A method for producing fumaric acid by metabolically engineering escherichia coli

Abstract

The application discloses a method for producing fumaric acid by metabolically engineering escherichia coli, and belongs to the fermentation field. In the escherichia coli E.coli FMME-N-5, fumarase gene, fumarate reductase gene, aspartase gene and lactate dehydrogenase gene are knocked out, and phosphoenolpyruvate carboxylase gene and succinate dehydrogenase coding gene are overexpressed. The obtained strain E.coli FMME-N-5 (△fumB△frdBC△aspA△ldhA)-ppc-sdhCDAB is used for fermenting and producing fumaric acid. In a 7.5L fermenter, the fermentation lasts for 65h, the yield of fumaric acid reaches 45g / L, the production intensity is 0.69g / L / h, the yield of fumaric acid to glucose is 0.52g / g, the byproduct formic acid does not accumulate, the accumulation amounts of succinic acid, lactic acid and acetic acid are all lower than 1g / L, and the method has certain industrial production potential.

Description

TECHNICAL FIELD

[0001] The application relates to a method for producing fumaric acid by metabolic engineering of Escherichia coli, and belongs to the field of fermentation engineering. BACKGROUND

[0002] Fumaric acid is an important intermediate of fine chemical products. Fumaric acid has a special structure and can undergo various chemical reactions, such as esterification, hydrogenation, isomerization and the like, and is widely used in the fields of chemicals, food and medicine. In chemical production, unsaturated polyester produced from fumaric acid as a raw material has very high chemical corrosion resistance and heat resistance; fumaric acid ester and styrene can be used as raw materials to produce a good lubricating oil pour point depressant. In food production, fumaric acid as an important food additive, when reacted with sodium hydroxide to form sodium salt, is a commonly used acid taste regulator. Food-grade fumaric acid is weakly acidic and is mainly used in the preparation of candies, beverages and jellies. In the field of medicine, fumaric acid and fumaric acid ester are important intermediates and precursors for drug production, which are used in drug production, cancer research and neuroimmunology. Fumaric acid is a high-quality nanometer drug carrier component, and the drug carried by it has a long drug release period, stable drug effect and significant inhibition effect on many cancer diseases. Fumaric acid has been listed by the US Department of Energy as one of the top ten architectural compounds with priority development value.

[0003] Fumaric acid can be produced by isomerization of malic acid, which can be obtained by conversion of maleic anhydride, which can be obtained by catalytic oxidation of gaseous hydrocarbon compounds in turn. This way of producing fumaric acid from malic acid is limited by reaction equilibrium. The conversion is carried out at high temperature, which produces by-products, and the yield is lower than the equilibrium yield. Maleic acid isomerase can catalyze the conversion of malic acid to fumaric acid, and there is no other by-product in the equilibrium product, and the highest conversion rate of fumaric acid can reach 95%. Microbial fermentation method for producing fumaric acid is a promising production direction to replace the chemical method based on petrochemical raw materials. Rhizopus oryzae, Rhizopus arrhizus and Rhizopus nigricans can achieve high yield and high production intensity of fumaric acid, and the yield and production intensity can reach 0.85 g / g and 4.25 g / L / h. However, the large size of the Rhizopus ball in the fermentation process affects the dissolved oxygen and thus limits the yield; and the genetic manipulation tools for modifying Rhizopus are relatively scarce. In recent years, with the diversity and high efficiency of gene editing tools, researchers have begun to use metabolic engineering strategies in model microorganisms such as Escherichia coli and yeast to improve fumaric acid.

