Use of the gene NjTPS21 in catalyzing preparation of sesquiterpenes

Abstract

The application relates to the technical field of genetic engineering, and particularly relates to application of NjTPS21 gene in catalyzing preparation of sesquiterpenes. The sesquiterpenes are at least one of the following: Aciphyllene, Cyclohexane, Aristolene, (+)-Calarene, alpha-Panasinsene, gamma-Maaliene, Aromadendrene, Alloaromadendrene, Selina-5,11-diene, Valerena-4,7(11)-diene, beta-caryophyllene, beta-Guaiene, (+)-Ledene and beta-Cyclogermacrane. The application discloses that NjTPS21 can catalyze at least 14 kinds of structurally different sesquiterpenes by using a substrate FPP, which lays a foundation for obtaining the monomer compounds in a large scale through synthetic biology in the future. Meanwhile, the gene can also be used as a molecular marker for quality evaluation and breeding research of Gastrodia elata Bl. medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the NjTPS21 gene in the catalytic preparation of sesquiterpenoid compounds. Background Technology

[0002] Terpenes are a large class of natural products found in nature. Among them, sesquiterpenes are of great value in the fields of medicine, fragrance, and agriculture due to their diverse chemical structures and significant biological activities (such as antibacterial, anti-inflammatory, and anticancer effects). Many sesquiterpenes are the core pharmacodynamic components of traditional medicinal plants. Nardostachys jatamansi, as an important traditional Chinese medicine, is believed to have unique sedative and anti-anxiety effects closely related to its rich content of complex sesquiterpene volatile oil components.

[0003] Currently, the main route for obtaining these sesquiterpenoids still relies on direct extraction from plants. However, this method is limited by the long growth cycle of plants, low content, and significant influence from environmental factors, as well as the complex composition of the extracts and the difficulty in separation and purification. Chemical synthesis, on the other hand, faces challenges such as cumbersome steps, low yield, and high cost due to the complex skeleton and multiple chiral centers of sesquiterpenes. In recent years, synthetic biology has provided a revolutionary strategy for the sustainable and efficient production of plant natural products. Its core is to achieve heterologous production of target compounds by reconstructing biosynthetic pathways in microorganisms (such as Escherichia coli and yeast). The key to the success of this strategy lies in identifying and acquiring functional genes encoding key catalytic enzymes, especially terpene synthase (TPS) genes. TPS is a core enzyme that determines the carbon skeleton structure and diversity of terpenoids, catalyzing the formation of a wide variety of monoterpenes and sesquiterpenes from common precursors (such as farnesyl pyrophosphate, FPP).

[0004] Although some plant TPS genes have been cloned and functionally identified, our understanding of the TPS gene family in *Nardostachys jatamansi*, an important medicinal plant, remains very limited. In current technologies, many TPS genes produce relatively single products, while "multifunctional" TPS enzymes capable of catalyzing the production of more than ten different sesquiterpenes from a single FPP substrate are relatively rare, and their catalytic mechanisms are more complex and interesting. Such multifunctional enzymes are key to the chemical diversity of plants, but also represent a current research challenge and gap. The lack of specificity regarding *Nardostachys jatamansi* sources, especially the functional understanding of multifunctional TPS genes, severely hinders the analysis of the biosynthetic network of medicinal sesquiterpenes from the source, and also limits the development of using metabolic engineering to directionally modify microorganisms for the green and customizable production of these high-value compounds.

[0005] Therefore, cloning a new TPS gene from Nardostachys jatamansi and systematically elucidating its catalytic function can not only fill the gap in our understanding of the genetic basis of Nardostachys jatamansi's secondary metabolic pathways and deepen our understanding of the "one enzyme, multiple yields" phenomenon in plants, but also provide an indispensable and potentially superior key gene element for the subsequent construction of engineered strains that produce high yields of single or specific combinations of sesquiterpenes through synthetic biology techniques.

