Cytochrome P450 enzyme mutant and hydroxylation application thereof
By introducing specific mutations at key sites of the cytochrome P450 BM3 enzyme, its catalytic activity and selectivity were optimized, solving the problems of low catalytic activity and insufficient selectivity in the 6β-hydroxylation reaction of steroidal compounds, and achieving efficient biosynthesis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENYANG PHARMA UNIV
- Filing Date
- 2026-02-02
- Publication Date
- 2026-05-12
AI Technical Summary
Existing P450 enzymes exhibit low catalytic activity and insufficient selectivity in the 6β-hydroxylation of steroidal compounds, making it difficult to meet the needs of industrial production.
By introducing single or multiple mutations at specific sites of cytochrome P450 BM3 enzymes, unique mutants such as W72F, L75A, and L82A can be formed, thereby optimizing their catalytic activity and selectivity.
It significantly improves the conversion rate and selectivity of 6β-hydroxylation of steroidal compounds, provides an efficient biosynthetic pathway, and overcomes the shortcomings of existing technologies.
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Figure CN122012422A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of enzyme engineering and genetic engineering, specifically to a mutant based on cytochrome P450 enzyme and its application in catalyzing the 6β-hydroxylation of specific steroid compounds. Background Technology
[0002] Steroid drugs are widely used clinically to treat various diseases due to their important pharmacological activities, including anti-inflammatory, anticancer, anti-allergic, and contraceptive effects. The biological activity of these drugs is closely related to their molecular structure, and achieving regio- and stereoselective hydroxylation of the steroid skeleton is one of the key steps in regulating their pharmacological functions. Chemical hydroxylation methods typically suffer from harsh reaction conditions, poor selectivity, and severe environmental pollution. In contrast, biocatalysis using cytochrome P450 (CYP) enzymes offers significant advantages such as mild reaction conditions and high regio- and stereoselectivity, and has become an important strategy for synthesizing high-value-added steroid drugs.
[0003] Cytochrome P450 BM3 is a compound derived from Bacillus megaterium (Bacillus megaterium). Bacillus megaterium Naturally occurring fusion-type monooxygenases (P450) have attracted much attention due to their high electron transfer efficiency and fast catalytic rate. Recently, the P450 BM3 mutant LG23, obtained through directed evolution, has been reported to perform 7β-hydroxylation on various steroid substrates (such as testosterone and nandrolone). However, for the 6β-hydroxylation reaction, which is also of great value in the synthesis of steroid drugs, existing P450 enzymes generally suffer from low catalytic activity, narrow substrate spectrum, and insufficient selectivity, making it difficult to meet the needs of industrial production.
[0004] Currently, no publicly available technology discloses a P450 mutant capable of efficiently 6β-hydroxylating specific steroid substrates such as 1,4-androsadienedione (ADD) and 4-androsadienedione (4AD) through single- or multi-point mutation. Therefore, there is an urgent need to develop a novel P450 enzyme mutant capable of efficiently and selectively catalyzing the 6β-hydroxylation of steroidal compounds (such as 1,4-androsadienedione) to overcome the current technological bottlenecks. Summary of the Invention
[0005] The purpose of this invention is to provide a cytochrome P450 enzyme mutant and its application in catalyzing the 6β-hydroxylation of specific steroidal compounds (such as ADD, 4AD, etc.), aiming to solve the technical problems of low activity and poor selectivity of existing P450 enzymes in catalyzing the 6β-hydroxylation of the aforementioned specific steroidal substrates. The mutant is based on the LG23 backbone, and by introducing specific substitutions at amino acid residues at positions 72, 75, 78, 82, 85, 87, 88, 181, and 437, unique single- or multi-site mutants are formed. This combination of mutation sites is not found in any publicly available technology for the 6β-hydroxylation of the aforementioned specific steroidal substrates, and experiments have confirmed that it exhibits unique and excellent 6β-hydroxylation performance for these substrates.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: The present invention provides a cytochrome P450 BM3 mutant, which is based on the P450 BM3 mutant LG23 (with the amino acid sequence shown in SEQ ID NO: 2) and involves single or multiple mutations at amino acid residues 72, 75, 78, 82, 85, 87, 88, 181 and 437.
