LU102870B1 - Recombinant corynebacterium strain for modifying 5'-end sequence of 4-hydroxy-tetrahydrodipicolinate synthase (hts) gene and use thereof - Google Patents

Abstract

The present disclosure relates to a construction method of a recombinant Corynebacterium strain for modifying a 5'-end sequence of a 4-hydroxy-tetrahydrodipicolinate synthase (HTS) and use thereof. The recombinant Corynebacterium strain of the present disclosure replaces a nucleoside sequence from nucleosides -19 to -12 before a start codon ATG in the 5'-end sequence of a HTS gene in a host Corynebacterium strain to reduce the stability of a secondary structure of the 5'-end sequence of the HTS gene, to allow for easier transcription or expression thereof. Compared with an original strain, the recombinant Corynebacterium strain is improved in the synthesis efficiency of L-lysine and further the yield of L-lysine, and production cost is reduced; moreover, there is no conflict with the existing modification method for high L-lysine production, but there is still a need to further verify whether the method mutually facilitates the existing modification method.

Description

RECOMBINANT CORYNEBACTERIUM STRAIN FOR MODIFYING S'-END HU102670 SEQUENCE OF 4-HYDROXY-TETRAHYDRODIPICOLINATE SYNTHASE (HTS)

GENE AND USE THEREOF TECHNICAL FIELD

[01] The present disclosure relates to a recombinant Corynebacterium strain for modifying a 5'-end sequence of a 4-hydroxy-tetrahydrodipicolinate synthase (HTS) gene and use thereof, and belongs to the technical field of bioengineering.

BACKGROUND ART

[02] L-Lysine is a basic unit of protein as a raw material for the synthesis of human hormones, enzymes and antibodies, and participates in metabolism and various physiological activities of human body. The L-lysine is an essential amino acid for the human body and is generally used in various amino acid infusion formulas. L-lysine was originally separated from protein hydrolysates, and then from chemical synthesis and enzymatic methods, and was first produced in Japan using microbial fermentation in 1960. The L-lysine is produced by raw materials such as various starch-hydrolyzed saccharides or molasses through fermentation with mutants of Brevibacterium and Corynebacterium. Feed-grade lysine is obtained through separation, concentration, evaporation, crystallization and drying, and food-grade lysine and pharmaceutical-grade lysine are obtained by refining. In China, research on lysine strain breeding and fermentation has begun since the mid-1960s, but it was difficult to industrialize due to low yields. It was not until the industrialization of lysine in the world in the late 1970s and early 1980s that research in China made a breakthrough. At present, most of the lysine- producing enterprises in the world adopt the fermentation method, which produce L-lysine, and have developed basically mature production processes.

[03] The microorganisms used for L-lysine production are from a plurality of genuses, such as Corynebacterium, Bacillus, and Escherichia. However, wild-type strains have poor ability to produce L-lysine, produce a plurality of metabolic by-products, and are difficult to realize the preparation of high-purity and high-yield L-lysine. Therefore, it is usually necessary to obtain strains that produce high-yield L-lysine. At present, methods for obtaining high-yield L-lysine strains mainly include mutation screening and breeding or genetic engineering and breeding. Mutation screening and breeding refers to the induction of unspecified gene site mutations in strains through ultraviolet irradiation or other external conditions, and then screening to obtain high-yield strains. This method lacks directionality, and is difficult to control gene mutation sites, with poor expectations of strain performance. Genetic engineering and breeding is to optimize the breeding of strains through specific genetic modification methods, such as introducing 102870 beneficial enzyme genes with high enzyme activity by increasing copies or site-directed mutation, or knocking out unfavorable genes to make enzyme activity/expression disappear. At present, Corynebacterium glutamicum CICC 23604 has been widely used in industrial fermentation to produce various amino acids, and its products have been certified by the FDA as "generally regarded as safe" (GRAS). Therefore, the use of metabolic engineering methods to construct recombinant C. glutamicum is an effective way to produce food-safe L-lysine.

[04] The 5'-end sequence of a gene generally refers to a DNA sequence located upstream of the 5'-end of a structural gene, which usually contains RNA polymerase recognition, binding, and transcriptional start sites. It contains a conserved sequence required for the RNA polymerase specific binding and transcription initiation. The conserved sequence is not transcribed by itself, and its characteristics are initially identified by mutations that can increase or decrease the gene transcription rate. Promoters are generally located inside the 5'-end sequence of the gene, and their length varies with biological species, generally no more than 200 bp. They are typical cis- acting elements that combine with transcription factors (trans-acting factors) to regulate the level, location, and pattern of gene expression (Chinese Patent No. CN109385424A). Replacement of the 5'-end sequence of the gene can be used to regulate gene expression in a specific way, such as conditional expression or overexpression thereof. PCR-based gene targeting achieves the chromosomal integration of the upstream regulatory sequence of the open reading frame (ORF) through homologous recombination, which can change the genome stably (Chinese Patent No. CN111655860A).

[05] Although there have been a plurality of reports on increasing the L-lysine yield, it is still necessary to develop new methods for increasing the L-lysine yield.

SUMMARY

[06] In view of the shortcomings in the prior art, the present disclosure provides a construction method of a recombinant Corynebacterium strain for modifying a 5'-end sequence of a 4-hydroxy-tetrahydrodipicolinate synthase (H7S) gene and use thereof. The recombinant Corynebacterium strain of the present disclosure is obtained by modification of a host Corynebacterium strain by genetic engineering. The specific strategy is to replace part of a nucleotide sequence of a 5'-end sequence of a HTS gene with a 5'-end sequence of a gene to obtain a recombinant Corynebacterium strain with high L-lysine yield.

[07] Term description:

[08] The HTS is an enzyme widely present in various bacteria and other microorganisms that is important in synthesis of the L-lysine, and can catalyze formation of L-aspartate-4- 1 semialdehyde to dihydrodipicolinic acid. HU102870

[09] The technical solutions of the present disclosure are as follows:

[10] A recombinant Corynebacterium strain for modifying a 5'-end sequence of a HTS gene is provided, where in a host Corynebacterium strain, > GAAGGTAA2, a nucleoside sequence from nucleosides -19 to -12 before a start codon ATG in the 5'-end sequence of the HTS gene, is replaced with "*’TGTGGTAT- to reduce the stability of a secondary structure of the 5'-end sequence of the HTS gene, to allow for easier transcription or expression thereof.

