Terpene synthase rri tps1 synthesizing gamma-humulene in roxburgh rose and its encoding product and application

Abstract

The present application relates to the technical field of genetic engineering, and aims to provide a terpenoid synthase RriTPS1 for synthesizing gamma-humulene in Rosa roxburghii, a coding product and application thereof, a coding gene of a terpenoid synthase RriTPS1 for synthesizing gamma-humulene is cloned and functionally identified from a plant of the family Rosaceae, and the coded protein is a protein with the same function obtained by substitution, deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in Seq ID No. 2. The terpenoid synthase gene RriTPS1 is connected with an expression vector pCold-TF to construct a recombinant plasmid capable of being expressed in Escherichia coli, and then the recombinant plasmid is transformed into Escherichia coli to obtain a genetically engineered cell capable of heterogeneously synthesizing gamma-humulene. The genetically engineered cell constructed in the present application has the advantages of safety and stability, short production cycle and high product yield, and can be used for subsequent large-scale production of gamma-humulene by a bacterial system, thereby providing an efficient and feasible technical method for solving the problem of huge market demand for terpenoid components.

Description

[0001] This invention relates to the field of genetic engineering technology, and in particular to RriTPS1, a terpene synthase for synthesizing γ-humulene from prickly pear, its encoded products, and applications. Background Technology

[0002] Terpenes are the most structurally diverse family of natural products, playing an indispensable role in the life activities of most organisms. Their molecular skeleton is based on isoprene (C5) as the basic building block, and they can be classified into monoterpenes (C5+, C6+, C7+, C8+, C9 ... 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), sesquiterpenes (C 25 ), triterpenes (C 30 ) and tetraterpenes (C 40 Sesquiterpenoids, such as sesquiterpenes, are composed of three isoprene units containing 15 carbon atoms. Their complex and diverse structures endow them with rich biological activities, thus they are widely used in clinical drug development. They have extremely high application value and economic potential, and have attracted much attention from the scientific research and industry sectors.

[0003] prickly pear ( Rosa roxbungi The prickly pear, a perennial shrub belonging to the genus Rosa in the family Rosaceae, is also known as the prickly pear, mountain king fruit, Buddha's fruit, and thorny pomegranate. As a unique medicinal and edible plant in western my country, it possesses high nutritional and medicinal value, being rich in prickly pear polysaccharides, terpenoids, vitamins, and flavonoids. Terpenoids are an important class of bioactive metabolites in prickly pear, and terpenoid synthases, as key enzymes catalyzing the biosynthesis of terpenoids, are crucial nodes in the terpenoid metabolism process. Currently, the prickly pear terpenoid synthase gene RriTPS1 has not been isolated and identified, and its application in the synthesis of γ-humulene has not yet been observed. Summary of the Invention

[0004] The purpose of this invention is to develop the function of the prickly pear sesquiterpene synthase gene, and to provide the γ-humulene synthase gene RriTPS1, its encoded product, and its applications.

[0005] To achieve the above objectives, the present invention discloses the prickly pear terpene synthase gene RriTPS1, wherein the gene RriTPS1 has a nucleotide sequence as shown in SEQ ID NO. 1 or a nucleotide sequence as shown in SEQ ID NO. 1 with one or more nucleotides substituted, deleted or added and expressing the same functional protein.

[0006] The sequence of SEQ ID NO.1 is as follows: Without affecting its activity, the amino acid sequence shown in SEQ ID NO 2 is obtained by substituting, deleting, or adding one or more amino acids.

[0007] The sequence of SEQ ID NO.2 is as follows: MSVPDLVSELAQTPNITAPPDVTRRSANYSPSIWGDHFLSYASIEAADGKSKKHIQDLKEELKRMIMAPAKRPSQKLHFIDHIQRLGVSYHFEDEIDQILQQVHREEEYDDLCTTALSFRLLRQQGYNISCNMFNKFKD DDGRFKESLIDDVLGLLSLYEASHLQMPGEDILNDALTFTTAHLESAAHRLSSSSLLWKQVTHALYQPLWQGMARIEARHYLSIYQEDDSHNETLLNFAKLDFNTVQKVHQKELSEITRWWKDLDFVTKLPFARDKVVE AYFWALAVYFQPEYYFARIVLAKAVAIITVIDDIYDVHGTYEELESFTEAIERWNISVVDQLPDYMKVCYEGLLSFFTELEESLANKGILYRLHYAREAFKAQVRAYFQEAKWLKQKYTPTMEEYMSVERDASFFMVAV VSFVGMGVIVTKDSMDWVFNEPKISKAASIIGRLMNDLVSHKFEQKREHIASAVECYMKQYGVTDEEAEVELTKQVNDAWQDINEEWLNATSIPRPLLLLILNFARSTEVLYKGEDVFTHSENVLKGHVVSLFIEPVPI This invention discloses specific primers for the γ-humulene terpene synthase gene RriTPS1, including an upstream primer as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4.

