JP2002281993A - Method for producing shikimic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To economically produce shikimic acid by using a microorganism. SOLUTION: The objective shikimic acid is produced by utilizing a microorganism that belongs to the genus Citrobacter and secrets the shikimic acid extracellularly.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Shikimic acid is an optically active substance having three asymmetric carbon atoms, and is used as a raw material for pharmaceuticals.

[0002]

2. Description of the Related Art Conventionally, shikimic acid is extracted from plants, synthesized from quinic acid or saccharides (Chemical Review 65 435 (1965)), and produced by fermentation using Escherichia coli (Journal Biotechnology). logical
Chemistry (Journal Biological Chemistry) 191 3
15 (1951)), a method of producing by fermentation with recombinant Escherichia coli (Journal of American Chemical Society) 121 160
3 (1999)). Further, production of shikimic acid by a microorganism belonging to the genus Citrobacter is disclosed in
No. 00-78967.

[0003]

The method of extracting from plants is as follows.
The shikimic acid content in the plant is low and purification is difficult. The synthesis method using quinic acid or a saccharide as a raw material has problems that the raw material price is high and the synthesis step is long. Furthermore, the production method by fermentation has a problem that there are many by-products.

[0004] In addition, the fermentation method using microorganisms belonging to the genus Citrobacter also has a problem that the accumulated concentration of shikimic acid is not sufficient.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors have searched for microorganisms other than Escherichia coli that have the potential to produce shikimic acid, and discovered microorganisms that secrete shikimic acid outside the cells. As a result of extensive studies, it was found that a mutant strain of a microorganism having specific properties secreted shikimic acid outside the cells. In a normal fermentation method, that is, a method of adding a carbon source and a nitrogen source and the like to produce a product while the microorganisms grow, it is natural that shikimic acid is produced and accumulated outside the cells, Unlike ordinary fermentation methods, using bacterial cells or cultures obtained by culturing, or their processed products, the growth of microorganisms is effectively stopped, and shikimic acid is removed from a carbon source under conditions where only enzymatic reactions are performed. It is also included in the present invention to produce and secrete extracellularly.

[0006] The secretion of shikimic acid outside the cells means that shikimic acid is produced in the medium of the culture solution, that is, the culture supernatant obtained by removing microorganisms from the culture solution by centrifugation or filtration. This indicates that shikimic acid is produced and accumulated therein.

The present inventors have found that a mutant strain of a microorganism suitable for improving the efficiency of shikimic acid production is a mutant strain resistant to a p-hydroxybenzoic acid analog, and have completed the present invention. That is, the present invention is a method for producing shikimic acid using a microorganism having a property of secreting shikimic acid outside the cells and having a property of being resistant to a p-hydroxybenzoic acid analog.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the present invention may be any microorganism as long as it has a property of secreting shikimic acid outside the cells and a property of being resistant to a p-hydroxybenzoic acid analog. Since shikimic acid is a metabolic intermediate of the aromatic amino acid biosynthesis system, it is desirable that the aromatic amino acid biosynthesis is strong. Preferably, it is a microorganism belonging to the genus Citrobacter, more preferably, Citrobacter freundi. A representative shikimate secreting strain used as a parent strain of the present invention is Citrobacter freundii 4AA-12 (FERM BP-6722). This mutant strain obtained 5-fluorotryptophan resistance and o-fluorophenylalanine resistance from the Citrobacter Freundi IFO13545 strain and became an aromatic amino acid-producing strain, OFPR36-158 strain, as a parent strain. This is a shikimate-secreting mutant.

[0009] The induction of the mutant strain is carried out by irradiating the parent strain with ultraviolet light,
Alternatively, after treatment with a mutagen (eg, N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), a shikimate-secreting mutant can be obtained by a known method. For example, the parent strain is grown on a well-growing agar medium such as a natural medium after the mutagenesis treatment.
Minimal medium containing these amino acids and vitamins, replicating to a minimal medium containing the species (L-tyrosine, L-phenylalanine, L-tryptophan) and p-aminobenzoic acid and a minimal medium containing these amino acids and vitamins Obtain colonies that grew only on top.

The p-hydroxybenzoic acid analog used in the present invention is a structural analog of p-hydroxybenzoic acid, which is a compound that inhibits the growth of microorganisms. Any substance having such properties may be used, but examples thereof include 4-hydroxy-3-methoxybenzoic acid, m-hydroxybenzoic acid,
o-Hydroxybenzoic acid and the like.

The p-hydroxybenzoic acid analog resistant strain is
A mutant strain capable of growing at a concentration at which the growth of a parent strain of a microorganism is inhibited by the addition of the compound to a medium is indicated.

A representative shikimate secreting strain resistant to the p-hydroxybenzoic acid analog used in the present invention is Citrobacter Freundi U-5 (FERM P-
18267) strains. This mutant is a mutant obtained using Citrobacter Freundi 4AA-12 (FERM BP-6722) as a parent strain.

The induction of the mutant strain is carried out by irradiating the parent strain with ultraviolet rays,
Alternatively, after treatment with a mutagenic agent (for example, N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), the cells are grown in a medium to which a p-hydroxybenzoic acid analog at a concentration at which the parent strain cannot grow is added. Microorganisms can be obtained.

The culture method in the fermentation method of the present invention will be described. The medium for shikimic acid production includes a carbon source, a nitrogen source,
A normal medium containing inorganic ions and, if necessary, other organic trace components can be used.

