Mutant succinate dehydrogenase, recombinant microorganism, and method of making and use thereof

Abstract

The present application relates to the field of microbial technology, in particular to succinate dehydrogenase mutant, recombinant microorganism and its preparation method and application. The present application finds that the yield and conversion rate of products such as lysine taking aspartic acid as a synthetic precursor can be significantly improved by reducing the expression and enzyme activity of succinate dehydrogenase. The succinate dehydrogenase mutant provided by the present application can weaken succinate dehydrogenase, promote the accumulation of products such as lysine taking aspartic acid as a synthetic precursor, and the yield and conversion rate of lysine of the recombinant microorganism expressing the mutant are significantly improved compared with the original strain. The mutant and the recombinant microorganism provide an advantageous modification target, mutant and strain resource for the breeding of aspartic acid, its derivatives and aspartic acid family amino acid production strains.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to succinate dehydrogenase mutants, recombinant microorganisms, their preparation methods, and applications. Background Technology

[0002] Aspartic acid and aspartic acid family amino acids have wide applications in feed, food, medicine, and chemical industries. Among them, L-lysine is a basic essential amino acid with the molecular formula C6H2O. 14 N₂O₂ is a white or nearly white crystalline powder. It darkens at 210℃ and decomposes at 224.5℃. It is readily soluble in water, slightly soluble in alcohol, and insoluble in ether. L-Lysine is widely used in animal feed, pharmaceuticals, and the food industry, with approximately 90% of L-lysine used in the feed industry and 10% in the food and pharmaceutical industries. As an animal feed additive, L-lysine promotes the absorption of other amino acids by animals, thereby improving feed quality. Therefore, improving the production efficiency of L-lysine is of great significance.

[0003] Currently, the most common method for producing L-lysine is microbial fermentation, which offers advantages such as low raw material costs, mild reaction conditions, and ease of large-scale production. Escherichia coli, due to its rapid growth rate, clear genetic background, simple culture conditions, and mature metabolic engineering techniques, is widely used in industrial fermentation and can be used to produce L-amino acids, nucleotides, and other organic acids.

[0004] Succinate dehydrogenase (SDH) is an enzyme in the tricarboxylic acid cycle that reversibly catalyzes the interconversion of succinate and fumarate. Depending on the microbial species, SDH is composed of 3 or 4 subunits; modification of any one subunit can alter its function. SDHA encodes a flavoprotein subunit, which participates in the synthesis of fumarate from succinate. Chinese patent CN101563453B discloses that weakening the SDHA gene weakens SDH, resulting in high production of glutamate and its family of amino acids by *U. bromelain*. Chinese patent CN106191155B discloses that weakening the expression of the SDHA gene significantly increases the production of polyketide compounds in *E. coli*.

[0005] Currently, there are no reports on whether the weakening of the flavoprotein subunit encoding gene sdhA affects the accumulation of aspartic amino acids such as L-lysine. Summary of the Invention

[0006] The primary objective of this invention is to provide the application of succinate dehydrogenase in increasing the yield and conversion rate of aspartic acid or aspartic acid family amino acids.

[0007] A second objective of this invention is to provide a succinate dehydrogenase mutant and its applications.

[0008] A third objective of this invention is to provide a recombinant microorganism.

[0009] A fourth objective of this invention is to provide a method for producing aspartic acid, its derivatives, or aspartic acid family amino acids.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides any of the following applications of succinate dehydrogenase or its inhibitor, succinate dehydrogenase encoding gene or its inhibitor, and biological materials containing succinate dehydrogenase encoding gene or said inhibitor:

[0012] (1) Application in increasing the yield and / or conversion rate of aspartic acid, its derivatives or aspartic amino acids in microorganisms.

[0013] (2) Application in the construction of microorganisms for the production of aspartic acid, its derivatives or aspartic amino acids;

[0014] (3) Application in the fermentation production of aspartic acid, its derivatives or aspartic acid family amino acids.

[0015] This invention has found that reducing the expression and activity of succinate dehydrogenase in microorganisms can significantly increase the yield and conversion rate of aspartic acid, its derivatives, and aspartic group amino acids.

[0016] In this invention, the aspartic acid derivative is a substance synthesized by modifying aspartic acid or using aspartic acid as a precursor. The aspartic acid group amino acids include lysine, threonine, methionine, homoserine, and isoleucine.

[0017] The succinate dehydrogenase described in this invention may be one or more of the subunits of succinate dehydrogenase.

[0018] Specifically, the subunits of succinate dehydrogenase include SdhA, SdhB, SdhC, and SdhD.

[0019] Preferably, the succinate dehydrogenase is the flavoprotein subunit of succinate dehydrogenase (SdhA), and its encoding gene is sdhA.

[0020] In this invention, the microorganism is preferably Enterobacteriaceae, more preferably Escherichiae, and most preferably Escherichia coli.

[0021] For wild-type Escherichia coli, the amino acid sequence of its flavoprotein subunit is shown in SEQ ID NO.6.

[0022] In this invention, the inhibitor is a protein, DNA, RNA, compound, or composition capable of inhibiting the expression and / or enzyme activity of succinate dehydrogenase.

[0023] In this invention, the biological materials include, but are not limited to, recombinant DNA, vectors, and host cells (including microbial cells).

[0024] Specifically, the above-described application is achieved by reducing the expression and / or activity of the succinate dehydrogenase.

[0025] Secondly, the present invention provides a succinate dehydrogenase mutant, using the amino acid sequence of wild-type succinate dehydrogenase from Escherichia coli as a reference sequence, wherein the succinate dehydrogenase mutant contains a mutation in which alanine at position 530 is replaced by an amino acid other than alanine.

[0026] Through years of research, the inventors discovered that mutating amino acids at specific positions in the flavoprotein subunit of succinate dehydrogenase can weaken succinate dehydrogenase, thereby increasing the production of aspartic acid amino acids such as lysine.

[0027] Preferably, the amino acid sequence of wild-type Escherichia coli succinate dehydrogenase is used as a reference sequence, wherein the succinate dehydrogenase mutant contains a mutation in which alanine at position 530 is replaced by glycine, isoleucine, histidine, glutamic acid or threonine.

[0028] As a preferred embodiment of the present invention, the present invention provides a succinate dehydrogenase mutant having an amino acid sequence as shown in any of SEQ ID NO. 1-5.

[0029] Those skilled in the art should understand that adding a tagged protein to the N-terminus or C-terminus of the above-mentioned protein mutant sequence or fusing it with other proteins to form a fusion protein, without changing the activity of the above-mentioned mutant protein itself, the tagged protein or fusion protein is also within the scope of protection of this invention.

[0030] Thirdly, the present invention provides a nucleic acid molecule encoding the succinate dehydrogenase mutant.

[0031] Based on the amino acid sequence of the protein mutant provided above, those skilled in the art can obtain the sequence of the nucleic acid it encodes. Due to the degeneracy of codons, there is more than one nucleic acid sequence encoding the above amino acid sequence, and all nucleic acids capable of encoding the above protein mutant are within the protection scope of this invention.

[0032] As one embodiment of the present invention, the nucleic acid molecule encoding the succinate dehydrogenase mutant has a nucleotide sequence as shown in any of SEQ ID NO. 8-12.

[0033] Fourthly, the present invention provides biological materials containing the nucleic acid molecules, wherein the biological material is recombinant DNA, a vector, or a host cell.

[0034] The recombinant DNA includes, but is not limited to, expression cassettes. The expression cassette is a recombinant nucleic acid molecule obtained by linking elements upstream or downstream of the nucleic acid to drive its transcription and expression.

[0035] The vector may be an expression vector or a cloning vector, including but not limited to plasmid vectors, phage vectors, transposons, etc.

[0036] Fifthly, the present invention provides a recombinant microorganism, wherein the expression and / or enzyme activity of succinate dehydrogenase are reduced compared with the starting strain.

[0037] Preferably, the reduction in expression and / or enzyme activity is achieved through a combination of one or more of the following methods (1) and (2):

[0038] (1) Insert, delete or replace one or more bases in the gene encoding succinate dehydrogenase to reduce the expression and / or activity of succinate dehydrogenase.

[0039] (2) Replace the transcriptional or translational regulatory element of the gene encoding succinate dehydrogenase with a less active regulatory element to reduce the expression and / or activity of succinate dehydrogenase.

[0040] The transcriptional regulatory elements mentioned in (2) above include promoters, enhancers, 5'-UTRs, terminators, etc. Translational regulatory elements include ribosome binding sites, etc.

[0041] Preferably, the recombinant microorganism expresses the succinate dehydrogenase mutant and does not express the succinate dehydrogenase present in its originating strain; or, the start codon of the encoding gene for succinate dehydrogenase in the recombinant microorganism is replaced with a start codon having weaker activity.

[0042] In one embodiment of the present invention, the succinate dehydrogenase encoding gene in the starting strain is mutated to the encoding gene of the succinate dehydrogenase mutant.

[0043] In another embodiment of the present invention, the start codon of the succinate dehydrogenase encoding gene in the starting strain is replaced with GTG instead of ATG.

[0044] The recombinant microorganisms described in this invention are preferably Enterobacteriaceae, more preferably Escherichiae, and most preferably Escherichia coli.

[0045] In this invention, the starting strain is a strain capable of accumulating aspartic acid, its derivatives, or aspartic acid family amino acids.

