Protein for catalyzing zingerone to synthesize zingerone epoxide as well as related biological material and application thereof

Abstract

The invention discloses a protein for catalyzing zingerone to synthesize zingerone epoxide as well as a related biological material and application thereof. The protein protected by the invention is lilac pinnatifida albicans zingerone epoxidase CYP76S105. According to the embodiment of the invention, the clove pinnatifida CYP76S105 is expressed in the yeast to obtain a yeast particle extracting solution containing the CYP76S105, and the yeast particle extracting solution has the function of catalyzing zingiberone to synthesize zingiberone epoxide (zingiberone epoxide). The method fills up the research blank of hechidone modification of the syringa pinnatifida, provides a foundation for in-vitro production of a hechidone epoxy compound, provides a feasible scheme for protecting wild agilawood resources, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological genetic engineering, and particularly relates to a protein for catalyzing the synthesis of zerumbone epoxide from zerumbone, and related biological materials and applications thereof. Background Art

[0002] Syringa pinnatifolia Hemsl. is a plant of the genus Syringa in the family Oleaceae. Its peeled and dried woody roots, stems and thick branches are used as Mongolian medicine mountain aloeswood, which has effects such as suppressing Heyi, clearing heat and relieving pain. Modern pharmacological research also shows that this medicine has multiple effects such as myocardial protection, analgesia, anti-tumor, antibacterial, anti-inflammatory and antioxidant. Due to the slow growth of Syringa pinnatifolia and the vulnerability of its ecological resources, wild Syringa pinnatifolia was listed as a rare and protected species in the last century, and its resources are scarce and the supply of medicinal materials is significantly insufficient.

[0003] Chemical research on Syringa pinnatifolia shows that sesquiterpenoid components are important pharmacodynamic components of mountain aloeswood medicinal materials and have good activity against myocardial ischemia, and zerumbone and its derivatives in Syringa pinnatifolia account for a relatively large proportion in the sesquiterpenoids of mountain aloeswood. Therefore, using the method of biosynthesis to synthesize zerumbone and its derivatives in Syringa pinnatifolia in vitro is of great significance for the protection of wild resources of Syringa pinnatifolia. At the same time, the research on the biosynthesis of zerumbone and its derivatives in Syringa pinnatifolia also provides basic data for the molecular mechanism of the synthesis of volatile components in Syringa plants. However, at present, there are no reports on the research related to the biosynthesis and modification of sesquiterpenoids in Syringa pinnatifolia. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to synthesize Syringa pinnatifolia sesquiterpenoids in vitro and / or how to synthesize derivatives of zerumbone in Syringa pinnatifolia and / or how to obtain the epoxide of zerumbone in Syringa pinnatifolia.

[0005] To solve the above technical problem, the present invention first provides a protein, and the protein can be a protein of any one of the following A1), A2) or A3):

[0006] A1) A protein with an amino acid sequence of Sequence 2 in the sequence listing;

[0007] A2) A fusion protein obtained by fusing a protein tag at the carboxyl terminus and / or amino terminus of the protein shown in A1);

[0008] A3) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the sequence listing, having the same function, derived from A1) or A2), or having more than 80% identity with the protein shown in A1) or A2).

[0009] The protein described above can be derived from Syringa pinnatifolia Hemsl.

[0010] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained through biological expression.

[0011] In the above-mentioned protein, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein by using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein-tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0012] In the above-mentioned protein, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined by using homology search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0013] In the above-mentioned protein, the identity of more than 80% can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0014] To solve the above technical problems, the present invention also provides any one of the following applications of the protein described above:

[0015] M1) The application of the protein in catalyzing the formation of hopane-type sesquiterpene derivatives from hopane-type sesquiterpenes;

[0016] M2) The application of the protein in the production, preparation, or development of products containing hopane-type sesquiterpene derivatives;

[0017] M3) The application of the protein in the production, preparation, or development of products related to Syringa pinnatifolia sesquiterpenes;

