LU102871B1 - Method for improving l-lysine yield of corynebacterium glutamicum recombinant strain - Google Patents

Abstract

The present disclosure relates to a method for improving L-lysine yield of a Corynebacterium glutamicum recombinant strain. The Corynebacterium glutamicum recombinant strain of the present disclosure replaces a nucleoside sequence from nucleosides -58 to -49 before a start codon ATG in the 5'-end sequence of a 4-hydroxy-tetrahydrodipicolinate reductase (HTR) gene in a host Corynebacterium glutamicum strain to reduce the stability of a secondary structure of the 5'-end sequence of the HTR gene, to allow for easier transcription or expression thereof. Compared with an original strain, the Corynebacterium glutamicum recombinant 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

METHOD FOR IMPROVING L-LYSINE YIELD OF CORYNEBACTERIUM LU102871

GLUTAMICUM RECOMBINANT STRAIN TECHNICAL FIELD

[01] The present disclosure relates to a method for improving L-lysine yield of a Corynebacterium glutamicum recombinant strain, and belongs to the technical field of bioengineering.

BACKGROUND ART

[02] L-Lysine, as a white or nearly-white free-flowing crystalline powder, is one of the important components of protein, and is also one of the eight essential amino acids that humans and animals cannot synthesize by themselves. The L-lysine is also called "the first essential amino acid" due to lacking in food generally. L-Lysine has various physiological functions, such as balancing the composition of amino acids, regulating the metabolic balance in the body, improving the absorption and utilization of cereal proteins for the body and promoting the growth and development of the body. Therefore, the L-lysine is widely used in the fields of feed additives, food fortifiers and pharmaceuticals. Due to strong market demand, the L-lysine has now become the second largest amino acid variety after glutamic acid, with an annual output of about 3.5 million tons. The L-type stereospecificity of amino acids determines that the production of amino acids by fermentation method is simpler and faster than the process of chemical synthesis. 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 by fermentation 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 LU102871 breeding is to optimize the breeding of strains through specific genetic modification methods, such as introducing 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 method for improving L-lysine yield of a Corynebacterium glutamicum recombinant strain. The Corynebacterium glutamicum recombinant strain of the present disclosure is obtained by modification of a host Corynebacterium glutamicum strain by genetic engineering. The specific strategy is to replace part of a nucleotide sequence of a 5'-end sequence of a 4-hydroxy- tetrahydrodipicolinate reductase (H7R) gene with a 5'-end sequence of a gene to obtain a Corynebacterium glutamicum recombinant strainwith high L-lysine yield.

[07] Term description: 1

[08] The HTR is an enzyme widely present in various bacteria and other microorganisms that LU102871 is important in synthesis of the L-lysine, and can catalyze formation of dihydrodipicolinic acid to hexahydrodipicolinic acid.

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

[10] A Corynebacterium glutamicum recombinant strain for modifying a 5'-end sequence of a HTR gene is provided, where in a host Corynebacterium glutamicum strain, **ACGTCTAGAC- 4 a nucleoside sequence from nucleosides -58 to -49 before a start codon ATG in the 5'-end sequence of the HTR gene, is replaced with S¥TGTGGTATAA™ to reduce the stability of a secondary structure of the 5'-end sequence of the HTR gene, to allow for easier transcription or expression thereof.

[11] According to the present disclosure, preferably, the host Corynebacterium glutamicum strain may be C. glutamicum CICC23604 or C. glutamicum CGMCC1.15647.

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

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

[14] According to the present disclosure, preferably, the nucleotide sequence of the HTR gene may be a nucleotide sequence having a sequence identity of >99.3% to SEQ ID NO. 2 or SEQ ID NO. 9.

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

[16] According to the present disclosure, preferably, the 5'-end sequence of the HTR gene may be a nucleotide sequence having a sequence identity of >98.0% to SEQ ID NO. 3 or SEQ ID NO. 10.

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

[18] step 1, synthesizing a nucleotide sequence of upstream homologous arm- BTGTGGTATAA*-downstream homologous arm, where the upstream homologous arm and the downstream homologous arm are 500-600 bp nucleoside sequences “*ACGTCTAGAC# from nucleosides -58 to -49 before and after a start codon ATG in the 5'-end sequence of HTR gene, respectively;

[19] step 2, ligating the nucleotide sequence of the upstream homologous arm- °°TGTGGTATAA“°-downstream homologous arm to a pK19mobsacB vector to construct a replacement vector;

[20] step 3, transforming the replacement vector into host Corynebacterium glutamicum 2 strain competent cells, and screening a kanamycin-resistant positive transformant to obtain a LU102871 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 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 Corynebacterium glutamicum recombinant strain that has undergone two homologous single crossovers.

[22] According to the present disclosure, preferably, the nucleotide sequence of the upstream homologous arm-*TGTGGTATAA*-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] FI: 5'"-ATGATTGAACAAGATGGATTGC-3', as shown in SEQ ID NO. 15, and

[25] RI1:5-TCAGAAGAACTCGTCAAGAAGGCG-3', as shown in SEQ ID NO. 16.

