Method for preparing P-gp inhibitor Chrysosporazine C through biological enzyme catalysis

Abstract

The invention provides an application of a Cry7 protein with an amino acid sequence as shown in SEQ ID NO: 1 in preparation of a P-gp inhibitor Chrysosporazine C, and the Cry7 protein has a nucleotide sequence as shown in SEQ ID NO: 2. The formula of the Chrysosporazine C is as shown in a formula II in the specification. The invention also provides a method for preparing a P-gp inhibitor Chrysosporazine C by utilizing a biological enzyme catalyst, which comprises the following steps: purifying and expressing Cry7 protein with an amino acid sequence shown as SEQ ID NO: 1, and carrying out catalytic fermentation by utilizing the Cry7 protein by taking Chrysosporazine D shown as a formula I as a substrate, so as to obtain the Chrysosporazine C. The invention also provides a method for preparing the P-gp inhibitor Chrysosporazine C. The invention also provides a method for preparing the P-gp inhibitor Chrysosporazine C. The invention also provides a method for preparing the P-gp inhibitor Chrysosporazine C. The invention also provides a method for preparing the P-gp inhibitor Chrysosporazine C. The method has the advantages that the production route is simple, the operation period is short, and the Chrysosporazine D can be converted into the Chrysosporazine C with higher biological activity in a short period of time.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a method for preparing P-gp inhibitor Chrysosporazine C by using biological enzyme catalysis. BACKGROUND

[0002] Natural products have unique structures and wide biological activities, and are an important source of new drug discovery. However, it is difficult to separate active compounds by traditional separation methods or organic synthesis methods due to problems such as long time consumption and harsh reaction conditions. Biological enzyme catalysis uses recombinant enzymes as catalysts, and has the characteristics of mild conditions and environmental friendliness. Through the mediation of a biological catalytic carrier, natural active small molecules can be obtained in a short period of time.

[0003] Fungi have mild culture conditions and grow rapidly, and are often used as a carrier for biological catalysis and fermentation. When used for biological catalysis, they can act as a host and work together with exogenous biological catalysts to catalyze the formation of alkaloids and flavonoids and other types of natural products.

[0004] P-glycoprotein (P-gp) overexpression is one of the main causes of drug resistance to many drugs, including antitumor drugs. P-gp can cause drug resistance by twisting and extruding drugs out of cells. In order to solve the problem of multiple drug resistance caused by P-gp overexpression, P-gp inhibitors derived from natural products have received extensive attention.

[0005] Chrysosporazines, a kind of phenylpropanoid piperazine alkaloids derived from fungi, have been found to have good P-gp inhibitory activity. Five new phenylpropanoid piperazine compounds, named Chrysosporazine A-E, were isolated and identified from the fungus Chrysosporium sp. CMB-F214 derived from the gastrointestinal tract of marine fish in Australia. Systematic pharmacological activity evaluation showed that the IC 50 (half maximal inhibitory concentration) and FR (resistance fold) of Chrysosporazine D were significantly greater than those of Chrysosporazine C, i.e., Chrysosporazine C had more excellent biological activity. Moreover, Chrysosporazine C did not show cytotoxicity at high concentrations (30 μM), and its P-gp inhibitory activity against human colorectal cancer cells was even stronger than that of verapamil, indicating that the biological activity of Chrysosporazine C was significantly better than that of Chrysosporazine D.

[0006] Therefore, how to prepare Chrysosporazine C with higher biological activity is a problem to be solved at present. SUMMARY

[0007] The present application aims to overcome the above-mentioned deficiencies in the prior art, and provides a method for preparing P-gp inhibitor Chrysosporazine C by using a biological enzyme catalyst.

[0008] The first object of the present application is to provide the use of Cry7 protein with an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of P-gp inhibitor Chrysosporazine C.

[0009] The second object of the present application is to provide the use of a gene with a nucleotide sequence as shown in SEQ ID NO: 2 in the preparation of P-gp inhibitor Chrysosporazine C. cry7

[0010] The third object of the present application is to provide a recombinant vector.

[0011] The fourth object of the present application is to provide a recombinant cell.

[0012] The fifth object of the present application is to provide the use of the above-mentioned recombinant vector and / or recombinant cell in the preparation of P-gp inhibitor Chrysosporazine C.

[0013] The sixth object of the present application is to provide a method for preparing P-gp inhibitor Chrysosporazine C by using a biological enzyme catalyst.

[0014] The seventh object of the present application is to provide Chrysosporazine C prepared by the above-mentioned method.

[0015] In order to achieve the above-mentioned objects, the present application is realized by the following scheme: The present application claims the use of Cry7 protein with an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of P-gp inhibitor Chrysosporazine C; The chemical formula of Chrysosporazine C is as shown in formula II;

[0016] Formula II.

