Novel santalene synthase gene as well as encoding protein and application thereof

Abstract

The invention discloses a novel santalene synthase gene as well as an encoding protein and application thereof. The amino acid sequence of the santalene synthetase is as shown in SEQ ID NO. 2. The invention relates to a method for carrying out functional verification on a new sandalene synthase gene encoding enzyme from sandalwood and carrying out synthesis of sandalene and bergamoene in escherichia coli and tobacco leaves. According to the invention, the enzyme can be massively prepared by utilizing escherichia coli, is applied to synthesis of santalene and bergamoene, can be further applied to synthesis of bergamoene or santalene, and has important significance; the heterologous synthesis of the high-value santalene and the bergamoene in plants can also be realized, and the method has a wide industrial practical prospect and a large-scale development potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology and plant gene engineering and relates to a new santalene synthase gene and its encoding protein and application. Background Art

[0002] Sandalwood is a small tropical tree of the genus Santalum in the family Santalaceae. Wild resources are mainly distributed in India, and are now widely cultivated in Australia, Indonesia and southern China. The high-quality essential oil in the heartwood of sandalwood mainly includes (Z)-α-santalol and (Z)-β-santalol, accounting for more than 80% of the total essential oil content; at the same time, it contains lower levels of α-santalene, β-santalene, trans-α-bergamoten e, santalone, santalal, etc. Sandalwood essential oil is widely used in the fragrance industry for its fragrance, and is used in the medical field for its anti-inflammatory, antibacterial and anti-tumor activities. Due to the overexploitation of wild resources, the International Union for Conservation of Nature has listed it as an endangered species.

[0003] In recent years, the biosynthetic pathways of the main components of sandalwood essential oil have been basically clarified. Isopentenylpyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) produced by the mevalonate pathway (MVA pathway) in the plant cytoplasm are synthesized into the common precursor FPP of sesquiterpenoids under the catalysis of farnesyl pyrophosphate synthase (FPS), and then santalene synthase (SaSSy) catalyzes the synthesis of α-santalene, trans-α-bergamotene, epi-β-santalene, and β-santalene; cytochrome P450 monooxygenase (SaCYP736A167) catalyzes these santalene isomers to synthesize the main components of essential oil, (Z)-α-santalol, trans-α-bergamotol, epi-β-santalol, and (Z)-β-santalol. SaSSY is a key enzyme in this pathway and plays a vital role in the synthesis of essential oils.

[0004] Under natural conditions, it takes about 6-8 years for sandalwood to form heartwood, and 30-40 years for economic benefits to be realized. Driven by market demand and considerable economic value, wild sandalwood resources have been severely cut down and resources are in short supply. In order to explore solutions to the problem of increasingly scarce sandalwood resources, in recent years, certain progress has been made in the production of santalene and santalol using yeast heterologous chassis. The santalene synthase gene was first identified in several species of the genus Santalum, including S. album, S. austrocaledonicum, and S. spicatum. In addition, studies have reported that santalene synthesis genes exist in plants such as Cinnamomu m camphora, Curcuma wenyujin, and Clausenalansium. The products of these santalene synthases are not completely the same, especially the products catalyzed by the santalene synthase of sandalwood species are not very specific. In addition to α / β-santalene, there are also low-content isomers such as epi-β-santalene and trans-α-bergamotene. Therefore, it is of great significance to discover more santalene synthase genes and further use metabolic engineering or biosynthesis technology to synthesize and apply different products. Summary of the invention

[0005] The present invention has discovered a new santalene synthase gene (named SaSSY2) of a sandalwood species through research, which can be expressed in large quantities using genetically engineered Escherichia coli bacteria, and the enzyme has good catalytic synthesis of α-santalene, trans-α-bergamotene, epi-β-santalene, β-santalene and sabinene, especially containing a higher content of bergamotene, accounting for 49.58-58.91%, which is higher than the total content of santalene.

[0006] The present invention first provides a new santalene synthase gene, which is derived from sandalwood (S.album), and the amino acid sequence is shown in SEQ ID NO.2. The homology with the amino acid sequence of SaSSY is 91.97% ( Figure 1 The present invention further provides the use of the novel santalene synthase (SaSSY2) in the preparation of α-santalene, trans-α-bergamotene, epi-β-santalene, β-santalene and sabinene. The present invention provides a gene SaSSY2 encoding the novel santalene synthase. Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] The present invention provides a recombinant expression vector comprising the gene. Preferably, the recombinant expression vector uses a pET28a vector as a skeleton. The present invention further provides a genetically engineered bacterium comprising the recombinant expression vector. Preferably, the genetically engineered bacterium uses an Escherichia coli host cell; the Escherichia coli strain used as the host cell can be selected from Rossetta 2 (DE3).

