JP2000245459A - Production of tetrahydrofolic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method that can efficiently produce tetrahydrofolic acid through the fermentation process and provide a microorganism that can be used in this fermentation process. SOLUTION: A microorganism that has the resistance to folic acid analogues, for example, trimethoprim or sulfaguanidine, and is capable of producing tetrahydrofolic acid, for example, a Coryne form bacteria is cultured to accumulate the tetrahydrofolic acid in the cell bodies of the microorganism whereby the objective tetrahydrofolic acid is produced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing tetrahydrofolate and a microorganism capable of producing tetrahydrofolate. Tetrahydrofolate has an effect of improving the reproduction efficiency of sows, and can be used for feed additives for sows and feeds containing the feed additives. In addition, derivatives obtained from tetrahydrofolic acid are also useful for medicines and the like.

[0002]

2. Description of the Related Art Folic acid is generally produced by a chemical synthesis method. This chemically synthesized folic acid is in an oxidized form and does not act as a coenzyme as it is. Usually, after absorption into the living body, it is converted to 7,8-dihydrofolate by dihydrofolate dehydrogenase, which is further enzymatically reduced to supplement reduced tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5MF). Expresses the action as an enzyme.

As a method for producing tetrahydrofolic acid, a method for diastereoselectively hydrogenating folic acid is known. For example, in a process for diastereoselectively hydrogenating folic acid to tetrahydrofolic acid, a solution of 20 bar or more in a buffered aqueous solution of pH 3 to 12 in the presence of a fixed optically active rhodium (I) complex of an organic diphosphine. H
₍₂₎ A method for diastereoselectively hydrogenating folate to tetrahydrofolate, characterized in that folate is hydrogenated at a pressure and a temperature exceeding 60 ° C. (JP-A-6-9635). .

[0004] Methods for producing tetrahydrofolate using microorganisms include the genus Corynebacterium, the genus Bacillus,
A method using a microorganism belonging to the genus Lactococcus is known (Japanese Patent Application Laid-open No. Hei 7-47911). Although these methods can simplify the process as compared to chemical synthesis requiring a plurality of steps, the efficiency is not sufficient.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above viewpoints, and has as its object to provide a method for efficiently producing tetrahydrofolic acid by a fermentation method and a microorganism which can be used in the method.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, have found that a microorganism resistant to a folate analog has a high tetrahydrofolate-producing ability. Was completed.

That is, the present invention is characterized in that a microorganism having tolerance to folate analogs and capable of producing tetrahydrofolate is cultured in a medium, and tetrahydrofolate is produced and accumulated in the cells of the microorganism. It is a manufacturing method of.

[0008] Examples of the microorganism include microorganisms belonging to coryneform bacteria. In the present invention, examples of the folate analog include trimethoprim, sulfaguanidine, or both of them.

[0009] Examples of the microorganism used in the method of the present invention include a transformant into which a trimethoprim resistance gene has been introduced.

[0010] Examples of the microorganism include a transformant into which a trimethoprim resistance gene has been introduced.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The microorganism used in the present invention is a microorganism having folate analog resistance and having tetrahydrofolate-producing ability. Here, "folate analog resistance" refers to the ability of the microorganism of the present invention to proliferate when cultured in a medium containing a folate analog at a concentration at which a wild strain cannot grow. For example, a microorganism that forms a colony when cultured at 31.5 ° C. for 3 to 7 days in a medium containing 600 to 900 μg / ml of trimethoprim has resistance to trimethoprim. In addition, a microorganism that forms a colony when cultured at 31.5 ° C. for 3 to 7 days in a medium containing 400 to 500 μg / ml of sulfaguanidine has resistance to sulfaguanidine.

Further, “tetrahydrofolate-producing ability” refers to
The microorganism used in the method of the present invention is a microorganism which is taxonomically equivalent to the microorganism and does not have folate analog resistance and more tetrahydrofolate, preferably 15 mg or more per 100 g of dried cells, more preferably 20m
The ability to accumulate more than g.

