CN115975963B - Method for synthesizing hydroxylated flavone compound by using escherichia coli P450 enzyme whole cell catalysis and application thereof - Google Patents
Method for synthesizing hydroxylated flavone compound by using escherichia coli P450 enzyme whole cell catalysis and application thereof Download PDFInfo
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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
Technical Field
The invention relates to 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.
Background
Cytochrome P450 enzymes are enzymes with heme as prosthetic groups, and widely exist in organisms such as animals, plants, archaea, bacteria, eukaryotes and the like. In mammals such as humans, P450 enzymes are widely involved in the degradation of heterologous substances, detoxification, drug metabolism, and synthesis of steroid hormones and vitamins. In plants, P450 enzymes catalyze oxidation reactions such as regional or stereo hydroxylation, epoxidation, dealkylation, dehalogenation, and the like of a range of natural products (e.g., terpenes, polyenes, glycopeptides, alkaloids, fatty acids, steroids). Because of the wide substrate spectrum and variety of catalytic reactions, a large number of P450 enzymes are increasingly being used in the microbiology field to synthesize valuable natural products or drugs. In the past, an extracellular enzymatic catalysis method is mostly adopted, the method usually needs to purify P450 enzyme and coenzyme, and expensive cofactor NAD (P) H and cofactor circulatory system are additionally added, so that the catalytic synthesis cost is high. Compared to extracellular enzymatic catalysis methods, whole cell catalysis methods are alternatives with low catalytic costs.
Flavonoids are a class of polyphenolic compounds which are widely present in plants and which have a basic parent 2-phenyl chromone (C6-C3-C6 structure) and can be classified as flavones, flavonols, isoflavones, etc. according to the parent structure (figure 1). The flavonoid compound has various biological activities such as antioxidation, antivirus, anti-tumor, antibiosis and the like, and has wide application in the field of food and medicine. However, the poor water solubility and instability of flavonoids have limited their use in medicine. The hydroxylation reaction not only can improve the solubility and stability of the flavonoid compounds, but also can enhance the biological activity of the flavonoid compounds and enrich the types of the flavonoid compounds.
Compared with chemical hydroxylation, biocatalytic hydroxylation is an environment-friendly method for obtaining hydroxylated flavone compounds. Coli is the most commonly used expression system and efficient whole cell transformation system for recombinant proteins, while P450 enzymes involved in hydroxylation reactions are poorly soluble and difficult to express in prokaryotes. It has been found that CYP107P2 (Pandey et al, enzyme microb.technology 48 (4-5), 386-392.2011), CYP107Y1 (Pandey et al, enzyme microb.technology 48 (4-5), 386-392.2011), P450BM3 (Chu et al, microb.cell face.15 (1), 135.2016), CYP105D7 (Liu et al, J.mol.catalyst.B: enzyme.132, 91-97.2016) and CYP105A5 (bearings et al, catalysts 12 (10), 1157.2022) can hydroxylate specific flavonoid compounds, but have low conversion rates. Therefore, the development of P450 enzymes which can be efficiently expressed in prokaryotes is of great importance for the synthesis of flavonoids.
Disclosure of Invention
The invention provides a cytochrome P450 enzyme mutant P450sca-2, which takes an amino acid sequence shown in SEQ ID NO.1 as a starting sequence, and carries out any one of the following improvements:
(1) Mutating arginine at position 88 of the amino acid sequence shown in SEQ ID NO.1 to alanine;
(2) Mutating serine at position 96 of the amino acid sequence shown in SEQ ID NO.1 into alanine;
(3) Arginine at position 88 of the amino acid sequence shown in SEQ ID NO.1 was mutated to alanine, and serine at position 96 was mutated to alanine.
The invention also provides genes encoding the mutants.
The invention also provides recombinant escherichia coli for 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 further 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 further expresses a redox partner gene; the redox partner gene comprises a combination of any one of flavodoxin reductase Fpr (SEQ ID NO. 20) of escherichia coli, flavodoxin Fld (SEQ ID NO. 15) of escherichia coli, fldA (SEQ ID NO. 16) of escherichia coli, fldB (SEQ ID NO. 17) of escherichia coli, flavodoxin YkuN (SEQ ID NO. 18) of bacillus subtilis and YkuP (SEQ ID NO. 19) of bacillus subtilis.
