# Patent

## Title
Process for producing L-glutamic acid

## Abstract

L-glutamic acid is produced by culturing a microorganism in a liquid medium containing L-glutamic acid at a saturation concentration, wherein the microorganism can metabolize a carbon source at a specific pH, and also has an ability to accumulate L-glutamic acid in the medium in an amount which exceeds the saturation concentration of L-glutamic acid, wherein the pH of the medium is controlled so that L-glutamic acid precipitates.

## Background
The present invention is a continuation under 35 U.S.C. §120 of U.S. patent application Ser. No. 11/297,383, filed Dec. 9, 2005, now U.S. Pat. No. 7,354,744 which claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2003-165545, filed Jun. 10, 2003, which is a continuation under 35 U.S.C. §120 of PCT/JP2004/008140, filed Jun. 10, 2004, the entireties of which are incorporated by reference. Also, the Sequence Listing filed electronically herewith is hereby incorporated by reference (File name: US-256_Seq_List_Copy—1; File size: 83 KB; Date recorded: Feb. 19, 2008).


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for producing L-glutamic acid by fermentation. L-glutamic acid is widely used as a raw material in the production of seasonings and so forth.
2. Description of the Related Art
L-glutamic acid is mainly produced by fermentation using an L-glutamic acid-producing bacterium of the so-called coryneform bacterium belonging to the genus Brevibacterium, Corynebacterium or Microbacterium or mutant strains thereof. Moreover, methods utilizing a microorganism belonging to the genus Bacillus, Streptomyces, Penicillium, Pseudomonas, Arthrobacter, Serratia, Candida, or Aerobacter aerogenes (currently Enterobacter aerogenes), a mutant strain of Escherichia coli, or the like are known. Furthermore, also known are methods of producing L-glutamic acid using a microorganism belonging to the genus Klebsiella, Erwinia, or Pantoea (U.S. Pat. No. 6,197,559), and methods of producing L-glutamic acid using an Enterobacter bacterium (U.S. Pat. No. 6,331,419).
Furthermore, various techniques for improving L-glutamic acid-producing ability by enhancing activities of L-glutamic acid biosynthetic enzymes through the use of recombinant DNA techniques have been disclosed. For example, it was reported that the introduction of a gene encoding citrate synthase derived from Escherichia coli or Corynebacterium glutamicum was effective for enhancing L-glutamic acid-producing ability in bacterium belonging to the genus Corynebacterium or Brevibacterium (Japanese Patent Publication (JP 7-121228 B). In addition, JP 61-268185 A discloses a cell harboring recombinant DNA containing a glutamate dehydrogenase gene derived from Corynebacterium bacteria. Furthermore, JP 63-214189 A discloses a technique for increasing L-glutamic acid-producing ability by amplifying genes encoding glutamate dehydrogenase, isocitrate dehydrogenase, aconitate hydratase, and a citrate synthase.
L-glutamic acid production has been considerably increased by the aforementioned breeding of microorganisms or improving production methods. However, in order to respond to increased demand in the future, the development of methods which provide more efficient production of L-glutamic acid at a lower cost are still necessary, and therefore, still represent a need in the art.
Methods for L-glutamic acid fermentation while precipitating L-glutamic acid, which accumulates in culture broth, have been developed (EP 1078989 A). Because the usual L-glutamic acid-producing bacteria cannot grow under acidic conditions, L-glutamic acid fermentation was conventionally performed under neutral conditions. Contrary to such conventional techniques, microorganisms which could produce L-glutamic acid under acidic conditions were screened, and it has been reported that L-glutamic acid can be produced and accumulated in the medium while precipitating the L-glutamic acid by culturing the obtained microorganism (Enterobacter agglomerans) in a liquid medium in which the pH is controlled so that L-glutamic acid is precipitated.
Furthermore, methods are known for producing L-glutamic acid by culturing such an L-glutamic acid-producing bacterium that can grow under acidic conditions as described above in a medium having a total content of organic acids that inhibit growth of the bacterium in an amount that does not inhibit the growth of the bacterium (European Patent Application Laid-open No. 1233070) and for producing L-glutamic acid by culturing such a bacterium as described above at a first pH optimal for growth of the microorganism and then culturing the bacterium at a second pH optimal for L-glutamic acid production by the microorganism and lower than the first pH (European Patent Application Laid-open No. 1233068). Furthermore, a method is known for producing and accumulating L-glutamic acid in a medium while precipitating the L-glutamic acid in the medium, wherein crystals of L-glutamic acid are made to exist in the medium while L-glutamic acid concentration in the medium is lower than the concentration at which natural crystallization of L-glutamic acid occurs (European Patent Application Laid-open No. 1233069).
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for more efficiently producing L-glutamic acid compared with prior art techniques by using a bacterium having an ability to produce L-glutamic acid such as a bacterium belonging to the genus Pantoea. 
The inventors of the present invention found that if a high L-glutamic acid-producing ability was imparted to a bacterium belonging to the genus Pantoea, acetoin and 2,3-butanediol are also produced together with L-glutamic acid. Furthermore, they considered that if it became possible to suppress the production of these by-products, the yield of L-glutamic acid per unit amount of the main raw material (sugar) would be improved. Thus, the inventors of present invention conducted various research and, as a result, found that if pantothenic acid was added to a medium, the by-production of acetoin and 2,3-butanediol was reduced, and as a result, the fermentation yield of L-glutamic acid was improved. Thus, they accomplished the present invention.
That is, the objects of the present invention are as follows. It is an object of the present invention to provide a method for producing L-glutamic acid comprising culturing a microorganism belonging to the genus Pantoea in a medium which contains pantothenic acid, wherein the pH of the medium is controlled so to induce precipitation of L-glutamic acid, and collecting said L-glutamic acid from said medium.
It is a further object of the invention to provide the method as described above, wherein said microorganism is able to metabolize a carbon source in a second medium which contains L-glutamic acid at a saturation concentration and has an ability to cause accumulation of L-glutamic acid in said second medium, wherein said second medium is at a second pH.
It is a further object of the invention to provide the method as described above, wherein the microorganism is Pantoea ananatis. 
It is a further object of the invention to provide the method as described above, wherein said pantothenic acid is a pantothenic acid salt, and the concentration of said salt is at least 1 mg/L.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a restriction map of a DNA fragment of pTWVEK101 derived from Pantoea ananatis. 
FIG. 2 shows construction of a plasmid pMWCPG containing genes gltA, ppc and gdhA.
FIG. 3 shows construction of a plasmid RSF-Tet containing the replication origin of the wide host range plasmid RSF1010 and tetracycline resistance gene.
FIG. 4 shows construction of a plasmid RSFCPG containing the replication origin of the wide host range plasmid RSF1010, tetracycline resistance gene, gltA gene, ppc gene and gdhA gene.
FIG. 5 shows construction of a plasmid pSTVCB containing the gltA gene.
FIG. 6 is an explanatory chart showing the principle of improvement of L-glutamic acid yield provided by addition of pantothenic acid.
FIG. 7 is a graph showing a relationship between concentrations of calcium pantothenate added to a medium and fermentation yield of L-glutamic acid.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the present invention will be explained in detail.
The present invention provides a method for producing L-glutamic acid by fermentation by culturing a microorganism in a medium which contains pantothenic acid, wherein the pH of the medium is controlled so to induce precipitation of L-glutamic acid, and wherein said microorganism is able to metabolize a carbon source at a second pH in a second medium which contains L-glutamic acid at a saturation concentration, and wherein said microorganism has an ability to cause accumulation of L-glutamic acid in said second medium at said second pH, and wherein said L-glutamic acid accumulates in an amount exceeding the amount corresponding to the saturation concentration of L-glutamic acid (henceforth also referred to as an “L-glutamic acid-accumulating microorganism”).
The above L-glutamic acid-accumulating microorganism can be obtained, for example, as follows. A sample containing microorganisms is inoculated into a liquid medium containing 1) L-glutamic acid at a saturation concentration and 2) a carbon source at a specific pH.A strain that is able to metabolize the carbon source is then selected. Although the specific pH is not particularly limited, it is usually about 5.0 or less, preferably about 4.5 or less, further preferably about 4.3 or less. The L-glutamic acid-accumulating microorganism is used for production of L-glutamic acid by fermentation with accompanying precipitation of the L-glutamic acid. If the pH is too high, it becomes difficult for the microorganism to produce L-glutamic acid in an amount sufficient for precipitation. Therefore, the pH is preferably in the aforementioned range.
If the pH of an aqueous solution containing L-glutamic acid is lowered, the solubility of L-glutamic acid significantly falls when the pH is about equal to the pKa of the γ-carboxyl group, or about 4.25 at 25° C. The solubility is the lowest at the isoelectric point (pH 3.2), and the amount of L-glutamic acid which exceeds the saturation concentration precipitates. While it depends on the composition of the medium, L-glutamic acid is dissolved in an amount of 10-20 g/L at pH 3.2, 30-40 g/L at pH 4.0 and 50-60 g/L at pH 4.7, at about 30° C. Usually the pH does not need to be 3.0 or lower, because the L-glutamic acid precipitating effect reaches its upper limit when the pH falls below a certain value. However, the pH may be 3.0 or less.
In addition, the expression that a microorganism “can metabolize a carbon source” or “is able to metabolize a carbon source” means that the microorganism can proliferate or can consume a carbon source even though it cannot proliferate, that is, it indicates that the microorganism catabolizes a carbon source such as sugars or organic acids. Specifically, for example, if a microorganism proliferates when it is cultured in a liquid medium containing L-glutamic acid at a saturation concentration at pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, most preferably about pH 4.0, at an appropriate temperature, for example, 28° C., 37° C. or 50° C., for 2 to 4 days, then this is a microorganism that can metabolize the carbon source in the medium. Furthermore, for example, if a microorganism consumes a carbon source even though the microorganism does not proliferate, when it is cultured in a synthetic liquid medium containing L-glutamic acid at a saturation concentration at pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, most preferably about pH 4.0, at an appropriate temperature, for example, 28° C., 37° C. or 50° C., for 2 to 4 days, then this is a microorganism that can metabolize the carbon source in the medium.
