Use of a self-sufficient p450 enzyme catalyzing intermolecular c-s bond coupling and mutants thereof

Abstract

The application discloses a kind of self-sufficient P450 enzyme and mutant application catalyzing intermolecular C-S bond coupling, belong to enzyme engineering and organic synthesis field.The application is by directed evolution, the key active site of P450 enzyme is mutated, especially F424, V118 and C385 residue.The mutant constructed in the application significantly improves the activity of C-S bond coupling, especially V118A / C385H / F424P mutant is excellent on different aryl mercaptan substrate.The microbial cell expressing mutant can be used as whole-cell catalyst for C-S bond coupling reaction, and lays a solid foundation for industrial application.

Description

TECHNICAL FIELD

[0001] The application relates to an application of a self-sufficient P450 enzyme and mutants for catalyzing intermolecular C-S bond coupling, and belongs to the field of enzyme engineering and organic synthesis. BACKGROUND

[0002] In the field of organic synthesis and drug research, the coupling reaction of intermolecular C-S bond is a key step for constructing sulfur-containing organic compounds, especially the skeleton of a bioactive molecule. However, traditional catalytic methods are often limited by low efficiency, high energy consumption and poor product selectivity, which is difficult to meet the needs of complex molecule synthesis. As a kind of biological catalyst with diverse functions in nature, cytochrome P450 enzyme provides new possibilities for organic synthesis due to its unique catalytic mechanism and wide substrate adaptability.

[0003] However, natural P450 enzymes are often limited in catalyzing the coupling reaction of intermolecular C-S bond due to insufficient catalytic activity, low selectivity for specific substrates or the need for additional cofactor support. SUMMARY

[0004] The application aims to solve the problems of insufficient catalytic activity and low C-S bond formation efficiency of P450 enzymes in catalyzing the coupling reaction of C-S bond in the prior art, and provides a P450 enzyme and whole-cell catalyst for efficiently catalyzing such reactions and improving product conversion rate and turnover number.

[0005] The application provides a P450 enzyme mutant, which is a P450 enzyme mutant with enhanced catalytic ability for the coupling reaction of C-S bond by mutating the V118, C385 and F424 sites of a P450 enzyme parent.

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

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

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

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

[0010] The application also provides a recombinant plasmid containing the gene.

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

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

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

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

[0015] In an embodiment, the cell catalyst contains a recombinant E. coli cell expressing the P450 enzyme mutant.

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

[0017] The present application also provides a method for preparing the cell catalyst, which is to culture the recombinant E. coli in a culture medium, and collect bacterial cells with catalytic activity of the P450 enzyme as a whole-cell catalyst.

[0018] The present application also provides a method for improving catalytic activity and / or turnover efficiency of a P450 enzyme on an aryl mercaptan substrate, which is to mutate valine at position 118 to alanine, cysteine at position 385 to histidine and / or phenylalanine at position 424 to proline.

[0019] The present application also provides application of the P450 enzyme mutant and / or the cell catalyst in a C-S bond coupling reaction.

[0020] Advantages:

[0021] (1) The present application successfully obtains a P450 enzyme mutant capable of efficiently catalyzing a C-S bond coupling reaction through a directed evolution strategy, especially a V118A / C385H / F424P mutant, which has a significantly improved catalytic activity compared with a wild-type P450 enzyme without an additional cofactor, thereby providing an efficient and reliable catalytic tool for a C-S bond coupling reaction.

[0022] (2) The V118A / C385H / F424P mutant exhibits excellent conversion rate and turnover number on various aryl mercaptan substrates, which not only widens the application range of the P450 enzyme, but also provides a new and efficient catalyst selection for the field of organic synthesis.

[0023] (3) The P450 enzyme mutant of the present application has simple and efficient preparation method, and is easy to mass production, which lays a solid foundation for industrial application. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to the specific embodiments of the present application combined with the drawings, in which

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

[0026] Figure 2 is the SDS-PAGE electrophoresis analysis result of the expression product obtained by the shake flask induction fermentation of the recombinant E.coli in Example 1 of the present application; wherein, the upward arrow represents supernatant, and the downward arrow represents precipitate.

[0027] Figure 3 is the result of screening the random combination mutant in the 96-well plate in Example 2 of the present application, wherein P450 SH is the starting sequence with C385H mutation.

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

[0029] The present application will be further described below in combination with the drawings and specific embodiments, so that the person skilled in the art can better understand the present application and implement it, but the embodiments are not as a limitation on the present application.

[0030] In the following examples, E.coli BL21(DE3) is purchased from Bei Na Biological, and pET-28a(+) plasmid is purchased from Novagen company.

[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 10.0 g·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 10.0 g·L -1 , agar powder 15 g·L -1 , ampicillin 100 μg·L -1 .

[0034] 5X stock preparation: Na2HP04(34 g), KH2P04(15 g) and NaCl (2.5 g) were dissolved in 1 L water and autoclaved.

