CN116640752A - Aconitate hydratase mutant and application thereof - Google Patents

Aconitate hydratase mutant and application thereof Download PDF

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CN116640752A
CN116640752A CN202210141296.3A CN202210141296A CN116640752A CN 116640752 A CN116640752 A CN 116640752A CN 202210141296 A CN202210141296 A CN 202210141296A CN 116640752 A CN116640752 A CN 116640752A
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gly
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王亚迪
胡丹
张孟娟
赵津津
李岩
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Langfang Meihua Bio Technology Development Co Ltd
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01003—Aconitate hydratase (4.2.1.3)

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Abstract

The invention relates to the technical field of microorganisms, in particular to aconitate hydratase mutants and application thereof. The aconitate hydratase mutant provided by the invention has an amino acid sequence shown in any one of SEQ ID NO. 1-5. The aconitate hydratase mutant provided by the invention obviously reduces the enzyme activity of aconitate hydratase, and can enable oxaloacetate to flow to the synthesis path of aspartic acid and downstream metabolites thereof more. The recombinant microorganism constructed by the aconitate hydratase mutant has obviously improved lysine synthesis capability, obviously improved lysine yield and conversion rate, and better growth performance.

Description

Aconitate hydratase mutant and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to aconitate hydratase mutants and application thereof.
Background
L-lysine is basic essential amino acid with molecular formula of C 6 H 14 N 2 O 2 The appearance is white or almost white crystalline powder, darkens at 210 ℃, decomposes at 224.5 ℃, is easily soluble in water, slightly soluble in alcohol, and insoluble in ether. L-lysine is widely used in the fields of animal feed, medicine and food industry, of which about 90% is used in the feed industry and 10% is used in the food and medicine industry. The L-lysine is added into the animal feed additive to promote the organism to absorb other amino acids, thereby improving the quality of the feed.
At present, the most commonly used production method of L-lysine is a microbial fermentation method, and the microbial fermentation method has the advantages of low raw material cost, mild reaction conditions, easiness in realizing large-scale production and the like. Coli (Escherichia coli) has the advantages of high growth speed, clear genetic background, simple culture condition, mature metabolic engineering means and the like, is widely applied to the field of industrial fermentation, and can be used for producing L-amino acid, nucleotide, other organic acids and the like.
Aconitate hydratase (Aconitate hydratase A) is an enzyme in the tricarboxylic acid cycle that catalyzes two chemical reactions, namely conversion of citric acid to aconitic acid and conversion of aconitic acid to isocitric acid, respectively, whereas citric acid requires one enzyme catalytic step synthesis with oxaloacetic acid as a precursor, while oxaloacetic acid is also a precursor for synthesis of aspartic acid, whereas lysine is synthesized with aspartic acid as a precursor. Therefore, consumption of oxaloacetate can be reduced by weakening down metabolism of citric acid, which is advantageous for entrance of oxaloacetate into lysine synthesis pathway, thereby improving lysine yield. At present, the prior art reports on weakening aconitate hydratase expression, but no mutant with reduced enzyme activity obtained by mutating aconitate hydratase has been reported.
Disclosure of Invention
The invention aims to provide aconitate hydratase mutants and application thereof. Another object of the present invention is to provide a recombinant microorganism expressing the aconitate hydratase mutant and its use.
Specifically, the invention provides the following technical scheme:
the invention provides an aconitate hydratase mutant, which has an amino acid sequence shown in any one of SEQ ID NO. 1-5.
Aconitate hydratase mutants with sequences shown in SEQ ID No.1-5 are obtained by mutating 522 th amino acid into histidine (H), glutamic acid (E), isoleucine (I), tryptophan (W) and glycine (G) on the basis of aconitate hydratase of wild type escherichia coli.
The amino acid sequence of aconitate hydratase of wild type escherichia coli MG1655 is shown as SEQ ID NO. 6.
The invention discovers that mutation of 522 th amino acid of aconitate hydratase into the amino acid can obviously reduce the catalytic activity of aconitate hydratase, but can not lead to complete inactivation of the aconitate hydratase, the reduction of aconitate hydratase activity can lead oxaloacetic acid to be used for synthesizing aspartic acid more, enhance the metabolic flux of an aspartic acid pathway, further be beneficial to improving the synthesis of aspartic acid and metabolic products taking aspartic acid as precursors, and the residual aconitate hydratase activity can maintain the requirements of tricarboxylic acid circulation and bacterial growth, and the mutation can not lead to obvious inhibition of bacterial growth.
It will be appreciated by those skilled in the art that the addition of a tag protein to the N-or C-terminus of the aconitate hydratase mutant sequence or the fusion of the tag protein with other proteins to form a fusion protein will not significantly alter the activity of the aconitate hydratase mutant without altering the structure of the aconitate hydratase mutant itself, and therefore, the tagged protein or fusion protein is also within the scope of the invention.
The invention also provides a nucleic acid molecule encoding the aconitate hydratase mutant described above.
Based on the amino acid sequence of the aconitate hydratase mutants, one skilled in the art can determine the nucleotide sequence of the nucleic acid molecule encoding the mutants. Based on the degeneracy of the codons, more than one of the sequences of the above-mentioned nucleic acid molecules, all nucleic acid molecules capable of encoding the above-mentioned aconitate hydratase mutants are within the scope of the invention.
The invention also provides a biological material containing the nucleic acid molecule, wherein the biological material is an expression cassette, a vector or a host cell.
Wherein, the expression cassette may be a recombinant DNA molecule obtained by ligating elements for driving transcription and translation of the gene upstream or downstream of the gene.
The vector may be an expression vector or a cloning vector, including but not limited to, plasmid vectors, phage vectors, viral vectors, transposons, and the like.
The host cell may be a microbial cell.
