Recombinant escherichia coli THRS-6 and method for synthesizing L-threonine by using recombinant escherichia coli THRS-6

Abstract

The invention relates to recombinant escherichia coli THRS-6 and a method for synthesizing L-threonine by using the recombinant escherichia coli THRS-6. The invention belongs to the technical field of bioengineering, and relates to an aspartic acid kinase thrAmut and pyridine nucleotide transhydrogenase pntAB gene derived from escherichia coli, an aspartic acid dehydrogenase aspDH gene derived from pseudomonas aeruginosa, a hemoglobin vgb gene derived from vitreoscilla and a transcriptional regulation factor irrE gene derived from radiococcus. The recombinant escherichia coli is used as a substrate, the ldhA gene is knocked out to further modify the recombinant escherichia coli, the recombinant escherichia coli THRS-6 with glucose as the substrate is obtained, the THRS-6 is used for fermentation, and the yield and conversion rate of the L-threonine are obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Escherichia coli THRS-6 and a method for using the recombinant Escherichia coli THRS-6 to synthesize L-threonine. Background Art

[0002] Threonine is an essential amino acid for mammals, mainly used in medicine, chemical reagents, food fortifiers, feed additives, etc. Because threonine contains hydroxyl groups in its structure, it has a water-retaining effect on human skin. It combines with oligosaccharide chains and plays an important role in protecting cell membranes. It can promote phospholipid synthesis and fatty acid oxidation in the body. Threonine preparations have the medicinal effects of promoting human development and resisting fatty liver, and are a component of compound amino acid infusions. At the same time, threonine is the second limiting amino acid in pig feed and the third limiting amino acid in poultry feed. In recent years, as the market share of L-threonine has gradually increased, its commercial value has also been valued by people.

[0003] At present, the main methods for producing L-threonine are chemical synthesis, protein hydrolysis and microbial fermentation. Among them, microbial fermentation has low production cost, high production intensity and low environmental pollution, making it the most widely used method for industrial production of L-threonine. At present, the strains producing L-threonine mainly include Escherichia coli, Corynebacterium glutamicum and Serratia marcescens. Tianjin Institute of Industrial Microbiology has removed the L-threonine metabolic bottleneck through in vitro metabolic bottleneck identification, which has increased the L-threonine production by 30%. Qiao Jianjun of Tianjin University used THRD as the starting strain and improved the L-threonine production through two-stage carbon allocation and cofactor generation strategies. The strain produced 70.8g / L L-threonine in 40h, and the sugar-acid conversion rate was 40.4%. Wang Xiaoyuan of Jiangnan University transformed Escherichia coli TWF001 through metabolic engineering. After 36h of shake flask culture, it can produce 15.85g / L L-threonine, and the sugar-acid conversion rate is 53%. Sang Yup Lee et al. used the system biology method to construct an engineered strain from E coli W3100 (1acI-) to produce 82.4 g / L of L-threonine in 50 h of fermentation, with a sugar-acid conversion rate of 39.3%. The Tianjin Institute of Industrial Microbiology successfully screened an L-threonine mutant strain that could produce 123.61 g / L of L-threonine by constructing an L-threonine biosensor.

[0004] At present, there is still a lot of room for improvement in the yield and sugar-acid conversion rate of efficient de novo synthesis of threonine using Escherichia coli. Exploring new engineering targets to further increase L-threonine production is a goal that people are constantly working on. Expanding the L-threonine synthesis pathway by introducing exogenous genes can further increase the carbon flux of L-threonine synthesis. In addition, enhancing the stress resistance of the strain (resistance to high osmotic pressure, etc.) is expected to further increase the yield of L-threonine. Enhancing the strain's oxygen utilization rate can further increase the supply of intracellular energy and use it for product synthesis. This multi-dimensional engineering strategy has important guiding significance for the construction of recombinant strains that efficiently synthesize L-threonine. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a recombinant Escherichia coli THRS-6, a construction method thereof and a method for synthesizing L-threonine. Based on multi-dimensional metabolic engineering transformation and optimization strategies, a recombinant Escherichia coli strain that efficiently synthesizes L-threonine is obtained.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a recombinant Escherichia coli THRS-6, wherein the recombinant Escherichia coli THRS-6 is an Escherichia coli THRS-6 strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 31695;

