Recombinant escherichia coli for efficiently producing glycollic acid and construction method of recombinant escherichia coli

Abstract

The invention discloses escherichia coli recombinant bacteria with high glycolic acid yield and a construction method of the escherichia coli recombinant bacteria, and belongs to the technical field of bioengineering. Lactate dehydrogenase ldhA, phosphate transacetylase pta, acetokinase ackA and pyruvate oxidase poxB are knocked out in escherichia coli, key enzymes isocitrate lyase aceA and glyoxylate reductase ghrA in a glycollic acid synthesis path are over-expressed in a genome level, conversion of carbon flow to glycollic acid is enhanced, and the glycolic acid is converted into glycolic acid. On the basis, malic acid synthase AaceB, malic acid synthase GglcB and glycollic acid oxidase glcDEF are knocked out, a branch metabolic pathway is blocked, an efficient strain which is free of plasmids and resistance genes and does not need to induce glycollic acid production is constructed, the accumulation amount of L-valine fermented in a 5L fermentation tank for 48 hours reaches 51.2 g / L, the yield is 0.6 g / g, and the production intensity is 1.06 g / L / h.

Description

Technical Field

[0001] The invention relates to a recombinant Escherichia coli capable of efficiently producing glycolic acid and a construction method thereof, belonging to the technical field of bioengineering. Background Art

[0002] Glycolic acid (Glycolate, GlcN), also known as glycolic acid or glycolic acid, is the smallest α-hydroxy acid with a relative molecular mass of 76.05 and a molecular formula of C 2 H 4 O 3 . Glycolic acid is the simplest aliphatic hydroxy acid with active hydroxyl and carboxyl groups and a small molecular weight. Its strong penetrability and water solubility make it widely used in chemical cleaning, daily chemical industry, medicine, biodegradable materials and other fields. At present, glycolic acid is mainly used in chemical cleaning, daily chemical industry and new biodegradable materials. In addition, glycolic acid is also used as an ingredient for fungicides, adhesives, petroleum demulsifiers, welding agents and coatings, and for the synthesis of various pesticides and chemical additives. In recent years, with the increasing maturity of new processes and the expansion of production scale, the application field of glycolic acid has continued to expand. However, due to the limitation of the total demand for glycolic acid, the market concentration of the glycolic acid industry is relatively high. Although glycolic acid has a certain production scale at this stage, the production process is relatively backward and the product quality is not high.

[0003] In recent years, researchers at home and abroad have systematically studied the production of glycolic acid by microorganisms using metabolic engineering, and the production strains are mainly Escherichia coli. Among them, Deng Yu et al. used E. coli MG1655 as the starting strain, and used rational metabolic engineering methods and modular pathway engineering strategies to optimize the production of glycolic acid to 68g / L; Zhang Xueli et al. used E. coli ATCC8739 as the chassis to construct a production strain that can use acetic acid as a substrate, with a yield of 73.3g / L. However, the above strains all require the use of expensive IPTG as an inducer, which is not conducive to industrial production. Therefore, constructing a plasmid-free, resistance-free, and induction-free glycolic acid production strain has become an urgent problem to be solved. Summary of the invention

[0004] To solve the above problems, the purpose of the present invention is to provide an engineered Escherichia coli strain that produces high levels of glycolic acid, and a method for producing glycolic acid using the engineered strain. The present invention achieves efficient production of glycolic acid by knocking out byproduct pathway genes in Escherichia coli cells and overexpressing key enzymes in the glycolic acid synthesis pathway (isocitrate lyase aceA, glyoxylate reductase ghrA), laying the foundation for the industrial production of glycolic acid.

[0005] The first technical solution provided by the present invention is a recombinant Escherichia coli for efficiently producing glycolic acid. The recombinant Escherichia coli is a recombinant Escherichia coli in which lactate dehydrogenase ldhA, phosphate acetyltransferase pta, acetate kinase ackA, isocitrate dehydrogenase icd, malate synthase A aceB, malate synthase G glcB, aldehyde dehydrogenase aldA and glycolate dehydrogenase glcDEF are knocked out, and isocitrate lyase aceA and glyoxylate reductase ghrA are overexpressed.

