Recombinant Escherichia coli for producing caffeic acid and its application

Abstract

The present invention discloses a recombinant Escherichia coli for producing caffeic acid and its application, belonging to the technical field of genetic engineering and bioengineering. The present invention heterologously expresses the tyrosine ammonia lyase FjTAL of Flavobacterium johnsonii and the endogenous hpaBC of Escherichia coli BL21 (DE3) in Escherichia coli, thereby realizing the synthesis of caffeic acid; and by overexpressing the tyrosine ammonia lyase TcTAL derived from Trichosporon dermatitidis, the accumulation of intermediates in the synthesis of caffeic acid is reduced; by knocking out the competitive pathway for the synthesis of tyrosine, a precursor of caffeic acid synthesis, strengthening the synthesis of cofactors and overexpressing polyphenol transporters, the yield of caffeic acid is significantly increased. The caffeic acid yield of the engineered strain constructed by the present invention can reach 775.7 mg / L, providing a new method for the industrial production of caffeic acid and its derivatives.

Description

Technical Field

[0001] The invention relates to a recombinant Escherichia coli for producing caffeic acid and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] Caffeic acid (3,4-dihydroxycinnamic acid) is a naturally occurring phenylpropanoid compound with numerous important medicinal properties. It is widely used as an antioxidant and anti-inflammatory agent. Furthermore, its derivative, rosmarinic acid, also possesses important antioxidant, antithrombotic, antibacterial, and antiviral activities.

[0003] The primary method for producing caffeic acid is plant extraction, but the process is energy-intensive and environmentally unfriendly. Chemical synthesis of caffeic acid typically uses 3,4-dihydroxybenzaldehyde and malonic acid as substrates, toluene as a solvent, and pyridine as a catalyst, which is environmentally unfriendly and produces low yields. Therefore, green, sustainable microbial fermentation methods are being considered for producing caffeic acid. The synthesis of caffeic acid can be divided into two stages: the first is the synthesis of tyrosine from glucose or glycerol via the glycolysis pathway, the pentose phosphate pathway, and the shikimate pathway; the second is the sequential deamination and hydroxylation of tyrosine to produce caffeic acid. While research has been conducted on the biosynthesis of caffeic acid, its yield remains limited by issues such as excessive intermediate accumulation, limited cofactor synthesis, and poor product tolerance. Therefore, how to synthesize caffeic acid biologically and achieve high yields is an urgent issue that needs to be addressed. Summary of the Invention

[0004] The present invention provides a recombinant Escherichia coli for synthesizing caffeic acid, wherein at least one of the following improvements is made to the starting strain of Escherichia coli:

[0005] (1) Expression of tyrosine ammonia lyase from Flavobacterium johnsoniae and endogenous 4-hydroxyphenylacetate-3-monooxygenase from Escherichia coli BL21(DE3);

[0006] (2) Expression of tyrosine ammonia lyase from Trichosporon cutaneum;

[0007] (3) Overexpression of FAD synthase from endogenous Escherichia coli BL21(DE3) and chorismate mutase from Zymomonas mobilis;

[0008] (4) Overexpression of efflux proteins from Escherichia coli.

[0009] In one embodiment, the recombinant E. coli also expresses a 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant aroG from E. coli.fbr , to relieve phenylalanine feedback inhibition; the gene aroG fbr The nucleotide sequence is shown in SEQ ID NO.3.

[0010] In one embodiment, the tyrosine ammonia lyase has an amino acid sequence shown in Genbank accession number: WP_012023194.1, and the nucleotide sequence of its encoding gene FjTAL is shown in SEQ ID NO.1; the nucleotide sequence of the 4-hydroxyphenylacetic acid-3-monooxygenase encoding gene hpaBC is shown in SEQ ID NO.2.

[0011] In one embodiment, the nucleotide sequence of the tyrosine ammonia lyase encoding gene TcTAL is shown as SEQ ID NO.4.

