CN117448356A - Method for synthesizing estriol by utilizing cytochrome P450 enzyme recombinant bacteria - Google Patents
Method for synthesizing estriol by utilizing cytochrome P450 enzyme recombinant bacteria Download PDFInfo
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- CN117448356A CN117448356A CN202311405388.9A CN202311405388A CN117448356A CN 117448356 A CN117448356 A CN 117448356A CN 202311405388 A CN202311405388 A CN 202311405388A CN 117448356 A CN117448356 A CN 117448356A
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- estriol
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- 229960001348 estriol Drugs 0.000 title claims abstract description 36
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 title claims abstract description 32
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 title claims abstract description 32
- 241000894006 Bacteria Species 0.000 title claims abstract description 28
- PROQIPRRNZUXQM-ZXXIGWHRSA-N estriol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H]([C@H](O)C4)O)[C@@H]4[C@@H]3CCC2=C1 PROQIPRRNZUXQM-ZXXIGWHRSA-N 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 48
- 239000000758 substrate Substances 0.000 claims abstract description 14
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims abstract description 13
- 229960005309 estradiol Drugs 0.000 claims abstract description 13
- 229930182833 estradiol Natural products 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000000855 fermentation Methods 0.000 claims description 16
- 230000004151 fermentation Effects 0.000 claims description 16
- -1 estriol compound Chemical class 0.000 claims description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 6
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- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 abstract description 10
- 241000741989 Streptomyces nanshensis Species 0.000 abstract description 2
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- 239000003814 drug Substances 0.000 description 8
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- DNXHEGUUPJUMQT-CBZIJGRNSA-N Estrone Chemical compound OC1=CC=C2[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 DNXHEGUUPJUMQT-CBZIJGRNSA-N 0.000 description 1
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- 208000019255 Menstrual disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- TUZYXOIXSAXUGO-UHFFFAOYSA-N Pravastatin Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CC(O)C=C21 TUZYXOIXSAXUGO-UHFFFAOYSA-N 0.000 description 1
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- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000029849 luteinization Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
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- 210000003635 pituitary gland Anatomy 0.000 description 1
- TUZYXOIXSAXUGO-PZAWKZKUSA-N pravastatin Chemical compound C1=C[C@H](C)[C@H](CC[C@@H](O)C[C@@H](O)CC(O)=O)[C@H]2[C@@H](OC(=O)[C@@H](C)CC)C[C@H](O)C=C21 TUZYXOIXSAXUGO-PZAWKZKUSA-N 0.000 description 1
- 229960002965 pravastatin Drugs 0.000 description 1
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- 238000007086 side reaction Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P33/14—Hydroxylating at 16 position
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The invention discloses a method for synthesizing estriol by utilizing cytochrome P450 enzyme recombinant bacteria, which belongs to the technical field of biology, and comprises the steps of firstly constructing a CYP154C34 gene and a reduction partner RhFRED gene co-expression vector from Streptomyces nanshaensis, and expressing in escherichia coli to obtain the cytochrome P450 enzyme recombinant bacteria; after culturing, the substrate estradiol is fed by a resting cell method for biological conversion, and an estriol product is obtained. This reaction is strictly regioselective and stereoselective, yielding a single product. The method obviously improves the added value of the product and has higher industrialization prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for synthesizing an estriol compound by utilizing a cytochrome P450 enzyme recombinant bacterium, in particular to a method for obtaining the estriol compound by utilizing a novel cytochrome P450 enzyme CYP154C34 and a reduction partner RhFRED coexpression recombinant bacterium to carry out bioconversion.
Background
Cytochrome P450 enzymes act as biocatalysts, are capable of catalyzing unactivated hydrocarbon bonds in compounds under mild conditions, and have stringent regioselectivity and stereoselectivity. It has great application potential in the fields of environmental pollutant degradation, fine chemical synthesis and medicine synthesis biology. With the great development of DNA sequencing technology, the accumulation of a large amount of microbial genome data information, and the development of new P450 enzymes with industrial application value are becoming a focus of attention. The existing industrial success cases of synthesis of drug intermediates such as Artemisia annua acid, pravastatin and the like and drugs through the catalysis of P450 enzyme lead people to have more confidence in the industrial synthesis of more types of drugs catalyzed by the P450 enzyme.
