Hydroxylase and application thereof in synthesis of 14-deoxyandrographolide

Abstract

The invention discloses a hydroxylase and an application of the hydroxylase in synthesis of 14-deoxidized andrographolide. The hydroxylase is a protein CYP72A399, and the amino acid sequence of the protein CYP72A399 is as shown in SEQ ID No: 2. Experiments prove that the protein CYP72A399 can be used as hydroxylase to catalyze neoandrographolide aglycone to generate 14-deoxidized andrographolide. Therefore, the protein CYP72A399 has important theoretical and practical significance on synthesis of the 14-deoxidized andrographolide and cultivation of high-quality andrographis paniculata. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a hydroxylase and application thereof in synthesizing 14-deoxyandrographolide. Background Art

[0002] Andrographis paniculata (Burm.f.) Wall.ex Nees in Wallich is a traditional Chinese medicine from the Acanthaceae family. Its main active ingredients are diterpenoid lactone compounds, which are often used to treat sore throat, influenza, respiratory tract infections, etc. Andrographis paniculata is not only used as a Chinese herbal medicine in Chinese medicine clinical practice, but also developed into a variety of preparations for clinical use, such as Andrographis paniculata tablets and Andrographis paniculata capsules for clinical use, and Andrographolide, the active ingredient of Andrographis paniculata, has been developed into Andrographolide pills and its sulfonated derivatives (the main ingredient of Xiyanping injection). In addition to its main active ingredients, andrographolide and dehydroandrographolide, other lactone components also have good pharmacological activity. With the continuous research on natural products in Andrographis paniculata, the pharmacological activities of other andrographolide components are constantly being discovered. Among them, 14-deoxyandrographolide has good antibacterial, antifungal, antimalarial activity, chronic alcoholism and other pharmacological activities. Its derivatives have also been shown to have good pharmacological activity, such as 3-O-sulfate-14-deoxyandrographolide has good pharmacological activity.

[0003] Cytochrome P450 (CYP450 or CYP450, CYP450 for short) is a class of membrane proteins containing heme, which is widely present in various organisms. Cytochrome P450 can catalyze various types of reactions, such as hydroxylation, epoxidation, isomerization, dealkylation, etc. With the separation and functional identification of CYP450 in plants, the catalytic function of plant CYP450 genes has been continuously discovered. However, few CYP450s related to the biosynthesis of hemiaran-type diterpenes have been reported in recent years. The hydroxylation of the C3 position of andrographolide components is a key step in the natural products such as andrographolide and 14-deoxyandrographolide, but proteins with the hydroxylation modification function at this position have not yet been reported. Summary of the invention

[0004] The purpose of the present invention is to synthesize 14-deoxyandrographolide.

[0005] The present invention firstly protects the application of protein CYP72A399, which can be at least one of the following c1)-c6):

[0006] c1) as hydroxylase;

[0007] c2) preparing a product having hydroxylase activity;

[0008] c3) synthesizing 14-deoxyandrographolide;

[0009] c4) preparing a product for synthesizing 14-deoxyandrographolide;

[0010] c5) catalyzing new andrographolide aglycones;

[0011] c6) preparing a product for catalyzing the production of new andrographolide aglycones.

[0012] In the above application, the protein CYP72A399 can be a1) or a2) or a3):

[0013] a1) the amino acid sequence is the protein shown in SEQ ID No: 2;

[0014] a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in SEQ ID NO: 2;

[0015] a3) A protein having hydroxylase activity obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in a1) or a2).

[0016] The specific labels of the proteins in a2) above are shown in Table 1.

[0017] Table 1. Sequences of tags

[0018] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagII 8 WSHQ c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0019] In the protein described in a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0020] The protein in a3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0021] The coding gene of the protein in a3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID No: 1, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0022] The present invention also protects the use of a nucleic acid molecule encoding any of the above-mentioned proteins CYP72A399, which may be at least one of the following c1)-c6):

[0023] c1) as hydroxylase;

[0024] c2) preparing a product having hydroxylase activity;

[0025] c3) synthesizing 14-deoxyandrographolide;

[0026] c4) preparing a product for synthesizing 14-deoxyandrographolide;

[0027] c5) catalyzing new andrographolide aglycones;

[0028] c6) preparing a product for catalyzing the production of new andrographolide aglycones.

