Cytochrome p450 bm3 mutants and uses thereof

Abstract

The application belongs to the technical field of biology and relates to a cytochrome P450 BM3 mutant and application thereof. The mutant is obtained by implementing F87A, F81I and A82F site-directed mutation on the amino acid sequence shown in the sequence table SEQ ID NO. 2. The cytochrome P450 BM3 mutant provided by the application exhibits high catalytic activity to the unnatural substrate epothilone D, and realizes efficient conversion of the epothilone D. The mutant can catalyze the hydroxylation reaction of the epothilone D at multiple sites (such as C-21 and C-26) to generate at least four different derivatives, thereby providing a valuable compound library for drug screening.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a cytochrome P450 BM3 mutant and its applications. Background Technology

[0002] Epothilones are macrocyclic lactones produced by myxobacteria. Their mechanism of action is similar to paclitaxel, inhibiting tumor cell division by stabilizing microtubules and exhibiting potent antitumor activity. Epothilone D, as an important member of this class, has attracted considerable attention due to its pharmacological activity. However, natural epothilones suffer from high toxicity and poor water solubility, limiting their clinical application. Structural modification of the epothilone core, particularly hydroxylation, is an effective strategy for obtaining low-toxicity, high-activity derivatives.

[0003] Currently, the preparation of epothilone derivatives mainly relies on complex total chemical synthesis or semi-synthetic methods, which suffer from problems such as cumbersome steps, low overall yield, difficulty in controlling regio and stereoselectivity, high cost, and significant environmental pollution. For example, the total synthesis of epothilone B requires more than 20 steps, with an overall yield of only 8.9%. Biocatalysis has advantages such as mild reaction conditions, high selectivity, and environmental friendliness, but natural enzymes are difficult to catalyze non-natural substrates.

[0004] Cytochrome P450 monooxygenases are a class of potent catalysts that catalyze the hydroxylation of inert CH bonds, participating in natural product synthesis, steroid synthesis, xenobiotic metabolism, and drug degradation. Cytochrome P450 BM3 (CYP102A1) is derived from Bacillus megaterium (…). Bacillus megaterium This enzyme, due to the natural fusion of its heme and reductase domains within a single peptide chain, exhibits extremely high electron transfer efficiency. Furthermore, it possesses outstanding advantages such as high catalytic efficiency, good soluble expression, and ease of engineering modification, making it considered an ideal biocatalytic platform. However, the natural substrate of wild-type P450 BM3 is a long-chain fatty acid, exhibiting almost no catalytic activity towards epothilone D. Summary of the Invention

[0005] The purpose of this invention is to solve the problems existing in the prior art and to provide a cytochrome P450 BM3 mutant and its application in the preparation of epothilone D derivatives.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The present invention provides a P450 BM3 mutant, which is obtained by performing site-directed mutations of F87A, F81I and A82F on the amino acid sequence shown in SEQ ID NO. 2.

[0008] Furthermore, the present invention provides a DNA molecule comprising a nucleotide sequence encoding the cytochrome P450BM3 mutant or its complementary sequence.

[0009] Furthermore, the present invention provides a recombinant plasmid containing the aforementioned DNA molecule.

[0010] Furthermore, the present invention provides a recombinant strain containing the aforementioned recombinant plasmid.

[0011] Furthermore, the present invention provides the use of the cytochrome P450 BM3 mutant, the DNA molecule, the recombinant plasmid, or the recombinant strain in the preparation of one selected from 21-OH-epotoxin D, 26-OH-epotoxin D, epotoxin B, and 21-OH-epotoxin B by catalyzing the hydroxylation reaction of epotoxin D.

[0012] Furthermore, the present invention also provides a method for preparing the epothilone D derivative, comprising: using epothilone D as a substrate, catalyzing the hydroxylation reaction of epothilone D with the cytochrome P450 BM3 mutant to obtain a product mixture containing multiple hydroxylated derivatives.

[0013] Furthermore, the reaction is carried out in a buffer system containing an NADPH regeneration system (such as glucose / glucose dehydrogenase).

