CN115975963A - Method for synthesizing hydroxylated flavone compound by using escherichia coli P450 enzyme whole-cell catalysis and application - Google Patents
Method for synthesizing hydroxylated flavone compound by using escherichia coli P450 enzyme whole-cell catalysis and application Download PDFInfo
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Abstract
The invention discloses a method for synthesizing a hydroxylated flavone compound by utilizing escherichia coli P450 enzyme whole-cell catalysis and application, belonging to the technical field of genetic engineering. The invention screens out P450sca-2 of the high-yield hydroxylated flavone compound mut The sca-2 is optimized through reduction partner engineering, enzyme engineering and whole cell catalysis condition mut The efficiency of R88A/S96A for catalyzing naringenin to synthesize eriodictyol reaches 77 percent; catalytic dihydrokaempferolThe efficiency of the finished dihydroquercetin reaches 66 percent; the efficiency of catalyzing apigenin to synthesize luteolin reaches 32%; the efficiency of catalyzing daidzein to synthesize 7,3',4' -trihydroxy isoflavone reaches 75%, and a new strategy is provided for improving the biosynthesis of hydroxylated compounds.
Description
Technical Field
The invention relates to a method for synthesizing a hydroxylated flavone compound by utilizing escherichia coli P450 enzyme whole-cell catalysis and application, belonging to the technical field of genetic engineering.
Background
Cytochrome P450 enzymes are enzymes taking heme as a prosthetic group 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 heterologous substance degradation, detoxification, drug metabolism, and synthesis of steroid hormones and vitamins. In plants, P450 enzymes can catalyze a series of oxidation catalytic reactions of natural products (such as terpenes, polyenes, glycopeptides, alkaloids, fatty acids and steroids) such as regional or three-dimensional hydroxylation, epoxidation, dealkylation, dehalogenation and the like. Due to the wide substrate spectrum and the diversity of catalytic reactions of P450 enzymes, a large number of P450 enzymes are increasingly being used in the field of microbial pharmaceuticals to synthesize valuable natural products or drugs. In the past, an extracellular enzymatic catalysis method is mostly adopted, P450 enzyme and coenzyme are often required to be purified, expensive cofactor NAD (P) H and cofactor circulating system are additionally required to be added, and the catalytic synthesis cost is high. Compared to extracellular enzymatic catalysis methods, whole-cell catalysis methods are an alternative with low catalytic costs.
Flavonoids are a class of polyphenol compounds widely present in plants, which have a basic parent nucleus of 2-phenylchromone (C6-C3-C6 structure), and can be classified into flavones, flavonols, flavanones, flavanonols, isoflavones, etc. according to the parent structure (fig. 1). The flavonoid compound has multiple biological activities of oxidation resistance, virus resistance, tumor resistance, bacteria resistance and the like, and is widely applied in the field of food and medicine. However, the poor and unstable water solubility of flavone compounds limits their use in medicine. The hydroxylation reaction can not only improve the solubility and stability of the flavonoid compounds, but also enhance the biological activity of the flavonoid compounds and enrich the types of the flavonoid compounds.
Compared with chemical hydroxylation reaction, the biological catalytic hydroxylation reaction is an environment-friendly method for obtaining the hydroxylated flavone compound. Coli is the most common expression system and efficient whole-cell transformation system for recombinant proteins, and the P450 enzyme participating in hydroxylation reaction has poor solubility and difficult expression in prokaryotes. It has been found that the expression of 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: enzyme 132, 91-97.2016) and CYP105A5 (Subedi et al, catalysis 12 (10), 1157.2022) in Escherichia coli can hydroxylate a specific flavone compound, but has problems of low conversion rate and the like. Therefore, the development of P450 enzyme capable of being efficiently expressed in prokaryotes for synthesizing flavonoids is of great significance.
