METHOD FOR PRODUCING POLY-gamma-GLUTAMIC ACID AND MICROORGANISM USED IN THE PRODUCTION METHOD

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to a method for producing poly-γ-glutamic acid and a microorganism used in the method.

Description of the Related Art

Poly-γ-glutamic acid is known to be the main substance which causes stringiness of fermented soybeans (natto), and various uses thereof are possible in many fields, including foods, cosmetics, medical products, and so forth. Poly-γ-glutamic acid is mainly produced by culturing microorganisms which have an ability to produce poly-γ- glutamic acid, for example, strains of the genus Bacillus, and collecting poly-γ-glutamic acid from the culture (refer to Gekkan Soshiki Baiyo (Monthly Tissue Culture), 16, 10, 369-372, 1990).

Methods for increasing the amount of poly-γ-glutamic acid produced in microbial fermentation have hitherto been developed, and include utilizing a strain which has modified metabolic systems and produces a large amount of poly-γ-glutamic acid. These modified metabolic systems might be involved in the synthesis and decomposition of poly-γ-glutamic acid, and include, for example, a mutant strain which has low ammonia productivity (Japanese Patent Laid-open (Kokai) No..8- 154616), a mutant strain wherein glutamate synthase activity is eliminated or reduced (Japanese Patent Laid-open No. 2000-333690), and so forth. During extended cultures, poly-γ-glutamic acid can decompose, which reduces the accumulation of poly-γ-glutamic acid To alleviate this problem, methods have been developed using a mutant strain wherein the activity of poly- γ-glutamic acid decomposing enzyme encoded by the ywtD gene is eliminated or reduced (Japanese Patent Laid-open No. 2003-235566, U.S. Patent Published Application No. 20030175936), and a mutant strain wherein poly-γ-glutamic acid decomposing enzyme encoded by pghA gene (ywrD gene) is eliminated or reduced (Japanese Patent Laid-open No. 2003-230384).

It has been reported that as the amount of the macromolecule poly-γ-glutamic acid increases during the course of fermentation, the fermentation liquor becomes a pseudoplastic fluid and hence demonstrates fluidity of a non-Newtonian fluid due to an increase in the viscosity of the fermentation liquor, and thus the speed of oxygen transfer and the specific oxygen uptake rate of microorganisms in the medium are reduced (Biotechnology and Bioengineering, 82, 3, 299-305, 2003). It is also known that aeration of the medium is a very important factor in the production of poly-γ-glutamic acid by microbial fermentation. However, the breeding of strains showing a greater ability to produce poly-γ-glutamic acid under reduced oxygen conditions has not been reported to date.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for more efficiently producing poly-γ-glutamic acid by fermentation compared with conventional techniques and a microorganism useful for said production.

The present inventors found that the ability of the Bacillus microorganism to produce poly-γ-glutamic acid was improved when resistance to an electron transport system inhibitor was imparted to the strain. The present invention was accomplished based on these findings.

It is an object of the present invention to provide a method for producing poly-γ- glutamic acid comprising culturing in a medium a microorganism belonging to the genus Bacillus which has an ability to produce poly-γ-glutamic acid and is resistant to an electron transport system inhibitor and collecting the poly-γ-glutamic acid from the medium.

It is a further object of the present invention to provide the method for producing poly-γ-glutamic acid as described above, wherein the electron transport system inhibitor comprises a substance selected from the group consisting of a NADH-ubiquinone reductase inhibitor, a succinate-ubiquinone reductase inhibitor, a ubiquinol-cytochrome c reductase inhibitor, a cytochrome c oxidase inhibitor, and combinations thereof.

It is a further object of the present invention to provide the method for producing poly-γ-glutamic acid as described above, wherein the electron transport system inhibitor comprises a substance selected from the group consisting of thenoyltrifluoroacetone (TTFA), capsaicin, sodium azide, lidocaine, hydroxylamine, o-methyl hydroxylamine and

combinations thereof.