[0029] In the above application, the nucleic acid molecule encoding any of the above proteins CYP72A399 can be a DNA molecule as shown in b1) or b2) or b3) or b4) below:

[0030] b1) a DNA molecule whose coding region is shown in SEQ ID NO: 1;

[0031] b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 1;

[0032] b3) a DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2), derived from Andrographis paniculata and encoding any of the above-mentioned proteins CYP72A399;

[0033] b4) A DNA molecule derived from Andrographis paniculata and encoding any of the above-mentioned proteins CYP72A399 which hybridizes with the DNA molecule specified in b1) or b2) under stringent conditions.

[0034] The 75% or more identity may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.

[0035] The stringent conditions are hybridization and membrane washing twice at 68°C in a 2×SSC, 0.1% SDS solution, each for 5 min, and hybridization and membrane washing twice at 68°C in a 0.5×SSC, 0.1% SDS solution, each for 15 min.

[0036] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0037] The present invention also protects the use of an expression cassette, a recombinant vector, a recombinant microorganism, a recombinant microsome or a transgenic cell line containing any of the above-mentioned nucleic acid molecules, which may be at least one of the following c1) to c6):

[0038] c1) as hydroxylase;

[0039] c2) preparing a product having hydroxylase activity;

[0040] c3) synthesizing 14-deoxyandrographolide;

[0041] c4) preparing a product for synthesizing 14-deoxyandrographolide;

[0042] c5) catalyzing new andrographolide aglycones;

[0043] c6) preparing a product for catalyzing the production of new andrographolide aglycones.

[0044] In the above application, the recombinant vector may be a recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID NO: 1 into the multiple cloning site of the expression vector.

[0045] The expression vector may be a yeast expression vector pESC-Trp.

[0046] The recombinant vector may be a recombinant plasmid pESC-CYP. The recombinant plasmid pESC-CYP may be a recombinant plasmid obtained by replacing a small DNA fragment between restriction endonucleases BamHI and SalI of the yeast expression vector pESC-Trp with a nucleotide sequence shown in SEQ ID No: 1.

[0047] In the above application, the recombinant microorganism may be a recombinant microorganism obtained by introducing any of the above recombinant vectors into a starting microorganism.

[0048] The starting microorganism may be yeast, bacteria, algae or fungi.

[0049] The yeast may specifically be Saccharomyces cerevisiae WAT11.

[0050] The recombinant microorganism may specifically be recombinant Saccharomyces cerevisiae WAT11-pESC-CYP, which is obtained by introducing the recombinant plasmid pESC-CYP into Saccharomyces cerevisiae WAT11.

[0051] In the above application, the recombinant microsomes can be prepared by treating the fermentation product of any of the above recombinant microorganisms with polyethylene glycol and NaCl.

[0052] In any of the above applications, in c1) or c2), the hydroxylase can hydroxylate the C3 position.

[0053] In any of the above applications, in c3) or c4), the synthesis of 14-deoxyandrographolide uses neoandrographolide aglycone as a substrate.

[0054] The present invention also protects a method for synthesizing 14-deoxyandrographolide, comprising the following steps: using any of the above-mentioned proteins CYP72A399 or any of the above-mentioned recombinant microsomes to catalyze the new andrographolide aglycone, thereby obtaining 14-deoxyandrographolide.

[0055] In the above method, the reaction system of "using any of the above-mentioned proteins CYP72A399 or any of the above-mentioned recombinant microsomes to catalyze the production of neoandrographolide aglycone" can be composed of pH 7.5, 50mM Tris-HCl buffer, NADPH, FAD, FMN, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, DTT, any of the above-mentioned recombinant microsomes and neoandrographolide aglycone. Neoandrographolide aglycone is used as a substrate. In the reaction system, the concentrations of NADPH, FAD, FMN, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, DTT, recombinant microsomes and neoandrographolide aglycone can be 500μM, 5μM, 5μM, 4mM, 4mM, 2mM, 500μg / mL and 20mM, respectively.

[0056] In the above method, the reaction condition of "using any of the above-mentioned proteins CYP72A399 or any of the above-mentioned recombinant microsomes to catalyze the production of new andrographolide aglycone" can be 30° C. for 4 hours.

