AaTPS48 and its coding gene and application

Abstract

This invention relates to the terpene synthase AaTPS48 from Artemisia argyi, its encoding gene, and its applications. It provides an important foundation for utilizing genetic engineering techniques to increase the content of the active components myrcene and (trans)-β-farnesene in Artemisia argyi, or to directly produce myrcene and (trans)-β-farnesene. The technical solution is that the terpene synthase AaTPS48 is a protein of the following a) or b) or c): a) the amino acid sequence of the protein shown in SEQ ID No. 1; b) a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 1; c) a protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1. The AaTPS48 protein of this invention can catalyze both GPP to form myrcene and FPP to form (trans)-β-farnesene, playing an important role in the biosynthesis of myrcene, (trans)-β-farnesene, and other terpene compounds in Artemisia argyi.

Description

I. Technical Field

[0001] This invention relates to the field of medicinal plant genetic engineering, and in particular to a terpene synthase AaTPS48 from Artemisia argyi, its encoding gene, and its applications. II. Background Technology

[0002] Artemisia argyi Levl. et Vant., a plant belonging to the genus Artemisia in the family Asteraceae, is a dried leaf typically harvested in summer before flowering and when the leaves are lush. It is pungent and bitter in taste, warm in nature, and slightly toxic. It is believed to have effects such as regulating qi and blood, dispelling cold and dampness, warming the meridians, stopping bleeding, and calming the fetus. In my country, Artemisia argyi is mainly produced in Henan, Hubei, Hunan, Anhui, Shandong, and Hebei provinces. The "Tangyin Bei'ai" and "Nanyang Ai" from Henan, the "Qi Ai" from Hebei, and the "Qi Ai" from Hubei are all renowned for their superior quality. The volatile oil in Artemisia argyi leaves is the main material basis for its therapeutic effects, exhibiting significant antibacterial, antiviral, anti-inflammatory, orifice-opening, swelling-reducing, analgesic, antitussive, and antiasthmatic effects.

[0003] Myrcene, also known as geraniol, is renowned for its medicinal properties, including analgesic, antioxidant, anti-inflammatory, antibacterial, and melanin-inhibiting and antioxidant effects. Myrcene is also an important industrial raw material, widely used in the perfume industry as a precursor to various high-value synthetic fragrances, and can also be used to synthesize vitamin E. Furthermore, myrcene exhibits high polymerization reactivity, enabling it to polymerize into high-performance rubber materials, thus possessing significant application value.

[0004] (trans)-β-farnesene, also known as (trans)-β-farnesene, is a major component of the volatile oil in Artemisia argyi. As an aphid alarm pheromone, (trans)-β-farnesene not only attracts aphids from their habitats to actively contact pesticides, reducing pesticide usage, but also attracts aphid predators, avoiding pesticide use. Recent research indicates that (trans)-β-farnesene is a specific activator of mosquito odor receptors and can be used as a highly effective synergist for repelling and killing mosquitoes, playing an important role as an insect pheromone in plant disease prevention.

[0005] Myrcene is an acyclic monoterpene compound, while (trans)-β-farnesene belongs to the acyclic sesquiterpenes. It is a universal substrate for terpenes, isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP), generated via the cytoplasmic mevalonic acid (MVA) pathway and the plastid 2-methyl-D-erythritol-4-phosphate (MEP) pathway. From IPP, the monoterpene precursor geranyl diphosphate (GPP) and the sesquiterpene precursor farnesyl diphosphate (FPP) are produced. The terpene synthase AaTPS48 in Artemisia argyi can catalyze both the formation of myrcene from GPP and the formation of (trans)-β-farnesene from FPP. Developing new drugs from the active ingredients of traditional Chinese medicine is a promising approach; however, the slow growth of plants, the limited content of active ingredients in plants, and the cumbersome extraction and separation processes that easily cause environmental pollution significantly limit its development. Exploring and elucidating the biosynthetic pathways and regulatory mechanisms of terpenoids in Artemisia argyi contributes to providing a theoretical basis for the formation of medicinal quality and opens up broad application possibilities for using biotechnology to increase the content of target components or directly produce active ingredients. The cloning and functional verification of the Artemisia argyi terpenoid synthase AaTPS48 gene provides an important foundation for using genetic engineering technology to increase the content of the active components myrcene and (trans)-β-farnesene in Artemisia argyi or to directly produce myrcene and (trans)-β-farnesene. Prior to the publication of this invention, no Artemisia argyi terpenoid synthase AaTPS48 gene and its amino acid sequence mentioned in this invention have been publicly disclosed or reported. III. Summary of the Invention