On the basis of the aconitate hydratase mutant, the invention provides a recombinant microorganism which expresses the aconitate hydratase mutant.
Preferably, the recombinant microorganism does not express aconitate hydratase possessed by its starting strain.
Further preferably, in the recombinant microorganism, the gene encoding aconitate hydratase is replaced with the nucleic acid molecule encoding the aconitate hydratase mutant described above.
The recombinant microorganism described above is a bacterium of the genus Escherichia, preferably Escherichia coli (Escherichia coli).
The starting strain mentioned above refers to a starting strain for replacing the gene encoding aconitate hydratase with the gene encoding aconitate hydratase mutant, namely: the recombinant microorganism can be obtained by replacing the gene encoding aconitate hydratase of the original strain with the gene encoding aconitate hydratase mutant.
The recombinant microorganism obtained by mutating aconitate hydratase encoding genes in the initial strain into genes encoding aconitate hydratase mutants has the advantages that the yield and the conversion rate of aspartic acid or metabolic products taking aspartic acid as precursors are obviously improved compared with the initial strain, and the recombinant microorganism has better growth performance.
Preferably, the starting strain is E.coli capable of synthesizing and accumulating lysine.
As one embodiment of the invention, the starting strain is lysine-producing Escherichia coli MHZ-0914 obtained by mutagenesis, and the strain is preserved in China general microbiological culture Collection center (CGMCC, address: north Xielu No.1, 3 of the Beijing Chaoyang area, post code 100101 of the national academy of sciences of China) at 1/6/1 of 2021, and the preservation number is CGMCC No.22648, and the classification is named Escherichia coli.
The invention also provides a construction method of the recombinant microorganism, which comprises the following steps: and replacing a gene encoding aconitate hydratase in the original strain of the recombinant microorganism with a gene encoding the aconitate hydratase mutant.
The substitution of the above genes can be achieved by conventional means in the art, for example: and replacing a gene encoding aconitate hydratase on a chromosome of the original strain with a gene encoding the aconitate hydratase mutant by adopting a homologous recombination method.
The invention further provides the use of any one of said aconitate hydratase mutants or said nucleic acid molecules or said biological material or said recombinant microorganism as follows:
(1) Use in the construction of a production strain for the production of aspartic acid or a metabolite of aspartic acid as a synthetic precursor;
(2) Use in the fermentative production of aspartic acid or metabolites with aspartic acid as synthesis precursor;
(3) Use in increasing the yield and/or conversion of aspartic acid or a metabolite having aspartic acid as a synthetic precursor.
In the above (1), the production strain is preferably E.coli.
Preferably, the metabolic product of aspartic acid as a synthetic precursor according to the present invention is lysine.
The present invention provides a method for fermenting aspartic acid or a metabolite having aspartic acid as a synthetic precursor, the method comprising: culturing the recombinant microorganism to obtain a culture, and separating and extracting the culture to obtain aspartic acid or a metabolite taking the aspartic acid as a synthesis precursor.
Specifically, the method comprises the following steps: inoculating the recombinant microorganism into a seed culture medium for seed culture to obtain seed liquid, inoculating the seed liquid into a fermentation culture medium for culture to obtain fermentation liquor, and separating and extracting the fermentation liquor to obtain aspartic acid or a metabolite taking the aspartic acid as a synthesis precursor.
Preferably, the metabolite with aspartic acid as a synthetic precursor is lysine.
Preferably, the fermentation medium comprises the following components: 55-65g/L of glucose, 8-12g/L of molasses, 35-45g/L of ammonium sulfate, 8-12g/L of corn steep liquor, 1-2g/L of monopotassium phosphate, 0.5-1.5g/L of magnesium sulfate heptahydrate, 0.02-0.04g/L of ferric sulfate, 0.02-0.04g/L of manganese sulfate and 0-30g/L of calcium carbonate.
The invention has the beneficial effects that: the aconitate hydratase mutant provided by the invention obviously reduces the enzyme activity of aconitate hydratase, and can enable oxaloacetate to flow to the synthesis path of aspartic acid and downstream metabolites thereof more. The recombinant microorganism constructed by the aconitate hydratase mutant has obviously improved lysine synthesis capability, obviously improved lysine yield and conversion rate, and better growth performance.
Detailed Description
The amino acid sequence of the aconitate hydratase mutant provided by the invention is shown in any one of SEQ ID NO. 1-5.
The invention also provides a recombinant microorganism expressing the aconitate hydratase mutant, and the recombinant microorganism is preferably recombinant escherichia coli. The recombinant escherichia coli is obtained by mutating an original aconitate hydratase coding gene in an original strain into a coding gene of the aconitate hydratase mutant.
The present invention also provides a method for producing lysine by fermentation using the recombinant microorganism, the method comprising: culturing the recombinant microorganism to obtain a culture, and separating and extracting the culture to obtain lysine.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
The names and sequences of the primers involved in the following examples are shown in Table 1.
TABLE 1 primer sequences
Example 1 contains the mutant Gene acnA S522H Construction of recombinant strains of (2)
In this example, escherichia coli MHZ-0914 (with the preservation number of CGMCC No. 22648) is used as an initial strain, and aconitate hydratase mutant (AcnA) expressed as SEQ ID No.1 is provided S522H ) The amino acid sequence of aconitic acid hydratase of the escherichia coli MHZ-0914 is shown as SEQ ID NO.6 through sequencing analysis.
The construction method of the recombinant escherichia coli comprises the following steps:
(1) pTargetF-N20 (acnA S522H) plasmid and Donor DNA construction
Step 1: using pTF-acnA-sgRNA-F/pTF-acnA-sgRNA-R as primers, using plasmid pTargetF as template (Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al, appl. EnvironMicrobiol, 2015), amplifying to obtain a linear plasmid of pTF with N20, assembling this linear plasmid using a seamless assembly ClonExpress kit at 37 ℃, then transforming Trans1-T1 competent cells to obtain plasmid pTargetF-N20 (acnA S522H), and subjecting the plasmid to PCR identification and sequencing verification;