[0017] The present application also claims the use of a gene with a nucleotide sequence as shown in SEQ ID NO: 2 in the preparation of P-gp inhibitor Chrysosporazine C; cry7 ​​The Chrysosporazine C has a chemical formula as shown in Formula II.

[0018] Formula II.

[0019] The gene encodes a Cry7 protein with an amino acid sequence as shown in SEQ ID NO: 1. cry7 The gene encodes a Cry7 protein with an amino acid sequence as shown in SEQ ID NO: 1.

[0020] The present application also claims a recombinant vector, which is a vector containing a gene with a nucleotide sequence as shown in SEQ ID NO: 2. cry7 The gene encodes a Cry7 protein with an amino acid sequence as shown in SEQ ID NO: 1.

[0021] Preferably, the vector is pRSF and pKW-2u-pyrO-4g.

[0022] Preferably, the vector is pRSF and pKW-2u-pyrO-4g.

[0023] Preferably, the vector is pRSF and pKW-2u-pyrO-4g. S11. Amplifying a gene with a nucleotide sequence as shown in SEQ ID NO: 2 by PCR to obtain a target gene fragment. cry7 S11. Amplifying a gene with a nucleotide sequence as shown in SEQ ID NO: 2 by PCR to obtain a target gene fragment. S12. Connecting the linearized vector with the target gene fragment obtained in step S11 and screening, to obtain the recombinant vector.

[0024] The present application also claims a recombinant cell, which is a cell containing any of the above-mentioned recombinant vectors.

[0025] Preferably, the recombinant vector is a pKW-2u-pyrO-4g vector containing a gene with a nucleotide sequence as shown in SEQ ID NO: 2. cry7 Preferably, the recombinant vector is a pKW-2u-pyrO-4g vector containing a gene with a nucleotide sequence as shown in SEQ ID NO: 2.

[0026] Preferably, the cell is a LO8030 cell. Aspergillus nidulans Preferably, the cell is a LO8030 cell.

[0027] The present application also claims the use of any of the above-mentioned recombinant vectors and / or the above-mentioned recombinant cells in the preparation of a P-gp inhibitor Chrysosporazine C. The Chrysosporazine C has a chemical formula as shown in Formula II.

[0028] Formula II.

[0029] The present application also claims a method for preparing P-gp inhibitor Chrysosporazine C by using biological enzyme catalyst, comprising the following steps: S1. Purifying the Cry7 protein expressed by the amino acid sequence as shown in SEQ ID NO: 1 by using any of the above-mentioned recombinant vectors and / or the above-mentioned recombinant cells; S2. Using the purified Cry7 protein expressed by step S1 to react with Chrysosporazine D as shown in Formula I as the substrate, so as to obtain Chrysosporazine C as shown in Formula II;

[0030] Formula I;

[0031] Formula II.

[0032] Preferably, step S1 is purifying the Cry7 protein expressed by the amino acid sequence as shown in SEQ ID NO: 1 by using the above-mentioned recombinant cells.

[0033] More preferably, the purifying the Cry7 protein expressed by the amino acid sequence as shown in SEQ ID NO: 1 by using the above-mentioned recombinant cells is specifically catalytic fermentation of the above-mentioned recombinant cells to purify the Cry7 protein expressed by the amino acid sequence as shown in SEQ ID NO: 1.

[0034] Preferably, the concentration of the substrate in step S2 is 1-2 mM, and the concentration of the purified Cry7 protein expressed is 20-25 μM.

[0035] More preferably, the concentration of the substrate in step S2 is 1 mM, and the concentration of the purified Cry7 protein expressed is 25 μM.

[0036] Preferably, when the purified Cry7 protein expressed by step S1 is used to react in step S2, ascorbic acid, α-ketoglutaric acid and ferrous sulfate need to be added.

[0037] More preferably, the final concentration of ascorbic acid in the reaction system is 4-6 mM, the final concentration of α-ketoglutaric acid in the reaction system is 4-6 mM, and the final concentration of ferrous sulfate in the reaction system is 4-6 mM.

[0038] Further preferably, the final concentration of ascorbic acid in the reaction system is 5 mM, the final concentration of α-ketoglutaric acid in the reaction system is 5 mM, and the final concentration of ferrous sulfate in the reaction system is 5 mM.

[0039] The application also claims the Chrysosporazine C prepared by any of the above-mentioned methods.