[0004] Currently, the efficiency of Escherichia coli in fermenting to produce fumaric acid is low, and there are usually by-products such as lactic acid, formic acid and acetic acid in the fermentation broth, the product is decomposed and utilized during the fermentation process, the substrate glucose cannot be tolerated at high concentration, the glucose is absorbed too fast to cause metabolic imbalance, and the activity of key enzyme for fumaric acid production is low; in order to obtain a high-performance production strain, it is usually necessary to use traditional breeding methods, combined with various omics analysis and molecular biology modification. At present, the use of molecular modification methods to improve the yield of fumaric acid has achieved certain results, and the conventional modification involves knocking out fumarase gene fumABC, fumarate reductase gene frdABCD and aspA gene encoding aspartate, etc. to block the downstream decomposition of fumaric acid. And overexpressing citrate synthase encoding gene cs and genes carAB and argI involved in urea cycle to strengthen fumaric acid accumulation. The highest yield of fumaric acid produced by Escherichia coli fermentation reported so far is 41.5g / L, and the production strain overexpresses phosphoenolpyruvate carboxylase encoding gene ppc and acetaldehyde cycle operon aceBA on the basis of knocking out fumABC gene, uses glycerol as fermentation substrate, and the production intensity is 0.51g / L / h, and the yield is 0.44g / g, but a large amount of by-products are accumulated during the fermentation process, and the accumulation amount of acetic acid reaches 8.7g / L. How to improve the yield of fumaric acid while avoiding the accumulation of fermentation by-products is a technical problem that needs to be solved at present. SUMMARY

[0005] In order to solve the above problems, the application provides an Escherichia coli engineering strain for efficiently producing fumaric acid, which knocks out fumarase gene (fumB), fumarate reductase gene (frdBC), aspartate enzyme gene (aspA) and lactate dehydrogenase gene (ldhA) in the host strain Escherichia coli FMME-N-5 by using Red homologous recombination method, and overexpresses phosphoenolpyruvate carboxylase gene (ppc) from high-yield succinic acid engineering strain in the research room and succinate dehydrogenase gene (sdhCDAB) for strengthening electron transfer and converting succinic acid to fumaric acid, so as to realize efficient production of fumaric acid under limited oxygen condition.

[0006] The first object of the application is to provide an Escherichia coli engineering strain for efficiently producing fumaric acid. The Escherichia coli engineering strain knocks out one or more of fumarase gene fumB, fumarate reductase gene frdBC, aspartate enzyme gene aspA and lactate dehydrogenase gene ldhA in Escherichia coli, and overexpresses phosphoenolpyruvate carboxylase gene ppc from high-yield succinic acid engineering strain in the research room and succinate dehydrogenase gene sdhCDAB for strengthening electron transfer.

[0007] Further, the nucleotide sequence of the fumarase gene fumB is shown as SEQ ID NO. 1; the nucleotide sequence of the fumarate reductase gene frdBC is shown as SEQ ID NO. 2; the nucleotide sequence of the aspartase gene aspA is shown as SEQ ID NO. 3; the nucleotide sequence of the lactate dehydrogenase gene ldhA is shown as SEQ ID NO. 4; the nucleotide sequence of the phosphoenolpyruvate carboxylase gene ppc is shown as SEQ ID NO. 5, and the nucleotide sequence of the succinate dehydrogenase gene sdhCDAB is shown as SEQ ID NO. 6.

[0008] Further, the phosphoenolpyruvate carboxylase gene ppc is overexpressed by the vector PCDR, and the succinate dehydrogenase gene sdhCDAB is overexpressed by the vector pEM.

[0009] Further, the host of the engineered Escherichia coli strain is Escherichia coli FMME-N-5, which was preserved in the China Center for Type Culture Collection on August 27, 2020, at Wuhan University in Wuhan, China, and has the preservation number CCTCC NO: M20200454, and has been disclosed in patent CN112239738B.

[0010] A second object of the present application is to provide a construction method of the engineered Escherichia coli strain, and the steps of the construction method are as follows:

[0011] The gene knockout frame fragments of fumB, frdBC, aspA and ldhA are respectively constructed, and the gene knockout frame fragments are sequentially transferred into the host bacteria with the pKD46 plasmid to obtain the strains with the target genes knocked out;

[0012] The gene fragments of the phosphoenolpyruvate carboxylase gene ppc and the succinate dehydrogenase gene sdhCDAB are amplified, the gene fragments are respectively connected to the corresponding expression vectors, and then the expression vectors with the gene fragments connected are transferred into the strains of step (1) to obtain the recombinant Escherichia coli.