It is a further object of the present invention to provide the method for producing poly-γ-glutamic acid as described above, wherein said microorganism is Bacillus subtilis.

It is a further object of the present invention to provide a microorganism belonging to the genus Bacillus which has an ability to produce poly-γ-glutamic acid and is modified so to be resistant to an electron transport system inhibitor.

It is a further object of the present invention to provide the microorganism as described above, which is Bacillus subtilis.

According to the present invention, an efficient method for producing poly-γ- glutamic acid is provided. According to the present invention, a novel bacterium having resistance to an electron transport system inhibitor is also provided, and utilization of this bacterium enables efficient production of poly-γ-glutamic acid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventors of the present invention theorized that, because the poly-γ-glutamic acid synthesis reaction requires ATP; therefore, production of poly-γ-glutamic acid is greatly affected by the efficiency of ATP production by oxidative phosphorylation coupled with the electron transport system when there is an increase in viscosity of the fermentation liquor and a reduction in the oxygen transfer velocity. <1> A microorganism having an ability to produce poly-γ-glutamic acid

In this specification, the "ability to produce poly-γ-glutamic acid" means an ability to produce and secrete poly-γ-glutamic acid.

Examples of a microorganism belonging to the genus Bacillus which has an ability to produce poly-γ-glutamic acid used in the present invention include, for example, Bacillus subtilis, Bacillus licheniformis, Bacillus anthracis, Bacillus megaterium, and so forth.

More specifically, for example, Bacillus subtilis NBRC 3335, Bacillus subtilis NBRC 3336 (M. Kunioka and A. Goto, Appl. Microbiol. Biotechnol., 40, 867-872, 1994), Bacillus subtilis NBRC 16449, Bacillus licheniformis ATCC 9945 (F. A. Troy, J. Biol. Chem., 248, 305-315 (1973)) and so forth, and commercially available natto-producing bacteria usually used to produce fermented soybeans such as Miyagino, Takahashi, Asahikawa, Matsumura, and Naruse are encompassed.

The NBRC 3335, NBRC 3336 and NBRC 16449 strains can be purchased from

the National Institute of Technology and Evaluation (NITE), Department of Biotechnology, NITE Biological Resource Center (NBRC) (postal code: 292-0818, 2-5-8, Kazusa Kamatari, Kisarazu-shi, Chiba-ken). The ATCC 9945 strain can be purchased from the American Type Culture Collection (address: P.O. Box 1549, Manassas, VA 20108, United States of America).

Furthermore, the microorganism of the present invention may belong to the genus Bacillus which has an improved poly-γ-glutamic acid-producing ability, and is obtained from any of the bacterial strains described above by breeding. The Bacillus microorganism of the present invention which has improved poly-γ-glutamic acid- producing ability can be obtained by, for example, suppressing expression of the glutamate synthase gene (gltA gene) by disrupting the gene (Japanese Patent Laid-open No. 2000-333690). Furthermore, a Bacillus microorganism which has improved poly-γ- glutamic acid-producing ability can also be obtained by reducing or eliminating the decomposition of poly-γ-glutamic acid (Japanese Patent Laid-open No. 2003-235566, U.S. Patent Published Application No. 20030175936, Japanese Patent Laid-open No. 2003- 230384).

<2> A method for obtaining a microorganism which is modified to be resistant to an electron transport system inhibitor

The bacterium of the present invention is obtained by imparting resistance to an electron transport system inhibitor to a Bacillus bacterium which has an ability to produce and secrete poly-γ-glutamic acid. The phrase "resistance to an electron transport system inhibitor" means that the bacterium permits growth of a microorganism in a medium containing an electron transport system inhibitor, wherein the inhibitor is at such a concentration that a Bacillus bacterium which is not resistant to the electron transport system inhibitor, for example, a wild-type strain or a non-modified strain of Bacillus bacterium, cannot grow. This phrase can also mean that the growth rate of a microorganism which is modified to be resistant is faster than that of a wild-type strain or a non-modified strain of a Bacillus bacterium when both are cultured in a medium containing an electron transport system inhibitor. Specifically, for example, when a Bacillus bacterium is inoculated into an agar medium suitable for growth of the Bacillus bacterium, for example, LB agar medium, which also contains an electron transport system inhibitor, and the bacterium is cultured at an optimum temperature, if the Bacillus