In one embodiment, the recombinant E.coli co-expresses a gene encoding the P450sca-2 mutant derived from C.carbophilus (Streptomyces carbophilus), a gene encoding a flavoprotein derived from E.coli Fld, and a gene encoding a flavoprotein reductase derived from E.coli Fpr using an expression vector.
In one embodiment, the flavoprotein has the amino acid sequence shown as SEQ ID NO. 2; the flavin oxidation protein reductase has an amino acid sequence shown as 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 (DE 3), C41 (DE 3) or C43 (DE 3).
The invention also provides a method for synthesizing the hydroxylated flavone compound by whole cell catalysis, which comprises the steps of taking the recombinant escherichia coli as a cell catalyst, and reacting for at least 1-12h at 20-37 ℃ 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 obtained by culturing the recombinant E.coli at 35-37℃to OD 600 The value is 0.6-0.8, and the isopropyl-beta-D-thiogalactoside is added and then is continuously cultured for 12-20 hours at 20-30 ℃ to prepare the compound; wherein the concentration of isopropyl-beta-D-thiogalactoside is 0.1-1mM.
In one embodiment, the fermented broth is centrifuged at 8000rpm at 4℃for 10-20min, the cells are collected, and the cells are washed with potassium phosphate buffer at pH 8.0. After the washing, the washed cells were resuspended in potassium phosphate (containing 5% -10% v/v glycerol) at pH 8.0 to obtain a cell catalyst.
In one embodiment, the reaction system further comprises a potassium phosphate buffer at a concentration of 50-100 mM.
In one embodiment, the flavonoid compounds include, but are not limited to, one or more of naringenin, dihydrokaempferol, kaempferol, daidzein, apigenin.
The invention also provides application of the genetically engineered bacterium in producing eriodictyol (naringenin is used as a substrate), dihydroquercetin (dihydrokaempferol is used as a substrate), quercetin (kaempferol is used as a substrate), luteolin (apigenin is used as a substrate) and 7,3',4' -trihydroxyisoflavone (daidzein is used as a substrate) in various hydroxylated flavone compounds.
The beneficial effects are that:
(1) The invention selects the high-yield hydroxylated flavone compound P450sca-2 through screening mut And obtaining sca-2 by protein engineering mut R88A/S96A mutant enhances P450sca-2 mut Is a catalyst activity of (a).
(2) Screening to obtain endogenous flavopropoxide Fld and flavopropoxide reductase Fpr of Escherichia coli to improve P450sca-2 by reduction partner engineering mut Whole cell catalytic activity.
(3) The invention passes through the optimized P450sca-2 mut The efficiency of catalyzing naringenin to synthesize eriodictyol by the whole-cell catalytic system is 77%; the efficiency of catalyzing the dihydrokaempferol to synthesize the dihydroquercetin is 66%; the efficiency of catalyzing apigenin to synthesize luteolin is 32%; the efficiency of catalyzing the synthesis of 7,3',4' -trihydroxyisoflavone by daidzein is 75%.
Drawings
FIG. 1 shows the skeleton structure and main classification of flavone compounds.
FIG. 2 is a graph showing the effect of P450 enzymes and gene combinations on the production of hydroxylated flavone compounds.
FIG. 3 is a schematic representation of redox partner engineering enhancement of P450sca-2 mut Catalytic efficiency.
FIG. 4 shows an enzyme engineering increase in P450sca-2 mut Catalytic efficiency.
FIG. 5 is a diagram of P450sca-2 mut Sca-2 mut Analysis of R88A/S96A interaction with substrate.
FIG. 6 shows an optimized increase in P450sca-2 in whole cell conditions mut R88A/S96A ability to synthesize hydroxylated flavones.
FIG. 7 is P450sca-2 mut The R88A/S96A is applied to the synthesis of other hydroxylated flavonoid compounds.
Detailed Description
Materials and methods
LB medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract, and sterilizing at 121deg.C for 15min.