[0013] A third object of the present application is to provide the application of the engineered Escherichia coli strain in the fermentation production of fumaric acid. The application is to perform aerobic-limited oxygen two-stage fermentation in the fermentation medium by using the engineered Escherichia coli strain to obtain the fermentation liquor containing fumaric acid.

[0014] Further, the fermentation medium used in the fermentation contains glucose 40-50 g / L, Na2HPO3.5H2O 20-50 mM, KHCO3 30-50 mM, Na2HPO4.12H2O 15.11 g / L, KH2PO4 3 g / L NH4Cl 1 g / L, NaCl 0.5 g / L, 1 mL of trace element solution per L of culture medium; trace element solution: FeCl3.6H2O 2.4 g / L, CoCl2.6H2O 0.3 g / L, CuCl2 0.15 g / L, ZnCl2.4H2O 0.3 g / L, NaMnO4 0.3 g / L, H3BO3 0.075 g / L, MnCl2.4H2O 0.495 g / L, dissolved in 0.1 M HCl to prepare.

[0015] Further, the aerobic-limited oxygen two-stage fermentation is carried out when the OD of the bacteria is 40-50. 600 The aerobic stage is converted to the limited oxygen stage.

[0016] Further, the conversion of the aerobic stage to the limited oxygen stage is carried out by adjusting the dissolved oxygen from 20-30% to 50-60% or adding 10-20 g / L of bicarbonate.

[0017] Further, in the limited oxygen stage, the glucose concentration is controlled to be 5-10 g / L.

[0018] Further, the inoculation amount of the aerobic-limited oxygen two-stage fermentation is 5-10% by volume percentage; and the fermentation temperature is 34-38°C.

[0019] Further, the fermentation time of the aerobic-limited oxygen two-stage fermentation is 60-70 h.

[0020] Further, in the anaerobic stage, a pH neutralizer is added, and the pH neutralizer is one or a mixture of several of Na2CO3, K2CO3, NaOH, KOH, CaCO3, and MgCO3.

[0021] Further, in the aerobic-limited oxygen two-stage fermentation process, an osmotic pressure protective agent is added, and the osmotic pressure protective agent is one or a mixture of several of proline, methionine, betaine, and cysteine.

[0022] In the present application, the phosphoenolpyruvate carboxylase ppc can catalyze the production of oxalic acid from phosphoenolpyruvic acid, promote the synthesis of product precursors, and have a significant beneficial effect on the main metabolic pathway of fumaric acid production; the succinate dehydrogenase sdhCDAB can catalyze the conversion of succinic acid to fumaric acid, greatly improving the yield of fumaric acid.

[0023] The beneficial effects of the present application are:

[0024] The present application adopts Red homologous recombination technology to knock out the related enzymes coded by the by-products affecting the generation of fumaric acid, including fumarase, fumarate reductase, aspartase and lactate dehydrogenase, which can significantly reduce the accumulation of by-products while not affecting the growth rate of the bacteria, and is beneficial to the accumulation of fumaric acid; meanwhile, overexpression of phosphoenolpyruvate carboxylase and overexpression of succinate dehydrogenase effectively improve the yield of fumaric acid. The production intensity and capacity of the E. coli engineering strain E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA)-ppc-sdhCDAB constructed by the present application are higher than the reported E. coli strains for producing fumaric acid by using glucose, and the yield of fumaric acid reaches 45 g / L after 65 h of fermentation, the yield of fumaric acid is 0.52 g / g of glucose, and the production intensity is 0.69 g / L / h; meanwhile, the accumulation amount of by-products in the fermentation process is significantly lower than that of the prior art, among which formic acid does not accumulate, and the accumulation amounts of succinic acid, lactic acid and acetic acid are all lower than 1 g / L, which is beneficial to the industrialized production of fumaric acid. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Verification gel for knocking out fumarase gene (lanes 1-4 are parallel experiments);