[0008] The present invention also discloses a recombinant expression vector of the above-mentioned γ-humulene terpene synthase gene RriTPS1, wherein the recombinant expression vector is pCold-TF / RriTPS1.

[0009] The present invention also discloses the application of the above-mentioned γ-humulene terpene synthase gene RriTPS1 in the preparation of sesquiterpene compound γ-humulene, including transfecting the prickly pear sesquiterpene synthase gene RriTPS1 into cells, expressing prickly pear sesquiterpene synthase in host cells, and the prickly pear sesquiterpene synthase promoting the synthesis of sesquiterpene compound γ-humulene.

[0010] The sesquiterpene compound is γ-humulene.

[0011] As an improvement, the beneficial effects of this invention are as follows: The terpene synthase gene RriTPS1 provided by this invention is the first to be cloned and prepared from the prickly pear plant, and the product is a single product γ-humulene. RriTPS1 is a key regulatory gene in the γ-humulene synthesis pathway. This enzyme can use farnesyl pyrophosphate (FPP) as a substrate to cyclize and synthesize the macrocyclic sesquiterpene γ-humulene. Using the gene provided by this invention, the sesquiterpene component γ-humulene can be synthesized in large quantities through genetic engineering technology, providing an effective method to meet the huge market demand for γ-humulene. Attached Figure Description

[0012] Figure 1 Agarose gel electrophoresis image of γ-humulene synthase RriTPS1; Figure 2 This is a multiple sequence comparison diagram of γ-humulene synthase RriTPS1; Figure 3 Predictive analysis of the structural and functional domains of γ-humulene synthase RriTPS1; Figure 4 Secondary structure prediction for γ-humulene synthase RriTPS1; Figure 5 Predicted tertiary structure of γ-humulene synthase RriTPS1; Figure 6 Phylogenetic tree of γ-humulene synthase RriTPS1; Figure 7 SDS-polyacrylamide gel electrophoresis image of γ-humulene synthase; Figure 8 GC-MS analysis of the engineered Escherichia coli extract containing γ-humulene synthase RriTPS1; Figure 9 GC-MS analysis of FPP formation products catalyzed by the protein of γ-humulene terpene synthase RriTPS1; Detailed Implementation

[0013] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. Identical components are indicated by the same reference numerals. It should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings, while the terms "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively.

[0014] To make the content of this invention easier to understand, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Identical components are indicated by the same reference numerals.

[0015] Obtaining and bioinformatics analysis of the cDNA sequence of the gene encoding γ-humulene synthase RriTPS1: Total RNA was extracted from *Rhizopus rosa-sinensis* using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Vinozan Biotechnology Co., Ltd.). The procedure was described in the kit's instruction manual. RNA was obtained and then reverse transcribed into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit. PCR amplification was performed using specific upstream and downstream primers RriTPS1-F / RriTPS1-R (as shown in Table 1) to obtain the full-length cDNA sequence of the RriTPS1 gene (e.g., Seq ID No. 1). Table 1. Specific upstream and downstream primer base sequences RriTPS1-F Seq ID No. 3 ATGTCTGTCCCTGATTTAGTGTC RriTPS1-R Seq ID No. 4 TTATATGGGGACGGGTTCGATAA The open reading frame (ORF) of the gene encoding the γ-humulene synthase RriTPS1 is 1671 bp (Seq ID No. 1). Figure 1 As shown, encoding 556 amino acids (Seq ID No. 2) with a molecular weight of 64.4 kDa, the amino acid sequence encoded by this γ-humulene synthase RriTPS1 gene contains typical DDXXD and NSE / DTE conserved motifs, such as... Figure 2 As shown.

[0016] Protein domain analysis using NCBI's Conserved Domain Database (CDD) indicates that RriTPS1 belongs to the Isoprenoid-Biosyn-C1 superfamily and is an isoprenoid biosynthetic enzyme. The analysis results are as follows: Figure 3 As shown.

[0017] Analysis of the secondary structure of the RiTPS1 protein using the SOPMA online software revealed that α-helixes accounted for 68.53%, β-turns for 2.16%, and regular coils for 29.32%, indicating that α-helixes are the dominant form of the protein's secondary structure. Figure 4 As shown.

