A hydroxylase and its use in the synthesis of 14-deoxoandrographolide

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a hydroxylase and application thereof in synthesis of 14-deoxyandrographolide. BACKGROUND

[0002] Andrographis paniculata (Burm. f.) Wall. ex Nees in Wallich is a traditional Chinese medicine from Acanthaceae, and its main effective component is a halophylic diterpene lactone compound, which is often used for treating sore throat, common cold, respiratory tract infection, etc. Andrographis paniculata is not only used as a Chinese medicinal material in clinical treatment of traditional Chinese medicine, but also is developed into various preparations for clinical use, such as andrographolide tablets and andrographolide capsules. Andrographolide, an effective component of andrographis paniculata, is developed into andrographolide dripping pills and sulfonated derivatives (main component of Xiyanping injection). In addition to the main active components andrographolide and dehydroandrographolide, other lactone components also have good pharmacological activity. With the continuous research on natural products in andrographis paniculata, the pharmacological activity of other andrographolide components is continuously discovered. 14-deoxyandrographolide has good antibacterial, antifungal, antimalarial activity, chronic alcoholism and other pharmacological activities, and its derivatives are also proved to have good pharmacological activity, such as 3-O-sulfate-14-deoxyandrographolide has good pharmacological activity.

[0003] Cytochrome P450 (cytochrome P450 or CYP450, simply CYP450) is a kind of heme-containing membrane protein, which widely exists 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 is continuously discovered. However, few CYP450s related to the biosynthesis of halophylic diterpene lactones have been reported in recent years. The hydroxylation of C3 position of andrographolide components is a key step for the synthesis of natural products such as andrographolide and 14-deoxyandrographolide. However, the protein with the function of hydroxylation modification at this position has not been reported. SUMMARY

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

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

[0006] c1) as a hydroxylase;

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

[0008] c3) synthesizing 14-deoxoandrographolide;

[0009] c4) preparing products for synthesizing 14-deoxoandrographolide;

[0010] c5) catalyzing neoandrographolide aglycone;

[0011] c6) preparing products for catalyzing neoandrographolide aglycone.

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

[0013] a1) a protein with an amino acid sequence as shown in SEQ ID No: 2;

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

[0015] a3) a protein with hydroxylase activity obtained by substitution and / or deletion and / or addition of one or several amino acid residues to the protein as shown in a1) or a2).

[0016] In the above-mentioned a2), the tag is specifically shown in Table 1.

[0017] Table 1. Sequence of the tag

[0018] Tag Residue Sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

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

[0020] The protein in the above-mentioned a3) can be artificially synthesized, or can be obtained by first synthesizing its encoding gene and then performing biological expression.

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

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

[0023] c1) as a hydroxylase;

[0024] c2) preparing products with hydroxylase activity;

[0025] c3) synthesizing 14-deoxoandrographolide;

[0026] c4) preparing products for synthesizing 14-deoxoandrographolide;

[0027] c5) catalyzing neoandrographolide aglycone;

[0028] c6) preparing products for catalyzing neoandrographolide aglycone.

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

[0030] b1) a DNA molecule encoding region as shown in SEQ ID NO: 1;

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

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

[0033] b4) a DNA molecule derived from Andrographis paniculata and encoding any of the above-mentioned protein CYP72A399, which hybridizes to the DNA molecule defined in b1) or b2) under stringent conditions.

[0034] The 75% or more identity can 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 in a solution of 2xSSC, 0.1% SDS at 68℃, and washing the membrane twice for 5 min each time in a solution of 0.5xSSC, 0.1% SDS at 68℃, and then washing the membrane twice for 15 min each time in a solution of 0.5xSSC, 0.1% SDS at 68℃.

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

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

[0038] c1) as a hydroxylase;

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

[0040] c3) synthesizing 14-deoxoandrographolide;

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

[0042] c5) catalyzing neandrographolide aglycone;

[0043] c6) preparing a product for catalyzing neandrographolide aglycone.

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

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

[0046] The recombinant vector can be specifically a recombinant plasmid pESC-CYP, which is a recombinant plasmid obtained by replacing the small DNA fragment between the restriction enzymes BamHI and SalI of the yeast expression vector pESC-Trp with the nucleotide sequence shown in SEQ ID NO: 1.

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

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

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

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

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

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

[0053] In any of the above-mentioned applications, in c3) or c4), the synthesis of 14-deoxoandrographolide is performed using neandrographolide aglycone as a substrate.

[0054] The present application also protects a method for synthesizing 14-deoxoandrographolide, comprising the following steps: using any of the above-mentioned protein CYP72A399 or any of the above-mentioned recombinant microsomes to catalyze the andrographolide aglycone, so as to obtain 14-deoxoandrographolide.

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

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

[0057] In the above method, the "obtaining 14-deoxoandrographolide" can be achieved 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-deoxoandrographolide.