[0046] Preferably, the starting strain is an Escherichia coli capable of accumulating lysine.

[0047] As one embodiment of the present invention, the starting strain is a lysine-producing Escherichia coli MHZ-0914 obtained by mutagenesis. This strain was deposited on June 1, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 22648, and classified as Escherichia coli.

[0048] Sixthly, the present invention provides any of the following applications of the succinate dehydrogenase mutant, the nucleic acid molecule, the biological material, or the recombinant microorganism:

[0049] (1) Application in increasing the yield and / or conversion rate of aspartic acid, its derivatives or aspartic amino acids in microorganisms.

[0050] (2) Application in the construction of microorganisms for the production of aspartic acid, its derivatives or aspartic amino acids;

[0051] (3) Application in the fermentation production of aspartic acid, its derivatives or aspartic acid family amino acids.

[0052] In a seventh aspect, the present invention also provides a method for constructing the recombinant microorganism described above, comprising: inserting, deleting or replacing one or more bases in the coding gene for succinate dehydrogenase in the starting strain to reduce the expression and / or enzyme activity of succinate dehydrogenase.

[0053] Alternatively, the transcriptional or translational regulatory element of the gene encoding the succinate dehydrogenase may be replaced with a less active regulatory element to reduce its expression and / or enzyme activity.

[0054] Preferably, the preparation method includes: mutating the gene encoding succinate dehydrogenase in the starting strain to the gene of the succinate dehydrogenase mutant described above, or, it includes: replacing the start codon of the gene encoding succinate dehydrogenase in the recombinant microorganism with a start codon that has weaker activity.

[0055] Eighthly, the present invention provides a method for producing aspartic acid, its derivatives or aspartic group amino acids, comprising: culturing the recombinant microorganisms described above.

[0056] Specifically, the method for producing aspartic acid, its derivatives, or aspartic acid family amino acids includes: inoculating the recombinant microorganisms into an activation culture medium for cultivation, selecting activated strains and inoculating them into a seed culture medium for seed culture, and then transferring the seed culture into a fermentation culture medium for fermentation.

[0057] Preferably, the fermentation medium comprises the following components: glucose 55-65 g / L, molasses 5-15 g / L, ammonium sulfate 35-45 g / L, corn steep liquor 8-12 g / L, potassium dihydrogen phosphate 1.5-2 g / L, magnesium sulfate heptahydrate 0.5-1.5 g / L, ferric sulfate 0.01-0.05 g / L, manganese sulfate 0.02-0.05 g / L, calcium carbonate 10-30 g / L, and pH 6.8-7.2.

[0058] Preferably, the seed culture medium comprises the following components: glucose 15-25 g / L, ammonium sulfate 3-5 g / L, corn steep liquor 1-3 g / L, potassium dihydrogen phosphate 2-4 g / L, magnesium sulfate heptahydrate 0.2-0.6 g / L, ferric sulfate 0.005-0.02 g / L, manganese sulfate 0.005-0.02 g / L, pH 6.8-7.2.

[0059] The aspartic acid group amino acid described in this invention is preferably lysine.

[0060] The beneficial effects of this invention are as follows: This invention discovers that by reducing the expression and activity of succinate dehydrogenase, the yield and conversion rate of aspartic amino acids such as lysine can be significantly improved. The succinate dehydrogenase mutant provided by this invention can weaken succinate dehydrogenase, promote the synthesis of aspartic acid, and thus promote the accumulation of aspartic amino acids such as lysine. The lysine yield and conversion rate of recombinant microorganisms expressing this mutant are significantly improved compared with the starting strain. This mutant and recombinant microorganism provide favorable modification targets, mutants, and strain resources for the breeding of aspartic acid, its derivatives, and aspartic amino acid producing strains. Detailed Implementation

[0061] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0062] The primer names and sequences involved in the following examples are shown in Table 1.

[0063] Table 1 Primer sequences

[0064]

[0065] The starting strain used in the following examples is a lysine-producing Escherichia coli MHZ-0914 obtained through mutagenesis. This strain was deposited on June 1, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 22648, and classified as Escherichia coli.

[0066] Unless otherwise specified, the techniques or conditions described in the following examples shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0067] Example 1: Mutant gene sdhA A530G Construction of recombinant strains

[0068] 1. pTargetF-N20(sdhA) A530G Plasmid and Donor DNA Construction

[0069] Step 1: Using pTF-sdhA-sgRNA-F / pTF-sdhA-sgRNA-R as primers (Table 1) and plasmid pTargetF as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), a linear plasmid containing N20 was amplified. This linear plasmid was assembled at 37°C using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain plasmid pTargetF-N20(sdhA A530G ), PCR identification and sequencing verification;

[0070] Step 2: Using the MG1655 genome as a template, select sdhA A530 -UF / sdhA A530G -UR primer pair, amplified the upstream homologous arm ①, and selected sdhA A530G -DF / sdhA A530 -DR primer pair, amplified downstream homologous arm ②, using ① and ② as templates, selected sdhA A530 -UF / sdhA A530 -DR primer pair, amplified Donor DNA.

[0071] 2. Preparation and electroporation of competent cells

[0072] Step 1: Electrotransfer the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into CGMCC No. 22648 competent cells (the transformation method and competent cell preparation method are both based on Molecular Cloning III);

[0073] Step 2: Pick a single colony and place it in 5 mL of LB medium containing kanamycin and a final concentration of 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500. 650 Electrocompetent cells were prepared after 0.4 μL of the solution (the method for preparing competent cells is described in Molecular Cloning III).

[0074] Step 3: Use the pTargetF-N20(sdhA) obtained in step 1 A530G The plasmid and Donor DNA were simultaneously electroporated into pCas competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), plated on LB plates containing spectinomycin and kanamycin, and incubated at 30°C until single colonies were visible.

[0075] 3. Validation of recombinant strains

[0076] Step 1: Use primer pair sdhA A530G -F1 / sdhA A530 -R was used to verify the above single colonies using colony PCR;

[0077] Step 2: Use primer pair sdhA to identify the correctly identified strain by PCR. A530 -F / sdhA A530 -R amplification, the amplification product is sent for sequencing.

[0078] 4. Loss of constructing related plasmids

[0079] Step 1: Select a single colony that has been correctly sequenced and inoculate it into a 5 mL LB tube containing kanamycin and a final concentration of 0.5 mM IPTG. Incubate overnight at 30°C and then streak it onto an LB agar plate containing kanamycin.

[0080] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20 plasmid has been lost.

[0081] Step 3: Pick positive colonies that have lost the pTargetF-N20 plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42°C for 8 hours, then streak them onto LB plates and incubate overnight at 37°C.

[0082] Step 4: Pick a single colony and spot it onto LB agar plates containing kanamycin and those without. If the colony cannot grow on the LB agar plate containing kanamycin but grows on the LB agar plate without antibiotic, it indicates that the pCas plasmid has been lost, and MHZ-0914-1(sdhA) is obtained. A530G ) strain.

[0083] Example 2 Mutant gene sdhA A530I Construction of recombinant strains

[0084] Following the method in Example 1, a recombinant strain with a mutation of isoleucine at amino acid position 530 of succinate dehydrogenase was obtained, and the strain was named MHZ-0914-2.

[0085] Example 3 Mutant gene sdhA A530H Construction of recombinant strains

[0086] Following the method in Example 1, a recombinant strain with a mutation of histidine at amino acid position 530 of succinate dehydrogenase was obtained, and the strain was named MHZ-0914-3.

[0087] Example 4 Mutant gene sdhA A530E Construction of recombinant strains

[0088] Following the method in Example 1, a recombinant strain with a mutation at amino acid position 530 of succinate dehydrogenase to glutamic acid was obtained, and the strain was named MHZ-0914-4.

[0089] Example 5 Mutant gene sdhA A530T Construction of recombinant strains

[0090] Following the method in Example 1, a recombinant strain with a mutation of amino acid 530 in succinate dehydrogenase to threonine was obtained, and the strain was named MHZ-0914-5.

[0091] Example 6 Lysine Fermentation Experiment

[0092] The culture medium used in the lysine fermentation experiment is as follows:

[0093] Seed activation medium: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 18 g / L agar powder, adjust pH to 7.0.

[0094] Seed culture medium: glucose 20 g / L, ammonium sulfate 4 g / L, corn steep liquor 2.0 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.4 g / L, ferric sulfate 0.01 g / L, manganese sulfate 0.01 g / L, adjust pH to 7.0.

[0095] 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, adjust pH to 7.0.

[0096] The lysine fermentation method is as follows:

[0097] 1. Seed activation: Take the strain to be verified from the cryopreservation tube, streak it on the seed activation medium, and incubate at 37℃ for 12h;

[0098] 2. Seed culture: Pick one loop of activated seeds from the plate and inoculate it into a 500 mL Erlenmeyer flask containing 20 mL of seed culture medium. Incubate at 33 °C and 220 r / min for 7 h with shaking.

[0099] 3. Fermentation culture: Inoculate 2 mL of seed culture into a 500 mL Erlenmeyer flask containing 30 mL of fermentation medium, and culture at 37 °C and 220 r / min for 12 h with shaking. Perform three replicates for each strain.

[0100] 4. OD 600 Measurement: Take 100 μl of fermentation broth, dilute it appropriately, and use a spectrophotometer to detect the OD at a wavelength of 600 nm. Perform three replicates for each strain, calculate the average value, and the detected OD is... 600 As shown in Table 2.

