An enzyme for synthesizing perilla alcohol or its derivative and application thereof

Abstract

The application provides an enzyme for synthesizing white camphene or a derivative thereof and an application thereof, and first proposes a white camphene synthesis enzyme in bacteria, an amino acid sequence of which is shown as SEQ ID NO:1, which can be used for biosynthesizing white camphene in vitro or in bacteria, and has the advantages of being more economical and environmentally friendly compared with synthesizing white camphene by chemical synthesis or extracting white camphene from plants in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to an enzyme for synthesizing calarene or a derivative thereof and application thereof. BACKGROUND

[0002] Terepenoids are one of the most abundant and diverse secondary metabolites in plants, and the basic unit is isoprene (C5H8). Terepenoids have a wide range of applications in practical applications, and can be developed as natural fragrances, fuels, food additives, therapeutic agents, metabolic regulators and insecticides. In addition, clinical studies have shown that certain terepenoids have neuroprotective, memory-enhancing, anti-inflammatory, respiratory function-improving, heart-stimulating and sedative-hypnotic pharmacological effects.

[0003] Calarene is a sesquiterpene with biological activity, which is usually extracted from plants (such as Kadsura heteroclite stems), and has toxicity to root-knot nematodes (Meloidogyne incognita), maize weevils (Sitophilus zeamais) and mosquitoes, which has attracted attention in the field of plant control. However, the extraction of calarene mainly depends on plant sources, and the process is usually complex 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 prospect.

[0004] Compared with chemical synthesis or plant extraction, biosynthesis of sesquiterpenes has the advantages of economy and environmental protection. However, there is no report on the related synthetic enzymes for biosynthesis of calarene. Therefore, it is necessary to develop an enzyme capable of synthesizing calarene or a derivative thereof. SUMMARY

[0005] The present application aims to provide an enzyme for synthesizing calarene or a derivative thereof and application thereof, and to screen an enzyme for synthesizing calarene or a derivative thereof, which uses farnesyl pyrophosphatase as a substrate and obtains the product calarene or a derivative thereof by enzyme catalysis, so as to avoid a complex chemical synthesis process and provide a new way for biosynthesis of the product calarene or a derivative thereof.

[0006] In a first aspect of the present application, an enzyme for synthesizing calarene or a derivative thereof is provided, and the amino acid sequence of the enzyme for synthesizing calarene or a derivative thereof is shown in SEQ ID NO: 1.

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

[0008] In a third aspect of the present application, an enzyme expression vector for synthesizing erilenes or derivatives thereof is provided, wherein the expression vector comprises the nucleic acid molecule.

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

[0010] As a specific embodiment, the preparation method of the enzyme expression vector for synthesizing erilenes or derivatives thereof comprises:

[0011] obtaining a target gene fragment of the nucleic acid molecule of the enzyme for synthesizing erilenes or derivatives thereof;

[0012] The target gene fragment and the pET28a expression vector are subjected to double enzyme digestion with NdeI and NotI, followed by enzyme ligation to obtain the expression vector pET28a-orf2064.

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

[0014] The starting strain of the engineered bacterium comprises but is not limited to all bacteria comprising MVA and MEP pathways, and as an example, can be Escherichia coli and Streptomyces.

[0015] In a fifth aspect of the present application, the enzyme for synthesizing erilenes or derivatives thereof, the nucleic acid molecule, the expression vector, the recombinant bacterium or the engineered cell line are applied to synthesis of erilenes or derivatives thereof.

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

[0017] In a seventh aspect of the present application, a method for synthesizing erilenes or derivatives thereof by catalyzing farnesyl pyrophosphin is provided, wherein the method comprises:

[0018] using the enzyme for synthesizing erilenes or derivatives thereof to catalyze the synthesis of erilenes or derivatives thereof with farnesyl pyrophosphin as a substrate;

[0019] or using the recombinant bacterium or the engineered cell line to synthesize erilenes or derivatives thereof in vivo.

