CN115820581A - Cytochrome P450 mutant enzyme, recombinant bacterium, preparation method and application - Google Patents
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Abstract
The invention discloses a cytochrome P450 mutant enzyme, a recombinant bacterium, a preparation method and application, and belongs to the technical field of biology. The recombinant strain is a co-expression vector of a CYP154C2 (M191F/V285L) mutant enzyme gene and a reduction partner RhFRED gene, and is expressed in escherichia coli; after the culture, the substrates testosterone and androstenedione are fed by a resting cell method for biotransformation to obtain a 2 alpha-position hydroxylation product. The cytochrome P450 mutant enzyme and the recombinant bacterium have strict regioselectivity and stereoselectivity, and a single product is generated. The conversion rate of converting testosterone and androstenedione is improved by about 10 times compared with wild type, the utilization rate of raw materials and energy is improved, the production cost of the 2 alpha-hydroxylated steroid compound is reduced, and the application value in industrial production is higher.
Description
Technical Field
The invention belongs to the technical field of synthetic biology, and particularly relates to a technology for synthesizing a 2 alpha-hydroxylated steroid compound by using cytochrome P450 enzyme, specifically to a technology for synthesizing a 2 alpha-hydroxylated steroid compound by using cytochrome P450 enzyme CYP154C2 (M191F/V285L) The mutant and a co-expression recombinant bacterium of a reduction partner RhFRED are subjected to biotransformation to obtain 2 alpha-hydroxy steroid compounds such as 2 alpha-hydroxy testosterone, 2 alpha-hydroxy androstenedione and the like.
Background
Compared with a chemical catalyst, the biological catalyst cytochrome P450 has many advantages which cannot be compared with the chemical catalyst, for example, cytochrome P450 enzyme can catalyze carbon-hydrogen bonds which are not activated in a compound under relatively mild conditions, has very strict regioselectivity and stereoselectivity in the aspect of selectivity, and has wide application prospect in the field of synthesis of fine chemicals and medicines. Today, the rapid development of DNA sequencing technology, the increasing and accumulation of massive microbial genome sequencing data, and the search for the yet-to-be-discovered P450 enzyme with industrial application value is becoming a focus. The successful industrial case that the intermediates of the drugs such as hydrocortisone, pravastatin and the like and the drugs are synthesized by P450 enzyme catalysis has made people have more confidence that the P450 enzyme can catalyze more different types of drugs for industrial synthesis.
The steroid drugs are the second main class of drugs which are only second to antibiotics in current clinical application drugs, and most of the steroid drugs are compounds with new biological activity obtained by modifying the structure of natural steroid compounds. The structural modification reaction of the steroid compound comprises desaturation, alcohol oxidation, ring breaking, epoxidation and the like. Structural modifications to steroids may cause significant changes in the physicochemical and pharmaceutical properties of the compound. For example, the novel prednisone derivatives 15 β,17, 20 β, 21-tetrahydroxypregna-4-ene-3, 11-dione and 15 β,17, 20 β, 21-tetrahydroxypregna-1,4-diene-3, 11-dione, as well as 15 β -hydroxylated variants, are all potential candidates for drug design and process development.
Disclosure of Invention
The invention aims to provide a cytochrome P450 mutant enzyme, a recombinant bacterium, a preparation method and application of synthesizing a 2 alpha-hydroxylated steroid compound by using the cytochrome P450 mutant enzyme.
The purpose of the invention is realized by the following technical scheme:
a cytochrome P450 mutant enzyme, wherein the nucleotide sequence of the cytochrome P450 mutant enzyme is shown as SEQ ID NO. 3.
A recombinant bacterium comprising the cytochrome P450 mutant enzyme.
Further, the recombinant bacterium is prepared by the following method:
cloning CYP154C2-RhFRED gene without His label from pET28B-CYP154C2-RhFRED (patent No. CN 110885868B) by using seamless cloning technology, introducing into the region between Nco I and Xho I of pET28a to obtain pET28a-CYP154C2-RhFRED, and mutating M191F and V285L respectively by using site-directed mutagenesis technology to obtain pET28a-CYP154C2 (M191F/V285L) -RhFRED. And transferring the plasmid into escherichia coli BL21 (DE 3) to construct and obtain a recombinant bacterium. The gene sequence of pET28a is shown in SEQ ID NO.1, and pET28a-CYP154C2 (M191F/V285L) The sequence of the-RhFRED gene is shown as SEQ ID NO.2, wherein CYP154C2 (M191F/V285L) The gene sequence is shown as SEQ ID NO.3, and the RhFRED gene sequence is shown as SEQ ID NO. 4.
