Lysine production method, mutant, and recombinant microorganism and application

Abstract

The application relates to the technical field of microbial engineering, and particularly discloses a lysine production method, a mutant, a recombinant microorganism and applications. The lysine production method comprises the step of carrying out fermentation culture by using a recombinant microorganism. Compared with a starting strain, the recombinant microorganism has reduced aspartate-tRNA ligase activity, and preferably also has reduced isocitrate dehydrogenase activity. The recombinant microorganism is used for preparing lysine, so that the yield of lysine can be improved.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and more specifically, to a method for producing lysine, mutants and recombinant microorganisms and their applications. Background Technology

[0002] L-Lysine is a basic essential amino acid with the molecular formula C6H12O. 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. It 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 helps the body absorb other amino acids, thereby improving feed quality. Therefore, the production of lysine has broad prospects.

[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 for the production of L-amino acids, nucleotides, and other organic acids. *E. coli* synthesizes L-lysine starting with L-aspartic acid, through multiple enzymatic reactions. The yield of L-lysine is related to the enzyme activity and metabolic flux distribution in the biosynthetic pathway. Enhancing the enzyme activity in the terminal synthesis pathway, altering the direction of the central metabolic flux, and increasing the supply of reducing power all contribute to high L-lysine production in *E. coli*.

[0004] Aspartate-tRNA ligase (argS) is a member of the aminoacyl-tRNA synthetase family, which performs translation by covalently linking amino acids to specific tRNA molecules. In *E. coli*, aspartate-tRNA ligase mainly participates in the ligation reaction between L-aspartate and tRNA (Asp). This process involves two steps: first, L-aspartate, as a substrate, is activated by ATP to form Asp-AMP; then, Asp-AMP is transferred to the acceptor terminus of tRNA (Asp) to form L-aspartate-tRNA. asp L-Aspartic acid is a precursor for the synthesis of L-lysine. Currently, there are no reports on whether decreased aspartate-tRNA ligase activity affects the accumulation of L-lysine and other aspartic acid group amino acids in *E. coli*. Summary of the Invention

[0005] The purpose of this invention is to provide a method for increasing lysine production.

[0006] The technical solution of the present invention is as follows:

[0007] A method for producing lysine includes a step of fermentation culture with recombinant microorganisms, said recombinant microorganisms having reduced aspartate-tRNA ligase activity compared to the starting strain.

[0008] This invention has found that reducing the activity of aspartate-tRNA ligase can increase lysine production.

[0009] The reduction can be achieved using conventional techniques in the field of metabolic engineering, such as altering the promoter regulatory region, replacing the start codon, or changing the amino acid sequence.

[0010] Preferably, the reduction in aspartate-tRNA ligase activity is achieved by altering the promoter regulatory region, replacing the start codon, or mutating the amino acid sequence; more preferably, the reduction in aspartate-tRNA ligase activity is achieved by mutating alanine at position 275 of the amino acid sequence of the aspartate-tRNA ligase to valine.

[0011] The present invention has found that amino acid substitution at a specific position in the aspartate-tRNA ligase gene can reduce its enzyme activity, thereby increasing lysine production.

[0012] In the lysine production method of the present invention, the recombinant microorganism expresses a mutated aspartate-tRNA ligase, the amino acid sequence of which is shown in SEQ ID NO.21.

[0013] Preferably, in the lysine production method of the present invention, the recombinant microorganism has reduced isocitrate dehydrogenase activity compared to the starting strain.

[0014] The present invention also found that the weakening of aspartate-tRNA ligase and the modification of isocitrate dehydrogenase have a combined enhancing effect.

[0015] More preferably, the reduction in isocitrate dehydrogenase activity is achieved by carrying D398E and D410E mutations in the isocitrate dehydrogenase.

[0016] The mutant isocitrate dehydrogenase has amino acids at positions 398 and 410 where aspartic acid is mutated to glutamic acid.

[0017] Further preferably, the recombinant microorganism expresses a mutated isocitrate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.23.

