A method for improving l-threonine production and conversion rate by combined mutation of gltA gene and promoter region

Abstract

The application discloses a method for improving L-threonine yield and conversion rate by gltA gene and promoter region combined mutation, and belongs to the technical field of genetic engineering. The method comprises the following steps: constructing a recombinant plasmid pECgRNA-gltA-N20, constructing a gltA homologous fragment containing a point mutation, constructing a strain containing a point mutation gltA, and fermenting. The strain containing the point mutation gltA is transformed into Escherichia coli competent cells by using the plasmid pEcCAS, positive single clones are picked, and a recombinant strain is obtained. The gltA-PM fragment and the recombinant plasmid pECgRNA-N20-gltA are transformed into the recombinant strain competent cells, and a double-mutation strain is obtained. The double-mutation strain is a strain simultaneously carrying a start codon mutation and an upstream promoter region mutation, and the L-threonine yield and conversion rate of the double-mutation strain are superior to those of a strain containing only a single mutation.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region. Background Technology

[0002] L-Threonine is an essential amino acid that humans and animals cannot synthesize on their own and must obtain from external sources. It is the second largest limiting amino acid in pig feed after lysine, and the third largest limiting amino acid in poultry feed. Its market demand is growing at a rate of 20% annually. Besides the feed industry, L-Threonine has significant applications in the food and pharmaceutical industries.

[0003] Currently, the main methods for producing L-threonine include chemical synthesis, protein hydrolysis, and microbial fermentation. Among these, microbial fermentation has become the mainstream method for industrial production due to its advantages such as simple process, short production cycle, low production cost, and low pollution. Escherichia coli used in microbial fermentation is currently the main strain for L-threonine fermentation because of its clear genetic background, mature gene editing tools, and short fermentation cycle.

[0004] L-Threonine belongs to the aspartic acid family of amino acids, and its biosynthesis uses oxaloacetate as a precursor. During the process of glucose entering the tricarboxylic acid cycle via glycolysis, a large amount of carbon skeleton is released in the form of carbon dioxide, resulting in carbon loss. Therefore, reducing the flux of the tricarboxylic acid cycle and increasing the level of oxaloacetate synthesis is an effective strategy to reduce carbon loss and increase L-Threonine production. Citrate synthase (encoded by gltA) is the first rate-limiting enzyme in the tricarboxylic acid cycle and a key target for regulating this pathway.