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

[27] 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.

[28] 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.

[29] In the present disclosure, after a conditioned medium is used for screening and verification in step 4, PCR amplification technology may be used for further verification.

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

[31] According to the present disclosure, preferably, the use may be intended to inoculate the Corynebacterium glutamicum recombinant 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;

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

[33] 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)»SO4, 0.34 g/L. L-leucine, 2 g/L. KH:PO4, 1.5 g/L 3

MgSO4-7H20, and 0.001 g/L biotin. LU102871

[34] According to the present disclosure, preferably, the seed culture may be conducted at 200-220 rpm and 28-30°C for 18-25 h; the fermentation culture may be conducted at 200-220 rpm and 28-30°C.

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

[36] 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 HTR gene of the 5'-end sequence to result in easier transcription or expression thereof by replacing “.ACGTCTAGAC™, a nucleoside sequence from nucleosides -58 to -49 before a start codon ATG in the 5'-end sequence of the HTR gene, with *°TGTGGTATAA°

[37] Beneficial effects:

[38] The present disclosure provides a Corynebacterium glutamicum recombinant strain for modifying a 5'-end sequence of a HTR gene, where “*ACGTCTAGAC”°, a nucleoside sequence from nucleosides -58 to -49 before a start codon ATG in the 5'-end sequence of the HTR gene, is replaced with *"TGTGGTATAA“°. Compared with an original strain, the Corynebacterium glutamicum recombinant 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

[39] 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.

[40] Microbial source:

[41] 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 HTR is shown in SEQ ID NO. 1, the nucleotide sequence of the HTR gene is shown in SEQ ID NO. 2, and the 5'-end sequence of the HTR gene is shown in SEQ ID NO. 3.

[42] C. glutamicum CGMCC1.15647 was purchased from the China General Microbiological Culture Collection Center (CGMCC) and deposited with an accession number of 4

CGMCC1.15647, where the amino acid sequence of HTR is shown in SEQ ID NO. 8, the LU102871 nucleotide sequence of the HTR gene is shown in SEQ ID NO. 9, and the 5'-end sequence of the HTR gene is shown in SEQ ID NO. 10.

[43] The amino acid sequence shown in SEQ ID NO. 1 is 100% identical to that shown in SEQ ID NO. 8, the nucleotide sequence shown in SEQ ID NO. 2 is 99.3% identical to that shown in SEQ ID NO. 9, and the nucleotide sequence shown in SEQ ID NO. 3 is 98.0% identical to that shown in SEQ ID NO. 10.

[44] 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.

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

[46] 1.1 C. glutamicum CICC23604

[47] For C. glutamicum CICC23604, a nucleotide sequence of the upstream homologous arm- SBTGTGGTATAA*-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 -58 to -49, *#ACGTCTAGAC”-°, before a start codon ATG in the 5'-end sequence of C. glutamicum CICC23604 HTR 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-|ACGTCTAGAC-*- downstream homologous arm was shown in SEQ ID NO. 6, and the nucleotide sequence of the designed and synthesized upstream homologous arm-*TGTGGTATAA*-downstream homologous arm was shown in SEQ ID NO. 7. The sequence shown in SEQ ID NO. 7 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated between restriction sites of Hind III 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 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-HTR1.

[48] 1.2 C. glutamicum CGMCC1.15647

[49] For C. glutamicum CGMCC1.15647, a nucleotide sequence of the upstream homologous arm-"TGTGGTATAA“*-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 -58 to -49, “JACGTCTAGAC-°, before a start codon ATG in the 5'-end sequence of C. glutamicum CGMCC1.15647 HTR gene, LU102871 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 SEQ ID NO. 12; the nucleotide sequence of the original upstream homologous arm-- SBACGTCTAGAC“*-downstream homologous arm was shown in SEQ ID NO. 13, and the nucleotide sequence of the designed and synthesized upstream homologous arm- BTGTGGTATAA*-downstream homologous arm was shown in SEQ ID NO. 14. The sequence shown in SEQ ID NO. 14 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated between restriction sites of Hind III and EcoR I of the pK19mobsacB vector (GenBank: LC257601.1). The ligation product was transformed into E. coli DH5a competent cells, and 200 uL 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-HTR2.

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

[51] (1) A single colony of C. glutamicum was picked and inoculated in 10 mL of seed culture medium at 37°C and 220 r/min for overnight culture;

[52] 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; [S3] (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;

[54] (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; [S5] (4) after ice bath, the bacterial suspension was centrifuged at 4°C and 5,000 r/min for 5min to collect the cells; [S6] (5) the centrifuged cells were washed thrice with pre-cooled electroporation buffer (ETM);

[57] each liter of electroporation buffer (1,000 mL) was composed of: 91 g of sorbitol, 91 g of mannitol, and 100 mL of glycerol;

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

[59] (7) 100 pL of the prepared competent cells were dispensed into a tube and stored at - 80°C for later use.