[0018] In the lysine production method of the present invention, the starting strain is Escherichia coli.

[0019] The present invention also provides an aspartate-tRNA ligase mutant, which uses the amino acid sequence of wild-type aspartate-tRNA ligase as a reference sequence, wherein the aspartate-tRNA ligase mutant contains a mutation in which alanine at position 275 is replaced by valine; preferably, the amino acid sequence of the aspartate-tRNA ligase mutant is shown in SEQ ID NO.21.

[0020] The present invention also provides a recombinant microorganism expressing the above-mentioned aspartate-tRNA ligase mutant;

[0021] Preferably, the recombinant microorganism also expresses an isocitrate dehydrogenase mutant containing D398E and D410E mutations;

[0022] And / or, the starting strain of the recombinant microorganism is Escherichia coli.

[0023] The present invention further provides any of the following applications of the above-mentioned recombinant microorganisms:

[0024] (1) Application in the fermentation production of lysine;

[0025] (2) Application in the genetic breeding of microorganisms for the production of lysine;

[0026] (3) Application in increasing the yield of lysine produced by fermentation.

[0027] The beneficial effects of this invention are at least as follows:

[0028] This invention applies a mutated aspartate-tRNA ligase to E. coli fermentation, resulting in a significant increase in lysine yield. The mutated isocitrate dehydrogenase, with amino acids at positions 398 and 410 both mutated from aspartic acid to glutamic acid, can itself promote lysine yield. When combined with the mutated aspartate-tRNA ligase, it has a synergistic effect. This mutation can be applied not only to lysine but also to other compounds using aspartic acid as a precursor. Detailed Implementation

[0029] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the following examples, they are performed according to the techniques or conditions described in the literature in the art, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] The primer names and sequences involved in the embodiments of this invention are shown in Table 1.

[0031] Table 1 Primer sequences (SEQ ID No. 1-19)

[0032]

[0033]

[0034] Example 1: Mutant gene aspS (A275V) Construction of recombinant strains

[0035] The amino acid sequence of aspS is shown in SEQ ID No. 20. (A275V) The amino acid sequence is shown in SEQ ID No. 21.

[0036] (1) Construction of pTargetF-N20 plasmid and Donor DNA

[0037] Step 1: Using pTF-aspS-sgRNA-F / pTF-aspS-sgRNA-R as primers (all primer sequences are shown in 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), the pTF 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 (aspS A275V). PCR identification and sequencing verification were performed.

[0038] Step 2: Using the E. coli MG1655 genome (NC_000913) as a template, the upstream homologous arm ① was amplified using the aspS-UF / aspS-UR primer pair, and the downstream homologous arm ② was amplified using the aspS-DF / aspS-DR primer pair. Using ① and ② as templates, the Donor DNA was amplified using the aspS-UF / aspS-DR primer pair.

[0039] (2) Preparation and electroporation of competent cells

[0040] Step 1: Electrotransfer the pCas plasmid (see Multigene Editing in the Escherichiacoli 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);

[0041] The originating strain, CGMCC No. 22648, is a lysine-producing Escherichia coli strain 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 the accession number CGMCC No. 22648, and classified as Escherichia coli.

[0042] Step 2: Pick a single colony and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 r / min until the OD 650 is 0.4. Then prepare electroporation competent cells (refer to Molecular Cloning III for the preparation method of competent cells).

[0043] Step 3: Electroporate the pTargetF-N20 plasmid and Donor DNA obtained in (1) into pCas competent cells (electroplation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30°C until single colonies are visible.

[0044] (3) Validation of recombinant strains

[0045] Step 1: Use primer pair aspS-F1 / aspS-R to perform colony PCR verification on the above single colonies;

[0046] Step 2: Amplify the PCR-identified strains using primer pairs aspS-F / aspS-R, and send the amplification products for sequencing.

[0047] (4) Construction of related plasmids lost

[0048] 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.

[0049] 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.

[0050] Step 3: Pick positive colonies that have lost pTargetF-N20 plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0051] 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 the strain CGMCC No.22648-aspS is obtained. (A275V) .