[0006] Against this backdrop, this application completed the cloning and functional verification of a novel TPS gene, NjTPS21, from Nardostachys jatamansi, and discovered that it can catalyze the production of at least 14 sesquiterpenes, which lays an important foundation for research and application in related fields. Summary of the Invention

[0007] The purpose of this invention is to provide NjTPS21 The application of genes in the catalytic preparation of sesquiterpenoids, used to reveal NjTPS21 The gene-encoded protein can use FPP as a substrate to specifically catalyze the generation of various sesquiterpene compounds.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The application of the NjTPS21 gene in the production of sesquiterpenoid compounds, wherein the nucleotide sequence of the NjTPS21 gene is shown in SEQ ID NO.1; and the amino acid sequence of the protein expressed by the NjTPS21 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the sesquiterpene compound is at least one of the following: Aciphyllene, Cyclohexane, Aristolene, (+)-Calarene, α-Panasinsene, γ-Maaliene, Aromadendrene, Alloaromadendrene, Selina-5,11-diene, Valerena-4,7(11)-diene, β-caryophyllene, β-Guaiene, (+)-Ledene, β-Cyclogermacrane.

[0010] Furthermore, NjTPS21 catalyzes the production of sesquiterpenoids by FPP.

[0011] A method for catalyzing the production of sesquiterpenoids by FPP using the NjTPS21 gene includes the following steps: S1: Cloning of the NjTPS21 gene; Take a sample of Nardostachys jatamansi, grind it and extract total RNA; use the extracted total RNA as a template to reverse transcribe cDNA; amplify cDNA by PCR, and recover and purify the DNA fragment after PCR to obtain DNA fragments; S2: Construct the pET28a(+)-NjTPS21 prokaryotic expression vector; Homologous arm sequences of the pET-28a(+) vector were added to both ends of the DNA fragment obtained in the first step by PCR to obtain the NjTPS21 gene with homologous arms; the empty pET-28a(+) vector was linearized by double digestion with BamHI and HindIII restriction enzymes, and the product was recovered to obtain the linearized pET-28a(+) vector; the NjTPS21 gene with homologous arms was recombined with the linearized pET-28a(+) vector to obtain the pET-28a(+)-NjTPS21 recombinant plasmid; S3: Prokaryotic expression and FPP substrate supply in E. coli; The recombinant plasmid pET-28a(+)-NjTPS21 and the helper plasmid capable of producing FPP were simultaneously transformed into competent cells of Escherichia coli C41; an inducer was added to induce expression; the expression products were collected and sesquiterpenoid compounds were isolated.

[0012] Furthermore, in S1, PCR amplification of cDNA includes: Primer design was performed, and the designed primer sequences are as follows: The forward primer is: ATGGACAGCTACCTTAATGC; The reverse primer is: TTAACTCGGGACGCTCTCT.

[0013] 6. The method according to claim 4, characterized in that, in S2, the homologous arm sequences of the pET-28a(+) vector are added to both ends of the DNA fragment obtained in the first step by PCR, comprising: Add the homologous arm “CAGCAAATGGGTCGCGGATCC” next to the BamH I site of the pET-28a(+) vector to the upstream primer, and add the homologous arm “CTCGAGTGCGGCCGCAAGCTT” next to the Hind III site to the downstream primer.

[0014] 7. The method according to claim 5, wherein in step S3, the auxiliary plasmid capable of high FPP production is pMevT-MBIS.

[0015] 8. The method according to claim 5, wherein in S3, the sesquiterpene compound is at least one of the following: Aciphyllene, Cyclohexane, Aristolene, (+)-Calarene, α-Panasinsene, γ-Maaliene, Aromadendrene, Alloaromadendrene, Selina-5,11-diene, Valerena-4,7(11)-diene, β-caryophyllene, β-Guaiene, (+)-Ledene, β-Cyclogermacrane.

[0016] 9. A recombinant plasmid for producing sesquiterpenoid compounds, characterized in that the recombinant plasmid is inserted with the NjTPS21 gene containing the nucleotide sequence shown in SEQ ID NO: 1.