[0006] In one embodiment of the present invention, the Gene IDs of the genes encoding lactate dehydrogenase ldhA, phosphate acetyltransferase pta, acetate kinase ackA, and isocitrate dehydrogenase icd are 946315, 946778, 946775, and 945702, respectively.

[0007] In one embodiment of the present invention, the amino acid sequence of isocitrate lyase aceA is any one of SEQ ID NO.1 to SEQ ID NO.3, and the amino acid sequence of glyoxylate reductase ghrA is any one of SEQ ID NO.4 to SEQ ID NO.6.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the isocitrate lyase aceA gene is any one of SEQ ID NO.8 to SEQ ID NO.10, and the nucleotide sequence encoding the glyoxylate reductase gene ghrA gene is any one of SEQ ID NO.11 to SEQ ID NO.13.

[0009] In one embodiment of the present invention, the nucleotide sequence of the promoter fragment of the isocitrate lyase gene aceA is such as SEQ ID NO.7, and the nucleotide sequence of the promoter fragment of the glyoxylate reductase gene ghrA is such as SEQ ID NO.7.

[0010] In one embodiment of the present invention, the host of the recombinant Escherichia coli is E. coli ATCC8739.

[0011] The second technical solution provided by the present invention is a method for constructing a recombinant Escherichia coli that efficiently produces glycolic acid. The method comprises knocking out lactate dehydrogenase ldhA, phosphate acetyltransferase pta, acetate kinase ackA, isocitrate dehydrogenase icd, malate synthase A aceB, malate synthase G glcB, aldehyde dehydrogenase aldA and glycolate dehydrogenase glcDEF of Escherichia coli, and simultaneously overexpressing isocitrate lyase aceA and glyoxylate reductase ghrA.

[0012] In one embodiment of the present invention, the isocitrate lyase gene aceA is expressed through genome integration.

[0013] In one embodiment of the present invention, the glyoxylate reductase gene ghrA is expressed by genome integration.

[0014] In one embodiment of the present invention, the Gene IDs of the genes encoding lactate dehydrogenase ldhA, phosphate acetyltransferase pta, acetate kinase ackA, and isocitrate dehydrogenase icd are 946315, 946778, 946775, and 945702, respectively.

[0015] In one embodiment of the present invention, the nucleotide sequence encoding the isocitrate lyase aceA gene is any one of SEQ ID NO.1-SEQ ID NO.3, and the nucleotide sequence encoding the glyoxylate reductase gene ghrA gene is any one of SEQ ID NO.4-SEQ ID NO.6.

[0016] In one embodiment of the present invention, the nucleotide sequence of the promoter fragment of the isocitrate lyase gene aceA is such as SEQ ID NO.7, and the nucleotide sequence of the promoter fragment of the glyoxylate reductase gene ghrA is such as SEQ ID NO.7.

[0017] In one embodiment of the present invention, the host of the recombinant Escherichia coli is E. coli ATCC8739.

[0018] The third technical solution provided by the present invention is a method for preparing glycolic acid, wherein the method comprises introducing the recombinant Escherichia coli described in the first technical solution into a fermentation system to ferment and generate glycolic acid.

[0019] In one embodiment of the present invention, the fermentation system contains: 5.6 g / L dipotassium hydrogen phosphate, 2.4 g / L potassium dihydrogen phosphate, 15 g / L glucose, 20 g / L corn steep liquor, 2 g / L sodium L-glutamate, and 0.25 g / L magnesium sulfate heptahydrate.

[0020] In one embodiment of the present invention, the recombinant Escherichia coli is inoculated into the fermenter at an inoculation rate of 10%.

[0021] In one embodiment of the present invention, during the fermentation process, the residual sugar in the fermentation system is controlled at 0-5 g / L, the dissolved oxygen is above 30%, the pH is maintained at 7.0 by NaOH, the temperature is at 37° C., and the fermentation is performed for at least 48 hours.