The microorganism capable of producing tetrahydrofolate may be any microorganism that produces tetrahydrofolate. For example, a microorganism belonging to the genus Coryneform, the genus Bacillus, the genus Lactococcus, the genus Saccharomyces, the genus Candida, the genus Aspergillus, etc. Is mentioned. Among these, coryneform bacteria are preferred. Coryneform bacteria include bacteria that were previously classified into the genus Brevibacterium, but are now integrated into the genus Corynebacterium (Int. J. Syst.
cteriol., 41, 255 (1981)), as well as bacteria of the genus Brevibacterium, which is closely related to the genus Corynebacterium. Specific examples of the above microorganisms include, for example, Corynebacterium glutamicum (formerly Br
evibacterium lactofermentum) ATCC13869), Corynebacterium ammoniagenes (Corynebacterium
(Former Brevibacterium) ammoniagenes ATCC6871),
Brevibacterium flavu
m ATCC13826, Corynebacterium glutamicum ATCC13032, ATCC1306
0), Bacillus subtilis ATC
C13952, IFO3009, IFO13169, etc.), Lactococcus
Lactis (Lactococcus lactis subsp. Cremoris ATCC1
Bacteria such as 9257); Saccharomyces cerevisiae (IFO2044, IFO2375, etc.), Candida utilis (formerly Torulopsis)
utilis ATCC9226); and filamentous fungi such as Aspergillus oryzae (Aspergillus oryzae IFO30104 and the like) and Aspergillus niger (Aspergillus niger IFO4414 and the like).

A microorganism having folate analog resistance can be obtained by mutagenizing a microbial cell, inoculating it into a medium containing a folate analog, for example, trimethoprim or sulfaguanidine or both, and isolating the growing mutant strain. Can be. The mutagenesis treatment can be performed by ultraviolet irradiation, or by treatment with a mutagen used in the usual mutagenesis treatment, such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrous acid. . A suitable concentration of the folate analog to be added to the medium is determined, for example, by applying the mutated cells to a plate medium having various concentrations of folate analog resistance and selecting conditions under which a small number of colonies appear. Can be. Specifically, for trimethoprim, 600-
About 900 μg / ml, 4 for sulfaguanidine
A concentration of about 00 to 500 μg / ml is preferred.

[0015] The tetrahydrofolate productivity of the folate analog-resistant mutant obtained as described above may be determined, for example, by comparing Pediococcus acidil (Pediococcus acidil).
actici) It can be analyzed by a microbioassay using ATCC8081 strain. That is, a folate analog-resistant strain is inoculated on a minimal medium plate, and after a colony is grown, Pediococcus sp.
Tetrahydrofolate productivity and production can be examined by overlaying a soft agar minimal medium containing a culture solution of Acidilactici ATCC8081 strain, and by the presence or absence and size of the halo.

The microorganism having folate analog resistance can also be obtained by transforming the microorganism with a recombinant DNA containing a gene expressing folate analog resistance, preferably a multicopy vector. As a gene expressing folate analog resistance, for example, a trimethoprim resistance gene derived from Brevibacterium lactofermentum is known (JP-A-61-152289).
No.). Brevibacterium lactofermentum AJ12296 harboring a mutant multicopy number plasmid vector pAJ226cop containing the same gene has been deposited as FERM P-8821 with the Biotechnology Industrial Research Institute, Ministry of International Trade and Industry, Ministry of International Trade and Industry. When a coryneform bacterium is transformed with pAJ226cop, a trimethoprim-resistant strain is obtained.

In order to introduce the recombinant DNA into the coryneform bacterium, the transformation may be carried out according to the transformation method reported so far. For example, a method to increase the permeability of DNA by treating recipient cells with calcium chloride, as reported for Escherichia coli K-12 (Mandel, M. and H
iga, A., J. Mol. Biol., 53, 159 (1970)) and a method for preparing competent cells from cells in the growth stage and introducing DNA as described in Bacillus subtilis. (Duncan, CH, Wilson, GAand Young, F.
E., Gene, 1, 153 (1977)). Alternatively, the cells of a DNA recipient, such as those known for Bacillus subtilis, actinomycetes and yeast, are transformed into protoplasts or spheroplasts that readily incorporate the recombinant DNA and the recombinant DNA is introduced into the DNA recipient. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168,
111 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nat
ure, 274, 398 (1978); Hinnen, A., Hicks, JBand Fink,
Natl. Acad. Sci. USA, 75 1929 (1978)). The electric pulse method (Japanese Patent Laid-Open No. 2-2077)
No. 91) is also effective.

