Protein derived from olea europaea and related biological materials and application thereof
By expressing the epoxygenase gene CYP76S63 of olive olive olive olive olive epoxidation in vitro, the research gap in olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive olive derivatives are promoted, and the application potential is important.
Patent Information
- Application Number
- CN202410114709.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-26
- Publication Date
- 2025-07-29
AI Technical Summary
There is a gap in research on gingerone modification enzymes in oil olives in the prior art, and the lack of methods to catalyze hopane-type sesquiterpenes and gingerone derivatives, which limits their application in the development of fragrances, pesticides and drugs.
The olive olive gingerone epoxy enzyme gene CYP76S63 was cloned and expressed. The protein that catalyzes the epoxygenation of gingerone epoxide was obtained by recombinant expression in yeast. The in vitro biosynthesis of gingerone was achieved using DNA recombination technology and yeast expression vector.
The successful catalyzing of ginger ketone epoxides in the formation of ginger ketone epoxides in the form of ginger ketone derivatives has provided a basis for the in vitro biosynthesis of ginger ketone derivatives, filled the gap in the research on P450 modification in olives, and has broad application prospects.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to proteins derived from Olea europaea, as well as related biological materials and applications thereof. Background Art
[0002] Olea europaea Linn. is a plant of the genus Olea in the family Oleaceae, and is distributed in subtropical regions around the world. People in the Mediterranean region use extracts of Olea europaea leaves to treat diseases such as fever and malaria. Modern pharmacological studies also show that the volatile oil of Olea europaea leaves contains rich terpene components and has pharmacological activities such as antioxidant, antibacterial, and inhibiting the proliferation of tumor cells. However, there are no reports on the biosynthesis of terpene components in Olea europaea.
[0003] Zingiberenone is a humulane-type sesquiterpenoid compound with activities such as anti-myocardial ischemia and anti-cancer. Cytochrome P450 is a class of monooxygenases encoded by a supergene family. Zingiberenone can be modified by P450 in plants to produce various zingiberenone derivatives, which have important values in the fields of fragrance, pesticide, and drug development. However, there are no reports on whether there is a P450 enzyme in Olea europaea that can modify zingiberenone. Summary of the Invention
[0004] The technical problem to be solved by the present invention is how to obtain derivatives of humulane-type sesquiterpenoids and / or how to obtain epoxides of humulane-type sesquiterpenoids and / or how to obtain zingiberenone derivatives and / or how to obtain zingiberenone epoxides.
[0005] To solve the above technical problem, the present invention first provides any one of the following applications of a protein:
[0006] M1) The application of the protein in catalyzing the formation of derivatives of humulane-type sesquiterpenoids from humulane-type sesquiterpenoids;
[0007] M2) The application of the protein in the production, preparation or development of products containing humulane-type sesquiterpenoid derivatives;
[0008] M3) The application of the protein in the production, preparation or development of products related to zingiberenone derivatives, wherein the zingiberenone derivative is zingiberenone epoxide;
[0009] The protein can be any one of the following proteins A1), A2) or A3):
[0010] A1) A protein with an amino acid sequence of Sequence 2 in the sequence listing;
[0011] A2) A fusion protein obtained by fusing a protein tag at the carboxyl terminus and / or amino terminus of the protein shown in A1);
[0012] A3) A protein which is obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing, has the same function, is derived from A1) or A2), or has an identity of more than 80% with the protein shown in A1) or A2).
[0013] In the above application, the protein may be derived from Olea europaea Linn.
[0014] The above protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression.
[0015] In the above protein, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein by using DNA in vitro 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.
[0016] In the above protein, identity refers to the identity of amino acid sequences. The identity of amino acid sequences 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, 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.
[0017] In the above 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.
[0018] In the above application, the hopane-type sesquiterpene may be zerumbone. The derivative of the hopane-type sesquiterpene may be a zerumbone derivative. The zerumbone derivative may be zerumbone epoxide. The zerumbone epoxide is a compound with the chemical structural formula shown in Formula 1:
[0019]
[0020] The protein described above also falls within the scope of protection of the present invention.
[0021] To solve the above technical problems, the present invention also provides biological materials related to the protein described above, and the biological materials can be any one of the following D1) to D6):
[0022] D1) A nucleic acid molecule encoding the protein described above;
[0023] D2) An expression cassette containing the nucleic acid molecule described in D1);
[0024] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);
[0025] 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);
[0026] D5) A nucleic acid molecule that promotes or enhances the expression of the protein described above;
[0027] D6) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in D5).