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

[61] First, the fragment concentration of the replacement vector pK19mobsacB-HTR1 or 6 pK19mobsacB-HTR2 was determined by using a nucleic acid ultramicrospectrophotometer. LU102871 After reaching a concentration of 300 pg/mL, the replacement vector was electroporated at a 1,800 V electric shock for 5 ms for transformation into C. glutamicum CICC23604 competent 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.

[62] 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.

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

[64] (1) Primary screening by kanamycin resistance

[65] 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;

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

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

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

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

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

[71] Table 1 PCR amplification system

[72] Reagent Volume (nL) 2xHiFi-PCRmaster 10 F1 (10 pmol/L) 1 R1 (10 pmol/L) 1 Template 1 ddH:O 7

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

[74] 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.

[75] A strain with a specific band at 795 bp was selected as a strain with homologous single 7 crossover for further verification. LU102871

[76] (2) Secondary screening on sucrose plate [771 The above correctly verified strains with homologous single crossover were inoculated into antibiotic-free liquid LBG medium, respectively, and passed naturally for three generations, where each generation was cultured for 24 h; finally, 200 uL 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;

[78] where, for the transformant of C. glutamicum CICC23604, the primer sequences for the PCR amplification were as follows:

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

[80] R2:5-TTACAGGCCTAGGTAATGCTCA-3' (SEQ ID NO. 18).

[81] The 3'-end sequence of the F2 primer contained a replaced site ** TGTGGTATAA;

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

[83] Table 2 PCR amplification system 84 0000000000000 Reagent Volume (nL) 2xHiFi-PCRmaster 10 F2 (10 pmol/L) 1 R2 (10 umol/L) 1 Template 1 ddH2O 7

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

[86] 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;

[87] a specific band at about 817 bp was picked, ligated to a pMD18-T vector, and sequenced using vector primers; after verification, a Corynebacterium glutamicum recombinant strain HTR1 that completed two homologous single crossovers was obtained.

[88] For the transformant of C. glutamicum CGMCC1.15647, the primer sequences for the PCR amplification were as follows:

[89] F3:5-TTCTGAACGGGTTGTGGTATAA-3" (SEQ ID NO. 19), and

[90] R3:5-TTACAGGCCTAGGTAATGCTCA-3' (SEQ ID NO. 20).

[91] The 3'-end sequence of the F3 primer contained a replaced site **TGTGGTATAA;

[92] a system of the PCR amplification was as follows: 8

[93] Table 3 PCR amplification system LU102871 2 à : Reagent Volume (uL) 2xHiFi-PCRmaster 10 F3 (10 pmol/L) 1 R3 (10 umol/L) 1 Template 1 ddH2O 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] a specific band at about 817 bp was picked, ligated to a pMD18-T vector, and sequenced using vector primers; after verification, a Corynebacterium glutamicum recombinant strain HTR2 that completed two homologous single crossovers was obtained.

[98] Example 5: Stability verification of recombinant strains

[99] The Corynebacterium glutamicum recombinant strains HTR1 and HTR2 that were screened and verified as correct above were subcultured, respectively. First, a single colony was picked from the plate and inoculated in the antibiotic-free liquid LBG medium for 12 h, and then 1% by volume of inoculum was passed for 30 consecutive generations. The genome was extracted from the last generation of bacterial suspension, and F2 and R2, F3 and R3 were used as primers for colony PCR verification. The results showed that a specific gene band with a length of about 817 bp could be amplified in the amplification of Corynebacterium glutamicum recombinant strains HTR1 and HTR2 by using primers F2 and R2 and F3 and R3, respectively, which was consistent with the theoretical value. This demonstrated that the replaced - SBTGTGGTATAA™ had been successfully integrated into the genomes of Corynebacterium glutamicum recombinant strains HTR1 and HTR2 and existed stably.

[100] Herein, a system of the PCR amplification was as follows:

[101] Table 4 PCR amplification system

[102] Reagent Volume (nL) 2xHiFi-PCRmaster 10 Upstream primer (10 pmol/L) 1 Downstream primer (10 pmol/L) 1 Template 1 ddH2O 7

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

[104] 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 9 storage at 4°C. LU102871

[105] Example 6: L-lysine fermentation test

[106] The above prepared Corynebacterium glutamicum recombinant strains HTR1 and HTR2 were inoculated into 100 mL of LBG medium (5 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and 10 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 KHz:PO4, 1.5 g/L MgSO4-7H20, and 0.001 g/L biotin) for fermentation culture for 48 h, respectively; both Corynebacterium glutamicum recombinant 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 5 and 6.