[0006] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide an Artemisia argyi terpene synthase AaTPS48 and its encoding gene and application, which provides an important foundation for using genetic engineering technology to increase the content of active ingredients myrcene and (trans)-β-farnesene in Artemisia argyi or to directly produce myrcene and (trans)-β-farnesene.

[0007] The technical solution provided by this invention is that the Artemisia argyi terpene synthase AaTPS48 is a protein that is either a), b), or c) of the following:

[0008] a) The amino acid sequence is that of the protein shown in SEQ ID No. 1;

[0009] b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 1;

[0010] c) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1.

[0011] SEQ ID No.1 consists of 575 amino acid residues.

[0012] To facilitate the purification of the protein in a), a tag as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein represented by SEQ ID No. 1 in the sequence listing.

[0013] Table 1. Sequence of Labels

[0014] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0015] The protein in c) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0016] The proteins mentioned in c) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0017] The gene encoding the protein in c) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in positions 1-1728 of SEQ ID No. 2, and / or by performing a missense mutation on one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0018] A second objective of this invention is to provide biomaterials related to the AaTPS48 protein.

[0019] The biomaterial related to the AaTPS48 protein provided by this invention is any one of the following A1) to A12):

[0020] A1) A nucleic acid molecule encoding the AaTPS48 protein;

[0021] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0022] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0023] A4) A recombinant vector containing the expression cassette described in A2);

[0024] A5) Recombinant microorganisms containing the nucleic acid molecules described in A1);

[0025] A6) Recombinant microorganisms containing the expression cassette described in A2);

[0026] A7) Recombinant microorganisms containing the recombinant vector described in A3);

[0027] A8) Recombinant microorganisms containing the recombinant vector described in A4);

[0028] A9) Transgenic plant cell lines containing the nucleic acid molecules described in A1);

[0029] A10) A transgenic plant cell line containing the expression cassette described in A2);

[0030] A11) Transgenic plant cell lines containing the recombinant vector described in A3);

[0031] A12) Transgenic plant cell lines containing the recombinant vector described in A4).

[0032] In the above-mentioned biological materials, the nucleic acid molecule described in A1) is a gene as shown in 1), 2), or 3) below:

[0033] 1) Its coding sequence is the cDNA molecule shown in positions 1-1728 of SEQ ID No. 2;

[0034] 2) A cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the AaTPS48 protein;

[0035] 3) A cDNA molecule or genomic DNA molecule that hybridizes to the nucleotide sequence defined in 1) or 2) under strict conditions and encodes the AaTPS48 protein.

[0036] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0037] Those skilled in the art can readily mutate the nucleotide sequence encoding AaTPS48 of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence encoding AaTPS48, provided they encode AaTPS48 and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention.

[0038] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0039] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0040] In the above-mentioned biological materials, the stringent conditions are: hybridization and washing twice at 68°C in a solution of 2×SSC and 0.1% SDS, each time for 5 min; followed by hybridization and washing twice at 68°C in a solution of 0.5×SSC and 0.1% SDS, each time for 15 min; or hybridization and washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0041] In the aforementioned biological materials, the expression cassette (AaTPS48 gene expression cassette) containing a nucleic acid molecule encoding AaTPS48, as described in A2), refers to DNA capable of expressing AaTPS48 in host cells. This DNA may include not only a promoter to initiate AaTPS48 transcription but also a terminator to terminate AaTPS48 transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0042] In the aforementioned biological materials, the carrier may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0043] In the above-mentioned biological materials, the microorganisms may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0044] Of the aforementioned biological materials, the transgenic plant cell lines do not include propagation materials.

[0045] A third objective of this invention is to provide new uses for the AaTPS48 protein.