The microorganism that can metabolize a carbon source includes a microorganism that can grow in the aforementioned liquid medium. The expression that a microorganism “can grow” means that it can proliferate, or it can produce L-glutamic acid even though it cannot proliferate. Specifically, for example, if a microorganism proliferates when it is cultured in a liquid medium containing L-glutamic acid at a saturation concentration at pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, most preferably about pH 4.0, at an appropriate temperature, for example, 28° C., 37° C. or 50° C., for 2 to 4 days, then this is a microorganism that can grow in the medium. Furthermore, for example, if a microorganism increases the amount of L-glutamic acid in a synthetic liquid medium even though the microorganism does not proliferate, when the microorganism is cultured in the synthetic liquid medium containing L-glutamic acid at a saturation concentration at pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, most preferably about pH 4.0, at an appropriate temperature, for example, 28° C., 37° C. or 50° C., for 2 to 4 days, then this is a microorganism that can grow in the medium.
The selection of a microorganism as described above may be repeated two or more times under the same conditions, or by changing the pH or the concentration of L-glutamic acid. Selection at an early stage can be performed in a medium containing L-glutamic acid at a concentration lower than the saturation concentration, and subsequent selection can be performed in a medium containing L-glutamic acid at the saturation concentration. Furthermore, strains with favorable properties, such as a superior proliferation rate, may be selected.
The L-glutamic acid-accumulating microorganism has an ability to cause accumulation of an amount of L-glutamic acid which exceeds the saturation concentration of L-glutamic acid in a liquid medium, in addition to the properties described above. The pH of the aforementioned liquid medium is preferably the same as or close to that of the medium used for screening a microorganism having the aforementioned properties. Usually, a microorganism becomes more sensitive to L-glutamic acid at a high concentration as the pH falls. Therefore, it is preferred that the pH is not low in view of resistance to L-glutamic acid, but a low pH is preferred for production of L-glutamic acid with accompanying precipitation. To satisfy these conditions, the pH can be in the range of 3 to 5, preferably 4 to 5, more preferably 4 to 4.7, further preferably 4 to 4.5, particularly preferably 4.0 to 4.3.
Examples of the L-glutamic acid-accumulating microorganism or breeding materials thereof include, but are not limited to, microorganisms belonging to the genus Pantoea, Enterobacter, Klebsiella, Serratia, Erwinia, Escherichia, Corynebacterium, Brevibacterium, Alicyclobacillus, Bacillus, Saccharomyces, or the like. Of these, microorganisms belonging to the genus Pantoea are preferred. Hereinafter, the microorganism of the present invention will be explained mainly for microorganisms belonging to the genus Pantoea. However, the microorganism is not limited to those belonging to the genus Pantoea, and those belonging to other genera can be similarly used.
An example of a microorganism belonging to the Pantoea includes, but is not limited to, Pantoea ananatis, preferably Pantoea ananatis AJ13355. This strain was isolated from soil in Iwata-shi, Shizuoka, Japan, and can proliferate in a medium containing L-glutamic acid and a carbon source at low pH.
The Pantoea ananatis AJ13355 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary) on Feb. 19, 1998 and received an accession number of FERM P-16644. It was then converted to an international deposit under the provisions of Budapest Treaty on Jan. 11, 1999 and received an accession number of FERM BP-6614.
The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13355 strain. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth (see the examples section).
Although the strains AJ13356 and AJ13601 that were derived from AJ13355 strain were also deposited at the aforementioned depository as Enterobacter agglomerans, they are described as Pantoea ananatis in this specification.
The L-glutamic acid-accumulating microorganism may originally have L-glutamic acid-producing ability, or may have L-glutamic acid-producing ability imparted or increased by breeding through mutagenesis, recombinant DNA techniques, or the like.
The L-glutamic acid-producing ability can be imparted or increased by, for example, increasing an activity of an enzyme that catalyzes a biosynthetic reaction of L-glutamic acid. The L-glutamic acid-producing ability can also be increased by decreasing or eliminating activity of an enzyme that catalyzes a reaction which branches off from the biosynthetic pathway of L-glutamic acid, and generates a compound other than L-glutamic acid.
Examples of the enzyme that catalyzes the biosynthetic reaction of L-glutamic acid include, but are not limited to, glutamate dehydrogenase (hereinafter, also referred to as “GDH”), glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase (hereafter, also referred to as “CS”), phosphoenolpyruvate carboxylase (hereinafter, also referred to as “PEPC”), pyruvate dehydrogenase, pyruvate kinase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase, and so forth. Of these enzymes, one or any combination of CS, PEPC, and GDH are preferred. Furthermore, it is preferred that the activities of all the three of the enzymes CS, PEPC, and GDH, are enhanced in the L-glutamic acid-accumulating microorganism. In particular, CS from Brevibacterium lactofermentum is preferred, because it is not subject to inhibition by α-ketoglutaric acid, L-glutamic acid, and NADH.
In order to enhance the activity of CS, PEPC or GDH, for example, a gene encoding CS, PEPC or GDH can be cloned on an appropriate plasmid and transformed into a host microorganism. The copy number of the gene encoding CS, PEPC, or GDH (hereinafter, abbreviated as “gltA gene”, “ppc gene”, and “gdhA gene”, respectively) in the transformant can be increased, resulting in an increase in the activity of CS, PEPC, or GDH.
The cloned gltA, ppc, and gdhA genes are introduced into the aforementioned starting parent strain solely or randomly in combination. When two or three kinds of the genes are introduced, they may be cloned on one plasmid and introduced into the host, or separately cloned onto two or three different plasmids that can coexist, and then introduced into the host.
Two or more genes encoding the same enzyme, but derived from different microorganisms, may be introduced into the same host.
The plasmids described above are not particularly limited so long as they are autonomously replicable in a microorganism belonging to, for example, the genus Pantoea or the like. Examples of these plasmids include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218, pACYC177, pACYC184, and so forth. Vectors of phage DNA can also be used for introducing the aforementioned genes.
Transformation can be performed by, for example, the method of D. M. Morrison (Methods in Enzymology, 68, 326 (1979)), wherein permeability of recipient bacterium cells is increased by treating the cells with calcium chloride (Mandel M. and Higa A., J. Mol. Biol., 53, 159 (1970)), electroporation (Miller J. H., “A Short Course in Bacterial Genetics”, Cold Spring Harbor Laboratory Press, U.S.A., 1992), or the like.
The activity of CS, PEPC or GDH can also be increased by allowing multiple copies of the gltA gene, the ppc gene, or the gdhA gene to be present on chromosomal DNA of the aforementioned starting parent strain. Multiple copies of the gltA gene, the ppc gene, or the gdhA gene may be introduced into the chromosomal DNA by homologous recombination. In order to introduce multiple copies of these genes into the chromosomal DNA of a bacterium belonging to the genus Pantoea, sequences can be used which are present on the chromosomal DNA in multiple copy number, such as a repetitive DNA and inverted repeats present at the end of a transposable element. Alternatively, multiple copies of the gltA gene, the ppc gene, or the gdhA gene can be introduced into the chromosomal DNA by incorporating them into a transposon and transferring it. As a result, the copy number of gltA gene, the ppc gene, or the gdhA gene in a transformant strain is increased, and thus the activity of CS, PEPC, or GDH is increased.
As organisms used as a source of the gltA gene, the ppc gene, or the gdhA gene of which copy number is to be increased, any organism can be used so long as it has activity of CS, PEPC, or GDH. Examples of the organism preferably include, but are not limited to, bacteria belonging to the genus Pantoea, Enterobacter, Klebsiella, Erwinia, Serratia, Escherichia, Corynebacterium, Brevibacterium, or Bacillus. Specifically, Escherichia coli, Brevibacterium lactofermentum and so forth are encompassed by the present invention. The gltA gene, the ppc gene, and the gdhA gene can be obtained from chromosomal DNA of the microorganisms described above.
The gltA gene, the ppc gene, and the gdhA gene can be obtained using a mutant strain which is deficient in the activity of CS, PEPC, or GDH so that a DNA fragment is isolated that supplements its auxotrophy from the chromosomal DNA of the aforementioned microorganism. Furthermore, since the nucleotide sequences of these genes from bacteria belonging to the genera Escherichia and Corynebacterium are known (Biochemistry, 22, pp. 5243-5249, (1983); J. Biochem., 95, pp. 909-916, (1984); Gene, 27, pp. 193-199, (1984); Microbiology, 140, pp. 1817-1828, (1994); Mol. Gen. Genet., 218, pp. 330-339, (1989); Molecular Microbiology, 6, pp. 317-326, (1992)), they can also be obtained by PCR utilizing primers which have been synthesized based on each nucleotide sequence and using the chromosomal DNA as a template. It is known that, in enterobacteria such as bacteria belonging to the genus Enterobacter or Klebsiella, introduction of a gltA gene from a coryneform bacterium is more effective for enhancing the L-glutamic acid-producing ability when compared with that of a gltA gene from a bacterium of the same species (European Patent Application Laid-open No. 0999282). The strains of Pantoea ananatis described herein are described as Enterobacter agglomerans. 
The activity of CS, PEPC, or GDH can also be increased by enhancing the expression of the gltA gene, the ppc gene, or the gdhA gene, besides the aforementioned amplification of the genes. For example, the expression can be enhanced by replacing a promoter for the gltA gene, the ppc gene, or the gdhA gene with a stronger promoter. For example, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter and PL promoter of the lamda phage, and so forth are known as strong promoters. The gltA gene, the ppc gene and the gdhA gene which have had their respective promoters replaced are then cloned into a plasmid and introduced into the host microorganism, or introduced into the chromosomal DNA of the host microorganism using repetitive DNA, inverted repeat, transposon, or the like.
