Recombinant bacterium for efficiently synthesizing caffeic acid as well as construction method and application of recombinant bacterium

Abstract

The invention discloses a recombinant bacterium for efficiently synthesizing caffeic acid as well as a construction method and application of the recombinant bacterium, and relates to the technical field of genetic engineering. It is found that fre, mdaB, hisP and gltL genes in tyrosine producing bacteria are specifically reduced or knocked out, then caffeic acid synthetic pathway enzyme expression vectors are transferred, the production capacity of caffeic acid of the strains can be remarkably improved, the production capacity can be improved by 11.5%-20.4% on the original basis, and when the tyrosine producing bacteria are applied to caffeic acid production, the yield of caffeic acid can be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a recombinant bacterium that efficiently synthesizes caffeic acid, its construction method, and its application. Background Technology

[0002] Caffeic acid (3,4-dihydroxycinnamic acid), an important natural polyphenol, is widely found in coffee beans, apples, olives, and various medicinal plants. It has attracted considerable attention due to its multiple biological activities, including antioxidant, anti-inflammatory, anticancer, and antibacterial properties. Furthermore, caffeic acid derivatives such as caffeic acid phenethyl ester (CAPE) show great potential in pharmacology due to their higher bioavailability.

[0003] In related technologies, caffeic acid is mainly derived from natural extraction and chemical synthesis, but it is expensive. Based on this, researchers have proposed using engineered microorganisms to synthesize caffeic acid de novo or via enzyme-catalyzed precursor synthesis, using glucose as a starting material. For example, using *Saccharomyces cerevisiae* as the starting strain, caffeic acid can be integrated into its genome. RgTAL Gene, HpaB Genes and HpaC Genes, and regulate steps that restrict precursor synthesis within the cell (e.g., knockout). Aro3 and / or Aro10 By using genetic engineering, stable and high-yielding brewer's yeast strains can be obtained, enabling one-step synthesis of glucose into caffeic acid without the need for additional exogenous precursors. However, in practical applications, the yield of caffeic acid still falls short of actual demand.

[0004] Therefore, there is an urgent need to find a recombinant bacterium that can efficiently synthesize caffeic acid in order to further increase caffeic acid production. Summary of the Invention

[0005] The first aspect of this invention aims to provide a method for specifically reducing or knocking out [certain features]. fre , mdaB , hisP and gltL Application of substances containing at least one gene in the fermentation synthesis of caffeic acid by tyrosine-producing bacteria.

[0006] A second aspect of the present invention is to provide a recombinant bacterium.

[0007] The third aspect of this invention aims to provide a method for constructing recombinant bacteria.

[0008] The fourth aspect of this invention aims to provide the application of recombinant bacteria in the preparation of caffeic acid.

[0009] The fifth aspect of this invention is to provide a method for preparing caffeic acid.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides the use of a substance that specifically reduces or knocks out at least one gene from A1 to A4 in the fermentation synthesis of caffeic acid by tyrosine-producing bacteria, wherein: A1) NAD(P)H-dependent flavin reductase gene; A2) NADPH-dependent quinone reductase gene; A3) Genes of histidine / lysine / arginine / ornithine transport proteins that bind to ATP; A4) Gene for ATP-binding glutamate / aspartate transport protein.

[0011] According to some embodiments of the present invention, the NAD(P)H-dependent flavin reductase gene (NAD(P)H-flavinreductase, abbreviated as NAD(P)H) is used for... fre The Locus tag for the gene is b3844.

[0012] According to some embodiments of the present invention, the NADPH-dependent quinone reductase gene (NADPH: quinone oxidoreductase) MdaB , referred to as MdaB The Locus tag for the gene is b3028.

[0013] According to some embodiments of the present invention, the ATP-binding histidine / arginine / ornithine ABC transporter / histidine ABC transporter, ATP-binding subunit, is referred to as... hisP The Locus tag for the gene is b2306.

[0014] According to some embodiments of the present invention, the ATP-binding glutamate / aspartate ABC transporter ATP-binding subunit (abbreviated as...) gltL The Locustag for the gene is b0652.

[0015] It is understood that the Locus tag is a gene identifier that can be queried on the NCBI website (https: / / www.ncbi.nlm.nih.gov / gene / ).

[0016] According to some embodiments of the present invention, the specificity reduction or knockout freGenetic substances include targeted fre The sgRNA of the gene. Preferably, the sgRNA comprises a nucleotide sequence as shown in SEQ ID NO: 7.

