New applications of deoxyartemisinin B synthase and its isoenzyme A0A2U1PPI9

Abstract

The present invention discloses the application of deoxyartemisinin B synthase and its isoenzyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydrodeoxyartemisinin B from dihydroartemisinin acid. In the biosynthetic pathway of artemisinin, the present invention for the first time discloses the process of the biosynthesis of dihydrodeoxyartemisinin B from dihydroartemisinin acid. Through bioinformatics analysis and in vitro activity verification of candidate enzymes, it is shown that both deoxyartemisinin B synthase and its isoenzyme A0A2U1PPI9 have the new function of catalyzing the biosynthesis of dihydrodeoxyartemisinin B from dihydroartemisinin acid, enriching the route of the artemisinin biosynthetic pathway and providing a theoretical basis for the analysis of the terminal biosynthetic pathway of artemisinin and the heterologous synthesis of artemisinin compounds.

Description

Technical Field

[0001] The present invention relates to the field of biosynthesis, and particularly to the application of deoxyarteannuin B synthase (DBS) and its isoenzyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydro-epi-deoxyarteannuin B (DHDB) from dihydroartemisinic acid (DHAA). Background Art

[0002] Artemisinin (ART), which has excellent antimalarial activity, is mainly used for the symptom control of vivax malaria and falciparum malaria and the treatment of chloroquine-resistant malaria. With the continuous in-depth research, it has been found that ART and its analogs have various pharmacological effects such as anti-tumor, anti-inflammatory and immunomodulatory effects. For example, dihydroartemisinin has the function of interfering with the plasma membrane and mitochondria and has the effect of relieving symptoms and treating early systemic lupus erythematosus; ART can target the mitochondrial protease LONP1, promote the binding of LONP1 to its substrate CYP11A1, accelerate the degradation of CYP11A1, and then inhibit the synthesis of ovarian androgens, reduce the androgen level in patients with PCOS, and improve the menstrual cycle and ovarian polycystic changes; artesunate targets MD2 and can effectively inhibit the expression of fibrosis genes in cardiac fibroblasts, inhibit their proliferation, migration and contraction, reduce collagen deposition, and improve the cardiac function of heart failure mice. The continuous discovery of new pharmacological activities has gradually increased the market demand for ART. However, the content of ART in Artemisia annua is extremely low (0.01-1.00%, dry weight). Although the total chemical synthesis of ART has been achieved, the process is complex, the yield is low and the environmental pollution is serious, and industrial production has not been carried out. Therefore, the market supply problem of ART has been attracting much attention.

[0003] Since the terminal biosynthetic pathway of ART has not been elucidated, it is still not possible to directly produce ART using synthetic biology methods. By mining the Artemisia annua transcriptome database and performing bioinformatics analysis, ZL 202410006829.6 screened an active enzyme DBS that can catalyze the biosynthesis of deoxyarteannuin B (DB) from artemisinic acid (AA). However, studies have shown that the autoxidation of AA generates artemisinin B (ART-B), while the autoxidation of dihydroartemisinic acid (DHAA) generates ART. At the same time, some researchers added the substrate DHAA to Artemisia annua cells and found the generation of the product ART through biotransformation, indicating that DHAA is the direct precursor compound of ART, while AA is the precursor compound of ART-B. Through research, the present invention found that the active enzyme DBS and its isoenzyme A0A2U1PPI9 can catalyze the biosynthesis of DHDB from DHAA, providing a theoretical basis for the analysis of the terminal biosynthetic pathway of ART and new ideas and methods for producing ART compounds using synthetic biology methods. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a new application of deoxyarteannuin B synthase and its isoenzyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydro-deoxyarteannuin B from dihydroartemisinic acid, providing a theoretical basis for the analysis of the terminal biosynthetic pathway of artemisinin.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides the application of deoxyarteannuin B synthase (DBS) or its isoenzyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydro-epi-deoxyarteannuin B (DHDB) from dihydroartemisinic acid (DHAA).

