JPS6234400B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-phenylalanine by fermentation. Conventionally, known methods for producing L-phenylalanine by fermentation include methods using microorganisms belonging to the genus Brevibacterium and methods using microorganisms belonging to the genus Corynebacterium. In contrast, the present inventors have demonstrated that L-
As a result of research to develop a method for producing phenylalanine, we found that a mutant strain of Escherichia microorganisms that has tyrosine auxotrophy, phenylalanine analog resistance, tryptophan analog resistance, and 2-thiazolealanine resistance discovered a strain capable of producing significant amounts of L-phenylalanine. This invention was completed based on this knowledge. The phenylalanine analog referred to in the present invention is
O-, m- or p-fluorophenylalanine,
O-, m- or p-aminophenylalanine, β
-Phenylserine, cyclohexylalanine, α
-Amino-β phenylethanesulfonic acid, O-,
m- or p-bromophenylalanine, β-2-
Thienylalanine, β-3-thienylalanine,
β-2-furylalanine, β-2-virolalanine, 1-cyclopentene-1-alanine, 1-
Cyclohexene-1-alanine, 2-amino-4
-Methyl-4-hexenoic acid, S-(1,2-dichlorovinyl)-cysteine, β-4-pyridylalanine, β-2-pyridylalanine, β-4-pyrazolalanine, p-nitro-phenylalanine etc. In addition, tryptophan analogs as used in the present invention include 5-fluorotryptophan, 6-fluorotryptophan, 7-azatryptophan, methyltryptophan, naphthylalanine,
Indole acrylic acid, naphthylacrylic acid, β
-(2-benzothienyl)alanine, indole, styryl-acetic acid, tryptazone, etc. Specifically, the microorganism used in the method of the present invention is, for example, Escherichia coli-AJ11377 (FERM-
P5037). This strain is Escherichia coli
This is a mutant strain that has undergone mutation from K-12 and has been endowed with tyrosine auxotrophy, phenylalanine analog resistance, tryptophan analog resistance, and 2-thiazolealanine resistance. Table 1 shows the results of examining the resistance of this strain to 2-thiazolealanine, p-fluorophenylalanine, which is an example of a phenylalanine analog, and 5-fluorotryptophan, which is an example of a tryptophan analog. Shown in Tables 2 and 3. As p-fluorophenylalanine, DL
-p-fluorophenylalanine was used, DL-5-fluorotryptophan was used as 5-fluorophenylalanine, and DL-2-thiazolealanine was used as 2-thiazolealanine. p-Fluorophenylalanine, 5-fluorotryptophan, and 2-thiazolealanine were dissolved in a minimal medium with the following composition to each concentration shown in each table, and Escherichia coli K-12, Escherichia coli K-12,
After inoculating with Escherichia coli AJ11377,
The bacteria were cultured at 31°C for 24 hours and the growth of the bacteria was investigated. In addition, since Escherichia coli AJ11377 requires L-tyrosine for growth, 100 γ/ml of L-tyrosine was added to the minimal medium. Minimum medium composition: glucose 0.5g/dl, ammonium sulfate 0.1g/dl, monopotassium phosphate 0.846
g/dl, sodium citrate 0.05g/dl, potassium hydroxide 0.226g/dl, magnesium sulfate heptahydrate 0.04g/dl, FeSO ₄ 7H ₂ O1mg/dl, MnSO ₄
Contains 1 mg/dl of 4H ₂ O and 1000 g/dl of thiamine hydrochloride.

【table】

【table】

[Table] Drug resistance as used in the present invention refers to a case where the specific growth rate in the presence of a drug is higher than that of the parent strain Escherichia coli K-12 under the above culture conditions. In addition, the specific growth rate is the amount of bacterial growth (minus the amount of inoculated bacteria) when no chemicals are added.
And so. Growth was measured by absorbance at 570 nm. The culture medium for producing L-phenylalanine is not particularly limited, and a conventional medium containing a carbon source, a nitrogen source, an inorganic salt and, if necessary, an organic micronutrient is used. Carbon sources include carbohydrates (glucose, fructose, hydrolysates of starch, cellulose, etc.), organic acids (acetic acid, citric acid, etc.), alcohols (glycerin, ethanol, methanol, etc.), or hydrocarbons (normal paraffin, etc.). ) can be used. As a nitrogen source, ammonium sulfate, ammonium nitrate, ammonium phosphate, ammonium chloride, ammonia gas, etc.
As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and other trace metal salts are used as necessary. As organic micronutrients, if there is a nutritional requirement, appropriate amounts of corresponding amino acids, vitamins, fatty acids, organic basic substances, etc. are added, and if necessary, amino acids, vitamins, soybean hydrolyzate are added as growth promoting substances. , yeast extract, peptone, casein hydrolyzate, etc. Culture conditions may be the usual methods, PH5 or higher,
9. Temperature 20℃ to 45℃, 20℃ to 45℃ under aerobic conditions.
It is sufficient to culture for 96 hours. If the pH drops during culture, add calcium carbonate after sterilization separately, or neutralize with alkali such as aqueous ammonia or ammonia gas. In addition, when an organic acid is used as a carbon source, the increase in pH is neutralized with a mineral acid or an organic acid. Example 1 Glucose 5g/dl, ammonium sulfate, 2.5
g/dl, potassium phosphate 0.2g/dl, magnesium sulfate heptahydrate 0.1g/dl, yeast extract 0.05g/dl
dl, L-tyrosine 100mg/dl, thiamine hydrochloride
1000γ/, FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・
An aqueous medium with a composition of 4H ₂ O 1mg/dl, calcium carbonate 2.5g/dl (separate sterilization added) and pH 7.0 was added to the
Dispense 20ml into a ml flask and add Escherichia.
One loop of E. coli AJ11377 and the parent strain K-12 was planted and cultured at 31°C for 72 hours. The amount of L-phenylalanine accumulated in AJ11377 at the end of fermentation is 250 mg/dl, and that of K-12 is 10 mg/dl.
It was hot.

Claims (1)

[Claims] 1. Cultivate a microorganism that belongs to the genus Escherichia, has tyrosine auxotrophy, phenylalanine analog resistance, tryptophan analog resistance, and 2-thiazolealanine resistance, and has the ability to produce L-phenylalanine, and produce it in a medium. A method for producing L-phenylalanine by a fermentation method, which is characterized by collecting accumulated L-phenylalanine.