CN116103213A

CN116103213A - Method for producing fumaric acid by metabolic engineering of escherichia coli

Info

Publication number: CN116103213A
Application number: CN202310047372.9A
Authority: CN
Inventors: 刘立明; 孟欣; 陈修来; 刘佳; 高聪; 郭亮; 宋伟; 吴静
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-01-31
Filing date: 2023-01-31
Publication date: 2023-05-12

Abstract

The invention discloses a method for producing fumaric acid by metabolic engineering of escherichia coli, and belongs to the field of fermentation. The invention knocks out fumaric acid enzyme gene, fumaric acid reductase gene, aspartic acid enzyme gene and lactic acid dehydrogenase gene in E.coli FMME-N-5, and overexpresses phosphoenolpyruvate carboxylase gene and succinic acid dehydrogenase encoding gene. The strain E.coli FMME-N-5 (delta fumB delta frdBC delta aspA delta ldhA) -ppc-sdhCDA is used for fermenting and producing fumaric acid, the fumaric acid is fermented for 65 hours on a 7.5L fermentation tank, the yield of the fumaric acid reaches 45g/L, the production intensity is 0.69g/L/h, the yield of the fumaric acid to glucose is 0.52g/g, the by-product formic acid does not accumulate, and the accumulation amount of succinic acid, lactic acid and acetic acid is lower than 1g/L, so that the strain has certain industrial production potential.

Description

Method for producing fumaric acid by metabolic engineering of escherichia coli

Technical Field

The invention relates to a method for producing fumaric acid by metabolic engineering of escherichia coli, belonging to the field of fermentation engineering.

Background

Fumaric acid is an important intermediate for fine chemical products. Fumaric acid has a special structure, can generate various chemical reactions, such as esterification, hydrogenation, isomerization and the like, and is widely applied to the fields of chemicals, foods, medicines and the like. In the production of chemicals, unsaturated polyester produced by taking fumaric acid as a raw material has very high chemical corrosion resistance and heat resistance; the lubricant pour point depressant with good performance can be produced by using fumaric acid ester, styrene and the like as raw materials. In food production, fumaric acid is an important food additive, and sodium salt prepared by reacting fumaric acid with sodium hydroxide is a commonly used sour regulator. Food-grade fumaric acid is weakly acidic and is widely used for preparing candies, beverages, jellies and the like. In the pharmaceutical field, fumaric acid and fumaric acid esters are used as important intermediates and precursors for the production of pharmaceuticals, in the fields of pharmaceutical production, cancer research, neuroimmunology and the like. Fumaric acid is a high-quality nano drug carrier component, and the drug carried by the fumaric acid has long slow release period and stable drug effect and has remarkable inhibiting effect on a plurality of cancer diseases. Fumaric acid has been listed by the U.S. department of energy as one of the ten major framework compounds of preferential development value.

Fumaric acid can be produced by malic acid isomerisation, which can be obtained by conversion of maleic anhydride, which can be obtained by sequential catalytic oxidation of gaseous hydrocarbon compounds. This manner of isomerising malic acid to fumaric acid is limited by the equilibrium of the reaction. The conversion is carried out at high temperatures, by-products are produced, the yield of which is lower than the equilibrium yield. The maleic acid isomerase can catalyze the conversion of malic acid into fumaric acid, no other byproducts exist in the balanced product, and the highest conversion rate of fumaric acid can reach 95%. The microbial fermentation method for producing fumaric acid is a promising production direction for replacing chemical methods based on petrochemical raw materials. Rhizopus oryzae, rhizopus arrhizus, rhizopus nigrus and the like can realize high yield and high production strength of fumaric acid, and the yield and the production strength can reach 0.85g/g and 4.25g/L/h. However, too large a pellet of rhizopus during fermentation affects dissolved oxygen to limit yield; and genetic manipulation tools for engineering rhizopus are relatively lacking. In recent years, with the diversity and high efficiency of development of gene editing tools, researchers have begun to increase fumaric acid in microorganisms of the E.coli, yeast and other modes by means of metabolic engineering strategies.