[0017] 10. A genetically engineered bacterium for producing sesquiterpenoid compounds, characterized in that the host cell of the engineered bacterium contains a recombinant expression plasmid carrying the NjTPS21 gene, the sequence listing of which is shown in SEQ ID NO: 1; the host cell of the engineered bacterium also contains an auxiliary plasmid capable of producing FPP.

[0018] The present invention has at least the following beneficial effects: This invention discloses that NjTPS21 can catalyze the generation of at least 14 structurally different sesquiterpenes using the substrate FPP. This discovery lays the foundation for the large-scale acquisition of these monomeric compounds through synthetic biology techniques in the future. Furthermore, this gene can also serve as a molecular marker for quality evaluation and breeding research of Nardostachys jatamansi. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Comparative chromatograms of multiple sesquiterpenoid compounds in the NjTPS21 catalytic product; Figure 2 Comparison of mass spectrometry identification of Aciphyllene; Figure 3 Comparison of mass spectrometry identification of Cyclohexane; Figure 4Comparison of mass spectrometry identification of Aristolene; Figure 5 Comparison of mass spectrometry identification of Calarene; Figure 6 Comparison of mass spectrometry identification of α-Panasinsene; Figure 7 Comparison of mass spectrometry identification of γ-Maalien; Figure 8 Comparison of mass spectrometry identification of Aromadendrene; Figure 9 Comparison of mass spectrometry identification of Alloaromadendrene; Figure 10 Comparison of mass spectrometry identification of Selina-5,11-diene; Figure 11 Comparison of mass spectrometry identification of Valerena-4,7(11)-diene; Figure 12 Comparison of mass spectrometry identification of β-caryophyllene; Figure 13 Comparison of mass spectrometry identification of β-Guaiene; Figure 14 Comparison of mass spectrometry identification of (+)-Ledene; Figure 15 Comparison of mass spectrometry identification of β-Cyclogermacrane. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the 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 merely illustrative and not intended to limit the invention.

[0022] Example 1: This example is mainly used to disclose the nucleotide sequence SEQ ID NO.1 and amino acid sequence SEQ ID NO.2 of NjTPS21.

[0023] SEQ ID NO.1: NjTPS21 nucleotide sequence SEQ ID NO.2: NjTPS21 amino acid sequence MDSYLNASSAPPPKKNMQEPVRPIANYHPSVWGNQFLKYASNPKQSDGGA EEQHEQLKEALRKKLVVNVANERAGEQLTLIDAIQRLGVAYQFETEIDVV LNNQLQLLNNQDDDLHMVSLRFRLLRQHGHNVSCGVFGKFKDIEGRFKEC LMDDVRGLLSLYESTHMRLHKEDILEEALEFTTTHLEQVVKSPLSGSVLA SQVVHALNMPIRKGLTRIEARHFIPIYQQDESHDETLLKFAKLDFNMLQK VHQREVADITMWWKDLNVSEKLPYARDRAVECYFWILGVYFEPQYSRARR ILTKVICMTSLIDDTYDSYGTFEELILFTDAIQRWDVNAKNQLPEYMRHI FGELLDVYGAMEEELSKEGISYRVDYAKQIMKQLVTAYNHEAIWYHDGYV PTLEEYLEVALVSCGYMMLATTSFVGMGVTAVTKQALDWVSSKPLMVQAS SIINRLADDKVGHEFEQQRGHVVSGVECYMKQHNATKEEVLVEFNRRITS AWKDMNQECLHPLPVPMHLLERVLNLARFMNIFYKDEDCYTHSNTRMKGI ITSILIESVPS Example 2: This example mainly discloses the cloning of the NjTPS21 gene from Nardostachys jatamansi, and demonstrates through specific experiments that it can catalyze the production of various sesquiterpenoid compounds.

