Enzyme for synthesizing calamenene or derivative thereof and application of enzyme

Abstract

The invention provides an enzyme for synthesizing calamenene or a derivative thereof and application of the enzyme, the calamenene synthetase in bacteria is provided for the first time, the amino acid sequence of the calamenene synthetase is shown as SEQ ID NO: 1, the calamenene synthetase can be used for biologically synthesizing the calamenene in vitro or in the bacteria, and compared with the prior art that the calamenene is extracted through chemical synthesis or plants, the yield of the calamenene synthetase is greatly improved. And the method has the advantages of being more economical and environment-friendly.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to an enzyme for synthesizing candecene or its derivatives and application thereof. Background Art

[0002] Terpenoids are one of the most abundant and diverse secondary metabolites in plants, and their basic unit is isoprene (C5H8). Terpenoids have a wide range of uses in practical applications and can be developed into natural flavors, fuels, food additives, therapeutic agents, metabolic regulators, and pesticides. In addition, clinical studies have shown that some terpenoids have pharmacological effects such as neuroprotection, memory enhancement, antibacterial and anti-inflammatory, respiratory function improvement, heart stimulation, and sedative and hypnotic effects.

[0003] Calarene is a biologically active sesquiterpene, usually extracted from plants (such as the stems of Kadsura heteroclite). It is toxic to Meloidogyne incognita, Sitophilus zeamais and mosquitoes, and has attracted attention in the field of plant control. However, the extraction of calarene mainly depends on plant sources, and this process is usually complicated and has a low yield, which restricts the in-depth study of calarene and makes it difficult to fully develop its potential medicinal value and application prospects.

[0004] Compared with chemical synthesis or plant extraction, biosynthesis of sesquiterpenes shows more economical and environmental advantages. However, no relevant synthases that can be used for the biosynthesis of candelene have been reported. Therefore, it is necessary to develop an enzyme that can synthesize candelene or its derivatives. Summary of the invention

[0005] The present invention aims to provide an enzyme for synthesizing cancene or its derivatives and its application, screen out an enzyme for synthesizing cancene or its derivatives, take farnesyl pyrophosphate as substrate, utilize enzyme catalysis to obtain product cancene or its derivatives, avoid complicated chemical synthesis process, and provide a new way for biosynthesizing product cancene or its derivatives.

[0006] In a first aspect of the present invention, an enzyme for synthesizing cancene or its derivatives is provided. The amino acid sequence of the enzyme for synthesizing cancene or its derivatives is shown in SEQ ID NO:1.

[0007] In the second aspect of the present invention, a nucleic acid molecule encoding the enzyme for synthesizing candecene or its derivatives is provided, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0008] In the third aspect of the present invention, an enzyme expression vector for synthesizing candecene or its derivatives is provided, wherein the expression vector comprises a nucleic acid molecule encoding the enzyme.

[0009] Furthermore, the expression vector comprises one of a prokaryotic expression vector and a viral vector.

[0010] As a specific embodiment, the method for preparing the enzyme expression vector for synthesizing candecene or its derivatives comprises:

[0011] Obtaining the target gene fragment of the nucleic acid molecule of the enzyme for synthesizing candecene or its derivatives;

[0012] The target gene fragment and the pET28α expression vector were double-digested with NdeI and NotI, and then ligated to obtain the expression vector pET28α-orf2064.

[0013] In the fourth aspect of the present invention, a recombinant bacterium or an engineered cell line comprising the expression vector is provided.

[0014] The starting strains of the engineered bacteria include but are not limited to all bacteria containing MVA and MEP pathways, and as examples, may be Escherichia coli and Streptomyces.

[0015] In the fifth aspect of the present invention, there is provided an application of the enzyme for synthesizing cancene or its derivatives, or the nucleic acid molecule, or the expression vector, or the recombinant bacteria or engineered cell line in synthesizing cancene or its derivatives.