[0046] This invention provides the use of AaTPS48 protein as a terpene synthase or in the synthesis of terpene compounds.

[0047] This invention provides the use of AaTPS48 protein as a catalyst for the formation of myrcene from gerany pyrophosphate (GPP) or for the formation of (trans)-β-farnesene from farnesyl pyrophosphate (FPP).

[0048] A fourth objective of this invention is to provide new uses for the aforementioned related biomaterials.

[0049] This invention provides the application of the aforementioned biomaterials in the preparation of terpene synthases or the synthesis of terpene compounds.

[0050] This invention provides the application of the aforementioned biomaterials in catalyzing the formation of myrcene from gerany pyrophosphate (GPP) or the formation of (trans)-β-farnesene from farnesyl pyrophosphate (FPP).

[0051] In the above applications,

[0052] The terpenoids are monoterpenoids and / or sesquiterpenoids;

[0053] The monoterpene compound is myrcene, and the sesquiterpene compound is (trans)-β-farnesene.

[0054] The final objective of this invention is to provide a method for synthesizing terpenoid compounds.

[0055] The method for synthesizing terpenoids provided by the present invention includes the following steps: mixing AaTPS48 protein, substrate and enzyme buffer, reacting to obtain terpenoids.

[0056] In the above method,

[0057] The mass ratio of the AaTPS48 protein to the substrate is 2:1;

[0058] In the above method,

[0059] The enzyme buffer solution is composed of HEPES, MgCl2, PMSF, and DTT.

[0060] The concentration of HEPES in the enzyme buffer is 50 mM.

[0061] The concentration of MgCl2 in the enzyme buffer is 10 mM;

[0062] The concentration of PMSF in the enzyme buffer is 1 mM;

[0063] The concentration of DTT in the enzyme buffer is 5 mM;

[0064] The pH value of the enzyme buffer is 7.0.

[0065] In the above method,

[0066] The substrate is gerany pyrophosphate or farnesyl pyrophosphate;

[0067] The terpenoids are monoterpenoids and / or sesquiterpenoids;

[0068] The monoterpene compound is myrcene, and the sesquiterpene compound is (trans)-β-farnesene.

[0069] This invention cloned the AaTPS48 gene from Artemisia argyi cDNA. This gene is the first key enzyme gene obtained from Artemisia argyi that can catalyze the synthesis of monoterpenes by GPP and sesquiterpenes by FPP. Experiments have demonstrated that the AaTPS48 protein of this invention can catalyze both the formation of myrcene by GPP and the formation of (trans)-β-farnesene by FPP. It plays an important role not only in the biosynthesis of myrcene, (trans)-β-farnesene, and other terpenes in Artemisia argyi, but also has significant theoretical and practical implications for regulating and producing plant terpenes and cultivating high-quality Artemisia argyi. IV. Description of the attached drawings

[0070] Figure 1 This is an agarose gel electrophoresis image of the Artemisia argyi AaTPS48 gene clone of this invention. M represents Trans2K DNA Marker (nucleic acid molecular weight standard, with bands from top to bottom being 2000, 1000, 750, 500, 250, and 100 bp).

[0071] Figure 2 This invention uses polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the AaTPS48 protein expressed in *E. coli*. Lane 1 represents the protein molecular weight standard, with bands from top to bottom at 180, 135, 100, 75, 63, 48, and 35 kDa. Lane 2 represents the target protein expressed by the recombinant plasmid pET32a::AaTPS48, with the arrow indicating the target protein. Lane 3 represents the negative control empty vector plasmid pET32a.

[0072] Figure 3 GC-MS analysis of the AaTPS48 enzymatic reaction product of this invention. Figure 3 A is the extracted ion chromatogram of the product formed by the standard Myrcene and the AaTPS48 catalytic substrate GPP; Figure 3 B is the extracted ion chromatogram of the product formed by the standard (E)-β-Farnesene and the AaTPS48 catalytic substrate FPP; Figure 3 C is the mass spectrum of the standard Myrcene. Figure 3 D is the mass spectrum of the product formed by the AaTPS48 catalytic substrate GPP; Figure 3 E is the mass spectrum of the standard (E)-β-Farnesene. Figure 3 F is the mass spectrum of the product formed by the AaTPS48 catalytic substrate FPP.

