CN113493500A - Glutamic acid efflux protein mutant and application thereof - Google Patents

Glutamic acid efflux protein mutant and application thereof Download PDF

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CN113493500A
CN113493500A CN202011267094.0A CN202011267094A CN113493500A CN 113493500 A CN113493500 A CN 113493500A CN 202011267094 A CN202011267094 A CN 202011267094A CN 113493500 A CN113493500 A CN 113493500A
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朱蕾蕾
聂志华
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Abstract

The invention discloses a glutamic acid efflux protein mutant and application thereof. The invention obtains an efflux mutant with improved glutamic acid efflux capability by an directed evolution method, and provides a method for producing L-glutamic acid, which comprises the following steps: expressing the glutamic acid efflux protein mutant in a recipient bacterium to obtain a recombinant bacterium; and carrying out fermentation culture on the recombinant bacteria to obtain glutamic acid from fermentation liquor. The invention lays a foundation for enriching the understanding of the glutamic acid discharge and improving the yield of the glutamic acid in production.

Description

Glutamic acid efflux protein mutant and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, and particularly relates to a glutamic acid efflux protein mutant and application thereof.
Background
The history of C.glutamicum began in the 50's of the 20 th century, when Japanese scientists Kinoshita and Udaka isolated this bacterium and discovered its ability to secrete large amounts of glutamic acid. Later, bacterial fermentation was used to produce glutamate, completely replacing chemical synthesis with biological processes. Subsequently, the bacterium is continuously studied by scientists and utilized its unique physiological properties to improve the production of glutamic acid. In addition, Corynebacterium glutamicum has now become the industrial host for the production of various amino acids. In the amino acid market, the market for L-glutamic acid, which is widely used as a flavor enhancer, is large in scale, and the total amount of L-glutamic acid produced per year is about 321 ten thousand tons, which accounts for about 50% of the total amount of amino acid produced. Therefore, it is necessary to improve the yield of glutamic acid.
However, with the rapid development of molecular biology in recent years, the application of molecular biology technology in the field of glutamic acid research is becoming more and more extensive, and the mechanism of a large amount of glutamic acid fermentation is continuously revealed. A novel glutamic acid efflux channel protein, namely an efflux protein coded by the mscCG2 gene, is found in Corynebacterium glutamicum by an Zhengping subject group of the institute of Tianjin Industrial biotechnology of Chinese academy of sciences in 2018, has 3 transmembrane domains, is positioned at a cytoplasmic membrane, is a mechanically sensitive channel protein, and has only 23% of amino acid similarity compared with MscCG. At present, the research and molecular modification on the exo-efflux protein are less, and no literature for improving the exo-efflux capability of the exo-efflux protein through mutation is found. Therefore, the method is necessary to improve the yield of the glutamic acid by carrying out gene modification on the efflux protein and screening the L-glutamic acid corynebacterium strain with stronger capacity of efflux the glutamic acid.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a glutamic acid efflux protein mutant with improved efflux capability, a coding gene and related application thereof.
In a first aspect, the invention claims glutamate efflux protein mutants.
The glutamic acid efflux protein mutant claimed by the invention is obtained by carrying out point mutation on MscCG2 glutamic acid efflux protein, and the mutation site contains (or is) part or all of the following: 60, 149, 165, 269 and 279 from the N-terminus.
Preferably, the amino acid sequence of the glutamate efflux protein mutant has more than 95% identity with the sequence only containing each mutation site.
Further, the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: bit 165 from the N-terminus.
Further, the glutamate efflux protein mutant can be any one of the following:
(A1) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: position 165 from the N-terminus (corresponding to mutant M1);
(A2) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: 165 th and 60 th from the N-terminus (corresponding to mutant M2);
(A3) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: position 149 from the N-terminus (corresponding to mutant M4);
(A4) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: position 60 from the N-terminus (corresponding to mutant M5);
(A5) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: 269 th from the N-terminus (corresponding to mutant M6);
(A6) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: position 279 from the N-terminus (corresponding to mutant M7);
(A7) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: 165 th and 269 th from the N-terminus (corresponding to mutant M8);
(A8) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: 165 th and 279 th from the N-terminus (corresponding to mutant M9);
(A9) the glutamate efflux protein mutant is a protein obtained by point mutation of at least the following amino acid residues (or the following amino acid residues) of the MscCG2 glutamate efflux protein: 165, 269 and 279 from the N-terminus (corresponding to mutant M10);
wherein the MscCG2 glutamate efflux protein can be an MscCG2 glutamate efflux protein from Corynebacterium glutamicum Z188.