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

[0021] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages:

[0022] The application provides a catalytic product and application thereof, and the application screens an enzyme for synthesizing white camphene or a derivative thereof, can farnesyl pyrophosphin as a substrate, and obtains the product white camphene or a derivative thereof by using enzyme catalysis, so that a complex chemical synthesis process can be avoided, and a new way for biologically synthesizing the product white camphene or a derivative thereof is provided. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0024] Figure 1 It is the experimental result graph of the embodiment 2 of the present application, wherein, the graph A is the SDS-PAGE detection result of the recombinant protein ORF2064 obtained in the embodiment 2; the graph B is the gas chromatography detection result of the experimental group a and the control group b-c in the embodiment 2; the graph C is the mass spectrum detection result of the 2 experimental groups a in the embodiment 1; the graph D is the standard GC-MS spectrum of white camphene (from the National Institute of Standards and Technology Library (NIST08).

[0025] Figure 2 It is the experimental result graph of the embodiment 3 of the present application, wherein, the graph A is the gas chromatography detection result of the fermentation product of the engineering strain based on the E. coli BL21 (DE3) in the embodiment 3. The graph B is the mass spectrum detection result of the fermentation product of the engineering strain based on the E. coli BL21 (DE3) in the embodiment 3. The graph C is the gas chromatography detection result of the engineering strain based on the Streptomyces avermitilis NRRL8165 in the embodiment 3. The graph D is the mass spectrum detection result of the engineering strain based on the Streptomyces avermitilis NRRL8165 in the embodiment 3. DETAILED DESCRIPTION

[0026] The advantages and various effects of the present application will be more clearly presented by the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0027] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the present application belongs. If there is a conflict, the present specification takes precedence.

[0028] Unless otherwise specifically explained, various materials, reagents, instruments and equipment that are used in the present application are commercially available or are obtained by conventional methods.

[0029] The "comprise" or "include" in the present application is an open description, containing the specified components or steps, and other specified components or steps that do not materially affect.

[0030] The "comprise" or "include" in the present application is an open description, containing the specified components or steps, and other specified components or steps that do not materially affect.

[0031] The embodiments of the first aspect of the present application provide a use of an enzyme with an amino acid sequence as shown in SEQ ID NO: 1 in synthesis of white camphene or its derivative.

[0032] The present application first proposes a white camphene synthase (ORF2064) in bacteria, and the amino acid sequence thereof is shown in SEQ ID NO: 1.

[0033] MDKNAIGTRIRRVGKAGVRDGVRDPVHEHLRRTQDSLATPFVVRENSAARDVFAASVQWMQEHALLTGERRQWLEKCDIGALLGLTNPQVDRRLLRLAGDWYVWLYAFDDGVCDEALTGAQAWDMAHLTLRLKRSVTGGAPGDGPEDNYARALSDLRLRIAEHATPAQLVRWTEAVRDYLNGQLWETTYRATGRIPAVDDYITMRESASGCLSCFALLPVLNRYHLPEEVRA HPDVEQLSRSANRIIAWDNDLFSYLKELGDHSAVANLITAVARERRCDIAAAITYARKQRDGELATFLRAEKRVARHLGVEGRYYIADLKNWVSGSLEFHRTSRRFVSAPRVVHL (SEQ ID NO: 1)

[0034] The sequence is obtained by sequencing and translating the amino acid sequence after amplifying the fragment containing the white camphene synthase gene from strain S. exfoliatus UC5319 using primers CK21 (347) F / CK21R.

[0035] The nucleotide sequence encoding the white camphene 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] Strain Streptomyces exfoliatus UC5319 is described in the article “Seo M J, Zhu D, Endo S, et al. Genome Mining in Streptomyces. Elucidation of the Role of Baeyer Villiger 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 application.