[0057] In the above method, the "obtaining 14-deoxyandrographolide" can be obtained by extracting the catalytic product. Specifically, an equal volume of ethyl acetate is added to the catalytic product for extraction, and the ethyl acetate phase is collected. The ethyl acetate phase contains 14-deoxyandrographolide.

[0058] After a large number of experiments, the inventors of the present application obtained the CYP72A399 gene with a nucleotide sequence as shown in SEQ ID NO: 1 from the Andrographis paniculata genome. The CYP72A399 gene encodes the protein CYP72A399. The amino acid sequence of the protein CYP72A399 is shown in SEQ ID No: 2. It was further proved through experiments that the protein CYP72A399 can act as a hydroxylase to catalyze the production of 14-deoxyandrographolide from the neoandrographolide aglycone. It can be seen that the protein CYP72A399 has important theoretical and practical significance for the synthesis of 14-deoxyandrographolide and the cultivation of high-quality Andrographis paniculata. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1This is the UPLC-TOF analysis of the reaction products catalyzed by CYP72A399 microsomes and control microsomes in step 3 of the embodiment. Wherein, A is the total ion map of the reaction products catalyzed by CYP72A399 microsomes and control microsomes, Standard of 2 is the 14-deoxyandrographolide standard, 3+CYP72A399 is the reaction group of CYP72A399 microsomes with neoandrographolide aglycone as substrate, and 3+empty vector is the reaction group of control microsomes with neoandrographolide aglycone as substrate; B is the mass spectrum corresponding to the peak 14-deoxyandrographolide of the reaction product catalyzed by CYP72A399 microsomes; C is the standard mass spectrum of 14-deoxyandrographolide.

[0060] Figure 2 Schematic diagram of protein CYP72A399 catalyzing the production of 14-deoxyandrographolide from andrograpanin. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0062] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0063] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0064] In the following examples, the yeast expression vector pESC-Trp is a product of Agilent. Histidine-deficient medium was purchased from Beijing Fanjinuo Technology Co., Ltd. Neoandrographolide aglycone is a product of Beijing Rongcheng Xinde Technology Development Co., Ltd., with a catalog number of BT-CXL10. 14-Deoxyandrographolide standard is a product of Beijing Rongcheng Xinde Technology Development Co., Ltd., with a catalog number of BT-CXL04.

[0065] The chemical structural formula of the new andrographolide aglycone is shown in formula (I):

[0066]

[0067] The chemical structure of 14-deoxyandrographolide is shown in formula (II):

[0068]

[0069] The solutes and their concentrations of YPL medium are 1% yeast extract, 2% peptone and 2% galactose, the solvent is water, and the pH value is natural.

[0070] In the following examples, Saccharomyces cerevisiae WAT11 is recorded in the following document: Philippe, etc. Cloning, Yeast Expression, and Characterization of the Coupling of Two Distantly Related Arabidopsis thaliana NADPH-Cytochrome P450 Reductases with P450 CYP73A5*. [J] JBC, Vol. 272, 19176-19186, 1997; the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0071] Example: Protein CYP72A399 catalyzes the production of 14-deoxyandrographolide from Andrograpanin

[0072] 1. Discovery of the CYP72A399 gene

[0073] After a large number of experiments, the inventors of the present application found a CYP72A399 gene in the genome of Andrographis paniculata with a nucleotide sequence as shown in SEQ ID No: 1. The CYP72A399 gene encodes a protein CYP72A399. The amino acid sequence of the protein CYP72A399 is shown in SEQ ID No: 2.

[0074] 2. Preparation of Microsomes and Microsome Solutions

[0075] 1. Construction of recombinant plasmid pESC-CYP

[0076] The small DNA fragment between the restriction endonucleases BamHI and SalI of the yeast expression vector pESC-Trp was replaced with the nucleotide sequence shown in SEQ ID No: 1 to obtain the recombinant plasmid pESC-CYP.

[0077] The recombinant plasmid pESC-CYP expressed protein CYP72A399.

[0078] 2. Obtaining recombinant Saccharomyces cerevisiae

[0079] The recombinant plasmid pESC-CYP was introduced into Saccharomyces cerevisiae WAT11 to obtain recombinant Saccharomyces cerevisiae, which was named WAT11-pESC-CYP.

[0080] The yeast expression vector pESC-Trp was introduced into Saccharomyces cerevisiae WAT11 to obtain recombinant Saccharomyces cerevisiae, named WAT11-pESC, which was used as a control strain.

