A method for synthesizing 5-hydroxyferulic acid using recombinant Escherichia coli whole cells

Abstract

The present invention discloses a recombinant Escherichia coli and a method for synthesizing 5-hydroxyferulic acid from ferulic acid, belonging to the field of biotechnology. In the present invention, the 4-hydroxyphenylacetic acid-3-hydroxylase gene derived from Pseudomonas aeruginosa is heterologously expressed in Escherichia coli, and a recombinant bacterium capable of directly converting ferulic acid into 5-hydroxyferulic acid is successfully constructed. The whole-cell catalysis can produce 5-hydroxyferulic acid with a yield of up to 1.97 g / L and a productivity of 91% within 24 h. The recombinant bacterium and the whole-cell catalysis system provided by the present invention have simple processes and are environmentally friendly, laying a certain foundation for industrial production.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing 5-hydroxyferulic acid using recombinant Escherichia coli whole cells, belonging to the field of biotechnology. Background Art

[0002] 5-Hydroxyferulic acid, also known as 3-methylcaffeic acid, is a cinnamic acid derivative with one more hydroxyl group in its structure than ferulic acid, indicating stronger antioxidant ability. 5-Hydroxyferulic acid is a key intermediate in various lignin synthesis pathways, present in various plants such as poplar, and can also be detected in fruits and vegetables such as pears and cacti. Analogous to ferulic acid, 5-hydroxyferulic acid with strong antioxidant properties has great commercial application potential in industries such as food, cosmetics, and pharmaceuticals.

[0003] Currently, 5-hydroxyferulic acid is mainly synthesized by chemical methods, mainly including: synthesizing 5-hydroxyferulic acid using 5-hydroxyvanillin and malonic acid as substrates; generating 5-hydroxyferulic acid through multiple chemical reactions of 3,4,5-trihydroxybenzaldehyde and acetic anhydride; synthesizing 5-hydroxyferulic acid from 5-ethynyl-3-methoxy-1,2-benzenediol and hydrogen cyanide; or demethylating sinapic acid to obtain 5-hydroxyferulic acid. These methods all have problems such as large substrate toxicity, high requirements for reaction conditions, such as high temperature and pressure, metal ions, and strong acids and bases, and the generation of other toxic by-products, which increases the difficulty of downstream separation and purification and is not conducive to environmental protection.

[0004] In plants, 5-hydroxyferulic acid is mainly obtained through the phenylpropanoid compound synthesis pathway, where ferulic acid-5-hydroxylase (F5H) catalyzes ferulic acid to generate 5-hydroxyferulic acid. F5H belongs to the monooxygenase of the CYP84 subfamily in the cytochrome P450 superfamily. The vast majority of F5H exists in plants, has a transmembrane structure, and P450 enzymes require redox partners to assist, making it difficult to express in conventional model strains. So far, only functional expression has been achieved in Saccharomyces cerevisiae, and the optimized specific enzyme activity is 2.6 ± 0.4 pmol / sec / mg protein. 5-Hydroxyferulic acid can also be achieved by catalyzing 5-hydroxyconiferyl aldehyde with cinnamaldehyde dehydrogenase. Among them, the substrate 5-hydroxyconiferyl aldehyde is also obtained by catalyzing coniferyl aldehyde with F5H in plants, and the reaction steps are cumbersome and prone to environmental pollution. Therefore, there is an urgent need to find a green synthesis method with simple reaction steps. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant bacterium for converting and producing 5-hydroxyferulic acid, which catalytically generates 5-hydroxyferulic acid from ferulic acid in one step. The present invention also provides an optimized whole cell reaction condition to maximize the efficiency of extracellular conversion of ferulic acid.

[0006] In the early stage of the experiment, it was found that 4-hydroxyphenylacetate-3-hydroxylase is a type of two-component flavin-dependent monooxygenase, which consists of an oxidation component and a reduction component, and can achieve the hydroxylation of 4-hydroxyphenylacetic acid analogs. The present invention relates to a method for catalytic synthesis of 5-hydroxyferulic acid using this enzyme. The whole-cell catalysis method adopted in the present invention uses whole cells as catalysts. The cells are easy to culture and can be reused, with the advantages of being easy to prepare and having low cost. Moreover, the whole-cell catalytic reaction is mild, and there is no need to add some toxic organic substances such as strong acids and strong bases or high temperature and high pressure during the reaction process. After the reaction, the cells and the liquid containing the product can be directly separated by centrifugal filtration, with few operation steps and simple process.