[0022] The fourth technical solution provided by the present invention is the use of the recombinant Escherichia coli described in the first technical solution, the method described in the second technical solution, or the method described in the third technical solution in the production of glycolic acid or products containing glycolic acid.

[0023] The technical effects of the present invention are as follows:

[0024] The present invention knocks out byproduct pathway genes in Escherichia coli cells and overexpresses key enzymes in the glycolic acid synthesis pathway (isocitrate lyase aceA, glyoxylate reductase ghrA), and the recombinant Escherichia coli constructed can use glucose as a substrate to obtain high glycolic acid in a short time. The fermentation process of the strain is easy to operate. In a 5L fermenter, the yield of 48h fermentation can reach 51.2g / L, and the production intensity is 1.06g / L / h. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of the transformation strategy of E.coli ATCC8739 of the present invention;

[0026] Figure 2 The results are shown in the fed-batch fermentation of the engineered strain GA-6 of the present invention in a 5 L fermenter. DETAILED DESCRIPTION

[0027] The following reference Figures 1-2 , the preferred embodiments of the present invention are described. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0028] Test method:

[0029] Cell concentration determination: Take an appropriate amount of fermentation broth and dilute it, and use a UV spectrophotometer to measure the OD at a wavelength of 600nm. 600 express.

[0030] Method for detecting glycolic acid and byproducts: centrifuge the fermentation broth at 12000r / min for 10min, dilute the supernatant to a certain multiple, and determine the content of glycolic acid and byproduct acetic acid by high performance liquid chromatography (HPLC). The HPLC detection conditions are: the chromatographic column is Aminex HPX-87H (300mm×7.8mm) chromatographic column, the mobile phase is 5mmol / L dilute sulfuric acid, the flow rate is 0.6mL / min, the column temperature is 52℃, the detector is an ultraviolet detector, and the wavelength is 210nm.

[0031] Conversion rate = (yield × fermentation liquid volume) / total sugar consumption in fermentation %.

[0032] Production intensity: glycolic acid production per unit time, production intensity = production (g / L) / fermentation time (h).

[0033] The culture medium involved in the embodiment:

[0034] LB medium: 10 g / L NaCl, 5 g / L yeast powder, 10 g / L peptone.

[0035] Primary seed culture medium: 10g / L NaCl, 5g / L yeast powder, 10g / L peptone, 1g / L sodium glutamate.

[0036] Secondary seed culture medium: 10g / L NaCl, 5g / L yeast powder, 10g / L peptone, 1g / L sodium glutamate, 1g / L glucose.

[0037] Fermentation medium: 5.6 g / L dipotassium hydrogen phosphate, 2.4 g / L potassium dihydrogen phosphate, 15 g / L glucose, 20 g / L corn steep liquor, 2 g / L sodium L-glutamate, 0.25 g / L magnesium sulfate heptahydrate.

[0038] Plasmids, strains and primers involved in the examples:

[0039] Table 1 Plasmid information

[0040]

[0041] Table 2 Strain information

[0042]

[0043] Table 3 Primers and sequences

[0044]

[0045]

[0046]

[0047] The above-mentioned pTarget plasmid, pCas9 plasmid, E. coli ATCC8739 strain and E. coli TOP10 strain are all commercial plasmids and strains.

[0048] Example 1 Construction of an engineered strain GA-1 in which the ldhA, pta, ackA, and poxB genes were deleted from the genome

[0049] Using E. coli ATCC8739 as the starting strain, the engineered strain GA-1 with ldhA, pta, ackA and poxB knocked out was constructed. The gene editing operations were all completed using CRISPR-Cas9 gene editing technology.