At present, the efficiency of producing fumaric acid by escherichia coli fermentation is low, byproducts such as lactic acid, formic acid, acetic acid and the like are usually present in fermentation liquid, and the problems of unbalanced metabolism, low key enzyme activity in fumaric acid production and the like caused by the fact that the products are decomposed and utilized in the fermentation process and the glucose and glucose which are substrates with high concentration cannot be tolerated are absorbed too fast; in order to obtain high-performance production strains, a method combining traditional breeding means, various histology analyses and molecular biology transformation is generally required. At present, a certain effect is achieved by improving the yield of fumaric acid through a molecular modification means, and conventional modification involves knocking out a fumaric acid enzyme gene fumABC, a fumaric acid reductase gene frdABCD, an aspA gene encoding aspartic acid and the like so as to block downstream decomposition of fumaric acid. And, the citric acid synthase encoding gene cs, genes carbb and argI involved in urea cycle, and the like are overexpressed to enhance fumaric acid accumulation. The reported highest yield of fumaric acid produced by escherichia coli fermentation is 41.5g/L, the production strain overexpresses a phosphoenolpyruvate carboxylase coding gene ppc and a glyoxylate cycle operon aceBA on the basis of knocking out a fumABC gene, glycerol is used as a fermentation substrate, the production strength is 0.51g/L/h, the yield is 0.44g/g, a large amount of byproducts are accumulated in the fermentation process, and the accumulation amount of acetic acid reaches 8.7g/L. How to avoid accumulation of fermentation byproducts while improving the yield of fumaric acid is a technical problem to be solved at present.

Disclosure of Invention

In order to solve the problems, the invention provides an escherichia coli engineering strain for efficiently producing fumaric acid, which adopts a Red homologous recombination method to knock out a fumaric acid enzyme gene (fumB), a fumaric acid reductase gene (frdBC), an aspartase gene (aspA) and a lactic acid dehydrogenase gene (ldhA) coded in host escherichia coli FMME-N-5, and overexpresses a phosphoenolpyruvate carboxylase gene (ppc) and a succinic dehydrogenase gene (sdhCDAB) for enhancing electron transfer and succinic acid conversion fumaric acid, thereby realizing the efficient production of the fumaric acid under the oxygen-limited condition.

The first object of the invention is to provide an escherichia coli engineering strain for efficiently producing fumaric acid. The engineering strain of the escherichia coli knocks out one or more of fumB, frdBC, aspA and ldhA of the escherichia coli, and overexpresses the phosphoenolpyruvate carboxylase gene ppc and the sdhCDB of the enhanced electron transfer, which are derived from the engineering strain of the high-yield succinic acid in the research laboratory.

Further, the nucleotide sequence of the fumB gene is shown as SEQ ID NO. 1; the nucleotide sequence of the fumaric acid reductase gene frdBC is shown as SEQ ID NO. 2; the nucleotide sequence of aspA gene aspA is shown in SEQ ID NO. 3; the nucleotide sequence of the lactate dehydrogenase gene ldhA is shown in 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 succinic dehydrogenase gene sdhCDAB is shown as SEQ ID NO. 6.

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

Further, the host of the E.coli engineering strain is E.coli FMME-N-5, which is preserved in China center for type culture Collection (China university of Wuhan, china, with a preservation number of CCTCCNO: M20200454) in the year 2020, and is disclosed in patent CN 112239738B.

The second object of the invention is to provide a construction method of the escherichia coli engineering strain, which comprises the following steps:

constructing gene knockout frame fragments of fumB, frdBC, aspA and ldhA respectively; sequentially transferring the gene knockout frame fragments into host bacteria with a pKD46 plasmid, and screening to obtain a strain for knocking out a target gene;

amplifying to obtain gene fragments of a phosphoenolpyruvate carboxylase gene ppc and a succinic dehydrogenase gene sdhCDAB; and (3) respectively connecting the gene segments to corresponding expression vectors, and transferring the expression vectors connected with the gene segments into the strain in the step (1) to obtain the recombinant escherichia coli.