[0026] Figure 2 Verification gel for knocking out fumarate reductase gene (lanes 1-5 are parallel experiments);

[0027] Figure 3 Verification gel for knocking out aspartase gene (lanes 1-4 are parallel experiments);

[0028] Figure 4 Verification gel for knocking out lactate dehydrogenase gene (lanes 1-5 are parallel experiments);

[0029] Figure 5 Verification gel for expressing phosphoenolpyruvate carboxylase gene (lanes 1-4 are parallel experiments);

[0030] Figure 6 Verification gel for expressing succinate dehydrogenase gene (lanes 1-10 are parallel experiments);

[0031] Figure 7 Vector map for expressing phosphoenolpyruvate carboxylase gene;

[0032] Figure 8 Vector map for expressing succinate dehydrogenase gene;

[0033] Figure 9The results of the fed-batch fermentation of the E. coli engineering strain E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA)-ppc-sdhCDAB in a fermenter. 1# is E. coli FMME-N-5; 2# is E. coli FMME-N-5 (△fumB); 3# is E. coli FMME-N-5 (△fumB△frdBC); 4# is E. coli FMME-N-5 (△fumB△frdBC△aspA); 5# is E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA); 6# is E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA)-ppc-sdhCDAB. DETAILED DESCRIPTION

[0034] The present application is further described in conjunction with the following specific examples, so that those skilled in the art can better understand the present application and implement it. The listed examples are not intended to limit the present application.

[0035] Relevant nucleotide sequence information in the sequence listing:

[0036] The sequence information of SEQ ID NO. 1 is the nucleotide sequence of fumarase gene fumB;

[0037] The sequence information of SEQ ID NO. 2 is the nucleotide sequence of fumarase gene frdBC;

[0038] The sequence information of SEQ ID NO. 3 is the nucleotide sequence of aspartase gene aspA;

[0039] The sequence information of SEQ ID NO. 4 is the nucleotide sequence of lactate dehydrogenase gene ldhA;

[0040] The sequence information of SEQ ID NO. 5 is the nucleotide sequence of phosphoenolpyruvate carboxylase gene ppc of the high-yield succinic acid E. coli;

[0041] The sequence information of SEQ ID NO. 6 is the nucleotide sequence of succinate dehydrogenase gene sdhCDAB from Paracoccus denitrificans;

[0042] Determination of cell concentration:

[0043] An appropriate amount of fermentation broth was neutralized with 2 mol / L hydrochloric acid, and the cell density was determined by the absorbance value at 600 nm wavelength detected by a spectrophotometer.

[0044] Determination of glucose:

[0045] Preparation of fermentation broth: The fermentation broth was centrifuged at 12000 r / min for 5 min to obtain the supernatant. The supernatant was diluted to an appropriate multiple, and the glucose concentration of the fermentation broth was detected by M-100 biological sensor analyzer.

[0046] Determination of organic acid:

[0047] High performance liquid chromatography: The fermentation broth was centrifuged at 12000 r / min for 5 min to obtain the supernatant. After dilution to an appropriate multiple, the yields of fumaric acid, succinic acid, formic acid, lactic acid and acetic acid were detected by high performance liquid chromatography (HPLC). The instrument was Water se2695 reverse phase high performance liquid chromatograph, the chromatographic column was Bio-Rad HPX87H; the mobile phase was 5 mmol / L H2SO4; the flow rate was set to 0.6 mL / min; the detection wavelength was 210 nm, and the column temperature was 35°C.

[0048] Construction of expression vector pCDR: The pCDR plasmid was obtained by modifying the promoter region of pCDM4 (purchased from addgene, #49796). The gene sequence of the pCDR plasmid is shown in SEQ ID NO. 6 in the sequence listing of patent CN110951660B on page 6.