[0024] The specific steps are as follows: 1.1 Total RNA extraction and reverse transcription The spikenard sample was thoroughly ground in liquid nitrogen for RNA extraction. During RNA extraction, the spatula, mortar, and pestle were sterilized with alcohol to inactivate RNase and other contaminants, and dedicated enzyme-free EP tubes and pipette tips were used. The extraction method followed the FastPure Plant Universal Total RNA Isolation Kit instructions. Reverse transcription was performed according to the HiScriptIII All-in-one RT SuperMix Perfect for qPCR kit instructions.

[0025] 1.2 RT-PCR gene cloning Based on the NjTPS21 gene and its full-length ORF sequence from third-generation transcriptome sequencing, cloning primers were designed using Primer5 software. These primers were synthesized at Chengdu Qingke Biotechnology Co., Ltd. and stored at -20°C. The gene fragment was amplified from cDNA using Phanta Max Super-Fidelity DNA Polymerase. The reaction systems used for gene cloning are shown in Table 1, and the reaction procedures are shown in Table 2.

[0026] Table 1 RT-PCR cloning reaction system

[0027] Table 2. TPS21 gene amplification program

[0028] After the PCR reaction was completed, the above reaction solution was subjected to electrophoresis detection. The target band was excised and recovered according to the FastPure Gel DNAExtraction Mini Kit instructions, and the concentration of the product was determined.

[0029] 1.3 Construction of candidate gene prokaryotic expression vectors In the above NjTPS21 The upstream primer was modified by adding a homologous arm at the BamHI restriction site of pET-28a(+) “CAGCAAATGGGTCGCGGATCC” to its 5' end, and the downstream primer was modified by adding a homologous arm at the HindIII restriction site of pET-28a(+) “CTCGAGTGCGGCCGCAAGCTT” to its 5' end. The recombinant primers were synthesized by Chengdu Qingke Company. Using the gel recovery product under section “1.2” as a template, the target gene was amplified a second time using primers containing homologous arms and Phanta Max Super-Fidelity DNA Polymerase. The target band was gel-cleaved and its concentration was determined according to the above instructions.

[0030] The pET-28a(+) vector was double-digested using BamHI and HindIII restriction enzymes from Takara Pharmaceuticals, Japan. The digestion reaction system is shown in Table 3. The reaction system was incubated at 30°C for 5 min and then at 37°C for 15 min. The digestion products were recovered by gel extraction to obtain the linearized pET-28a(+) vector.

[0031] Following the instructions of the ClonExpress Ultra One Step Cloning Kit V2, the target gene with homologous arms was homologously recombinated with the linearized pET-28a(+) vector. The reaction system is shown in Table 4. The recombination product was transformed into DH5α by heat shock, plated, and incubated overnight at 37 °C. Single colonies were picked for colony PCR detection, and positive colonies were sent to Kexin Technology Co., Ltd. for sequencing.

[0032] Table 3 Enzyme digestion reaction system

[0033] Table 4 Recombination Reaction System

[0034] 1.4 Prokaryotic expression of Escherichia coli The recombinant plasmid pMevT-MBIS, which produces FPP (this plasmid is fused with E. coli), was used. atoB, idi, ispA Genes, and brewer's yeast HMG-CoA, MVD1, ERG8, ERG12 and N-terminal modified tHMGR The gene (which can produce high levels of FPP in prokaryotes) was simultaneously transformed into C41 competent cells along with the pET28a(+)-NjTPS21 recombinant plasmid successfully constructed under section "1.3". The transformation system was as follows: after incubating on ice for 20-30 min, heat-shocked at 42 ℃ for 90 s, followed by incubation on ice for 3-5 min, then added 200 μL of LB liquid medium, and cultured at 37 ℃ with shaking for 1 h. The culture was then plated onto LB agar plates containing kanamycin and chloramphenicol and incubated overnight at 37 ℃. The next day, 5-10 single colonies were picked and cultured in 5 mL of LB liquid medium overnight at 37 ℃ and 200 r / min. The following day, 2 mL of bacterial culture was transferred to 50 mL of NZY medium (5 g / L NaCl, 5 g / L Yeast Extract, 10 g / L Casein Hydrolysate, 1 g / L MgSO4, pH=7.0). When the OD600 reached 0.8, the medium was transferred to 16 ℃ and incubated at 200 r / min for 10 min, followed by overnight induction with 1 mM IPTG. The next day, approximately 5 mL of the bacterial culture was collected for GC-MS analysis. A blank control was prepared by co-transforming pMevT-MBIS with the pET28a(+) empty vector.