Steroid drugs are the second most commonly used drugs next to antibiotics, most of which are compounds with novel biological activities obtained by structural modification of natural steroid compounds. The estradiol can be modified to obtain the estriol medicine. Estriol has smaller estrogenic activity, about 6 times of estrone in oral activity, no carcinogenesis and no change in-vivo estradiol level after administration. Estriol has selective action on the vagina and cervical canal without affecting the uterine entity and endometrium. Animal experiments show that the estriol has stronger effect on the angulation of the vaginal epithelium than the estradiol, can promote the hyperplasia of the vaginal epithelium, the keratosis of surface cells, the angiogenesis of mucous membrane and the healing of injury of the vaginal epithelium, but has weaker effect on the weight gain of the uterus of a mouse; meanwhile, the estriol can strengthen the function of cervical cells, enable cervical muscle fibers to proliferate and increase the elasticity and softness of the cervix. In addition, estriol has feedback inhibition effect on hypothalamus and pituitary gland, but does not inhibit ovulation, and only has obvious influence on luteal phase, so that the estriol can be used as auxiliary medicine for mid-term induced labor and induced abortion and for treating various menstrual diseases. Estriol also has obvious effect on hematopoietic system, can reduce permeability and fragility of blood vessel, and can be used for treating various hemorrhages. The composition also has the function of rapidly increasing peripheral leucocytes, and generally takes effect 1-3 days after administration, but has shorter action time and is effective for reducing leucocytes caused by radiotherapy and chemotherapy.
Disclosure of Invention
The invention aims to disclose a method for synthesizing an estriol compound by utilizing cytochrome P450 enzyme recombinant bacteria.
The aim of the invention is realized by the following technical scheme:
a cytochrome P450 enzyme has a coding gene of CYP154C34 gene, and the CYP154C34 gene sequence is shown in SEQ ID NO. 3.
The recombinant strain comprises a coding gene of the cytochrome P450 enzyme, a RhFRED gene and a host cell, wherein the coding gene of the cytochrome P450 enzyme is a CYP154C34 gene, the CYP154C34 gene sequence is shown as SEQ ID NO.3, and the RhFRED gene sequence is shown as SEQ ID NO. 4.
A method for synthesizing an estriol compound by utilizing cytochrome P450 enzyme recombinant bacteria specifically comprises the following steps:
and (3) performing bioconversion on a substrate estradiol by using fermentation liquor of cytochrome P450 enzyme recombinant bacteria to obtain estriol.
Further, the cytochrome P450 enzyme recombinant bacterium fermentation broth for coexpression of the CYP154C34 gene and the RhFRED gene is prepared by the following method:
connecting the pET28a vector with the CYP154C34 gene and the RhFRED gene by using a seamless cloning technology to obtain a recombinant plasmid pET28a-CYP154C34-RhFRED, transferring the plasmid into escherichia coli BL21 (DE 3) to construct recombinant escherichia coli, namely cytochrome P450 enzyme recombinant bacteria, and coexpression of the CYP154C34 gene and the RhFRED gene by the cytochrome P450 enzyme recombinant bacteria; obtaining a bacterial fermentation broth for co-expressing CYP154C34 gene and RhFRED gene through screening, pre-culturing and expanding culturing; the gene sequence of pET28a is shown as SEQ ID NO.1, the gene sequence of pET28a-CYP154C34-RhFRED is shown as SEQ ID NO.2, the gene sequence of CYP154C34 is shown as SEQ ID NO.3, and the gene sequence of RhFRED is shown as SEQ ID NO. 4.
Further, the conditions for obtaining estriol by bioconversion of substrate estradiol by using fermentation broth of cytochrome P450 enzyme recombinant bacteria co-expressing CYP154C34 gene and RhFRED gene are as follows:
centrifugally re-suspending the fermentation liquor of the recombinant bacteria in a buffer solution, keeping the pH value at 7.4, adding an estradiol substrate for bioconversion, and extracting and separating the fermentation liquor after oscillating reaction for 24 hours at 25 ℃ to obtain an estriol compound; the concentration of the substrate in the buffer was 100. Mu.M.