[0005] Chinese patents CN109852572B (A method for increasing L-threonine production by knocking out the PTS system in *E. coli*), CN116622600A (A method for constructing a threonine-producing strain), and CN117965574A (A mutant of a key enzyme in L-threonine synthesis) all utilize genetic engineering to modify the gltA gene to increase L-threonine production, providing valuable references for the expression regulation of citrate synthase. However, the modification strategies in these three patents are all concentrated at a single level. For example, Chinese patent CN109852572B only regulates gltA expression at the transcriptional level by replacing the promoter (Ptrc); and Chinese patent CN116622600A only weakens gltA gene expression at the translational level by mutating the start codon (ATG to GTG). Chinese patent CN117965574A optimizes only the ribosome binding site (RBS) in the L-threonine synthesis key enzyme mutant to regulate the translation rate. Each of the three patents mentioned above employs a single modification method to the gltA gene, resulting in limited improvement in L-threonine production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for enhancing L-threonine yield and conversion rate through joint mutation of the gltA gene and its promoter region. Using CRISPR-Cas9 technology, a specific mutation is introduced into an L-threonine fermentation strain, specifically by mutating the start codon of the gltA gene from the highly efficient ATG to the less efficient TTG. During the construction process, a double-mutant strain carrying both the start codon mutation and the upstream promoter region mutation was also obtained. Enzyme activity and fermentation tests confirmed that the L-threonine yield and conversion rate of this double-mutant strain were superior to those of the strain containing only the start codon mutation, thus yielding a mutant strain with significantly enhanced L-threonine fermentation capacity.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region includes the following steps: constructing a recombinant plasmid pECgRNA-gltA-N20, constructing a gltA homologous fragment containing a point mutation, constructing a strain containing a gltA point mutation, and fermentation; The recombinant plasmid pECgRNA-gltA-N20 was constructed by searching for the gltA gene in the E. coli genome sequence, selecting the N20 sequence immediately downstream of the PAM sequence from the gltA gene, and annealing forward and reverse oligonucleotides to form dsDNA with BsaI sticky ends; the dsDNA was ligated with the linearized plasmid digested with BsaI, transformed into competent E. coli cells, single clones were picked, identified as positive clones, and the plasmid was extracted to obtain the recombinant plasmid pECgRNA-gltA-N20; In the constructed recombinant plasmid pECgRNA-gltA-N20, the Escherichia coli genome sequence is the Escherichia coli MG1655 genome sequence; The gene sequence number of the gltA gene is NC_000913.3; The nucleotide sequence of the N20 sequence is shown in SEQ ID No. 1 of the sequence listing; The nucleotide sequence of the positive oligonucleotide is shown in SEQ ID No. 2 in the sequence listing; The nucleotide sequence of the reverse oligonucleotide is shown in SEQ ID No. 3 in the sequence listing; The annealing procedure is as follows: denaturation at 95°C for 5 minutes, followed by cooling to 16°C at a rate of 0.12°C / s and holding for 10 minutes; The competent Escherichia coli cells are Escherichia coli DH5α competent cells; The heat shock method was used when transforming the cells into competent E. coli cells. The construction of the gltA homologous fragment containing point mutations was carried out using the E. coli genome sequence as a template. Forward primers P1, reverse primers P2, forward primers P3, and reverse primers P4 were designed based on the gltA gene sequence. The target fragment was amplified using forward primers P1 / reverse primers P2 and forward primers P3 / reverse primers P4 to obtain two fragments. Overlap extension PCR was performed using forward primers P1 / reverse primers P4 to obtain the gltA-PM fragment. In the construction of the gltA homologous fragment containing point mutations, the E. coli genome sequence is the E. coli Thr-0 genome sequence; The E. coli Thr-0 genome sequence is as follows: E.coli W3110 / thrA* / lysC* / Ptrc-ThrABC / △ lysA / △metA / △tdh / Ptrc-ppc Genome sequence; When designing forward primer P3 based on the gltA gene sequence, a synonymous mutation was introduced in the coding region downstream of the N20 target sequence by designing forward primer P3, which mutated the base G to the base C at position 334 in the gltA-M fragment; The purpose of introducing synonymous mutations in the coding region downstream of the N20 target sequence is to disrupt the Cas9 target site recognition sequence without changing the amino acid sequence of citrate synthase, thereby preventing the Cas9 protein expressed by plasmid pEcCAS from repeatedly cutting the edited site of the genome after editing, and ensuring the stability of the genome of the recombinant strain. The nucleotide sequence of the forward primer P1 is shown in SEQ ID No. 4 of the sequence listing; The nucleotide sequence of the reverse primer P2 is shown in SEQ ID No. 5 of the sequence listing; The nucleotide sequence of the forward primer P3 is shown in SEQ ID No. 6 of the sequence listing; The nucleotide sequence of the reverse primer P4 is shown in SEQ ID No. 7 of the sequence listing; The nucleotide sequence of the gltA-PM fragment is shown in SEQ ID No. 9 of the sequence listing; In the gltA-PM fragment, the 216th position is mutated from base A to base C, and the 299th position is mutated from base A to base T; The process for constructing a strain containing the point mutation gltA involved transforming the plasmid pEcCAS, carrying a kanamycin resistance marker from the CRISPR knockout system, into competent E. coli cells. Positive single clones were selected to obtain the recombinant strain Thr-0 / pEcCAS. The gltA-PM fragment and the recombinant plasmid pECgRNA-N20-gltA were then co-transformed into competent cells of the recombinant strain Thr-0 / pEcCAS. PCR verification was performed, and positive transformants capable of amplifying the gltA-PM fragment were selected. In the strain containing the point mutation gltA, the competent Escherichia coli cells are Escherichia coli Thr-0 competent cells; The *E. coli* Thr-0 competent cells are... E.coli W3110 / thrA* / lysC* / Ptrc-ThrABC / △ lysA / △metA / △tdh / Ptrc-ppc competent cells; Electroporation was used when transforming plasmid pEcCAS into competent E. coli cells and when co-transforming the gltA-PM fragment with recombinant plasmid pECgRNA-N20-gltA into recombinant strain Thr-0 / pEcCAS competent cells. The fermentation process involves activating the double mutant strain to the logarithmic phase and then inoculating it into a fermentation medium for fermentation. In the fermentation process, the fermentation medium is formulated as follows: 44-46 g / L glucose, 0.15-0.17 mL / L phosphate, 0.38-0.42 g / L magnesium sulfate, 4.8-5.2 g / L yeast extract, 2.8-3.2 g / L potassium dihydrogen phosphate, 4.8-5.2 g / L ammonium sulfate, 9.5-10.5 g / L manganese sulfate, 1.8-2.2 g / L zinc sulfate, 9-11 mg / L ferrous sulfate, and 18-22 g / L calcium carbonate; The fermentation temperature is 36-38℃ and the rotation speed is 200-240 rpm.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: Unlike existing technologies, this invention proposes for the first time a dual modification strategy combining promoter mutation and start codon substitution of the gltA gene. Promoter mutation reduces expression at the transcriptional level, while start codon substitution precisely regulates translation. The synergistic effect of this combined modification significantly improves both threonine yield and sugar-acid conversion rate, achieving synergistic optimization of yield and conversion rate. Compared with existing strains, this strain significantly improves L-threonine yield and substrate conversion rate. After 18 hours of fermentation, the L-threonine yield reaches 26.23±0.15 g / L, and the conversion rate reaches 58.30±0.34%, thus effectively reducing production costs and demonstrating promising industrial application prospects. Detailed Implementation