[0101] Lysine concentration determination: Centrifuge 2 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and use HPLC to detect the L-lysine content in the fermentation broth of recombinant bacteria and control bacteria. Perform three replicates for each strain and calculate the average value. The detected lysine concentrations are shown in Table 2.

[0102] Enzyme activity assay: Succinate dehydrogenase activity was determined by measuring the reduction of 2,6-dichloroindophenol (DCIP). The specific method is described in the reference Kurokawa T, Sakamoto J. Purification and characterization of succinate:menaquinone oxidoreductase from Corynebacterium glutamicum[J]. Archives of Microbiology, 2005, 183(5):317. Three replicates were performed for each strain, and the average value was calculated. The detected succinate dehydrogenase activity is shown in Table 2.

[0103] Table 2. Lysine production, growth, and enzyme activity of the recombinant strains.

[0104] strain L-Lysine (g / L) Sugar-acid conversion rate % <![CDATA[OD 600 ]]> Enzyme activity U / mg CGMCC No. 22648 18.8 28.2 15.2 1.36 <![CDATA[MHZ-0914-1(sdhA A530G )]]> 22.4 33.6 15.0 1.24 <![CDATA[MHZ-0914-2(sdhA A530I )]]> 22.0 33.1 14.9 1.21 <![CDATA[MHZ-0914-3(sdhA A530H )]]> 22.1 33.2 14.7 1.21 <![CDATA[MHZ-0914-4(sdhA A530E )]]> 23.3 33.9 14.5 1.19 <![CDATA[MHZ-0914-5(sdhA A530T )]]> 19.8 29.7 14.8 1.22

[0105] Fermentation results showed that after the amino acid at position 530 of the protein encoded by the sdhA gene was mutated from alanine (A) to glycine (G), isoleucine (I), histidine (H), glutamic acid (E), and threonine (T), the activity of succinate dehydrogenase in the mutant strains decreased to some extent, and the final OD was comparable to that of the original strain. However, the lysine yield increased in all mutant strains, with the mutation to glutamic acid (E) showing the best effect. MHZ-0914-4(sdhA) A530E The recombinant strain produced 4.5 g / L more L-lysine than the original strain, and the conversion rate was 5.7% higher.

[0106] Example 7 Construction of recombinant strains with start codon substitution

[0107] Based on the enzyme activity detection results of the above mutants, it is speculated that other attenuation methods of the sdhA gene may also have the effect of increasing lysine production and conversion rate. Therefore, further attenuation methods of the sdhA gene were carried out.

[0108] Following the method in Example 1, the succinate dehydrogenase start codon was mutated from ATG to GTG. The nucleotide sequence of the substituted sdhA gene is shown in SEQ ID NO.7, and the primers used are shown in Table 1 (sdhA). A1G -UF、sdhA A1G -UR、sdhA A1G -DF、sdhA A1G -DR、sdhA A1G -R、sdhA A1G-F), to obtain the recombinant strain MHZ-0914-6(sdhA) with the succinate dehydrogenase start codon mutated from ATG to GTG. A1G The strain was named MHZ-0914-6.

[0109] Example 8 Lysine Fermentation Experiment

[0110] The performance of the recombinant strain constructed in Example 7 was tested according to the method of Example 6. The lysine concentration and growth were obtained as shown in Table 3.

[0111] Table 3 Lysine production and growth detection of recombinant strains

[0112] strain L-Lysine (g / L) Conversion rate % <![CDATA[OD 600 ]]> CGMCC No. 22648 18.8 28.2 15.2 MHZ-0914-6 24.1 34.9 13.4

[0113] The results showed that the expression level of succinate dehydrogenase in the obtained MHZ-0914-6 strain decreased by approximately 70-80% compared to the original strain (with slight variations in different culture media), while the lysine production was significantly increased. The L-lysine production of the recombinant strain MHZ-0914-6 was 5.3 g / L higher than that of the original strain, and the conversion rate was increased by 6.7%. This example further illustrates that weakening succinate dehydrogenase can promote lysine production in Escherichia coli.