[0049] Construction of expression vector pEM: The plasmid pEM is obtained by replacing the T5 promoter and the multiple cloning site Amp R ,f1 replication site of the plasmid pQE-80l-kan with the plasmid pETM6. (See patent CN113293120A specification part page 4, paragraph 0055 and table 1, third row for details).

[0050] Example 1: Construction of E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA)-ldhA-sdhCDAB strain

[0051] 1. Construction of E. coli FMME-N-5 competent cells

[0052] Escherichia coli FMME-N-5 is a succinic acid-producing strain constructed by the present experiment, which was preserved in China Center for Type Culture Collection on August 27, 2020, at Wuhan University in Wuhan, China, with the preservation number CCTCC NO: M20200454, and has been disclosed in patent CN112239738B. 100 μL of the preserved bacterial solution was transferred from the glycerol tube to 25 mL / 100 mL of liquid LB medium, and incubated at 37°C, 200 rpm overnight. 800 μL of the activated bacterial solution was transferred to 50 mL / 250 mL of liquid LB medium and incubated for 1.5-2 h. When the OD reached 0.5-0.6, the culture was stopped and transferred to a 4°C ice bath for 20 min. Then the supernatant was removed by centrifugation, and 25 mL of 0.1M CaCl2 was added to the ice bath for 15-20 min. Centrifugation at 5000 rpm for 5 min removed the supernatant, and 1 mL of 30% glycerol and 0.1M CaCl2 was added. The solution was aliquoted into sterile EP tubes, each containing 100 μL, and stored at -40°C for later use.

[0053] 2. Knockout of fumarase gene fumB

[0054] The knockout primers QCfumB-F and QCfumB-R (Table 2) were designed according to the fumB gene sequence of Escherichia coli MG1655 in the NCBI database, and the knockout frame of fumB was amplified and gel recovered using pKD4 plasmid as the template. Note: The pKD4 plasmid contains two FRT sites (which can be folded under the action of the Flp recombinase to eliminate the DNA sequence between the FRT sites), and the coding gene of kanamycin (kan, as a selection pressure for gene knockout) between the two FRT sites, i.e., FRT-kan-FRT. When knocking out the gene, the primers are designed to amplify the FRT-kan-FRT DNA fragment. It should be noted that the two amplification primers designed contain 39-49 bp of the target gene upstream and downstream, respectively, i.e., the final DNA fragment obtained is "39-49 bp upstream of the target gene-FRT-kan-FRT-39-49 bp downstream of the target gene", which is called the knockout frame of the target gene. The fumB knockout frame was introduced into the E. coli FMME-N-5 competent cells containing the pKD46 plasmid by electroporation (the electroporation voltage and time were 1800V and 5ms, respectively). The electroporated competent cells were plated on LB solid medium containing kanamycin (50 μg / mL) and incubated upside down for 12-24 h. After single colonies grew on the plate, the positive transformants were screened using the verification primers QCfumB-test-F and QCfumB-test-R (Table 2).

[0055] The pCP20 plasmid was transformed into positive transformants to eliminate the kanamycin resistance gene, and then verified using the QCfumB-test-F and QCfumB-test-R primers. The electrophoresis band size of the successfully knocked-out transformants was 500 bp, and the electrophoresis band size of the control group without knocking out was 2147 bp. Figure 2 The results showed that the fumB gene was successfully knocked out in FMME-N-5 in vivo, and the E. coli FMME-N-5 (△fumB) strain was obtained.

[0056] 3. Knockout of fumarate reductase gene frdBC

[0057] The knockout primers QCfrdBC-F and QCfrdBC-R (Table 2) were designed according to the frdBC gene sequence of Escherichia coli MG1655 in the NCBI database, and the knockout frame of frdBC was amplified and gel recovered using the pKD4 plasmid as a template. The knockout frame of fumB was introduced into the E. coli FMME-N-5 (△fumB) competent cells containing the pKD46 plasmid by electroporation (the electroporation voltage and time were 1800 V and 5 ms, respectively). The electroporated competent cells were plated on LB solid medium containing kanamycin (50 g / mL) and incubated for 12-24 h. After single colonies grew on the plate, the positive transformants were screened using the verification primers QCfrdBC-test-F and QCfrdBC-test-R (Table 2).