[11] According to the present disclosure, preferably, the host Corynebacterium strain may be Corynebacterium glutamicum, and more preferably C. glutamicum CICC23604 or C.

glutamicum CGMCC1.15647.

[12] According to the present disclosure, preferably, the amino acid sequence of the HTS may be shown in SEQ ID NO. 1 or SEQ ID NO. 8.

[13] According to the present disclosure, preferably, the amino acid sequence of the HTS may be an amino acid sequence having a sequence identity of >99.7% to SEQ ID NO. 1 or SEQ ID NO. 8.

[14] According to the present disclosure, preferably, the nucleotide sequence of the HTS gene may be shown in SEQ ID NO. 2 or SEQ ID NO. 9.

[15] According to the present disclosure, preferably, the nucleotide sequence of the HTS gene may be a nucleotide sequence having a sequence identity of >99.7% to SEQ ID NO. 2 or SEQ ID NO. 9.

[16] According to the present disclosure, preferably, the 5'-end sequence of the HTS gene may be shown in SEQ ID NO. 3 or SEQ ID NO. 10.

[17] A construction method of the foregoing recombinant Corynebacterium strain is provided, including the following steps:

[18] step 1, synthesizing a nucleotide sequence of upstream homologous arm-""TGTGGTAT-

2.downstream homologous arm, where the upstream homologous arm and the downstream homologous arm are 500-600 bp nucleoside sequences from nucleosides -19 to -12 before and after a start codon ATG in the 5'-end sequence of HTS gene, respectively;

[19] step 2, ligating the nucleotide sequence of the upstream homologous arm- 1’TGTGGTAT-?-downstream homologous arm to a pKl9mobsacB vector to construct a replacement vector;

[20] step 3, transforming the replacement vector into host Corynebacterium competent cells, and screening a kanamycin-resistant positive transformant to obtain a recombinant strain that has undergone a first homologous single crossover; and

[21] step 4, after natural passage of the recombinant strain that has undergone the first 2 homologous single crossover, screening colonies that are capable of growing on a 10% sucrose 102870 medium but not on a kanamycin-resistant medium, and verifying the colonies to obtain a recombinant Corynebacterium strain that has undergone two homologous single crossovers.

[22] According to the present disclosure, preferably, the nucleotide sequence of the upstream homologous arm-""’TGTGGTAT-”-downstream homologous arm in step 2 may be ligated between restriction sites of Hind III and EcoR I of the pK19mobsacB vector.

[23] According to the present disclosure, preferably, in step 3, kanamycin-resistant gene primers may be used to screen the kanamycin-resistant positive transformant by PCR amplification technology, where sequences of the primers are as follows:

[24] Fl: 5-ATGATTGAACAAGATGGATTGC-3' (SEQ ID NO.15), and

[25] RI1:5-TCAGAAGAACTCGTCAAGAAGGCG-3' (SEQ ID NO.16).

[26] According to the present disclosure, preferably, in step (4), the verifying may be conducted by the PCR amplification, where the PCR amplification has a primer sequence as follows:

[27] F2:5-AAATGAGGGAATGTGGTAT-3' (SEQ ID NO.17),

[28] R2: 5'-TTATAGAACTCCAGCTTTTTTCA-3' (SEQ ID NO.18)

[29] Further preferably, a system of the PCR amplification may include: 10 pL of 2xHiFi- PCRmaster, 1 uL of 10 umol/L upstream primer, 1 pL of 10 pmol/L. downstream primer, 1 pL of template, and 7 uL of ddH2O;

[30] a program of the PCR amplification may be: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 4°C.

[31] According to the present disclosure, preferably, the medium used in step 4 may be an LBG medium: 5 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl.

[32] Use of the foregoing recombinant Corynebacterium strain in the production of L-lysine is provided.

[33] According to the present disclosure, preferably, the use may be intended to inoculate the recombinant Corynebacterium strain into a liquid LBG medium for seed culture, and thereafter inoculate 2-5% by volume of inoculum onto a fermentation medium for fermentation culture;

[34] the LBG medium may include: 5 g/L glucose, 10 g/L peptone, 5 g/L. yeast extract, and g/L NaCl;

[35] the fermentation medium may include: 100 g/L glucose, 20 g/L peptone, 30 mL/L corn steep liquor, 5 g/L urea, 25 g/L (NH4)2SO4, 0.34 g/L L-leucine, 2 g/L KHz:PO4, 1.5 g/L MgSO4:7H,0, and 0.001 g/L biotin.

[36] According to the present disclosure, preferably, the seed culture may be conducted at 3

200-220 rpm and 28-30°C for 18-25 h; the fermentation culture may be conducted at 200-220 102870 rpm and 28-30°C.

[37] The technical principle of the present disclosure is as follows:

[38] The 5'-end sequence of the gene contains a promoter region sequence of the gene, and the secondary structure of the promoter region sequence can affect the promoter efficiency, and further influence the expression activity of post-promoter genes. The present disclosure can reduce the stability of the secondary structure of the HTS gene of the 5'-end sequence to result in easier transcription or expression thereof by replacing ""GAAGGTAA-, a nucleoside sequence from nucleosides -19 to -12 before a start codon ATG in the 5'-end sequence of the HTS gene, with ""TGTGGTAT-"?,

[39] Beneficial effects:

[40] The present disclosure provides a recombinant Corynebacterium strain for modifying a 5'-end sequence of a HTS gene, where "’"GAAGGTAA?, a nucleoside sequence from nucleosides -19 to -12 before a start codon ATG in the 5'-end sequence of the HTS gene, is replaced with "PTGTGGTAT-2. Compared with an original strain, the recombinant Corynebacterium strain is improved in the synthesis efficiency of L-lysine and further the yield of L-lysine, and production cost is reduced; moreover, there is no conflict with the existing modification method for high L-lysine production, but there is still a need to further verify whether the method mutually facilitates the existing modification method.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[41] Technical solutions of the present disclosure will be further described below with reference to examples, but the protection scope of the present disclosure is not limited thereto. All reagents and chemicals involved in the examples are common commercial products, unless otherwise specified; all experimental methods involved in the examples are conventional technical means in the art, unless otherwise specified.

