Construction method and application of P450 fusion protein

Abstract

The invention provides a construction method and application of a P450 fusion protein. The construction method comprises the following steps: step (1), fusing abamectin oxidation type P450 Ema1 with a Rhodococcus P450RhF-RPs reductase structural domain by adopting a connecting peptide to obtain Ema1-Rhf; and (2) carrying out point mutation on the Ema1-Rhf, and constructing the P450 fusion protein. According to the invention, redox partners derived from Rhodococcus are connected with a structural domain of P450 Ema1 through a designed connecting peptide; the cytochrome P450 monooxygenase mutant further improves the enzyme activity in the process of catalyzing abamectin to be converted into methylamino abamectin, the maximum conversion rate can be improved by 2.2 times through determination, and the mutant achieves 90% conversion rate within 39.8 hours, which is far better than the expression of an unfused system. And the method has certain significance on industrial production of the emamectin benzoate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering and enzyme catalysis, in particular to a construction method of P450 fusion protein and application thereof. BACKGROUND

[0002] Cytochrome P450 monooxygenases (CYP450s) are widely distributed and are a superfamily of proteins containing heme B. The heme, as a co-factor of P450 enzymes, is connected to an absolutely conserved cysteine, which makes its reduced state bind to carbon monoxide to produce a characteristic absorption peak at 450 nm, hence the name. P450 enzymes have excellent catalytic diversity and are mainly involved in the metabolism of exogenous substances and the biosynthesis of natural products in organisms. They can catalyze various types of chemical reactions with structurally diverse organic compounds as substrates. P450 enzymes can selectively oxidize C-H bonds in substrate molecules under mild conditions, and thus have high practical value and a long history of application in the production of fine chemicals, chemical intermediates and drug molecules.

[0003] Avermectin is a class of natural macrolide compounds produced by fermentation of Streptomyces avermitilis, including A1a, A1b, B1a, B1b and other active isomers. Among them, avermectin B1a is the most valuable natural precursor due to its excellent biological activity. Its core application fields are concentrated in agricultural pest control and animal parasitic pest control, and it has low toxicity to non-target organisms in the environment, making it an indispensable green biopesticide in modern agriculture. On this basis, semi-synthetic derivatives obtained by structural modification further expand its application potential. Among them, emamectin, as a key derivative of avermectin, is obtained by oxidative modification of the C4" position of avermectin B1a and the introduction of a carbamate structure. Its insecticidal activity is more than 100 times higher than that of natural avermectin, and its mammalian toxicity is significantly reduced and its environmental compatibility is better. It has become the world's leading commercial insecticide.

[0004] Natural cytochrome P450 enzymes catalyze electron transfer mediated by redox partners (RPs), while the transient interaction between exogenous RPs and P450s results in low electron transfer efficiency, which restricts the catalytic efficiency. In the prior art, there are problems such as low conversion rate, long reaction time, and insufficient enzyme stability in the biocatalytic conversion of avermectin B1a. For example, in a non-fusion system, the conversion rate is only 36% within 72 hours, which cannot meet the needs of industrial production. Although there are strategies for constructing fusion enzymes, there is a lack of rational design methods based on structural prediction and electron tunneling geometry standards, which cannot precisely optimize the linker sequence and length, resulting in limited activity improvement of fusion enzymes. SUMMARY

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a construction method of P450 fusion protein and its application, which is used to solve the technical problems of low catalytic efficiency and long reaction period in the existing oxidation reaction of aminomethylavermectin.

[0006] To achieve the above-mentioned purpose and other related purposes, the present application provides a construction method of P450 fusion protein, comprising the following steps:

[0007] Step (1): using a linker to fuse avermectin oxidized P450 Ema1 and Rhodococcus P450 RhF-RPs reductase domain to obtain Ema1-Rhf;

[0008] Step (2): point mutation is performed on Ema1-Rhf to construct a P450 fusion protein.