[0016] In the sixth aspect of the present invention, a catalytic product is provided, comprising at least one of the enzyme for synthesizing candecene or its derivatives, the nucleic acid molecule, the expression vector, the recombinant bacteria or the engineered cell line.

[0017] In a seventh aspect of the present invention, a method for catalyzing farnesyl pyrophosphate to synthesize candelene or its derivatives is provided, the method comprising:

[0018] Using farnesyl pyrophosphate as a substrate, the enzyme for synthesizing cancene or its derivatives is used to catalyze the synthesis of cancene or its derivatives;

[0019] Alternatively, the recombinant bacteria or engineered cell system is used to synthesize the product aconene or its derivatives in vivo.

[0020] Furthermore, the temperature of the catalytic reaction is 25-35°C.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] The present invention provides a catalytic product and application thereof. The present invention screens out an enzyme for synthesizing cancene or its derivatives, and can use farnesyl pyrophosphate as a substrate to obtain the product cancene or its derivatives by utilizing enzyme catalysis, thereby avoiding a complex chemical synthesis process and providing a new approach for the biosynthesis of the product cancene or its derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 Figure 1 is the experimental result diagram of Example 2 of the present invention, wherein Figure A is the SDS-PAGE detection result of the recombinant protein ORF2064 obtained in Example 2; Figure B is the gas chromatography detection result of the experimental group a and the control group bc in Example 2; Figure C is the mass spectrometry detection result of the experimental group a in Example 1; Figure D is the standard GC-MS spectrum of calendula (derived from the National Institute of Standards and Technology Library (NIST08).

[0025] Figure 2 Figure 3 is an experimental result diagram of Example 3 of the present invention, wherein Figure A is the gas chromatography detection result of the fermentation product of the engineered strain based on Escherichia coli BL21 (DE3) in Example 3. Figure B is the mass spectrometry detection result of the fermentation product of the engineered strain based on Escherichia coli BL21 (DE3) in Example 3. Figure C is the gas chromatography detection result of the engineered strain based on Streptomyces avermitilis NRRL8165 in Example 3. Figure D is the mass spectrometry detection result of the engineered strain based on Streptomyces avermitilis NRRL8165 in Example 3. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0029] The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0030] The term “include” or “comprising” described in the present invention is an open description, which contains the specified components or steps described and other specified components or steps that will not be substantially affected.

[0031] An embodiment of the first aspect of the present invention provides use of an enzyme having an amino acid sequence as shown in SEQ ID NO: 1 in synthesizing cancene or its derivatives.

[0032] The present invention proposes for the first time an aconene synthase (ORF2064) in bacteria, the amino acid sequence of which is shown in the following SEQ ID NO: 1.

[0033] MDKNAIGTRIRRVGKAGVRDGVRDPVHEHLRRTQDSLATPFVVRENSAARDVFAASVQWMQEHALLTGERRQWLEKCDIGALLGLTNPQVDRRLLRLAGDWYVWLYAFDDGVCDEA LTGAQAWDMAHLTLRLKRSVTGGAPGDGPEDNYARALSDLRLRIAEHATPAQLVRWTEAVRDYLNGQLWETTYRATGRIPAVDDYITMRESASGCLSCFALLPVLNRYHLPEEVRA HPDVEQLSRSANRIIAWDNDLFSYLKELGDHSAVANLITAVARERRCDIAAAITYARKQRDGELATFLRAEKRVARHLGVEGRYYIADLKNWVSGSLEFHRTSRRFVSAPRVVHL(SEQ ID NO:1)

[0034] The sequence was obtained by amplifying a fragment containing the candelene synthase gene from strain S. exfoliatus UC5319 using primers CK21(347)F / CK21R, sequencing the fragment and translating it into an amino acid sequence.

[0035] The nucleotide sequence encoding the cancerene synthase is shown in SEQ ID NO.2.