[0008] The present invention also provides a method for preparing santalene and bergamotene, using the enzyme encoded by the gene SaSSY2, taking farnesyl pyrophosphate as a substrate, and catalyzing the synthesis of santalene and bergamotene under the action of dithiothreitol, MgCl2 and glycerol. Preferably, the amount of the catalyst in the initial catalytic reaction system is 10.0 μg / mL, the substrate concentration is 2 μg / mL, the concentrations of dithiothreitol and MgCl2 are 5 mM and 10 mM respectively, and the volume concentration of glycerol is 10%.

[0009] The present invention also provides a method for heterologously synthesizing santalene and bergamotene in plants. The gene is used to construct an Agrobacterium engineering vector in vitro, or the gene is transiently expressed in tobacco leaves to synthesize santalene and bergamotene.

[0010] The invention verifies the function of a new santalene synthase gene encoding enzyme from sandalwood, and invents and applies a method for synthesizing santalene and bergamotene in Escherichia coli and tobacco leaves. It is found that the enzyme from sandalwood can catalyze FPP in a prokaryotic expression system to generate trans-α-bergamotene (49.58%), α-santalene (30.09%), β-santalene (15.46%), epi-β-santalene (1.42%) and sabinene (3.18%). Trans-α-bergamotene (58.91%), α-santalene (28.05%), β-santalene (11.88%) and epi-β-santalene (1.16%) are synthesized heterologously using tobacco. According to the peak area calculation, the total contents of santalene and bergamotene reach 41.09-46.97% and 49.58-58.91% respectively. The present invention can realize the large-scale preparation of the enzyme by using Escherichia coli and apply it to the synthesis of santalene and bergamotene, and can further be applied to the synthesis of bergamotene or santalene, which has important significance; and can also realize the heterologous synthesis of high-value santalene and bergamotene in plants, which has broad industrial practical prospects and potential for large-scale development. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is an amino acid sequence alignment of santalene synthase (SaSSY) and a novel santalene synthase (SaSSY2);

[0012] Figure 2This is the in vitro enzymatic activity of the protein expressed by the new santalene synthase gene (SaSSY2) in Escherichia coli. A, total ion peak of the product detected by GC-MS; B, comparison of the relative content of different products; C, mass spectra corresponding to different peaks (left), mass spectra of the corresponding database standard substances (right);

[0013] Figure 3 These are the functional characteristics of the new santalene synthase gene (SaSSY2) expressed in tobacco leaves. A, total ion peak diagram of the product detected by GC-MS; B, comparison of the relative contents of different products; C, mass spectra corresponding to different peaks;

[0014] Figure 4 This is a comparison of the total content of santalene and bergamotene synthesized by in vitro enzymatic reaction of proteins expressed in Escherichia coli and heterologous synthesis in tobacco leaves. DETAILED DESCRIPTION

[0015] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0016] Embodiment 1:

[0017] 1. Full-length cloning of SaSSY2 gene

[0018] Total RNA from the stems of Santalum album was extracted using a plant RNA extraction kit. Promega M-MLV reverse transcriptase was used for reverse transcription to obtain cDNA. Primers (upstream primer: 5'-ATGGATTCTTCCACCGCCACCGCCA-3'; downstream primer: 5'-TCAGTCGAGAGGAATAGGATCGATCA-3') were designed based on the candidate SaSSY2 gene sequence, and high-fidelity PCR reaction was performed using cDNA as a template. PCR reaction system: 10×PCR buffer 5μL, 1μL of upstream and downstream primers (10μM), 1μL of cDNA, 0.5μL of high-fidelity KOD enzyme, and water was added to make up 50μL. PCR reaction parameters were: 95℃ denaturation for 5min, 95℃ denaturation for 30sec, 60℃ annealing for 30sec, 72℃ extension for 2min, 30 cycles; 70℃ extension for 10min. After the PCR reaction was completed, the product was subjected to agarose gel electrophoresis, the gel was cut to recover the target band, and cloned into the pMD18-T vector (Takara). Positive clones were screened on LB solid plates containing 100 μg / mL ampicillin and sent to BGI for sequencing verification. The gene SaSSY2 with the nucleotide sequence shown in SEQ ID NO.1 was successfully obtained. The amino acid sequence of the enzyme encoded by the gene is shown in SEQ ID NO.2. The amino acid sequence of the santalene synthase (SaSSY) and the new santalene synthase (SaSSY2) are compared as shown in the following figure. Figure 1 shown.

