In vitro synthesis of terpene aroma substances using the OfTPS7 gene

Abstract

The present invention discloses a method for utilizing OfTPS7 A method for in vitro synthesis of terpene aroma substances and screening of key terpene synthase genes from Osmanthus fragrans OfTPS7 The key terpene synthase OfTPS7 was expressed using a prokaryotic expression system. The purified OfTPS7 protein was subjected to in vitro enzymatic activity experiments using geranyl diphosphate (GPP) as a substrate. The products were detected using headspace solid-phase microextraction combined with GC-MS. The results showed that OfTPS7 can catalyze GPP to produce terpene aroma substances such as linalool, β-myrcene, and D-limonene.

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 OfTPS7 gene. Background Art

[0002] In recent years, the flavor and fragrance industry has experienced rapid growth, and the market for essential oils from traditional Chinese aromatic plants holds enormous potential. Domestic essential oil sales are expected to exceed 10.5 billion yuan in 2022, and since 2016, they have been widely used in the food, cosmetics, and healthcare markets at an annual growth rate of over 20%. Osmanthus fragrans is not only one of China's ten famous flowers but also a significant traditional aromatic plant. Due to its diverse cultivars, extremely short flowering periods, and complex extraction processes, direct extraction of aromatic compounds from petals suffers from drawbacks such as low yield, low purity, and short production cycles. Targeted and efficient biosynthesis of osmanthus fragrans aromatic compounds offers a new path to overcome this bottleneck. In recent years, the biosynthesis industry has flourished. Biosynthesized fragrance products can be directly added to foods, health supplements, pharmaceuticals, and high-end cosmetics, and can also serve as raw materials for fine chemicals and biopharmaceuticals. These products are closely linked to the development of the healthcare industry and offer significant economic and social benefits. Compared to traditional plant extraction and chemical synthesis, biosynthesis offers advantages such as low pollution, low energy consumption, and high atom economy, enabling precise, standardized, and year-round industrial production. The key to achieving green manufacturing is to discover high-quality aroma-producing genes and establish a biosynthesis technology system for terpenoid aroma substances. Studies have shown that linalool and its oxides, β-myrcene, D-limonene and other terpene compounds are the most important aroma-active substances in osmanthus essential oil and fresh flowers, and play a key role in the fragrance and aroma 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 in floral fragrance that are abundant and diverse in variety. They also have health benefits such as relieving anxiety, sterilization, and anti-oxidation, and are widely used in medicine, food, cosmetics and other fields.

[0003] Terpene synthase (TPS) is a key enzyme in the final step of terpene synthesis, catalyzing the conversion of a variety of substrates into monoterpenes, sesquiterpenes, and diterpenes. Variations in the TPS gene structure can result in varying catalytic functions and activities, contributing significantly to the structural diversity of terpenes in nature. TPS exhibits diverse functions, catalyzing a variety of substrates into monoterpenes, diterpenes, and sesquiterpenes. For example, HcTPS7 catalyzes geranyl diphosphate (GPP) to produce sabinene, α-terpenes, γ-terpenes, α-pinene, and other products. Therefore, TPS is a key determinant of the diverse production of terpenes in plants. Summary of the Invention

[0004] The present invention aims to screen out a key terpene synthase gene OfTPS7 from osmanthus fragrans, and synthesize terpene aroma substances in vitro by prokaryotic expression of the key terpene synthase OfTPS7.

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

[0006] The use of OfTPS7 protein in catalyzing geranyl diphosphate to synthesize terpene aroma substances, the amino acid sequence of the OfTPS7 protein is shown in SEQ ID NO.2, and the terpene aroma substances include linalool, β-myrcene and D-limonene.

[0007] A method for synthesizing terpene aroma compounds in vitro using the OfTPS7 gene includes expressing the OfTPS7 protein in vitro and using the OfTPS7 protein to catalyze the synthesis of terpene aroma compounds from geranyl diphosphate. Specifically, a prokaryotic expression vector containing the OfTPS7 gene is transformed into Escherichia coli, cultured and induced to express the OfTPS7 protein. Terpene aroma compounds such as linalool, β-myrcene, and D-limonene are synthesized from geranyl diphosphate using the OfTPS7 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 GCMS analysis of the products of OfTPS7-catalyzed GPP. In the figure, 1 is β-myrcene, 2 is D-limonene, and 3 is linalool. DETAILED DESCRIPTION

[0009] Example 1 Screening and cloning of the key terpene synthase gene OfTPS7

[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 OfTPS7.

[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 thoroughly to fully lyse the sample.

[0013] (2) Place 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 of TRizon Reagent, cap the tube, shake vigorously for 15 seconds, and let it stand at room temperature for 2 minutes;

[0015] (4) Centrifuge at 4°C and 12,000 rpm for 10 min. The sample will separate 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 and mix thoroughly by inversion; then 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 return the adsorption column to the collection tube;

[0018] (7) Add 700 μL of Buffer RW1 to the adsorption column and centrifuge at 12,000 rpm for 20 s. Discard the waste liquid in the collection tube and return the adsorption column to 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 return the adsorption column to 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] Based on the sequence information of the terpene synthase gene OfTPS7 in the 'Boye Yingui' transcriptome, specific primers (upstream primer 5'-ATGGCAGTTTGCAATTTTGTTCCAC-3', downstream primer 5'-TCAATTATGTTCTTCTTGGTTCCAT-3') were designed using Primer 5.0. Cloning was performed using the 'Boye Yingui' cDNA as a template. The PCR reaction system and procedure are shown in Table 1. The PCR product was purified using 1.5% agarose gel and recovered using the same agarose gel DNA recovery kit. The recovered product was ligated into a circle with the pTOPO-TA vector. The ligation product was heat-shocked and transformed into Escherichia coli DH5α, cultured overnight at 37°C, and single clones were tested using the M13F and M13R universal vector primers. Positive clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Correctly sequenced clones were shaken and stored in a -80°C freezer. The CDS sequence of the OfTPS7 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 TPS7 protein