The activity of CS, PEPC, or GDH can also be increased by replacing the promoter of the gltA gene, the ppc gene, or the gdhA gene on the chromosome with a stronger promoter (see WO87/03006 and Japanese Patent Application Laid-open No. 61-268183), or inserting a strong promoter upstream of the gene coding sequence (see Gene, 29, pp. 231-241 (1984)). Specifically, homologous recombination can be performed between the gltA gene, the ppc gene, or the gdhA gene for which the promoter is replaced with a stronger one or DNA containing a part thereof, and the corresponding gene on the chromosome.
Examples of the enzyme that catalyzes the reaction which branches off from the biosynthetic pathway of the L-glutamic acid and generates a compound other than L-glutamic acid include α-ketoglutarate dehydrogenase (hereinafter, also referred to as “αKGDH”), isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and so forth. Of these enzymes, αKGDH is preferred.
In order to decrease or eliminate the activities of the aforementioned enzymes in a microorganism belonging to the genus Pantoea or the like, mutations for decreasing or eliminating the intracellular activity of the enzymes can be introduced into the genes of the aforementioned enzymes by a usual mutagenesis treatment method or a genetic engineering method.
Examples of a mutagenesis treatment method include, for example, methods utilizing irradiation with X-rays or ultraviolet rays, methods utilizing treatment with a mutagenesis agent such as N-methyl-N′-nitro-N-nitrosoguanidine, and so forth. The mutation may be introduced into the coding region for the enzyme or a region which regulates expression, such as a promoter.
Examples of the genetic engineering methods include, for example, methods utilizing gene recombination, transduction, cell fusion, and so forth. For example, a drug resistance gene is inserted into a cloned target gene to prepare a gene that has lost its function (defective gene). Subsequently, this defective gene is introduced into a host microorganism, and the target gene on the chromosome is replaced with the aforementioned defective gene by utilizing homologous recombination (gene disruption).
The decrease of intracellular activity of the target enzyme in a cell and the degree of the decrease can be confirmed by measuring the enzyme activity of a cell extract or a purified fraction thereof obtained from a candidate strain and comparing it with that of a wild-type strain. For example, the αKGDH activity can be measured by the method of Reed et al. (Reed L. J. and Mukherjee B. B., Methods in Enzymology, 13, pp. 55-61 (1969)).
Depending on the target enzyme, a target mutant strain can be selected based on the phenotype of the mutant strain. For example, a mutant strain wherein the αKGDH activity is eliminated or decreased cannot proliferate or shows a markedly reduced proliferation rate in a minimal medium containing glucose or a minimal medium containing acetic acid or L-glutamic acid as the sole carbon source under aerobic culture conditions. However, normal proliferation occurs even under the same conditions by adding succinic acid or lysine, methionine, and diaminopimelic acid to a minimal medium containing glucose. By utilizing these phenomena as indicators, a mutant strain with decreased αKGDH activity or which is deficient in the activity can be selected.
A method for preparing an αKGDH gene-deficient strain of Brevibacterium lactofermentum by utilizing homologous recombination is described in detail in WO95/34672. Similar methods can be applied to other microorganisms.
Furthermore, techniques such as the cloning of genes and digestion and ligation of DNA, transformation, and so forth are described in detail in Molecular Cloning, 2nd Edition, Cold Spring Harbor Press (1989), and so forth.
A specific example of a mutant strain deficient in αKGDH activity or with decreased αKGDH activity obtained as described above includes Pantoea ananatis AJ13356. Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary) on Feb. 19, 1998 and received an accession number of FERM P-16645. It was then converted to an international deposit under the provisions of Budapest Treaty on Jan. 11, 1999 and received an accession number of FERM BP-6615. The Pantoea ananatis AJ13356 is deficient in αKGDH activity as a result of disruption of the αKGDH-E1 subunit gene (sucA).
When Pantoea ananatis, which is an example of the microorganism used in the present invention, is cultured in a medium containing a saccharide, mucus is extracellularly secreted, occasionally resulting in low operation efficiency. Therefore, when Pantoea ananatis which secretes mucus is used, it is preferable to use a mutant strain that secretes less mucus as compared with a wild-type strain. Examples of mutagenesis treatment include, for example, methods utilizing irradiation with X-ray or ultraviolet ray, methods utilizing treatment with a mutagenesis agent such as N-methyl-N′-nitro-N-nitrosoguanidine, and so forth. A mutant strain with decreased secretion of mucus can be selected by inoculating mutagenized bacterial cells in a medium containing a saccharide, for example, an LB medium plate containing 5 g/L of glucose, culturing them while tilting the plate about 45 degrees and selecting a colony that does not show a flow of mucus.
In the present invention, the impartation or enhancement of L-glutamic acid-producing ability and the impartation of other desirable properties such as reducing mucus secretion as described above can be carried out in any order.
The nucleotide sequence of the sucA gene of Pantoea ananatis as a gene used for the breeding of such L-glutamic acid-producing bacteria as described above and the amino acid sequence of the αKGDH-E1 subunit encoded by the gene are shown SEQ ID NO: 1 and SEQ ID NO: 3.
Furthermore, the nucleotide sequence of the plasmid RSFCPG containing the gltA gene, gdhA gene, and ppc gene derived from Escherichia coli (see Reference Example 1) is shown in SEQ ID NO: 8. In SEQ ID NO: 8, the coding regions of the gltA gene, gdhA gene, and ppc gene are shown at nucleotide numbers 1770 to 487 (encoded by the complementary strand), 2598 to 3941, and 7869 to 5218 (encoded by the complementary strand), respectively. The amino acid sequences of CS, GDH, and PEPC encoded by these genes are shown in SEQ ID NOS: 9, 10, and 11, respectively. Furthermore, the nucleotide sequence of plasmid pSTVCB containing the gltA gene derived from Brevibacterium lactofermentum (see Reference Example 1) and the amino acid sequence of CS encoded by this gene are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively.
CS, GDH, and PEPC may include, besides the wild-type sequences, sequences which have substitution, deletion, insertion, addition, or inversion of one or several amino acid residues that do not substantially degrade the activities of the enzymes. Although the number of “several” amino acid residues referred to herein differs depending on positions in the three-dimensional structures of the proteins or types of amino acid residues, it may be specifically between 2 to 30, preferably between 2 to 20, more preferably between 2 to 10. Therefore, changes to CS, GDH, or PEPC such as those described above are typically conservative changes so as to maintain the activity of CS, GDH, or PEPC. Substitution changes include those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Examples of amino acids which may be substituted for an original amino acid in a CS, GDH, or PEPC protein and which are regarded as conservative substitutions include: Ala substituted with ser or thr; arg substituted with gln, his, or lys; asn substituted with glu, gln, lys, his, or asp; asp substituted with asn, glu, or gln; cys substituted with ser or ala; gln substituted with asn, glu, lys, his, asp, or arg; glu substituted with asn, gln, lys, or asp; gly substituted with pro; his substituted with asn, lys, gln, arg, or tyr; ile substituted with leu, met, val, or phe; leu substituted with ile, met, val, or phe; lys substituted with asn, glu, gln, his, or arg; met substituted with ile, leu, val, or phe; phe substituted with trp, tyr, met, ile, or leu; ser substituted with thr or ala; thr substituted with ser or ala; trp substituted with phe or tyr; tyr substituted with his, phe, or trp; and val substituted with met, ile, or leu.
Examples of DNA coding for substantially the same protein or peptide as CS, GDH, or PEPC include DNA hybridizable with an open reading frame (ORF) of the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 8, or a probe that can be prepared from the nucleotide sequence under stringent conditions and encodes a protein having the activity of CS, GDH, or PEPC. The “stringent conditions” referred to herein include conditions under which so-called specific hybrid is formed, and non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value. However, for example, stringent conditions may include conditions under which DNAs having high homology, for example, DNAs having homology of not less than 50%, preferably not less than 70%, more preferably not less than 90%, most preferably not less than 95% hybridize with each other, but DNAs having homology lower than the above do not hybridize with each other. Alternatively, stringent conditions include conditions whereby DNAs hybridize with each other at a salt concentration typically used during washing in Southern hybridization, i.e., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, at 60° C.
The ORF of the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 8 or a partial sequence thereof can also be used as the probe. Such a probe can be prepared by PCR using oligonucleotides based on the nucleotide sequence of SEQ ID NO: 8 or 12 as primers and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 8 or 12 or a partial nucleotide sequence thereof as a template. When a DNA fragment having a length of about 300 bp is used as the probe, the washing conditions for the hybridization can be, for example, 2×SSC and 0.1% SDS at 50° C.
It is sufficient that the deletion-type sucA gene used for gene disruption has homology to such a degree that it causes homologous recombination with the sucA gene on a chromosomal DNA of an objective microorganism. Such homology is preferably not less than 85%, more preferably not less than 90%, particularly preferably not less than 95%. Moreover, DNAs hybridizable under stringent conditions may cause homologous recombination.
Specific examples of such a strain obtained as described above include the AJ13601 strain derived from the aforementioned Pantoea ananatis AJ13355 strain. This strain was obtained by selecting a low mucus-producing strain from the AJ13355 strain, disrupting the αKGDH gene, introducing the gltA, ppc, and gdhA genes derived from Escherichia coli, and the gltA gene derived from Brevibacterium lactofermentum, selecting a strain which is resistant to L-glutamic acid at high concentration and low pH, and selecting a strain which shows superior growth and L-glutamic acid-producing ability.
