New application of artemisinin B synthase

Abstract

The invention discloses a new application of artemisinin B synthase (ARTBs), and particularly discloses a new application of the artemisinin B synthase (ARTBs). According to the artemisinin B synthase ARTBs and the application thereof, the artemisinin B synthase ARTBs is obtained through screening by mining transcriptome data of Artemisia annua, and functional verification shows that the artemisinin B synthase ARTBs has the biological function of catalyzing deoxidized artemisinin B to biosynthesize artemisinin B or catalyzing dihydrodeoxidized artemisinin B to biosynthesize dihydroartemisinin B; a saccharomyces cerevisiae engineering strain for producing ART-B and a precursor compound DB thereof is constructed in saccharomyces cerevisiae BY4741, so that a theoretical basis is provided for producing ART-B and DHART-B by adopting a synthetic biological method, the route of an artemisinin biosynthetic pathway is enriched, and a theoretical basis is provided for analysis of an ART terminal biosynthetic pathway.

Description

Technical Field

[0001] The present invention relates to the field of biosynthesis, and specifically relates to a new application of a class of arteannuin B synthases (ARTBs) in catalyzing the biosynthesis of arteannuin B (ART-B) from deoxyarteannuin B (DB) or catalyzing the biosynthesis of dihydroarteannuin B (DHART-B) from dihydrodeoxyarteannuin B (DHDB). Background Art

[0002] Artemisia annua L. is an annual herbaceous plant of the genus Artemisia in the Compositae family and is the only natural source of the antimalarial drug artemisinin (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, so industrial production has not been carried out. Therefore, the market supply problem of ART has always attracted much attention.

[0003] The arteannuin compound ART-B is also a sesquiterpene lactone compound present in Artemisia annua. Studies have shown that ART-B can be used to prepare immunosuppressive drugs and has potential value in the drug development for the prevention and treatment of autoimmune diseases and inflammatory diseases. ART-B also shows activity against the SARS-CoV-2 virus, with a half-maximal effective concentration of 10.28 μM, indicating that it may have certain potential in anti-coronavirus treatment. ART-B has obvious cytotoxicity to P388 mouse leukemia suspension cells and human liver cancer cells SMMC-7721. At the same time, there are also studies showing that ART-B can reverse the drug resistance of A549 / cisplatin cells by activating the MAPK / P53 signaling pathway, increasing the sensitivity of non-small cell lung cancer cells to cisplatin, and thus synergistically playing a role in the treatment of non-small cell lung cancer with cisplatin.

[0004] Since the terminal biosynthetic pathway of ART has not been elucidated, it is still not possible to directly produce ART compounds by synthetic biology methods. Summary of the Invention

[0005] To solve the above technical problems, the purpose of the present invention is to provide a new application of a class of arteannuin B synthases (ARTBs) in catalyzing the biosynthesis of arteannuin B from deoxyarteannuin B or catalyzing the biosynthesis of dihydroarteannuin B from dihydrodeoxyarteannuin B, so as to provide a theoretical basis for the analysis of the terminal biosynthetic pathway of ART.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides the use of a class of arteannuin B synthases (ARTBs) in catalyzing the biosynthesis of arteannuin B (ART-B) from deoxyarteannuin B (DB) or catalyzing the biosynthesis of dihydroarteannuin B (DHART-B) from dihydrodeoxyarteannuin B (DHDB).

[0008] The present invention first found through literature retrieval that there is a parthenolide synthase (PTS) in the biosynthetic pathway of parthenolide that can catalyze the biosynthesis of parthenolide (PT) from costunolide (CO). At the same time, PT is a sesquiterpene lactone compound, and both parthenium and Artemisia annua belong to the Compositae family. Therefore, it is speculated that there is also an active enzyme similar to PTS in Artemisia annua that can carry out catalytic reactions with similar functions to achieve the biosynthesis of ART-B. Then, using PTS as a bait to Blast in the Artemisia annua transcriptome database, oxidases homologous to PTS are retrieved from the Blast results. Finally, the functions of the screened candidate enzymes are verified, and a class of oxidases belonging to the cytochrome P450 family is obtained. This class of oxidases belonging to the cytochrome P450 family has the biological functions of catalyzing the biosynthesis of arteannuin B (ART-B) from deoxyarteannuin B (DB) and catalyzing the biosynthesis of dihydroarteannuin B (DHART-B) from dihydrodeoxyarteannuin B (DHDB). The oxidase with the above biological functions is named arteannuin B synthase (ARTBs).

