CN115975963B - Methods and applications of whole-cell catalytic synthesis of hydroxylated flavonoids using Escherichia coli P450 enzyme - Google Patents

Abstract

The invention discloses a method for synthesizing a hydroxylated flavone compound by using escherichia coli P450 enzyme whole cell catalysis and application thereof, belonging to the technical field of genetic engineering. The invention screens out P450sca-2 with high yield of the hydroxylated flavone compound _mut Optimizing the conditions of reduction partner engineering, enzyme engineering and whole cell catalysis to obtain sca-2 _mut The efficiency of synthesizing eriodictyol by catalyzing naringenin by R88A/S96A reaches 77%; the efficiency of catalyzing the dihydrokaempferol to synthesize the dihydroquercetin reaches 66 percent; the efficiency of catalyzing apigenin to synthesize luteolin reaches 32%; the efficiency of catalyzing daidzein to synthesize 7,3',4' -trihydroxyisoflavone reaches 75%, and a new strategy is provided for improving biosynthesis of hydroxylated compounds.

Description

Method for whole-cell catalytic synthesis of hydroxylated flavonoids using Escherichia coli P450 enzyme Laws and applications

Technical field

The invention relates to a method and application of whole-cell catalytic synthesis of hydroxylated flavonoid compounds using Escherichia coli P450 enzyme, and belongs to the technical field of genetic engineering.

Background technique

Cytochrome P450 enzymes are a type of enzyme that uses heme as a prosthetic group and are widely found in animals, plants, archaea, bacteria, eukaryotes and other organisms. In mammals such as humans, P450 enzymes are widely involved in the degradation of xenobiotics, detoxification, drug metabolism, and the synthesis of steroid hormones and vitamins. In plants, P450 enzymes can catalyze the regional or three-dimensional hydroxylation, epoxidation, and dealkylation of a series of natural products (such as terpenes, polyenes, glycopeptides, alkaloids, fatty acids, and steroids). Oxidative catalytic reactions such as base and dehalogenation. Because P450 enzymes have a wide substrate spectrum and diversity of catalytic reactions, a large number of P450 enzymes have been gradually applied to the field of microbial pharmaceuticals to synthesize valuable natural products or drugs. Previous studies mostly used extracellular enzymatic catalysis methods, which often require the purification of P450 enzymes and coenzymes, and the addition of expensive cofactor NAD(P)H and cofactor recycling systems, resulting in high catalytic synthesis costs. Compared with extracellular enzymatic catalytic methods, whole-cell catalytic methods are a low-cost catalytic alternative.

Flavonoids are a class of polyphenolic compounds widely found in plants. They have a basic parent core, 2-phenylchromone (C6-C3-C6 structure). According to the parent structure, they can be divided into flavones, flavonols, dihydroflavones, Dihydroflavonols, isoflavones, etc. (Figure 1). Flavonoids have various biological activities such as antioxidant, antiviral, antitumor, and antibacterial, and are widely used in the field of food and medicine. However, the poor water solubility and instability of flavonoids limit their medicinal applications. The hydroxylation reaction can not only improve the solubility and stability of flavonoids, but also enhance their biological activity and enrich the types of flavonoids.

Compared with chemical hydroxylation, biocatalytic hydroxylation is an environmentally friendly method to obtain hydroxylated flavonoids. Escherichia coli is the most commonly used expression system for recombinant proteins and an efficient whole-cell transformation system. However, the P450 enzyme involved in the hydroxylation reaction has poor solubility in prokaryotes and is difficult to express. It is currently found that CYP107P2 (Pandey et al., Enzyme Microb. Technol. 48 (4-5), 386-392.2011), CYP107Y1 (Pandey et al., Enzyme Microb. Technol. 48 (4-5), 386-392.2011), P450BM3 (Chu et al., Microb. Cell Fact. 15(1), 135.2016), CYP105D7 (Liu et al., J. Mol. Catal. B: Enzym. 132, 91-97.2016) and CYP105A5 ( Subedi et al., Catalysts 12(10), 1157.2022) can hydroxylate specific flavonoid compounds, but there are problems such as low conversion rate. Therefore, it is of great significance to develop P450 enzymes that can be efficiently expressed in prokaryotes to synthesize flavonoids.