An application of the recombinant bacterium in synthesizing 2 alpha-hydroxylated steroid compounds.
Further, the application specifically comprises:
expanding and culturing the recombinant bacteria to the bacterial liquid OD 600 When the value reaches 0.8 and above, adding IPTG and 5-aminolevulinic acid salt to induce the bacteria to express protein, simultaneously reducing the temperature to 22 ℃, continuously culturing for 20h, and centrifugally collecting the bacteria;
suspending the collected thallus in 50mM phosphate buffer solution, keeping pH at 7.4, adding steroid substrate for biotransformation, performing oscillation reaction at 25 deg.C, and extracting and separating the fermentation broth to obtain 2-hydroxy steroid compound.
Further, the final concentration of IPTG in TB medium was 0.1mM and the concentration of 5-aminolevulinate in TB medium was 0.5mM.
Furthermore, the concentration of the substrate in the phosphate buffer solution is 100 mu M, and the substrate is a steroid compound such as testosterone or androstenedione.
Further, phosphate buffer consisted of phosphate at a final concentration of 50mM (pH = 7.4) and glycerol at a volume of 10% of the phosphate buffer.
Further, the substrate is testosterone, and the obtained 2-hydroxylated steroid compound is 2 alpha-hydroxytestosterone through biotransformation.
Further, the substrate is androstenedione, and the 2-hydroxylated steroid compound obtained through biotransformation is 2 alpha-hydroxyandrostenedione.
The advantages and innovation points of the invention are as follows: (1) CYP154C2 according to the invention (M191F/V285L) The recombinant bacteria of the novel enzyme are novel enzymes which are not disclosed, can efficiently convert steroid compounds such as testosterone, androstenedione and the like into 2 alpha-position hydroxylation products, and the products are mainly prepared by a chemical synthesis method in the industry at present; (2) CYP154C2 (M191F/V285L) The enzyme has strict regioselectivity and stereoselectivity on steroid compounds, only produces 2 alpha-hydroxy steroid, and does not produce other isomers, so that the separation efficiency can be improved; (3) the catalytic reaction system prepared by the invention can perform selective oxidation reaction under mild conditions, and has very wide application prospect in the field of biological drug synthesis compared with the traditional chemical reaction with more complex process flow; (4) by utilizing the biotransformation of the recombinant bacteria, side reaction products can be obviously reduced, and the target product is easy to separate, thereby achieving high efficiency. (5) The catalytic system constructed by the invention has higher conversion efficiency on specific steroid compounds, improves the 2 alpha position hydroxylation conversion rate of testosterone and androstenedione compared with the wild type by nearly ten times, greatly improves the utilization efficiency of raw materials and energy, and can generate more objective economic benefit.
Drawings
FIG. 1 shows CYP154C2 (M191F/V285L) A scheme for bioconversion with a substrate;
FIG. 2 is an HPLC chromatogram of an experimental product, wherein FIG. 2A is example 1 and FIG. 2B is example 2;
Detailed Description
The present invention will be further described in detail with reference to examples. The embodiments are provided to facilitate a better understanding of the invention and are not intended to limit the invention.
Example 1
FIG. 1 shows CYP154C2 (M191F/V285L) The flow chart of the biotransformation with the substrate specifically comprises the following steps:
1. preparing mutant enzyme gene and co-expressing recombinant escherichia coli:
the pET28a plasmid was linearized with restriction enzymes NcoI and XhoI to give a linearized vector.
The sequence of the CYP154C2-RhFRED coding gene CYP154C2-RhFRED is shown in SEQ ID NO.5, the seamless cloning technology can be utilized to clone the CYP154C2-RhFRED gene without the His label from pET28B-CYP154C2-RhFRED (patent number: CN 110885868B) through PCR, and the sequence of the used forward primer is SEQ ID NO.6, which specifically comprises the following steps: 5'-AGGAGATATACCATGacgactcgtatcgccctggac-3', the reverse primer sequence is SEQ ID NO.7, which specifically comprises: 5'-GGTGGTGGTGCTCGAGtcagagtcgcagggccagccg-3'.
