New application of deoxyartemisinin B synthase and isoenzyme A0A2U1PPI9 thereof

Abstract

The invention discloses a deoxyartemisinin B synthase and an application of an isoenzyme A0A2U1PPI9 of the deoxyartemisinin B synthase in catalysis of dihydroartemisinin acid for biosynthesis of dihydrodeoxyartemisinin B. The invention further discloses a preparation method of the deoxyartemisinin B synthase. The invention discloses a process for biosynthesizing dihydrodeoxidized artemisinin B from dihydroartemisinin for the first time in a biosynthesis pathway of artemisinin, and bioinformatics analysis and in-vitro activity verification of candidate enzymes show that both the deoxidized artemisinin B synthase and the isoenzyme A0A2U1PPI9 have a new function of catalyzing the biosynthesis of dihydrodeoxidized artemisinin B from dihydroartemisinin, so that the application of the deoxidized artemisinin B synthase and the isoenzyme A0A2U1PPI9 to the biosynthesis of dihydrodeoxidized artemisinin B is developed. Lines of artemisinin biosynthetic pathways are enriched, and a theoretical basis is provided for analysis of terminal biosynthetic pathways of artemisinin and heterologous synthesis of artemisinin compounds.

Description

Technical Field

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

[0002] Artemisinin (ART) has excellent antimalarial activity and is mainly used for symptom control of Plasmodium vivax malaria and falciparum malaria and the treatment of chloroquine-resistant malaria. With the continuous deepening of research, people have found that ART and its analogs have multiple pharmacological effects such as anti-tumor, anti-inflammatory and immunomodulatory. For example, dihydroartemisinin has the function of interfering with the surface membrane and mitochondria, and has the effect of relieving symptoms and treating early systemic lupus erythematosus; ART can target mitochondrial protease LONP1, promote the binding of LONP1 with its substrate CYP11A1, accelerate the degradation of CYP11A1, and then inhibit the synthesis of ovarian androgens, reduce the androgen level of PCOS patients, 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 chemical total synthesis of ART has been achieved, the process is complicated, the yield is low and the environmental pollution is serious. Industrial production has not been carried out, so the market supply of ART has always attracted much attention.

[0003] Since the terminal biosynthetic pathway of ART has not been elucidated, it is still impossible to directly produce ART using synthetic biology methods. ZL 202410006829.6 Through mining the transcriptome database of Artemisia annua and bioinformatics analysis, an active enzyme DBS was screened out that can catalyze the biosynthesis of deoxyarteannuin B (DB) from artemisinic acid (AA). However, studies have shown that AA is automatically oxidized to produce artemisinin B (ART-B), while dihydroartemisinic acid (DHAA) is automatically oxidized to produce ART. At the same time, some researchers added the substrate DHAA into Artemisia annua cells and found that the product ART was generated through biotransformation, that is, DHAA is the direct precursor compound of ART, and AA is the precursor compound of ART-B. The present invention has found through research that the active enzyme DBS and its isozyme A0A2U1PPI9 can catalyze the biosynthesis of DHAA into DHDB. This application provides a theoretical basis for the analysis of the ART terminal biosynthetic pathway and provides 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 deoxyartemisinin B synthase and its isozyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydroartemisinic acid into dihydrodeoxyartemisinin B, so as to provide 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 a first aspect, the present invention provides the use of deoxyarteannuin B synthase (DBS) or its isozyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydrodeoxyartemisinic acid (DHAA) into dihydro-epi-deoxyarteannuin B (DHDB).

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

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

[0009] The present invention finds through bioinformatics analysis that the amino acid sequences of DBS and A0A2U1PPI9 have a certain degree of conservation, that is, the active enzyme A0A2U1PPI9 is ​​an isozyme of DBS.

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

[0011] The base sequence of the gene encoding the isozyme A0A2U1PPI9 is ​​shown in SEQ ID NO.2.

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

[0013] In a second aspect, the present invention further provides the use of isozyme A0A2U1PPI9 in the construction of a chassis strain producing artemisinin compounds, and the amino acid sequence of the isozyme A0A2U1PPI9 is ​​shown in SEQ ID NO.1.

