CN113278572A - Recombinant corynebacterium for modifying 5' -terminal sequence of HTS gene and application thereof - Google Patents

Abstract

The invention relates to a recombinant corynebacterium for modifying a 5' terminal sequence of an HTS gene and application thereof. The recombinant corynebacterium of the invention replaces nucleotide sequences from-19 to-12 before the initiation codon ATG in the 5 'end sequence of HTS gene in corynebacterium host bacteria, reduces the stability of the secondary structure of the 5' end sequence of HTS gene, and makes it easier to be transcribed or expressed. Compared with the original strain, the recombinant corynebacterium has certain improvement on the synthesis efficiency of L-lysine, further improves the yield of the L-lysine, reduces the production cost, has no conflict with the existing high-yield L-lysine modification method, but still needs to be further verified whether the recombinant corynebacterium has the mutual promotion effect with the existing modification method.

Description

Recombinant corynebacterium for modifying 5' -terminal sequence of HTS gene and application thereof

Technical Field

The invention relates to a recombinant corynebacterium for modifying a 5' terminal sequence of an HTS gene and application thereof, belonging to the technical field of biological engineering.

Background

L-lysine is a basic unit constituting protein, is a raw material for synthesizing human body hormone, enzyme and antibody, participates in human body metabolism and various physiological activities, is essential amino acid for human body, and is basically available in various amino acid transfusion formulas. L-lysine was first isolated from a protein hydrolysate, and then a chemical synthesis method and an enzymatic method were invented, and was first produced by a microbial fermentation method in Japan in 1960. The L-lysine is prepared from various starch hydrolyzed sugar or molasses by fermenting with variant strains of Brevibacterium and Corynebacterium, separating, concentrating, evaporating, crystallizing, drying to obtain feed-grade lysine, and refining to obtain food-grade and pharmaceutical-grade lysine. The technology for producing glutamic acid by fermentation is comprehensively popularized in China in 1965, and scientific research and production of fermented lysine are driven since then. At present, the domestic lysine acid production rate and the conversion rate exceed 20g/dL and 60 percent.

Microorganisms for producing L-lysine include various species such as Corynebacterium, Bacillus, Escherichia, etc., but wild-type strains have poor L-lysine producing ability, many metabolic byproducts, and it is difficult to achieve the production of L-lysine with high purity and high yield. Therefore, it is generally desired to obtain a strain having a high L-lysine yield. At present, methods for obtaining strains with high L-lysine yield mainly comprise mutation screening breeding or genetic engineering breeding. The mutation screening breeding refers to inducing a strain to generate nonspecific gene site mutation by ultraviolet irradiation or other external condition stimulation, and then screening to obtain a high-yield strain. Genetic engineering breeding is to optimize the breeding strains by means of well-defined genetic engineering, for example, by introducing a beneficial enzyme gene with high enzymatic activity by increased copy or site-directed mutagenesis, or by knocking out an unfavorable gene to eliminate enzymatic activity/expression. Currently, Corynebacterium glutamicum CICC23604 has been widely used in industrial fermentation for producing various amino acids, and the product is approved as "general regulated as safe" (GRAS) by FDA. Therefore, the construction of the recombinant corynebacterium glutamicum by using a metabolic engineering means is an effective way for producing food safety-grade L-lysine.

The 5 'end sequence of a gene generally refers to a DNA sequence located upstream of the 5' end of a structural gene and generally comprising a transcription site recognized, bound and initiated by RNA polymerase, which contains conserved sequences required for RNA polymerase specific binding and transcription initiation, and which is not transcribed per se. Its properties were originally identified by mutations that increase or decrease the transcription rate of the gene. The promoter is generally located in the 5' end sequence of the gene, has a length which is different according to the organism, generally does not exceed 200bp, is a typical cis-acting element, and is combined with a transcription factor (trans-acting factor) to regulate the level, the position and the mode of gene expression (Chinese patent document CN 109385424A). Gene 5' end sequence substitutions may be used to modulate gene expression in a particular manner, such as their conditional expression or overexpression. PCR-based gene targeting, and chromosomal integration of regulatory sequences upstream of the Open Reading Frame (ORF) by homologous recombination, can stably alter the genome (chinese patent document CN 111655860A).

Although there are many reports on the improvement of L-lysine productivity, there is still a need to develop a novel method for improving L-lysine productivity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a recombinant corynebacterium for modifying a 5' terminal sequence of an HTS gene and application thereof. The recombinant corynebacterium of the invention is obtained by modifying corynebacterium host bacteria through genetic engineering, and the specific strategy is to replace partial nucleotide sequence in 5 'end sequence of 4-hydroxy-tetrahydrodipicolinate synthase (HTS) gene by using a gene 5' end sequence replacement method to obtain the recombinant corynebacterium with high L-lysine yield.

Description of terms:

4-hydroxy-tetrahydrodipicolinate synthase (HTS): the enzyme catalyzing aspartate semialdehyde to form dihydropyridine dicarboxylic acid, which plays an important role in the synthesis of L-lysine, is widely present in various bacterial microorganisms.

The technical scheme of the invention is as follows:

a recombinant corynebacterium for modifying the 5 'end sequence of HTS gene features that the nucleotide sequence from-19 to-12 before the ATG start codon in the 5' end sequence of HTS gene is used in the corynebacterium host^-19GAAGGTAA^-12Is replaced by^-19TGTGGTAT^-12And the stability of the secondary structure of the 5' end sequence of the HTS gene is reduced, so that the HTS gene is easier to be transcribed or expressed.

Preferably according to the invention, the corynebacterium host bacterium is corynebacterium glutamicum; more preferably Corynebacterium glutamicum CICC23604 or Corynebacterium glutamicum CGMCC 1.15647.

Preferably according to the invention, the amino acid sequence of the HTS is SEQ ID NO.1 or SEQ ID NO. 8.

Preferably, according to the invention, the amino acid sequence of the HTS has an amino acid sequence with a sequence identity of at least 99.7% with the sequence of SEQ ID No.1 or SEQ ID No. 8.

Preferably, according to the invention, the nucleotide sequence of the HTS gene is SEQ ID NO.2 or SEQ ID NO. 9.