bacterium forms a colony within two days whereas a wild-type strain or a non-modified strain of the Bacillus bacterium does not form a colony after two days, the Bacillus bacterium is considered to be resistant to the electron transport system inhibitor. The aforementioned optimum temperature is a temperature at which the bacterium shows favorable growth when the bacterium is cultured in a medium which does not contain an electron transport system inhibitor. Alternatively, a strain that forms a colony within two days on an agar medium which contains an electron transport system inhibitor, for example, thenoyltrifluoroacetone (TTFA) or capsaicin, at a concentration of 0.1 to 10 mM, is considered to be resistant to the electron transport system inhibitor.

A Bacillus bacterium which is resistant to an electron transport system inhibitor can be obtained by, for example, separating and isolating a mutant strain that can grow in a medium containing an electron transport system inhibitor. This separation and isolation procedure can be repeated using more than one electron transport system inhibitor to obtain multi-resistant strains to plural electron transport system inhibitors.

The electron transport system of microorganisms constitutes a NADH- ubiquinone reductase complex, a succinate-ubiquinone reductase complex, an ubiquinol- cytochrome c reductase complex, and a cytochrome c oxidase complex, and inhibitors specific to each complex are known (Handbook of Enzyme Inhibitors, Ed. by Helmward Zollner, Wiley-VCH, 1999). For example, examples of the NADH -ubiquinone reductase inhibitor include rotenone, capsaicin, amytal, and acrinol; examples of the succinate- ubiquinone reductase inhibitor include antimycin A and cinnerizine; examples of the ubiquinol-cytochrome c reductase inhibitor include antimycin A, diphenylamine, and thenoyltrifluoroacetone (TTFA); and examples of the cytochrome c oxidase inhibitor include sodium azide, lidocaine, potassium cyanide, hydroxylamine, and o-methyl hydroxylamine. Among these inhibitors, TTFA, capsaicin, sodium azide, lidocaine, hydroxylamine, o-methyl hydroxylamine and combinations thereof are preferably used. Hereinafter, the method for obtaining Bacillus bacteria which is resistant to various electron transport system inhibitors will be described.

A Bacillus bacterium which is resistant to an electron transport system inhibitor can be obtained by culturing a Bacillus bacterium in a medium containing an electron transport system inhibitor at a concentration which inhibits growth of the bacterium, and selecting a strain which is able to grow. Growth inhibition referred to herein means a delay of growth and/or an arrest of growth. Selection may be performed one time, or

two or more times. The amount of the electron transport system inhibitor added to the medium is not particularly limited so long as it is present at a concentration that inhibits growth of a parent strain, and this concentration may vary depending on the type of the inhibitor. However, a concentration of 0.1 to 10 mM is generally sufficient.

Prior to selection, the Bacillus bacterium may be subjected to a mutagenesis treatment. Typical mutagenesis treatments include ultraviolet irradiation or a treatment with a conventional artificial mutagenesis agent such as N-methyl-N'-nitro-N- nitrosoguanidine (NTG). Selection of an electron transport system inhibitor resistant strain may be performed for one kind of electron transport system inhibitor, or may be performed for two or more kinds of electron transport system inhibitors. Furthermore, the selection may be performed one time, or two or more times for one kind of electron transport system inhibitor.

A Bacillus bacterium which is resistant to an electron transport system inhibitor and is obtained as described above, can grow even in the presence of an electron transport system inhibitor when the inhibitor is at a concentration at which the parent strain cannot grow.