[0040] Compared with the prior art, the application has the following beneficial effects: The application provides an application of a Cry7 protein with an amino acid sequence shown in SEQ ID NO: 1 in preparation of a P-gp inhibitor Chrysosporazine C; the Chrysosporazine C has a chemical formula shown in formula II; and the application provides a method for preparing the P-gp inhibitor Chrysosporazine C by using a biological enzyme catalyst, which comprises the following steps: purifying the Cry7 protein with the amino acid sequence shown in SEQ ID NO: 1, taking Chrysosporazine D with a chemical formula shown in formula I as a substrate, and reacting by using the Cry7 protein to obtain the Chrysosporazine C with the chemical formula shown in formula II. The method has a simple production route, a short operation period, and can convert Chrysosporazine D into Chrysosporazine C with higher biological activity in a short period. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 It is an agarose gel electrophoresis result chart in the template DNA extraction process in Example 1; Figure 2 It is an agarose gel electrophoresis result chart of an amplification product in the target gene acquisition process in Example 1; Figure 3 It is an enzyme digestion verification result chart in the recombinant plasmid construction process in Example 1; Figure 4 It is an LC-MS analysis chart in Example 2; Figure 5 It is an SDS-PAGE gel electrophoresis result chart of the purified Cry7 protein in Example 3; Figure 6 It is an HPLC chart after incubation of each group in Example 3. DETAILED DESCRIPTION

[0042] The application will be further described in detail below in combination with the drawings and specific examples in the specification, and the examples are only used to explain the application and are not used to limit the scope of the application. In the following examples, the test methods used are conventional methods unless otherwise specified, and the materials, reagents and the like used are reagents and materials that can be obtained through commercial channels unless otherwise specified.

[0043] Strains used in the examples of the application Chrysosporium sp. CMB-F214Elbanna A H, Khalil Z G, Bernhardt P V, et al. Chrysosporazines A–E: P-Glycoprotein inhibitory piperazines from an Australian marine fish gastrointestinal tract-derived fungus, Chrysosporium sp. CMB-F214[J]. Organic Letters, 2019, 21(19): 8097-8100. The strain was obtained from the corresponding author of the prior art.

[0044] The pKW-2u-pyrO-4g plasmid used in the embodiments of the present application is derived from the prior art: Jinyu Hu, Farzaneh, et al. Heterologous biosynthesis of elsinochrome A sheds light on the formation of the photosensitive perylenequinone system. [J]. Chemical Science, 2019. DOI:10.1039 / c8sc02870b.

[0045] The pRSF plasmid used in the embodiments of the present application is derived from the prior art: Butt, Tauseef R et al. SUMO fusion technology for difficult-to-express proteins. [J]. Protein expression and purification vol. 43, 1 (2005): 1-9. DOI:10.1016 / j.pep.2005.03.016 Example 1 Construction of recombinant plasmid pKW-2u-pyrO-4g-cry7 I. Experimental methods 1. Acquisition of target gene Based on Chrysosporium sp . CMB-F214, the whole genome thereof was extracted as template DNA for PCR amplification of the target fragment, as follows: S1. The Chrysosporiumsp. CMB-F214 (wild type strain described in the existing paper DOI: 10.1021 / acs.orglett.9b03094) was inoculated on YPD {containing 1 g of Peptone (OXOID, LP0137), 0.5 g of Yeast extract (OXOID, LP0021B) per 50 mL of water, and 2.5 mL of 40% glucose (w / v) was added after sterilization} culture plates, and the bacteria were collected after 4 days of culture at 25°C; S2. 0.5 g of the bacteria collected in step S1 was freeze-dried in liquid nitrogen, and the crushed bacteria were ground with a grinding rod. 400 μL of PL2 buffer (containing 10 mM Tris, 10 mM EDTA and 0.9 g of SDS per 90 mL of water) was added, and after being mixed well, 0.1 μg of RNase A (BioFroxx, 1341MG100) was added. After being placed in a 70°C metal bath at 450 rpm for 20 min, it was cooled to 25°C to obtain a cooled solution; S3. 400 μL of PL3 buffer (containing 2.8 M potassium acetate per 100 mL of water) was added to the cooled solution obtained in S2, and after being mixed well, 400 μL of chloroform:isopropanol mixture (chloroform and isopropanol were mixed uniformly at a volume ratio of 24:1) was added. After being mixed well, it was centrifuged at 14000 rpm at 25°C for 4 min, and the supernatant was collected; S4. 600 μL of the supernatant obtained in step S3 was transferred to a 1.5 mL EP tube, 500 μL of isopropanol was added, and after being mixed well by inverting, it was placed at 25°C for 8 min, and then centrifuged at 14000 rpm at 25°C for 10 min. The supernatant was discarded, 400 μL of 70% ethanol (v / v) was added to the precipitate, and after being mixed well by inverting, it was centrifuged at 14000 rpm at 25°C for 1 min. The supernatant was discarded, and the tube was inverted and placed in the ventilation port of the biosafety cabinet to dry. Then 60 μL of TE buffer was added, and after being mixed well by shaking, the nucleic acid concentration was measured to be 98 ng / μL using a nanodrop (KAIAO, K5600), and gel electrophoresis was performed to determine the purity. The gel electrophoresis band was bright and there was no impurity band, and there was no RNA residue. Thus, the template DNA was obtained Chrysosporium sp. CMB-F214 gDNA).

