In-vitro synthesis of terpene aroma substances by using OfTPS6 gene

Abstract

The invention discloses a method for synthesizing terpene aroma substances in vitro by using an OfTPS6 gene, which comprises the following steps: screening out a key terpene synthase gene OfTPS6 from osmanthus fragrans, expressing the key terpene synthase OfTPS6 by using a prokaryotic expression system, carrying out an in-vitro enzyme activity experiment on the purified OfTPS6 protein by using GPP as a substrate, detecting a product by using headspace solid-phase microextraction in combination with GC-MS (Gas Chromatography-Mass Spectrometry), and detecting the terpene aroma substances by using an OfTPS6 gene. The result proves that the OfTPS6 can be used for catalyzing the GPP to generate terpene aroma substances such as linalool, beta-myrcene, alpha-pinene, cis-beta-ocimene, D-limonene and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for synthesizing terpene aroma substances in vitro by utilizing the OfTPS6 gene. Background Art

[0002] In recent years, the flavor and fragrance industry has developed rapidly, and the essential oil market of traditional Chinese aromatic plants has great potential. In 2022, domestic essential oil sales will exceed 10.5 billion yuan, and since 2016, it has been widely used in the food, cosmetics and health care markets at an annual growth rate of more than 20%. Osmanthus is not only one of the top ten famous flowers in China, but also an important traditional aromatic plant. Studies have shown that terpene compounds such as linalool and its oxides, β-myrcene, cis-β-ocimene, and D-limonene are the most important aroma active substances in osmanthus essential oil and fresh flowers, and play a key role in the fragrance and fragrance intensity of the product. Terpene compounds are a class of hydrocarbon compounds formed with isoprene (C5) as the basic structural unit. They are key aroma active substances with abundant and diverse content in floral fragrance. They also have health care values ​​such as relieving anxiety, sterilization, and anti-oxidation, and are widely used in medicine, food, cosmetics and other fields.

[0003] Terpene synthase (TPS) is the last key enzyme in the synthesis of terpenes, which can catalyze a variety of substrates to produce monoterpenes, sesquiterpenes and diterpenes. Changes in the TPS gene structure can produce different catalytic functions and activities, which is an important reason for the structural diversity of terpene compounds in nature. TPS has diverse functions and can catalyze a variety of substrates to produce monoterpenes, diterpenes and sesquiterpenes. For example, HcTPS7 catalyzes geranyl diphosphate (GPP) to produce sabinene, α-terpene, γ-terpene, α-pinene and other products. Therefore, TPS is the determining factor in the production of a variety of terpene compounds in plants. Summary of the invention

[0004] The present invention aims to screen out a key terpene synthase gene OfTPS6 from osmanthus fragrans, and synthesize terpene aroma substances in vitro by prokaryotic expression of the key terpene synthase OfTPS6.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The use of OfTPS6 protein in catalyzing geranyl diphosphate to synthesize terpene aroma substances, the amino acid sequence of the OfTPS6 protein is shown in SEQ ID NO.2, and the terpene aroma substances include linalool, β-myrcene, α-pinene, cis-β-ocimene, and D-limonene.

[0007] The method for synthesizing terpene aroma substances in vitro using the OfTPS6 gene comprises expressing the OfTPS6 protein in vitro, and using the OfTPS6 protein to catalyze geranyl diphosphate to synthesize terpene aroma substances. Specifically, a prokaryotic expression vector containing the OfTPS6 gene is transformed into Escherichia coli, the culture is expanded and the expression of the OfTPS6 protein is induced, and the OfTPS6 protein is used to catalyze geranyl diphosphate to synthesize terpene aroma substances such as linalool, β-myrcene, α-pinene, cis-β-ocimene, and D-limonene. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The products of OfTPS6-catalyzed GPP were detected by GCMS. In the figure, 1 is β-myrcene, 2 is D-limonene, 3 is α-pinene, 4 is cis-β-basil, and 5 is linalool. DETAILED DESCRIPTION

[0009] Example 1 Screening and cloning of the key terpene synthase gene OfTPS6

[0010] The inventors identified a key terpene synthase gene from the genome and transcriptomes of 'Boye Yingui' at different flowering stages, and named the gene OfTPS6.

[0011] 1. RNA extraction from petals of 'Boye Yingui'

[0012] (1) Weigh 0.2 g of sample, grind it thoroughly in liquid nitrogen and place it in a 2 mL enzyme-free centrifuge tube. Quickly add 1 mL of TRlzon Reagen and mix well to fully lyse the sample.

[0013] (2) Leave at room temperature for 5 min to allow the protein-nucleic acid complex to completely separate;

[0014] (3) Add chloroform at a ratio of 200 μL chloroform per 1 ml TRizon Reagent, cap the tube, shake vigorously for 15 seconds, and leave at room temperature for 2 minutes;

[0015] (4) Centrifuge at 4°C and 12,000 rpm for 10 min. The sample is then divided into three layers: a red organic phase, an intermediate layer, and an upper colorless aqueous phase. The RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube.

