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

Abstract

The invention relates to a recombinant escherichia coli THRS and a method for synthesizing L-threonine by using the recombinant escherichia coli THRS. The invention belongs to the technical field of bioengineering, and provides a recombinant escherichia coli THRS which overexpresses encoding genes of ppc, aspC, aspA, thrAmutBC, asd, rhtC, rhtA, asdtm and thrAmut, knockout an encoding gene of fnr and knockout an encoding gene of iclR, the recombinant escherichia coli THRS takes glucose as a substrate for fermentation, and the yield and conversion rate of L-threonine are obviously improved; the L-threonine strain has the advantages that the L-threonine strain does not contain exogenous plasmids, all modification sites are integrated onto a genome, higher stability is achieved, antibiotics do not need to be added into a fermentation culture medium of the strain, and the produced L-threonine product is free of antibiotic residues and higher in safety.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Escherichia coli THRS and a method for using the recombinant Escherichia coli THRS 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, and has become 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. Sang YupLee et al. used the system biology method to construct an engineering strain from E coli W3100 (lacI-) to produce 82.4g / L of L-threonine in 50h fermentation, and the sugar-acid conversion rate was 39.3%. Qiao Jianjun of Tianjin University used THRD as the starting strain and improved the production of L-threonine through two-stage carbon allocation and cofactor generation strategies. The strain produced 70.8g / L of 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%. Tianjin Institute of Industrial Microbiology successfully screened out an L-threonine mutant strain by constructing an L-threonine biosensor that can produce 123.61g / L L-threonine.

[0004] At present, there is still much room for improvement in the yield and sugar-acid conversion rate of efficient de novo synthesis of threonine using Escherichia coli. In addition, the method of overexpressing key genes through plasmids requires the addition of antibiotics during the fermentation process to maintain the stability of the plasmid, which not only increases safety risks but also increases fermentation costs. Therefore, the development of a plasmid-free and efficient L-threonine synthesis recombinant strain is of great significance for industrial production. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a recombinant Escherichia coli THRS, a construction method thereof and a method for synthesizing L-threonine. Based on a system metabolic engineering transformation and optimization strategy, 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, which is Escherichia coli, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee 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.

[0008] The second aspect of the present invention provides a method for constructing a recombinant Escherichia coli THRS according to the first aspect, the method comprising the following steps:

[0009] (1) PCR amplify the upstream homology arm and downstream homology arm of the pseudogene yeeL from the genome of Escherichia coli;

[0010] (2) PCR amplification of the ppc gene driven by the Trc promoter from the Escherichia coli genome;

[0011] (3) Fusing the upstream and downstream homology arms of yeeL with the ppc fragment driven by the Trc promoter to obtain the U-ppc-D fragment;

[0012] (4) The obtained fusion fragment U-ppc-D and the vector containing yeeL-sgRNA were transformed into the basic strain to obtain a recombinant strain in which the pseudogene yeeL was knocked out and the ppc driven by the Trc promoter was integrated at the yeeL site;

[0013] (5) Based on the recombinant strains obtained in the previous round, the same method as steps (1) to (4) above was repeated to construct a recombinant strain with rhtC integrated at the yjhE site; a recombinant strain with aspC integrated at the yblE site; a recombinant strain with aspA integrated at the ycdN site; and a recombinant strain with thrA integrated at the fliN site. mut Recombinant strains of BC; Recombinant strains with asd integrated at the yeeP site; Recombinant strains with rhtA integrated at the yjiP site; Recombinant strains with asdtm integrated at the yncI site; Recombinant strains with thrA integrated at the mbhA site mut Recombinant strains overexpressing ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mutRecombinant strains of

[0014] (6) Overexpression of ppc, aspC, aspA, and thrA obtained in step (5) mut BC, asd, rhtC, rhtA, asdtm and thrA mut Based on the recombinant strain of , fnr and iclR were knocked out to obtain the recombinant strain THRS in which fnr and iclR were knocked out;

[0015] Preferably, CRISPR Cas9 gene editing is used;

[0016] Preferably, the basic strain is Escherichia coli K-12;

[0017] Preferably, the ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut 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, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 respectively;

[0018] Preferably, the nucleic acid sequences of the fnr encoding gene and the iclR encoding gene are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively;

[0019] Preferably, the ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut The coding gene is expressed by the replaced promoter;

[0020] Preferably, the replaced promoter includes Trc promoter; the sequence of the Trc promoter is shown as SEQ ID NO.1.

