3-isopropylmalate dehydratase mutant and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a 3-isopropyl malate dehydratase mutant and application thereof. The 3-isopropyl malate dehydratase mutant is obtained by mutating two sites in the sequence of isopropyl malate dehydratase into other amino acids. The present application provides two mutant sites of isopropyl malate dehydratase, and further finds that after the two sites of isopropyl malate dehydratase in the strain are mutated, the yield of L-valine is significantly improved, and the yield of byproduct leucine is significantly reduced, which has important significance in the field of improving the yield of L-valine produced by the strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a 3-isopropylmalate dehydratase mutant and its applications. Background Technology

[0002] L-valine, chemically known as L-α-aminoisovaleric acid, has the molecular formula C5H12H2O. 11 NO2 has a relative molecular mass of 117.15. L-valine is a white crystalline or crystalline powder, odorless, with a bitter taste. Its solubility in water is 88.5 g / L at 25°C and 96.2 g / L at 50°C. It is insoluble in cold ethanol, ether, and acetone. L-valine has an isoelectric point of 5.96 and a melting point of 315°C.

[0003] L-valine is one of the eight essential amino acids for the human body and one of the three branched-chain amino acids (including valine, leucine, and isoleucine). Due to its unique structure and function, it plays a particularly important role in human metabolism. L-valine has wide applications in the pharmaceutical, food, and feed industries. In the pharmaceutical industry, L-valine is used as a major component in amino acid infusions and comprehensive amino acid preparations, and can be used to treat liver failure and central nervous system dysfunction. In the food industry, L-valine is used as a food additive, nutritional supplement, and flavoring agent. Furthermore, L-valine can also be used in amino acid functional beverages and sports drinks, promoting muscle growth, strengthening liver function, and reducing muscle fatigue. In the feed industry, L-valine plays a significant role in promoting milk secretion from animal mammary glands.

[0004] Currently, there are three methods for producing L-valine: extraction, chemical synthesis, and microbial fermentation. Extraction and chemical synthesis are difficult to scale up industrially due to limited raw material sources, high production costs, and environmental pollution. Microbial fermentation, on the other hand, offers advantages such as low raw material costs, mild reaction conditions, and ease of large-scale production, making it the most common method for L-valine production. However, the fermentation performance of current L-valine strains remains relatively poor, and the high levels of leucine as a byproduct result in low conversion rates, failing to meet the demands of large-scale industrial production. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides a 3-isopropylmalate dehydratase mutant and its application.

[0006] In a first aspect, the present invention provides a 3-isopropylmalate dehydratase mutant, wherein the 3-isopropylmalate dehydratase mutant is obtained by mutating the 248th and / or 396th amino acids of isopropylmalate dehydratase to other amino acids.

[0007] Furthermore, the amino acid sequence of the isopropyl malate dehydrase includes the sequence shown in SEQ ID NO.1.

[0008] Furthermore, the mutation into other amino acids is as follows:

[0009] The mutation can result in one of the following: leucine, valine, isoleucine, serine, or alanine.

[0010] Furthermore, the 248th amino acid of isopropyl malate dehydratase is mutated from methionine (M) to leucine (L), valine (V), or isoleucine (I). Specifically, the corresponding base is mutated from ATG to CTG, GTG, or ATA.

[0011] Furthermore, the 396th amino acid of isopropyl malate dehydratase is mutated from threonine (T) to serine (S), alanine (A), or isoleucine (I). Specifically, the corresponding base is mutated from ACC to AGC, GCC, or ATC.

[0012] The present invention further provides a nucleic acid encoding the 3-isopropylmalate dehydratase mutant.

[0013] In a second aspect, the present invention provides a recombinant microorganism in which the 248th and / or 396th amino acids of the isopropyl malate dehydrase are mutated to any other arbitrary amino acid.

[0014] Furthermore, the isopropyl malate dehydrase in the recombinant microorganism comprises an amino acid sequence as shown in any of SEQ ID NO. 3-9.

[0015] Furthermore, the isopropyl malate dehydrase in the recombinant microorganism is encoded by a nucleotide sequence as shown in any of SEQ ID NO. 10-16.

[0016] Furthermore, the recombinant microorganism is based on Corynebacterium, preferably one or more of Corynebacterium glutamicum, Corynebacterium pingeri, or Corynebacterium flavum.