[0028] Among the above biological materials, the nucleic acid molecule described in D1) can be the coding gene of the protein shown in the following d1) or d2):
[0029] d1) A DNA molecule whose coding sequence is the nucleotide of Sequence 1 in the sequence listing;
[0030] d2) A DNA molecule that hybridizes with the cDNA or DNA molecule defined in d1) and encodes a protein with the same function.
[0031] To solve the above technical problems, the present invention also provides any one of the following applications of the biological materials described above:
[0032] N1) The application of the biological material in catalyzing the formation of hopane - type sesquiterpene derivatives from hopane - type sesquiterpenes;
[0033] N2) The application of the biological material in the production, preparation or development of products containing hopane - type sesquiterpene derivatives;
[0034] N3) The application of the biological material in the production, preparation or development of products related to zerumbone derivatives, and the zerumbone derivative is zerumbone epoxide.
[0035] In the above application, the hopane-type sesquiterpene may be zerumbone. The derivative of the hopane-type sesquiterpene may be a zerumbone derivative. The zerumbone derivative may be zerumbone epoxide. The zerumbone epoxide is a compound with the chemical structural formula of Formula 1:
[0036]
[0037] To solve the above technical problems, the present invention also provides a method for preparing the protein described above, comprising the following steps: expressing the coding gene of the protein described above in a eukaryotic microorganism to obtain the protein.
[0038] In the above method, the expression includes introducing the coding gene of the protein described above into a recipient microorganism to obtain a recombinant microorganism expressing the coding gene, culturing the recombinant microorganism, and expressing to obtain the protein; the eukaryotic microorganism or the recipient microorganism is yeast.
[0039] In the above biological material, the expression cassette containing a nucleic acid molecule in B2) refers to DNA that can express the protein described in the above application in a host cell. This DNA not only includes a promoter that initiates the transcription of the protein-coding gene, but also includes a terminator that terminates the transcription of the protein-coding gene. Further, the expression cassette may further 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.
[0040] The recombinant expression vector containing the protein-coding gene expression cassette can be constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile 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 the 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 nopaline synthase gene Nos of the Agrobacterium tumefaciens Ti plasmid gene and the 3' untranslated regions transcribed by plant genes (such as soybean storage protein genes) have similar functions. When constructing a plant expression vector using the gene of the present invention, 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 have the same reading frame as the coding sequence to ensure the correct translation of the entire sequence.
[0041] Among the above biological materials, the recombinant microorganism can specifically be yeast, bacteria, algae, and fungi.
[0042] The present invention discloses the sequence and function of an olea europaea zerumbone epoxidase gene CYP76S63 and its encoded protein, and finds that olea europaea CYP76S63 has the function of catalyzing the synthesis of zerumbone epoxide from zerumbone. The present invention fills the research gap in the modification of zerumbone by P450 in olea europaea, provides a basis for the in vitro production of zerumbone epoxide, provides a feasible scheme for the in vitro biosynthesis of zerumbone derivatives, and has good research potential and broad application prospects. Description of the Drawings
[0043] Figure 1 Schematic diagram of the yeast recombinant expression vector of the olea europaea zerumbone epoxidase gene CYP76S63.
[0044] Figure 2 Total ion chromatogram of the product of the olea europaea zerumbone epoxidase CYP76S63 protein catalyzing zerumbone and the standard zerumbone epoxide. The upper figure is the total ion chromatogram of the product of the olea europaea zerumbone epoxidase CYP76S63 protein catalyzing zerumbone; the lower figure is the total ion chromatogram of the standard zerumbone epoxide; the ordinate is the relative abundance; the abscissa is the time.
[0045] Figure 3 The mass spectrometry diagram of the catalytic product of oleander zerumbone epoxidase CYP76S63 and the mass spectrometry diagram of the standard zerumbone epoxide. The upper diagram is the spectrum of the standard zerumbone epoxide; the lower diagram is the mass spectrometry diagram of the catalytic product of oleander-derived zerumbone epoxidase CYP76S63 in the present invention; the vertical axis is the relative abundance, and the horizontal axis is the mass-to-charge ratio (m / z). Specific embodiments
[0046] The present invention will be further described in detail below in conjunction with specific embodiments. 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 limit the present invention in any way.