[0114] 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 fall within the scope of protection claimed by the present invention. sequence list <110> Meihua (Shanghai) Biotechnology Co., Ltd. <120> Succinate dehydrogenase mutants, recombinant microorganisms, their preparation methods and applications <130> KHP211117881.0 <160> 39 <170> SIPOSequenceListing 1.0 <210> 1 <211> 588 <212> PRT <213> Artificial Sequence <400> 1 Met Lys Leu Pro Val Arg Glu Phe Asp Ala Val Val Ile Gly Ala Gly 1 5 10 15 Gly Ala Gly Met Arg Ala Ala Leu Gln Ile Ser Gln Ser Gly Gln Thr 20 25 30 Cys Ala Leu Leu Ser Lys Val Phe Pro Thr Arg Ser His Thr Val Ser 35 40 45 Ala Gln Gly Gly Ile Thr Val Ala Leu Gly Asn Thr His Glu Asp Asn 50 55 60 Trp Glu Trp His Met Tyr Asp Thr Val Lys Gly Ser Asp Tyr Ile Gly 65 70 75 80 Asp Gln Asp Ala Ile Glu Tyr Met Cys Lys Thr Gly Pro Glu Ala Ile 85 90 95 Leu Glu Leu Glu His Met Gly Leu Pro Phe Ser Arg Leu Asp Asp Gly 100 105 110 Arg Ile Tyr Gln Arg Pro Phe Gly Gly Gln Ser Lys Asn Phe Gly Gly 115 120 125 Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala Asp Arg Thr Gly His Ala 130 135 140 Leu Leu His Thr Leu Tyr Gln Gln Asn Leu Lys Asn His Thr Thr Ile 145 150 155 160 Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val Lys Asn Gln Asp Gly Ala 165 170 175 Val Val Gly Cys Thr Ala Leu Cys Ile Glu Thr Gly Glu Val Val Tyr 180 185 190 Phe Lys Ala Arg Ala Thr Val Leu Ala Thr Gly Gly Ala Gly Arg Ile 195 200 205 Tyr Gln Ser Thr Thr Asn Ala His Ile Asn Thr Gly Asp Gly Val Gly 210 215 220 Met Ala Ile Arg Ala Gly Val Pro Val Gln Asp Met Glu Met Trp Gln 225 230 235 240 Phe His Pro Thr Gly Ile Ala Gly Ala Gly Val Leu Val Thr Glu Gly 245 250 255 Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn Lys His Gly Glu Arg Phe 260 265 270 Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp Leu Ala Gly Arg Asp Val 275 280 285 Val Ala Arg Ser Ile Met Ile Glu Ile Arg Glu Gly Arg Gly Cys Asp 290 295 300 Gly Pro Trp Gly Pro His Ala Lys Leu Lys Leu Asp His Leu Gly Lys 305 310 315 320 Glu Val Leu Glu Ser Arg Leu Pro Gly Ile Leu Glu Leu Ser Arg Thr 325 330 335 Phe Ala His Val Asp Pro Val Lys Glu Pro Ile Pro Val Ile Pro Thr 340 345 350 Cys His Tyr Met Met Gly Gly Ile Pro Thr Lys Val Thr Gly Gln Ala 355 360 365 Leu Thr Val Asn Glu Lys Gly Glu Asp Val Val Val Pro Gly Leu Phe 370 375 380 Ala Val Gly Glu Ile Ala Cys Val Ser Val His Gly Ala Asn Arg Leu 385 390 395 400 Gly Gly Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Ala Gly 405 410 415 Leu His Leu Gln Glu Ser Ile Ala Glu Gln Gly Ala Leu Arg Asp Ala 420 425 430 Ser Glu Ser Asp Val Glu Ala Ser Leu Asp Arg Leu Asn Arg Trp Asn 435 440 445 Asn Asn Arg Asn Gly Glu Asp Pro Val Ala Ile Arg Lys Ala Leu Gln 450 455 460 Glu Cys Met Gln His Asn Phe Ser Val Phe Arg Glu Gly Asp Ala Met 465 470 475 480 Ala Lys Gly Leu Glu Gln Leu Lys Val Ile Arg Glu Arg Leu Lys Asn 485 490 495 Ala Arg Leu Asp Asp Thr Ser Ser Glu Phe Asn Thr Gln Arg Val Glu 500 505 510 Cys Leu Glu Leu Asp Asn Leu Met Glu Thr Ala Tyr Ala Thr Ala Val 515 520 525 Ser Gly Asn Phe Arg Thr Glu Ser Arg Gly Ala His Ser Arg Phe Asp 530 535 540 Phe Pro Asp Arg Asp Asp Glu Asn Trp Leu Cys His Ser Leu Tyr Leu 545 550 555 560 Pro Glu Ser Glu Ser Met Thr Arg Arg Ser Val Asn Met Glu Pro Lys 565 570 575 Leu Arg Pro Ala Phe Pro Pro Lys Ile Arg Thr Tyr 580 585 <210> 2 <211> 588 <212> PRT <213> Artificial Sequence <400> 2 Met Lys Leu Pro Val Arg Glu Phe Asp Ala Val Val Ile Gly Ala Gly 1 5 10 15 Gly Ala Gly Met Arg Ala Ala Leu Gln Ile Ser Gln Ser Gly Gln Thr 20 25 30 Cys Ala Leu Leu Ser Lys Val Phe Pro Thr Arg Ser His Thr Val Ser 35 40 45 Ala Gln Gly Gly Ile Thr Val Ala Leu Gly Asn Thr His Glu Asp Asn 50 55 60 Trp Glu Trp His Met Tyr Asp Thr Val Lys Gly Ser Asp Tyr Ile Gly 65 70 75 80 Asp Gln Asp Ala Ile Glu Tyr Met Cys Lys Thr Gly Pro Glu Ala Ile 85 90 95 Leu Glu Leu Glu His Met Gly Leu Pro Phe Ser Arg Leu Asp Asp Gly 100 105 110 Arg Ile Tyr Gln Arg Pro Phe Gly Gly Gln Ser Lys Asn Phe Gly Gly 115 120 125 Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala Asp Arg Thr Gly His Ala 130 135 140 Leu Leu His Thr Leu Tyr Gln Gln Asn Leu Lys Asn His Thr Thr Ile 145 150 155 160 Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val Lys Asn Gln Asp Gly Ala 165 170 175 Val Val Gly Cys Thr Ala Leu Cys Ile Glu Thr Gly Glu Val Val Tyr 180 185 190 Phe Lys Ala Arg Ala Thr Val Leu Ala Thr Gly Gly Ala Gly Arg Ile 195 200 205 Tyr Gln Ser Thr Thr Asn Ala His Ile Asn Thr Gly Asp Gly Val Gly 210 215 220 Met Ala Ile Arg Ala Gly Val Pro Val Gln Asp Met Glu Met Trp Gln 225 230 235 240 Phe His Pro Thr Gly Ile Ala Gly Ala Gly Val Leu Val Thr Glu Gly 245 250 255 Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn Lys His Gly Glu Arg Phe 260 265 270 Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp Leu Ala Gly Arg Asp Val 275 280 285 Val Ala Arg Ser Ile Met Ile Glu Ile Arg Glu Gly Arg Gly Cys Asp 290 295 300 Gly Pro Trp Gly Pro His Ala Lys Leu Lys Leu Asp His Leu Gly Lys 305 310 315 320 Glu Val Leu Glu Ser Arg Leu Pro Gly Ile Leu Glu Leu Ser Arg Thr 325 330 335 Phe Ala His Val Asp Pro Val Lys Glu Pro Ile Pro Val Ile Pro Thr 340 345 350 Cys His Tyr Met Met Gly Gly Ile Pro Thr Lys Val Thr Gly Gln Ala 355 360 365 Leu Thr Val Asn Glu Lys Gly Glu Asp Val Val Val Pro Gly Leu Phe 370 375 380 Ala Val Gly Glu Ile Ala Cys Val Ser Val His Gly Ala Asn Arg Leu 385 390 395 400 Gly Gly Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Ala Gly 405 410 415 Leu His Leu Gln Glu Ser Ile Ala Glu Gln Gly Ala Leu Arg Asp Ala 420 425 430 Ser Glu Ser Asp Val Glu Ala Ser Leu Asp Arg Leu Asn Arg Trp Asn 435 440 445 Asn Asn Arg Asn Gly Glu Asp Pro Val Ala Ile Arg Lys Ala Leu Gln 450 455 460 Glu Cys Met Gln His Asn Phe Ser Val Phe Arg Glu Gly Asp Ala Met 465 470 475 480 Ala Lys Gly Leu Glu Gln Leu Lys Val Ile Arg Glu Arg Leu Lys Asn 485 490 495 Ala Arg Leu Asp Asp Thr Ser Ser Glu Phe Asn Thr Gln Arg Val Glu 500 505 510 Cys Leu Glu Leu Asp Asn Leu Met Glu Thr Ala Tyr Ala Thr Ala Val 515 520 525 Ser Ile Asn Phe Arg Thr Glu Ser Arg Gly Ala His Ser Arg Phe Asp 530 535 540 Phe Pro Asp Arg Asp Asp Glu Asn Trp Leu Cys His Ser Leu Tyr Leu 545 550 555 560 Pro Glu Ser Glu Ser Met Thr Arg Arg Ser Val Asn Met Glu Pro Lys 565 570 575 Leu Arg Pro Ala Phe Pro Pro Lys Ile Arg Thr Tyr 580 585 <210> 3 <211> 588 <212> PRT <213> Artificial Sequence <400> 3 Met Lys Leu Pro Val Arg Glu Phe Asp Ala Val Val Ile Gly Ala Gly 1 5 10 15 Gly Ala Gly Met Arg Ala Ala Leu Gln Ile Ser Gln Ser Gly Gln Thr 20 25 30 Cys Ala Leu Leu Ser Lys Val Phe Pro Thr Arg Ser His Thr Val Ser 35 40 45 Ala Gln Gly Gly Ile Thr Val Ala Leu Gly Asn Thr His Glu Asp Asn 50 55 60 Trp Glu Trp His Met Tyr Asp Thr Val Lys Gly Ser Asp Tyr Ile Gly 65 70 75 80 Asp Gln Asp Ala Ile Glu Tyr Met Cys Lys Thr Gly Pro Glu Ala Ile 85 90 95 Leu Glu Leu Glu His Met Gly Leu Pro Phe Ser Arg Leu Asp Asp Gly 100 105 110 Arg Ile Tyr Gln Arg Pro Phe Gly Gly Gln Ser Lys Asn Phe Gly Gly 115 120 125 Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala Asp Arg Thr Gly His Ala 130 135 140 Leu Leu His Thr Leu Tyr Gln Gln Asn Leu Lys Asn His Thr Thr Ile 145 150 155 160 Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val Lys Asn Gln Asp Gly Ala 165 170 175 Val Val Gly Cys Thr Ala Leu Cys Ile Glu Thr Gly Glu Val Val Tyr 180 185 190 Phe Lys Ala Arg Ala Thr Val Leu Ala Thr Gly Gly Ala Gly Arg Ile 195 200 205 Tyr Gln Ser Thr Thr Asn Ala His Ile Asn Thr Gly Asp Gly Val Gly 210 215 220 Met Ala Ile Arg Ala Gly Val Pro Val Gln Asp Met Glu Met Trp Gln 225 230 235 240 Phe His Pro Thr Gly Ile Ala Gly Ala Gly Val Leu Val Thr Glu Gly 245 250 255 Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn Lys His Gly Glu Arg Phe 260 265 270 Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp Leu Ala Gly Arg Asp Val 275 280 285 Val Ala Arg Ser Ile Met Ile Glu Ile Arg Glu Gly Arg Gly Cys Asp 290 295 300 Gly Pro Trp Gly Pro His Ala Lys Leu Lys Leu Asp His Leu Gly Lys 305 310 315 320 Glu Val Leu Glu Ser Arg Leu Pro Gly Ile Leu Glu Leu Ser Arg Thr 325 330 335 Phe Ala His Val Asp Pro Val Lys Glu Pro Ile Pro Val Ile Pro Thr 340 345 350 Cys His Tyr Met Met Gly Gly Ile Pro Thr Lys Val Thr Gly Gln Ala 355 360 365 Leu Thr Val Asn Glu Lys Gly Glu Asp Val Val Val Pro Gly Leu Phe 370 375 380 Ala Val Gly Glu Ile Ala Cys Val Ser Val His Gly Ala Asn Arg Leu 385 390 395 400 Gly