[0073] Figure 4 The present invention relates to the fermentation production of Myrcene by yeast strain MD-Aa48 (obtained by introducing recombinant plasmid pESC-Leu::AaTPS48 into yeast strain MD). V. Detailed Implementation Methods

[0074] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0075] Unless otherwise specified, the experimental methods used in this invention are all common knowledge to those skilled in the art. Various buffer solutions, test reagents, etc., can be obtained commercially or prepared by conventional experimental methods unless otherwise specified.

[0076] The following experiments Q5 2×Master Mix and BamHI restriction endonuclease are products of New England Biolabs.

[0077] The Rapid Universal Plant RNA Extraction Kit is a product of Beijing Huayueyang Biotechnology Co., Ltd.

[0078] TransScriptⅡTwo-Step RT-PCR Super Mix, pEASY-Blunt Zero Cloning Kit, Trans2K DNA Marker, pEASY-Basic Seamless Cloning and Assembly Kit, E. coli competent cells Trans1-T1 and Transetta(DE3), pET32a(+) vector, and ProteinIso Ni-NTA Resin are products of Beijing TransGen Biotech Co., Ltd.

[0079] Rainbow 180 broad-spectrum protein marker is a product of Solarbio.

[0080] Geranyl pyrophosphate (GPP) is a product of Sigma-Aldrich, with catalog number G6772 and CAS number 763-10-0.

[0081] Farnesyl pyrophosphate (FPP) is a product of Sigma-Aldrich, with catalog number F6892 and CAS number 13058-04-3.

[0082] Myrcene is a product of Sigma-Aldrich, catalog number 64643, CAS number 123-35-3;

[0083] (trans)-β-farnesene ((E)-β-farnesene) is a product of Yuanye Company, with product catalog number S25161 and CAS number 18794-84-8.

[0084] I. Cloning of the full-length cDNA sequence of the Artemisia argyi AaTPS48 gene

[0085] 1. Extraction of total RNA

[0086] The total RNA was extracted from Artemisia argyi leaves according to the instructions of the Rapid Universal Plant RNA Extraction Kit from Beijing Huayueyang Biotechnology Co., Ltd.

[0087] 2. Synthesis of first-strand cDNA

[0088] The reverse transcription reaction was performed according to the instructions of the TransScript II Two-Step RT-PCR Super Mix first-strand cDNA synthesis kit from Beijing TransGen Biotech Co., Ltd. The reverse transcription reaction system is shown in Table 1.

[0089] Table 1 Reverse transcription reaction system

[0090]

[0091] (1) The reverse transcription reaction was carried out under the following conditions: 25℃ for 10 min, 50℃ for 30 min; and inactivated by heating at 85℃ for 5 s.

[0092] (4) cDNA samples should be stored at -20℃.

[0093] 3. Primer design

[0094] Based on the transcriptome data of Artemisia argyi leaves, the open reading frame (ORF) sequence of the AaTPS48 gene was obtained, and cloning primers AaTPS48-F and AaTPS48-R were designed accordingly. The primer sequences are as follows:

[0095] AaTPS48-F: 5'-ATGTCAACTACTATTCCTGTTTCTAGT-3';

[0096] AaTPS48-R: 5'-TTAGACAACCATAGGGTGAACGAAG-3'.

[0097] 4. PCR amplification

[0098] Using the cDNA obtained in step 2 as a template, a high-fidelity enzyme was employed. Q5 PCR amplification was performed using 2×Master Mix, AaTPS48-F, and AaTPS48-R primers to obtain the PCR amplification products, as shown in the following figures. Figure 1As shown, the PCR amplification products were sequenced. The PCR reaction procedure is as follows:

[0099] PCR reaction program: 98℃ pre-denaturation for 1 min; 98℃ for 20 s, 55℃ for 20 s, 72℃ for 90 s, 40 cycles; 72℃ extension for 10 min.

[0100] Sequencing results showed that the sequence of the PCR amplification product is shown in SEQ ID No.2. The gene shown in SEQ ID No.2 is named AaTPS48, which encodes a protein consisting of 575 amino acid residues. The protein is named AaTPS48, and the amino acid sequence of the protein is SEQ ID No.1.