[107] Table 5 Average L-lysine yields of Corynebacterium glutamicum recombinant strain HTRI1 and original strain at different times

[108] Fermentation time Strain 12h 24h 36h 48 h Recombinant strain HTR1 3.9 g/L 26.9 g/L 43.0 g/L 47.5 g/L Original strain CICC23604 4.3 g/L 18.2 g/L 39.9 g/L 41.1 g/L

[109] Table 6 Average L-lysine yields of Corynebacterium glutamicum recombinant strain HTR2 and original strain at different times

[110] Fermentation time Strain 12h 24h 36h 48 h Recombinant strain PC2 0.02 g/L 0.56 g/L 0.88 g/L 0.98 g/L Original strain CGMCC1.15647 0.03 g/L 0.25 g/L 0.48 g/L 0.44 g/L

[111] According to the results, compared with the original strain, the content of L-lysine in the fermentation broth of the Corynebacterium glutamicum recombinant strain HTR1 reached 47.5 g/L 48 h after fermentation, which was 15.6% higher than that of the original strain; the content of L-lysine in the fermentation broth of the Corynebacterium glutamicum recombinant strain HTR2 reached 0.98 g/L, which was 2.23 times that of the original strain. This indicated that replacement of the nucleoside sequence **ACGTCTAGAC”° from nucleosides -58 to -49 before the start codon ATG in the 5'-end sequence of the HTR gene with S*TGTGGTATAA™ could increase the acid production level of L-lysine fermentation, which is also a new way to increase the L-lysine yield.

[112] Stability analysis was conducted on sequences before and after replacement of nucleoside sequence from nucleosides -58 to -49 before the start codon ATG in the 5'-end LU102871 sequences (SEQ ID NO. 3 and SEQ ID NO. 10) of the HTR 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 from nucleosides -58 to -49 before the start codon ATG in the 5'-end sequence of the HTR gene of C. glutamicum CICC23604 was replaced with *°TGTGGTATAA-°, the minimum free energy of the 5'-end sequence was increased from -9.20 kcal/mol to -6.50 kcal/mol; after the nucleoside sequence from nucleosides -58 to -49 before the start codon ATG in the 5'-end sequence of the HTR gene of C. glutamicum CGMCC1.15647 was replaced with TGTGGTATAA™, the minimum free energy was increased from -12.40 kcal/mol to -9.70 kcal/mol.

This indicated that the sequence replacement could reduce the stability of the 5'-end sequence, which in turn makes the HTR gene easier to be transcribed and translated and ultimately increases the level of acid production by amino acid fermentation. 11

SEQUENCE LISTING LU102871 <110> Qilu University of Technology Zhucheng Dongxiao Biotechnology Co., Ltd. <120> METHOD FOR IMPROVING L-LYSINE YIELD OF CORYNEBACTERIUM

GLUTAMICUM RECOMBINANT STRAIN <130> HKJU202109573 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HTR of CICC23604 <400> 1 Met Gly Ile Lys Val Gly Val Leu Gly Ala Lys Gly Arg Val Gly Gln 1 5 10 15 Thr Ile Val Ala Ala Val Asn Glu Ser Asp Asp Leu Glu Leu Val Ala Glu Ile Gly Val Asp Asp Asp Leu Ser Leu Leu Val Asp Asn Gly Ala 40 45 12

Glu Val Val Val Asp Phe Thr Thr Pro Asn Ala Val Met Gly Asn Leu 50 55 60 Glu Phe Cys Ile Asn Asn Gly Ile Ser Ala Val Val Gly Thr Thr Gly 65 70 75 80 Phe Asp Asp Ala Arg Leu Glu Gln Val Arg Ala Trp Leu Glu Gly Lys 85 90 95 Asp Asn Val Gly Val Leu Ile Ala Pro Asn Phe Ala Ile Ser Ala Val 100 105 110 Leu Thr Met Val Phe Ser Lys Gln Ala Ala Arg Phe Phe Glu Ser Ala 115 120 125 Glu Val Ile Glu Leu His His Pro Asn Lys Leu Asp Ala Pro Ser Gly 130 135 140 Thr Ala Ile His Thr Ala Gln Gly Ile Ala Ala Ala Arg Lys Glu Ala 145 150 155 160 Gly Met Asp Ala Gln Pro Asp Ala Thr Glu Gln Ala Leu Glu Gly Ser 165 170 175 13

Arg Gly Ala Ser Val Asp Gly Ile Pro Val His Ala Val Arg Met Ser LU102871 180 185 190 Gly Met Val Ala His Glu Gln Val Ile Phe Gly Thr Gln Gly Gln Thr 195 200 205 Leu Thr Ile Lys Gln Asp Ser Tyr Asp Arg Asn Ser Phe Ala Pro Gly 210 215 220 Val Leu Val Gly Val Arg Asn Ile Ala Gln His Pro Gly Leu Val Val 225 230 235 240 Gly Leu Glu His Tyr Leu Gly Leu 245 <210> 2 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of HTR of CICC23604 <400> 2 atgggaatca aggttggegt tctcggagec aaaggecgtg ttggtcaaac tattgtggea 60 gcagtcaatg agtccgacga tctggagctt gttgcagaga tcggcgtega cgatgatttg 120 agccttctgg tagacaacgg cectgaagtt gtcettgact tcaccactec taacgetgtg 180 14 atgggcaacc tggagttctg catcaacaac ggcatttctg cggttgttgg aaccacggge 240 tttgatgatg ctcgtttgga gcagettcec gcctgecttg aaggaaaaga caatgtcggt 300 gttetgatcg cacctaactt tgetatetet gcggtettga ceatggtett ticcaagecag 360 gctgeccgct tettcgaate agetgaagtt attgagetge accaccccaa caagctggat 420 gcaccttcag gcaccgcgat ccacactect caggecattg ctgeggeacg caaagaagca 480 ggcatggacg cacagccaga tgcgaccgag caggcacttg aggetteccg tggcgcaage 540 gtagatggaa tcccagttca cgcagtccgc atgtceggea tggttgctca cgageaagtt 600 atctttggca cccagggtca gaccttgacc atcaagcagg actcctatga tcgeaactca 660 tttecaccag gtgtettggt gggtetgcgc aacattgcac agcacccagg cctagtegta 720 ggacttgagc attacctagg cctgtaa 747 <210> 3 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> 5'-end DNA sequence of HTR of CICC23604 <400> 3 aacggtcagt taggtatgga tatcagcacc tictgaatgg gtacgtctag actggtggec 60 gtttgaaaaa ctcttcgece cacgaaaatg aaggagaata 100