[0035] 1.5 GC-MS Detection Detection was performed using a Shimadzu triple quadrupole gas chromatography-mass spectrometry (GC-MS) system-TQ8050 NX. Headspace sampling was employed, with an injection volume of 5 mL of bacterial culture. GC conditions were: Shimadzu SH-Polar Wax (60 m × 0.25 mm × 0.25 μm), injection port temperature 250 ℃, split injection mode (split ratio 5:1), and constant linear velocity carrier gas control mode (linear velocity 25.5 cm·s). −1 The carrier gas was helium with a purity of 99.999%, and the purge flow rate was set to 3.0 mL / min. −1 The column temperature was programmed, with an initial temperature of 40 °C, held for 5 min, and then increased at 3 °C / min. −1 The temperature was raised to 250 °C and held for 15 min; the column equilibration time was 3.0 min. Mass spectrometry conditions: ionization was electron impact ionization (EI) with an ionization energy of 70 eV, ion source temperature of 200 °C, interface temperature of 250 °C, and argon as the collision gas; the mass spectrometry acquisition mode was full scan with a scan mass number range of 35-500 amu, the detector voltage was +0.1 kV relative to the tuning result, and the solvent delay time was 1 min. HS-SPME conditions: After equilibration at 50 °C for 5 min, the headspace vial containing the sample was transferred to the extraction apparatus. The SPME arrow solid-phase extraction head was inserted into the headspace vial through a PTFE headspace septum without contacting the sample. Extraction and adsorption were performed at 50 °C for 15 min. After the autosampler removed the extraction head, it was quickly inserted into the GC-MS / MS injection port, and desorption was performed at 250 °C for 2 min. The solid phase extraction head is a composite of three materials: DVB / CWR / PDMS, with a film thickness of 120 μm and a length of 20 mm. The solid phase extraction head is aged at 250℃ for 3 min before and after sample injection.

[0036] Data were analyzed using a Shimadzu GC-MS solution (Ver 4.53) workstation and the NIST20 mass spectrometry library. The target product was further confirmed by comparison with standards using retention time and mass spectra.

[0037] 2. Results 2.1 Construction of pET28a(+)-NjTPS21 prokaryotic expression vector Using cDNA reverse transcribed from total RNA of Nardostachys jatamansi as a template, PCR amplification was performed using specific primers, and finally, a clone with a complete ORF was cloned. NjTPS21 The gene was homologously recombined into the BamHI and HandIII sites of the pET-28a(+) expression vector. NjTPS21 The primers and their annealing temperatures for the genes are shown in Table 5.

[0038] Table 5 NjTPS21 Primers and annealing temperature for genes

[0039] 2.2 NjTPSs Functional verification The pET28a(+)-NjTPS21 constructed above was co-transformed with pMevT-MBIS into C41 Escherichia coli and fermented. The products were analyzed by GC-MS (Table 6, mass spectra of the products are in the appendix). The results show that: NjTPS21 can catalyze the formation of 14 sesquiterpenoid products from FPP, including Aciphyllene, Cyclohexane, Aristolene, (+)-Calarene, α-Panasinsene, γ-Maaliene, Aromadendrene, Alloaromadendrene, Selina-5,11-diene, Valerena-4,7(11)-diene, β-caryophyllene, β-Guaiene, (+)-Ledene, and β-Cyclogermacrane (see...). Figure 1 and Figures 2-15 ).

[0040] Table 6. Catalyst products of NjTPS21

[0041] Note: All samples were analyzed using chromatographic columns and analytical conditions specified by Shimadzu; all products were retrieved and analyzed from the NIST database, and the similarity scores were all above 85.