[0009] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0010] The culture medium components involved in this invention are as follows: The components of LB solid medium are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and pH 7.0.

[0011] The components of LB liquid medium are: Tryptone 10g / L, Yeast Extract 5g / L, NaCl 10g / L, pH 7.0.

[0012] The LBS solid culture medium consists of: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, sucrose 10% (w / v, i.e., 100 g / L), agar 20 g / L, and pH 7.0.

[0013] The LBS liquid culture medium consists of: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, sucrose 10% (w / v, i.e., 100 g / L), pH 7.0.

[0014] Example 1 1. Construct the recombinant plasmid pECgRNA-gltA-N20 (The purpose of constructing the recombinant plasmid pECgRNA-gltA-N20 is to use the recombinant plasmid pECgRNA-gltA-N20 to locate the gltA position in the genome, guide the Cas cutting protein to cut the DNA, achieve homologous recombination, and achieve the goal of gltA gene mutation.) Based on the E. coli MG1655 genome sequence published by NCBI, the gltA gene was located (https: / / www.ncbi.nlm.nih.gov / gene / 945323) (the gene sequence number of the gltA gene is NC_000913.3). The N20 sequence was then constructed based on this sequence. The nucleotide sequence of the N20 sequence is shown in SEQ ID No. 1 of the sequence listing, and its downstream end is immediately adjacent to the PAM sequence (NGG). Adaptor sequences for BsaI sticky ends were added to the 5' end of the N20 sequence. The forward oligonucleotide (N20-gltA-F) was augmented with TAGT (nucleotide sequence shown in SEQ ID No. 2 of the sequence listing), and the reverse oligonucleotide (N20-gltA-R) was augmented with AAAC (nucleotide sequence shown in SEQ ID No. 3 of the sequence listing). After synthesizing these two oligonucleotides, annealing was performed to form double-stranded DNA (dsDNA) with BsaI sticky ends. The annealing procedure is as follows: denature at 95℃ for 5 min, cool to 16℃ at a rate of 0.12℃ / s and hold for 10 min, and then store the product at -20℃ for later use.