[0017] According to some embodiments of the present invention, the specificity reduction or knockout mdaB Genetic substances include targeted mdaB The sgRNA of the gene. Preferably, the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 14.

[0018] According to some embodiments of the present invention, the specificity reduction or knockout hisP Genetic substances include targeted hisP The sgRNA of the gene. Preferably, the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 21.

[0019] According to some embodiments of the present invention, the specificity reduction or knockout gltL Genetic substances include targeted gltL The sgRNA of the gene. Preferably, the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 28.

[0020] According to some embodiments of the present invention, the tyrosine-producing bacteria express an enzyme in the caffeic acid synthesis pathway.

[0021] According to some embodiments of the present invention, the caffeic acid synthesis pathway enzyme includes at least one of tyrosine lyase and 3-hydroxylase.

[0022] According to some embodiments of the present invention, the 3-hydroxylase is selected from Pseudomonas aeruginosa (… Pseudomonas aeruginosa PaHpaB (GenBank PKG21040.1) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa PaHpaC (GenBank PKG21041.1), Enterobacter hopterii ( Enterobacter hormaechei EhHpaC (GenBank PJG38871.1), luminescent bacteria ( Photorhabdus luminescens ) PlHpaC (GenBank AAO17198.1), Salmonella Enterobacter ( Salmonella enterica SeHpaC (GenBank GAR62209.1), Burkholderia cepacia ( Chitinasiproducens palmae CpHpaC (GenBank SDV46364.1), Escherichia coli EcHpaC (GenBank ACT46002.1), or Klebsiella pneumoniae ( Klebsiella pneumoniaeIt is a heterozygote of KpHpaC (GenBank CDO16164.1).

[0023] According to some embodiments of the present invention, the 3-hydroxylase is a hybrid of PaHpaB or its mutant and SeHpaC.

[0024] According to some embodiments of the present invention, the PaHpaB mutant is a mutant in which Ala at position 211 is replaced by Trp (A211W), Ser at position 210 is replaced by Ala (S210A), or Ser at position 210 is replaced by Gly (S210G).

[0025] According to some embodiments of the present invention, the tyrosine-producing bacterium is Escherichia coli.

[0026] According to some embodiments of the present invention, the *Escherichia coli* is any one of K12-derived bacteria, BL21(DE3) bacteria, BL21(DE3)-derived bacteria, BREL606 bacteria, W bacteria, or DH1 bacteria. Tyrosine-producing *Escherichia coli* is particularly preferred.

[0027] According to some embodiments of the present invention, the overexpression of Cra is achieved by any of B1) to B5): B1) Introduce a vector containing the coding gene of Cra into the starting bacteria; B2) Increase the copy number of the gene encoding Cra on the chromosome; B3) Alter the promoter sequence of the gene encoding Cra on the chromosome; B4) The strong promoter is operatively linked to the coding gene of Cra; B5) Change the nucleotide sequence encoding the Cra.

[0028] According to some embodiments of the present invention, B1) the vector is an expression vector. The expression vector is capable of normal replication and expression in host cells.

[0029] According to some embodiments of the present invention, the vectors include, but are not limited to, pETDuet series expression vectors, pACYCDuet series expression vectors, pRSFDuet series expression vectors, pCOLADuet series expression vectors, or pCDFDuet series expression vectors.

[0030] According to some embodiments of the present invention, B3) includes replacing the original promoter of the gene encoding Cra on the chromosome with a strong promoter.

[0031] According to some embodiments of the present invention, the strong promoter includes at least one of the P37 promoter, P8 promoter, J23100 promoter, J23119 promoter, P21285 promoter, M1-93 promoter, and PL1118 promoter. The strong promoter can be recognized by the recombinant bacteria.

[0032] According to some embodiments of the present invention, the changes in B5) include codon optimization.

[0033] According to some embodiments of the present invention, the tyrosine-producing bacteria are E. coli TYR-14B1 (P cra ::P37).

[0034] A second aspect of the present invention provides a recombinant bacterium, said recombinant bacterium being obtained by knocking out or knocking down at least one gene among A1) to A4) starting from a tyrosine-producing bacterium, wherein: A1) NAD(P)H-dependent flavin reductase gene; A2) NADPH-dependent quinone reductase gene; A3) Genes of histidine / lysine / arginine / ornithine transport proteins that bind to ATP; A4) Gene for ATP-binding glutamate / aspartate transport protein.