Gly Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Ala Gly 405 410 415 Leu His Leu Gln Glu Ser Ile Ala Glu Gln Gly Ala Leu Arg Asp Ala 420 425 430 Ser Glu Ser Asp Val Glu Ala Ser Leu Asp Arg Leu Asn Arg Trp Asn 435 440 445 Asn Asn Arg Asn Gly Glu Asp Pro Val Ala Ile Arg Lys Ala Leu Gln 450 455 460 Glu Cys Met Gln His Asn Phe Ser Val Phe Arg Glu Gly Asp Ala Met 465 470 475 480 Ala Lys Gly Leu Glu Gln Leu Lys Val Ile Arg Glu Arg Leu Lys Asn 485 490 495 Ala Arg Leu Asp Asp Thr Ser Ser Glu Phe Asn Thr Gln Arg Val Glu 500 505 510 Cys Leu Glu Leu Asp Asn Leu Met Glu Thr Ala Tyr Ala Thr Ala Val 515 520 525 Ser His Asn Phe Arg Thr Glu Ser Arg Gly Ala His Ser Arg Phe Asp 530 535 540 Phe Pro Asp Arg Asp Asp Glu Asn Trp Leu Cys His Ser Leu Tyr Leu 545 550 555 560 Pro Glu Ser Glu Ser Met Thr Arg Arg Ser Val Asn Met Glu Pro Lys 565 570 575 Leu Arg Pro Ala Phe Pro Pro Lys Ile Arg Thr Tyr 580 585 <210> 4 <211> 588 <212> PRT <213> Artificial Sequence <400> 4 Met Lys Leu Pro Val Arg Glu Phe Asp Ala Val Val Ile Gly Ala Gly 1 5 10 15 Gly Ala Gly Met Arg Ala Ala Leu Gln Ile Ser Gln Ser Gly Gln Thr 20 25 30 Cys Ala Leu Leu Ser Lys Val Phe Pro Thr Arg Ser His Thr Val Ser 35 40 45 Ala Gln Gly Gly Ile Thr Val Ala Leu Gly Asn Thr His Glu Asp Asn 50 55 60 Trp Glu Trp His Met Tyr Asp Thr Val Lys Gly Ser Asp Tyr Ile Gly 65 70 75 80 Asp Gln Asp Ala Ile Glu Tyr Met Cys Lys Thr Gly Pro Glu Ala Ile 85 90 95 Leu Glu Leu Glu His Met Gly Leu Pro Phe Ser Arg Leu Asp Asp Gly 100 105 110 Arg Ile Tyr Gln Arg Pro Phe Gly Gly Gln Ser Lys Asn Phe Gly Gly 115 120 125 Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala Asp Arg Thr Gly His Ala 130 135 140 Leu Leu His Thr Leu Tyr Gln Gln Asn Leu Lys Asn His Thr Thr Ile 145 150 155 160 Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val Lys Asn Gln Asp Gly Ala 165 170 175 Val Val Gly Cys Thr Ala Leu Cys Ile Glu Thr Gly Glu Val Val Tyr 180 185 190 Phe Lys Ala Arg Ala Thr Val Leu Ala Thr Gly Gly Ala Gly Arg Ile 195 200 205 Tyr Gln Ser Thr Thr Asn Ala His Ile Asn Thr Gly Asp Gly Val Gly 210 215 220 Met Ala Ile Arg Ala Gly Val Pro Val Gln Asp Met Glu Met Trp Gln 225 230 235 240 Phe His Pro Thr Gly Ile Ala Gly Ala Gly Val Leu Val Thr Glu Gly 245 250 255 Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn Lys His Gly Glu Arg Phe 260 265 270 Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp Leu Ala Gly Arg Asp Val 275 280 285 Val Ala Arg Ser Ile Met Ile Glu Ile Arg Glu Gly Arg Gly Cys Asp 290 295 300 Gly Pro Trp Gly Pro His Ala Lys Leu Lys Leu Asp His Leu Gly Lys 305 310 315 320 Glu Val Leu Glu Ser Arg Leu Pro Gly Ile Leu Glu Leu Ser Arg Thr 325 330 335 Phe Ala His Val Asp Pro Val Lys Glu Pro Ile Pro Val Ile Pro Thr 340 345 350 Cys His Tyr Met Met Gly Gly Ile Pro Thr Lys Val Thr Gly Gln Ala 355 360 365 Leu Thr Val Asn Glu Lys Gly Glu Asp Val Val Val Pro Gly Leu Phe 370 375 380 Ala Val Gly Glu Ile Ala Cys Val Ser Val His Gly Ala Asn Arg Leu 385 390 395 400 Gly Gly Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Ala Gly 405 410 415 Leu His Leu Gln Glu Ser Ile Ala Glu Gln Gly Ala Leu Arg Asp Ala 420 425 430 Ser Glu Ser Asp Val Glu Ala Ser Leu Asp Arg Leu Asn Arg Trp Asn 435 440 445 Asn Asn Arg Asn Gly Glu Asp Pro Val Ala Ile Arg Lys Ala Leu Gln 450 455 460 Glu Cys Met Gln His Asn Phe Ser Val Phe Arg Glu Gly Asp Ala Met 465 470 475 480 Ala Lys Gly Leu Glu Gln Leu Lys Val Ile Arg Glu Arg Leu Lys Asn 485 490 495 Ala Arg Leu Asp Asp Thr Ser Ser Glu Phe Asn Thr Gln Arg Val Glu 500 505 510 Cys Leu Glu Leu Asp Asn Leu Met Glu Thr Ala Tyr Ala Thr Ala Val 515 520 525 Ser Glu Asn Phe Arg Thr Glu Ser Arg Gly Ala His Ser Arg Phe Asp 530 535 540 Phe Pro Asp Arg Asp Asp Glu Asn Trp Leu Cys His Ser Leu Tyr Leu 545 550 555 560 Pro Glu Ser Glu Ser Met Thr Arg Arg Ser Val Asn Met Glu Pro Lys 565 570 575 Leu Arg Pro Ala Phe Pro Pro Lys Ile Arg Thr Tyr 580 585 <210> 5 <211> 588 <212> PRT <213> Artificial Sequence <400> 5 Met Lys Leu Pro Val Arg Glu Phe Asp Ala Val Val Ile Gly Ala Gly 1 5 10 15 Gly Ala Gly Met Arg Ala Ala Leu Gln Ile Ser Gln Ser Gly Gln Thr 20 25 30 Cys Ala Leu Leu Ser Lys Val Phe Pro Thr Arg Ser His Thr Val Ser 35 40 45 Ala Gln Gly Gly Ile Thr Val Ala Leu Gly Asn Thr His Glu Asp Asn 50 55 60 Trp Glu Trp His Met Tyr Asp Thr Val Lys Gly Ser Asp Tyr Ile Gly 65 70 75 80 Asp Gln Asp Ala Ile Glu Tyr Met Cys Lys Thr Gly Pro Glu Ala Ile 85 90 95 Leu Glu Leu Glu His Met Gly Leu Pro Phe Ser Arg Leu Asp Asp Gly 100 105 110 Arg Ile Tyr Gln Arg Pro Phe Gly Gly Gln Ser Lys Asn Phe Gly Gly 115 120 125 Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala Asp Arg Thr Gly His Ala 130 135 140 Leu Leu His Thr Leu Tyr Gln Gln Asn Leu Lys Asn His Thr Thr Ile 145 150 155 160 Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val Lys Asn Gln Asp Gly Ala 165 170 175 Val Val Gly Cys Thr Ala Leu Cys Ile Glu Thr Gly Glu Val Val Tyr 180 185 190 Phe Lys Ala Arg Ala Thr Val Leu Ala Thr Gly Gly Ala Gly Arg Ile 195 200 205 Tyr Gln Ser Thr Thr Asn Ala His Ile Asn Thr Gly Asp Gly Val Gly 210 215 220 Met Ala Ile Arg Ala Gly Val Pro Val Gln Asp Met Glu Met Trp Gln 225 230 235 240 Phe His Pro Thr Gly Ile Ala Gly Ala Gly Val Leu Val Thr Glu Gly 245 250 255 Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn Lys His Gly Glu Arg Phe 260 265 270 Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp Leu Ala Gly Arg Asp Val 275 280 285 Val Ala Arg Ser Ile Met Ile Glu Ile Arg Glu Gly Arg Gly Cys Asp 290 295 300 Gly Pro Trp Gly Pro His Ala Lys Leu Lys Leu Asp His Leu Gly Lys 305 310 315 320 Glu Val Leu Glu Ser Arg Leu Pro Gly Ile Leu Glu Leu Ser Arg Thr 325 330 335 Phe Ala His Val Asp Pro Val Lys Glu Pro Ile Pro Val Ile Pro Thr 340 345 350 Cys His Tyr Met Met Gly Gly Ile Pro Thr Lys Val Thr Gly Gln Ala 355 360 365 Leu Thr Val Asn Glu Lys Gly Glu Asp Val Val Val Pro Gly Leu Phe 370 375 380 Ala Val Gly Glu Ile Ala Cys Val Ser Val His Gly Ala Asn Arg Leu 385 390 395 400 Gly Gly Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Ala Gly 405 410 415 Leu His Leu Gln Glu Ser Ile Ala Glu Gln Gly Ala Leu Arg Asp Ala 420 425 430 Ser Glu Ser Asp Val Glu Ala Ser Leu Asp Arg Leu Asn Arg Trp Asn 435 440 445 Asn Asn Arg Asn Gly Glu Asp Pro Val Ala Ile Arg Lys Ala Leu Gln 450 455 460 Glu Cys Met Gln His Asn Phe Ser Val Phe Arg Glu Gly Asp Ala Met 465 470 475 480 Ala Lys Gly Leu Glu Gln Leu Lys Val Ile Arg Glu Arg Leu Lys Asn 485 490 495 Ala Arg Leu Asp Asp Thr Ser Ser Glu Phe Asn Thr Gln Arg Val Glu 500 505 510 Cys Leu Glu Leu Asp Asn Leu Met Glu Thr Ala Tyr Ala Thr Ala Val 515 520 525 Ser Thr Asn Phe Arg Thr Glu Ser Arg Gly Ala His Ser Arg Phe Asp 530 535 540 Phe Pro Asp Arg Asp Asp Glu Asn Trp Leu Cys His Ser Leu Tyr Leu 545 550 555 560 Pro Glu Ser Glu Ser Met Thr Arg Arg Ser Val Asn Met Glu Pro Lys 565 570 575 Leu Arg Pro Ala Phe Pro Pro Lys Ile Arg Thr Tyr 580 585 <210> 6 <211> 588 <212> PRT <213> Artificial Sequence <400> 6 Met Lys Leu Pro Val Arg Glu Phe Asp Ala Val Val Ile Gly Ala Gly 1 5 10 15 Gly Ala Gly Met Arg Ala Ala Leu Gln Ile Ser Gln Ser Gly Gln Thr 20 25 30 Cys Ala Leu Leu Ser Lys Val Phe Pro Thr Arg Ser His Thr Val Ser 35 40 45 Ala Gln Gly Gly Ile Thr Val Ala Leu Gly Asn Thr His Glu Asp Asn 50 55 60 Trp Glu Trp His Met Tyr Asp Thr Val Lys Gly Ser Asp Tyr Ile Gly 65 70 75 80 Asp Gln Asp Ala Ile Glu Tyr Met Cys Lys Thr Gly Pro Glu Ala Ile 85 90 95 Leu Glu Leu Glu His Met Gly Leu Pro Phe Ser Arg Leu Asp Asp Gly 100 105 110 Arg Ile Tyr Gln Arg Pro Phe Gly Gly Gln Ser Lys Asn Phe Gly Gly 115 120 125 Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala Asp Arg Thr Gly His Ala 130 135 140 Leu Leu His Thr Leu Tyr Gln Gln Asn Leu Lys Asn His Thr Thr Ile 145 150 155 160 Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val Lys Asn Gln Asp Gly Ala 165 170 175 Val Val Gly Cys Thr Ala Leu Cys Ile Glu Thr Gly Glu Val Val Tyr 180 185 190 Phe Lys Ala Arg Ala Thr Val Leu Ala Thr Gly Gly Ala Gly Arg Ile 195 200 205 Tyr Gln Ser Thr Thr Asn Ala His Ile Asn Thr Gly Asp Gly Val Gly 210 215 220 Met Ala Ile Arg Ala Gly Val Pro Val Gln Asp Met Glu Met Trp Gln 225 230 235 240 Phe His Pro Thr Gly Ile Ala Gly Ala Gly Val Leu Val Thr Glu Gly 245 250 255 Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn Lys His Gly Glu Arg Phe 260 265 270 Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp Leu Ala Gly Arg Asp Val 275 280 285 Val Ala Arg Ser Ile Met Ile Glu Ile