[42] Microbial source:

[43] C. glutamicum CICC23604 was purchased from the China Center of Industrial Culture Collection (CICC) and deposited with an accession number of CICC23604, where the amino acid sequence of HTS is shown in SEQ ID NO. 1, the nucleotide sequence of the HTS gene is shown in SEQ ID NO. 2, and the 5'-end sequence of the HTS gene is shown in SEQ ID NO. 3.

[44] C. glutamicum CGMCC1.15647 was purchased from the China General Microbiological Culture Collection Center (CGMCC) and deposited with an accession number of CGMCC1.15647, where the amino acid sequence of HTS is shown in SEQ ID NO. 8, the nucleotide sequence of the HTS gene is shown in SEQ ID NO. 9, and the 5'-end sequence of the 4

HTS gene is shown in SEQ ID NO. 10. HU102870

[45] The amino acid sequence shown in SEQ ID NO. 1 is 99.7% identical to that shown in SEQ ID NO. 8, the nucleotide sequence shown in SEQ ID NO. 2 is 99.7% identical to that shown in SEQ ID NO. 9, and the nucleotide sequence shown in SEQ ID NO. 3 is 100% identical to that shown in SEQ ID NO. 10.

[46] The corn steep liquor involved in the examples is a special corn steep liquor for fermentation and may be purchased from Zhucheng Dongxiao Biotechnology Co., Ltd.

[47] Example 1: Synthesis of homologous arm gene containing a nucleotide sequence to be replaced and construction of replacement vector

[48] 1.1 C. glutamicum CICC23604

[49] For C. glutamicum CICC23604, a nucleotide sequence of the upstream homologous arm- ‘PTGTGGTAT'2-downstream homologous arm was synthesized, where the upstream homologous arm and the downstream homologous arm were 500-600 bp nucleoside sequences before and after a nucleoside sequence from nucleosides -19 to -12, ""GAAGGTAA"?, before a start codon ATG in the 5'-end sequence of C. glutamicum CICC23604 HTS gene, respectively; herein, the nucleotide sequence of the upstream homologous arm was shown in SEQ ID NO. 4, and the nucleotide sequence of the downstream homologous arm was shown in SEQ ID NO. 5; the nucleotide sequence of the original upstream homologous arm-""GAAGGTAA"2- downstream homologous arm was shown in SEQ ID NO. 6, and the nucleotide sequence of the designed and synthesized upstream homologous arm-""TGTGGTAT-'2-downstream homologous arm was shown in SEQ ID NO. 7. The nucleotide sequence shown in SEQ ID NO. 7 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated between restriction sites of HindIII and EcoR I of the pK19mobsacB vector (GenBank: LC257601.1). The ligation product was transformed into Escherichia coli DH5a competent cells, and 200 pL. of transformation buffer was spread on a solid LB plate supplemented with kanamycin (final concentration of 50 ug/mL) using a sterilized spreader; the plate was incubated overnight in an incubator at 37°C to screen and obtain positive transformants, which were used for the extraction and storage of the replacement vector pK19mobsacB-HTS1.

[50] 1.2 C. glutamicum CGMCC1.15647 [S1] For C. glutamicum CGMCC1.15647, a nucleotide sequence of the upstream homologous arm-""TGTGGTAT-?-downstream homologous arm was synthesized, where the upstream homologous arm and the downstream homologous arm were 500-600 bp nucleoside sequences before and after a nucleoside sequence from nucleosides -19 to -12, ""GAAGGTAA"?, before a start codon ATG in the 5'-end sequence of C. glutamicum CGMCC1.15647 HTS gene, respectively; herein, the nucleotide sequence of the upstream homologous arm was shown in

SEQ ID NO. 11, and the nucleotide sequence of the downstream homologous arm was shown in 102870 SEQ ID NO. 12; the nucleotide sequence of the original upstream homologous arm- PYGAAGGTAA2-downstream homologous arm was shown in SEQ ID NO. 13, and the nucleotide sequence of the designed and synthesized upstream homologous arm-"°TGTGGTAT-

12.downstream homologous arm was shown in SEQ ID NO. 14. The nucleotide sequence shown in SEQ ID NO. 14 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated between restriction sites of HindIII and EcoRI of the pK19mobsacB vector (GenBank: LC257601.1). The ligation product was transformed into Æ coli DHS5a competent cells, and 200 pL of transformation buffer was spread on a solid LB plate supplemented with kanamycin (final concentration of 50 pg/mL) using a sterilized spreader; the plate was incubated overnight in an incubator at 37°C to screen and obtain positive transformants, which were used for the extraction and storage of the replacement vector pK19mobsacB-HTS2.

[S2] Example 2: Preparation of C. glutamicum CICC23604/CGMCC1.15647 competent cells

[53] (1) A single colony of C. glutamicum was picked and inoculated in 10 mL of seed culture medium at 37°C and 220r/min for overnight culture; [S4] the seed culture medium (1,000 mL) was composed of: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, and 91 g of sorbitol; [SS] (2) 1 mL of the above bacterial suspension was transferred to 100 mL of seed culture medium, and cultured to OD600 = 0.9 at 37°C and 220 r/min; [S6] (3) the bacterial suspension was transferred to a 100 mL centrifuge tube, and placed on ice bath for 15-20 min to stop the growth of the cells;

[57] (4) after ice bath, the bacterial suspension was centrifuged at 4°C and 5,000 r/min for 5min to collect the cells; [S8] (5) the centrifuged cells were washed thrice with pre-cooled electroporation buffer (ETM); [S9] each liter of electroporation buffer (1,000 mL) was composed of: 91 g of sorbitol, 91 g of mannitol, and 100 mL of glycerol;

[60] (6) after washing, the cells were resuspended with 1,000 uL of electroporation buffer to obtain competent cells;

[61] (7) 100 pL of the prepared competent cells were dispensed into a tube and stored at - 80°C for later use.