[0015] The pECgRNA plasmid was digested with BsaI at 37℃ for 2 h. The digestion product was purified by gel extraction to obtain a linearized plasmid, which was ligated with the above dsDNA at 16℃ for 30 min. The ligation product was then transformed into E. coli DH5α competent cells. The transformation method was as follows: E. coli DH5α competent cells were thawed on ice (90 μL after thawing), 10 μL of ligation product was added, and the cells were incubated on ice for 30 min, then heat-shocked at 37℃ for 5 min, immediately incubated on ice for 2 min, and 500 μL of LB medium was added. The cells were then incubated at 37℃ and 1000 rpm for 45 min. After resuscitation, centrifuge at 5000 rpm for 5 min and discard 500 μL of supernatant. Mix the remaining 100 μL of bacterial culture by pipetting and spread it onto LB solid medium containing 50 μg / mL spectinomycin. Incubate overnight at 37°C. Pick single clones and inoculate them into LB liquid medium containing 50 μg / mL spectinomycin for amplification. DNA sequencing identifies positive clones. Extract the plasmid to obtain the recombinant plasmid pECgRNA-N20-gltA. Store the recombinant plasmid pECgRNA-N20-gltA with glycerol for later use.

[0016] 2. Constructing gltA homologous fragments containing point mutations Based on the gltA gene sequence, forward primer P1, reverse primer P2, forward primer P3, and reverse primer P4 were designed. The specific primer sequences are as follows: P1: The nucleotide sequence is shown in SEQ ID No. 4 of the sequence listing; P2: The nucleotide sequence is shown in SEQ ID No. 5 of the sequence listing; P3: The nucleotide sequence is shown in SEQ ID No. 6 of the sequence listing; P4: The nucleotide sequence is shown in SEQ ID No. 7 of the sequence listing; Using the high-threonine-producing strain Escherichia coli Thr-0 (i.e. E.coli W3110 / thrA* / lysC* / Ptrc-ThrABC / △lysA / △metA / △tdh / Ptrc-ppcUsing genomic DNA as a template, forward primer P1, reverse primer P2, forward primer P3, and reverse primer P4 were designed based on the gltA gene sequence. When designing forward primer 3, a synonymous mutation was introduced in the coding region downstream of the N20 target sequence (the gltA-M fragment and the gltA-PM fragment, i.e., the 334th position of SEQ ID No. 8 and SEQ ID No. 9 in the sequence listing, was mutated from base G to base C). This disrupted the target site recognition sequence of Cas9 without changing the amino acid sequence of citrate synthase, thus preventing the Cas9 protein expressed by plasmid pEcCAS from repeatedly cutting the edited site of the genome after editing, and ensuring the stability of the genome of the obtained recombinant strain. The target fragment was then amplified using forward primer P1 / reverse primer P2 and forward primer P3 / reverse primer P4, yielding DNA products of 333 bp and 404 bp in length, respectively. The two fragments had an 18 bp overlap at the promoter mutation site. After separation and purification by agarose gel electrophoresis, the purified fragments were used as a mixed template for overlap extension PCR using forward primer P1 / reverse primer P4, successfully amplifying a fusion fragment of 719 bp. Sequencing of this fusion fragment revealed two sequence types: one was the expected single-mutant fragment (a mutation from base A to base T at position 299), named gltA-M; the other had an additional mutation in the promoter region (a mutation from base A to base C at position 216), forming a double-mutant fragment, named gltA-PM. The nucleotide sequence of the gltA-M fragment is shown in SEQ ID No. 8 of the sequence listing, and the nucleotide sequence of the gltA-PM fragment is shown in No. 9. The ratio of gltA-M fragments to gltA-PM fragments is 3:1.

[0017] 3. Construct a strain containing the point mutation gltA The plasmid pEcCAS, carrying a kanamycin resistance marker from the CRISPR knockout system, was electroporated into L-threonine-producing Escherichia coli Thr-0 competent cells. After screening on LB solid medium containing 50 μg / mL kanamycin, positive single clones were selected and cultured to obtain the recombinant strain Thr-0 / pEcCAS.