[0008] Preferably, the recombinant E. coli THRS-6 overexpresses aspartate kinase thrA from E. coli mut and pyridine nucleotide transhydrogenase pntAB gene, aspartate dehydrogenase aspDH gene from Pseudomonas aeruginosa, hemoglobin vgb gene from Vitreoscilla, and transcriptional regulator irrE gene from Radiodurans radiodurans, and the ldhA gene was knocked out;

[0009] Preferably, the aspartate kinase thrA mut The glycine at position 433 of the gene was mutated to arginine;

[0010] Preferably, the thrA mut The nucleic acid sequences of the coding genes of pntAB, aspDH, vgb and irrE are shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively;

[0011] Preferably, the nucleic acid sequence of the gene encoding ldhA is shown in SEQ ID NO. 7;

[0012] Preferably, overexpression of thrA mut The coding genes of pntAB, aspDH, vgb and irrE are based on Escherichia coli THRS as the starting strain, and the pseudogene tdcC of the starting strain is knocked out by gene editing and the thrA mut knock out the pseudogene ilvN of the starting strain and integrate the coding gene of pntAB at the ilvN site; knock out the pseudogene tfaD of the starting strain and integrate the coding gene of aspDH at the tfaD site; knock out the pseudogene rph of the starting strain and integrate the coding gene of vgb at the rph site; knock out the gene bcsB of the starting strain and integrate the coding gene of irrE at the bcsB site;

[0013] Preferably, the gene encoding ldhA is knocked out by inactivating or completely deleting it in the recombinant Escherichia coli genome by gene editing; the gene editing method includes CRISPR Cas9 gene editing;

[0014] Preferably, the starting strain of the recombinant Escherichia coli THRS-6 comprises recombinant Escherichia coli THRS;

[0015] The Escherichia coli THRS is an Escherichia coli THRS strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31694;

[0016] Preferably, the thrA mut , pntAB , aspDH , vgb , and irrE encoding genes are expressed from the replaced promoter;

[0017] Preferably, the replacement promoter includes Trc promoter; preferably, the sequence of the Trc promoter is shown as SEQ ID NO. 1.

[0018] The second aspect of the present invention provides a method for constructing the recombinant Escherichia coli THRS-6 according to the first aspect, the method comprising the following steps:

[0019] (1) PCR amplified the upstream homology arm and downstream homology arm of the pseudogene tdcC from the genome of Escherichia coli K-12 W3110;

[0020] (2) PCR amplification of thrA driven by the Trc promoter from the Escherichia coli genome mut Gene;

[0021] (3) The upstream and downstream homology arms of tdcC and thrA driven by Trc promoter mut Fragment fusion to obtain U-thrA mut -D fragment;

[0022] (4) The obtained fusion fragment U-thrA mut -D and the vector containing tdcC-sgRNA were transformed into recombinant E. coli THRS, resulting in the knockout of pseudogene tdcC and the integration of thrA driven by Trc promoter at the tdcC site. mut Recombinant strains of

[0023] (5) Based on the recombinant strain obtained in the previous round, the same method as steps (1) to (4) above was repeated to construct a recombinant strain with pntAB integrated at the ilvN site; a recombinant strain with aspDH integrated at the tfaD site; a recombinant strain with vgb integrated at the rph site; a recombinant strain with irrE integrated at the bcsB site; and a recombinant strain with thrA overexpression. mut , pntAB, aspDH, vgb and irrE recombinant strains;

[0024] (6) The overexpressed thrA obtained in step (5) mut ldhA was knocked out based on the recombinant strains of pntAB, aspDH, vgb and irrE to obtain the recombinant Escherichia coli THRS-6 with ldhA knocked out;

[0025] Preferably, CRISPR Cas9 gene editing is used;

[0026] Preferably, the recombinant Escherichia coli THRS is an Escherichia coli THRS strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31694.