[0017] The present invention further provides the application of the 3-isopropylmalate dehydratase mutant in increasing the yield of branched-chain amino acids in microorganisms.

[0018] Furthermore, the microorganism includes one or more of Corynebacterium glutamicum, Corynebacterium pingeri, or Bacillus flavus; the branched-chain amino acid is L-valine.

[0019] The present invention further provides the application of the recombinant microorganism in the production of branched-chain amino acids, preferably L-valine.

[0020] The present invention has the following beneficial effects:

[0021] This invention reveals that mutations at amino acid positions 248 or 396 in isopropyl malate dehydratase from microorganisms significantly increase L-valine yield and conversion rate compared to the starting strain, while drastically decreasing L-leucine yield, while maintaining overall good production performance. In particular, simultaneous mutations at both sites resulted in a valine yield of 8.7 g / L, an increase of 2.8 g / L (47.5%) compared to the starting strain; however, the yield of the byproduct leucine was 0.3 g / L, a decrease of 85.7% compared to the starting strain.

[0022] The isopropyl malate dehydratase provided by this invention is applicable to various Corynebacterium glutamicum and can be used to regulate the production of branched-chain amino acids such as valine, isoleucine, and leucine or their derivatives. Detailed Implementation

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0024] The primer names and sequences involved in the embodiments are shown in Table 1.

[0025] Table 1 shows the primers used in the examples.

[0026]

[0027]

[0028] Example 1

[0029] Starting with MHZ-1012-2, the leuC gene in MHZ-1012-2 was mutated to encode the 3-isopropylmalate dehydratase mutant shown in SEQ ID NO.10-12, and 3-isopropylmalate dehydratase mutant strain 2-LeuC was constructed sequentially. M248L 2-LeuC M248V 2-LeuC M248I The specific construction method is as follows:

[0030] 1. Plasmid pK18mobsacB-leuC M248L Construction

[0031] Using Phusion superfidelity polymerase (New England BioLabs), with the genome of the starting strain MHZ-1012-2 as a template, and leuC M248L -UP-1F / leuC M248L Using UP-1R as primers, recombinant fragment UP-1 was prepared, and leuC was used as the primer. M248L -DN-2F / leuC M248LUsing DN-2R as primers, recombinant fragment DN-1 was prepared; using plasmid pk18-mob-sacB as a template, and leuC... M248L -pk18-3F / leuC M248L -pk18-3R was used as a primer to obtain fragment pk18-1, which was purified using an agarose gel extraction kit (Tiangen). The reaction was then carried out according to the Gibson assembly kit configuration system, as shown in Table 2.

[0032] Table 2 Gibson Assembly Reaction System

[0033]

[0034]

[0035] The prepared reaction mixture was incubated at 37°C for 30 min. 10 μL of the incubator was then transformed into Trans1T1 competent cells (TransGen Biotech). Single clones were picked, and colony PCR was used to confirm the correct insertion fragment. Further enzyme digestion confirmed the presence of a positive clone containing the pK18mobsacB fragment. Finally, the plasmid was sent to Genewiz Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was named pK18mobsacB-leuC. M248L .

[0036] 2. Plasmid pK18mobsacB-leuC M248V pK18mobsacB-leuC M248I Construction

[0037] Following the same method as step 1 above, primer leuC M248L Replace -UP-1R with leuC respectively M248V -UP-1R、leuC M248I -UP-1R, primer leuC M248L -DN-2F should be replaced with leuC. M248V -DN-2F、leuC M248I -DN-2F, the constructed plasmids were named pK18mobsacB-leuC respectively. M248V pK18mobsacB-leuC M248I .

[0038] 3. 3-Isopropylmalate dehydratase mutant strain 2-LeuC M248L 2-LeuC M248V 2-LeuC M248I Construction

[0039] The recombinant plasmid pK18mobsacB-leuC obtained by the methods described in steps 1 and 2 above is used. M248L pK18mobsacB-leuC M248V pK18mobsacB-leuC M248I Transformants were transferred into the starting strain MHZ-1012-2 and recombinants were selected on selective medium containing 15 mg / L kanamycin. The culture temperature was 30°C, and the culture was inverted. The selected transformants were cultured overnight in ordinary liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m / s). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the target mutant strain, which was named 2-LeuC. M248L 2-LeuC M248V 2-LeuC M248I .