Arg Glu Gly Arg Gly Cys Asp 290 295 300 Gly Pro Trp Gly Pro His Ala Lys Leu Lys Leu Asp His Leu Gly Lys 305 310 315 320 Glu Val Leu Glu Ser Arg Leu Pro Gly Ile Leu Glu Leu Ser Arg Thr 325 330 335 Phe Ala His Val Asp Pro Val Lys Glu Pro Ile Pro Val Ile Pro Thr 340 345 350 Cys His Tyr Met Met Gly Gly Ile Pro Thr Lys Val Thr Gly Gln Ala 355 360 365 Leu Thr Val Asn Glu Lys Gly Glu Asp Val Val Val Pro Gly Leu Phe 370 375 380 Ala Val Gly Glu Ile Ala Cys Val Ser Val His Gly Ala Asn Arg Leu 385 390 395 400 Gly Gly Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Ala Gly 405 410 415 Leu His Leu Gln Glu Ser Ile Ala Glu Gln Gly Ala Leu Arg Asp Ala 420 425 430 Ser Glu Ser Asp Val Glu Ala Ser Leu Asp Arg Leu Asn Arg Trp Asn 435 440 445 Asn Asn Arg Asn Gly Glu Asp Pro Val Ala Ile Arg Lys Ala Leu Gln 450 455 460 Glu Cys Met Gln His Asn Phe Ser Val Phe Arg Glu Gly Asp Ala Met 465 470 475 480 Ala Lys Gly Leu Glu Gln Leu Lys Val Ile Arg Glu Arg Leu Lys Asn 485 490 495 Ala Arg Leu Asp Asp Thr Ser Ser Glu Phe Asn Thr Gln Arg Val Glu 500 505 510 Cys Leu Glu Leu Asp Asn Leu Met Glu Thr Ala Tyr Ala Thr Ala Val 515 520 525 Ser Ala Asn Phe Arg Thr Glu Ser Arg Gly Ala His Ser Arg Phe Asp 530 535 540 Phe Pro Asp Arg Asp Asp Glu Asn Trp Leu Cys His Ser Leu Tyr Leu 545 550 555 560 Pro Glu Ser Glu Ser Met Thr Arg Arg Ser Val Asn Met Glu Pro Lys 565 570 575 Leu Arg Pro Ala Phe Pro Pro Lys Ile Arg Thr Tyr 580 585 <210> 7 <211> 1767 <212> DNA <213> Artificial Sequence <400> 7 gtgaaattgc cagtcagaga atttgatgca gttgtgattg gtgccggtgg cgcaggtatg 60 cgcgcggcgc tgcaaatttc ccagagcggc cagacctgtg cgctgctctc taaagtcttc 120 ccgacccgtt cccataccgt ttctgcgcaa ggcggcatta ccgttgcgct gggtaatacc 180 catgaagata actgggaatg gcatatgtac gacaccgtga aagggtcgga ctatatcggt 240 gaccaggacg cgattgaata tatgtgtaaa accgggccgg aagcgattct ggaactcgaa 300 cacatgggcc tgccgttctc gcgtctcgat gatggtcgta tctatcaacg tccgtttggc 360 ggtcagtcga aaaacttcgg cggcgagcag gcggcacgca ctgcggcagc agctgaccgt 420 accggtcacg cactgttgca cacgctttat cagcagaacc tgaaaaacca caccaccatt 480 ttctccgagt ggtatgcgct ggatctggtg aaaaaccagg atggcgcggt ggtgggttgt 540 accgcactgt gcatcgaaac cggtgaagtg gtttatttca aagcccgcgc taccgtgctg 600 gcgactggcg gagcagggcg tatttatcag tccaccacca acgcccacat taacaccggc 660 gacggtgtcg gcatggctat ccgtgccggc gtaccggtgc aggatatgga aatgtggcag 720 ttccacccga ccggcattgc cggtgcgggc gtactggtca ccgaaggttg ccgtggtgaa 780 ggcggttatc tgctgaacaa acatggcgaa cgttttatgg agcgttatgc gccgaacgcc 840 aaagacctgg cgggccgtga cgtggttgcg cgttccatca tgatcgaaat ccgtgaaggt cgcggctgtg atggtccgtg ggggccacac gcgaaactga aactcgatca cctgggtaaa gaagttctcg aatcccgtct gccgggtatc ctggagcttt cccgtacctt cgctcacgtc gatccggtga aagagccgat tccggttatc ccaacctgtc actacatgat gggcggtatt ccgaccaaag ttaccggtca ggcactgact gtgaatgaga aaggcgaaga tgtggttgtt ccgggactgt ttgccgttgg tgaaatcgct tgtgtatcgg tacacggcgc taaccgtctg ggcggcaact cgctgctgga cctggtggtc tttggtcgcg cggcaggtct gcatctgcaa gagtctatcg ccgagcaggg cgcactgcgc gatgccagcg agtctgatgt tgaagcgtct ctggatcgcc tgaaccgctg gathering cgtaacggtg aagatccggt ggcgatccgt aaagcgctgc aagaatgtat gcagcataac ttctcggtct tccgtgaagg tgatgcgatg 1440 gcgaaagggc ttgagcagtt gaaagtgatc cgcgagcgtc tgaaaaatgc ccgtctggat 1500 gacacttcca gcgagttcaa cacccagcgc gttgagtgcc tggaactgga taacctgatg 1560 gaaacggcgt atgcaacggc tgtttctgcc aacttccgta ccgaaagccg tggcgcgcat 1620 agccgcttcg acttcccgga tcgtgatgat gaaaactggc tgtgccactc cctgtatctg 1680 ccagagtcgg aatccatgac gcgccgaagc gtcaacatgg aaccgaaact gcgcccggca 1740 ttcccgccga agattcgtac ttactaa 1767 <210> 8 <211> 1767 <212> DNA <213> Artificial Sequence <400> 8 atgaaattgc cagtcagaga atttgatgca gttgtgattg gtgccggtgg cgcaggtatg 60 cgcgcggcgc tgcaaatttc ccagagcggc cagacctgtg cgctgctctc taaagtcttc 120 ccgacccgtt cccataccgt ttctgcgcaa ggcggcatta ccgttgcgct gggtaatacc 180 catgaagata actgggaatg gcatatgtac gacaccgtga aagggtcgga ctatatcggt 240 gaccaggacg cgattgate tatgtgtaa accgggccgg aagcgattct ggaactcga cacatgggcc tgccgttctc gcgtctcgat gatggtcgta tctatcaacg tccgtttggc 360 ggtcagtcga aaaacttcgg cggcgagcag gcggcacgca ctgcggcagc agctgaccgt 420 accggtcacg cactgttgca cacgctttat cagcagaacc tgaaaaacca caccaccatt ttctccgagt ggtatgcgct ggatctggtg aaaaaccagg atggcgcggt ggtgggttgt 540 accgcactgt gcatcgaaac cggtgaagtg gtttatttca aagcccgcgc taccgtgctg gcgactggcg gagcagggcg tatttatcag tccaccacca acgcccacat taacaccggc gacggtgtcg gcatggctat ccgtgccggc gtaccggtgc aggatatgga aatgtggcag 720 ttccacccga ccggcattgc cggtgcgggc gtactggtca ccgaaggttg ccgtggtgaa 780 ggcggttatc tgctgaacaa acatggcgaa cgttttatgg agcgttatgc gccgaacgcc 840 aaagacctgg cgggccgtga cgtggttgcg cgttccatca tgatcgaaat ccgtgaaggt cgcggctgtg atggtccgtg ggggccacac gcgaaactga aactcgatca cctgggtaaa gaagttctcg aatcccgtct gccgggtatc ctggagcttt cccgtacctt cgctcacgtc 1020 gatccggtga aagagccgat tccggttatc ccaacctgtc actacatgat gggcggtatt 1080 ccgaccaaag ttaccggtca ggcactgact gtgaatgaga aaggcgaaga tgtggttgtt 1140 ccgggactgt ttgccgttgg tgaaatcgct tgtgtatcgg tacacggcgc taaccgtctg 1200 ggcggcaact cgctgctgga cctggtggtc tttggtcgcg cggcaggtct gcatctgcaa 1260 gagtctatcg ccgagcaggg cgcactgcgc gatgccagcg agtctgatgt tgaagcgtct 1320 ctggatcgcc tgaaccgctg gaacaataat cgtaacggtg aagatccggt ggcgatccgt 1380 aaagcgctgc aagaatgtat gcagcataac ttctcggtct tccgtgaagg tgatgcgatg 1440 gcgaaagggc ttgagcagtt gaaagtgatc cgcgagcgtc tgaaaaatgc ccgtctggat 1500 gacacttcca gcgagttcaa cacccagcgc gttgagtgcc tggaactgga taacctgatg 1560 gaaacggcgt atgcaacggc tgtttctggc aacttccgta ccgaaagccg tggcgcgcat 1620 agccgcttcg acttcccgga tcgtgatgat gaaaactggc tgtgccactc cctgtatctg 1680 ccagagtcgg aatccatgac gcgccgaagc gtcaacatgg aaccgaaact gcgcccggca 1740 ccagagtcgg aatccatgac gcgccgaagc gtcaacatgg aaccgaaact gcgcccggca 1740 ttcccgccga agattcgtac ttactaa 1767 ttcccgccga agattcgtac ttactaa 1767 <210> 9<210> 9 <211> 1767<211> 1767 <212> DNA<212> DNA <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 9<400> 9 atgaaattgc cagtcagaga atttgatgca gttgtgattg gtgccggtgg cgcaggtatg 60 atgaaattgc cagtcagaga atttgatgca gttgtgattg gtgccggtgg cgcaggtatg 60 cgcgcggcgc tgcaaatttc ccagagcggc cagacctgtg cgctgctctc taaagtcttc 120 cgcgcggcgc tgcaaatttc ccagagcggc cagacctgtg cgctgctctc taaagtcttc 120 ccgacccgtt cccataccgt ttctgcgcaa ggcggcatta ccgttgcgct gggtaatacc 180 ccgacccgtt cccataccgt ttctgcgcaa ggcggcatta ccgttgcgct gggtaatacc 180 [[ID=accgcactgt gcatcgaaac cggtgaagtg gtttatttca aagcccgcgc taccgtgctg gcgactggcg gagcagggcg tatttatcag tccaccacca acgcccacat taacaccggc gacggtgtcg gcatggctat ccgtgccggc gtaccggtgc aggatatgga aatgtggcag 720 ttccacccga ccggcattgc cggtgcgggc gtactggtca ccgaaggttg ccgtggtgaa 780 ggcggttatc tgctgaacaa acatggcgaa cgttttatgg agcgttatgc gccgaacgcc 840 aaagacctgg cgggccgtga cgtggttgcg cgttccatca tgatcgaaat ccgtgaaggt cgcggctgtg atggtccgtg ggggccacac gcgaaactga aactcgatca cctgggtaaa gaagttctcg aatcccgtct gccgggtatc ctggagcttt cccgtacctt cgctcacgtc gatccggtga aagagccgat tccggttatc ccaacctgtc actacatgat gggcggtatt ccgaccaaag ttaccggtca ggcactgact gtgaatgaga aaggcgaaga tgtggttgtt ccgggactgt ttgccgttgg tgaaatcgct tgtgtatcgg tacacggcgc taaccgtctg ggcggcaact cgctgctgga cctggtggtc tttggtcgcg cggcaggtct gcatctgcaa gagtctatcg ccgagcaggg cgcactgcgc gatgccagcg agtctgatgt tgaagcgtct 1320 ctggatcgcc tgaaccgctg gaacaataat cgtaacggtg aagatccggt ggcgatccgt 1380 aaagcgctgc aagaatgtat gcagcataac ttctcggtct tccgtgaagg tgatgcgatg 1440 gcgaaagggc ttgagcagtt gaaagtgatc cgcgagcgtc tgaaaaatgc ccgtctggat 1500 gacacttcca gcgagttcaa cacccagcgc gttgagtgcc tggaactgga taacctgatg 1560 gaaacggcgt atgcaacggc tgtttctatc aacttccgta ccgaaagccg tggcgcgcat 1620 agccgcttcg acttcccgga tcgtgatgat gaaaactggc tgtgccactc cctgtatctg 1680 ccagagtcgg aatccatgac gcgccgaagc gtcaacatgg aaccgaaact gcgcccggca 1740 ttcccgccga agattcgtac ttactaa 1767 <210> 10 <211> 1767 <212> DNA <213> Artificial Sequence <400> 10 atgaaattgc cagtcagaga atttgatgca gttgtgattg gtgccggtgg cgcaggtatg 60 cgcgcggcgc tgcaaatttc ccagagcggc cagacctgtg cgctgctctc taaagtcttc 120 ccgacccgtt cccataccgt ttctgcgcaa ggcggcatta ccgttgcgct gggtaatacc 180 240. catgaagata actgggaatg gcatatgtac gacaccgtga aagggtcgga ctatatcggt gaccaggacg cgattgate tatgtgtaa accgggccgg aagcgattct ggaactcga cacatgggcc tgccgttctc gcgtctcgat gatggtcgta tctatcaacg tccgtttggc 360 ggtcagtcga aaaacttcgg cggcgagcag gcggcacgca ctgcggcagc agctgaccgt 420 accggtcacg cactgttgca cacgctttat cagcagaacc tgaaaaacca