[0027] The third aspect of the present invention provides a method for biosynthesizing L-threonine, wherein the method uses the recombinant Escherichia coli THRS-6 according to the first aspect of the present invention for fermentation;

[0028] Preferably, the method for biosynthesizing L-threonine comprises the following steps:

[0029] 1) inoculating the recombinant Escherichia coli THRS-6 on a seed culture medium for culturing to obtain a seed solution;

[0030] 2) inoculating the seed liquid obtained in step 1) into a fermentation medium, performing aerobic fermentation, and obtaining L-threonine from the fermentation liquid;

[0031] Preferably, the seed culture medium comprises the following components: corn steep liquor powder 4-10 g / L, glucose 10-30 g / L, yeast powder 2.5-10 g / L, KH 2 PO 4 1-3g / L, magnesium sulfate 1-3g / L, FeSO 4 7H 2 O 10-30mg / L and MnSO 4 ·H 2 O 10-30mg / L, Biotin 10-30mg / L;

[0032] Preferably, the fermentation medium comprises the following components: glucose 10-30 g / L, potassium dihydrogen phosphate 1-5 g / L, yeast powder 1-5 g / L, betaine 0.5-2 g / L, magnesium sulfate 0.5-2 g / L, FeSO 4 7H 2 O 2-20mg / L, ammonium sulfate 2-20g / L, MnSO 4 ·H 2 O 2-20mg / L, corn steep liquor powder 2-20g / L and vitamin B1 2-20mg / L.

[0033] Beneficial effect: The recombinant Escherichia coli THRS-6 prepared by the present invention overexpresses thrA mut , pntAB, aspDH, vgb and irrE encoding genes, and knock out the encoding gene of ldhA. The recombinant Escherichia coli THRS-6 can use glucose as a substrate, and the recombinant Escherichia coli THRS-6 is used for fermentation, and the L-threonine yield and conversion rate are significantly improved, laying a foundation for the industrial production of L-threonine. In the embodiment of the present invention, the recombinant Escherichia coli THRS-6 is used, glucose is used as a substrate, and fermented in a 5L fermentation tank for 48h, 125.3g / L threonine can be produced, and the sugar acid conversion rate reaches 42%, indicating that the recombinant strain for efficiently synthesizing L-threonine of the present invention has a wide range of industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are the fermentation parameters of the THRS-6 strain in a 5-L fermenter. The squares represent the residual glucose in the fermentation broth at each sampling point (g / L), the circles represent the biomass of the strain at each sampling point (OD600), and the triangles represent the L-threonine production in the fermentation broth at each sampling point (g / L).

[0035] A recombinant Escherichia coli THRS, wherein the recombinant Escherichia coli THRS is an Escherichia coli THRS strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31694.

[0036] A recombinant Escherichia coli THRS-6, wherein the recombinant Escherichia coli THRS-6 is an Escherichia coli (Escherichiacoli) THRS-6 strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31695. DETAILED DESCRIPTION

[0037] The following is a detailed description of a recombinant Escherichia coli THRS-6 provided by the present invention, its construction method and its method for synthesizing L-threonine in conjunction with the examples and drawings. It is understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0038] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] Example 1 Recombinant Escherichia coli THRS-6 and its construction method

[0040] In this example, Escherichia coli THRS was used as the starting strain, and the pseudogene tdcC of the starting strain was knocked out by gene editing and the thrA was integrated at the tdcC site. mut The coding gene of the pntAB gene is integrated at the ilvN site by knocking out the pseudogene ilvN of the starting strain; and the coding gene of the aspDH gene is integrated at the tfaD site by knocking out the pseudogene tfaD of the starting strain; the coding gene of the vgb gene is integrated at the rph site by knocking out the pseudogene rph of the starting strain; the coding gene of the irrE gene is integrated at the bcsB site by knocking out the ldhA gene of the starting strain.