[0058] The pCP20 plasmid was transformed into positive transformants to eliminate the kanamycin resistance gene, and then verified using the QCfrdBC-test-F and QCfrdBC-test-R primers. The electrophoresis band size of the successfully knocked-out transformants was 506 bp, and the electrophoresis band size of the control group without knocking out was 1916 bp. Figure 3 The E. coli FMME-N-5 (△fumB△frdBC) strain was obtained.

[0059] 4. Knockout of aspartase gene aspA

[0060] Knockout primers QCaspA-F and QCaspA-R (Table 2) were designed according to the aspA gene sequence of Escherichia coli MG1655 in NCBI database, and the knockout cassette of aspA was amplified using pKD4 plasmid as template and gel recovered. The knockout cassette of aspA was introduced into E. coli FMME-N-5 (△fumB△frdBC) competent cells containing pKD46 plasmid by electroporation (the voltage and time of electroporation were 1800 V and 5 ms, respectively). The electroporated competent cells were spread on LB solid medium plates containing kanamycin (50 μg / mL) and incubated upside down for 12-24 h. After single colonies grew on the plates, positive transformants were screened using verification primers QCaspA-test-F and QCaspA-test-R (Table 2).

[0061] The pCP20 plasmid was transformed into the positive transformants to eliminate the kanamycin resistance gene, and verification was performed using primers QCaspA-test-F and QCaspA-test-R. The electrophoretic band size of the successfully knocked out transformants was 510 bp, and the electrophoretic band size of the control group without knockout was 1937 bp. Figure 5 ) The E. coli FMME-N-5 (△fumB△frdBC△aspA) strain was obtained.

[0062] 5. Knockout of lactate dehydrogenase gene ldhA

[0063] Knockout primers QCldhA-F and QCldhA-R (Table 2) were designed according to the ldhA gene sequence of Escherichia coli MG1655 in NCBI database, and the knockout cassette of ldhA was amplified using pKD4 plasmid as template and gel recovered. The knockout cassette of ldhA was introduced into E. coli FMME-N-5 (△fumB△frdBC△aspA) competent cells containing pKD46 plasmid by electroporation (the voltage and time of electroporation were 1800 V and 5 ms, respectively). The electroporated competent cells were spread on LB solid medium plates containing kanamycin (50 μg / mL) and incubated upside down for 12-24 h. After single colonies grew on the plates, positive transformants were screened using verification primers QCldhA-test-F and QCldhA-test-R (Table 2).

[0064] The pCP20 plasmid was transformed into the positive transformants to eliminate the kanamycin resistance gene, and verification was performed using primers QCldhA-test-F and QCldhA-test-R. The electrophoretic band size of the successfully knocked out transformants was 500 bp, and the electrophoretic band size of the control group without knockout was 1490 bp. Figure 4). The E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA) strain was obtained.

[0065] 6. Construction of expression vector pCDR-ppc

[0066] The phosphoenolpyruvate carboxylase gene ppc used in the present application was derived from the succinic acid-producing engineered E. coli in our laboratory, and the genomic DNA of the engineered E. coli was extracted.

[0067] According to the published genomic information sequence, primer pairs PCDR-ppc-F and PCDR-ppc-R (Table 2) were designed, respectively, and the ppc gene was amplified by using the genomic DNA of the succinic acid-producing Actinobacillus succinogenes as a template, a standard PCR amplification system and a program.