[0019] 2. Construction of SaSSY2 gene recombinant vector and engineered bacteria

[0020] Primers for amplifying the complete coding reading frame were designed based on the coding sequence of the SaSSY2 gene, and restriction endonuclease sites were introduced on the upstream and downstream primers (BamHI for the upstream and NotI for the downstream), respectively. Specifically, the upstream primer is: 5'-CGCGGATCCATGGATTCTTCCACCGCCACCGCCA-3', and the downstream primer is: 5'-TCAGTCGAGAGGAATAGGATCGATCAGCGGCCGCTTTA-3'. The full-length gene with restriction sites was amplified using the plasmid as a template. PCR reaction system: 10×PCR buffer 5μL, 1μL of upstream and downstream primers, 1μL of cDNA, 0.5μL of high-fidelity KOD enzyme, and 50μL of water was added to make up. The PCR reaction parameters were: first, denaturation at 95℃ for 5min; second, denaturation at 95℃ for 30sec, annealing at 60℃ for 30sec, extension at 72℃ for 2min, 30 cycles; and finally extension at 70℃ for 10min. After PCR amplification, the product was subjected to agarose gel electrophoresis, and the target band was recovered by cutting the gel. The In-fusion method was used to connect to the pET-28a vector, transform DH5a, screen the single clone in LB+Kana (kanamycin), send it for sequencing to verify the correct construction of the vector, and expand the bacterial solution to extract the pET28a recombinant plasmid-pET28a-SSY2 plasmid. Take 2μl of pET28a-SSY2 plasmid, transfer it into 100μl Rossetta 2 (DE3) competent cells by heat shock method, and screen the positive clones on 50μg / m L Kana, 100μg / ml Chl (chloramphenicol) LB solid plates to obtain the engineered bacteria Rossetta 2 (DE3) / pET28a-SSY2.

[0021] 3. Protein Induction Expression and Purification

[0022] The engineered bacteria Rossetta 2 (DE3) / pET28a-SSY2 clone obtained in step 2 was picked into 3 ml LB liquid medium containing 50 μg / mL Kana and 100 μg / mL Chl, and cultured overnight at 37 ° C in a shaking incubator. Added into LB liquid medium containing corresponding antibiotics at a ratio of 1:100, cultured at 37 ° C, and the OD of the bacterial solution was expanded. 600=0.6-0.8; add 1m M IPTG, induce at 16℃ for 24 hours, and collect the cells by centrifugation. Resuspend the cells with 45ml lysis buffer (50mM NaH2PO4, pH 8.0, 300mM NaCl), and place on ice for 30min; ultrasonically disrupt for 0.5h, add imidazole with a final concentration of 10mM and 1ml protein purification filler, shake slowly on a small shaker for 0.5-1h, and mount the protein on the filler; desalt and elute with a PD-10 column to obtain the SaSS Y2 recombinant enzyme solution.

[0023] 4. Analysis of the catalytic properties of the recombinant enzyme SaSSY2 in vitro

[0024] The prepared SaSSY2 recombinant enzyme solution (concentration of 50 mg / ml, volume of 10 μL) was added with Tris-HCl (pH 7.0) buffer, 2 μg substrate FPP, 10 mM MgCl2, 5 mM DTT, and 100 μL glycerol to form a reaction system of 1 ml. The mixture was fully reacted at 30°C for 2 hours, and n-hexane was added to extract the reaction product. The product was qualitatively analyzed by GC-MS. Chromatographic conditions: GC-2010 Shimadzu gas chromatograph was used; the chromatographic column was HP-5; carrier gas: N2, purge flow rate was 3 mL / min, no split; the column box started at 40°C, retained for 2 minutes, then heated to 220°C at a rate of 7°C / min, retained for 5 minutes; the injection port temperature was 250°C; the detector temperature was 250°C. Mass spectrometry data were collected in 45-220 full scan mode. The product was analyzed by comparing with the NIST database ( Figure 2 ) Calculate the relative contents of santalen and bergamotene based on the peak areas (e.g. Figure 4 The results showed that the enzyme derived from sandalwood could catalyze FPP in the prokaryotic expression system to produce trans-α-bergamotene (49.58%), α-santalene (30.09%), β-santalene (15.46%), epi-β-santalene (1.42%) and sabinene (3.18%).

[0025] 5. Transient expression of SaSSY2 in Nicotiana bentamiana to obtain metabolites