[0041] In this example, the overexpression of thrA mutThe coding genes of pntAB, aspDH, vgb and irrE are all expressed by the Trc promoter. The Trc promoter sequence in this embodiment is SEQ ID NO. 1. The present invention does not specifically limit the gene editing method. In this embodiment, CRISPR Cas9 is used for gene editing.

[0042] In this example, the overexpression of thrA mut The coding gene of pntAB comes from Escherichia coli, the coding gene of overexpression of aspDH comes from Pseudomonas aeruginosa, the coding gene of overexpression of vgb comes from Vitreoscilla, and the coding gene of overexpression of irrE comes from Radiodurans deinococcus.

[0043] Integrating thrA mut The thrA mut The glycine at position 433 of thrA is mutated to arginine. mut The complete coding gene sequences of pntAB, aspDH, vgb and irrE are shown in SEQ ID NOs.2 to 6; the sequence of the ldhA gene is shown in SEQ ID NO.7.

[0044] The starting strain of the recombinant Escherichia coli described in this embodiment, or also referred to as the basic strain, is recombinant Escherichia coli THRS, and the Escherichia coli THRS is an Escherichia coli THRS strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 31694.

[0045] This embodiment also provides a method for constructing the recombinant E. coli THRS-6, which uses the CRISPR Cas9 method for gene editing, including the following steps: (1) PCR amplifying the upstream homology arm and downstream homology arm of the pseudogene tdcC from the genome of E. coli K-12 W3110; (2) PCR amplifying the thrA gene driven by the Trc promoter from the genome of E. coli mut gene; (3) the upstream and downstream homologous arms of tdcC and thrA driven by Trc promoter mut Fragment fusion to obtain U-thrA mut -D fragment; (4) the obtained fusion fragment U-thrA mut -D and the vector containing tdcC-sgRNA were transformed into recombinant E. coli THRS, resulting in the knockout of pseudogene tdcC and the integration of thrA driven by Trc promoter at the tdcC site. mut(5) Based on the recombinant strain obtained in the previous round, the same method as steps (1) to (4) was used and repeated, except that the corresponding primers for each site to be integrated and each target gene were used to construct a recombinant strain with pntAB integrated at the ilvN site; a recombinant strain with aspDH integrated at the tfaD site; a recombinant strain with vgb integrated at the rph site; and a recombinant strain with irrE integrated at the bcsB site. Finally, a recombinant strain overexpressing thrA was obtained. mut , pntAB, aspDH, vgb and irrE recombinant strain; and (6) the overexpression thrA obtained in step (5) mut Based on the recombinant strains of pntAB, aspDH, vgb and irrE, ldhA was knocked out to obtain the recombinant strain with ldhA knocked out.

[0046] In this example, the primers shown in Table 1 were used to construct the recombinant E. coli THRS-6.

[0047] Table 1 Primer information

[0048]

[0049]

[0050] The thrA mut The nucleic acid sequences of the coding genes of pntAB, aspDH, vgb and irrE are shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively;

[0051] The nucleic acid sequence of the gene encoding ldhA is shown in SEQ ID NO. 7.

[0052] The specific steps of the construction method of the recombinant Escherichia coli are:

[0053] 1. Fusion fragment U-thrA mut -D build

[0054] Using the primers tdcC-UF, tdcC-UR, tdcC-DF and tdcC-DR in Table 1, the upstream and downstream homology arm fragments on both sides of the tdcC gene were amplified from the Escherichia coli K-12 genome to obtain fragments tdcC1 and tdcC2;

[0055] The thrA gene was amplified from the Escherichia coli genome using primers thrA-F and thrA-R in Table 1 to obtain fragment thrA mut ;

[0056] The fragments tdcC1, thrA mut , tdcC2 for fusion PCR to obtain the fusion fragment U-thrA mut -D.

[0057] 2. Construction of tdcC-sgRNA recombinant plasmid

[0058] Using primers pGRB-F and pGRB-R, linearized vector L-pGRB was obtained from vector pGRB by PCR, and the designed sgRNA was connected to the linearized vector L-pGRB to construct the recombinant plasmid tdcC-sgRNA.

