Genetically engineered bacteria overexpressing phosphoenolpyruvate carboxylase and its application in production of alpha-ketoglutaric acid

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase and application of the genetically engineered bacterium in production of alpha-ketoglutaric acid. The construction method of the genetically engineered bacterium comprises the following steps: on the basis of an original Escherichia coli, first, knocking out a pyruvate metabolic bypass gene, then, knocking out an alpha-ketoglutaric acid metabolic bypass gene, and finally, strengthening expression of a ppc gene, so that the genetically engineered bacterium is obtained. The genetically engineered bacterium can efficiently utilize glucose to produce alpha-ketoglutaric acid, and greatly improves the yield of alpha-ketoglutaric acid.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to genetically engineered bacteria that overexpress phosphoenolpyruvate carboxylase and their application in the production of α-ketoglutarate. Background Technology

[0002] α-Ketoglutaric acid (α-KG) is an important short-chain carboxylic acid molecule widely used in food, pharmaceuticals, cosmetics, and animal feed. Furthermore, as a key dicarboxylic acid in the tricarboxylic acid cycle and amino acid metabolism, α-ketoglutaric acid plays a crucial role in amino acid formation and nitrogen transport.

[0003] Currently, the main methods for synthesizing α-KG include chemical synthesis, enzymatic methods, and microbial fermentation. Industrially, chemical synthesis is the primary method, using diethyl succinate and diethyl oxalate as raw materials. This method involves condensation, hydrolysis, distillation concentration, and crystallization to synthesize α-KG, achieving a maximum yield of 75%. However, this method generates numerous byproducts during its complex multi-step synthesis process, including highly toxic substances such as cyanide, heavy metal pollutants, and explosives. This not only increases the difficulty of separation and reduces productivity but also raises the risks associated with production.

[0004] Compared with chemical synthesis and enzymatic catalysis, microbial fermentation has advantages such as low cost, high yield, low pollution, and few byproducts, and has great potential for industrial production. Current research on fermentation production of α-ketoglutarate mainly focuses on *Agropyron lipolytica* and *Gnaphalium affine*, with some strains showing potential for industrial production. However, problems remain, including excessively long fermentation cycles (>70 hours), low yields, and high production costs. Compared with yeast, *Escherichia coli* (E. coli) has a faster growth rate and stronger metabolic capacity. However, there is limited research on metabolic engineering of *E. coli* for α-ketoglutarate production; therefore, the potential of *E. coli* for α-ketoglutarate production needs further exploration. Summary of the Invention

[0005] In view of the above, it is necessary to provide a genetically engineered bacterium that overexpresses phosphoenolpyruvate carboxylase and its application in the production of α-ketoglutarate. The genetically engineered bacterium constructed in this invention optimizes the glucose utilization pathway, produces oxaloacetic acid from a large amount of phosphoenolpyruvate, enters the tricarboxylic acid cycle, strengthens the target sites related to the α-KG synthesis pathway, and knocks out the byproduct competing bypass, thereby obtaining a highly efficient pathway for the synthesis of α-KG. This enables the efficient utilization of glucose to produce α-ketoglutarate, greatly increasing the yield of α-ketoglutarate, while shortening the production cycle and reducing production costs.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A genetically engineered bacterium that overexpresses phosphoenolpyruvate carboxylase, wherein the construction method of the genetically engineered bacterium includes: first knocking out the pyruvate metabolism bypass gene, then knocking out the α-ketoglutarate metabolism bypass gene, and finally enhancing the expression of the ppc gene, thereby obtaining the genetically engineered bacterium.

[0008] Furthermore, the original Escherichia coli was E. coli MG1655.

[0009] Furthermore, the pyruvate metabolism bypass genes are ldhA and ackA genes, and the α-ketoglutarate metabolism bypass genes are gdhA and sucA genes.