[0036]

[0037] CK21(347)F: GGAATTCCATATGGACAAGAACGCGATT (SEQ ID NO: 3);

[0038] CK21R: ATAAGAATGCGGCCGCTCAGAGGTGCACGACGCGAGG (SEQ ID NO: 4);

[0039] The strain Streptomyces exfoliatus UC5319 is recorded in the article "Seo MJ, Zhu D, Endo S, et al. Genome Mining in Streptomyces. Elucidation of the Role of BaeyerVilliger Monooxygenases and Non-Heme Iron-Dependent Dehydrogenase / Oxygenases in the Final Steps of the Biosynthesis of Pentalenolactone and Neopentalenolactone [J]. 2011." The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

[0040] In some embodiments, the use includes: use of an enzyme having an amino acid sequence as shown in SEQ ID NO: 1 to synthesize candecene or its derivatives in vitro or in bacteria.

[0041] The inventors of the present application first proposed that the above-mentioned candecene synthase can be used to synthesize candecene, including but not limited to in vitro incubation or fermentation of engineered strains that overexpress or heterologously express the candecene synthase.

[0042] As an example, in an in vitro reaction, farnesyl diphosphate (FPP) was used as a substrate to form candelene under the catalysis of the enzyme shown in SEQ ID NO: 1.

[0043]

[0044] In some embodiments, the candecene derivatives include but are not limited to candecene oxide.

[0045] It can be understood that the use of the enzyme with the amino acid sequence shown in SEQ ID NO: 1 provided by the present invention to synthesize candecene and further obtain its derivatives or downstream metabolites on this basis is also covered by the protection scope of the present invention.

[0046] As an example, the following shows compound 1: nardoaristol; compound 2: 1(10)-aristolen-9β-ol; compound 3: kanshone C; compound 4: 3-hydroxylkanshone H; compound 5: 3-oxokanshone H; compound 6: kanshone H; compound 7: (-)-aristolone; compound 8: (-)-(14β,15β)-aristolone; compound 9: 1-hydroxylaristolone; compound 10: 1(10)-aristolen-12-al; compound 11: kanshone G (kanshone G); Compound 12: 9β-debilon (9β-aristolochia ketone); Compound 13: debilon (aristolochia ketone); Compound 14: nardostachone (nardostachone); Compound 15: 1(10)-aristolen-2-one (1(10)-aristolan-2-one); Compound 16: aristolanhydride; Compound 17: kanshone F (kanshone F).

[0047]

[0048] In some embodiments, the bacteria include: Escherichia coli and Streptomyces.

[0049] According to a second aspect of the present invention, an embodiment provides an engineered strain, wherein the engineered strain overexpresses or heterologously expresses an enzyme as shown in SEQ ID NO:1.

[0050] In some embodiments, the engineered strain is used to synthesize candecene or its derivatives.

[0051] Strain source

[0052] S. exfoliatus UC5319 is recorded in the article “Seo MJ, Zhu D, Endo S, et al. Genome Mining in Streptomyces. Elucidation of the Role of BaeyerVilliger Monooxygenases and Non-Heme Iron-Dependent Dehydrogenase / Oxygenases in the Final Steps of the Biosynthesis of Pentalenolactone and Neopentalenolactone[J]. 2011.”, which is available to the public from the applicant and can only be used to repeat the experiments of the present invention.

[0053] S. avermitilis NRRL8165 is described in “Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M., and Omura, S. (2003) Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis, Nat Biotechnol 21, 526-531.”, which is available to the public from the applicant and can only be used to repeat the experiments of the present invention.

[0054] The following is a detailed description of an enzyme for synthesizing candelay or its derivatives and its application in combination with examples and experimental data.

[0055] Example 1 Construction of enzyme expression vector and engineering bacteria for synthesizing calendula or its derivatives

[0056] 1. Amplify a fragment containing the candelene synthase gene from strain S. exfoliatus UC5319 using the primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0057] 2. The target gene fragment and pET28α expression vector were placed at 37°C and double digested with NdeI and NotI. The reaction system was as follows:

[0058] orf2064 fragment double enzyme digestion system:

[0059] Table 2

[0060] pET28α double restriction enzyme digestion system:

[0061] Table 3

[0062]

[0063] After double digestion reaction was carried out at 37°C for 3 h using NdeI and NotI restriction endonucleases, Loading Buffer was added to terminate the reaction, and the double digestion product was purified and recovered according to the instructions of the gel recovery kit.