[0052] Example 2 Mutant gene icd (D398E、D410E) Construction of recombinant strains

[0053] The amino acid sequence of icd is shown in SEQ ID No. 22. (D398E、D410E) The amino acid sequence is shown in SEQ ID No. 23.

[0054] Following the method in Example 1, using CGMCC No. 22648 as the starting strain and pTF-icd-sgRNA-F, pTF-icd-sgRNA-R, icd-UF, icd-UR, icd-DF, icd-DR, icd-F1, icd-F2, icd-F, and icd-R as primers, a strain with isocitrate dehydrogenase whose amino acid sequences at positions 398 and 410 are both mutated from aspartic acid to glutamic acid was obtained. This strain was named CGMCC No. 22648-icd. (D398E (D410E).

[0055] Example 3 includes the mutant gene icd (D398E、D410E) Construction of recombinant strains

[0056] Referring to the method in Example 1, using CGMCC No. 22648-aspS (A275V) Using pTF-icd-sgRNA-F, pTF-icd-sgRNA-R, icd-UF, icd-UR, icd-DF, icd-DR, icd-F1, icd-F2, icd-F, and icd-R as primers, a strain with isocitrate dehydrogenase whose amino acid sequences at positions 398 and 410 are both mutated from aspartic acid to glutamic acid was obtained. This strain was named CGMCC No. 22648-aspS. (A275V) -icd (D398E、D410E) .

[0057] Example 4 Lysine Fermentation Experiment

[0058] 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.

[0059] 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.

[0060] 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.

[0061] (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;

[0062] (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. Culture at 33℃ and 220 r / min for 7 h with shaking.

[0063] (3) Fermentation culture: Inoculate 2 mL of seed liquid 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.

[0064] 4. OD600 determination: Take 100 μl of fermentation broth, dilute it appropriately, and use a spectrophotometer to detect OD at a wavelength of 600 nm. Perform three replicates for each strain and calculate the average value. The detected OD600 values ​​are shown in Table 2.

[0065] 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.

[0066] Table 2 Lysine production and growth detection of recombinant strains

[0067]

[0068] Fermentation results showed that the three recombinant strains of this invention had varying degrees of improvement in lysine yield and conversion rate compared with the original strain CGMCC No.22648, and the differences were significant (P<0.05). The OD600 remained basically unchanged, indicating that the mutation sites of this invention have a positive effect.

[0069] Both aspartate-tRNA ligase and isocitrate dehydrogenase showed positive effects, and the combined modification of both had a more significant effect than the modification of either alone. The recombinant strain CGMCC No.22648-icd obtained by the combined modification (D398E , D410E) -aspS (A275V) L-lysine production increased by 4.2 g / L compared to the starting strain, and the conversion rate increased by 7.0%. From a metabolic perspective, the decrease in aspartate-tRNA ligase activity was conducive to aspartate accumulation, and the weakening of isocitrate dehydrogenase reduced the carbon flux entering the TCA cycle, allowing more carbon flux to enter the aspartate pathway, which was beneficial to lysine accumulation.