[0018] M4) The application of the protein in the production, preparation, or development of products related to Aquilaria sinensis sesquiterpenes

[0019] Application of the protein in the production, preparation or development of products related to zerumbone derivatives of Syringa pinnatifolia

[0020] In the above application, the hopane sesquiterpene or the Syringa pinnatifolia sesquiterpene or the Aquilaria sinensis sesquiterpene may be zerumbone. The derivative of the hopane sesquiterpene compound and / or the Syringa pinnatifolia sesquiterpene and / or the Aquilaria sinensis sesquiterpene may be a zerumbone derivative. The zerumbone derivative may be a zerumbone epoxide. The zerumbone epoxide may be zerumbone epoxide. The zerumbone epoxide is a compound with the chemical structural formula shown in Formula 2:

[0021]

[0022] To solve the above technical problems, the present invention also provides a biological material related to the protein described above. The biological material may be any one of the following D1) to D6):

[0023] D1) A nucleic acid molecule encoding the protein described above;

[0024] D2) An expression cassette containing the nucleic acid molecule described in D1);

[0025] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0026] D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0027] D5) A nucleic acid molecule that promotes or enhances the expression of the protein described above;

[0028] D6) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in D5).

[0029] In the above biological material, the nucleic acid molecule described in D1) may be the coding gene of the protein shown in the following d1) or d2):

[0030] d1) A DNA molecule with a coding sequence being the nucleotide of Sequence 1 in the sequence listing;

[0031] d2) A DNA molecule that hybridizes with the cDNA or DNA molecule defined in d1) and encodes a protein with the same function.

[0032] Among the above biological materials, the expression cassette containing a nucleic acid molecule as described in B2) refers to DNA that can express the protein described in the above application in a host cell. This DNA may not only include a promoter that initiates the transcription of the protein-coding gene, but also include a terminator that terminates the transcription of the protein-coding gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters.

[0033] Existing plant expression vectors can be used to construct recombinant expression vectors containing the expression cassette of the protein-coding gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant particle bombardment, etc. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (from CAMBIA), etc. The plant expression vector may also contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the genes of the Agrobacterium crown gall induction (Ti) plasmid (such as the nopaline synthase gene Nos) and the 3'-untranslated regions transcribed from plant genes (such as soybean storage protein genes) have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence.

[0034] Among the above biological materials, the recombinant microorganism can specifically be yeast, bacteria, algae and fungi.

[0035] To solve the above technical problems, the present invention also provides any one of the following applications of the above-mentioned biological materials:

[0036] N1) The application of the biological material in catalyzing the formation of hopane-type sesquiterpene derivatives from hopane-type sesquiterpenes;

[0037] N2) The application of the biological material in the production, preparation or development of products containing hopane-type sesquiterpene derivatives;

[0038] N3) The application of the biological material in the production, preparation or development of products related to Syringa pinnatifolia sesquiterpenes;

[0039] N4) The application of the biological material in the production, preparation or development of products related to Aquilaria sinensis sesquiterpenes

[0040] N5) Use of the said biological material in the production, preparation or development of products related to zerumbone derivatives of Syringa pinnatifolia

[0041] In the above application, the hopane-type sesquiterpene or the Syringa pinnatifolia sesquiterpene or the Aquilaria sinensis sesquiterpene may be zerumbone. The derivative of the hopane-type sesquiterpene and / or the Syringa pinnatifolia sesquiterpene and / or the Aquilaria sinensis sesquiterpene may be a zerumbone derivative. The zerumbone derivative may be a zerumbone epoxide. The zerumbone epoxide may be zerumbone epoxide. The zerumbone epoxide is a compound with the chemical structural formula

[0042] The compound of Formula 2:

[0043]

[0044] In order to solve the above technical problems, the present invention also provides any one of the following products containing the above-mentioned protein and / or the above-mentioned biological material:

[0045] P1. Products for the production, preparation or development of derivatives of hopane-type sesquiterpenes or Syringa pinnatifolia sesquiterpenes or Aquilaria sinensis sesquiterpenes;