[0014] In a third aspect, the present invention also provides a catalytic synthesis method of dihydrodeoxyartemisinin B, using dihydroartemisinic acid as a substrate and utilizing the deoxyartemisinin B synthase or its isozyme A0A2U1PPI9 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] The present invention discloses for the first time the process of DHAA biosynthesis of DHDB in the biosynthetic pathway of ART. Through bioinformatics analysis and in vitro activity verification of candidate enzymes, it is shown that DBS and its isozyme A0A2U1PPI9 both have the new function of catalyzing the biosynthesis of DHAA into DHDB, enriching the circuit 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 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 active enzyme DBS as bait;

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

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

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

[0021] Figure 5 The amount of product generated when active enzyme DBS and A0A2U1PPI9 catalyzed the biosynthesis of DHDB from DHAA (n=3, *** indicates significant difference at the P<0.001 level);

[0022] Figure 6 HPLC detection of the time-dependent changes of DHAA biosynthesis DHDB catalyzed by active enzyme DBS;

[0023] Figure 7 The amount of product produced when the active enzyme DBS catalyzes the biosynthesis of DHDB from DHAA changes with the reaction time;

[0024] Figure 8 HPLC detection of the temperature-dependent changes of DHAA biosynthesis DHDB catalyzed by active enzyme DBS;

[0025] Fig. 9 When the active enzyme DBS catalyzes the biosynthesis of DHDB from DHAA, the amount of product produced changes with the reaction temperature;

[0026] Fig.10 Kinetic parameters of the biosynthesis of DHDB from DHAA catalyzed by the active enzyme DBS. DETAILED DESCRIPTION

[0027] The present invention is further described below by means of specific implementation methods. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to 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 number: ZL 202410006829.6). The amino acid sequence of DBS was used as bait to screen candidate enzymes with high homology to its amino acid sequence in the Artemisia annua transcriptome database (NCBI) Blast. The homologous enzymes screened were analyzed by phylogenetic tree using MGGA11 and iTOL analysis software ( Figure 1 ). We found that the enzyme A0A2U1PPI9 is ​​in the same branch as DBS, and the amino acid sequence is highly conserved ( Figure 2), so we speculated that the candidate enzyme A0A2U1PPI9 may have the same biological function as the active enzyme DBS, and subsequently performed functional verification on it.

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

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

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

[0033] In the clean bench, take 10 μL of pESC-His glycerol bacteria and add it to 5 mL of LB liquid medium containing ampicillin (Amp) resistance, and culture it at 37°C and 220 rpm until the OD 600 =0.6, and then the plasmid pESC-His was extracted using the Qingke plasmid extraction kit.

[0034] The concentration of the extracted plasmid was measured by ultra-micro biodetector, and then the reaction system was prepared according to Table 1 to perform double enzyme digestion on the plasmid pESC-His, and the prepared system was placed in a 37°C water bath for 2 hours. After the end, the enzyme digestion reaction system was tested by 1% agarose gel electrophoresis, and the gel blocks of corresponding molecular weight were cut and stored in 1.5mL EP tubes. Finally, the gel recovery kit (Sangong) was used to recover the plasmid after double enzyme digestion.

[0035] Table 1. Configuration of double restriction enzyme digestion system for plasmid pESC-His

[0036]

[0037] b: Amplification of target gene A0A2U1PPI9

[0038] RNA was extracted from Artemisia annua plants using a plant total RNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd.), and cDNA was obtained by reverse transcription using a reverse transcription kit (TOYOBO). Primers for the candidate enzyme DBS were designed according to the plasmid pESC-His and restriction sites SalI and BamHI (Table 2), and then the candidate enzyme was PCR amplified. The amplification system and PCR amplification program are shown in Tables 3 and 4. After the PCR amplification, the reaction system was tested by 1% agarose gel electrophoresis, and the gel blocks of the corresponding molecular weight were cut and stored in a 1.5 mL EP tube, and the amplified target gene was recovered using a gel recovery kit (Sangon).

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

[0040]

[0041] Table 3. PCR amplification system of candidate enzyme A0A2U1PPI9

[0042]

[0043] Table 4. PCR amplification procedure 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 "Hieff Clone Universal One Step Cloning Kit" was used for homologous recombination, and the recombination was carried out at 50°C for 30 minutes. After the recombination was completed, it was immediately transformed into E. coli DH5α competent cells. The next day, a single clone was picked from the transformed resistance plate for colony PCR verification.