[0012] In one embodiment, the chorismate mutase has the amino acid sequence shown in Genbank Accession No.: AAA27684.1.

[0013] In one embodiment, the gene encoding the FAD synthetase is the ribF gene; the gene encoding the chorismate mutase is the tyrC gene; the nucleotide sequences of the ribF and tyrC genes are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0014] In one embodiment, the efflux protein has an amino acid sequence shown in Genbank accession number: ARH97076.1, and the nucleotide sequence of its encoding gene ycjP is shown in SEQ ID NO.7.

[0015] In one embodiment, there is an RBS shown in SEQ ID NO.8 between the FjTAL and hpaBC.

[0016] In one embodiment, the FjTAL and hpaBC genes are expressed from plasmid pACYCDuet or pCDFDuet-1.

[0017] In one embodiment, the aroG mutant gene aroG is expressed in plasmid pETDuet-1. fbr .

[0018] In one embodiment, the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) ΔtyrR with the tyrR gene knocked out as a host.

[0019] In one embodiment, the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) ΔtyrRΔcrrΔptsGΔpheA in which the genes tyrR, ptsG, crr and pheA are knocked out as a host; the genes ptsG, crr and pheA are the encoding genes of the glucose-specific phosphoenolpyruvate phosphate transport system IIBC component, IIA component and chorismate mutase, respectively.

[0020] The present invention also provides a method for producing caffeic acid, wherein the method utilizes the recombinant Escherichia coli to ferment and produce caffeic acid.

[0021] In one embodiment, the recombinant E. coli is inoculated into a fermentation medium and cultured at 37°C until the OD 600 The cell proliferation rate was 0.6±0.1, and IPTG was added to a final concentration of 0.1 mM. The cells were induced at 30°C and 220 r / min for at least 48 h.

[0022] In one embodiment, the culture time is 48-72 hours.

[0023] In one embodiment, the fermentation medium contains: glucose, glycerol, (NH4)2SO4, K2HPO4·3H2O, KH2PO4, MgSO4·7H2O, sodium citrate, vitamin B1, and yeast extract.

[0024] The present invention also provides the use of the recombinant Escherichia coli in producing caffeic acid and its derivatives.

[0025] In one embodiment, the derivatives include but are not limited to chlorogenic acid, rosmarinic acid, and caffeic acid phenethyl ester.

[0026] Beneficial effects:

[0027] (1) The present invention heterologously expresses the tyrosine ammonia lyase FjTAL of Flavobacterium johnsonii and the endogenous hpaBC of E. coli BL21(DE3) in E. coli to achieve higher yields of caffeic acid synthesis. On this basis, the connection mode and expression intensity of FjTAL and hpaBC are optimized to further increase the yield of caffeic acid.

[0028] (2) The present invention reduces the accumulation of intermediates in caffeic acid synthesis by overexpressing tyrosine ammonia lyase TcTAL derived from Trichosporon cutaneum.

[0029] (3) The present invention also enhances the synthesis of cofactor FAD by knocking out the competitive pathway for tyrosine synthesis and overexpressing endogenous ribF in Escherichia coli BL21 (DE3), thereby increasing the caffeic acid yield to 585.8 mg / L.

[0030] (4) The present invention also provides a new use of the endogenous efflux protein ycjP of Escherichia coli BL21(DE3) in increasing caffeic acid production. By overexpressing ycjP to promote caffeic acid efflux, the accumulation of caffeic acid is further increased, so that the accumulation of caffeic acid can reach 775.7 mg / L under shake flask fermentation conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the metabolic process of heterologous caffeic acid synthesis in Escherichia coli.

[0032] Figure 2 The caffeic acid chromatogram of Escherichia coli CA05 in fermentation medium (A) and the chromatogram of caffeic acid standard (B).

[0033] Figure 3 The graph shows the production of different recombinant E. coli in fermentation medium. DETAILED DESCRIPTION

[0034] (1) Culture medium

[0035] Seed culture medium (LB): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L; solid culture medium was supplemented with 2% (mass fraction) agar powder.

