Nardostachyos root and rhizome sesquiterpene synthase gene NjTPS15 and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a nardostachyos root and rhizome sesquiterpene synthase gene NjTPS15 and application thereof. Comprising the application of the NjTPS15 gene in the preparation of the sesquiterpene compound and the application of the NjTPS15 protein expressed by the NjTPS15 gene in the preparation of the sesquiterpene compound. The cDNA sequence of the NjTPS15 gene is successfully cloned from rhizoma nardostachyos through RT-PCR (Reverse Transcription-Polymerase Chain Reaction), then a fragment is constructed on a pET28a (+) prokaryotic expression vector through a homologous recombination method, the recombinant pET28a (+) / NjTPS15 is converted to an escherichia coli chassis strain for producing FPP (Factor Phosphorus Protein), the NjTPS15 sesquiterpenoid synthase gene of successfully converted escherichia coli is induced to be expressed, and the gene expression of the NjTPS15 sesquiterpenoid synthase is obtained. According to the invention, beta-Patchoulene, alpha-Guaiene, alpha-Patchoulene, gamma-gumulene, Humulene, delta-Guaiene, alpha-Selinine, (-)-7-epi-alpha-Selinene, Patchouli alcohol and Pogostole can be simultaneously produced in the escherichia coli for the first time, and the method has the advantages that the method is simple and convenient to operate, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a nard sesquiterpenoid synthase gene NjTPS15 and an application thereof. Background Art

[0002] Nardostachys jatamansi (D.on) DC. is a plant of the genus Nardostachys in the family Patrinaceae. The 2020 edition of the Chinese Pharmacopoeia lists its medicinal parts as its roots and rhizomes, which have the effects of regulating qi, alleviating pain, and relieving depression and invigorating the spleen. Nardostachys jatamansi is also a common aromatic herb used in other traditional medicines, such as India and Japan. Its essential oil extract has broad application value in the daily chemical industry, such as the globally popular "nardostachys jatamansi essential oil."

[0003] Chemical analysis shows that nardus is rich in sesquiterpenoids such as β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchoulialcohol, and Pogostole. These components also constitute the material basis for the medicinal effects of nardus and the essential oil of nardus. Due to the complex composition, the above components are difficult to synthesize chemically and are mainly extracted and separated from nature. However, their content in nature is low, and obtaining them is a great challenge. Using modern biotechnology, enzyme-catalyzed green synthesis is a potential effective strategy. As sesquiterpenoid alkanes, the key step in the synthesis of sesquiterpenoid alkanes is the catalysis of the farnesyl pyrophosphate (FPP) substrate by terpene synthases (TPS). A search has revealed several TPS enzymes capable of synthesizing sesquiterpenoid alkanes from plants, animals, and microorganisms. However, no TPS enzyme has been reported to catalyze the production of these compounds, particularly one capable of producing all of them simultaneously. Currently, sesquiterpenoids such as β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole are primarily isolated and extracted from plants. However, their low concentrations and complex composition make their isolation and extraction difficult. Regarding their biosynthesis, genes involved in the upstream synthesis steps leading to the production of the precursor compound FPP have been identified. However, the key enzymes of TPS that can catalyze the biosynthesis of sesquiterpenoids such as β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole from FPP have not yet been elucidated.

[0004] To this end, based on research, we proposed a sesquiterpenoid synthase gene NjTPS15 of the present invention and its application Summary of the Invention

[0005] The present invention aims to provide a nard sesquiterpenoid synthase gene NjTPS15 and an application thereof, identify a TPS key enzyme that can simultaneously catalyze the biosynthesis of sesquiterpenoid compounds such as β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole from FPP, and provide a reference for the green heterologous synthesis of these compounds.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides the use of the NjTPS15 gene in the preparation of sesquiterpene compounds. The nucleotide sequence of the NjTPS15 gene is as shown in SEQ ID NO.1, or a nucleotide sequence with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.1.

[0008] Furthermore, the sesquiterpene compound includes at least one of the following: β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole.

[0009] The present invention provides the use of the NjTPS15 protein expressed by the NjTPS15 gene in the preparation of sesquiterpene compounds, wherein the amino acid sequence of the NjTPS15 protein is as shown in SEQ ID NO.2 or an amino acid sequence with the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.2.

[0010] Furthermore, the sesquiterpene compound includes at least one of the following: β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole.