The amino acid sequence of the MscCG2 glutamic acid efflux protein from Corynebacterium glutamicum Z188 is specifically shown in SEQ ID No. 1.
In the glutamic acid efflux protein mutant, the 60 th point mutation from the N-terminal is G60C, the 149 th point mutation is F149L, the 165 th point mutation is L165P, the 269 th point mutation is I269V, and the 279 th point mutation is F279L.
For amino acid substitutions, the following nomenclature is used: original amino acid (wild type), position (i.e. position in SEQ ID No. 1), substituted amino acid. Accordingly, substitution of proline for the original leucine amino acid at position 165 of SEQ ID No.1 is "L165P".
In a specific embodiment of the present invention, the glutamate efflux protein mutant is specifically any one of the following:
(a1) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: L165P (corresponding to mutant M1, amino acid sequence shown in SEQ ID No. 2);
(a2) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: L165P, G60C (corresponding to mutant M2, the amino acid sequence is shown in SEQ ID No. 3);
(a3) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: F149L (corresponding to mutant M4, amino acid sequence shown in SEQ ID No. 4);
(a4) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: G60C (corresponding to mutant M5, the amino acid sequence is shown as SEQ ID No. 5);
(a5) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: I269V (corresponding mutant M6, amino acid sequence shown in SEQ ID No. 6);
(a6) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: F279L (corresponding to mutant M7, amino acid sequence shown in SEQ ID No. 7);
(a7) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: L165P, I269V (corresponding to mutant M8, amino acid sequence shown in SEQ ID No. 8);
(a8) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: L165P, F279L (corresponding to mutant M9, the amino acid sequence is shown in SEQ ID No. 9);
(a9) the glutamic acid efflux protein mutant is a protein obtained by point mutation of amino acid residues of the following sites of MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1: L165P, I269V, F279L (corresponding to mutant M10, amino acid sequence shown in SEQ ID No. 10);
in a second aspect, the invention claims biological material related to glutamate efflux protein mutants.
The biological material related to the glutamic acid efflux protein mutant can be any one of the following materials:
(I) a nucleic acid molecule encoding said mutant glutamate efflux protein;
(II) expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.
Wherein the nucleic acid molecule for coding the MscCG2 glutamic acid efflux protein from Corynebacterium glutamicum is a DNA molecule shown in SEQ ID No. 11.
Further, the nucleic acid molecule encoding the glutamate efflux protein mutant is specifically any one of the following:
(B1) a DNA molecule shown as SEQ ID No.12 (corresponding to mutant M1);
(B2) a DNA molecule shown as SEQ ID No.13 (corresponding to mutant M2);
(B3) a DNA molecule shown as SEQ ID No.14 (corresponding to mutant M4);
(B4) a DNA molecule shown as SEQ ID No.15 (corresponding to mutant M5);
(B5) a DNA molecule shown as SEQ ID No.16 (corresponding to mutant M6);
(B6) a DNA molecule shown as SEQ ID No.17 (corresponding to mutant M7);
(B7) a DNA molecule shown as SEQ ID No.18 (corresponding to mutant M8);
(B8) a DNA molecule shown as SEQ ID No.19 (corresponding to mutant M9);
(B9) a DNA molecule shown as SEQ ID No.20 (corresponding to mutant M10);
in a specific embodiment of the invention, the recombinant vector is a recombinant plasmid obtained by cloning the nucleic acid molecule encoding the glutamate efflux protein mutant between SacI and BamHI of the multiple cloning site of pTRCmob vector.
The recombinant bacterium can be corynebacterium glutamicum containing the nucleic acid molecule.
In a third aspect, the invention claims the use of a glutamate efflux protein mutant or biomaterial as described hereinbefore in any one of:
(C1) producing glutamic acid;
(C2) the glutamic acid yield is improved;
(C3) the glutamic acid efflux capacity is improved;
(C4) producing monosodium glutamate, spices or food additives;
(C5) preparing salt substitute, nutritional supplement or biochemical reagent.