TB medium: 12g/L peptone, 5g/L glycerol, 24g/L yeast extract, 17mM potassium dihydrogen phosphate, 72mM dipotassium hydrogen phosphate, and sterilizing at 121deg.C for 15min.
Whole cell catalysis:
(1) Culturing recombinant Escherichia coli at 35-37deg.C to OD 600 The value is 0.6-0.8, and after adding isopropyl-beta-D-thiogalactoside, culturing is continued for 12-20h at 20-30 ℃. Wherein the concentration of isopropyl-beta-D-thiogalactoside is 0.1-1mM.
(2) Centrifuging the fermentation broth obtained in the step (1) at 4 ℃ and 8000rpm for 10-20min, collecting thalli, and washing the thalli with potassium phosphate buffer with pH of 8.0. After the washing, the washed cells were resuspended in potassium phosphate (containing 5% -10% v/v glycerol) at pH 8.0.
(3) Carrying out hydroxylation reaction of flavone compounds by using the bacterial suspension obtained in the step (2), wherein a whole-cell reaction system comprises (based on the final concentration): 50g/L of bacterial cells and 100mg/L of flavonoid compounds; the flavonoids include naringenin, dihydrokaempferol, kaempferol, daidzein and apigenin. The whole cell reacts at 20-37 ℃ for at least 1-12h, after the reaction is finished, a proper amount of reaction liquid is taken out, the ethyl acetate with the same volume is added for extraction, and 3' -hydroxylated flavone compound is obtained after extraction and separation and is analyzed and detected by high performance liquid chromatography.
High performance liquid chromatography:
(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 phase chromatography column ZORBAX Eclipse XDB-C18 (5 μm, 4.6X1250 mm, agilent, USA); column temperature is 40 ℃; flow rate: 0.8mL/min; elution procedure: 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) The wavelength was detected using an ultraviolet detector at 290nm.
Calculation of catalytic efficiency: the concentration of the product (mg/L)/the concentration of the substrate (mg/L). Times.100%.
Example 1: screening for P450 enzymes with high production of hydroxylated flavone compounds
CYP105P2 (SEQ ID NO. 4) derived from Streptomyces coelicolor (Streptomyces peucetius), CYP105D7 (SEQ ID NO. 5) derived from Streptomyces avermitilis (Streptomyces avermitilis), CYP105AB3 (Q87W/T115A/H132L/R191W/G294D, abbreviated P450 moxA) derived from actinomycetes (Nonomuraea recticatena) were selected mut The method comprises the steps of carrying out a first treatment on the surface of the SEQ ID No. 6), CYP105A1 from Streptomyces griseus (Streptomyces griseolus) (R73A/R84A; abbreviated CYP105A1 mut The method comprises the steps of carrying out a first treatment on the surface of the SEQ ID NO. 7), CYP105A3 (G52S/T85F/F89I/T119S/P159A/V194N/D269E/T323A/N363Y/E370V) derived from carbon chain mould (Streptomyces carbophil); abbreviated as P450sca-2 mut The method comprises the steps of carrying out a first treatment on the surface of the SEQ ID NO. 1), synthesizing the genes (nucleotide sequences are respectively shown as SEQ ID NO.8-SEQ ID NO. 12) through codon optimization of escherichia coli, respectively subcloning the genes into Nde I and Xho I sites of pRSFDuet-1 to respectively obtain recombinant plasmids pRSF-105P2, pRSF-105D7 and pRSF-moxA mut 、pRSF-105A1 mut And pRSF-sca-2 mut . Since the CYP105 family is a typical three-component P450 enzyme, requiring transfer of electrons from an electron donor NAD (P) H to the P450 enzyme active center, ferredoxin CamB (SEQ ID NO. 13) and ferredoxin reductase CamA (SEQ ID NO. 14) of Pseudomonas putida (Pseudomonas putida) were selected as reduction chaperones. Synthesizing the ferrosilicon reductase gene CamA and the ferrosilicon gene CamB after codon optimization, and subcloning the CamA and the CamB genes into recombinant plasmids pRSF-105P2, pRSF-105D7 and pRSF-moxA mut 、pRSF-105A1 mut And pRSF-sca-2 mut Between the Nco I and Sac I cleavage sites, recombinant plasmids pRSF-105P2-CamA-CamB, pRSF-105D7-CamA-CamB, pRSF-moxA were obtained mut -CamA-CamB、pRSF-105A1 mut -CamA-CamB and pRSF-sca-2 mut -CamA-CamB. The 5 recombinant plasmids are respectively transformed into large plasmids5 recombinant strains, designated HFLA-1 to HFLA-5, respectively, were obtained from the E.coli expression host C41 (DE 3) (FIG. 2).