Mixing the PCR product and the linearized vector in a certain proportion
And (3) reacting at 37 ℃ for 30min under the catalysis of the recombinase to obtain a recombinant plasmid pET28a-CYP154C2-RhFRED. The recombinant plasmid pET28a-CYP154C2 is obtained by taking pET28a-CYP154C2-RhFRED as a template and utilizing the site-directed mutagenesis technology (M191F) -RhFRED, the forward primer sequence used is SEQ ID No. 8: 5'-CCACCGTCTCCGCGAAGATCCCGGCGAGC-3', the reverse primer sequence is SEQ ID NO.9, which specifically comprises: 5'-GCTCGCCGGGATCTTCGCGGAGACGGTGG-3'. Similarly, the directional mutation technology is used for preparing pET28a-CYP154C2 (M191F) Obtaining recombinant plasmid pET28a-CYP154C2 by taking RhFRED as a template (M191F/V285L) -RhFRED, the forward primer sequence used is SEQ ID No. 10: 5'-AGCGGATCAGGAGGTGGGAGGTCGG-3', the reverse primer sequence is SEQ ID NO.11The body is as follows: 5'-CCGACCTCCCACCTCCTGATCCGCT-3'. The recombinant plasmid pET28b-CYP154C2 (M191F/V285L) Introducing RhFRED into competent cells of Escherichia coli BL21 (DE 3), and screening single clones by using LB solid culture medium containing kanamycin to obtain recombinant Escherichia coli co-expressed by multiple enzymes.
2. Pre-culture and scale-up culture of recombinant E.coli:
inoculating the coexpression recombinant Escherichia coli into LB liquid culture medium, culturing at 37 deg.C and 220rpm under pH of 7.4-7.6 for 16 hr to obtain seed solution of recombinant Escherichia coli. Inoculating the seed solution into a TB culture medium for amplification culture and fermentation, wherein the volume ratio of the seed solution to the TB culture medium is 1; after inoculation, the recombinant Escherichia coli is continuously cultured for 3-4h at 37 ℃ and the rotating speed of a shaking table of 220rpm/min until the OD of the bacterial liquid 600 And (3) when the value reaches 0.8, adding IPTG and 5-aminolevulinic acid salt to induce the thalli to express a large amount of protein, simultaneously reducing the temperature to 22 ℃, continuously culturing for 20h, and centrifugally collecting the thalli. The concentration of IPTG in TB medium was 0.1mM and the concentration of 5-aminolevulinic acid salt in TB medium was 0.5mM.
3. Biotransformation to synthesize 2 alpha-hydroxytestosterone:
the fermentation broth was centrifuged at low speed to collect the cells, and the collected cells were resuspended in phosphate buffer consisting of phosphate at a final concentration of 50mM and 10vol% glycerol, maintaining pH at 7.4. Adding testosterone, carrying out oscillation reaction at 25 ℃ for 24 hours, adding equal volume of ethyl acetate for extraction, collecting and combining ethyl acetate layers, and completely volatilizing ethyl acetate to obtain a product containing 2 alpha-hydroxytestosterone. The volume ratio of the fermentation liquid before centrifugation to the phosphate buffer solution is 5:1 (for example, 100ml of phosphate buffer solution is added to 500ml of the centrifuged thallus of the fermentation liquid), and the concentration of the testosterone in the phosphate buffer solution is 100 mu M.
Example 2
And (3) replacing the substrate in the step 3 of the example 1 with androstenedione, wherein the concentration of the androstenedione in a phosphate buffer solution is 100 mu M, and the rest conditions are unchanged to finally obtain the 2 alpha-hydroxyandrostenedione.
Example 3
To the 2 α -hydroxytestosterone obtained in example 1 or the 2 α -hydroxyandrostenedione obtained in example 2, 100 μ L of methanol was added for dissolution and HPLC detection analysis was performed. In the invention, the HPLC analysis adopts a Waters E2695 high performance liquid chromatography system of Singapore and a COSMOSIL packed column, the mobile phase adopts 35 vt-100 vt% methanol-water gradient elution for 10min, the detection wavelength is 240nm, and the flow rate is 1ml/min.
Fig. 2 is an HPLC profile of an experimental sample, wherein fig. 2A is a profile of a standard of testosterone (below) and a profile of a product containing 2 α -hydroxytestosterone obtained after 24 hours of reaction (above), retention times of testosterone and 2 α -hydroxytestosterone are 9.521min and 8.321min, respectively, and a conversion rate of the biocatalysis is calculated to be 67% according to a peak integral area of 2 α -hydroxytestosterone/(a peak integral area of 2 α -hydroxytestosterone + a peak integral area of testosterone) in the experimental sample; fig. 2B shows a standard of androstenedione (below) and a product spectrum containing 2 α -hydroxyandrostenedione obtained after 24 hours of reaction (above), the retention time of androstenedione and 2 α -hydroxyandrostenedione is 9.061min and 7.635min, respectively, and the conversion rate of biocatalysis is 39% calculated according to the peak integral area of 2 α -hydroxyandrostenedione/(peak integral area of 2 α -hydroxyandrostenedione + peak integral area of androstenedione) in the experimental sample. The results all show that the obtained product is a unique product and the conversion efficiency is extremely high, which is improved by nearly 10 times compared with the wild-type CYP154C2, thereby indicating the stricter substrate selectivity and the high catalytic efficiency.
