Zingiberone epoxidase protein in ligustrum sinense as well as biological material and application of zingiberone epoxidase protein

Abstract

The invention discloses zingiberone epoxidase protein in ligustrum sinense as well as a biological material and application of the zingiberone epoxidase protein. The amino acid sequence of the zingiberone epoxidase protein CYP76S110 to be protected in the ligustrum sinense is 2 in a sequence table, and the coding nucleotide sequence of the zingiberone epoxidase protein CYP76S110 is 1 in the sequence table. According to the embodiment of the invention, after the coding gene of the CYP76S110 protein is introduced into yeast for expression, an obtained recombinant yeast microsome solution containing the CYP76S110 protein can be used for catalyzing zingiberone to synthesize zingiberone epoxide (zingiberone epoxide), and the recombinant yeast microsome solution can be used for catalyzing zingiberone to synthesize zingiberone epoxide (zingiberone epoxide). The method can be applied to in-vitro biosynthesis of the zingiberone derivative.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological genetic engineering, and particularly relates to zerumbone epoxidase protein in Ligustrum sinense Lour., its biological materials and applications. Background Art

[0002] Ligustrum sinense Lour. is a plant of the genus Ligustrum in the family Oleaceae, mainly used for garden ornamentation. The bark and leaves of Ligustrum sinense Lour. can be used as medicine, with the efficacy of clearing heat and reducing fire, and can treat toothache, hematemesis, oral ulcers, sore throat, etc. Modern pharmacological studies have shown that the extracts from Ligustrum sinense Lour. can protect the erythrocyte membrane against hemolysis induced by peroxyl free radicals.

[0003] Zerumbone is a sesquiterpene compound of the humulane type, with activities such as anti-myocardial cell hypoxia and anti-oral squamous cell carcinoma. Cytochrome P450 is a class of monooxygenases encoded by a supergene family. Through the modification of P450 in plants, zerumbone can produce various zerumbone derivatives, which have important values in the fields of spices, pesticides and drug development. However, there is no report on whether the P450 enzyme in Ligustrum sinense Lour. can catalyze zerumbone. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to obtain derivatives of sesquiterpene compounds of the humulane type and / or how to obtain epoxides of sesquiterpene compounds of the humulane type and / or how to obtain zerumbone derivatives and / or how to obtain zerumbone epoxides.

[0005] To solve the above technical problem, the present invention first provides a protein, and the protein can be the 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 above-mentioned protein can be derived from Ligustrum sinense Lour.

[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 on 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 zerumbone derivatives, and the zerumbone derivative is zerumbone epoxide.

[0018] In the above applications, the hopane-type sesquiterpene can be zerumbone. The derivatives of hopane-type sesquiterpenes can be zerumbone derivatives. The zerumbone derivative can be zerumbone epoxide, and the zerumbone epoxide can be a compound with the chemical structural formula of Formula 2:

[0019]

[0020] To solve the above technical problems, the present invention also provides biomaterials related to the above-mentioned protein, and the biomaterials can be any one of the following D1) to D6):

[0021] D1) A nucleic acid molecule encoding the above-mentioned protein;

[0022] D2) An expression cassette containing the nucleic acid molecule described in D1);

[0023] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0024] 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);

[0025] D5) A nucleic acid molecule that promotes or enhances the expression of the above-mentioned protein;

[0026] D6) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in D5).

[0027] Among the above-mentioned biomaterials, the nucleic acid molecule described in D1) can be the coding gene of the protein shown in the following d1) or d2):

[0028] d1) A DNA molecule whose coding sequence is the nucleotide of Sequence 1 in the sequence listing;

[0029] d2) A DNA molecule that hybridizes with the cDNA or DNA molecule defined in d1) and encodes a protein with the same function.

[0030] To solve the above technical problems, the present invention also provides any one of the following applications of the above-mentioned biomaterials:

[0031] N1) Application of the biomaterial in catalyzing the formation of hopane-type sesquiterpene derivatives from hopane-type sesquiterpenes;

[0032] N2) Application of the biomaterial in the production, preparation or development of products of hopane-type sesquiterpene derivatives;

[0033] N3) Application of the biomaterial in the production, preparation or development of products related to zerumbone derivatives, and the zerumbone derivative is zerumbone epoxide.