Shake flask fermentation of HFLA-1 to HFLA-5 recombinant strains using TB culture when recombinant E.coli is cultured to OD at 37 ℃ 600 At a value of 0.6-0.8, 1mM isopropyl-. Beta. -D-thiogalactoside was added and the culture was continued at 25℃for 20 hours. After the fermentation was completed, the fermentation broth was centrifuged at 8000rpm at 4℃for 10min, and the cells were collected and washed with potassium phosphate buffer at pH 8.0. After the washing, the washed cells were resuspended in potassium phosphate (10% v/v glycerol) at pH 8.0 to give the whole cell catalyst. The whole cell catalyzed reaction system is (based on final concentration): 50g/L of bacterial cells and 100mg/L of naringenin. The results are shown in FIG. 2, recombinant strain HFLA-5 (containing recombinant plasmid pRSF-sca-2 mut CamA-CamB) can produce 20.3mg/L eriodictyol, which are recombinant strains HFLA-2 (containing recombinant plasmid pRSF-105D 7-CamA-CamB), HFLA-3 (containing recombinant plasmid pRSF-moxA) mut CamA-CamB), HFLA-4 (containing recombinant plasmid pRSF-105A 1) mut -CamA-CamB) yield 3.9, 1.3, 1.8 times.
Example 2: reduction partner engineering to increase P450sca-2 mut Catalytic efficiency of (2)
For three-component P450 enzymes, the efficiency of electron transfer is critical for whole cell catalysis. To screen out suitable reduction partners, flavoprotein Fld (SEQ ID NO. 15), fldA (SEQ ID NO. 16), fldB (SEQ ID NO. 17) and flavoprotein YkuN (SEQ ID NO. 18), ykuP (SEQ ID NO. 19) from Bacillus subtilis are selected to be combined with flavodoxin reductase Fpr (SEQ ID NO. 20) from Escherichia coli, respectively, to obtain 5 groups of reduction partner combinations; selecting a combination of ferredoxin Fdx _1499 and ferredoxin reductase FdR _0978 derived from synechococcus (Synechococcus elongates) as a group 6 reduction partner; 6 sets of reduction partners were assembled into the recombinant plasmid pRSF-sca-2 constructed in example 1, respectively mut Recombinant plasmid pRSF-sca-2 is obtained between the Nco I and Sac I cleavage sites mut -Fld-Fpr，pRSF-sca-2 mut -FldA-Fpr，pRSF-sca-2 mut -FldB-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 P450BM3 reduction domain portion (SEQ ID NO. 21) of Bacillus megaterium (Bacillus megaterium) was selected as group 7 reduction partner for fusion to sca-2 mut Construction of recombinant plasmid pRSF-sca-2 at C-terminus mut -BM3. The above 7 recombinant plasmids were transformed into C41 (DE 3) to obtain recombinant strains HFLA-6 to HFLA-12, respectively.
Shake flask fermentation experiments were performed on recombinant strains HFLA-6 to HFLA-12 under the same conditions as in example 1, and after fermentation, the fermentation broth was centrifuged at 8000rpm at 4℃for 10min, and the cells were collected and washed with potassium phosphate buffer at pH 8.0. After the washing, the washed cells were resuspended in potassium phosphate (10% v/v glycerol or 10% w/v glucose) at pH 8.0 to give the whole cell catalyst. The whole cell catalyzed reaction system is (based on final concentration): 50g/L of bacterial cells and 100mg/L of naringenin. As a result, as shown in FIG. 3, recombinant strain HFLA-7 (containing recombinant plasmid pRSF-sca-2 in whole cell catalytic system containing 10% v/v glycerol mut The most potent eriodictyol-producing strain (38.6 mg/L) was the control strain HFLA-5 (containing recombinant plasmid pRSF-sca-2) mut -CamA-CamB) yield 1.9 times.