[0064] 3. After being cut by the same restriction endonuclease, the double-digestion products have the same sticky ends and can be connected to a complete plasmid by DNA ligase. The orf2064 gene and pET28α double-digestion products with the same sticky ends are placed in the same PCR tube. The ligation reaction uses a 10μL system, and the target gene and plasmid digestion products are added in a ratio of 3:1, mixed with 1μL T4 DNA ligase, and connected overnight at 16°C. The successfully connected plasmid is named pET28α-orf2064.

[0065] 4. Transform the ligation product into E. coli DH5α competent cells. The transformation steps are as follows:

[0066] a. First adjust the temperature of the constant temperature water bath to 42°C.

[0067] b. Take out a tube (100 μL) of competent bacteria from the -80°C ultra-low temperature freezer, immediately melt it with your fingers, then insert it into ice and place it in an ice bath for 10 minutes.

[0068] c. Add 10 μL of the ligation product pET28α-orf2064, shake gently and place on ice for 20 minutes.

[0069] d. Shake it gently and place it in a 42℃ water bath for 90 seconds of heat shock, then quickly return it to ice and let it stand for 5 minutes.

[0070] e. Add 900 μL of LB medium without antibiotics into each of the above tubes and mix gently. Then fix them on the spring frame of the shaker and shake them at 37 degrees Celsius for 50 minutes.

[0071] f. Add 300 μl of the above transformation mixture taken out from the clean bench to each solid LB plate culture dish containing appropriate antibiotics, and evenly apply it using a glass coating rod that has been burned with an alcohol lamp and cooled.

[0072] g. Place the marked culture dish in a 37°C constant temperature incubator for 30-60 minutes. After the liquid on the surface has penetrated into the culture medium, turn it upside down and place it in a 37°C constant temperature incubator overnight.

[0073] 5. Identify the recombinant plasmid of the grown single colony: Pick the single colony grown on the above LB solid medium, inoculate it into 5 mL of LB medium containing kanamycin, and incubate it at 37°C and 200 r·min. -1 The cells were cultured overnight under the same conditions, and part of the bacterial solution was taken to preserve the strain in a glycerol tube. An appropriate amount of bacterial solution was taken to extract the recombinant plasmid according to the instructions of the plasmid extraction kit. The extracted recombinant plasmid was double-digested with NdeI and NotI, which was the same as the double-digestion system of pET28α. The products after double-digestion were analyzed by 1% agarose gel electrophoresis for the target gene fragment and the vector fragment. After double-digestion identification, the recombinant plasmids that were positive for double-digestion were sent to Shanghai Biotech for sequence determination, and the recombinant expression vector pET28α-orf2064 was successfully obtained.

[0074] Example 2: Synthesis of Acorene in vitro

[0075] 1. Obtaining recombinant protein ORF2064

[0076] The recombinant expression vector pET28α-orf2064 prepared in Example 1 was transferred into Escherichia coli BL21 (DE3) for expression and purification. The SDS-PAGE image of the obtained recombinant protein is shown in Figure 1 A.

[0077] 2. In vitro reaction

[0078] 1 mL of buffer (50 mM PIPES, 15 mM MgSO) containing 1 μM recombinant ORF2064 and 20 μM FPP 4 , 100 mM NaCl, 5 mM β-mercaptoethanol and 20% glycerol (v / v, pH 6.8), covered with 1 mL of n-hexane, reacted at 30°C for 16 hours, and then quenched with 25 μL of EDTA (500 mM), as experimental group a.