[0035] According to some embodiments of the present invention, the tyrosine-producing bacterium is Escherichia coli.

[0036] According to some embodiments of the present invention, the tyrosine-producing bacteria are E. coli TYR-14B1 (P cra ::P37).

[0037] According to some embodiments of the present invention, the recombinant bacteria contains a caffeic acid synthesis pathway enzyme expression vector.

[0038] According to some embodiments of the present invention, the caffeic acid synthesis pathway enzyme expression vector includes the pZEA-RgTAL-PaHpaB-SeHpaC expression vector.

[0039] A third aspect of the present invention provides a method for constructing recombinant bacteria as described in the second aspect, comprising: Knock out or knock down at least one gene from A1 to A4 in the starting strain capable of tyrosine synthesis, and then transfer it into the caffeic acid synthesis pathway enzyme expression vector to obtain the product, wherein: A1) NAD(P)H-dependent flavin reductase gene; A2) NADPH-dependent quinone reductase gene; A3) Genes of histidine / lysine / arginine / ornithine transport proteins that bind to ATP; A4) Gene for ATP-binding glutamate / aspartate transport protein.

[0040] A fourth aspect of the invention provides the use of the recombinant bacteria as described in the second aspect in the preparation of caffeic acid.

[0041] A fifth aspect of the present invention provides a method for preparing caffeic acid, comprising inoculating a recombinant bacterium as described in the second aspect into a fermentation broth, and obtaining the caffeic acid after fermentation.

[0042] According to some embodiments of the present invention, the temperature of the fermentation treatment is 37±2℃.

[0043] According to some embodiments of the present invention, the fermentation treatment time is 24-96 hours.

[0044] According to some embodiments of the present invention, the fermentation broth contains 2-3 g / L yeast extract, 10-15 g / L Na2HPO4•7H2O, 1-4 g / L KH2PO4, 0.1-0.8 g / L NaCl, 0.5-1.5 g / L NH4Cl, 15-25 g / L glycerol, and 5-15 mL / L trace element solution.

[0045] According to some embodiments of the present invention, the trace element solution comprises: 8~12 g / L FeSO4•7H2O, 1~1.5 g / L CaCl2, 1.5~2.5 g / L ZnSO4•7H2O, 0.2~0.8 g / L MnSO4•4H2O, 0.5~1.5 g / L CuSO4•5H2O, and 0.05~0.15 g / L (NH4)6Mo7O. 24 • 4H2O, 0.1~0.3 g / L Na2B4O7•10H2O and 8~12 ml / L 35%HCl.

[0046] According to some embodiments of the present invention, the method further includes the step of separating and purifying caffeic acid from the fermentation broth obtained after the fermentation treatment.

[0047] The recombinant bacteria for efficient synthesis of caffeic acid, its construction method, and its application, as described in this invention, have at least the following beneficial effects: This invention discovered that knocking out tyrosine-producing bacteria... fre , mdaB , hisP or gltL Genes help to further increase caffeic acid production.

[0048] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This refers to the biosynthetic pathway of caffeic acid. Detailed Implementation

[0050] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0051] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0052] In the description of this invention, "and / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0053] In other embodiments where specific conditions are not specified, the procedures shall be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0054] Inventive concept: In previous studies, the inventors’ team used CRISPRa activation screening technology (Advanced Biotechnology 2024, 2:15) to activate and screen endogenous transcription factors in E. coli with the goal of improving E. coli’s tolerance to caffeic acid and its ability to synthesize caffeic acid, and identified the relevant genes. Figure 1 The biosynthetic pathway of caffeic acid was demonstrated.

[0055] Based on this, the present invention uses tyrosine-producing bacteria E. coli TYR-14B1 (P craUsing P37 (AdvancedBiotechnology 2024, 2:15) as the starting strain, different gene knockout strains were constructed. At the same time, the caffeic acid synthesis pathway plasmid pZEA-RgTAL-PaHpaB-SeHpaC (expressing RgTAL and PaHpaB / SeHpaC hybrids, AdvancedBiotechnology (2024) 2:15) was transformed into the above gene knockout strains, and the caffeic acid synthesis effect was tested to screen recombinant strains that can synthesize caffeic acid efficiently.

[0056] Example 1 fre gene knockout This embodiment uses tyrosine-producing bacteria. E. coli TYR-14B1 (P cra Using P37 (AdvancedBiotechnology 2024, 2:15) as the starting strain, a [system / mechanism] was constructed. fre Gene knockout strains, including those in this example. fre The sequences of the primers used for gene knockout are shown in Table 1.