[0040] In some embodiments, the use comprises: the use of an enzyme with an amino acid sequence as set forth in SEQ ID NO: 1 to synthesize a eremophilene or a derivative thereof in vitro or in bacteria.

[0041] The present inventors first proposed that the above-mentioned eremophilene synthase can be used to synthesize eremophilene, including but not limited to in vitro incubation or fermentation of an engineered strain overexpressing or heterologously expressing the eremophilene synthase.

[0042] As an example, in an in vitro reaction, eremophilene is formed using farnesyl diphosphate (FPP) as a substrate under the catalysis of an enzyme with an amino acid sequence as set forth in SEQ ID NO: 1.

[0043]

[0044] In some embodiments, the eremophilene derivative includes but is not limited to eremophilene oxide.

[0045] It can be understood that the use of an enzyme with an amino acid sequence as set forth in SEQ ID NO: 1 to synthesize eremophilene and further obtain derivatives or downstream metabolites thereof on this basis is also covered in the protection scope of the present application.

[0046] As examples, the following are shown: 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; Compound 12: 9β-debilon; Compound 13: debilon; Compound 14: nardostachone; Compound 15: 1(10)-aristolen-2-one; Compound 16: aristolanhydride; Compound 17: kanshone F.

[0047]

[0048] In some embodiments, the bacteria comprise: E. coli and Streptomyces.

[0049] In some embodiments, the engineered strain is used for synthesizing a compound selected from the group consisting of: nardoaristol; 1(10)-aristolen-9β-ol; kanshone C; 3-hydroxylkanshone H; 3-oxokanshone H; kanshone H; (-)-aristolone; (-)-(14β,15β)-aristolone; 1-hydroxylaristolone; 1(10)-aristolen-12-al; kanshone G; 9β-debilon; debilon; nardostachone; 1(10)-aristolen-2-one; aristolanhydride; and kanshone F.

[0050] In some embodiments, the engineered strain is used for synthesizing a compound selected from the group consisting of: nardoaristol; 1(10)-aristolen-9β-ol; kanshone C; 3-hydroxylkanshone H; 3-oxokanshone H; kanshone H; (-)-aristolone; (-)-(14β,15β)-aristolone; 1-hydroxylaristolone; 1(10)-aristolen-12-al; kanshone G; 9β-debilon; debilon; nardostachone; 1(10)-aristolen-2-one; aristolanhydride; and kanshone F.

[0051] Strain origin

[0052] S. exfoliatus UC5319 is described in the article "Seo M J, Zhu D, Endo S, et al. Genome Mining in Streptomyces. Elucidation of the Role of Baeyer Villiger 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 application.

[0053] S. avermitilis NRRL8165 is described in the article "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 application.

[0054] The enzyme for synthesizing eremophilene or its derivatives and its application will be described in detail below in combination with examples and experimental data.

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

[0056] 1. The primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4 is used to amplify a fragment containing the eremophilene synthase gene from strain S. exfoliatus UC5319;

[0057] 2. The target gene fragment and pET28a expression vector are subjected to double enzyme digestion with Ndel and Notl at 37°C, and the reaction system is as follows:

[0058] Double enzyme digestion system of orf2064 fragment:

[0059] Table 2

[0060] pET28α double enzyme cutting system:

[0061] Table 3

[0062]

[0063] After the double enzyme cutting reaction was carried out for 3h at 37°C using Ndel and Notl restriction endonuclease, the reaction was terminated by adding Loading Buffer, and the double enzyme cutting product was purified and recovered according to the instruction of the gel recovery kit.

[0064] 3、After the same restriction enzyme cutting, the double enzyme cutting product has the same sticky end, and can be connected into a complete plasmid by DNA ligase. The orf2064 gene and the pET28a double enzyme cutting product containing the same sticky end were placed in the same PCR tube, and the connection reaction was carried out using a 10μL system. The target gene and the plasmid enzyme cutting product were added in a ratio of 3:1, mixed with 1μL T4 DNA ligase, and connected at 16°C overnight. The successfully connected plasmid was named pET28a-orf2064.