[0007] The present invention provides a recombinant bacterium, which expresses 4-hydroxyphenylacetate-3-hydroxylase derived from Pseudomonas aeruginosa. The 4-hydroxyphenylacetate-3-hydroxylase consists of two parts, hpaB and hpaC. The nucleotide sequence of the gene encoding hpaB is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding hpaC is shown in SEQ ID NO.2.

[0008] In one embodiment of the present invention, the gene encoding hpaB and the gene encoding hpaC are constructed on the same plasmid or separately constructed on two different plasmids.

[0009] In one embodiment of the present invention, the gene encoding hpaB and the gene encoding hpaC are constructed on the same plasmid, and in the order of gene expression, they are the gene encoding hpaB and the gene encoding hpaC in sequence.

[0010] In one embodiment of the present invention, one or more of plasmid pETDuet-1, pRSFDuet-1, pACYCDuet-1, or pET28a is used as the expression vector.

[0011] In one embodiment of the present invention, Escherichia coli is used as the expression host.

[0012] In one embodiment of the present invention, E. coli BL21(DE3) is used as the expression host.

[0013] The present invention provides a whole-cell catalyst, which comprises the above-mentioned recombinant bacterium.

[0014] The present invention also provides a method for producing 5-hydroxyferulic acid. The method is to ferment and produce 5-hydroxyferulic acid in a reaction system containing ferulic acid by using the above-mentioned recombinant bacterium or the above-mentioned whole-cell catalyst.

[0015] In one embodiment of the present invention, the buffer in the reaction system is selected from one of 20 - 100 mM potassium phosphate buffer, Tris-HCl buffer, citrate buffer, or HEPES buffer, with a pH of 5 - 9.

[0016] In one embodiment of the present invention, the reaction system further contains one or more of DMSO, glycerol, glucose, Tween 80, and Triton X100.

[0017] In one embodiment of the present invention, the reaction system is composed of ferulic acid at 1 - 10 g / L, whole-cell catalyst at 0.5 - 1.25 g / L, DMSO at 0 - 5%, glycerol at 0 - 20%, glucose at 0 - 20%, and Tween 80 or Triton X100 at 0 - 2%.

[0018] In one embodiment of the present invention, the reaction system is composed of ferulic acid at 1 - 10 g / L, whole-cell catalyst at 0.5 - 1.25 g / L, glycerol at 5 - 20%, and Tween 80 or Triton X100 at 0 - 2%.

[0019] In one embodiment of the present invention, the reaction conditions for production are conversion for 4 - 24 h under the conditions of a temperature of 20 - 40°C and a rotation speed of 110 - 220 rpm.

[0020] In one embodiment, the preparation method of the whole-cell catalyst is to inoculate the above-mentioned recombinant bacterium into an activation medium to obtain a seed solution, and then inoculate the seed solution into an induction medium. When the OD 600 reaches 0.4 - 0.8, IPTG is added for induction culture for 8 - 15 h.

[0021] In one embodiment, the activation medium includes peptone at 5 - 15 g / L, yeast extract at 3 - 6 g / L, and NaCl at 5 - 15 g / L.

[0022] In one embodiment, the induction medium includes one of LB medium, TB medium, SOC medium, SC medium, or M9 medium.

[0023] The present invention also provides the application of the above-mentioned recombinant bacterium, the above-mentioned whole-cell catalyst, or the above-mentioned method in the preparation of a product containing 5-hydroxyferulic acid.

[0024] Beneficial effects:

[0025] The present invention realizes the expression of 4-hydroxyphenylacetic acid-3-hydroxylase in Escherichia coli, constructs a recombinant bacterium expressing 4-hydroxyphenylacetic acid-3-hydroxylase, and uses the recombinant bacterium of the present invention to produce 5-hydroxyferulic acid with ferulic acid as a substrate. Within 24 hours, the yield of 5-hydroxyferulic acid produced by whole-cell catalysis can reach 1.97 g / L, and the yield is 91%. The establishment of this whole-cell conversion system solves the problems of cumbersome steps and environmental pollution in the chemical synthesis of 5-hydroxyferulic acid, realizes mild reaction conditions, pollution-free, one-step production of 5-hydroxyferulic acid, and lays a certain theoretical foundation for subsequent industrial production. Description of the Drawings