[0057] Table 1:

[0058] This embodiment fre The specific experimental method for gene knockout is as follows.

[0059] 1. Build knockout fre sgRNA expression plasmid Using F1 / F2 primers and pTargetB plasmid (Fronters Microbiology 2018, 9:1623) as a template, PCR amplification was performed. The plasmid was digested with DpnI at 37 °C for 2 hours. The linearized plasmid was recovered and chemically transformed into... E. coli In DH5α, the sample was spread onto spectinomycin resistance plates and incubated overnight at 37 °C. Single colonies growing on the plate were picked, and the plasmid from these colonies was extracted; this constitutes the knockout. fre sgRNA expression plasmid pTargetB- fre .

[0060] 2. Obtain fre Target practice footage Using F2 / F3 as primers, E. coli Using DH5α as a template, the gene was amplified by PCR. fre The 500 bp upstream homologous arm used for knockout; using F4 / F5 as primers, E. coli Using DH5α as a template, the gene was amplified by PCR. freThe 500 bp downstream homologous arm used for knockout; using F3 / F6 as primers and the aforementioned upstream and downstream homologous arms as templates, PCR amplification yielded a 1000 bp [missing information]. fre Target practice footage.

[0061] 3. fre Gene knockout (1) Obtaining arabinose-induced strains: First, pCas9* was chemically converted into tyrosine-producing bacteria previously constructed in the laboratory. E. coli TYR-14B1(P cra In ::P37) (Advanced Biotechnology 2024, 2:15), a single strain was picked and inoculated into a test tube and cultured overnight at 30°C and 220 rpm.

[0062] The seed culture obtained from the overnight culture was then inoculated into a shake flask containing 50 mL of LB medium and cultured at 30 °C and 220 rpm until the OD600 reached 0.4–0.5. 0.5 mL of 1 M arabinose was added to the shake flask for induction, and the culture was continued at 30 °C and 220 rpm for 1 h to obtain the final product.

[0063] (2) Electroconversion: The induced bacterial culture was collected into centrifuge tubes to prepare electrocompetent cells. Then, 500 ng of the constructed pTargetB- fre Expression plasmid and 500 ng of the above fre The target fragment is simultaneously added to competent cells for electroconversion.

[0064] After electroconversion, the samples were first revived and cultured in a shaker at 30 °C and 220 rpm for 1 hour. Then, 10 μL of 1M arabinose was added to induce the reaction for 1 hour before being spread onto LB plates containing kanamycin and spectinomycin resistance. The plates were then incubated upside down in a 30 °C incubator for 24-36 h.

[0065] (3) Screening for positive clones: After the above culture, single colonies grown on the plates were selected for PCR verification. Then, the single colonies that were correctly verified by PCR were inoculated into 1 mL of LB medium, 5 μL of 0.5 M IPTG was added, and the culture was carried out overnight at 30 ℃ and 220 rpm.

[0066] Streak the overnight culture onto kanamycin-resistant LB agar plates and incubate inverted at 30 °C overnight. Then, simultaneously spot the overnight cultured single colonies onto LB agar plates containing kanamycin and spectinomycin, and incubate inverted at 30 °C overnight. Select single colonies that grow on kanamycin-resistant plates but not on kanamycin + spectinomycin-resistant plates (single colonies with pTargetF removed) and inoculate them into 1 mL of LB medium, incubating overnight at 37 °C and 220 rpm.

[0067] The bacterial culture that had been incubated overnight was streaked onto antibiotic-free LB agar plates and incubated upside down at 37 °C overnight. Then, single colonies from the overnight culture were simultaneously spotted onto both kanamycin-resistant and antibiotic-free LB agar plates and incubated upside down at 37 °C overnight.

[0068] Select single colonies that grow on antibiotic-free plates but not on kanamycin-resistant plates to obtain single colonies from which both pCas9* plasmid and pTargetB plasmid have been removed.

[0069] Example 2 mdaB gene knockout This embodiment uses tyrosine-producing bacteria. E. coli TYR-14B1 (P cra Using P37 (AdvancedBiotechnology 2024, 2:15) as the starting strain, a [system / mechanism] was constructed. mdaB Gene knockout strains, including those in this example. mdaB The sequences of the primers used for gene knockout are shown in Table 2.