[0101] II. Obtaining Artemisia argyi AaTPS48 protein

[0102] 1. Construction of recombinant vectors

[0103] Using the pEASY-Basic Seamless Cloning and Assembly Kit from Beijing TransGen Biotech Co., Ltd., the DNA fragment shown in positions 1-1728 of SEQ ID No. 2 was constructed into the BamHI restriction site of the pET32a(+) vector (TransGen Biotech Co., Ltd.), while keeping other sequences of the pET32a(+) vector unchanged, to obtain the recombinant plasmid pET32a::AaTPS48. The primer sequences are as follows (underlined sequences indicate vector homologous regions):

[0104] AaTPS48-32a-F:

[0105] 5'- CCATGGCTGATATCGGA ATGTCAACTACTATTCCTGTTTCTAGT-3';

[0106] AaTPS48-32a-R:

[0107] 5'- ACGGAGCTCGAATTCGG GACAACCATAGGGTGAACGAAG-3'.

[0108] 2. Obtaining recombinant bacteria

[0109] The recombinant plasmid pET32a::AaTPS48 was transformed into the Escherichia coli expression strain Transetta(DE3) (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain the recombinant pET32a::AaTPS48 strain; at the same time, the empty pET32a vector without the target gene was transformed into the Escherichia coli expression strain Transetta(DE3) as a control strain.

[0110] 3. Obtaining recombinant protein AaTPS48

[0111] Recombinant pET32a::AaTPS48 bacteria and control bacteria were inoculated separately into 5 mL of LB broth (containing 100 mg / L ampicillin) and cultured overnight at 37°C with shaking. The next day, the culture was diluted 1:40 and added to 200 mL of LB broth, and cultured at 37°C with shaking until OD reached. 600 When the concentration reaches 0.6-0.8, transfer the culture to 16℃ and shake for 1.5 hours. Add IPTG to a final concentration of 0.4 mM and continue culturing at 16℃ for 18 hours to induce target protein expression. Centrifuge the bacterial culture at 8000g for 5 min, discard the supernatant, and collect the recombinant pET32a::AaTPS48 bacteria and control bacteria. Store them at -80℃ for later use.

[0112] 4. Extraction of recombinant protein AaTPS48

[0113] Proteins were extracted from the cells of recombinant pET32a::AaTPS48 and control bacteria. The specific steps were as follows: The cells of recombinant pET32a::AaTPS48 and control bacteria were resuspended in 5 mL of pre-chilled HEPES buffer (50 mM HEPES, 1 mM PMSF, 5 mM DTT, pH 7.0); the cells were then sonicated in an ice bath (10% power, 1 s sonication, 3 s interval, for 16 min, repeated once); and centrifuged at 10000 g, 4 °C for 30 min to obtain the supernatant of recombinant pET32a::AaTPS48 and the supernatant of control bacteria, which were the protein solutions.

[0114] The supernatant of the recombinant bacteria was subjected to SDS-PAGE, and the results are as follows: Figure 2 As shown in the figure, the AaTPS48 recombinant protein is approximately 85 kDa, which is consistent with expectations.

[0115] III. Enzymatic Activity Analysis of Recombinant Protein AaTPS48

[0116] 1. Enzymatic activity

[0117] (1) Preparation of enzyme-catalyzed reaction system

[0118] Take the protein solution from step 1, section "4", and perform an enzymatic reaction. The supernatant from the control bacteria serves as the control group. Based on the different substrates, the samples are divided into two groups, each with a control. The enzymatic reaction systems for each group are as follows:

[0119] GPP Group:

[0120] The total enzymatic reaction volume was 300 μL. 293 μL of supernatant from the recombinant bacteria and 293 μL of supernatant from the control bacteria were added, along with 5.5 μL of gerany pyrophosphate (GPP) and 1.5 μL of MgCl2. The mixture was then sealed with 300 μL of n-hexane and incubated at 30 °C for 2 hours. After incubation, the reaction was terminated by vortexing for 30 seconds. The mixture was extracted twice with n-hexane, and the n-hexane layers were combined and dried under nitrogen. The extract was then dissolved in 100 μL of n-hexane for GC-MS analysis.