[0021] The third aspect of the present invention provides a method for biosynthesizing L-threonine, wherein the method uses the recombinant Escherichia coli THRS according to the first aspect of the present invention for fermentation;

[0022] Preferably, the method for biosynthesizing L-threonine comprises the following steps:

[0023] 1) inoculating the recombinant Escherichia coli THRS on a seed culture medium for culturing to obtain a seed solution;

[0024] 2) inoculating the seed liquid obtained in step 1) into a fermentation medium, performing aerobic fermentation, and obtaining L-threonine from the fermentation liquid;

[0025] 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;

[0026] 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.

[0027] Beneficial effect: The present invention provides a method for overexpressing ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut The recombinant Escherichia coli THRS of the coding gene of fnr and knocking out the coding gene of iclR is fermented with glucose as a substrate, 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 is used to ferment glucose as a substrate in a 5L fermentation tank for 48 hours, and 100.3g / L threonine can be produced, and the sugar-acid conversion rate reaches 30%; in addition, the strain of the present invention does not contain exogenous plasmids, and all the transformation sites are integrated into the genome, which has higher stability in actual production, and no antibiotics need to be added to the fermentation medium of the strain, and the L-threonine product produced by the strain has no antibiotic residues, and has higher safety in the use of feed additives. The strain has good prospects for industrial utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the gel electrophoresis diagram of the fusion fragment U-ppc-D, channel 1: Marker; channel 2: fragment yeeL1; channel 3: fragment yeeL2; channels 4 and 5: successfully connected U-ppc-D fragments.

[0029] Figure 2 This is the map of the yeeL-sgRNA recombinant plasmid.

[0030] Figure 3 This is the gel electrophoresis diagram of the successful integration of U-ppc-D fragment (2000bp is the original band, 4000bp is the successfully integrated band), channel A: Maker; channels D, H, and L are the control primers yeeL-UF and yeeL-DR bands; channels B, C, E, F, G, I, J, K, M, N, O, P, and Q are all colony bands that need to be screened after electroporation, among which channels I, K, and P are the bands of successful integration of U-ppc-D fragment, and the strain corresponding to channel P was selected for subsequent transformation.

[0031] Figure 4 This is the map of the fnr-sgRNA recombinant plasmid.

[0032] Figure 5 It is the change of biomass of THRS strain during fermentation in 5L fermenter.

[0033] Figure 6 It is the change of residual glucose content during the fermentation of THRS strain in 5L fermenter.

[0034] Figure 7 The figure shows the change of L-threonine production during the fermentation of THRS strain in a 5L fermenter.

[0035] A recombinant Escherichia coli THRS, wherein the recombinant Escherichia coli THRS is Escherichia coli, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 20, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 31694. DETAILED DESCRIPTION

[0036] The following is a detailed description of a recombinant Escherichia coli THRS provided by the present invention, its construction method and its method for synthesizing L-threonine in conjunction with the examples and drawings. It is to be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0037] 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.

[0038] Example 1 Recombinant Escherichia coli THRS and its construction method

[0039] In this example, Escherichia coli K-12 is used as the starting strain, and the starting strain lacks lacI and tdh genes. By gene editing, the pseudogene yeeL of the starting strain is knocked out and the coding gene of ppc is integrated at the yeeL site; the pseudogene yjhE of the starting strain is knocked out and the coding gene of rhtC is integrated at the yjhE site; the pseudogene yblE of the starting strain is knocked out and the coding gene of aspC is integrated at the yblE site; the pseudogene ycdN of the starting strain is knocked out and the coding gene of aspA is integrated at the ycdN site; the pseudogene fliN of the starting strain is knocked out and the coding gene of thrA is integrated at the fliN site. mut The coding gene of BC; knock out the pseudogene yeeP of the starting strain and integrate the coding gene of asd at the yeeP site; knock out the pseudogene yjiP of the starting strain and integrate the coding gene of rhtA at the yjiP site; knock out the pseudogene yncI of the starting strain and integrate the coding gene of asdtm at the yncI site; knock out the pseudogene mbhA of the starting strain and integrate thrA at the mbhA site mut and knocking out the fnr and iclR genes of the starting strain.