[0026] Figure 1 LC-MS / MS diagram of the product 5-hydroxyferulic acid in the whole-cell reaction solution, A: Standard: 5-hydroxyferulic acid, B: Sample. Detailed Embodiments

[0027] The following will describe the implementation scheme of the present invention in detail in combination with embodiments. However, those skilled in the art will understand that the specific material ratios, process conditions and their results described in the following embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0028] (I) Culture Medium

[0029] TB culture medium: 12 g / L peptone, 24 g / L yeast extract, 4 g / L glycerol, 2.31 g / L KH2PO4, 16.43 g / L K2HPO4·3H2O; pH 7.0;

[0030] LB culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl; pH 7.0;

[0031] SOC culture medium: 20 g / L peptone, 5 g / L yeast extract, 0.5 g / L NaCl, 0.186 g / L KCl, 2.033 g / L MgCl2·6H2O, 3.6 g / L glucose; pH 7.0;

[0032] SB culture medium: 30 g / L peptone, 20 g / L yeast extract, 10 g / L MoPs; pH 7.0;

[0033] M9 culture medium: 6.78 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L NaCl, 0.241 g / L MgSO4, 1.0 g / L NH4Cl, 0.011 g / L CaCl2, 4 g / L glucose; pH 7.0.

[0034] (II) Detection Method

[0035] Detection method for the content of 5-hydroxyferulic acid: The high-performance liquid chromatography detection method is as follows: The chromatographic column is an Amethyst C18-H (4.6×250 mm, 5 μm) reverse-phase chromatographic column, the detection wavelength is 322 nm, the column temperature is 30 °C, the mobile phase is methanol: 10% acetic acid aqueous solution = 38:42 (v:v), and the flow rate is 0.8 mL·min -1 , and the running time for a single sample is 12 min.

[0036] Calculation of the yield of 5-hydroxyferulic acid: The actual molar amount of 5-hydroxyferulic acid in the system / the total molar amount of ferulic acid in the system * 100%

[0037] Example 1 Construction of recombinant bacteria of 4-hydroxyphenylacetic acid-3-hydroxylase

[0038] Using the genome of Pseudomonas aeruginosa as a template, in the presence of DNA polymerase, using the primer pairs of hpaB (SEQ ID NO.3 and SEQ ID NO.4) and the primer pairs of hpaC (SEQ ID NO.5 and SEQ ID NO.6), PCR amplification of the target gene is carried out to obtain the gene fragments of hpaB and hpaC of the 4-hydroxyphenylacetic acid-3-hydroxylase two-component. The corresponding gene fragments and the plasmid pETDuet-1 are double-digested with BamHI and HindIII and Nde I and Kpn I respectively, and the two gene fragments are sequentially ligated to the plasmid pETDuet-1 with a ligase, and introduced into the competent cells of Escherichia coli E. coli BL21(DE3) by heat shock method to obtain the recombinant bacteria BL21(DE3) / pETDuet-hpaBC.

[0039] Similarly, using the genome of Pseudomonas aeruginosa as a template, in the presence of DNA polymerase, using the primer pairs of hpaB (SEQ ID NO.7 and SEQ ID NO.8) and the primer pairs of hpaC (SEQ ID NO.9 and SEQ ID NO.10) to amplify the gene fragments of hpaB and hpaC. The corresponding gene fragments and the plasmid pETDuet-1 are double-digested with Nde I and Kpn I and BamHI and HindIII respectively, and the two gene fragments are sequentially ligated to the plasmid pETDuet-1 with a ligase, and introduced into the competent cells of E. coli BL21(DE3) by heat shock method to obtain the recombinant bacteria BL21(DE3) / pETDuet-hpaCB.

[0040] Example 2 Comparison of the catalytic synthesis of 5-hydroxyferulic acid by recombinant bacteria BL21(DE3) / pETDuet-hpaBC and BL21(DE3) / pETDuet-hpaCB

[0041] (1) Preparation of whole cells: Pick single colonies of the recombinant bacteria BL21(DE3) / pETDuet-hpaBC and BL21(DE3) / pETDuet-hpaCB in Example 1 into 5 mL of TB liquid medium (containing antibiotics), and shake overnight at 37 °C with a rotation speed of 220 rpm. The next day, inoculate the bacterial solution into 30 mL of LB medium at 2% (v / v) respectively, and continue to culture at 37 °C with a rotation speed of 220 rpm. When OD 600 is between 0.4 and 0.8, add 0.2 mM IPTG, and induce expression at 20 °C. After culturing for 9 h, collect the bacterial solution, centrifuge to obtain the cells, and resuspend them with buffer to obtain wet cells.