By culturing the L-glutamic acid-accumulating microorganism in a liquid medium that is adjusted to a pH that allows precipitation of L-glutamic acid, L-glutamic acid can be produced and accumulated while it is precipitated. The “conditions that allow precipitation of L-glutamic acid produced by the microorganism” referred to herein means conditions that allow precipitation of L-glutamic acid when the L-glutamic acid-accumulating microorganism produces and accumulates L-glutamic acid. Although the pH of these conditions may vary depending on the L-glutamic acid-producing ability of the microorganism, it is usually 3 to 5, preferably 4.5 or less, more preferably 4 or less when the microorganism is a bacterium belonging to the genus Pantoea. 
Furthermore, as for the aforementioned pH condition that allows precipitation of L-glutamic acid, the pH is determined on conditions which allow the L-glutamic acid-accumulating microorganism to metabolize a carbon source in a liquid medium containing L-glutamic acid at a saturation concentration, and exhibit an ability to cause accumulation of L-glutamic acid in the medium in an amount which exceeds the saturation concentration of L-glutamic acid in the medium at that pH.
When the L-glutamic acid-accumulating microorganism is cultured in a medium under the aforementioned conditions, the amount of L-glutamic acid which accumulates can be increased by adding pantothenic acid to the medium. This is presumably because the secondary production of acetoin and 2,3-butanediol is reduced by the addition of pantothenic acid in the medium, and as a result, the fermentation yield of L-glutamic acid is improved.
The pantothenic acid to be contained in the medium is preferably added as a pantothenic acid salt. The amount of the pantothenic acid salt is preferably 1 mg/L or more, more preferably 4 mg/L or more, particularly preferably 8 mg/L or more. The type of pantothenic acid salt is not particularly limited, and examples include calcium salt, sodium salt, and so forth.
Pantothenic acid may be present in the medium during the whole culture process, or only during part of the process. For example, when the method of the present invention comprises the step of proliferating the L-glutamic acid accumulating microorganism and the step of allowing production of L-glutamic acid, pantothenic acid may be present in the medium during at least the step of allowing production of L-glutamic acid, and pantothenic acid may or may not be contained in the medium during the step of proliferating the L-glutamic acid-accumulating microorganism. Furthermore, as for the step of allowing production of L-glutamic acid, the amount of pantothenic acid may not necessarily be within the aforementioned range during the entire time period of this step. That is, pantothenic acid may be present so that the amount is within the aforementioned range during an early part of the step, and the amount may be reduced as the culture progresses. Additional pantothenic acid may also be added intermittently.
Known methods of producing L-glutamic acid using an L-glutamic acid-accumulating microorganism while precipitating L-glutamic acid can be used, except that a medium containing pantothenic acid is used (for example, JP 2001-333769 A (EP 1078989 A), JP 2002-238591 A (EP 1233070 A), JP 2002-238592 A (EP 1233068 A), JP 2002-238593 A (EP 1233069 A)).
For example, one of the preferred embodiments of the method of the present invention is to produce L-glutamic acid by culturing an L-glutamic acid-accumulating microorganism in a medium containing a pantothenic acid salt and having a pH of 5.0 or less, and in which the total content of organic acids that inhibit growth of the microorganism is an amount that does not inhibit the growth of the microorganism (see Japanese Patent Laid-open No. 2002-238591 A (European Patent Laid-open No. 1233070)). In this embodiment, the organic acid that inhibits growth of a microorganism at a pH of a medium means an organic acid that has an inhibitory effect on the growth of the microorganism when it exists at a certain concentration (usually 0.5 g/L or more) in the medium at the pH, and it is usually an organic acid having 1-3 carbons, i.e., formic acid, acetic acid, or propionic acid.
The total content of the organic acid is preferably 0.4 g/L or less, more preferably 0.3 g/L or less, even more preferably 0.2 g/L or less.
Another preferred embodiment of the method of the present invention is to produce an L-glutamic acid which includes culturing an L-glutamic acid-accumulating microorganism at a first pH optimal for growth of the microorganism and then culturing the microorganism at a second pH optimal for production of L-glutamic acid by the microorganism and lower than the first pH, and in which the L-glutamic acid-accumulating bacterium is cultured in a medium containing pantothenic acid during at least the culture at the second pH (see Japanese Patent Laid-open No. 2002-238592 (European Patent Laid-open No. 1233068)).
Another preferred embodiment of the method of the present invention is to produce an L-glutamic acid which includes culturing an L-glutamic acid-accumulating microorganism at a first pH at which growth of the microorganism is not inhibited by organic acids contained in the medium, and then culturing the microorganism at a second pH which is optimal for production of L-glutamic acid by the microorganism and which is lower than the first pH, and wherein the L-glutamic acid-accumulating bacterium is cultured in the medium containing pantothenic acid during at least the culture at the second pH (see Japanese Patent Laid-open No. 2002-238591 (European Patent Laid-open No. 1233070)).
It was found that an L-glutamic acid-producing bacterium was generally inhibited by an organic acid under acidic conditions, whereas it could consume an organic acid under neutral conditions (see Japanese Patent Laid-open No. 2002-238591 (European Patent Laid-open No. 1233070)). Based on this property, by growing the cells at a neutral pH and then changing the pH to be acidic to produce L-glutamic acid, it becomes possible to obtain higher productivity and it also becomes possible to use various materials as a sugar source.
In this embodiment, the “organic acid” means an organic acid that has an inhibitory effect on the growth of the microorganism when it exists at a certain concentration (usually 0.5 g/L or more) in a medium at the second pH, and the organic acid usually has 1-3 carbons, i.e., formic acid, acetic acid, or propionic acid.
The first pH and the second pH are selected so that they meet the properties of the chosen L-glutamic acid-producing bacterium. These pH values can easily be measured by those skilled in the art. For example, the pH at which inhibition of growth of a microorganism is not caused by an organic acid in a medium can be determined by culturing an L-glutamic acid-producing bacterium in a medium containing an organic acid adjusted to various pH values, measuring cell amounts based on absorbance or the like, and comparing the cell amounts with cell amounts of the L-glutamic acid-producing bacterium cultured under the same conditions but in the absence of the organic acid. The pH which is suitable for the production of L-glutamic acid refers to the pH at which L-glutamic acid accumulates in a medium, which is determined by culturing an L-glutamic acid-producing bacterium in media at various pH values. Specifically, it can be determined by measuring amounts of L-glutamic acid which has accumulated in media at various pH values and comparing them.
The first pH is not particularly limited so long as growth of the microorganism is not inhibited by the organic acid in the medium, and it is usually 5.0 to 8.0.
The second pH is preferably a pH at which the produced L-glutamic acid precipitates, and such pH is usually 3.0 to 5.0. Reducing productivity by the accumulation of L-glutamic acid at a high concentration can be obviated by performing the culture at the pH at which the produced L-glutamic acid precipitates.
The first pH and the second pH may not be strictly constant during the culture so long as the advantage of the present invention can be obtained, and they may fluctuate.
The L-glutamic acid-producing bacterium produces L-glutamic acid even at the first pH, and therefore pH is lowered by the produced L-glutamic acid. Therefore, the culture at the first pH is preferably performed while maintaining pH of the medium at the first pH by adding an alkalizing substance to the medium.
Although the alkalizing substance is not particularly limited so long as it does not adversely affect the growth of the L-glutamic acid-producing bacterium or L-glutamic acid production, ammonia gas is preferred.
The pH of the medium may be lowered from the first pH to the second pH by adding an acidic substance. However, pH is lowered by production of L-glutamic acid by the L-glutamic acid-producing bacterium during the culture as described above. Therefore, it is preferable to lower the pH of the medium from the first pH to the second pH by controlling the amount of alkalizing substance which is added, because the addition of the acidic substance can be omitted.
The culture at the first pH may be continued until the organic acid in the medium is depleted. “Depletion” means that the amount of the organic acid decreases to a level at which growth of the L-glutamic acid-producing bacterium is not inhibited during the culture at the second pH. Such a level of the organic acid can be easily measured by those skilled in the art. For example, the level can be determined by culturing an L-glutamic acid-producing bacterium in media containing an organic acid at various concentrations at the second pH, measuring cell amounts of the L-glutamic acid-producing bacterium, and comparing the cell amounts with cell amounts of the L-glutamic acid-producing bacterium cultured under the same conditions but in the absence of the organic acid. Generally, as the second pH becomes lower, the level of the organic acid also becomes lower.
A further preferred embodiment of the method of the present invention is to produce L-glutamic acid by fermentation by culturing an L-glutamic acid-accumulating bacterium in a medium whereby the pH is controlled so that L-glutamic acid which is produced is precipitated to cause accumulation of L-glutamic acid in the medium while precipitating the L-glutamic acid, wherein crystals of L-glutamic acid are induced to exist in the medium during a period wherein the L-glutamic acid concentration in the medium is lower than the concentration at which natural crystallization of L-glutamic acid occurs, and the medium contains pantothenic acid (see Japanese Patent Laid-open No. 2002-238593 (European Patent Laid-open No. 1233069)). The “natural crystallization” means that, when a microorganism having an ability to produce L-glutamic acid accumulates L-glutamic acid, the L-glutamic acid concentration in the medium exceeds the saturation concentration of L-glutamic acid, and thus L-glutamic acid naturally precipitates in the medium.