[0040] In this embodiment, the overexpression of ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut The coding genes 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, and this embodiment uses CRISPR Cas9 for gene editing.

[0041] In this embodiment, the overexpression of ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, and thrA mut The encoding gene of comes from Escherichia coli, and the encoding gene for overexpressing asdtm comes from Tistrella mobilis.

[0042] Integrating thrA mut BC, thrA mut The thrAmut BC and thrA mut The amino acid sequence encoded by the gene has a glycine mutation at position 433 to an arginine. The complete encoding gene sequence of asdtm described in this example is shown in SEQ ID NO.2.

[0043] This embodiment also provides a method for constructing the recombinant Escherichia coli THRS, 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 yeeL from the genome of Escherichia coli K-12; (2) PCR amplifying the ppc gene driven by the Trc promoter from the Escherichia coli genome; (3) fusing the upstream and downstream homology arms of yeeL and the ppc fragment driven by the Trc promoter to obtain a U-ppc-D fragment; (4) transforming the obtained fusion fragment U-ppc-D and a vector containing yeeL-sgRNA into the recombinant Escherichia coli K-12; 2. Obtain a recombinant strain in which the pseudogene yeeL is knocked out and ppc driven by the Trc promoter is integrated at the yeeL site; (5) Using the recombinant strain obtained in the previous round as the basis for construction, the same method as in steps (1) to (4) is used and repeated. The only difference is that the corresponding primers for each site to be integrated and each target gene are used to construct a recombinant strain in which rhtC is integrated at yjhE; a recombinant strain in which aspC is integrated at the yblE site; a recombinant strain in which aspA is integrated at the ycdN site; and a recombinant strain in which thrA is integrated at the fliN site. mut Recombinant strains of BC; Recombinant strains with asd integrated at the yeeP site; Recombinant strains with rhtA integrated at the yjiP site; Recombinant strains with asdtm integrated at the yncI site; Recombinant strains with thrA integrated at the mbhA site mut Recombinant strains overexpressing ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut (6) the recombinant strain obtained in step (5) that overexpresses ppc, aspC, aspA, and thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut Based on the recombinant strain, fnr and iclR were knocked out to obtain the recombinant strain with fnr and iclR knocked out.

[0044] In this example, the primers shown in Table 1 were used to construct the recombinant E. coli THRS.

[0045] Table 1 Primer information

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] The ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut 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, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 respectively;

[0052] The nucleic acid sequences of the fnr encoding gene and the iclR encoding gene are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

[0053] The specific steps of the construction method of the recombinant Escherichia coli THRS are:

[0054] 1. Construction of fusion fragment U-ppc-D

[0055] Using the primers yeeL-UF, yeeL-UR, yeeL-DF and yeeL-DR in Table 1, the upstream and downstream homology arm fragments on both sides of the yeeL gene were amplified from the Escherichia coli K-12 genome to obtain fragments yeeL1 and yeeL2;

[0056] Using primers ppc-F and ppc-R in Table 1, the ppc gene was amplified from the E. coli genome to obtain fragment ppc;

[0057] The fragments yeeL1, ppc, and yeeL2 were subjected to fusion PCR to obtain the fusion fragment U-ppc-D, which was verified by gel electrophoresis. The verification results are shown in Figure 1 .

[0058] 2. Construction of yeeL-sgRNA recombinant plasmid

[0059] Using primers pGRB-F and pGRB-R, linearized vector L-pGRB was obtained by PCR from vector pGRB. The designed sgRNA was connected with linearized vector L-pGRB to construct recombinant plasmid yeeL-sgRNA. The verification results can be found in Figure 2 .

