Mutant and method for producing L-glutamic acid by fermentation

SUMMARY OF THE INVENTION
1. Field of the Invention
The present invention concerns a mutant useful in producing L-glutamic acid by fermentation, as well as a method of producing L-glutamic acid by fermentation using such a mutant. L-glutamic acid is an amino acid used in foods and medicaments.
2. Discussion of the Background
L-glutamic acid has conventionally been produced by fermentation using glutamic acid-producing bacteria and mutants thereof, such as those of the genus Brevibacterium, Corynebacterium or Microbacterium ("Amino Acid Fermentation," Gakkai Shuppan Center, pp. 195 to 215 (1986)). Other known methods of producing L-glutamic acid by fermentation include those employing microorganisms of the genera Bacillus, Streptomyces or Penicillium (U.S. Pat. No. 3,220,929) and microorganisms of the genera Pseudomonas, Arthrobacter, Serratia or Candida (U.S. Pat. No. 3,563,857). Even though such conventional methods produce significant amounts of L-glutamic acid, a more efficient and less expensive method of producing L-glutamic acid is desired in order to meet the ever-increasing demand.
Escherichia coli is potentially an excellent L-glutamic acid-producing bacterium, in view of (a) its high growth rate and (b) the availability of sufficient genetic information. However, the reported amount of L-glutamic acid production by Escherichia coli is as low as 2.3 g/l (J. Biochem., Vol. 50, pp. 164 to 165 (1961)). Recently, a mutant exhibiting a deficient or reduced .alpha.-ketoglutarate dehydrogenase (hereinafter referred to as .alpha.-KGDH) was reported to have the ability to produce large amounts of L-glutamic acid (French Patent Application Laid-Open No. 2680178).
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to enhance the L-glutamic acid-producing ability of strains belonging to the genus Escherichia.
A further object of the present invention is to provide a method of producing L-glutamic acid more efficiently and at a lower cost than prior methods.
These and other objects, which will become apparent during the following detailed description of the preferred embodiments, have been surprisingly provided by a mutant of Escherichia coli, the .alpha.-KGDH activity of which is deficient or reduced, and the phosphoenolpyruvate carboxylase (hereinafter referred to as PPC) and glutamate dehydrogenase (hereinafter referred to as GDH) activities of which are amplified.

BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
FIG. 1 shows the procedure for constructing pBR-sucAB;
FIG. 2 shows the procedure for disrupting the sucA gene on Escherichia coli W3110 chromosomal DNA; and
FIG. 3 shows the procedure for constructing pGK.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Accordingly, the present invention includes a mutant of the genus Escherichia which produces L-glutamic acid, the .alpha.-KGDH activity of which is deficient or reduced, and the PPC and GDH activities of which are amplified; and a method of producing L-glutamic acid by fermentation comprising (a) culturing in a liquid culture medium a mutant of the genus Escherichia which produces L-glutamic acid, the .alpha.-KGDH activity of which is deficient or reduced and the PPC and GDH activities of which are amplified, (b) accumulating L-glutamic acid in the culture medium, and (c) recovering L-glutamic acid therefrom.
In accordance with the present invention, a mutant of the genus Escherichia which exhibits deficient, reduced and/or amplified activity refers to deficient, reduced and/or amplified activity relative to the starting strain (e.g., E. coli K-12 (ATCC 10798), W3110 (ATCC 27325), B (ATCC 11303) or W (ATCC 9637)), a wild-type (naturally-occurring) strain or a non-pathogenic strain of the genus Escherichia.
The present invention is detailed below.
(1) Derivation of a mutant of the genus Escherichia exhibiting deficient or reduced .alpha.-KGDH activity
Any non-pathogenic strains of the genus Escherichia may be employed as a starting parent strain to be used in preparation of the present mutant. Examples of such non-pathogenic strains include:
(1) Escherichia coli K-12 (ATCC 10798)
(2) Escherichia coli W3110 (ATCC 27325)
(3) Escherichia coli B (ATCC 11303)
(4) Escherichia coli W (ATCC 9637)
A mutant of the genus Escherichia which produces L-glutamic acid and which has deficient or reduced .alpha.-KGDH activity may be prepared in accordance with known procedures (e.g., Maniatis et al, "Molecular Cloning," Cold Spring Harbor Laboratories, Cold Spring Harbor, Mass. (1989)), or may be prepared as follows.
A non-pathogenic starting parent strain of the genus Escherichia (e.g., one of those mentioned above) may be exposed to X-radiation, ultraviolet light, or one or more mutagenic agents such as N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as NG) to introduce a mutation in the genetic material of the parent strain.
Alternatively, gene engineering technology, for example, gene recombination, gene transformation or cell fusion, may be used to efficiently introduce the intended mutation. A method of obtaining an .alpha.-KGDH-deficient mutant by gene recombination is conducted as follows.
Based on the known nucleotide sequence of the E. coli .alpha.-ketoglutarate decarboxylase gene (hereinafter referred to as the sucA gene; see Euro. J. Biochem., Vol. 141, pp. 351 to 359 (1984)), primers corresponding to nucleotide sequences at or near the 5'- and 3'-termini may be designed and synthesized. Thereafter, the sucA gene is amplified by PCR using the chromosomal DNA as a template. Into the amplified sucA gene, a drug-resistance gene is inserted to obtain a sucA gene whose function is lost. Subsequent homologous recombination replaces the sucA gene on the chromosome with a sucA gene whose function is lost as a result of insertion of the drug-resistance gene.
After subjecting the parent strain to mutagenic treatment, the intended mutants may be screened by known techniques for identifying such mutants by their biological properties or by the procedures illustrated below.
A mutant exhibiting deficient or reduced .alpha.-KGDH activity is either not able to grow or is able to grow only at a significantly reduced growth rate under aerobic conditions in a minimum culture medium containing glucose as the carbon source. However, even under such conditions, normal growth is possible by either (a) adding succinic acid or (b) lysine plus methionine to the minimum culture medium containing glucose. On the other hand, anaerobic conditions allow the mutant to grow even in the minimum culture medium containing only glucose as a carbon source (Molec. Gen. Genetics, Vol. 105, pp. 182 to 190 (1969)). Based on these findings, the desired mutants can be screened and/or identified.
An example of the mutants thus obtained having deficient or reduced .alpha.-KGDH activity is Escherichia coli W3110 sucA::Km.sup.r.
A mutant whose .alpha.-KGDH activity is deficient or reduced is more useful in view of its enhanced ability to produce L-glutamic acid when it further has either reduced L-glutamic acid-degrading activity or constitutive expression of the ace operon (i.e., malate synthase (aceB)-isocitrate lyase (aceA)isocitrate dehydrogenase kinase/phosphatase (aceK)). These properties are discussed in French Patent Application Laid-open No. 2680178. As a matter of course, properties already known to be effective for improving L-glutamic acid-productivity, such as various types of auxotrophy, antimetabolite resistance and antimetabolite sensitivity, are also desirable for enhancing L-glutamic acid production ability.
A mutant having a reduced ability to degrade L-glutamic acid may be isolated as a mutant which either cannot grow or can grow only slightly in a minimum culture medium containing L-glutamic acid as the sole carbon source (e.g., instead of glucose) or as a sole nitrogen source (e.g., instead of ammonium sulfate). However, as a matter of course, when an auxotroph is employed for the derivation, the minimum essential amount of the nutrient required for growth of the auxotroph may be added to the culture medium.
A mutant in which the expression of the ace operon is constitutive may be obtained as a strain whose parent strain is a phosphoenolpyruvate synthase-deficient strain and which can grow in a minimum culture medium containing lactic acid as the carbon source, but which cannot grow in a minimum culture medium containing pyruvic acid (and optional, further containing acetic acid) as the carbon source. Alternatively, a mutant which constitutively expresses the ace operon can be obtained as a strain which shows a higher growth rate than that of its parent strain, the .alpha.-KGDH activity of which is deficient or reduced under aerobic conditions (J. Bacteriol., Vol. 96, pp. 2185 to 2186 (1968)).
Examples of the mutants described above include Escherichia coli AJ 12628 (FERM BP-3854) and Escherichia coli AJ 12624 (FERM BP-3853).
Escherichia coli AJ 12628 is a mutant which constitutively expresses the ace operon and has a reduced .alpha.-KGDH activity and a reduced ability to degrade L-glutamic acid. Escherichia coli AJ 12624 is a mutant having reduced .alpha.-KGDH activity and a reduced ability to degrade L-glutamic acid (French Patent Application Laid-open No. 2680178).