[0016] (5) Add an equal volume of 70% ethanol (prepared with RNase-free water) to the aqueous solution obtained, invert and mix; add the entire solution to the RNA adsorption column;

[0017] (6) Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0018] (7) Add 700 μL of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0019] (8) Add 500 μL of Buffer RW2 to the adsorption column, centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0020] (9) Repeat step 8 and place the adsorption column at room temperature for several minutes to dry thoroughly;

[0021] (10) Place the adsorption column in a new enzyme-free centrifuge tube, add 30-50 μL RNase-Free Water, leave at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, collect the RNA solution, and store at -80°C.

[0022] 2. Design primers and amplify target genes

[0023] According to the sequence information of the terpene synthase gene OfTPS6 in the transcriptome of 'Boye Yingui', specific primers (upstream primer 5'-ATGGAGTGTACAATGAGGAGTATTT-3', downstream primer 5'-TCAATTAATAGGAGTGAAGAACAAA-3') were designed using Primer5.0, and cloning was performed using the cDNA of 'Boye Yingui' as a template. The PCR reaction system and procedure are shown in Table 1. The PCR product was purified using 1.5% gel agar, and the product was recovered. The experimental method was referred to the agarose gel DNA recovery kit. The recovered product was connected to the pTOPO-TA vector to form a circle, and the connection product was transformed into Escherichia coli DH5α by heat shock, cultured at 37°C overnight, and the M13F and M13R vector universal primers were used to detect the positive monoclonal clones. The positive monoclonal clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the monoclonal clones with correct sequencing were shaken and preserved in a -80°C refrigerator. The CDS sequence of the OfTPS6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0024] Table 1 PCR reaction system and procedure

[0025]

[0026] Example 2: In vitro catalytic synthesis of terpene aroma substances by TPS6 protein

[0027] 1. Expression vector construction

[0028] (a) Vector linearization: Select Pet-21b vector and use NdeI and XhoI to double-digest to obtain a linearized vector;

[0029] (b) Homologous arm cloning of target fragment: using the correctly sequenced plasmid (pTOPO-TA-OfTPS6) as a template, CEDesign V1.04 was used to design homologous arm primers containing restriction sites (forward primer: 5'-AAGTTGATGCACATATGGAGTGTACAATGAGGAGCATG-3', reverse primer: 5'-cagccggatcCTCGAGTTAATTAATAGGAGTGAAGAACAAAGCC-3'), PCR amplification was performed according to Table 1, the product was gel agar purified, and recovered by DNA gel recovery kit;

[0030] (c) Homologous recombination: The linearized vector and the recovered product of the homology arm clone of the target fragment were mixed in a certain ratio, and reacted at 37°C for 30 minutes under the catalysis of the homologous recombination enzyme ExnaseII to connect the target fragment to the vector to form a circular plasmid. The recombination reaction system is shown in Table 2.

[0031] Table 2 Homologous recombination reaction system

[0032]

[0033] (d) Transformation of recombinant plasmid into Escherichia coli DH5α: The recombinant plasmid was transferred into Escherichia coli DH5α using the heat shock method and cultured in an incubator at 37°C overnight. The vector universal primers were used for single clone positive detection. The positive single clone was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the correct plasmid was returned for sequencing.

[0034] 2. Prokaryotic expression

[0035] The successfully constructed positive plasmid was transformed into Rosetta2 (DE3) cells, and 5 μl of the transformed positive bacteria was added to 10 ml of LB liquid medium containing Amp for overnight culture, and then expanded at a ratio of 1:100 to OD 600 The value was 0.6-0.8, IPTG was added to a final concentration of 1 M, and the culture was continued in a 16°C shaker at 200 rpm for 14-16 hours.

[0036] 3. Protein purification and concentration

[0037] After induction, centrifuge at 4°C and 4000rpm for 20 minutes, discard the supernatant, resuspend the precipitate with 1×PBS containing DTT and PMSF, add lysozyme with a final concentration of 1mg / ml, and place for 30 minutes; use high-pressure crushing, 750-850pa high-pressure crushing for 10 minutes, centrifuge at 4°C to take the supernatant. Transfer the supernatant to a chromatography column containing Ni-NTA Agarose (the column was rinsed with Lysis buffer 3 times before use), pass through the nickel column 5 times, wash the impurities with 10-15 column volumes of Wash buffer, and then elute the target protein with 10-15 column volumes of Eluate buffer, 2ml each time, for a total of 5 times, and collect the target protein.

[0038] The collected target protein was added into a 30KDa ultrafiltration tube and concentrated by centrifugation at 4°C and 4000 rpm for 15 minutes. 5 μl of the concentrated protein was used for gel running and the rest was used for in vitro enzyme activity experiments.