[0010] Furthermore, the nucleotide sequence of the ldhA gene is shown in SEQ ID No. 1, the nucleotide sequence of the ackA gene is shown in SEQ ID No. 2, the nucleotide sequence of the gdhA gene is shown in SEQ ID No. 3, the nucleotide sequence of the sucA gene is shown in SEQ ID No. 4, and the nucleotide sequence of the ppc gene is shown in SEQ ID No. 5.

[0011] Furthermore, knocking out the ldhA gene includes the following steps:

[0012] (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the ldhA gene, and the above fragments were fused by overlap PCR to obtain the knockout fragment of the ldhA gene.

[0013] (2) After transforming E. coli DH5α by PCR reaction using primers F-ldhA-N20 and R-ldhA-N20, the pTargetF-ldhA plasmid was extracted;

[0014] (3) Electroporate the knockout fragment of the ldhA gene and the pTargetF-ldhA plasmid into the original Escherichia coli competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG1.

[0015] Furthermore, knocking out the ackA gene includes the following steps:

[0016] (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the ackA gene, and the above fragments were fused by overlap PCR to obtain the knockout fragment of the ackA gene.

[0017] (2) After transforming E. coli DH5α by PCR reaction using primers F-ackA-N20 and R-ackA-N20, the pTargetF-ackA plasmid was extracted;

[0018] (3) Electroporate the knockout fragment of the ackA gene and the pTargetF-ackA plasmid into AKG1 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG2.

[0019] Furthermore, knocking out the gdhA gene includes the following steps:

[0020] (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the gdhA gene, and the above fragments were fused by overlapping PCR to obtain the knockout fragment of the gdhA gene.

[0021] (2) After transforming E. coli DH5α by PCR reaction using primers F-gdhA-N20 and R-gdhA-N20, the pTargetF-gdhA plasmid was extracted;

[0022] (3) Electroporate the knockout fragment of the gdhA gene and the pTargetF-gdhA plasmid into AKG2 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG3.

[0023] Furthermore, knocking out the sucA gene includes the following steps:

[0024] (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the sucA gene, and the two fragments were fused by overlapping PCR to obtain the knockout fragment of the sucA gene.

[0025] (2) After transforming E. coli DH5α by PCR reaction using primers F-sucA-N20 and R-sucA-N20, the pTargetF-sucA plasmid was extracted;

[0026] (3) Electroporate the knockout fragment of the sucA gene and the pTargetF-sucA plasmid into AKG3 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG4.

[0027] Furthermore, the enhancement of ppc gene expression includes the following steps:

[0028] (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the ygh X gene; primers F-ppc and R-ppc were designed according to the ppc gene to amplify the ppc insert fragment, and the promoter Ptrc was designed in the downstream primer of the upstream homologous arm of the ygh X gene and the upstream primer of the ppc gene; the above fragments were fused by overlap PCR to obtain the integrated fragment of the ppc gene;

[0029] (2) After transforming E. coli DH5α by PCR reaction using primers F-ygh X-N20 and R-ygh X-N20, the pTargetF-ygh X plasmid was extracted;

[0030] (3) Electroporate the integrated fragment of the ppc gene and the pTargetF-ygh X plasmid into AKG4 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG5, which is the genetically engineered bacterium.

[0031] The present invention also provides the application of the above-mentioned genetically engineered bacteria in the production of α-ketoglutarate.

[0032] Furthermore, the method for producing α-ketoglutarate by the genetically engineered bacteria includes: inoculating the genetically engineered bacteria into a fermentation medium, fermenting at 37°C for 4 hours, adding IPTG, and then continuing fermentation at 30°C.

[0033] Furthermore, the fermentation medium comprises the following components at the following concentrations: Na₂HPO₄·12H₂O 16.0-18.0 g / L, KH₂PO₄ 2.0-4.0 g / L, NaCl 0.4-0.6 g / L, NH₄Cl 0.9-1.1 g / L, MgSO₄ 1.5-2.5 mM, CaCl₂ 0.05-0.15 mM, glucose 9.0-11.0 g / L, yeast extract 1.5-2.5 g / L, and glycine 1.5-2.5 g / L.