[0046] Using the whole genome of Chrysosporium sp. CMB-F214 as the template DNA Chrysosporium sp .Cry7 gene) was amplified by PCR using the gDNA of CMB-F214 as template, according to the amplification system shown in Table 1 combined with the PCR reaction program shown in Table 2, the amplification product was collected, and the length was confirmed by agarose gel electrophoresis, the target fragment band was collected and cut with an agarose gel recovery kit (OMEGA, D2500-02) for gel recovery, thereby obtaining the amplification fragment carrying the target gene (nucleotide sequence as shown in SEQ ID NO: 2 cry7 gene) ; wherein the nucleotide sequence as shown in SEQ ID NO: 2 cry7 gene encodes the Cry7 protein with the amino acid sequence as shown in SEQ ID NO: 1. cry7

[0047] Table 1 Amplification system

[0048] Table 2 PCR reaction program

[0049] The information of cry7 F (SEQ ID NO: 3) and cry7 R (SEQ ID NO: 4) is shown in Table 3.

[0050] Table 3 Information of cry7 F and cry7 R

[0051] 2. Construction of recombinant plasmid The recombinant yeast plasmid BJ5464-cry7 was constructed according to the frozen yeast transformation kit (ZYMO Research, T2001), and the specific process was as follows: ​After 2 μL of target gene with a concentration of 55 ng / μL and 2 μL of pKW-2u-pyrO-4g linear plasmid digested by PacI (NEB R0547S) for 10 h were mixed uniformly on ice, 50 μL of yeast competent cells BJ5464 was added, and then 400 μL of solution 3 buffer (derived from frozen yeast transformation kit) was added. After mixing uniformly by inversion, incubation was performed at 30°C, with 3 cycles (1 min standing, 350 rpm / min reaction for 15 min). After incubation, the mixture was uniformly smeared on a ura auxotrophic Drop Out culture plate (containing 6.7 g of amino acid-free yeast nitrogen source (SIGMA, Y0626), 20 g of glucose, 1.4 g of yeast auxotrophic medium supplement (SIGMA, Y2001), 76 mg of histidine, 76 mg of tryptophan, 380 mg of leucine, and 20 g of medium agar) per L, and incubation was performed at 30°C until colonies grew on the culture plate. The transformants were picked up to obtain the pre-transformed recombinant yeast plasmid BJ5464-cry7.

[0052] After 50 μL of E. coli competent cells (NEB, C3020K) were placed in a centrifuge tube, 3 μL of the pre-transformed recombinant yeast plasmid BJ5464-cry7 was added and mixed uniformly on ice, and then transferred to a pre-cooled electroporation cup. The electroporation cup was inserted into the electroporation instrument, and after the buzzer sounded, the electroporation cup was taken out, 300 μL of LB liquid medium was added, and the mixture was mixed uniformly. The suspension in the electroporation cup was transferred to a 1.5 mL EP tube, which was incubated at 37°C and 100 rpm for 1 h. Then, the mixture was uniformly smeared on an LB (ampicillin-resistant) solid plate, which was incubated at 37°C for 12 h. Then, the transformants on the LB (ampicillin-resistant) solid plate were selected and incubated at 37°C and 180 rpm for 10 h. The plasmid was extracted, and a digestion system was prepared according to the instructions using high-fidelity enzyme Pvul (NEB, R3150S). The plasmid was digested and verified by agarose gel electrophoresis. The correct plasmid was the recombinant plasmid pKW-2u-pyrO-4g-cry7.

[0053] II. Experimental results The agarose gel electrophoresis result graph during the template DNA extraction process is shown in Figure 1 The results show that the electrophoresis band is bright and there is no impurity band, indicating that there is no RNA residue.

[0054] The agarose gel electrophoresis result graph of the amplification product during the target gene acquisition process is shown in Figure 2 The results show that a bright band appears at 1000 bp in the electrophoresis result, indicating that the target fragment is successfully amplified.

[0055] The digestion verification result graph during the construction of the recombinant plasmid is shown inFigure 3 As shown, Figure 3 In the diagram, A represents the simulated enzyme digestion result, lane MW represents the marker lane, lane 1 represents the simulated electrophoresis result after enzyme digestion of the empty vector (i.e., pKW-2u-pyrO-4g plasmid), and lane 2 represents the simulated electrophoresis result after enzyme digestion of the recombinant plasmid pKW-2u-pyrO-4g-cry7. Figure 3 B in the figure represents the agarose gel electrophoresis result during the enzyme digestion verification process.

[0056] The results showed that the agarose gel electrophoresis results during the enzyme digestion verification process were consistent with the electrophoresis simulation results of the recombinant plasmid pKW-2u-pyrO-4g-cry7 in the enzyme digestion simulation results, indicating that the recombinant plasmid pKW-2u-pyrO-4g-cry7 was successfully constructed.