[0027] 1. Expression vector construction

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

[0029] (b) Homologous arm cloning of the target fragment: Using the correctly sequenced plasmid (pTOPO-TA-OfTPS7) as a template, homologous arm primers containing restriction enzyme cleavage sites were designed using CEDesign V1.04 (forward primer: 5'-AAGTTGATGCACATATGAATACCCAAATTCCTGCTTCAGC-3', reverse primer: 5'-cagccggatcCTCGAGTTATGGTATGGGTTGAACTAATAAC-3'). PCR amplification was performed according to Table 1, and the product was gel-purified and recovered using a 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. Under the catalysis of the homologous recombinase Exnase II, the reaction was carried out at 37°C for 30 minutes 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 E. coli DH5α: The recombinant plasmid was transformed into E. 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 sequencing-corrected plasmid was returned.

[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 When the value is 0.6-0.8, add IPTG with a final concentration of 1 M, place in a 16°C shaker at 200 rpm and continue induction culture for 14-16 hours.

[0036] 3. Protein purification and concentration

[0037] After induction, centrifuge at 4000 rpm for 20 minutes at 4°C, discard the supernatant, resuspend the pellet in 1× PBS containing DTT and PMSF, add lysozyme to a final concentration of 1 mg / ml, and let it sit for 30 minutes. High-pressure crushing was performed at 750-850 Pa for 10 minutes, and the supernatant was collected by centrifugation at 4°C. The supernatant was transferred to a chromatography column containing Ni-NTA Agarose (the column was rinsed three times with Lysis Buffer before use). After passing through the nickel column five times, impurities were washed with 10-15 column volumes of Wash Buffer, and the target protein was eluted with 10-15 column volumes of Eluate Buffer, 2 ml each time, for a total of five times to collect the target protein.

[0038] The collected target protein was added to 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 gels are used to examine purified proteins. The main steps are as follows: Prepare a 1.00 mm PAGE gel using a 7.5% PAGE gel rapid cup kit. Add 5 μl of protein loading buffer to the purified protein and denature it at 99°C for 5 minutes before loading the sample. During the stacking gel phase, set the current to 15-20 mA. Once the sample enters the separating gel, reduce the current to 25-30 mA. Continue running the gel until it reaches approximately 1 cm from the bottom.

[0041] Coomassie Brilliant Blue Staining and Destaining: Carefully remove the completed PAGE gel and place it on a glass dish. Add Coomassie Brilliant Blue Rapid Staining Solution, submerging the gel in the staining solution. Stain on a shaker for 2 hours. After staining, discard the staining solution, rinse once with Coomassie Brilliant Blue Rapid Destaining Solution, rinse twice with distilled water, and destain overnight with Coomassie Brilliant Blue Rapid Destaining Solution. SDS-PAGE gel analysis indicates that the protein is expressed and its molecular weight is consistent with the expected value. It can be used for in vitro enzyme activity assays.

[0042] 5. In vitro enzyme function verification

[0043] Add 60 μg OfTPS7 protein, 2 mM GPP substrate, 50 mM HEPES, 75 mM MgCl2, 5 mM DTT, 5% glycerol to a 25 ml glass bottle, 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: A DB-5MS column (30 m × 0.25 mm × 0.25 μm, Themo Scientific, Bellefonte, PA, USA) was used, and the carrier gas was high-purity helium (99.999%) with a split ratio of 20:1 and a flow rate of 1 ml / min. The ion source and inlet temperatures were 280°C and 230°C, respectively, and the transfer line temperature was 250°C.

[0045] The temperature program was as follows: 40°C for 3 min, then increased to 120°C at a rate of 3°C / min and held for 3 min, then increased to 220°C at a rate of 8°C / min and held for 2 min. MS conditions were 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 substances were identified based on standard samples and the NIST mass spectrum database (NIST 2017). The identification results were based on the mass spectrum data of each component with a matching degree of >80% with the mass spectrum data of the standard substances provided in the NIST (2017) spectrum database. At the same time, the identification results were based on the use of normal alkane standard samples (C8–C 30 ) was calculated to further confirm the qualitative results. The RI value was calculated 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 n-alkanes immediately before and after the aroma substance X / min; C RT is the number of carbon atoms in normal alkanes corresponding to the time of the substance to be tested.

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

[0048] The purified OfTPS7 protein was subjected to in vitro enzyme activity experiments using GPP as a substrate. The products were detected using headspace solid phase microextraction combined with GC-MS. The results showed that OfTPS7 could catalyze GPP to produce the main product linalool and a small amount of β-myrcene and D-limonene ( Figure 1 ).

Claims

1. The use of TPS7 protein in catalyzing the synthesis of terpene aroma substances from geranyl diphosphate, characterized in that: The amino acid sequence of the OfTPS7 protein is shown in SEQ ID NO. 2, and the terpene aroma substance is β-myrcene and / or D-limonene.

2. Utilize OfTPS7 A method for synthesizing terpene aroma substances in vitro, characterized in that: The method comprises expressing the OfTPS7 protein in vitro, and utilizing the OfTPS7 protein to catalyze geranyl diphosphate to synthesize terpene aroma substances. The coding gene sequence of the OfTPS7 protein is shown in SEQ ID NO.1, and the terpene aroma substances are β-myrcene and / or D-limonene.

3. The method according to claim 2, characterized in that will contain OfTPS7 The prokaryotic expression vector of the gene was transformed into Escherichia coli, expanded and cultured to induce the expression of OfTPS7 protein.

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