[0007] Through in vitro activity verification, the present invention found that DBS and its isoenzyme A0A2U1PPI9 have the function of catalyzing the biosynthesis of DHDB from DHAA.

[0008] The present invention further investigated the enzymatic properties of DBS. The results showed that the reaction time for DBS to catalyze the biosynthesis of DHDB from DHAA was 1 - 64 h, preferably 8 - 40 h, and the reaction temperature was 20 - 60 °C, preferably 30 - 60 °C; the optimal reaction time was 24 h, the optimal reaction temperature was 50 °C, and the K of DBS catalyzing the biosynthesis of DHDB from DHAAm The value is 205.17 μM, K cat / K m The value is 1.0×10 -2 min -1 .μM -1 .

[0009] Through bioinformatics analysis, the present invention discovers that the amino acid sequences of DBS and A0A2U1PPI9 have a certain degree of conservation, that is, the active enzyme A0A2U1PPI9 is an isoenzyme of DBS.

[0010] The amino acid sequence of the isoenzyme A0A2U1PPI9 described in the present invention is shown in SEQ ID NO.1.

[0011] For the gene encoding the isoenzyme A0A2U1PPI9, its base sequence is shown in SEQ ID NO.2.

[0012] The heterologous expression of the isoenzyme A0A2U1PPI9 described in the present invention specifically refers to the heterologous expression in prokaryotes and eukaryotes.

[0013] In the second aspect, the present invention also provides the application of the isoenzyme A0A2U1PPI9 in the construction of chassis strains for producing artemisinin-like compounds. The amino acid sequence of the isoenzyme A0A2U1PPI9 is shown in SEQ ID NO.1.

[0014] In the third aspect, the present invention also provides a method for catalytic synthesis of dihydrodeoxyartemisinin B. Using dihydroartemisinic acid as a substrate, the deoxyartemisinin B synthase or its isoenzyme A0A2U1PPI9 described above is used to catalyze the biosynthesis of dihydrodeoxyartemisinin B from dihydroartemisinic acid.

[0015] Compared with the prior art, the present invention has the following excellent effects:

[0016] In the biosynthetic pathway of ART, the present invention for the first time discloses the process of biosynthesis of DHDB from DHAA. Through bioinformatics analysis and in vitro activity verification of candidate enzymes, it is shown that both DBS and its isoenzyme A0A2U1PPI9 have the new function of catalyzing the biosynthesis of DHDB from DHAA, enriching the routes of the ART biosynthetic pathway and providing a theoretical basis for the analysis of the terminal biosynthetic pathway of ART and the heterologous synthesis of ART-like compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Phylogenetic tree analysis of candidate enzymes obtained by Blast in the Artemisia annua transcriptome database using the active enzyme DBS as a bait;

[0018] Figure 2Amino acid sequence alignment of active enzyme DBS and A0A2U1PPI9;

[0019] Figure 3 HPLC detection of the catalysis of DHAA by active enzyme A0A2U1PPI9;

[0020] Figure 4 HPLC detection of the catalysis of DHAA by active enzyme DBS;

[0021] Figure 5 Product formation amounts of active enzyme DBS and A0A2U1PPI9 during the biosynthesis of DHDB from DHAA (n = 3, *** indicates significant difference at P < 0.001 level);

[0022] Figure 6 HPLC detection of the biosynthesis of DHDB from DHAA catalyzed by active enzyme DBS over time;

[0023] Figure 7 Product formation amounts of the biosynthesis of DHDB from DHAA catalyzed by active enzyme DBS over reaction time;

[0024] Figure 8 HPLC detection of the biosynthesis of DHDB from DHAA catalyzed by active enzyme DBS over temperature;

[0025] Figure 9 Product formation amounts of the biosynthesis of DHDB from DHAA catalyzed by active enzyme DBS over reaction temperature;

[0026] Figure 10 Kinetic parameters of the biosynthesis of DHDB from DHAA catalyzed by active enzyme DBS. Detailed implementation manners

[0027] The present invention will be further illustrated by specific implementation manners below. The following examples are specific implementation manners of the present invention, but the implementation manners of the present invention are not limited by the following examples.