<210> 4 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the upstream homologous arm of CICC23604 <400> 4 aaaaacaact cgcgtgaacg tttcgtgete ggcaacgcgg atgccagcga tcgacatatc 60 ggagtcacca acttgagect getgcttetg atccatcgac ggggaaccca acggeggeaa 120 agcagtgggg gaaggggagt tggtggactc tgaaccagtg gectctgaag tggtaggega 180 cggggcagcea tctgaaggeg tecgagttet getgaccggg ttageggttt cagtttctgt 240 cacaactgga gcaggactag cagaggttgt aggcgttgag cegettccat cacaagcact 300 taaaagtaaa gaggcggaaa ccacaagcgc caaggaacta cctgeggaac gggeggtgaa 360 gggcaactta agtctcatat ttcaaacata gttccacctg tgtgattaat ccccagaacg 420 gaacaaactg atgaacaatc gttaacaaca cagaccaaaa cggtcagtta ggtatggata 480 tcagcacctt ctgaatgggt 500 <210> 5 <211> 500 <212> DNA <213> Artificial Sequence

16

<220> <223> DNA sequence of the downstream homologous arm of CICC23604 <400> 5 tggteggcgt ttgaaaaact cticgeccea cgaaaatgaa ggagaataat gggaatcaag 60 gttggcegttc tcggagecaa aggecgtgtt ggtcaaacta ttgtggeage agtcaatgag 120 tccgacgatc tegagcttet tgcagagate ggegtcgacg atgatttgag ccttctggta 180 gacaacgecg ctgaagttgt cettgacttc accactecta acgctetgat gggcaacctg 240 gagttctgea tcaacaacgg catttctgcg gttgttggaa ccacgggcett tgatgatget 300 cetttegagc aggttcgege ctgecttgaa ggaaaagaca atetcggtet tctgatcgca 360 cctaactttg ctatctctge getettgacc atggtctttt ccaagcagec tgecegettc 1420 ttcgaatcag ctgaagttat tgagctgcac caccccaaca agetggatge accttcagge 1480 accgcgatce acactgetca 500 <210> 6 <211> 1010 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the original fragment of CICC23604 before recombinant <400> 6 17 aaaaacaact cgcgtgaacg titcgtgctc ggcaacgcgg atgccagcga tcgacatatc 60 LU102871 ggagtcacca acttgagect getgettetg atccatcgac ggggaaccca acggeggeaa 120 agcagtgggg gaaggggagt tggtggactc tgaaccagtg gectctgaag tggtaggcga 180 cggggcagca tetgaaggeg tgegagttgt getgaccggg ttageggttt cagtttetgt 240 cacaactgga gcaggactag cagaggttgt aggcgttgag cegettccat cacaagcact 300 taaaagtaaa gaggcggaaa ccacaagcgc caaggaacta cctecggaac gggeggtgaa 360 gggcaactta agtctcatat ttcaaacata gttccacctg tetgattaat ccccagaacg 420 gaacaaactg atgaacaatc gttaacaaca cagaccaaaa cggtcagtta ggtatggata 480 tcagcacctt ctgaatgggt acgtctagac tggtgggegt ttgaaaaact cttcgeccca 540 cgaaaatgaa ggagaataat gggaatcaag gttggcgttc tcggagccaa aggeegtgtt 600 ggtcaaacta ttgtggcage agtcaatgag tccgacgate tggagcettgt tgcagagate 660 ggcgtegacg atgatttgag ccttctggta gacaacggeg ctgaagttgt cgttgacttc 720 accactccta acgctetgat gggcaacctg gagttctgea tcaacaacgg catttctgeg 780 gttgttggaa ccacggectt tgatgatect cgtttggage agettcgcec ctggettgaa 840 ggaaaagaca atgtcggtgt tctgatcgea cctaactttg ctatetctge ggtgttgace 900 atggtctttt ccaagcaggc tgeccgette ttcgaatcag ctgaagttat tgagetgcac 960 caccccaaca agctggatgc accttcagge accgegatee acactectca 1010