The medium used for culturing the microorganism of the present invention is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and other organic components as required. As the carbon source, sugars such as glucose, lactose, galactose, hydrolysates of fructose and starch, alcohols such as glycerol and sorbitol, and organic acids such as fumaric acid, citric acid and succinic acid can be used.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

The organic micronutrients include vitamin B ₁ ,
It is desirable to add required substances such as biotin or yeast extract in an appropriate amount. In addition to these, if necessary,
Potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. are added in small amounts.

By adding paraaminobenzoic acid, oxidized folate and / or nucleic acid to the medium, the amount of tetrahydrofolate produced in the microbial cells obtained by culturing may be increased. Nucleic acids include guanosine, inosine, xanthine, 5'-guanylic acid, 5 '
-Inosinic acid, 5'-xanthylic acid, guanosine-5 '
-Diphosphate, guanosine-5'-triphosphate, and the like. The amount of these additives is such that the intracellular production of tetrahydrofolate is increased as compared with the case where they are not added, for example, 1 mg / L to 1 g / L, preferably 10 to 100 mg. / L. If the amount is too small, the effect of the addition will not be exhibited, and if it is too large, the growth of microorganisms may be inhibited.

As the culturing method, the conditions usually used for culturing these microorganisms can be used as they are. For 12 hours to 5 days.

The collection of tetrahydrofolate from the culture
It may be carried out in the same manner as the production of tetrahydrofolate by a known fermentation method.Specifically, lysozyme, enzymatic treatment of pahain, etc., by a method such as hot water treatment, can be collected from cell crushed cells or cell extract. it can.

When tetrahydrofolate is used as a feed for swine, a cell-containing culture solution obtained by culturing the microorganism of the present invention may be used as it is, or the cells separated by an appropriate method may be spray-dried or the like. May be orally administered as it is. The cells obtained by culturing the microorganism of the present invention are subjected to crushing treatment or extraction treatment after the cells are once separated from the culture solution by an appropriate method. If there is no problem and the crushing treatment or the extraction treatment is not hindered, the culture solution can be subjected to the crushing treatment or the extraction treatment as it is or after being concentrated. The cells to be crushed or extracted may be either live cells or sterilized cells.

The method of crushing is not particularly limited, and may be, for example, any of a conventionally known mechanical method and a method using an enzyme. As the mechanical method, the method itself can be a conventional method. For example, the cells may be crushed by a glass bead using "beads beater" (manufactured by BioSpec), and the cells may be crushed by pressure. Crushing may be performed, or cells may be crushed using an ultrasonic crusher or the like. Even when microbial cells are disrupted using an enzyme, the method itself can be performed by a conventional method.For example, a cultured cell is heat-sterilized as it is, and then a cell wall lysing enzyme is added to the cell to lyse the cell wall of the cell. Decompose. The enzyme used at this time may be any as long as it has the ability to decompose and crush the cell wall.
As those having such ability, conventionally known lysozyme, protease, zymolyase and the like can be mentioned as typical examples. Enzyme treatment conditions can of course follow known methods.

There is no particular limitation on the method of extracting the microorganism cells, for example, autolysis or 90 ° C to 12 ° C.
Extraction can be performed by heating the cells in hot water at a temperature of 0 ° C.

The crushed bacterial cell or the bacterial cell extract thus prepared is orally administered to a sow as it is, or after being appropriately concentrated or dried or in a form to which an appropriate additive is added. Further, since folate is scarcely present in the cell wall, fragments of the remaining cell wall may be removed from the disrupted bacterial cells.

[0028]

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

[0029]

Example 1 Acquisition of a Folate Analog-Resistant Mutant The cells of Brevibacterium lactofermentum ATCC 13869 strain cultured in the CM-2G medium shown in Table 1 were transformed into N-methyl-N′-nitro-N-nitrosoguanidine. (NTG) 50mM
In solution (250μg / ml) dissolved in phosphate buffer (pH 7.0)
Treated for 10 minutes. Mutagenized cells were added to a minimal agar medium containing 600 μg / ml of trimethoprim or 500 μg / ml of sulfaguanidine, or both (compositions are shown in Table 2).
). After culturing at 31.5 ° C. for 3 to 7 days, the grown colonies were picked and isolated. These mutants are
The strains were named o.5, No.6 and No.7.