step 2: the MHZ-0914 genome is used as a template, an acnAS522-UF/acnAS522H-UR primer pair is selected for amplification to obtain an upstream homology arm (1), an acnAS522H-DF/acnAS522-DR primer pair is selected for amplification to obtain a downstream homology arm (2), and the acnAS522-UF/acnAS522-DR primer pair is selected for amplification by using (1) and (2) as templates to obtain the Donor DNA.
(2) Competent cell preparation and electrotransformation
Step 1: the pCas plasmid (Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System, jiang Y, chen B, et al appl. EnvironMicrobiol, 2015) was electrotransferred into CGMCC No.22648 competent cells (both transformation and competent preparation methods refer to molecular clone III);
step 2: single colonies from step 1 were picked up and cultured to OD at 30℃at 200r/min in 5mL LB medium containing kanamycin and arabinose at a final concentration of 10mM 650 Electrotransformation competent cells were prepared after 0.4 (competent preparation methods reference molecular clone III);
step 3: the pTargetF-N20 (acnA S522H) plasmid constructed in (1) and the Donor DNA were simultaneously electrotransferred into cells with pCas competence (electrotransfer conditions: 2.5GV,200Ω, 25. Mu.F), plated on LB plates containing spectinomycin and kanamycin, and cultured at 30℃until single colonies were visible.
(3) Recombinant verification
Step 1: performing colony PCR verification on the single colony obtained in the step 3 of the step (2) by using a primer pair acnAS522H-F1/acnAS 522-R;
step 2: the PCR identification of the correct strain was amplified using the primer pair acnAS522-F/acnAS522-R and the amplified product was sequenced.
(4) Construction of recombinant strains with related plasmid loss
Step 1: picking the single colony which is verified to be correct by sequencing in the step (3), inoculating the single colony into 5mL of LB culture medium containing kanamycin and IPTG with the final concentration of 0.5mM, culturing overnight at 30 ℃, and streaking on an LB plate containing kanamycin;
step 2: selecting the single colony obtained in the step 1, and culturing the single colony on a LB plate containing kanamycin and spectinomycin and a LB plate only containing kanamycin at 30 ℃ overnight, if the single colony cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, wherein the single colony indicates that the pTargetF-N20 plasmid is lost;
step 3: selecting positive colonies lost by pTargetF-N20 plasmid, inoculating to an antibiotic-free LB medium, culturing at 42 ℃ for 8 hours, streaking on an LB plate, and culturing at 37 ℃ overnight;
step 4: picking single colony to be on the LB plate containing kanamycin and the non-resistant LB plate, if the colony can not grow on the LB plate containing kanamycin, the colony grows on the non-resistant LB plate, which shows that pCas plasmid is lost, and CGMCC No.22648-acnA is obtained S522H Compared with CGMCC No.22648 strain, the strain has mutated acnA encoding gene to make its encoding sequence shown in SEQ ID NO.1 aconitate hydratase mutant.
Example 2 containing the mutant Gene acnA S522E Construction of recombinant strains of (2)
In this example, escherichia coli MHZ-0914 (with the preservation number of CGMCC No. 22648) is used as an initial strain, and aconitate hydratase mutant (AcnA) expressed as SEQ ID No.2 is provided S522E ) The construction method of the recombinant E.coli of (2) refers to the method of example 1, and the obtained recombinant strain is named CGMCC No.22648-acnA S522E 。
Example 3 mutant Gene acnA S522I Recombinant strain construction of (2)
In this example, escherichia coli MHZ-0914 (with the preservation number of CGMCC No. 22648) is used as an initial strain, and aconitate hydratase mutant (AcnA) expressed as SEQ ID No.3 is provided S522I ) The construction method of the recombinant E.coli of (2) refers to the method of example 1, and the obtained recombinant strain is named CGMCC No.22648-acnA S522I 。
Example 4 mutant Gene acnA S522W Recombinant strain construction of (2)
In this example, escherichia coli MHZ-0914 (with the preservation number of CGMCC No. 22648) is used as an initial strain, and aconitate hydratase mutant (AcnA) expressed as SEQ ID No.4 is provided S522W ) The construction method of the recombinant E.coli of (2) refers to the method of example 1, and the obtained recombinant strain is named CGMCC No.22648-acnA S522W 。
Example 5 mutant Gene acnA S522G Recombinant strain construction of (2)
In this example, escherichia coli MHZ-0914 (with the preservation number of CGMCC No. 22648) is used as an initial strain, and aconitate hydratase mutant (AcnA) expressed as SEQ ID No.5 is provided S522G ) The construction method of the recombinant E.coli of (2) refers to the method of example 1, and the obtained recombinant strain is named CGMCC No.22648-acnA S522G 。
EXAMPLE 6 lysine fermentation experiment
The recombinant strains constructed in examples 1 to 5 were subjected to lysine fermentation experiments, and the medium formulation used in the lysine fermentation process was as follows:
seed activation medium: 10g/L peptone, 10g/L NaCl, 5g/L yeast powder and 18g/L agar powder, and adjusting the pH value to 7.0.
Seed culture medium: glucose 20g/L, ammonium sulfate 4g/L, corn steep liquor 2.0g/L, monopotassium phosphate 3g/L, magnesium sulfate heptahydrate 0.4g/L, ferric sulfate 0.01g/L and manganese sulfate 0.01g/L, and adjusting the pH to 7.0.
Fermentation medium: glucose 60g/L, molasses 10g/L, ammonium sulfate 40g/L, corn steep liquor 10g/L, potassium dihydrogen phosphate 1.6g/L, magnesium sulfate heptahydrate 1.0g/L, ferric sulfate 0.03g/L, manganese sulfate 0.03g/L, calcium carbonate 25g/L, and pH adjusted to 7.0.
The lysine fermentation method comprises the following steps:
1. seed activation: taking the strain to be verified from the freezing tube, streaking and activating on a seed activation culture medium, and culturing for 12 hours at 37 ℃;
2. seed culture: the plate activated seeds 1 are picked and looped into a 500mL triangular flask filled with 20mL of seed culture medium, and subjected to shaking culture at 33 ℃ and 220r/min for 7h to obtain seed liquid;
3. fermentation culture: 2mL of seed solution is inoculated into a 500mL triangular flask filled with 30mL of fermentation medium, and is subjected to shaking culture for 12h at 37 ℃ and 220r/min, and three strains are parallel to each other, so that fermentation liquid is obtained.
4、OD 600 And (3) measuring: diluting 100 μl of fermentation broth by a proper multiple, detecting OD at 600nm wavelength with spectrophotometer, performing three parallels for each strain, calculating average value, and detecting OD 600 As shown in table 2.