[0040] Example 2: Construction of 3-isopropylmalate dehydratase mutant strain 2-LeuC T396S 2-LeuC T396A 2-LeuC T396I

[0041] Starting with MHZ-1012-2, the leuC gene in MHZ-1012-2 was mutated to encode the 3-isopropylmalate dehydratase mutant shown in SEQ ID NO.13-15, and 3-isopropylmalate dehydratase mutant strain 2-LeuC was constructed sequentially. T396S 2-LeuC T396A 2-LeuC T396I The specific construction method is as follows.

[0042] 1. Plasmid pK18mobsacB-leuC T396S pK18mobsacB-leuC T396A pK18mobsacB-leuC T396I Construction

[0043] According to step 1 of Example 1 above, the plasmid pK18mobsacB-leuC M248L The same method was used to construct the plasmids, with primers replaced sequentially to complete the construction. The constructed plasmids were named pK18mobsacB-leuC.T396S pK18mobsacB-leuC T396A pK18mobsacB-leuC T396I .

[0044] 2,3-Isopropylmalate dehydratase mutant strain 2-LeuC T396S 2-LeuC T396A 2-LeuC T396I Construction

[0045] The recombinant plasmid pK18mobsacB-leuC obtained by the method described in step 1 above. T396S pK18mobsacB-leuC T396A pK18mobsacB-leuC T396I Transformants were transferred into the starting strain MHZ-1012-2 and recombinants were selected on selective medium containing 15 mg / L kanamycin. The culture temperature was 30°C, and the culture was inverted. The selected transformants were cultured overnight in ordinary liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m / s). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the target mutant strain, which was named 2-LeuC. T396S 2-LeuC T396A 2-LeuC T396I .

[0046] Example 3: Construction of 3-Isopropylmalate Dehydratase Mutant Strain 2-LeuC M248I,T396A

[0047] Starting with MHZ-1012-2, the leuC gene in MHZ-1012-2 was mutated to encode the 3-isopropylmalate dehydratase mutant shown in SEQ ID NO.9, thus constructing the 3-isopropylmalate dehydratase mutant strain 2-LeuC. M248I,T396A The specific construction method is as follows.

[0048] The recombinant plasmid pK18mobsacB-leuC obtained by the method described in Example 2 above. T396A The recombinant bacteria 2-LeuC obtained by constructing the strain using the method described in Example 1 above. M248IIn this study, recombinants were selected on selective medium containing 15 mg / L kanamycin. The culture temperature was 30°C, and the culture was inverted. The selected transformants were then cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm on a rotary shaker. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ mcg / L). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the target mutant strain, named 2-LeuC. M248I,T396A .

[0049] Example 4: Verification of the effect of strain fermentation in producing L-valine

[0050] This embodiment verifies the mutant strains constructed in Examples 1-3 through fermentation. The specific methods are as follows:

[0051] 1. Culture medium

[0052] Seed culture medium: 15 g / L soybean meal extract, 20 g / L glucose, 7 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 2 g / L urea, balance water, pH 7.2.

[0053] Fermentation medium: soybean meal extract 15 g / L, glucose 60 g / L, ammonium sulfate 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 2 g / L, calcium carbonate 40 g / L, vitamin C B3 15mg / L, V H 50 μg / L, V B1 • HCl 100 μg / L, balance water, pH 7.2.

[0054] 2. Production of L-valine by shake-flask fermentation

[0055] (1) Seed culture: Pick one loop of slant seeds and inoculate them into a 500 mL Erlenmeyer flask containing 50 mL of seed culture medium. Culture at 30 °C and 220 r / min for 10-12 h with shaking.

[0056] (2) Fermentation culture: 5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of fermentation medium and cultured at 30 °C and 220 r / min for 48 h with shaking.

[0057] (3) Centrifuge 1 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and detect L-valine and L-leucine in the fermentation broth by HPLC. At the same time, the OD value of the fermentation broth at 562 nm was detected by spectrophotometry. The results are shown in the table below.

[0058] Table 3 Results of bacterial growth and product content detection

[0059]

[0060] Note: * indicates a significant difference compared to the starting strain. All strain names above omit 2-LeuC, retaining only the mutation mode.