[62] Example 3: Electroporation of replacement vector into C. glutamicum competent cells

[63] First, the fragment concentration of the replacement vector pK19mobsacB-HTS1 or pK19mobsacB-HTS2 was determined by using a nucleic acid ultramicrospectrophotometer.

After reaching a concentration of 300 pg/mL, the replacement vector was electroporated at a 6

1,800 V electric shock for 5 ms for transformation into C. glutamicum CICC23604 competent 20 cells and C. glutamicum competent cells, respectively; after the resulting cells were resuscitated and cultured at 30°C for 1 h using a liquid resuscitation medium, 100 pL of the cell suspension was spread on the LB solid medium supplemented with 25 pg/ml kanamycin and cultured at 37°C for two days; kanamycin-resistant transformants of C. glutamicum CICC23604 and C. glutamicum CGMCC1.15647 were screened, respectively.

[64] Herein, the liquid resuscitation medium (1,000 mL) was composed of: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 91 g of sorbitol, and 69.4 g of mannitol.

[65] Example 4: Culture and identification of positive recombinant strains

[66] (1) Primary screening by a kanamycin resistance plate

[67] Single colonies on the kanamycin-resistant plate in Example 3 were picked and inoculated in liquid LBG media supplemented with 25 pg/ml. kanamycin, respectively; the genome was extracted as template DNA, and kanamycin-resistant gene primers were used for PCR amplification and verification. A band of interest at 795 bp indicated a positive transformant;

[68] where the LBG medium included: 5 g/L glucose, 10 g/L. peptone, 5 g/L. yeast extract, and 10 g/L NaCl;

[69] sequences of the kanamycin-resistant gene primers were as follows:

[70] F1:5-ATGATTGAACAAGATGGATTGC-3' (SEQ ID NO.15), and

[71] RI: 5-TCAGAAGAACTCGTCAAGAAGGCG-3' (SEQ ID NO.16).

[72] A system of the PCR amplification was as follows:

[73] Table 1 PCR amplification system

I Reagent Volume (pL) 2xHiFi-PCRmaster 10 F1 (10 pmol/L) 1 R1 (10 pmol/L) 1 Template 1 ddH,O 7

[75] A program of the PCR amplification was as follows:

[76] initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 4°C.

[77] À strain with a specific band at 795 bp was selected as a strain with homologous single crossover for further verification.

[78] (2) Secondary screening on sucrose plate

[79] The above correctly verified strains with homologous single crossover were inoculated 7 into antibiotic-free liquid LBG medium, respectively, and passed naturally for three generations. 02870 where each generation was cultured for 24 h; finally, 200 pL of bacterial suspension was spread onto solid LBG medium supplemented with 10% sucrose (without antibiotics); after 18-24 h, colonies were picked and inoculated onto the kanamycin-resistant (25 pg/mL) solid LBG medium for culture. Colonies that could grow on 10% sucrose LBG medium but could not grow on the kanamycin-resistant LBG medium were screened; the genome was extracted and verified by PCR for verification;

[80] where, the primer sequences for the PCR amplification were as follows:

[81] F2:5-AAATGAGGGAATGTGGTAT-3' (SEQ ID NO.17), and

[82] R2:5-TTATAGAACTCCAGCTTTTTTCA-3' (SEQ ID NO.18).

[83] The 3'-end sequence of the F2 primer contained a replaced site *'TGTGGTAT-'%;

[84] a system of the PCR amplification was as follows:

[85] Table 2 PCR amplification system

[86] Reagent Volume (pL) 2xHiFi-PCRmaster 10 F2 (10 pmol/L) 1 R2 (10 pmol/L) 1 Template 1 ddH,O 7

[87] A program of the PCR amplification was as follows:

[88] initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 4°C;

[89] a specific band at about 936 bp was picked, separately ligated to a pMD18-T vector, and sequenced using vector primers; after verification, recombinant Corynebacterium strains HTS1 and HTS2 that completed two homologous single crossovers were obtained.

[90] Example 5: Stability verification of recombinant Corynebacterium glutamicum

[91] The screened and verified recombinant Corynebacterium glutamicum HTS1 and HTS2 were subcultured separately; a single colony on the plate was selected and inoculated in a liquid LBG medium without antibiotics for 12 h, and subcultured for 30 generations according to an inoculation amount of 1% by volume; the last generation of bacterial solution was selected for genome extraction, and colony PCR verification was conducted using F2 and R2 as primers. The results show that a specific gene band can be amplified using the primers F2 and R2, with a size of about 936 bp that is line with the theoretical value. It is proved that the replaced - l’TGTGGTAT‘? has been successfully integrated into the genomes of the recombinant Corynebacterium glutamicum HTS1 and HTS2, and exists stably; 8

. . LU102870

[92] where, a system of the PCR amplification was as follows:

[93] Table 3 PCR amplification system

[94] Reagent Volume (uL) 2xHiFi-PCRmaster 10 F2 (10 pmol/L) 1 R2 (10 umol/L) 1 Template 1 ddH20 7

[95] A program of the PCR amplification was as follows:

[96] initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 4°C;

[97] Example 6: L-lysine fermentation test

[98] The above prepared recombinant Corynebacterium strains HTS1 and HTS2 were inoculated into 100 mL of LBG medium (5 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and g/L NaCl) for seed culture at 220 rpm and 30°C for 20 h, respectively; thereafter, 2% by volume of inoculum was inoculated into 100 mL of fermentation medium (100 g/L. glucose, 20 g/L peptone, 30 mL/L corn steep liquor, 5 g/L urea, 25 g/L. (NH4)2SO4, 0.34 g/L L-leucine, 2 g/L KHzPO4, 1.5 g/L MgSO04-7H;0, and 0.001 g/L biotin) for fermentation culture for 48 h; both recombinant Corynebacterium strains were sampled every 12 h, and the content of L-lysine in the fermentation broth was determined by the SBA-40C biosensor analyzer (manufactured by the Biology Institute of Shandong Academy of Sciences). The results are shown in Tables 4 and 5.