[0070] Table 2:

[0071] This embodiment mdaB The gene knockout method was performed as described in Example 1, the difference being the primers used, where F7 / F8 were used for PCR amplification. mdaB The target sequence (N20) was used to construct an sgRNA expression plasmid; F9 / F10, F11 / F12, and F9 / F12 were used for PCR amplification of the upstream and downstream homologous arms and the targeting fragment, respectively.

[0072] The selected single colonies were validated, and the results were consistent with expectations. mdaB Gene knockout strain.

[0073] Example 3 hisP gene knockout This embodiment uses tyrosine-producing bacteria. E. coli TYR-14B1 (P craUsing P37 (AdvancedBiotechnology 2024, 2:15) as the starting strain, a [system / mechanism] was constructed. hisP Gene knockout strains, including those in this example. hisP The sequences of the primers used for gene knockout are shown in Table 3.

[0074] Table 3:

[0075] This embodiment hisP The gene knockout method was performed as described in Example 1, the difference being the primers used, where F13 / F14 were used for PCR amplification. hisP The target sequence was used to construct an sgRNA expression plasmid; F15 / F16, F17 / F18, and F15 / F18 were used for PCR amplification of the upstream and downstream homologous arms and the targeting fragment, respectively.

[0076] The selected single colonies were validated, and the results were consistent with expectations. hisP Gene knockout strain.

[0077] Example 4 gltL gene knockout This embodiment uses tyrosine-producing bacteria. E. coli TYR-14B1 (P cra Using P37 (AdvancedBiotechnology 2024, 2:15) as the starting strain, a [system / mechanism] was constructed. gltL Gene knockout strains, including those in this example. gltL The sequences of the primers used for gene knockout are shown in Table 4.

[0078] Table 4:

[0079] This embodiment gltL The gene knockout method was performed according to Example 1, the difference being the primers used, where F19 / F20 were used for PCR amplification. hisP The target sequence was used to construct an sgRNA expression plasmid; F21 / F22, F23 / F24, and F21 / F24 were used for PCR amplification of the upstream and downstream homologous arms and the targeting fragment, respectively.

[0080] The selected single colonies were validated, and the results were consistent with expectations. gltL Gene knockout strain.

[0081] Example 5 Construction of Recombinant Strains In this embodiment, the gene knockout strains obtained in Examples 1-4 above were used as recipient strains to construct different recombinant Escherichia coli strains that produce caffeic acid. Specific experiments are as follows: The caffeic acid synthesis pathway plasmid pZEA-RgTAL-PaHpaB-SeHpaC (expressing RgTAL and PaHpaB / SeHpaC hybrids, Advanced Biotechnology (2024) 2:15) was transformed into the gene knockout strains obtained in Examples 1-4 above and their originating tyrosine-producing bacteria, respectively. E. coli TYR-14B1 (P cra In ::P37) (Advanced Biotechnology (2024) 2:15), different recombinant Escherichia coli producing caffeic acid were constructed, namely TYR-14B1(P cra ::P37, ∆ fre Recombinant Escherichia coli, TYR-14B1 (P cra ::P37, ∆ mdaB Recombinant Escherichia coli, TYR-14B1 (P cra ::P37, ∆ hisP Recombinant Escherichia coli, TYR-14B1 (P cra ::P37, ∆ gltL Recombinant Escherichia coli and TYR-14B1 (P cra ::P37) Recombinant Escherichia coli.

[0082] Example of detection: This test example examines the ability of the recombinant Escherichia coli constructed above to synthesize caffeic acid. The specific method is as follows: The recombinant E. coli samples were inoculated onto LB agar plates, and fresh single colonies were picked and placed in 5 mL of LB liquid medium. The plates were incubated at 37°C and 200 rpm for approximately 14 h. A certain volume of the seed culture was then transferred to 50 mL of fermentation medium to initiate OD200. 600 It is 0.1.

[0083] Each liter of fermentation medium contains 2.5 g yeast extract, 12.8 g Na₂HPO₄•7H₂O, 3 g KH₂PO₄, 0.5 g NaCl, 1.0 g NH₄Cl, 20 g glycerol, 10 mL trace element solution, and the remainder water. Each liter of trace element solution contains: 10 g FeSO₄•7H₂O, 1.35 g CaCl₂, 2.2 g ZnSO₄•7H₂O, 0.58 g MnSO₄•4H₂O, 1 g CuSO₄•5H₂O, and 0.1 g (NH₄)₆Mo₇O. 24• 4H2O, 0.2 g Na2B4O7•10H2O and 35% (wt) HCl (10 ml). The prepared trace element solution was sterilized by filtration through a 2 μm filter.