[0034] Furthermore, the final concentration of the IPTG is 0.05 mM.

[0035] Furthermore, during the fermentation process, 500 g / L of glucose is added to control the glucose concentration in the fermentation broth to be maintained at 0.5-3.0 g / L, and ammonia water is added to control the pH of the fermentation broth to 7.0.

[0036] The present invention has the following beneficial effects:

[0037] This invention enhances the expression of the phosphoenolpyruvate carboxylase gene ppc in *E. coli* by knocking out the ldhA gene encoding lactate dehydrogenase and the ackA gene encoding acetate kinase in the bypass pathway, thereby improving glucose utilization efficiency. Furthermore, by knocking out the gdhA gene encoding glutamate dehydrogenase and the sucA gene encoding α-ketoglutarate dehydrogenase, the α-ketoglutarate synthesis pathway is further optimized, effectively improving the ability of recombinant *E. coli* strains to synthesize α-ketoglutarate from glucose, showing promise for industrial production of α-ketoglutarate. Attached Figure Description

[0038] Figure 1 This is a high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 1 of this invention.

[0039] Figure 2 This is a high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 2 of this invention.

[0040] Figure 3 This is a high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 3 of this invention.

[0041] Figure 4 This is a high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 4 of this invention.

[0042] Figure 5 This is a high-performance liquid chromatogram obtained from the detection of the fermentation broth in Example 2 of the present invention. Detailed Implementation

[0043] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0044] In this invention, unless otherwise specified, all reagents and consumables used are purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and techniques used are conventional methods and techniques in the field.

[0045] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0046] The fermentation medium used in the following examples has the following formulation: Na2HPO4·12H2O 16.0-18.0 g / L, KH2PO4 2.0-4.0 g / L, NaCl 0.4-0.6 g / L, NH4Cl 0.9-1.1 g / L, MgSO4 1.5-2.5 mM, CaCl2 0.05-0.15 mM, glucose 9.0-11.0 g / L, yeast extract 1.5-2.5 g / L, and glycine 1.5-2.5 g / L.

[0047] For example, the fermentation medium is formulated as follows (Formula 1): Na2HPO4·12H2O 16.0 g / L, KH2PO4 2.0 g / L, NaCl 0.4 g / L, NH4Cl 0.9 g / L, MgSO4 1.5 mM, CaCl2 0.05 mM, glucose 9.0 g / L, yeast extract 1.5 g / L, and glycine 1.5 g / L.

[0048] For example, the fermentation medium can also be formulated as (Formula 2): Na2HPO4·12H2O 18.0 g / L, KH2PO4 4.0 g / L, NaCl 0.6 g / L, NH4Cl 1.1 g / L, MgSO4 2.5 mM, CaCl2 0.15 mM, glucose 11.0 g / L, yeast extract 2.5 g / L, glycine 2.5 g / L.

[0049] For example, the fermentation medium can also be formulated as (Formula 3): Na2HPO4·12H2O 17.0 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L, MgSO4 2.0 mM, CaCl2 0.10 mM, glucose 10.0 g / L, yeast extract 2.0 g / L, and glycine 2.0 g / L.

[0050] The following examples specifically use fermentation medium (Formula 2) for fermentation culture.

[0051] Furthermore, the original Escherichia coli used in the following examples is E. coli MG1655.

[0052] Example 1

[0053] This embodiment provides a genetically engineered bacterium that overexpresses phosphoenolpyruvate carboxylase. The construction method of the genetically engineered bacterium is as follows:

[0054] 1. Knockout of the ldhA gene

[0055] (1) Using the E.coli MG1655 genome as a template, upstream homologous arm primers (F-ldhA-up, R-ldhA-up) and downstream homologous arm primers (F-ldhA-down, R-ldhA-down) were designed based on the upstream and downstream sequences of the ldhA gene. The upstream and downstream homologous arm fragments were amplified by PCR. The above fragments were fused by overlapping PCR to obtain the ldhA gene knockout fragment.