The third object of the invention is to provide the application of the escherichia coli engineering bacteria in the aspect of producing fumaric acid by fermentation. The application is that the escherichia coli engineering strain is adopted to perform aerobic-oxygen limiting two-stage fermentation in a fermentation medium to obtain fermentation liquor containing fumaric acid.

Further, the fermentation medium used for the fermentation contains 40-50g/L glucose and Na ₂ HPO ₃ ·5H ₂ O20-50mM，KHCO ₃ 30-50mM，Na ₂ HPO ₄ ·12H ₂ O15.11g/L、KH ₂ PO ₄ 3g/LNH ₄ Cl1g/L and NaCl0.5g/L, wherein each L of culture medium contains 1mL of trace element liquid; microelement liquid: feCl ₃ ·6H ₂ O2.4g/L、CoCl ₂ ·6H ₂ O0.3g/L、CuCl ₂ 0.15g/L、ZnCl ₂ ·4H ₂ O0.3g/L、NaMnO ₄ 0.3g/L、H ₃ BO ₃ 0.075g/L、MnCl ₂ ·4H ₂ O0.495g/L, dissolved in 0.1 MHCl.

Further, the aerobic-limited oxygen two-stage fermentation is performed in the OD of the thallus ₆₀₀ When=40-50, the aerobic phase is shifted to the limiting phase.

Further, the conversion of the aerobic stage to the oxygen limiting stage is carried out by adjusting the dissolved oxygen content to 50-60% or adding 10-20g/L bicarbonate.

Further, in the oxygen limiting stage, the glucose concentration is controlled to be 5-10g/L.

Further, the inoculum size of the aerobic-limited two-stage fermentation is 5-10% by volume percent; the fermentation temperature is 34-38 ℃.

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

Further, in the anaerobic stage, a pH neutralizer is added, wherein the pH neutralizer is Na ₂ CO ₃ 、K ₂ CO ₃ 、NaOH、KOH、CaCO ₃ 、MgCO ₃ One or more of the above materials are mixed.

Further, in the aerobic-anaerobic two-stage fermentation process, an osmotic pressure protective agent is added, wherein the osmotic pressure protective agent is one or a mixture of more of proline, methionine, betaine and cysteine.

In the invention, the phosphoenolpyruvate carboxylase ppc can catalyze phosphoenolpyruvic acid to produce oxalic acid acetic acid, promote the synthesis of a product precursor and have obvious beneficial effects on the main metabolic pathway of fumaric acid generation; the succinic dehydrogenase sdhCDA can catalyze the conversion of succinic acid into fumaric acid, and the yield of fumaric acid is greatly improved.

The invention has the beneficial effects that:

the invention adopts Red homologous recombination technology to knock out related enzymes which influence the encoding of byproducts generated by fumaric acid, including fumaric acid enzyme, fumaric acid reductase, aspartic acid enzyme and lactic acid dehydrogenase, which can obviously reduce the accumulation of byproducts while not influencing the growth speed of thalli, and is beneficial to the accumulation of fumaric acid; meanwhile, the phosphoenolpyruvate carboxylase is overexpressed and the succinate dehydrogenase is overexpressed, so that the yield of fumaric acid is effectively improved. The E.coli engineering strain E.coli FMME-N-5 (delta fumB delta frdBC delta aspA delta ldhA) -ppc-sdhCDA production strength and productivity are higher than those of the reported E.coli production strain by glucose, the yield of accumulated fumaric acid reaches 45g/L after fermentation for 65h, the yield of the fumaric acid is 0.52g/g glucose, and the production strength is 0.69g/L/h; meanwhile, the accumulation amount of byproducts in the fermentation process is obviously lower than that in the prior art, wherein formic acid is not accumulated, and the accumulation amounts of succinic acid, lactic acid and acetic acid are all lower than 1g/L, so that the method is favorable for industrial production of fumaric acid.