18

<210> 7 <211> 1010 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the recombinant fragment of CICC23604 <220> <221> misc feature <222> (501)..(510) <223> The original nucleotide sequence ACGTCTAGAC is replaced with

TGTGGTATAA <400> 7 aaaaacaact cgcgtgaacg titcgtgctc ggcaacgcgg atgccagcga tcgacatatc 60 ggagtcacca acttgagect getgcttetg atccatcgac ggggaaccca acggeggeaa 120 agcagtgggg gaaggggagt tggtggactc tgaaccagtg gectetgaag tggtaggega 180 cegggcagca tctgaaggeg tecgagttet getgaccggg ttagcgettt cagtttctet 240 cacaactgga gcaggactag cagaggttgt aggcgttgag ccgcttccat cacaagcact 300 taaaagtaaa gaggcggaaa ccacaagcgc caaggaacta cctgeggaac gggeggtgaa 360 gggcaactta agtctcatat ttcaaacata gttccacctg tetgattaat ccccagaacg 420 gaacaaactg atgaacaatc gttaacaaca cagaccaaaa cggtcagtta ggtatggata 480 tcagcacctt ctgaatgggt tetggtataa tggtggecgt ttgaaaaact cttcgcecca 540 19 cgaaaatgaa ggagaataat gggaatcaag gttggcgttc tcggagccaa aggeegtgtt 600 ggtcaaacta ttgtggcage agtcaatgag tccgacgate tggagcettgt tgcagagate 660 ggcgtegacg atgatttgag ccttctggta gacaacggeg ctgaagttgt cgttgacttc 720 accactecta acgetgtgat gggeaacctg gagttctgca tcaacaacgg catttctgeg 780 gttgttggaa ccacggectt tgatgatect cgtttggage aggttcgege ctggettgaa 840 ggaaaagaca atgtcggtgt tctgatcgca cctaactttg ctatctetge getettgacc 900 atggtctttt ccaagcaggc tgcccgcttc ttcgaatcag ctgaagttat tgagetgcac 960 caccccaaca agctggatge accttcagge accgcgatec acactectca 1010 <210> 8 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HTR of CGMCC1.15647 <400> 8 Met Gly Ile Lys Val Gly Val Leu Gly Ala Lys Gly Arg Val Gly Gln 1 5 10 15 Thr Ile Val Ala Ala Val Asn Glu Ser Asp Asp Leu Glu Leu Val Ala

20

Glu Ile Gly Val Asp Asp Asp Leu Ser Leu Leu Val Asp Asn Gly Ala 40 45 Glu Val Val Val Asp Phe Thr Thr Pro Asn Ala Val Met Gly Asn Leu 50 55 60 Glu Phe Cys Ile Asn Asn Gly Ile Ser Ala Val Val Gly Thr Thr Gly 65 70 75 80 Phe Asp Asp Ala Arg Leu Glu Gln Val Arg Ala Trp Leu Glu Gly Lys 85 90 95 Asp Asn Val Gly Val Leu Ile Ala Pro Asn Phe Ala Ile Ser Ala Val 100 105 110 Leu Thr Met Val Phe Ser Lys Gln Ala Ala Arg Phe Phe Glu Ser Ala 115 120 125 Glu Val Ile Glu Leu His His Pro Asn Lys Leu Asp Ala Pro Ser Gly 130 135 140 Thr Ala Ile His Thr Ala Gin Gly Ile Ala Ala Ala Arg Lys Glu Ala 145 150 155 160 21

Gly Met Asp Ala Gln Pro Asp Ala Thr Glu Gln Ala Leu Glu Gly Ser LU102871 165 170 175 Arg Gly Ala Ser Val Asp Gly Ile Pro Val His Ala Val Arg Met Ser 180 185 190 Gly Met Val Ala His Glu Gln Val Ile Phe Gly Thr Gln Gly Gln Thr 195 200 205 Leu Thr Ile Lys Gln Asp Ser Tyr Asp Arg Asn Ser Phe Ala Pro Gly 210 215 220 Val Leu Val Gly Val Arg Asn Ile Ala Gln His Pro Gly Leu Val Val 225 230 235 240 Gly Leu Glu His Tyr Leu Gly Leu 245 <210> 9 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of HTR of CGMCC1.15647 <400> 9 atgggaatca aggttggcet tctcggagec aaaggecgtg ttggtcaaac tattgtggea 60 22 gcagtcaatg agtccgatga tctggagctt gttgcagaga tcggegtega cgatgatttg 120 agccttctgg tagacaacgg cectgaagtt gtegttgact tcaccactec taacgetgtg 180 atgggtaacc tggagttctg catcaataac ggeatttctg cggttgttgg aaccacggge 240 ttcgatgatg ctcgtttgga geaggttcge geetggettg aaggaaaaga caatgteggt 300 gttctgatcg cacctaactt tgetatetet gcggtettga ceatggtett ttccaagcag 360 gctgcecect tettcgaate agctgaagtt attgagetge accaccccaa caagetggat 420 gcaccticag gcaccgcgat ccacactget cagggeattg ctgeggeacg caaagaagea 1480 ggcatggacg cacagccaga tgcgaccgag caggeacttg agggttceeg tggegeaage 540 gtagatggaa tcccagttca cgeagtcege atgtccggea tggttgetea cgageaagtt 600 atctttggca cccaggggca gaccttgace atcaagcagg actectatga tcgeaactca 660 titgcaccag gtgtettggt gggtgtgcgc aacattgcac agcacccagg cctagtegta 720 ggacttgagc attacctagg cctgtaa 747 <210> 10 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> 5'-end DNA sequence of HTR of CGMCC1.15647

