JPS61128896A - Production of l-tyrosine by fermentation method - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as hormone, etc. more inexpensively, by cultivating aerobically a bacterium belonging to the genus Berevibacterium, having reduced pyruvate kinase activity, capable of producing L-tyrosine, in a liquid medium. CONSTITUTION:A bacterium (e.g., Brevibacterium flavum ATCC14067, etc.) belonging to the genus Brevibacterium, having reduced pyruvate kinase activity, capable of producing L-tyrosine, is cultivated aerobically in a liquid medium comprising glucose, ammonium sulfate, urea, potassium primary phosphate, agar, etc., L-tyrosine is formed and accumulated in the culture solution, and L-tyrosin is collected from it.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] <Industrial Application Field> The present invention relates to a method for producing L-tyrosine (hereinafter referred to as tyrosine) by a fermentation method. Tyrosine is used as a precursor of adrenal medullary hormone, thyroxine, melanin, alkaloids, etc. Conventional methods for producing tyrosine by fermentation include methods using mutant strains of Brevibacterium bacteria that require L-phenylalanine and are resistant to phenylalanine analogs (Agric, Blot, Ch.
em, , 37 (10) 2327-2336 (1
973) and Special Publication No. 49-38837) are known.

<Problems to be Solved by the Present Invention> The problems to be solved by the present invention are to produce tyrosine at a lower cost.

[Structure of the invention]

<Means for Solving the Problems> The present inventors conducted research to develop a method for producing tyrosine at a lower cost by fermentation, and found that Brevibacterium bacteria have the ability to produce tyrosine as well as gilpine. When combined with the property of reducing acid kinase activity, it was found that the tyrosine-producing bacteria can produce even more tyrosine than conventional tyrosine-producing bacteria that do not have a reduction in pyruvate kinase activity. This Q2 invention was completed as a result of further research based on this knowledge.

The microorganisms used in the tyrosine production method of the present invention are:
It belongs to the genus Brevibacterium and is a mutant strain having reduced acid kinase activity and properties necessary for tyrosine production, such as resistance to m-70 lophenylalanine. In addition to these properties, production ability can be further improved by imparting requirements for L-phenylalanine, resistance to other phenylalanine analogs, resistance to tyrosine analogs, etc.

The parent strain of the mutant strain of the present invention is a microorganism of the genus Brevibacterium known as a so-called L-glutamic acid producing bacterium, and examples thereof include the following strains.

Brevibacterium 7 Lapum ATCC14067 Previbacterium divariotum ATCC14020 Brevibacterium lactofermentum ATcc13
869 Brech Hacterium roserum ATCC1382
5. The mutant strains used in the present invention can be obtained by mutating the above-mentioned strains as parent strains to reduce pyruvate kinase activity and impart tyrosine production ability. The mutation operation can be carried out using conventional methods, such as ultraviolet irradiation or N-
This can be carried out by treatment with a chemical agent such as methyl-N'-nitro-N-nitrosoguanidine (hereinafter abbreviated as NG) or nitrous acid.

A mutant strain with reduced pyruvate kinase activity can be obtained by mutating the parent strain, measuring the pyruvate kinase activity of the mutated cells, and selecting (Japanese Patent Application Laid-Open No. 170487-1987).

The following is an example of the strain used in the present invention, Previpacterium frapum AJ-12179 (FERM-P'IQI3).
) is shown below. That is, the patent application 1970-170
Previbacterium frapum AJ-11955 (FERM-P66
65) as a parent strain, from which a phenylalanine analog resistant strain was derived. Brevibacterium flavum AJ
-11955u treated with 150 μW of NG at 30°C for 15 minutes fc (survival rate 2, 0%). Next, D, an analogue of phenylalanine, was added at a concentration of 800 μg Anl at which the parent strain could not grow on the synthetic medium shown in Table 1.
A plate medium was prepared by adding L-m-fluorophenylalanine, and the mutation-treated AJ-11955 strain was applied thereto. After standing at 30°C for 10 days, a strain that grew as a colony, that is, a mutant strain resistant to m-fluorophenylalanine, was obtained. Among them, mutant strain AJ- with excellent tyrosine production ability was collected.
12179 (FERM-P 7Qt3) (hirupate kinase reduction, m-)'lorophenylalanine resistance,
5-(2-aminoethyl)-L-cysteine resistance) was collected. This mutant strain is a m-fluorophenylalanine-resistant tyrosine-producing mutant strain AJ-12180 (FERM-P) which has pyruvate kinase activity as shown in Examples.
7'//4) produced 1.7 times more cytyrosine.

Next, the mutant strain AJ-12179 obtained by this operation
The results of comparing the pyruvate kinase activity and degree of resistance to m-fluorophenylalanine with the parent strain AJ-11955, the wild strain ATCC-14067, and the tyrosine-producing strain AJ-12180 having pyruvate kinase activity are shown in Tables 3 and 3, respectively. Shown in the table.