[0050] Taking knocking out the ldhA gene as an example, the specific method is as follows:

[0051] (1) Using the genome of E. coli GA-0 strain as a template, primers P1 and P2, P3 and P4 were used to amplify the upper and lower homologous arms of the ldhA gene, and then primers P1 and P4 were used to fuse the upper and lower arms into the ldhA knockout cassette;

[0052] (2) Using the pTarget plasmid as a template and P5 and P6 as primers, the linearized fragment was amplified and transformed into the cloning host E. coli TOP10 by homologous recombination to extract the pTarget-ldhA plasmid;

[0053] (3) The verified pTarget-ldhA plasmid and ldhA knockout box were electroporated into GA-0 containing the pCas9 plasmid, and after electroporation, they were spread on LB plates containing kanamycin and spectinomycin and cultured at 30°C;

[0054] (4) Perform colony PCR using validation primers P7 and P8, and sequence and screen to obtain the correct transformants;

[0055] (5) The correct transformants were transferred to a medium containing 0.1% IPTG to eliminate the pTarget plasmid, and the correct transformants were obtained by streaking on a plate;

[0056] (6) The correct transformants were transferred to the culture medium and cultured at 42°C to eliminate the pCas9 plasmid, and the correct transformants were obtained by streaking on the plate;

[0057] (7) Knock out the pta, ackA and poxB genes in the same manner as above to obtain E. coli GA-1.

[0058] Example 2: Construction of an engineered strain GA-2 with the icd gene knocked out in the genome

[0059] The E. coli GA-1 constructed in Example 1 was used as the starting strain, and the icd gene encoding isocitrate dehydrogenase on the genome was knocked out to construct the engineered strain E. coli GA-2. The gene editing operations were all completed using CRISPR-Cas9 gene editing technology.

[0060] The specific method is as follows:

[0061] (1) Using the genome of E. coli GA-0 strain as a template, primers P9 and P10, P11 and P12 were used to amplify the upper and lower homologous arms of the icd gene, and then primers P9 and P12 were used to fuse the upper and lower arms into an icd knockout cassette;

[0062] (2) Using the pTarget plasmid as a template and P13 and P14 as primers, the linearized fragment was amplified and transformed into the cloning host E. coli TOP10 by homologous recombination to extract the pTarget-icd plasmid;

[0063] (3) The verified pTarget-icd plasmid and ldhA knockout box were electroporated into GA-1 containing the pCas9 plasmid, and after electroporation, they were spread on LB plates containing kanamycin and spectinomycin and cultured at 30°C;

[0064] (4) Use verification primers P15 and P16 to perform colony PCR and sequence screening to obtain correct transformants;

[0065] (5) The correct transformants were transferred to a medium containing 0.1% IPTG to eliminate the pTarget plasmid, and the correct transformants were obtained by streaking on a plate;

[0066] (6) The correct transformants were transferred to the culture medium and cultured at 42°C to eliminate the pCas9 plasmid, and the correct transformants were obtained by streaking on the plate;

[0067] (7) Obtain E. coli GA-2.

[0068] Example 3: Construction of an engineered strain GA-3 with aceB and glcB genes knocked out in the genome

[0069] Using E. coli GA-2 constructed in Example 2 as the starting strain, the aceB gene encoding malate synthase A and the glcB gene encoding malate synthase G on the genome were knocked out to construct the engineered strain E. coli GA-3. The gene editing operations were all completed using CRISPR-Cas9 gene editing technology.

[0070] Taking knocking out aceB as an example, the specific method is as follows:

[0071] (1) Using the genome of E. coli GA-0 strain as a template, primers P17 and P18, P19 and P20 were used to amplify the upper and lower homologous arms of the aceB gene, and then primers P17 and P20 were used to fuse the upper and lower arms into the aceB knockout cassette;

[0072] (2) Using the pTarget plasmid as a template and P21 and P22 as primers, the linearized fragment was amplified and transformed into the cloning host E. coli TOP10 by homologous recombination to extract the pTarget-aceB plasmid;

[0073] (3) The verified pTarget-aceB plasmid and aceB knockout box were electroporated into GA-2 containing the pCas9 plasmid, and after electroporation, they were spread on LB plates containing kanamycin and spectinomycin and cultured at 30°C;

[0074] (4) Use verification primers P23 and P24 to perform colony PCR and sequence screening to obtain correct transformants;

[0075] (5) The correct transformants were transferred to a medium containing 0.1% IPTG to eliminate the pTarget plasmid, and the correct transformants were obtained by streaking on a plate;

[0076] (6) The correct transformants were transferred to the culture medium and cultured at 42°C to eliminate the pCas9 plasmid, and the correct transformants were obtained by streaking on the plate;

[0077] (7) Knock out the glcB gene in the same manner as above to obtain E. coli GA-3.