In a fourth aspect, the invention claims a method for producing glutamic acid and/or increasing the production of glutamic acid and/or increasing the efflux capability of glutamic acid.
The method for producing glutamic acid and/or improving the yield of glutamic acid claimed by the invention can comprise the following steps: expressing the glutamic acid efflux protein mutant in a receptor bacterium to obtain a recombinant bacterium; and carrying out fermentation culture on the recombinant bacteria to obtain glutamic acid from fermentation liquor.
Further, expression of the mutant glutamate efflux protein in the recipient bacterium can be achieved by introducing the "nucleic acid molecule encoding the mutant glutamate efflux protein" as described above into the recipient bacterium.
Still further, the "nucleic acid molecule encoding the glutamate efflux protein mutant" may be introduced into the recipient bacterium in the form of a recombinant vector.
In a specific embodiment of the invention, the recombinant vector is specifically a recombinant plasmid obtained by cloning the nucleic acid molecule encoding the glutamate efflux protein mutant between SacI and BamHI of a multiple cloning site of pTRCmob vector.
Further, the recipient bacterium is corynebacterium glutamicum.
In the method, the culture medium of the fermentation culture can be CGXII culture medium, and the culture conditions can be 30 ℃ and 220r/min for 24 h.
In the specific embodiment of the invention, the recombinant strain is inoculated in CGXII seed culture medium, cultured for 12h at 30 ℃ and 220r/min and then cultured by using initial OD600Inoculating CGX with an inoculum size of 0.15II, culturing the strain in a fermentation medium at 30 ℃ and 220r/min for 24 hours.
In the present invention, the glutamic acid is specifically L-glutamic acid.
The invention obtains the efflux mutant with improved glutamic acid efflux capability by a directed evolution method. The efflux capacity of the efflux mutant is improved compared with that of the wild type. The glutamic acid efflux protein mutant obtained by the invention is more beneficial to improving the yield of glutamic acid, and lays a foundation for enriching the understanding of the efflux of the glutamic acid and improving the yield of glutamic acid in production.
Drawings
FIG. 1 is a graph comparing efflux mutants to wild-type glutamate production when cultured in 25ml CGXII fermentation medium.
The reference MscG 2 in FIG. 1 is a wild-type control, and "g/L glutamic acid" on the ordinate refers to the amount of L-glutamic acid produced per L fermentation broth.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Gene cloning of glutamate efflux protein
MscCG2 glutamic acid efflux protein target gene is cloned from a genome of corynebacterium glutamicum, the gene sequence is shown as SEQ ID NO.11, and then the gene is connected to a pTRCmob vector, sequencing is carried out to determine that the vector is successfully constructed to obtain a recombinant plasmid, and the recombinant plasmid is named as pTRCmob-mscG 2.
The structure of pTRCmob-mscG 2 is described as: the DNA fragment shown in SEQ ID NO.11 is cloned between SacI and BamHI of pTRCmob vector to obtain recombinant plasmid. SEQ ID No.11 is a target gene of a wild MscCG2 glutamic acid efflux protein, and codes the wild MscCG2 glutamic acid efflux protein shown in SEQ ID No. 1.