[0059] 3. Overexpression of thrA mut Construction of recombinant Escherichia coli

[0060] The recombinant plasmid tdcC-sgRNA and fusion fragment U-thrA mut -D was transformed into recombinant E. coli THRS, and the transformants were screened by colony PCR using primers tdcC-UF and tdcC-DR to confirm the fusion fragment U- thrA mut -D was successfully integrated into the tdcC site, and 2 mM arabinose was added and cultured at 30 °C for 12 h. The recombinant plasmid tdcC-sgRNA was removed to obtain the recombinant strain.

[0061] 4. Based on the recombinant strain obtained in the previous round, the same method as described in steps 1 to 3 above was used, except that the primers in Table 1 corresponding to each site to be integrated and each target gene were used to construct the recombinant strain integrating pntAB, aspDH, vgb, and irrE in sequence, and finally the recombinant strain overexpressing thrA was obtained. mut , pntAB, aspDH, vgb and irrE recombinant strains.

[0062] 5. Using the primers ldhA-UF, ldhA-UR, ldhA-DF and ldhA-DR in Table 1, the upstream and downstream homology arm fragments on both sides of the ldhA gene were amplified from the Escherichia coli K-12 genome to obtain fragments ldhA1 and ldhA2;

[0063] Using the primers ldhA-UF and ldhA-DR in Table 1, the fragments ldhA1 and ldhA2 were subjected to fusion PCR to obtain the fusion fragment U-ldhA-D.

[0064] 6. Construction of ldhA-sgRNA recombinant plasmid

[0065] Using primers pGRB-F and pGRB-R, linearized vector L-pGRB was obtained from vector pGRB by PCR, and the designed sgRNA was connected to the linearized vector L-pGRB to construct the recombinant plasmid ldhA-sgRNA.

[0066] 7. Construction of recombinant Escherichia coli with ldhA gene knockout

[0067] The recombinant plasmid ldhA-sgRNA and fusion fragment U-ldhA-D were transformed into the overexpression thrA mut , pntAB, aspDH, vgb and irrE recombinant strains were obtained. The primers ldhA-UF and ldhA-DR were used to perform colony PCR to screen transformants. The fusion fragment U-ldhA-D was successfully integrated into the ldhA site. 2 mM arabinose was added and cultured at 30 °C for 12 h. The recombinant plasmid ldhA-sgRNA was removed and the overexpression of thrA was obtained. mut , pntAB, aspDH, vgb and irrE, and recombinant Escherichia coli THRS-6 with the ldhA gene knocked out.

[0068] The recombinant Escherichia coli THRS-6 obtained above is an Escherichia coli THRS-6 strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31695.

[0069] Example 2 Biosynthesis of L-threonine using recombinant Escherichia coli THRS-6

[0070] The recombinant strain THRS-6 constructed in Example 1 was inoculated into a seed culture medium for seed culture, and then the seed culture was transferred to a fermentation culture medium for culture at an inoculation rate of 20%. After the inoculation, aerobic fermentation was performed. The temperature of the aerobic fermentation was 37° C. and the dissolved oxygen was 30%. During the aerobic fermentation, after the base sugar was exhausted, the residual sugar was controlled at 0~1 g / L by adding glucose.

[0071] 1.5 L seed tank process control

[0072] a. Adjust the temperature to 37℃, pH 7.0, speed to 500rpm, air volume to 0.3m 3 / h, the whole process temperature is controlled at 37℃, the tank pressure is 0.05~0.08MPa, and the culture cycle is 10h;

[0073] b. Transplantation standard: OD600:12-15.

[0074] c. Seed culture medium is corn syrup powder 5g / L, glucose 20g / L, yeast powder 5g / L, KH2 PO 4 2g / L, magnesium sulfate 1g / L, FeSO 4 7H 2 O 20mg / L, MnSO 4 ·H 2 O 20mg / L, biotin 30mg / L.