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 is improved by any one of the following steps:
(1) Mutating arginine at position 88 of the amino acid sequence shown in SEQ ID NO.1 into alanine;
(2) The serine at the 96 th site of the amino acid sequence shown in SEQ ID NO.1 is mutated into alanine;
(3) The arginine at the 88 th position of the amino acid sequence shown in SEQ ID NO.1 is mutated into alanine, and the serine at the 96 th position is mutated into alanine.
The invention also provides genes encoding the mutants.
The invention also provides recombinant escherichia 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 further expresses a ferredoxin reductase gene CamA (SEQ ID No. 14) and a ferredoxin gene CamB (SEQ ID No. 13).
In one embodiment, the escherichia 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 Flad (SEQ ID No. 15) derived from Escherichia coli, fladA (SEQ ID No. 16) derived from Escherichia coli, fldB (SEQ ID No. 17) derived from Escherichia coli, flavodoxin YkuN (SEQ ID No. 18) derived from Bacillus subtilis and YkuP (SEQ ID No. 19) derived from Bacillus subtilis.
In one embodiment, the recombinant escherichia coli co-expresses the gene encoding the c.thermophilus-derived P450sca-2 mutant, the escherichia coli-derived flavodoxin gene Fld, and the escherichia coli-derived flavodoxin reductase gene Fpr using an expression vector.
In one embodiment, the flavodoxin has the amino acid sequence shown as SEQ ID No. 2; the flavodoxin 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 escherichia 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 takes the recombinant escherichia coli as a cell catalyst to react 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 the recombinant E.coli cultured at 35-37 ℃ to OD 600 Adding isopropyl-beta-D-thiogalactoside, and culturing at 20-30 deg.C for 12-20 hr to obtain the final product with a value of 0.6-0.8; wherein the concentration of isopropyl-beta-D-thiogalactoside is 0.1-1mM.
In one embodiment, the fermented broth is centrifuged at 8000rpm for 10-20min at 4 deg.C to collect the bacterial cells, and the cells are washed with pH 8.0 potassium phosphate buffer. After completion of the washing, the washed cells were resuspended with potassium phosphate (containing 5% -10% of glycerol) at pH 8.0 to obtain a cell catalyst.
In one embodiment, the reaction system further contains potassium phosphate buffer at a concentration of 50 to 100 mM.
In one embodiment, the flavone compounds include, but are not limited to, one or more of naringenin, dihydrokaempferol, kaempferol, daidzein, apigenin.
The invention also provides application of the genetic engineering bacteria in producing various hydroxylated flavone compounds of eriodictyol (taking naringenin as a substrate), dihydroquercetin (taking dihydrokaempferol as a substrate), quercetin (taking kaempferol as a substrate), luteolin (taking apigenin as a substrate) and 7,3',4' -trihydroxyisoflavone (taking daidzein as a substrate).
Has the advantages that:
(1) The invention obtains the high-yield hydroxylated flavone compound P450sca-2 by screening mut And obtaining sca-2 by protein engineering mut Enhancement of P450sca-2 by R88A/S96A mutant mut The catalytic activity of (2).
(2) Screening out Escherichia coli endogenous flavodoxin Fld and flavodoxin reductase Fpr to improve P450sca-2 by reduction chaperone engineering mut Whole cell catalytic activity.
(3) The invention is realized by optimized P450sca-2 mut The full-cell catalytic system catalyzes the naringenin to synthesize the eriodictyol with the efficiency of 77 percent; the efficiency of catalyzing the dihydrokaempferol to synthesize the dihydroquercetin is 66 percent; the efficiency of catalyzing apigenin to synthesize luteolin is 32%; the efficiency of catalyzing daidzein to synthesize 7,3',4' -trihydroxy isoflavone is 75%.
Drawings
FIG. 1 shows the skeleton structure and the main classification of flavone compounds.
FIG. 2 shows the effect of P450 enzymes and gene combinations on the production of hydroxylated flavonoids.
FIG. 3 shows oxidationEngineering of reduction partners to increase P450sca-2 mut Catalytic efficiency.
FIG. 4 shows the improvement of P450sca-2 by enzyme engineering mut Catalytic efficiency.