[99] Table 4 Average L-lysine yields of recombinant Corynebacterium strain HTS1 and original strain at different times

[100] Fermentation time Strain 12h 24h 36h 48 h Recombinant strain HTS1 4.2 g/L 17.8 g/L 42.3 g/L 43.9 g/L Original strain CICC23604 4.0 g/L 18.2 g/L 40.3 g/L 41.1 g/L

[101] Table 5 Average L-lysine yields of recombinant Corynebacterium strain HTS2 and original strain at different times

[102] Fermentation time Strain 12h 24h 36h 48 h Recombinant strain HTS2 0.03 g/L 0.47 g/L 0.83 g/L 1.02 g/L Original strain CGMCC1.15647 0.03 g/L 0.25 g/L 0.48 g/L 0.44 g/L

[103] According to the results, compared with the original strain, the content of L-lysine in the 9 fermentation broth of the recombinant Corynebacterium strain HTS1 reached 43.9 g/L 48 h after 02870 fermentation, which was 6.8% higher than that of the original strain; the content of L-lysine in the fermentation broth of the recombinant Corynebacterium strain HTS2 reached 1.02 g/L, which was 2.32 times that of the original strain. This indicated that replacement of the nucleoside sequence "’GAAGGTAA-” from nucleosides -19 to -12 before the start codon ATG in the 5'- end sequence of the HTS gene of C. glutamicum with "“’TGTGGTAT-? could increase the acid production level of L-lysine fermentation, which is also a new way to increase the L-lysine yield.

[104] Stability analysis was conducted on sequences before and after replacement of nucleoside sequence from nucleosides -19 to -12 before the start codon ATG in the 5'-end sequences (SEQ ID NO. 3 and SEQ ID NO. 10) of the HTS gene of C. glutamicum CICC23604 and C. glutamicum CGMCC1.15647 using RNAfold web server (http://rna.tbi.univie.ac.at/cgi- bin/RNA WebSuite/RNAfold.cgi). According to the results, after the nucleosides sequence - PYGAAGGTAA? from nucleosides -19 to -12 before the start codon ATG in the 5'-end sequence of the HTS gene of C. glutamicum CICC23604 was replaced with "*’TGTGGTAT-?, the minimum free energy of the 5'-end sequence was increased from -44.6 kcal/mol to -44.2 kcal/mol. This indicated that the sequence replacement could reduce the stability of the 5'-end sequence, which in turn makes the HTS gene easier to be transcribed and translated and ultimately increases the level of acid production by amino acid fermentation.

SEQUENCE LISTING LU102870 <110> Qilu University of Technology Zhucheng Dongxiao Biotechnology Co., Ltd. <120> RECOMBINANT CORYNEBACTERIUM STRAIN FOR MODIFYING 5'-END

SEQUENCE OF 4-HYDROXY-TETRAHYDRODIPICOLINATE SYNTHASE (HTS) GENE AND

USE

THEREOF <130> HKJU202109571 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> HTS of 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 Ile Ala Ala Gly Arg Glu Val Ala Ala Tyr Leu Val Asp Lys Gly Leu 40 45 Asp Ser Leu Val Leu Ala Gly Thr Thr Gly Glu Ser Pro Thr Thr Thr 11

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 12

<211> 906 LU102870 <212> DNA <213> Artificial Sequence <220> <223> HTS gene of Corynebacterium glutamicum CICC23604 <400> 2 atgagcacag gtttaacagc taagaccgga gtagagcact tcggeaccgt tggagtagca 60 atggttactc cattcacgga atccggagac atcgatatcg ctgetggccg cgaagtcgeg 120 gcttatttgg ttgataaggg cttggattct ttggttctcg cgggeaccac tggtgaatce 180 ccaacgacaa ccgecgetga aaaactagaa ctgetcaagg cegttcgtga ggaagttggg 240 gatcgggcga agctcatcge cggtgtcgga accaacaaca cgeggacate tgtggaactt 300 gcggaagcetg ctgettetge tggegeagac ggecttttag ttgtaactee ttattactce 360 aagccgagcc aagagggatt getggegeac ttcggtgeaa ttgetgecage aacagagett 420 ccaatttgtc tctatgacat tcctggtcgg tcaggtattc caattgagtc tgataccatg 480 agacgcctga gtgaattacc tacgattttg gcggtcaagg acgccaaggg tgacctegtt 540 gcagccacgt cattgatcaa agaaacggga cttecctget attcaggcga tgacccacta 600 aaccttgttt gecttecttt gggcggatea getttcattt ccgtaattgg acatgcagec 660 cccacagcat tacgtgagtt gtacacaagc ttcgaggaag gegacctegt ccgtgcgegg 720 gaaatcaacg ccaaactatc accgctggta gctgcccaag gtegettggg tggagtcage 780 ttggcaaaag ctectctecg tctgcaggec atcaacgtag gagatecteg acttccaatt 840 atggctccaa atgagcagga acttgaggct ctccgagaag acatgaaaaa agctggagtt 900 ctataa 906 <210> 3 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> 5'-end sequence of the HTS gene of Corynebacterium glutamicum

CICC23604

13

<400> 3 tttggctgta aaagacagcee gtaaaaacct cttectcatg tcaattettc ttatcggaat 60 gtgecttggg cgattgttat gcaaaagttg ttaggttttt tgcggggtte tttaacccece 120 aaatgaggga agaaggtaac cttgaactct 150 <210> 4 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> the upstream homologous arm of Corynebacterium glutamicum CICC23604 <400> 4 getgettaat gcectegaag aaaaacttgg cgatgaaccg aatecacttt taaggaaaaa 60 gcaggctcgt caagcagete gegetgtget gcccaacect acagagteca gaatcgtggt 120 gtctggaaac ttecgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180 cgtcgaaatc cgcgaagtag cggtaggatg tttaagaaag ctgcaggtag cagcgccaac 240 tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagecce 300 gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atatagttaa 360 gacaacattt ttggctgtaa aagacagccg taaaaaccte ttgctcatet caattgttct 420 tatcggaatg tggettgggc gattgttatg caaaagttgt taggtttttt gcgggettgt 480 ttaaccccca aatgagggaa 500 <210> 5 <211> 500 <212> DNA <213> Artificial Sequence <220> 14

<223> the downstream homologous arm of Corynebacterium glutamicum LU102870 CICC23604