Contents of the invention

The invention provides a cytochrome P450 enzyme mutant P450sca-2, which is based on the amino acid sequence shown in SEQ ID NO. 1 as the starting sequence, and has made any of the following improvements:

(1) Mutation of arginine at position 88 of the amino acid sequence shown in SEQ ID NO. 1 to alanine;

(2) Mutation of serine at position 96 of the amino acid sequence shown in SEQ ID NO. 1 to alanine;

(3) Mutate the arginine at position 88 of the amino acid sequence shown in SEQ ID NO. 1 to alanine, and mutate the serine at position 96 to alanine.

The invention also provides genes encoding said mutants.

The present invention also provides recombinant E. coli expressing the cytochrome P450 enzyme mutant P450sca-2 or the cytochrome P450 enzyme shown in SEQ ID NO.1.

In one embodiment, the recombinant E. coli also expresses the ferredoxin reductase gene CamA (SEQ ID NO. 14) and the ferredoxin gene CamB (SEQ ID NO. 13).

In one embodiment, the E. coli also expresses a redox chaperone gene; the redox chaperone gene includes E. coli flavodoxin reductase Fpr (SEQ ID NO. 20) and E. coli-derived flavin Oxidized protein Fld (SEQ ID NO. 15), E. coli-derived FldA (SEQ ID NO. 16), E. coli-derived FldB (SEQ ID NO. 17), Bacillus subtilis-derived flavin oxidation protein YkuN (SEQ ID NO. .18), a combination of any protein from YkuP (SEQ ID NO. 19) derived from Bacillus subtilis.

In one embodiment, the recombinant E. coli uses an expression vector to co-express the gene encoding the P450sca-2 mutant derived from Streptomyces carbophilus, the E. coli-derived flavin oxidation protein gene Fld and The gene Fpr encoding flavin oxidizing protein reductase derived from Escherichia coli.

In one embodiment, the flavin oxidized protein has an amino acid sequence as shown in SEQ ID NO.2; the flavoxid protein reductase has an amino acid sequence as shown in SEQ ID NO.3.

In one embodiment, the expression vector is pRSFDuet-1.

In one embodiment, the E. coli includes, but is not limited to, BL21(DE3), C41(DE3) or C43(DE3).

The present invention also provides a method for whole-cell catalytic synthesis of hydroxylated flavonoid compounds, which uses the recombinant Escherichia coli as a cell catalyst and reacts at 20-37°C for at least 1-12 hours in a reaction system containing a substrate.

In one embodiment, the substrate includes, but is not limited to, one or more of naringenin, dihydrokaempferol, kaempferol, daidzein or apigenin.

In one embodiment, the cell catalyst is to culture the recombinant Escherichia coli at 35-37°C until the OD ₆₀₀ value is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside and then culture it at 20 Prepared by continuing to culture at -30°C for 12-20 hours; the concentration of isopropyl-β-D-thiogalactopyranoside is 0.1-1mM.

In one embodiment, the fermented fermentation broth is centrifuged at 4°C and 8000 rpm for 10-20 minutes, the bacterial cells are collected, and the bacterial cells are washed with potassium phosphate buffer at pH 8.0. After washing, resuspend the washed cells in potassium phosphate with pH 8.0 (containing 5%-10% v/v glycerol) to obtain the cell catalyst.

In one embodiment, the reaction system also contains potassium phosphate buffer with a concentration of 50-100 mM.

In one embodiment, the flavonoid compound includes, but is not limited to, one or more of naringenin, dihydrokaempferol, kaempferol, daidzein, and apigenin.

The invention also provides that the genetically engineered bacteria are capable of producing eriodictyol (using naringenin as a substrate), dihydroquercetin (using dihydrokaempferol as a substrate), and quercetin (using kaempferol as a substrate). ), luteolin (with apigenin as substrate) and 7,3',4'-trihydroxyisoflavones (with daidzein as substrate) in various hydroxylated flavonoid compounds.