23

<400> 10 LU102871 aacggtcagt taggtatgga tatcagcacc ttctgaacgg gtacgtctag actggtgggc 60 gtttgaaaaa ctcttcgccc cacgaaaatg aaggagcata 100 <210> 11 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the upstream homologous arm of CGMCC1.15647 <400> 11 aaaaacaact cgcgtgaacg titcgtgctc ggcaacgcgg atgccagcga tcgacatatc 60 ggagtcacca acttgagcct gctecttctg atccatcgac ggegaaccca acggcagcaa 120 agcagtgggg gaaggggagt tggtggactc tgaaccagtg gectetgaag tggtaggega 180 cggggceagea tetgaaggeg tgegagttgt getgaccggg ttageggttt cagttictgt 240 cacaactgga gcaggactag cagaggttgt aggcgttgag ccgettccat cacaagcact 300 taaaagtaaa gaggcggaaa ccacaagege caaggaacta cctgeggaac gggeggtgaa 360 gggcaactta agtctcatat ttcaaacata gttccacctg tgtgattaat ccctagaacg 420 gaacaaactg atgaacaatc gttaacaaca cagaccaaaa cggtcagtta ggtatggata 480 tcagcacctt ctgaacgggt 500

24

<210> 12 LU102871 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence ofthe downstream homologous arm of CGMCC1.15647 <400> 12 tggtgggcegt ttgaaaaact cttcgcccca cgaaaatgaa ggagcataat gggaatcaag 60 ettggcettc tcggagccaa aggecgtgtt ggtcaaacta ttgtggcagc agtcaatgag 120 tecgatgatc tggagettgt tgcagagatc ggegtegacg atgatttgag cettetggta 180 gacaacgecg ctgaagttgt cgttgacttc accactecta acgetgtgat gggtaacctg 240 gagttctgea tcaataacgg catttctecg gttgttggaa ccacggectt cgatgatget 300 cgtitggage aggttegegc ctggcttgaa ggaaaagaca atgtcggtet tetgatcgea 360 cctaactttg ctatctctge ggtgttgace atggtetttt ccaageagge tgcecgette 1420 ttcgaatcag ctgaagttat tgagctgcac caccccaaca agetggatge accttcagge 1480 accgcgatcc acactectca 500 <210> 13 <211> 1010 <212> DNA <213> Artificial Sequence <220>

<223> DNA sequence of the original fragment of CGMCC1.15647 before LU102871 recombinant <400> 13 aaaaacaact cgcgtgaacg tttcgtgetc ggcaacgcgg atgccagcga tcgacatatc 60 ggagtcacca acttgagect getgcttetg atccatcgac ggggaaccca acggcageaa 120 agcagtgggg gaaggggagt tggtggactc tgaaccagtg gectetgaag tggtaggega 180 cegggcagca tctgaaggeg tecgagttet getgaccggg ttagcgettt cagtttctet 240 cacaactgga gcaggactag cagagettet aggcgttgag ccgettceat cacaagcact 300 taaaagtaaa gaggcggaaa ccacaagcgc caaggaacta cctgeggaac gggeggtgaa 360 gggcaactta agtctcatat ttcaaacata gttccacctg tgtgattaat ccctagaacg 420 gaacaaactg atgaacaatc gttaacaaca cagaccaaaa cggtcagtta ggtatggata 480 tcagcacctt ctgaacgggt acgtctagac tggtgggcgt ttgaaaaact cticgcccca 540 cgaaaatgaa ggagcataat gggaatcaag gttggegttc tcggagecaa aggecgtgtt 600 ggtcaaacta ttgtggcage agtcaatgag tccgatgate tegagcttet tgcagagatc 660 ggcgtcgacg atgatttgag ccttctggta gacaacggeg ctgaagttgt cgttgacttc 720 accactccta acgctetgat gggtaacctg gagttctgca tcaataacgg catttetgeg 780 gttgttggaa ccacgggett cgatgatgcet cgtttggage aggticgegc ctggettgaa 840 ggaaaagaca atgtcggtgt tctgatcgea cctaactttg ctatctetge getettgacc 900

26 atggtctttt ccaagcaggc tgeccegette ttcgaatcag ctgaagttat tgagctgcac 960 LU102871 caccccaaca agetggatge accttcagge accgcgatee acactgetea 1010 <210> 14 <211> 1010 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the recombinant fragment of CGMCC1.15647 <220> <221> misc_feature <222> (501)..(510) <223> The original nucleotide sequence ACGTCTAGAC is replaced with

