Self-sufficiency P450 enzyme for catalyzing intermolecular CS bond coupling and application of mutant

Abstract

The invention discloses a self-sufficiency P450 enzyme for catalyzing intermolecular C-S bond coupling and application of a mutant, and belongs to the field of enzyme engineering and organic synthesis. According to the invention, through directed evolution, key active sites of P450 enzyme, especially F424, V118 and C385 residues, are mutated. According to the mutant constructed by the invention, the C-S bond coupling activity is remarkably improved, and particularly, the V118A / C385H / F424P mutant has excellent performance on different aryl mercaptan substrates. Microbial cells expressing the mutant can be used as a whole-cell catalyst for a C-S bond coupling reaction, and a solid foundation is laid for industrial application.

Description

Technical Field

[0001] The invention relates to a self-sufficient P450 enzyme for catalyzing CS bond coupling between molecules and application of a mutant thereof, and belongs to the field of enzyme engineering and organic synthesis. Background Art

[0002] In the field of organic synthesis and drug development, the coupling reaction of intermolecular CS bonds is a key step in the construction of sulfur-containing organic compounds, especially biologically active molecular skeletons. However, traditional catalytic methods are often limited by low efficiency, high energy consumption, and poor product selectivity, making it difficult to meet the needs of complex molecular synthesis. Cytochrome P450 enzymes are a class of functionally diverse biocatalysts in nature. Their unique catalytic mechanism and wide substrate adaptability provide new possibilities for organic synthesis.

[0003] However, when natural P450 enzymes catalyze intermolecular CS bond coupling reactions, their applications are often limited by insufficient catalytic activity, low selectivity for specific substrates, or the need for additional cofactor support. Summary of the invention

[0004] The present invention aims to solve the problems of insufficient catalytic activity and low CS bond formation efficiency of P450 enzymes in catalyzing CS bond coupling reactions in the prior art, and to provide a P450 enzyme and a whole-cell catalyst that can efficiently catalyze such reactions and improve product conversion rate and turnover number.

[0005] The invention provides a P450 enzyme mutant, which is obtained by mutating the V118, C385 and F424 sites of a P450 enzyme parent and has enhanced catalytic ability for CS bond coupling reaction.

[0006] In one embodiment, the P450 enzyme mutant is based on the sequence shown in SEQ ID NO.1, with the valine at position 118 mutated to alanine, the cysteine ​​at position 385 mutated to histidine, and the phenylalanine at position 424 mutated to proline.

[0007] In one embodiment, the amino acid sequence of the P450 enzyme mutant is shown in SEQ ID NO.2.

[0008] The present invention also provides a gene encoding the P450 enzyme mutant.

[0009] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.4.

[0010] The invention also provides a recombinant plasmid comprising the gene.

[0011] In one embodiment, the plasmid includes but is not limited to pET-28a(+), pET-28b(+) or pET-20b(+).

[0012] The invention also provides a host cell carrying the gene or recombinant plasmid.

[0013] In one embodiment, the host cell includes, but is not limited to, a bacterial cell or a fungal cell.

[0014] The present invention also provides a cell catalyst having the catalytic activity of the P450 enzyme mutant.

[0015] In one embodiment, the cellular catalyst comprises a recombinant E. coli cell expressing the P450 enzyme mutant.

[0016] In one embodiment, the recombinant Escherichia coli uses E. coli BL21 (DE3) as a host and pET 28a (+) as an expression vector to express the P450 enzyme mutant.

[0017] The present invention also provides a method for preparing the cell catalyst, which comprises culturing the recombinant Escherichia coli in a culture medium and collecting bacterial cells with P450 enzyme catalytic activity as whole cell catalysts.

[0018] The present invention also provides a method for improving the catalytic activity and / or turnover efficiency of P450 enzymes for aromatic thiol substrates, which comprises mutating the valine at position 118 of the P450 enzyme to alanine, mutating the cysteine ​​at position 385 to histidine and / or mutating the phenylalanine at position 424 to proline.

[0019] The present invention also provides the use of the P450 enzyme mutant and / or the cell catalyst in participating in CS bond coupling reaction.

[0020] Beneficial effects:

[0021] (1) The present invention successfully obtained a P450 enzyme mutant that can efficiently catalyze the CS bond coupling reaction through a directed evolution strategy, especially the V118A / C385H / F424P mutant. Its catalytic activity is significantly improved compared with the wild-type P450 enzyme without the need for additional cofactors, providing an efficient and reliable catalytic tool for the CS bond coupling reaction.