5. Lysine concentration measurement: 2mL of the fermentation broth was centrifuged (12000 rpm,2 min), the supernatant was collected, the content of L-lysine in the fermentation broth of the recombinant bacteria and the control bacteria was measured by HPLC, three bacteria were used in parallel, and the average value was calculated, and the measured lysine concentration was shown in Table 2.
6. Enzyme activity determination: aconitate hydratase activity assay was carried out using a aconitate enzyme assay kit from Biovision company (product number K716-100). The sample preparation method is as follows: the cells were collected by centrifugation at 12000rpm at 4℃for 10min, and the cells were dissolved in a pre-chilled buffer, sonicated for 20s, and the supernatant was collected for enzyme activity assay. Three strains were prepared in parallel, the average value was calculated, and the enzyme activities of aconitate hydratase detected are shown in Table 2.
TABLE 2 lysine production, growth and enzyme Activity detection of recombinant strains
Strain L-lysine (g/L) Sugar acid conversion% OD 600 Enzyme activity U/mg
CGMCC No.22648 18.9 28.6 15.1 2.49
CGMCC No.22648-acnA S522H 21.5 32.9 14.8 2.18
CGMCC No.22648-acnA S522E 22.0 33.8 15.0 2.15
CGMCC No.22648-acnA S522I 21.4 32.8 14.9 2.19
CGMCC No.22648-acnA S522W 22.6 34.8 14.4 1.98
CGMCC No.22648-acnA S522G 20.5 31.1 14.9 2.32
Fermentation results show that 522 th ammonia of acnA gene coding protein in original strainAfter the mutation of the amino acid from serine (S) to histidine (H), glutamic acid (E), isoleucine (I), tryptophan (W) and glycine (G), the activity of aconitate hydratase of the obtained recombinant strain was reduced to some extent as compared with that of the enzyme of the starting strain (same amino acid sequence as AcnA of wild-type E.coli MG 1655), and the activity was significantly different (P < 0.05). The final OD of the recombinant strain is equivalent to that of the original strain, but the lysine yield and the conversion rate are both obviously improved (P is less than 0.05), wherein the effect of mutation of 522 th amino acid into histidine (H), glutamic acid (E), isoleucine (I) and tryptophan (W) is obviously better than that of mutation into glycine (G), the effect of mutation of 522 th amino acid into tryptophan (W) is optimal, and the recombinant strain CGMCC No.22648-acnA S522W The L-lysine yield of the strain is improved by 3.7g/L compared with the original strain, and the conversion rate is improved by 6.2 percent compared with the original strain.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
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Asp Pro Ile Glu Thr Ala Ile Lys Lys His Asp Leu Thr Val Gly Ala
515 520 525
Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val
530 535 540
Lys Thr Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu
545 550 555 560
Ala Gly Asn Met Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp
565 570 575
Arg Lys Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln
580 585 590
Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605
Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly Ile Asn
610 615 620
Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp Ser Thr Tyr Ile
625 630 635 640
Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Thr Pro Ala Pro Val
645 650 655
Glu Asp Ile His Gly Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val
660 665 670
Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro
675 680 685
Ala Gly Arg Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn
690 695 700
Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr
705 710 715 720
Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu Gly
725 730 735
Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser Ile Tyr Asp
740 745 750
Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro Leu Ala Val Ile Ala
755 760 765
Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly
770 775 780
Pro Arg Leu Leu Gly Ile Arg Val Val Ile Ala Glu Ser Phe Glu Arg
785 790 795 800
Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815
Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys
820 825 830
Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845
Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro Cys Arg
850 855 860
Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp Gly
865 870 875 880
Ile Leu His Tyr Val Ile Arg Asn Met Leu Lys
885 890
<210> 2
<211> 891
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Ser Ser Thr Leu Arg Glu Ala Ser Lys Asp Thr Leu Gln Ala Lys
1 5 10 15
Asp Lys Thr Tyr His Tyr Tyr Ser Leu Pro Leu Ala Ala Lys Ser Leu
20 25 30
Gly Asp Ile Thr Arg Leu Pro Lys Ser Leu Lys Val Leu Leu Glu Asn
35 40 45
Leu Leu Arg Trp Gln Asp Gly Asn Ser Val Thr Glu Glu Asp Ile His
50 55 60
Ala Leu Ala Gly Trp Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala
65 70 75 80
Tyr Arg Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala
85 90 95
Val Val Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly
100 105 110
Asp Thr Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125
His Ser Val Thr Val Asp Arg Phe Gly Asp Asp Glu Ala Phe Glu Glu
130 135 140
Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg Tyr Val Phe Leu
145 150 155 160
Lys Trp Gly Lys Gln Ala Phe Ser Arg Phe Ser Val Val Pro Pro Gly
165 170 175
Thr Gly Ile Cys His Gln Val Asn Leu Glu Tyr Leu Gly Lys Ala Val
180 185 190
Trp Ser Glu Leu Gln Asp Gly Glu Trp Ile Ala Tyr Pro Asp Thr Leu
195 200 205
Val Gly Thr Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu
210 215 220
Gly Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln
225 230 235 240
Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr Gly
245 250 255
Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu Thr Val Thr
260 265 270
Gln Met Leu Arg Lys His Gly Val Val Gly Lys Phe Val Glu Phe Tyr
275 280 285
Gly Asp Gly Leu Asp Ser Leu Pro Leu Ala Asp Arg Ala Thr Ile Ala
290 295 300
Asn Met Ser Pro Glu Tyr Gly Ala Thr Cys Gly Phe Phe Pro Ile Asp
305 310 315 320
Ala Val Thr Leu Asp Tyr Met Arg Leu Ser Gly Arg Ser Glu Asp Gln
325 330 335
Val Glu Leu Val Glu Lys Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn
340 345 350
Pro Gly Asp Glu Pro Ile Phe Thr Ser Thr Leu Glu Leu Asp Met Asn
355 360 365
Asp Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp Arg Val
370 375 380
Ala Leu Pro Asp Val Pro Lys Ala Phe Ala Ala Ser Asn Glu Leu Glu
385 390 395 400
Val Asn Ala Thr His Lys Asp Arg Gln Pro Val Asp Tyr Val Met Asn
405 410 415
Gly His Gln Tyr Gln Leu Pro Asp Gly Ala Val Val Ile Ala Ala Ile
420 425 430
Thr Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Ala Ala Gly
435 440 445
Leu Leu Ala Lys Lys Ala Val Thr Leu Gly Leu Lys Arg Gln Pro Trp
450 455 460
Val Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu
465 470 475 480
Ala Lys Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn Leu
485 490 495
Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro
500 505 510
Asp Pro Ile Glu Thr Ala Ile Lys Lys Glu Asp Leu Thr Val Gly Ala
515 520 525
Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val
530 535 540
Lys Thr Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu
545 550 555 560
Ala Gly Asn Met Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp
565 570 575
Arg Lys Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln
580 585 590
Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605
Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly Ile Asn
610 615 620
Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp Ser Thr Tyr Ile
625 630 635 640
Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Thr Pro Ala Pro Val
645 650 655
Glu Asp Ile His Gly Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val
660 665 670
Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro
675 680 685
Ala Gly Arg Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn
690 695 700
Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr
705 710 715 720
Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu Gly
725 730 735
Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser Ile Tyr Asp
740 745 750
Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro Leu Ala Val Ile Ala
755 760 765
Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly
770 775 780
Pro Arg Leu Leu Gly Ile Arg Val Val Ile Ala Glu Ser Phe Glu Arg
785 790 795 800
Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815
Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys
820 825 830
Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845
Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro Cys Arg
850 855 860
Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp Gly
865 870 875 880
Ile Leu His Tyr Val Ile Arg Asn Met Leu Lys
885 890
<210> 3
<211> 891
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Met Ser Ser Thr Leu Arg Glu Ala Ser Lys Asp Thr Leu Gln Ala Lys
1 5 10 15
Asp Lys Thr Tyr His Tyr Tyr Ser Leu Pro Leu Ala Ala Lys Ser Leu
20 25 30
Gly Asp Ile Thr Arg Leu Pro Lys Ser Leu Lys Val Leu Leu Glu Asn
35 40 45
Leu Leu Arg Trp Gln Asp Gly Asn Ser Val Thr Glu Glu Asp Ile His
50 55 60
Ala Leu Ala Gly Trp Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala
65 70 75 80
Tyr Arg Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala
85 90 95
Val Val Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly
100 105 110
Asp Thr Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125
His Ser Val Thr Val Asp Arg Phe Gly Asp Asp Glu Ala Phe Glu Glu
130 135 140
Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg Tyr Val Phe Leu
145 150 155 160
Lys Trp Gly Lys Gln Ala Phe Ser Arg Phe Ser Val Val Pro Pro Gly
165 170 175
Thr Gly Ile Cys His Gln Val Asn Leu Glu Tyr Leu Gly Lys Ala Val
180 185 190
Trp Ser Glu Leu Gln Asp Gly Glu Trp Ile Ala Tyr Pro Asp Thr Leu
195 200 205
Val Gly Thr Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu
210 215 220
Gly Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln
225 230 235 240
Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr Gly
245 250 255
Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu Thr Val Thr
260 265 270
Gln Met Leu Arg Lys His Gly Val Val Gly Lys Phe Val Glu Phe Tyr
275 280 285
Gly Asp Gly Leu Asp Ser Leu Pro Leu Ala Asp Arg Ala Thr Ile Ala
290 295 300
Asn Met Ser Pro Glu Tyr Gly Ala Thr Cys Gly Phe Phe Pro Ile Asp
305 310 315 320
Ala Val Thr Leu Asp Tyr Met Arg Leu Ser Gly Arg Ser Glu Asp Gln
325 330 335
Val Glu Leu Val Glu Lys Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn
340 345 350
Pro Gly Asp Glu Pro Ile Phe Thr Ser Thr Leu Glu Leu Asp Met Asn
355 360 365
Asp Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp Arg Val
370 375 380
Ala Leu Pro Asp Val Pro Lys Ala Phe Ala Ala Ser Asn Glu Leu Glu
385 390 395 400
Val Asn Ala Thr His Lys Asp Arg Gln Pro Val Asp Tyr Val Met Asn
405 410 415
Gly His Gln Tyr Gln Leu Pro Asp Gly Ala Val Val Ile Ala Ala Ile
420 425 430
Thr Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Ala Ala Gly
435 440 445
Leu Leu Ala Lys Lys Ala Val Thr Leu Gly Leu Lys Arg Gln Pro Trp
450 455 460
Val Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu
465 470 475 480
Ala Lys Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn Leu
485 490 495
Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro
500 505 510
Asp Pro Ile Glu Thr Ala Ile Lys Lys Ile Asp Leu Thr Val Gly Ala
515 520 525
Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val
530 535 540
Lys Thr Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu
545 550 555 560
Ala Gly Asn Met Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp
565 570 575
Arg Lys Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln
580 585 590
Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605
Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly Ile Asn
610 615 620
Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp Ser Thr Tyr Ile
625 630 635 640
Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Thr Pro Ala Pro Val
645 650 655
Glu Asp Ile His Gly Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val
660 665 670
Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro
675 680 685
Ala Gly Arg Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn
690 695 700
Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr
705 710 715 720
Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu Gly
725 730 735
Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser Ile Tyr Asp
740 745 750
Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro Leu Ala Val Ile Ala
755 760 765
Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly
770 775 780
Pro Arg Leu Leu Gly Ile Arg Val Val Ile Ala Glu Ser Phe Glu Arg
785 790 795 800
Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815
Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys
820 825 830
Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845
Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro Cys Arg
850 855 860
Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp Gly
865 870 875 880
Ile Leu His Tyr Val Ile Arg Asn Met Leu Lys
885 890
<210> 4
<211> 891
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Met Ser Ser Thr Leu Arg Glu Ala Ser Lys Asp Thr Leu Gln Ala Lys
1 5 10 15
Asp Lys Thr Tyr His Tyr Tyr Ser Leu Pro Leu Ala Ala Lys Ser Leu
20 25 30
Gly Asp Ile Thr Arg Leu Pro Lys Ser Leu Lys Val Leu Leu Glu Asn
35 40 45
Leu Leu Arg Trp Gln Asp Gly Asn Ser Val Thr Glu Glu Asp Ile His
50 55 60
Ala Leu Ala Gly Trp Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala
65 70 75 80
Tyr Arg Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala
85 90 95
Val Val Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly
100 105 110
Asp Thr Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125
His Ser Val Thr Val Asp Arg Phe Gly Asp Asp Glu Ala Phe Glu Glu
130 135 140
Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg Tyr Val Phe Leu
145 150 155 160
Lys Trp Gly Lys Gln Ala Phe Ser Arg Phe Ser Val Val Pro Pro Gly
165 170 175