[0009] Based on the oxidized P450 Ema1, the present application provides a self-sufficient cytochrome P450 fusion protein with high electron transfer efficiency, strong catalytic activity and good stability, and simultaneously provides an efficient construction method and an industrial application scheme thereof.

[0010] The technical scheme adopted by the present application is fusion enzyme core component and linker design. The fusion enzyme core component is: taking avermectin oxidized P450 Ema1 as the catalytic domain, and selecting the redox partner (containing ferredoxin domain and ferredoxin reductase domain) of Rhodococcus P450 RhF-RPs reductase domain as the electron transfer domain.

[0011] Preferably, the linker is selected from any one of Linker1~6, and the amino acid sequences of Linker1~6 are shown in SEQ ID NO: 1~6 in the following table: .

[0012] Preferably, in step (1), the amino acid sequence of Ema1-Rhf is shown in SEQ ID NO: 7~12 in the following table: .

[0013] More preferably, in step (2), the mutation sites of Ema1-Rhf are selected from one or any combination of M212A, T392H, P427C, C383S, C590T and S550C.

[0014] More preferably, the linker is Linker4, and the amino acid sequences of the P450 fusion proteins obtained after mutation of each mutation site are shown in SEQ ID NO: 13~18 in the following table: .

[0015] The Linker4 adopts an 8-amino acid length sequence "GEPAARAVS", which can stabilize the functional conformation of the fusion enzyme and keep the heme and Fe2S2 cluster at a high efficient electron tunneling distance of 14-18 Å.

[0016] The application further provides a P450 fusion protein constructed by the construction method.

[0017] The application further provides an expression method of the P450 fusion protein, which comprises the following steps: constructing a recombinant expression plasmid containing a gene of the P450 fusion protein; introducing the constructed recombinant expression plasmid into a host cell; culturing the host cell to obtain a genetically engineered bacterium; and making the gene of the P450 fusion protein expressed.

[0018] Preferably, the starting vector of the recombinant expression plasmid is pRSF-duet.

[0019] Preferably, the host cell is E. coli BL21 (DE3).

[0020] The application further provides a recombinant vector containing the gene encoding the P450 fusion protein. The recombinant vector comprises a polynucleotide operably linked to a control sequence suitable for directing expression in a host cell. Preferably, the recombinant vector takes plasmid pRSF-duet as a backbone.

[0021] The application further provides a genetically engineered bacterium obtained by the expression method.

[0022] The application further provides a genetically engineered bacterium containing the gene encoding the P450 fusion protein. Specifically, the genetically engineered bacterium is prepared by the following method: transforming a recombinant vector containing the gene encoding the P450 fusion protein and taking plasmid pRSF-duet as a backbone into a host bacterium to obtain a genetically engineered bacterium containing the recombinant vector. Preferably, E. coli BL21 (DE3) is used as the host bacterium.

[0023] The application further provides an application of the genetically engineered bacterium in preparing ivermectin.

[0024] Under the guidance of AlphaFold3 structure prediction and efficient electron tunneling geometry standard, we rationally designed a series of fusion structures with different connecting sequences and lengths. Molecular dynamics simulation showed that the 8-amino acid linker length of Linker4 was optimal for enzyme activity, as it stabilized the functional conformation, minimized structural fluctuations, and maintained a good distance between the heme and Fe2S2 cluster, thereby achieving efficient electron transfer. The cytochrome P450 fusion protein further improved the enzyme activity of cytochrome P450 monooxygenase in the process of catalyzing the synthesis of emamectin, an important intermediate of emamectin benzoate.

[0025] Preferably, the wet bacteria or pure enzyme extracted after ultrasonic disruption of the wet bacteria obtained by fermentation culture of the genetically engineered bacteria is used as a catalyst, and the reaction system is composed of the reaction substrate of avermectin in a buffer solution with a pH of 7-8.5 to synthesize 4''-oxo-avermectin.

[0026] Preferably, the reaction system further comprises one or more of coenzyme NADH, glucose dehydrogenase GDH, and glucose;

[0027] Preferably, the buffer solution is a phosphate buffer solution. More preferably, the phosphate buffer solution is a mixture of K2HPO4 and KH2PO4 with a concentration of 100 mM.