According to a preferred embodiment of the invention, the nucleotide sequence of the HTS gene has a nucleotide sequence with a sequence identity of 99.7% or more with SEQ ID NO.2 or SEQ ID NO. 9.

Preferably, according to the invention, the 5' end sequence of the HTS gene is SEQ ID NO.3 or SEQ ID NO. 10.

The construction method of the recombinant corynebacterium comprises the following steps:

(1) synthesis of upstream homology arm-^-19TGTGGTAT^-12Nucleotide sequence of downstream homology arm, wherein upstream homology arm and downstream homology arm are nucleotide sequences from-19 to-12 before ATG (initiation codon) in 5' end sequence of HTS gene^{- 19}GAAGGTAA^-12A nucleotide sequence with the length of 500-600 bp is respectively arranged in front of and behind the nucleotide sequence;

(2) the upstream homology arm^-19TGTGGTAT^-12The nucleotide sequence of the downstream homology arm was ligated into the pK19mobsacB vector, constructing a replacement vector;

(3) transforming the replacement vector into a corynebacterium host strain competent cell, and screening a positive transformant with kanamycin resistance to obtain a recombinant strain with first homologous single exchange;

(4) and after natural passage of the recombinant bacteria subjected to the first homologous single exchange, screening bacterial colonies which can grow on a 10% sucrose culture medium but cannot grow on a kanamycin-resistant culture medium, and verifying to obtain the recombinant corynebacteria subjected to the two homologous single exchanges.

Preferably according to the invention, the upstream homology arm in step (2)^-19TGTGGTAT^-12The nucleotide sequence of the downstream homology arm is ligated between Hind III, EcoR I cleavage sites of the pK19mobsacB vector.

Preferably, in step (3), the kanamycin-resistant gene primers are used to screen for kanamycin-resistant positive transformants by using PCR amplification technology, and the sequences of the primers are as follows:

F1：5′-ATGATTGAACAAGATGGATTGC-3′，

R1：5′-TCAGAAGAACTCGTCAAGAAGGCG-3′。

preferably, the verification in step (4) is performed by using a PCR amplification technique, and the primer sequences amplified by PCR are as follows:

F2：5′-AAATGAGGGAATGTGGTAT-3′，

R2：5′-TTATAGAACTCCAGCTTTTTTCA-3′。

further preferably, the PCR amplification system is: 2 XHiFi-PCRmaster 10 μ L, 10 μmol/L forward primer 1 μ L, 10 μmol/L reverse primer 1 μ L, template 1 μ L, ddH₂O 7μL；

The procedure of PCR amplification is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

Preferably according to the invention, the medium used in step (4) is LBG medium: 5g/L of glucose, 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl.

The use of the recombinant coryneform bacterium as described above for the production of L-lysine.

According to the invention, the application is that the recombinant corynebacterium is inoculated into the liquid LBG culture medium for seed culture, and then the recombinant corynebacterium is inoculated into the fermentation culture medium for fermentation culture according to the inoculum size of 2-5% by volume percentage;

the LBG medium: 5g/L of glucose, 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl;

the fermentation medium comprises: 100g/L glucose, 20g/L peptone, 30mL/L corn steep liquor, 5g/L urea, (NH)₄)₂SO₄25g/L, L-leucine 0.34g/L, KH₂PO₄ 2g/L，MgSO₄·7H₂O1.5g/L and biotin 0.001 g/L.

According to the preferable selection of the invention, the seed culture conditions are 200-220 rpm and 28-30 ℃ for 18-25 h; the fermentation culture conditions are 200-220 rpm and 28-30 ℃.

The technical principle of the invention is as follows:

the 5' end sequence of the gene comprises a promoter region sequence of the gene, and the secondary structure of the promoter region sequence can influence the promoter efficiency of the promoter and further influence the expression activity of the gene after the promoter. The invention relates to a method for preparing a HTS gene by a nucleotide sequence from-19 to-12 before an initiation codon ATG in a 5' end sequence of an HTS gene^-19GAAGGTAA^-12Is replaced by^-19TGTGGTAT^-12The stability of the secondary structure of the 5' end sequence of the HTS gene can be reduced, so that the HTS gene can be transcribed or expressed more easily.

Has the advantages that:

the invention provides a recombinant corynebacterium for modifying 5 'end sequence of HTS gene, which is a nucleotide sequence from-19 to-12 before ATG (initiation codon) in 5' end sequence of HTS gene^-19GAAGGTAA^-12Is replaced by^-19TGTGGTAT^-12Compared with the original strain, the recombinant corynebacterium has certain improvement on the synthesis efficiency of the L-lysine, further improves the yield of the L-lysine, reduces the production cost, has no conflict with the existing high-yield L-lysine modification method, but still needs to be further verified whether the recombinant corynebacterium has the mutual promotion effect with the existing modification method.

Detailed Description

The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto. Reagents and medicines involved in the examples are all common commercial products unless otherwise specified; the experimental methods mentioned in the examples are all conventional technical means in the field unless otherwise specified.

The microbial source is as follows:

corynebacterium glutamicum CICC23604, purchased from China center for culture Collection of industrial microorganisms, with strain number CICC23604, wherein the amino acid sequence of 4-hydroxy-tetrahydrodipicolinate synthase (HTS) is SEQ ID NO.1, the nucleotide sequence of HTS gene is SEQ ID NO.2, and the 5' end sequence of HTS gene is SEQ ID NO. 3.

Corynebacterium glutamicum CGMCC1.15647 purchased from China general microbiological culture Collection center with the strain number of CGMCC1.15647, wherein the amino acid sequence of 4-hydroxy-tetrahydrodipicolinate synthase (HTS) is SEQ ID NO.8, the nucleotide sequence of HTS gene is SEQ ID NO.9, and the 5' end sequence of HTS gene is SEQ ID NO. 10.

The consistency of the amino acid sequences of SEQ ID NO.1 and SEQ ID NO.8 is 99.7%, the consistency of the nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.9 is 99.7%, and the consistency of the nucleotide sequences of SEQ ID NO.3 and SEQ ID NO.10 is 100%.

The corn steep liquor referred to in the examples is corn steep liquor specially used for fermentation, and is available from biotech ltd.