[0075] 2.5 L fermentation tank fermentation process control

[0076] a. Adjust the temperature to 37℃, pH7.0, initial speed to 300rpm, air volume to 0.3m3 / h, the whole process temperature to 37℃, tank pressure to 0.05~0.08MPa;

[0077] c.DO control: 0h, air volume 0.3m 3 / h, 300rpm, tank pressure 0.05MPa;

[0078] d. When DO drops below 30%, the dissolved oxygen level is controlled at 30% by adjusting the ventilation volume and stirring speed until the fermentation ends;

[0079] e. The fermentation medium is glucose 20g / L, potassium dihydrogen phosphate 2g / L, yeast powder 3g / L, betaine 1g / L, magnesium sulfate 1g / L, FeSO 4 7H 2 O 10mg / L, ammonium sulfate 4.5g / L, MnSO 4 ·H 2 O 10mg / L, corn steep liquor powder 8g / L, vitamin B1 10mg / L.

[0080] 3. Determination method of L-threonine:

[0081] 1) Sample treatment: Take 1 mL of fermentation liquid after 48 hours of fermentation, centrifuge at 12000 rpm, centrifuge for 10 minutes to remove the bacteria and take the supernatant. Dilute the supernatant appropriately with deionized water and then filter it through a filter membrane with a pore size of 0.22 μm.

[0082] 2) Analytical method: OPA pre-column derivatization

[0083] 3) Chromatographic conditions:

[0084] (1) Chromatographic column: Chromatographic column C18 (250×4.6) mm

[0085] (2) Column temperature: 40°C

[0086] (3) Mobile phase A: Weigh 3.01 g of anhydrous sodium acetate into a beaker, add ultrapure water to dissolve and dilute to 1 L, then add 200 μL of triethylamine, and adjust the pH to 7.20±0.05 with 5% acetic acid; add 5 mL of tetrahydrofuran after filtration, mix and filter with a 0.22 μm inorganic filter membrane, then place in an ultrasonic cleaning pot to exhaust for 20 min and set aside.

[0087] Mobile phase B: Weigh 3.01 g of anhydrous sodium acetate into a beaker; add ultrapure water to dissolve and dilute to 200 mL; adjust the pH to 7.20±0.05 with 5% acetic acid; then add 400 mL of acetonitrile and 400 mL of methanol to the solution, mix and filter, place in an ultrasonic cleaning pot to exhaust for 20 minutes, and set aside.

[0088] (4) Flow rate: 1.0 mL / min;

[0089] (5) UV detector: 338 nm;

[0090] (6) Column temperature: 40°C.

[0091] 4. Verification of the production performance of THRS-6 strain in a 5L fermenter

[0092]

[0093] By continuing to transform the strain THRS with a certain threonine production, through the above-mentioned gene overexpression and knockout steps, the threonine production of the modified strain THRS-6 was increased by 24.9% compared with the original strain THRS, and the conversion rate was increased by 12%, providing a good foundation for further industrial applications.

[0094] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A recombinant Escherichia coli THRS-6, characterized in that: The recombinant Escherichia coli THRS-6 is an Escherichia coli THRS-6 strain, which was deposited in the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit number CGMCC No. 31695.

2. The recombinant Escherichia coli THRS-6 according to claim 1, characterized in that The recombinant Escherichia coli THRS-6 overexpresses aspartate kinase from Escherichia coli thrA mut and pyridine nucleotide transhydrogenase pntAB Gene, Aspartate dehydrogenase from Pseudomonas aeruginosa aspD Gene, hemoglobin from Vitreoscilla vgb Genes and transcriptional regulators from Deinococcus radiodurans iR Gene and knockout oeLh Gene.

3. The recombinant Escherichia coli THRS-6 according to claim 2, characterized in that Aspartate kinase thrA mut The glycine at position 433 of the gene was mutated to arginine; Said thrA mut , pntAB , aspD , vgb and iR The nucleic acid sequences of the encoding genes are shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively; Said oeLh The nucleic acid sequence of the encoding gene is shown in SEQ ID NO.

7.