[0007] Specifically, the mutant is selected from any of the following: W72F, L75A, L75F, L78A, L82A, L82C, L82G, L82T, G85A, G87A, G87V, L181A, L437, G85S / S88L, G87A / S88T, G87A / S88T / Q188L.
[0008] The present invention provides a DNA molecule that encodes a nucleotide sequence or a complementary sequence thereof of a cytochrome P450 enzyme mutant as described above.
[0009] The present invention provides a recombinant expression plasmid containing DNA molecules as described above.
[0010] The present invention provides a genetically engineered bacterium containing the recombinant expression plasmid as described above.
[0011] This invention provides the application of the cytochrome P450 enzyme mutant, the DNA molecule, the recombinant expression plasmid, or the genetically engineered bacteria in catalyzing the generation of 6β-hydroxylated products from steroidal compounds.
[0012] This invention also provides a method for catalytically generating 6β-hydroxylated products from steroidal compounds, comprising: (1) Prepare recombinant expression plasmids encoding the P450 BM3 mutant; (2) The recombinant expression plasmid was transferred into the host strain to obtain the genetically engineered bacteria; (3) Using steroidal compounds as substrates, genetically engineered bacteria are used to carry out whole-cell catalytic reactions to generate the corresponding 6β-hydroxylated products.
[0013] The steroidal compound is selected from the following:
[0014] The whole-cell catalytic reaction was carried out in genetically engineered bacteria containing the mutant under conditions of pH 7.0-8.0, temperature 16℃-25℃, and reaction time 10-24 hours.
[0015] The beneficial effects of this invention are: This invention uses the amino acid sequence shown in SEQ ID NO. 2 as an initial template and, through rational design, performs site-directed mutagenesis at key sites, successfully obtaining a series of novel mutants capable of efficiently and selectively catalyzing the 6β-hydroxylation of various steroidal compounds. Compared to the parent enzyme LG23, the mutants obtained in this invention exhibit significantly improved selectivity for the 6β-hydroxylation of the aforementioned substrates and a substantial increase in conversion rate. This effectively overcomes the problems of insufficient activity and selectivity of P450 enzymes in this reaction in existing technologies, providing a powerful tool for the green and efficient biosynthesis of 6β-hydroxylated steroidal drugs. Attached Figure Description
[0016] Figure 1 This is a schematic diagram of the reaction in which the P450 BM3 mutant catalyzes the formation of 6β-OH-ADD from ADD via whole-cell catalysis. Figure 2 The HPLC chromatogram of ADD catalyzed by the mutant L82A; Figure 3 For product 6β-OH-ADD 1 H NMR spectrum; Figure 4 For product 6β-OH-ADD 13 C NMR spectrum. Detailed Implementation
[0017] The specific embodiments of the present invention will be further described below with reference to examples. It should be noted that the specific embodiments described herein are for illustrative and explanatory purposes only and do not constitute a limitation thereof. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.
[0018] The following explanations will cover some of the terms and materials used in the embodiments to facilitate understanding by those skilled in the art.
[0019] LB liquid medium: Weigh 10g tryptone, 10g sodium chloride, and 5g yeast extract, add an appropriate amount of purified water and bring the volume to 1 L. Adjust the pH to 7.4 with NaOH solution and dilute HCl, and sterilize at 121℃ for 20 min. LB solid medium: Add 2.0g agar powder to the LB liquid medium and autoclave.
[0020] TB liquid medium: Weigh 24g yeast extract and 12g tryptone, measure 4mL glycerol, dissolve in purified water and bring the volume to 900mL; separately weigh 2.31g potassium dihydrogen phosphate and 12.54g dipotassium hydrogen phosphate, dissolve in purified water and bring the volume to 100mL; sterilize the above solutions at 121℃ for 20min, then add 10mL buffer solution to 90mL of medium in a clean bench, mix well and obtain TB liquid medium.
[0021] Example 1 Construction and expression of the LG23 recombinant strain: In this embodiment, the P450 BM3 mutant LG23 (Li A, Acevedo-Rocha CG, D'Amore L, et al. Regio-and stereoselective steroid hydroxylation at C7 bycytochrome P450 monooxygenase mutants[J]. Angew Chem Int Ed, 2020, 59(29):12499-12505. DOI: 10.1002 / ange.202003139.) reported in the literature was used as the parental template. Its encoding gene was cloned into the pRSFDuet-1 vector, and a recombinant plasmid was constructed as the template plasmid for subsequent mutant construction. The nucleotide sequence of the parental template LG23 is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.