Example 1: homologous arm gene synthesis containing replacement nucleotide sequence and construction of replacement vector

1.1 Corynebacterium glutamicum CICC23604

Synthesis of upstream homology arm for C.glutamicum CICC23604^-19TGTGGTAT^-12Nucleotide sequence of downstream homology arm, upstream homology arm and downstream homology arm being nucleotide sequences from-19 to-12 before ATG initiation codon in 5' end sequence of HTS gene^-19GAAGGTAA^-12The nucleotide sequence of 500bp length is respectively arranged in front of and behind the upstream homology arm, wherein the nucleotide sequence of the upstream homology arm is SEQ ID NO.4, the nucleotide sequence of the downstream homology arm is SEQ ID NO.5, and the original upstream homology arm^{- 19}GAAGGTAA^-12The nucleotide sequence of the downstream homology arm is SEQ ID NO.6, the upstream homology arm being designed for synthesis^{- 19}TGTGGTAT^-12The nucleotide sequence of the downstream homology arm is SEQ ID No. 7. The nucleotide sequence of SEQ ID NO.7 was synthesized by Shanghai Bioengineering Co., Ltd and ligated between Hind III and EcoRI cleavage sites of pK19mobsacB vector (GenBank: LC 257601.1). The ligation products were transformed into E.coli DH 5. alpha. competent cells, 200. mu.L of the transformation solution was spread on solid LB plates containing kanamycin antibiotic (50. mu.g/mL final concentration) using a sterilized spreader, cultured overnight in an incubator at 37 ℃ and screened for positive transformants for extraction and preservation of the replacement vector pK19mobsacB-HTS 1.

1.2 Corynebacterium glutamicum CGMCC1.15647

Synthesis of upstream homology arm for Corynebacterium glutamicum CGMCC1.15647^-19TGTGGTAT^-12Nucleotide sequence of downstream homology arm, upstream homology arm and downstream homology arm being nucleotide sequences from-19 to-12 before ATG initiation codon in 5' end sequence of HTS gene^-19GAAGGTAA^-12The nucleotide sequence of 500bp length is respectively arranged in front of and behind the upstream homology arm, wherein the nucleotide sequence of the upstream homology arm is SEQ ID NO.11, the nucleotide sequence of the downstream homology arm is SEQ ID NO.12, and the original upstream homology arm^-19GAAGGTAA^-12The nucleotide sequence of the downstream homology arm is SEQ ID NO.13, the upstream homology arm being designed for synthesis^{- 19}TGTGGTAT^-12The nucleotide sequence of the downstream homology arm is SEQ ID No. 14. The nucleotide sequence of SEQ ID NO.14 was synthesized by Shanghai Bioengineering Co., Ltd and ligated between Hind III and EcoR I cleavage sites of pK19mobsacB vector (GenBank: LC 257601.1). The ligation products were transformed into E.coli DH 5. alpha. competent cells, 200. mu.L of the transformation solution was spread on solid LB plates containing kanamycin antibiotic (50. mu.g/mL final concentration) using a sterilized spreader, cultured overnight in an incubator at 37 ℃ and screened for positive transformants for extraction and preservation of the replacement vector pK19mobsacB-HTS 2.

Example 2: preparation of Corynebacterium glutamicum CICC23604/CGMCC1.15647 competent cell

(1) Picking single corynebacterium glutamicum colony, inoculating the single corynebacterium glutamicum colony in 10mL of seed culture medium, culturing at 37 ℃ at 220r/min overnight;

seed medium (1000mL), composition as follows: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 91g of sorbitol;

(2) transferring 1mL of the above bacterial solution into 100mL of seed culture medium, culturing at 37 deg.C and 220r/min to OD₆₀₀＝0.9；

(3) Transferring the bacterial liquid to a 100mL centrifuge tube, and carrying out ice bath for 15-20min to stop the growth of thalli;

(4) centrifuging at 4 deg.C and 5000r/min for 5min after ice bath, and collecting thallus;

(5) the centrifuged cells were washed 3 times with pre-cooled electrotransfer buffer (ETM);

electrotransfer buffer (1000mL), composition per liter as follows: 91g of sorbitol, 91g of mannitol and 100mL of glycerol;

(6) after washing, resuspending the thallus with 1000 μ L of electrotransfer buffer solution to obtain competent cells;

(7) the prepared competent cells are subpackaged in 100 mu L/tube and stored at-80 ℃ for later use.

Example 3: replacement vector electrotransformation of corynebacterium glutamicum competent cells

Firstly, a nucleic acid ultramicro spectrophotometer is utilized to measure the concentration of a replacement vector pK19mobsacB-HTS1 or pK19mobsacB-HTS2 fragment, 1800V electric shock is carried out for 5ms after the concentration reaches 300 mu g/mL, the electric transformation is carried out, the electric transformation is respectively carried out on the electric transformation to corynebacterium glutamicum CICC23604 competent cells and corynebacterium glutamicum CGMCC1.15647 competent cells, the obtained cells are recovered and cultured for 1h at 30 ℃ by using a liquid recovery culture medium, 100 mu L of the electric transformation is taken to be coated on an LB solid culture medium containing 25 mu g/mL kanamycin, the electric transformation is carried out for 2 days at 37 ℃, and corynebacterium glutamicum CICC23604 transformants with kanamycin resistance and corynebacterium glutamicum CGMCC1.15647 transformants with kanamycin resistance are respectively screened.

Wherein the liquid recovery culture medium comprises the following components per 1000 mL: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 91g of sorbitol and 69.4g of mannitol.

Example 4: culture and identification of positive recombinant bacteria

(1) Kanamycin-resistant flat plate primary screen

Selecting single colonies on the kanamycin-resistant plate in example 3, respectively inoculating the single colonies in a liquid LBG culture medium containing 25 mug/mL kanamycin, extracting a genome as a template DNA, and performing PCR amplification by using a kanamycin-resistant gene primer for verification, wherein a target band at 795bp is a positive transformant;

wherein the LBG medium: 5g/L of glucose, 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl;

the kanamycin resistance gene primer sequences are as follows:

F1：5′-ATGATTGAACAAGATGGATTGC-3′，

R1：5′-TCAGAAGAACTCGTCAAGAAGGCG-3′；

the PCR amplification system is as follows:

TABLE 1 PCR amplification System

The PCR amplification procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

Strains with specific bands at 795bp were selected for further validation as strains with homologous single crossover.