[0034] In the above application, the hopane-type sesquiterpenoid may be zerumbone. The derivative of the hopane-type sesquiterpenoid may be a zerumbone derivative. The zerumbone derivative may be zerumbone epoxide, and the zerumbone epoxide may be a compound with the chemical structural formula of Formula 2:

[0035]

[0036] In the above biological material, the expression cassette containing the nucleic acid molecule described 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 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.

[0037] An existing plant expression vector can be used to construct a recombinant expression vector 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 guide the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the genes of Agrobacterium crown gall-inducing (Ti) plasmids (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 translational enhancers or transcriptional 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.

[0038] In the above biological material, the recombinant microorganism may specifically be yeast, bacteria, algae and fungi.

[0039] To solve the above technical problems, the present invention also provides a method for preparing the protein described above, including the following steps: expressing the coding gene of the protein described above in a microorganism to obtain the protein.

[0040] In the above method, the expression may include 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 the protein. The eukaryotic microorganism or the recipient microorganism may be yeast.

[0041] The present invention provides a zerumbone epoxidase gene CYP76S110 from Ligustrum sinense. The present invention fills the blank in the research of P450 enzymes in Ligustrum sinense, provides a basis for the production of zerumbone epoxide by synthetic biological methods, and has good research and development potential and application prospects.

[0042] The present invention discloses the sequence and function of a zerumbone epoxidase gene CYP76S110 from Ligustrum sinense and its encoded protein, and finds that CYP76S110 of Ligustrum sinense has the function of catalyzing the synthesis of zerumbone epoxide from zerumbone. The present invention fills the blank in the research of P450 modification of zerumbone in Ligustrum sinense, 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. Brief Description of the Drawings

[0043] Figure 1 It is a schematic structural diagram of a yeast recombinant expression vector for the zerumbone epoxidase gene CYP76S110 of Ligustrum sinense.

[0044] Figure 2 It is the total ion chromatogram of the product catalyzed by the zerumbone epoxidase CYP76S110 protein of Ligustrum sinense and the standard zerumbone epoxide. The upper figure is the total ion chromatogram of the product catalyzed by the zerumbone epoxidase CYP76S110 protein; 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 It is the mass spectrum of the product catalyzed by the zerumbone epoxidase CYP76S110 of Ligustrum sinense and the mass spectrum of the standard zerumbone epoxide. The upper figure is the spectrum of the standard zerumbone epoxide; the lower figure is the mass spectrum of the product catalyzed by the zerumbone epoxidase CYP76S110 from Ligustrum sinense in the present invention; the ordinate is the relative abundance, and the abscissa is the mass-to-charge ratio. Detailed 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 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] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0048] The expression strain WAT11 Saccharomyces cerevisiae competent cells used in the embodiments of the present invention are products of Beijing Coolaber Technology Co., Ltd., with the product number CC311.

[0049] The NADPH in the embodiments of the present invention is from: Beijing Solarbio Science & Technology Co., Ltd., N8100-1005mg;

[0050] glucose-6-phosphate is from: Beijing Solarbio Science & Technology Co., Ltd., D8090-1g;

[0051] glucose-6-phosphate dehydrogenase is from: Beijing Solarbio Science & Technology Co., Ltd., G8020-1000U;

[0052] flavin adenine dinucleotide (FAD) is from: Sigma-Aldrich, F6625-25MG;

[0053] Riboflavin 5′-monophosphate (FMN) is from: Sigma-Aldrich, F2253-25MG;

[0054] Zerumbone, with the CAS number 471-05-6, is from: Shanghai Yuanye Bio-Technology Co., Ltd., B29167-10mg.

[0055] In the embodiments of the present invention: galactose is from Shanghai Macklin Biochemical Co., Ltd., with the product number D810318-500g; the yeast extract powder is a product of Thermo Scientific, with the product number LP0021B; peptone is from Shanghai Shengong Biological Engineering Co., Ltd., with the product number A505247-0500.