In the mutant thus obtained which exhibits deficient or reduced .alpha.-KGDH activity, the biosynthesis of L-glutamic acid via a-ketoglutaric acid in the TCA cycle is improved, resulting in an enhanced ability to produce L-glutamic acid. Also, the productivity of L-glutamic acid is increased in both (1) the mutant exhibiting deficient or reduced e.alpha.-KGDH activity and significantly lower ability to degrade the produced L-glutamic acid and (2) the mutant further constitutively expressing the ace operon, whereby its growth is improved.
(2) Derivation of a mutant of the genus Escherichia having amplified PPC activity and GDH activity
In the examples described below, a mutant of the genus Escherichia having amplified PPC and GDH activities was obtained from a starting parent strain having (1) the ability to produce L-glutamic acid and (2) deficient or reduced .alpha.-KGDH activity. It is also possible to use a wild strain of the genus Escherichia as the parent strain to first obtain a mutant having amplified PPC and GDH activities, then breed a mutant therefrom which exhibits deficient or reduced .alpha.-KGDH activity.
Accordingly, the starting parent strain used to prepare a mutant having amplified PPC and GDH activities is preferably (a) a mutant of the genus Escherichia which produces L-glutamic acid and which exhibits deficient or reduced .alpha.-KGDH activity, or (b) a non-pathogenic wild-type strain of the genus Escherichia. Examples of such strains include Escherichia coli W3100 sucA::Km.sup.r, Escherichia coli AJ 12628 (FERM BP-3854) and Escherichia coli AJ 12624 (FERM BP-3853) (L-glutamic acid-producing mutants whose .alpha.-KGDH activity is deficient or reduced); Escherichia coli K-12 (ATCC 10798), Escherichia coli W3110 (ATCC 27325), Escherichia coli B (ATCC 11303) and Escherichia coli W (ATCC 9637) (non-pathogenic wild strains of the genus Escherichia).
In order to amplify PPC and GDH activities, the genes coding for PPC and GDH are cloned on an appropriate plasmid, which is then used to transform the starting parent strain employed as a host. The copies of the genes coding for PPC and GDH (hereinafter referred to as the ppc gene and the gdhA gene, respectively) in the transformed cells are increased, resulting in amplified PPC and GDH activities.
The ppc and gdhA genes may be cloned into a single plasmid to be introduced into the starting parent strain employed as the host, or may be cloned separately into two types of plasmid which are compatible in the starting parent strain.
Alternatively, the amplification of PPC and GDH activities may be conducted by allowing the ppc and gdhA genes to be present as multicopies on the chromosomal DNA of the starting parent strain employed as the host. In order to introduce the ppc and gdhA genes as multicopies into the chromosomal DNA of the genus Escherichia, homologous recombination is applied utilizing a target sequence present as a multicopy on the chromosomal DNA. The sequence present as the multicopy may be a repetitive DNA and an inverted repeat present at the terminal of insertion sequence. Alternatively, as described in Japanese Patent Application Laid-open No. 2-109985, the ppc and gdhA genes may be cloned on a transposon, which is then transposed, thereby introducing the multicopy into the chromosomal DNA. The number of copies of the ppc and gdhA genes in the transformed cells are thus increased, resulting in amplification of PPC and GDH activities.
In addition to the gene amplification techniques described above, the amplification of PPC and GDH activities may also be conducted by replacing the promoters of the ppc and gdhA genes with those having higher potencies. For example, the lac promoter, trp promoter, trc promoter, tac promoter and the lambda phage P.sub.R and P.sub.L promoters are known to be strong promoters. By enhancing expression of the ppc and/or gdhA genes, the PPC and/or GDH activities may be amplified.
The ppc and gdhA genes can be obtained by isolating the genes which are complementary with regard to auxotrophy of the mutants which are either PPC- or GDH-deficient. Alternatively, since the nucleotide sequences of these genes of Escherichia coli are known (J. Biochem., Vol. 95, pp. 909 to 916 (1984); Gene, Vol. 27, pp. 193 to 199 (1984)), the primers may be designed and synthesized based on the nucleotide sequences, and then the genes may be obtained by PCR, using the chromosomal DNA as a template.
(3) Production of L-glutamic acid by fermentation using an L-glutamic acid-producing mutant of the genus Escherichia which exhibits deficient or reduced .alpha.-KGDH activity and which has amplified PPC and GDH activities
For the purpose of producing L-glutamic acid by fermentation using an L-glutamic acid-producing mutant of the genus Escherichia which exhibits deficient or reduced .alpha.-KGDH activity and which has amplified PPC and GDH activities, a standard culture medium containing one or more carbon sources, one or more nitrogen sources, essential and/or non-essential inorganic salts and, if necessary or desired, organic trace nutrients such as amino acids and vitamins and may be employed in accordance with standard culture methods. The carbon sources and the nitrogen sources employed in the culture medium may be any of those catabolized by the mutant employed.
The carbon sources may include saccharides and organic acids. Suitable saccharides may include glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate and molasses. Suitable organic acids may include acetic acid and citric acid. Any of the carbon sources may be employed independently or in combination with other carbon sources.
The nitrogen sources may include ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, and nitrate salts such as sodium nitrate and potassium nitrate.
The organic trace nutrients may include amino acids, vitamins, fatty acids and nucleic acids, as they are or as contained in peptone, casamino acid, yeast extract, soy protein hydrolysate and the like. In cases of using an auxotroph, the nutrient required by the auxotroph should be supplemented in the culture medium.
Suitable inorganic salts may include alkali metal and alkaline earth metal phosphate salts, other biologically acceptable magnesium salts and calcium salts, biologically acceptable iron and manganese salts, and the like.
Cultivation/fermentation is conducted (preferably with aeration) at a temperature of from 20.degree. to 45.degree. C. and at a pH of from 5 to 9. The pH is preferably controlled such that it is maintained in the given range (5-9). When the pH is controlled during cultivation, calcium carbonate or an alkali (such as ammonia gas) may be added to the culture medium to neutralize the acid(s) produced by the culture (which may result in lowering the pH to 5 or less). After culturing for a length of time from 10 hours to 4 days, a significant amount of L-glutamic acid is accumulated in the culture medium.
L-glutamic acid in the culture medium after cultivation may be recovered by any known method. For example, the cells may be removed from the culture. The culture medium may then be concentrated and the L-glutamic acid precipitated (e.g., by adding acid to reduce its solubility). Alternatively, the cell-free culture medium may be subjected to ion exchange chromatography to obtain the L-glutamic acid.
The present invention is further described in the following examples, which are given for illustration of the present invention, and are not intended to be limiting thereof. Additional features of the present invention will become readily apparent in the course of the following descriptions of exemplary embodiments.
EXAMPLE 1
(1) Cloning of the sucA and dihydrolipoamide succinyl transferase gene of Escherichia coli
The nucleotide sequences of the sucA and dihydrolipoamide succinyl transferase genes (hereinafter, the dihydrolipoamide succinyl transferase gene is referred to as the sucB gene) of Escherichia coli K12 are known. The known nucleotide sequences of the sucA and sucB genes are disclosed in Euro. J. Biochem., Vol. 141, pp. 351 to 374 (1984), and are also shown below as SEQ ID NO:7 in the Sequence Listing. The nucleotide sequence from the 327th through the 3128th residues corresponds to the ORF (open reading frame) of the sucA gene, while the nucleotide sequence from the 3143rd through the 4357th residues corresponds to the ORF of the sucB gene. Based on the nucleotide sequences reported, primers were synthesized (SEQ ID NOS:1-4), and the sucA and sucB genes were amplified by PCR, employing the chromosomal DNA of Escherichia coli W3110 as a template.
The synthetic primers used to amplify the sucA gene had the nucleotide sequences of SEQ ID NOS:1-2. Sequence ID No. 1 corresponds to the sequence consisting of the 45th through 65th base residues in the nucleotide sequence figure of the sucA gene described in Euro. J. Biochem., Vol. 141, p. 354 (1984). It also corresponds to the sequence consisting of bases 45-65 of SEQ ID NO:7.
Sequence ID No. 2 corresponds to the sequence consisting of the 3173rd through 3193rd base residues in the nucleotide sequence figure of the sucB gene as shown in Euro. J. Biochem., Vol. 141, p. 364 (1984). It also corresponds to the sequence consisting of bases 3173-3193 of SEQ ID NO:7.