Thr Gly Ile Cys His Gln Val Asn Leu Glu Tyr Leu Gly Lys Ala Val
180 185 190
Trp Ser Glu Leu Gln Asp Gly Glu Trp Ile Ala Tyr Pro Asp Thr Leu
195 200 205
Val Gly Thr Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu
210 215 220
Gly Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln
225 230 235 240
Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr Gly
245 250 255
Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu Thr Val Thr
260 265 270
Gln Met Leu Arg Lys His Gly Val Val Gly Lys Phe Val Glu Phe Tyr
275 280 285
Gly Asp Gly Leu Asp Ser Leu Pro Leu Ala Asp Arg Ala Thr Ile Ala
290 295 300
Asn Met Ser Pro Glu Tyr Gly Ala Thr Cys Gly Phe Phe Pro Ile Asp
305 310 315 320
Ala Val Thr Leu Asp Tyr Met Arg Leu Ser Gly Arg Ser Glu Asp Gln
325 330 335
Val Glu Leu Val Glu Lys Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn
340 345 350
Pro Gly Asp Glu Pro Ile Phe Thr Ser Thr Leu Glu Leu Asp Met Asn
355 360 365
Asp Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp Arg Val
370 375 380
Ala Leu Pro Asp Val Pro Lys Ala Phe Ala Ala Ser Asn Glu Leu Glu
385 390 395 400
Val Asn Ala Thr His Lys Asp Arg Gln Pro Val Asp Tyr Val Met Asn
405 410 415
Gly His Gln Tyr Gln Leu Pro Asp Gly Ala Val Val Ile Ala Ala Ile
420 425 430
Thr Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Ala Ala Gly
435 440 445
Leu Leu Ala Lys Lys Ala Val Thr Leu Gly Leu Lys Arg Gln Pro Trp
450 455 460
Val Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu
465 470 475 480
Ala Lys Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn Leu
485 490 495
Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro
500 505 510
Asp Pro Ile Glu Thr Ala Ile Lys Lys Trp Asp Leu Thr Val Gly Ala
515 520 525
Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val
530 535 540
Lys Thr Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu
545 550 555 560
Ala Gly Asn Met Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp
565 570 575
Arg Lys Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln
580 585 590
Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605
Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly Ile Asn
610 615 620
Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp Ser Thr Tyr Ile
625 630 635 640
Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Thr Pro Ala Pro Val
645 650 655
Glu Asp Ile His Gly Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val
660 665 670
Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro
675 680 685
Ala Gly Arg Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn
690 695 700
Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr
705 710 715 720
Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu Gly
725 730 735
Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser Ile Tyr Asp
740 745 750
Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro Leu Ala Val Ile Ala
755 760 765
Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly
770 775 780
Pro Arg Leu Leu Gly Ile Arg Val Val Ile Ala Glu Ser Phe Glu Arg
785 790 795 800
Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815
Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys
820 825 830
Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845
Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro Cys Arg
850 855 860
Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp Gly
865 870 875 880
Ile Leu His Tyr Val Ile Arg Asn Met Leu Lys
885 890
<210> 5
<211> 891
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Met Ser Ser Thr Leu Arg Glu Ala Ser Lys Asp Thr Leu Gln Ala Lys
1 5 10 15
Asp Lys Thr Tyr His Tyr Tyr Ser Leu Pro Leu Ala Ala Lys Ser Leu
20 25 30
Gly Asp Ile Thr Arg Leu Pro Lys Ser Leu Lys Val Leu Leu Glu Asn
35 40 45
Leu Leu Arg Trp Gln Asp Gly Asn Ser Val Thr Glu Glu Asp Ile His
50 55 60
Ala Leu Ala Gly Trp Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala
65 70 75 80
Tyr Arg Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala
85 90 95
Val Val Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly
100 105 110
Asp Thr Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125
His Ser Val Thr Val Asp Arg Phe Gly Asp Asp Glu Ala Phe Glu Glu
130 135 140
Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg Tyr Val Phe Leu
145 150 155 160
Lys Trp Gly Lys Gln Ala Phe Ser Arg Phe Ser Val Val Pro Pro Gly
165 170 175
Thr Gly Ile Cys His Gln Val Asn Leu Glu Tyr Leu Gly Lys Ala Val
180 185 190
Trp Ser Glu Leu Gln Asp Gly Glu Trp Ile Ala Tyr Pro Asp Thr Leu
195 200 205
Val Gly Thr Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu
210 215 220
Gly Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln
225 230 235 240
Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr Gly
245 250 255
Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu Thr Val Thr
260 265 270
Gln Met Leu Arg Lys His Gly Val Val Gly Lys Phe Val Glu Phe Tyr
275 280 285
Gly Asp Gly Leu Asp Ser Leu Pro Leu Ala Asp Arg Ala Thr Ile Ala
290 295 300
Asn Met Ser Pro Glu Tyr Gly Ala Thr Cys Gly Phe Phe Pro Ile Asp
305 310 315 320
Ala Val Thr Leu Asp Tyr Met Arg Leu Ser Gly Arg Ser Glu Asp Gln
325 330 335
Val Glu Leu Val Glu Lys Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn
340 345 350
Pro Gly Asp Glu Pro Ile Phe Thr Ser Thr Leu Glu Leu Asp Met Asn
355 360 365
Asp Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp Arg Val
370 375 380
Ala Leu Pro Asp Val Pro Lys Ala Phe Ala Ala Ser Asn Glu Leu Glu
385 390 395 400
Val Asn Ala Thr His Lys Asp Arg Gln Pro Val Asp Tyr Val Met Asn
405 410 415
Gly His Gln Tyr Gln Leu Pro Asp Gly Ala Val Val Ile Ala Ala Ile
420 425 430
Thr Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Ala Ala Gly
435 440 445
Leu Leu Ala Lys Lys Ala Val Thr Leu Gly Leu Lys Arg Gln Pro Trp
450 455 460
Val Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu
465 470 475 480
Ala Lys Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn Leu
485 490 495
Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro
500 505 510
Asp Pro Ile Glu Thr Ala Ile Lys Lys Gly Asp Leu Thr Val Gly Ala
515 520 525
Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val
530 535 540
Lys Thr Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu
545 550 555 560
Ala Gly Asn Met Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp
565 570 575
Arg Lys Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln
580 585 590
Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605
Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly Ile Asn
610 615 620
Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp Ser Thr Tyr Ile
625 630 635 640
Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Thr Pro Ala Pro Val
645 650 655
Glu Asp Ile His Gly Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val
660 665 670
Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro
675 680 685
Ala Gly Arg Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn
690 695 700
Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr
705 710 715 720
Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu Gly
725 730 735
Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser Ile Tyr Asp
740 745 750
Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro Leu Ala Val Ile Ala
755 760 765
Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly
770 775 780
Pro Arg Leu Leu Gly Ile Arg Val Val Ile Ala Glu Ser Phe Glu Arg
785 790 795 800
Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815
Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys
820 825 830
Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845
Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro Cys Arg
850 855 860
Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp Gly
865 870 875 880
Ile Leu His Tyr Val Ile Arg Asn Met Leu Lys
885 890
<210> 6
<211> 891
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Met Ser Ser Thr Leu Arg Glu Ala Ser Lys Asp Thr Leu Gln Ala Lys
1 5 10 15
Asp Lys Thr Tyr His Tyr Tyr Ser Leu Pro Leu Ala Ala Lys Ser Leu
20 25 30
Gly Asp Ile Thr Arg Leu Pro Lys Ser Leu Lys Val Leu Leu Glu Asn
35 40 45
Leu Leu Arg Trp Gln Asp Gly Asn Ser Val Thr Glu Glu Asp Ile His
50 55 60
Ala Leu Ala Gly Trp Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala
65 70 75 80
Tyr Arg Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala
85 90 95
Val Val Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly
100 105 110
Asp Thr Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125
His Ser Val Thr Val Asp Arg Phe Gly Asp Asp Glu Ala Phe Glu Glu
130 135 140
Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg Tyr Val Phe Leu
145 150 155 160
Lys Trp Gly Lys Gln Ala Phe Ser Arg Phe Ser Val Val Pro Pro Gly
165 170 175
Thr Gly Ile Cys His Gln Val Asn Leu Glu Tyr Leu Gly Lys Ala Val
180 185 190
Trp Ser Glu Leu Gln Asp Gly Glu Trp Ile Ala Tyr Pro Asp Thr Leu
195 200 205
Val Gly Thr Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu
210 215 220
Gly Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln
225 230 235 240
Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr Gly
245 250 255
Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu Thr Val Thr
260 265 270
Gln Met Leu Arg Lys His Gly Val Val Gly Lys Phe Val Glu Phe Tyr
275 280 285
Gly Asp Gly Leu Asp Ser Leu Pro Leu Ala Asp Arg Ala Thr Ile Ala
290 295 300
Asn Met Ser Pro Glu Tyr Gly Ala Thr Cys Gly Phe Phe Pro Ile Asp
305 310 315 320
Ala Val Thr Leu Asp Tyr Met Arg Leu Ser Gly Arg Ser Glu Asp Gln
325 330 335
Val Glu Leu Val Glu Lys Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn
340 345 350
Pro Gly Asp Glu Pro Ile Phe Thr Ser Thr Leu Glu Leu Asp Met Asn
355 360 365
Asp Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp Arg Val
370 375 380
Ala Leu Pro Asp Val Pro Lys Ala Phe Ala Ala Ser Asn Glu Leu Glu
385 390 395 400
Val Asn Ala Thr His Lys Asp Arg Gln Pro Val Asp Tyr Val Met Asn
405 410 415
Gly His Gln Tyr Gln Leu Pro Asp Gly Ala Val Val Ile Ala Ala Ile
420 425 430
Thr Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Ala Ala Gly
435 440 445
Leu Leu Ala Lys Lys Ala Val Thr Leu Gly Leu Lys Arg Gln Pro Trp
450 455 460
Val Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu
465 470 475 480
Ala Lys Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn Leu
485 490 495
Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro
500 505 510
Asp Pro Ile Glu Thr Ala Ile Lys Lys Ser Asp Leu Thr Val Gly Ala
515 520 525
Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val
530 535 540
Lys Thr Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu
545 550 555 560
Ala Gly Asn Met Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp
565 570 575
Arg Lys Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln
580 585 590
Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605
Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly Ile Asn
610 615 620
Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp Ser Thr Tyr Ile
625 630 635 640
Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Thr Pro Ala Pro Val
645 650 655
Glu Asp Ile His Gly Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val
660 665 670
Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro
675 680 685
Ala Gly Arg Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn
690 695 700
Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr
705 710 715 720
Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu Gly
725 730 735
Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser Ile Tyr Asp
740 745 750
Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro Leu Ala Val Ile Ala
755 760 765
Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly
770 775 780
Pro Arg Leu Leu Gly Ile Arg Val Val Ile Ala Glu Ser Phe Glu Arg
785 790 795 800
Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815
Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys
820 825 830
Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845
Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro Cys Arg
850 855 860
Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp Gly
865 870 875 880
Ile Leu His Tyr Val Ile Arg Asn Met Leu Lys
885 890
<210> 7
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
tcctaggtat aatactagtt gggcctcaag cggcaaccag ttttagagct agaaatagc 59
<210> 8
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
actagtatta tacctaggac tgagctagct gtcaag 36
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ggacatcagt atcagttacc 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atgttttttg attgccgttt 20
<210> 11
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
aaacggcaat caaaaaacat gatttaaccg tcggtgcggt 40
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
ctcttttttg attgccgttt 20
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
aaacggcaat caaaaaagag gatttaaccg tcggtgcggt 40
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gccttttttg attgccgttt 20
<210> 15
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
aaacggcaat caaaaaaggc gatttaaccg tcggtgcggt 40
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gatttttttg attgccgttt 20
<210> 17
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
aaacggcaat caaaaaaatc gatttaaccg tcggtgcggt 40
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ccattttttg attgccgttt 20
<210> 19
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
aaacggcaat caaaaaatgg gatttaaccg tcggtgcggt 40
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
accgtaggta tcggatcgtg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
aaacggcaat caaaaaacat 20
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
aaacggcaat caaaaaagag 20
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
aaacggcaat caaaaaaggc 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
aaacggcaat caaaaaaatc 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
aaacggcaat caaaaaatgg 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggatcgcgtt gcactgcccg 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
atcccccagc attgcgagga 20