[0070] 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> Langfang Meihua Biotechnology Development Co., Ltd. <120> A method for producing lysine, mutants and recombinant microorganisms and their applications <130> KHP211125947.2 <160> twenty three <170> SIPOSequenceListing 1.0 <210> 1 <211> 59 <212> DNA <213> Artificial Sequence <400> 1 tcctaggtat aatactagtt gaatttactc agatcgatgg ttttagagct agaaatagc 59 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <400> 2 actagtatta tacctaggac tgagctagct gtcaag 36 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gctgaccaaa gccacgccgg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 ttccgcaaag gtcattaccg 20 <210> 5 <211> 41 <212> DNA <213> Artificial Sequence <400> 5 cggtaatgac ctttgcggaa gtagaacgcc gttatggttc t 41 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tgcgcggcag tacgatccag 20 <210> 7 <211> twenty three <212> DNA <213> Artificial Sequence <400> 7 cggtaatgac ctttgcggaa gta 23 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tacctcgacc tgcgtcgtcc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 aagtcgataa cccacagcgg 20 <210> 10 <211> 59 <212> DNA <213> Artificial Sequence <400> 10 tcctaggtat aatactagtc tttcatgacg gcaaacaatg ttttagagct agaaatagc 59 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <400> 11 actagtatta tacctaggac tgagctagct gtcaag 36 <210> 12 <211> twenty one <212> DNA <213> Artificial Sequence <400> 12 tgatgttatc gcctgtatga a 21 <210> 13 <211> 38 <212> DNA <213> Artificial Sequence <400> 13 catttcagca gcttagcgcc ttccatcaga cgttcgaa 38 <210> 14 <211> 54 <212> DNA <213> Artificial Sequence <400> 14 gaaggcgcta agctgctgaa atgttcagag tttggtgaag cgatcatcga aaac 54 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 ttacaccggt taagtctggt 20 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <400> 16 ttcgaacgtc tgatggaa 18 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <400> 17 aaatgttcag agtttggtga a 21 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 ggcggtgaac tgatcgacg 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 acaaatgaag tggatagtaa 20 <210> 20 <211> 590 <212> PRT <213> Artificial Sequence <400> 20 Met Arg Thr Glu Tyr Cys Gly Gln Leu Arg Leu Ser His Val Gly Gln 1 5 10 15 Gln Val Thr Leu Cys Gly Trp Val Asn Arg Arg Arg Asp Leu Gly Ser 20 25 30 Leu Ile Phe Ile Asp Met Arg Asp Arg Glu Gly Ile Val Gln Val Phe 35 40 45 Phe Asp Pro Asp Arg Ala Asp Ala Leu Lys Leu Ala Ser Glu Leu Arg 50 55 60 Asn Glu Phe Cys Ile Gln Val Thr Gly Thr Val Arg Ala Arg Asp Glu 65 70 75 80 Lys Asn Ile Asn Arg Asp Met Ala Thr Gly Glu Ile Glu Val Leu Ala 85 90 95 Ser Ser Leu Thr Ile Ile Asn Arg Ala Asp Val Leu Pro Leu Asp Ser 100 105 110 Asn His Val Asn Thr Glu Glu Ala Arg Leu Lys Tyr Arg Tyr Leu Asp 115 120 125 Leu Arg Arg Pro Glu Met Ala Gln Arg Leu Lys Thr Arg Ala Lys Ile 130 135 140 Thr Ser Leu Val Arg Arg Phe Met Asp Asp His Gly Phe Leu Asp Ile 145 150 155 160 Glu Thr Pro Met Leu Thr Lys Ala Thr Pro Glu Gly Ala Arg Asp Tyr 165 170 175 Leu Val