<400> 5 ccttgaactc tatgagcaca ggtttaacag ctaagaccgg agtagagcac ttcggcaccg 60 ttegagtagc aatggttact ccattcacgg aatccggaga catcgatate getgetggee 120 gcgaagtcec ggcttatttg gttgataagg gettggattc tttggttctec gecgggeacca 180 ctggtgaatc cccaacgaca accgccectg aaaaactaga actgetcaag gccgttcgtg 240 aggaagttgg ggatcgggcg aagctcatcg ccggtgtcgg aaccaacaac acgeggacat 300 ctgtggaact tgcggaagcet getgettetg ctggegeaga cggectttta gttgtaacte 360 cttattactc caagecgagc caagagggat tgctggegea cttcggtgea attectgcag 1420 caacagaggt tccaatttgt ctctatgaca ttcctggtcg gtcaggtatt ccaattgagt 480 ctgataccat gagacgcctg 500

<210> 6

<211> 1008

<212> DNA

<213> Artificial Sequence

<220>

<223> the original upstream and downstream homologous arm of CICC23604

<400> 6 getgettaat gegetggaag aaaaacttgg cgatgaaccg aatgcacttt taaggaaaaa 60 gcaggctcgt caagcagete gegetgtget gcccaacect acagagteca gaatcgtggt 120 gtctggaaac ttccgeacct ggaggcattt cattggcate cgagccagtg aacatgcaga 180 cgtcgaaatc cgcgaagtag cggtaggatg tttaagaaag ctgcaggtag cagcgccaac 240 tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagecce 300 gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atatagttaa 360 gacaacattt ttggctgtaa aagacagccg taaaaaccte ttectcatet caattettet 420 tatcggaatg tggcttgggc gattgttatg caaaagttet taggtttttt gcggggttgt 480 ttaaccccca aatgagggaa gaaggtaacc ttgaactcta tgagcacagg tttaacaget 540 aagaccggag tagagcactt cggcaccett ggagtagcaa tggttactce attcacggaa 600 tccggagaca tcgatatcge tgetggecge gaagtegegg cttatttggt tgataaggge 660 LU102870 ttggattctt tggttctcge gggcaccact getgaatecc caacgacaac cgecgetgaa 720 aaactagaac tgctcaaggce cgttcgtgag gaagttgggg atcgggegaa getcatcgee 780 ggtgtcggaa ccaacaacac geggacatct gtggaacttg cggaagetge tgettetget 840 ggcgcagacg gecttttagt tgtaactcct tattactcca agccgagcca agagggattg 900 ctggcgceact tecggtecaat tectgcagca acagaggtte caatttetet ctatgacatt 960 cctggtcggt caggtattec aattgagtct gataccatga gacgectg 1008 <210> 7 <211> 1008 <212> DNA <213> Artificial Sequence <220> <223> the designed and synthesized upstream and downstream homologous arm <220> <221> misc feature <222> (501)..(508) <223> The original nucleotide sequence GAAGGTAA is replaced with

TGTGGTAT <400> 7 getgettaat gcectegaag aaaaacttgg cgatgaaccg aatecacttt taaggaaaaa 60 gcaggctcgt caagcagete gegetgtget gcccaacect acagagteca gaatcgtggt 120 gtctggaaac ttecgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180 cgtcgaaatc cgcgaagtag cggtaggatg tttaagaaag ctgcaggtag cagcgccaac 240 tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagecce 300 gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atatagttaa 360 gacaacattt ttggctgtaa aagacagccg taaaaaccte ttectcatet caattettet 420 tatcggaatg tggettgggc gattgttatg caaaagttgt taggtttttt gcgggettgt 480 ttaaccccca aatgagggaa tgtggtatee ttgaactcta tgagcacagg tttaacaget 540 16 aagaccggag tagagcactt cggcaccgtt ggagtagcaa tggttactce attcacggaa 600 LU102870 tccggagaca tcgatatcge tgetggecge gaagtegegg cttatttggt tgataaggge 660 ttggattctt tggttctcge gggcaccact getgaatecc caacgacaac cgecgetgaa 720 aaactagaac tectcaaggc cgticgtgag gaagttgggg atcgggegaa getcatcgee 780 ggtgtcggaa ccaacaacac geggacatct gtggaacttg cggaagetge tgettetget 840 ggcgcagacg gecttttagt tgtaactect tattactcca agccgagcca agagggattg 900 ctggcgceact tcggtgcaat tectgcagca acagaggtte caatttetet ctatgacatt 960 cctggtcggt caggtattce aattgagtct gataccatga gacgectg 1008 <210> 8 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> HTS of 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

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

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

17

Leu Val Val Thr Pro Tyr Tyr Ser Lys Pro Ser Gln Glu Gly Leu Leu LU102870 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> Artificial Sequence <220> <223> HTS gene of Corynebacterium glutamicum CGMCC1.15647 18

<400> 9 LU102870 atgagcacag gtttaacagc taagaccgga gtagagcact tcggcaccet tegagtagca 60 atggttactc cattcacgga atccggagac atcgatatcg ctgetggccg cgagetcgeg 120 gcttatttgg ttgataaggg cttggattct ttggttetcg cgggcaccac tggtgaatce 180 ccaacgacaa ctgccgctga aaaactagaa ctgetcaagg cegttcgtga ggaagttggg 240 gatcgggcga agctcatcge cggtetcgga accaacaaca cgcegacatc tgtggaactt 300 gcggaagctg ctgettetge tggcgcagac ggecttttag ttgtaactec ttattactce 360 aagccgagcc aagagggatt gctggcgcac ttcggtgcaa ttgctgcagc aacagaggtt 420 ccaatttgtc tctatgacat tcctggtcgg tcaggtattc caattgagtc tgataccatg 480 agacgcctga gtgaattacc tacgattttg gcggtcaagg acgccaaggg tgacctegtt 540 gcagccacgt cattgatcaa agaaacggga cttecctget attcaggcga tgacccacta 600 aaccttgttt gecttgcttt gggcggatca ggtttcattt ccgtaattgg acatgcagec 660 cccacagcat tacgtgagtt gtacacaagc ttcgaggaag gegacctegt ccgtgcgegg 720 gaaatcaacg ccaaactatc accgetggta getgeccaag gtegettggg tggagtcage 780 ttggcaaaag ctectctecg tetgecagggce atcaacgtag gagatecteg acttccaatt 840 atggctccaa atgagcagga acttgaggct ctccgagaag acatgaaaaa agctggagtt 900 ctataa 906 <210> 10 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> 5'-end sequence of the HTS gene of Corynebacterium glutamicum