[0009] The class of arteannuin B synthases described in the present invention includes 8 oxidases, namely:

[0010] A0A2U1MQZ2 (ARTBs-1), the amino acid sequence is shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.9.

[0011] A0A2U1MEK0 (ARTBs-2), the amino acid sequence is shown in SEQ ID NO.2, and its coding sequence is shown in SEQ ID NO.10.

[0012] A0A2U1QCU0 (ARTBs-3), the amino acid sequence is shown in SEQ ID NO.3, and its coding sequence is shown in SEQ ID NO.11.

[0013] K7N8B9 (ARTBs-4), the amino acid sequence is shown in SEQ ID NO.4, and its coding sequence is shown in SEQ ID NO.12.

[0014] A0A2U1MLH1 (ARTBs-5), the amino acid sequence is shown in SEQ ID NO.5, and its coding sequence is shown in SEQ ID NO.13.

[0015] A0A2U1NH79 (ARTBs-6), the amino acid sequence is shown in SEQ ID NO.6, and its coding sequence is shown in SEQ ID NO.14.

[0016] K7N8J2 (ARTBs-7), the amino acid sequence is shown in SEQ ID NO.7, and its coding sequence is shown in SEQ ID NO.15.

[0017] A0A2U1NKI2 (ARTBs-8), the amino acid sequence is shown in SEQ ID NO.8, and its coding sequence is shown in SEQ ID NO.16.

[0018] The heterologous expression of a class of artemisinin B synthases described in the present invention is specifically the expression in prokaryotes and eukaryotes.

[0019] Preferably, the prokaryotes include Escherichia coli and Agrobacterium; the eukaryotes include yeast systems, insect systems, and fungi.

[0020] More preferably, the yeast system is Saccharomyces cerevisiae BY4741.

[0021] In a second aspect, the present invention provides the application of a class of artemisinin B synthases in the construction of Saccharomyces cerevisiae engineering strains for the production of artemisinin B or dihydroartemisinin B, and the amino acid sequences of the artemisinin B synthases are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and / or SEQ ID NO.8.

[0022] The present invention has constructed Saccharomyces cerevisiae engineering strains for the production of ART-B and its precursor compound DB in Saccharomyces cerevisiae BY4741, and their shake flask yields are 910.6 μg / L and 30.7 μg / L respectively, realizing the fermentative production of ART-B.

[0023] Thirdly, the present invention provides an application of a class of arteannuin B synthases in the construction of chassis strains for producing arteannuin compounds in prokaryotic or eukaryotic systems, and the amino acid sequences of the arteannuin B synthases are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and / or SEQ ID NO.8.

[0024] Compared with the prior art, the present invention has the following excellent effects:

[0025] Through mining the transcriptome data of Artemisia annua, the present invention screened a class of arteannuin B synthases (Arteannuin B synthase, ARTBs). After functional verification, this class of arteannuin B synthases has the biological functions of catalyzing the biosynthesis of arteannuin B (Arteannuin B, ART-B) from deoxyarteannuin B (Deoxyarteannuin B, DB) and catalyzing the biosynthesis of dihydroarteannuin B (Dihydroarteannuin B, DHART-B) from dihydrodeoxyarteannuin B (Dihydrodeoxyarteannuin B, DHDB). At the same time, a Saccharomyces cerevisiae engineering strain for producing ART-B and its precursor compound DB was constructed in Saccharomyces cerevisiae BY4741, providing a theoretical basis for the production of ART-B and DHART-B by synthetic biology methods, enriching the routes of the ART biosynthetic pathway, and providing a theoretical basis for the analysis of the ART terminal biosynthetic pathway. Description of the Drawings

[0026] Figure 1 Speculative route map for the biosynthesis of arteannuin B from deoxyarteannuin B;

[0027] Figure 2 Phylogenetic tree analysis of the oxidase obtained by Blast in the Artemisia annua transcriptome database using PTS as a bait and PTS;

[0028] Figure 3 Amino acid sequence alignment of the candidate enzyme and PTS;

[0029] Figure 4 Expression plasmid map of the candidate enzyme;

[0030] Figure 5 Integration plasmid map of the candidate enzyme;

[0031] Figure 6 LC-MS detection of the candidate enzyme catalyzing DB;