[0022] The nucleotide sequence (SEQ ID NO. 1) of the aforementioned parental template is as follows:
[0023] The amino acid sequence (SEQ ID NO. 2) of the aforementioned parental template is as follows:
[0024] Specifically: The nucleotide sequence of the parental template was cloned into the pRSFDuet-1 vector. Not I and Hind Between restriction enzyme sites (III), the gene was named pRSFDuet-1-LG23. pRSFDuet-1-LG23 was transformed into *E. coli* DH5α or BL21(DE3) competent cells to obtain recombinant strains, which were then used for gene cloning and heterologous expression, respectively. The specific steps are as follows: (1) Take the prepared competent cells out of the -80℃ freezer and place them on ice to thaw naturally; (2) Under aseptic conditions, add 2 μL of recombinant plasmid to 100 μL of competent cells, gently tap the tube wall to mix, and let stand in an ice bath for 30 min; at the same time, preheat the hot water bath to 42℃. (3) Heat shock the mixture at 42°C for 70 seconds, then quickly transfer it back to ice and continue the ice bath for 2 minutes; (4) Add 1 mL of LB liquid culture medium under aseptic conditions and incubate at 37°C and 220 rpm for 1 h with shaking. (5) Centrifuge the bacterial solution at 3500 rpm for 5 min, discard the supernatant, spread the remaining bacterial solution evenly on LB solid plates containing kanamycin resistance, and incubate upside down in a constant temperature incubator at 37℃ for 12 h to 16 h.
[0025] Induction of recombinant bacterial expression: Single colonies in good growth condition from the above plates were inoculated into 25 mL of TB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C with shaking at 220 rpm until OD200 was achieved. 600 All values reached 0.6~0.8; IPTG was added to a final concentration of 0.2mM, and the cells were cultured at 25℃ and 220rpm for 20h with shaking; the induced bacterial culture was centrifuged at 4℃ and 4000rpm for 10min, the supernatant was discarded, and the cells were washed twice with 45mL of 0.1M KPi buffer (pH 8.0); the cells were resuspended in 5mL of 0.1M KPi buffer (pH 8.0) and stored at -80℃ for later use.
[0026] Example 2 Construction of cytochrome P450 enzyme mutants: Using the pRSFDuet-1-LG23 recombinant plasmid as a template, mutant primers were designed to perform mutant PCR, and the corresponding mutant recombinant plasmid was obtained, thereby constructing the cytochrome P450 enzyme mutant.
[0027] The cytochrome P450 enzyme mutants are all based on the amino acid sequence shown in SEQ ID NO. 2, with single or multiple mutations at amino acid residues 72, 75, 78, 82, 85, 87, 88, 181 and 437.
[0028] Specifically, the amino acid mutation is any one of the following: W72F, L75A, L75F, L78A, L82A, L82C, L82G, L82T, G85A, G87A, G87V, L181A, L437, G85S / S88L, G87A / S88T, G87A / S88T / Q188L.
[0029] Site-directed mutagenesis experiments were performed according to the method described in the Fast Mutagenesis System site-directed mutagenesis kit (TransGenBiotech, Beijing, China). Mutants were constructed by PCR amplification of full-length plasmids using the 2×TransStart FastPfuFlyPCR SuperMix polymerase premix recommended in the method. The sequences encoding the mutated amino acids and the homologous sequences used for DNA assembly were introduced using PCR primers (BGI, Beijing, China).
[0030] The PCR reaction system is as follows: The total reaction volume is 50 μL, with 25 μL of 2×TransStart FastPfu FlyPCRSuperMix, 20 μL of ddH2O, 1 μL each of forward and reverse primers, and 2 μL of template added.
[0031] The sequences of the upstream and downstream mutation primers used in this embodiment are shown in the table below: Table 1: Upstream and downstream mutant primer sequences
[0032] Note: G87A-F2 and G87A-R are upstream and downstream primers for constructing the double mutant G87A / S88T based on the single mutant S88T.