The pyruvate kinase activity was measured using Agric.

Biol, Chem, 48, (5) 118
9-1197 (1984). Also m-
The degree of resistance to 70 lophenylalanine was investigated as follows. Dissolve DL-m-70 lophenylalanine in the synthetic medium shown in Table 3 to the concentration shown in Table 3 to make a plate medium (diameter 8.5 mm), and add Approximately 107 bacteria grew in the medium shown on the cloth.
After inoculation, culturing was carried out at 30°C for 4 days, and the number of colonies produced was determined.

As shown in Tables 3 and 3, strain AJ-12179 was found to have both the properties of reduced pyruvate kinase and resistance to m-70 lophenylalanine.

In the present invention, resistance to m-70 lophenylalanine means that approximately 1 microorganism is added to the above medium containing 30004/ml of DL-m-70 lophenylalanine under the above culture conditions.
0:=7 refers to the case where 1000 or more colonies are generated after 4 days of inoculation and culturing at 30°C.

The culture medium for tyrosine production is particularly limited and is a conventional medium containing carbon sources, nitrogen sources, inorganic salts and, if necessary, organic micronutrients.

Carbon sources include carbohydrates (glucose, fructose or starch, hydrolysates of cellulose, molasses, etc.);
Organic acids (acetic acid, citric acid, etc.), alcohols (glycerin, ethanol, etc.), or hydrocarbons (normal paraffin, etc.) can be used. As nitrogen sources, ammonium sulfate, urea, ammonium nitrate, ammonium phosphate, Ammonium chloride, ammonia gas, and others are used as necessary, and inorganic salts such as phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and other trace metal salts are used as necessary.As organic trace nutrients, nutritional requirements are used. If necessary, add appropriate amounts of the relevant amino acids, vitamins, fatty acids, organic basic substances, etc., and if necessary, add amino acids, vitamins, flavor liquid (registered trademark, soybean hydrolyzate), yeast as growth promoting substances. Extracts, benotone, casamino acids, etc. can be used.

The culture may be carried out in a conventional manner under aerobic conditions at a pH of 5 to 9 and a temperature of 20 to 40°C for 24 to 72 hours. If - decreases during culture, calcium carbonate should be separately sterilized and added, or neutralized with an alkali such as aqueous ammonia or ammonia gas. In addition, when an organic acid is used as a carbon source, an increase in - is neutralized with a mineral acid or an organic acid.

Tyrosine can be isolated and collected using conventional methods.

It was confirmed that the Rf value on the paper chromatogram and the biological activity value determined by the microbial quantitative method of the obtained product matched those of the tyrosine specimen, and it was identified as tyrosine.

Tyrosine was determined using Leuconostoc mesenteroides (
It was carried out according to the microbial quantification method using ATCC 8042).

Examples will be described below.

Example 1 20ml of tyrosine production medium with the composition shown in Section 5 below
into 500 tnl flasks, one platinum loopful of each of the microorganisms shown in Table 6 was inoculated into each flask, and the flasks were incubated at 30°C for 72 hours.
Cultured with shaking for hours. The amount of tyrosine produced in each culture solution was as shown in Table 6.

Table - Synthetic medium composition Glucose 209/Ammonium sulfate 5 Urea 2 Potassium phosphate II Magnesium sulfate
lg ferrous sulfate 101n9/manganese sulfate 8 d-biotin 50μVL vitamin B,・
Hydrochloride 2000μVt agar
20 g/l pH6.6 Second coat Pyruvate kinase activity of bacterial strain ATCC140
67 (wild strain) 100AJ-119550,
2 AJ-1218098 AJ-121790,1 (q) Tertiary coat M-70 phenylalanine resistance of strain 0
+ + + (g) represents the number of colonies of 1000 or more, and ←) represents the case where the number of colonies is 0.

Fourth coat Seed culture medium glucose 209/ ammonium sulfate 1og7 urine
element 3 17/l phosphoric acid 1
Potassium I Vt Magnesium Sulfate
0.4Vt ferrous sulfate 10rI
vt manganese sulfate s mytd-biotin 300μ! 9/l vitamin B1/hydrochloride
100 μg/l PH7.3 Fifth coat Tyrosine production medium Glucose 130g/2 Ammonium sulfate 259/l Fumaric acid
12 fJ/acetic acid 3 mVt monopotassium phosphate 1 g/magnesium sulfate 19/ ferrous sulfate
10 crows

Claims (1)

[Claims] A microorganism belonging to the genus Brevibacterium and having reduced pyruvate kinase activity and L-tyrosine producing ability is cultured aerobically in a liquid medium to produce and accumulate L-tyrosine in the culture solution. L- characterized by collecting
Tyrosine production method