[0011] The present invention provides a method for preparing sesquiterpene compounds using the NjTPS15 gene, comprising the following steps:

[0012] Step 1: Cloning the NjTPS15 gene

[0013] Take the root sample of Nardostachys japonica, grind it quickly and thoroughly in liquid nitrogen, extract RNA, and reverse transcribe the RNA into cDNA;

[0014] Using cDNA as template, upstream primers and downstream primers were used for amplification;

[0015] Step 2: Construction of pET-28a(+) / NjTPS15 recombinant vector

[0016] Add a homology arm to the BamH I restriction enzyme cleavage site at the 5' end of the upstream primer; add a homology arm to the Hind III restriction enzyme cleavage site at the downstream primer;

[0017] Using the recovered product as a template, primers containing homology arms were used to amplify the target gene a second time, and the NjTPS15 band was excised and recovered;

[0018] The pET-28a(+) vector was double-digested with BamH I and Hind III endonucleases, and the digestion products were recovered by gel electrophoresis to obtain the linearized pET-28a(+) vector.

[0019] The NjTPS15 gene with homology arms was homologously recombined with the linearized pET-28a(+) vector, and the recombinant product was transformed into Escherichia coli DH5α by the heat shock method to construct the pET-28a(+) / NjTPS15 recombinant vector;

[0020] Step 3: Converting to FPP chassis E. coli and fermentation

[0021] The FPP-producing recombinant plasmid pMevT-MBIS, which is fused with the atoB, idi, and ispA genes of Escherichia coli, and the HMG-CoA, MVD1, ERG8, ERG12, and N-terminally modified tHMGR genes of Saccharomyces cerevisiae, was simultaneously transformed into Escherichia coli C41 competent cells together with the pET28a(+) / NjTPS15 recombinant plasmid.

[0022] The competent E. coli C41 cells are transferred into a fermentation broth to obtain a sesquiterpene product through fermentation.

[0023] Furthermore, the upstream primer is: ATGTCAATTATTATTGCAACAAACG; the downstream primer is: TTATATGGATACGGGATCTACGA.

[0024] Furthermore, the homology arm at the BamH I restriction enzyme cleavage site is: CAGCAAATGGGTCGCGGATCC; the homology arm at the Hind III restriction enzyme cleavage site is: CTCGAGTGCGGCCGCAAGCTT.

[0025] Furthermore, the fermentation-derived sesquiterpene products include β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole.

[0026] The present invention has at least the following beneficial effects:

[0027] The present invention characterizes for the first time the unique role of the NjTPS15 gene of Nardostachys rapa in the biosynthesis pathways of β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol and Pogostole. The cDNA sequence of the NjTPS15 gene was successfully cloned from Nardostachys rapa by RT-PCR. The fragment was then constructed into the pET28a(+) prokaryotic expression vector by homologous recombination. The recombinant pET28a(+) / NjTPS15 was transformed into an FPP-producing Escherichia coli chassis strain, and the expression of the NjTPS15 sesquiterpenoid synthase gene in the transformed Escherichia coli was induced. For the first time, the simultaneous production of β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole was achieved in Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of 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 paying any creative work.

[0029] Figure 1GC-MS chromatograms of NjTPS15, blank control, and Patchouli alcohol standard;

[0030] Figure 2 Mass spectrum of β-Patchoulene, a product of NjTPS15 (left: mass spectrum of the target component, right: mass spectrum from the NIST database);

[0031] Figure 3 Mass spectrum of NjTPS15 product α-Guaiene (left: target component mass spectrum, right: NIST database mass spectrum)

[0032] Figure 4 Mass spectrum of NjTPS15 product α-Patchoulene (left: target component mass spectrum, right: NIST database mass spectrum)

[0033] Figure 5 Mass spectrum of γ-gurjunene, a product of NjTPS15 (left: mass spectrum of the target component, right: mass spectrum from the NIST database)

[0034] Figure 6 Mass spectrum of Humulene, a product of NjTPS15 (left: target component mass spectrum, middle: NIST database mass spectrum, right: standard mass spectrum)

[0035] Figure 7 Mass spectrum of NjTPS15 product Δ-Guaiene (left: target component mass spectrum, right: NIST database mass spectrum)