Example 3: enzyme engineering to improve sca-2 mut Catalytic efficiency of (2)
Multiple sequence alignment was performed with CYP105A1 (uniProtKB: P18326), CYP105A3 (uniProtKB: Q59831), CYP105AB3 (GenBank: AXG 58041.1), CYP105D4 (SEQ ID NO. 22), CYP105D5 (SEQ ID NO. 23), CYP105D7 (uniProtKB: Q82518) and CYP105P2 (uniProtKB: Q70AS 3), and sca-2 was selected mut The mutants were designed with 6 amino acids (Arg 77, arg88, arg93, gly95, ser96 and Arg 197) around the substrate binding pocket. Recombinant strain HFLA-7 constructed in example 2 (containing plasmid pRSF-sca-2) mut -Fld-Fpr) as a template to construct a recombinant plasmid pRSF-sca-2 containing a mutation mut R77A-Fld-Fpr、pRSF-sca-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. And the 7 plasmids are respectively processedTransformation into C41 (DE 3) gave R77A, R88A, R93A, G A, S96A, R197A and R88A/S96A recombinant strains.
TABLE 1 strains and characteristics
The recombinant strains (R77A, R88A, R93A, G95A, S A, R197A and R88A/S96A) were subjected to shake flask fermentation under the same conditions as in example 1 and whole cell preparation under the following conditions (in terms of final concentration): 50g/L of bacterial cells and 100mg/L of naringenin. As a result, the strain was compared with HFLA-7 strain (containing the plasmid pRSF-sca-2 mut Fld-Fpr), recombinant strain R88A strain (containing plasmid pRSF-sca-2 mut R88A-Fld-Fpr) and S96A strains (containing plasmid pRSF-sca-2) mut S96A-Fld-Fpr) can synthesize 60.9mg/L and 49.0mg/L eriodictyol; the improvement is 58% and 27% respectively. Strain R88A/S96A (containing plasmid pRSF-sca-2 mut R88A/S96A-Fld-Fpr) can synthesize 67.2mg/L eriodictyol, 74% higher than HFLA-7 strain.
For sca-2 mut And sca-2 mut R88A/S96A performs homology modeling and molecular docking with small molecule naringenin, although sca-2 mut Arginine at position 88 and serine at position 96 did not directly interact with naringin, but when arginine at position 88 was mutated to alanine and serine at position 96 was mutated to alanine, the number of hydrogen build-up around the substrate increased (FIG. 4) and the hydrophobic interaction was enhanced (FIG. 5).
Example 4: full cell condition optimization to improve the capability of synthesizing hydroxylated flavone
The R88A/S96A strain constructed in example 3 was subjected to whole cell condition optimization, and the whole cell catalyzed reaction system was (based on the final concentration): 50g/L of bacterial cells and 100mg/L of naringenin. When the reaction pH was set to 8.0, the effect of the reaction temperature (20 ℃, 25 ℃,30 ℃, 37 ℃,40 ℃) on whole cell catalysis was examined; the effect of the reaction pH (6.0, 7.0, 8.0 and 9.0) on whole cell catalysis was examined when the reaction temperature was set to 37 ℃. Other reaction conditions were the same as in example 3. As a result, as shown in FIG. 6, the optimal reaction condition for the R88A/S96A strain was pH 8.0, and the reaction temperature was 37 ℃. Under this reaction condition, 71.3mg/L eriodictyol can be produced using naringin at a final concentration of 100mg/L.
The P450 enzyme is an enzyme with heme as a prosthetic group, and the activity of the P450 enzyme can be improved by enhancing intracellular heme supply when the P450 enzyme is expressed. Whereas commercial E.coli did not have heme transporter, direct addition of heme was not available to examine addition of different final concentrations (50,100,200,300 and 400 mg/L) of heme precursor 5-aminolevulinic acid and different final concentrations (5, 10,20,30 and 40 mg/L) of FeSO 4 Effects on whole cell activity. 100mg/L of 5-amino acid levulinic acid and 20mg/L of FeSO are added to the culture medium 4 When whole cell catalysis is used, 77.3mg/L eriodictyol can be produced.