[0068] The ppc amplified by PCR was recovered by agarose gel electrophoresis, and the recovered product was double-digested with restriction enzymes Kpnl and Xhol for 3 h, respectively, using the expression vector pCDR. The digested products were recovered by agarose gel electrophoresis, and the sizes of the DNA and the linearized plasmid were 2652 bp and 3958 bp, respectively. Then, T4 DNA ligase was used for overnight ligation at 16°C, and the ligation product was transformed into JM109 competent cells. Single colonies were picked, and PCR was performed using primer pairs PCDR-ppc-test-F and PCDR-ppc-test-R. The positive transformants were sequenced, and the results were correct, indicating that the expression vector was successfully constructed. The plasmid was named pCDR-ppc.

[0069] 7. Construction of expression vector pEM-sdhCDAB

[0070] The succinate dehydrogenase gene sdhCDAB used in the present application was derived from Paracoccus denitrificans, and the genomic DNA of Paracoccus denitrificans was extracted.

[0071] According to the published genomic information sequence, primer pairs pEM-sdhCDAB-F and pEM-sdhCDAB-R (Table 2) were designed, respectively, and the sdhCDAB gene was amplified by using the genomic DNA of Paracoccus denitrificans as a template, a standard PCR amplification system and a program.

[0072] PCR amplification of sdhCDAB, agarose gel electrophoresis of nucleic acid was used to cut and recover the gel, the recovered product and expression vector pEM were respectively digested with restriction enzymes BamHI and SacI for 3h, the enzyme digestion products were recovered by agarose gel electrophoresis, the size of DNA and linearized plasmid was 3276bp and 5221bp respectively, then T4 DNA ligase was used for overnight ligation at 16℃, transformed into JM109 competent cells, single colonies were picked, PCR verification was performed using pEM-sdhCDAB-test-F and pEM-sdhCDAB-test-R primers, positive transformants were sequenced, alignment was correct, proving that the expression vector was successfully constructed, and the plasmid was named pEM-sdhCDAB.

[0073] 8. Overexpression of phosphoenolpyruvate carboxylase gene ppc and succinate dehydrogenase gene sdhCDAB

[0074] The two plasmids pCDR-ppc and pEM-sdhCDAB obtained above were electrochemically transformed into E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA) competent cells, and coated on a double-antibiotic plate containing streptomycin sulfate and ampicillin, and the obtained transformants were the genetically engineered E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA)-ppc-sdhCDAB of the application.

[0075] Example 2: Fed-batch fermentation in a fermenter to produce fumaric acid

[0076] E. coli FMME-N-5 and each recombinant strain constructed in Example 1 were respectively subjected to aerobic-oxygen-limited two-stage fermentation to produce fumaric acid.

[0077] LB medium, activation condition: 37℃ incubator for 12h. The fermentation medium contains: glucose 50g / L, Na2HPO3·5H2O 20mM, KHCO350mM, Na2HPO4·12H2O 15.11g / L, KH2PO43g / L NH4Cl 1g / L, NaCl 0.5g / L, trace element solution 1mL / L; trace element solution: FeCl3·6H2O 2.4g / L, CoCl2·6H2O 0.3g / L, CuCl2 0.15g / L, ZnCl2·4H2O 0.3g / L, NaMnO4 0.3g / L, H3BO3 0.075g / L, MnCl2·4H2O 0.495g / L, solvent: 0.1M HCl. After activation, the single colony of each strain was inoculated into liquid LB medium, and the seed liquid was obtained by incubation at 37℃, 200rpm for 8h. The initial liquid volume of the fermenter was 4L, and the seed liquid was inoculated into the fermenter at a rate of 10%(v / v). The aerobic fermentation conditions were as follows: the culture temperature was 37℃, the rotation speed was controlled at 600rpm, the aeration rate was 1vvm, the ammonia water was used to control the pH at 7.0, and the dissolved oxygen was controlled at 20% level. When the cell concentration grew to OD 600 =45, the rotation speed was reduced to 100rpm, 800g / L glucose was added to control the concentration in the fermentation broth below 10g / L, and the pH was controlled at 6.3-6.5 by adding sodium carbonate. The fermentation period was 65h.