[0014] Compared with the prior art, the present invention has the following significant advantages:

[0015] (1) High catalytic activity: The cytochrome P450 BM3 mutant provided by the present invention exhibits high catalytic activity against the non-natural substrate epothilone D, realizing the efficient conversion of epothilone D;

[0016] (2) Regioselectivity: This mutant can catalyze the hydroxylation of epothilone D at multiple sites (such as C-21 and C-26) to generate at least four different derivatives, providing a valuable compound library for drug screening;

[0017] (3) Green and efficient: This method uses biocatalysis to replace traditional chemical synthesis. The reaction steps are simple and the conditions are mild. It does not require the use of precious metal catalysts and harsh reaction conditions, which is in line with the principles of green chemistry.

[0018] (4) Low cost: The reaction uses the NADPH cycle system, which greatly reduces the cost of coenzymes and makes large-scale bio-preparation possible. Attached Figure Description

[0019] Figure 1A schematic diagram of the reaction pathway of epothilone D catalyzed by the cytochrome P450 mutant BM3-B3B9 and the structure of the main product.

[0020] Figure 2 A comparison of the conversion rates of epothilone D catalyzed by the cytochrome P450 mutant BM3-B3B9 and other cytochrome P450 BM3 mutants in the comparative example;

[0021] Figure 3 High-performance liquid chromatography (HPLC) chromatograms of the reaction of cytochrome P450 mutant BM3-B3B9 with epothilone D catalyzed by the cytochrome P450 mutant BM3-B3B9; (a) reaction group; (b) control group.

[0022] Figure 4 LC-MS spectra of the products of the epothilone D reaction catalyzed by the cytochrome P450 mutant BM3-B3B9; where (a) Epo-2, [M+H] + =524.2677; (b) Epo-3, [M+H] + =508.2728; (c) Epo-4, [M+H] + =508.2728; (d) Epo-5, [M+H] + =508.2728;

[0023] Figure 5 The 1H NMR spectrum of Epo-2, the product of the reaction of epothilone D catalyzed by the cytochrome P450 mutant BM3-B3B9;

[0024] Figure 6 The 1H NMR spectrum of Epo-3, the product of the reaction of epothilone D catalyzed by the cytochrome P450 mutant BM3-B3B9;

[0025] Figure 7 The 1H NMR spectrum of Epo-4, the product of the cytochrome P450 mutant BM3-B3B9 catalyzing the reaction of epothilone D;

[0026] Figure 8 The 1H NMR spectrum of Epo-5, the product of the reaction of epothilone D catalyzed by the cytochrome P450 mutant BM3-B3B9. Detailed Implementation

[0027] The technical solutions in this embodiment will be clearly and completely described below with reference to the embodiments of this application. The present invention will be further illustrated through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0028] Example 1: Construction of a recombinant strain containing the cytochrome P450 mutant BM3-B3B9

[0029] From Bacillus megaterium ( Bacillus megatherium Using a plasmid of the wild-type cytochrome P450 BM3 gene as a template, iterative site-directed mutagenesis was performed using three rounds of overlap extension PCR targeting key sites F87, F81, and A82 amino acids near its substrate binding pocket. In each round, a validated mutant plasmid from the previous step was used as a template, and primers designed to contain specific mutation sites (F87A, F81I, A82F) were used for amplification, resulting in the triple mutant plasmid pET28a-BM3-B3B9 containing the F87A, F81I, and A82F mutations.

[0030] The first round of mutation (introduction of F87A) specifically involved designing a pair of specific primers containing the F87A mutation site (ttt→gca, Phe→Ala) using the pET-28a plasmid containing the wild-type cytochrome P450 BM3 gene as a template. High-fidelity DNA polymerase 2×Phanta Flash Master Mix was used for PCR to amplify the linearized full-length plasmid fragment. The PCR product was then treated with DpnI restriction endonuclease to specifically digest the methylated template plasmid. The remaining product was transformed into... E. coli DH5α competent cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37 °C for 12 h. Single clones were picked from the plates and sequenced. The recombinant plasmid with correct sequencing results was the F87A single-point mutant plasmid.

[0031] The second round of mutation (introduction of F81I) specifically involved designing specific primers containing the F81I mutation sites (ttt→att, Phe→Ile) using the validated F87A mutant plasmid as a template. The above PCR, DpnI digestion, transformation, and sequencing validation procedures were repeated to finally obtain a double mutant plasmid (F87A / F81I) containing both F87A and F81I mutations.

[0032] The third round of mutation (introduction of A82F) involved designing specific primers containing the A82F mutation sites (gca→ttt, Ala→Phe) using the F87A / F81I double mutant plasmid as a template. This mutation process was repeated, and after sequencing verification, a triple mutant plasmid containing mutations at all three target sites (F87A, F81I, and A82F) was successfully obtained and named pET28a-BM3-B3B9.