[0046] P2. Products for production, preparation or development;

[0047] P3. Products for the production, preparation or development of zerumbone derivatives of Syringa pinnatifolia

[0048] In the above products, the derivative of the hopane-type sesquiterpene and / or the Syringa pinnatifolia sesquiterpene and / or the Aquilaria sinensis sesquiterpene may be a zerumbone derivative. The zerumbone derivative may be a zerumbone epoxide. The zerumbone epoxide may be zerumbone epoxide. The zerumbone epoxide is a compound with the chemical structural formula of Formula 2:

[0049]

[0050] The present invention provides a Syringa pinnatifolia zerumbone epoxidase gene CYP76S105. The present invention fills the research gap in the modification of zerumbone of Syringa pinnatifolia, provides a basis for the in vitro production of zerumbone epoxide, and has good research and development potential and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the PCR amplification electrophoresis diagram of the Syringa pinnatifolia zerumbone epoxidase gene CYP76S105, where M represents the marker and 1 is the PCR amplification electrophoresis diagram of CYP76S105.

[0052] Figure 2 Schematic diagram of the recombinant yeast expression vector for zerumbone epoxidase gene CYP76S105 of Syringa pinnatifolia

[0053] Figure 3 Ion current chromatograms of the product of zerumbone catalyzed by zerumbone epoxidase CYP76S105 protein of Syringa pinnatifolia, substrate FPP, and the standard zerumbone epoxide. The upper chromatogram is the total ion current chromatogram of the product of zerumbone catalyzed by CYP76S105 protein; the middle chromatogram is the ion current chromatogram of the standard zerumbone epoxide; the lower chromatogram is the ion current chromatogram of the substrate FPP; the vertical axis is relative abundance, and the horizontal axis is time (min).

[0054] Figure 4 Mass spectra of the product catalyzed by zerumbone epoxidase CYP76S105 of Syringa pinnatifolia and the mass spectrum of the standard zerumbone epoxide. The left spectrum is the mass spectrum of the product catalyzed by CYP76S105; the right spectrum is the spectrum of the standard zerumbone epoxide. The vertical axis is relative abundance, and the horizontal axis is mass-to-charge ratio (m / z).

[0055] Figure 5 Schematic diagram of the conversion of zerumbone to zerumbone epoxide catalyzed by CYP76S105 of Syringa pinnatifolia Detailed implementation manners

[0056] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0057] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0058] In the embodiments of the present invention, NADPH is sourced from: Solarbio Science & Technology Co., Ltd., Beijing, N8100 - 1005 mg;

[0059] glucose-6-phosphate is sourced from: Solarbio Science & Technology Co., Ltd., Beijing, D8090 - 1 g;

[0060] Glucose-6-phosphate dehydrogenase is sourced from Beijing Solarbio Science & Technology Co., Ltd., G8020-1000U;

[0061] Flavin adenine dinucleotide (FAD) is sourced from Sigma-Aldrich, F6625-25MG;

[0062] Riboflavin 5′-monophosphate (FMN) is sourced from Sigma-Aldrich, F2253-25MG;

[0063] Zerumbone, with CAS number 471-05-6, is sourced from Shanghai Yuanye Bio-Technology Co., Ltd., B29167-10mg.

[0064] In the examples of the present invention: Galactose is sourced from Shanghai Macklin Biochemical Co., Ltd., product number D810318-500g; Yeast extract powder is a product of Thermo Scientific, product number LP0021B; Peptone is from Shanghai Shengong Biological Engineering Co., Ltd., product number A505247-0500.

[0065] The CAS number of the zerumbone epoxide standard in the examples of the present invention is 22471-70-1, and it is prepared according to the method in the following literature. The public can obtain it from the applicant and it is only used to repeat the present invention. Relevant literature: Zhang R, Feng X, Su G, Mu Z, Zhang H, Zhao Y, Jiao S, Cao L, Chen S, Tu P, Chai X. Bioactive Sesquiterpenoids from the Peeled Stems of Syringa pinnatifolia. J Nat Prod. 2018 Aug 24;81(8):1711-1720.