[0042] (2) Whole-cell catalysis: The total volume of the reaction system is 1 mL, which contains 1 g / L ferulic acid, wet cell OD 600 = 60 and potassium phosphate buffer pH 7.2 100 mM. React at 30 °C with a rotation speed of 220 rpm for 12 h, take samples and centrifuge at high speed, filter the supernatant through a membrane and put it into a liquid-phase injection vial, which is the sample to be detected. Subsequently, use high-performance liquid chromatography to detect the content of 5-hydroxyferulic acid in the sample. The results show that BL21(DE3) / pETDuet-hpaBC and BL21(DE3) / pETDuet-hpaCB produce 0.32 g / L and 0.23 g / L of 5-hydroxyferulic acid respectively.

[0043] Example 3 Effects of different media on the catalytic synthesis of 5-hydroxyferulic acid by recombinant strains

[0044] Pick a single colony of BL21(DE3) / pETDuet-hpaBC on a solid plate into 5 mL of LB liquid medium (containing antibiotics), shake overnight at 37 °C and 220 rpm as the seed solution. The next day, inoculate the seed solution into 30 mL of each of the 5 media, namely LB, SOC, TB, SB, and M9, at 2% (v / v), and culture at 37 °C with a rotation speed of 220 rpm. When OD 600 is between 0.4 and 0.8, add 0.2 mM IPTG, and lower the temperature to 20 °C for induction. Take samples every 1 - 2 h to detect OD 600 , and take samples at 9, 13, 17, and 21 h of induction for detection, and collect the cells. Perform whole-cell catalysis of 5-hydroxyferulic acid according to the method in step (2) of Example 2, and determine the content of 5-hydroxyferulic acid. The results are as follows:

[0045] Table 1 Effects of different induction durations on the production of 5-hydroxyferulic acid

[0046]

[0047] The results showed that the yield of 5-hydroxyferulic acid decreased with the increase of the culture time of the recombinant bacterium. When the culture time of the recombinant bacterium was 9 h, the yield of 5-hydroxyferulic acid was the highest. Among them, the viability of the recombinant bacterium in M9 medium was much higher than that in other media. SOC medium was not conducive to the culture of the recombinant bacterium, while the effects of LB, TB, and SB media were similar.

[0048] Example 4 Catalytic synthesis of 5-hydroxyferulic acid by whole-cell catalyst under different catalytic reaction conditions

[0049] According to the method of Example 3, first culture the BL21(DE3) / pETDuet-hpaBC seed solution in LB medium, and then transfer it to TB medium for induction expression for 12 h, and collect the thalli.

[0050] 1. Optimization of reaction temperature: The catalytic reaction system was 10 mL, which contained 5% (w / v) wet cells, 10% (v / v) glycerol, 4% (v / v) DMSO, 1% (v / v) Tween 80, 2 g / L ferulic acid, and 100 mM potassium phosphate buffer at pH 7.5. The reaction was carried out at 20, 25, 30, 35, and 40 °C at a rotation speed of 220 rpm. The yield of 5-hydroxyferulic acid was as follows:

[0051] Table 2 Effects of different temperatures on the whole-cell catalytic reaction

[0052] Temperature (°C) 20 25 30 35 40 5-Hydroxyferulic acid concentration (g / L) 0.54 0.65 0.84 0.89 1.17

[0053] It shows that the higher the reaction temperature, the higher the yield of 5-hydroxyferulic acid. At the reaction temperature of 40 °C, the yield of 5-hydroxyferulic acid was about 2.2 times that at 20 °C. Therefore, the whole-cell catalytic reaction temperature was selected as 40 °C.

[0054] 2. Optimization of DMSO concentration: The catalytic reaction system was 10 mL, which contained 5% (w / v) wet cells, 10% (v / v) glycerol, 1% (v / v) Tween 80, 2 g / L ferulic acid, and 100 mM potassium phosphate buffer at pH 7.5. DMSO was added at 0, 1, 2, 3, 4, and 5% (v / v) respectively, and the reaction was carried out at 30 °C and a rotation speed of 220 rpm. The yield of 5-hydroxyferulic acid was measured, and the results were as follows:

[0055] Table 3 Effects of different concentrations of DMSO on the whole-cell catalytic reaction

[0056]

[0057]

[0058] It is shown that the concentration of DMSO has little effect on the yield of 5-hydroxyferulic acid. Therefore, DMSO can be not added in the whole-cell catalyzed reaction.