Drawings

FIG. 1 is a diagram of a knocked-out fumarate gene-verifying gel (lanes 1-4 are in parallel);

FIG. 2 is a diagram of a knock-out fumaric acid reductase gene-verifying gel (lanes 1-5 are in parallel);

FIG. 3 is a schematic diagram of a knockdown aspartase gene-verifying gel (lanes 1-4 are in parallel);

FIG. 4 is a map of the knockdown lactate dehydrogenase gene-verifying gel (lanes 1-5 are in parallel);

FIG. 5 is a diagram of a gene-validated gel for expression of phosphoenolpyruvate carboxylase (lanes 1-4 are in parallel);

FIG. 6 is a gel diagram of gene expression of succinate dehydrogenase (lanes 1-10 are in parallel experiments);

FIG. 7 is a map of a gene vector expressing phosphoenolpyruvate carboxylase;

FIG. 8 is a map of a gene vector expressing succinate dehydrogenase;

FIG. 9 shows the results of fed-batch fermentation of E.coli engineering strain E.coli FMME-N-5 (. DELTA.fumB. DELTA.frdBC. DELTA.aspA. DELTA.ldhA) -ppc-sdhCDA fermentor. Wherein # 1 is E.coliFMME-N-5;2# is E.coliFMME-N-5 (. DELTA.fumB); 3# is E.coliFMME-N-5 (ΔfumB ΔfrdBC); 4# is E.coliFMME-N-5 (ΔfumB ΔfrdBC ΔaspA); 5# is E.coliFMME-N-5 (ΔfumB ΔfrdBC ΔaspA ΔldhA); no.6 is E.coli FMME-N-5 (. DELTA.fumB. DELTA.frdBC. DELTA.aspA. DELTA.ldhA) -ppc-sdhCDA.

Detailed Description

The present invention will be further described with reference to specific examples, which are not intended to limit the invention so that those skilled in the art may better understand the invention and practice it.

Related nucleotide sequence information in the sequence table:

the sequence information of SEQ ID NO.1 is the nucleotide sequence of fumB of the fumaric acid enzyme gene;

the sequence information of SEQ ID NO.2 is the nucleotide sequence of the fumarase gene frdBC;

SEQ ID NO.3 sequence information is the nucleotide sequence of the aspA gene;

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

SEQ ID NO.5 sequence information is the nucleotide sequence of the phosphoenolpyruvate carboxylase gene ppc of high-yield succinic acid escherichia coli;

the SEQ ID NO.6 sequence information is the nucleotide sequence of a succinic dehydrogenase gene sdhCDAB derived from Paracoccus denitrificans;

measurement of cell concentration:

and (3) neutralizing a proper amount of fermentation liquor with 2mol/L hydrochloric acid, and expressing the cell density by using a light absorption value detected at a wavelength of 600nm of a spectrophotometer.

Determination of glucose:

pretreatment of fermentation liquor: taking fermentation liquor 12000r/min, centrifuging for 5min, and taking supernatant. Diluting to a proper multiple, and detecting the glucose concentration of the fermentation broth by using an M-100 biosensing analyzer.

Determination of organic acids:

high performance liquid chromatography: pretreatment of fermentation liquor: taking fermentation liquor 12000r/min, centrifuging for 5min, and taking supernatant. After dilution by a suitable factor, the yields of fumaric acid, succinic acid, formic acid, lactic acid, acetic acid were measured by High Performance Liquid Chromatography (HPLC). The instrument is a Waterse2695 reversed-phase high performance liquid chromatograph, and the chromatographic column adopts Bio-RadHPX87H; mobile phase 5mmol/LH ₂ SO ₄ The method comprises the steps of carrying out a first treatment on the surface of the The flow rate was set at 0.6mL/min; the detection wavelength was 210nm and the column temperature was 35 ℃.

Construction of the expression vector pCDR: the pCDR plasmid was a plasmid obtained by engineering the promoter region of pCDM4 (purchased from addgene, # 49796). The gene sequence of the pCDR plasmid is shown as SEQ ID NO.6 of the 6 th page of the sequence Listing of the patent CN 110951660B.