[0036] Fermentation medium: 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH₄)₂SO₄, 3 g / L K₂HPO₄·3H₂O, 2 g / L KH₂PO₄, 2 g / L MgSO₄·7H₂O, 1 g / L sodium citrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. 250 g / L of glucose was sterilized separately and mixed thoroughly before inoculation.

[0037] (II) PCR reaction system and amplification conditions: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 20 ng of template DNA, 25 μL of 2× Phanta Max Master Mix, and double-distilled water to 50 μL. Amplification conditions: 95°C pre-incubation for 3 min, followed by 30 cycles (95°C for 15 s, 55°C for 15 s, and 72°C for 15 s), followed by extension at 72°C for 10 min.

[0038] (III) Preparation of competent E. coli: Streak the BL21 (DE3) glycerol tube onto the corresponding LB plate and culture at 37°C overnight (about 12 hours). After 12 hours, pick out flat, round, and large-growing bacteria and inoculate them into a 50 mL shake flask containing 5 mL LB medium. Incubate at 37°C and 220 rpm for about 8-10 hours. Transfer the inoculum to a 250 mL conical flask containing 50 mL LB at 1% inoculum. Incubate at 37°C and 220 rpm for about 2 hours until the OD reaches 0.600 =0.6-0.8; transfer the bacterial solution to a 50 mL centrifuge tube and place on ice for about 10-15 minutes; centrifuge at 4000 rpm and 4°C for 5 minutes, and remove the supernatant; add 5 mL of solution A to resuspend; centrifuge at 4000 rpm and 4°C for 5 minutes, and remove the supernatant; add 5 mL of solution B to resuspend the bacteria, and aliquot into 100 μL portions and store at -80°C.

[0039] (IV) Transformation of E. coli: Thaw competent E. coli cells on ice; add 10 μL of the recombinant product to 100 μL of competent cells and let it stand on ice for 30 min; heat shock the culture in a 42°C water bath for 45 s and let it stand on ice for 2 min; add 1 mL of LB medium and shake the culture at 37°C, 220 rpm for 60 min; centrifuge at 4000 rpm for 2 min, remove 900 μL of the supernatant, resuspend the cells in the remaining medium, and spread them on plates with the corresponding resistance.

[0040] (V) Extraction of caffeic acid: After fermentation, 500 μL of the fermentation broth was added with an equal volume of methanol. The mixture was vigorously shaken and mixed, and then centrifuged at 14,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm organic phase filter membrane and the product was detected using a Shimadzu LC-20A high performance liquid chromatograph.

[0041] (VI) HPLC determination of caffeic acid: Chromatographic separation was performed using a Thermo Fisher C18 column (4.6 mm × 250 mm, 5 μm); the column oven temperature was set at 40°C; the injection volume was 10 μL; the mobile phases were: phase A: ultrapure water (added with 0.1% trifluoroacetic acid), phase B: acetonitrile (added with 0.1% trifluoroacetic acid); the total flow rate was 1 mL / min, and the elution mode was gradient elution: 0-10 min, phase B: 10-60%; 10-20 min, phase B: 60-80%; 20-22 min, phase B: 80-10%; 22-25 min, phase B: 10%; detector wavelength: 323 nm.

[0042] (VI) Strain information is shown in Table 1:

[0043] Table 1 Strains and genes involved in the present invention

[0044]

[0045] Example 1: Optimizing the connection mode of FjTAL and HpaBC to improve the synthesis efficiency of caffeic acid