[0032] Figure 7 LC-MS detection of the candidate enzyme catalyzing DHDB

[0033] Figure 8 Expression plasmid map of the active enzyme DBS

[0034] Figure 9 Integrated plasmid map of the active enzyme DBS

[0035] Figure 10 Standard curve of DB

[0036] Figure 11 LC-MS detection of the engineered Saccharomyces cerevisiae strain producing DB by flask fermentation

[0037] Figure 12 Plasmid map of the engineered Saccharomyces cerevisiae strain producing ART-B

[0038] Figure 13 Standard curve of ART-B

[0039] Figure 14 LC-MS detection of the engineered Saccharomyces cerevisiae strain producing ART-B by flask fermentation Detailed implementation manners

[0040] The present invention will be further described below through specific implementation manners. 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.

[0041] Example 1: Screening of the active enzyme

[0042] Based on the chemical structures of DB and ART-B, it is speculated that the process of catalyzing DB to biosynthesize ART-B is an oxidation reaction, so the participation of oxidase is required ( Figure 1 in A). Based on this, a literature search was conducted and it was found that in the biosynthetic pathway of PT, PTS can catalyze the biosynthesis of PT from CO ( Figure 1 in B). Since both Tanacetum parthenium and Artemisia annua belong to the Compositae family, and the chemical structures of compound DB and CO, ART-B and PT are similar, and they are all oxidation reactions occurring at double bonds ( Figure 1 ), it is speculated that there may be a class of oxidases with high homology to PTS in Artemisia annua that can catalyze the biosynthesis of ART-B from DB. In the present invention, the amino acid sequence of PTS was used as a bait to conduct a Blast in the Artemisia annua transcriptome database (NCBI), and then the results obtained from the Blast were subjected to phylogenetic tree analysis with PTS ( Figure 2 ), and the oxidases in the same branch as PTS were screened from the results of the phylogenetic tree analysis as candidate enzymes ( Figure 2 ), and then the amino acid sequences of the candidate enzymes were aligned with PTS ( Figure 3)。

[0043] The results showed that: as shown in the Blast result of Figure 2 , using PTS as a bait, many related oxidases were Blast in the Artemisia annua transcriptome database. Through phylogenetic tree analysis, it was found that 10 oxidases were relatively close to the distribution of PTS, and their protein ID numbers were: A0A2U1MQZ2, A0A2U1MEK0, A0A2U1QCU0, K7N8B9, A0A2U1MLH1, A0A2U1NH79, K7N8J2, A0A2U1NKI2, A0A2U1KMP3 and A0A2U1LDA9. From Figure 3 the amino acid sequence alignment results, it can be seen that except for the candidate enzymes A0A2U1KMP3 and A0A2U1LDA9, the other 8 candidate enzymes have high conservation and sequence similarity with PTS. Heterologous expression and functional verification were carried out on the 8 candidate enzymes A0A2U1MQZ2 (ARTBs-1), A0A2U1MEK0 (ARTBs-2), A0A2U1QCU0 (ARTBs-3), K7N8B9 (ARTBs-4), A0A2U1MLH1 (ARTBs-5), A0A2U1NH79 (ARTBs-6), K7N8J2 (ARTBs-7) and A0A2U1NKI2 (ARTBs-8).

[0044] Example 2: Heterologous expression and functional verification of candidate enzymes in Saccharomyces cerevisiae BY4741

[0045] ⑴ Extraction and double digestion of plasmid pESC-His

[0046] 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 at 37 °C and 220 rpm until OD 600 = 0.6, then extract the plasmid pESC-His using the Tsingke plasmid extraction kit. The plasmid map of pESC-His is as shown in Figure 4 -A.

[0047] Use a ultra-micro biological detector to measure the concentration of the extracted plasmid, and then prepare the reaction system according to Table 1 for double digestion of the 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 store it in a 1.5 mL EP tube. Finally, use a gel recovery kit (Sangon) to recover the plasmid after double digestion.