(2) Sucrose flat plate double screen

Respectively inoculating the strains which are verified to be correct and have homologous single exchange into a liquid LBG culture medium without antibiotics, carrying out natural passage for 3 times, carrying out 24-hour culture in each generation, finally coating 200 mu L of bacterial liquid on an LBG solid culture medium (without antibiotics) containing 10% of sucrose, selecting bacterial colonies, carrying out dibbling on the LBG solid culture medium with kanamycin resistance (25 mu g/mL) for culture, screening bacterial colonies which can grow on the LBG culture medium with 10% of sucrose but cannot grow on the LBG culture medium with kanamycin resistance, extracting genomes, and verifying by adopting PCR amplification;

wherein the primer sequences for PCR amplification are as follows:

F2：5′-AAATGAGGGAATGTGGTAT-3′，

R2：5′-TTATAGAACTCCAGCTTTTTTCA-3′；

the 3' terminal sequence of the F2 primer contains a site after substitution^-19TGTGGTAT^-12；

The PCR amplification system is as follows:

TABLE 2 PCR amplification System

The PCR amplification procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

Specific bands at 936bp are selected and respectively connected to pMD18-T vectors, vector primers are adopted for sequencing, and recombinant corynebacterium glutamicum HTS1 and HTS2 which complete two times of homologous single exchange are obtained after verification.

Example 5: stability verification of recombinant Corynebacterium glutamicum

The screened and verified recombinant corynebacterium glutamicum HTS1 and HTS2 are subjected to subculture respectively, a single colony on a plate is selected and inoculated into a liquid LBG culture medium without antibiotics for 12 hours, then continuous subculture is carried out for 30 generations according to the inoculum size of 1% by volume percentage, the last generation of bacterial liquid is taken for genome extraction, and colony PCR verification is carried out by taking F2 and R2 as primers. The results show that the use of primers F2 and R2 can amplify a specific gene band of about 936bp, which is consistent with the theoretical value, demonstrating that the substitution is^-19TGTGGTAT^-12Has successfully integrated and stably exists on the genomes of recombinant corynebacterium glutamicum HTS1 and HTS 2;

wherein, the PCR amplification system comprises the following components:

TABLE 3 PCR amplification System

The PCR amplification procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

Example 6: l-lysine fermentation test

The recombinant corynebacterium glutamicum HTS1 and HTS2 prepared above were inoculated into 100mL of BG culture medium (glucose 5g/L, peptone 10g/L, yeast extract 5g/L, NaCl10g/L), respectively, and seed-cultured at 30 ℃ and 220rpm for 20 hours, after which it was inoculated into 100mL of fermentation medium (glucose 100g/L,peptone 20g/L, corn steep liquor 30mL/L, urea 5g/L, (NH)₄)₂SO₄25g/L, L-leucine 0.34g/L, KH₂PO₄2g/L，MgSO₄·7H₂O1.5g/L, biotin 0.001g/L) was cultured for 48 hours, samples were taken every 12 hours, and the content of L-lysine in the fermentation broth was measured by biosensing analyzer SBA-40C (biological research, manufactured by institute of sciences, Shandong province), with the results shown in tables 4 and 5.

TABLE 4 average production of L-lysine from various time points of recombinant Corynebacterium glutamicum HTS1 and of the original strain

TABLE 5 average production of L-lysine from various time points of recombinant Corynebacterium glutamicum HTS2 and the original strain

The results show that the L-lysine content in the recombinant Corynebacterium glutamicum HTS1 fermentation broth reaches 43.9g/L and is increased by 6.8% compared with the original strain after 48h of fermentation, and the L-lysine content in the recombinant Corynebacterium glutamicum HTS2 fermentation broth reaches 1.02g/L which is 2.32 times of that of the original strain, which indicates that the nucleotide sequence from the front-19 to the-12 of the ATG of the initiation codon in the 5' end sequence of the Corynebacterium glutamicum HTS gene^-19GAAGGTAA^-12Is replaced by^-19TGTGGTAT^-12The fermentation level of the L-lysine can be improved, and the method is a novel method for improving the yield of the L-lysine.

The stability analysis of the sequences before and after the replacement of the nucleotide sequence from the position-19 to the position-12 before the initiation codon ATG in the 5 ' end sequence of the HTS gene in the Corynebacterium glutamicum CICC23604 and CGMCC1.15647 (SEQ ID NO.3 and SEQ ID NO.10) was performed by using RNAfold web server (http:// rna.tbi.univie.ac.at/cgi-bin/RNAwebsuite/RNAfold.cgi), and the results showed that the sequences before and after the replacement of the nucleotide sequence from the position-19 to the position-12 before the initiation codon ATG in the 5 ' end sequence of the HTS gene in the Corynebacterium glutamicum CICC23604 and CGMCC1.15647 were stable, and the results showed that the sequences after the replacement of the nucleotide sequence from the position-19 to the position-12 before the initiation codon ATG in the 5 ' end sequence of the HTS gene in the Corynebacterium glutamicum CICC23604 and CGMCC1.15647Nucleotide sequence^-19GAAGGTAA^-12Is replaced by^-19TGTGGTAT^-12Then, the minimum free energy (minimum free energy) of the 5' -terminal sequence was increased from-44.6 kcal/mol to-44.2 kcal/mol. This indicates that sequence replacement can reduce the stability of the 5' terminal sequence, which in turn makes the HTS gene easier to be transcribed and translated, and ultimately increases the acid production level of amino acid fermentation.

SEQUENCE LISTING

<110> university of Qilu Industrial science

ZHUCHENG DONGXIAO BIOTECHNOLOGY Co.,Ltd.