4. The recombinant Escherichia coli THRS-6 according to claim 2 or 3, characterized in that Overexpression thrA mut , pntAB , aspD , vgb and iR The coding gene of the Escherichia coli THRS is used as the starting strain, and the pseudogene of the starting strain is knocked out by gene editing. tC and in tC Site integration thrA mut The coding gene of Knock out the pseudogenes of the starting strain ilvN and in ilvN Site integration pntAB The coding gene of Knock out the pseudogenes of the starting strain df and in df Site integration aspD The coding gene of Knock out the pseudogenes of the starting strain r and in r Site integration vgb Knock out the gene encoding the starting strain bxb and in bxb Site integration iR The coding gene of Knockout oeLh The coding gene is inactivated or completely deleted in the recombinant E. coli genome by gene editing; The Escherichia coli THRS is an Escherichia coli THRS strain, which was deposited in the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit number CGMCC No. 31694.

5. The recombinant Escherichia coli THRS-6 according to claim 4, characterized in that Said thrA mut , pntAB , aspD , vgb and iR The coding gene is expressed by the replaced promoter; The replacement promoter includes Trc promoter; the sequence of Trc promoter is shown in SEQ ID NO.

1.

6. The method for constructing the recombinant Escherichia coli THRS-6 according to any one of claims 1 to 5, characterized in that: The steps include: (1) PCR amplification of pseudogenes from the genome of Escherichia coli K-12 tC The upstream homology arm and the downstream homology arm; (2) PCR amplification of the Trc promoter-driven gene from the Escherichia coli genome thrA mut Gene; (3) tC The upstream and downstream homology arms and Trc promoter drive thrA mut Fragment fusion to obtain U-thrA mut -D fragment; (4) The obtained fusion fragment U-thrA mut -D and the vector containing tdcC-sgRNA were transformed into recombinant E. coli THRS to knock out the pseudogene tC and in tC Integration site driven by the Trc promoter thrA mut Recombinant strains of (5) Based on the recombinant strain obtained in the previous round, the same method as steps (1) to (4) above was repeated to construct the ilvN Site integration pntAB Recombinant strains of exist df Site integration aspD Recombinant strains of exist r Site integration vgb Recombinant strains of exist bxb Site integration iR Recombinant strains overexpressing thrA mut , pntAB , aspD , vgb and iR Recombinant strains of (6) Overexpression obtained in step (5) thrA mut , pntAB , aspD , vgb and iR Knockout based on the recombinant strain ldhA, Get knockout oeLh Recombinant Escherichia coli THRS-6.

7. The construction method according to claim 6, characterized in that: Using CRISPR Cas9 gene editing method.

8. The construction method according to claim 6 or 7, characterized in that: The recombinant Escherichia coli THRS is an Escherichia coli THRS strain, which was deposited in the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, with the deposit number CGMCC No.31694.

9. A method for biosynthesizing L-threonine, characterized in that: The recombinant Escherichia coli THRS-6 according to any one of claims 1 to 5 is used for fermentation.

10. The method according to claim 9, characterized in that The following steps are involved: 1) inoculating the recombinant Escherichia coli THRS-6 on a seed culture medium for culturing to obtain a seed solution; 2) inoculating the seed liquid obtained in step 1) into a fermentation medium, performing aerobic fermentation, and obtaining L-threonine from the fermentation liquid; The seed culture medium comprises the following components in concentrations: 4-10 g / L corn steep liquor dry powder, 10-30 g / L glucose, 2.5-10 g / L yeast powder, 1-3 g / L KH2PO4, 1-3 g / L magnesium sulfate, 10-30 mg / L FeSO4·7H2O and 10-30 mg / L MnSO4·H2O, and 10-30 mg / L biotin; The fermentation medium comprises components with the following concentrations: 10-30 g / L of glucose, 1-5 g / L of potassium dihydrogen phosphate, 1-5 g / L of yeast powder, 0.5-2 g / L of betaine, 0.5-2 g / L of magnesium sulfate, 2-20 mg / L of FeSO4·7H2O, 2-20 g / L of ammonium sulfate, 2-20 mg / L of MnSO4·H2O, 2-20 g / L of corn steep liquor dry powder and 2-20 mg / L of vitamin B1.

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