[0033] The recombinant plasmid pET28a-BM3-B3B9 and E. coliBL21(DE3) competent cells were mixed and incubated on ice for 30 min, then heat-shocked in a 42°C water bath for 45 seconds, and then quickly returned to ice for 2 min. Liquid culture medium was added to allow the cells to recover for 50 min, and the cells were spread on LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 12 h. The single colonies that grew were the P450 BM3-B3B9 recombinant strain.

[0034] Table 1. Sequences of primers used in the three rounds of mutation.

[0035]

[0036] The nucleotide sequence of wild-type cytochrome P450 BM3 is shown in SEQ ID NO.1.

[0037] The amino acid sequence of wild-type cytochrome P450 BM3 is shown in SEQ ID NO.2.

[0038] The triple mutant plasmid with correct sequencing results has the nucleotide sequence shown in SEQ ID NO.3.

[0039] The triple mutant plasmid with correct sequencing results has the amino acid sequence shown in SEQ ID NO.4.

[0040] Example 2: Purification to obtain soluble BM3-B3B9 protein

[0041] Single clones were picked from the plates and cultured in a constant temperature shaker at 37 ℃ and 220 rpm until the logarithmic growth phase. They were then transferred to shake flasks at a 1:100 ratio and cultured until OD (October Expiratory Time) was reached. 600 The concentration of the drug was 0.6-0.8. IPTG (0.2 mM) and 5-aminolevulinic acid (5-ALA) were added to a final concentration of 0.5 mM as precursors for heme synthesis. Expression was induced for 20 hours in a constant-temperature shaker at 18°C ​​and 180 rpm. After culture, the bacterial cells were collected by centrifugation at 8000 rpm for 8 min, yielding cells containing the BM3-B3B9 protein.

[0042] The bacterial cells were resuspended in Lysis buffer and then sonicated for 30 min until the bacterial suspension became clear and watery. The supernatant was collected by centrifuging the bacterial suspension at 4 °C and 10,000 rpm for 60 min. The supernatant was mixed with Ni-NTA agarose gel at a volume ratio of 50:1 and incubated with gentle shaking at 4 °C for 1 h. The mixture was then transferred to a protein separation column and allowed to flow out completely. Wash buffer was added to remove contaminating proteins, and Elution buffer was added to elute the target protein. The eluted target protein was desalted with desalting buffer to remove imidazole and concentrated to 58 mg / mL using an ultrafiltration tube to obtain soluble BM3-B3B9 protein.

[0043] The lysis buffer is specifically composed of: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% Glycerol, and pH 8.0.

[0044] The wash buffer consists of: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% Glycerol, and pH 8.0.

[0045] The elution buffer is specifically composed of: 50 mM NaH2PO4, 300 mM NaCl, 250 mM Mimidazole, 10% Glycerol, and pH 8.0.

[0046] The desalting buffer is specifically composed of: 50 mM NaH2PO4, 300 mM NaCl, 10% Glycerol, and pH 7.4.

[0047] Example 3: Preparation of Epothilone D Derivatives by Establishing the BM3-B3B9 Protease Reaction System

[0048] The reaction pathway of epothilone D catalyzed by mutant BM3-B3B9 and the structure of the main product are as follows: Figure 1 As shown.

[0049] 1. Small-scale pre-reaction in 100 μL:

[0050] Add 1 μL of epothilone D, 4 μL of soluble P450 BM3-B3B9 protein, 1 μL of glucose, and 1 μL of NADP. +Add 2 μL GDH and 1 μL NADPH to an Erlenmeyer flask, and finally add desalting buffer to bring the reaction mixture to a final volume of 100 μL. Incubate the reaction mixture in a 30°C water bath for 6 h. After the reaction is complete, add 200 μL methanol to terminate the reaction. Centrifuge the terminated reaction product at 12000 rpm for 10 min. After centrifugation, collect the supernatant to obtain the reaction product mixture.