[0058] The present inventors have obtained the CYP72A399 gene with the nucleotide sequence as shown in SEQ ID NO: 1 from the Andrographis genome through a large number of experiments, and the CYP72A399 gene encodes the protein CYP72A399. The amino acid sequence of the protein CYP72A399 is as shown in SEQ ID No: 2. It is further proved through experiments that the protein CYP72A399 can catalyze the andrographolide aglycone to generate 14-deoxoandrographolide as a hydroxylase. Therefore, the protein CYP72A399 has important theoretical and practical significance for synthesizing 14-deoxoandrographolide and cultivating high-quality Andrographis. The present application has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1UPLC-TOF analysis of reaction products catalyzed by CYP72A399 microsomes and control microsomes in Example Step 3. Wherein, A is the total ion chromatogram of reaction products catalyzed by CYP72A399 microsomes and control microsomes, Standard of 2 is 14-deoxoandrographolide standard, 3+CYP72A399 is the reaction group of CYP72A399 microsomes with andrographolide aglycone as substrate, 3+empty vector is the reaction group of control microsomes with andrographolide aglycone as substrate; B is the mass spectrum of reaction product peak 14-deoxoandrographolide catalyzed by CYP72A399 microsomes; C is the mass spectrum of 14-deoxoandrographolide standard.

[0060] Figure 2 Schematic diagram of protein CYP72A399 catalyzing andrographolide aglycone (Andrograpanin) to generate 14-deoxoandrographolide. DETAILED DESCRIPTION

[0061] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

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

[0063] In the quantitative test in the following examples, three repeated experiments were set up, and the average value was taken.

[0064] In the following examples, the yeast expression vector pESC-Trp is a product of Agilent Company. The histidine-deficient medium is purchased from Beijing Fankeno Technology Co., Ltd. Andrographolide aglycone is a product of Beijing Rongcheng Xinde Technology Development Co., Ltd., with product catalog number BT-CXL10. 14-deoxoandrographolide standard is a product of Beijing Rongcheng Xinde Technology Development Co., Ltd., with product number BT-CXL04.

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

[0066]

[0067] The chemical structural formula of 14-deoxoandrographolide is shown as formula (II):

[0068]

[0069] The solute of YPL medium and its concentration 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 described 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 biological material is available from the applicant and is only used for repeating the relevant experiments of the present application and cannot be used for other purposes.

[0071] Example, Protein CYP72A399 Catalyzes the Generation of 14-Deoxyandrographolide from Andrograpanin

[0072] I. Discovery of CYP72A399 Gene

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

[0074] II. Preparation of Microsomes and Microsomal Solution

[0075] 1. Construction of Recombinant Plasmid pESC-CYP

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

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

[0078] 2. Obtaining of Recombinant Saccharomyces cerevisiae

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

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

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

[0082] (1) A single colony of the 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 a bacterial solution 1.

[0083] (2) After step (1) was completed, the bacterial solution 1 was centrifuged at 3000 g for 5 min to collect bacterial bodies 1.

[0084] (3) After step (2) was completed, the bacterial bodies 1 were resuspended with sterilized distilled water and then centrifuged at 3000 g for 5 min to collect bacterial bodies 2.

[0085] (4) After step (3) was completed, the bacterial bodies 2 were resuspended with sterilized distilled water and then centrifuged at 3000 g for 5 min to collect bacterial bodies 3.

[0086] (5) After step (4) was completed, 50 mL of YPL medium (for inducing expression of exogenous genes) was added to the bacterial bodies 3, which were then cultured at 30°C and 150 rpm for 12-16 h to obtain a fermentation product.

[0087] 4. Preparation of microsomes

[0088] (1) The fermentation product obtained in step 3 was centrifuged at 3000 g for 5 min to collect bacterial bodies A, which were then resuspended with 5 mL of TEK solution (i.e., the YPL medium and the TEK solution used in step 3 (5) had a volume ratio of 10:1) and placed at room temperature for 5 min to obtain a 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, and the solvent was water, with a pH value of 7.4.

[0090] (2) After step (1) was completed, the cell suspension 1 was centrifuged at 3000 g for 10 min to collect bacterial bodies B, which were then resuspended with 500 μL of ice-bath TESB solution (i.e., the YPL medium and the TESB solution used in step 3 (5) had a volume ratio of 100:1) to obtain a 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, and the solvent was water, with a pH value of 7.4.

[0092] (3) After step (2) is completed, take the cell suspension 2, freeze and high-pressure break the cells 3 times, then centrifuge at 12000g for 20 min, and collect the supernatant.

[0093] (4) After step (3) is completed, add PEG4000 and NaCl (5g PEG4000 and 0.44g NaCl per 50mL supernatant) to the supernatant to obtain a mixture; then centrifuge the mixture at 12000rpm for 20 min in an ice bath, and collect the precipitate. The precipitate is the microsomes.

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

[0095] The solute and its concentration of the TEG solution are 50mM Tris-HCl, 1mM EDTA and 20% (v / v) glycerol, the solvent is water, and the pH value is 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 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 control microsome solution.

[0098] The only difference between the CYP72A399 microsomes and the control microsomes is that the latter expresses the protein CYP72A399.