The synthetic primers used to amplify the sucB gene had the nucleotide sequences of SEQ ID NOS:3-4. Sequence ID No. 3 corresponds to the sequence consisting of the 2179th through 2198th base residues in the nucleotide sequence figure of the sucA gene as shown in Euro. J. Biochem., Vol. 141, p. 354 (1984). It also corresponds to the sequence consisting of bases 2179-2198 of SEQ ID NO:7.
Sequence ID No. 4 corresponds to the sequence consisting of the 4566th-4591st base residues in the nucleotide sequence figure of the sucB gene shown in Euro. J. Biochem., Vol. 141, p. 364 (1984). It also corresponds to the sequence consisting of bases 4566-4591 of SEQ ID NO:7. The sucA gene and the sucB gene form an operon.
The chromosomal DNA of Escherichia coli W3110 was recovered by a standard method (Seibutsukogaku Jikkensho, ed. by Nippon Seibutsu Kogaku Kai, pp. 97 to 98, Baifukan (1992)) .
PCR was carried out under standard conditions, as described on page 8 of PCR Technology (Henry Erlich, ed., Stockton Press (1989)).
Both ends of the PCR products thus produced were converted into blunt ends using T4 DNA polymerase, and were subsequently cloned into a vector pBR322 at the EcoRV site. The plasmid obtained by cloning the sucA gene into pBR322 was designated as pBR-sucA, and that constructed with the sucB gene was designated as pBR-sucB. The plasmids thus obtained were introduced into Escherichia coli JM109 and the transformant was cultured. Restriction maps were then constructed and compared with the restriction maps of the sucA and sucB genes reported, thereby confirming that the genes which had been cloned were the sucA and sucB genes.
As shown in FIG. 1, pBR-sucB was digested with KpnI and EcoRI to prepare a DNA fragment containing the sucB gene. pBR-sucA was digested with KpnI and EcoRI to prepare a large fragment. Both fragments were ligated using T4 DNA ligase to produce pBR-sucAB. (2) Disruption of the sucA gene on the chromosomal DNA of
Escherichia coli W3110
FIG. 2 outlines the disruption of the sucA gene on the chromosomal DNA of Escherichia coli W3110.
pBR-sucAB was digested with KpnI and T4 DNA polymerase was used to obtain blunt ends. On the other hand, pUC4K (purchased from Pharmacia) was digested with PstI to prepare a DNA fragment containing a kanamycin-resistance gene, which was converted to have blunt ends using T4 DNA polymerase. Both fragments were ligated using T4 DNA ligase to obtain pBR-sucA::Km.sup.r. From this plasmid, a HindIII-EcoRI fragment containing the kanamycin-resistance gene was cut out as a linear DNA, which was used to transform Escherichia coli JC7623 (thr-1, ara-14, leuB6, .DELTA.(gpt-proA)62, lacY1, tsx-23, supE44, galK2, .lambda..sup.-, rac.sup.-, sbcB 15, hisG4, rfbD1, recB21, recC22, rpsL31, kdgK51, xyl-5, mtl-1, argE3, thi-1), obtained from the Escherichia coli Genetic Stock Center (at Yale University, USA). Strains in which the sucA gene on the chromosomal DNA was replaced with the sucA gene into which the kanamycin-resistance gene (sucA::Km.sup.r) had been inserted were screened on L medium (bactotrypton 10 g/l, yeast extract 5 g/l, NaCl 5 g/, agar 15 g/l, pH 7.2), supplemented with 25 g/ml of kanamycin. Since Escherichia coli JC7623 possesses recB-, recC- and sbcB- genes, recombination can be achieved at a high frequency, even if the transformation is conducted using linear DNA.
From each of twelve (12) kanamycin-resistant strains thus obtained, the chromosomal DNA was prepared and digested with KpnI. By Southern hybridization using a DNA fragment containing the sucA gene as a probe, all 12 strains were confirmed to be strains in which the sucA gene on the chromosomal DNA was replaced with the sucA gene into which the kanamycin-resistance gene had been inserted. While a wild strain exhibits two bands at 5.2 Kb and 6.2 Kb due to the presence of a KpnI site in the DNA fragment containing the sucA gene, when a 2.6 Kb EcoRI-HindIII fragment containing the sucA gene of pBR-sucA was used as the probe in the Southern hybridization, strains having the sucA gene into which the kanamycin-resistance gene has been inserted exhibit only one band at 11.4 Kb due to the disruption of the KpnI site upon introduction of the kanamycin-resistance gene. The kanamycin-resistant Escherichia coli JC7623 (sucA::Km.sup.r ) thus obtained was then infected with P1 phage, and the phage lysate was prepared.
Escherichia coli W3110 was then transduced with the sucA::Km.sup.r. Transduction with P1 phage was conducted by a standard method (Seibutsu-kogaku Jikkensho, Nippon Seibutsu Kogaku Kai, ed., pp. 75 to 76, Baifukan (1992)). One representative strain of the kanamycin-resistant strains isolated was designated as W3110 sucA::Km.sup.r.
The .alpha.-KGDH activities of the strain W3110 sucA::Km.sup.r and Escherichia coli W3110 were determined according to the method described by Reed et al ("Methods in Enzymology," Vol. 13, pp. 55 (1969)). The results are shown in Table 1. No .alpha.-KGDH activity was detected in Escherichia coli W3110 sucA::Km.sup.r. Thus, Escherichia coli W3110 sucA::Km.sup.r is a strain whose .alpha.-KGDH activity is deficient.
TABLE 1______________________________________ W3110 W3110-sucA::Km.sup.r______________________________________.alpha.-KGDH activity 3.70 Not detected______________________________________ (Unit of activity = micromoles/mg protein/min)
(3) Cloning the gdhA gene of Escherichia coli W3110
Similar to the cloning of the sucA and sucB genes, PCR was used to clone the gdhA gene. Based on the nucleotide sequence of the gdhA gene reported by Fernando et al, primers for PCR were synthesized. The nucleotide sequence of the gdhA gene is disclosed in Gene, Vol. 27, pp. 193 to 199 (1984), and is also shown here as SEQ ID NO:8 in the Sequence Listing. The nucleotide sequences of the primers are shown as SEQ ID NOS:5-6.
Sequence ID No. 5 corresponds to the sequence from the -191st through the -171st residues in the nucleotide sequence figure of the gdhA gene shown in Gene, Vol. 27, p.195 (1984), and it also corresponds to the sequence from the 3rd through the 23rd residues in SEQ ID NO:8. Sequence ID No. 6 corresponds to the sequence consisting of the 1687th through the 1707th residues in the nucleotide sequence figure of the gdhA gene shown in Gene, Vol. 27, p.195, (1984), and it also corresponds to the sequence consisting of the 1880th through the 1900th residues in SEQ ID NO:8.
Using the synthetic primers, the gdhA gene was amplified by PCR, employing the chromosomal DNA of Escherichia coli W3110 as a template. PCR products thus obtained were purified and converted to have blunt ends using T4 DNA polymerase, then ligated to pBR322 digested with EcoRV to obtain a plasmid pBRGDH.
(4) Construction of a plasmid having the ppc and gdhA genes
FIG. 3 shows the procedure for the construction of a plasmid having the ppc and gdhA genes. The plasmid pS2, in which a 4.4 Kb SalI fragment containing the entire ppc gene derived from Escherichia coli K-12 has been inserted into the SalI site of pBR322 (J. Biochem., Vol. 9, pp. 909 to 916 (1984)), was digested with HindIII and both ends were blunted using T4 DNA polymerase. On the other hand, a DNA fragment containing the gdhA gene synthesized by PCR was blunt-ended using T4 DNA polymerase. Subsequently, both fragments were ligated using T4 DNA ligase. The plasmid thus obtained was used to transform a GDH-deficient strain, Escherichia coli PA 340 (thr-1, fhuA2, leuB6, lacYl, supE44, gal-6, .lambda..sup.-, gdh- 1, hisG1, rfbD1, galP63, .DELTA.(gltB-F), rpsL19, malT1(lambdaR), yl-7mtl-2, argH1, thi-1), obtained from the Escherichia coli Genetic Stock Center (at Yale University, USA). An ampicillin-resistant strain which lost its glutamic acid requirement for growth was isolated. From this strain, a plasmid was prepared and the corresponding restriction map was constructed, whereby it was confirmed that the ppc and gdhA genes were present on this plasmid. This plasmid was designated as pGK.