FIG. 5 shows P450sca-2 mut And sca-2 mut And (3) analyzing the interaction of R88A/S96A and the substrate.
FIG. 6 shows the improvement of P450sca-2 by whole cell condition optimization mut R88A/S96A has the capability of synthesizing hydroxylated flavone.
FIG. 7 shows P450sca-2 mut Application of R88A/S96A in synthesizing other hydroxylated flavonoids compounds.
Detailed Description
Materials and methods
LB culture medium: 10g/L peptone, 10g/L sodium chloride and 5g/L yeast extract, and sterilizing at 121 ℃ for 15min.
TB culture medium: 12g/L peptone, 5g/L glycerin, 24g/L yeast extract, 17mM potassium dihydrogen phosphate, 72mM dipotassium hydrogen phosphate, sterilizing at 121 ℃ for 15min.
Whole-cell catalysis:
(1) Culturing recombinant Escherichia coli at 35-37 deg.C to OD 600 The value is 0.6-0.8, adding isopropyl-beta-D-thiogalactoside, and culturing at 20-30 deg.C for 12-20 hr. Wherein the concentration of isopropyl-beta-D-thiogalactoside is 0.1-1mM.
(2) Centrifuging the fermentation liquid obtained in the step (1) at 4 ℃ and 8000rpm for 10-20min, collecting thalli, and washing the thalli with a potassium phosphate buffer solution with the pH of 8.0. After completion of the washing, the washed cells were resuspended with potassium phosphate (containing 5% -10% v/v of glycerol) at pH 8.0.
(3) Carrying out hydroxylation reaction on the flavone compound by using the bacterial suspension obtained in the step (2), wherein a whole-cell reaction system comprises (by final concentration): 50g/L of thallus cells and 100mg/L of flavonoid compounds; the flavonoids include naringenin, dihydrokaempferol, kaempferol, daidzein and apigenin. Reacting whole cell at 20-37 deg.C for at least 1-12h, taking out appropriate amount of reaction solution, adding equal volume of ethyl acetate for extraction, extracting, separating to obtain 3' hydroxylated flavone compound, and analyzing and detecting with high performance liquid chromatography.
High performance liquid chromatography analysis:
(1) Mobile phase: phase A is ultrapure water containing 0.1% trifluoroacetic acid, phase B is methanol containing 0.1% trifluoroacetic acid
(2) And (3) chromatographic column: reverse chromatography column ZORBAX Eclipse XDB-C18 (5 μm, 4.6X 250mm, agilent, USA); the column temperature was 40 ℃; flow rate: 0.8mL/min; elution procedure: 0-1min,10% B;1-10min,10% -40% by weight of B;10-20min,40% -60% by weight of B;20-23min,60% by weight B;23-25min,60% -10% B;25-27min,10% B.
(3) The wavelength was detected at 290nm using an ultraviolet detector.
Calculation of catalytic efficiency: concentration of product (mg/L)/concentration of substrate (mg/L). Times.100%.