To make crystals of L-glutamic acid exist in the medium means artificially providing the crystals of L-glutamic acid in the medium. Examples of include adding crystals, dissolving a certain amount of L-glutamic acid in the medium at the start of the culture, and decreasing the pH during the culture to forcibly precipitate crystals, and so forth. The amount of crystals made to exist in the medium is usually 0.01 to 10 g/L. Furthermore, the period where the crystals are made to exist is preferably a period where the amount of accumulated L-glutamic acid in the medium increases to a concentration around the saturation concentration (for example, when pH is 4.5 or higher, 25 g/L or more). The amount of L-glutamic acid crystals that exist in the medium and the concentration of L-glutamic acid can be measured by methods well known to those skilled in the art. Crystals of L-glutamic acid are measured after the medium is left standing, and the crystals are collected by decantation. The concentration of L-glutamic acid in the medium is a concentration of dissolved L-glutamic acid. When crystals precipitate in the medium, the concentration of L-glutamic acid means the L-glutamic acid concentration which is measured in a clear solution obtained by separating solid content from the medium by centrifugation (or filtration).
Making the crystals of L-glutamic acid exist in the medium is preferably done by the addition of the crystals of L-glutamic acid.
Crystals of L-glutamic acid include those of α-form and β-form (H. Takahashi, T. Takenishi, N. Nagashima, Bull. Chem. Soc. Japan, 35, 923 (1962); J. D. Bernal, Z. Krist., 78, 363 (1931); S. Hirokawa, Acta Cryst., 8, 637 (1955)). When crystals of the α-form are to be obtained, the crystals to be added are preferably those of the α-form.
The preferred amount of crystals to be added varies depending on the conditions, including the form of the crystals and so forth, and it is usually 0.2 g/L or more for the α-form crystals. If the crystals are added in the aforementioned amount or a larger amount, α-form crystals can be obtained with good reproducibility. Crystals of α-form can be handled more easily than compared with crystals of the β-form in view of the morphology thereof.
As the medium used for the present invention, a usual nutrient medium containing a carbon source, nitrogen source, and inorganic salts as well as organic trace amount nutrients such as amino acids and vitamins as required can be used, except that it contains pantothenic acid, and the pH is adjusted to satisfy the predetermined conditions. Either a synthetic medium or natural medium may be used. Any carbon source and nitrogen source that can be used by the chosen strain may be used in the medium.
Saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate, and molasses can be used as the carbon source. In addition, organic acids such as acetic acid and citric acid may be used alone or in combination with another carbon source.
Ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, nitrates, and so forth are used as the nitrogen source.
Amino acids, vitamins, fatty acids, nucleic acids, and those containing these substances such as peptone, casamino acid, yeast extract, and soybean protein decomposition products are used as the organic trace nutrients. When an auxotrophic mutant strain that requires an amino acid and so forth for metabolization or growth is used, the required nutrient must be supplemented.
Phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and so forth are used as mineral salts.
As for the culture method, an aeration culture at 20 to 42° C. is usually performed provided that the pH is controlled to be a predetermined value, preferably 3 to 5.
After completion of the culture, the L-glutamic acid which has precipitated in the culture can be collected by centrifugation, filtration, or the like. L-Glutamic acid dissolved in the medium can be also collected by known methods. For example, the L-glutamic acid can be isolated by concentrating the culture broth to crystallize it or isolated by ion exchange chromatography or the like. It is also possible to crystallize L-glutamic acid dissolved in the medium and then collect the crystallized L-glutamic acid along with the L-glutamic acid precipitated during the culture.
When the L-glutamic acid exceeds the saturation concentration and precipitates, the concentration of L-glutamic acid dissolved in the medium is maintained at a constant level. Therefore, the effect of a high concentration of L-glutamic acid on microorganisms can be reduced. Accordingly, it also becomes possible to breed a microorganism which has even better L-glutamic acid-producing ability. Furthermore, since L-glutamic acid is precipitated as crystals, acidification of the culture broth by accumulation of L-glutamic acid is suppressed, and therefore the amount of alkali which needs to be used for maintaining the pH of the culture can significantly be reduced.
EXAMPLES
Hereafter, the present invention will be more specifically explained with reference to the following non-limited examples.
Reference Example 1
<1> Screening of Microorganism Having L-Glutamic Acid Resistance in Acidic Environment
Screening of a microorganism having L-glutamic acid resistance in an acidic environment was performed as follows. One (1) g each of about 500 samples obtained from nature including soil, fruits, plant bodies, river water, and so forth was suspended in 5 mL of sterilized water, and 200 μL thereof was applied on 20 mL of solid medium adjusted to pH 4.0 with HCl. The composition of the medium was as follows: 3 g/L of glucose, 1 g/L of ammonium sulfate, 0.2 g/L of magnesium sulfate heptahydrate, 0.5 g/L of potassium dihydrogenphosphate, 0.2 g/L of sodium chloride, 0.1 g/L of calcium chloride dihydrate, 0.01 g/L of ferrous sulfate heptahydrate, 0.01 g/L of manganese sulfate tetrahydrate, 0.72 mg/L of zinc sulfate dihydrate, 0.64 mg/L of copper sulfate pentahydrate, 0.72 mg/L of cobalt chloride hexahydrate, 0.4 mg/L of boric acid, 1.2 mg/L of sodium molybdate dihydrate, 50 μg/L of biotin, 50 μg/L of calcium pantothenate, 50 μg/L of folic acid, 50 μg/L of inositol, 50 μg/L of niacin, 50 μg/L of p-aminobenzoic acid, 50 μg/L of pyridoxine hydrochloride, 50 μg/L of riboflavin, 50 μg/L of thiamin hydrochloride, 50 mg/L of cycloheximide, and 20 g/L of agar.
The media plated with the above samples were incubated at 28° C., 37° C., or 50° C. for 2 to 4 days, and 378 colony-forming strains were obtained.
Subsequently, each of the strains obtained as described above was inoculated in a test tube of 16.5 cm in length and 14 mm in diameter containing 3 mL of liquid medium (adjusted to pH 4.0 with HCl) which contained L-glutamic acid at a saturation concentration, and cultured at 28° C., 37° C., or 50° C. for 24 hours to 3 days with shaking. Then, the strains which grew were selected. The composition of the aforementioned medium was follows: 40 g/L of glucose, 20 g/L of ammonium sulfate, 0.5 g/L of magnesium sulfate heptahydrate, 2 g/L of potassium dihydrogenphosphate, 0.5 g/L of sodium chloride, 0.25 g/L of calcium chloride dihydrate, 0.02 g/L of ferrous sulfate heptahydrate, 0.02 g/L of manganese sulfate tetrahydrate, 0.72 mg/L of zinc sulfate dihydrate, 0.64 mg/L of copper sulfate pentahydrate, 0.72 mg/L of cobalt chloride hexahydrate, 0.4 mg/L of boric acid, 1.2 mg/L of sodium molybdate dihydrate, and 2 g/L of yeast extract.
Thus, 78 strains of microorganisms which showed L-glutamic acid resistance in an acidic environment were successfully obtained.
<2> Selection of the Strains Showing a Superior Growth Rate from Microorganisms Having L-Glutamic Acid Resistance in an Acidic Environment
The various microorganisms having L-glutamic acid resistance in an acidic environment obtained as described above are each inoculated into a test tube of 16.5 cm in length and 14 mm in diameter containing 3 mL of medium (adjusted to pH 4.0 with HCl) obtained by adding 20 g/L of glutamic acid and 2 g/L of glucose to M9 medium (Sambrook, J., Fritsh, E. F. and Maniatis, T., “Molecular Cloning”, Cold Spring Harbor Laboratory Press, U.S.A., 1989), and the turbidity of the medium was measured over time, so to select strains showing a favorable growth rate. As a result, the AJ13355 strain was obtained from soil in Iwata-shi, Shizuoka, Japan which showed a favorable growth rate. This strain was determined to be Enterobacter agglomerans based on its bacteriological properties described above. Enterobacter agglomerans includes those re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, and so forth on the basis of nucleotide sequence analysis of 16S rRNA or the like, and the AJ13355 strain is classified into Pantoea ananatis among these.
<3> Acquisition of a Strain with Reduced Mucus Secretion from Pantoea ananatis AJ13355 Strain
Since the Pantoea ananatis AJ13355 strain secretes mucus extracellularly when cultured in a medium containing a saccharide, operation efficiency is not favorable. Therefore, a strain with reduced mucus secretion was obtained by the ultraviolet irradiation method (Miller, J. H. et al., “A Short Course in Bacterial Genetics; Laboratory Manual”, p. 150, 1992, Cold Spring Harbor Laboratory Press, U.S.A.).
The Pantoea ananatis AJ13355 strain was irradiated with an ultraviolet ray for 2 minutes at a position 60 cm away from a 60-W ultraviolet lamp and cultured in LB medium overnight to fix the mutation. The mutagenized strain was diluted and inoculated in LB medium containing 5 g/L of glucose and 20 g/L of agar so that about 100 colonies per plate emerged, and was cultured at 30° C. overnight while the plate was tilted at about 45 degrees, and then 20 colonies without mucus flow were selected.
As a strain for which no revertant emerged even after subculturing 5 times in LB medium containing 5 g/L of glucose and 20 g/L of agar, and which showed growth equivalent to the parent strain in LB medium, LB medium containing 5 g/L of glucose and M9 medium (Sambrook, J. et al., Molecular Cloning, 2nd Edition, Cold Spring Harbor Press, U.S.A., 1989) supplemented with 20 g/L of L-glutamic acid and 2 g/L of glucose and adjusted to pH 4.5 with HCl, the SC17 strain was selected from the strains selected above.
<4> Construction of Glutamic Acid-Producing Bacterium from Pantoea ananatis SC17 Strain
(1) Preparation of αKGDH Deficient Strain from Pantoea ananatis SC17 Strain
A strain that was deficient in αKGDH and had enhanced L-glutamic acid biosynthetic system was prepared from the Pantoea ananatis SC17 strain.
(i) Cloning of αKGDH Gene (Hereinafter, Referred to as “sucAB”) of Pantoea Ananatis AJ13355 Strain
The sucAB gene of the Pantoea ananatis AJ13355 strain was cloned by selecting a DNA fragment complementing the acetic acid-unassimilating property of the αKGDH-E1 subunit gene (hereafter, referred to as “sucA”)-deficient strain of Escherichia coli from chromosomal DNA of the Pantoea ananatis AJ13355 strain.
The chromosomal DNA of the Pantoea ananatis AJ13355 strain was isolated by a method usually employed for extracting chromosomal DNA from Escherichia coli (Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, pp. 97-98, Baifukan, 1992). The pTWV228 (resistant to ampicillin), which is a commercial product of Takara Shuzo Co., Ltd, and was used as a vector.