Beneficial effects:

(1) The present invention selects the high-yield hydroxylated flavonoid compound P450sca-2 _mut and obtains the sca-2 _mut R88A/S96A mutant through protein engineering to enhance the catalytic activity of P450sca-2 _mut .

(2) Through reducing partner engineering, the endogenous flavin oxidation protein Fld and flavin oxidation protein reductase Fpr of Escherichia coli were screened to improve the whole-cell catalytic activity of P450sca-2 _mut .

(3) The efficiency of the present invention in catalyzing the synthesis of eriodictyol from naringenin through the optimized P450sca-2 _mut whole-cell catalytic system is 77%; the efficiency of catalyzing the synthesis of dihydrokaempferol into dihydroquercetin is 66%; and the efficiency of catalyzing apigenin The efficiency of synthesizing luteolin is 32%; the efficiency of catalyzing the synthesis of 7,3',4'-trihydroxyisoflavones from daidzein is 75%.

Description of the drawings

Figure 1 shows the skeleton structure and main classification of flavonoid compounds.

Figure 2 shows the effect of P450 enzyme and gene combinations on the production of hydroxylated flavonoid compounds.

Figure 3 shows redox chaperone engineering to improve the catalytic efficiency of P450sca-2 _mut .

Figure 4 shows enzyme engineering to improve the catalytic efficiency of P450sca-2 _mut .

Figure 5 shows the interaction analysis between P450sca-2 _mut and sca-2 _mut R88A/S96A and substrate.

Figure 6 shows the optimization of whole cell conditions to improve the ability of P450sca-2 _mut R88A/S96A to synthesize hydroxylated flavonoids.

Figure 7 shows the application of P450sca-2 _mut R88A/S96A in the synthesis of other hydroxylated flavonoids.

Detailed ways

Materials and Methods

LB medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract, sterilized at 121°C for 15 minutes.

TB culture medium: 12g/L peptone, 5g/L glycerol, 24g/L yeast extract, 17mM potassium dihydrogen phosphate, 72mM dipotassium hydrogen phosphate, sterilized at 121°C for 15 minutes.

Whole cell catalysis:

(1) Cultivate the recombinant E. coli at 35-37°C until the OD ₆₀₀ value is 0.6-0.8. Add isopropyl-β-D-thiogalactopyranoside and continue culturing at 20-30°C for 12-20 hours. Among them, the concentration of isopropyl-β-D-thiogalactopyranoside is 0.1-1mM.

(2) Centrifuge the fermentation broth obtained in step (1) at 4°C and 8000 rpm for 10-20 minutes, collect the bacterial cells, and wash the bacterial cells with potassium phosphate buffer at pH 8.0. After washing, resuspend the washed cells in potassium phosphate with pH 8.0 (containing 5%-10% v/v glycerol).

(3) Use the bacterial suspension obtained in step (2) to carry out the hydroxylation reaction of flavonoid compounds. The whole cell reaction system includes (based on the final concentration): bacterial cells 50g/L, flavonoids 100mg/L; flavonoids Includes naringenin, dihydrokaempferol, kaempferol, daidzein and apigenin. The whole cell reaction is carried out at 20-37°C for at least 1-12 hours. After the reaction is completed, take an appropriate amount of the reaction solution and add an equal volume of ethyl acetate for extraction. After extraction and separation, the 3'hydroxylated flavonoid compound is obtained and analyzed by high performance liquid chromatography. Carry out analysis and testing.

High performance liquid chromatography analysis:

(1) Mobile phase: Phase A is ultrapure water containing 0.1% trifluoroacetic acid, and phase B is methanol containing 0.1% trifluoroacetic acid.