[0018] The constructed gltA-M and gltA-PM fragments were co-electrotransformed with the recombinant plasmid pECgRNA-N20-gltA into competent cells of the recombinant strain Thr-0 / pEcCAS. The specific procedure was as follows: 100 μL of competent cells were thawed on ice, and 100 ng of the recombinant plasmid pECgRNA-N20-gltA and 400 ng of either the gltA-M or gltA-PM fragment were added sequentially. After gentle mixing, the mixture was transferred to a pre-chilled 2 mm Bio-rad electrotransfer cuvette for electrotransfer. The electrotransfer parameters were 2.5 kV, 25 μF, and 200 Ω. Immediately after electrotransfer, 900 μL of LB liquid medium was added to the cuvette, and the mixture was mixed by pipetting and transferred to a sterile centrifuge tube. The cells were then incubated at 1000 rpm and 37°C for 45 min. After culturing, centrifuge at 5000 rpm for 5 min to remove 900 μL of supernatant. Mix the remaining 100 μL of bacterial culture by pipetting and spreading it onto LB solid medium containing 50 μg / mL spectinomycin and 50 μg / mL kanamycin. After screening with spectinomycin and kanamycin double antibiotic plates, colony PCR was performed on single colonies using forward primer P1 / reverse primer P4. Those that could amplify a 719 bp DNA fragment (nucleotide sequence as shown in SEQ ID No. 8 and No. 9 in the sequence listing) were considered potential positive transformants. The PCR products were sequenced, and the target mutant strain was obtained by sequence alignment screening.

[0019] The obtained single-mutant and double-mutant strains were inoculated into LB liquid medium containing 20 mM rhamnose and 50 μg / mL kanamycin, respectively, and cultured overnight at 37°C with shaking to induce the elimination of plasmid pECgRNA-N20-gltA. The bacterial culture was then transferred to LBS liquid medium and cultured overnight at 37°C with shaking to induce the elimination of plasmid pEcCAS. The bacterial culture was then spread onto LBS solid medium, and single colonies were picked and inoculated onto LB solid plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin, and no antibiotics, respectively. Colonies that did not grow on antibiotic-containing plates but grew on antibiotic-free plates were selected. Further sequencing was used to verify the target gene sequence. After confirmation, mutant strains were obtained and named Thr-0gltA-M (a single-mutant strain containing the gltA-M fragment) and Thr-0 gltA-PM (a double-mutant strain containing the gltA-PM fragment), respectively.

[0020] 4. Detection of citrate synthase activity Thr-0, Thr-0 gltA-M, and Thr-0 gltA-PM strains were inoculated into LB liquid medium and cultured at 37°C and 220 rpm until the mid-log phase (OD2). 600(For a concentration of 0.6), take 1 mL of bacterial culture and centrifuge at 8000 rpm for 10 min at 4℃ to collect the bacterial cells. Wash the bacterial cells twice with pre-cooled 50 mM Tris-HCl solution (pH 8.0), and centrifuge to collect the precipitate. Resuspend the bacterial cells in 1 mL of 50 mM Tris-HCl solution (pH 8.0) containing 1 mM PMSF, and sonicate on ice (200 W power, 3 s sonication, 5 s interval, total time 10 min). Centrifuge the lysate at 12000 rpm for 20 min at 4℃, and collect the supernatant as the crude enzyme solution. The total protein concentration of the crude enzyme solution is determined using the Bradford method to calculate the specific activity of the enzyme, thus eliminating the influence of bacterial cell quantity differences on the enzyme activity results. For the assay, bovine serum albumin (BSA) was used as the standard, and standard solutions of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL were prepared. The crude enzyme solution was diluted 10-fold, and 20 μL of each solution was added to 200 μL of Bradford working solution. After mixing, the solution was incubated at room temperature for 5 min, and the absorbance was measured at 595 nm. The protein concentration was calculated based on the standard curve. Three parallel experiments were performed for each sample, and the average value was taken. The total protein concentration of the crude enzyme solution from the wild-type strain Thr-0 was 3.21 ± 0.18 mg / mL, the total protein concentration from the single mutant strain Thr-0 gltA-M was 3.15 ± 0.15 mg / mL, and the total protein concentration from the double mutant strain Thr-0gltA-PM was 3.18 ± 0.16 mg / mL. There was no significant difference among the three.