[0065] 4、The connection product was transformed into E. coli DH5a competent cells, and the transformation steps were as follows:

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

[0067] b、Take one tube (100μL) of competent bacteria from the ultra-low temperature freezer at -80°C, immediately warm it up with your fingers, then insert it into ice for 10 minutes of ice bath.

[0068] c、Add 10μL of connection product pET28a-orf2064, gently shake and place on ice for 20 minutes.

[0069] d、Gently shake it and insert it into a 42°C water bath for 90s of heat shock, then quickly put it back into ice and stand for 5 minutes.

[0070] e、Add 900μL of LB medium without antibiotic to each tube, mix gently, then fix it on the spring rack of the shaking bed, and shake it at 37°C for 50 minutes.

[0071] f、Add 300μl of the above transformation mixture taken from the clean bench to the solid LB plate culture dish containing the appropriate antibiotic, and use a glass coating rod that has been burned by an alcohol lamp and cooled to evenly coat it.

[0072] g. Label the coated dishes and place them in a 37°C incubator for 30-60 minutes. After the liquid on the surface has penetrated into the medium, place the dishes upside down in a 37°C incubator overnight.

[0073] 5. Single colony identification: pick a single colony from the LB solid medium and inoculate it into 5 mL of LB medium containing kanamycin. Incubate the medium at 37°C, 200 r / min overnight. Take a portion of the bacterial solution and store it in a glycerol tube. Extract the recombinant plasmid from the rest of the solution according to the instructions of the plasmid extraction kit. Digest the extracted recombinant plasmid with Ndel and Notl, and analyze the target gene fragment and the vector fragment by 1% agarose gel electrophoresis. Send the recombinant plasmid that is positive in the double digestion to Shanghai Biosci & Tech Co., Ltd. for sequence determination. The recombinant expression vector pET28α-orf2064 is successfully obtained. -1

[0074] Example 2. Synthesis of nerolidol in vitro

[0075] 1. Obtaining of recombinant protein ORF2064

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

[0077] 2. In vitro reaction

[0078] 1 mL of buffer solution (50 mM PIPES, 15 mM MgSO4, 100 mM NaCl, 5 mM β- mercaptoethanol and 20% glycerol (v / v), pH 6.8) containing 1 μM recombinant ORF2064 and 20 μM FPP, which is covered with 1 mL of n-hexane, is reacted at 30°C for 16 hours. Then, 25 μL of EDTA (500 mM) is added to quench the reaction, which is taken as experimental group a.

[0079] Meanwhile, control groups b and c are set up. The above-mentioned in vitro reaction is carried out using boiled recombinant protein (boiled ORF2064) as control group b, and using standard FPP as control group c.

[0080] 3. Product detection

[0081] After the reaction system of step 1.2 is extracted with n-hexane and concentrated, further GC-MS analysis is carried out. The detection results are shown in Figure 1 B and Figure 1 C. In addition Figure 1 ​D is the standard spectrum of calendula (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 using an HP5MS capillary column (30 m × 0.25 mm) in positive ion mode at 70 eV electron impact (EI), 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 a hold at 280°C for 2 min.

[0083] Samples were analyzed on a Shimadzu SPD-M20A / LC-20AT using a Thermo Scientific C18 reversed-phase HPLC column (250 × 4.6 mm, 5 μm). Mobile phase A: water; mobile phase B: acetonitrile; UV detection at λ: 210 nm, elution program: 80% B, flow rate: 0.8 mL / min, elution duration: 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 Candecene in Escherichia coli

[0087] 1. Engineered Escherichia coli BL21(DE3) strain heterologously expressing 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 candelilla 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 transformed 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, incubated overnight at 37°C. Then 1% (v / v) of the culture was transferred to fresh 2xYT medium, inoculated at 37°C until OD 600 0.4-0.6 was reached. 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] III. Experimental results

[0095] The resulting fermentation broth was extracted and concentrated, and further analyzed by HPLC and GC-MS (same detection conditions as in Example 2), and the results are shown in Figure 2 A and Figure 2 B. The results are in agreement with the products obtained from the in vitro reaction of Example 2.