[0033] Reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 20 s, 55℃ annealing for 20 s, 57℃ extension for 75 s, for a total of 25 cycles from denaturation to extension; 72℃ for a further extension for 10 min, and PCR products stored at 4℃.
[0034] The PCR products were treated with DMT enzyme to digest the original DNA template. 1 µL of DMT enzyme was added to the amplification product, mixed well, and incubated at 37°C for 1 h.
[0035] The product after incubation was transformed into E. coli DH5α competent cells using the chemical transformation method (TransGen Biotech, Beijing, China). The specific steps are as follows: (1) Take DH5a competent cells and place them on ice to thaw. Add 2 μL to 5 μL of the above-mentioned DMT enzyme digestion product and incubate on ice for 30 min. (2) Heat shock at 42℃ for 60s, followed immediately by an ice bath for 2min; (3) Add 500 μL of antibiotic-free LB medium, and then revive in a shaker at 37°C and 220 rpm for 1 h; (4) After the recovery is complete, take 100 μL of bacterial culture and spread it on an LB plate containing 50 μg / mL kanamycin resistance. Place the plate in a 37°C incubator and incubate overnight for about 12 to 16 hours. (5) Select a single colony of a transformant with good growth and inoculate it into 5 mL of LB liquid medium containing Kana resistance. Incubate at 37°C and 220 r / min for 12 h. (6) Take about 3 mL of activated bacterial solution and use Tiangen Rapid Plasmid Mini-Prep Kit (DP105) to extract plasmids for sequencing.
[0036] Among them, mutants with multiple point mutations are obtained by superimposing mutations on the basis of single-point mutants using the same method as above, resulting in multi-mutants with multiple point mutations.
[0037] Subsequently, the mutant was induced to express using the same method as the template described above, and the resulting bacterial cells were frozen at -20°C.
[0038] Example 3 Whole-cell catalytic reactions and activity assays: This embodiment uses ADD as an example substrate and employs a whole-cell catalytic reaction to catalyze the 6β-hydroxylation of ADD using the aforementioned cytochrome P450 enzyme mutant, generating the product 6β-OH-ADD. Figure 1 As shown.
[0039] ADD is androsta-1,4-diene-3,17-dione, with the following structural formula:
[0040] 6β-OH-ADD is the 6β-hydroxylation product of ADD catalyzed by the above-mentioned cytochrome P450 enzyme mutant, with the following structural formula:
[0041] It should be noted that the mutant provided in this embodiment is not limited to catalyzing the formation of 6β-OH-ADD from ADD. This mutant can also catalyze the 6β-hydroxylation of other steroidal compounds to generate the corresponding target products.
[0042] Take the frozen bacterial cells mentioned above, add 900 μL of 0.1 M KPi buffer (pH 8.0) containing 10% (v / v) glycerol to each tube, and fully resuspend the bacterial cells.
[0043] Transfer 500 μL of the resuspended bacterial solution to a short test tube and add 1 mM of steroidal substrate (100 mM substrate stock solution dissolved in DMF). Place the reaction system at 25 °C and 220 rpm and shake for 24 h. After the reaction is complete, add an equal volume of ethyl acetate, shake thoroughly, and repeat the extraction three times. Combine the organic phases and dry by rotary evaporation. Dissolve the solution completely in methanol, then filter through a 0.22 μm organic microporous membrane and perform activity detection by HPLC.
[0044] The results of the mutant activity test are shown in Table 2: Table 2: Results of mutant activity test
[0045] Note: Conversion and selectivity are calculated using the peak areas of the substrate and product in HPLC. The conversion is calculated as: (Total product peak area / (Total product peak area + Substrate peak area)) × 100%; the selectivity is calculated as: (Target product peak area / Total product peak area) × 100%.
[0046] As can be seen from the activity test results in Table 2, the P450 BM3 mutant in this example exhibits significantly better performance than the parental template LG23 in catalyzing the 6β-hydroxylation of steroidal compounds.