[0046] With reference to the construction process of strain Nar-17LM1 in "Fermentation and Metabolic Pathway Optimization to De Novo Synthesize (2S)-Naringenin in Escherichia coli", the tyrR gene of Escherichia coli BL21(DE3) was knocked out to obtain E. coli BL21(DE3)ΔtyrR, which was used as the starting strain for synthesizing caffeic acid. The synthesized FjTAL sequence (nucleotide sequence shown in SEQ ID NO.1) was used as a template and the FjTAL fragment was PCR amplified with primer pair F1 / R1. The hpaBC fragment (nucleotide sequence shown in SEQ ID NO.2) was PCR amplified with primer pair F2 / R2 using the E. coli BL21(DE3) genome as a template. The pCDFDuet-1 vector was used as a template and primers FP1 / RP1 were used for amplification, and the product was purified. The fragment FjTAL, fragment hpaBC and vector pCDFDuet-1 backbone were recombined by the Gibson assembly method to obtain a recombinant vector. The recombinant vector was transformed into Escherichia coli JM109, and the plasmid was extracted and sequenced for verification to obtain the correct recombinant vector pCDFDuet-FjTAL-RBS-HpaBC, in which the RBS sequence between FjTAL and hpaBC was AAGGAGATATACC (as shown in SEQ ID NO.8).

[0047] Plasmid pMD-tyrA fbr -aroG fbr (published in "Fermentation and Metabolic Pathway Optimization to De Novo Synthesize (2S)-Naringenin in Escherichia coli") was used as a template and primer pair F3 / R3 was used to amplify aroG fbr The fragment (nucleotide sequence is shown in SEQ ID NO.3) was amplified using primers FP2 / RP2 using pETDuet-1 vector as template and the product was purified. fbr The recombinant vector was recombined with the vector pETDuet-1 backbone to obtain the recombinant vector, which was transformed into Escherichia coli JM109. The plasmid was extracted and sequenced to verify the correct recombinant vector pETDuet-aroG fbr The recombinant vectors pCDFDuet-FjTAL-RBS-HpaBC and pETDuet-aroG fbrThe engineered strain CA01 was transformed into Escherichia coli BL21 (DE3) ΔtyrR. The engineered strain was cultured in a seed medium at 37°C and 220 rpm for 12 hours to obtain a seed solution. The seed solution was then inoculated into a fermentation medium containing 50 μg / mL ampicillin, 50 μg / mL kanamycin, and 37 μg / mL chloramphenicol at a final concentration of 2%. After culturing at 37°C and 220 rpm for 3 hours (OD 600 The final concentration of IPTG was 0.1 mM. The synthesis of caffeic acid was induced and carried out at 30°C and 220 rpm for 48 h from the time of inoculation into the fermentation medium. Liquid chromatography confirmed that the engineered strain CA01 could produce caffeic acid. Figure 3 As shown, the caffeic acid production in the fermentation broth of CA01 was detected to be 99.1 mg / L.

[0048] Example 2: Optimizing FjTAL and HpaBC Strength to Improve Caffeic Acid Synthesis Efficiency

[0049] In order to optimize the copy number of the key genes FjTAL and hpaBC for caffeic acid synthesis, the pCDFDuet-FjTAL-RBS-HpaBC constructed in Example 1 of the recombinant vector was used as a template and primers F1 / R2 were used to amplify the fragment FjTAL-RBS-HpaBC; the pACYCDuet-1 vector was used as a template and primers FP1 / RP1 were used to amplify and purify the product. The fragment FjTAL-RBS-HpaBC and the vector pACYCDuet-1 backbone were recombined by the Gibson assembly method to obtain a recombinant vector, which was transformed into Escherichia coli JM109, the plasmid was extracted and sequenced to obtain the correct recombinant vector pACYCDuet-FjTAL-RBS-HpaBC. The recombinant vector pACYCDuet-FjTAL-RBS-HpaBC and the pETDuet-aroG constructed in Example 1 were combined. fbr The engineered strain CA05 was transformed into Escherichia coli BL21(DE3)ΔtyrR. The engineered strain was cultured in a seed medium at 37°C and 220 rpm for 12 hours to obtain a seed solution, which was then inoculated at a 2% inoculum into a fermentation medium containing a final concentration of 50 μg / mL ampicillin, 50 μg / mL kanamycin, and 37 μg / mL chloramphenicol. After culturing at 37°C and 220 rpm for 3 hours (OD 600 The final concentration of IPTG was 0.1 mM. The synthesis of caffeic acid was induced at 30°C and 220 rpm for 48 h from the time of inoculation into the fermentation medium. Figure 3As shown, the caffeic acid production in the fermentation broth of CA05 was detected to be 200.1 mg / L.