[0056] (2) After transforming E. coli DH5α by PCR reaction using primers F-ldhA-N20 and R-ldhA-N20, the pTargetF-ldhA plasmid was extracted;

[0057] (3) The knockout fragment of the ldhA gene and the plasmid pTargetF-ldhA were electroporated into E.coli MG1655 competent cells. Positive strains were screened and then the plasmid was eliminated to obtain strain AKG1. The positive strain was verified by PCR and electrophoresis to confirm that the ldhA gene knockout was successful.

[0058] 2. Knockout of the ackA gene

[0059] (1) Using the E.coli MG1655 genome as a template, upstream homologous arm primers (F-ackA-up, R-ackA-up) and downstream homologous arm primers (F-ackA-down, R-ackA-down) were designed based on the upstream and downstream sequences of its ackA gene, and the upstream and downstream homologous arm fragments were amplified by PCR. The above fragments were fused by overlapping PCR to obtain the knockout fragment of the ackA gene.

[0060] (2) After transforming E. coli DH5α by PCR reaction using primers F-ackA-N20 and R-ackA-N20, the pTargetF-ackA plasmid was extracted;

[0061] (3) Electroporate the knockout fragment of the ackA gene and the pTargetF-ackA plasmid into AKG1 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG2. The positive strains are verified by PCR and electrophoresis to confirm that the ackA gene knockout was successful.

[0062] 3. Knockout of the gdhA gene

[0063] (1) Using the E.coli MG1655 genome as a template, upstream homologous arm primers (F-gdhA-up, R-gdhA-up) and downstream homologous arm primers (F-gdhA-down, R-gdhA-down) were designed based on the upstream and downstream sequences of the gdhA gene. The upstream and downstream homologous arm fragments were amplified by PCR, and the above fragments were fused by overlapping PCR to obtain the gdhA gene knockout fragment.

[0064] (2) After transforming E. coli DH5α by PCR reaction using primers F-gdhA-N20 and R-gdhA-N20, the pTargetF-gdhA plasmid was extracted;

[0065] (3) Electroporate the knockout fragment of the gdhA gene and the pTargetF-gdhA plasmid into AKG2 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG3. The positive strains are verified by PCR and electrophoresis to confirm that the gdhA gene knockout was successful.

[0066] 4. Knockout of the sucA gene

[0067] (1) Using the E.coli MG1655 genome as a template, upstream homologous arm primers (F-sucA-up, R-sucA-up) and downstream homologous arm primers (F-sucA-down, R-sucA-down) were designed based on the upstream and downstream sequences of the sucA gene. The upstream and downstream homologous arm fragments were amplified by PCR, and the above fragments were fused by overlapping PCR to obtain the sucA gene knockout fragment.

[0068] (2) After transforming E. coli DH5α by PCR reaction using primers F-sucA-N20 and R-sucA-N20, the pTargetF-sucA plasmid was extracted;

[0069] (3) Electroporate the knockout fragment of the sucA gene and the pTargetF-sucA plasmid into AKG3 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG4. The positive strains are verified by PCR and electrophoresis to confirm that the sucA gene knockout was successful.

[0070] 5. Enhance the expression of the ppc gene

[0071] (1) Using the E. coli MG1655 genome as a template, primers for the upstream homologous arm (F-yghX-up, R-yghX-up) and primers for the downstream homologous arm (F-yghX-do, R-yghX-do) were designed based on the upstream and downstream sequences of the ygh X gene, and the upstream and downstream homologous arm fragments were amplified by PCR; primers F-ppc and R-ppc were designed based on the ppc gene to amplify the ppc insert fragment, and the promoter Ptrc was designed in the downstream primer of the upstream homologous arm of the ygh X gene and the upstream primer of the ppc gene; the above fragments were fused by overlap PCR to obtain the integrated fragment of the ppc gene;

[0072] (2) After transforming E. coli DH5α by PCR reaction using primers F-ygh X-N20 and R-ygh X-N20, the pTargetF-ygh X plasmid was extracted;

[0073] (3) Electroporate the integrated fragment of the ppc gene and the pTargetF-ygh X plasmid into AKG4 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG5. The strain AKG5 is the genetically engineered bacterium. The integration of the ppc gene was successfully verified by PCR and electrophoresis of the positive strain.