[0057] Example 2: Heterologous expression of recombinant plasmid pKW-2u-pyro-4g-cry7 I. Experimental Methods The recombinant plasmid pKW-2u-pyro-4g-cry7 obtained in Example 1 was heterologously expressed into... Aspergillus nidulans The specific steps in LO8030 cells are as follows: S1. Experimental group: Take 3 μL of the recombinant plasmid pKW-2u-pyrO-4g-cry7 (5000ng / μL) prepared in Example 1 and 3 μL of hvm3-11 plasmid (expressing Chrysosporazine D, chemical formula as shown in Formula I) (hvm3-11 plasmid is described in the prior art DOI: 10.1021 / acs.orglett.3c04310), drop them onto the side wall of a 15mL EP tube, and then use 91μL STC buffer {STC buffer (1L): 218.6g of sorbitol, 10mM Tris-HCl (pH 7.5), 1.47g CaCl2·2H2O} to flush the plasmid on the side wall of the EP tube to the bottom of the EP tube;

[0058] Formula I; S2. Add 125 μL of [amount] to each of the experimental groups obtained in step S1. Aspergillus nidulansLO8030 protoplast (recorded in prior art: Chiang Y M, Ahuja M, Oakley C E, et al. Development of genetic dereplication strains in Aspergillus nidulans results in the discovery of aspercryptin [J]. Angewandte Chemie International Edition, 2016, 55(5): 1662-1665.) was placed on ice for 10-15 min to obtain protoplast mixture.

[0059] S3. 250 μL of 60% (w / v) PEG 4000 {60% PEG (100 mL): 60 g of PEG 4000, 0.555 g of CaCl2, 5 mL of 1 M Tris-HCl (pH 7.5)} was added to the protoplast mixture obtained in step S2, mixed evenly, then 500 μL of 60% (w / v) PEG 4000 was added again (repeated 2 times), placed at 25°C for 20 min, then 500 μL of STC buffer was added 3 times in turn, mixed evenly, to obtain protoplast mixture 1.

[0060] S4. The protoplast mixture 1 obtained in step S3 was evenly distributed on 3 SMM solid plates, dried and placed at 37°C to grow spores (colonies).

[0061] The spores grown on the SMM solid plate were preserved with 40% glycerol by mass fraction, and a single colony on the SMM solid plate was picked and streaked to a new SMM solid plate, which was placed at 37°C for 2 days. Then 3 mL of 0.1% Tween 80 by mass volume ratio was used to wash the spores grown on the plate, and was inoculated into 50 mL of GMM liquid medium, which was placed at 37°C, 180 rpm for 18 h. Then 135 μL of cyclopentanone was added, and the culture was placed at 25°C, 180 rpm for 2 days. Then 65 μL of cyclopentanone was added and cultured for 1 day. The cell layer and the medium layer were obtained by reduced pressure filtration. The SMM solid plate (agar plate) consists of 20 g / L glucose, 220 g / L D-sorbitol, 40 mL / L 50× Nitrate Salts, and 2 mL / L Trace elements. The Nitrate Salts contain 0.6 g / mL NaNO3, 0.052 g / mL KCl, 0.052 g / mL MgSO4·7H2O, and 0.152 g / mL KH2PO4. The Trace elements contain 22 g / L ZnSO4·7H2O, 11 g / L H3BO3, 5 g / L MnCl2·4H2O, 5 g / L FeSO4·7H2O, 1.6 g / L CoCl2·5H2O, 1.6 g / L CuSO4·5H2O, and 1.1 g / L (NH4)6Mo7O. 24 • 4H2O and 60 g / L Na4EDTA.

[0062] The difference between GMM liquid medium and SMM solid plate is that GMM liquid medium does not contain 220 g / L D-sorbitol, while the other components are exactly the same (excluding agar).

[0063] S5. Extract the culture medium layer obtained in step S4 with ethyl acetate, collect the organic solution and concentrate it, collect the concentrate for LC-MS analysis, and plot the HPLC detection chromatogram of the experimental group.

[0064] The conditions for LC-MS analysis are set as follows: LC-MS analysis was performed using a liquid chromatography-time-of-flight mass spectrometer (Shimadzu LCMS-IT-TOF) equipped with a photo-diode array (PDA) detector, which was able to obtain HRESI spectra of positive and negative ions. Chromatographic column: Kinetex C18 column (2.6μm; 2.1mm × 100mm; Phenomenex); Mobile phase: 0.1% formic acid aqueous solution as phase A, and methanol as phase B; Flow rate: 0.5 mL / min; The elution procedure during LC-MS analysis is shown in Table 4.

[0065] Table 4 Elution Procedure

[0066] The difference between the negative group and the experimental group is that: in step S1, the recombinant plasmid pKW-2u-pyrO-4g-cry7 (5000 ng / μL) prepared in Example 1 is not added, only the hvm3-11 plasmid (5000 ng / μL) is added, and the rest of the processing is exactly the same, and the negative group HPLC detection spectrum is obtained.