[0048] d: Cloning of fragment pESC-His-A0A2U1PPI9, recombination into plasmid pCDF-Ty2 and integration into yeast BY4741 genome

[0049] According to the EcoRI and SacI restriction sites of plasmid pCDF-Ty2, the His3 promoter-CYC1 terminator fragment in pESC-His-A0A2U1PPI9 was cloned and recombined into the pCDF-Ty2 plasmid using the method of step (1)-c in Example 1, named pCDF-Ty2-His-A0A2U1PPI9. Then, the Ty2F-Ty2R fragment in pCDF-Ty2-His-A0A2U1PPI9 was cloned, and finally the cloned fragment was integrated into the genome of Saccharomyces cerevisiae BY4741, and the Saccharomyces cerevisiae strain with the integrated target gene was screened on SD-His YNB solid culture 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 solution reaches 600=0.5. Take 1 mL of bacterial solution and inoculate it into YPD liquid medium induced by galactose: glucose (9:1) for induction expression, and culture it at 30℃ and 220rpm for 96h. Collect the bacterial solution, centrifuge it at 8000rpm and 4℃ for 15min, discard the supernatant, and add an appropriate amount of protein buffer to resuspend the precipitate. Use a high-pressure cell disruptor (25Pa) to break the resuspended solution, collect the bacterial solution and use it immediately for downstream experiments or store it at -20℃ for later use.

[0052] ⑶ Functional verification of active enzyme A0A2U1PPI9 catalyzing DHAA biosynthesis into DHDB

[0053] Establish an in vitro enzyme catalytic reaction system: take a 2mL EP tube, add active enzyme DBS 10μM, substrate DHAA 400μM, auxiliary factor NADPH 1mM in sequence, and make up to 1.0mL with PBS solution. React in the dark at 30℃ and 220rpm for 16h. Boiled DBS is used as the control group. After the reaction, add 800μL of ethyl acetate to the EP tube to terminate the reaction, centrifuge at 25℃ and 12000rpm for 2min, and collect the upper organic phase. Repeat the extraction 3 times, combine the organic phases, concentrate to dryness, and then add 100μL of methanol (HPLC grade) for HPLC (209nm). The HPLC detection conditions are shown in Table 5.

[0054] The results show that: Figure 3 The HPLC test results show that the active enzyme A0A2U1PPI9 generates a product after catalyzing DHAA. By comparing with the DHDB standard, the peak time is the same, and the enzyme catalysis product is determined to be DHDB. That is, A0A2U1PPI9 has the function of catalyzing the biosynthesis of DHDB from DHAA.

[0055] Table 5. HPLC elution conditions

[0056]

[0057]

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

[0059] Example 3: Functional study of active enzyme DBS catalyzing DHAA biosynthesis of DHDB

[0060] ⑴ Fermentation of active enzyme DBS

[0061] In the previous research work of this study, active enzyme DBS has been heterologously expressed in Saccharomyces cerevisiae BY4741 (patent number: ZL 202410006829.6), and the glycerol stock of the relevant strains has been stored in a -80℃ refrigerator. Take 10μL of the strain and inoculate it into 5mL SD-His YNB liquid culture medium, and culture it at 220rpm and 30℃ until the bacterial liquid OD reaches 600 =0.5. Take 1 mL of bacterial solution and inoculate it into YPD liquid medium induced by galactose: glucose (9:1) for induction expression, and culture it at 30℃ and 220rpm for 96h. Collect the bacterial solution, centrifuge it at 8000rpm and 4℃ for 15min, discard the supernatant, and add an appropriate amount of protein buffer to resuspend the precipitate. Use a high-pressure cell disruptor (25Pa) to break the resuspended solution, collect the bacterial solution and use it immediately for downstream experiments or store it at -20℃ for later use.

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

[0063] Establish an in vitro enzyme catalytic reaction system: take a 2mL EP tube, add active enzyme DBS 10μM, substrate DHAA 400μM, auxiliary factor NADPH 1mM in sequence, and make up to 1.0mL with PBS solution. React in the dark at 30℃ and 220rpm for 16h. Boiled DBS is used as the control group. After the reaction, add 800μL of ethyl acetate to the EP tube to terminate the reaction, centrifuge at 25℃ and 12000rpm for 2min, and collect the upper organic phase. Repeat the extraction 3 times, combine the organic phases, concentrate to dryness, and then add 100μL of methanol (HPLC grade) for HPLC (209nm). The HPLC detection conditions are shown in Table 5.

[0064] The results show that: Figure 4 The HPLC test results show that active enzyme DBS generates products after catalyzing DHAA. By comparing with the DHDB standard, the peak time is the same, and the enzyme catalysis product is determined to be DHDB. That is, DBS has the function of catalyzing the biosynthesis of DHDB from DHAA.