[0036] Figure 8 Mass spectrum of NjTPS15 product α-Selinine (left: target component mass spectrum, right: NIST database mass spectrum)

[0037] Figure 9 Mass spectrum of the NjTPS15 product (-)-7-epi-α-selinene (left: target component mass spectrum, right: NIST database mass spectrum)

[0038] Figure 10 Mass spectrum of NjTPS15 product Patchouli alcohol (left: target component mass spectrum, middle: NIST database mass spectrum, right: standard mass spectrum);

[0039] Figure 11 Mass spectrum of the NjTPS15 product Pogostole (left: target component mass spectrum, right: NIST database mass spectrum). DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] The present invention characterizes for the first time the unique role of the NjTPS15 gene of Nardostachys rapa in the biosynthesis pathways of β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol and Pogostole. We successfully cloned the cDNA sequence of the NjTPS15 gene from Nardostachys grandiflora by RT-PCR, and then constructed the fragment into the pET28a(+) prokaryotic expression vector by homologous recombination. The recombinant pET28a(+) / NjTPS15 was transformed into an FPP-producing Escherichia coli chassis strain, inducing the expression of the NjTPS15 sesquiterpenoid synthase gene in the successfully transformed Escherichia coli. For the first time, we achieved the simultaneous production of β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole in Escherichia coli.

[0042] Example 1

[0043] This example discloses the NjTPS15 gene. The NjTPS15 gene sequence contains 1677 nucleotides and encodes a protein of 558 amino acids. The sequence is as follows:

[0044] SEQ ID NO.1 nucleotide

[0045] ATGTCAATTATTATTGCAACAACGGTACTGAGCATCCAATTTTT

[0046] CGTCCATAGCAAATTTTCCACCAAGTTTATGGGGCAATCTTTTC

[0047] ACTTCATTCTCTATGGATAATCAGGCTAGGGAAATATATGCTAAA

[0048] GAACATGAAGGTTTAAAAGAAAAAGTGAGAGTGATGTTGTTAG

[0049] ATACAACAAATTACAAAATTTCAGAGAAAATCAATTTCATAAAC

[0050] ACAGTGGAAAGATTAGGTGTATTCATATCATTTTGAGAAAGAGAT

[0051] TGAAGAACTACTTCATCAAATGTTTGATGCTCATTCTAAACTCCT

[0052] AGATGATATTCAAGAATTTGATTTGTTCACTTTGGGAATTTACTT

[0053] CAGGATTCTAAGGCAACATGGTTATAAAATCTCTTGTGATGTTTT

[0054] CAATAAGTTGAAAGATAGCAATGGCGAATTCAAGGACGAACTT

[0055] AAAGATGATGTGAATGGTATGCTAAGTTTGTATGAAGCAACACA

[0056] TGTAAGAACACATGGAGAAAATATTTTAGATGAAGCTCTCATTT

[0057] ATACAAAAGCTCAACTTGAATCCATGGCCGCTGCAAGTTTAAGC

[0058] CCATTTCTCGCGAAGCAAGTTAAGCATGCTTTGATACAAGCTCT

[0059] CCACAAAGGGATCCCAAGAATCGAAGCACGTAATTATATCTCTG

[0060] TTTACGAAGAAGATCCTAACAATTGATTTGTTATTGAGGTTC

[0061] TCAAAGATAGATTTCAATCTAGTACAAATGATTCACAAGCAAGA

[0062] ATTTGCGATACCTTTAGGTGGTGGAAAGATTTGGAGTTCGAAT

[0063] CGAAACTATCTTTTGCAAGGAATAGAGTGGTGGAAGCCTACTTA

[0064] TGGACTCTTAGCGCGTACTACGAACCAAAATACTCTTCCGCTCG

[0065] GATTATATTAGTCAAACTAATGGTTATAATATCTGTTACGGATGAC

[0066] ACATATGATGCATATGGTACATTAGATGAACTTCAACTTTTTACA

[0067] GATGCAGTACAAAGGTTGGATATGAGTTCTATCAATCAACTTCC

[0068] AGATTACATGAAGACCATCTATAAAGCTCTCCTAGATCTTTTCGA

[0069] CGAAATAGAAGATCGATTATCGAAGCATGAAACTGATCATTCTT

[0070] ACCGCGTTGCTTATGCGAAATATGTGTATAAAGAGATCGTTAGGT

[0071] GCTACGATATGGAGTACAAATGGTTCAACAAAAATTACGTGCCG

[0072] GCATTTGAAGAATATATGCAGAAAGCGTTAGTCACATCAGGTAA

[0073] CCGTTTGCTCATAACGTTTTCCTTTCTGGGAATGGACGAAGTAG

[0074] CAACTATTCAAGCGTTCGAGTGGGTAAAAAGTAATGCCAAAAT

[0075] GATAGTCTCTTCCAATAAAGTATTACGACTTATTGATGACATAAT

[0076] GAGTCACGAGGAAGAGGATGAAAGGGGACATGTTGCAACAGG

[0077] GATTGAATGCTTTGTAAAAGAACATGGACTAACTAGGGAAGAG

[0078] GTTATCGTTGAATTTCATAAGAGGATTGATGATGCTTGGAAGGAT

[0079] ATAAATGAGGAATTTATAACGCCAAATAATTTACCGATTGAGATA

[0080] CTTACGCGTGTTCTAAACCTTACAAGAATTGGAGATGTTGTTTA

[0081] CAAGTATGATGACGGGTATACTCATCCGGAGAAAGCGTTGAAA

[0082] GATCACATCATATCGTTGTTCGTAGATCCCGTATCCATATAA

[0083] Amino acids of SEQ ID NO.2

[0084] MSIIIATNGTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEH

[0085] EGLKEKVRVMLLDTTNYKISEKINFINTVERLGVSYHFEKEIEELL

[0086] HQMFDAHSKLLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKD

[0087] SNGEFKDELKDDVNGMLSLYEATHVRTHGENILDEALIYTKAQLE

[0088] SMAAASLSPFLAKQVKHALIQALHKGIPRIEARNYISVYEEDPNKN

[0089] DLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRV <00几千0195>VEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDEL

[0091] QLFTDAVQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHET

[0092] DHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKAL

[0093] VTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLID

[0094] DIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKD

[0095] INEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHII

[0096] SLFVDPVSI

[0097] Example 2

[0098] The specific technical solutions are as follows:

[0099] 1. Cloning of the NjTPS15 gene from Nardostachys rapa

[0100] Nardostachys rhizome samples were quickly and thoroughly ground in liquid nitrogen for RNA extraction. During RNA extraction, pestles, mortars, and other instruments were sterilized with alcohol to inactivate contaminants such as RNases. Special enzyme-free extraction consumables, such as EP tubes and pipette tips, were used. RNA tested and found to meet requirements was then reverse-transcribed into cDNA according to the HiScript III All-in-one RT SuperMix Perfect for qPCR kit instructions.

[0101] Using cDNA as a template, "ATGTCAATTATTATTGCAACAAACG" as the upstream primer and "TTATATGGATACGGGATCTACGA" as the downstream primer, the ORF of the NjTPS15 gene was amplified from the cDNA using a super-fidelity enzyme. The reaction system and protocol are shown in Tables 1 and 2, respectively.

[0102] After PCR amplification, the target product was recovered according to the instructions of the FastPure Gel DNA Extraction Mini Kit.

[0103] Table 1 RT-PCR cloning reaction system

[0104]

[0105] Table 2 Amplification procedures

[0106]

[0107] 2. Construction of pET-28a(+) / NjTPS15 recombinant vector

[0108] Add the homology arm "CAGCAAATGGGTCGCGGATCC" to the BamHI restriction site in pET-28a(+) to the 5' end of the upstream primer, and add the homology arm "CTCGAGTGCGGCCGCAAGCTT" to the HindIII restriction site of the downstream primer. Using the gel-recovered product as a template, perform a secondary amplification of the target gene using primers containing the homology arm according to the above PCR system and procedure. Recover the NjTPS15 band from the gel.

[0109] The pET-28a(+) vector was double-digested using BamH I and Hind III endonucleases. The digestion reaction system is shown in Table 3. The digestion product was recovered by gel chromatography to obtain a linearized pET-28a(+) vector.

[0110] According to the instructions of the homologous recombination kit ClonExpress Ultra One Step Cloning Kit V2, the NjTPS15 gene with homology arms was homologously recombined with the linearized pET-28a(+) vector. The reaction system is shown in Table 4. The recombinant product was transformed into Escherichia coli DH5α by the heat shock method, plated, and cultured overnight at 37°C. Single colonies were picked for colony PCR detection, and positive plaques were sent for sequencing.