Construction of expression vector pEM: plasmid pEM is based on plasmid pETM6 using the T5 promoter of plasmid pQE-80l-kan and the multiple cloning site Amp ^R The f1 replication site is replaced. (see in particular page 4, paragraph 0055 and 0057 of the description part of patent CN113293120A, table 1, third line))。

Example 1: construction of the coliFMME-N-5 (DeltafumB DeltafrdBC DeltaaspA DeltaldhA) -ldhA-sdhDAB Strain

Construction of E.coli FMME-N-5 competent cells

Coli FMME-N-5 is a high-yield succinic acid strain constructed by the experiment and is preserved in China center for type culture Collection (China university of Wuhan, china with a preservation number of CCTCCNO: M20200454) in the year of 8 and 27 of 2020, and is disclosed in patent CN 112239738B. 100. Mu.L of the deposited bacterial liquid was transferred from the glycerol tube to 25mL/100mL of liquid LB, and the culture was activated at 37℃and 200rpm overnight. Transferring 800 mu L of activated bacterial liquid into 50mL/250mL of liquid LB for culturing for 1.5-2h, stopping culturing when the OD is 0.5-0.6, and transferring to an ice bath at 4 ℃ for 20min. The supernatant was then removed by centrifugation and 25ml of 0.1M NaCl was added ₂ Ice bath for 15-20 min. Centrifuging at 5000rpm for 5min, removing supernatant, adding 1mL30% glycerol and 0.1MCaCl ₂ Split charging into sterile EP tubes, each containing 100 μl, and storing at-40deg.C.

2. Knocking out fumB (fumB) of fumaric acid enzyme gene

The knockout primers QCUMB-F and QCUMB-R (Table 2) were designed based on the fuMB gene sequence of Escherichia coli MG1655 in NCBI database, and the knockout frame of fuMB was amplified using pKD4 plasmid as a template and gel was recovered. Remarks: the pKD4 plasmid contains two FRT sites (DNA sequences that can be folded by the action of a invertase to eliminate the DNA sequence between the FRT sites) between which is the gene encoding kanamycin (kan, the selection pressure for gene knockout), namely FRT-kan-FRT. In knocking out the gene, primers were designed to amplify a DNA fragment of FRT-kan-FRT. It should be noted that the two amplification primers designed contained 39-49bp upstream and downstream of the target gene, respectively, i.e., the DNA fragment we finally obtained was "39-49 bp upstream of the target gene-FRT-kan-FRT-39-49 bp downstream of the target gene", and this DNA fragment was called the knockout box of the target gene. The knockout cassette of fumB was introduced into e.coli fmme-N-5 competent cells containing pKD46 plasmid by electrotransformation (electrotransformation voltage and time 1800V and 5ms, respectively). Electrotransferred competent cells were plated on LB solid medium plates containing kanamycin (50 g/mL) and cultured upside down for 12-24h. After the plates developed single colonies, positive transformants were screened using the verification primers QcfumB-test-F and QcfumB-test-R (Table 2).

The pCP20 plasmid was transformed into positive transformants to eliminate kanamycin resistance gene, and verified using QCfumB-test-F and QCfumB-test-R primers, the size of the electrophoresis band of the successfully knocked-out transformants was 500bp, and the size of the electrophoresis band of the non-knocked-out control group was 2147bp (FIG. 2). The results showed that the fumB gene was successfully knocked out in FMME-N-5 to give E.coli FMME-N-5 (ΔfumB) strain.

3. Knocking out the fumaric acid reductase gene frdBC

The knockout primers QcfrdBC-F and QcfrdBC-R (Table 2) were designed based on the frdBC gene sequence of Escherichia coli MG1655 in NCBI database, and the frdBC knockout frame was amplified and gel recovered using the pKD4 plasmid as a template. The knockout cassette of fumB was introduced into competent cells of e.collfmme-N-5 (Δfumb) containing pKD46 plasmid by electrotransformation (electrotransformation voltage and time of 1800V and 5ms, respectively). Electrotransferred competent cells were plated on LB solid medium plates containing kanamycin (50 g/mL) and cultured upside down for 12-24h. After the plates had grown to a single colony, positive transformants were screened using the validation primers QcfrdBC-test-F and QcfrdBC-test-R (Table 2).