[0050] Table 2 Primer sequences used in this example

[0051]

[0052]

[0053] Example 3: Increasing the synthesis of the precursor tyrosine to increase caffeic acid production

[0054] Referring to the construction method of strain Nar-17LM3 in "Fermentation and Metabolic Pathway Optimization to De NovoSynthesize(2S)-Naringenin in Escherichia coli", the genes tyrR, crr, ptsG and pheA of Escherichia coli BL21(DE3) were knocked out to construct Escherichia coli BL21(DE3)ΔtyrRΔcrrΔptsGΔpheA.

[0055] In order to reduce the accumulation of intermediates in caffeic acid synthesis and increase the flux of caffeic acid synthesis, the synthesized TcTAL was used as a template (nucleotide sequence as shown in SEQ ID NO.4), and the primer pair F4 / R4 was used to amplify the fragment and purify and recover it. The fragment TcTAL and the pCDFDuet-1 backbone were recombined by the Gibson assembly method to obtain a recombinant vector, which was transformed into Escherichia coli JM109. The plasmid was extracted and sequenced to verify the correct recombinant vector pCDF-TcTAL. Then the recombinant vector pCDF-TcTAL, the recombinant plasmid pACYCDuet-FjTAL-RBS-HpaBC constructed in Example 2 and pETDuet-aroG fbr The engineered strain was transformed into Escherichia coli BL21 (DE3) ΔtyrRΔcrrΔptsGΔpheA to obtain recombinant Escherichia coli CA09. The engineered strain was cultured in a seed medium at 37°C and 220 rpm for 12 hours to obtain a seed solution, which was then inoculated into a fermentation medium containing a final concentration of 50 μg / mL ampicillin, 50 μg / mL kanamycin, and 37 μg / mL chloramphenicol at a 2% inoculum volume. After culturing at 37°C and 220 rpm for 3 hours (OD 600 0.6±0.1), IPTG was added to a final concentration of 0.1 mM, and the synthesis of caffeic acid was induced at 30°C and 220 r / min. The fermentation was carried out for 72 hours from the time of inoculation into the fermentation medium. Figure 3As shown, the caffeic acid production of CA09 was 380.8 mg / L.

[0056] Table 3 Primer sequences used in this example

[0057]

[0058] Example 4: Optimizing cofactor synthesis efficiency to promote caffeic acid synthesis

[0059] To promote the synthesis of the E. coli cofactor FAD, the ribF fragment (nucleotide sequence shown in SEQ ID NO. 5) was amplified using the E. coli BL21 (DE3) genome as a template with primer pair F5 / R5, and the fragment was purified. The recombinant vector pCDF-TcTAL constructed in Example 3 was used as a template, and the primer pair FP3 / RP3 was used to amplify and purify the fragment. The purified ribF fragment and the pCDF-TcTAL backbone were recombined by Gibson assembly to obtain a recombinant vector. The recombinant vector was transformed into E. coli JM109, and the plasmid was extracted and sequenced to obtain the correct recombinant vector pCDF-TcTAL-ribF.

[0060] In order to further relieve the feedback inhibition of tyrosine synthesis, the synthetic tyrC gene (nucleotide sequence shown in SEQ ID NO.6) was used as a template, primer pair F6 / R6 was used to amplify and purify the fragment tyrC; the recombinant plasmid pETDuet-aroG constructed in Example 2 was used. fbr As a template, primer pair FP4 / RP4 was used to amplify and purify the plasmid backbone fragment. The fragment tyrC and pETDuet-aroG were assembled by Gibson method. fbr The backbone fragments were recombined to obtain a recombinant vector, which was transformed into Escherichia coli JM109. The plasmid was extracted and sequenced to verify the correct recombinant vector pETDuet-aroG. fbr -tyrC.