[0047] 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 the field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.
[0048] The sources of the reagents in the embodiments of the present invention are as follows:
[0049] SD-His liquid dropout medium is from Beijing Fannuoji Technology Co., Ltd.;
[0050] SD-His dropout solid medium is from Beijing Fannuoji Technology Co., Ltd.;
[0051] Galactose is from Shanghai Macklin Biochemical Co., Ltd., product number D810318-500g; yeast extract is from Thermo Scientific, product number LP0021B; peptone is from Shanghai Shengong Biological Engineering Co., Ltd., product number A505247-0500;
[0052] Zerumbone, with a CAS number of 471-05-6, is from Shanghai Yuanye Bio-Technology Co., Ltd., B29167-10mg;
[0053] The CAS number of the zerumbone epoxide standard is 22471-70-1. It was prepared according to the method in the following literature and can be obtained from the applicant for the sole purpose of repeating 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.
[0054] NADPH is sourced from: Solarbio Science & Technology Co., Ltd., Beijing;
[0055] glucose-6-phosphate is sourced from: Solarbio Science & Technology Co., Ltd., Beijing,;
[0056] glucose-6-phosphate dehydrogenase is sourced from: Solarbio Science & Technology Co., Ltd., Beijing, G8020-1000U;
[0057] flavin adenine dinucleotide (FAD) is sourced from: Sigma-Aldrich, F6625-25MG;
[0058] Riboflavin 5′-monophosphate (FMN) is sourced from: Sigma-Aldrich, F2253-25MG.
[0059] Example 1: Expression and Functional Analysis of the Olea europaea Zerumbone Epoxidase Gene CYP76S63
[0060] 1. Obtaining the Olea europaea Zerumbone Epoxidase Gene CYP76S63
[0061] Based on the sequence of the zerumbone epoxidase gene in Syringa pinnatifolia, the sequence information of the homologous gene CYP76S63 in the Olea europaea genome was retrieved. The full-length gene sequence is shown as Sequence 1 in the sequence listing, and the corresponding protein sequence is shown as Sequence 2 in the sequence listing.
[0062] The sequence of Sequence 1 is as follows (5’-3’):
[0063]
[0064] The sequence of Sequence 2 is as follows:
[0065] MDFQLFLLVLFSVIWACIFFITSNSKHRKSAKLPPGPYPFPIIGNILQLGENPHQSLAKLSQTYGPLMHLKMGYRETIVVSSPEMAKIVLQKYDQTFSSRTVPVTLESVEHGKFSMGFMPVDNQWRKLRRILKEQMFSLQRLDASQGIRREKLQKLYEYVNQCRVNGHAVDIAEATFTTSLNTISSTLFSVDFADFDSDSSQEFKDVVWNVMKCIGSPNLADYFPVLKYVDPQGISRRTKFYFGKLYAILDSIIDQRLISRGTLEKNDLLEALLDLYQKPEPELSRNEIRHILMDLFFGSTDTVPSTVEWAMAELLRNPKTMSRAKKELSDVIGEHGVIQESDISKLPYLQALVKETHRLHPVAPLLVPHKAEADVEINGYIVPKNARILVNTWATSRDSNIWLSPDSFMPERFLDRKIDFNGQDFKFIPFGAGRRMCPGLPLANRVVHVMLATLIHNFDWKLEEGLKPEEIDMSEKFTTTLHKAIPLKAFPIKL。
[0066] 2. Construction of the CYP76S63 yeast recombinant expression vector
[0067] Through in vitro gene synthesis, a recombinant plasmid containing the CYP76S63 gene shown in Sequence 1 was obtained, and subsequent PCR reactions were carried out using this as a template. The online software NEBcutter 2.0 was used to analyze and select restriction enzyme sites that exist in the yeast expression vector pESC-His but not in the inserted gene (CYP76S63 gene) sequence. Primers with restriction enzyme digestion and ligation sites (BamHI and SalI) were designed, and adapters with restriction enzyme sites were added to both ends of the full-length sequence of the CYP76S63 gene by PCR amplification. The primers for the CYP76S63 gene with restriction enzyme sites are as follows:
[0068]
[0069] The PCR program was set as: 98°C for 10 s, 68°C for 2 min, for 35 cycles. The PCR reaction system for the CYP76S63 gene with added adapters is as follows.