[0048] Table 1. Configuration table of double digestion system of plasmid pESC-His

[0049]

[0050]

[0051] ① The plasmid extraction process is as follows:

[0052] Add 250 μL of Buffer BL to the adsorption column (the adsorption column has been previously placed in the collection tube), centrifuge at 4 °C and 13,500 rpm for 1 min to activate the silica gel membrane;

[0053] Take the cultured bacterial solution, centrifuge at 25 °C and 3,500 rpm for 10 min, and discard the supernatant;

[0054] Add 250 μL of Buffer S1 to resuspend the bacterial cells, and vortex until there are no cell clumps;

[0055] Add 250 μL of Buffer S2, gently invert up and down 6 - 8 times to fully lyse the bacterial cells;

[0056] Add 350 μL of Buffer S3, gently invert up and down 6 - 8 times, and centrifuge at 25 °C and 13,500 rpm for 10 min;

[0057] Transfer the supernatant to the adsorption column, centrifuge at 25 °C and 13,500 rpm for 1 min, discard the waste liquid, and place the adsorption column back into the collection tube;

[0058] Add 700 μL of Buffer W2 to the adsorption column, centrifuge at 25 °C and 13,500 rpm for 1 min, discard the waste liquid, and repeat the operation once;

[0059] Place the adsorption column back into the collection tube and centrifuge at 25 °C and 13,500 rpm for 2 min;

[0060] Take out the adsorption column, place it in a clean 1.5 mL EP tube, and let it stand at 25 °C for 5 min to allow the residual ethanol to fully evaporate;

[0061] Add 35 μL of dd H 2 O (pre - heated at 65 °C), let it stand at 25 °C for 5 min, and centrifuge at 25 °C and 13,500 rpm for 2 min;

[0062] At this time, the solution at the bottom of the EP tube is the extracted plasmid, which can be immediately used for downstream experiments or stored in a - 20 °C refrigerator for later use.

[0063] ② The gel extraction process is as follows:

[0064] Use a clean surgical blade to cut out the agarose gel block of the target fragment, place it in a 1.5 mL EP tube, and weigh it;

[0065] Add an appropriate amount of Buffer B2 according to the ratio of adding 500 μL of Buffer B2 to every 100 mg of agarose.

[0066] Place it in a water bath at 65 °C for 5 - 10 min until the gel block completely melts.

[0067] Transfer all the melted solution to the adsorption column, centrifuge at 25 °C and 9000 rpm for 30 s, pour out the liquid in the collection tube, and put the adsorption column into the same collection tube.

[0068] Add 300 μL of Buffer B2 to the adsorption column, centrifuge at 25 °C and 10000 rpm for 30 s, pour out the liquid in the collection tube, and put the adsorption column into the same collection tube.

[0069] Add 500 μL of Wash Solution to the adsorption column, centrifuge at 25 °C and 10000 rpm for 30 s, pour out the liquid in the collection tube, put the adsorption column into the same collection tube, and repeat the operation once.

[0070] Put the empty adsorption column and the collection tube into the centrifuge, centrifuge at 25 °C and 10000 rpm for 1 min.

[0071] Take out the adsorption column, put it into a clean 1.5 mL EP tube, and let it stand at 25 °C for 5 min to fully volatilize the residual ethanol.

[0072] Add 20 μL of dd H 2 O (preheated at 65 °C) to the center of the adsorption membrane, let it stand at 25 °C for 2 min, and centrifuge at 25 °C and 10000 rpm for 1 min.

[0073] Place the obtained DNA recovery solution in a -20 °C refrigerator for storage for later use or immediately use it in the following experiment.

[0074] The results showed that the plasmid pESC-His digested by SalI and BamHI was obtained through plasmid extraction, double digestion and gel recovery, as Figure 5 shown by the detection result of the dextran gel electrophoresis in -A.

[0075] ⑵ Amplification of the target gene

[0076] Total RNA of Artemisia annua was extracted using a Total RNA Extraction Kit (Nanjing Novoprotein Biological Technology Co., Ltd.), and cDNA was obtained by reverse transcription using a Reverse Transcription Kit (TOYOBO). Primers for 8 candidate enzymes were designed according to plasmid pESC-His and restriction enzyme sites XhoI and BamHI (Table 2), and then the candidate enzymes were amplified by PCR respectively. The amplification system and PCR amplification program are shown in Table 3 and Table 4. After the PCR amplification, the reaction system was detected by 1% agarose gel electrophoresis, and the gel block corresponding to the molecular weight was cut and stored in a 1.5 mL EPP tube. The amplified target gene was recovered using a Gel Extraction Kit (Sangon).

[0077] Table 2. Primer list for PCR amplification of candidate enzymes

[0078]

[0079] Table 3. PCR amplification system of candidate enzyme DBS

[0080]

[0081] Table 4. PCR amplification program of candidate enzyme DBS

[0082]

[0083] Note: Denaturation, renaturation, and extension were carried out for 33 cycles in total.