[0059] 3. Optimization of Tween 80 concentration: The catalytic reaction system was 10 mL, which contained 5% (w / v) wet cells, 10% (v / v) glycerol, 4% (v / v) DMSO, 2 g / L ferulic acid, and 100 mM potassium phosphate buffer at pH 7.5. Tween 80 was added at 0, 0.5, 1, 1.5% (v / v) respectively, and the reaction was carried out at 30 °C with a rotation speed of 220 rpm. The yield of 5-hydroxyferulic acid was measured after 12 h, and the results were as follows:

[0060] Table 4 Effects of different concentrations of Tween 80 on the whole-cell catalyzed reaction

[0061] Tween80 (%) 0 0.5 1 1.5 5-Hydroxyferulic acid concentration (g / L) 0.68 1.02 0.84 0.74

[0062] It is shown that when the concentration of Tween 80 is 0.5%, the yield of 5-hydroxyferulic acid is the highest, about 1.5 times that without adding Tween 80. Therefore, the addition amount of Tween 80 in the whole-cell catalyzed reaction system is 0.5% (v / v).

[0063] 4. Optimization of glycerol concentration: The catalytic reaction system was 10 mL, which contained 5% (w / v) wet cells, 4% (v / v) DMSO, 1% (v / v) Tween 80, 2 g / L ferulic acid, and 100 mM potassium phosphate buffer at pH 7.5. Glycerol was added at 0, 5, 10, 15, 20% (v / v) respectively, and the reaction was carried out at 30 °C with a rotation speed of 220 rpm. The yield of 5-hydroxyferulic acid was measured after 12 h, and the results were as follows:

[0064] Table 5 Effects of different concentrations of glycerol on the whole-cell catalyzed reaction

[0065] Glycerol (%) 0 5 10 15 20 5-Hydroxyferulic acid concentration (g / L) 0.30 1.16 0.84 1.06 1.03

[0066] It is shown that glycerol has a great influence on the yield of 5-hydroxyferulic acid. When the glycerol content is 5% (v / v), it is 3.9 times that of the control 0% (v / v). Therefore, the addition amount of glycerol in the whole-cell catalyzed reaction system is 5%.

[0067] 5. Optimization of reaction pH: The catalytic reaction system was 10 mL, which contained 5% (w / v) wet cells, 10% (v / v) glycerol, 4% (v / v) DMSO, 1% (v / v) Tween 80, and 2 g / L ferulic acid. It was supplemented to 10 mL with 100 mM potassium phosphate buffer at pH 5.5, 6, 6.5, 7, 7.5, 8, 8.5 respectively. The reaction was carried out at 30 °C with a rotation speed of 220 rpm. The yield of 5-hydroxyferulic acid was measured after 12 h, and the results were as follows:

[0068] Table 6 Effects of different pH values on the whole-cell catalyzed reaction

[0069] pH 5.5 6 6.5 7 7.5 8 8.5 5-Hydroxyferulic acid concentration (g / L) 0.48 0.68 0.98 1.03 0.84 0.77 0.58

[0070] It shows that pH has a great influence on the yield of 5-hydroxyferulic acid. The yield of 5-hydroxyferulic acid in the pH 7 system is about 2.1 times that in the pH 5.5 system. Therefore, the pH of the whole-cell catalyzed reaction system is 7.

[0071] 6. Optimization of the wet cell addition amount: The catalyzed reaction system is 10 mL, which contains wet cell contents of 2.5, 5, 7.5, 10, 12.5, 15% (w / v), glycerol 10% (v / v), DMSO 4% (v / v), Tween 80 1% (v / v), and ferulic acid 2 g / L. It is supplemented to 10 mL with 100 mM, pH 7.5 potassium phosphate buffer. The reaction is carried out at 30 °C and a rotation speed of 220 rpm. The yield of 5-hydroxyferulic acid is measured after 12 h. The results are as follows:

[0072] Table 7 Effects of different cell concentrations on the whole-cell catalyzed reaction

[0073] Wet cells (%) 2.5 5 7.5 10 12.5 5-Hydroxyferulic acid concentration (g / L) 0.56 0.84 1.16 1.14 1.46

[0074] It shows that in the whole-cell catalyzed reaction, the higher the concentration of the whole-cell catalyst, the higher the yield of 5-hydroxyferulic acid. However, when the cell content > 5% (w / v), the yield of 5-hydroxyferulic acid changes little with the change of cell concentration. Therefore, the wet cell content used in the whole-cell catalyzed reaction is 5% (w / v).