The pCP20 plasmid was transformed into positive transformants to eliminate kanamycin resistance gene, and verified using QCfrdBC-test-F and QCfrdBC-test-R primers, the size of the electrophoresis band of the successfully knocked-out transformants was 506bp, and the size of the electrophoresis band of the non-knocked-out control group was 1916bp (FIG. 3). The E.coli FMME-N-5 (ΔfumB ΔfrdBC) strain was obtained.

4. Knocking out aspartase gene aspA

The knock-out primers QCospA-F and QCospA-R (Table 2) were designed based on the aspA gene sequence of Escherichia coli MG1655 in NCBI database, and the knock-out box of aspA was amplified and gel recovered using the pKD4 plasmid as a template. The knockout cassette of aspA was introduced into E.coli FMME-N-5 (. DELTA.fumB.DELTA.frdBC) competent cells containing the pKD46 plasmid by electrotransformation (1800V and 5ms for electrotransformation voltage and time, respectively). Electrotransferred competent cells were plated on LB solid medium plates containing kanamycin (50 g/mL) and cultured upside down for 12-24h. After the plates developed single colonies, positive transformants were screened using the verification primers QCospA-test-F and QCospA-test-R (Table 2).

The pCP20 plasmid was transformed into positive transformants to eliminate kanamycin resistance gene, and verified using QCIA-test-F and QCIA-test-R primers, the size of the electrophoresis band of the successfully knocked-out transformants was 510bp, and the size of the electrophoresis band of the non-knocked-out control group was 1937bp (FIG. 5). The E.coli FMME-N-5 (ΔfumB ΔfrdBC ΔaspA) strain was obtained.

5. Knock-out lactate dehydrogenase gene ldhA

The knockout primers QCldhA-F and QCldhA-R (Table 2) were designed based on the ldhA gene sequence of Escherichia coli MG1655 in NCBI database, and the knockout frame of ldhA was amplified using pKD4 plasmid as a template and gel recovery was performed. The knock-out box of ldhA was introduced into E.coli FMME-N-5 (. DELTA.fumB. DELTA.frdBC. DELTA.aspA) competent cells containing the pKD46 plasmid by electrotransformation (electrotransformation voltage and time of 1800V and 5ms, respectively). Electrotransferred competent cells were plated on LB solid medium plates containing kanamycin (50 g/mL) and cultured upside down for 12-24h. After the plates developed single colonies, positive transformants were screened using the verification primers QCldhA-test-F and QCldhA-test-R (Table 2).

The pCP20 plasmid was transformed into positive transformants to eliminate the kanamycin resistance gene, and verified using QCldhA-test-F and QCldhA-test-R primers, the size of the electrophoresis band of the successfully knocked-out transformants was 500bp, and the size of the electrophoresis band of the non-knocked-out control group was 1490bp (FIG. 4). The E.coli FMME-N-5 (ΔfumB ΔfrdBC ΔaspA ΔldhA) strain was obtained.

6. Construction of expression vector pCDR-ppc

The phosphoenolpyruvate carboxylase gene ppc used in the culture is derived from engineering escherichia coli which produces succinic acid in high yield in the research laboratory, and genomic DNA of the engineering escherichia coli is extracted.

According to the published genome information sequences, primer pairs PCDR-ppc-F and PCDR-ppc-R (Table 2) are respectively designed, and the ppc gene is obtained by using the extracted genome DNA of actinobacillus succinogenes as a template and adopting a standard PCR amplification system and a standard PCR amplification program.

And (3) carrying out gel cutting recovery on the ppc obtained by PCR amplification by agarose gel nucleic acid electrophoresis, carrying out double enzyme cutting on the recovered product and an expression vector pCDR by using restriction enzymes KpnI and XhoI for 3 hours, carrying out gel recovery on the enzyme-cut product by agarose gel nucleic acid electrophoresis, respectively carrying out DNA and linearization plasmid sizes of 2652 and 3958bp, then carrying out overnight connection by using T4DNA ligase at 16 ℃, transforming into JM109 competent cells, picking single colonies, carrying out PCR verification by using PCDR-ppc-test-F and PCDR-ppc-test-R primers, sequencing positive transformants, comparing correctly, and proving that the construction of the expression vector is successful, and the plasmid is named pCDR-ppc.