[0056] The CAS number of the zerumbone epoxide standard in the embodiments of the present invention is 22471-70-1, and it is sourced from Beijing BTRK Biomedicine Technology Co., Ltd.

[0057] Example 1: Yeast Eukaryotic Expression of the Zerumbone Epoxidase Gene CYP76S110 in Ligustrum sinense

[0058] 1. Construction of the Yeast Expression Vector pESC-His-CYP76S110

[0059] Based on the sequence of the zerumbone epoxidase gene in Syringa pinnatifolia, the sequence information of the homologous gene CYP76S110 in the genome of Ligustrum sinense 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. Through in vitro gene synthesis, a recombinant plasmid containing the CYP76S110 gene shown in Sequence 1 was obtained, and it was used as a template for subsequent PCR reactions. The NEBcutter 2.0 online software was used to analyze and select restriction enzyme sites that exist in the yeast expression vector pESC-His (preserved 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 tanshinone synthesis in Salvia miltiorrhiza. Nat Commun. 2021 Jan 29;12(1):685.doi:10.1038 / s41467-021-20959-1) but do not exist in the inserted gene (CYP76S110 gene) sequence. 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 CYP76S110 gene sequence through PCR amplification. The primers with restriction enzyme sites (BamHI and SalI) designed for CYP76S110 are as follows:

[0060]

[0061] The PCR reaction system for adding linkers to CYP76S110 is as follows:

[0062]

[0063] The PCR program is set as follows:

[0064]

[0065] The obtained PCR product was subjected to gel recovery using the GeneJET Gel Extraction Kit (Thermo Scientific, USA) to obtain a purified product.

[0066] For vector linearization, the blank pESC-His vector was double-digested with the restriction endonucleases BamHI and SalI to obtain a linearized empty vector; meanwhile, the purified product was double-digested with the restriction endonucleases BamHI and SalI to obtain the double-digested product of the target fragment. The digestion conditions were both 37 °C for 3 h. The digestion system was as follows:

[0067]

[0068] Using -Basic Seamless Cloning and Assembly Kit (TransGen Biotech Co., Ltd., Beijing) instruction manual, the linearized vector and the double-digested product of the target fragment were ligated to insert the gene and the vector, obtaining a 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 an ice bath and kept for 2 min, and then 100 μL of the product was added to LB liquid medium and cultured on a shaker at 37 °C and 200 rpm for 1 h. 100 μL of the product was taken and spread on an LB solid medium containing 100 mg / L ampicillin, and single colonies were picked and cultured after overnight incubation at 37 °C to obtain the recombinant yeast expression vector plasmid pESC-His-CYP76S110. Through sequencing analysis, it was found that the recombinant expression vector pESC-His-CYP76S110 contained the sequence of the CYP76S110 gene shown in Sequence 1 in the Sequence Listing and could express the CYP76S110 protein shown in Sequence 2 in the Sequence Listing. After confirming the correct sequence, the pESC-His-CYP76S110 plasmid was extracted and its concentration was measured. The schematic diagram of the pESC-His-CYP76S110 recombinant vector is as Figure 1 shown.

[0069] The sequence of Sequence 1 in the Sequence Listing is as follows (5'-3'):

[0070]

[0071] The sequence 2 in the sequence listing is as follows:

[0072] MDSQLFLLVLFSITWACIFFLTSYSKHKKSGKLPPGPYPFPIIGNILQLGKNPYRSLTKLSLTYGPLMYLKIGYRETIVVSSPEVAKVVLRKYDQTFSSRPVPVALESMEHGKFSMAWLPVDNQWRKLRRIQKEQMFSLQRLEAGQGIRREKLHKLRDYLNQCCVDGHAVDIAEATFTTSLNIISSTLFSVDFADYNSNSSQELKDVVWNVMKCIGSPNLADFFPLLKHVDPQGISRRTKFYFGKLYAILDGIIDQRLKSRGTLEKNDLLEALLDLYQKHEPELSRNEIKHILMDLFLGGTDTSPVTVEWAMVELLRNPKKMLKAKSELRDVVGENGVIQESDISKLPYLQAIVKETHRLHPIAPLLVPHKAVADVEINGYIVPKNAQILVNIWASSRDSNIWSSPDSFMPERFLDLKTDYNDQDFKFIPFGAGRRICPGLPLAHRVVHGMLAALIHNFDWRLEEGLRPEEIDMSEEYTTTMHKAIPLKAFPIKL。