[0111] Table 3 Enzyme digestion reaction system

[0112]

[0113] Table 4 Recombination reaction system

[0114]

[0115] The NjTPS15 gene sequence contains 1677 nucleotides and encodes a protein of 558 amino acids (see Example 1 above).

[0116] 3. E. coli and fermentation for FPP production

[0117] The FPP-producing recombinant plasmid pMevT-MBIS (which incorporates the atoB, idi, and ispA genes of Escherichia coli and the HMG-CoA, MVD1, ERG8, ERG12, and N-terminally modified tHMGR genes of Saccharomyces cerevisiae) was simultaneously transformed into E. coli C41 competent cells along with the previously constructed pET28a(+) / NjTPS15 recombinant plasmid. The transformation system was as follows: ice-bath for 20-30 minutes, heat shock at 42°C for 90 seconds, ice-bath for 3-5 minutes, addition of 200 μL of LB liquid medium, incubation at 37°C with shaking for 1 hour, plating on LB plates containing kanamycin and chloramphenicol, and incubation at 37°C overnight. The next day, 5-10 single colonies were picked and transferred to 5 mL of LB liquid medium and incubated at 37°C at 200 rpm overnight. The next day, transfer 2 mL of the bacterial culture to 50 mL of NZY medium (5 g / L NaCl, 5 g / L Lyeast Extract, 10 g / L Casein Hydrolysate, 1 g / L MgSO4, pH 7.0). Cultivate to an OD600 of 0.8, then incubate at 16°C at 200 rpm for 10 minutes. Induce overnight with 1 mM IPTG. The next day, remove approximately 5 mL of the bacterial culture for GC-MS analysis.

[0118] GC-MS analysis was performed using a Shimadzu triple quadrupole gas chromatograph-mass spectrometer (GCMS-TQ8050 NX) with headspace injection of 5 mL of bacterial solution. GC conditions included a Shimadzu SH-PolarWax (60 m × 0.25 mm × 0.25 μm) inlet at 250°C, split injection with a split ratio of 5:1, and constant linear velocity (25.5 cm·s). The carrier gas was helium with a purity of 99.999%, with a purge flow rate of 3.0 mL / min. The column temperature was programmed, starting at 40°C for 5 min, then increasing at 3°C / min to 250°C and holding for 15 min. The column equilibration time was 3.0 min. Mass spectrometry conditions: Ionization was performed using electron impact ionization (EI) with an ionization energy of 70 eV, an ion source temperature of 200°C, an interface temperature of 250°C, and argon as the collision gas. The mass spectrometer acquisition mode was full scan, with a mass range of 35–500 amu. The detector voltage was +0.1 kV relative to the tuning result, and a solvent delay time of 1 minute. HS-SPME conditions: The headspace vial containing the sample was equilibrated at 50°C for 5 minutes before being transferred to the extraction apparatus. The SPME arrow solid-phase extraction tip was inserted into the headspace vial through a polytetrafluoroethylene headspace septum without contacting the sample. Extraction and adsorption were performed at 50°C for 15 minutes. The tip was then removed from the autosampler and quickly inserted into the GC-MS / MS inlet for desorption at 250°C for 2 minutes. The solid-phase extraction tip was a DVB / CWR / PDMS composite with a membrane thickness of 120 μm and a length of 20 mm. The tip was conditioned at 250°C for 3 minutes before and after injection. The data were analyzed using Shimadzu GC-MS solution (Ver4.53) workstation and NIST20 mass spectral library.

[0119] The fermentation broth co-transformed with pMevT-MBIS and pET28a(+) empty vector was used as a blank control. Sesquiterpenoid products including β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchoulialcohol and Pogostole were detected in the fermentation broth transformed with the pET28a(+) / NjTPS15 recombinant plasmid, as shown in Table 5. Figures 1 to 10 These results indicate that NjTPS15 can catalyze FPP to form the above 10 sesquiterpenoid products.