Further, the buffer A was formulated as 40mM dipotassium hydrogen phosphate, 10mM potassium dihydrogen phosphate and 10vol% glycerol.
Further, the pre-culture and the expansion culture are specifically as follows:
adding the constructed recombinant escherichia coli into an LB (LB) culture medium, and culturing for 16 hours at 37 ℃ to obtain seed liquid of the recombinant escherichia coli; inoculating the seed solution into a Terrific Broth culture medium, wherein the volume ratio of the seed solution to the Terrific Broth culture medium is 1:100, and performing expansion culture at 37 ℃ under the condition of 180r/min of shaking table rotation speed until the bacterial liquid OD 600 The value reaches 0.8, then IPTG is added to induce the bacteria to express protein, the temperature is reduced to 22 ℃ at the same time, the culture is continued for 20 hours, and the bacteria are collected centrifugally. The final concentration of IPTG in the Terrific Broth medium was 0.1mM.
The beneficial effects of the invention are as follows: (1) the CYP154C34 is a novel enzyme which is not disclosed, recombinant bacteria of the novel enzyme can convert estradiol compounds to generate estriol compounds, and the products are mainly obtained from chemical synthesis methods in industry at present; (2) CYP154C34 enzyme has strict regioselectivity and stereoselectivity on steroid compounds, and can produce the only product, namely estriol, so that the separation and extraction efficiency can be improved; (3) the catalytic system constructed by the invention can selectively perform oxidation reaction under mild conditions, and has great application value in the field of biological pharmacy compared with the complex process flow of the traditional chemical reaction; (4) the biotransformation by utilizing recombinant bacteria can greatly reduce side reaction products, and the desired products can be easily separated, thus achieving high efficiency. (5) The conversion rate of the catalytic system constructed by the invention to the estradiol for catalyzing and producing the estriol is about 95%, the utilization efficiency of raw materials and energy sources is improved, and the catalytic system has a huge market prospect.
Drawings
FIG. 1 is a flow chart of CYP154C34 and substrate bioconversion;
fig. 2 is an HPLC profile of the experimental sample.
Detailed Description
The invention is further illustrated in detail below with reference to examples. The embodiments are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.
Example 1
FIG. 1 is a flow chart of the bioconversion of CYP154C34 with a substrate, specifically comprising the steps of:
1. preparing coexpression recombinant escherichia coli:
the pET28a plasmid was linearized with NdeI and XhoI as a pair of restriction enzymes.
The coding gene sn6073 sequence of CYP154C34 is shown in SEQ ID NO.3, the template is the whole genome of Streptomyces nanshaensis SCSIO01066, and the forward primer sequence is shown in SEQ ID NO.5 specifically: 5'-CGCGCGGCAGCCATATGatgagcccctacgccgaaccc-3' the reverse primer sequence used is SEQ ID NO.6, specifically: 5'-GCAGCACgccgccgtgcagccg-3'.
SEQ ID NO.3:
atgagcccctacgccgaacccgcttctcccggctcctcgtccaccccttcttcgtccaccccttccgcggcctccggcacggccggcgacggcaccgctcccgcgggccacgaaggctgccccatcgtcatcgacccgatggtggcccgcctcgacgaggagacccgcctgctgcgcgacgccggcgccctcacgcgtatcgaactgctcggcgtaccggcctggtcgatcacccggcacgcggacgcccggcggctgctgacggaccgccgtctggtcaaggacatcgggcagtggaacctgtggcgcagcggcgaggtcacccacgagtggccgctgatcggcatgatcgactccggccgctcgatgttcaccgtggacggcgccgagcaccgccggctgcggacgaagaccgcgcaggccgtcacgccccgccgcctggagaatctgcgtcccgtcatcgagagcatcacgcaccggctgctggacgatctggaggcgcagggcgccgagggccccgtggacctcaagtccgtcttcgcgctgccgctgccgatgtccgtgatcagcgcgctcatgggcgtcgacccggcgctccatccgcggctgcacacgctctacaaggcgttcttctcgatgctcacgccgcaggacgagcggctcgcggtcatcgacgaactggacggcatcttcacggacatggtccgggcccgcaccgccgagccgcgcgacgatctgaccagtgcgctgatcctcgcggacgagggcggcgagccgctgagcgaggaggaggtcgtcggcaacctcaaggcgatggtcgccgcggggcacgagacgacgatcggtctgatcctcaactccgtacgggcgctgctcacccacaccgaccaactggagacggtgctcaagggcgaggccggctgggacgcggtgatcgaggagaccctgcgctgggacaccccgaccacgcatctgctgatgcgcttcgcgacggaagacatcgaggtcggcggcggggtcatccgcgagggcgagggcgtggtgatctcgtaccgcgcgatcggctgggacaccgagcaacacggcccggacgccgaccggttcgacatcacacggcccacccgcaaccgccatatgaccttcggccacggcccgcacatctggccgggcgcggcgctctcgcggctggaggcgggcatcgccctgcccgcgctcttcgaccgcttccccggcatctccttcgccgtgcccgtcgaggagatcgtcaaccagccggtgctgacgcagaacgacctccggtcctttcccgtacggctgcacggcggctga
The coding gene RhFRED has a RhFRED sequence shown as SEQ ID NO.4, and is obtained by PCR, the template is pET28b-RhFRED plasmid, and the forward primer sequence is SEQ ID NO.7 specifically: 5'-acggcggcGTGCTGCACCGCCATCAACCG-3' the reverse primer sequence used is SEQ ID NO.8, specifically: 5'-GGTGGTGGTGCTCGAGTCAGAGGCGCAGGGCCAGCC-3'.
Mixing PCR product and linearization carrier in certain proportion, and inAnd (3) under the catalysis of the II recombinase, reacting for 30min at 37 ℃ to obtain recombinant plasmid pET28a-CYP154C34-RhFRED, wherein the gene sequence of pET28a-CYP154C34-RhFRED is shown as SEQ ID NO. 2. Transferring the recombinant plasmid pET28b-CYP154C2-RhFRED into competent cells of escherichia coli BL21 (DE 3), and screening out monoclonal by using an LB solid medium containing kanamycin to obtain recombinant escherichia coli with multienzyme coexpression.
2. Pre-culture and expansion culture of recombinant E.coli:
inoculating the co-expression recombinant escherichia coli into an LB liquid culture medium, and culturing for 16 hours at the temperature of 37 ℃ and the rotating speed of 220rpm under the pH value of 7.4-7.6 to obtain seed liquid of the recombinant escherichia coli. Inoculating the seed solution into a Terrific Broth culture medium for expansion culture and fermentation; the volume ratio of the seed solution to the Terrific Broth culture medium is 1:100, and after inoculation, the recombinant escherichia coli is continuously cultured at 37 ℃ until the bacterial solution OD 600 The value reaches 0.8, IPTG is added to induce the thalli to express a large amount of protein, the temperature is reduced to 22 ℃ at the same time, the rotating speed is reduced to 180rpm, the thalli are continuously cultured for 20 hours, and the thalli are centrifugally collected. The concentration of IPTG in the Terrific Broth medium was 0.1mM.
3. Bioconversion of estriol synthesis:
suspending the collected thalli in a buffer solution A, wherein the mass ratio of the fermentation liquor to the buffer solution A before centrifugation is 5:1, the pH value is kept at 7.4, a substrate estradiol is added for biological conversion, and after 24 hours of shaking reaction at 25 ℃, the fermentation liquor is extracted and separated to obtain the estriol compound. The concentration of the substrate in buffer A was 100. Mu.M. The buffer solution A has a formula of 40mM of dipotassium hydrogen phosphate, 10mM of potassium dihydrogen phosphate and 10% of glycerol by volume. Wherein, the method for extracting and separating the fermentation broth to obtain the estriol compound comprises the following steps: adding equal volume of ethyl acetate for extraction, collecting and combining ethyl acetate layers, and obtaining estriol after ethyl acetate is completely volatilized.
Example 2
After adding 100. Mu.l of methanol to the estriol obtained in example 1 for dissolution, the estriol was subjected to high performance liquid chromatography. HPLC analysis in the invention adopts a Waters E2695 high performance liquid chromatography system and a COSMIL packed column of Singapore, mobile phase adopts 35-100 vol% methanol-water gradient elution for 20min, detection wavelength is 280m, and flow rate is 1ml/min.