Pro Ser Arg Val His Lys Gly Lys Phe Tyr Ala Leu Pro Gln 180 185 190 Ser Pro Gln Leu Phe Lys Gln Leu Leu Met Met Ser Gly Phe Asp Arg 195 200 205 Tyr Tyr Gln Ile Val Lys Cys Phe Arg Asp Glu Asp Leu Arg Ala Asp 210 215 220 Arg Gln Pro Glu Phe Thr Gln Ile Asp Val Glu Thr Ser Phe Met Thr 225 230 235 240 Ala Pro Gln Val Arg Glu Val Met Glu Ala Leu Val Arg His Leu Trp 245 250 255 Leu Glu Val Lys Gly Val Asp Leu Gly Asp Phe Pro Val Met Thr Phe 260 265 270 Ala Glu Ala Glu Arg Arg Tyr Gly Ser Asp Lys Pro Asp Leu Arg Asn 275 280 285 Pro Met Glu Leu Thr Asp Val Ala Asp Leu Leu Lys Ser Val Glu Phe 290 295 300 Ala Val Phe Ala Gly Pro Ala Asn Asp Pro Lys Gly Arg Val Ala Ala 305 310 315 320 Leu Arg Val Pro Gly Gly Ala Ser Leu Thr Arg Lys Gln Ile Asp Glu 325 330 335 Tyr Gly Asn Phe Val Lys Ile Tyr Gly Ala Lys Gly Leu Ala Tyr Ile 340 345 350 Lys Val Asn Glu Arg Ala Lys Gly Leu Glu Gly Ile Asn Ser Pro Val 355 360 365 Ala Lys Phe Leu Asn Ala Glu Ile Ile Glu Asp Ile Leu Asp Arg Thr 370 375 380 Ala Ala Gln Asp Gly Asp Met Ile Phe Phe Gly Ala Asp Asn Lys Lys 385 390 395 400 Ile Val Ala Asp Ala Met Gly Ala Leu Arg Leu Lys Val Gly Lys Asp 405 410 415 Leu Gly Leu Thr Asp Glu Ser Lys Trp Ala Pro Leu Trp Val Ile Asp 420 425 430 Phe Pro Met Phe Glu Asp Asp Gly Glu Gly Gly Leu Thr Ala Met His 435 440 445 His Pro Phe Thr Ser Pro Lys Asp Met Thr Ala Ala Glu Leu Lys Ala 450 455 460 Ala Pro Glu Asn Ala Val Ala Asn Ala Tyr Asp Met Val Ile Asn Gly 465 470 475 480 Tyr Glu Val Gly Gly Gly Ser Val Arg Ile His Asn Gly Asp Met Gln 485 490 495 Gln Thr Val Phe Gly Ile Leu Gly Ile Asn Glu Glu Glu Gln Arg Glu 500 505 510 Lys Phe Gly Phe Leu Leu Asp Ala Leu Lys Tyr Gly Thr Pro Pro His 515 520 525 Ala Gly Leu Ala Phe Gly Leu Asp Arg Leu Thr Met Leu Leu Thr Gly 530 535 540 Thr Asp Asn Ile Arg Asp Val Ile Ala Phe Pro Lys Thr Thr Ala Ala 545 550 555 560 Ala Cys Leu Met Thr Glu Ala Pro Ser Phe Ala Asn Pro Thr Ala Leu 565 570 575 Ala Glu Leu Ser Ile Gln Val Val Lys Lys Ala Glu Asn Asn 580 585 590 <210> 21 <211> 590 <212> PRT <213> Artificial Sequence <400> 21 Met Arg Thr Glu Tyr Cys Gly Gln Leu Arg Leu Ser His Val Gly Gln 1 5 10 15 Gln Val Thr Leu Cys Gly Trp Val Asn Arg Arg Arg Asp Leu Gly Ser 20 25 30 Leu Ile Phe Ile Asp Met Arg Asp Arg Glu Gly Ile Val Gln Val Phe 35 40 45 Phe Asp Pro Asp Arg Ala Asp Ala Leu Lys Leu Ala Ser Glu Leu Arg 50 55 60 Asn Glu Phe Cys Ile Gln Val Thr Gly Thr Val Arg Ala Arg Asp Glu 65 70 75 80 Lys Asn Ile Asn Arg Asp Met Ala Thr Gly Glu Ile Glu Val Leu Ala 85 90 95 Ser Ser Leu Thr Ile Ile Asn Arg Ala Asp Val Leu Pro Leu Asp Ser 100 105 110 Asn His Val Asn Thr Glu Glu Ala Arg Leu Lys Tyr Arg Tyr Leu Asp 115 120 125 Leu Arg Arg Pro Glu Met Ala Gln Arg Leu Lys Thr Arg Ala Lys Ile 130 135 140 Thr Ser Leu Val Arg Arg Phe Met Asp Asp His Gly Phe Leu Asp Ile 145 150 155 160 Glu Thr Pro Met Leu Thr Lys Ala Thr Pro Glu Gly Ala Arg Asp Tyr 165 170 175 Leu Val Pro Ser Arg Val His Lys Gly Lys Phe Tyr Ala Leu Pro Gln 180 185 190 Ser Pro Gln Leu Phe Lys Gln Leu