Furthermore, it is also possible to further improve poly-γ-glutamic acid productivity of a Bacillus bacterium by imparting mutations for resistance to various kinds of antibiotics in addition to imparting resistance to an electron transport system inhibitor. For example, it is reported that a mutation for streptomycin resistance causes a mutation in the ribosome protein S12 and improves substance production of microorganisms (J. Shima, A. Hesketh, S. Okamoto, S. Kawamoto, K. Ochi, J. Bacteriol., 178:7276-7284, 1996). It is also revealed that SmpA of Staphylococcus epidermis, which functions as a transport protein for erythoromycin, has similarity with YwtA of Bacillus subtilis, which was suggested to function as a transporter of poly-γ-glutamic acid (Y. Urushibata, S. Tokuyama, Y. Tahara, J. Bacteriol., 184: 337-343, 2002). Therefore, the ability to produce poly-γ-glutamic acid may further be improved by imparting a mutation for streptomycin and/or erythromycin resistance to a bacterium belonging to the genus Bacillus.

<3> Production of poly-γ-glutamic acid by using a microorganism belonging to the genus Bacillus

By culturing a Bacillus microorganism which has the ability to produce poly-γ-

glutamic acid and is resistant to an electron transport system inhibitor, a marked amount of poly-γ-glutamic acid can be produced and secreted into a culture broth. It is estimated that production and secretion of poly-γ-glutamic acid can be improved by imparting resistance to an electron transport system inhibitor, because the efficiency of ATP production by oxidative phosphorylation coupled with the electron transport system will most likely not decrease under conditions of reduced velocity of oxygen transfer to the medium due to the impartation of the resistance.

A medium useful for production of poly-γ-glutamic acid can be a conventional medium which contains a carbon source, nitrogen source, inorganic ions, and other organic trace nutrient sources, as required. It is particularly preferable to add glutamic acid or a metal salt thereof, for example, sodium glutamate, potassium glutamate, or the like to the medium, because it provides efficient production of poly-γ-glutamic acid. Specific examples of medium ingredients include appropriate combinations of the following.

First, as the carbon source, glucose, fructose, sucrose, maltose, raw sugars, molasses (for example, beet molasses, sweet potato molasses), various starches (for example, tapioca, sago, sweet potato, potato, maize starches), or saccharified solutions thereof or suitable combinations of two or more of these can be used. The carbon source can be obtained by using acids or enzymes and so forth.

Furthermore, as the nitrogen source, glutamic acid, sodium glutamate, potassium glutamate, soy sauce koji, or an extract thereof, soy sauce fermentation products such as soy sauce and sediment of soy sauce or a mixture thereof, organic nitrogen sources such as peptone, soybean meal, corn steep liquor, yeast extract, meat extract, soybean itself or defatted soybean, powder, grain, or extract thereof, and urea, inorganic nitrogen sources such as ammonium salts of sulfuric acid, nitric acid, hydrochloric acid, carbonic acid, and so forth, ammonia gas and aqueous ammonia, appropriate combinations of two or more kinds thereof and so forth can be used.

In addition to the aforementioned carbon sources and nitrogen sources, various inorganic salts necessary for growth of microorganisms can be used and include, for example, sulfates, hydrochlorides, phosphates, and acetates of calcium, potassium, sodium, magnesium, manganese, iron, copper, zinc etc., amino acids, vitamins, and so forth. Amino acids which can be used as required include, in addition to the aforementioned glutamic acid, aspartic acid, alanine, leucine, phenylalanine, histidine etc.. As the

vitamins, biotin, thiamine, etc. can be used.

Furthermore, medium materials for solid culture which can be used include, for example, cooked soybeans, barley, wheat, buckwheat, maize, or a mixture thereof, or any of these which also contain glutamic acid, or a metal salt thereof can be preferably used.

In order to culture a microorganism belonging to the genus Bacillus, the aforementioned medium is sterilized by a typical method, for example, by heating to 110 to 140°C for 8 to 15 minutes, and then adding the microorganism the medium. In the case of a liquid culture, the culture is preferably performed under aerobic conditions, such as by shaking, aerating, and stirring, or the like. For such a culture, the culture temperature is 25 to 50°C, preferably 37 to 42°C.