(5) Introduction of pS2, pBRGDH and pGK into .alpha.-KGDH-deficient strain Escherichia coli W3100 sucA::Km.sup.r, and evaluation of L-glutamic acid production by the transformants
In separate procedures, the .alpha.-KGDH-deficient strain Escherichia coli W3100 sucA::Km.sup.r was transformed with each of pS2, pBRGDH and pGK. Each of the transformed strains was inoculated into a 500-ml shaker flask containing 20 ml of the culture medium having the composition shown in Table 2. Each of the cultures was then cultivated at 37.degree. C. until the glucose in the culture medium was consumed completely. The results are shown in Table 3.
TABLE 2______________________________________Component Concentration (g/l)______________________________________Glucose 40(NH.sub.4).sub.2 SO.sub.4 20KH.sub.2 PO.sub.4 1MgSO.sub.4.7H.sub.2 O 1FeSO.sub.4.7H.sub.2 O 0.01MnSO.sub.4.5H.sub.2 O 0.01Yeast extract 2Thiamine hydrochloride 0.01CaCO.sub.3 50______________________________________
TABLE 3______________________________________Strain Accumulated L-glutamic acid (g/l)______________________________________W3110 sucA::Km.sup.r 19.2W3110 sucA::Km.sup.r /pS2 19.9W3110 sucA::Km.sup.r /pBRGDH 2.8W3110 sucA::Km.sup.r /pGK 23.3(AJ 12949)______________________________________
Although the transformed strain having amplified PPC activity (W3110 sucA::Km.sup.r /pS2) exhibited slightly reduced growth as compared with the host strain, W3110 sucA::Km.sup.r, it accumulated L-glutamic acid in an amount similar to that accumulated by the host strain. The strain having amplified GDH activity (W3110 sucA::Km.sup.r /pBRGDH) exhibited quite poor growth, and the amount of the accumulated L-glutamic acid was surprisingly smaller than that accumulated by the host strain W3110 sucA::Km.sup.r.
On the contrary, the transformed strain in which both of PPC and GDH activities were amplified simultaneously (W3110 sucA::Km.sup.r /pGK) exhibited growth similar to that of the host strain, while also providing an increased amount of accumulated L-glutamic acid. Escherichia coli W3110 sucA::Km.sup.r /pGK, designated as AJ 12949, was originally deposited under the accession number FERM P-14039 on Dec. 28, 1993, at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan. The deposit was converted into a deposit under the Budapest Treaty under the accession number FERM BP4881 on Nov. 11, 1994.
The host strain W3110 sucA::Km.sup.r can be obtained by curing the plasmid from the deposited strain AJ 12949 without damaging the cell. The plasmid may be lost from AJ 12949 spontaneously, or may be cured in a curing procedure (Bact. Rev., Vol. 36, p. 361 to 405 (1972)). An example of the curing procedure is as follows.
The strain AJ 12949 is inoculated in an L-broth medium (10 g/l Bactotrypton, 5 g/l yeast extract, 5 g/l NaCl, pH 7.2), and cultivated at 40.degree. C. overnight. The culture broth is diluted appropriately and spread onto the L-medium. After incubating it at 37.degree. C. overnight, the colonies formed are transferred to the L-medium containing 100 .mu.g/ml of ampicillin, then ampicillin-sensitive colonies are isolated. The strain thus obtained is W3110 sucA::Km.sup.r.
SUMMARY
The present method and mutant provide increased productivity of L-glutamic acid, as well as the efficient and low-cost production of L-glutamic acid.
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 11(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc ="synthetic DNA"(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: 1..21(D) OTHER INFORMATION: /note="Primer for amplification ofsucA gene of Escherichia coli"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:ACGCGCAAGCGTCGCATCAGG21(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc ="synthetic DNA"(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: 1..21(D) OTHER INFORMATION: /note="Primer for amplification ofsucA gene of Escherichia coli"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:ATCGGCTACGAATTCAGGCAG21(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: 1..20(D) OTHER INFORMATION: /note="Primer for amplification ofsucB gene of Escherichia coli"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:CCGGTCGCGGTACCTTCTTC20(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: 1..26(D) OTHER INFORMATION: /note="Primer for amplification ofsucB gene of Escherichia coli"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:CGTAGACCGAATTCTTCGTATCGCTT26(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: 1..21(D) OTHER INFORMATION: /note="Primer for amplification ofgdhA gene of Escherichia coli"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GGGTGGCAAAGCTTTAGCGTC21(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: 1..21(D) OTHER INFORMATION: /note="Primer for amplification ofgdhA gene of Escherichia coli"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TCGAGAAGCATGCATTATATA21(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4623 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(vi) ORIGINAL SOURCE:(A) ORGANISM: Escherichia coli(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 327..3128(D) OTHER INFORMATION: /note="Method of featuredetermination: E"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 3143..4357(D) OTHER INFORMATION: /note="Method of featuredetermination: E"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:TCGATGTTGTTGCAACGTAATGCGTAAACCGTAGGCCTGATAAGACGCGCAAGCGTCGCA60TCAGGCAACCAGTGCCGGATGCGCGTGAACGCCTTATCCGGCCTACAAGTCATTACCCGT120AGGCCTGATAAGCGCAGCGCATCAGGCGTAACAAAGAAATGCAGGAAATCTTTAAAAACT180GCCCCTGACACTAAGACAGTTTTTAAAGGTTCCTTCGCGAGCCACTACGTAGACAAGAGC240TCGCAAGTGAACCCCGGCACGCACATCACTGTGCGTGGTAGTATCCACGGCGAAGTAAGC300ATAAAAAAGATGCTTAAGGGATCACGATGCAGAACAGCGCTTTGAAAGCCTGG353MetGlnAsnSerAlaLeuLysAlaTrp15TTGGACTCTTCTTACCTCTCTGGCGCAAACCAGAGCTGGATAGAACAG401LeuAspSerSerTyrLeuSerGlyAlaAsnGlnSerTrpIleGluGln10152025CTCTATGAAGACTTCTTAACCGATCCTGACTCGGTTGACGCTAACTGG449LeuTyrGluAspPheLeuThrAspProAspSerValAspAlaAsnTrp303540CGTTCGACGTTCCAGCAGTTACCTGGTACGGGAGTCAAACCGGATCAA497ArgSerThrPheGlnGlnLeuProGlyThrGlyValLysProAspGln455055TTCCACTCTCAAACGCGTGAATATTTCCGCCGCCTGGCGAAAGACGCT545PheHisSerGlnThrArgGluTyrPheArgArgLeuAlaLysAspAla606570TCACGTTACTCTTCAACGATCTCCGACCCTGACACCAATGTGAAGCAG593SerArgTyrSerSerThrIleSerAspProAspThrAsnValLysGln758085GTTAAAGTCCTGCAGCTCATTAACGCATACCGCTTCCGTGGTCACCAG641ValLysValLeuGlnLeuIleAsnAlaTyrArgPheArgGlyHisGln9095100105CATGCGAATCTCGATCCGCTGGGACTGTGGCAGCAAGATAAAGTGGCC689HisAlaAsnLeuAspProLeuGlyLeuTrpGlnGlnAspLysValAla110115120GATCTGGATCCGTCTTTCCACGATCTGACCGAAGCAGACTTCCAGGAG737AspLeuAspProSerPheHisAspLeuThrGluAlaAspPheGlnGlu125130135ACCTTCAACGTCGGTTCATTTGCCAGCGGCAAAGAAACCATGAAACTC785ThrPheAsnValGlySerPheAlaSerGlyLysGluThrMetLysLeu140145150GGCGAGCTGCTGGAAGCCCTCAAGCAAACCTACTGCGGCCCGATTGGT833GlyGluLeuLeuGluAlaLeuLysGlnThrTyrCysGlyProIleGly155160165GCCGAGTATATGCACATTACCAGCACCGAAGAAAAACGCTGGATCCAA881AlaGluTyrMetHisIleThrSerThrGluGluLysArgTrpIleGln170175180185CAGCGTATCGAGTCTGGTCGCGCGACTTTCAATAGCGAAGAGAAAAAA929GlnArgIleGluSerGlyArgAlaThrPheAsnSerGluGluLysLys190195200CGCTTCTTAAGCGAACTGACCGCCGCTGAAGGTCTTGAACGTTACCTC977ArgPheLeuSerGluLeuThrAlaAlaGluGlyLeuGluArgTyrLeu205210215GGCGCAAAATTCCCTGGCGCAAAACGCTTCTCGCTGGAAGGCGGTGAC1025GlyAlaLysPheProGlyAlaLysArgPheSerLeuGluGlyGlyAsp220225230GCGTTAATCCCGATGCTTAAAGAGATGATCCGCCACGCTGGCAACAGC1073AlaLeuIleProMetLeuLysGluMetIleArgHisAlaGlyAsnSer235240245GGCACCCGCGAAGTGGTTCTCGGGATGGCGCACCGTGGTCGTCTGAAC1121GlyThrArgGluValValLeuGlyMetAlaHisArgGlyArgLeuAsn250255260265GTGCTGGTGAACGTGCTGGGTAAAAAACCGCAAGACTTGTTCGACGAG1169ValLeuValAsnValLeuGlyLysLysProGlnAspLeuPheAspGlu270275280TTCGCCGGTAAACATAAAGAACACCTCGGCACGGGTGACGTGAAATAC1217PheAlaGlyLysHisLysGluHisLeuGlyThrGlyAspValLysTyr285290295CACATGGGCTTCTCGTCTGACTTCCAGACCGATGGCGGCCTGGTGCAC1265HisMetGlyPheSerSerAspPheGlnThrAspGlyGlyLeuValHis300305310CTGGCGCTGGCGTTTAACCCGTCTCACCTTGAGATTGTAAGCCCGGTA1313LeuAlaLeuAlaPheAsnProSerHisLeuGluIleValSerProVal315320325GTTATCGGTTCTGTTCGTGCCCGTCTGGACAGACTTGATGAGCCGAGC1361ValIleGlySerValArgAlaArgLeuAspArgLeuAspGluProSer330335340345AGCAACAAAGTGCTGCCAATCACCATCCACGGTGACGCCGCAGTGACC1409SerAsnLysValLeuProIleThrIleHisGlyAspAlaAlaValThr350355360GGGCAGGGCGTGGTTCAGGAAACCCTGAACATGTCGAAAGCGCGTGGT1457GlyGlnGlyValValGlnGluThrLeuAsnMetSerLysAlaArgGly365370375TATGAAGTTGGCGGTACGGTACGTATCGTTATCAACAACCAGGTTGGT1505TyrGluValGlyGlyThrValArgIleValIleAsnAsnGlnValGly380385390TTCACCACCTCTAATCCGCTGGATGCCCGTTCTACGCCGTACTGTACT1553PheThrThrSerAsnProLeuAspAlaArgSerThrProTyrCysThr395400405GATATCGGTAAGATGGTTCAGGCCCCGATTTTCCACGTTAACGCGGAC1601AspIleGlyLysMetValGlnAlaProIlePheHisValAsnAlaAsp410415420425GATCCGGAAGCCGTTGCCTTTGTGACCCGTCTGGCGCTCGATTTCCGT1649AspProGluAlaValAlaPheValThrArgLeuAlaLeuAspPheArg430435440AACACCTTTAAACGTGATGTCTTCATCGACCTGGTGTCGTACCGCCGT1697AsnThrPheLysArgAspValPheIleAspLeuValSerTyrArgArg445450455CACGGCCACAACGAAGCCGACGAGCCGAGCGCAACCCAGCCGCTGATG1745HisGlyHisAsnGluAlaAspGluProSerAlaThrGlnProLeuMet460465470TATCAGAAAATCAAAAAACATCCGACACCGCGCAAAATCTACGCTGAC1793TyrGlnLysIleLysLysHisProThrProArgLysIleTyrAlaAsp475480485AAGCTGGAGCAGGAAAAAGTGGCGACGCTGGAAGATGCCACCGAGATG1841LysLeuGluGlnGluLysValAlaThrLeuGluAspAlaThrGluMet490495500505GTTAACCTGTACCGCGATGCGCTGGATGCTGGCGATTGCGTAGTGGCA1889ValAsnLeuTyrArgAspAlaLeuAspAlaGlyAspCysValValAla510515520GAGTGGCGTCCGATGAACATGCACTCTTTCACCTGGTCGCCGTACCTC1937GluTrpArgProMetAsnMetHisSerPheThrTrpSerProTyrLeu525530535AACCACGAATGGGACGAAGAGTACCCGAACAAAGTTGAGATGAAGCGC1985AsnHisGluTrpAspGluGluTyrProAsnLysValGluMetLysArg540545550CTGCAGGAGCTGGCGAAACGCATCAGCACGGTGCCGGAAGCAGTTGAA2033LeuGlnGluLeuAlaLysArgIleSerThrValProGluAlaValGlu555560565ATGCAGTCTCGCGTTGCCAAGATTTATGGCGATCGCCAGGCGATGGCT2081MetGlnSerArgValAlaLysIleTyrGlyAspArgGlnAlaMetAla570575580585GCCGGTGAGAAACTGTTCGACTGGGGCGGTGCGGAAAACCTCGCTTAC2129AlaGlyGluLysLeuPheAspTrpGlyGlyAlaGluAsnLeuAlaTyr590595600GCCACGCTGGTTGATGAAGGCATTCCGGTTCGCCTGTCGGGTGAAGAC2177AlaThrLeuValAspGluGlyIleProValArgLeuSerGlyGluAsp605610615TCCGGTCGCGGTACCTTCTTCCACCGCCACGCGGTGATCCACAACCAG2225SerGlyArgGlyThrPhePheHisArgHisAlaValIleHisAsnGln620625630TCTAACGGTTCCACTTACACGCCGCTGCAACATATCCATAACGGGCAG2273SerAsnGlySerThrTyrThrProLeuGlnHisIleHisAsnGlyGln635640645GGCGCGTTCCGTGTCTGGGACTCCGTACTGTCTGAAGAAGCAGTGCTG2321GlyAlaPheArgValTrpAspSerValLeuSerGluGluAlaValLeu650655660665GCGTTTGAATATGGTTATGCCACCGCAGAACCACGCACTCTGACCATC2369AlaPheGluTyrGlyTyrAlaThrAlaGluProArgThrLeuThrIle670675680TGGGAAGCGCAGTTCGGTGACTTCCCCAACGGTGCGCAGGTGGTTATC2417TrpGluAlaGlnPheGlyAspPheProAsnGlyAlaGlnValValIle685690695GACCAGTTCATCTCCTCTGGCGAACAGAAATGGGGCCGGATGTGTGGT2465AspGlnPheIleSerSerGlyGluGlnLysTrpGlyArgMetCysGly700705710CTGGTGATGTTGCTGCCGCACGGTTACGAAGGGCAGGGGCCGGAGCAC2513LeuValMetLeuLeuProHisGlyTyrGluGlyGlnGlyProGluHis715720725TCCTCCGCGCGTCTGGAACGTTATCTGCAACTTTGTGCTGAGCAAAAC2561SerSerAlaArgLeuGluArgTyrLeuGlnLeuCysAlaGluGlnAsn730735740745ATGCAGGTTTGCGTACCGTCTACCCCGGCACAGGTTTACCACATGCTG2609MetGlnValCysValProSerThrProAlaGlnValTyrHisMetLeu750755760CGTCGTCAGGCGCTGCGCGGGATGCGTCGTCCGCTGGTCGTGATGTCG2657ArgArgGlnAlaLeuArgGlyMetArgArgProLeuValValMetSer765770775CCGAAATCCCTGCTGCGTCATCCGCTGGCGGTTTCCAGCCTCGAAGAA2705ProLysSerLeuLeuArgHisProLeuAlaValSerSerLeuGluGlu780785790CTGGCGAACGGCACCTTCCTGCCAGCCATCGGTGAAATCGACGAGCTT2753LeuAlaAsnGlyThrPheLeuProAlaIleGlyGluIleAspGluLeu795800805GATCCGAAGGGCGTGAAGCGCGTAGTGATGTGTTCTGGTAAGGTTTAT2801AspProLysGlyValLysArgValValMetCysSerGlyLysValTyr810815820825TACGACCTGCTGGAACAGCGTCGTAAGAACAATCAACACGATGTCGCC2849TyrAspLeuLeuGluGlnArgArgLysAsnAsnGlnHisAspValAla830835840ATTGTGCGTATCGAGCAACTCTACCCGTTCCCGCATAAAGCGATGCAG2897IleValArgIleGluGlnLeuTyrProPheProHisLysAlaMetGln845850855GAAGTGTTGCAGCAGTTTGCTCACGTCAAGGATTTTGTCTGGTGCCAG2945GluValLeuGlnGlnPheAlaHisValLysAspPheValTrpCysGln860865870GAAGAGCCGCTCAACCAGGGCGCATGGTACTGCAGCCAGCATCATTTC2993GluGluProLeuAsnGlnGlyAlaTrpTyrCysSerGlnHisHisPhe875880885CGTGAAGTGATTCCGTTTGGGGCTTCTCTGCGTTATGCAGGCCGCCCG3041ArgGluValIleProPheGlyAlaSerLeuArgTyrAlaGlyArgPro890895900905GCCTCCGCCTCTCCGGCGGTAGGGTATATGTCCGTTCACCAGAAACAG3089AlaSerAlaSerProAlaValGlyTyrMetSerValHisGlnLysGln910915920CAACAAGATCTGGTTAATGACGCGCTGAACGTCGAATAAATAAAGGATA3138GlnGlnAspLeuValAsnAspAlaLeuAsnValGlu*925930CACAATGAGTAGCGTAGATATTCTGGTCCCTGACCTGCCTGAATCCGTA3187MetSerSerValAspIleLeuValProAspLeuProGluSerVal151015GCCGATGCCACCGTCGCAACCTGGCATAAAAAACCCGGCGACGCAGTC3235AlaAspAlaThrValAlaThrTrpHisLysLysProGlyAspAlaVal202530GTACGTGATGAAGTGCTGGTAGAAATCGAAACTGACAAAGTGGTACTG3283ValArgAspGluValLeuValGluIleGluThrAspLysValValLeu354045GAAGTACCGGCATCAGCAGACGGCATTCTGGATGCGGTTCTGGAAGAT3331GluValProAlaSerAlaAspGlyIleLeuAspAlaValLeuGluAsp505560GAAGGTACAACGGTAACGTCTCGTCAGATCCTTGGTCGCCTGCGTGAA3379GluGlyThrThrValThrSerArgGlnIleLeuGlyArgLeuArgGlu657075GGCAACAGCGCCGGTAAAGAAACCAGCGCCAAATCTGAAGAGAAAGCG3427GlyAsnSerAlaGlyLysGluThrSerAlaLysSerGluGluLysAla80859095TCCACTCCGGCGCAACGCCAGCAGGCGTCTCTGGAAGAGCAAAACAAC3475SerThrProAlaGlnArgGlnGlnAlaSerLeuGluGluGlnAsnAsn100105110GATGCGTTAAGCCCGGCGATCCGTCGCCTGCTGGCTGAACACAATCTC3523AspAlaLeuSerProAlaIleArgArgLeuLeuAlaGluHisAsnLeu115120125GACGCCAGCGCCATTAAAGGCACCGGTGTGGGTGGTCGTCTGACTCGT3571AspAlaSerAlaIleLysGlyThrGlyValGlyGlyArgLeuThrArg130135140GAAGATGTGGAAAAACATCTGGCGAAAGCCCCGGCGAAAGAGTCTGCT3619GluAspValGluLysHisLeuAlaLysAlaProAlaLysGluSerAla145150155CCGGCAGCGGCTGCTCCGGCGGCGCAACCGGCTCTGGCTGCACGTAGT3667ProAlaAlaAlaAlaProAlaAlaGlnProAlaLeuAlaAlaArgSer160165170175GAAAAACGTGTCCCGATGACTCGCCTGCGTAAGCGTGTGGCAGAGCGT3715GluLysArgValProMetThrArgLeuArgLysArgValAlaGluArg180185190CTGCTGGAAGCGAAAAACTCCACCGCCATGCTGACCACGTTCAACGAA3763LeuLeuGluAlaLysAsnSerThrAlaMetLeuThrThrPheAsnGlu195200205GTCAACATGAAGCCGATTATGGATCTGCGTAAGCAGTACGGTGAAGCG3811ValAsnMetLysProIleMetAspLeuArgLysGlnTyrGlyGluAla210215220TTTGAAAAACGCCACGGCATCCGTCTGGGCTTTATGTCCTTCTACGTG3859PheGluLysArgHisGlyIleArgLeuGlyPheMetSerPheTyrVal225230235AAAGCGGTGGTTGAAGCCCTGAAACGTTACCCGGAAGTGAACGCTTCT3907LysAlaValValGluAlaLeuLysArgTyrProGluValAsnAlaSer240245250255ATCGACGGCGATGACGTGGTTTACCACAACTATTTCGACGTCAGCATG3955IleAspGlyAspAspValValTyrHisAsnTyrPheAspValSerMet260265270GCGGTTTCTACGCCGCGCGGCCTGGTGACGCCGGTTCTGCGTGATGTC4003AlaValSerThrProArgGlyLeuValThrProValLeuArgAspVal275280285GATACCCTCGGCATGGCAGACATCGAGAAGAAAATCAAAGAGCTGGCA4051AspThrLeuGlyMetAlaAspIleGluLysLysIleLysGluLeuAla290295300GTCAAAGGCCGTGACGGCAAGCTGACCGTTGAAGATCTGACCGGTGGT4099ValLysGlyArgAspGlyLysLeuThrValGluAspLeuThrGlyGly305310315AACTTCACCATCACCAACGGTGGTGTGTTCGGTTCCCTGATGTCTACG4147AsnPheThrIleThrAsnGlyGlyValPheGlySerLeuMetSerThr320325330335CCGATCATCAACCCGCCGCAGAGCGCAATTCTGGGTATGCACGCTATC4195ProIleIleAsnProProGlnSerAlaIleLeuGlyMetHisAlaIle340345350AAAGATCGTCCGATGGCGGTGAATGGTCAGGTTGAGATCCTGCCGATG4243LysAspArgProMetAlaValAsnGlyGlnValGluIleLeuProMet355360365ATGTACCTGGCGCTGTCCTACGATCACCGTCTGATCGATGGTCGCGAA4291MetTyrLeuAlaLeuSerTyrAspHisArgLeuIleAspGlyArgGlu370375380TCCGTGGGCTTCCTGGTAACGATCAAAGAGTTGCTGGAAGATCCGACG4339SerValGlyPheLeuValThrIleLysGluLeuLeuGluAspProThr385390395CGTCTGCTGCTGGACGTGTAGTAGTTTAAGTTTCACCTGCACTGTAGA4387ArgLeuLeuLeuAspVal400405CCGGATAAGGCATTATCGCCTTCTCCGGCAATTGAAGCCTGATGCGACGCTGACGCGTCT4447TATCAGGCCTACGGGACCACCAATGTAGGTCGGATAAGGCGCAACGCCGCATCCGACAAG4507CGATGCCTGATGTGACGTTTAACGTGTCTTATCAGGCCTACGGGTGACCGACAATGCCCG4567GAAGCGATACGAAATATTCGGTCTACGGTTTAAAAGATAACGATTACTGAAGGATG4623(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 933 