[0066] The expression strain WAT11 Saccharomyces cerevisiae competent cells used in the examples of the present invention are products of Beijing Coolaber Technology Co., Ltd., product number CC311.

[0067] Example 1: Full-length cloning of the zerumbone epoxidase gene CYP76S105 from Syringa pinnatifolia

[0068] 1. Extraction of RNA from Syringa pinnatifolia

[0069] Add 1 mL of Trizol reagent into a 2 mL centrifuge tube. Then, grind the material sample into fine powder under liquid nitrogen and quickly add it to the centrifuge tube containing Trizol reagent. Vortex to mix well. Centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to another centrifuge tube. Add chloroform approximately 1 / 5 of the volume of the supernatant into the centrifuge tube, shake vigorously for 15 s, let it stand at room temperature for 2 - 3 min, and then centrifuge at 12,000 rpm for 15 min. Pipette the upper aqueous phase into another centrifuge tube, add isopropanol half of the volume of the aqueous phase into this centrifuge tube, invert to mix well and let it stand at room temperature for 10 min, and then centrifuge at 12,000 rpm for 10 min. After centrifugation, discard the supernatant. A small amount of white precipitate can be seen at the bottom of the tube. Wash the inner lid, tube wall and precipitate in the centrifuge tube with 75% ethanol and centrifuge at 12,000 rpm for 5 min, discard the supernatant. Then, change the 75% ethanol in the previous step to absolute ethanol and repeat once. Finally, dry the absolute ethanol to prevent interference with the next reaction, and detect the RNA integrity by 1% agarose gel electrophoresis.

[0070] 2. cDNA Reverse Transcription of *Syringa pinnatifolia*

[0071] Use a pipette to aspirate 10 μL of the RNA obtained in step 1 and add 2 μL of Oligo(dT) 18 , centrifuge and mix well, then react at 70 °C for 10 min, and then ice-bath for 2 min. Subsequently, add 4 μL of 5×M-MLV reverse transcriptase buffer, 1 μL of recombinant RNase inhibitor, 2 μL of dNTP, 1 μL of M-MLV reverse transcriptase (TAKARA, 200 U / μL) to the solution obtained in the previous step, react at 42 °C for 60 min, maintain at 70 °C for 15 min, and finally store the obtained cDNA at -20 °C in the refrigerator.

[0072] 3. Cloning of the Full-Length Sequence of *Syringa pinnatifolia* CYP76S105

[0073] The primers used for the full-length cloning of CYP76S105 are:

[0074] Forward primer: 5’-ATGGATTCCCAATTGTTCCTGC-3’;

[0075] Reverse primer: 5’-TCACAATTTGATTGGAAAAGCCTTGAG-3’.

[0076] The PCR reaction system used for the full-length cloning of CYP76S105 is:

[0077]

[0078] The PCR reaction program used for the full-length cloning of CYP76S105 is:

[0079]

[0080] Agarose gel electrophoresis:

[0081] Dilute the agarose gel powder to 100 ml with 1× concentration of TAE buffer at a concentration of 1.5%, and dissolve it by heating in a microwave oven. Add a drop of Ethidium bromide (EB) in a fume hood, shake well, and pour it into the gel plate with a comb inserted in the fume hood. Wait for it to cool and solidify before use. During electrophoresis, pipette an appropriate amount of PCR product (or other samples for electrophoresis). If it is a non-pre-stained product, add an appropriate concentration of loading buffer. Also use 1× concentration of TAE buffer for electrophoresis. Set the voltage to 200 V and the current to 400 mA. Stop electrophoresis when the bands run to about three-quarters. Observe and take pictures using a gel electrophoresis imager. Recover the target bands by gel extraction to obtain purified products. The gel extraction is performed using the Thermo Scientific GeneJET Gel Extraction Kit.