[0021] The activity of citrate synthase in the crude enzyme solution was then identified using the DTNB continuous monitoring method. The reaction system is shown in Table 1.

[0022] Table 1: Reaction system for citrate synthase activity assay

[0023] The above components were mixed and added to a quartz cuvette, and preheated at 37°C for 5 min. 50 μL of 10 mM oxaloacetic acid was added to initiate the reaction. After rapid mixing, the absorbance was continuously monitored at a wavelength of 412 nm. Records were taken every 10 s for 3 min. Three replicates were set up for each sample group, and the average value was taken. The detection results are shown in Table 2.

[0024] Table 2: Results of Citrate Synthase Activity Detection

[0025] The results in the table above show that the crude enzyme solution of Thr-0 gltA-PM has the lowest citrate synthase activity and relative enzyme activity.

[0026] 5. Threonine shake-flask fermentation experiment Thr-0, Thr-0 gltA-M, and Thr-0 gltA-PM strains were spread onto LB solid medium, and pea-sized streaks were inoculated into LB liquid medium in shake tubes. The cultures were incubated at 37°C for 4 hours to reach the logarithmic growth phase, yielding activated bacterial suspensions. These suspensions were then transferred to four-baffle shake flasks and sealed with gauze. Each shake flask contained 30 mL of the activated bacterial suspension. The formulation of the shake flask fermentation medium is shown in Table 3. The activated bacterial suspensions were then inoculated into the shake flasks to allow the initial OD to be determined. 600 The concentration was set to 0.1, and then shake-flask fermentation was carried out.

[0027] Table 3: Shake Flask Fermentation Medium Formulation

[0028] Shake-flask fermentation conditions: 37℃, initial pH 6.8, rotation speed 220 rpm, culture period 18 h. The fermentation results of L-threonine are shown in Table 4.

[0029] Table 4: Fermentation results of L-threonine

[0030] Note: The conversion rate is the conversion rate from glucose to L-threonine. The calculation formula is: Conversion rate (%) = (L-threonine yield / total sugar concentration) × 100%; Total sugar concentration = Initial sugar concentration - Residual sugar concentration at the end of fermentation.

[0031] The results showed that the L-threonine conversion rate of the gltA mutant strain was improved compared with that of the original strain Thr-0. The conversion rate of the double mutant strain Thr-0 gltA-PM was significantly improved, indicating that this strain can effectively reduce production costs and has good prospects for industrial application.

Claims

1. A method for improving L-threonine yield and conversion rate through combined mutation of the gltA gene and its promoter region, characterized in that, Includes the following steps: Construct the recombinant plasmid pECgRNA-gltA-N20, construct the gltA homologous fragment containing the point mutation, construct the strain containing the gltA point mutation, and ferment; The process for constructing a strain containing the point mutation gltA involved transforming the plasmid pEcCAS, carrying a kanamycin resistance marker from the CRISPR knockout system, into competent E. coli cells. Positive single clones were selected to obtain the recombinant strain Thr-0 / pEcCAS. The gltA-PM fragment and the recombinant plasmid pECgRNA-N20-gltA were then co-transformed into competent cells of the recombinant strain Thr-0 / pEcCAS. PCR verification was performed, and positive transformants capable of amplifying the gltA-PM fragment were selected. The nucleotide sequence of the gltA-PM fragment is shown in SEQ ID No. 9 of the sequence listing.

2. The method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region according to claim 1, characterized in that, The recombinant plasmid pECgRNA-gltA-N20 was constructed by searching for the gltA gene in the E. coli genome sequence, selecting the N20 sequence that is immediately downstream of the PAM sequence from the gltA gene, and forming dsDNA with BsaI sticky ends by annealing forward and reverse oligonucleotides. After ligating dsDNA with a linearized plasmid digested with BsaI, the ligation was performed into competent E. coli cells. Single clones were picked, identified as positive clones, and the plasmid was extracted to obtain the recombinant plasmid pECgRNA-gltA-N20.