[0061] The results showed that the L-valine yield of the starting strain MHZ-1012-2 was only 5.9 g / L, while that of the 3-isopropylmalate dehydratase mutant strain 2-LeuC M248L 2-LeuC M248V 2-LeuC M248I 2-LeuC T396S 2-LeuC T396A 2-LeuC T396I 2-LeuC M248I,T396A The yield and conversion rate of L-valine were significantly increased compared to the original strain, while the yield of L-leucine decreased significantly, and overall good growth performance was still maintained. Among them, the α-isopropylmalate synthase mutant strain 2-LeuC... M248I ,T396A The most significant improvement was observed in the valine yield, which was 8.7 g / L, an increase of 2.8 g / L (47.5%) compared to the original strain. Conversely, the yield of the byproduct leucine was only 0.3 g / L, a decrease of 85.7% compared to the original strain. This demonstrates that the 3-isopropylmalate dehydratase mutant and the 3-isopropylmalate dehydratase mutant strain provided by this invention significantly promote the yield of the target product valine and significantly reduce the yield of the byproduct leucine. This 3-isopropylmalate dehydratase mutant and its recombinant microorganism provide a reference for the construction of production strains that produce valine, isoleucine, and derivatives using valine as precursors.

[0062] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention. sequence list <110> Langfang Meihua Biotechnology Development Co., Ltd. <120> A 3-isopropylmalate dehydratase mutant and its application <130> KHP211123189.8 <160> 36 <170> SIPOSequenceListing 1.0 <210> 1 <211> 481 <212> PRT <213> Artificial Sequence <400> 1 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Met Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Thr Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 2 <211> 1446 <212> DNA <213> Artificial Sequence <400> 2 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg catgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccacctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 3 <211> 481 <212> PRT <213> Artificial Sequence <400> 3 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Leu Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Thr Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 4 <211> 481 <212> PRT <213> Artificial Sequence <400> 4 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Val Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Thr Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 5 <211> 481 <212> PRT <213> Artificial Sequence <400> 5 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Ile Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Thr Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 6 <211> 481 <212> PRT <213> Artificial Sequence <400> 6 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Met Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Ser Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 7 <211> 481 <212> PRT <213> Artificial Sequence <400> 7 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Met Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Ala Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 8 <211> 481 <212> PRT <213> Artificial Sequence <400> 8 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Met Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Ile Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 9 <211> 481 <212> PRT <213> Artificial Sequence <400> 9 Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu 1 5 10 15 Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly 20 25 30 Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr 35 40 45 Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg 50 55 60 His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu 65 70 75 80 Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg 85 90 95 Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg 100 105 110 Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly 115 120 125 Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp 130 135 140 Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile 145 150 155 160 Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu 165 170 175 Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro 180 185 190 Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly 195 200 205 Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile 210 215 220 Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile 225 230 235 240 Glu Ala Gly Ala Arg Ala Gly Ile Ile Ala Pro Asp Gln Thr Thr Phe 245 250 255 Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp 260 265 270 Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr 275 280 285 Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile 290 295 300 Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val 305 310 315 320 Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu 325 330 335 Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp 340 345 350 Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile 355 360 365 Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala 370 375 380 Asp Gly Met Arg Met Met Val Val Pro Ser Ser Ala Trp Ile Lys Gln 385 390 395 400 Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala 405 410 415 Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp 420 425 430 Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe 435 440 445 Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala 450 455 460 Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp 465 470 475 480 Ile <210> 10 <211> 1446 <212> DNA <213> Artificial Sequence <400> 10 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg cctgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccacctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 11 <211> 1446 <212> DNA <213> Artificial Sequence <400> 11 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg cgtgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccacctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 12 <211> 1446 <212> DNA <213> Artificial Sequence <400> 12 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg cataatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccacctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 13 <211> 1446 <212> DNA <213> Artificial Sequence <400> 13 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg catgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccagctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 14 <211> 1446 <212> DNA <213> Artificial Sequence <400> 14 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg catgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccgcctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 15 <211> 1446 <212> DNA <213> Artificial Sequence <400> 15 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg catgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccatctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 16 <211> 1446 <212> DNA <213> Artificial Sequence <400> 16 atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60 tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120 ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180 cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240 ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300 accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360 cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420 gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480 ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540 accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600 ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660 ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720 gaagctggcg cacgtgccgg cataatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780 ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840 ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900 accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960 ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020 tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080 ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140 cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccgcctg gatcaagcaa 1200 gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260 gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320 gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380 gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440 atctaa 1446 <210> 17 <211> 44 <212> DNA <213> Artificial Sequence <400> 17 acaacgtcgt gactgggaaa acccaggcat caagactggc tcac 44 <210> 18 <211> 42 <212> DNA <213> Artificial Sequence <400> 18 gtggtttggt ctggggcgat caggccggca cgtgcgccag ct 42 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <400> 19 agctggcgca cgtgccggcc tgatcgcccc agaccaaacc ac 42 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <400> 20 cgtaatcatg tcatagctgt ttcctcagcg ccagcgtcgg tgaag 45 <210> twenty one <211> 44 <212> DNA <213> Artificial Sequence <400> twenty one gtgagccagt cttgatgcct gggttttccc agtcacgacg ttgt <210> 22 <211> 45 <212> DNA <213> Artificial Sequence <400> 22 cttcaccgac gctggcgctg aggaaacagc tatgacatga ttacg <210> 23 <211> 42 <212> DNA <213> Artificial Sequence <400> 23 gtggtttggt ctggggcgat cacgccggca cgtgcgccag ct <210> 24 <211> 42 <212> DNA <213> Artificial Sequence <400> 24 agctggcgca cgtgccggcg tgatcgcccc agaccaaacc ac <210> 25 <211> 42 <212> DNA <213> Artificial Sequence <400> 25 gtggtttggt ctggggcgat tatgccggca cgtgcgccag ct <210> 26 <211> 42 <212> DNA <213> Artificial Sequence <400> 26 agctggcgca cgtgccggca taatcgcccc agaccaaacc ac 42 <210> 27 <211> 44 <212> DNA <213> Artificial Sequence <400> 27 acaacgtcgt gactgggaaa acccgatgca cgcatgacca tgtg 44 <210> 28 <211> 44 <212> DNA <213> Artificial Sequence <400> 28 cggcctcttg cttgatccag ctggaggaag gcacgaccat catg 44 <210> 29 <211> 44 <212> DNA <213> Artificial Sequence <400> 29 catgatggtc gtgccttcct ccagctggat caagcaagag gccg 44 <210> 30 <211> 45 <212> DNA <213> Artificial Sequence <400> 30 cgtaatcatg tcatagctgt ttccatatct gcaggtgagg acagg 45 <210> 31 <211> 44 <212> DNA <213> Artificial Sequence <400> 31 cacatggtca tgcgtgcatc gggttttccc agtcacgacg ttgt 44 <210> 32 <211> 45 <212> DNA <213> Artificial Sequence <400> 32 cctgtcctca cctgcagata tggaaacagc tatgacatga ttacg 45 <210> 33 <211> 44 <212> DNA <213> Artificial Sequence <400> 33 cggcctcttg cttgatccag gcggaggaag gcacgaccat catg 44 <210> 34 <211> 44 <212> DNA <213> Artificial Sequence <400> 34 catgatggtc gtgccttcct ccgcctggat caagcaagag gccg 44 <210> 35 <211> 44 <212> DNA <213> Artificial Sequence <400> 35 cggcctcttg cttgatccag atggaggaag gcacgaccat catg 44 <210> 36 <211> 44 <212> DNA <213> Artificial Sequence <400> 36 catgatggtc gtgccttcct ccatctggat caagcaagag gccg 44