[0082] 4. Vector ligation and transformation

[0083] Use the pEASY-Blunt Zero Cloning Kit for vector ligation. Take 4 μL of the purified product obtained in step 3, add 1 μL of the pEASY-Blunt Zero Cloning Vector, gently mix, and react at room temperature for 30 min to obtain the ligation product. Then place the centrifuge tube on ice. Thaw the Trans1-T1 competent cells (TransGen Biotech, CD501-02) on ice in advance. The centrifuge tubes used are also placed on ice to cool down. When the competent cells are just thawed, quickly aliquot them at 50 μL per tube, and add the ligation product to the competent cells. Mix well and incubate on ice for 30 min. Then perform heat shock at 42 °C for 30 s and immediately place on ice for 2 min. After that, add 450 μL of liquid LB medium without ampicillin to the product after transformation into cells, and culture it at 200 rpm and 37 °C on a shaker for 1 h. Take it out and centrifuge at 2000 rpm for 4 min. Aspirate 300 μL of the supernatant medium, and mix the remaining and spread it on the LB solid medium containing ampicillin, and culture it overnight at 37 °C.

[0084] 5 Monoclonal culture and verification

[0085] Pick the monoclonal colonies grown on the overnight culture medium and culture them in 1 ml of LB medium containing ampicillin at 200 rpm and 37 °C for 6 h. Take 1 μL of the bacterial solution for PCR verification. The reaction system is as follows:

[0086]

[0087] PCR reaction program for bacterial liquid:

[0088]

[0089] After determining that the bacterial liquid has a target-sized band (about 2000 bp, Figure 1 in lane 1 of the middle electrophoresis), the recombinant plasmid pEASY-CYP76S105 is obtained. Through sequencing analysis, it is found that the recombinant plasmid pEASY-CYP76S105 contains the sequence of the CYP76S105 gene shown in Sequence 1 in the sequence listing, and the amino acid sequence of the CYP76S105 protein is shown in Sequence 2 in the sequence listing.

[0090] The sequence of Sequence 1 is as follows (5'-3'):

[0091]

[0092] The sequence of Sequence 2 is as follows:

[0093] MDSQLFLLVLFSITWACIFFLTSNSKHRKSGKLPPGPYPFPIIGNILQLGKNPHQS

[0094] LAKLSQTYGPLMYLKMGYTETVVVSSPEIAKVVLQKYDQIFSSRTVPVALESVKHG

[0095] KFSMGFLPVDNQWRKLRRIQKEQTFSLQKLEAGQGIRREKLHKLRDYVNQCCVDG

[0096] HAVDIAEATFTTSLNIISSTLFSVDFADYNSDSSQELKDVVRNVMKCIGSPNLADYFP

[0097] LLKYVDPQGISRRTKFYFGKLYAILDSIIDQRLKSRGTLERNDLLEALLDLNQKHEPE

[0098] LSRNEIKHILMDLFLGGTDTSPATVEWAMVELLRNPKKMSKAKSELRDVIGENGVIQ

[0099] ESDISKLPYLQAIVKETHRLHPIAPLLVPHKAVADVEINGYIVPKNAQILVNIWASSRD

[0100] SNIWSSPESFMPERFLDLKTDYNGQDFKFIPFGAGRRICPGLPLAHRVVHVMLATLIH

[0101] NFDWRLEEGLKPEEIDMSEEYTTTMHKAIPLKAFPIKL。

[0102] Example 2: Yeast Eukaryotic Expression of the Zingiberene Epoxidase Gene CYP76S105 from Syringa pinnatifolia

[0103] 1. Construction of Yeast Recombinant Expression Vector

[0104] Using the full-length CYP76S105 gene obtained in Example 1 as a template, the NEBcutter 2.0 online software was used to analyze and select restriction enzyme sites that exist in the yeast expression vector pESC-His but not in the CYP76S105 gene. Primers with restriction enzyme ligation sites (BamHI and SalI) were designed, and linkers with restriction enzyme sites were added to both ends of the full-length CYP76S105 gene sequence. The primers with restriction enzyme sites (BamHI and SalI) designed for CYP76S105 are as follows.