(2) Chromatographic column: reverse chromatography column ZORBAX Eclipse XDB-C18 (5μm, 4.6×250mm, Agilent, USA); column temperature is 40°C; flow rate: 0.8mL/min; elution program: 0-1min, 10% B; 1-10min, 10%-40%B; 10-20min, 40%-60%B; 20-23min, 60%B; 23-25min, 60%-10%B; 25-27min, 10%B .

(3) Use a UV detector with a detection wavelength of 290nm.

Calculation of catalytic efficiency: product concentration (mg/L)/substrate concentration (mg/L) × 100%.

Example 1: Screening of P450 enzymes with high yield of hydroxylated flavonoid compounds

Select CYP105P2 (SEQ ID NO. 4) derived from Streptomyces peucetius, CYP105D7 (SEQ ID NO. 5) derived from Streptomyces avermitilis, and Nonomuraea recticatena. CYP105AB3 (Q87W/T115A/H132L/R191W/G294D, abbreviated as P450moxA _mut ; SEQ ID NO. 6), CYP105A1 (R73A/R84A; abbreviated as CYP105A1 _mut ; SEQ ID NO. 6) derived from Streptomyces griseolus .7), CYP105A3 (G52S/T85F/F89I/T119S/P159A/V194N/D269E/T323A/N363Y/E370V; abbreviated as P450sca-2 _mut ; SEQ ID NO.1) derived from Streptomycescarbophil, The above genes were synthesized through E. coli codon optimization (the nucleotide sequences are shown in SEQ ID NO. 8-SEQ ID NO. 12 respectively), and the above genes were subcloned into the Nde I and Xho I positions of pRSFDuet-1. point, and the recombinant plasmids pRSF-105P2, pRSF-105D7, pRSF-moxA _mut , pRSF-105A1 _mut and pRSF-sca-2 _mut were obtained respectively. Since the CYP105 family is a typical three-component P450 enzyme and requires a reducing chaperone to transfer electrons from the electron donor NAD(P)H to the active center of the P450 enzyme, ferredoxin from Pseudomonas putida was chosen. CamB (SEQ ID NO. 13) and ferredoxin reductase CamA (SEQ ID NO. 14) serve as reduction chaperones. The codon-optimized ferredoxin reductase gene CamA and ferredoxin gene CamB were synthesized, and the CamA and CamB genes were subcloned into the recombinant plasmids pRSF-105P2, pRSF-105D7, pRSF-moxA _mut , and pRSF-105A1 Between the Nco I and Sac I restriction sites of _mut and pRSF-sca-2 _mut , the recombinant plasmids pRSF-105P2-CamA-CamB, pRSF-105D7-CamA-CamB, pRSF-moxA _mut -CamA-CamB, and pRSF were obtained -105A1 _mut -CamA-CamB and pRSF-sca-2 _mut -CamA-CamB. The above five recombinant plasmids were transformed into E. coli expression host C41 (DE3) to obtain five recombinant strains, respectively named HFLA-1 to HFLA-5 (Figure 2).

Use TB culture for shake flask fermentation of the HFLA-1 to HFLA-5 recombinant strains. When the recombinant E. coli is cultured at 37°C until the OD ₆₀₀ value is 0.6-0.8, add 1mM isopropyl-β-D-thiogalacto After glycoside, culture was continued at 25°C for 20 h. After the fermentation is completed, the fermentation broth is centrifuged at 4°C and 8000 rpm for 10 minutes, the bacterial cells are collected, and the bacterial cells are washed with potassium phosphate buffer at pH 8.0. After washing, resuspend the washed cells in potassium phosphate with pH 8.0 (containing 10% v/v glycerol) to serve as the whole cell catalyst. The reaction system catalyzed by whole cells is (based on final concentration): bacterial cells 50g/L, naringenin 100mg/L. The results are shown in Figure 2. The recombinant strain HFLA-5 (containing the recombinant plasmid pRSF-sca-2 _mut -CamA-CamB) can produce 20.3 mg/L eriodictyol, respectively. The recombinant strain HFLA-2 (containing the recombinant plasmid pRSF- 105D7-CamA-CamB), HFLA-3 (containing recombinant plasmid pRSF-moxA _mut -CamA-CamB), HFLA-4 (containing recombinant plasmid pRSF-105A1 _mut -CamA-CamB), 3.9, 1.3, and 1.8 times the production.