[0061] The recombinant vectors pCDF-TcTAL-ribF, pETDuet-aroG fbr -tyrC and pACYCDuet-FjTAL-RBS-HpaBC constructed in Example 2 were transformed into Escherichia coli BL21 (DE3) ΔtyrRΔcrrΔptsGΔpheA to obtain recombinant Escherichia coli CA19. The engineered strain was cultured in a seed medium at 37°C and 220 r / min for 12 hours to obtain a seed solution, which was then transferred to a fermentation medium containing ampicillin at a final concentration of 50 μg / mL, kanamycin at a final concentration of 50 μg / mL, kanamycin at a final concentration of 37 μg / mL, and chloramphenicol at a final concentration of 37°C and 220 r / min. 6000.6±0.1), IPTG was added to a final concentration of 0.1 mM, and the synthesis of caffeic acid was induced at 30°C and 220 r / min. The fermentation was carried out for 72 hours from the time of inoculation into the fermentation medium. Figure 3 As shown, the caffeic acid production of CA19 was 585.8 mg / L.

[0062] Table 4 Primer sequences used in this example

[0063]

[0064] Example 5: Overexpression of efflux proteins to increase caffeic acid production

[0065] In order to reduce the cytotoxicity of caffeic acid and increase the efflux efficiency of caffeic acid, the Escherichia coli BL21 (DE3) genome was used as a template, and the primer pair F7 / R7 was used to amplify the fragment ycjP (nucleotide sequence shown in SEQ ID NO.7), and the fragment was purified; the pACYCDuet-FjTAL-RBS-HpaBC constructed in Example 2 of the recombinant vector was used as a template, and the primer pair FP5 / RP5 was used to amplify and purify the plasmid backbone fragment. The fragment ycjP and the pACYCDuet-FjTAL-RBS-HpaBC backbone fragment were recombined by the Gibson assembly method to obtain a recombinant vector, which was transformed into Escherichia coli JM109, and the plasmid was extracted and sequenced to obtain the correct recombinant vector pACYCDuet-FjTAL-RBS-HpaBC-ycjP. Then the recombinant vector pACYCDuet-FjTAL-RBS-HpaBC-ycjP and the pETDuet-aroG constructed in Example 4 were combined. fbr -tyrC and pCDF-TcTAL-ribF were transformed into Escherichia coli BL21 (DE3) ΔtyrRΔcrrΔptsGΔpheA to obtain recombinant Escherichia coli CA24. The engineered strain was cultured in a seed medium at 37°C and 220 r / min for 12 hours to obtain a seed solution, which was then inoculated into a fermentation medium containing a final concentration of 50 μg / mL ampicillin, 50 μg / mL kanamycin, and 37 μg / mL chloramphenicol at a 2% inoculum volume. After culturing at 37°C and 220 r / min for 3 hours, the OD 600 0.6±0.1), IPTG was added to a final concentration of 0.1 mM, and the synthesis of caffeic acid was induced at 30°C and 220 r / min. The fermentation was carried out for 72 hours from the time of inoculation into the fermentation medium. Figure 3 As shown, the caffeic acid production of CA24 was 775.7 mg / L.

[0066] All primer sequences are listed in Table 5.

[0067] Table 5 Primer sequences used in this example

[0068]

[0069] Comparative Example 1:

[0070] The specific implementation method is the same as Example 1, except that FjTAL and hpaBC were connected to the plasmid pCDFDuet-1 in the order of hpaBC-RBS-FjTAL to construct the recombinant plasmid pCDF-hpaBC-RBS-FjTAL. Fermentation was carried out under the same conditions as Example 1. The results showed that the caffeic acid production of the constructed recombinant bacteria was only 36.0 mg / L.

[0071] Comparative Example 2:

[0072] The specific implementation method is the same as Example 1, except that FjTAL and hpaBC were connected to the plasmid pCDFDuet-1 with the connecting peptide GGGS. The fermentation was carried out under the same conditions as Example 1. The results showed that the caffeic acid yield of the constructed recombinant bacteria was only 50.0 mg / L.