[0121] FPP Group:

[0122] The total enzymatic reaction volume was 300 μL. 293 μL of supernatant from the recombinant bacteria and 293 μL of supernatant from the control bacteria were added, along with 5.5 μL of gerany pyrophosphate (FPP) and 1.5 μL of MgCl2. The mixture was then sealed with 300 μL of n-hexane and incubated at 30 °C for 2 hours. After incubation, the reaction was terminated by vortexing for 30 seconds. The mixture was extracted twice with n-hexane, and the n-hexane layers were combined and dried under nitrogen. The extract was then dissolved in 100 μL of n-hexane for GC-MS analysis.

[0123] (2) GC-MS analysis

[0124] The target compound was detected by gas chromatography-mass spectrometry (GC-MS): The GC-MS analysis system was a Thermo TRACE1310 / TSQ 8000 gas chromatograph with a TG-5MS capillary column (30 m × 0.25 mm × 0.25 μm); the temperature program was as follows: initial column temperature 50 °C, increasing at 5 °C / min. -1 The temperature was increased to 85°C and maintained for 3 minutes; then increased at a rate of 2°C / min. -1 The rate increases to 100℃; at 4℃·min -1 The rate increases to 200℃; at 20℃·min -1 The injection rate was increased to 300℃ and held for 5 min. The injection port temperature was 250℃, the carrier gas was He, and the carrier gas flow rate was 1 mL / min. -1 The sample was injected via split injection at a volume of 1.0 μL, a split ratio of 10:1, and a split flow rate of 10 mL / min. -1 The mass spectrometry conditions were as follows: ionization mode: EI, electron energy: 70 eV; ion source temperature: 250℃, transfer line temperature: 250℃. The scanning mode was full scan, with a detection range of 40–400 m / z; solvent delay: 3 min.

[0125] GC-MS analysis results are as follows Figure 3As shown: Compared with the empty vector enzymatic reaction control, the AaTPS48 recombinant protein can catalyze both the formation of myrcene from GPP and the formation of (trans)-β-farnesene from FPP.

[0126] IV. Fermentation of Myrcene using Artemisia argyi AaTPS48 and yeast strain

[0127] I. Strain Construction

[0128] 1. Construction of eukaryotic expression vectors

[0129] Using the pEASY-Uni Seamless Cloning and Assembly Kit from Beijing TransGen Biotechnology Co., Ltd., the DNA fragment shown in positions 1-1728 of SEQ ID No. 2 was constructed into the BamHI restriction site of the pESC-Leu vector (Agilent Technologies), while keeping other sequences of the pESC-Leu vector unchanged, to obtain the recombinant plasmid pESC-Leu::AaTPS48. The primer sequences are as follows (underlined sequences indicate vector homologous regions):

[0130] AaTPS48-Leu-F:5'- AAGGAGAAAAAACCCCG ATGTCAACTACTATTCCTGTTTCTAGT-3';

[0131] AaTPS48-Leu-R:5'- AGTGAGTCGTATTACGG GACAACCATAGGGTGAACGAAG-3'.

[0132] 2. Preparation of competent yeast cells

[0133] SD-Ura solid plates: SD-Ura + 2% glucose + 2% agar; without agar, it becomes the corresponding liquid culture medium (SD-Ura liquid culture medium);

[0134] SD-Ura-Leu solid plates: SD-Ura-Leu + 2% glucose + 2% agar; without agar, it becomes the corresponding liquid culture medium (SD-Ura-Leu liquid culture medium).

[0135] MD yeast (MD yeast is derived from yeast strain CEN.PK2-1D, genotypes of which are: MATα, URA3-52, TRP1-289, LEU2-3112, HIS3Δ1, MAL2-8C, SUC2; MD yeast genotypes are: CEN.PK2-1D, YPRC△15URA3-P) GAL1 -ERG10-T TPI1 -PGAL10 -ERG13-T PGI -P GAL1 -tHMG1-T ADH1 -P GAL10 -tHMG1-T CYC1 -P GAL1 -tHMG1-T FBA1 -P GAL10 -ERG12-T PDC1 -P GAL1 -ERG8-T RPS2 -P GAL10 -ERG19-T TDH1 -P GAL1 -IDI1-T CCW12 -P GAL10 -ERG20 F96W / N127W -T RPL9A Spread SD-Ura solid plates and incubate upside down at 30°C for 48-72 hours.