CGMCC1.15647 <400> 10 titgectgta aaagacagec gtaaaaacct citgctcatg tcaattgtte ttatcggaat 60 gtggcttggg cgattgttat gcaaaagttg ttaggttttt tgeggggttg tttaacccec 120 aaatgaggga agaaggtaac cttgaactct 150

19

<210> 11 LU102870 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> the upstream homologous arm of Corynebacterium glutamicum CGMCC1.15647 <400> 11 gctecttaat gcectegaag aaaaacttgg cgatgaaccg aatgcaattt taaggaaaaa 60 gcaggctegt caagcagctc gegetgtget gcccaacgct acagagtcca gaatcgtagt 120 gtctggaaac ttecgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180 cgttgaaatc cecgaagtag cggtagaatg tttaagaaag ctgcaggtag cagcgccaac 240 tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagecce 300 gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atataggtaa 360 gacaacattt ttggctgtaa aagacagccg taaaaaccte ttgctcatet caattgttct 420 tatcggaatg tggcttgggc gattgttatg caaaagttet taggtttttt gcggggttgt 480 ttaaccccca aatgagggaa 500 <210> 12 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> the downstream homologous arm of Corynebacterium glutamicum CGMCC1.15647 <400> 12 ccttgaactc tatgagcaca ggtttaacag ctaagaccgg agtagagcac ttcggcaccg 60 ttggagtagc aatggttact ccattcacgg aatccggaga catcgatatc getgetggee 120 gcgagctegc ggcttatttg gttgataagg gettggatte tttggttctc gcgggcacca 180 ctggtgaatc cccaacgaca acteccectg aaaaactaga actectcaag geegttegtg 240 LU102870 aggaagttgg ggatcgggcg aagctcatcg ccggtgtegg aaccaacaac acgeggacat 300 ctgtggaact tgcggaagcet getgettetg ctggegeaga cggectttta gttgtaacte 360 cttattactc caagccgagc caagagggat tgetggegea cttcggtgea attectgcag 420 caacagaggt tccaatttgt ctctatgaca ttcctggtcg gtcaggtatt ccaattgagt 480 ctgataccat gagacgcctg 500 <210> 13 <211> 1008 <212> DNA <213> Artificial Sequence <220> <223> the original upstream and downstream homologous arm of

CGMCC1.15647 <400> 13 gctecttaat gcectegaag aaaaacttgg cgatgaaccg aatgcaattt taaggaaaaa 60 gcaggctegt caagcagctc gegetgtget gcccaacgct acagagtcca gaatcgtagt 120 gtctggaaac ttecgcacct ggaggcattt cattggcatg cgagccagtg aacatgcaga 180 cgttgaaatc cecgaagtag cggtagaatg tttaagaaag ctgcaggtag cagcgccaac 240 tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaagecce 300 gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atataggtaa 360 gacaacattt ttggctgtaa aagacagccg taaaaaccte ttgctcatet caattgttct 420 tatcggaatg tggcttgggc gattgttatg caaaagttet taggtttttt gcggggttgt 480 ttaaccccca aatgagggaa gaaggtaacc ttgaactcta tgagcacagg tttaacaget 540 aagaccggag tagagcactt cggcaccett ggagtagcaa tggttactce attcacggaa 600 tccggagaca tcgatatcge tgetggeege gagctcgcgg cttatttggt tgataaggge 660 ttggattctt tggttctcge gggcaccact getgaatecc caacgacaac tgeegetgaa 720 aaactagaac tgctcaaggce cgttcgtgag gaagttgggg atcgggegaa getcatcgee 780 ggtgtcggaa ccaacaacac geggacatct gtggaacttg cggaagetge tgettctget 840 ggcgcagacg gecttttagt tgtaactcct tattactcca agccgagcca agagggattg 900 ctggcgcact teggtgcaat tectgcagca acagaggttc caatttgtct ctatgacatt 960

21 cctggtcggt caggtattce aattgagtct gataccatga gacgectg 1008 LU102870 <210> 14 <211> 1008 <212> DNA <213> Artificial Sequence <220> <223> the designed and synthesized upstream and downstream homologous arm <220> <221> misc_feature <222> (501)..(508) <223> The original nucleotide sequence GAAGGTAA is replaced with

TGTGGTAT <400> 14 getgettaat gegetggaag aaaaacttgg cgatgaaccg aatgcaattt taaggaaaaa 60 gcaggctegt caagcagctc gegetgtget gecccaacgcet acagagtcca gaatcgtagt 120 gtctggaaac ttccgeacct ggaggcattt cattggcate cgagccagtg aacatgcaga 180 cgttgaaatc cgecgaagtag cggtagaatg tttaagaaag ctgcaggtag cagcgccaac 240 tgttttcggt gattttgaga ttgaaacttt ggcagacgga tcgcaaatgg caacaageccc 300 gtatgtcatg gacttttaac gcaaagctca cacccacgag ctaaaaattc atataggtaa 360 gacaacattt ttggctgtaa aagacagccg taaaaacctc ttectcatet caattettet 420 tatcggaatg tggcttgggc gattgttatg caaaagttgt taggtttttt gcggggttgt 480 ttaaccccca aatgagggaa tgtggtatee ttgaactcta tgagcacagg tttaacaget 540 aagaccggag tagagcactt cggcaccett ggagtagcaa tggttactce attcacggaa 600 tccggagaca tcgatatcge tgetggeege gagctcgcgg cttatttggt tgataaggge 660 ttggattctt tggttctcge gggcaccact getgaatecc caacgacaac teccectgaa 720 aaactagaac tgctcaaggce cgttcgtgag gaagttgggg atcgggegaa getcatcgee 780 ggtgtcggaa ccaacaacac geggacatct gtggaacttg cggaagetge tgettetget 840 ggcgcagacg gecttttagt tgtaactcct tattactcca agccgagcca agagggattg 900 22 ctggcgceact tcggtgcaat tectgcagca acagaggtte caatttetet ctatgacatt 960 LU102870 cctggtcggt caggtattec aattgagtct gataccatga gacgectg 1008