[0070]
[0071] The obtained PCR product was subjected to gel recovery using the GeneJET Gel Extraction Kit (Thermo Scientific, USA) to obtain a purified product.
[0072] For vector linearization, the blank pESC-His vector (stored in our laboratory, related literature: Ma Y, Cui G, Chen T, Ma X, Wang R, Jin B, Yang J, Kang L, Tang J, Lai C, Wang Y, Zhao Y, Shen Y, Zeng W, Peters RJ, Qi X, Guo J, Huang L. Expansion within the CYP71D subfamily drives the heterocyclization of tanshinones synthesis in Salvia miltiorrhiza. Nat Commun. 2021 Jan 29;12(1):685. doi: 10.1038 / s41467-021-20959-1. The public can obtain it from the applicant and is only used to repeat this invention) was double-digested with the restriction endonucleases BamHI and SalI to obtain a linearized empty vector; at the same time, the purified product was double-digested with the restriction endonucleases BamHI and SalI to obtain a double-digested product of the target fragment. The digestion conditions were both 37 °C for 3 h. The digestion system was as follows:
[0073]
[0074]
[0075] Use -Basic Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing) Instruction Manual. 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 competent cells Trans1-T1 (TransGen Biotech, CD501-02). 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. Subsequently, LB liquid medium was added 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. Single colonies were picked and cultured to obtain the recombinant yeast expression vector plasmid pESC-His-CYP76S63. Through sequencing analysis, it was found that the recombinant expression vector pESC-His-CYP76S63 contained the sequence of the CYP76S63 gene shown in Sequence 1 in the Sequence Listing and could express the CYP76S63 protein shown in Sequence 2 in the Sequence Listing. After confirming the correct sequence, the pESC-His-CYP76S63 plasmid was extracted and its concentration was measured. The schematic diagram of the pESC-His-CYP76S63 recombinant vector is as shown in Figure 1 shown.
[0076] 2. Obtaining, Transformation and Induction of Recombinant Yeast
[0077] According to the instruction manual of Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA), the pESC-His-CYP76S63 vector obtained in Step 1 above was transformed into the WAT11 yeast strain (product of Beijing Coolaber Technology Co., Ltd., catalog number CC311), and SD-His defective medium was used to screen the successfully transformed single colonies. The obtained colonies were expanded in a liquid SD-His defective medium on a shaker at 30 °C and 200 rpm to 50 mL. When the OD 600nm reached about 1, the medium was removed by low-speed centrifugation, and the medium was converted to 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-CYP76S63).
[0078] 3. Preparation of Microsomes from WAT11 / pESC-His-CYP76S63 Recombinant Yeast
[0079] 3.1 Prepare the following buffers required for the microsome preparation step:
[0080] TE buffer: 50 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0);
[0081] TEK buffer: TE buffer containing 0.1 M potassium chloride;
[0082] TESB buffer: TE buffer containing 0.6 M sorbitol;
[0083] TEG: TE buffer containing 20% glycerol;
[0084] 3.2 Preparation of pESC-His-CYP76S63 yeast microsomes
[0085] Take the culture solution of the induced recombinant yeast (WAT11 / pESC-His-CYP76S63) obtained in step 3.1, centrifuge at 4000 rpm at low temperature, discard the supernatant, add 5 mL of TEK buffer to resuspend, place at room temperature for 5 min, centrifuge at 4000 rpm at low temperature for 10 min, and discard the supernatant. Add TESB buffer to the bacterial cells to resuspend, and break them by freezing and high pressure for 3 times, then centrifuge at 12000 rpm at low temperature for 20 min. Take the supernatant, add PEG4000 with a final concentration of 10%, dissolve it in an ice bath, centrifuge at 12000 rpm at low temperature for 20 min, and then discard the supernatant. Dissolve it with TE buffer to obtain the WAT11 / pESC-His-CYP76S6 recombinant yeast microsome solution. The WAT11 / pESC-His-CYP76S63 recombinant yeast microsome solution contains CYP76S63 protein (the amino acid sequence is sequence 2 in the sequence listing).