[0105]

[0106] The PCR reaction system for adding linkers to CYP76S105 is as follows:

[0107]

[0108] The PCR reaction program is as follows:

[0109]

[0110] The obtained PCR product was subjected to gel recovery using the GeneJET Gel Extraction Kit (Thermo Scientific, USA) to obtain a purified product.

[0111] For vector linearization, the blank pESC-His vector (preserved in this laboratory, related literature: Guo J, Zhou YJ, Hillwig ML, Shen Y, Yang L, Wang Y, Zhang X, Liu W, Peters RJ, Chen X, Zhao ZK, Huang L. CYP76AH1 catalyzes turnover of miltiradiene intanshinones biosynthesis and enables heterologous production of ferruginol in yeasts. Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):12108-13. doi:10.1073 / pnas.1218061110. Epub 2013 Jun 28. Available to the public from the applicant for the sole purpose of reproducing this invention) was digested with the restriction enzymes BamHI and SalI to obtain a linearized empty vector; at the same time, the purified product was digested with the restriction enzymes BamHI and SalI to obtain a double-digested product of the target fragment. The digestion conditions were both digestion at 37°C for 3 h, and the digestion system is as follows:

[0112]

[0113] Use -According to the instruction manual of the -Basic Seamless Cloning and Assembly Kit (TransGen Biotech Co., Ltd., Beijing), the double-digested products of the linearized vector and the target fragment were used for the ligation of the inserted gene and the vector to obtain the ligation product. The ligation product was transformed into Escherichia coli Trans1-T1 competent cells. After ice-bathing for 30 min, it was heat-shocked in a 42 °C water bath for 30 s, quickly transferred to the ice bath and kept for 2 min, and then added with LB liquid medium and cultured on a shaker at 37 °C and 200 rpm for 1 h. 100 μL of the product was spread on an LB solid medium containing 100 mg / L ampicillin and cultured overnight at 37 °C. Then, single colonies were picked and cultured to obtain the recombinant yeast expression vector plasmid pESC-His-CYP76S105. Through sequencing analysis, it was found that the recombinant expression vector pESC-His-CYP76S105 contained the sequence of the CYP76S105 gene shown in Sequence 1 in the Sequence Listing and could express the CYP76S105 protein shown in Sequence 2 in the Sequence Listing. After confirming that the sequence was correct, the pESC-His-CYP76S105 plasmid was extracted and its concentration was measured. The schematic diagram of the pESC-His-CYP76S105 recombinant vector is as Figure 2 shown.

[0114] 2. Yeast transformation and induction of pESC-His-CYP76S105

[0115] According to the instruction manual of the Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA), the pESC-His-CYP76S105 vector constructed in Step 1 above was transformed into the WAT11 yeast strain, and single colonies successfully transformed were screened using an SD-His defective medium (Beijing PanNuoJi Technology Co., Ltd.). The obtained colonies were expanded and cultured to 50 mL in a liquid SD-His defective medium (Beijing PanNuoJi Technology Co., Ltd.) on a shaker at 30 °C and 200 rpm. When the OD 600nm reached about 1, the medium was removed by low-speed centrifugation, and the medium was converted into an induction medium in which glucose in the medium was replaced with galactose at the same concentration (2% galactose, 1% yeast extract powder, 2% peptone, 0.8% agar powder, and the rest was water), and cultured overnight on a shaker at 30 °C and 200 rpm to obtain the culture solution of the induced recombinant yeast (WAT11 / pESC-His-CYP76S105).