[0018] Using the SWISS-MODEL program (http: / / swissmodel.expasy.org / ), homology modeling and tertiary structure prediction were performed based on the amino acid sequence of RriTPS1. The obtained tertiary structure is as follows: Figure 5 As shown in the figure. The RriTPS1 protein model used template 5ily.1.A, with a model score of 0.79 and sequence homology of 46.00%. A Neighbor-joining phylogenetic tree was constructed using MEGA11 software with 1000 bootstrap repetitions. Phylogenetic relationships were analyzed between 45 functionally identified plant terpene synthase gene sequences and the amino acid sequence of *RriTPS1* from *Prickly Pear*. The results are shown in the figure. Figure 6 As shown, the RriTPS1 gene of prickly pear belongs to the terpene synthase a family (TPS-a), and it is most closely related to the PdTPS2 gene of almond. PdTPS2 (MK426620.1) is a gene that has been reported to have catalytic function. Therefore, the RriTPS1 gene is predicted to have the ability to catalyze the biosynthesis of terpenes.

[0019] RNA was extracted from prickly pear using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Winozan Biotechnology Co., Ltd.). The procedure was described in the kit's instruction manual. RNA was obtained and reverse transcribed into cDNA using the SMARTTMRACE cDNA Amplification Kit. RriTPS1-F(Xho) and RriTPS1-R(Hind) (as shown in Table 4) were used as primers.

[0020] Table 2 shows the amplification of the target gene using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase. The reaction system is as follows: <![CDATA[ddH2O]]> Up to 50 μL 2×Phanta Max Buffer 25 μL dNTP Mix 1 μL RriTPS1-F(Xho) 2 μL RriTPS1-R(Hind) 2 μL Phanta Max Super-Fidelity DNA Polymerase Prickly Pear cDNA 1 μL 1 μL Table 3. After centrifuging and mixing the above system, amplify the target gene according to the procedure in the table below. 95 ℃ 3 min 1 95 ℃ 15 sec 58 ℃ 15 sec 35 72 ℃ 1 min 72 ℃ 5 min 1 16 ℃ 2 min 1 After the reaction, the product band size was detected by 1% agarose gel electrophoresis, and the target fragment was recovered and purified by gel extraction. Restriction endonucleases were used. Xho Ⅰ and Hind III. The pCold-TF vector was double-digested with enzymes. The underlined regions in the primer sequences represent the corresponding restriction sites (as shown in Table 4). The digestion system was incubated at 37°C for 2 h, followed by detection by 1% agarose gel electrophoresis and recovery of the purified vector fragment. Homologous recombination ligation was performed between the purified PCR product and the digested and recovered pCold-TF vector using recombinase. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar containing 0.34 mg / mL ampicillin. The cells were incubated overnight at 37°C. After single colonies emerged, single colonies were picked for PCR identification and enzyme digestion verification. Positive clones were screened and further sent to a sequencing platform for DNA sequence identification.

[0021] Table 4. Base sequences of specific upstream and downstream primers RriTPS1-F(Xho) atggagctcggtaccctcgagATGTCTGTCCCTGATTTAGTGTC RriTPS1-R(Hind) agactgcaggtcgacaagcttTTATATGGGGACGGGTTCGATAA

[0022] Plasmids were extracted from the correctly sequenced recombinant pCold-TF / RriTPS1 strain and transformed into the *E. coli* expression strain BL21(DE3). Screening was performed using LB agar plates supplemented with ampicillin (1.0 mg / mL). Single colonies were randomly selected for colony PCR verification. Verified single colonies were inoculated into centrifuge tubes containing 1.5 mL of ampicillin-containing LB liquid medium and incubated overnight at 37°C with shaking. The activated bacteria were then inoculated at a 1:100 ratio into 100 mL of LB liquid medium and incubated at 37°C with shaking until OD (out of control) was reached. 600The value was approximately 0.5. 2 mL of bacterial culture was used as a control, and the remainder was added to 98 μL of 0.5 mM IPTG. The mixture was induced overnight at 16°C. The relevant protein purification buffers were prepared using a His-tagged protein purification kit. Bacterial cultures induced at low temperature with Binding Buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 10 mM imidazole) and Soluble Binding Buffer (2.0 mM Tris-HCl pH 7.9, 500 mM NaCl, 500 mM imidazole) were centrifuged at 12,000 rpm for 10 min at 4 °C, the supernatant was discarded, and the precipitate was resuspended in 5 mL of bacterial lysis buffer. The bacterial cells were then sonicated on ice for 10 min. 100 μL was reserved as a control, and the remaining samples were centrifuged at 12,000 rpm for 3 min at 4 °C. The supernatant was collected, diluted with an equal volume of Binding Buffer, loaded onto the column, and the flow-through was collected. The column was washed with 45 mL of Soluble Binding Buffer to remove impurities. Elution was performed using 3 mL of Soluble Binding Buffer, and the elution peak was collected as the purified protein. An equal volume of glycerol was added, mixed well, and stored at -20°C. Protein solubility was analyzed by 10% SDS-PAGE following the relevant procedures. The purified RriTPS1 recombinant protein contained a trigger factor, the size of which was as expected. Figure 7 As shown.