7. Construction of the expression vector pEM-sdhCDAB

The succinic dehydrogenase gene sdhCDAB used in the present invention is derived from Paracoccus denitrificans, and genomic DNA of Paracoccus denitrificans is extracted.

According to the published genome information sequences, primer pairs pEM-sdhCBA-F and pEM-sdhCBA-R (Table 2) are respectively designed, and the sdhCBA gene is obtained by taking the genomic DNA of the extracted paracoccus denitrificans as a template and adopting a standard PCR amplification system and a standard PCR amplification program.

And (3) carrying out double enzyme digestion on the recovered product and an expression vector pEM by utilizing restriction enzymes BamHI and SacI respectively, carrying out gel recovery on the enzyme digestion product by adopting agarose gel nucleic acid electrophoresis, carrying out overnight connection at the temperature of T4DNA ligase of 3276bp and 5221bp respectively on DNA and linearization plasmid, transforming into JM109 competent cells, picking single colonies, carrying out PCR verification by using pEM-sdhCDA-test-F and pEM-sdhCDA-test-R primers, sequencing positive transformants, and comparing correctly, thereby proving that the expression vector is constructed successfully and the plasmid is named pEM-sdhCDA.

8. Overexpression of the phosphoenolpyruvate carboxylase gene ppc and the succinate dehydrogenase gene sdhCDAB

The two plasmids pCDR-ppc and pEM-sdhCDA obtained above are electrochemically transformed into E.coli FMME-N-5 (delta fumB delta frdBA delta ldhA) competent cells, and the competent cells are coated on a double-resistant plate containing streptomycin sulfate and ampicillin to obtain the transformant which is the escherichia coli genetic engineering bacterium E.coli FMME-N-5 (delta fumB delta frdBC delta aspA delta ldhA) -ppc-sdhCDA.

Example 2: fed-batch fermentation of fermentation tank to produce fumaric acid

E.coli FMME-N-5 and each recombinant strain constructed in example 1 were subjected to two-stage aerobic-anaerobic fermentation to produce fumaric acid.

Plate activation medium and activation culture conditions: the plate activation medium is LB medium, and the activation conditions are as follows: the incubator was inverted at 37℃for 12 hours. The fermentation medium used for fermentation contains: glucose 50g/L, na ₂ HPO ₃ ·5H ₂ O20mM、KHCO ₃ 50mM、Na ₂ HPO ₄ ·12H ₂ O15.11g/L、KH ₂ PO ₄ 3g/LNH ₄ Cl1g/L, naCl0.5g/L and trace element liquid 1mL/L; microelement liquid: feCl ₃ ·6H ₂ O2.4g/L、CoCl ₂ ·6H ₂ O0.3g/L、CuCl ₂ 0.15g/L、ZnCl ₂ ·4H ₂ O0.3g/L、NaMnO ₄ 0.3g/L、H ₃ BO ₃ 0.075g/L、MnCl ₂ ·4H ₂ O0.495g/L, 0.1MHCl as solvent. After each strain was activated on a plate, a single colony was picked up in a liquid LB seed medium, and cultured at 37℃for 8 hours at 200rpm to obtain a seed solution. The initial liquid loading amount of the fermentation tank is 4L, the seed liquid is inoculated into the fermentation tank according to the inoculation amount of 10% (v/v), and the fermentation conditions in the aerobic stage are as follows: the culture temperature is 37 ℃, the rotating speed is controlled at 600rpm, the ventilation amount is 1vvm, the pH value is controlled at 7.0, the dissolved oxygen in the whole aerobic stage is controlled at 20 percent, and when the concentration of the thalli grows to OD ₆₀₀ =45, conversion to oxygen-limited phase: the ventilation is reduced to 0.2vvm, the stirring rotation speed is 100rpm, 800g/L glucose is added, the feeding rate is controlled to control the concentration in the fermentation liquor to be lower than 10g/L, the pH=6.3-6.5 is controlled by sodium carbonate in the oxygen limiting stage, and the fermentation period is 65h.