Example 2 screening of glutamate efflux protein mutants with improved efflux Capacity
In order to improve the efflux ability of the above-mentioned glutamate efflux protein, the recombinant construct of example 1 was usedThe plasmid pTRCmob-mscCG2 was used as a template, a primer (upstream primer: 5'-CAGACCATGGAATTCGAGCTCATGATTTTAGGCGTACCCATTC-3'; downstream primer: 5'-AGGTCGACTCTAGAGGATCCCTAAGGGGTGGACGTCGGCG-3') was designed, and Mn was added2+The objective gene is randomly mutated by a PCR method. After the mutant product is electrically transferred to a corynebacterium glutamicum competent cell and cultured overnight at 30 ℃ to obtain a recombinant bacterium, grown monoclonals are respectively inoculated to a 96-well plate (containing 150 mu l of LBG culture medium and 25mg/ml Kana) by using a sterile toothpick, and are subjected to shake culture at 30 ℃ and 800rpm for 45 hours, and then the yield of the glutamic acid is detected. And analyzing the detection result, and finally screening 4 mutant strains with improved glutamic acid efflux, wherein the mutant strains are named as M1, M2, M8 and M9. A wild type is used as a template, a primer (an upstream primer: 5'-GGTGTTGCGCTTGGTCTTGGCGCACAGTCGCTG-3'; a downstream primer: 5'-CAGCGACTGTGCGCCAAGACCAAGCGCAACACC-3') is designed, phenylalanine at position 149 is mutated into leucine, a mutation product is electrically transferred into a corynebacterium glutamicum competent cell, and the corynebacterium glutamicum competent cell is cultured overnight at 30 ℃ to obtain a recombinant strain which is named as M4. M2 is used as a template, a primer (an upstream primer: 5'-CAGCGGTATTTTTATGCTCATTGAGGATCAATACGG-3'; a downstream primer: 5'-CCGTATTGATCCTCAATGAGCATAAAAATACCGCTG-3') is designed, the proline at position 165 is subjected to reversion and mutation to leucine, a mutation product is electrically transferred to a corynebacterium glutamicum competent cell, and the corynebacterium glutamicum competent cell is cultured overnight at 30 ℃ to obtain a recombinant strain named as M5. M8 is used as a template, a primer (an upstream primer: 5'-CAGCGGTATTTTTATGCTCATTGAGGATCAATACGG-3'; a downstream primer: 5'-CCGTATTGATCCTCAATGAGCATAAAAATACCGCTG-3') is designed, the proline at position 165 is subjected to reversion and mutation to leucine, a mutation product is electrically transferred to a corynebacterium glutamicum competent cell, and the corynebacterium glutamicum competent cell is cultured overnight at 30 ℃ to obtain a recombinant strain named as M6. M9 is used as a template, a primer (an upstream primer: 5'-CAGCGGTATTTTTATGCTCATTGAGGATCAATACGG-3'; a downstream primer: 5'-CCGTATTGATCCTCAATGAGCATAAAAATACCGCTG-3') is designed, the proline at position 165 is subjected to reversion and mutation to leucine, a mutation product is electrically transferred to a corynebacterium glutamicum competent cell, and the corynebacterium glutamicum competent cell is cultured overnight at 30 ℃ to obtain a recombinant strain named as M7. M9 was used as a template to design a primer (forward primer: 5'-CCTGAGATGAAGGGCGTCTCTGCATTCGAACCT-3'; reverse primer: 5 ' -AGGTTCGAATGCAGAGACGCCC)TTCATCTCAGG-3'), mutating isoleucine at position 269 to valine, electrically transferring the mutation product to Corynebacterium glutamicum competent cells, and culturing at 30 deg.C overnight to obtain recombinant strain named M10.
The specific amino acid position of each mutant strain is subjected to gene sequencing, and the specific amino acid sequence and gene sequence after mutation are shown in Table 1 in detail.
TABLE 1 mutant amino acid sites
Figure BDA0002776418020000071
Note: amino acid substitutions in the table, using the following nomenclature: original amino acid (wild type), position (i.e. position in SEQ ID No. 1), substituted amino acid.
Example 3 expression of efflux proteins of glutamic acid and efflux mutants thereof in Corynebacterium glutamicum
Inoculating loop Positive bacteria of wild type and 9 mutants shown in Table 1 obtained in example 2 were scraped off and inoculated in 10mL of CGXII seed medium, cultured at 30 ℃ and 220rpm in a constant temperature shaking incubator (Shanghai Zhichu instruments Co., Ltd., Shanghai, China) for 12 hours, and then cultured at the starting OD600The strain was inoculated to an inoculum size of 0.15 into 25ml of CGXII fermentation medium (into a 500ml Erlenmeyer flask) and cultured on a constant temperature shaker (Shanghai Zhichu apparatus Co., Ltd., Shanghai, China) at 30 ℃ and 220 rpm. After 24h of culture, the fermentation broth was collected by centrifugation, and the L-glutamic acid yield in the fermentation broth was measured with an SBA-40D biosensor analyzer (institute of biological sciences, Shandong, China, Proc. of science, Shandong, province).