Leu Met Met Ser Gly Phe Asp Arg 195 200 205 Tyr Tyr Gln Ile Val Lys Cys Phe Arg Asp Glu Asp Leu Arg Ala Asp 210 215 220 Arg Gln Pro Glu Phe Thr Gln Ile Asp Val Glu Thr Ser Phe Met Thr 225 230 235 240 Ala Pro Gln Val Arg Glu Val Met Glu Ala Leu Val Arg His Leu Trp 245 250 255 Leu Glu Val Lys Gly Val Asp Leu Gly Asp Phe Pro Val Met Thr Phe 260 265 270 Ala Glu Val Glu Arg Arg Tyr Gly Ser Asp Lys Pro Asp Leu Arg Asn 275 280 285 Pro Met Glu Leu Thr Asp Val Ala Asp Leu Leu Lys Ser Val Glu Phe 290 295 300 Ala Val Phe Ala Gly Pro Ala Asn Asp Pro Lys Gly Arg Val Ala Ala 305 310 315 320 Leu Arg Val Pro Gly Gly Ala Ser Leu Thr Arg Lys Gln Ile Asp Glu 325 330 335 Tyr Gly Asn Phe Val Lys Ile Tyr Gly Ala Lys Gly Leu Ala Tyr Ile 340 345 350 Lys Val Asn Glu Arg Ala Lys Gly Leu Glu Gly Ile Asn Ser Pro Val 355 360 365 Ala Lys Phe Leu Asn Ala Glu Ile Ile Glu Asp Ile Leu Asp Arg Thr 370 375 380 Ala Ala Gln Asp Gly Asp Met Ile Phe Phe Gly Ala Asp Asn Lys Lys 385 390 395 400 Ile Val Ala Asp Ala Met Gly Ala Leu Arg Leu Lys Val Gly Lys Asp 405 410 415 Leu Gly Leu Thr Asp Glu Ser Lys Trp Ala Pro Leu Trp Val Ile Asp 420 425 430 Phe Pro Met Phe Glu Asp Asp Gly Glu Gly Gly Leu Thr Ala Met His 435 440 445 His Pro Phe Thr Ser Pro Lys Asp Met Thr Ala Ala Glu Leu Lys Ala 450 455 460 Ala Pro Glu Asn Ala Val Ala Asn Ala Tyr Asp Met Val Ile Asn Gly 465 470 475 480 Tyr Glu Val Gly Gly Gly Ser Val Arg Ile His Asn Gly Asp Met Gln 485 490 495 Gln Thr Val Phe Gly Ile Leu Gly Ile Asn Glu Glu Glu Gln Arg Glu 500 505 510 Lys Phe Gly Phe Leu Leu Asp Ala Leu Lys Tyr Gly Thr Pro Pro His 515 520 525 Ala Gly Leu Ala Phe Gly Leu Asp Arg Leu Thr Met Leu Leu Thr Gly 530 535 540 Thr Asp Asn Ile Arg Asp Val Ile Ala Phe Pro Lys Thr Thr Ala Ala 545 550 555 560 Ala Cys Leu Met Thr Glu Ala Pro Ser Phe Ala Asn Pro Thr Ala Leu 565 570 575 Ala Glu Leu Ser Ile Gln Val Val Lys Lys Ala Glu Asn Asn 580 585 590 <210> 22 <211> 416 <212> PRT <213> Artificial Sequence <400> 22 Met Glu Ser Lys Val Val Val Pro Ala Gln Gly Lys Lys Ile Thr Leu 1 5 10 15 Gln Asn Gly Lys Leu Asn Val Pro Glu Asn Pro Ile Ile Pro Tyr Ile 20 25 30 Glu Gly Asp Gly Ile Gly Val Asp Val Thr Pro Ala Met Leu Lys Val 35 40 45 Val Asp Ala Ala Val Glu Lys Ala Tyr Lys Gly Glu Arg Lys Ile Ser 50 55 60 Trp Met Glu Ile Tyr Thr Gly Glu Lys Ser Thr Gln Val Tyr Gly Gln 65 70 75 80 Asp Val Trp Leu Pro Ala Glu Thr Leu Asp Leu Ile Arg Glu Tyr Arg 85 90 95 Val Ala Ile Lys Gly Pro Leu Thr Thr Pro Val Gly Gly Gly Ile Arg 100 105 110 Ser Leu Asn Val Ala Leu Arg Gln Glu Leu Asp Leu Tyr Ile Cys Leu 115 120 125 Arg Pro Val Arg Tyr Tyr Gln Gly Thr Pro Ser Pro Val Lys His Pro 130 135 140 Glu Leu Thr Asp Met Val Ile Phe Arg Glu Asn Ser Glu Asp Ile Tyr 145 150 155 160 Ala Gly Ile Glu Trp Lys Ala Asp Ser Ala Asp Ala Glu Lys Val Ile 165 170 175 Lys Phe Leu Arg Glu Glu Met Gly Val Lys Lys Ile Arg Phe Pro Glu 180 185 190 His Cys Gly Ile Gly Ile Lys Pro Cys Ser Glu Glu Gly Thr Lys Arg 195 200 205 Leu Val Arg Ala Ala Ile Glu Tyr Ala Ile Ala Asn Asp Arg Asp