[0084] (3) Homologous recombination and transformation of plasmid and target gene

[0085] Homologous recombination was carried out using the "Hieff Clone Universal One Step Cloning Kit" at 50 °C for 30 min, and immediately transformed into Escherichia coli DH5α competent cells after the recombination. The next day, monoclonal colonies were picked from the transformed resistant plates for colony PCR verification.

[0086] (4) Cloning of the expression fragment His3 promoter - Cyc1 terminator of candidate enzyme, recombination into the integration plasmid pCDF-ty2, and integration into the genome of Saccharomyces cerevisiae BY4741

[0087] According to the integration plasmid pCDF-ty2, SacI and EcoRI restriction enzyme sites were selected, and primers were designed (Table 5) to clone the His3 promoter - CYC1 terminater fragment in pESC-His-gene ( Figure 4 -B,C), and recombined into the pCDF-ty2 plasmid using the method in step (3) of Example 2 ( Figure 5In A), named pCDF-ty2-His-gene( Figure 5 In B). Then design primers (Table 5) to clone the ty2 F - ty2 R fragment in pCDF-ty2-His-gene( Figure 5 In C). Finally, integrate the cloned fragment into the genome of Saccharomyces cerevisiae BY4741 and screen on the corresponding defective (SD-His) YNB medium.

[0088] Table 5. Primer list for cloning and integrating fragments

[0089]

[0090]

[0091] ⑸ Heterologous expression of candidate enzymes in Saccharomyces cerevisiae BY4741

[0092] In a laminar flow hood, pipette 10 μL of the successfully integrated strain from step (4) and add it to 5 mL of YPD medium with glucose as the carbon source. Culture at 30 °C and 220 rpm until the OD value of the bacterial liquid reaches 0.4 - 0.6. Take 1 mL of the bacterial liquid and inoculate it into the galactose-induced YPD medium for expression. After culturing at 30 °C and 220 rpm for 96 hours, harvest. Use a high-pressure cell crusher (25 Pa) to lyse the bacterial liquid, and collect the bacterial liquid immediately for downstream experiments or store it at -20 °C for later use.

[0093] ⑹ Function verification of candidate enzymes

[0094] Establish an in vitro enzyme-catalyzed reaction system: Take a 2 mL EP tube and add 10 μM of the candidate protein, the substrate DB or DHDB, and 1 mM of the cofactor NADPH in sequence. Make up to 1.0 mL with PBS solution and react in the dark at 30 °C and 220 rpm for 30 min. The boiled candidate protein is used as a 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. Repeat the extraction 3 times, combine the organic phases, concentrate to dryness, and then add 100 μL of methanol (mass spectrometry grade) for LC-MS detection. The LC-MS detection conditions are shown in Table 6.

[0095] The results show that: From Figure 6 the LC-MS detection results in A, it can be seen that products are generated after the candidate enzymes catalyze DB. By comparing with the ART-B standard product, it is found that the peak emergence time is the same, and the product ion peak is the same as the ART-B standard product ion peak( Figure 6 In B, C), it is determined that the candidate enzymes can catalyze the biosynthesis of ART-B from DB. From Figure 7From the LC-MS detection results of A, it can be seen that products were generated after the candidate enzymes catalyzed DHDB. By comparing with the DHART-B standard product, it was found that the peak emergence times were the same, and the product ion peaks were the same as those of the DHART-B standard product ( Figure 7 in B and C), it was determined that the candidate enzymes could catalyze the biosynthesis of DHART-B from DHDB. From Figure 6 and Figure 7 the detection results, it can be seen that the 8 candidate enzymes all had the same biological function, but different enzyme activities. Among them, the candidate enzyme ARTBs-1 had the strongest catalytic activity and was used for the subsequent application in the engineering strain of Saccharomyces cerevisiae for producing ART-B.

[0096] Table 6. Elution conditions of LC-MS

[0097]

[0098]

[0099] A: ddH 2 0; B: Acetonitrile (LC-MS grade).