[0067] The difference between the control group and the experimental group is that: in step S1, the recombinant plasmid pKW-2u-pyrO-4g-cry7 (5000 ng / μL) prepared in Example 1 and the hvm3-11 plasmid (5000 ng / μL) are not added, and the rest of the processing is exactly the same, and the blank group HPLC detection spectrum is obtained.

[0068] II. Experimental results The LC-MS analysis spectrum is shown in Figure 4 The results show that: there is no absorption peak of the compound in the spectrum of the blank group; the HPLC spectrum of the negative group (i.e. only expressing the hvm3-11 plasmid to obtain Chrysosporazine D compound) contains only the absorption peak of the compound Chrysosporazine D (the number 1 in the spectrum corresponds to the compound); and in the HPLC spectrum of the experimental group (i.e. co-expressed with Cry7), the absorption peak of the compound Chrysosporazine D (the absorption peak numbered 1) disappears, and the absorption peak of the compound 2 appears, indicating that after co-expressing the hvm3-11 plasmid and the recombinant plasmid pKW-2u-pyrO-4g-cry7 prepared in Example 1, the Chrysosporazine D expressed by the hvm3-11 plasmid is converted to compound 2 by the Cry7 protein expressed by the recombinant plasmid pKW-2u-pyrO-4g-cry7.

[0069] Example 3 Purification of Cry7 protein and in vitro activity test I. Protein purification 1. Experimental method S1. According to the purpose gene acquisition step described in step 1 of Example 1, replace cry7 F (SEQ ID NO: 3) and cry7 R (SEQ ID NO: 4) in the amplification system shown in Table 1 with cry7 F' (SEQ ID NO: 5) and cry7 R' (SEQ ID NO: 6), and the rest is the same, to obtain an amplification fragment 1 (including cry7 the gene and the homologous arm for linking to the PRSF vector) carrying the target gene (the nucleotide sequence is shown as SEQ ID NO: 2 cry7 2. The difference between the control group and the experimental group is that: in step S1, the recombinant plasmid pKW-2u-pyrO-4g-cry7 (5000 ng / μL) prepared in Example 1 and the hvm3-11 plasmid (5000 ng / μL) are not added, and the rest of the processing is exactly the same, and the blank group HPLC detection spectrum is obtained. cry7The gene encodes a Cry7 protein with an amino acid sequence as shown in SEQ ID NO: 1; wherein the information of cry7 F' and cry7 R' is shown in Table 6.

[0070] Table 6 Information of cry7 F' and cry7 R'

[0071] Then the amplified fragment 1 is connected to the PRSF vector (kanamycin resistance) by homologous recombination (pRSF is recorded in prior art DOI: 10.1016 / j.pep.2005.03.016) to obtain a recombinant PRSF vector, which is then transformed into an E. coli TOP10 strain (Kangtai Life, KTSM103L), and after plating, a single colony is picked and inoculated in 5 mL of LB medium, which is cultured at 37°C for 8 h, and then 1 mL of liquid is taken for sequencing in a clean bench environment. After sequencing, the plasmid is extracted using a plasmid extraction kit (Vazyme, DP103-03) according to the kit instructions, and the extracted plasmid is transformed into BL21 (DE3) (Kangtai Life, KTSM113L) for plating, and a single colony is picked and inoculated in 100 mL of LB liquid medium (kanamycin resistance), which is cultured at 37°C, 220 rpm to OD 600 between 0.6 and 0.8, 0.2 M IPTG is added, and the induction is performed at 16°C, 220 rpm for 16 h, and the crude enzyme solution is collected; S2. The crude enzyme solution obtained in step S1 is centrifuged at 3600 rpm, 16°C for 10 min, the precipitate is collected and resuspended in 50 mL of Buffer A (containing 1 M NaCl, 25 mM Tris 8.0 and 25 mM imidazole) to obtain a resuspension liquid, then 0.1% (v / v) PMSF and 0.33% (v / v) β-mercaptoethanol are added to the resuspension liquid, and the mixture is vortexed and then broken by a bacteria breaking instrument to obtain a crude protein solution; S3. The crude protein solution obtained in step S2 is centrifuged at 4°C, 12000 rpm for 25 min, and the supernatant is collected for elution and purification to obtain the purified Cry7 protein, and the purity of the purified Cry7 protein is detected by SDS-PAGE gel electrophoresis; Wherein the elution and purification use a nickel column (Yonglian Biotechnology, Shanghai) as follows: The eluate was collected and concentrated to 3 mL after being digested by ULP1 protease for 3 h, then diluted to 30 mL by Buffer C (containing 100 mM NaCl and 25 mM Tris 8.0), and a Buffer C protein solution was obtained; Then, the new nickel column was sequentially washed by 50 mL of Buffer B, 50 mL of Buffer A, 30 mL of the Buffer C protein solution, 40 mL of Buffer C, and 50 mL of Buffer B, the eluate was collected and concentrated to 1 mL, then chromatography was performed by using a molecular sieve gel column, Buffer D (containing 100 mM NaCl, 25 mM Tris 8.0, and 5 mM DTT) was used as the buffer for the molecular sieve gel column, and the protein after chromatography was collected, which was the purified Cry7 protein.