[0099] III. UPLC-QTOF analysis of the protein CYP72A399

[0100] 1. Prepare the reaction system. The reaction system is 500μL, which is composed of pH7.5, 50mM Tris-HCl buffer, NADPH, FAD, FMN, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, DTT, the microsome solution (CYP72A399 microsome solution or control microsome solution) obtained in step two, and neoeonifide aglycone. Neoeonifide aglycone is used as the substrate. The concentrations of NADPH, FAD, FMN, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, DTT, microsomes and neoeonifide aglycone in the reaction system are 500μM, 5μM, 5μM, 4mM, 4mM, 2mM, 500μg / mL and 20mM, respectively.

[0101] 2. After step 1, the reaction system was placed at 30℃ for 4 hours (for catalysis) to obtain a reaction product.

[0102] 3. After step 2, an equal volume of ethyl acetate was added to the reaction product for extraction, and the ethyl acetate phase was collected.

[0103] 4. After step 3, the ethyl acetate phase and 14-deoxoandrographolide standard were respectively subjected to UPLC-QTOF analysis.

[0104] The detection results are shown in Figure 1 . The results show that, compared with the control microsomes, the ethyl acetate phase of the reaction system added with CYP72A399 microsomes produces a different product at 16.5 min (retention time) in the total ion chromatogram, and the product is completely consistent with the mass spectrum of the 14-deoxoandrographolide standard, and thus the product is identified as 14-deoxoandrographolide.

[0105] Therefore, the protein CYP72A399 can be used as a hydroxylase to catalyze the generation of 14-deoxoandrographolide from neotanshinlactone aglycone (hydroxylation of C3 of neotanshinlactone aglycone), and a specific schematic diagram is shown in Figure 2 .

[0106] The above has described the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without unnecessary experiments. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by using conventional techniques known in the art, which deviates from the range disclosed in the present application.

Claims

1. Use of a protein CYP72A399 for at least one of the following c1) - c4): c1) as a hydroxylase; c2) for preparing a product having a hydroxylase activity; c3) for catalyzing synthesis of 14-deoxoandrographolide using andrographolide aglycone as a substrate; c4) for preparing a product for catalyzing synthesis of 14-deoxoandrographolide using andrographolide aglycone; said protein CYP72A399 is a1) or a2): a1) a protein having an amino acid sequence as set forth in SEQ ID No: 2; a2) a fusion protein obtained by linking a tag to the N-terminus or / and C-terminus of a protein as set forth in SEQ ID No: 2; in said c1) or c2), said hydroxylase hydroxylates C3 of andrographolide aglycone.

2. Use of a nucleic acid molecule encoding the protein CYP72A399 as set forth in claim 1 for c2) or c4): c2) for preparing a product having a hydroxylase activity; c4) for preparing a product for catalyzing synthesis of 14-deoxoandrographolide using andrographolide aglycone; in said c2), said hydroxylase hydroxylates C3 of andrographolide aglycone; said nucleic acid molecule is a DNA molecule as set forth in b1) or b2): b1) a DNA molecule having a coding region as set forth in SEQ ID No: 1; b2) a DNA molecule having a nucleotide sequence as set forth in SEQ ID No:

1.

4. Use of an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the nucleic acid molecule as set forth in claim 2 or 3 for c2) or c4): c2) for preparing a product having a hydroxylase activity; c4) for preparing a product for catalyzing synthesis of 14-deoxoandrographolide using andrographolide aglycone; in said c2), said hydroxylase hydroxylates C3 of andrographolide aglycone; said recombinant vector is a recombinant plasmid obtained by inserting the DNA molecule as set forth in SEQ ID No: 1 into a multiple cloning site of an expression vector.

5. A recombinant vector containing the nucleic acid molecule as set forth in claim 2 or 3.

6. A recombinant microorganism obtained by introducing the recombinant vector as set forth in claim 5 into a starting microorganism.

7. Use of a recombinant microsome containing the protein CYP72A399 as set forth in claim 1 for c2) or c4): c2) for preparing a product having a hydroxylase activity; c4) for preparing a product for catalyzing synthesis of 14-deoxoandrographolide using andrographolide aglycone; in said c2), said hydroxylase hydroxylates C3 of andrographolide aglycone; said recombinant microsome is prepared by treating a fermentation product of the recombinant microorganism as set forth in claim 4 or 6 with polyethylene glycol and NaCl.

8. A recombinant microsome containing the protein CYP72A399 as set forth in claim 1.

9. A method for synthesizing 14-deoxoandrographolide, comprising the step of catalyzing andrographolide aglycone using the protein CYP72A399 as set forth in claim 1 or the recombinant microsome as set forth in claim 7 or 8, thereby obtaining 14-deoxoandrographolide. ​ ​ ​ ​ ​ 3. Use of a nucleic acid molecule according to claim 2, characterized in that: ​ ​ ​ ​ ​ ​ ​ 5. Use according to claim 4, characterized in that: ​ 6. Use according to claim 4, characterized in that: ​ ​ ​ ​ ​ 8. Use according to claim 7, characterized in that: ​ ​

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