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

[0066] From the results of Examples 2 and 3, it can be seen that both active enzymes DBS and A0A2U1PPI9 can catalyze the biosynthesis of DHDB from DHAA, and the amino acid sequences of DBS and A0A2U1PPI9 are somewhat conservative ( Figure 2), that is, the active enzyme A0A2U1PPI9 is ​​an isozyme of DBS. In order to explore the catalytic efficiency of the above two active enzymes in catalyzing the biosynthesis of DHDB from DHAA, we statistically calculated the production of their product DHDB. First, a standard curve of DHDB was established, and then the production of the product of each enzyme-catalyzed reaction was calculated. The catalytic efficiency of the active enzymes DBS and A0A2U1PPI9 was compared based on the production of the product DHDB.

[0067] The results showed that the amount of DHDB produced by the active enzyme DBS catalyzing the biosynthesis of DHAA was 32.11 mg / L, and the amount of DHDB produced by the active enzyme A0A2U1PPI9 catalyzing the biosynthesis of DHAA was 13.20 mg / L ( Figure 5 ), the activity of DBS in catalyzing the biosynthesis of DHDB from DHAA is 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.

[0068] Example 5: Optimal time study of DBS-catalyzed DHAA biosynthesis of DHDB

[0069] The optimal reaction time of DBS catalyzing the biosynthesis of DHDB from DHAA was investigated by the single variable method, the reaction temperature was 30°C, and the concentration of the substrate DHAA was 400 μM. The reaction time ranges were 1, 8, 16, 24, 32, 40, 48, 56 and 64 h, with 3 parallels in each group. An in vitro activity experiment on DBS was established according to the method of Example 3-⑵, the reaction was terminated after different reaction times, and the optimal reaction time of the active enzyme DBS catalyzing the biosynthesis of DHDB from DHAA was determined according to the amount of product DHDB generated.

[0070] The results show that: Figure 6 and Figure 7 As shown, the optimal reaction time for DBS-catalyzed DHAA biosynthesis of DHDB is 24 h.

[0071] Example 6: Investigation of the Optimal Temperature for the Biosynthesis of DHDB by DHAA Catalyzed by DBS

[0072] The optimal reaction temperature of DBS catalyzing the biosynthesis of DHDB from DHAA was investigated by the single variable method. The reaction time was 24h and the concentration of substrate DHAA was 400μM. The reaction temperature range was 20, 30, 40, 50 and 60℃, with 3 parallels in each group. In vitro activity experiments on DBS were established at different temperatures, and the optimal reaction temperature of active enzyme DBS catalyzing the biosynthesis of DHDB from DHAA was determined according to the amount of product DHDB generated.

[0073] The results show that: Figure 8 and Fig. 9It can be seen that the optimal reaction temperature for DBS-catalyzed DHAA biosynthesis of DHDB is 50℃.

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

[0075] According to the 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 24h, and the optimal reaction temperature is 50°C. Therefore, the kinetic parameters of the biosynthesis of DHDB from DHAA catalyzed by the active enzyme DBS are calculated under the optimal conditions. The concentrations of the substrate DHAA are 0, 0.01, 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, 1.50 and 2.00 mM, respectively. The in vitro activity is verified under the conditions of the optimal reaction time and the optimal reaction temperature. Different concentrations of substrates are added respectively, and 3 parallels are set for each group. After the reaction is completed, the reaction rate of DBS corresponding to different substrate concentrations is calculated based on the amount of product DHDB generated and the reaction time. The Michaelis-Menten equation curve is drawn according to the substrate concentration and the reaction rate, and the K of DBS catalyzing the biosynthesis of DHDB from DHAA is obtained. m value.

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

Claims

1. Application of deoxyartemisinin B synthase or its isozyme A0A2U1PPI9 in catalyzing the biosynthesis of dihydrodeoxyartemisinin B from dihydroartemisinic acid.

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, preferably 8-40 h, and the reaction temperature is 20-60°C, preferably 30-60°C.

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

4. The use according to claim 1, characterized in that: The amino acid sequence of the isozyme A0A2U1PPI9 is ​​shown in SEQ ID NO.

1.

5. The use according to claim 4, characterized in that: The base sequence of the gene encoding the isozyme A0A2U1PPI9 is ​​shown in SEQ ID NO.

2.

6. The use according to claim 4, characterized in that: The heterologous expression of the isozyme A0A2U1PPI9 is ​​specifically heterologous expression in prokaryotes and eukaryotes.

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

1.

8. 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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