Example 2: Reducing chaperone engineering to improve catalytic efficiency of P450 sca-2 _mut

For three-component P450 enzymes, the efficiency of electron transfer is critical for whole-cell catalysis. In order to screen out suitable reduction partners, flavin oxidation proteins Fld (SEQ ID NO. 15), FldA (SEQ ID NO. 16), FldB (SEQ ID NO. 17) derived from Escherichia coli and flavin oxidation proteins derived from Bacillus subtilis were selected. The flavodoxin proteins YkuN (SEQ ID NO. 18) and YkuP (SEQ ID NO. 19) were respectively combined with the flavodoxin reductase Fpr (SEQ ID NO. 20) derived from Escherichia coli to obtain 5 sets of reducing partner combinations. ; Select ferredoxin Fdx_1499 and ferredoxin reductase FdR_0978 derived from Synechococcus elongates as the sixth group of reducing partner combinations; respectively assemble the six groups of reducing partner combinations into the recombinant plasmid pRSF- constructed in Example 1 Recombinant plasmids pRSF-sca-2 _{mut -Fld} -Fpr, pRSF-sca-2 _mut -FldA-Fpr, pRSF-sca-2 _mut -FldB- were obtained between the Nco _I and Sac I restriction sites of sca-2 mut. Fpr, pRSF-sca-2 _mut -YkuN-Fpr, pRSF-sca-2 _mut -YkuP-Fpr and pRSF-sca-2 _mut -Fdx_1499-FdR_0978. In addition, the P450 BM3 reduction domain part of Bacillus megaterium (SEQ ID NO. 21) was selected as the seventh group of reduction partners and fused to the C terminus of sca-2 _mut to construct the recombinant plasmid pRSF-sca-2 _mut -BM3 . The above seven recombinant plasmids were transformed into C41 (DE3) to obtain recombinant strains HFLA-6 to HFLA-12.

The recombinant strains HFLA-6 to HFLA-12 were subjected to shake flask fermentation experiments. The fermentation conditions were the same as in Example 1. After the fermentation was completed, the fermentation liquid was centrifuged at 4°C and 8000 rpm for 10 min. The cells were collected and buffered with potassium phosphate at pH 8.0. Wash the bacteria with liquid. After the washing is completed, resuspend the washed cells in potassium phosphate with pH 8.0 (containing 10% v/v glycerol or 10% w/v glucose) to become the whole cell catalyst. The reaction system catalyzed by whole cells is (based on final concentration): bacterial cells 50g/L, naringenin 100mg/L. The results are shown in Figure 3. In the whole-cell catalytic system containing 10% v/v glycerol, the recombinant strain HFLA-7 (containing the recombinant plasmid pRSF-sca-2 _mut -Fld-Fpr) has the strongest ability to produce eriodictyol. (38.6mg/L), which is 1.9 times the production of the control strain HFLA-5 (containing the recombinant plasmid pRSF-sca-2 _mut -CamA-CamB).

Example 3: Enzyme engineering to improve catalytic efficiency of sca-2 _mut

Choose CYP105A1(UniProtKB:P18326), CYP105A3(UniProtKB:Q59831), CYP105AB3(GenBank:AXG58041.1), CYP105D4(SEQ ID NO.22), CYP105D5(SEQ IDNO.23), CYP105D7(UniProtKB :Q82518) Perform multiple sequence alignment with CYP105P2 (UniProtKB:Q70AS3), and select 6 amino acids (Arg77, Arg88, Arg93, Gly95, Ser96 and Arg197) around the sca-2 _mut substrate binding pocket to design mutants. Using the recombinant strain HFLA-7 constructed in Example 2 (containing plasmid pRSF-sca-2 _mut -Fld-Fpr) as a template, the mutation-containing recombinant plasmids pRSF-sca-2 _mut R77A-Fld-Fpr and pRSF-sca- were constructed. 2 _mut R88A-Fld-Fpr, pRSF-sca-2 _mut R93A-Fld-Fpr, pRSF-sca-2 _mut G95A-Fld-Fpr, pRSF-sca-2 _mut S96A-Fld-Fpr, pRSF-sca-2 _mut R197A-Fld-Fpr, pRSF-sca-2 _mut R88A/S96A-Fld-Fpr. The above seven plasmids were transformed into C41(DE3) to obtain R77A, R88A, R93A, G95A, S96A, R197A and R88A/S96A recombinant strains.