[0035] M9-N buffer preparation: 200 mL of 5X stock was diluted to 1 L, 1 mL of CaCl2(0.1 M), 2 mL of MgS04(1.0 M) were added and pH was adjusted to 7.4 or 8.2 with 40% NaOH to get 1X stock, i.e. M9-N buffer.

[0036] Relative TOF: Relative TOF of mutant (Mutant) with respect to wild type enzyme (WT) was calculated as follows:

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

[0038] Wherein, TOF (Turnover Frequency) can be expressed as:

[0039] TOF = Product amount / (Enzyme concentration x time); wherein, reaction time is limited to 30 minutes.

[0040] Turnover Number (TTN): It represents the number of substrate molecules converted to product molecules per enzyme molecule in 1 h under optimal conditions (i.e. when enzyme is saturated with substrate). The formula for TTN is:

[0041] 1 h-TTN = Substrate converted to product molecules / Enzyme molecules.

[0042] Example 1: Cloning and expression of P450 enzyme and its mutants

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

[0044] Specific primers were designed and mutations were introduced by PCR technique. Subsequently, the PCR products were digested with Dpnl enzyme and 10 μL of the PCR product was used to transform 50 μL of E. coli BL21(DE3). The transformed cells were grown in 5 mL of Luria-Bertani medium containing kanamycin overnight. The next day, 2 mL of the preculture was inoculated into 50 mL of Luria-Bertani medium containing kanamycin and grown at 37°C, 220 rpm for 2.5 hours. 5-aminolevulinic acid (ALA), FeS04, and isopropyl-β-D-thiogalactopyranoside (IPTG) were added to a final concentration of 0.5 mM, respectively, and then expressed at 22°C, 130 rpm for 20-24 hours. After expression, the cells were collected by centrifugation and resuspended in M9-N buffer, adjusted to OD 60 32, pH 7.5, to obtain whole-cell catalysts.

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

[0046] After saturation saturation mutation (SSM) and combinatorial mutation to generate libraries at V118, T278, C385H, F424, etc. sites, respectively, single colonies were randomly picked from kanamycin LB agar plates (LB Kan) and grown in 300 μL of LB medium containing 0.1 mg / mL kanamycin in sterilized 96-well deep plates. Each plate contained six wells inoculated with single colonies expressing the parent enzyme and two sterile wells. The cultures were grown at 37°C, 250 rpm for 8-12 hours. Subsequently, the cultures were transferred to LB medium containing 0.1 mg / mL kanamycin and grown at 37°C, 120 rpm for 3 hours. After that, the 96-well plates were cooled in an ice bath, IPTG and ALA were added to a final concentration of 0.5 mM, respectively, and then expressed at 16°C, 180 rpm for 20-22 hours. The cells were collected by centrifugation and the supernatant was discarded, and 360 μL of M9-N (pH 7.5) was added to each well. After the cells were completely resuspended by shaking, the reaction was initiated by adding thiothiol and ethyl diazoacetate (EDA) to a final concentration of 20 mM. The plates were 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] The cell concentration of the whole-cell resuspension was adjusted to OD 600 = 30. Then, 370 μL of the resuspension was aliquoted into 1.5 mL centrifuge tubes, and thiothiol and EDA were added to a final concentration of 20 mM, respectively, to initiate the reaction.

[0049] The reaction was carried out at 50°C and 120 rpm for 1-12 hours. Periodically, samples were taken, internal standard was added to the reaction solution, and supercritical chromatography analysis was performed to quantify the product.

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

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

[0052] Figure 4

[0053] Table 1 Comparison of the relative turnover efficiency (TOF) of P450 enzyme and mutant V118A / C385H / F424P

[0054]

[0055] Comparative Example 1:

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

[0057] Table 2 Comparison of the relative turnover efficiency of different mutants

[0058]

[0059]

[0060] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.​​

Claims

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

2. The gene encoding the P450 enzyme mutant of claim 1.

3. A recombinant plasmid containing the gene described in claim 2.

4. A host cell carrying the gene of claim 2 or the recombinant plasmid of claim 3.

5. Recombinant Escherichia coli, characterized in that, by E. coli Using BL21(DE3) as the host, the P450 enzyme mutant described in claim 1 was expressed using pET 28a(+) as the expression vector.

6. A cell catalyst, characterized in that, It contains the recombinant Escherichia coli cells as described in claim 5.

7. The method for preparing the cell catalyst of claim 6, characterized in that, The recombinant Escherichia coli was cultured in a culture medium, and bacterial cells with P450 enzyme catalytic activity were collected as whole-cell catalysts.

8. A method for improving the catalytic activity and / or turnover efficiency of P450 enzymes for arylthiol substrates, characterized in that, Based on the sequence shown in SEQ ID NO.1, valine at position 118 was mutated to alanine, cysteine ​​at position 385 was mutated to histidine, and phenylalanine at position 424 was mutated to proline.

9. The use of the P450 enzyme mutant of claim 1, or the recombinant Escherichia coli of claim 5, or the cell catalyst of claim 6 in participating in the CS bond coupling reaction.

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