[0078] Example 4: Construction of an engineered strain GA-4 with ghrA gene integrated into the genome

[0079] The E. coli GA-3 constructed in Example 3 was used as the starting strain, and the glyoxylate reductase encoding gene ghrA was integrated, and its coding sequence could be any one of the glyoxylate reductase encoding gene nucleotide sequences of SEQ ID NO.8 to SEQ ID NO.10, and its promoter sequence was SEQ ID NO.7, to construct the engineering strain E. coli GA-4. The gene editing operations were all completed using CRISPR-Cas9 gene editing technology.

[0080] The specific method is as follows:

[0081] (1) Using the genome of E. coli GA-0 strain as a template, primers P25 and P26, P27 and P28 were used to amplify the upper and lower homologous arms of the yahj locus, and primers P29 and P30 were used to amplify ghrA (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.8), and then primers P25 and P28 were used to fuse the upper and lower arms and the ghrA gene into a yahj::ghrA integration frame;

[0082] (2) Using the pTarget plasmid as a template and P31 and P32 as primers, the linearized fragment was amplified and transformed into the cloning host E. coli TOP10 by homologous recombination to extract the pTarget-yahj plasmid;

[0083] (3) The verified pTarget-yahj plasmid and yahj::ghrA integration frame were electroporated into GA-3 containing the pCas9 plasmid, and after electroporation, they were spread on LB plates containing kanamycin and spectinomycin and cultured at 30°C;

[0084] (4) Use verification primers P33 and P34 to perform colony PCR and sequence screening to obtain correct transformants;

[0085] (5) The correct transformants were transferred to a medium containing 0.1% IPTG to eliminate the pTarget plasmid, and the correct transformants were obtained by streaking on a plate;

[0086] (6) The correct transformants were transferred to the culture medium and cultured at 42°C to eliminate the pCas9 plasmid, and the correct transformants were obtained by streaking on the plate;

[0087] (7) Obtain E. coli GA-4.

[0088] Example 5: Construction of an engineered strain GA-5 with aceA gene integrated into the genome

[0089] Using E. coli GA-4 constructed in Example 4 as the starting strain, the isocitrate lyase encoding gene aceA was integrated, whose coding sequence can be any one of SEQ ID NO.11 to SEQ ID NO.13, and whose promoter sequence is SEQ ID NO.7, to construct the engineering strain E. coli GA-5. The gene editing operations were all completed using CRISPR-Cas9 gene editing technology.

[0090] The specific method is as follows:

[0091] (1) Using the genome of E. coli GA-0 strain as a template, primers P35 and P36, P37 and P38 were used to amplify the upper and lower homologous arms of the yghx locus, and primers P39 and P40 were used to amplify aceA (the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.11), and then primers P35 and P38 were used to fuse the lower arm and aceA gene to form a yghx::aceA integration frame;

[0092] (2) Using the pTarget plasmid as a template and P41 and P42 as primers, the linearized fragment was amplified and transformed into the cloning host E. coli TOP10 by homologous recombination to extract the pTarget-yahj plasmid;

[0093] (3) The verified pTarget-yahj plasmid and yahj::ghrA integration frame were electroporated into GA-4 containing the pCas9 plasmid, and after electroporation, they were spread on LB plates containing kanamycin and spectinomycin and cultured at 30°C;

[0094] (4) Colony PCR was performed using validation primers P43 and P44, and sequencing was performed to screen for correct transformants;

[0095] (5) The correct transformants were transferred to a medium containing 0.1% IPTG to eliminate the pTarget plasmid, and the correct transformants were obtained by streaking on a plate;

[0096] (6) The correct transformants were transferred to the culture medium and cultured at 42°C to eliminate the pCas9 plasmid, and the correct transformants were obtained by streaking on the plate;

[0097] (7) Obtain E. coli GA-5.