Wherein, the solvent of the CGXII seed culture medium is water, and the solutes and the concentrations are as follows: 5g/L glucose, 20g/L ammonium sulfate, 5g/L urea, 1g/L monopotassium phosphate, 1.3g/L dipotassium phosphate, 80g/L MOPS, 0.01g/L calcium chloride, 0.25g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.01g/L manganese sulfate, 0.001g/L zinc sulfate, 0.2mg/L copper sulfate, 0.02mg/L nickel chloride, 0.03g/L dihydroxybenzoic acid, 2.5. mu.g/L biotin, 0.1mg/L Thiamine HCL VB 1.
The solvent of the CGXII fermentation medium is water, and the solutes and the concentrations are as follows: 80g/L glucose, 20g/L ammonium sulfate, 5g/L urea, 1g/L monopotassium phosphate, 1.3g/L dipotassium phosphate, 80g/L MOPS, 0.01g/L calcium chloride, 0.25g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.01g/L manganese sulfate, 0.001g/L zinc sulfate, 0.2mg/L copper sulfate, 0.02mg/L nickel chloride, 0.03g/L dihydroxybenzoic acid, 0.5. mu.g/L biotin, 0.1mg/L Thiamine HCL VB 1.
The detection results are shown in figure 1, and figure 1 is cultured in 25ml CGXII fermentation medium, and the results show that the yield of the efflux mutant glutamic acid is 20-50% higher than that of the wild type glutamic acid.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and variations be included within the scope of the invention as claimed and not departing from the spirit thereof.
Sequence listing
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Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Cys Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Phe Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Pro Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Ile Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Phe Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 4
<211> 334
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Ile Ile Ala Ser Thr Asp Thr Glu Ser Ile Val Thr Asp Val Ser
1 5 10 15
Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Gly Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Leu Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Leu Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Ile Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Phe Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 5
<211> 334
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Ile Ile Ala Ser Thr Asp Thr Glu Ser Ile Val Thr Asp Val Ser
1 5 10 15
Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Cys Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Phe Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Leu Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Ile Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Phe Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 6
<211> 334
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Ile Ile Ala Ser Thr Asp Thr Glu Ser Ile Val Thr Asp Val Ser
1 5 10 15
Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Gly Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Phe Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Leu Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Val Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Phe Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 7
<211> 334
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Ile Ile Ala Ser Thr Asp Thr Glu Ser Ile Val Thr Asp Val Ser
1 5 10 15
Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Gly Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Phe Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Leu Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Ile Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Leu Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 8
<211> 334
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Ile Ile Ala Ser Thr Asp Thr Glu Ser Ile Val Thr Asp Val Ser
1 5 10 15
Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Gly Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Phe Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Pro Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Val Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Phe Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 9
<211> 334
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Ile Ile Ala Ser Thr Asp Thr Glu Ser Ile Val Thr Asp Val Ser
1 5 10 15
Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Gly Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Phe Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Pro Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Ile Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Leu Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 10
<211> 334
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Ile Ile Ala Ser Thr Asp Thr Glu Ser Ile Val Thr Asp Val Ser
1 5 10 15
Asn Trp Trp Asp Asn Pro Leu Thr Gln Asp Trp Leu Ile Asn Lys Pro
20 25 30
Ile Ser Ile Ala Ile Thr Ile Val Val Ala Phe Phe Ala His Trp Leu
35 40 45
Leu Arg Lys Leu Ile Thr Lys Ala Val Glu Arg Gly Val Arg Lys Pro
50 55 60
