-13 and ATCC21851 which is the parent strain of the mutant K81 and the BPS-13 strain, and their PC activity and PK activity. The βFP sensitivity was determined by applying the bacterial strain to a minimal agar medium having the composition shown in Table 1 containing various concentrations of βFP, and growing at 30 ° C for 4 days. The PC activity and PK activity were determined using the crude enzyme extract of the cells and the Journal of Biochemistry (J.
Biochem.), 66 (3), 297-311 (1969), Agricultural Biological Chemistry (Agric.Bic
l. Chem.), 48 (5), 1189-1197 (1984).

Glucose _{30g /, MgSO 4 · 7H 2} O0.5g /, FeSO 4 · 7H 2
_{O10mg /, KH 2 PO 4 1g} /, MnSO 4 · 4H 2 O1mg /, ammonium sulfate 4g /, urea 2 g /, biotin 50 [mu] g /, p-aminobenzoic acid 2.5 mg /, thiamine hydrochloride 1 mg /, salt
50 mg /, L-tyrosine 50 mg /, L-phenylalanine
Inoculate the strain into a medium (pH 7.2) with a composition of 50 mg /
The cells were collected after shaking culture for 24 hours. The cells were washed twice with a 0.2% potassium chloride aqueous solution, and then washed with a 0.1 M Tris-HCl buffer (pH
After suspension in 7.5), the cells were disrupted by sonication. After centrifugation, the supernatant was dialyzed overnight against the same buffer to obtain a crude enzyme solution.
The enzyme activity in Table 2 is shown as a relative value when the specific activity per protein amount in the crude enzyme extract was calculated and the value of the parent strain was set to 100.

(2) L-tyrosine-producing bacterium L-tyrosine with reduced PC activity disclosed in Japanese Patent Application No. 63-241688.
Tyrosine-producing bacterium Corynebacterium glutamicum K77
The strain (FERM BP-2062) was used as a parent strain. The modification treatment was performed in the same manner as in (1) of Example 1, and an L-tyrosine-producing bacterium Corynebacterium glutamicum K79 strain with reduced PK activity was obtained by isolating a βFP-sensitive mutant strain.
In the same manner as in Example 1 (1), βFP of the parent strain K77 and the mutant strain K79 and T6 (ATCC21851 / pCDS-CM1) which is the parent strain of the K77 strain were used.
Table 3 shows the results of measuring the sensitivity, PC activity and PK activity.

K79 strain was transferred to FERM BP-2368 on April 1, 1989
Has been deposited as

(3) L-phenylalanine-producing bacterium L-protein having reduced PC activity disclosed in Japanese Patent Application No. 63-241688.
Phenylalanine producing bacterium Corynebacterium glutamicum K78 (FERM BP-2063) was used as a parent strain. Mutagenesis was carried out in the same manner as in (1) of Example 1, and an L-phenylalanine-producing bacterium Corynebacterium glutamicum K80 strain having reduced PK activity was obtained by isolating a βFP-sensitive strain. In the same manner as in Example 1 (1), the parent strain K78 and the mutant strain K80 and the T17 strain (ATCC21851 / pEaro
Table 4 shows the results of measurement of βFP sensitivity, PC activity and PK activity of G-pheA3). The K80 strain was deposited with MIC as FERM BP-2369 on April 1, 1989.

Example 2. (1) L-tryptophan production test Corynebacterium glutamicum K81 (FERM BP-24)
09) in 20 ml of seed medium (glucose 2%, polypeptone,
1.5%, yeast extract 1.5%, salt 0.25%, urea 0.1%, L
-Tyrosine 200 mg /, L-phenylalanine 200 mg /,
Inoculate 300 ml Erlenmeyer flask containing pH 7.2)
For a period of time, the cells were shake-cultured on a rotary shaker at 210 rpm. 2 ml of the seed culture was inoculated into a 300 ml Erlenmeyer flask containing 20 ml of a fermentation medium having the following composition, and cultured for 72 hours in the same manner as in the seed culture. As a control, parent strain BPS-13 (FERM
BP-1777) and the ATCC21851 strain were similarly cultured. After culture
The culture solution was subjected to paper chromatography, and after ninhydrin was colored, the amount of L-tryptophan produced was measured by colorimetric quantification.

 The results are shown in Table 5.

Composition of fermentation medium: glucose 6%, KH ₂ PO ₄ 0.05%, K ₂ HP
O ₄ 0.05%, MgSO ₄ .7H ₂ O 0.025%, ammonium sulfate 2
%, Biotin _{30μg /, MnSO 4 · 7H 2} O10mg /, corn
0.5% steep liquor, 2% CaCO ₃ (pH 7.2) (2) L-tyrosine production test Corynebacterium glutamicum K79 (FERM BP-23
68) in 20 ml of seed medium (glucose 2%, polypeptone 1.
5%, yeast extract 1.5%, salt 0.25%, urea 0.1%, pH 7.
The mixture was inoculated into a 300 ml Erlenmeyer flask containing 2), and cultured with shaking at 30 ° C. for 24 hours on a rotary shaker at 210 rpm.
2 ml of this seed culture solution contains 300 ml of a fermentation medium having the following composition:
The mixture was inoculated into a ml Erlenmeyer flask and cultured for 72 hours in the same manner as in the seed culture. Parental strain K77 (FERM BP-2062) as a control
Was also cultured in the T6 strain (ATCC21851 / pCDS-CM1). After cultivation, 50 μL of 6N NaOH solution is added to 1 ml of the culture solution, and
After heating for 1 minute to completely dissolve the precipitated L-tyrosine, the culture solution was subjected to paper chromatography, and after ninhydrin was developed, the amount of L-tyrosine produced was measured by colorimetric quantification.

 The results are shown in Table 6.

Composition of fermentation medium: glucose 6%, KH ₂ PO ₄ 0.05%, K ₂ HP
O ₄ 0.05%, MgSO ₄ .7H ₂ O 0.025%, ammonium sulfate 2
%, Biotin _{30μg /, MnSO 4 · 7H 2} O10mg /, corn
0.5% steep liquor, 2% CaCO ₃ (pH 7.2) (3) L-phenylalanine production test Corynebacterium glutamicum K80 (FERM BP-23
69) and parent strain K78 (FERM BP-2063) and T17 strain (ATCC218).
51 / pEaroG-pheA3) was cultured in the same manner as in Example 2, (2). After the culture, the culture solution was subjected to paper chromatography, and after ninhydrin was colored, the amount of L-phenylalanine produced was measured by colorimetric quantification.

 Table 7 shows the results.

Effect of the Invention By using the coryneform glutamic acid-producing bacterium of the present invention, L-tryptophan, L-tyrosine or L-phenylalanine-producing bacterium of the conventional strain can be produced at a higher yield.
-Fermentative production of tryptophan, L-tyrosine or L-phenylalanine.

Claims (1)

(57) [Claims] 1. A microorganism which belongs to a coryneform glutamic acid-producing bacterium, has a phosphoenolpyruvate carboxylase activity and a pyruvate kinase activity reduced or deleted, and has an ability to produce L-tryptophan, L-tyrosine or L-phenylalanine. And cultured in L-
Production of L-tryptophan, L-tyrosine or L-phenylalanine, which comprises producing and accumulating tryptophan, L-tyrosine or L-phenylalanine, and collecting L-tryptophan, L-tyrosine or L-phenylalanine from the culture. Law.