[0028] Example 1: Screening of active enzyme A0A2U1PPI9

[0029] It is known that active enzyme DBS can catalyze AA to generate DB (Patent No.: ZL 202410006829.6). Using the amino acid sequence of DBS as a bait, Blast in the Artemisia annua transcriptome database (NCBI) to screen candidate enzymes with highly homologous amino acid sequences. Use MGGA11 and iTOL analysis software to perform phylogenetic tree analysis on the screened homologous enzymes ( Figure 1 ). We found that enzyme A0A2U1PPI9 is in the same branch as DBS and has a high degree of amino acid sequence conservation ( Figure 2), so we speculate that the candidate enzyme A0A2U1PPI9 may have the same biological function as the active enzyme DBS, and its function will be verified subsequently.

[0030] Example 2: Functional study on the biosynthesis of DHDB by the active enzyme A0A2U1PPI9 catalyzing DHAA

[0031] ⑴ Heterologous expression of the active enzyme A0A2U1PPI9 in Saccharomyces cerevisiae BY4741

[0032] a: Extraction and double digestion of plasmid pESC-His

[0033] Take 10 μL of glycerol bacteria of pESC-His in a laminar flow hood and add it to 5 mL of LB liquid medium containing Ampicillin (Amp) resistance. Culture it at 37 °C and 220 rpm until OD 600 = 0.6, and then extract plasmid pESC-His using the Tsingke plasmid extraction kit.

[0034] Use a ultra-micro biological detector to measure the concentration of the extracted plasmid, and then prepare a reaction system according to Table 1 for double digestion of plasmid pESC-His. Place the prepared system in a 37 °C water bath for 2 h. After completion, perform 1% agarose gel electrophoresis on the enzyme digestion reaction system, cut the gel block corresponding to the molecular weight and save it in a 1.5 mL EP tube. Finally, use a gel extraction kit (Sangon Biotech) to extract the plasmid after double digestion.

[0035] Table 1. Configuration table of the double digestion system of plasmid pESC-His

[0036]

[0037] b: Amplification of the target gene A0A2U1PPI9

[0038] Extract RNA from Artemisia annua plants using a plant total RNA extraction kit (Nanjing Novozymes Biotech Co., Ltd.), and reverse transcribe it to obtain cDNA using a reverse transcription kit (TOYOBO). Design primers for the candidate enzyme DBS according to plasmid pESC-His and the restriction enzyme sites SalI and BamHI (Table 2), and then perform PCR amplification on the candidate enzyme. The amplification system and PCR amplification program are shown in Tables 3 and 4. After PCR amplification, perform 1% agarose gel electrophoresis on the reaction system, cut the gel block corresponding to the molecular weight and save it in a 1.5 mL EP tube, and use a gel extraction kit (Sangon Biotech) to extract the amplified target gene.

[0039] Table 2. Primer list for PCR amplification of the candidate enzyme A0A2U1PPI9

[0040]

[0041] Table 3. PCR Amplification System for Candidate Enzyme A0A2U1PPI9

[0042]

[0043] Table 4. PCR Amplification Program for Candidate Enzyme A0A2U1PPI9

[0044]

[0045] Note: Denaturation, renaturation, and extension were performed for 33 cycles in total.

[0046] c: Homologous Recombination and Transformation of Plasmid pESC-His and Target Gene A0A2U1PPI9

[0047] The homologous recombination was carried out using the "Hieff Clone Universal One Step Cloning Kit" kit at 50 °C for 30 min. Immediately after the recombination, it was transformed into competent Escherichia coli DH5α cells. The next day, monoclonal colonies were picked from the transformed resistant plates for colony PCR verification.