Claims

1. A 3-isopropylmalate dehydratase mutant, characterized in that, The amino acid sequence of the 3-isopropylmalate dehydratase mutant is shown in any one of SEQ ID NO. 3-5 or 9.

2. A nucleic acid, characterized in that, The nucleic acid is used to encode the 3-isopropylmalate dehydratase mutant of claim 1.

3. A recombinant microorganism, characterized in that, The amino acid sequence of the isopropyl malate dehydrase in the recombinant microorganism is shown in any one of SEQ ID NO. 3-5 or 9.

4. The recombinant microorganism according to claim 3, characterized in that, The isopropyl malate dehydrase in the recombinant microorganism is encoded by a nucleotide sequence as shown in any of SEQ ID NO. 10-12 or 16.

5. The recombinant microorganism according to claim 3 or 4, characterized in that, The recombinant microorganisms were derived from Corynebacterium strain.

6. The recombinant microorganism according to claim 5, characterized in that, The recombinant microorganisms are derived from one or more of Corynebacterium glutamicum, Corynebacterium pingeri, or Bacillus flavus.

7. The application of the 3-isopropylmalate dehydratase mutant of claim 1 in increasing the yield of branched-chain amino acids in microorganisms; wherein the microorganism is Corynebacterium glutamicum and the branched-chain amino acid is L-valine.

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