The 2 alpha-hydroxylated steroid compound obtained by the catalytic system is expected to have better application value in the field of biological pharmacy, and compared with the disadvantages of complex process flow and multiple byproducts of traditional chemical synthesis, the product obtained by the catalytic system is single, the catalytic efficiency is extremely high, and better economic benefit and environmental benefit are achieved.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should all embodiments be exhaustive. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Claims (10)
1. A cytochrome P450 mutant enzyme is characterized in that the nucleotide sequence of the cytochrome P450 mutant enzyme is shown as SEQ ID NO. 3.
2. A recombinant bacterium comprising the cytochrome P450 mutant enzyme according to claim 1.
3. The recombinant bacterium according to claim 2, which is prepared by the following method:
introducing CYP154C2-RhFRED gene into pET28a plasmid to obtain pET28a-CYP154C2-RhFRED, and respectively mutating M191F and V285L by using site-directed mutagenesis technology to obtain pET28a-CYP154C2 (M191F/V285L) -RhFRED; pET28a-CYP154C2 (M191F/V285L) Transferring the-RhFRED plasmid into escherichia coli BL21 (DE 3) to obtain a recombinant strain; wherein the gene sequence of the pET28a plasmid is shown in SEQ ID NO.1, and pET28a-CYP154C2 (M191F/V285L) The sequence of the-RhFRED gene is shown as SEQ ID NO.2, CYP154C2 (M191F/V285L) The gene sequence is shown as SEQ ID NO.3, and the RhFRED gene sequence is shown as SEQ ID NO. 4.
4. Use of a recombinant bacterium according to any one of claims 2 to 3 for the synthesis of 2 α -hydroxylated steroids.
5. The application according to claim 4, characterized in that it is in particular:
expanding and culturing the recombinant bacteria to the bacterial liquid OD 600 When the value reaches 0.8 and above, adding IPTG and 5-aminolevulinic acid salt to induce the bacteria to express protein, simultaneously reducing the temperature to 22 ℃, continuing culturing, and centrifugally collecting the bacteria;
suspending the collected thallus in 50mM phosphate buffer solution, keeping pH at 7.4, adding steroid substrate for biotransformation, oscillating reaction at 25 deg.C, extracting and separating the fermentation broth to obtain 2-hydroxy steroid compound.
6. The use of claim 5, wherein the method for the expanded culture of the recombinant bacteria comprises:
adding the recombinant bacteria into an LB culture medium, and culturing for 16h at 37 ℃ to obtain a seed solution of the recombinant escherichia coli; inoculating the seed solution into a TB culture medium, wherein the volume ratio of the seed solution to the TB culture medium is 1 600 The value reaches 0.8 and above.
7. Use according to claim 5, wherein the final concentration of IPTG in TB medium is 0.1mM and the concentration of 5-aminolevulinate in TB medium is 0.5mM.
8. Use according to claim 5, characterized in that the phosphate buffer consists of phosphate at a final concentration of 50mM, pH =7.4 and glycerol at a volume of 10% of the phosphate buffer.
9. Use according to claim 4, wherein the substrate is testosterone and the 2-hydroxylated steroid compound obtained by biotransformation is 2 α -hydroxytestosterone.
10. Use according to claim 1, characterized in that the substrate is androstenedione and the 2-hydroxylated steroid compound obtained by biotransformation is 2 α -hydroxyandrostenedione.
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| CN110885868A (en) * | 2019-11-15 | 2020-03-17 | 浙江理工大学 | Method for synthesizing 2 α -hydroxylated steroid compound by using cytochrome P450 enzyme |
| CN113980883A (en) * | 2021-12-02 | 2022-01-28 | 浙江理工大学 | Recombinant escherichia coli for high-yield hydroxylated steroid bulk drug and application thereof |
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|---|---|---|---|---|
| CN110885868A (en) * | 2019-11-15 | 2020-03-17 | 浙江理工大学 | Method for synthesizing 2 α -hydroxylated steroid compound by using cytochrome P450 enzyme |
| CN113980883A (en) * | 2021-12-02 | 2022-01-28 | 浙江理工大学 | Recombinant escherichia coli for high-yield hydroxylated steroid bulk drug and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| QIANWEN, WANG;BINGBING, MA;SHINYA, FUSHINOBU;CHUNFANG, ZHANG;LIAN-HUA, XU: "Regio- and stereoselective hydroxylation of testosterone by a novel cytochrome P450 154C2 from Streptomyces avermitilis.", vol. 522, no. 2, 31 December 2020 (2020-12-31) * |
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