[0116] 3. Preparation of pESC-His-CYP76S105 yeast microsomes

[0117] 3.1 Preparation of the buffer solutions required for the microsome preparation steps is as follows:

[0118] TE buffer: 50 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0)

[0119] TEK buffer: TE buffer containing 0.1 M potassium chloride

[0120] TESB buffer: TE buffer containing 0.6 M sorbitol

[0121] TEG: TE buffer containing 20% glycerol

[0122] 3.2 Preparation of pESC-His-CYP76S105 yeast microsomes

[0123] Take the induced recombinant yeast (WAT11 / pESC-His-CYP76S105) culture solution obtained in step 3.1, centrifuge at 4000 rpm at low temperature, discard the supernatant, add 5 mL of TEK buffer to resuspend, let stand at room temperature for 5 min, centrifuge at 4000 rpm at low temperature for 10 min, and discard the supernatant. Add TESB buffer to resuspend the cell pellet, disrupt by high-pressure freezing 3 times, and centrifuge at 12000 rpm at low temperature for 20 min. Take the supernatant, add PEG4000 with a final concentration of 10%, dissolve on ice bath, centrifuge at 12000 rpm at low temperature for 20 min, and then discard the supernatant. Dissolve with TE buffer to obtain the WAT11 / pESC-His-CYP76S105 recombinant yeast microsome solution. The WAT11 / pESC-His-CYP76S105 recombinant yeast microsome solution contains CYP76S105 protein (the amino acid sequence is sequence 2 in the sequence listing).

[0124] Example 3. Functional verification of the curcumin epoxidase gene CYP76S105 of Syringa pinnatifolia

[0125] 1. In vitro catalytic reaction

[0126] The catalytic system (1 mL) contains the following components: 50 mM Tris-HCl (pH 7.5), 1 mM nicotinamide adenine dinucleotide phosphate (NADPH), 5 mM glucose-6-phosphate, 1 active unit of glucose-6-phosphate dehydrogenase, 10 μM flavin adenine dinucleotide (FAD), 10 μM Riboflavin 5′-monophosphate (FMN), 0.5 mg of the recombinant yeast microsome solution (containing CYP76S105 protein) obtained in Example 2, and 100 μM of the zerumbone substrate. Incubate and catalyze at 30 °C on a shaker at 200 rpm for 3 h, extract with n-hexane, and detect the product by GC / MS.

[0127] 2. Detection of catalytic products

[0128] The GC / MS system used includes Thermo Fisher TRACE 1310 GC (TG-5MS chromatographic column) and TSQ8000 Triple Quadrupole MS. The reaction conditions are as follows: initially hold at 100 °C for 3.5 min; heat to 170 °C at a rate of 20 °C / min; heat to 230 °C at a rate of 5 °C / min; heat to 300 °C at a rate of 30 °C / min and hold for 3 min. The inlet temperature is 250 °C, the split flow rate is 30 mL / min, and the carrier gas flow rate is 1 mL / min. The transfer line and ion source temperatures are 300 °C and 260 °C respectively, and the EI mode is used.

[0129] The results of the catalytic detection show that the total ion current chromatogram results ( Figure 3 ) and the mass spectrometry chromatogram results ( Figure 4 ) both show that CYP76S105 has the function of catalyzing zerumbone to generate zerumbone epoxide ( Figure 5 ). In the present invention, CYP76S105 derived from Syringa pinnatifolia has the function of catalyzing the synthesis of zerumbone epoxide from zerumbone, and can be applied to the biosynthesis of sesquiterpenoids of Syringa pinnatifolia or Aquilaria sinensis.

[0130] The chemical structural formula of zerumbone is as Formula 1 below:

[0131]

[0132] The chemical structural formula of zerumbone epoxide is as follows in Formula 2:

[0133]

[0134] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application is intended to cover any variations, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. A protein, characterized in that: The protein is a protein of any one of the following A1), A2) or A3): A1) A protein with an amino acid sequence being the sequence 2 in the sequence listing; A2) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of the protein shown in A1); A3) A protein which is derived from A1) or A2) or has an identity of more than 80% with the protein shown in A1) or A2), and is obtained by substitution and / or deletion and / or addition of one or several amino acid residues to the amino acid sequence shown in the sequence listing 2 and has the same function.