[0060] 3. Construction of recombinant Escherichia coli overexpressing the ppc gene

[0061] The recombinant plasmid yeeL-sgRNA and fusion fragment U-ppc-D were transformed into E. coli K-12, and the primers yeeL-UF and yeeL-DR were used to perform colony PCR to screen the transformants and confirm that the fusion fragment U-ppc-D was successfully integrated into the yeeL site. The verification results can be found in Figure 3 , add 2 mM arabinose and culture at 30°C for 12 h, remove the recombinant plasmid yeeL-sgRNA, and obtain the recombinant strain.

[0062] 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 integration of aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm, thrA mut The recombinant strain overexpressing ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut of the recombinant strains.

[0063] 5. Using the primers fnr-UF, fnr-UR, fnr-DF and fnr-DR in Table 1, the upstream and downstream homology arm fragments on both sides of the fnr gene were amplified from the Escherichia coli K-12 genome to obtain fragments fnr1 and fnr2;

[0064] Using primers fnr-UF and fnr-DR in Table 1, fragments fnr1 and fnr2 were subjected to fusion PCR to obtain fusion fragment U-fnr-D.

[0065] 6. Construction of fnr-sgRNA recombinant plasmid

[0066] Using primers pGRB-F and pGRB-R, linearized vector L-pGRB was obtained by PCR from vector pGRB. The designed sgRNA was connected with linearized vector L-pGRB to construct recombinant plasmid fnr-sgRNA. The verification results can be found in Figure 4 .

[0067] 7. Construction of recombinant Escherichia coli with knockout of fnr gene

[0068] The recombinant plasmid fnr-sgRNA and fusion fragment U-fnr-D were transformed into the overexpression plasmid ppc, aspC, aspA, and thrA obtained in step 4 above. mutBC, asd, rhtC, rhtA, asdtm and thrA mut The recombinant strain was prepared and the primers fnr-UF and fnr-DR were used for colony PCR screening of transformants to confirm that the fusion fragment U-fnr-D was successfully integrated into the fnr site. 2 mM arabinose was added and cultured at 30°C for 12 h. The recombinant plasmid fnr-sgRNA was removed to obtain the recombinant strain.

[0069] 8. Based on the recombinant strain obtained in step 7, the same method as described in steps 5 to 7 was used, except that the primers in Table 1 corresponding to the target gene iclR were used to construct a recombinant strain with iclR knocked out, and finally ppc, aspC, aspA, and thrA overexpression were obtained. mut BC, asd, rhtC, rhtA, asdtm and thrA mut And recombinant Escherichia coli THRS with fnr and iclR knocked out.

[0070] The recombinant Escherichia coli THRS obtained above is Escherichia coli, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 20, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31694.

[0071] Example 2 Biosynthesis of L-threonine using recombinant Escherichia coli THRS

[0072] The recombinant Escherichia coli THRS 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.

[0073] 1.5 L seed tank process control

[0074] a. Adjust the temperature to 37℃, pH 7.0, speed to 500rpm, air volume to 0.3m3 / h, the whole process temperature to 37℃, tank pressure to 0.05~0.08MPa, and the culture period to 10h;

[0075] b. Transplantation standard: OD600:12-15.

[0076] c. Seed culture medium is corn syrup powder 5g / L, glucose 20g / L, yeast powder 5g / L, KH 2 PO 4 2g / L, magnesium sulfate 1g / L, FeSO4 7H 2 O 20mg / L, MnSO 4 ·H 2 O 20 mg / L;

[0077] 2.5 L fermentation tank fermentation process control

[0078] a. Adjust the temperature to 37℃, pH7.0, initial speed to 300rpm, air volume to 0.3m 3 / h, the whole process temperature is controlled at 37℃, and the tank pressure is 0.05~0.08MPa;

[0079] c.DO control: 0h, air volume 0.3m 3 / h, 300rpm, tank pressure 0.05MPa;

[0080] 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;

[0081] 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.

[0082] 3. Determination method of L-threonine:

[0083] 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.

[0084] 2) Analytical method: OPA pre-column derivatization

[0085] 3) Chromatographic conditions:

[0086] (1) Chromatographic column: Chromatographic column C18 (250×4.6) mm

[0087] (2) Column temperature: 40°C

[0088] (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.

[0089] 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.