[0047] Under the same reaction conditions, multiple mutants not only maintained high conversion rates (e.g., L82A and L82T achieved 99.8% conversion), but more importantly, their 6β-hydroxylation selectivity was significantly improved. For example, the L75A mutant showed a selectivity of 95.1%, L82A 87.2%, and W72F 85.4%, while the parental LG23 showed a selectivity of only 50.2%. This indicates that by rationally mutating key sites such as 72, 75, 82, 85, 87, and 88 on the basis of LG23, the enzyme's active pocket was successfully reshaped, effectively guiding the reaction toward 6β-hydroxylation and significantly inhibiting the formation of byproducts.
[0048] Figure 2 The HPLC chromatogram of ADD catalyzed by mutant L82A is shown in Table 2. Figure 2It is evident that the mutant L82A not only exhibits higher conversion rates but also improves the selectivity for the product 6β-OH ADD. Compared to the template LG23, its conversion rate increases from 75.9% to 87.2%, and its selectivity increases from 50.2% to 73.7%, demonstrating highly efficient 6β-hydroxylation performance for ADD.
[0049] Example 4 Structural identification of the product: The catalytic reaction system of Example 3 was scaled up. After the reaction was completed, the reaction solution was transferred to a separatory funnel and extracted twice with an equal volume of ethyl acetate. The two organic phase extracts were combined and placed in a rotary evaporator flask. The mixture was then evaporated to dryness under reduced pressure to obtain a crude extract.
[0050] The extract was fully dissolved in an appropriate amount of methanol, filtered through a 0.22 μm organic filter membrane, and used as the sample for semi-preparative high-performance liquid chromatography (HPLC). The conversion product was separated and purified by semi-preparative HPLC, and the collected eluent was rotary evaporated to dryness to obtain the purified solid product.
[0051] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 H NMR and carbon spectroscopy (H NMR) 13 The obtained product was characterized by C1NMR. 1 HNMR and 13 C NMR spectra as follows Figures 3-4 As shown, it was confirmed to be 6β-OH-ADD.
[0052] In summary, this invention successfully constructed a series of novel mutants based on the amino acid sequence template shown in SEQ ID NO. 2. By introducing specific amino acid substitutions at key sites, the catalytic performance of cytochrome P450 enzymes for the 6β-hydroxylation of various steroidal compounds was significantly improved, exhibiting good conversion rate and 6β-hydroxylation selectivity. This provides a novel biomanufacturing pathway for the synthesis of key intermediates in 6β-hydroxylated steroidal drugs (such as anti-inflammatory drugs, cardiovascular drugs, and novel hormone drugs).
[0053] While the present invention has been disclosed through preferred embodiments, it is not intended to limit the invention. Any person skilled in the art can make various modifications and variations to the above embodiments without departing from the spirit and scope of the invention.
Claims
1. A cytochrome P450 enzyme mutant, characterized in that, The mutant is generated by single-point or multi-point mutation in the amino acid sequence shown in SEQ ID NO. 2, and the mutant is selected from any of the following: W72F, L75A, L75F, L78A, L82A, L82C, L82G, L82T, G85A, G87A, G87V, L181A, L437, G85S / S88L, G87A / S88T, G87A / S88T / Q188L.
2. A DNA molecule, characterized in that, The DNA molecule encodes the nucleotide sequence of the cytochrome P450 enzyme mutant as described in claim 1, or its complementary sequence.
3. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid contains the DNA molecule as described in claim 2.
4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain the recombinant expression plasmid as described in claim 3.
5. The use of the cytochrome P450 enzyme mutant of claim 1, the DNA molecule of claim 2, the recombinant expression plasmid of claim 3, or the genetically engineered bacteria of claim 4 in catalyzing the generation of 6β-hydroxylated products from steroidal compounds.
6. A method for catalytically generating 6β-hydroxylated products from steroidal compounds, characterized in that, include: Prepare recombinant expression plasmids encoding the P450BM3 mutant; The recombinant expression plasmid was transferred into a host strain to obtain genetically engineered bacteria; Using steroidal compounds as substrates, whole-cell catalytic reactions are carried out to generate the corresponding 6β-hydroxylated products.
7. The method as described in claim 6, characterized in that, The steroidal compound is one of the following compounds: 。 8. The method as described in claim 6, characterized in that, The whole-cell catalytic reaction was carried out in genetically engineered bacteria containing the mutant under conditions of pH 7.0-8.0, temperature 16℃-25℃, and reaction time 10-24 hours.