[0086] 4. Functional verification of the oleander zerumbone epoxidase gene CYP76S63
[0087] 4.1 In vitro catalytic reaction of CYP76S63
[0088] The catalytic system (1 mL) contains the following components: 50 mM Tris-HCl (pH 7.5), 1 mM NADPH, 5 mM glucose-6-phosphate, 1 active unit of glucose-6-phosphate dehydrogenase, 10 μM FAD, 10 μM FMN, 0.5 mg of the recombinant yeast microsome solution obtained in step 3 (containing CYP76S63 protein), and 100 μM 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.
[0089] 4.2 Detection of catalytic products
[0090] The GC / MS system used included a Thermo Fisher TRACE 1310 GC (TG-5MS chromatographic column) and a TSQ 8000 Triple Quadrupole MS. The reaction conditions were as follows: initially maintained at 100 °C for 3.5 min; heated to 170 °C at a rate of 20 °C / min; heated to 230 °C at a rate of 5 °C / min; heated to 300 °C at a rate of 30 °C / min and maintained for 3 min. The inlet temperature was 250 °C, the split flow rate was 30 mL / min, and the carrier gas (N2) flow rate was 1 mL / min. The transfer line and ion source temperatures were 300 °C and 260 °C respectively, and the EI mode was used.
[0091] The preparation of microsomes using the above method and the catalytic detection results showed that the total ion current chromatogram results ( Figure 2 ) and the mass spectrometry chromatogram results ( Figure 3 ) both showed that CYP76S63 had the effect of catalyzing zerumbone to generate zerumbone epoxide. Therefore, CYP76S63 derived from Olea europaea in the present invention has the function of catalyzing the synthesis of zerumbone epoxide from zerumbone, is an epoxidase, and can be applied to the in vitro biosynthesis of zerumbone derivatives. The chemical structural formula of the zerumbone derivative zerumbone epoxide is shown in Formula 1:
[0092]
[0093] The above has detailed the present invention. 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 intends to cover any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.
Claims
1. Use of any one of the following proteins: M1) Use of the protein in catalyzing the production of hopane - type sesquiterpene derivatives from hopane - type sesquiterpenes; M2) Use of the protein in the production, preparation or development of products containing hopane - type sesquiterpene derivatives; M3) Use of the protein in the production, preparation or development of products related to zerumbone derivatives, where the zerumbone derivative is zerumbone epoxide; The protein is one of the following A1), A2) or A3): A1) A protein with an amino acid sequence as shown in Sequence 2 in the Sequence Listing; A2) A fusion protein obtained by fusing a protein tag to the carboxyl - terminal and / or amino - terminal of the protein shown in A1); 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).
2. The application according to claim 1, characterized in that: The protein is derived from Olea europaea.
3. The application according to claim 1 or 2, characterized in that: The hopane - type sesquiterpene is zerumbone; the hopane - type sesquiterpene derivative is a zerumbone derivative; the zerumbone derivative is zerumbone epoxide, and the zerumbone epoxide is a compound with the chemical structural formula shown in Formula 1:
4. The protein according to any one of claims 1 - 3.
5. A biomaterial related to the protein according to claim 4, characterized in that: The biological material is any one of the following D1) to D6): D1) A nucleic acid molecule encoding the protein according to claim 4; 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 according to claim 4; 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 as the nucleotide shown in 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. Use of any one of the following biological materials according to claim 5 or 6: N1) Use of the biological material in catalyzing the production of hopane - type sesquiterpene derivatives from hopane - type sesquiterpenes; N2) Use of the biological material in the production, preparation or development of products containing hopane - type sesquiterpene derivatives; N3) Use of the biological material in the production, preparation or development of products related to zerumbone derivatives, where the zerumbone derivative is zerumbone epoxide.
8. The application according to claim 7, characterized in that: The hopane - type sesquiterpenoid is zerumbone; the derivative of the hopane - type sesquiterpenoid is a zerumbone derivative; the zerumbone derivative is zerumbone epoxide, and the zerumbone epoxide is a compound with the chemical structural formula of Formula 1:
9. A method for preparing the protein according to claim 1, comprising the following steps: expressing the coding gene of the protein according to claim 1 in a eukaryotic microorganism to obtain the protein.
10. The method according to claim 9, characterized in that: The expression includes introducing the coding gene of the protein according to claim 1 into a recipient microorganism to obtain a recombinant microorganism expressing the coding gene, culturing the recombinant microorganism, and expressing to obtain the protein; the eukaryotic microorganism or the recipient microorganism is yeast.