Example 1: p450 enzyme for screening high-yield hydroxylated flavone compound
CYP105P2 (SEQ ID NO. 4) derived from Streptomyces bauctii (Streptomyces peuceteus), CYP105D7 (SEQ ID NO. 5) derived from Streptomyces avermitilis (Streptomyces avermitilis), CYP105AB3 (Q87W/T115A/H132L/R191W/G294D) derived from actinomycetes (Nonomuraea rececticatena), abbreviated as P450moxA, were selected mut (ii) a SEQ ID NO. 6), CYP105A1 (R73A/R84A; abbreviated CYP105A1 mut (ii) a SEQ ID NO. 7), CYP105A3 (G52S/T85F/F89I/T119S/P159A/V194N/D269E/T323A/N363Y/E370V, derived from a carbon chain-philic mold (Streptomyces carbophil); abbreviated as P450sca-2 mut (ii) a SEQ ID NO. 1), synthesizing the gene (nucleotide sequences are respectively shown as SEQ ID NO.8-SEQ ID NO. 12) through codon optimization of escherichia coli, subcloning the gene into Nde I and Xho I sites of pRSFDuet-1 respectively to obtain recombinant plasmids pRSF-105P2, pRSF-105D7 and pRSF-moxA respectively mut 、pRSF-105A1 mut And pRSF-sca-2 mut . Since the CYP105 family is a typical three-component P450 enzyme requiring the transfer of electrons from the 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 the reduction chaperones. Ferredoxin after codon optimization of synthesisThe genes of the white reductase gene CamA and the ferredoxin gene CamB are subcloned into recombinant plasmids pRSF-105P2, pRSF-105D7 and pRSF-moxA mut 、pRSF-105A1 mut And pRSF-sca-2 mut The recombinant plasmids pRSF-105P2-CamA-CamB, pRSF-105D7-CamA-CamB and pRSF-moxA were obtained between the restriction sites of Nco I and Sac I mut -CamA-CamB、pRSF-105A1 mut -CamA-CamB and pRSF-sca-2 mut -CamA-CamB. The 5 recombinant plasmids are respectively transformed into an escherichia coli expression host C41 (DE 3) to obtain 5 recombinant strains which are respectively named as HFLA-1 to HFLA-5.
The HFLA-1 to HFLA-5 recombinant strains were subjected to shake flask fermentation using TB culture when recombinant E.coli was grown to OD at 37 ℃ 600 When the value is 0.6-0.8, the incubation is continued for 20h at 25 ℃ after addition of 1mM isopropyl-beta-D-thiogalactoside. After completion of the fermentation, the fermentation broth was centrifuged at 8000rpm at 4 ℃ for 10min, and the cells were collected and washed with a pH 8.0 potassium phosphate buffer. After completion of the washing, the washed cells were resuspended with potassium phosphate (10% v/v in glycerol) at pH 8.0 to obtain a whole cell catalyst. The reaction system of whole-cell catalysis is (by final concentration): 50g/L of bacterial cells and 100mg/L of naringenin. As a result, as shown in FIG. 2, the recombinant strain HFLA-5 (containing the recombinant plasmid pRSF-sca-2) mut -CamA-CamB) capable of producing 20.3mg/L eriodictyol, respectively, 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-105A1 mut -CamA-CamB) yield 3.9, 1.3, 1.8 times.
Example 2: engineering of reduction partners to increase P450sca-2 mut Catalytic efficiency of
For three-component P450 enzymes, the efficiency of electron transfer is critical for whole-cell catalysis. In order to screen out suitable reduction partners, flavodoxin Fla (SEQ ID NO. 15), fla (SEQ ID NO. 16), fladB (SEQ ID NO. 17) derived from Escherichia coli and flavodoxin YkuN (SEQ ID NO. 18), ykuP (SEQ ID NO. 19) derived from Bacillus subtilis were selected to be combined with flavodoxin reductase Fpr (SEQ ID NO. 20) derived from Escherichia coli, respectively, to obtain 5 groups of reduction partnersCombining; selecting as group 6 combination of reducing partners ferredoxin Fdx _1499 and ferredoxin reductase FdR _0978 derived from Synechococcus elongatus (Synechococcus elongatus); the 6 groups of reduction partners were assembled in combination into the recombinant plasmid pRSF-sca-2 constructed in example 1, respectively mut The recombinant plasmid pRSF-sca-2 was obtained between the Nco I and Sac I cleavage sites of (1) 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 to be fused to sca-2 mut The C-terminal of (a) was constructed as a recombinant plasmid pRSF-sca-2 mut -BM3. The above 7 recombinant plasmids were transformed into C41 (DE 3) to obtain recombinant strains HFLA-6 to HFLA-12, respectively.