[0072] 2. Experimental results The SDS-PAGE gel electrophoresis result of the purified Cry7 protein is shown in Figure 5 The results show that only one bright electrophoresis band appears in the lane corresponding to the purified Cry7 protein, with a size of 37.2 kDa, indicating that the protein is successfully purified, and there is no other impurity protein in the purified Cry7 protein.

[0073] II. In vitro activity test 1. Experimental method The experimental group (Cry7+1) setting and treatment included the following steps: S1. Take 1.5 mL of an EP tube, mix 1 mM of Chrysosporazine D (chemical formula as formula I), 5 mM of ascorbic acid, 5 mM of alpha-ketoglutaric acid, 5 mM of FeSO4·7H2O, and 25 μM of purified Cry7 protein in 50 mM Tris-HCl buffer (pH 7.8), and then supplement the total volume to 200 μL (the reaction system is 200 μL) by using 50 mM Tris-HCl buffer (pH 7.8), to obtain a mixed solution; S2. The mixed solution obtained in step S1 was incubated in a 37°C incubator for 18 h, after incubation, 200 μL of acetonitrile was used for extraction, and the organic layer was collected for LC-MS analysis to obtain an HPLC spectrum; the LC-MS condition settings are shown in Example 2.

[0074] The control group (Boilded Cry7+1) is: the purified Cry7 protein in step S1 is replaced with an equal amount of boiled inactivated Cry7 protein, and the rest of the treatment is unchanged, and incubation and LC-MS analysis are performed.

[0075] The negative control group (1 in Tris-HCl) is: no purified Cry7 protein is added in step S1, and the rest of the treatment is unchanged, and incubation and LC-MS analysis are performed.

[0076] 2. Experimental results The HPLC chromatogram of each group after incubation is shown in Figure 6 The results show that in the HPLC chromatogram of the negative control group (1 in Tris-HCl), only the absorption peak of Chrysosporazine D appears (position 1 in the figure); in the control group (Boilded Cry7+1), only the absorption peak of Chrysosporazine D appears (position 1 in the figure), indicating that the inactivated Cry7 protein cannot promote the conversion of Chrysosporazine D to other substances; and in the HPLC chromatogram of the experimental group (Cry7+1), no absorption peak of Chrysosporazine D appears, but a new compound appears (position 2 in the figure), indicating that the purified Cry7 protein (amino acid sequence shown in SEQ ID NO: 1) can promote the conversion of Chrysosporazine D to a new compound.

[0077] Example 4 Identification of compounds I. Experimental method S1. The fermentation system of S4 in step one of Example 2 is expanded to 4L, as follows: The strain (LO8030 strain co-expressing hvm3-11 and pKW-pyrO-4g-cry7 plasmid) after preservation with glycerol of mass fraction 40% in S4 of Example 2 step one is divided into 12 plates, and the 50 mL of GMM liquid medium in Example 2 step one S4 is expanded to 4L of GMM medium, and the other culture conditions are expanded by the same ratio for culture. After the culture is completed, 4L of ethyl acetate is used to extract the fermentation broth, and the organic layer 1 is collected. Aspergillus nidulans At the same time, the experimental group (Cry7+1) in the in vitro activity test in step two of Example 3 is expanded by ten times by the same ratio, and is treated according to the same treatment method. Organic layer 2 is collected.

[0078]

[0079] ​Mixing organic layer 1 and organic layer 2, after rotary evaporation, sample mixing was performed using silica gel with a particle size of 230-400 mesh, followed by preliminary separation using an automatic column machine (Simei Technology, SepaBean Machine), and elution was performed according to the elution program shown in Table 7 using dichloromethane as mobile phase A and methanol as mobile phase B, to obtain the eluted sample; Table 7 Elution program

[0080] The remaining column machine conditions are as follows: Collection wavelength range: 220-800 nm Collection volume: 30 mL / tube S2. Take 1 mL of the eluted sample obtained in step S1 from each test tube and perform LC-MS detection (the LC-MS condition settings are shown in Example 2), collect all test tubes containing target compound 2 (the position 2 absorption peak appearing in Examples 2 and 3 corresponds to the compound), concentrate to obtain a sub-component containing the sample, then dissolve with 1 mL of methanol and transfer to a 1.5 mL EP tube, and use semi-preparative high-performance liquid chromatography to purify the sample.