[0028] Preferably, the reaction substrate avermectin is dissolved in an equal mixture of DMSO and Tween 40

[0029] As described above, the present application has the following beneficial effects: the redox partners derived from Rhodococcus are connected to the domains of P450 Ema1 through a designed linker peptide; the cytochrome P450 monooxygenase mutant further improves the enzyme activity in the process of catalyzing the conversion of avermectin to emamectin, and the conversion rate can be increased by 2.2 times at most, and the mutant achieves a conversion rate of 90% within 39.8 hours, far exceeding the performance of the unfused system. The industrial production of emamectin, a key intermediate of eamectin, has certain significance. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Comparison example diagram of the catalytic oxidation of emamectin electron transfer system shown as avermectin B1a.

[0031] Figure 2 Conversion rate comparison diagram of single-point mutation based on the fusion enzyme in Example 3.

[0032] Figure 3The graph showing the consumption of NADH in Example 4 indicates the electron transfer rate of the P450 enzyme system. The fusion enzyme exhibits much higher electron transfer efficiency than the individual expression components. The observed electron transfer rate is increased by 2.8 times, confirming that the fusion design strategy successfully alleviates the key bottleneck in P450 application - the transfer of limited electrons.

[0033] Figure 4 The graph showing the time-conversion curve of emamectin benzoate in Example 5. DETAILED DESCRIPTION

[0034] The present application is described in detail by specific specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the specification. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details in the specification based on different views and applications without departing from the spirit of the present application.

[0035] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application all use conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0036] Example 1

[0037] Obtaining of cytochrome P450 fusion protein mutants:

[0038] The recombinant expression vector pRSF duet-Ema1 containing the cytochrome P450 Ema1 coding gene (synthesized by Beijing Qikexin Biotechnology Co., Ltd.) was used as a template to design primer sequences for PCR, and a redox partner from Rhodococcus was amplified by PCR and point mutations were made on this basis. The designed primer sequences are shown in Table 1.

[0039] The PCR (20 μL) amplification system was as follows: 2x PCR buffer solution 10 μL, upper and lower primers 0.8 μL each, template plasmid 0.2 μL, dNTP 0.8 μL, high-fidelity enzyme 0.2 μL, and ddH2O was added to make up to 20 μL.

[0040] The PCR amplification program was as follows: 95°C pre-denaturation for 2 min, 95°C denaturation for 30 s, 63°C annealing for 30 s, 72°C extension for 1 min per kb, 25 cycles, 72°C extension for 10 min, and 4°C storage.

[0041] After PCR, 5 μL of the amplified product was subjected to nucleic acid gel electrophoresis analysis, and the obtained target band of the clear PCR product was added to 2 μL of Dpn I endonuclease, which was incubated at 37 °C for 2 h to digest the template. The obtained product was purified using a PCR purification kit, and C115-02 cloning reagent (ClonExpress Ultra One-Step Cloning Kit from Vazyme) was used for reaction at 50 °C for 15 min, followed by heating at 80 °C for 20 min to achieve heat inactivation. The treated product was transformed into chemically competent Escherichia coli BL21 (DE3), which was spread on LB medium containing 50 μg / mL kanamycin and incubated at 37 °C overnight. The bacterial cells were collected to obtain transformants containing cytochrome P450 fusion protein mutants. Single colonies were selected and sent to Beijing Qikong Biotechnology Co., Ltd. for sequencing to verify the introduced mutations. After verification, subsequent reactions were performed.

[0042] Table 1. Primer table .

[0043] Example 2

[0044] Comparison of activities of wild-type cytochrome P450 fusion protein and its mutants

[0045] Culture medium preparation: Prepare an appropriate amount of 100 mL TB medium, and sterilize it by high-pressure steam at 121 °C for 20 min. After cooling, it is ready for use.