<120> recombinant corynebacterium for modifying 5' terminal sequence of HTS gene and application thereof

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 301

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CICC23604

<400> 1

Met Ser Thr Gly Leu Thr Ala Lys Thr Gly Val Glu His Phe Gly Thr

1 5 10 15

Val Gly Val Ala Met Val Thr Pro Phe Thr Glu Ser Gly Asp Ile Asp

20 25 30

Ile Ala Ala Gly Arg Glu Val Ala Ala Tyr Leu Val Asp Lys Gly Leu

35 40 45

Asp Ser Leu Val Leu Ala Gly Thr Thr Gly Glu Ser Pro Thr Thr Thr

50 55 60

Ala Ala Glu Lys Leu Glu Leu Leu Lys Ala Val Arg Glu Glu Val Gly

65 70 75 80

Asp Arg Ala Lys Leu Ile Ala Gly Val Gly Thr Asn Asn Thr Arg Thr

85 90 95

Ser Val Glu Leu Ala Glu Ala Ala Ala Ser Ala Gly Ala Asp Gly Leu

100 105 110

Leu Val Val Thr Pro Tyr Tyr Ser Lys Pro Ser Gln Glu Gly Leu Leu

115 120 125

Ala His Phe Gly Ala Ile Ala Ala Ala Thr Glu Val Pro Ile Cys Leu

130 135 140

Tyr Asp Ile Pro Gly Arg Ser Gly Ile Pro Ile Glu Ser Asp Thr Met

145 150 155 160

Arg Arg Leu Ser Glu Leu Pro Thr Ile Leu Ala Val Lys Asp Ala Lys

165 170 175

Gly Asp Leu Val Ala Ala Thr Ser Leu Ile Lys Glu Thr Gly Leu Ala

180 185 190

Trp Tyr Ser Gly Asp Asp Pro Leu Asn Leu Val Trp Leu Ala Leu Gly

195 200 205

Gly Ser Gly Phe Ile Ser Val Ile Gly His Ala Ala Pro Thr Ala Leu

210 215 220

Arg Glu Leu Tyr Thr Ser Phe Glu Glu Gly Asp Leu Val Arg Ala Arg

225 230 235 240

Glu Ile Asn Ala Lys Leu Ser Pro Leu Val Ala Ala Gln Gly Arg Leu

245 250 255

Gly Gly Val Ser Leu Ala Lys Ala Ala Leu Arg Leu Gln Gly Ile Asn

260 265 270

Val Gly Asp Pro Arg Leu Pro Ile Met Ala Pro Asn Glu Gln Glu Leu

275 280 285

Glu Ala Leu Arg Glu Asp Met Lys Lys Ala Gly Val Leu

290 295 300

<210> 2

<211> 906

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CICC23604

<400> 2

atgagcacag gtttaacagc taagaccgga gtagagcact tcggcaccgt tggagtagca 60

atggttactc cattcacgga atccggagac atcgatatcg ctgctggccg cgaagtcgcg 120

gcttatttgg ttgataaggg cttggattct ttggttctcg cgggcaccac tggtgaatcc 180

ccaacgacaa ccgccgctga aaaactagaa ctgctcaagg ccgttcgtga ggaagttggg 240

gatcgggcga agctcatcgc cggtgtcgga accaacaaca cgcggacatc tgtggaactt 300

gcggaagctg ctgcttctgc tggcgcagac ggccttttag ttgtaactcc ttattactcc 360

aagccgagcc aagagggatt gctggcgcac ttcggtgcaa ttgctgcagc aacagaggtt 420

ccaatttgtc tctatgacat tcctggtcgg tcaggtattc caattgagtc tgataccatg 480

agacgcctga gtgaattacc tacgattttg gcggtcaagg acgccaaggg tgacctcgtt 540

gcagccacgt cattgatcaa agaaacggga cttgcctggt attcaggcga tgacccacta 600

aaccttgttt ggcttgcttt gggcggatca ggtttcattt ccgtaattgg acatgcagcc 660

cccacagcat tacgtgagtt gtacacaagc ttcgaggaag gcgacctcgt ccgtgcgcgg 720

gaaatcaacg ccaaactatc accgctggta gctgcccaag gtcgcttggg tggagtcagc 780

ttggcaaaag ctgctctgcg tctgcagggc atcaacgtag gagatcctcg acttccaatt 840

atggctccaa atgagcagga acttgaggct ctccgagaag acatgaaaaa agctggagtt 900

ctataa 906

<210> 3

<211> 150

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CICC23604

<400> 3

tttggctgta aaagacagcc gtaaaaacct cttgctcatg tcaattgttc ttatcggaat 60

gtggcttggg cgattgttat gcaaaagttg ttaggttttt tgcggggttg tttaaccccc 120

aaatgaggga agaaggtaac cttgaactct 150

<210> 4

<211> 500

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CICC23604

<400> 4

gctgcttaat gcgctggaag aaaaacttgg cgatgaaccg aatgcacttt taaggaaaaa 60

gcaggctcgt caagcagctc gcgctgtgct gcccaacgct acagagtcca gaatcgtggt 120

gtctggaaac ttccgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180

cgtcgaaatc cgcgaagtag cggtaggatg tttaagaaag ctgcaggtag cagcgccaac 240

tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagccc 300

gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atatagttaa 360

gacaacattt ttggctgtaa aagacagccg taaaaacctc ttgctcatgt caattgttct 420

tatcggaatg tggcttgggc gattgttatg caaaagttgt taggtttttt gcggggttgt 480

ttaaccccca aatgagggaa 500

<210> 5

<211> 500

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CICC23604

<400> 5

ccttgaactc tatgagcaca ggtttaacag ctaagaccgg agtagagcac ttcggcaccg 60

ttggagtagc aatggttact ccattcacgg aatccggaga catcgatatc gctgctggcc 120

gcgaagtcgc ggcttatttg gttgataagg gcttggattc tttggttctc gcgggcacca 180

ctggtgaatc cccaacgaca accgccgctg aaaaactaga actgctcaag gccgttcgtg 240

aggaagttgg ggatcgggcg aagctcatcg ccggtgtcgg aaccaacaac acgcggacat 300

ctgtggaact tgcggaagct gctgcttctg ctggcgcaga cggcctttta gttgtaactc 360

cttattactc caagccgagc caagagggat tgctggcgca cttcggtgca attgctgcag 420

caacagaggt tccaatttgt ctctatgaca ttcctggtcg gtcaggtatt ccaattgagt 480

ctgataccat gagacgcctg 500

<210> 6

<211> 1008

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CICC23604

<400> 6

gctgcttaat gcgctggaag aaaaacttgg cgatgaaccg aatgcacttt taaggaaaaa 60

gcaggctcgt caagcagctc gcgctgtgct gcccaacgct acagagtcca gaatcgtggt 120

gtctggaaac ttccgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180

cgtcgaaatc cgcgaagtag cggtaggatg tttaagaaag ctgcaggtag cagcgccaac 240

tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagccc 300

gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atatagttaa 360

gacaacattt ttggctgtaa aagacagccg taaaaacctc ttgctcatgt caattgttct 420

tatcggaatg tggcttgggc gattgttatg caaaagttgt taggtttttt gcggggttgt 480

ttaaccccca aatgagggaa gaaggtaacc ttgaactcta tgagcacagg tttaacagct 540

aagaccggag tagagcactt cggcaccgtt ggagtagcaa tggttactcc attcacggaa 600

tccggagaca tcgatatcgc tgctggccgc gaagtcgcgg cttatttggt tgataagggc 660

ttggattctt tggttctcgc gggcaccact ggtgaatccc caacgacaac cgccgctgaa 720

aaactagaac tgctcaaggc cgttcgtgag gaagttgggg atcgggcgaa gctcatcgcc 780

ggtgtcggaa ccaacaacac gcggacatct gtggaacttg cggaagctgc tgcttctgct 840

ggcgcagacg gccttttagt tgtaactcct tattactcca agccgagcca agagggattg 900

ctggcgcact tcggtgcaat tgctgcagca acagaggttc caatttgtct ctatgacatt 960

cctggtcggt caggtattcc aattgagtct gataccatga gacgcctg 1008

<210> 7

<211> 1008

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (501)..(508)