[0074] The nucleotide sequence of the ldhA gene is shown in SEQ ID No. 1, the nucleotide sequence of the ackA gene is shown in SEQ ID No. 2, the nucleotide sequence of the gdhA gene is shown in SEQ ID No. 3, the nucleotide sequence of the sucA gene is shown in SEQ ID No. 4, and the nucleotide sequence of the ppc gene is shown in SEQ ID No. 5.

[0075] The sequences of the above primers are shown in Table 1 below.

[0076] Table 1 Primer sequences and gene sequence list

[0077]

[0078] Example 2

[0079] This embodiment provides a method for producing α-ketoglutaric acid using the genetically engineered bacteria (strain AKG5) described in Example 1. The method is as follows: the genetically engineered bacteria are inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 5% (v / v). After fermentation at 37°C for 4 hours, IPTG is added to a final concentration of 0.05 mM, and fermentation continues at 30°C. During fermentation, 500 g / L of glucose is added continuously to control the glucose concentration of the fermentation broth to 0.5-3.0 g / L, and the pH of the fermentation broth is controlled to 7.0 by adding ammonia. Fermentation is stopped when the pH reaches 0, yielding the fermentation broth.

[0080] Comparative Examples 1-5

[0081] Comparative Examples 1-5 aim to compare the effects of different genetic modification strategies on the synthesis capacity of α-ketoglutarate. Compared to Example 2, the methods for producing α-ketoglutarate in Comparative Examples 1-5 differ in that the fermentation strains used in Comparative Examples 1-5 are the original *E. coli* MG1655 from Example 1 and the intermediate strains AKG1-AKG4 used in the construction process, respectively; all other production processes are the same.

[0082] Detection methods

[0083] 1. Sample pretreatment: Take 5 mL of fermentation broth from each fermenter, place it in a centrifuge tube, and centrifuge at 10,000 rpm for 10 minutes. Collect the supernatant, filter it through a 0.22 μm microporous membrane, and collect the filtrate for subsequent analysis.

[0084] 2. Detection: Quantitative analysis was performed using high-performance liquid chromatography (HPLC). Instrument and Column: HPLC system equipped with a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm). Analytical Conditions: Mobile phase: 5 mM sulfuric acid aqueous solution; flow rate: 0.6 mL / min; column temperature: 35℃. Detector: Ultraviolet detector; detection wavelength: 210 nm.

[0085] The high-performance liquid chromatograms obtained from the detection are as follows: Figure 1-5 As shown. Among them, Figure 1 The image shows the high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 1. Figure 2 The image shows the high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 2. Figure 3 The image shows the high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 3. Figure 4 The high-performance liquid chromatogram obtained from the fermentation broth of Comparative Example 4 is shown below. Figure 5 The image shows the high-performance liquid chromatogram obtained from the fermentation broth in Example 2. The corresponding detection data results are shown in Table 2 below.

[0086] Table 2. Effects of different strains on the synthesis capacity of α-ketoglutarate

[0087]

[0088] Combination Figure 1-5 As shown in Table 2, the genetically engineered strain AKG5 obtained by using E. coli MG1655 as the starting strain, knocking out the pyruvate metabolism bypass gene, then knocking out the α-ketoglutarate metabolism bypass gene, and finally enhancing the expression of the ppc gene, has a strong α-ketoglutarate synthesis capacity. Compared with the starting strain, the α-ketoglutarate content produced by fermentation for 24 hours is increased by 318.3%.