Examples of the carbon source include glucose, fructose, hydrolysates of starch and cellulose, sugars such as molasses, organic acids such as fumaric acid, citric acid and succinic acid, and alcohols such as methanol, ethanol and glycerol. 0.1 to 4.0% of an organic ammonium salt such as ammonium acetate, an inorganic ammonium salt such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonia gas, aqueous ammonia, urea, etc. as a nitrogen source. As the organic trace component, the required substance such as biotin is 0.00%.
A medium containing 0001% to 0.1%, and optionally 0 to 5% as needed, such as corn steep liquor, peptone, yeast extract, etc., is preferably used. In addition to these, potassium phosphate, magnesium sulfate, calcium chloride, sodium chloride, zinc sulfate, copper sulfate,
Preferably, iron, etc., is added as a minor component. Preferably, an antifoaming agent is also added to stabilize the culture conditions.

The culture is usually performed under aerobic conditions. During the culture,
The pH of the medium is adjusted to 3-8, the temperature is adjusted to 20-40 ° C,
Preferable results can be obtained by shaking or aeration and stirring culture for 24 to 144 hours.

[0017]

The present invention will be described below in detail with reference to examples.

Reference Example 1 (Isolation of Shikimate Secreting Mutant) The cells of Citrobacter freundii OFPR36-158 were treated with N-methyl-N'-nitro-N-nitrosoguanidine by a conventional method (30).
After 0 μg / ml) and 30 ° C. for 10 minutes, the cells were appropriately diluted, applied to the medium shown in Table 1, and cultured at 30 ° C. for 2 days. Mutant-treated Citrobacter Freundi OFPR36-158 colonies grown on the medium shown in Table 1 were replicated in the usual manner to the medium (plate medium) shown in Tables 2 and 3 and were incubated at 30 ° C. for 3 days. Cultured. Colonies (4AA-12) that did not grow on the medium of Table 2 but grew on the medium of Table 3
Strain (FERM BP-6722) was isolated.

[0019]

[Table 1]

[0020]

[Table 2]

[0021]

[Table 3] Reference Example 2 (Isolation of mutant resistant to p-hydroxybenzoic acid analog) Citrobacter Freundi 4AA-12 (FERM BP-6722)
Were treated with N-methyl-N'-nitro-N-nitrosoguanidine (300 μg / ml) at 30 ° C.
After that, the cells were appropriately diluted, applied to a plate medium in which 4-hydroxy-3-methoxybenzoic acid was added to the medium shown in Table 2 at a concentration of 10 mM, and cultured at 30 ° C. for 4 days.

The grown mutant colonies of the mutant strain of Citrobacter fruindi 4AA-12 which had been subjected to mutation treatment were purified using the medium shown in Table 2, and the obtained colonies were isolated to obtain shikimic acid production candidate strains.

Example 1 (Culture of mutant strain resistant to p-hydroxybenzoic acid analog and production of shikimic acid by fermentation method) The strains shown in Table 5 were respectively subjected to the medium shown in Table 1 (agar was removed from the medium shown in Table 2). After shaking for 24 hours at 30 ° C. in a liquid medium, the culture was preliminarily cultured at 115 ° C. for 10 minutes in a 500-ml Erlenmeyer flask containing 50 ml of the medium having the composition shown in Table 4 and sterilized at 180 rpm at an amplitude of 30 cm. The cells were cultured under the conditions for 100 hours.

After the cultivation, an HPLC method using the shikimic acid concentration in the filtrate from which the cells and calcium carbonate were removed (column: Shimadzu SCR-101H, mobile phase: 0.1 M phosphoric acid 1 mL / min., Detection: Two
54 nm), the results shown in Table 5 were obtained.

[0025]

[Table 4]

[0026]

[Table 5] Reference Example 3 (resistance of 4-hydroxy-3-methoxybenzoic acid-resistant mutant to 4-hydroxy-3-methoxybenzoic acid) p-hydroxybenzoic acid at the concentration shown in Table 6 was added to the medium shown in Table 3. The strains shown in Table 6 were applied to the medium so
The cells were cultured at 0 ° C. for 4 days. Table 6 shows the results. It was shown that the p-hydroxybenzoic acid analog resistant mutant U-5 has acquired resistance to the p-hydroxybenzoic acid analog, 4-hydroxy-3-methoxybenzoic acid.

[0027]

[Table 6] Example 2 A supernatant of 1 liter of the culture solution obtained in Example 1 was passed through a cation exchange resin Livatit S100 (manufactured by Rohm and Haas Co.), the flow-through fraction was collected, and the anion exchange resin Diaion WA30 (Mitsubishi) 0.1 NN
The fraction was desorbed with aOH, and the fraction containing shikimic acid was collected, passed again through Livatit S100, and the flow-through fraction was concentrated to 120 mL. The solution was extracted three times with 100 mL of n-heptanol containing 0.3 M of tridodecylamine and then back-extracted into an aqueous phase by adding oleic acid to 0.15 M. The aqueous solution was treated with activated carbon, concentrated and crystallized, and further recrystallized with isopropanol to obtain a shikimic acid crystal having a purity of 97.5% or more.
9 g were obtained.

[0028]

According to the method of the present invention, shikimic acid can be produced more economically as compared with the existing method.

Claims (5)

[Claims] (1) It has the property of secreting shikimic acid outside the cells,
A method for producing shikimic acid using a microorganism having a property resistant to a p-hydroxybenzoic acid analog. 2. It has the property of secreting shikimic acid outside the cells,
And culturing a microorganism having a property resistant to p-hydroxybenzoic acid analog to accumulate shikimic acid in the culture solution, isolating and collecting shikimic acid from the culture solution,
A method for producing shikimic acid. 3. The method for producing shikimic acid according to claim 1, wherein the microorganism belongs to the genus Citrobacter. 4. The method for producing shikimic acid according to claim 3, wherein the microorganism belonging to the genus Citrobacter is Citrobacter freundii. 5. The method for producing shikimic acid according to claim 1, wherein the p-hydroxybenzoic acid analog is 4-hydroxy-3-methoxybenzoic acid.