Ser 210 215 220 Val Thr Leu Val His Lys Gly Asn Ile Met Lys Phe Thr Glu Gly Ala 225 230 235 240 Phe Lys Asp Trp Gly Tyr Gln Leu Ala Arg Glu Glu Phe Gly Gly Glu 245 250 255 Leu Ile Asp Gly Gly Pro Trp Leu Lys Val Lys Asn Pro Asn Thr Gly 260 265 270 Lys Glu Ile Val Ile Lys Asp Val Ile Ala Asp Ala Phe Leu Gln Gln 275 280 285 Ile Leu Leu Arg Pro Ala Glu Tyr Asp Val Ile Ala Cys Met Asn Leu 290 295 300 Asn Gly Asp Tyr Ile Ser Asp Ala Leu Ala Ala Gln Val Gly Gly Ile 305 310 315 320 Gly Ile Ala Pro Gly Ala Asn Ile Gly Asp Glu Cys Ala Leu Phe Glu 325 330 335 Ala Thr His Gly Thr Ala Pro Lys Tyr Ala Gly Gln Asp Lys Val Asn 340 345 350 Pro Gly Ser Ile Ile Leu Ser Ala Glu Met Met Leu Arg His Met Gly 355 360 365 Trp Thr Glu Ala Ala Asp Leu Ile Val Lys Gly Met Glu Gly Ala Ile 370 375 380 Asn Ala Lys Thr Val Thr Tyr Asp Phe Glu Arg Leu Met Asp Gly Ala 385 390 395 400 Lys Leu Leu Lys Cys Ser Glu Phe Gly Asp Ala Ile Ile Glu Asn Met 405 410 415 <210> 23 <211> 416 <212> PRT <213> Artificial Sequence <400> 23 Met Glu Ser Lys Val Val Val Pro Ala Gln Gly Lys Lys Ile Thr Leu 1 5 10 15 Gln Asn Gly Lys Leu Asn Val Pro Glu Asn Pro Ile Ile Pro Tyr Ile 20 25 30 Glu Gly Asp Gly Ile Gly Val Asp Val Thr Pro Ala Met Leu Lys Val 35 40 45 Val Asp Ala Ala Val Glu Lys Ala Tyr Lys Gly Glu Arg Lys Ile Ser 50 55 60 Trp Met Glu Ile Tyr Thr Gly Glu Lys Ser Thr Gln Val Tyr Gly Gln 65 70 75 80 Asp Val Trp Leu Pro Ala Glu Thr Leu Asp Leu Ile Arg Glu Tyr Arg 85 90 95 Val Ala Ile Lys Gly Pro Leu Thr Thr Pro Val Gly Gly Gly Ile Arg 100 105 110 Ser Leu Asn Val Ala Leu Arg Gln Glu Leu Asp Leu Tyr Ile Cys Leu 115 120 125 Arg Pro Val Arg Tyr Tyr Gln Gly Thr Pro Ser Pro Val Lys His Pro 130 135 140 Glu Leu Thr Asp Met Val Ile Phe Arg Glu Asn Ser Glu Asp Ile Tyr 145 150 155 160 Ala Gly Ile Glu Trp Lys Ala Asp Ser Ala Asp Ala Glu Lys Val Ile 165 170 175 Lys Phe Leu Arg Glu Glu Met Gly Val Lys Lys Ile Arg Phe Pro Glu 180 185 190 His Cys Gly Ile Gly Ile Lys Pro Cys Ser Glu Glu Gly Thr Lys Arg 195 200 205 Leu Val Arg Ala Ala Ile Glu Tyr Ala Ile Ala Asn Asp Arg Asp Ser 210 215 220 Val Thr Leu Val His Lys Gly Asn Ile Met Lys Phe Thr Glu Gly Ala 225 230 235 240 Phe Lys Asp Trp Gly Tyr Gln Leu Ala Arg Glu Glu Phe Gly Gly Glu 245 250 255 Leu Ile Asp Gly Gly Pro Trp Leu Lys Val Lys Asn Pro Asn Thr Gly 260 265 270 Lys Glu Ile Val Ile Lys Asp Val Ile Ala Asp Ala Phe Leu Gln Gln 275 280 285 Ile Leu Leu Arg Pro Ala Glu Tyr Asp Val Ile Ala Cys Met Asn Leu 290 295 300 Asn Gly Asp Tyr Ile Ser Asp Ala Leu Ala Ala Gln Val Gly Gly Ile 305 310 315 320 Gly Ile Ala Pro Gly Ala Asn Ile Gly Asp Glu Cys Ala Leu Phe Glu 325 330 335 Ala Thr His Gly Thr Ala Pro Lys Tyr Ala Gly Gln Asp Lys Val Asn 340 345 350 Pro Gly Ser Ile Ile Leu Ser Ala Glu Met Met Leu Arg His Met Gly 355 360 365 Trp Thr Glu Ala Ala Asp Leu Ile Val Lys Gly Met Glu Gly Ala Ile 370 375 380 Asn Ala Lys Thr Val Thr Tyr Asp Phe Glu Arg Leu Met Glu Gly Ala 385 390 395 400 Lys Leu Leu Lys Cys Ser Glu Phe Gly Glu Ala Ile Ile Glu Asn Met 405 410 415