caccaccatt ttctccgagt ggtatgcgct ggatctggtg aaaaaccagg atggcgcggt ggtgggttgt 540 accgcactgt gcatcgaaac cggtgaagtg gtttatttca aagcccgcgc taccgtgctg gcgactggcg gagcagggcg tatttatcag tccaccacca acgcccacat taacaccggc gacggtgtcg gcatggctat ccgtgccggc gtaccggtgc aggatatgga aatgtggcag 720 ttccacccga ccggcattgc cggtgcgggc gtactggtca ccgaaggttg ccgtggtgaa 780 ggcggttatc tgctgaacaa acatggcgaa cgttttatgg agcgttatgc gccgaacgcc 840 aaagacctgg cgggccgtga cgtggttgcg cgttccatca tgatcgaaat ccgtgaaggt 900 cgcggctgtg atggtccgtg ggggccacac gcgaaactga aactcgatca cctgggtaaaa 960 gaagttctcg aatcccgtct gccgggtatc ctggagcttt cccgtacctt cgctcacgtc 1020 gatccggtga aagagccgat tccggttatc ccaacctgtc actacatgat gggcggtatt 1080 ccgaccaaag ttaccggtca ggcactgact gtgaatgaga aaggcgaaga tgtggttgtt 1140 ccgggactgt ttgccgttgg tgaaatcgct tgtgtatcgg tacacggcgc taaccgtctg 1200 ggcggcaact cgctgctgga cctggtggtc tttggtcgcg cggcaggtct gcatctgcaa 1260 gagtctatcg ccgagcaggg cgcactgcgc gatgccagcg agtctgatgt tgaagcgtct 1320 ctggatcgcc tgaaccgctg gaacaataat cgtaacggtg aagatccggt ggcgatccgt 1380 aaagcgctgc aagaatgtat gcagcataac ttctcggtct tccgtgaagg tgatgcgatg 1440 gcgaaagggc ttgagcagtt gaaagtgatc cgcgagcgtc tgaaaaatgc ccgtctggat 1500 gacacttcca gcgagttcaa cacccagcgc gttgagtgcc tggaactgga taacctgatg 1560 gaaacggcgt atgcaacggc tgtttctcac aacttccgta ccgaaagccg tggcgcgcat 1620 agccgcttcg acttcccgga tcgtgatgat gaaaactggc tgtgccactc cctgtatctg 1680 ccagagtcgg aatccatgac gcgccgaagc gtcaacatgg aaccgaaact gcgcccggca 1740 ttcccgccga agattcgtac ttactaa 1767 <210> 11 <211> 1767 <212> DNA <213> Artificial Sequence <400> 11 atgaaattgc cagtcagaga atttgatgca gttgtgattg gtgccggtgg cgcaggtatg 60 cgcgcggcgc tgcaaatttc ccagagcggc cagacctgtg cgctgctctc taaagtcttc 120 ccgacccgtt cccataccgt ttctgcgcaa ggcggcatta ccgttgcgct gggtaatacc 180 catgaagata actgggaatg gcatatgtac gacaccgtga aagggtcgga ctatatcggt 240 gaccaggacg cgattgaata tatgtgtaaa accgggccgg aagcgattct ggaactcgaa 300 cacatgggcc tgccgttctc gcgtctcgat gatggtcgta tctatcaacg tccgtttggc 360 ggtcagtcga aaaacttcgg cggcgagcag gcggcacgca ctgcggcagc agctgaccgt 420 accggtcacg cactgttgca cacgctttat cagcagaacc tgaaaaacca caccaccatt ttctccgagt ggtatgcgct ggatctggtg aaaaaccagg atggcgcggt ggtgggttgt 540 accgcactgt gcatcgaaac cggtgaagtg gtttatttca aagcccgcgc taccgtgctg gcgactggcg gagcagggcg tatttatcag tccaccacca acgcccacat taacaccggc gacggtgtcg gcatggctat ccgtgccggc gtaccggtgc aggatatgga aatgtggcag 720 ttccacccga ccggcattgc cggtgcgggc gtactggtca ccgaaggttg ccgtggtgaa 780 ggcggttatc tgctgaacaa acatggcgaa cgttttatgg agcgttatgc gccgaacgcc 840 aaagacctgg cgggccgtga cgtggttgcg cgttccatca tgatcgaaat ccgtgaaggt cgcggctgtg atggtccgtg ggggccacac gcgaaactga aactcgatca cctgggtaaa gaagttctcg aatcccgtct gccgggtatc ctggagcttt cccgtacctt cgctcacgtc gatccggtga aagagccgat tccggttatc ccaacctgtc actacatgat gggcggtatt ccgaccaaag ttaccggtca ggcactgact gtgaatgaga aaggcgaaga tgtggttgtt ccgggactgt ttgccgttgg tgaaatcgct tgtgtatcgg tacacggcgc taaccgtctg 1200 ggcggcaact cgctgctgga cctggtggtc tttggtcgcg cggcaggtct gcatctgcaa 1260 gagtctatcg ccgagcaggg cgcactgcgc gatgccagcg agtctgatgt tgaagcgtct 1320 ctggatcgcc tgaaccgctg gaacaataat cgtaacggtg aagatccggt ggcgatccgt 1380 aaagcgctgc aagaatgtat gcagcataac ttctcggtct tccgtgaagg tgatgcgatg 1440 gcgaaagggc ttgagcagtt gaaagtgatc cgcgagcgtc tgaaaaatgc ccgtctggat 1500 gacacttcca gcgagttcaa cacccagcgc gttgagtgcc tggaactgga taacctgatg 1560 gaaacggcgt atgcaacggc tgtttctgaa aacttccgta ccgaaagccg tggcgcgcat 1620 agccgcttcg acttcccgga tcgtgatgat gaaaactggc tgtgccactc cctgtatctg 1680 ccagagtcgg aatccatgac gcgccgaagc gtcaacatgg aaccgaaact gcgcccggca 1740 ttcccgccga agattcgtac ttactaa 1767 <210> 12 <211> 1767 <212> DNA <213> Artificial Sequence <400> 12 60. atgaaattgc cagtcagaga atttgatgca gttgtgattg gtgccggtgg cgcaggtatg cgcgcggcgc tgcaaatttc ccagagcggc cagacctgtg cgctgctctc taaagtcttc ccgacccgtt cccataccgt ttctgcgcaa ggcggcatta ccgttgcgct gggtaatacc 180 240. catgaagata actgggaatg gcatatgtac gacaccgtga aagggtcgga ctatatcggt gaccaggacg cgattgate tatgtgtaa accgggccgg aagcgattct ggaactcga cacatgggcc tgccgttctc gcgtctcgat gatggtcgta tctatcaacg tccgtttggc 360 ggtcagtcga aaaacttcgg cggcgagcag gcggcacgca ctgcggcagc agctgaccgt 420 accggtcacg cactgttgca cacgctttat cagcagaacc tgaaaaacca caccaccatt ttctccgagt ggtatgcgct ggatctggtg aaaaaccagg atggcgcggt ggtgggttgt 540 accgcactgt gcatcgaaac cggtgaagtg gtttatttca aagcccgcgc taccgtgctg gcgactggcg gagcagggcg tatttatcag tccaccacca acgcccacat taacaccggc gacggtgtcg gcatggctat ccgtgccggc gtaccggtgc aggatatgga aatgtggcag 720 ttccacccga ccggcattgc cggtgcgggc gtactggtca ccgaaggttg ccgtggtgaa 780 ggcggttatc tgctgaacaa acatggcgaa cgttttatgg agcgttatgc gccgaacgcc 840 aaagacctgg cgggccgtga cgtggttgcg cgttccatca tgatcgaaat ccgtgaaggt 900 cgcggctgtg atggtccgtg ggggccacac gcgaaactga aactcgatca cctgggtaaaa 960 gaagttctcg aatcccgtct gccgggtatc ctggagcttt cccgtacctt cgctcacgtc 1020 gatccggtga aagagccgat tccggttatc ccaacctgtc actacatgat gggcggtatt 1080 ccgaccaaag ttaccggtca ggcactgact gtgaatgaga aaggcgaaga tgtggttgtt 1140 ccgggactgt ttgccgttgg tgaaatcgct tgtgtatcgg tacacggcgc taaccgtctg 1200 ggcggcaact cgctgctgga cctggtggtc tttggtcgcg cggcaggtct gcatctgcaa 1260 gagtctatcg ccgagcaggg cgcactgcgc gatgccagcg agtctgatgt tgaagcgtct 1320 ctggatcgcc tgaaccgctg gaacaataat cgtaacggtg aagatccggt ggcgatccgt 1380 aaagcgctgc aagaatgtat gcagcataac ttctcggtct tccgtgaagg tgatgcgatg 1440 gcgaaagggc ttgagcagtt gaaagtgatc cgcgagcgtc tgaaaaatgc ccgtctggat 1500 gacacttcca gcgagttcaa cacccagcgc gttgagtgcc tggaactgga taacctgatg 1560 gaaacggcgt atgcaacggc tgtttctacc aacttccgta ccgaaagccg tggcgcgcat 1620 agccgcttcg acttcccgga tcgtgatgat gaaaactggc tgtgccactc cctgtatctg 1680 ccagagtcgg aatccatgac gcgccgaagc gtcaacatgg aaccgaaact gcgcccggca 1740 ttcccgccga agattcgtac ttactaa 1767 <210> 13 <211> 59 <212> DNA <213> Artificial Sequence <400> 13 tcctaggtat aatactagtc gtatctatca acgtccgttg ttttagagct agaaatagc 59 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <400> 14 actagtatta tacctaggac tgagctagct gtcaag 36 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 cgcactgcgc gatgccagcg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 gccagaaaca gccgttgcat 20 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <400> 17 tatgcaacgg ctgtttctgg caacttccgt accgaaagcc 40 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 gatagaaaca gccgttgcat 20 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <400> 19 tatgcaacgg ctgtttctat caacttccgt accgaaagcc 40 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 gtgagaaaca gccgttgcat 20 <210> twenty one <211> 40 <212> DNA <213> Artificial Sequence <400> twenty one tatgcaacgg ctgtttctca caacttccgt accgaaagcc 40 <210> twenty two <211> 20 <212> DNA <213> Artificial Sequence <400> twenty two ttcagaaaca gccgttgcat 20 <210> twenty three <211> 40 <212> DNA <213> Artificial Sequence <400> twenty three tatgcaacgg ctgtttctga aaacttccgt accgaaagcc 40 <210> twenty four <211> 20 <212> DNA <213> Artificial Sequence <400> twenty four ccaagaaaca gccgttgcat 20 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <400> 25 tatgcaacgg ctgtttctac caacttccgt accgaaagcc 40 <210> 26 <211> twenty one <212> DNA <213> Artificial Sequence <400> 26 gctgggatct ttctctttta g 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 atgcaacggc tgtttctggc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <400> 28 atgcaacggc tgtttctatc 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 atgcaacggc tgtttctcac 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 atgcaacggc tgtttctgaa 20 <210> 31 <211> twenty one <212> DNA <213> Artificial Sequence <400> 31 atgcaacggc tgtttcttac c 21 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 cggcactcaa ccagccgggc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 aatcgcttgt gtatcggtac 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 acatgatttc atcctcgttc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 cacacacccc acaccacaac 20 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <400> 36 gtgaaattgc cagtcagaga atttgatgca gttgtgattg 40 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <400> 37 tgtgttcgag ttccagaatc 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <400> 38 cgtgtgcaac agtgcgtgac 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <400> 39 tttcacttct cgcaggagtc 20