TGTGGTATAA <400> 14 aaaaacaact cgcgtgaacg tttcgtgete ggcaacgcgg atgccagcga tcgacatatc 60 ggagtcacca acttgagcct getgettetg atccatcgac ggggaaccea acggecageaa 120 agcagtgggg gaagggoagt tggtggactc tgaaccagtg gectctgaag tggtaggcga 180 cggggceagea tetgaaggeg tgegagttgt getgaccggg ttageggttt cagtttetgt 240 cacaactgga gcaggactag cagaggttgt aggcgttgag ccgettccat cacaagcact 300 taaaagtaaa gaggcggaaa ccacaagcgc caaggaacta cctgeggaac gggeggtgaa 360 gggcaactta agtctcatat ttcaaacata gttccacctg tgtgattaat ccctagaacg 1420 27 gaacaaactg atgaacaatc gttaacaaca cagaccaaaa cggtcagtta ggtatggata 480 tcagcacctt ctgaacgggt tgtggtataa tggtgggcgt ttgaaaaact cttcgcecca 540 cgaaaatgaa ggagcataat gggaatcaag gttggegttc tcggagecaa aggecgtgtt 600 ggtcaaacta ttgtggcagc agtcaatgag tccgatgate tggagcettgt tgcagagatc 660 gecetcgacg atgatttgag ccttctggta gacaacggeg ctgaagttet cgttgacttc 720 accactccta acgctetgat gggtaacctg gagtictgca tcaataacgg catttctgeg 780 gttgttggaa ccacgggett cgatgatget cgtttggage aggticgegc ctggettgaa 840 ggaaaagaca atgtcggtgt tctgatcgea cctaactttg ctatctetge getettgacc 900 atggtctttt ccaagcaggce tgcccgcttc ttcgaatcag ctgaagttat tgagctgcac 960 caccccaaca agetggatge accttcagge accgcgatec acactgetea 1010 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer F1 <400> 15 atgattgaac aagatggatt gc 22

28

<210> 16 LU102871

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer R1

<400> 16 tcagaagaac tcgtcaagaa ggeg 24

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer F2

<400> 17 ttctgaatgg gttetggtat aa 22

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer R2

<400> 18 ttacaggcct aggtaatect ca 22 29

<210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer F3 <400> 19 ttctgaacgg gttgtggtat aa 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer R3 <400> 20 ttacaggcct aggtaatect ca 22

Claims (10)

WHAT IS CLAIMED IS:

1. A Corynebacterium glutamicum recombinant strain for modifying a 5'-end sequence of a 4-hydroxy-tetrahydrodipicolinate reductase (H7R) gene, wherein in a host Corynebacterium glutamicum strain, “*ACGTCTAGAC”°, a nucleoside sequence from nucleosides -58 to -49 before a start codon ATG in the S'-end sequence of the HTR gene, is replaced with - BTGTGGTATAA™ to reduce the stability of a secondary structure of the 5'-end sequence of the HTR gene, to allow for easier transcription or expression thereof.

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

3. The Corynebacterium glutamicum recombinant strain according to claim 1, wherein the HTR has an amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 8

4. The Corynebacterium glutamicum recombinant strainaccording to claim 1, wherein the HTR gene has a nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 9; preferably, the nucleotide sequence of the H7R gene is a nucleotide sequence having a sequence identity of >99.3% to SEQ ID NO. 2 or SEQ ID NO. 9.

5. The Corynebacterium glutamicum recombinant strainaccording to claim 1, wherein the S'-end sequence of the HTR gene is shown in SEQ ID NO. 3 or SEQ ID NO. 10; preferably, the 5'-end sequence of the HTR gene is a nucleotide sequence having a sequence identity of >98.0% to SEQ ID NO. 3 or SEQ ID NO. 10.

6. A construction method of the Corynebacterium glutamicum recombinant strainaccording to claim 1, comprising the following steps: step 1, synthesizing a nucleotide sequence of upstream homologous arm- BTGTGGTATAA*-downstream homologous arm, wherein the upstream homologous arm and the downstream homologous arm are 500-600 bp nucleoside sequences “SACGTCTAGAC™ from nucleosides -58 to -49 before and after a start codon ATG in the 5'-end sequence of HTR gene, respectively; 31 step 2, ligating the nucleotide sequence of the upstream homologous arnt#102871 BTGTGGTATAA*-downstream homologous arm to a pK19mobsacB vector to construct a replacement vector; step 3, transforming the replacement vector into host Corynebacterium glutamicum straincompetent 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 Corynebacterium glutamicum recombinant 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-*TGTGGTATAA- 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: S'"-ATGATTGAACAAGATGGATTGC-3', as shown in SEQ ID NO. 15, and R1: 5'-TCAGAAGAACTCGTCAAGAAGGCG-3', as shown 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 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 iii. the medium used in step 4 comprises an LBG medium: 5 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl.

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

32

9. The use according to claim 8, wherein the use is intended to inoculate th&102871 Corynebacterium glutamicum recombinant 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.

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