A recombinant Escherichia coli THRS-6 and method for synthesizing L-threonine using the same

Abstract

A recombinant Escherichia coli THRS-6 and a method for synthesizing L-threonine thereof. The present invention belongs to the technical field of bioengineering, and overexpresses aspartokinase thrA <supgt; mut < / supgt; and pyridine nucleotide transhydrogenase pntAB genes, aspartate dehydrogenase aspDH gene derived from Pseudomonas aeruginosa, hemoglobin vgb gene derived from Vitreoscilla sp., and transcriptional regulator irrE gene derived from Deinococcus radiodurans, and further modifies the recombinant Escherichia coli by knocking out ldhA genes, obtaining a recombinant Escherichia coli THRS-6 using glucose as a substrate. Fermentation is carried out using the THRS-6, and the yield and conversion rate of L-threonine are significantly 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 hours 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 great room for improvement in the de novo efficient synthesis of threonine by Escherichia coli in terms of yield and sugar-acid conversion rate. Exploring new engineering targets to further increase L-threonine production is an ongoing goal. 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 (such as high osmotic pressure tolerance) is expected to further increase the yield of L-threonine, and enhancing the oxygen utilization rate of the strain can further increase the intracellular energy supply and use it for product synthesis. This multi-dimensional engineering strategy has important guiding significance for constructing recombinant strains for efficient synthesis of L-threonine. Summary of the Invention

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

[0006] To achieve the above invention purpose, the present invention provides the following technical solutions:

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

[0008] Preferably, the recombinant Escherichia coli THRS-6 overexpresses aspartokinase thrA mut derived from Escherichia coli and pyridine nucleotide transhydrogenase pntAB genes, aspartate dehydrogenase aspDH gene derived from Pseudomonas aeruginosa, hemoglobin vgb gene derived from Vitreoscilla sp., and transcriptional regulator irrE gene derived from Deinococcus radiodurans, and knocks out the ldhA gene;

[0009] Preferably, the aspartokinase thrA mut has glycine at position 433 mutated to arginine;

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

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

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

[0013] Preferably, knocking out the coding gene of ldhA is inactivated or completely deleted in the recombinant Escherichia coli genome by gene editing; The gene editing method includes the gene editing method of CRISPR Cas9;

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

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

[0016] Preferably, the coding genes of thrA mut 、pntAB, aspDH, vgb, and irrE are expressed under the initiation of a replaced promoter;

[0017] Preferably, the replaced promoter includes the Trc promoter; Preferably, the sequence of the Trc promoter is as shown in 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 above, and the method includes the following steps:

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

[0020] (2) PCR amplify the thrA mut gene driven by the Trc promoter from the Escherichia coli genome;

[0021] (3) Fuse the upstream and downstream homologous arms of tdcC and the thrA fragment driven by the Trc promoter to obtain the U-thrA mut -D fragment; mut

[0022] (4) Transform the obtained fusion fragment U-thrA mut -D and the vector containing tdcC-sgRNA into the recombinant Escherichia coli THRS to obtain a recombinant strain in which the pseudogene tdcC is knocked out and thrA mut driven by the Trc promoter is integrated at the tdcC locus;

[0023] (5) Based on the recombinant strain obtained in the previous round, using and repeating the same methods as steps (1)-(4) above, recombinant strains with pntAB integrated at the ilvN locus, recombinant strains with aspDH integrated at the tfaD locus, recombinant strains with vgb integrated at the rph locus, and recombinant strains with irrE integrated at the bcsB locus were successively constructed; recombinant strains overexpressing thrA mut , pntAB, aspDH, vgb, and irrE were obtained;

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

[0025] Preferably, the gene editing method of CRISPR Cas9 is adopted;

[0026] Preferably, the recombinant Escherichia coli THRS is Escherichia coli THRS strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024, at the address of 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, and the method uses the recombinant Escherichia coli THRS-6 according to the first aspect of the present invention above for fermentation;

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

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

[0030] 2) Inoculate the seed liquid obtained in step 1) into a fermentation medium for aerobic fermentation to obtain L-threonine from the fermentation broth;

[0031] Preferably, the seed culture medium comprises components in the following concentrations: 4 - 10 g / L of dried corn steep liquor, 10 - 30 g / L of glucose, 2.5 - 10 g / L of yeast powder, 1 - 3 g / L of KH2PO4, 1 - 3 g / L of magnesium sulfate, 10 - 30 mg / L of FeSO4·7H2O, 10 - 30 mg / L of MnSO4·H2O, and 10 - 30 mg / L of biotin (Biotin);

[0032] Preferably, the fermentation culture medium comprises components in 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 dried corn steep liquor, and 2 - 20 mg / L of vitamin B1.