Fig. 2 is an HPLC profile of an experimental sample, which is a standard of estradiol and an estriol profile obtained after 24 hours of reaction, and the biocatalytic conversion rate is 94.7% calculated according to the area of integration of estriol peak/(area of integration of estriol peak+area of integration of estriol peak) in the experimental sample. This result shows that the obtained product is the only product and the conversion efficiency is high, indicating strict substrate selectivity and catalytic efficiency.
The estriol compound obtained by the catalytic system is expected to have better application value in the field of biological pharmacy, and compared with the complex process flow of traditional chemical synthesis and the disadvantages of multiple byproducts, the catalytic system has single product and extremely high catalytic efficiency, and has better economic benefit and environmental benefit.
Claims (7)
1. The cytochrome P450 enzyme is characterized in that the coding gene is CYP154C34 gene, and the CYP154C34 gene sequence is shown in SEQ ID NO. 3.
2. A recombinant bacterium of a cytochrome P450 enzyme is characterized by comprising a coding gene of the cytochrome P450 enzyme according to claim 1, a RhFRED gene and a host cell, wherein the coding gene of the cytochrome P450 enzyme is a CYP154C34 gene, the CYP154C34 gene sequence is shown as SEQ ID NO.3, and the RhFRED gene sequence is shown as SEQ ID NO. 4.
3. A method for synthesizing an estriol compound by utilizing cytochrome P450 enzyme recombinant bacteria is characterized by comprising the following steps:
bioconversion of the substrate estradiol to produce estriol using the fermentation broth of the recombinant cytochrome P450 enzyme bacterium of claim 2.
4. The method according to claim 1, wherein the fermentation broth of the recombinant cytochrome P450 enzyme is prepared by the following method:
connecting the pET28a vector with the CYP154C34 gene and the RhFRED gene together to obtain a recombinant plasmid pET28a-CYP154C34-RhFRED, transferring the plasmid into escherichia coli BL21 (DE 3) to construct a cytochrome P450 enzyme recombinant bacterium, and coexpression of the CYP154C34 gene and the RhFRED gene by the cytochrome P450 enzyme recombinant bacterium; obtaining a bacterial fermentation broth through screening, pre-culture and expansion culture; the gene sequence of pET28a is shown as SEQ ID NO.1, the gene sequence of pET28a-CYP154C34-RhFRED is shown as SEQ ID NO.2, the gene sequence of CYP154C34 is shown as SEQ ID NO.3, and the gene sequence of RhFRED is shown as SEQ ID NO. 4.
5. The method according to claim 4, wherein the conditions for obtaining estriol by bioconversion of substrate estradiol with fermentation broth of recombinant cytochrome P450 enzyme are:
centrifugally re-suspending the fermentation liquor of the recombinant bacteria in a buffer solution, keeping the pH value at 7.4, adding an estradiol substrate for bioconversion, and extracting and separating the fermentation liquor after oscillating reaction for 24 hours at 25 ℃ to obtain an estriol compound; the concentration of the substrate in the buffer was 100. Mu.M.
6. The method of claim 5, wherein the buffer is formulated as 40mM dipotassium hydrogen phosphate, 10mM potassium dihydrogen phosphate, and 10 vol.% glycerol.
7. The method according to claim 4, wherein the pre-culture and the expansion culture are specifically:
adding the constructed cytochrome P450 enzyme recombinant bacteria into an LB culture medium, and culturing for 16 hours at 37 ℃ to obtain seed liquid of the cytochrome P450 enzyme recombinant bacteria; inoculating the seed solution into a Terrific Broth culture medium, wherein the volume ratio of the seed solution to the Terrific Broth culture medium is 1:100, and performing expansion culture at 37 ℃ under the condition of 180r/min of shaking table rotation speed until the bacterial liquid OD 600 The value reaches 0.8, then IPTG is added to induce the bacteria to express protein, the temperature is reduced to 22 ℃ at the same time, the culture is continued for 20 hours, and the bacteria are collected centrifugally. The final concentration of IPTG in the Terrific Broth medium was 0.1mM.
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