[0098] Example 6: Construction of an engineered strain GA-6 with aldA and glcDEF genes knocked out from the genome

[0099] Using the E. coli GA-5 constructed in Example 5 as the starting strain, the aldehyde dehydrogenase aldA and the glycolate oxidase glcDEF were knocked out to construct the engineered strain E. coli GA-6. The gene editing operations were all completed using CRISPR-Cas9 gene editing technology.

[0100] Taking aldA knockout as an example, the specific method is as follows:

[0101] (1) Using the genome of E. coli GA-0 strain as a template, primers P45 and P46, P47 and P48 were used to amplify the upper and lower homologous arms of the aldA gene, and then primers P45 and P48 were used to fuse the upper and lower arms into an aldA knockout cassette;

[0102] (2) Using the pTarget plasmid as a template and P49 and P50 as primers, the linearized fragment was amplified and transformed into the cloning host E. coli TOP10 by homologous recombination to extract the pTarget-aldA plasmid;

[0103] (3) The verified pTarget-aldA plasmid and aldA knockout cassette were electroporated into GA-5 containing the pCas9 plasmid, and after electroporation, they were spread on LB plates containing kanamycin and spectinomycin and cultured at 30°C;

[0104] (4) Perform colony PCR using validation primers P51 and P24, and sequence and screen to obtain the correct transformants;

[0105] (5) The correct transformants were transferred to a medium containing 0.1% IPTG and cultured to eliminate the pTarget plasmid, and the correct transformants were obtained by streaking on a plate;

[0106] (6) The correct transformants were transferred to the culture medium and cultured at 42°C to eliminate the pCas9 plasmid, and the correct transformants were obtained by streaking on the plate;

[0107] (7) Knock out the glcDEF gene by the same steps as above to obtain GA-6.

[0108] Example 7: Fed-batch fermentation of recombinant strain E. coli GA-6 in a 5 L fermenter

[0109] The recombinant strain E. coli GA-5 prepared in Example 5 and the recombinant strain E. coli GA-6 prepared in Example 6 were used for fermentation according to the following tank loading process: frozen glycerol tube→slant activation→primary shake flask seeds→secondary shake flask seeds→fermenter.

[0110] (1) Slant activation: Streak the bacteria in the glycerol tube onto a slant medium and culture at 37°C for 20 to 24 hours.

[0111] (2) Primary shake flask seed culture: Wash the bacterial lawn on the slant medium with sterile water, inoculate 2% of the inoculum into a 500 mL Erlenmeyer flask containing 100 mL of culture medium, and culture at 200 r / min and 37°C for 6-8 h. The OD 600 Between 6.0 and 10.0.

[0112] (3) Secondary shake flask seed culture: The primary shake flask seed was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of secondary seed culture medium at a 2% inoculation rate, and cultured at 200 r / min and 37°C for 6-8 h. At 4.5 h, 100 μL of 50% ammonia water was added, and the OD 600 Between 8.0 and 10.0;

[0113] (4) 5 L Dibier parallel reactor fermentation: The initial liquid volume of the fermenter was 2.5 L, and the secondary seed liquid was inoculated into the fermentation medium at an inoculation rate of 10% (volume ratio) of the total volume. The temperature was 37°C, the initial ventilation volume was 1.5 vvm, and ammonia water was added during the fermentation process to control the pH at 6.9 ± 0.2, and the dissolved oxygen was controlled to be no less than 35%;

[0114] Fermentation medium: 5.6% potassium hydrogen phosphate, 2.4% potassium dihydrogen phosphate, 10% glucose, 20% corn steep liquor, 1.7% sodium L-glutamate, 0.25% magnesium sulfate heptahydrate, and 1 mL / L defoaming agent.