[0084] The inoculated culture medium was fermented in a shaker at 37°C and 200 rpm for 72 h. Samples were taken, and the concentration of caffeic acid was determined by HPLC.

[0085] The results of the detection of caffeic acid synthesized by recombinant Escherichia coli are shown in Table 5.

[0086] Table 5:

[0087] In the table: * indicates P <0.01.

[0088] As shown in the table above, knockout fre , mdaB , hisP and gltL It has a significant effect on promoting the synthesis of caffeic acid, which can increase the yield of caffeic acid by 11.5% to 20.4%.

[0089] In summary, this invention provides a recombinant bacterium for efficient synthesis of caffeic acid, its construction method, and its application. Specifically, this invention uses tyrosine-producing bacteria... E. coli TYR-14B1 (P cra ::P37) (Advanced Biotechnology 2024, 2:15) was used as the starting strain, and different genes were constructed (such as...) fre , mdaB , hisP and gltL The gene knockout strain was transformed into the caffeic acid synthesis pathway plasmid pZEA-RgTAL-PaHpaB-SeHpaC (expressing RgTAL and PaHpaB / SeHpaC hybrids, Advanced Biotechnology (2024) 2:15), and the caffeic acid synthesis effect was detected. The results showed that the caffeic acid production could be increased by 11.5%~20.4% on the original basis.

[0090] It should also be noted that, in addition to the expression vectors and tyrosine-producing strains described in this embodiment, other expression vectors and tyrosine-producing strains can also be used in this invention. Besides the fermentation method for producing caffeic acid mentioned in this embodiment, whole-cell catalysis can also be used to produce caffeic acid.

[0091] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of substances that specifically reduce or knock out at least one gene among A1) to A4) in the fermentation synthesis of caffeic acid by tyrosine-producing bacteria, wherein: A1) NAD(P)H-dependent flavin reductase gene; A2) NADPH-dependent quinone reductase gene; A3) Genes of histidine / lysine / arginine / ornithine transport proteins that bind to ATP; A4) ATP-binding glutamate / aspartate transport protein gene.

2. The application according to claim 1, characterized in that, The tyrosine-producing bacteria express an enzyme that synthesizes caffeic acid. Preferably, the caffeic acid synthesis pathway enzyme includes at least one of tyrosine lyase and 3-hydroxylase.

3. The application according to claim 1 or 2, characterized in that, The tyrosine-producing bacterium is Escherichia coli.

4. A recombinant bacterium, characterized in that, The recombinant bacteria are obtained by knocking out or downregulating at least one gene from A1 to A4, starting with a tyrosine-producing bacterium, wherein: A1) NAD(P)H-dependent flavin reductase gene; A2) NADPH-dependent quinone reductase gene; A3) Genes of histidine / lysine / arginine / ornithine transport proteins that bind to ATP; A4) ATP-binding glutamate / aspartate transport protein gene.

5. The recombinant bacteria according to claim 4, characterized in that, The tyrosine-producing bacterium is Escherichia coli; Preferably, the tyrosine-producing bacteria is E. coli TYR-14B1 (P cra ::P37).

6. The recombinant bacteria according to claim 4 or 5, characterized in that, The recombinant bacteria contain a caffeic acid synthesis pathway enzyme expression vector; Preferably, the caffeic acid synthesis pathway enzyme expression vector includes the pZEA-RgTAL-PaHpaB-SeHpaC expression vector.

7. A method for constructing recombinant bacteria as described in claim 6, characterized in that, include: Knock out or knock down at least one gene from A1 to A4) in the starting strain capable of tyrosine synthesis, and then transfer it into the caffeic acid synthesis pathway enzyme expression vector to obtain the product; wherein: A1) NAD(P)H-dependent flavin reductase gene; A2) NADPH-dependent quinone reductase gene; A3) Genes of histidine / lysine / arginine / ornithine transport proteins that bind to ATP; A4) ATP-binding glutamate / aspartate transport protein gene.

8. The use of the recombinant bacteria as described in any one of claims 4 to 6 in the preparation of caffeic acid.

9. A method for preparing caffeic acid, characterized in that, The recombinant bacteria as described in any one of claims 4 to 6 are inoculated into the fermentation broth and then fermented to obtain the product.

10. The method according to claim 9, characterized in that, The fermentation process was carried out at a temperature of 37±2℃.

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