The yield of fumaric acid in the supernatant of the fermentation broth was checked by High Performance Liquid Chromatography (HPLC). As shown in Table 1, E.coli FMME-N-5 (. DELTA.fumB. DELTA.frdBC. DELTA.aspA. DELTA.ldhA) -ppc-sdhCBA gave a yield of fumaric acid of 45g/L (FIG. 9), a yield of fumaric acid to glucose of 0.52g/g, a production strength of 0.69g/L/h, a yield of succinic acid as a by-product accumulated in the fermentation of 0.89g/L, a lactic acid yield of 0.86g/L and an acetic acid yield of 0.54g/L.

TABLE 1 fumaric acid yield

TABLE 2 Gene knockout primer and overexpressing primer sequences

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While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. An engineered strain of escherichia coli producing fumaric acid, characterized in that the engineered strain of escherichia coli is knocked out of fumB, frdBC, aspA and ldhA, and overexpresses ppc and sdhCDAB.

2. The escherichia coli engineering strain according to claim 1, wherein the nucleotide sequence of the fumB of the fumaric acid enzyme gene is shown as SEQ ID No. 1; the nucleotide sequence of the fumaric acid 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 in SEQ ID NO. 4; the nucleotide sequence of the phosphoenolpyruvate carboxylase gene ppc is shown as SEQ ID NO. 5; the nucleotide sequence of the succinic dehydrogenase gene sdhCDAB is shown in SEQ ID NO. 6.

3. The engineered strain of escherichia coli according to claim 2, wherein the phosphoenolpyruvate carboxylase gene ppc is overexpressed by the vector PCDR and the succinate dehydrogenase gene sdhCDAB is overexpressed by the vector pEM.

4. An engineered strain of escherichia coli as defined in any one of claims 1-3, wherein the host of the engineered strain of escherichia coli is escherichia coli FMME-N-5 with a collection number of cctccc No. M20200454.

5. A method of constructing the engineered strain of escherichia coli of claim 3, comprising the steps of:

(1) Respectively constructing gene knockout frames of fumB, frdBC, aspA and ldhA, sequentially transferring the gene knockout frame fragments into host bacteria with a pKD46 plasmid, and screening to obtain a strain for knocking out a target gene;

(2) Amplifying to obtain gene fragments of a phosphoenolpyruvate carboxylase gene ppc and a succinic dehydrogenase gene sdhCDAB; ligating the gene ppc to the vector PCDR, ligating the gene sdhCDAB to the vector pEM, and transferring the expression vector with the gene fragment into the strain of the step (1), thereby obtaining the E.coli engineering strain.

6. The use of the engineered strain of escherichia coli according to any one of claims 1-4 for producing fumaric acid, wherein the use is to perform aerobic-oxygen limited two-stage fermentation in a fermentation medium by using the engineered strain of escherichia coli to obtain a fermentation broth containing fumaric acid, the aerobic-oxygen limited two-stage fermentation comprising the following stages:

(1) And (3) an aerobic stage: maintaining ventilation of 0.5-2vvm and stirring rotation speed of 500-700rpm;

(2) Oxygen limiting stage: at the cell OD ₆₀₀ When the flow rate is 40-50, the ventilation rate is 0.1-0.3vvm, and the stirring speed is 50-200rpm.

7. The use according to claim 6, wherein the aerobic phase is inoculated in an amount of 5-10% by volume.

8. The use according to claim 6, wherein the dissolved oxygen in the aerobic stage is controlled to be 20-30%.

9. The use according to claim 6, characterized in that the glucose concentration is controlled to 5-10g/L during the oxygen limitation phase.

10. The use according to claim 6, wherein the fermentation temperature is 34-38 ℃.