[0039] 4. SDS-PAGE gel electrophoresis

[0040] SDS-PAGE gel is used to detect purified proteins. The main steps are as follows: Use a 7.5% PAGE gel fast paper cup kit to prepare a 1.00mm PAGE gel. Add 5μl of protein loading buffer to the purified protein, boil at 99℃ for 5 minutes to denature it, and then load the sample. During the concentration gel stage, the current is set to 15-20mA. After the sample enters the separation gel, the current is changed to 25-30mA. Continue running the gel until it is about 1cm from the bottom.

[0041] Coomassie Brilliant Blue staining and decolorization: Carefully pick out the PAGE gel after running and place it on a glass dish, add Coomassie Brilliant Blue fast staining solution, immerse the gel in the staining solution, and stain it on a shaker for 2 hours. After staining, pour out the staining solution, wash it once with Coomassie Brilliant Blue fast decolorizing solution, wash it twice with distilled water, and add Coomassie Brilliant Blue fast decolorizing solution to decolorize overnight. SDS-PAGE gel detection found that the protein can be expressed and the molecular weight is consistent with expectations, which can be used for in vitro enzyme activity experiments.

[0042] 5. In vitro enzyme function verification

[0043] In a 25 ml glass bottle, add 60 μg OfTPS6 protein, 2 mM GPP substrate, 50 mM HEPES, 75 mM MgCl2, 5 mM DTT, and 5% glycerol, and make up to 300 μl with ddH2O. React at 30°C for 2 hours. Then insert the extraction head (50 / 30 μm DVB / CAR / PDMS) into the reaction bottle and perform headspace extraction for 30 minutes. After the extraction, insert the extraction head into the GC-MS injection port for sample loading.

[0044] Gas chromatography-mass spectrometry conditions: DB-5MS column (30 m × 0.25 mm × 0.25 μm, Themo Scientific, Bellefonte, PA, USA), carrier gas was high-purity helium (99.999%), split ratio 20:1, flow rate 1 ml / min. The temperatures of the ion source and injection port were 280 °C and 230 °C, respectively, and the temperature of the transfer line was 250 °C.

[0045] Temperature program: 40°C for 3 min, then heated to 120°C at a rate of 3°C / min and kept for 3 min, then heated to 220°C at a rate of 8°C / min and kept for 2 min. MS conditions are as follows: EI (electron impact) ion source, electron impact energy 70 eV, positive ion scan mode, mass scan range m / z 40-450 amu.

[0046] Substance identification method: GC-MS raw data were processed and exported using Xcalibur software. Volatile substance identification was based on standard samples and the NIST mass spectrum database (NIST 2017). The identification results were based on a mass spectrum data of each component with a matching degree of >80% with the mass spectrum data of the standard substances provided in the spectrum library NIST (2017). At the same time, the Kovats' Retention Indices (RI) calculated using the normal alkane standard sample (C8–C30) under the same heating program were used for further qualitative confirmation. The RI value calculation formula is as follows: RI = (R TX -R T ) / (R T+1 -R T )×100+C RT ×100. Where: R TX is the retention time of the substance X to be tested / min; R T and R T+1 is the retention time of the normal alkanes immediately before and after the aroma substance X / min; C RT is the number of carbon atoms in normal alkane corresponding to the time of the substance to be tested.

[0047] Substance quantification method: internal standard quantification method is used. The content of each aroma component (μg / g FW) = peak area of ​​each component / peak area of ​​internal standard × content of internal standard (μg) / sample amount (g) × f (f is the correction factor of each component to the internal standard, f=1).

[0048] The purified OfTPS6 protein was used as a substrate for in vitro enzyme activity experiments. Headspace solid phase microextraction combined with GC-MS was used to detect the products. The results showed that OfTPS6 could catalyze GPP to produce the main product linalool and a small amount of byproducts such as β-myrcene, α-pinene, cis-β-ocimene and D-limonene ( Figure 1 )。

Claims

1. The use of OfTPS6 protein in catalyzing geranyl diphosphate to synthesize terpene aroma substances, characterized in that: The amino acid sequence of the OfTPS6 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The terpene aroma substances include linalool, β-myrcene, α-pinene, cis-β-ocimene and D-limonene.

3. Utilize OfTPS6 A method for synthesizing terpene aroma substances in vitro, characterized in that: The method comprises expressing OfTPS6 protein in vitro, and utilizing OfTPS6 protein to catalyze geranyl diphosphate to synthesize terpene aroma substances, wherein the coding gene sequence of the OfTPS6 protein is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that will contain OfTPS6 The prokaryotic expression vector of the gene was transformed into Escherichia coli, expanded and cultured to induce the expression of OfTPS6 protein.

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