[0079] Control groups b and c were set up at the same time. The boiled recombinant protein (boiled ORF2064) was used for the above in vitro reaction as control group b, and standard FPP was used as control group c.

[0080] 3. Product testing

[0081] The reaction system in step 1.2 was extracted and concentrated using n-hexane, and further analyzed by GC-MS. The test results are as follows Figure 1 B and Figure 1 As shown in C, in addition Figure 1D is the standard spectrum of Acorene (derived from the National Institute of Standards and Technology Library (NIST08).

[0082] Detection conditions: GC-MS analysis was performed on an Agilent 7890A / 5975C-GC / MSD under 70 eV electron impact (EI) using an HP5MS capillary column (30 m×0.25 mm) in positive ion mode, with a solvent delay of 3 min and a temperature program of 60°C for 2 min, followed by a temperature gradient of 60–280°C at 20°C / min for 11 min and 280°C for 2 min.

[0083] The samples were analyzed on a Shimadzu SPD-M20A / LC-20AT using a Thermo Scientific C18 reverse phase HPLC column (250×4.6 mm, 5 μm). Mobile phase A: water; mobile phase B: acetonitrile; UV detection λ: 210 nm, elution program as follows: 80% B, flow rate 0.8 mL / min, for 30 minutes.

[0084] 4. Test results:

[0085] like Figure 1 As shown in B, compared with the control groups b and c, the experimental group a showed a peak at 8.517 min. Figure 1 C and Figure 1 D identified the substance as calendula.

[0086] Example 3: Biosynthesis of Acorene in Escherichia coli

[0087] 1. Engineering strain Escherichia coli BL21(DE3) for heterologous expression of ORF2064

[0088] 1. Preparation of recombinant expression vector pET21a-orf2064

[0089] The target gene fragment of the nucleic acid molecule for obtaining the enzyme for synthesizing candecene or its derivatives is shown in SEQ ID NO.2;

[0090] The target gene fragment and the pET21α expression vector were double-digested with NdeI and NotI, and then ligated to obtain the expression vector pET28α-orf2064.

[0091] 2. The recombinant expression vector pET21a-orf2064 was transferred into Escherichia coli BL21 (DE3) to heterologously express ORF2064, thereby obtaining an engineered strain heterologously expressing ORF2064.

[0092] 2. Fermentation of engineered strains

[0093] LB medium, cultured overnight at 37°C. Then 1% (v / v) of the culture was transferred to fresh 2×YT medium and inoculated at 37°C until OD 600 IPTG was added to a final concentration of 0.1 mM and the culture was further incubated at 28 °C for 3-4 hours.

[0094] 3. Experimental Results

[0095] The obtained fermentation broth was extracted and concentrated, and further analyzed by HPLC and GC-MS (the detection conditions were the same as those in Example 2). The detection results were as follows: Figure 2 A and Figure 2 B. It is consistent with the product obtained by the in vitro reaction in Example 2.

[0096] After the fermented cane ene was purified by HPLC (same detection conditions as in Example 2), NMR analysis was performed, and the analysis results were as follows: 13 C NMR (100 MHz, CDCl 3 )δ144.3,120.5,36.9,36.8,33.6,30.0,29.9,27.4,25.9,23.1,21.0,19.7,18.7,16.7,16.2ppm; 1 H NMR (400 MHz, CDCl 3 )δ5.24(m,1H),2.23(m,1H),1.99-1.92(m,3H),1.76(m,1H),1.73(m,1H),1.42(m,2H),1.39 (m,1H),1.07(s,3H),1.02(s,3H),0.97(d,J=7.7Hz,6H),0.74(m,1H),0.56(d,J=9.2Hz,1H).

[0097] NMR analysis conditions: Carbon-13 and proton spectra were recorded on a Bruker AVANCE 400 MHz NMR spectrometer operating at 400 and 100 MHz, respectively. 1 H and 13 C, equipped with a 5 mm BBFO smart probe. All experiments were performed at 303 K in CDCl 3 Chemical shifts are reported in ppm and referenced to the solvent resonance signal (CDCl3: 1 Hδ ppm 7.26, 13 Cδ ppm 77.16).