[0030]

[Table 1] Table 1 成分 Component content グ ル コ ー ス Glucose 0.5 g / dl Yeast extract 1.0 g / dl Polypeptone 1.0 g / dl Sodium chloride 0.5 g / dl Agar 2.0 g / dl pH 7.0 ──────────────────

[0031]

[Table 2] Table 2 成分 Ingredient content 酵素 Enzyme sugar 0.5 g / _{dl (NH 4) 2 SO 4} 0.15 g / dl KH 2 PO 4 0.1 g / dl K 2 HPO 4 0.3 g / dl MgSO 4 · 7H 2 O 0.01 g / dl FeSO 4 · 7H 2 O 0.001g / dl MnSO 4・ NH ₂ O 0.001 g / dl Vitamin B ₁ hydrochloride 100 μg / L Biotin 30 μg / L Urea 0.15 g / dl CaCl ₂ 0.001 g / dl Agar 2.0 g / dl pH 7.0 ──────────── ──────

The degree of resistance to the trimethoprim and sulfaguanidine of the folate analog-resistant mutant strains isolated as described above was compared with that of the parent strain. The above mutant strain was used for CM-2G
The cells were cultured in a medium (having the composition shown in Table 1) at 31.5 ° C. for 24 hours, and the obtained cells were suspended in sterilized water. The suspension was treated with trimethoprim or sulfaguanidine at the concentrations shown in Tables 3 and 4. Table 3 and Table 4 show the degree of growth after inoculating a minimal agar plate medium (having the composition shown in Table 2) containing, and culturing at 31.5 ° C for 72 hours. The symbols in the table are as follows. +: Grows well. ±: grows. -: Does not grow.

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

Example 2 Production of Tetrahydrofolate <1> Culture of Folic Acid Analog-Resistant Mutant A 50 ml medium having the composition shown in Table 5 was dispensed into a 500 ml Sakaguchi flask, and sterilized by heat. Inoculate the analog-resistant mutant strains No. 5, No. 6, and No. 7 at 30 ° C. for 24 hours.
The cells were cultured with shaking for hours. 40 ml of the obtained seed culture was inoculated into a 1 L small fermenter containing 400 ml of a production medium having the composition shown in Table 6, and cultured at 30 ° C., 1000 rpm, and aeration rate of 200 ml / min for 24 hours. Adjustment of the pH and supply of the nitrogen source during the culture were performed using aqueous ammonia, and the pH was maintained at 4.5 to 7.5. After the culture, the cells were collected by centrifugation and freeze-dried to obtain a powder.

[0036]

[Table 5] Table 5 成分 Component content 酵素 Enzyme sugar 2.0 g / _{dl KH 2 PO 4 0.1 g /} dl MgSO 4 · 7H 2 O 0.04 g / dl FeSO 4 · 7H 2 O 0.001g / dl MnSO 4 · nH 2 O 0.001g / dl Mameko ^{(mameno) TN 1) 0.15 g} / dl Yeast extract 0.1 g / dl Vitamin B ₁ hydrochloride 200 μg / L biotin 50 μg / L GD-113 ²⁾ 0.003 ml / L pH 6.5 ────────────────── 1) : Ajinomoto Co., Inc. 2): Defoamer

[0037]

[Table 6] Table 6 成分 Ingredient content 酵素 Enzyme sugar 5.0 g / _{dl (NH 4) 2 SO 4} 0.5 g / dl H 3 PO 4 0.072g / dl KOH 0.36 g / dl MgSO 4 · 7H 2 O 0.04 g / dl Mameko (mameno) TN 0.1 g / dl FeSO 4 · 7H 2 O 0.001 g / dl MnSO ₄・ nH ₂ O 0.001 g / dl pABA 0.01 g / dl Sodium quartamate 0.5 g / dl Yeast extract 0.2 g / dl Vitamin B ₁ hydrochloride 400 μg / L Biotin 100 μg / L DL-methionine 0.02 g / dl GD-113 0.01ml / L pH 6.5 ──────────────────