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:MetGlnAsnSerAlaLeuLysAlaTrpLeuAspSerSerTyrLeuSer151015GlyAlaAsnGlnSerTrpIleGluGlnLeuTyrGluAspPheLeuThr202530AspProAspSerValAspAlaAsnTrpArgSerThrPheGlnGlnLeu354045ProGlyThrGlyValLysProAspGlnPheHisSerGlnThrArgGlu505560TyrPheArgArgLeuAlaLysAspAlaSerArgTyrSerSerThrIle65707580SerAspProAspThrAsnValLysGlnValLysValLeuGlnLeuIle859095AsnAlaTyrArgPheArgGlyHisGlnHisAlaAsnLeuAspProLeu100105110GlyLeuTrpGlnGlnAspLysValAlaAspLeuAspProSerPheHis115120125AspLeuThrGluAlaAspPheGlnGluThrPheAsnValGlySerPhe130135140AlaSerGlyLysGluThrMetLysLeuGlyGluLeuLeuGluAlaLeu145150155160LysGlnThrTyrCysGlyProIleGlyAlaGluTyrMetHisIleThr165170175SerThrGluGluLysArgTrpIleGlnGlnArgIleGluSerGlyArg180185190AlaThrPheAsnSerGluGluLysLysArgPheLeuSerGluLeuThr195200205AlaAlaGluGlyLeuGluArgTyrLeuGlyAlaLysPheProGlyAla210215220LysArgPheSerLeuGluGlyGlyAspAlaLeuIleProMetLeuLys225230235240GluMetIleArgHisAlaGlyAsnSerGlyThrArgGluValValLeu245250255GlyMetAlaHisArgGlyArgLeuAsnValLeuValAsnValLeuGly260265270LysLysProGlnAspLeuPheAspGluPheAlaGlyLysHisLysGlu275280285HisLeuGlyThrGlyAspValLysTyrHisMetGlyPheSerSerAsp290295300PheGlnThrAspGlyGlyLeuValHisLeuAlaLeuAlaPheAsnPro305310315320SerHisLeuGluIleValSerProValValIleGlySerValArgAla325330335ArgLeuAspArgLeuAspGluProSerSerAsnLysValLeuProIle340345350ThrIleHisGlyAspAlaAlaValThrGlyGlnGlyValValGlnGlu355360365ThrLeuAsnMetSerLysAlaArgGlyTyrGluValGlyGlyThrVal370375380ArgIleValIleAsnAsnGlnValGlyPheThrThrSerAsnProLeu385390395400AspAlaArgSerThrProTyrCysThrAspIleGlyLysMetValGln405410415AlaProIlePheHisValAsnAlaAspAspProGluAlaValAlaPhe420425430ValThrArgLeuAlaLeuAspPheArgAsnThrPheLysArgAspVal435440445PheIleAspLeuValSerTyrArgArgHisGlyHisAsnGluAlaAsp450455460GluProSerAlaThrGlnProLeuMetTyrGlnLysIleLysLysHis465470475480ProThrProArgLysIleTyrAlaAspLysLeuGluGlnGluLysVal485490495AlaThrLeuGluAspAlaThrGluMetValAsnLeuTyrArgAspAla500505510LeuAspAlaGlyAspCysValValAlaGluTrpArgProMetAsnMet515520525HisSerPheThrTrpSerProTyrLeuAsnHisGluTrpAspGluGlu530535540TyrProAsnLysValGluMetLysArgLeuGlnGluLeuAlaLysArg545550555560IleSerThrValProGluAlaValGluMetGlnSerArgValAlaLys565570575IleTyrGlyAspArgGlnAlaMetAlaAlaGlyGluLysLeuPheAsp580585590TrpGlyGlyAlaGluAsnLeuAlaTyrAlaThrLeuValAspGluGly595600605IleProValArgLeuSerGlyGluAspSerGlyArgGlyThrPhePhe610615620HisArgHisAlaValIleHisAsnGlnSerAsnGlySerThrTyrThr625630635640ProLeuGlnHisIleHisAsnGlyGlnGlyAlaPheArgValTrpAsp645650655SerValLeuSerGluGluAlaValLeuAlaPheGluTyrGlyTyrAla660665670ThrAlaGluProArgThrLeuThrIleTrpGluAlaGlnPheGlyAsp675680685PheProAsnGlyAlaGlnValValIleAspGlnPheIleSerSerGly690695700GluGlnLysTrpGlyArgMetCysGlyLeuValMetLeuLeuProHis705710715720GlyTyrGluGlyGlnGlyProGluHisSerSerAlaArgLeuGluArg725730735TyrLeuGlnLeuCysAlaGluGlnAsnMetGlnValCysValProSer740745750ThrProAlaGlnValTyrHisMetLeuArgArgGlnAlaLeuArgGly755760765MetArgArgProLeuValValMetSerProLysSerLeuLeuArgHis770775780ProLeuAlaValSerSerLeuGluGluLeuAlaAsnGlyThrPheLeu785790795800ProAlaIleGlyGluIleAspGluLeuAspProLysGlyValLysArg805810815ValValMetCysSerGlyLysValTyrTyrAspLeuLeuGluGlnArg820825830ArgLysAsnAsnGlnHisAspValAlaIleValArgIleGluGlnLeu835840845TyrProPheProHisLysAlaMetGlnGluValLeuGlnGlnPheAla850855860HisValLysAspPheValTrpCysGlnGluGluProLeuAsnGlnGly865870875880AlaTrpTyrCysSerGlnHisHisPheArgGluValIleProPheGly885890895AlaSerLeuArgTyrAlaGlyArgProAlaSerAlaSerProAlaVal900905910GlyTyrMetSerValHisGlnLysGlnGlnGlnAspLeuValAsnAsp915920925AlaLeuAsnValGlu930(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 405 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:MetSerSerValAspIleLeuValProAspLeuProGluSerValAla151015AspAlaThrValAlaThrTrpHisLysLysProGlyAspAlaValVal202530ArgAspGluValLeuValGluIleGluThrAspLysValValLeuGlu354045ValProAlaSerAlaAspGlyIleLeuAspAlaValLeuGluAspGlu505560GlyThrThrValThrSerArgGlnIleLeuGlyArgLeuArgGluGly65707580AsnSerAlaGlyLysGluThrSerAlaLysSerGluGluLysAlaSer859095ThrProAlaGlnArgGlnGlnAlaSerLeuGluGluGlnAsnAsnAsp100105110AlaLeuSerProAlaIleArgArgLeuLeuAlaGluHisAsnLeuAsp115120125AlaSerAlaIleLysGlyThrGlyValGlyGlyArgLeuThrArgGlu130135140AspValGluLysHisLeuAlaLysAlaProAlaLysGluSerAlaPro145150155160AlaAlaAlaAlaProAlaAlaGlnProAlaLeuAlaAlaArgSerGlu165170175LysArgValProMetThrArgLeuArgLysArgValAlaGluArgLeu180185190LeuGluAlaLysAsnSerThrAlaMetLeuThrThrPheAsnGluVal195200205AsnMetLysProIleMetAspLeuArgLysGlnTyrGlyGluAlaPhe210215220GluLysArgHisGlyIleArgLeuGlyPheMetSerPheTyrValLys225230235240AlaValValGluAlaLeuLysArgTyrProGluValAsnAlaSerIle245250255AspGlyAspAspValValTyrHisAsnTyrPheAspValSerMetAla260265270ValSerThrProArgGlyLeuValThrProValLeuArgAspValAsp275280285ThrLeuGlyMetAlaAspIleGluLysLysIleLysGluLeuAlaVal290295300LysGlyArgAspGlyLysLeuThrValGluAspLeuThrGlyGlyAsn305310315320PheThrIleThrAsnGlyGlyValPheGlySerLeuMetSerThrPro325330335IleIleAsnProProGlnSerAlaIleLeuGlyMetHisAlaIleLys340345350AspArgProMetAlaValAsnGlyGlnValGluIleLeuProMetMet355360365TyrLeuAlaLeuSerTyrAspHisArgLeuIleAspGlyArgGluSer370375380ValGlyPheLeuValThrIleLysGluLeuLeuGluAspProThrArg385390395400LeuLeuLeuAspVal405(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1937 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(vi) ORIGINAL SOURCE:(A) ORGANISM: Escherichia coli(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 194..1537(D) OTHER INFORMATION: /note="Method of featuredetermination: E"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:CCGGGTGGCAAAACTTTAGCGTCTGAGGTTATCGCAATTTGGTTATGAGATTACTCTCGT60TATTAATTTGCTTTCCTGGGTCATTTTTTTCTTGCTTACCGTCACATTCTTGATGGTATA120GTCGAAAACTGCAAAAGCACATGACATAAACAACATAAGCACAATCGTATTAATATATAA180GGGTTTTATATCTATGGATCAGACATATTCTCTGGAGTCATTCCTCAAC229MetAspGlnThrTyrSerLeuGluSerPheLeuAsn410415CATGTCCAAAAGCGCGACCCGAATCAAACCGAGTTCGCGCAAGCCGTT277HisValGlnLysArgAspProAsnGlnThrGluPheAlaGlnAlaVal420425430CGTGAAGTAATGACCACACTCTGGCCTTTTCTTGAACAAAATCCAAAA325ArgGluValMetThrThrLeuTrpProPheLeuGluGlnAsnProLys435440445TATCGCCAGATGTCATTACTGGAGCGTCTGGTTGAACCGGAGCGCGTG373TyrArgGlnMetSerLeuLeuGluArgLeuValGluProGluArgVal450455460465ATCCAGTTTCGCGTGGTATGGGTTGATGATCGCAACCAGATACAGGTC421IleGlnPheArgValValTrpValAspAspArgAsnGlnIleGlnVal470475480AACCGTGCATGGCGTGTGCAGTTCAGCTCTGCCATCGGCCCGTACAAA469AsnArgAlaTrpArgValGlnPheSerSerAlaIleGlyProTyrLys485490495GGCGGTATGCGCTTCCATCCGTCAGTTAACCTTTCCATTCTCAAATTC517GlyGlyMetArgPheHisProSerValAsnLeuSerIleLeuLysPhe500505510CTCGGCTTTGAACAAACCTTCAAAAATGCCCTGACTACTCTGCCGATG565LeuGlyPheGluGlnThrPheLysAsnAlaLeuThrThrLeuProMet515520525GGCGGTGGTAAAGGCGGCAGCGATTTCGATCCGAAAGGAAAAAGCGAA613GlyGlyGlyLysGlyGlySerAspPheAspProLysGlyLysSerGlu530535540545GGTGAAGTGATGCGTTTTTGCCAGGCGCTGATGACTGAACTGTATCGC661GlyGluValMetArgPheCysGlnAlaLeuMetThrGluLeuTyrArg550555560CACCTGGGCGCGGATACCGACGTTCCGGCAGGTGATATCGGGGTTGGT709HisLeuGlyAlaAspThrAspValProAlaGlyAspIleGlyValGly565570575GGTCGTGAAGTCGGCTTTATGGCGGGGATGATGAAAAAGCTCTCCAAC757GlyArgGluValGlyPheMetAlaGlyMetMetLysLysLeuSerAsn580585590AATACCGCCTGCGTCTTCACCGGTAAGGGCCTTTCATTTGGCGGCAGT805AsnThrAlaCysValPheThrGlyLysGlyLeuSerPheGlyGlySer595600605CTTATTCGCCCGGAAGCTACCGGCTACGGTCTGGTTTATTTCACAGAA853LeuIleArgProGluAlaThrGlyTyrGlyLeuValTyrPheThrGlu610615620625GCAATGCTAAAACGCCACGGTATGGGTTTTGAAGGGATGCGCGTTTCC901AlaMetLeuLysArgHisGlyMetGlyPheGluGlyMetArgValSer630635640GTTTCTGGCTCCGGCAACGTCGCCCAGTACGCTATCGAAAAAGCGATG949ValSerGlySerGlyAsnValAlaGlnTyrAlaIleGluLysAlaMet645650655GAATTTGGTGCTCGTGTGATCACTGCGTCAGACTCCAGCGGCACTGTA997GluPheGlyAlaArgValIleThrAlaSerAspSerSerGlyThrVal660665670GTTGATGAAAGCGGATTCACGAAAGAGAAACTGGCACGTCTTATCGAA1045ValAspGluSerGlyPheThrLysGluLysLeuAlaArgLeuIleGlu675680685ATCAAAGCCAGCCGCGATGGTCGAGTGGCAGATTACGCCAAAGAATTT1093IleLysAlaSerArgAspGlyArgValAlaAspTyrAlaLysGluPhe690695700705GGTCTGGTCTATCTCGAAGGCCAACAGCCGTGGTCTCTACCGGTTGAT1141GlyLeuValTyrLeuGluGlyGlnGlnProTrpSerLeuProValAsp710715720ATCGCCCTGCCTTGCGCCACCCAGAATGAACTGGATGTTGACGCCGCG1189IleAlaLeuProCysAlaThrGlnAsnGluLeuAspValAspAlaAla725730735CATCAGCTTATCGCTAATGGCGTTAAAGCCGTCGCCGAAGGGGCAAAT1237HisGlnLeuIleAlaAsnGlyValLysAlaValAlaGluGlyAlaAsn740745750ATGCCGACCACCATCGAAGCGACTGAACTGTTCCAGCAGGCAGGCGTA1285MetProThrThrIleGluAlaThrGluLeuPheGlnGlnAlaGlyVal755760765CTATTTGCACCGGGTAAAGCGGCTAATGCTGGTGGCGTCGCTACATCG1333LeuPheAlaProGlyLysAlaAlaAsnAlaGlyGlyValAlaThrSer770775780785GGCCTGGAAATGCCACAAAACGCTGCGCGCCTGGGCTGGAAAGCCGAG1381GlyLeuGluMetProGlnAsnAlaAlaArgLeuGlyTrpLysAlaGlu790795800AAAGTTGACGCACGTTTGCATCACATCATGCTGGATATCCACCATGCC1429LysValAspAlaArgLeuHisHisIleMetLeuAspIleHisHisAla805810815TGTGTTGAGCATGGTGGTGAAGGTGAGCAAACCAACTACGTGCAGGGC1477CysValGluHisGlyGlyGluGlyGluGlnThrAsnTyrValGlnGly820825830GCGAACATTGCCGGTTTTGTGAAGGTTGCCGATGCGATGCTGGCGCAG1525AlaAsnIleAlaGlyPheValLysValAlaAspAlaMetLeuAlaGln835840845GGTGTGATTTAAGTTGTAAATGCCTGATGGCGCTACGCTTATCAGGCCTACA1577GlyValIle*850AATGGGCACAATTCATTGCAGTTACGCTCTAATGTAGGCCGGGCAAGCGCAGCGCCCCCG1637GCAAAATTTCAGGCGTTTATGAGTATTTAACGGATGATGCTCCCCACGGAACATTTCTTA1697TGGGCCAACGGCATTTCTTACTGTAGTGCTCCCAAAACTGCTTGTCGTAACGATAACACG1757CTTCAAGTTCAGCATCCGTTAACTTTCTGCGGACTCACGCGCGCAGCACTATGCCAGTAA1817AGAAATCCCATTTGACTATTTTTTTGATAATCTTCTTCGCTTTCGAACAACTCGTGCGCC1877TTTCGAGAAGCAAGCATTATATAATGCCAGGCCAGTTCTTCTTCAATTGTCCCGTTTTGA1937(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 447 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:MetAspGlnThrTyrSerLeuGluSerPheLeuAsnHisValGlnLys151015ArgAspProAsnGlnThrGluPheAlaGlnAlaValArgGluValMet202530ThrThrLeuTrpProPheLeuGluGlnAsnProLysTyrArgGlnMet354045SerLeuLeuGluArgLeuValGluProGluArgValIleGlnPheArg505560ValValTrpValAspAspArgAsnGlnIleGlnValAsnArgAlaTrp65707580ArgValGlnPheSerSerAlaIleGlyProTyrLysGlyGlyMetArg859095PheHisProSerValAsnLeuSerIleLeuLysPheLeuGlyPheGlu100105110GlnThrPheLysAsnAlaLeuThrThrLeuProMetGlyGlyGlyLys115120125GlyGlySerAspPheAspProLysGlyLysSerGluGlyGluValMet130135140ArgPheCysGlnAlaLeuMetThrGluLeuTyrArgHisLeuGlyAla145150155160AspThrAspValProAlaGlyAspIleGlyValGlyGlyArgGluVal165170175GlyPheMetAlaGlyMetMetLysLysLeuSerAsnAsnThrAlaCys180185190ValPheThrGlyLysGlyLeuSerPheGlyGlySerLeuIleArgPro195200205GluAlaThrGlyTyrGlyLeuValTyrPheThrGluAlaMetLeuLys210215220ArgHisGlyMetGlyPheGluGlyMetArgValSerValSerGlySer225230235240GlyAsnValAlaGlnTyrAlaIleGluLysAlaMetGluPheGlyAla245250255ArgValIleThrAlaSerAspSerSerGlyThrValValAspGluSer260265270GlyPheThrLysGluLysLeuAlaArgLeuIleGluIleLysAlaSer275280285ArgAspGlyArgValAlaAspTyrAlaLysGluPheGlyLeuValTyr290295300LeuGluGlyGlnGlnProTrpSerLeuProValAspIleAlaLeuPro305310315320CysAlaThrGlnAsnGluLeuAspValAspAlaAlaHisGlnLeuIle325330335AlaAsnGlyValLysAlaValAlaGluGlyAlaAsnMetProThrThr340345350IleGluAlaThrGluLeuPheGlnGlnAlaGlyValLeuPheAlaPro355360365GlyLysAlaAlaAsnAlaGlyGlyValAlaThrSerGlyLeuGluMet370375380ProGlnAsnAlaAlaArgLeuGlyTrpLysAlaGluLysValAspAla385390395400ArgLeuHisHisIleMetLeuAspIleHisHisAlaCysValGluHis405410415GlyGlyGluGlyGluGlnThrAsnTyrValGlnGlyAlaAsnIleAla420425430GlyPheValLysValAlaAspAlaMetLeuAlaGlnGlyValIle435440445__________________________________________________________________________

Number	Name	Date
3663370	Kono et al.	May 1972
4347318	Miwa et al.	Aug 1982
4411991	Hirakawa et al.	Oct 1983
4729952	Hattori et al.	Mar 1988
4757009	Sano et al.	Jul 1988
5378616	Tujimoto et al.	Jan 1995

Number	Date	Country
8693	Jan 1981	JPX
8694	Jan 1981	JPX
20194	Feb 1983	JPX

Mutant and method for producing L-glutamic acid by fermentation

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