[0081] 3. Inducible expression of exogenous genes in recombinant Saccharomyces cerevisiae

[0082] (1) A single clone of recombinant Saccharomyces cerevisiae (WAT11-pESC-CYP or WAT11-pESC) was inoculated into 10 mL of histidine-deficient medium and cultured at 30°C and 250 rpm for 48 h to obtain bacterial solution 1;

[0083] (2) After completing step (1), take the bacterial solution 1, centrifuge at 3000 g for 5 min, and collect the bacterial cells 1;

[0084] (3) After completing step (2), take the bacterial cell 1, resuspend it with sterile distilled water, and then centrifuge it at 3000g for 5 minutes to collect the bacterial cell 2;

[0085] (4) After completing step (3), take the bacterial cell 2, resuspend it with sterile distilled water, and then centrifuge it at 3000g for 5 minutes to collect the bacterial cell 3;

[0086] (5) After completing step (4), add 50 mL of YPL medium (for the purpose of inducing exogenous gene expression) to the bacterial cell 3, and culture at 30° C. and 150 rpm for 12-16 h to obtain a fermentation product.

[0087] 4. Preparation of microsomes

[0088] (1) Take the fermentation product obtained in step 3, centrifuge at 3000g for 5 minutes, and collect bacterial cells A; then resuspend it with 5 mL of TEK solution (i.e., the volume ratio of YPL medium and TEK solution used in step 3 (5) is 10:1), and leave it at room temperature for 5 minutes to obtain cell suspension 1.

[0089] The solutes and their concentrations of the TEK solution were 50 mM Tris-HCl, 1 mM EDTA, and 100 mM KCl, the solvent was water, and the pH value was 7.4.

[0090] (2) After completing step (1), take cell suspension 1, centrifuge at 3000 g for 10 min, and collect bacterial cells B; then resuspend with 500 μL of ice-cold TESB solution (i.e., the volume ratio of YPL culture medium and TESB solution used in step 3 (5) is 100:1) to obtain cell suspension 2.

[0091] The solutes and their concentrations of the TESB solution were 50 mM Tris-HCl, 1 mM EDTA, and 600 mM Sorbitol, the solvent was water, and the pH value was 7.4.

[0092] (3) After completing step (2), take the cell suspension 2, freeze and high-pressure disrupt the cells three times, then centrifuge at 12000g for 20 minutes, and collect the supernatant.

[0093] (4) After completing step (3), PEG4000 and NaCl were added to the supernatant (5 g PEG4000 and 0.44 g NaCl were added to every 50 mL supernatant) to obtain a mixed solution; the mixed solution was then first ice-bathed for 15 min, and then centrifuged at 12000 rpm for 20 min to collect the precipitate. The precipitate is the microsome.

[0094] (5) After completing step (4), add 2 mL of TEG solution to the microsomes and dissolve them by blowing under ice bath to obtain a microsome solution with a concentration of about 25 mg / mL.

[0095] The solutes and concentrations of the TEG solution were 50 mM Tris-HCl, 1 mM EDTA, and 20% (v / v) glycerol, the solvent was water, and the pH value was 7.4.

[0096] When the recombinant Saccharomyces cerevisiae is WAT11-pESC-CYP, the obtained microsomes are named CYP72A399 microsomes, and the obtained microsome solution is the CYP72A399 microsome solution.

[0097] When the recombinant Saccharomyces cerevisiae is WAT11-pESC, the obtained microsomes are named control microsomes, and the obtained microsome solution is the control microsome solution.

[0098] The only difference of CYP72A399 microsomes compared to control microsomes was that the latter expressed the protein CYP72A399.

[0099] UPLC-QTOF analysis of protein CYP72A399

[0100] 1. Prepare the reaction system. The reaction system is 500 μL and consists of pH 7.5, 50 mM Tris-HCl buffer, NADPH, FAD, FMN, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, DTT, the microsomal solution obtained in step 2 (4(5)) (CYP72A399 microsomal solution or control microsomal solution) and neoandrographolide aglycone. Neoandrographolide aglycone is used as the substrate. In the reaction system, the concentrations of NADPH, FAD, FMN, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, DTT, microsomes and neoandrographolide aglycone are 500 μM, 5 μM, 5 μM, 4 mM, 4 mM, 2 mM, 500 μg / mL and 20 mM, respectively.