[0081] Collect the purified substance and perform nuclear magnetic resonance spectroscopy, and compare it with the nuclear magnetic resonance data of Chrysosporazine C in the prior art (Elbanna A H, Khalil Z G, Bernhardt P V, et al. Chrysosporazines A–E: P-Glycoprotein inhibitory piperazines from an Australian marine fish gastrointestinal tract-derived fungus, Chrysosporium sp. CMB-F214[J]. Organic Letters, 2019, 21(19):8097-8100.). Among them, semi-preparative liquid chromatography is performed using a Shimadzu gradient HPLC-15C system, which consists of two LC-20AP preparative liquid pumps (with static mixers), an SPDM20A diode array detector, and a CBM-20A system controller (with a standard Rheodyne sample inlet). The conditions during semi-preparative liquid chromatography are as follows: Chromatographic column: Welch Ultimate AQ-C18 column (10×250 mm, 5 μm); Mobile phase: methanol as mobile phase A, 0.1% (v / v) aqueous formic acid as mobile phase B; Flow rate: 3 mL / min; Injection volume: 200 μL; UV detection wavelength: 220-280 nm; The elution mode is isocratic elution.

[0082] II. Experimental results The comparison results of the nuclear magnetic data of the target compound 2 and Chrysosporazine C in the prior art are shown in Table 8.

[0083] Table 8 Comparison results of nuclear magnetic data

[0084] The results show that: in the methods shown in Example 2 and Example 3, the nuclear magnetic data of the new compound (target compound 2) produced are basically the same as the nuclear magnetic data of Chrysosporazine C in the prior art, indicating that the new compound obtained in the methods shown in Example 2 and Example 3 is Chrysosporazine C, and the methods shown in Example 2 and Example 3 can convert Chrysosporazine D into Chrysosporazine C. Chrysosporazine C has the chemical formula as shown in formula II.

[0085] Formula II.

[0086] Example 5 A method for preparing Chrysosporazine C The method for preparing Chrysosporazine C comprises the following steps: S1. Perform protein purification according to the method shown in step one of Example 3 to obtain the purified Cry7 protein; S2. Mix Chrysosporazine D (chemical formula as shown in formula I), ascorbic acid, α-ketoglutaric acid, FeSO4·7H2O and the purified Cry7 protein obtained in step S1 in Tris-HCl buffer (pH 7.8) to obtain a reaction system; wherein the total volume of the reaction system is 10 mL; The final concentration ratio of Chrysosporazine D, ascorbic acid, α-ketoglutaric acid, FeSO4·7H2O and the purified Cry7 protein obtained in step S1 in the reaction system is 1 mM: 5 mM: 5 mM: 5 mM: 25 μM; S3. The reaction system of step S2 was co-divided into 10 tubes, incubated in a 37℃ constant temperature box for 36h, after the incubation, extracted with a total of 10mL of ethyl acetate, collected the organic layer, after rotary evaporation, purified according to the method shown in step S4 of Example 4 using semi-preparative liquid chromatography, and the target compound was collected to obtain Chrysosporazine C.

[0087] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. For those skilled in the art, on the basis of the above description and ideas, other different forms of changes or variations can also be made, which do not need to be or cannot be exhaustively listed here. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of a Cry7 protein having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of a P-gp inhibitor Chrysosporazine C. The Chrysosporazine C has a chemical formula as shown in formula II. Formula II.

2. A nucleotide sequence as set forth in SEQ ID NO: 2 cry7 Use of the gene in the preparation of P-gp inhibitor Chrysosporazine C; The Chrysosporazine C has a chemical formula as shown in formula II. Formula II.

3. A recombinant vector, characterized in that, The recombinant vector is a vector containing a nucleotide sequence as shown in SEQ ID NO: 2 cry7 a gene.

4. The recombinant vector of claim 3, wherein, The vectors are pRSF and pKW-2u-pyrO-4g.

5. A recombinant cell, characterized in that, The recombinant cell is a cell containing the recombinant vector of any one of claims 3 or 4.

6. Use of the recombinant vector of any one of claims 3-4 and / or the recombinant cell of claim 5 in the preparation of a P-gp inhibitor Chrysosporazine C. The Chrysosporazine C has a chemical formula as shown in formula II. Formula II.

7. A method for preparing P-gp inhibitor Chrysosporazine C using a biological enzyme catalyst, characterized by, The method comprises the following steps: S1. Purifying the Cry7 protein having an amino acid sequence as shown in SEQ ID NO: 1 using the recombinant vector of any one of claims 3-4 and / or the recombinant cell of claim 5; S2. Using the purified Cry7 protein of step S1 to react with Chrysosporazine D having a chemical formula as shown in formula I, to obtain Chrysosporazine C having a chemical formula as shown in formula II. Formula I; Formula II.

8. The method of claim 7, wherein, The concentration of Chrysosporazine D in step S2 is 1-2 mM, and the concentration of the purified Cry7 protein is 20-25 μM.

9. The method of claim 7, wherein, In step S2, ascorbic acid, α-ketoglutaric acid and ferrous sulfate are also added.

10. Chrysosporazine C prepared by the method of any one of claims 7-9.

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