[0033] Beneficial effects: The recombinant Escherichia coli THRS-6 prepared by the present invention overexpresses the coding genes of thrA mut , pntAB, aspDH, vgb, and irrE, and knocks out the coding gene of ldhA. The recombinant Escherichia coli THRS-6 can use glucose as a substrate, and ferment using the recombinant Escherichia coli THRS-6, resulting in a significant increase in the yield and conversion rate of L-threonine, laying a foundation for the industrial production of L-threonine. In the examples of the present invention, using the recombinant Escherichia coli THRS-6 and glucose as a substrate, fermenting in a 5 L fermenter for 48 h can produce 125.3 g / L of threonine, and the sugar-acid conversion rate reaches 42%, indicating that the recombinant strain for highly efficient synthesis of L-threonine of the present invention has broad industrial application prospects. Description of the Drawings

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

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

[0036] A recombinant Escherichia coli THRS-6, which is Escherichia coli THRS-6 strain, was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31695. Detailed implementation manners

[0037] The following combines examples and drawings to provide 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. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0038] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained 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 through gene editing, the pseudogene tdcC of the starting strain was knocked out and the coding gene of thrA was integrated at the tdcC locus; the pseudogene ilvN of the starting strain was knocked out and the coding gene of pntAB was integrated at the ilvN locus; and the pseudogene tfaD of the starting strain was knocked out and the coding gene of aspDH was integrated at the tfaD locus; the pseudogene rph of the starting strain was knocked out and the coding gene of vgb was integrated at the rph locus; the gene bcsB of the starting strain was knocked out and the coding gene of irrE was integrated at the bcsB locus; the ldhA gene of the starting strain was knocked out. mut The coding genes of overexpressed thrA, pntAB, aspDH, vgb and irrE are all expressed under the initiation of the Trc promoter. The Trc promoter sequence in this example is SEQ ID NO.1. The present invention does not particularly limit the method of gene editing, and CRISPR Cas9 is selected for gene editing in this example.

[0041] In this example, the coding genes of overexpressed thrA mut and pntAB come from Escherichia coli, the coding gene of overexpressed aspDH comes from Pseudomonas aeruginosa, the coding gene of overexpressed vgb comes from Vitreoscilla, and the coding gene of overexpressed irrE comes from Deinococcus radiodurans.

[0042] In this example, the coding genes of overexpressed thrA mut and pntAB come from Escherichia coli, the coding gene of overexpressed aspDH comes from Pseudomonas aeruginosa, the coding gene of overexpressed vgb comes from Vitreoscilla, and the coding gene of overexpressed irrE comes from Deinococcus radiodurans.

[0043] Before integrating the thrA mut locus, it also includes mutating the glycine at position 433 of the thrA mut to arginine. The complete coding gene sequences of thrA mut , pntAB, aspDH, vgb, and irrE in this example are shown in SEQ ID NO.2 - 6; the sequence of the ldhA gene is shown in SEQ ID NO.7.

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

[0045] This example also provides a method for constructing the recombinant Escherichia coli THRS - 6. Gene editing is carried out using the CRISPR Cas9 method, including the following steps: (1) PCR amplify the upstream homologous arm and downstream homologous arm of the pseudogene tdcC from the genome of Escherichia coli K - 12 W3110; (2) PCR amplify the thrA mut gene driven by the Trc promoter from the Escherichia coli genome; (3) fuse the upstream and downstream homologous arms of tdcC and the thrA mut fragment driven by the Trc promoter to obtain the U - thrA mut -D fragment; (4) transform the obtained fusion fragment U - thrA mut -D and the vector containing tdcC - sgRNA into the recombinant Escherichia coli THRS to obtain a recombinant strain with the pseudogene tdcC knocked out and the thrA mut driven by the Trc promoter integrated at the tdcC locus; (5) using the recombinant strain obtained in the previous round as the construction basis, and repeating the same methods of steps (1) - (4) above, the only difference is using the corresponding primers for each integration site and each target gene, and successively constructing recombinant strains with pntAB integrated at the ilvN locus; recombinant strains with aspDH integrated at the tfaD locus; recombinant strains with vgb integrated at the rph locus; recombinant strains with irrE integrated at the bcsB locus; finally obtaining recombinant strains overexpressing thrA mut , pntAB, aspDH, vgb, and irrE; and (6) knocking out ldhA based on the recombinant strain overexpressing thrA mut , pntAB, aspDH, vgb, and irrE obtained in step (5) to obtain a recombinant strain with ldhA knocked out.

[0046] In this embodiment, to complete the construction of the recombinant Escherichia coli THRS-6, the primers shown in Table 1 were used.