2. The protein according to claim 1, wherein: The protein is derived from *Syringa pinnatifolia* Hemsl.

3. Any one of the following applications of the protein described in claim 1: M1) The application of the protein in catalyzing the formation of hopane-type sesquiterpenoid derivatives from hopane-type sesquiterpenoids; M2) The application of the protein in the production, preparation or development of products containing hopane-type sesquiterpenoid derivatives; M3) The application of the protein in the production, preparation or development of products related to *Syringa pinnatifolia* Hemsl. sesquiterpenoids; M4) The application of the protein in the production, preparation or development of products related to *Aquilaria sinensis* (Lour.) Spreng. sesquiterpenoids M5) The application of the protein in the production, preparation or development of products related to zerumbone derivatives of *Syringa pinnatifolia* Hemsl.

4. The application according to claim 3, wherein: The hopane-type sesquiterpenoid or the *Syringa pinnatifolia* Hemsl. sesquiterpenoid or the *Aquilaria sinensis* (Lour.) Spreng. sesquiterpenoid is zerumbone; the derivative of the hopane-type sesquiterpenoid compound and / or the *Syringa pinnatifolia* Hemsl. sesquiterpenoid and / or the *Aquilaria sinensis* (Lour.) Spreng. sesquiterpenoid is a zerumbone derivative; the zerumbone derivative is a zerumbone epoxide, the zerumbone epoxide is a compound with the chemical structural formula of formula 2:

5. A biomaterial related to the protein according to claim 1 or 2, characterized in that: The biological material is any one of the following D1) to D6): D1) A nucleic acid molecule encoding the protein described in claim 1; D2) An expression cassette containing the nucleic acid molecule described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A nucleic acid molecule that promotes or enhances the expression of the protein described in claim 1; D6) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in D5).

6. The biomaterial according to claim 5, wherein: The nucleic acid molecule described in D1) is the coding gene of the protein shown in the following d1) or d2): d1) A DNA molecule with a coding sequence being the nucleotide of sequence 1 in the sequence listing; d2) A DNA molecule that hybridizes with the cDNA or DNA molecule defined in d1) and encodes a protein with the same function.

7. Any one of the following applications of the biological material described in claim 5 or 6: N1) The application of the biological material in catalyzing the formation of hopane-type sesquiterpenoid derivatives from hopane-type sesquiterpenoids; N2) The application of the biological material in the production, preparation or development of products containing hopane-type sesquiterpenoid derivatives; N3) The application of the biological material in the production, preparation or development of products related to *Syringa pinnatifolia* Hemsl. sesquiterpenoids; Use of the biological material in the production, preparation or development of products related to Aquilaria sinensis sesquiterpenes Use of the biological material in the production, preparation or development of products related to zerumbone derivatives of Syringa pinnatifolia 8. The application according to claim 7, wherein: The hopane-type sesquiterpene or the Syringa pinnatifolia sesquiterpene or the Aquilaria sinensis sesquiterpene is zerumbone; the derivative of the hopane-type sesquiterpene and / or the Syringa pinnatifolia sesquiterpene and / or the Aquilaria sinensis sesquiterpene is a zerumbone derivative; the zerumbone derivative is a zerumbone epoxide, the zerumbone epoxide is zerumbone epoxide, and the zerumbone epoxide is a compound with the chemical structural formula of Formula 2:

9. Any of the following products containing the protein according to claim 1 or 2 and / or the biological material according to claim 5 or 6: P1. Products for the production, preparation or development of derivatives of hopane-type sesquiterpenes or Syringa pinnatifolia sesquiterpenes or Aquilaria sinensis sesquiterpenes; P2. Products for production, preparation or development; P3. Products for the production, preparation or development of zerumbone derivatives of Syringa pinnatifolia 10. The product according to claim 9, characterized in that: The derivative of the hopane-type sesquiterpene and / or the Syringa pinnatifolia sesquiterpene and / or the Aquilaria sinensis sesquiterpene is a zerumbone derivative; the zerumbone derivative is a zerumbone epoxide, the zerumbone epoxide is zerumbone epoxide, and the zerumbone epoxide is a compound with the chemical structural formula of Formula 2:

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