<223> replacement of the original nucleotide sequence GAAGGTAA by TGTGGTAT

<400> 7

gctgcttaat gcgctggaag aaaaacttgg cgatgaaccg aatgcacttt taaggaaaaa 60

gcaggctcgt caagcagctc gcgctgtgct gcccaacgct acagagtcca gaatcgtggt 120

gtctggaaac ttccgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180

cgtcgaaatc cgcgaagtag cggtaggatg tttaagaaag ctgcaggtag cagcgccaac 240

tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagccc 300

gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atatagttaa 360

gacaacattt ttggctgtaa aagacagccg taaaaacctc ttgctcatgt caattgttct 420

tatcggaatg tggcttgggc gattgttatg caaaagttgt taggtttttt gcggggttgt 480

ttaaccccca aatgagggaa tgtggtatcc ttgaactcta tgagcacagg tttaacagct 540

aagaccggag tagagcactt cggcaccgtt ggagtagcaa tggttactcc attcacggaa 600

tccggagaca tcgatatcgc tgctggccgc gaagtcgcgg cttatttggt tgataagggc 660

ttggattctt tggttctcgc gggcaccact ggtgaatccc caacgacaac cgccgctgaa 720

aaactagaac tgctcaaggc cgttcgtgag gaagttgggg atcgggcgaa gctcatcgcc 780

ggtgtcggaa ccaacaacac gcggacatct gtggaacttg cggaagctgc tgcttctgct 840

ggcgcagacg gccttttagt tgtaactcct tattactcca agccgagcca agagggattg 900

ctggcgcact tcggtgcaat tgctgcagca acagaggttc caatttgtct ctatgacatt 960

cctggtcggt caggtattcc aattgagtct gataccatga gacgcctg 1008

<210> 8

<211> 301

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC1.15647

<400> 8

Met Ser Thr Gly Leu Thr Ala Lys Thr Gly Val Glu His Phe Gly Thr

1 5 10 15

Val Gly Val Ala Met Val Thr Pro Phe Thr Glu Ser Gly Asp Ile Asp

20 25 30

Ile Ala Ala Gly Arg Glu Leu Ala Ala Tyr Leu Val Asp Lys Gly Leu

35 40 45

Asp Ser Leu Val Leu Ala Gly Thr Thr Gly Glu Ser Pro Thr Thr Thr

50 55 60

Ala Ala Glu Lys Leu Glu Leu Leu Lys Ala Val Arg Glu Glu Val Gly

65 70 75 80

Asp Arg Ala Lys Leu Ile Ala Gly Val Gly Thr Asn Asn Thr Arg Thr

85 90 95

Ser Val Glu Leu Ala Glu Ala Ala Ala Ser Ala Gly Ala Asp Gly Leu

100 105 110

Leu Val Val Thr Pro Tyr Tyr Ser Lys Pro Ser Gln Glu Gly Leu Leu

115 120 125

Ala His Phe Gly Ala Ile Ala Ala Ala Thr Glu Val Pro Ile Cys Leu

130 135 140

Tyr Asp Ile Pro Gly Arg Ser Gly Ile Pro Ile Glu Ser Asp Thr Met

145 150 155 160

Arg Arg Leu Ser Glu Leu Pro Thr Ile Leu Ala Val Lys Asp Ala Lys

165 170 175

Gly Asp Leu Val Ala Ala Thr Ser Leu Ile Lys Glu Thr Gly Leu Ala

180 185 190

Trp Tyr Ser Gly Asp Asp Pro Leu Asn Leu Val Trp Leu Ala Leu Gly

195 200 205

Gly Ser Gly Phe Ile Ser Val Ile Gly His Ala Ala Pro Thr Ala Leu

210 215 220

Arg Glu Leu Tyr Thr Ser Phe Glu Glu Gly Asp Leu Val Arg Ala Arg

225 230 235 240

Glu Ile Asn Ala Lys Leu Ser Pro Leu Val Ala Ala Gln Gly Arg Leu

245 250 255

Gly Gly Val Ser Leu Ala Lys Ala Ala Leu Arg Leu Gln Gly Ile Asn

260 265 270

Val Gly Asp Pro Arg Leu Pro Ile Met Ala Pro Asn Glu Gln Glu Leu

275 280 285

Glu Ala Leu Arg Glu Asp Met Lys Lys Ala Gly Val Leu

290 295 300

<210> 9

<211> 906

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC1.15647

<400> 9

atgagcacag gtttaacagc taagaccgga gtagagcact tcggcaccgt tggagtagca 60

atggttactc cattcacgga atccggagac atcgatatcg ctgctggccg cgagctcgcg 120

gcttatttgg ttgataaggg cttggattct ttggttctcg cgggcaccac tggtgaatcc 180

ccaacgacaa ctgccgctga aaaactagaa ctgctcaagg ccgttcgtga ggaagttggg 240

gatcgggcga agctcatcgc cggtgtcgga accaacaaca cgcggacatc tgtggaactt 300

gcggaagctg ctgcttctgc tggcgcagac ggccttttag ttgtaactcc ttattactcc 360

aagccgagcc aagagggatt gctggcgcac ttcggtgcaa ttgctgcagc aacagaggtt 420

ccaatttgtc tctatgacat tcctggtcgg tcaggtattc caattgagtc tgataccatg 480