[0047] Table 1 Primer Information

[0048]

[0049]

[0050] The said 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 coding gene of the said ldhA is shown in SEQ ID NO. 7.

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

[0053] 1. Construction of the fusion fragment U-thrA mut -D

[0054] Using the primers tdcC-U-F, tdcC-U-R, tdcC-D-F, and tdcC-D-R in Table 1, the upstream and downstream homologous arm fragments on both sides of the tdcC gene were amplified from the Escherichia coli K-12 genome respectively to obtain the fragments tdcC1 and tdcC2;

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

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

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

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

[0059] 3. Construction of the recombinant Escherichia coli overexpressing the thrA mut gene

[0060] The recombinant plasmid tdcC-sgRNA and the fusion fragment U-thrA mut -D were transformed into recombinant Escherichia coli THRS. Primers tdcC-U-F and tdcC-D-R were used for colony PCR to screen the transformants, and it was confirmed that the fusion fragment U-thrA mut -D was successfully integrated into the tdcC locus. The cells were cultured at 30 °C for 12 h with 2 mM arabinose added to remove the recombinant plasmid tdcC-sgRNA, and a recombinant strain was obtained.

[0061] 4. Using the recombinant strain obtained in the previous round as the construction basis, following the same methods described in steps 1 to 3 above, with the only difference being that the primers in Table 1 corresponding to each integration site and each target gene were used, recombinant strains integrating pntAB, aspDH, vgb, and irrE were constructed in sequence, and finally a recombinant strain overexpressing thrA mut , pntAB, aspDH, vgb, and irrE was obtained.

[0062] 5. Using the primers ldhA-U-F, ldhA-U-R, ldhA-D-F, and ldhA-D-R in Table 1, the upstream and downstream homologous 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-U-F and ldhA-D-R in Table 1, fragments ldhA1 and ldhA2 were subjected to fusion PCR to obtain the fusion fragment U-ldhA-D.

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

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

[0066] 7. Construction of recombinant Escherichia coli with the ldhA gene knocked out

[0067] The recombinant plasmid ldhA-sgRNA and the fusion fragment U-ldhA-D were transformed into the thrA-overexpressing strain obtained in step 4 above mut, a recombinant strain of pntAB, aspDH, vgb, and irrE. Primers ldhA-U-F and ldhA-D-R were used for colony PCR to screen for transformants, confirming that the fusion fragment U-ldhA-D was successfully integrated into the ldhA locus. Cultured at 30 °C for 12 h with 2 mM arabinose added, the recombinant plasmid ldhA-sgRNA was removed to obtain overexpressed thrA mut , pntAB, aspDH, vgb, and irrE, and the recombinant Escherichia coli THRS-6 with the ldhA gene knocked out.

[0068] The obtained recombinant Escherichia coli THRS-6 is the Escherichia coli THRS-6 strain, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on August 20, 2024. The deposit address is 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 the seed medium for seed culture, and then the seed culture was transferred to the fermentation medium for culture at an inoculation amount of 20%. After the inoculation, aerobic fermentation was carried out. The temperature of the aerobic fermentation was 37 °C, and the dissolved oxygen was 30%. During the aerobic fermentation process, after the bottom sugar was exhausted, the residual sugar was controlled at 0 - 1 g / L by feeding glucose.

[0071] 1. Process control in a 5 L seed tank

[0072] a. Adjust the temperature to 37 °C, pH 7.0, rotation speed 500 rpm, air volume 0.3 m 3 / h. The temperature was controlled at 37 °C throughout the process, the tank pressure was 0.05 - 0.08 MPa, and the culture period was 10 h;

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

[0074] c. The seed medium was corn steep liquor powder 5 g / L, glucose 20 g / L, yeast powder 5 g / L, KH2PO4 2 g / L, magnesium sulfate 1 g / L, FeSO4·7H2O 20 mg / L, MnSO4·H2O 20 mg / L, biotin 30 mg / L.

[0075] 2. Fermentation process control in a 5 L fermentation tank

[0076] a. Adjust the temperature to 37 °C, pH 7.0, initial rotation speed 300 rpm, air volume 0.3 m3 / h. The temperature was controlled at 37 °C throughout the process, and the tank pressure was 0.05 - 0.08 MPa;

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

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

[0079] e. The fermentation medium is glucose 20 g / L, potassium dihydrogen phosphate 2 g / L, yeast powder 3 g / L, betaine 1 g / L, magnesium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, ammonium sulfate 4.5 g / L, MnSO4·H2O 10 mg / L, corn steep liquor dry powder 8 g / L, vitamin B1 10 mg / L.