[0100] Example 3: Construction and fermentation production of the engineering strain of Saccharomyces cerevisiae producing DB

[0101] In the previous research of the applicant, the active enzyme deoxyarteannuin B synthase (DBS) has been discovered, which has the function of catalyzing the biosynthesis of DB from artemisinic acid (AA) (Patent No.: ZL202410006829.6). At the same time, the applicant has successfully constructed an engineering strain of Saccharomyces cerevisiae AA-1 for producing AA (Patent No.: 202410833259.8) in the previous stage. Therefore, on the basis of the previous research, first, the active enzyme DBS was recombined between the EcoRI and SacI restriction sites of the expression plasmid pESC-Trp according to the methods of (1)-(3) in Example 2 ( Figure 8 ), and then the expression fragment of the active enzyme DBS was integrated into the ty2 site of the engineering strain of Saccharomyces cerevisiae AA-1 according to the method of Example 2-(4) ( Figure 9 ). The engineering strain of Saccharomyces cerevisiae DB-1 producing DB was screened on the corresponding defective (SD-Leu+Ura+His+Trp) YNB medium. Finally, shake-flask fermentation was carried out at 30 °C and 220 rpm for 96 h, and the fermentation products were detected by LC-MS, a DB standard curve was established, and the content of the product DB was calculated. The primer list is shown in Table 7.

[0102] The results showed that as Figure 8 and Figure 9As shown, an engineered Saccharomyces cerevisiae strain for producing DB was constructed. A standard curve of DB was established, as Figure 10 shown. The standard curve of DB was y = 6550.4x - 6962.5, R 2 = 0.9995. The fermentation product was detected by LC-MS, and the production of the product DB was found ([[]] Figure 11 ). After calculation, the flask production of DB was 910.6 μg / L.

[0103] Table 7. Primer list for constructing engineered Saccharomyces cerevisiae strains for producing DB and ART-B

[0104]

[0105] Example 4: Construction and fermentation production of an engineered Saccharomyces cerevisiae strain producing ART-B

[0106] First, the candidate enzyme ARTBs-1 was recombined between the BamHI and SalI restriction sites of the expression plasmid pESC-Trp-DBS according to the method of Example 2 ( Figure 12 A therein), to obtain the expression fragment pESC-Trp-DBS + QZ2 (Trp1 promoter - Cyc1 terminator) ( Figure 12 B therein). The expression fragment was integrated into the ty2 locus of the strain DB-1 according to the method of Example 3 ( Figure 12 C, D therein). An engineered Saccharomyces cerevisiae strain producing ART-B was screened on the corresponding defective (SD-Leu+Ura+His+Trp) YNB medium. Finally, flask fermentation was carried out at 30 °C and 220 rpm for 96 h, and the fermentation product was detected by LC-MS. A standard curve of ART-B was established, and the content of the product ART-B was calculated. The primer list is shown in Table 7.

[0107] The results showed that: as Figure 12 shown, an engineered Saccharomyces cerevisiae strain for producing ART-B was constructed. A standard curve of ART-B was established, as Figure 13 shown. The standard curve of ART-B was y = 2006.1x + 13269, R 2 = 0.9991. The fermentation product was detected by LC-MS, and the production of the product ART-B was found ( Figure 14 ). After calculation, the flask production of ART-B was 30.7 μg / L.

Claims

1. Use of an artemisinin B synthase in catalyzing the biosynthesis of artemisinin B from deoxyartemisinin B or catalyzing the biosynthesis of dihydroartemisinin B from dihydrodeoxyartemisinin B, wherein the amino acid sequence of the artemisinin B synthase is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and / or SEQ ID NO.

8.

2. The use according to claim 1, characterized in that: The coding sequence of the artemisinin B synthase is shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and / or SEQ ID NO.

16.

3. The use according to claim 1 or 2, characterized in that: The heterologous expression of artemisinin B synthase specifically refers to expression in prokaryotes and eukaryotes.

4. The use according to claim 3, characterized in that: The prokaryotes include Escherichia coli and Agrobacterium; the eukaryotes include yeast systems, insect systems and fungi.

5. The use according to claim 4, characterized in that: The yeast system is Saccharomyces cerevisiae BY4741.

6. Use of a type of artemisinin B synthase in the construction of an engineered strain of Saccharomyces cerevisiae for producing artemisinin B or dihydroartemisinin B, wherein the amino acid sequence of the artemisinin B synthase is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and / or SEQ ID NO.

8.

7. The use of a class of artemisinin B synthase in the construction of a chassis strain for producing artemisinin-like compounds in a prokaryotic or eukaryotic system, wherein the amino acid sequence of the artemisinin B synthase is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and / or SEQ ID NO.8.

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