Claims

1. A method for producing lysine, characterized in that, The method includes a step of fermentation culture with recombinant microorganisms, wherein the starting strain of the recombinant microorganisms is Escherichia coli, and its accession number is CGMCC No. 22648; the recombinant microorganisms express a mutated aspartate-tRNA ligase, the amino acid sequence of which is shown in SEQ ID NO.

21.

2. The lysine production method according to claim 1, characterized in that, The recombinant microorganism also expresses a mutated isocitrate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

23.

3. An aspartate-tRNA ligase mutant, characterized in that, Using the sequence shown in SEQ ID NO.20 as a reference sequence, the aspartate-tRNA ligase mutant has a mutation in which alanine at position 275 is replaced by valine; the amino acid sequence of the aspartate-tRNA ligase mutant is shown in SEQ ID NO.

21.

4. A recombinant microorganism, characterized in that, The recombinant microorganism expresses the aspartate-tRNA ligase mutant as described in claim 3; the starting strain of the recombinant microorganism is Escherichia coli, and its preservation number is CGMCC No. 22648.

5. The recombinant microorganism according to claim 4, characterized in that, The recombinant microorganism also expresses an isocitrate dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO.

23.

6. Any of the following applications of the recombinant microorganism according to claim 4 or 5: (1) Application in the fermentation production of lysine; (2) Application in the genetic breeding of microorganisms for the production of lysine; (3) Application in increasing the yield of lysine produced by fermentation.

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