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

[0081] 1) Sample treatment: Take 1 mL of the fermentation broth after 48 h of fermentation, centrifuge at 12000 rpm for 10 min to remove the thallus 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) Analysis method: OPA pre-column derivation

[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, dissolve it with ultrapure water and make up the volume to 1 L, then add 200 μL of triethylamine, adjust the pH to 7.20±0.05 with 5% acetic acid; after suction filtration, add 5 mL of tetrahydrofuran, mix and then filter through a 0.22 μm inorganic filter membrane, and then put it into an ultrasonic cleaning pot to exhaust for 20 min for standby.

[0087] Mobile phase B: Weigh 3.01 g of anhydrous sodium acetate into a beaker; dissolve it with ultrapure water and make up the volume 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 this solution, mix and then filter, and put it into an ultrasonic cleaning pot to exhaust for 20 min for standby.

[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 strain THRS-6 in a 5L fermenter

[0092]

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

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

Claims

1. A recombinant Escherichia coli THRS-6, characterized in that, The recombinant Escherichia coli THRS-6 is Escherichia coli strain THRS-6, which was deposited at 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 aspartokinase derived from Escherichia coli thrA mut and pyridine nucleotide transhydrogenase pntAB genes, aspartate dehydrogenase derived from Pseudomonas aeruginosa aspDH genes, hemoglobin derived from Vitreoscilla vgb genes, and transcriptional regulator derived from Deinococcus radiodurans irrE genes, and knocks out ldhA genes.

3. The recombinant Escherichia coli THRS-6 according to claim 2, characterized in that, The aspartokinase thrA mut has glycine at position 433 mutated to arginine; The thrA mut 、 pntAB 、 aspDH 、 vgb and irrE nucleic acid sequences of the encoding genes are shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively; The ldhA 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 、 aspDH 、 vgb and irrE The coding genes of are based on Escherichia coli THRS as the starting strain, and through gene editing, the pseudogenes of the starting strain are knocked out tdcC and integrated at the tdcC site the thrA mut coding gene; Knock out the pseudogene of the starting strain ilvN and integrate at ilvN the locus pntAB with the encoding gene; Knock out the pseudogene of the starting strain tfaD and integrate at tfaD the locus aspDH encoding gene; Knock out the pseudogene of the starting strain rph and integrate at the rph site the coding gene of vgb ; knock out the gene bcsB of the starting strain and integrate at the bcsB site the coding gene of irrE ; Knockout ldhA The encoding gene of is inactivated or completely deleted by gene editing in the recombinant Escherichia coli genome; The Escherichia coli THRS is Escherichia coli strain THRS, which was deposited at 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, The said thrA mut , pntAB , aspDH , vgb and irrE encoding genes are expressed under the initiation of a replaced promoter; The replaced promoter includes the Trc promoter; the sequence of the Trc promoter is shown in SEQ ID NO.

1.

6. The construction method of recombinant Escherichia coli THRS-6 according to any one of claims 1-5, characterized in that, Comprising the following steps: (1) PCR amplify the upstream homologous arm and downstream homologous arm of the pseudogene from the genome of Escherichia coli K-12 tdcC ; (2) PCR amplify the gene driven by the Trc promoter from the Escherichia coli genome thrA mut gene; (3) Combine tdcC the upstream and downstream homologous arms of thrA mut fragment to obtain the U-thrA mut -D fragment; (4) The obtained fusion fragment U-thrA mut -D and the vector containing tdcC-sgRNA were transformed into recombinant Escherichia coli THRS to obtain the recombinant strain in which the pseudogene tdcC was knocked out and tdcC at the site was integrated with thrA mut driven by the Trc promoter; (5)Based on the recombinant strain obtained with the front wheel, using and repeating the same methods as in the above steps (1)-(4), recombinant strains integrated at the ilvN site were successively constructed; pntAB ​ at tfaD site integration aspDH recombinant strain; at rph site integration vgb recombinant strain; at bcsB site integration irrE recombinant strain; obtaining an overexpressed thrA mut , pntAB , aspDH , vgb and irrE recombinant strain; (6) Based on the recombinant strains overexpressing thrA mut , pntAB , aspDH , vgb and irrE , knockout ldhA, is carried out to obtain the recombinant Escherichia coli THRS-6 with ldhA knocked out.

7. The construction method according to claim 6, characterized in that, Adopting the gene editing method of CRISPR Cas9.

8. The construction method according to claim 6 or 7, characterized in that, The recombinant Escherichia coli THRS is Escherichia coli strain THRS, which was deposited at 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, Fermentation is carried out using the recombinant Escherichia coli THRS-6 according to any one of claims 1-5.

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

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