The dried bacterial cell powder 10 obtained as described above
The amount of tetrohydrofolate per 0 g was determined using Pediococcus acidilactici ATCC8081 according to Table 7.
And the folate content was quantified by the bioassay shown below. The cells obtained by culturing each mutant were freeze-dried, suspended in 0.2 MK ₂ HPO ₄ containing 2% ascorbic acid, and heat-treated at 120 ° C. for 5 minutes.
Enzyme treatment was performed at 37 ° C. for 24 hours using ncreas toluene (Difco). Next, after treating this at 120 ° C. for 5 minutes, 2%
Dilute with an ascorbic acid solution and use the medium for quantification (Folic Acid
Casei Medium (Difco)) and sterilized at 120 ° C. for 5 minutes. This is Pediococcus acidilactici
Inoculate ATCC8081, incubate at 31.5 ° C for 18 hours,
Folic acid was quantified by measuring the absorbance at 562 nm. As a standard, 5-CHO-tetrahydrofolate was used. Table 8 shows the results. As is evident from the results, the folate analog-resistant mutant produces 2-3 times more tetrahydrofolate than the wild-type strain. No. 5 strain, No. 6 strain and No. 7 strain were named Brevibacterium lactofermentum AJ13545, AJ13546 and AJ13547, respectively, and were deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry,
FERM P-17127, FERM P-17128, and FERM P, respectively
-17129 accession number.

[0039]

[Table 7] Table 7 成分 Component content Component content ─── ──────────────────────────────── Casamino acid 1.0 g / dl Calcium pantothenate 800 μg / L L-cysteine 75 mg / dl peraminobenzoic acid 0.1 mg / dl L-tryptophan 20 mg / dl biotin 2 μg / L L-aspartic acid 50 mg / dl K ₂ HPO ₄ 0.54 g / dl adenine sulfate 1.0 mg / dl MgSO ₄・ 7H ₂ O 0.04 g / dl uracil _{1.0 mg / dl FeSO 4 · 7H} 2 O 0.02 g / dl xanthine 2.0 mg / dl sodium citrate 6.2 g / dl glutathione 0.52 mg / dl of glucose 4.0 g / dl thiamine hydrochloride 400 [mu] g / L polysorbate 80 10 mg / dl riboflavin 1000 μg / L pyridoxine hydrochloride 4000 μg / L nicotinic acid 800 μg / L pH 6.5 ± 0.5 ───────────────────────────────── ──

[0040]

[Table 8]

[0041]

According to the present invention, tetrahydrofolic acid can be efficiently produced by a fermentation method.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shigetomasa Miyashiro 450, Omotomitsu, Orotomi-cho, Saga-gun, Saga Prefecture Inside the Kyushu Plant of Ajinomoto Co., Inc. Inside the Kyushu Plant of Ajinomoto Co., Inc. 1-1 Ajinomoto Co., Ltd., Fermentation Technology Research Laboratories, Ajinomoto Co., Ltd. (72) Inventor Yoko Kuwahara 1-1 Suzukicho, Kawasaki-ku, Kawasaki City, Kanagawa Prefecture, Japan Ajinomoto Co., Ltd. Inventor Kazuhiko Totsuka 1-15-1 Kyobashi, Chuo-ku, Tokyo Ajinomoto Co., Inc. (72) Inventor Yasuhiko Takeri 1-15-1 Kyobashi, Chuo-ku, Tokyo Ajinomoto Co. F Terms (reference) 4B024 AA10 BA80 CA01 DA05 GA11 GA25 4B064 AD08 CA02 CA19 CC24 DA11 4B065 AA01X AA01Y AA19X AA26X AA72X AB01 AC14 AC20 BA01 BA18 CA10 CA43

Claims (4)

[Claims] 1. A method for producing tetrahydrofolate, comprising culturing a microorganism having folate analog resistance and capable of producing tetrahydrofolate in a medium, and producing and accumulating tetrahydrofolate in cells of the microorganism. . 2. The method according to claim 1, wherein the microorganism belongs to a coryneform bacterium. 3. The method according to claim 1, wherein the folate analog is trimethoprim, sulfaguanidine, or both. 4. The method according to claim 1, wherein the microorganism is a transformant into which a trimethoprim resistance gene has been introduced.