Claims (10)

1. The aconitate hydratase mutant is characterized by having an amino acid sequence shown in any one of SEQ ID NO. 1-5.
2. A nucleic acid molecule encoding the aconitate hydratase mutant of claim 1.
3. A biological material comprising the nucleic acid molecule of claim 2, wherein the biological material is an expression cassette, a vector or a host cell.
4. A recombinant microorganism expressing the aconitate hydratase mutant of claim 1.
5. The recombinant microorganism according to claim 4, wherein the recombinant microorganism does not express aconitate hydratase possessed by its starting strain.
6. The recombinant microorganism according to claim 5, wherein the gene encoding aconitate hydratase is replaced with the nucleic acid molecule according to claim 2.
7. Recombinant microorganism according to any of claims 4 to 6, characterized in that it is a bacterium of the genus Escherichia, preferably Escherichia coli (Escherichia coli).
8. The method for constructing a recombinant microorganism according to any one of claims 4 to 7, comprising: replacing a gene encoding aconitate hydratase in an original strain of the recombinant microorganism with a gene encoding the aconitate hydratase mutant of claim 1.
9. Use of the aconitate hydratase mutant of claim 1 or the nucleic acid molecule of claim 2 or the biomaterial of claim 3 or the recombinant microorganism of any of claims 4-7, as follows:
(1) Use in the construction of a production strain for the production of aspartic acid or a metabolite of aspartic acid as a synthetic precursor;
(2) Use in the fermentative production of aspartic acid or metabolites with aspartic acid as synthesis precursor;
(3) Use in increasing the yield and/or conversion of aspartic acid or a metabolite having aspartic acid as a synthetic precursor;
preferably, the metabolite with aspartic acid as a synthetic precursor is lysine.
10. A method for fermenting aspartic acid or a metabolite having aspartic acid as a synthetic precursor, comprising: culturing the recombinant microorganism according to any one of claims 4 to 7 to obtain a culture, and separating and extracting the culture to obtain aspartic acid or a metabolite taking aspartic acid as a synthesis precursor;
preferably, the metabolite with aspartic acid as a synthetic precursor is lysine.
CN202210141296.3A 2022-02-16 2022-02-16 Aconitate hydratase mutant and application thereof Pending CN116640752A (en)

Priority Applications (1)

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CN202210141296.3A CN116640752A (en) 2022-02-16 2022-02-16 Aconitate hydratase mutant and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210141296.3A CN116640752A (en) 2022-02-16 2022-02-16 Aconitate hydratase mutant and application thereof

Publications (1)

Publication Number Publication Date
CN116640752A true CN116640752A (en) 2023-08-25

Family

ID=87621697

Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
CN (1) CN116640752A (en)

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