[0048] d: Cloning of Fragment pESC-His-A0A2U1PPI9, Recombination into Plasmid pCDF-Ty2, and Integration into the Genome of Yeast BY4741

[0049] According to the enzyme digestion sites of EcoRI and SacI selected for plasmid pCDF-Ty2, the His3promoter-CYC1 terminater fragment in pESC-His-A0A2U1PPI9 was cloned. It was recombined into the pCDF-Ty2 plasmid by the method of step (1)-c in Example 1 and named pCDF-Ty2-His-A0A2U1PPI9. Then the Ty2F-Ty2R fragment in pCDF-Ty2-His-A0A2U1PPI9 was cloned. Finally, the cloned fragment was integrated into the genome of Saccharomyces cerevisiae BY4741, and a Saccharomyces cerevisiae strain integrating the target gene was screened on the SD-His YNB solid medium.

[0050] ⑵ Fermentation of Active Enzyme A0A2U1PPI9

[0051] Take 10 μL of the above yeast strain and inoculate it into 5 mL of SD-His YNB liquid medium, and culture it at 220 rpm and 30 °C until the OD of the bacterial liquid 600= 0.5. Take 1 mL of the bacterial solution and inoculate it into a YPD liquid medium induced by galactose:glucose (9:1) for induced expression. Culture it at 30 °C and 220 rpm for 96 h. Collect the bacterial solution, centrifuge it at 8000 rpm and 4 °C for 15 min, discard the supernatant, and resuspend the precipitate with an appropriate amount of protein buffer. Use a high-pressure cell disruptor (25 Pa) to disrupt the resuspended solution, and collect the bacterial solution immediately for downstream experiments or store it at -20 °C for later use.

[0052] ⑶ Functional verification of the biosynthesis of DHDB catalyzed by active enzyme A0A2U1PPI9

[0053] Establish an in vitro enzymatic reaction system: Take a 2 mL EP tube, and sequentially add 10 μM of active enzyme DBS, 400 μM of substrate DHAA, and 1 mM of cofactor NADPH, and make up to 1.0 mL with PBS solution. React in the dark at 30 °C and 220 rpm for 16 h. Boiled DBS is used as the control group. After the reaction, add 800 μL of ethyl acetate to the EP tube to terminate the reaction, centrifuge at 25 °C and 12,000 rpm for 2 min, and collect the upper organic phase. Extract 3 times repeatedly, combine the organic phases, concentrate to dryness, and then add 100 μL of methanol (HPLC grade) for HPLC (209 nm). The HPLC detection conditions are shown in Table 5.

[0054] The results show that: From Figure 3 the HPLC detection results, it can be seen that there is a product formed after the active enzyme A0A2U1PPI9 catalyzes DHAA. By comparing with the DHDB standard product, it is found that the peak time is the same, and it is determined that the enzymatic reaction product is DHDB. That is, A0A2U1PPI9 has the function of catalyzing the biosynthesis of DHDB from DHAA.

[0055] Table 5. Elution conditions of HPLC

[0056]

[0057]

[0058] A: 0.1% trifluoroacetic acid aqueous solution; B: acetonitrile (HPLC grade).

[0059] Example 3: Functional study of the biosynthesis of DHDB catalyzed by active enzyme DBS

[0060] ⑴ Fermentation of active enzyme DBS

[0061] In the previous research work of this study, the active enzyme DBS has been heterologously expressed in Saccharomyces cerevisiae BY4741 (Patent No.: ZL 202410006829.6), and the glycerol bacteria of the relevant strains have been stored in a -80°C refrigerator. Take 10 μL of the strain and inoculate it into 5 mL of SD-His YNB liquid medium, and culture it at 220 rpm and 30°C until the OD of the bacterial liquid 600 = 0.5. Take 1 mL of the bacterial liquid and inoculate it into a galactose:glucose (9:1) induced YPD liquid medium for induced expression, and culture it at 30°C and 220 rpm for 96 h. Collect the bacterial liquid, centrifuge it at 8000 rpm and 4°C for 15 min, discard the supernatant, and add an appropriate amount of protein buffer to resuspend the precipitate. Use a high-pressure cell crusher (25 Pa) to break the resuspended liquid, and collect the bacterial liquid immediately for downstream experiments or store it at -20°C for later use.