[0098] Example 4: Biosynthesis of Acorene in Streptomyces

[0099] 1. Engineering strain Streptomyces avermitilis NRRL8165 heterologously expressing ORF2064

[0100] 1. Preparation of recombinant expression vector pIB139-orf2064

[0101] Obtaining the target gene fragment of the nucleic acid molecule of the enzyme for synthesizing candecene or its derivatives;

[0102] The target gene fragment and the pIB139 expression vector were double-digested with NdeI and NotI, and then ligated to obtain the expression vector pIB139-orf2064.

[0103] 2. The recombinant expression vector pIB139-orf2064 was transferred into Streptomyces S. avermitilis NRRL8165 to heterologously express ORF2064, thereby obtaining an engineered strain heterologously expressing ORF2064.

[0104] 2. Fermentation of engineered strains

[0105] The cells were cultured overnight in TSBY medium (3% tryptic soy broth, 10.3% sucrose, 0.5% yeast extract) at 28° C. Then 1% (v / v) of the culture was transferred to fresh SFM medium (2% soy flour, 2% mannitol) and cultured at 28° C. for 7 days.

[0106] 3. Experimental Results

[0107] The obtained fermentation broth was extracted and concentrated, and further analyzed by HPLC and GC-MS (the detection conditions were the same as those in Example 2). The detection results were as follows: Figure 2 C and Figure 2 D. The product obtained by fermentation in Example 3 is consistent.

[0108] It should be noted that in order to obtain enough candelay for NMR detection, the above-mentioned engineered strain based on Escherichia coli BL21 (DE3) can also carry a recombinant plasmid containing genes related to the mevalonate pathway (MVA pathway) (e.g., mvaE and mvaS genes from Enterococcus faecalis, and mvaK1, mvaD, mvaK2, idi genes from Streptococcus pneumoniae), which is used to increase the yield of precursor compounds of the substrate farnesyl pyrophosphate (FPP) of candelay synthase, i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The content recorded in this embodiment is sufficient to enable those skilled in the art to repeat the experimental results of the present invention.

[0109] In addition, it is understandable that the above-mentioned MVA pathway-related genes are only used to increase the production of cancene. In the absence of MVA pathway-related genes, for example, in the fermentation broth of an engineered strain based on Streptomyces avermitilis NRRL8165, cancene was also detected.

[0110] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0111] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0112] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0113] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An enzyme having an amino acid sequence as shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the enzyme according to claim 1, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

3. An expression vector comprising the nucleic acid molecule encoding the nucleic acid molecule of claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector includes one of a prokaryotic expression vector and a viral vector.

5. A recombinant bacterium or engineered cell line comprising the expression vector according to any one of claims 3-4.

6. An engineered strain, characterized in that: The engineered strain overexpresses or heterologously expresses the enzyme shown in SEQ ID NO:

1.

7. Use of the enzyme according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to any one of claims 3-4, the recombinant bacterium or engineered cell line according to claim 5, or the engineered strain according to claim 6 in synthesizing candelay or its derivatives.

8. The use according to claim 7, characterized in that: The substrate in the synthetic candecene or its derivative is farnesyl pyrophosphate.

9. A catalytic product, characterized in that: The invention comprises at least one of the enzyme according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to any one of claims 3-4, the recombinant bacteria or engineered cell line according to claim 5 and the engineered bacillus according to claim 6.

10. A method for synthesizing cancene or its derivatives by catalyzing farnesyl pyrophosphate, characterized in that: The method comprises: Using farnesyl pyrophosphate as a substrate, the enzyme according to claim 1 is used to catalyze the synthesis of the product candelene or its derivatives; Alternatively, the recombinant bacteria or engineered cell line according to claim 5 or the engineered strain according to claim 6 is used to synthesize the product aconene or its derivatives in vivo.

Images