The shake flask fermentation experiments were carried out on the recombinant strains HFLA-6 to HFLA-12 under the same conditions as in example 1, after completion of the fermentation, the fermentation broth was centrifuged at 8000rpm for 10min at 4 ℃ to collect the cells, and the cells were washed with potassium phosphate buffer at pH 8.0. After completion of the washing, the washed cells were resuspended with potassium phosphate (10% by volume of glycerol or 10% by volume of glucose) at pH 8.0 to obtain a whole cell catalyst. The reaction system of whole-cell catalysis is (by final concentration): 50g/L of bacterial cells and 100mg/L of naringenin. As a result, as shown in FIG. 3, in the whole-cell catalytic system containing 10% v/v of glycerol, the recombinant strain HFLA-7 (containing the recombinant plasmid pRSF-sca-2) mut Fld-Fpr) had the strongest ability to produce eriodictyol (38.6 mg/L), and was the control strain HFLA-5 (containing the recombinant plasmid pRSF-sca-2) mut -CamA-CamB) yield 1.9 times.
Example 3: enzymatic engineering to improve sca-2 mut Catalytic efficiency of
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) of the CYP105 family were selected for multiple sequence alignment, and sca-2 was selected mut 6 amino acids surrounding the substrate binding pocket (Arg 77, arg88, arg93, gly95, ser96 and Arg197 Design mutants. The recombinant strain HFLA-7 (containing plasmid pRSF-sca-2) constructed in example 2 mut -Fld-Fpr) as a template to construct a recombinant plasmid pRSF-sca-2 containing mutations 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 above 7 plasmids were transformed into C41 (DE 3) to obtain recombinant strains of R77A, R88A, R93A, G95A, S96A, R197A and R88A/S96A, respectively.
TABLE 1 strains and characteristics
The recombinant strains (R77A, R88A, R93A, G95A, S96A, R197A and R88A/S96A) were subjected to shake flask fermentation under the conditions and whole-cell preparation conditions as in example 1, and the reaction system of whole-cell catalysis was (in final concentration): 50g/L of bacterial cells and 100mg/L of naringenin. The results are shown in FIG. 4, which is consistent with the HFLA-7 strain (containing plasmid pRSF-sca-2) mut Fld-Fpr) recombinant strain R88A (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/S896A (containing plasmid pRSF-sca-2) mut R88A/S96A-Fld-Fpr) can synthesize 67.2mg/L eriodictyol, which is increased by 74% compared with HFLA-7 strain.
For sca-2 mut And sca-2 mut R88A/S896A was homologously modeled and molecularly docked with the small molecule naringenin, albeit sca-2 mut The middle arginine at position 88 and serine at position 96 did not directly react with naringenin, but when arginine at position 88 was mutated to alanine and serine at position 96 was mutated to alanine, the amount of hydrogen build-up around the substrate increased (FIG. 4) and the hydrophobic effect was enhanced (FIG. 5).
Example 4: optimizing and improving the capability of synthesizing hydroxylated flavone under the condition of whole cells
The R88A/S96A strain constructed in example 3 is subjected to whole-cell condition optimization, and the reaction system of whole-cell catalysis is as follows (by final concentration): 50g/L of thallus 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 reaction pH (6.0, 7.0, 8.0 and 9.0) on whole-cell catalysis was examined when the reaction temperature was set at 37 ℃. Other reaction conditions were the same as in example 3. As shown in FIG. 6, the optimum reaction conditions for the R88A/S96A strain were pH 8.0 and the reaction temperature was 37 ℃. Under the reaction conditions, 71.3mg/L of eriodictyol can be produced by using naringenin with the 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. Commercial E.coli, without the heme transporter, could not utilize the heme directly added, and examined the addition of the heme precursor 5-aminolevulinic acid at different final concentrations (50, 100,200,300 and 400 mg/L) and FeSO at different final concentrations (5, 10,20,30 and 40 mg/L) 4 Effect on whole cell activity. The 5-amino acid levulinic acid of 100mg/L and FeSO of 20mg/L are added into the culture medium 4 When the cell is full-cell catalyzed, 77.3mg/L eriodictyol can be produced.