3. The method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region according to claim 2, characterized in that, In the constructed recombinant plasmid pECgRNA-gltA-N20, the Escherichia coli genome sequence is the Escherichia coli MG1655 genome sequence; The gene sequence number of the gltA gene is NC_000913.3; The nucleotide sequence of the N20 sequence is shown in SEQ ID No. 1 of the sequence listing; The nucleotide sequence of the positive oligonucleotide is shown in SEQ ID No. 2 in the sequence listing; The nucleotide sequence of the reverse oligonucleotide is shown in SEQ ID No. 3 in the sequence listing; The annealing procedure is as follows: denaturation at 95°C for 5 minutes, followed by cooling to 16°C at a rate of 0.12°C / s and holding for 10 minutes; The competent Escherichia coli cells are Escherichia coli DH5α competent cells; The heat shock method was used when transforming the cells into competent E. coli cells.

4. The method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region according to claim 1, characterized in that, The construction of the gltA homologous fragment containing the point mutation was carried out using the E. coli genome sequence as a template. Forward primers P1, reverse primers P2, forward primers P3, and reverse primers P4 were designed based on the gltA gene sequence. The target fragment was amplified using forward primers P1 / reverse primers P2 and forward primers P3 / reverse primers P4 to obtain two fragments. Overlap extension PCR was performed using forward primers P1 / reverse primers P4 to obtain the gltA-PM fragment.

5. The method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region according to claim 4, characterized in that, In the construction of the gltA homologous fragment containing point mutations, the E. coli genome sequence is the E. coli Thr-0 genome sequence; The E. coli Thr-0 genome sequence is as follows: E. coli W3110 / thrA* / lysC* / Ptrc-ThrABC / △lysA / △metA / △tdh / Ptrc-ppc Genome sequence; When designing forward primer P3 based on the gltA gene sequence, a synonymous mutation was introduced in the coding region downstream of the N20 target sequence by designing forward primer P3, which mutated the base G to the base C at position 334 in the gltA-PM fragment. The nucleotide sequence of the forward primer P1 is shown in SEQ ID No. 4 of the sequence listing; The nucleotide sequence of the reverse primer P2 is shown in SEQ ID No. 5 of the sequence listing; The nucleotide sequence of the forward primer P3 is shown in SEQ ID No. 6 of the sequence listing; The nucleotide sequence of the reverse primer P4 is shown in SEQ ID No. 7 of the sequence listing; In the gltA-PM fragment, the 216th position is mutated from base A to base C, and the 299th position is mutated from base A to base T.

6. The method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region according to claim 1, characterized in that, In the strain containing the point mutation gltA, the competent Escherichia coli cells are Escherichia coli Thr-0 competent cells; The *E. coli* Thr-0 competent cells are... E. coli W3110 / thrA* / lysC* / Ptrc-ThrABC / △lysA / △metA / △tdh / Ptrc-ppc competent cells; Electroporation was used when transforming plasmid pEcCAS into competent E. coli cells, and when co-transforming the gltA-PM fragment with recombinant plasmid pECgRNA-N20-gltA into recombinant strain Thr-0 / pEcCAS competent cells.

7. The method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region according to claim 1, characterized in that, The fermentation process involves activating the double mutant strain to the logarithmic phase and then inoculating it into a fermentation medium for fermentation.

8. The method for improving L-threonine yield and conversion rate through joint mutation of the gltA gene and promoter region according to claim 7, characterized in that, In the fermentation process, the fermentation medium is formulated as follows: 44-46 g / L glucose, 0.15-0.17 mL / L phosphate, 0.38-0.42 g / L magnesium sulfate, 4.8-5.2 g / L yeast extract, 2.8-3.2 g / L potassium dihydrogen phosphate, 4.8-5.2 g / L ammonium sulfate, 9.5-10.5 g / L manganese sulfate, 1.8-2.2 g / L zinc sulfate, 9-11 mg / L ferrous sulfate, and 18-22 g / L calcium carbonate; The fermentation temperature is 36-38℃ and the rotation speed is 200-240 rpm.

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