agacgcctga gtgaattacc tacgattttg gcggtcaagg acgccaaggg tgacctcgtt 540

gcagccacgt cattgatcaa agaaacggga cttgcctggt attcaggcga tgacccacta 600

aaccttgttt ggcttgcttt gggcggatca ggtttcattt ccgtaattgg acatgcagcc 660

cccacagcat tacgtgagtt gtacacaagc ttcgaggaag gcgacctcgt ccgtgcgcgg 720

gaaatcaacg ccaaactatc accgctggta gctgcccaag gtcgcttggg tggagtcagc 780

ttggcaaaag ctgctctgcg tctgcagggc atcaacgtag gagatcctcg acttccaatt 840

atggctccaa atgagcagga acttgaggct ctccgagaag acatgaaaaa agctggagtt 900

ctataa 906

<210> 10

<211> 150

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC1.15647

<400> 10

tttggctgta aaagacagcc gtaaaaacct cttgctcatg tcaattgttc ttatcggaat 60

gtggcttggg cgattgttat gcaaaagttg ttaggttttt tgcggggttg tttaaccccc 120

aaatgaggga agaaggtaac cttgaactct 150

<210> 11

<211> 500

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC1.15647

<400> 11

gctgcttaat gcgctggaag aaaaacttgg cgatgaaccg aatgcaattt taaggaaaaa 60

gcaggctcgt caagcagctc gcgctgtgct gcccaacgct acagagtcca gaatcgtagt 120

gtctggaaac ttccgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180

cgttgaaatc cgcgaagtag cggtagaatg tttaagaaag ctgcaggtag cagcgccaac 240

tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagccc 300

gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atataggtaa 360

gacaacattt ttggctgtaa aagacagccg taaaaacctc ttgctcatgt caattgttct 420

tatcggaatg tggcttgggc gattgttatg caaaagttgt taggtttttt gcggggttgt 480

ttaaccccca aatgagggaa 500

<210> 12

<211> 500

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC1.15647

<400> 12

ccttgaactc tatgagcaca ggtttaacag ctaagaccgg agtagagcac ttcggcaccg 60

ttggagtagc aatggttact ccattcacgg aatccggaga catcgatatc gctgctggcc 120

gcgagctcgc ggcttatttg gttgataagg gcttggattc tttggttctc gcgggcacca 180

ctggtgaatc cccaacgaca actgccgctg aaaaactaga actgctcaag gccgttcgtg 240

aggaagttgg ggatcgggcg aagctcatcg ccggtgtcgg aaccaacaac acgcggacat 300

ctgtggaact tgcggaagct gctgcttctg ctggcgcaga cggcctttta gttgtaactc 360

cttattactc caagccgagc caagagggat tgctggcgca cttcggtgca attgctgcag 420

caacagaggt tccaatttgt ctctatgaca ttcctggtcg gtcaggtatt ccaattgagt 480

ctgataccat gagacgcctg 500

<210> 13

<211> 1008

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC1.15647

<400> 13

gctgcttaat gcgctggaag aaaaacttgg cgatgaaccg aatgcaattt taaggaaaaa 60

gcaggctcgt caagcagctc gcgctgtgct gcccaacgct acagagtcca gaatcgtagt 120

gtctggaaac ttccgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180

cgttgaaatc cgcgaagtag cggtagaatg tttaagaaag ctgcaggtag cagcgccaac 240

tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagccc 300

gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atataggtaa 360

gacaacattt ttggctgtaa aagacagccg taaaaacctc ttgctcatgt caattgttct 420

tatcggaatg tggcttgggc gattgttatg caaaagttgt taggtttttt gcggggttgt 480

ttaaccccca aatgagggaa gaaggtaacc ttgaactcta tgagcacagg tttaacagct 540

aagaccggag tagagcactt cggcaccgtt ggagtagcaa tggttactcc attcacggaa 600

tccggagaca tcgatatcgc tgctggccgc gagctcgcgg cttatttggt tgataagggc 660

ttggattctt tggttctcgc gggcaccact ggtgaatccc caacgacaac tgccgctgaa 720

aaactagaac tgctcaaggc cgttcgtgag gaagttgggg atcgggcgaa gctcatcgcc 780

ggtgtcggaa ccaacaacac gcggacatct gtggaacttg cggaagctgc tgcttctgct 840

ggcgcagacg gccttttagt tgtaactcct tattactcca agccgagcca agagggattg 900

ctggcgcact tcggtgcaat tgctgcagca acagaggttc caatttgtct ctatgacatt 960

cctggtcggt caggtattcc aattgagtct gataccatga gacgcctg 1008

<210> 14

<211> 1008

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (501)..(508)