Example 5: p450sca-2 mut Application in synthesizing other hydroxylated flavonoids compounds
The R88A/S896A recombinant strain constructed in example 3 was cultured in TB medium (supplemented with 5-aminolevulinic acid at a final concentration of 100mg/L and FeSO at a final concentration of 20 mg/L) 4 ) A shake flask fermentation was carried out under the same fermentation conditions as in example 1, and a whole-cell catalyst was prepared in the same manner as in example 1. Performing whole-cell catalysis with dihydrokaempferol, kaempferol, apigenin and daidzein with final concentration of 100mg/L as substrates. The whole-cell catalytic reaction system comprises: 100mg/L of substrate, 50g/L of bacterial cells, 37 ℃ of reaction temperature, 8.0 of reaction pH and 12 hours of reaction time. As shown in FIG. 7, the R88A/S896A recombinant strain produced 66.3mg/L dihydroquercetin, 5.7mg/L quercetin, 31.8mg/L luteolin, and 75.1 mg/L7, 3',4' -trihydroxyisoflavone, respectively.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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 (10)
1. The cytochrome P450 enzyme mutant is characterized in that an 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 into alanine;
(2) The serine at the 96 th site of the amino acid sequence shown in SEQ ID NO.1 is mutated into alanine;
(3) Arginine at position 88 of the amino acid sequence shown in SEQ ID NO.1 is mutated into alanine, and serine at position 96 is mutated into alanine.
2. A gene encoding the mutant of claim 1.
3. Recombinant Escherichia coli expressing the cytochrome P450 enzyme mutant of claim 1 or expressing the cytochrome P450 enzyme represented by SEQ ID No. 1.
4. The recombinant Escherichia coli according to claim 3, wherein ferredoxin reductase gene CamA and ferredoxin gene CamB are further expressed.
5. The recombinant E.coli of claim 3, wherein said E.coli further expresses a redox partner gene; the redox chaperone gene comprises a combination of flavodoxin reductase Fpr of escherichia coli and any one of flavodoxin Flad, fla, fldB and flavodoxin YkuN and YkuP derived from bacillus subtilis.
6. The recombinant Escherichia coli according to claim 3, wherein the cytochrome P450 enzyme mutant of claim 1, E.coli-derived flavodoxin and E.coli-derived flavodoxin reductase are co-expressed in the recombinant Escherichia coli using an expression vector; the flavodoxin has an amino acid sequence shown as SEQ ID NO. 2; the flavodoxin reductase has an amino acid sequence shown as SEQ ID NO. 3.
7. The recombinant E.coli of claim 6, wherein said expression vector is pRSFDuet-1.
8. The recombinant E.coli of any one of claims 3 to 7, wherein said E.coli includes but is not limited to BL21 (DE 3), C41 (DE 3) or C43 (DE 3).
9. A method for synthesizing hydroxylated flavone compound by whole cell catalysis is characterized in that recombinant Escherichia coli as claimed in any one of claims 3 to 8 is used as a cell catalyst to react for at least 1-12h at 20-37 ℃ in a reaction system containing a substrate; the substrate comprises one or more of naringenin, dihydrokaempferol, kaempferol, apigenin or daidzein.
10. Use of the recombinant escherichia coli of any one of claims 3 to 7 or the method of claim 9 for the production of a product comprising one or more hydroxylated flavone compounds; the hydroxylated flavone compounds include: eriodictyol, dihydroquercetin, quercetin, luteolin, or 7,3',4' -trihydroxyisoflavone.
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| "WP_000796310.1", 《NCBI-BLAST》 * |
| "WP_000807751.1", 《NCBI-BLAST》 * |
| LUAN LUONG CHU 等: "Hydroxylation of diverse flavonoids by CYP450 BM3 variants: biosynthesis of eriodictyol from naringenin in whole cells and its biological activities", 《MICROBIAL CELL FACTORIES》, vol. 15, no. 1 * |
| 张晓龙 等: "基于合成生物技术构建高效生物制造系统的研究进展", 《合成生物学》, vol. 2, no. 06 * |
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