[0022] (2) The V118A / C385H / F424P mutant exhibited excellent conversion rates and turnover numbers on a variety of aromatic thiol substrates, which not only broadened the application range of P450 enzymes but also provided a new and efficient catalyst option for the field of organic synthesis.

[0023] (3) The method for preparing the P450 enzyme mutant of the present invention is simple, efficient, and easy to scale up, thus laying a solid foundation for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to the specific embodiments of the present invention in combination with the accompanying drawings, wherein

[0025] Figure 1 It is a schematic diagram of enzyme catalysis prepared by the present invention.

[0026] Figure 2 This is the SDS-PAGE electrophoresis analysis result of the expression product obtained by shake flask induced fermentation of recombinant Escherichia coli in Example 1 of the present invention; wherein ↑ represents supernatant, and ↓ represents precipitate.

[0027] Figure 3 is the result of screening random combination mutants in 96-well plates in Example 2 of the present invention, wherein P450 SH It has a C385H mutation based on the starting sequence.

[0028] Figure 4 This is the GC chromatogram of the reaction liquid catalyzed by a substrate with a halogen group in Example 3 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] The E. coli BL21 (DE3) involved in the following examples was purchased from Beina Biotechnology, and the pET-28a (+) plasmid was purchased from Novagen.

[0031] The culture medium involved in the following examples is as follows:

[0032] LB liquid medium: yeast powder 5.0 g L -1 , Tryptone 10.0 g·L -1 、NaCl 10.0g·L -1 , ampicillin 100 μg·L -1 .

[0033] LB solid medium: yeast powder 5.0 g L -1 , Tryptone 10.0 g·L -1 、NaCl 10.0g·L -1 , agar powder 15g·L -1 , ampicillin 100 μg·L -1 .

[0034] Preparation of 5× stock solution: Dissolve Na2HPO4 (34 g), KH2PO4 (15 g) and NaCl (2.5 g) in 1 L of water and then autoclave.

[0035] Preparation of M9-N buffer: dilute 200 mL of 5X stock solution to 1 L, add 1 mL of CaCl2 (0.1 M), 2 mL of MgSO4 (1.0 M), and adjust the pH to 7.4 or 8.2 with 40% NaOH to obtain a 1X stock solution, i.e., M9-N buffer.

[0036] Relative turnover rate: The relative turnover rate (Relative TOF) of the mutant (Mutant) relative to the wild-type enzyme (WT) is calculated as follows:

[0037] Relative TOF (Relative TOF) = (Mutant TOF) / (WT TOF);

[0038] Among them, TOF (turnover frequency) can be expressed as:

[0039] TOF = product amount / (enzyme concentration×time); wherein the reaction time is limited to 30 minutes.

[0040] Turnover number (TTN): indicates the number of substrate molecules that can be catalyzed by each enzyme molecule to be converted into product molecules within 1 hour under the most suitable conditions (i.e. when the enzyme is saturated with substrate). The calculation formula of TTN is:

[0041] 1h-TTN = molecular weight of substrate converted to product / molecular weight of enzyme.

[0042] Example 1: Cloning and expression of P450 enzymes and their mutants

[0043] The starting sequence of the P450 enzyme is shown in SEQ ID NO. 1. The P450 enzyme and mutants were cloned and expressed using the pET 28a(+) expression vector (Novagen).

[0044] Specific primers were designed and mutations were introduced by PCR technology. Subsequently, the PCR product was digested with DpnI enzyme, and 10 μL of the PCR product was taken to transform 50 μL of E. coli BL21 (DE3). The transformed cells were cultured overnight in 5 mL of Luria-Bertani medium containing kanamycin. The next day, 2 mL of the preculture was inoculated into 50 mL of Luria-Bertani medium containing kanamycin and cultured with shaking at 37°C and 220 rpm for 2.5 hours. 5-Aminolevulinic acid (ALA), FeSO4 and isopropyl-β-D-thiogalactoside (IPTG) were added to a final concentration of 0.5 mM, respectively, and then expressed at 22°C and 130 rpm for 20-24 hours. After the expression was completed, the cells were collected by centrifugation, resuspended in M9-N buffer, and adjusted to OD 60 32, pH 7.5, and whole-cell catalysts were obtained.