[0078] The yield of fumaric acid in the supernatant of the fermentation broth was detected by high performance liquid chromatography (HPLC). As shown in Table 1, the yield of fumaric acid of E. coli FMME-N-5 (△fumB△frdBC△aspA△ldhA)-ppc-sdhCDAB after fermentation was 45g / L( Figure 9 ), the yield of fumaric acid to glucose reached 0.52g / g, the production intensity was 0.69g / L / h, the yield of succinic acid as a byproduct accumulated in the fermentation was 0.89g / L, the yield of lactic acid was 0.86g / L, and the yield of acetic acid was 0.54g / L.

[0079] Table 1 Yield of fumaric acid

[0080]

[0081] Table 2 Sequences of gene knockout primers and overexpression primers

[0082]

[0083]

[0084] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is to be understood that the application is not limited to those precise embodiments, and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope or spirit of the application. Therefore, the scope of the present application should be limited only by the appended claims.

Claims

1. An engineered E. coli bacterial strain for producing fumaric acid, characterized in that, The E. coli engineering strain is knocked out of fumarase gene fumB, fumarate reductase gene frdBC, aspartase gene aspA and lactate dehydrogenase gene ldhA, and overexpresses phosphoenolpyruvate carboxylase gene ppc and succinate dehydrogenase gene sdhCDAB; The nucleotide sequence of the fumarase gene fumB is shown as SEQ ID NO. 1; the nucleotide sequence of the fumarate reductase gene frdBC is shown as SEQ ID NO. 2; the nucleotide sequence of the aspartase gene aspA is shown as SEQ ID NO. 3; the nucleotide sequence of the lactate dehydrogenase gene ldhA is shown as SEQ ID NO. 4; the nucleotide sequence of the phosphoenolpyruvate carboxylase gene ppc is shown as SEQ ID NO. 5; and the nucleotide sequence of the succinate dehydrogenase gene sdhCDAB is shown as SEQ ID NO. 6; The host of the E. coli engineering strain is E. coli FMME-N-5, and the preservation number is CCTCC NO: M20200454.

2. The E. coli engineered strain of claim 1, characterized in that, The phosphoenolpyruvate carboxylase gene ppc is overexpressed by the vector pCDR, and the succinate dehydrogenase gene sdhCDAB is overexpressed by the vector pEM.

3. A method of constructing the engineered E. coli strain of claim 1 or 2, characterized in that, The steps of the method are: (1) respectively construct the gene knockout frame of fumB, frdBC, aspA and ldhA, and sequentially transfer the gene knockout frame fragments into the host bacteria with pKD46 plasmid to obtain the strain with the target gene knocked out; (2) amplify the gene fragments of phosphoenolpyruvate carboxylase gene ppc and succinate dehydrogenase gene sdhCDAB; connect the gene ppc to the vector pCDR, and connect the gene sdhCDAB to the vector pEM, and then transfer the expression vector with the gene fragments connected into the strain of step (1) to obtain the E. coli engineering strain.

4. Use of the E. coli engineered strain according to claim 1 or 2 for the production of fumaric acid, characterized in that, The application is to use the E. coli engineering strain to carry out aerobic-limited oxygen two-stage fermentation in a fermentation medium to obtain a fermentation liquor containing fumaric acid, and the aerobic-limited oxygen two-stage fermentation includes the following stages: (1) aerobic stage: maintain the ventilation amount at 0.5-2vvm, and the stirring speed at 500-700rpm; (2) Oxygen-limited stage: when the OD 600 of the bacteria is 40-50, the aeration rate is changed to 0.1-0.3 vvm and the stirring speed is 50-200 rpm.

5. Use according to claim 4, characterized in that, The inoculation amount of the aerobic stage is 5-10% by volume percentage.

6. Use according to claim 5, characterized in that, The dissolved oxygen in the aerobic stage is controlled at 20-30%.

7. Use according to claim 6, characterized in that, The glucose concentration is controlled at 5-10g / L in the limited oxygen stage.

8. Use according to claim 7, characterized in that, The fermentation temperature is 34-38℃.

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