[0096] After purification of the resulting ferulene from the fermentation broth using HPLC (same detection conditions as in Example 2), NMR analysis was performed, and the results are as follows: 13 C NMR (100 MHz, CDC13) δ 144.3, 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, 16.2 ppm; 1 H NMR (400 MHz, CDC13) δ 5.24 (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 = 7.7 Hz, 6H), 0.74 (m, 1H), 0.56 (d, J = 9.2 Hz, 1H).

[0097] NMR analysis conditions: Carbon-13 and proton spectra were recorded on a Bruker AVANCE 400 MHz NMR spectrometer, which operates at 400 and 100 MHz for 1 H and 13 C, equipped with a 5 mm BBFO smart probe. All experiments were performed in CDC13 at 303 K. Chemical shifts are reported in ppm and referenced to the solvent resonance signal (CDC13: 1 H δ ppm 7.26, 13 C δ ppm 77.16).

[0098] Example 4, Biosynthesis of ferulene in Streptomyces

[0099] I. The engineered strain of Streptomyces avermitilis NRRL8165 heterologously expressing ORF2064

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

[0101] obtaining the gene fragment of interest of the nucleic acid molecule of the enzyme for synthesizing white camphene or its derivatives;

[0102] The gene fragment of interest and the pIB139 expression vector were double-digested with Ndel and Notl, and then ligated to obtain the expression vector pIB139-orf2064.

[0103] 2. The recombinant expression vector pIB139-orf2064 was transformed into Streptomyces avermitilis NRRL8165 to heterologously express ORF2064, and the engineered strain heterologously expressing ORF2064 was obtained.

[0104] II. Fermentation of the engineered strain

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

[0106] III. Experimental results

[0107] The obtained fermentation broth was extracted, concentrated, and further analyzed by HPLC and GC-MS (under the same conditions as in Example 2), and the results are shown in Figure 2 C and Figure 2 D. The results of the fermentation in Example 3 are consistent.

[0108] It should be noted that in order to obtain sufficient white camphene for NMR detection, the above-mentioned engineered strain based on E. coli BL21 (DE3) can also carry a recombinant plasmid containing genes related to the mevalonate pathway (MVA pathway) (such as mvaE and mvaS genes from Enterococcus faecalis and mvaKl, mvaD, mvaK2, idi genes from Streptococcus pneumoniae) to increase the yield of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the precursors of the substrate farnesyl pyrophosphate (FPP) of the white camphene synthase. The content described in this example is sufficient to enable a person skilled in the art to repeat the experimental results of the present application.

[0109] In addition, it can be understood that the above-mentioned MVA pathway related genes are only used to improve the yield of gerberilene, and the presence of gerberilene is also detected in the fermentation broth of the engineered strain based on Streptomyces avermitilis NRRL8165 in the absence of MVA pathway related genes.

[0110] The above-mentioned embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above-mentioned embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principle of the present application should be equivalent replacement modes, and are all included in the protection scope of the present application.

[0111] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment.

[0112] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0113] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends 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 gene 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. An engineered cell line comprising the expression vector according to any one of claims 3-4.

6. The engineered cell line according to claim 5, characterized in that The engineered cell line is an engineered strain that overexpresses 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, and the engineered cell line according to any one of claims 5-6 in synthesizing cancerene.

8. The use according to claim 7, characterized in that The substrate in the synthesis of cancerene 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, and the engineered cell line according to any one of claims 5-6.

10. A method for synthesizing canepine 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 white corneum; Alternatively, the engineered cell line according to any one of claims 5 to 6 is used to synthesize the product cancerene in vivo.

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