[0045] Example 2: Reaction screening in 96-well plate whole cell format

[0046] After saturation mutation (SSM) and combinatorial mutation were performed on sites such as V118, T278, C385H, and F424 to generate libraries, single colonies were randomly selected from kanamycin LB agar plates (LBKan) and cultured in 300 μL LB medium containing 0.1 mg / mL kanamycin in a sterilized 96-well deep-well plate. Each plate contains six wells inoculated with single colonies expressing the parent enzyme and two sterile wells. Culture for 8-12 hours at 37°C and 250rpm. Subsequently, the culture was transferred to LB medium containing 0.1 mg / mL kanamycin and cultured for 3 hours at 37°C and 120rpm. Afterwards, the 96-well plate was cooled in an ice bath, IPTG and ALA were added to a final concentration of 0.5 mM, respectively, and then expressed for 20-22 hours at 16°C and 180rpm. The cells were collected by centrifugation, the supernatant was discarded, and 360 μL M9-N (pH 7.5) was added to each well. After the cells were completely resuspended by shaking, the reaction was initiated by adding 20 mM thiophenol and 20 mM ethyl diazoacetate (EDA) at a final concentration. The plate was sealed with aluminum foil tape and shaken at 120 rpm at 16°C overnight.

[0047] Example 3: Comparison of catalytic efficiency of P450 enzymes and their mutants

[0048] Adjust the cell concentration of the whole cell resuspension to OD 600 = 30. Then, 370 μL of the resuspension was dispensed into 1.5 mL centrifuge tubes, and thiophenol and EDA were added to a final concentration of 20 mM, respectively, to start the reaction.

[0049] The reaction was carried out at 50°C and 120 rpm for 1 to 12 hours. Samples were taken periodically, internal standards were added to the reaction solution, and supercritical fluid chromatography was performed to quantify the product.

[0050] The results show (Table 1) that, compared with the P450 enzyme parent, the relative turnover efficiency of the mutant (V118A / C385H / F424P) constructed in Example 2 was significantly improved in the reaction time of 30 min, reaching 13.6 times of the original.

[0051] In addition, P450 and its mutants also exhibited extraordinary catalytic efficiency in the presence of halogen groups, with a turnover number of up to 4399.

[0052] ( Figure 4 ).

[0053] Table 1 Comparison of relative turnover efficiency (relative TOF) of P450 enzymes and mutants V118A / C385H / F424P

[0054]

[0055] Comparative Example 1:

[0056] Specific implementation method The same as Examples 1-2, except that the mutants shown in Table 2 were also constructed, and the catalytic efficiency was measured according to the method of Example 3. The results are shown in Table 2, and the relative turnover efficiency was not significantly improved.

[0057] Table 2 Comparison of relative turnover rates of different mutants

[0058]

[0059]

[0060] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A P450 enzyme mutant, characterized in that: Compared with the P450 enzyme parent, the V118, C385 and / or F424 positions are mutated.

2. The P450 enzyme mutant according to claim 1, characterized in that Based on the sequence shown in SEQ ID NO.1, the valine at position 118 was mutated to alanine, the cysteine ​​at position 385 was mutated to histidine, and the phenylalanine at position 424 was mutated to proline.

3. A gene encoding the P450 enzyme mutant according to claim 1 or 2.

4. A recombinant plasmid containing the gene according to claim 3.

5. A host cell carrying the gene according to claim 3 or the recombinant plasmid according to claim 4.

6. A recombinant Escherichia coli, characterized in that The P450 enzyme mutant according to claim 1 or 2 is expressed using E. coli BL21 (DE3) as a host and pET 28a (+) as an expression vector.

7. A cell catalyst, characterized in that Containing the recombinant Escherichia coli cell according to claim 6.

8. A method for preparing the cell catalyst according to claim 7, characterized in that: The recombinant Escherichia coli is cultured in a culture medium, and bacterial cells with P450 enzyme catalytic activity are collected as whole-cell catalysts.

9. A method for improving the catalytic activity and / or turnover efficiency of a P450 enzyme towards an aromatic thiol substrate, characterized in that: The valine at position 118 of the P450 enzyme is mutated to alanine, the cysteine ​​at position 385 is mutated to histidine, and / or the phenylalanine at position 424 is mutated to proline.

10. Use of the P450 enzyme mutant according to claim 1 or 2, or the recombinant Escherichia coli according to claim 6, or the cell catalyst according to claim 7 in participating in a CS bond coupling reaction.

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