[0023] The recombinant pCold-TF / RriTPS1 plasmid was transformed into the MEV20 strain. Transformed colonies were screened using LB agar plates containing ampicillin (1.0 mg / mL), and single colonies were picked for verification. Verified single colonies were inoculated into 500 mL of TB medium containing the same antibiotic and cultured at 37°C with shaking until OD (occurrence limit). 600 The value was approximately 0.6. 0.5 mM IPTG was added, and the mixture was incubated at 16°C for 3 days. Petroleum ether extraction was performed, and the organic layer was collected and concentrated to 1 mL under reduced pressure to obtain the in vivo enzyme activity reaction sample. The sample was analyzed by GC-MS. The petroleum ether extract was further purified by silica gel column chromatography, eluting with n-hexane to yield the compound γ-humulene.

[0024] Prepare a 10× reaction buffer: 50 mM HEPES pH 7.4, 50 mM MgCl2, 1 M KCl, 20 mM MTT, and store at -20℃. Take 40 μL of purified protein, 40 μL of reaction buffer, 10 μL of FPP, and deionized water to a final volume of 400 μL. Mix all components thoroughly and incubate overnight at 30℃. Extract three times with an equal volume of petroleum ether, centrifuge at 12000 rpm for 10 min, dry with nitrogen, add 100 μL of petroleum ether, centrifuge at 12000 rpm for 10 min, and collect 80 μL of the supernatant for GC-MS analysis.

[0025] GC-MS chromatographic conditions: HP-5MS quartz capillary (30m×250μm×0.25μm); column temperature program settings: initial temperature 50℃, hold for 1 min; increase to 250℃ at 10℃ / min, hold for 2 min; split ratio 10:1, injection volume 5μL, carrier gas helium, helium flow rate 1 mL / min, column inlet pressure 40 kPa.

[0026] GC-MS mass spectrometry conditions: EI source, ion source temperature 250℃, electron energy 70 eV, mass range 35-550 amu.

[0027] In summary, this invention clones the gene encoding terpene synthase RriTPS1 from *Prickly pear*. By transferring the RriTPS1 gene into cells and expressing it in host cells, the synthesis of the macrocyclic sesquiterpene compound γ-humulene is promoted. This technology can be used to subsequently produce large quantities of γ-humulene through bacterial cells, providing an effective method to meet the huge market demand for terpene components. The gene provided by this invention can be used to increase the content of the terpene compound γ-humulene in *Prickly pear* through genetic engineering techniques.

[0028] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. RriTPS1, a terpene synthase for synthesizing γ-humulene from prickly pear, characterized in that, Includes the nucleotide sequence shown in SEQ ID NO.

1.

2. The product encoded by RriTPS1, a terpene synthase for synthesizing γ-humulene from *Rhizopus pilosa* according to claim 1, is characterized in that... It includes the amino acid sequence shown in SEQ ID NO.2, which, compared to the amino acid sequence shown in SEQ ID NO.2, is characterized by substitution, absence, or addition of one or more amino acids but expresses the same function.

3. The encoded product of RriTPS1, a terpene synthase for synthesizing γ-humulene from *Rhizopus pilosa* according to claim 2, is characterized in that... Specific primers for the terpene synthase RriTPS1, including γ-humulene, include an upstream primer as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No.

4.

4. The terpene synthase RriTPS1 for synthesizing γ-humulene from *Rhizopus pilosa* and its encoded product as described in claim 3, characterized in that, A recombinant expression vector containing the γ-humulene terpene synthase RriTPS1, wherein the recombinant expression vector is pCold-TF / RriTPS1.

5. The application of RriTPS1, a terpene synthase for synthesizing γ-humulene from prickly pear according to claim 1, is characterized in that, This includes the transfection of the γ-humulene terpene synthase RriTPS1 into cells and the expression of γ-humulene terpene synthase in host cells.

6. The application of RriTPS1, a terpene synthase for synthesizing γ-humulene from prickly pear according to claim 5, is characterized in that, The sesquiterpene compound is γ-humulene.

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