[0101] 2. After completing step 1, the reaction system is placed at 30° C. for 4 hours (for the purpose of catalysis) to obtain a reaction product.

[0102] 3. After completing step 2, add an equal volume of ethyl acetate to the reaction product for extraction, and collect the ethyl acetate phase.

[0103] 4. After completing step 3, perform UPLC-QTOF analysis on the ethyl acetate phase and the 14-deoxyandrographolide standard, respectively.

[0104] Test results are shown in Figure 1 The results showed that compared with the control microsomes, the ethyl acetate phase of the reaction system with CYP72A399 microsomes added produced a different product at 16.5 min (retention time) in the total ion diagram and the mass spectrum of the product was completely consistent with that of the 14-deoxyandrographolide standard. Therefore, the product was identified as 14-deoxyandrographolide.

[0105] It can be seen that protein CYP72A399 can act as a hydroxylase to catalyze the production of 14-deoxyandrographolide (hydroxylation of the C3 position of the neoandrographolide aglycone). The specific schematic diagram is shown in Figure 2 .

[0106] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. The application of protein CYP72A399 is at least one of the following c1)-c6): c1) as hydroxylase; c2) preparing a product having hydroxylase activity; c3) synthesizing 14-deoxyandrographolide; c4) preparing a product for synthesizing 14-deoxyandrographolide; c5) catalyzing new andrographolide aglycones; c6) preparing a product for catalyzing new andrographolide aglycones; The protein CYP72A399 is a1) or a2) or a3): a1) the amino acid sequence is the protein shown in SEQ ID No: 2; a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in SEQ ID NO: 2; a3) A protein having hydroxylase activity obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in a1) or a2).

2. The use of the nucleic acid molecule encoding the protein CYP72A399 of claim 1 is at least one of the following c1) to c6): c1) as hydroxylase; c2) preparing a product having hydroxylase activity; c3) synthesizing 14-deoxyandrographolide; c4) preparing a product for synthesizing 14-deoxyandrographolide; c5) catalyzing new andrographolide aglycones; c6) preparing a product for catalyzing the production of new andrographolide aglycones.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule as shown in b1) or b2) or b3) or b4) below: b1) a DNA molecule whose coding region is shown in SEQ ID NO: 1; b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 1; b3) a DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2), derived from Andrographis paniculata and encoding the protein CYP72A399 of claim 1; b4) A DNA molecule derived from Andrographis paniculata and encoding the protein CYP72A399 of claim 1 that hybridizes with the DNA molecule defined in b1) or b2) under stringent conditions.

4. Use of an expression cassette, a recombinant vector, a recombinant microorganism, a recombinant microsome or a transgenic cell line containing the nucleic acid molecule of claim 2 or 3, which is at least one of the following c1) to c6): c1) as hydroxylase; c2) preparing a product having hydroxylase activity; c3) synthesizing 14-deoxyandrographolide; c4) preparing a product for synthesizing 14-deoxyandrographolide; c5) catalyzing new andrographolide aglycones; c6) preparing a product for catalyzing the production of new andrographolide aglycones.

5. The use according to claim 4, characterized in that: The recombinant vector is a recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID NO: 1 into the multiple cloning site of the expression vector.

6. The use according to claim 4, characterized in that: The recombinant microorganism is a recombinant microorganism obtained by introducing the recombinant vector according to claim 5 into a starting microorganism.

7. The use according to claim 4, characterized in that: The recombinant microsomes are prepared by treating the fermentation product of the recombinant microorganism according to claim 4 or 6 with polyethylene glycol and NaCl.

8. The use according to claim 1, 2 or 4, characterized in that: In c1) or c2), the hydroxylase hydroxylates the C3 position.

9. The use according to claim 1, 2 or 4, characterized in that: In c3) or c4), the synthesis of 14-deoxyandrographolide is carried out using neoandrographolide aglycone as a substrate.

10. A method for synthesizing 14-deoxyandrographolide, comprising the following steps: using the protein CYP72A399 described in claim 1 or the recombinant microsomes described in claim 4 or 7 to catalyze the production of new andrographolide aglycones, thereby obtaining 14-deoxyandrographolide.

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