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of the kanamycin-resistant gene primer F1

<400> 15 atgattgaac aagatggatt gc 22

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of the kanamycin-resistant gene primer R1

<400> 16 tcagaagaac tcgtcaagaa ggcg 24

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> the primer sequences for the PCR amplification F2 23

<400> 17 aaatgaggga atgtggtat 19 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> the primer sequences for the PCR amplification R2 <400> 18 ttatagaact ccagcttttt tca 23

24

Claims (10)

WHAT IS CLAIMED IS:

1. A recombinant Corynebacterium strain for modifying a 5'-end sequence of a 4-hydroxy- tetrahydrodipicolinate synthase (HTS) gene, wherein in a host Corynebacterium strain, 7 GAAGGTAA"*, a nucleoside sequence from nucleosides -19 to -12 before a start codon ATG in the 5'-end sequence of the HTS gene, is replaced with "’TGTGGTAT-! to reduce the stability of a secondary structure of the 5'-end sequence of the H7S gene, to allow for easier transcription or expression thereof.

2. The recombinant Corynebacterium strain according to claim 1, wherein the host Corynebacterium strain is Corynebacterium glutamicum; preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum CICC23604 or Corynebacterium glutamicum CGMCC 1.15647.

3. The recombinant Corynebacterium strain according to claim 1, wherein the H7S gene has an amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 8; preferably, the amino acid sequence of the HTS is an amino acid sequence having a sequence identity of >99.7% to SEQ ID NO. 1 or SEQ ID NO. 8.

4. The recombinant Corynebacterium strain according to claim 1, wherein the H7S gene has a nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 9; preferably, the nucleotide sequence of the HTS gene is a nucleotide sequence having a sequence identity of >99.7% to SEQ ID NO. 2 or SEQ ID NO. 9.

5. The recombinant Corynebacterium strain according to claim 1, wherein the 5'-end sequence of the HTS gene is shown in SEQ ID NO. 3 or SEQ ID NO. 10.

6. A construction method of the recombinant Corynebacterium strain according to claim 1, comprising the following steps: step 1, synthesizing a nucleotide sequence of upstream homologous arm-""TGTGGTAT-?- downstream homologous arm, wherein the upstream homologous arm and the downstream homologous arm are 500-600 bp nucleoside sequences from nucleosides -19 to -12 before and after a start codon ATG in the 5'-end sequence of H7S gene, respectively;

step 2, ligating the nucleotide sequence of the upstream homologous arm-""TGTGGTAT-AJ102870 downstream homologous arm to a pK19mobsacB vector to construct a replacement vector; step 3, transforming the replacement vector into host Corynebacterium competent cells, and screening a kanamycin-resistant positive transformant to obtain a recombinant strain that has undergone a first homologous single crossover; and step 4, after natural passage of the recombinant strain that has undergone the first homologous single crossover, screening colonies that are capable of growing on a 10% sucrose medium but not on a kanamycin-resistant medium, and verifying the colonies to obtain a recombinant Corynebacterium strain that has undergone two homologous single crossovers.

7. The construction method according to claim 6, wherein one or more of the following conditions are met: i. the nucleotide sequence of the upstream homologous arm-""TGTGGTAT-!2-downstream homologous arm in step 2 is ligated between restriction sites of Hind III and EcoR I of the pK19mobsacB vector; ii. in step 3, kanamycin-resistant gene primers are used to screen the kanamycin-resistant positive transformant by PCR amplification technology, wherein sequences of the primers are as follows: F1: 5'-ATGATTGAACAAGATGGATTGC-3', set forth in SEQ ID NO.15, and R1: S'-TCAGAAGAACTCGTCAAGAAGGCG-3"', set forth in SEQ ID NO.16; a system of the PCR amplification comprises: 10 uL of 2xHiFi-PCRmaster, 1 uL of 10 umol/L upstream primer, 1 uL of 10 pmol/L downstream primer, 1 pL. of template, and 7 uL of ddH2O; a program of the PCR amplification is: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 4°C; iii. in step (4), the verifying is conducted by the PCR amplification, wherein the PCR amplification has a primer sequence as follows: F2: 5'-AAATGAGGGAATGTGGTAT-3, set forth in SEQ ID NO.17, R2: 5'-TTATAGAACTCCAGCTTTTTTCA-3/, set forth in SEQ ID NO.18; a system of the PCR amplification comprises: 10 uL of 2xHiFi-PCRmaster, 1 uL of 10 umol/L upstream primer, 1 uL of 10 pmol/L downstream primer, 1 pL. of template, and 7 uL of ddH2O; 26 a program of the PCR amplification is: initial denaturation at 95°C for 5 min; 30 cycles lf102870 denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 4°C; and iv, in step (4), the medium is an LBG medium comprising: 5 g/L. glucose, 10 g/L peptone, 5 g/L yeast extract and 10 g/L NaCl.

8. Use of the recombinant Corynebacterium strain according to claim 1 in the production of L-lysine.

9. The use according to claim 8, wherein the use is intended to inoculate the recombinant Corynebacterium strain into a liquid LBG medium for seed culture, and thereafter inoculate 2- 5% by volume of inoculum onto a fermentation medium for fermentation culture; the LBG medium comprises: 5 g/L glucose, 10 g/L. peptone, 5 g/L yeast extract, and 10 g/L NaCl; the fermentation medium comprises: 100 g/L glucose, 20 g/L. peptone, 30 mL/L corn steep liquor, 5 g/L urea, 25 g/L (NH4)2SO04, 0.34 g/L L-leucine, 2 g/L KH:PO4, 1.5 g/L MgSO4 7H20, and 0.001 g/L biotin.

10. The use according to claim 9, wherein the seed culture is conducted at 200-220 rpm and 28-30°C for 18-25 h; the fermentation culture is conducted at 200-220 rpm and 28-30°C.

27