The chromosomal DNA of the AJ13355 strain digested with EcoT221 and pTWV228 which had been digested with PstI were ligated using T4 ligase, and the ligation mixture was used to transform the sucA-deficient Escherichia coli JRG465 strain (Herbert, J. et al., Mol. Gen. Genetics, 105, 182 (1969)). A strain able to grow in an acetate minimal medium was selected from the transformant strains obtained above, and a plasmid was extracted from the obtained strain and designated pTWVEK101. The Escherichia coli JRG465 strain harboring pTWVEK101 recovered auxotrophy for succinic acid or L-lysine and L-methionine, besides the trait of acetic acid-unassimilating property. This suggests that pTWVEK101 contained the sucA gene of Pantoea ananatis. 
FIG. 1 shows a restriction enzyme map of a DNA fragment derived from Pantoea ananatis in pTWVEK101. In the nucleotide sequence of the hatched portion in FIG. 1, nucleotide sequences considered to be two full length ORFs and two nucleotide sequences considered to be partial sequences of ORFs were found. Amino acid sequences which are predicted to be encoded by these ORFs or the partial sequences are shown in SEQ ID NOS:2-5 starting from 5′ side. As a result of homology search, portions of the nucleotide sequences were determined to contain a 3′ end partial sequence of the succinate dehydrogenase iron-sulfur protein gene (sdhB), full length sucA and αKGDH-E2 subunit gene (sucB), and a 5′ end partial sequence of the succinyl CoA synthetase β subunit gene (sucC). A comparison of the amino acid sequences deduced from these nucleotide sequences with those derived from Escherichia coli (Eur. J. Biochem., 141, pp. 351-359 (1984); Eur. J. Biochem., 141, pp. 361-374 (1984); Biochemistry, 24, pp. 6245-6252 (1985)) showed that these amino acid sequences are very high homologus to each other. In addition, it was found that a cluster of sdhB-sucA-sucB-sucC was located on the chromosome of Pantoea ananatis, as in Escherichia coli (Eur. J. Biochem., 141, pp. 351-359 (1984); Eur. J. Biochem., 141, pp. 361-374 (1984); Biochemistry, 24, pp. 6245-6252 (1985)).
(ii) Acquisition of αKGDH-Deficient Strain Derived from Pantoea ananatis SC17 Strain
The homologous recombination was performed using the sucAB gene of Pantoea ananatis obtained as described above to obtain an αKGDH-deficient strain of Pantoea ananatis. 
After pTWVEK101 was digested with SphI to excise a fragment containing sucA, the fragment was blunt-ended with Klenow fragment (Takara Shuzo Co., Ltd.) and ligated with pBR322 which had been digested with EcoRI and blunt-ended with Klenow fragment, by using T4 DNA ligase (Takara Shuzo Co., Ltd.). The obtained plasmid was digested at the restriction enzyme BglII recognition site, located approximately at the center of sucA by using the enzyme, blunt-ended with Klenow fragment, and then ligated again by using T4 DNA ligase. It was thought that the sucA gene was unable to function due to the introduction of a frameshift mutation into sucA on the newly constructed plasmid during the above procedure.
The plasmid constructed as described above was digested with a restriction enzyme ApaLI, and subjected to agarose gel electrophoresis to recover a DNA fragment containing sucA into which the frameshift mutation was introduced and a tetracycline resistance gene derived from pBR322. The recovered DNA fragment was ligated again using T4 DNA ligase to construct a plasmid for disrupting the αKGDH gene.
The plasmid for disrupting the αKGDH gene obtained as described above was used to transform the Pantoea ananatis SC17 strain by electroporation (Miller, J. H., “A Short Course in Bacterial Genetics; Handbook”, p. 279, Cold Spring Harbor Laboratory Press, U.S.A., 1992), and a strain wherein sucA on the chromosome was replaced with a mutant sucA of the plasmid by homologous recombination was obtained using the tetracycline resistance as a marker. This strain was designated SC17sucA.
In order to confirm that the SC17sucA strain was deficient in αKGDH activity, the enzyme activity was measured by the method of Reed et al. (Reed, L. J. and Mukherjee, B. B., Methods in Enzymology, 13, pp. 55-61, (1969)) using cells of the strain cultured in LB medium to the logarithmic growth phase. As a result, αKGDH activity of 0.073 (ΔABS/min/mg protein) was detected from the SC17 strain, whereas no αKGDH activity was detected from the SC17sucA strain, and thus it was confirmed that the sucA was eliminated as intended.
(2) Enhancement of L-Glutamic Acid Biosynthesis System of Pantoea ananatis SC17sucA Strain
Subsequently, the citrate synthase gene, phosphoenolpyruvate carboxylase gene and glutamate dehydrogenase gene derived from Escherichia coli were introduced into the SC17sucA strain.
(i) Preparation of a Plasmid Having the gltA Gene, ppc Gene, and gdhA Gene, all Derived from Escherichia coli 
The procedures for preparing a plasmid having the gltA gene, the ppc gene, and the gdhA gene will be explained by referring to FIGS. 2 and 3.
A plasmid having the gdhA gene derived from Escherichia coli, pBRGDH (JP 7-203980 A), was digested with HindIII and SphI, and both ends were blunt-ended by treating with T4 DNA polymerase, and then the DNA fragment having the gdhA gene was purified and recovered. Separately, a plasmid having the gltA gene and ppc gene derived from Escherichia coli, pMWCP (WO97/08294), was digested with XbaI, and then both ends were blunt-ended with T4 DNA polymerase. This was mixed with the above purified DNA fragment having the gdhA gene and ligated using T4 ligase to obtain the plasmid pMWCPG, which is pMWCP with the addition of the gdhA gene (FIG. 2).
Concurrently, the plasmid pVIC40 (Japanese Patent Laid-open No. 8-047397) having the replication origin of the wide-host-range plasmid RSF1010 was digested with NotI, treated with T4 DNA polymerase, and digested with PstI. pBR322 was digested with EcoT14I, treated with T4 DNA polymerase and digested with PstI. Both products were mixed and ligated using T4 ligase to obtain a plasmid RSF-Tet having the replication origin of RSF1010 and the tetracycline resistance gene (FIG. 3).
Subsequently, pMWCPG was digested with EcoRI and PstI, and a DNA fragment having the gltA gene, the ppc gene, and the gdhA gene was purified and recovered. RSF-Tet was similarly digested with EcoRI and PstI, and a DNA fragment having the replication origin of RSF1010 was purified and recovered. Both products were mixed and ligated using T4 ligase to obtain a plasmid RSFCPG, which corresponded to RSF-Tet containing the gltA gene, the ppc gene, and the gdhA gene (FIG. 4). It was confirmed that the obtained plasmid RSFCPG expressed the gltA gene, the ppc gene, and the gdhA gene based on the supplementation of the auxotrophy of the gltA gene-, ppc gene-, or gdhA gene-deficient strain derived from Escherichia coli and measurement of each enzyme activity.
(ii) Preparation of Plasmid Having gltA Gene Derived from Brevibacterium lactofermentum 
A plasmid having the gltA gene derived from Brevibacterium lactofermentum was constructed as follows. PCR was performed using primers having nucleotide sequences SEQ ID NOS:6 and 7 which were prepared based on the nucleotide sequence of the Corynebacterium glutamicum gltA gene (Microbiology, 140, pp. 1817-1828 (1994)), and chromosomal DNA of Brevibacterium lactofermentum ATCC13869 was used as a template to obtain a gltA gene fragment of about 3 kb. This fragment was inserted into a plasmid pHSG399 (purchased from Takara Shuzo Co., Ltd.) which had been digested with SmaI to obtain a plasmid pHSGCB (FIG. 5). Subsequently, pHSGCB was digested with HindIII, and the excised gltA gene fragment of about 3 kb was inserted into the plasmid pSTV29 (purchased from Takara Shuzo Co., Ltd.) digested with HindIII to obtain a plasmid pSTVCB (FIG. 5). It was confirmed that the obtained plasmid pSTVCB expressed the gltA gene by measuring the enzyme activity in the Pantoea ananatis AJ13355 strain.
(iii) Introduction of RSFCPG and pSTVCB into SC17sucA Strain
The Pantoea ananatis SC17sucA strain was transformed with RSFCPG by electroporation to obtain a transformant SC17sucA/RSFCPG strain which was resistant to tetracycline. Furthermore, the SC17sucA/RSFCPG strain was transformed with pSTVCB by electroporation to obtain a transformant SC17sucA/RSFCPG+pSTVCB strain showing chloramphenicol resistance.
<4> Acquisition of a Strain with Improved Resistance to L-Glutamic Acid in a Low pH Environment
A strain with improved resistance to L-glutamic acid at high concentrations in a low pH environment (hereafter, also referred to as “strain with high-concentration Glu-resistance at low pH”) was isolated from the Pantoea ananatis SC17sucA/RSFCPG+pSTVCB strain.
The SC17sucA/RSFCPG+pSTVCB strain was cultured overnight at 30° C. in LBG medium (10 g/L of trypton, 5 g/L of yeast extract, 10 g/L of NaCl, 5 g/L of glucose), and the cells washed with saline was appropriately diluted and plated on an M9-E medium (4 g/L of glucose, 17 g/L of Na2HPO4.12H2O, 3 g/L of KH2PO4, 0.5 g/L of NaCl, 1 g/L of NH4Cl, 10 mM of MgSO4, 10 μM of CaCl2, 50 mg/L of L-lysine, 50 mg/L of L-methionine, 50 mg/L of DL-diaminopimelic acid, 25 mg/L of tetracycline, 25 mg/L of chloramphenicol, 30 g/L of L-glutamic acid, adjusted to pH 4.5 with aqueous ammonia) plate. A colony was obtained which emerged after culture at 32° C. for 2 days as a strain with high-concentration Glu-resistance at low pH.
For the obtained strain, the growth level in M9-E liquid medium was measured and L-glutamic acid-producing ability was tested in a 50-ml volume large test tube containing 5 ml of L-glutamic acid production test medium (40 g/L of glucose, 20 g/L of ammonium sulfate, 0.5 g/L of magnesium sulfate heptahydrate, 2 g/L of potassium dihydrogenphosphate, 0.5 g/L of sodium chloride, 0.25 g/L of calcium chloride dihydrate, 0.02 g/L of ferrous sulfate heptahydrate, 0.02 g/L of manganese sulfate tetrahydrate, 0.72 mg/L of zinc sulfate dihydrate, 0.64 mg/L of copper sulfate pentahydrate, 0.72 mg/L of cobalt chloride hexahydrate, 0.4 mg/L of boric acid, 1.2 mg/L of sodium molybdate dihydrate, 2 g/L of yeast extract, 200 mg/L of L-lysine hydrochloride, 200 mg/L of L-methionine, 200 mg/L of DL-α,ε-diaminopimelic acid, 25 mg/L of tetracycline hydrochloride, and 25 mg/L of chloramphenicol). A strain that exhibited the best growth level and the same L-glutamic acid-producing ability as that of its parent strain, the SC17/RSFCPG+pSTVCB strain, was designated Pantoea ananatis AJ13601. The AJ13601 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Aug. 18, 1999 and received an accession number of FERM P-17516. It was then converted to an international deposit under the provisions of Budapest Treaty on Jul. 6, 2000 and received an accession number of FERM BP-7207.
Example 1
The Pantoea ananatis AJ13601 strain was cultured in a medium containing calcium pantothenate (12 mg/L) and also in a medium not containing calcium pantothenate to investigate the production of L-glutamic acid.
Specifically, the culture was performed as follows. Cells of the Pantoea ananatis AJ13601 strain cultured at 30° C. for 14 hours in the LBG agar medium (10 g/L of trypton, 5 g/L of yeast extract, 10 g/L of NaCl, 15 g/L of agar) containing 25 mg/L of tetracycline hydrochloride and 25 mg/L of chloramphenicol were scraped from one plate and inoculated into 300 ml of seed culture medium having the following composition and contained in a 1 L-volume jar fermenter, and seed culture was performed under conditions of 34° C. and pH 6.0.
Composition of Seed Culture Medium:

