Claims

1. Use of reducing expression and / or enzyme activity of succinate dehydrogenase in increasing lysine production and / or conversion rate of Escherichia coli. wherein The reducing expression and / or enzyme activity of succinate dehydrogenase is mutating the gene encoding succinate dehydrogenase into a gene encoding a succinate dehydrogenase mutant, the amino acid sequence of which is shown in any one of SEQ ID NO. 1-5.

2. Use of reducing expression and / or enzyme activity of succinate dehydrogenase in constructing Escherichia coli for lysine production. wherein The reducing expression and / or enzyme activity of succinate dehydrogenase is mutating the gene encoding succinate dehydrogenase into a gene encoding a succinate dehydrogenase mutant, the amino acid sequence of which is shown in any one of SEQ ID NO. 1-5.

3. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli expresses a succinate dehydrogenase mutant, the amino acid sequence of which is shown in any one of SEQ ID NO. 1-5, and does not express the succinate dehydrogenase possessed by its starting strain.

4. Use of the recombinant Escherichia coli of claim 3 in any one of the following: (1) in increasing lysine production and / or conversion rate of Escherichia coli; (2) in constructing Escherichia coli for lysine production.

5. Use of the recombinant Escherichia coli of claim 3 in the fermentative production of lysine.

6. The method of constructing a recombinant E. coli of claim 3, wherein, comprising: mutating the gene encoding succinate dehydrogenase in the starting strain into a gene encoding a succinate dehydrogenase mutant, the amino acid sequence of which is shown in any one of SEQ ID NO. 1-5.

7. A method for producing lysine, characterized by, comprising: culturing the recombinant Escherichia coli of claim 3.

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