[0120] Table 5 Catalytic products of NjTPS15

[0121]

[0122]

[0123] Notes: 1. The Shimadzu SmartAroma Database can rapidly identify aroma compounds that cannot be confirmed by traditional methods by using retention time, chromatographic peaks, characteristic ions, and database spectral search and multiple comparisons. 2. The Shimadzu AART function (automatic adjustment of compound retention time) can automatically adjust the retention time of target compounds using retention indices and the retention time of n-alkanes. 3. Accurate retention times and retention indices can be obtained using Shimadzu-specified chromatographic columns and analytical conditions. Using the method files provided in the database as instrument conditions, compounds registered in the database can be accurately identified. 4. The product humulene was identified and qualitatively identified as humulene using the Shimadzu database. 5. Patchouli alcohol was accurately identified using purchased standards loaded into the Shimadzu aroma database using retention time, retention indices, characteristic ions, etc. 6. All of the above samples were analyzed using Shimadzu-specified chromatographic columns and analytical conditions. 7. All products were searched and analyzed using the NIST database, and the similarity scores were all above 85 points.

[0124] In combination with the above, it can be seen that: the present invention discovered a sesquiterpene synthase NjTPS15 from Nardostachys grandiflora for the first time; the present invention constructed a method for obtaining a pET28a(+) / NjTPS15 recombinant vector and a recombinant bacterium; the present invention provides an enzymatic conversion method for catalyzing FPP and simultaneously synthesizing β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol and Pogostole.

[0125] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions merely illustrate the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of NjTPS15 gene in the preparation of sesquiterpene compounds, characterized in that: The nucleotide sequence of the NjTPS15 gene is as shown in SEQ ID NO.1 or a nucleotide sequence having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The sesquiterpene compound includes at least one of the following species: β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole.

3. Application of NjTPS15 protein expressed by NjTPS15 gene in the preparation of sesquiterpene compounds, characterized in that: The amino acid sequence of the NjTPS15 protein is as shown in SEQ ID NO.2 or an amino acid sequence having the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.

2.

4. The use according to claim 3, characterized in that: The sesquiterpene compound includes at least one of the following species: β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol, and Pogostole.

5. A method for preparing sesquiterpene compounds using the NjTPS15 gene, characterized in that: The following steps are involved: Step 1: Cloning of NjTPS15 gene Take the rhizome samples of Nardostachys grandiflora, quickly grind them thoroughly in liquid nitrogen, extract RNA, and reverse transcribe the RNA into cDNA; Using cDNA as template, upstream primers and downstream primers were used for amplification; Step 2: Construction of pET-28a(+) / NjTPS15 recombinant vector Add a homology arm to the BamH I restriction site at the 5' end of the upstream primer; add a homology arm to the Hind III restriction site at the downstream primer; The recovered product was used as a template to perform secondary amplification of the target gene using primers containing homology arms, and the NjTPS15 band was excised and recovered; The pET-28a(+) vector was double-digested with BamH I and Hind III endonucleases, and the digestion products were recovered by gel to obtain the linearized pET-28a(+) vector; The NjTPS15 gene with homology arms was homologously recombined with the linearized pET-28a(+) vector, and the recombinant product was transformed into Escherichia coli DH5α by heat shock method to construct the pET-28a(+) / NjTPS15 recombinant vector; Step 3: Converting to FPP chassis E. coli and fermentation The FPP-producing recombinant plasmid pMevT-MBIS, which was fused with the atoB, idi, and ispA genes of Escherichia coli, the HMG-CoA, MVD1, ERG8, ERG12, and the N-terminally modified tHMGR gene of Saccharomyces cerevisiae, was transformed into Escherichia coli C41 competent cells together with the pET28a(+) / NjTPS15 recombinant plasmid. The competent cells of Escherichia coli C41 are transferred into a fermentation bacterial solution, and the sesquiterpene products are obtained by fermentation.

6. The method according to claim 5, characterized in that The upstream primer is: ATGTCAATTATTATTGCAACAAACG; the downstream primer is: TTATATGGATACGGGATCTACGA.

7. The method according to claim 5, characterized in that The homology arm at the BamH I restriction site is: CAGCAAATGGGTCGCGGATCC; the homology arm at the HindⅢ restriction site is: CTCGAGTGCGGCCGCAAGCTT.

8. The method according to claim 5, characterized in that The sesquiterpene products obtained by the fermentation include β-Patchoulene, α-Guaiene, α-Patchoulene, γ-gurjunene, Humulene, Δ-Guaiene, α-Selinine, (-)-7-epi-α-selinene, Patchouli alcohol and Pogostole.

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