[0046] Collection of wet bacterial cells: In the LB solid medium in Example 1 above, single colonies of uniform size were selected and inoculated into 10 mL kanamycin-resistant LB medium test tubes, which were incubated at 37 °C and 180 rpm for 8 h. Then 2 mL of fresh bacterial solution was taken from the incubated test tubes and inoculated into 100 mL kanamycin-resistant TB medium, which was incubated at 37 °C and 180 rpm until the OD600 was 0.4-0.6. After the OD600 was appropriate, 0.2 mM isopropyl-β-D-thiogalactoside (IPTG), 0.64 mM δ-aminolevulinic acid (δ-ALA), and 0.32 mM ferrous sulfate (Fe2SO4) were added, and the cytochrome P450 fusion protein expression was induced at 20 °C. After the culture reached the appropriate concentration, it was centrifuged at 4 °C and 8000 rpm for 10 min, and the supernatant was removed to collect the wet bacterial cells.

[0047] Cell disruption: Wash twice with 20 mM sodium phosphate buffer (pH 7.5) and resuspend in the same buffer at a ratio of approximately 1 gram of wet cells to 10 mL of buffer. Preparation of cell-free extract: Sonication was performed first (200 W, 1 second sonication time, 2-second interval, 100 cycles), followed by centrifugation at 4 °C, 10,000 × g for 30 minutes. The supernatant was collected for subsequent protein purification.

[0048] Protein purification: After centrifugation (4 °C, 10,000 g, 40 min) to clarify the lysate, purification was performed using immobilized metal affinity chromatography. The following buffers were used: wash buffer (50 mM phosphate buffer containing 0.3 M NaCl and 0.02 M imidazole, pH 7.5); elution buffer (50 mM phosphate buffer containing 0.3 M NaCl and 0.5 M imidazole, pH 7.5). Subsequently, the protein was desalted using a PD-10 desalting column (GE Healthcare) in 50 mM phosphate buffer (pH 7.5). Finally, the purified and desalted protein was stored in a solution containing 30% glycerol at -20 °C.

[0049] Protein Expression: Analytical-scale biooxidation of Ema1 against avermectin B1a was performed in 1.5 mL Eppendorf tubes containing 500 μL of reaction mixture. Each reaction typically consisted of 100 μL of purified Ema1 protein, 100 μL of redox partner cell-free extracts (e.g., 50 μL Fdx and 50 μL FdR), 1.0 mM NADH, 5 mM substrate, 5% (v / v) DMSO, 5% (v / v) Tween-40, 100 mM glucose, 1 U / mL GDH, and 100 mM sodium phosphate buffer (pH 7.5). For the Ema1-RhF fusion system, 100 μL of purified fusion protein was used instead of each individual component. The reaction mixture was incubated at 30°C with shaking at 800 rpm for 1 h.

[0050] After incubation, 200 μL of the reaction solution was extracted with 1.0 mL of ethyl acetate and vortexed at 1000 rpm for 10 min. Denatured proteins and insoluble substances were removed by centrifugation at 10,000 × g for 20 min at room temperature. The organic layer was dried on anhydrous Na₂SO₄ and evaporated to dryness at room temperature, then redissolved in 500 μL of HPLC-grade acetonitrile. The resulting solution was filtered through a 0.22 μm membrane and transferred to a sealed amber vial for HPLC analysis. The analytical results are shown in Table 2. The mutant F4 (Linker4), with a linker peptide length of 8 amino acid residues, showed a maximum conversion rate 2.2-fold higher than the non-fusion peptide.

[0051] Table 2. Amino acid sequences of Linker 1-6 and specific activities of purified Ema1 mutants .

[0052] Example 3

[0053] This invention involves mutating the selected M212A, T392H, and P427C sites at the Ema1 position, as well as the C383S, C590T, and S550C sites at electron transport positions. The results are as follows: Figure 2 The conversion rates shown are as follows: wild type 5g / l: 45%; T392H 5g / l: 76%; P427C 5g / l: 59%; C383S 5g / l: 58%; C590T 5g / l: 68%; S550C 5g / l: 70%; and M212A has the highest conversion rate at 90% (5g / l).