<223> replacement of the original nucleotide sequence GAAGGTAA by TGTGGTAT

<400> 14

gctgcttaat gcgctggaag aaaaacttgg cgatgaaccg aatgcaattt taaggaaaaa 60

gcaggctcgt caagcagctc gcgctgtgct gcccaacgct acagagtcca gaatcgtagt 120

gtctggaaac ttccgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180

cgttgaaatc cgcgaagtag cggtagaatg tttaagaaag ctgcaggtag cagcgccaac 240

tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagccc 300

gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atataggtaa 360

gacaacattt ttggctgtaa aagacagccg taaaaacctc ttgctcatgt caattgttct 420

tatcggaatg tggcttgggc gattgttatg caaaagttgt taggtttttt gcggggttgt 480

ttaaccccca aatgagggaa tgtggtatcc ttgaactcta tgagcacagg tttaacagct 540

aagaccggag tagagcactt cggcaccgtt ggagtagcaa tggttactcc attcacggaa 600

tccggagaca tcgatatcgc tgctggccgc gagctcgcgg cttatttggt tgataagggc 660

ttggattctt tggttctcgc gggcaccact ggtgaatccc caacgacaac tgccgctgaa 720

aaactagaac tgctcaaggc cgttcgtgag gaagttgggg atcgggcgaa gctcatcgcc 780

ggtgtcggaa ccaacaacac gcggacatct gtggaacttg cggaagctgc tgcttctgct 840

ggcgcagacg gccttttagt tgtaactcct tattactcca agccgagcca agagggattg 900

ctggcgcact tcggtgcaat tgctgcagca acagaggttc caatttgtct ctatgacatt 960

cctggtcggt caggtattcc aattgagtct gataccatga gacgcctg 1008

Claims (10)

1. A recombinant corynebacterium for modifying the 5 'end sequence of HTS gene features that the nucleotide sequence before-19-12 bits of ATG as start codon in the 5' end sequence of HTS gene is inserted in the host corynebacterium^-19GAAGGTAA^-12Is replaced by^{- 19}TGTGGTAT^-12And the stability of the secondary structure of the 5' end sequence of the HTS gene is reduced, so that the HTS gene is easier to be transcribed or expressed.

2. The recombinant coryneform bacterium according to claim 1, wherein said coryneform host bacterium is Corynebacterium glutamicum;

preferably, the corynebacterium glutamicum is corynebacterium glutamicum CICC23604 or corynebacterium glutamicum CGMCC 1.15647.

3. The recombinant coryneform bacterium according to claim 1, wherein the amino acid sequence of said HTS is SEQ ID No.1 or SEQ ID No. 8;

preferably, the amino acid sequence of the HTS has an amino acid sequence with a sequence identity of 99.7% or more with the sequence of SEQ ID No.1 or SEQ ID No. 8.

4. The recombinant coryneform bacterium according to claim 1, wherein the nucleotide sequence of said HTS gene is SEQ ID No.2 or SEQ ID No. 9;

preferably, the nucleotide sequence of the HTS gene is a nucleotide sequence with the sequence identity of SEQ ID NO.2 or SEQ ID NO.9 of more than or equal to 99.7%.

5. The recombinant coryneform bacterium according to claim 1, wherein the 5' -end sequence of said HTS gene is SEQ ID No.3 or SEQ ID No. 10.

6. The method of constructing recombinant coryneform bacteria according to claim 1, comprising the steps of:

(1) synthesis of upstream homology arm-^-19TGTGGTAT^-12Nucleotide sequence of downstream homology arm, wherein upstream homology arm and downstream homology arm are nucleotide sequences from-19 to-12 before ATG (initiation codon) in 5' end sequence of HTS gene^-19GAAGGTAA^-12A nucleotide sequence with the length of 500-600 bp is respectively arranged in front of and behind the nucleotide sequence;

(2) the upstream homology arm^-19TGTGGTAT^-12The nucleotide sequence of the downstream homology arm was ligated into the pK19mobsacB vector, constructing a replacement vector;

(3) transforming the replacement vector into a corynebacterium host strain competent cell, and screening a positive transformant with kanamycin resistance to obtain a recombinant strain with first homologous single exchange;

(4) and after natural passage of the recombinant bacteria subjected to the first homologous single exchange, screening bacterial colonies which can grow on a 10% sucrose culture medium but cannot grow on a kanamycin-resistant culture medium, and verifying to obtain the recombinant corynebacteria subjected to the two homologous single exchanges.

7. The construction method according to claim 6, wherein one or more of the following conditions are satisfied:

i. upstream homology arm in step (2)^-19TGTGGTAT^-12The nucleotide sequence of the downstream homology arm is ligated between Hind III, EcoR I cleavage sites of the pK19mobsacB vector;

screening positive transformants with kanamycin resistance in step (3) by using PCR amplification technology with kanamycin resistance gene primers having the following sequences:

F1：5′-ATGATTGAACAAGATGGATTGC-3′，

R1：5′-TCAGAAGAACTCGTCAAGAAGGCG-3′；

the PCR amplification system comprises: 2 XHiFi-PCRmaster 10 μ L, 10 μmol/L forward primer 1 μ L, 10 μmol/L reverse primer 1 μ L, template 1 μ L, ddH₂O 7μL；

The procedure of PCR amplification is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;

the verification in the step (4) is verified by adopting a PCR amplification technology, and the sequence of the primer amplified by the PCR is as follows:

F2：5′-AAATGAGGGAATGTGGTAT-3′，

R2：5′-TTATAGAACTCCAGCTTTTTTCA-3′；

the PCR amplification system comprises: 2 XHiFi-PCRmaster 10 μ L, 10 μmol/L forward primer 1 μ L, 10 μmol/L reverse primer 1 μ L, template 1 μ L, ddH₂O 7μL；

The procedure of PCR amplification is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;

the medium used in step (4) is LBG medium: 5g/L of glucose, 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl.

8. Use of the recombinant coryneform bacterium according to claim 1 for the production of L-lysine.

9. The use according to claim 8, wherein the recombinant corynebacterium is inoculated into a liquid LBG medium for seed culture, and then inoculated into a fermentation medium for fermentation culture in an inoculum size of 2-5% by volume;

the LBG medium: 5g/L of glucose, 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl;

the fermentation medium comprises: 100g/L glucose, 20g/L peptone, 30mL/L corn steep liquor, 5g/L urea, (NH)₄)₂SO₄25g/L, L-leucine 0.34g/L, KH₂PO₄ 2g/L，MgSO₄·7H₂O1.5g/L and biotin 0.001 g/L.

10. The use of claim 9, wherein the seed culture conditions are 200-220 rpm at 28-30 ℃ for 18-25 h; the fermentation culture conditions are 200-220 rpm and 28-30 ℃.