[0136] Yeast competent cells were prepared using the ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit:

[0137] (1) Pick a newly activated single colony from the SD-Ura plate and inoculate it into 10 mL of SD-Ura liquid medium. Incubate at 30°C with shaking until OD. 600 = Approximately 0.8-1.0;

[0138] (2) Centrifuge at 500g for 4 minutes at room temperature, and discard the supernatant;

[0139] (3) Add 10 mL of Frozen-EZ Solution 1 to suspend the bacterial cells, centrifuge at 500 g for 4 min at room temperature, and discard the supernatant;

[0140] (4) Add 1 mL of Frozen-EZ Solution 2 suspension and dispense 50 μL into sterile 1.5 mL EP tubes;

[0141] (5) Do not use liquid nitrogen to quickly freeze competent cells. Instead, slowly cool them to -70°C (4°C, 1 h; -20°C, 1 h; -40°C, 1 h; -70°C for storage).

[0142] 3. Constructing yeast strain MD-Aa48

[0143] (1) Take 0.2-1 μg of recombinant plasmid pESC-Leu::AaTPS48 (less than 5 μL) and mix it with 50 μL of MD competent cells;

[0144] (2) Add 500 μL of Frozen-EZ Solution 3 and mix vigorously;

[0145] (3) Incubate at 30℃ for 1-2 hours, mixing 2-3 times during the process;

[0146] (4) Take 50-150 μL of the incubated bacterial solution, spread it on the corresponding defective SD plate (SD-Ura-Leu), dry it, and then incubate it upside down at 30℃ for 48-96 h.

[0147] 4. Fermentation

[0148] (1) Pick a single colony that has grown on an SD-Ura-Leu solid plate and place it in 10 mL of SD-Ura-Leu liquid culture medium. Incubate at 30°C for 200 g for 48 h.

[0149] (2) Collect bacterial cells by centrifugation at 5000g for 5 min at room temperature, transfer them into 20mL of SD-Ura-Leu liquid medium (SD-Ura-Leu + 2% galactose), and induce culture at 30℃ for 200g for 72h.

[0150] 5. Extraction of fermentation products

[0151] The target components are terpenoids, which are lipid-soluble and readily soluble in ethyl acetate. Therefore, ethyl acetate was chosen as the solvent for extraction of the target terpenoids. The extraction steps are as follows:

[0152] (1) Collect the fermented bacterial liquid after fermentation and add an equal volume of ethyl acetate;

[0153] (2) Ultrasonic sterilization for 1 hour, with repeated shaking and oscillation during the period;

[0154] (3) Take the upper organic phase at room temperature (5000g) for 5 min, add an appropriate amount of anhydrous sodium sulfate (dried at 120℃ for 30 min), and shake while adding to remove the water from the extract;

[0155] (4) Concentrate to near dryness using a rotary evaporator.

[0156] (5) Take the concentrate, pass it through a 0.22μm PTFE syringe filter, store the filtrate in a liquid phase vial, seal it with a sealing film, and store it in a refrigerator at 4℃.

[0157] 5. GC-MS detection of fermentation products

[0158] The target compound was detected by gas chromatography-mass spectrometry (GC-MS): The GC-MS analysis system was a Thermo TRACE1310 / TSQ 8000 gas chromatograph with a TG-5MS capillary column (30 m × 0.25 mm × 0.25 μm); the temperature program was as follows: initial column temperature 50 °C, increasing at 5 °C / min. -1 The temperature was increased to 85°C and maintained for 3 minutes; then increased at a rate of 2°C / min. -1 The rate increases to 100℃; at 4℃·min -1 The rate increases to 200℃; at 20℃·min -1 The injection rate was increased to 300℃ and held for 5 min. The injection port temperature was 250℃, the carrier gas was He, and the carrier gas flow rate was 1 mL / min. -1 The sample was injected via split injection at a volume of 1.0 μL, a split ratio of 10:1, and a split flow rate of 10 mL / min. -1 The mass spectrometry conditions were as follows: ionization mode: EI, electron energy: 70 eV; ion source temperature: 250℃, transfer line temperature: 250℃. The scanning mode was full scan, with a detection range of 40–400 m / z; solvent delay: 3 min.