[0075] 7. Optimal reaction conditions: The catalyzed reaction system is 10 mL, which contains wet cell content 5% (w / v), glycerol 5% (v / v), Tween 80 0.5% (v / v), and ferulic acid 2 g / L. It is supplemented to 10 mL with 100 mM, pH 7 potassium phosphate buffer. The reaction is carried out at 40 °C and a rotation speed of 220 rpm. The yield of 5-hydroxyferulic acid is measured ( Figure 1 ) and the results are as follows:

[0076] Table 8 Yield of 5-hydroxyferulic acid under optimal conditions

[0077] Time (h) 8 12 16 24 5-Hydroxyferulic acid concentration (g / L) 1.23 1.50 1.75 1.97

[0078] Example 5 Effects of adding glucose or glycerol to the catalysis system on the whole-cell repeated batch conversion of ferulic acid

[0079] According to the method of Example 3, first culture the BL21(DE3) / pETDuet-hpaBC seed solution in LB medium, then transfer it to TB medium for induced expression for 12 h, and collect the cells to obtain the whole-cell catalyst. The catalytic reaction system is 10 mL, which contains 5% (w / v) wet cell content, 0.5% (v / v) Tween 80, 2 g / L ferulic acid, and is supplemented to 10 mL with 100 mM, pH 7 potassium phosphate buffer, which contains 5% (v / v) glycerol or 12.3% (g / v) glucose respectively, and react at 40 °C with a rotation speed of 220 rpm for 12 h. After the reaction, centrifuge to collect the cells for the next batch reaction, and measure the yield of 5-hydroxyferulic acid. The results without glycerol / glucose as a control are as follows:

[0080] Table 8 Influence of repeated batches of the whole-cell catalytic system on the conversion of ferulic acid

[0081] Reaction batch 1 2 3 Time (h) 12 12 12 Adding glycerol, 5-Hydroxyferulic acid concentration (g / L) 1.53 0.81 0.61 Adding glucose, 5-Hydroxyferulic acid concentration (g / L) 0.34 0.05 0.02 Control, 5-Hydroxyferulic acid concentration (g / L) 0.21 0 0

[0082] The results show that: in the catalytic system containing glycerol, the yield of 5-hydroxyferulic acid is significantly higher than that in the catalytic system containing the same molar amount of glucose. And after repeating 3 times, the yield of 5-hydroxyferulic acid is 0.61 g / L, which is about 40% of the first reaction. Adding glycerol to the whole-cell catalytic system is beneficial to the formation of 5-hydroxyferulic acid.

[0083] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for producing 5-hydroxyferulic acid, characterized in that, The method is to ferment and produce 5-hydroxyferulic acid in a reaction system containing ferulic acid by using recombinant bacteria or whole-cell catalysts; The recombinant bacteria use plasmid pETDuet-1 as an expression vector to express 4-hydroxyphenylacetic acid-3-hydroxylase, and the 4-hydroxyphenylacetic acid-3-hydroxylase is composed of hpaB and hpaC. The nucleotide sequence of the gene encoding hpaB is as shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding hpaC is as shown in SEQ ID NO.2; The whole-cell catalyst contains the recombinant bacteria described above.

2. The method according to claim 1, wherein The recombinant bacteria use Escherichia coli as an expression host.

3. The method according to claim 1, wherein The buffer in the reaction system is selected from one of 20-100 mM potassium phosphate buffer, Tris-HCl buffer, citrate buffer, or HEPES buffer, with a pH of 5-9.

4. The method according to claim 1 or 3, characterized in that The reaction system also contains one or more of DMSO, glycerol, glucose, Tween 80, and Triton X100.

5. The method according to claim 4, characterized in that, The reaction conditions for the fermentation production are conversion for 4-24 h under the conditions of a temperature of 20-40 °C and a rotation speed of 110-220 rpm.

6. Use of the method according to any one of claims 1 to 5 in the preparation of a product containing 5-hydroxyferulic acid.

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