[0042] The foregoing has shown and described 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 embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. NjTPS21 The application of genes in the production of sesquiterpenoids is characterized by, The nucleotide sequence of the NjTPS21 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein expressed by the NjTPS21 gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The sesquiterpene compound is at least one of the following: Aciphyllene, Cyclohexane, Aristolene, (+)-Calarene, α-Panasinsene, γ-Maaliene, Aromadendrene, Alloaromadendrene, Selina-5,11-diene, Valerena-4,7(11)-diene, β-caryophyllene, β-Guaiene, (+)-Ledene, β-Cyclogermacrane.

3. The application according to claim 1, characterized in that, NjTPS21 catalyzes the production of sesquiterpenoids via FPP.

4. A method of utilizing NjTPS21 A method for using genes to catalyze the production of sesquiterpenoids by FPP, characterized in that, Includes the following steps: S1: Cloning of the NjTPS21 gene; A sample of Nardostachys jatamansi was taken, ground, and total RNA was extracted. The extracted total RNA was used as a template for reverse transcription to obtain cDNA. cDNA was amplified by PCR, and the DNA fragments were recovered and purified after PCR. S2: Construct the pET28a(+)-NjTPS21 prokaryotic expression vector; Homologous arm sequences of the pET-28a(+) vector were added to both ends of the DNA fragment obtained in the first step by PCR to obtain the NjTPS21 gene with homologous arms; the empty pET-28a(+) vector was linearized by double digestion with BamHI and HindIII restriction enzymes, and the product was recovered to obtain the linearized pET-28a(+) vector; the gene with homologous arms was then... NjTPS21 The gene was recombinated with the linearized pET-28a(+) vector to obtain the pET-28a(+)-NjTPS21 recombinant plasmid; S3: Prokaryotic expression and substrate supply in E. coli; The recombinant plasmid pET-28a(+)-NjTPS21 and the helper plasmid capable of producing FPP were simultaneously transformed into competent cells of Escherichia coli C41; an inducer was added to induce expression; the expression products were collected and sesquiterpenoid compounds were isolated.

5. The method according to claim 4, characterized in that, In S1, PCR amplification of cDNA includes: Primer design was performed, and the designed primer sequences are as follows: The forward primer is: ATGGACAGCTACCTTAATGC; The reverse primer is: TTAACTCGGGACGCTCTCT.

6. The method according to claim 4, characterized in that, In step S2, homologous arm sequences of the pET-28a(+) vector are added to both ends of the DNA fragment obtained in step one by PCR, including: Add the homologous arm "CAGCAAATGGGTCGCGGATCC" next to the BamH I site of the pET-28a(+) vector to the upstream primer, and add the homologous arm "CTCGAGTGCGGCCGCAAGCTT" next to the Hind III site to the downstream primer.

7. The method according to claim 5, characterized in that, In S3, the helper plasmid that can produce high levels of FPP is pMevT-MBIS.

8. The method according to claim 5, characterized in that, In S3, the sesquiterpene compound is at least one of the following: Aciphyllene, Cyclohexane, Aristolene, (+)-Calarene, α-Panasinsene, γ-Maaliene, Aromadendrene, Alloaromadendrene, Selina-5,11-diene, Valerena-4,7(11)-diene, β-caryophyllene, β-Guaiene, (+)-Ledene, β-Cyclogermacrane.

9. A recombinant plasmid for producing sesquiterpenoid compounds, characterized in that, The recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO:

1. NjTPS21 Gene.

10. A genetically engineered bacterium for producing sesquiterpenoid compounds, characterized in that, The host cells of the engineered bacteria contain [a substance / organization]. NjTPS21 The recombinant expression plasmid of the gene, the sequence listing of the NjTPS21 gene is shown in SEQ ID NO: 1; the host cell of the engineered bacteria also contains an auxiliary plasmid capable of producing FPP.

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