[0159] GC-MS analysis results are as follows Figure 4 As shown: Yeast strains containing the pESC-Leu::AaTPS48 recombinant plasmid are able to synthesize myrcene.

[0160] V. Conclusion

[0161] This invention cloned the AaTPS48 gene from Artemisia argyi leaves. This gene is the first key enzyme gene obtained from Artemisia argyi that can catalyze both the formation of monoterpenes by GPP and the formation of sesquiterpenes by FPP. Experiments have demonstrated that the AaTPS48 protein of this invention can catalyze both the formation of the monoterpenoid myrcene by GPP and the formation of the sesquiterpenoid (trans)-β-farnesene by FPP. This not only plays an important role in the biosynthesis of terpenes such as myrcene and (trans)-β-farnesene in Artemisia argyi, but also has significant theoretical and practical implications for regulating and producing plant terpenes and cultivating high-quality Artemisia argyi.

[0162] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also fall within the protection scope of the invention, which is defined by the claims.

Claims

1. A type of Artemisia argyi terpenoid synthase AaTPS48, characterized in that, It is a protein of either a) or b): a) The amino acid sequence is that of the protein shown in SEQ ID No. 1; b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No.

1.

2. A biomaterial related to the Artemisia argyi terpenoid synthase AaTPS48 described in claim 1, characterized in that, It is any one of A1) to A12) below: A1) A nucleic acid molecule encoding the AaTPS48 protein; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) A recombinant vector containing the expression cassette described in A2); A5) Recombinant microorganisms containing the nucleic acid molecules described in A1); A6) Recombinant microorganisms containing the expression cassette described in A2); A7) Recombinant microorganisms containing the recombinant vector described in A3); A8) Recombinant microorganisms containing the recombinant vector described in A4).

3. The biomaterial related to Artemisia argyi terpenoid synthase AaTPS48 according to claim 2, characterized in that, A1) The nucleic acid molecule is a gene as shown in 1), 2), or 3) below: 1) Its coding sequence is the cDNA molecule shown in positions 1-1728 of SEQ ID No. 2; 2) Having 75% or more identity with the nucleotide sequence defined in 1), and encoding a cDNA molecule or genomic DNA molecule of the protein of claim 1; 3) Hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and to a cDNA molecule or genomic DNA molecule encoding the protein of claim 1.

4. The application of the Artemisia argyi terpene synthase AaTPS48 as described in claim 1 in the preparation of terpene compounds.

5. The use of the biomaterials related to the Artemisia argyi terpene synthase AaTPS48 as described in claim 2 or 3 in the preparation of terpene synthases or terpene compounds.

6. The use of the geraniol terpene synthase AaTPS48 of claim 1 in catalyzing the formation of myrcene from geraniol pyrophosphate (GPP) or the formation of (trans)-β-farnesene from farnesol pyrophosphate (FPP).

7. The use of the biomaterial related to the geraniol terpene synthase AaTPS48 as described in claim 2 or 3 in catalyzing the formation of myrcene from geraniol pyrophosphate (GPP) or the formation of (trans)-β-farnesene from farnesol pyrophosphate (FPP).

8. A method for synthesizing a terpene compound, characterized in that, The Artemisia argyi terpene synthase AaTPS48, the substrate, and the enzyme buffer described in claim 1 were mixed and reacted to obtain terpene compounds.

9. The method for synthesizing terpenoid compounds according to claim 8, characterized in that, The substrate is gerany pyrophosphate or farnesyl pyrophosphate; the terpenoid compound is a monoterpenoid compound and / or a sesquiterpenoid compound.

10. A method for the biosynthesis of terpenoid compounds, characterized in that, The terpene synthase AaTPS48 described in claim 1 is introduced into Saccharomyces cerevisiae to biosynthesize terpenoid compounds; the terpenoid compounds are monoterpenoid compounds and / or sesquiterpenoid compounds.

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