Furthermore, the pH of the medium is preferably adjusted by using sodium hydroxide, potassium hydroxide, ammonia, an aqueous solution thereof, or the like, and the culture is preferably performed at pH 5 to 9, more preferably at pH 6 to 8.

Furthermore, the culture period may typically be about 2 to 4 days. Also, in the case of a solid culture, a culture temperature of 25 to 50°C, preferably 37 to 42 ⁰ C, and a pH of 5 to 9, preferably 6 to 8, is employed during the culture, similar to the liquid culture. If the microorganism is cultured as described above, poly-γ-glutamic acid is secreted mainly to the outside of the cells and contained in the culture.

In order to separate and isolate poly-γ-glutamic acid from this culture, known methods can be used, for example, (1) a method of extracting and isolating poly-γ- glutamic acid from solid culture by using saline at a concentration of 20% or lower (Japanese Patent Laid-open No. 3-30648), (2) a precipitation method using copper sulfate (B.C. Throne, CC. Gomez, N.E. Noues and R.D. Housevright, J. Bacteriol., 68, 307, 1954), (3) an alcohol precipitation method (R.M. Vard, R.F. Anderson and F.K. Dean, Biotechnology and Bioengineering, 5, 41, 1963; S. Sawa, T. Murakawa, S. Murao, S. Omata, Noka (Journal of Japan Society for Bioscience, Biotechnology and Agrochemistry), 47, 159-165, 1973; H. Fujii, Noka, 37, 407-412, 1963 etc.), (4) a chromatography method using a crosslinked chitosan mold product as an adsorbent (Japanese Patent Laid-open No. 3-244392), (5) a molecular ultrafiltration method using a molecular ultrafiltration membrane, (6) a method consisting of an appropriate combination of the aforementioned methods (1) to (5), and so forth. Poly-γ-glutamic acid which is isolated and collected as described above may be made into a solution or a powder as required by known techniques such as by concentrating, hot-air drying, and

lyophilizing in a known manner.

Examples

The present invention will be explained more specifically with reference to the following examples. However, the scope of the present invention is not limited by these examples.

Example 1 : Acquisition of strains derived from Bacillus subtilis NBRC 16449 strain, which are resistant to various electron transport system inhibitors

The Bacillus subtilis NBRC 16449 which has the ability to produce poly-γ- glutamic acid was inoculated into 50 mL of LB medium (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of NaCl, pH 7.0) in a 500-mL Sakaguchi flask and cultured overnight at 30°C with shaking, and then the cells were collected.

The cells were washed with 0.1 M potassium phosphate buffer (pH 7.0), then suspended in a potassium phosphate buffer (pH 7.0) containing 500 mg/L of N-methyl- N'-nitro-N-nitrosoguanidine (NTG), and left at 30°C for 12 minutes. The cells which had been treated with NTG were washed 4 times with potassium phosphate buffer (pH 7.0), and a part of these cells were collected, applied to LB agar medium (LB medium containing 1.5% of agar) containing 0.61 mg/mL of capsaicin, and cultured at 30 ⁰ C for one to three days. The colonies that appeared were streaked on LB agar medium containing capsaicin at the same concentration as mentioned above, and cultured at 30°C.

Then, the colonies which appeared were each inoculated into 3 mL of a poly-γ- glutamic acid production medium (6% of glucose, 6% of ammonium sulfate, 0.4% of KH ₂ PO ₄ , 0.03% of magnesium sulfate, 0.001% of iron sulfate, 0.005% of manganese sulfate, 32 mL/L of hydrochloric acid-hydrolyzed soybean solution (containing 3.5% of total nitrogen), 4.5% of sodium glutamate, and adjusted to pH 7.0 with potassium hydroxide) containing 0.15 g of calcium carbonate in a test tube, and cultured at 37°C for three days with shaking. Then, the poly-γ-glutamic acid which was produced in the culture broth was quantified. Poly-γ-glutamic acid was quantified by using HPLC under the following conditions.