Sucrose
50
g/L



MgSO4•7H2O
0.4
g/L



KH2PO4
2.0
g/L



Yeast extract
4.0
g/L



FeSO4•7H2O
0.01
g/L



MnSO4•5H2O
0.01
g/L



L-Lysine hydrochloride
0.4
g/L



DL-Methionine
0.4
g/L



ε-Diaminopimelic acid
0.4
g/L



Tetracycline hydrochloride
25
mg/L



Chloramphenicol
25
mg/L










The pH was adjusted to 6.0 by adding ammonia gas during the culture. The seed culture was finished when depletion of the saccharide in the medium was observed, and the seed culture medium corresponding to 20% volume of the main culture medium was inoculated to 300 ml of the main culture medium contained in a 1 L-volume jar fermenter to perform the main culture under conditions of 34° C. and pH 4.5. The composition of the main culture medium is shown below.
Composition of Main Culture Medium:

















Glucose
50
g/L



(NH4)2SO4
5.0
g/L



MgSO4•7H2O
0.4
g/L



KH2PO4
6.0
g/L



NaCl
1.5
g/L



FeSO4•7H2O
0.01
g/L



MnSO4•5H2O
0.01
g/L



L-Lysine hydrochloride
0.8
g/L



DL-Methionine
0.6
g/L



DL-α,ε-Diaminopimelic acid
0.6
g/L



Tetracycline hydrochloride
25
mg/L



Chloramphenicol
25
mg/L



Calcium chloride dihydrate
0.75
g/L



Calcium pantothenate
12
mg/L









(added only for the culture with pantothenic acid)










The pH was adjusted to 4.5 by adding ammonia gas during the culture. After the saccharide in the medium was consumed and depleted, a 700 g/L of glucose aqueous solution was continuously added (5 ml/hr). When the L-glutamic acid concentration in the culture broth reached 45 g/L, 1.0 g/L of L-glutamic acid crystals were added to the medium as seed crystals to promote precipitation of L-glutamic acid in the culture broth.
After the main culture was performed for 50 hours, a marked amount of L-glutamic acid crystals were precipitated in the jar fermenter. Then, ammonia gas was added to raise pH to 6.0 and thereby dissolve all the L-glutamic acid crystals in the jar fermenter. Then, the amount of the produced L-glutamic acid was measured. The L-glutamic acid concentration was measured by using Automatic enzyme electrode analyzer As210 produced by Asahi Chemical Industry.
As a result, it was found that the L-glutamic acid fermentation yield was significantly improved by the addition of pantothenic acid, as shown in Table 1.