[0054] Example 4

[0055] Electron transfer rate of the P450 fusion protein system

[0056] Activity was determined under standard conditions using purified protein in 1 mM avermectin B1a, 0.5 mM NADH, 2.5% (v / v) DMSO, 2.5% (v / v) 231 Tween-40, and 100 mM potassium phosphate buffer (pH 7.5).

[0057] The results showed that when the above solution was added to a 96-well cell culture plate (200 μL per well) and NADH consumption was detected by enzyme-linked immunosorbent assay (ELISA), the results were as follows: Figure 3 As shown, this fusion enzyme exhibits higher electron transfer efficiency than the separately expressed components. The observed 2.8-fold increase in electron transfer rate confirms that the fusion design strategy successfully alleviates a key bottleneck in P450 applications—rate-limiting electron transport.

[0058] Example 5

[0059] The Practical Application Potential of Synthetic Emamectin

[0060] To assess its practical application potential, we conducted synthetic experiments using a GDH coupling system to regenerate NADH to drive the conversion of avermectin B1a to emamectin methylaminoavermectin. The conversion process of Emali-Fdx / FdR and the fusion variant F4 was monitored at pH 7.5, with product formation over time monitored by taking three samples at each time interval in a single experiment. Figure 4 The conversion curves for emamectin production show that the engineered variant achieved 90% conversion within 8 hours, while the non-fusion system required over 12 hours to reach only 32% conversion. These results indicate that the fusion protein is more efficient in accelerating emamectin synthesis, with a 2.2-fold increase in catalytic efficiency, and possesses strong potential for industrial applications.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method of constructing a P450 fusion protein, characterized by, The method comprises the following steps: Step (1): fusing avermectin oxidized P450 Ema1 with Rhodococcus P450 RhF-RP reductase domain by using a connecting peptide to obtain Ema1-Rhf; Step (2): performing point mutation on Ema1-Rhf to construct a P450 fusion protein.

2. The construction method of claim 1, wherein: In step (1), the connecting peptide is selected from any one of Linker1-6, and the amino acid sequences of the Linker1-6 are shown in SEQ ID NO: 1-6.

3. The method of construction of claim 2, wherein: In step (1), the amino acid sequence of Ema1-Rhf is shown in SEQ ID NO: 7-12.

4. The method of construction of claim 1, wherein: In step (2), the mutation sites of Ema1-Rhf are selected from one or any combination of M212A, T392H, P427C, C383S, C590T and S550C.

5. The method of construction of claim 4, wherein: The connecting peptide is linker4, and the amino acid sequences of the P450 fusion proteins obtained after mutation of the mutation sites are shown in SEQ ID NO: 13-18.

6. A P450 fusion protein constructed by the construction method of any one of claims 1-5.

7. A method of expressing a P450 fusion protein according to claim 6, characterized by: A recombinant expression plasmid containing a P450 fusion protein gene is constructed, the constructed recombinant expression plasmid is introduced into a host cell, the host cell is cultured to obtain a genetically engineered bacterium, and the P450 fusion protein gene is expressed.

8. The expression method of claim 4, wherein: The starting vector of the recombinant expression plasmid is pRSF-duet, and the host cell is E. coli BL21.

9. Use of the P450 fusion protein of claim 6 in the preparation of emamectin benzoate.

10. Use according to claim 9, characterized in that: The P450 fusion protein is expressed to obtain a genetically engineered bacterium, and the genetically engineered bacterium is fermented and cultured to obtain wet bacteria or pure enzyme extracted after ultrasonic disruption of the wet bacteria, which is used as a catalyst to synthesize emamectin benzoate in a reaction system composed of avermectin as a reaction substrate and a buffer solution with a pH of 7-8.5; the reaction system further comprises one or more of coenzyme NADH, glucose dehydrogenase GDH and glucose; and the buffer solution is a phosphate buffer.

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