Table 1 Bacterial strains and characteristics

The above recombinant strains (R77A, R88A, R93A, G95A, S96A, R197A and R88A/S96A) were subjected to shake flask fermentation. The fermentation conditions and whole cell preparation conditions were as in Example 1. The reaction system catalyzed by the whole cell was (according to the final concentration Calculation): bacterial cells 50g/L, naringenin 100mg/L. The results are shown in Figure 4. Compared with the HFLA-7 strain (containing plasmid pRSF-sca-2 _mut -Fld-Fpr), the recombinant strains R88A strain (containing plasmid pRSF-sca-2 _mut R88A-Fld-Fpr) and S96A The strain (containing plasmid pRSF-sca-2 _mut S96A-Fld-Fpr) can synthesize 60.9 mg/L and 49.0 mg/L eriodictyol; an increase of 58% and 27% respectively. Strain R88A/S96A (containing plasmid pRSF-sca-2 _mut R88A/S96A-Fld-Fpr) can synthesize 67.2 mg/L of eriodictyol, which is 74% higher than the HFLA-7 strain.

Homology modeling of sca-2 _mut and sca-2 _mut R88A/S96A was performed, and molecular docking was performed with the small molecule naringenin. Although arginine 88 and serine 96 in sca-2 _mut are not directly related to naringenin, However, when arginine at position 88 is mutated to alanine and serine at position 96 is mutated to alanine, the number of hydrogen bonds around the substrate increases (Figure 4) and the hydrophobic interaction is enhanced (Figure 5).

Example 4: Optimization of whole cell conditions to improve the ability to synthesize hydroxylated flavonoids

The whole cell conditions were optimized for the R88A/S96A strain constructed in Example 3. The reaction system catalyzed by the whole cell was (based on the final concentration): 50 g/L bacterial cells and 100 mg/L naringenin. When the reaction pH is set to 8.0, the effect of the reaction temperature (20°C, 25°C, 30°C, 37°C, 40°C) on whole-cell catalysis is examined; when the reaction temperature is set to 37°C, the reaction pH (6.0 , 7.0, 8.0 and 9.0) on whole-cell catalysis. Other reaction conditions are the same as Example 3. The results are shown in Figure 6. The optimal reaction conditions for the R88A/S96A strain were pH 8.0 and the reaction temperature was 37°C. Under these reaction conditions, naringenin with a final concentration of 100 mg/L can produce 71.3 mg/L of eriodictyol.

P450 enzymes are enzymes that use heme as a prosthetic group. When expressing P450 enzymes, enhancing the supply of intracellular heme can increase the activity of P450 enzymes. However, commercial E. coli does not have a heme transporter and cannot utilize directly added heme. We investigated the addition of heme precursor 5-aminolevulinic acid at different final concentrations (50, 100, 200, 300 and 400 mg/L) and different final concentrations (5, Effects of 10, 20, 30 and 40 mg/L) FeSO ₄ on whole cell viability. When 100 mg/L of 5-amino acid levulinic acid and 20 mg/L FeSO ₄ were finally added to the culture medium, 77.3 mg/L of eriodictyol could be produced by whole cell catalysis.