Pro Lys Thr Glu Val Pro Arg Phe Leu Val Lys Gly Gln Ala Lys Lys
65 70 75 80
Thr Asp Ile Pro Pro Glu Val Gln Ile Met Arg Lys Thr Gln Glu Gln
85 90 95
Arg Arg Gln Ala Arg Ile Arg Thr Leu Gly Ala Val Gly Lys Ser Ala
100 105 110
Val Ala Ile Phe Val Trp Thr Trp Ala Ala Leu Ala Ile Leu Thr Glu
115 120 125
Ile Gly Leu Asn Val Ala Pro Leu Ile Ala Ser Ala Gly Val Ala Gly
130 135 140
Val Ala Leu Gly Phe Gly Ala Gln Ser Leu Val Lys Asp Phe Leu Ser
145 150 155 160
Gly Ile Phe Met Pro Ile Glu Asp Gln Tyr Gly Val Gly Asp Thr Ile
165 170 175
Asp Val Gly Asp Gly Ile Ile Gly Asp Val Glu Asp Ile Ser Leu Arg
180 185 190
Thr Thr Thr Leu Arg Asp Leu Asp Gly Thr Val Trp Tyr Ile Arg Asn
195 200 205
Gly Glu Ile Leu Arg Val Gly Asn Phe Ser Asn Glu Tyr Ala Ile Ala
210 215 220
Arg Phe Glu Val Pro Val Gly Leu Ser Asn Asp Ser Asp Arg Ala Trp
225 230 235 240
Asp Val Ile Glu Asn Ser Phe Gln Glu Ala Val Lys Met Glu Ala Ile
245 250 255
Lys Asp Ser Val Ile Asp Thr Pro Glu Met Lys Gly Val Ser Ala Phe
260 265 270
Glu Pro Asp His Met Thr Leu Arg Gly Val Val Lys Thr Leu Pro Gly
275 280 285
Tyr Gln Trp Glu Val Gln Arg Tyr Val Tyr Ala Lys Val Leu Ser Asp
290 295 300
Met Gln Lys Glu Gly Ile Thr Thr Pro Tyr Pro His Gly Met Gly Gly
305 310 315 320
Val Gly Leu Ala Gln Gln Thr Leu Asp Lys Gly Asp Ala Asn
325 330
<210> 11
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgctcattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcatctct gcattcgaac ctgatcatat gacattccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 12
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgcccattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcatctct gcattcgaac ctgatcatat gacattccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 13
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgctgc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgcccattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcatctct gcattcgaac ctgatcatat gacattccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 14
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggtcttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgctcattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcatctct gcattcgaac ctgatcatat gacattccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 15
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgctgc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgctcattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcatctct gcattcgaac ctgatcatat gacattccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 16
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgctcattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcgtctct gcattcgaac ctgatcatat gacattccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 17
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgctcattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcatctct gcattcgaac ctgatcatat gacactccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 18
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgcccattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcgtctct gcattcgaac ctgatcatat gacattccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 19
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgcccattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcatctct gcattcgaac ctgatcatat gacactccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005
<210> 20
<211> 1005
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atgattatcg ctagtacaga tacagaaagt attgttaccg acgtttctaa ttggtgggat 60
aaccccctca cccaggattg gttgatcaat aagccaatca gcattgccat caccattgtt 120
gtggcgtttt ttgcccattg gttgttgcgc aagctcatca ccaaagccgt ggagcgcggc 180
gtgagaaagc cgccaaagac tgaagtcccg cggtttttag tcaagggtca ggcaaagaag 240
acagacattc cgcctgaagt ccagatcatg aggaagaccc aagagcagcg tcgacaagca 300
agaatccgca ctctgggtgc ggtgggcaag tccgctgtcg ccatttttgt gtggacgtgg 360
gcggcgctgg ccatcttgac tgaaatcgga cttaatgtgg cgcccctgat cgcctcggca 420
ggtgtggctg gtgttgcgct tggttttggc gcacagtcgc tggtgaagga ctttctcagc 480
ggtattttta tgcccattga ggatcaatac ggtgtcggcg acacgatcga tgtcggtgac 540
ggcattatcg gcgacgtgga ggacattagc ctgcgcacca ccacgttgcg cgatttggac 600
ggtaccgtgt ggtacatccg caacggtgaa attctgcgtg tgggtaactt ctccaatgag 660
tacgcgattg ctcgatttga ggtgccggtg gggctgtcca atgacagcga ccgcgcgtgg 720
gatgttattg aaaattcctt ccaggaggcc gtcaagatgg aggctattaa ggattccgtc 780
atcgacacac ctgagatgaa gggcgtctct gcattcgaac ctgatcatat gacactccgc 840
ggtgtggtca agacgctgcc cggttaccag tgggaggtgc agcgctacgt ctacgccaag 900
gtattgtccg acatgcaaaa agaaggcatc accacgccct atccacacgg catgggcgga 960
gtaggcctgg cacagcagac gctggataaa ggcgacgcca actaa 1005

Claims (10)

1. The glutamic acid efflux protein mutant is obtained by carrying out point mutation on MscCG2 glutamic acid efflux protein, and the mutation site contains part or all of the following: 60, 149, 165, 269 and 279 from the N-terminus.
2. The glutamate efflux protein mutant according to claim 1, wherein: the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: bit 165 from the N-terminus.