[0051] 2. Large-scale reaction in 20 mL

[0052] Add 20 μL epothilone D, 400 μL soluble BM3-B3B9 protein, 100 μL glucose, and 200 μL NADP. + Add 400 μL GDH and 100 μL NADPH to an Erlenmeyer flask, and finally add desalting buffer to bring the reaction mixture to a final volume of 20 mL. Incubate the reaction mixture in a constant temperature shaker at 30 °C and 60 rpm for 12 h. After the reaction is complete, add 30 mL of ethyl acetate to terminate the reaction and extract the product to obtain the reaction mixture.

[0053] The concentration of epothilone D (soluble in DMSO) was 20 mM, the concentration of the soluble P450 BM3-B3B9 protein was 58 mg / mL, the concentration of glucose was 1 M, and the concentration of NADP... + The concentration of GDH is 100 mM, the concentration of NADPH is 100 mM.

[0054] Example 4: Comparison of the catalytic activity of different P450 BM3 mutants against epothilone D

[0055] Using essentially the same conditions as the "small-scale pre-reaction" in Example 3, the catalytic activity of various mutants, including BM3F87A, A3A12, A4G10, B1B12, B3A5, and the BM3-B3B9 (B3B9) of this invention, on epothilone D was tested. The substrate conversion was determined by HPLC, and the results are as follows: Figure 3 As shown.

[0056] from Figure 3 It is evident that, under the same reaction conditions, the conversion rate of epothilone D by the BM3-B3B9 protein provided by this invention is much higher than that of other tested mutants, demonstrating the unique advantages and high efficiency of the BM3-B3B9 protein in catalyzing the hydroxylation reaction of epothilone D.

[0057] Example 5: Identification of the structure of epothilone D derivative

[0058] The crude product was separated and purified by preparative HPLC to obtain four main components (Epo-2, Epo-3, Epo-4, and Epo-5). The structures of the purified products were identified by liquid chromatography-mass spectrometry (LC-MS) and high-resolution nuclear magnetic resonance spectroscopy (NMR).

[0059] LC-MS analysis: such as Figure 4 As shown, the [M+H] of the four products + The peaks were 16 Da (m / z 507.2655) or 32 Da (m / z 523.2604) larger than the substrate epothilone D (m / z 491.2705), indicating that mono- or di-oxygen addition occurred.

[0060] NMR analysis: via 1 H NMR, 13 Spectral analysis using C NMR, COSY, HSQC, and HMBC methods ultimately determined:

[0061] (1) Epo-2: 21-OH-epothilone B (simultaneously undergoes C21 hydroxylation and C12-C13 epoxidation); such as Figure 5 As shown;

[0062] (2) Epo-3: 26-OH-epothilone D (the methyl group at C26 is hydroxylated to a hydroxymethyl group); e.g. Figure 6 As shown;

[0063] (3) Epo-4:epothilone B (C12-C13 double bond is epoxidized); e.g. Figure 7 As shown;

[0064] (4) Epo-5: 21-OH-epothilone D (the methyl group at C21 is hydroxylated to a hydroxymethyl group), such as Figure 8 As shown.

[0065] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A cytochrome P450 BM3 mutant, characterized in that, The mutant was obtained by performing site-directed mutations of F87A, F81I, and A82F on the amino acid sequence shown in SEQ ID NO.

2.

2. A DNA molecule, characterized in that, The DNA molecule contains a nucleotide sequence encoding the cytochrome P450BM3 mutant of claim 1 or its complementary sequence.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the DNA molecule as described in claim 2.

4. A recombinant bacterial strain, characterized in that, The recombinant strain contains the recombinant plasmid as described in claim 3.

5. Use of the cytochrome P450 BM3 mutant of claim 1, the DNA molecule of claim 2, the recombinant plasmid of claim 3, or the recombinant strain of claim 4 in catalyzing the hydroxylation of epothilone D to prepare a product selected from 21-OH-epothilone D, 26-OH-epothilone D, epothilone B, or 21-OH-epothilone B.

6. A method for preparing hydroxylated epothilone derivatives, characterized in that, The step of using epothilone D as a substrate, and in the presence of O2 and NADPH, catalyzing the hydroxylation reaction of the substrate using the cytochrome P450 BM3 mutant of claim 1; wherein the hydroxylated epothilone derivative is selected from 21-OH-epothilone D, 26-OH-epothilone D, epothilone B or 21-OH-epothilone B.

7. The method according to claim 6, characterized in that, The reaction was carried out in the presence of an NADPH regeneration system.

8. The method according to claim 7, characterized in that, The NADPH regeneration system contains glucose and NADP. + And glucose dehydrogenase.

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