[0062] ⑵ Functional verification of the active enzyme DBS catalyzing the biosynthesis of DHDB from DHAA

[0063] Establish an in vitro enzyme-catalyzed reaction system: Take a 2 mL EP tube, and sequentially add 10 μM of the active enzyme DBS, 400 μM of the substrate DHAA, 1 mM of the cofactor NADPH, and make up to 1.0 mL with PBS solution. React in the dark at 30°C and 220 rpm for 16 h. Boiled DBS is used as a control group. After the reaction is completed, add 800 μL of ethyl acetate to the EP tube to terminate the reaction, centrifuge at 25°C and 12,000 rpm for 2 min, and collect the upper organic phase. Repeat the extraction 3 times, combine the organic phases, concentrate to dryness, and then add 100 μL of methanol (HPLC grade) for HPLC (209 nm). The HPLC detection conditions are shown in Table 5.

[0064] The results show that: From Figure 4 the HPLC detection results, it can be seen that there is a product formed after the active enzyme DBS catalyzes DHAA. By comparing with the DHDB standard product, it is found that the peak emergence time is the same, and it is determined that the enzyme-catalyzed product is DHDB. That is, DBS has the function of catalyzing the biosynthesis of DHDB from DHAA.

[0065] Example 4: Comparison of the catalytic activities of the active enzyme DBS and A0A2U1PPI9 in catalyzing the biosynthesis of DHDB from DHAA

[0066] From the research results of Example 2 and Example 3, it can be known that both the active enzyme DBS and A0A2U1PPI9 can catalyze the biosynthesis of DHDB from DHAA, and the amino acid sequences of DBS and A0A2U1PPI9 have a certain degree of conservation( Figure 2), that is, the active enzyme A0A2U1PPI9 is an isoenzyme of DBS. To explore the catalytic efficiency of the above two active enzymes in catalyzing the biosynthesis of DHDB from DHAA, we statistically calculated the production amount of its product DHDB. First, a standard curve of DHDB was established, and then the production amount of the product of each enzyme-catalyzed reaction was calculated. The catalytic efficiencies of the active enzymes DBS and A0A2U1PPI9 were compared based on the production amount of the product DHDB.

[0067] The results showed that: the production amount of DHDB synthesized from DHAA by the active enzyme DBS was 32.11 mg / L, and the production amount of DHDB synthesized from DHAA by A0A2U1PPI9 was 13.20 mg / L( Figure 5 ), and the activity of DBS in catalyzing the biosynthesis of DHDB from DHAA was 2.43 times that of A0A2U1PPI9. Therefore, the enzymatic properties of the active enzyme DBS in catalyzing the biosynthesis of DHDB from DHAA were investigated subsequently.

[0068] Example 5: Investigation of the optimal time for DBS to catalyze the biosynthesis of DHDB from DHAA

[0069] The single variable method was used to investigate the optimal reaction time for DBS to catalyze the biosynthesis of DHDB from DHAA. The reaction temperature was 30 °C, and the concentration of the substrate DHAA was 400 μM. The investigation ranges of the reaction time were: 1, 8, 16, 24, 32, 40, 48, 56, and 64 h, and 3 parallels were set for each group. An in vitro activity experiment regarding DBS was established according to the method of Example 3-(2). The reaction was terminated after different reaction times, and the optimal reaction time for the active enzyme DBS to catalyze the biosynthesis of DHDB from DHAA was determined based on the production amount of the product DHDB.

[0070] The results showed that: as Figure 6 and Figure 7 shown, the optimal reaction time for DBS to catalyze the biosynthesis of DHDB from DHAA was 24 h.