[0026] According to the coding sequence of SaSSY2 gene, restriction endonuclease sites were introduced (upstream is EcoRⅠ, downstream is XbaⅠ), and the upstream primer was designed as: 5'-GCTTGATATCGAATTCATGGATTCTTCCACCGCCACCGCCA-3', and the downstream primer was designed as: 5'-CAGCGAATTATCTAGAGTCGAGAGGAATAGGATCGATCA-3'. Using the plasmid as a template, the ORF with restriction sites was amplified. The PCR reaction system was: 10×PCR buffer 5μL, 1μL of upstream and downstream primers, 1μL of cDNA, 0.5μL of high-fidelity KOD enzyme, and 50μL of water was added to make up. The PCR reaction parameters were: first, denaturation at 95℃ for 5min; secondly, denaturation at 95℃ for 30sec, annealing at 60℃ for 30sec, extension at 72℃ for 2min, 30 cycles; and finally extension at 70℃ for 10min. After PCR amplification, the product was subjected to agarose gel electrophoresis, and the target band was recovered by cutting the gel. The in-fusion method was used to connect to the pGreen 35S-GFP vector and verified by DNA sequencing of BGI. 5 μL of the obtained vector was added to 100 μL of Agrobacterium GV3101 competent cells, frozen in liquid nitrogen for 2 minutes, placed at 37°C for 30 minutes, added to 1 mL of LB liquid culture medium, cultured at 28°C for 2 hours, spread on LB plates containing 25 mg / L rifampicin, 25 mg / L gentamicin, and 50 mg / L kanamycin, and cultured at 28°C for 3 days. Pick a single clone of Agrobacterium and inoculate it into 2 mL of LB culture medium (containing 25 mg / L rifampicin, 25 mg / L gentamicin, and 50 mg / L kanamycin) and culture it overnight at 28°C and 200 rpm. The bacterial solution was centrifuged at 8000 rpm for 2 minutes, the precipitate was collected and resuspended in 10 mL of transformation solution (10 mM MES, 10 mM MgCl2, 0.2 mM acetosyringone), and injected into Nicotiana benthamiana leaves after standing at room temperature for 3 hours. After 5 days of greenhouse growth, infected leaves were collected for GC-MS analysis. The GC temperature was 60°C for 3 minutes, increased from 4°C / min to 240°C, and maintained at 240°C for 20 minutes. The scanning range was 0-240 m / z. As described above, santalene and bergamotene were detected, indicating that transient expression of the Sa SSY2 gene in Nicotiana benthamiana can synthesize santalene and bergamotene. Trans-α-bergamotene (58.91%), α-santalene (28.05%), β-santalene (11.88%), and epi-β-santalene (1.16%) were synthesized using tobacco heterologous Figure 3 ) According to the peak area calculation, the total contents of santalin and bergamotene reached 41.09-46.97% and 49.58-58.91% respectively ( Figure 4 ).

[0027] SEQ ID NO.1:

[0028]

[0029] SEQ ID NO.2:

[0030] MDSSTATAMRAPFIDHTDHVNLRTDNDSSENRRMGNYKPSIWNYDFLQSLAIRHNIMEERHLKLAEKLKGQVKFMFGAPMEPLAKLELVDVVQRLGLNHRFETEIKEALFSIYKDERNGWWFGHLHATSLRFRLLRQCGLFIPQDVFKTFQSKTGEFDMKLCDNVKGLLSLYEASFLGWRDENILDEAKAFATKYLKNAWENISQKWLAKRVKHALALPLHWRVPRIEARWFVKAYGEEENMNPTLLKLAKLDFNMVQSIHQEEIGELARWWVTTGLDKLAFARNNLLQSYMWSCAIASDPKFKLARETIVEIGSVLTVVDDAYDVYGSMDELDLYTNSVERWSCTEIDKLPNTLKLIFMSMFNKTNEVGLRVQHERGYSGITTFIKAWVEQCKSYQKEARWYHGGHTPPLEEYSLNGLVSIGFPLLLITGYVAIAENEAALDKVHPLPDLLHYSSLLSRLINDMGTSSDELERGDNLKSIQCYMNQTGASEKVAREHIKGIIEENWKILNECCFDQSQFQEPFVTFNLNSVRGSHFFYEFGDGFGVTDSWTKVDMKSVLIDPIPLD.

Claims

1. A santalene synthase, characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the santalene synthase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

4. Use of the santalene synthase according to claim 1 or the genes according to claims 2 and 3 in the preparation of santalene and / or bergamotene.

5. A recombinant expression vector comprising the gene according to claim 2 or 3.

6. The recombinant expression vector according to claim 5, characterized in that The pET28a vector was used as the backbone.

7. A genetically engineered bacterium comprising the recombinant expression vector according to claim 5 or 6.

8. The genetically engineered bacterium according to claim 7, characterized in that: The host cell used was Escherichia coli.

9. A method for preparing santalene and / or bergamotene, characterized in that: The santalene synthase according to claim 1 is used as a catalyst, farnesyl pyrophosphate is used as a substrate, and catalytic synthesis of santalene and bergamotene is carried out under the action of dithiothreitol, MgCl2 and glycerol.

10. The method according to claim 9, characterized in that The catalysis, the initial catalytic reaction system of the catalyst santalene synthase dosage is 10.0μg / mL, the substrate concentration is 2μg / mL, the concentrations of dithiothreitol and MgCl2 are 5mM and 10mM respectively, and the glycerol concentration is 10% (v / v).

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