3. The mutant glutamate efflux protein according to claim 1 or 2, wherein: the glutamate efflux protein mutant is any one of the following:
(A1) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 165 th bit from the N terminal;
(A2) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 165 th and 60 th bits from the N terminal;
(A3) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 149 th from the N-terminus;
(A4) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 60 th bit from the N terminal;
(A5) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 269 th from the N-terminus;
(A6) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 279 from the N terminus;
(A7) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 165 th and 269 th from the N-terminus;
(A8) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 165 th and 279 th positions from the N-terminus;
(A9) the glutamate efflux protein mutant is a protein obtained by point mutation of amino acid residues of at least the following sites of the MscCG2 glutamate efflux protein: 165, 269 and 279 from the N-terminus.
4. The mutant glutamate efflux protein according to any of claims 1-3, wherein: the MscCG2 glutamic acid efflux protein is an MscCG2 glutamic acid efflux protein from Corynebacterium glutamicum Z188.
5. The glutamate efflux protein mutant according to claim 4, wherein: the amino acid sequence of the MscCG2 glutamic acid efflux protein from Corynebacterium glutamicum Z188 is shown in SEQ ID No. 1.
6. The mutant glutamate efflux protein according to any of claims 1-5, wherein: in the glutamic acid efflux protein mutant, the 60 th point mutation from the N-terminal is G60C, the 149 th point mutation is F149L, the 165 th point mutation is L165P, the 269 th point mutation is I269V, and the 279 th point mutation is F279L.
7. The biological material related to the glutamate efflux protein mutant is any one of the following materials:
(I) a nucleic acid molecule encoding a glutamate efflux protein mutant according to any one of claims 1-6;
(II) expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.
8. The biomaterial of claim 7, wherein: the nucleic acid molecule for coding the MscCG2 glutamic acid efflux protein from Corynebacterium glutamicum at Z188 is a DNA molecule shown in SEQ ID No. 11;
further, the nucleic acid molecule encoding the glutamate efflux protein mutant is any one of the following:
(B1) DNA molecule shown in SEQ ID No. 12;
(B2) DNA molecule shown in SEQ ID No. 13;
(B3) DNA molecule shown in SEQ ID No. 14;
(B4) DNA molecule shown in SEQ ID No. 15;
(B5) DNA molecule shown in SEQ ID No. 16;
(B6) DNA molecule shown in SEQ ID No. 17;
(B7) DNA molecule shown in SEQ ID No. 18;
(B8) DNA molecule shown in SEQ ID No. 19;
(B9) DNA molecule shown in SEQ ID No. 20;
and/or
The recombinant bacterium is corynebacterium glutamicum containing the nucleic acid molecule.
9. Use of a glutamate efflux protein mutant or biological material of any one of claims 1-8, in any one of:
(C1) producing glutamic acid;
(C2) the glutamic acid yield is improved;
(C3) the glutamic acid efflux capacity is improved;
(C4) producing monosodium glutamate, spices or food additives;
(C5) preparing salt substitute, nutritional supplement or biochemical reagent.
10. A method for producing glutamic acid and/or increasing the production of glutamic acid and/or increasing the efflux capability of glutamic acid comprising the steps of: expressing the glutamic acid efflux protein mutant of any one of claims 1-6 in a recipient bacterium to obtain a recombinant bacterium; performing fermentation culture on the recombinant strain to obtain glutamic acid from fermentation liquor;
further, expressing the glutamate efflux protein mutant in the recipient bacterium is achieved by introducing the "nucleic acid molecule encoding the glutamate efflux protein mutant" as described in claim 7 or 8 into the recipient bacterium; and/or
Further, the recipient bacterium is corynebacterium glutamicum.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107746836A (en) * 2017-10-26 2018-03-02 江南大学 A kind of glutamine transaminage mutant expressed in an active
CN108342378A (en) * 2018-03-20 2018-07-31 福建师范大学 A kind of glutamic acid decarboxylase enzyme mutant and its encoding gene and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107746836A (en) * 2017-10-26 2018-03-02 江南大学 A kind of glutamine transaminage mutant expressed in an active
CN108342378A (en) * 2018-03-20 2018-07-31 福建师范大学 A kind of glutamic acid decarboxylase enzyme mutant and its encoding gene and application

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