Example 5: Application of P450 sca-2 _mut in the synthesis of other hydroxylated flavonoids

The R88A/S96A recombinant strain constructed in Example 3 was subjected to shake flask fermentation using TB medium (with the addition of 5-aminolevulinic acid at a final concentration of 100 mg/L and FeSO ₄ of 20 mg/L). The fermentation conditions were the same as in Example 1, and Whole cell catalyst was prepared according to the method of Example 1. Whole-cell catalysis was performed using dihydrokaempferol, kaempferol, apigenin, and daidzein at a final concentration of 100 mg/L as substrates. The whole-cell catalytic reaction system contains: substrate 100mg/L, bacterial cells 50g/L, reaction temperature 37°C, reaction pH 8.0, reaction time 12h. The results are shown in Figure 7. The R88A/S96A recombinant strain can produce 66.3mg/L dihydroquercetin, 5.7mg/L quercetin, 31.8mg/L luteolin and 75.1mg/L 7,3', respectively. 4'-Trihydroxyisoflavone.

Although the present invention has been disclosed above in terms of preferred embodiments, they are 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 protection scope of the present invention should be defined by the claims.

Claims (11)

1. Cytochrome P450 enzyme mutant, characterized by taking the amino acid sequence shown in SEQ ID NO.1 as the starting sequence and making any one of the following improvements: (1) Mutation of arginine at position 88 of the amino acid sequence shown in SEQ ID NO.1 to alanine; (2) Mutation of serine at position 96 of the amino acid sequence shown in SEQ ID NO. 1 to alanine; (3) Mute the arginine at position 88 of the amino acid sequence shown in SEQ ID NO. 1 to alanine, and mutate the serine at position 96 to alanine. 2. A gene encoding the mutant of claim 1. 3. Recombinant Escherichia coli expressing the cytochrome P450 enzyme mutant of claim 1. 4. The recombinant Escherichia coli according to claim 3, characterized in that the ferredoxin reductase gene CamA and the ferredoxin gene CamB are also expressed. 5. The recombinant Escherichia coli according to claim 3, wherein the Escherichia coli also expresses a redox chaperone gene; the redox chaperone gene is a gene encoding flavodoxin reductase Fpr of Escherichia coli and A combination of genes encoding any of the flavin oxidizing proteins Fld, FldA, and FldB derived from Escherichia coli and the flavin oxidizing proteins YkuN and YkuP derived from Bacillus subtilis. 6. The recombinant Escherichia coli according to claim 3, characterized in that the recombinant Escherichia coli uses an expression vector to co-express the cytochrome P450 enzyme mutant of claim 1, the flavin oxidation protein derived from Escherichia coli and Escherichia coli. The source of flavin oxidizing protein reductase; the flavin oxidizing protein has the amino acid sequence shown in SEQ ID NO.2; the flavin oxidizing protein reductase has the amino acid sequence shown in SEQ ID NO.3. 7. The recombinant Escherichia coli according to claim 6, wherein the expression vector is pRSFDuet-1. 8. The recombinant Escherichia coli according to any one of claims 3 to 7, characterized in that the Escherichia coli is BL21 (DE3), C41 (DE3) or C43 (DE3). 9. A method for whole-cell catalytic synthesis of hydroxylated flavonoid compounds, characterized in that the recombinant Escherichia coli according to any one of claims 3 to 7 is used as a cell catalyst, in a reaction system containing a substrate, at 20-37 °C for at least 1-12 h; the substrate is one or more of naringenin, dihydrokaempferol, kaempferol, apigenin or daidzein. 10. A method for whole-cell catalytic synthesis of hydroxylated flavonoid compounds, characterized in that the recombinant Escherichia coli according to claim 8 is used as a cell catalyst in a reaction system containing a substrate and reacted at 20-37°C for at least 1 -12 h; the substrate is one or more of naringenin, dihydrokaempferol, kaempferol, apigenin or daidzein. 11. Application of the recombinant Escherichia coli according to any one of claims 3 to 7 or the method according to any one of claims 9 to 10 in the production of products containing one or more hydroxylated flavonoid compounds; the hydroxylated flavonoid compound It is eriodictyol, dihydroquercetin, quercetin, luteolin or 7,3',4'-trihydroxyisoflavone.