[0090] (4) Flow rate: 1.0 mL / min;

[0091] (5) UV detector: 338 nm;

[0092] (6) Column temperature: 40°C.

[0093] Verification of the production performance of THRS strain in 4.5L fermenter

[0094]

[0095] From the data in the table above, we can see that the L-threonine production and conversion rate of the recombinant E. coli THRS have achieved a transformation from zero to high, and a high production and conversion rate have been obtained, which can better realize the industrial transformation of the strain. The fermentation parameters of the THRS strain in a 5L fermenter can be found in Figure 5-Figure 7 , which show the changes in biomass, residual glucose, and L-threonine production, respectively.

[0096] The present invention provides a method for transforming a model strain Escherichia coli to efficiently synthesize L-threonine from scratch, and based on a system metabolic engineering transformation and optimization strategy, obtains a recombinant Escherichia coli strain that efficiently synthesizes L-threonine. The recombinant Escherichia coli strain overexpresses ppc, aspC, aspA, thrA mut BC, asd, rhtC, rhtA, asdtm and thrA mut The invention discloses a recombinant Escherichia coli THRS which is a recombinant strain of Escherichia coli THRS. The recombinant Escherichia coli THRS is used for fermentation, and the L-threonine yield and conversion rate are significantly improved. The strain does not contain exogenous plasmids, and all the modified sites are integrated into the genome. The strain has higher stability in actual production, and antibiotics do not need to be added to the fermentation medium of the strain. The L-threonine product produced by the strain has no antibiotic residues, and has higher safety in the use of feed additives. The strain has good prospects for industrial utilization.

[0097] 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, characterized in that The recombinant Escherichia coli THRS is a Escherichia coli ), which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 20, 2024, with the deposit number CGMCC No. 31694.

2. The method for constructing a recombinant Escherichia coli THRS according to claim 1, characterized in that: The steps include: (1) PCR amplification of pseudogenes from the genome of Escherichia coli yeeL The upstream homology arm and the downstream homology arm; (2) PCR amplification of the Trc promoter-driven gene from the Escherichia coli genome ppc Gene; (3) yeeL The upstream and downstream homology arms and Trc promoter drive ppc The fragments were fused to obtain the U-ppc-D fragment; (4) The obtained fusion fragment U-ppc-D and the vector containing yeeL-sgRNA were transformed into the basic strain to knock out the pseudogene yeeL and in yeeL Integration site driven by the Trc promoter ppc 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 Yj Y Integration oeLh Recombinant strains of YbE Site integration asp The recombinant strain of C. Yj Y Site integration aspA Recombinant strains of exist fL Site integration thrA mut BC Recombinant strains of yeeP Site integration asd Recombinant strains of exist Yj Site integration oeLh Recombinant strains of exist yncI Site integration asdtm Recombinant strains of mbA Site integration thrA mut Recombinant strains overexpressing ppc , aspC , aspA , thrA mut BC , asd , oeLh , oeLh , asdtm and thrA mut Recombinant strains of (6) Overexpression obtained in step (5) ppc , aspC , aspA , thrA mut BC , asd , oeLh , oeLh , asdtm and thrA mut Knockout based on the recombinant strain fnr and iclR, Get knockout fnr and iC Recombinant Escherichia coli THRS.

3. The construction method according to claim 2, characterized in that: Using CRISPR Cas9 gene editing method.

4. The construction method according to claim 2 or 3, characterized in that: The basic strain is Escherichia coli K-12.

5. The construction method according to claim 4, characterized in that: Said ppc , aspC , aspA , thrA mut BC , asd , oeLh , oeLh , asdtm and thrA mut 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, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 respectively; Said fnr The coding genes and iC The nucleic acid sequences of the encoding genes are shown in SEQ ID NO. 11 and SEQ ID NO. 12 respectively.

6. The construction method according to claim 5, characterized in that: Said ppc , aspC , aspA , thrA mut BC , asd , oeLh , oeLh , asdtm and thrA mut 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.

7. A method for biosynthesizing L-threonine, characterized in that: The recombinant E. coli THRS according to claim 1 was used for fermentation.

8. The method according to claim 7, characterized in that The following steps are involved: 1) inoculating the recombinant Escherichia coli THRS 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.

Images