Column: Asahipak GF7M HQ (7.6 x 300 mm) + Asahipak GS-IG (7.6 x 50 mm) produced by Shodex

Mobile phase: 10 niM Tris-HCl buffer (pH 8.6), 10 g/1 of NaCl Flow rate: 0.9 mL/minute Detection: UV 220 nm Temperature: 50 ⁰ C Injection volume: 30 μL

The amount of poly-γ-glutamic acid was calculated from the area ratio by using poly-γ-glutamic acid having an average molecular weight of 30 kDa (produced by Ajinomoto) as a standard.

The ability of the strains to produce poly-γ-glutamic acid was evaluated for 50 strains of the capsaicin-resistant bacteria. Three strains which secreted poly-γ-glutamic acid in an amount greater than that of the parent Bacillus subtilis NBRC 16449 strain were selected and designated ClO, C20, and C32, respectively.

Then, the aforementioned cells which had been treated with NTG were applied to LB agar medium (LB medium containing 1.5% of agar) containing 1 mM thenoyltrifluoroacetone (TTFA), and TTFA-resistant strains were isolated. The ability of the strains to produce poly-γ-glutamic acid was evaluated for 50 strains of the TTFA- resistant bacteria in the same manner as described above. Three strains which secreted poly-γ-glutamic acid in an amount greater than that of the parent Bacillus subtilis NBRC 16449 strain were selected and designated T3, T28, and T25, respectively.

Furthermore, the aforementioned cells which had been treated with NTG were applied to LB agar medium (LB medium containing 1.5% of agar) containing 2.5 mM sodium azide, and sodium azide-resistant strains were isolated. The ability of the strains to produce poly-γ-glutamic acid of the 50 sodium-azide resistant strains was evaluated in the same manner as described above, and three strains which secrete poly-γ-glutamic acid in an amount greater than that of the parent Bacillus subtilis NBRC 16449 strain were selected and designated N20, N28, and N38, respectively.

The strains which were resistant to various electron transport system inhibitors, ClO, C20, C32, T3, T28, T25, N20, N28 and N38, were designated AJl 10393, AJl 10394, AJ110395, AJ110396, AJ110397, AJ110398, AJ110399, AJ110400 andAJ110401, respectively. They were deposited at the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on March 10, 2005 and given accession numbers of B-9007, B-9006, B-9005, B-9004, B-9003, B-9002,

B-9001, B-9000, B-8999, respectively. Then, the deposits were converted to international deposits under the provisions of the Budapest Treaty on May 10, 2005 with the same accession numbers.

Example 2: Production of poly-γ-glutamic acid by strains resistant to electron transport system inhibitors

The aforementioned nine strains obtained in Example 1 (ClO, C20, C32, T3, T28, T25, N20, N28 and N38) and the parent Bacillus subtilis NBRC 16449 strain were each inoculated into 3 mL of a preculture medium (2% of glucose, 0.4% of ammonium sulfate, 0.4% OfKH ₂ PO ₄ , 0.03% of magnesium sulfate, 0.001% of iron sulfate, 0.002% of manganese sulfate, 27 mL/L of hydrochloric acid-hydrolyzed soybean solution (containing 3.5% of total nitrogen), adjusted to pH 7.0 with potassium hydroxide) and cultured at 37°C for 16 hours.

2 mL of this preculture medium was inoculated into 60 mL of the aforementioned production medium which contained 3 g of calcium carbonate in a 500-mL Sakaguchi flask, and cultured at 37°C with shaking. The culture broth was sampled after 24 hours and 48 hours, and poly-γ-glutamic acid which had been produced was quantified by HPLC in the same manner as described in Example 1.

The amount of poly-γ-glutamic acid which had accumulated is shown in Table 1. Each strain which was resistant to an electron transport system inhibitor demonstrated secretion of poly-γ-glutamic acid in an amount greater than that of the parent strain, and thus it was demonstrated that the productivity of poly-γ-glutamic acid was improved by imparting resistance to an electron transport system inhibitor.

Table 1