[0073] Comparative Example 3:

[0074] The specific implementation method is the same as Example 5, except that the fragments ycjP are replaced with the endogenous aaeXAB genes encoding the p-hydroxybenzoic acid efflux system of Escherichia coli BL21 (DE3) (nucleotide sequence shown in SEQ ID NO. 9). The results showed that the caffeic acid yield was 419.4 mg / L.

[0075] Although the present invention has been disclosed above in terms of preferred embodiments, 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 synthesizing caffeic acid, characterized in that: The following improvements were made to the starting strain of E. coli: (1) Expression of tyrosine ammonia lyase FjTAL from Flavobacterium johnsoniae and endogenous 4-hydroxyphenylacetic acid-3-monooxygenase hpaBC from Escherichia coli BL21(DE3); (2) Expression of tyrosine ammonia lyase TcTAL from Trichosporon cutaneum; (3) Overexpression of FAD synthase from Escherichia coli and chorismate mutase from Zymomonas mobilis; (4) expressing a 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant from Escherichia coli; a gene encoding the 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant, aroG fbr The nucleotide sequence is shown in SEQ ID NO.3; (5) Overexpression of an efflux protein derived from Escherichia coli; the amino acid sequence of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsonii is shown in Genbank accession number: WP_012023194.1; the gene encoding the 4-hydroxyphenylacetic acid-3-monooxygenase hpaBC is shown in SEQ ID NO.2; the nucleotide sequence encoding the tyrosine ammonia lyase TcTAL derived from Trichosporon dermatitidis is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the FAD synthetase is shown in SEQ ID NO.5; the amino acid sequence of the chorismate mutase is shown in Genbank accession number: AAA27684.1; the amino acid sequence of the efflux protein is shown in Genbank accession number: ARH97076.

1.

2. The recombinant Escherichia coli according to claim 1, characterized in that There is an RBS shown in SEQ ID NO. 8 between the tyrosine ammonia lyase gene FjTAL and the 4-hydroxyphenylacetic acid-3-monooxygenase gene hpaBC.

3. The recombinant Escherichia coli according to claim 1, characterized in that The FjTAL and hpaBC genes were expressed using plasmid pACYCDuet or pCDFDuet-1.

4. The recombinant Escherichia coli according to claim 2, characterized in that The FjTAL and hpaBC genes were expressed using plasmid pACYCDuet or pCDFDuet-1.

5. The recombinant Escherichia coli according to any one of claims 1 to 4, characterized in that The aroG mutant gene aroG was expressed in plasmid pETDuet-1. fbr .

6. The recombinant Escherichia coli according to any one of claims 1 to 4, characterized in that The recombinant Escherichia coli is based on Escherichia coli BL21 (DE3) ΔtyrR in which the tyrR gene is knocked out; or The starting strain was Escherichia coli BL21(DE3)ΔtyrRΔcrrΔptsGΔpheA, in which tyrR, ptsG, crr and pheA were knocked out.

7. The recombinant Escherichia coli according to claim 5, characterized in that The recombinant Escherichia coli is based on Escherichia coli BL21 (DE3) ΔtyrR in which the tyrR gene is knocked out; or The starting strain was Escherichia coli BL21(DE3)ΔtyrRΔcrrΔptsGΔpheA, in which tyrR, ptsG, crr and pheA were knocked out.

8. A method for producing caffeic acid, characterized in that: Caffeic acid is produced by fermentation using the recombinant Escherichia coli according to any one of claims 1 to 7.

9. The method according to claim 8, characterized in that The recombinant E. coli was inoculated into the fermentation medium and cultured until OD 600 The concentration of glutathione was 0.5-0.7, IPTG was added for induction, and culture was carried out for at least 48 hours.

10. Use of the recombinant Escherichia coli according to any one of claims 1 to 7 in producing caffeic acid.

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