Example 5: p450sca-2 mut Application in synthesizing other hydroxylated flavonoid compounds
The R88A/S96A recombinant strain constructed in example 3 was cultured using TB medium (adding 5-aminolevulinic acid at a final concentration of 100mg/L and FeSO at 20mg/L 4 ) Shake flask fermentation was performed under the same conditions as in example 1, and a whole cell catalyst was prepared in the same manner as in example 1. The full cell catalysis is carried out by taking the dihydrokaempferol, kaempferol, apigenin and daidzein with the final concentration of 100mg/L as substrates. The whole cell catalytic reaction system comprises: substrate 100mg/L, bacterial cell 50g/L, reaction temperature 37 ℃, reaction pH 8.0, reaction time 12h. As shown in FIG. 7, the R88A/S96A recombinant strain can produce 66.3mg/L dihydroquercetin, 5.7mg/L quercetin, 31.8mg/L luteolin and 75.1 mg/L7, 3',4' -trihydroxyisoflavone, respectively.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (11)
1. The cytochrome P450 enzyme mutant is characterized in that the amino acid sequence shown in SEQ ID NO.1 is taken as a starting sequence, and any one of the following improvements is carried out:
(1) Mutating arginine at position 88 of the amino acid sequence shown in SEQ ID NO.1 to alanine;
(2) Mutating serine at position 96 of the amino acid sequence shown in SEQ ID NO.1 into alanine;
(3) Arginine at position 88 of the amino acid sequence shown in SEQ ID NO.1 was mutated to alanine, and serine at position 96 was mutated to alanine.
2. A gene encoding the mutant of claim 1.
3. Recombinant E.coli expressing the cytochrome P450 enzyme mutant according to claim 1.
4. The recombinant E.coli according to claim 3, wherein the ferredoxin reductase gene CamA and the ferredoxin gene CamB are also expressed.
5. The recombinant escherichia coli of claim 3, wherein the escherichia coli further expresses a redox partner gene; the redox partner gene is a combination of a gene for encoding flavodoxin reductase Fpr of escherichia coli, flavodoxin Fld, fldA, fldB derived from escherichia coli and a gene for encoding any one of flavodoxin YkuN and YkuP derived from bacillus subtilis.
6. The recombinant E.coli according to claim 3, wherein the recombinant E.coli co-expresses the cytochrome P450 enzyme mutant of claim 1, E.coli-derived flavoprotein oxide and E.coli-derived flavoprotein oxide reductase using an expression vector; the flavopropoxide has an amino acid sequence shown as SEQ ID NO. 2; the flavin oxidation protein reductase has an amino acid sequence shown as SEQ ID NO. 3.
7. The recombinant E.coli according to claim 6, wherein the expression vector is pRSFDuet-1.
8. The recombinant escherichia coli according to any one of claims 3-7, wherein the escherichia coli is BL21 (DE 3), C41 (DE 3) or C43 (DE 3).
9. A method for synthesizing a hydroxylated flavone compound by whole cell catalysis, which is characterized in that the recombinant escherichia coli according to any one of claims 3-7 is used as a cell catalyst, and the recombinant escherichia coli reacts at least 1-12h at 20-37 ℃ in a reaction system containing a substrate; the substrate is one or more of naringenin, dihydrokaempferol, kaempferol, apigenin or daidzein.
10. A method for synthesizing a hydroxylated flavone compound by whole cell catalysis, which is characterized in that the recombinant escherichia coli as defined in claim 8 is used as a cell catalyst, and the recombinant escherichia coli is reacted at 20-37 ℃ for at least 1-12h in a reaction system containing a substrate; the substrate is one or more of naringenin, dihydrokaempferol, kaempferol, apigenin or daidzein.
11. Use of a recombinant escherichia coli according to any one of claims 3 to 7 or a method according to any one of claims 9 to 10 for the production of a product comprising one or more hydroxylated flavone compounds; the hydroxylated flavone compound is eriodictyol, dihydroquercetin, quercetin, luteolin or 7,3',4' -trihydroxyisoflavone.
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