[0115] The ethanol acid production after 48 hours of fermentation was tested. The results showed that after 48 hours of fermentation, the ethanol acid production could reach 51.2 g / L, and the yield and production intensity reached 0.6 g / g and 1.06 g / L / h, respectively. In addition, the production performance of the GA05 strain was also tested, and its ethanol acid production, yield and production intensity were 45.1 g / L, 0.52 g / g and 0.94 g / L / h, respectively.

[0116] Example 8

[0117] For isocitrate lyase genes aceA from different sources and glyoxylate reductase genes ghrA from different sources, the enzyme activity data are shown in Table 1. Different combinations can be made with reference to the methods of Examples 4 to 6, and batch fed fermentation can be performed with reference to the method of Example 7.

[0118] Table 1 Specific enzyme activities of aceA and ghrA proteins from different sources

[0119]

[0120] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli for efficiently producing glycolic acid, characterized in that: The recombinant Escherichia coli is a recombinant Escherichia coli in which lactate dehydrogenase ldhA, phosphate acetyltransferase pta, acetate kinase ackA, isocitrate dehydrogenase icd, malate synthase A aceB, malate synthase G glcB, aldehyde dehydrogenase aldA and glycolate dehydrogenase glcDEF are knocked out, and isocitrate lyase aceA and glyoxylate reductase ghrA are overexpressed; the amino acid sequence of the isocitrate lyase aceA is any one of SEQ ID NO.1 to SEQ ID NO.3, and the amino acid sequence of the glyoxylate reductase ghrA is any one of SEQ ID NO.4 to SEQ ID NO.

6.

2. The recombinant Escherichia coli according to claim 1, characterized in that The Gene IDs of the genes encoding lactate dehydrogenase ldhA, phosphate acetyltransferase pta, acetate kinase ackA, and isocitrate dehydrogenase icd are 946315, 946778, 946775, and 945702, respectively.

3. The recombinant Escherichia coli according to claim 1, characterized in that The nucleotide sequence of the promoter fragment of the isocitrate lyase gene aceA is as shown in SEQ ID NO.7, and the nucleotide sequence of the promoter fragment of the glyoxylate reductase gene ghrA is as shown in SEQ ID NO.

7.

4. The recombinant Escherichia coli according to claim 1, characterized in that The host of the recombinant Escherichia coli is E. coli ATCC8739.

5. A method for constructing a recombinant Escherichia coli for efficiently producing glycolic acid, characterized in that: The method comprises the following steps: knocking out lactate dehydrogenase ldhA, phosphate acetyltransferase pta, acetate kinase ackA, isocitrate dehydrogenase icd, malate synthase A aceB, malate synthase G glcB, aldehyde dehydrogenase aldA and glycolate dehydrogenase glcDEF of Escherichia coli, and simultaneously overexpressing isocitrate lyase aceA and glyoxylate reductase ghrA; the nucleotide sequence encoding the isocitrate lyase aceA gene is any one of SEQ ID NO.1-SEQ ID NO.3, and the nucleotide sequence encoding the glyoxylate reductase gene ghrA gene is any one of SEQ ID NO.4-SEQ ID NO.

6.

6. The construction method according to claim 5, characterized in that: The isocitrate lyase gene aceA and the glyoxylate reductase gene ghrA are both expressed through genome integration.

7. A method for preparing glycolic acid, characterized in that: The method comprises introducing the recombinant Escherichia coli according to any one of claims 1 to 4 into a fermentation system to ferment and generate glycolic acid.

8. The method according to claim 7, characterized in that The recombinant Escherichia coli was inoculated into the fermentation system at an inoculation rate of 10%.

9. The method according to claim 7 or 8, characterized in that: During the fermentation process, the residual sugar in the fermentation system was controlled at 0-5 g / L, the dissolved oxygen was above 30%, the pH was maintained at 7.0 with NaOH, the temperature was at 37° C., and the fermentation was continued for at least 48 hours.

10. Use of the recombinant Escherichia coli according to any one of claims 1 to 4, the method according to claim 5 or 6, or the method according to any one of claims 7 to 9 in producing glycolic acid or a product containing glycolic acid.

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