TABLE 1







No addition
With addition of



of calcium
calcium pantothenate



pantothenate
(12 mg/L)


















L-Glutamic acid fermentation
40.2
53.3


yield (%)









The cause of the improvement of the L-glutamic acid yield provided by the addition of calcium pantothenate was investigated. As a result, reduction of acetoin, 2,3-butanediol and CO2 production was confirmed (Table 2). The acetoin and 2,3-butanediol concentrations were measured by using a gas chromatography apparatus GC1700 produced by Shimadzu with the following conditions.
Column Used:
DB-210 123-0233 produced by J & W Scientific, column length: 30 m, column diameter: 0.32 mm, film thickness: 5 μm
Measurement Conditions:
Temperature of vaporization chamber: 250° C.
Carrier gas: He
Pressure: 85.6 kPa
Total flow rate: 97.2 ml/min
Column flow rate: 0.93 ml/min
Linear velocity: 25.0 cm/sec
Purge flow rate: 3.0 ml/min
Split ratio: 100
Column temperature: 70° C.
Makeup gas: He
Makeup flow rate: 30.0 ml/min
H2 flow rate: 47 ml/min
Air flow rate: 400 ml/min
The discharged CO2 amount was measured using an Exhaust oxygen carbon dioxide meter Model EX-1562 produced by ABLE. The produced amounts of acetoin and 2,3-butanediol as well as the produced amount of CO2 calculated from these values are shown in Table 2 (all values are represented in terms of carbon amount). The measured values of CO2 discharged from the culture medium were 26.5% with no pantothenic acid and 27.6% with pantothenic acid.
When calcium pantothenate was added to the medium, the amounts of acetoin, 2,3-butanediol, and CO2 generated in association with the secondary production of the foregoing substances (two moles of CO2 is generated for 1 mole each of acetoin and 2,3-butanediol) were reduced by about 14.3% in terms of the carbon balance compared with when calcium pantothenate was not added. Because this value is substantially equivalent to the difference between the L-glutamic acid fermentation yields obtained with and without addition of calcium pantothenate, i.e., about 13.1%, it is considered that the yield improvement effect provided by the addition of calcium pantothenate was mainly caused by reduction of the secondary production of acetoin and 2,3-butanediol.









TABLE 2






No addition
With addition of


Secondary product
of calcium
calcium pantothenate


(%, in terms of carbon)
pantothenate
(12 mg/L)

















Acetoin + CO2 generated in
12.0
2.5


association with production of


acetoin


Butanediol + CO2 generated in
6.1
1.3


association with production of


butanediol









From the above results, the mechanism of the improvement of L-glutamic acid fermentation yield provided by the addition of pantothenic acid was estimated as follows. That is, it is considered that the L-glutamic acid fermentation yield was improved because insufficiency of coenzyme A (CoA) could be compensated by the addition of pantothenic acid. Pantothenic acid is contained in the structure of CoA in the form of pantetheine, and thus it is one of the constituents of CoA. CoA is used as a coenzyme in the process of converting pyruvic acid into acetyl-CoA in the metabolic pathway. On the other hand, acetoin and 2,3-butanediol are generated from pyruvic acid, and CO2 is discharged in association with the production of acetoin. When pantothenic acid was not added to the medium used for the culture of the L-glutamic acid-producing bacterium, acetoin and 2,3-butanediol accumulated in the medium. Therefore, it is considered that CoA was originally insufficient in the bacterium, and sufficient acetyl-CoA was not produced in the bacterium, which resulted in the secondary production of acetoin and 2,3-butanediol.
On the other hand, when sufficient amounts of CoA was supplied by the addition of pantothenic acid, the reaction caused by the pyruvate dehydrogenase complex (pyruvic acid→acetyl-CoA) was promoted, which reaction had served as a rate-limiting factor due to the insufficiency of CoA, and thus inflow of carbon into the TCA cycle was promoted. Thus, it is considered that the production of L-glutamic acid was promoted via α-ketoglutaric acid (αKG) as a result. Furthermore, when the production of acetoin and 2,3-butanediol decreases, CO2 generated in association with the production of these substances from pyruvic acid also decreases (2 moles of CO2 for 1 mole each of acetoin and 2,3-butanediol). Therefore, it is considered that this reduction also contributed to the improvement of the L-glutamic acid yield (FIG. 6).
On the basis of the above mechanism, it is considered that addition of a substance such as D-panto acid, β-alanine, or D-pantetheine instead of or in addition to the addition of pantothenic acid should provide comparable or more favorable improvement in the yield.
Example 2
The concentration of calcium pantothenate to be added to the medium was changed to 0 mg/L to 196 mg/L to examine the effect of calcium pantothenate concentration on the L-glutamic acid fermentation yield. The culture was performed in the same manner as in Example 1 except that the calcium pantothenate concentration was changed to 0, 1, 2, 4, 8, 12, 24, 48, 96, or 192 mg/L. The results are shown in FIG. 7. As confirmed from these results, the L-glutamic acid fermentation yield was positively improved depending on the calcium pantothenate concentration. Even when 1 mg/L of calcium pantothenate was added, the yield was improved by about 5% compared with no addition (0 mg/L). Because the yield improvement effect could be obtained depending on the addition concentration until the added calcium pantothenate concentration reached 12 mg/L, it is considered that the yield improvement effect can be obtained even with a concentration lower than 1 mg/L.
Example 3
The main culture was performed with addition of sodium pantothenate instead of calcium pantothenate. The culture conditions were the same as those used in Example 1. Sodium pantothenate was added at a concentration of 12.15 mg/L so that the molar concentration of pantothenic acid is equivalent to that obtained with the addition of 12 mg/L of calcium pantothenate. The results are shown in Table 3.
As shown by the results, equivalent L-glutamic acid fermentation yields were obtained with calcium pantothenate and sodium pantothenate. Thus, it was clarified that the factor of the yield improvement should be pantothenic acid itself, not the counter ion of pantothenic acid.










TABLE 3







Addition of calcium
Addition of sodium



pantothenate
pantothenate



(12 mg/L)
(12.15 mg/L)


















L-Glutamic acid
52.8
52.5


fermentation yield (%)









INDUSTRIAL APPLICABILITY
According to the present invention, L-glutamic acid can be more efficiently produced compared with conventional techniques by using a bacterium such as a bacterium belonging to the genus Pantoea. 
While the invention has been described with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.

## Claims

1. A method for producing L-glutamic acid comprising:
a) culturing a microorganism belonging to the genus Pantoea in a medium which contains pantothenic acid, wherein the pH of the medium is controlled to be 3 to 5 so to induce precipitation of L-glutamic acid, and
b) collecting said L-glutamic acid from said medium.

2. The method according to claim 1, wherein said microorganism is able to metabolize a carbon source in a second medium which contains L-glutamic acid at a saturation concentration and has the ability to cause accumulation of L-glutamic acid in said second medium, wherein said second medium is at pH 4.0 to 5.0.

3. The method according to claim 1, wherein the microorganism is Pantoea ananatis.

4. The method according to claim 2, wherein the microorganism is Pantoea ananatis.

5. The method according to claim 1, wherein said pantothenic acid is a pantothenic acid salt, and the concentration of said salt is at least 1 mg/L.

6. The method according to claim 2, wherein said pantothenic acid is a pantothenic acid salt, and the concentration of said salt is at least 1 mg/L.

7. The method according to claim 3, wherein said pantothenic acid is a pantothenic acid salt, and the concentration of said salt is at least 1 mg/L.

8. The method according to claim 4, wherein said pantothenic acid is a pantothenic acid salt, and the concentration of said salt is at least 1 mg/L.

9. The method according to claim 5, wherein said concentration of said salt is 4 mg/L or more.

10. The method according to claim 5, wherein said concentration of said salt is 8 mg/L or more.

11. The method according to claim 6, wherein said concentration of said salt is 4 mg/L or more.

12. The method according to claim 6, wherein said concentration of said salt is 8 mg/L or more.

13. The method according to claim 7, wherein said concentration of said salt is 4 mg/L or more.

14. The method according to claim 7, wherein said concentration of said salt is 8 mg/L or more.

15. The method according to claim 8, wherein said concentration of said salt is 4 mg/L or more.

16. The method according to claim 8, wherein said concentration of said salt is 8 mg/L or more.