Claims

1. A genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase, characterized in that, The method for constructing the genetically engineered bacteria includes: based on the original Escherichia coli MG1655, first knocking out the pyruvate metabolism bypass genes ldhA and ackA, then knocking out the α-ketoglutarate metabolism bypass genes gdhA and sucA, and finally enhancing the expression of the ppc gene to obtain the genetically engineered bacteria.

2. The genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase according to claim 1, characterized in that, The nucleotide sequence of the ldhA gene is shown in SEQ ID No. 1, the nucleotide sequence of the ackA gene is shown in SEQ ID No. 2, the nucleotide sequence of the gdhA gene is shown in SEQ ID No. 3, the nucleotide sequence of the sucA gene is shown in SEQ ID No. 4, and the nucleotide sequence of the ppc gene is shown in SEQ ID No.

5.

3. The genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase according to claim 1, characterized in that, Knocking out the ldhA gene includes the following steps: (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the ldhA gene, and the two fragments were fused by overlap PCR to obtain the knockout fragment of the ldhA gene. (2) After transforming E. coli DH5α by PCR reaction using primers F-ldhA-N20 and R-ldhA-N20, the pTargetF-ldhA plasmid was extracted; (3) Electroporate the knockout fragment of the ldhA gene and the pTargetF-ldhA plasmid into the original Escherichia coli competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG1.

4. The genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase according to claim 3, characterized in that, Knocking out the ackA gene includes the following steps: (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the ackA gene, and the above fragments were fused by overlapping PCR to obtain the knockout fragment of the ackA gene. (2) After transforming E. coli DH5α by PCR reaction using primers F-ackA-N20 and R-ackA-N20, the pTargetF-ackA plasmid was extracted; (3) Electroporate the knockout fragment of the ackA gene and the pTargetF-ackA plasmid into AKG1 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG2.

5. The genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase according to claim 4, characterized in that, Knocking out the gdhA gene includes the following steps: (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the gdhA gene, and the above fragments were fused by overlapping PCR to obtain the knockout fragment of the gdhA gene. (2) After transforming E. coli DH5α by PCR reaction using primers F-gdhA-N20 and R-gdhA-N20, the pTargetF-gdhA plasmid was extracted; (3) Electroporate the knockout fragment of the gdhA gene and the pTargetF-gdhA plasmid into AKG2 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG3.

6. The genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase according to claim 5, characterized in that, Knocking out the sucA gene includes the following steps: (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the sucA gene, and the above fragments were fused by overlap PCR to obtain the knockout fragment of the sucA gene. (2) After transforming E. coli DH5α by PCR reaction using primers F-sucA-N20 and R-sucA-N20, the pTargetF-sucA plasmid was extracted; (3) Electroporate the knockout fragment of the sucA gene and the pTargetF-sucA plasmid into AKG3 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG4.

7. The genetically engineered bacterium overexpressing phosphoenolpyruvate carboxylase according to claim 6, characterized in that, The enhancement of ppc gene expression includes the following steps: (1) Using the original Escherichia coli genome as a template, primers were designed to amplify the upstream and downstream homologous arm fragments of the ygh X gene; primers F-ppc and R-ppc were designed according to the ppc gene to amplify the ppc insert fragment, and the promoter Ptrc was designed in the downstream primer of the upstream homologous arm of the ygh X gene and the upstream primer of the ppc gene; the above fragments were fused by overlap PCR to obtain the integrated fragment of the ppc gene; (2) After transforming E. coli DH5α by PCR reaction using primers F-ygh X-N20 and R-ygh X-N20, the pTargetF-ygh X plasmid was extracted; (3) Electroporate the integrated fragment of the ppc gene and the pTargetF-ygh X plasmid into AKG4 competent cells, screen for positive strains, and then remove the plasmid to obtain strain AKG5, which is the genetically engineered bacterium.

8. The use of the genetically engineered bacteria as described in any one of claims 1-7 in the production of α-ketoglutaric acid.

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