[0071] Example 6: Investigation of the optimal temperature for DBS to catalyze the biosynthesis of DHDB from DHAA

[0072] The single variable method was used to investigate the optimal reaction temperature for DBS to catalyze the biosynthesis of DHDB from DHAA. The reaction time was 24 h, and the concentration of the substrate DHAA was 400 μM. The investigation ranges of the reaction temperature were: 20, 30, 40, 50, and 60 °C, and 3 parallels were set for each group. In vitro activity experiments regarding DBS were established at different temperatures, and the optimal reaction temperature for the active enzyme DBS to catalyze the biosynthesis of DHDB from DHAA was determined based on the production amount of the product DHDB.

[0073] The results showed that: as Figure 8 and Figure 9It can be seen that the optimal reaction temperature for DBS to catalyze the biosynthesis of DHDB from DHAA is 50 °C.

[0074] Example 7: Enzymatic kinetic parameters of DBS-catalyzed biosynthesis of DHDB from DHAA

[0075] According to the research results of Example 5 and Example 6, it is known that the optimal reaction time for DBS to catalyze the biosynthesis of DHDB from DHAA is 24 h, and the optimal reaction temperature is 50 °C. Therefore, under the optimal conditions, the kinetic parameters of the active enzyme DBS-catalyzed biosynthesis of DHDB from DHAA were calculated. The concentrations of the substrate DHAA were 0, 0.01, 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, 1.50, and 2.00 mM, respectively. In vitro activity verification was carried out under the conditions of the optimal reaction time and the optimal reaction temperature. Different concentrations of the substrate were added, and 3 parallels were set for each group. After the reaction was completed, the reaction rate of DBS corresponding to different substrate concentrations was calculated based on the production amount of the product DHDB and the reaction time. The Michaelis equation curve was plotted according to the substrate concentration and the reaction rate, and the K m value was obtained.

[0076] The results showed that: As Figure 10 can be seen, the K m value of DBS-catalyzed biosynthesis of DHDB from DHAA is 205.17 μM, and the K cat / K m value of DBS-catalyzed biosynthesis of DHDB from DHAA is 1.0×10 -2 min -1 .μM -1 .

Claims

1. Use of deoxyartemisinin B synthase or its isozyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydrodeoxyartemisinin B from dihydroartemisinic acid; the amino acid sequence of the isozyme A0A2U1PPI9 is ​​shown in SEQ ID NO.1; the base sequence of the gene encoding the isozyme A0A2U1PPI9 is ​​shown in SEQ ID NO.2; the amino acid sequence of the deoxyartemisinin B synthase is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The reaction time of the deoxyartemisinin B synthase catalyzing the biosynthesis of dihydroartemisinic acid into dihydrodeoxyartemisinin B is 1-64 h, and the reaction temperature is 20-60°C.

3. The use according to claim 2, characterized in that: The reaction time of the deoxyartemisinin B synthase catalyzing the biosynthesis of dihydroartemisinic acid into dihydrodeoxyartemisinin B is 8-40 h and the reaction temperature is 30-60°C.

4. The use according to claim 1, characterized in that: The deoxyartemisinin B synthase catalyzes the biosynthesis of dihydroartemisinic acid into dihydrodeoxyartemisinin B. Kcat / Km The value is 1.0×10 -2 min -1 .μM -1 .

5. The use according to claim 1, characterized in that: The heterologous expression of the isozyme A0A2U1PPI9 is ​​specifically heterologous expression in prokaryotes and eukaryotes.

6. Application of isozyme A0A2U1PPI9 in the construction of a chassis strain producing artemisinin-based compounds, the amino acid sequence of the isozyme A0A2U1PPI9 being shown in SEQ ID NO.

1.

7. A catalytic synthesis method of dihydrodeoxyartemisinin B, characterized in that: Using dihydroartemisinic acid as a substrate, the deoxyartemisinin B synthase or its isozyme A0A2U1PPI9 described in claim 1 is used to catalyze the biosynthesis of dihydrodeoxyartemisinin B from dihydroartemisinic acid.

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