[0073] 2. pESC-His-CYP76S110 yeast transformation and induction

[0074] According to the instructions of the Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA), the pESC-His-CYP76S110 vector constructed in step 1 above was transformed into the WAT11 yeast strain, and single clone colonies with successful transformation were screened using SD-His defective solid medium (Beijing FanNuoji Technology Co., Ltd.). The obtained colonies were cultured in a liquid SD-His defective medium (Beijing FanNuoji Technology Co., Ltd.) at 30 °C and 200 rpm on a shaker until the volume reached 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). The culture was induced overnight at 30 °C and 200 rpm on a shaker to obtain the culture solution of the induced recombinant yeast (WAT11 / pESC-His-CYP76S110).

[0075] 3. Preparation of pESC-His-CYP76S110 Yeast Microsomes

[0076] 3.1 Prepare the following buffers required for the microsome preparation steps:

[0077] TE buffer: 50 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0);

[0078] TEK buffer: TE buffer containing 0.1 M potassium chloride;

[0079] TESB buffer: TE buffer containing 0.6 M sorbitol;

[0080] TEG: TE buffer containing 20% glycerol.

[0081] 3.2 Preparation of pESC-His-CYP76S110 Yeast Microsomes

[0082] Take the culture solution of the induced recombinant yeast (WAT11 / pESC-His-CYP76S110) 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 cell pellet to resuspend, break by freezing and high pressure for 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 in an 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-CYP76S110 recombinant yeast microsome solution. The WAT11 / pESC-His-CYP76S110 recombinant yeast microsome solution contains CYP76S110 protein (the amino acid sequence is sequence 2 in the sequence listing).

[0083] Example 2. Functional Verification of the Zerumbone Epoxidase Gene CYP76S110 in Ligustrum sinense

[0084] 1. In Vitro Catalytic Reaction of CYP76S110

[0085] The catalytic system (1 mL) contains the following components: 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 obtained in Example 1 (containing CYP76S110 protein), 100 μM zerumbone substrate, and the rest is 50 mM Tris-HCl (pH 7.5) buffer. 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.

[0086] 2. Detection of catalytic products

[0087] Using zerumbone epoxide as the standard, a GC / MS system including Thermo Fisher TRACE 1310 GC (TG-5MS chromatographic column) and TSQ 8000 Triple Quadrupole MS was used. 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.

[0088] 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 CYP76S110 had the function of catalyzing zerumbone to generate zerumbone epoxide. Therefore, CYP76S110 derived from Ligustrum sinense Lour. 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.

[0089] The chemical structural formula of zerumbone is shown in Formula 1; the chemical structural formula of zerumbone epoxide is shown in Formula 2:

[0090]

[0091] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit 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 modifications, 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. 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 that is 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 obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 of the Sequence Listing, having the same function, derived from A1) or A2), or having an identity of more than 80% with the protein shown in A1) or A2).

2. The protein according to claim 1, characterized in that: The protein is derived from Ligustrum sinense Lour.

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 zerumbone derivatives, where the zerumbone derivative is zerumbone epoxide.

4. The application according to claim 3, wherein: The hopane-type sesquiterpenoid is zerumbone; the hopane-type sesquiterpenoid 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 2:

5. A biological material 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, characterized in that: 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 that is 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 zerumbone derivatives, where the zerumbone derivative is zerumbone epoxide.

8. The application according to claim 7, wherein: The hopane-type sesquiterpenoid is zerumbone; the hopane-type sesquiterpenoid 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 2:

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 microorganism to obtain the protein.

10. The method according to claim 9, wherein: 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.

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