Carboxylimonene synthetase SaBERS as well as coding gene and application thereof

Abstract

The invention discloses a bergamene synthetase SaBERS as well as a coding gene and application thereof. The amino acid sequence of the bergamotene synthetase is as shown in SEQ ID NO. 2. According to the invention, the function verification is carried out on the bergamene synthetase SaBERS from sandalwood. It is found that the sandalwood-sourced enzyme can be successfully expressed in a prokaryotic expression system to generate recombinant protein, and in an in-vitro enzymatic reaction system, the enzyme catalyzes FPP to generate alpha-bergamoene and beta-bergamoene. The contents of the two compounds calculated according to the peak area respectively reach 62.3 ng / ul and 13.4 ng / ul. According to the invention, the enzyme can be massively prepared by utilizing escherichia coli, is applied to synthesis of in-vitro bergamocene, can be further applied to synthesis of alpha-bergamocene or beta-bergamocene, and has wide industrial practical prospects and large-scale development potential.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a bergamotene synthase SaBERS, its encoding gene, and its applications. Background Technology

[0002] Plant terpenoids, as natural products, have long been widely used in the pharmaceutical and fragrance industries. Although thousands of terpenoids have been discovered in different plant species, their yields are typically low, and they are mostly induced by biotic and abiotic stresses. Their accumulation patterns vary with the seasons, or they may accumulate in specific tissues or be dynamically released. Many studies have shown that terpene synthases (TPSs) are crucial enzymes responsible for synthesizing structurally diverse terpenoids; they utilize substrates such as farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), or geranylgeranyl pyrophosphate (GPP) to generate monoterpenes, sesquiterpenes, or diterpenes. Increasing research is exploring the production of valuable terpenoids by transferring plant TPS genes into hosts such as bacteria or yeast. However, mining genes encoding the corresponding metabolite biosynthetic enzymes from plants is not only a breakthrough in basic research but also a key driver of innovation in agriculture, medicine, and biotechnology.

[0003] Sandalwood essential oil is widely used in the perfume industry and possesses anti-inflammatory, antibacterial, and antitumor properties. More than 100 terpenoid compounds have been isolated from the essential oil, with the main components being (…). Z (Z)-α-santalol and (Z)-β-santalol, other low-content components such as α-santalene, β-santalene, racemic α-bergamotene, ( E α-Santalol, sesquiterpenes, etc. The key enzyme in santalol biosynthesis, sandalene synthase (SaSSY), has been reported, catalyzing the synthesis of the major compounds α-santalene and β-santalene from the substrate FPP. However, the TPS gene for α-bergamotene synthesis has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a bergamotene synthase gene. SaBERS It differs from the previously reported SaSSY sequence by only one amino acid. It is expressed in large quantities in Escherichia coli to produce a recombinase, which catalyzes the synthesis of the main compound α-bergamerene from the substrate FPP, accounting for 82.2% of the relative content, with the remainder being β-bergamerene.

[0005] This invention first provides a bergamotene synthase derived from sandalwood ( Santalum album The amino acid sequence is shown in SEQ ID NO.2, and it encodes the bergimene synthase gene. SaBERS The nucleotide sequence is shown in SEQ ID NO. 1. It shares 98.97% homology with the SaSSY amino acid sequence, differing by only one amino acid at the enzyme active site. Figure 1 ).

[0006] The present invention also provides the bergamotene synthase SaBERS or its encoding gene. SaBERS Applications in the preparation of α-bergamerene and / or β-bergamerene.

[0007] The present invention also provides a bergamotene synthase gene comprising the aforementioned bergamotene synthase gene. SaBERS The recombinant expression vector. Preferably, the recombinant expression vector uses the pET28a vector as the backbone.

[0008] The present invention also provides a genetically engineered bacterium comprising the recombinant expression vector. Preferably, the host cell used for the genetically engineered bacterium is *Escherichia coli*; the *E. coli* strain used as the host cell can be Rosetta 2(DE3).

[0009] This invention also provides a method for preparing α-bergamotene and / or β-bergamotene, using bergamotene synthase SaBERS, with farnesyl pyrophosphate as a substrate, and catalyzing the synthesis of α-bergamotene and β-bergamotene in the presence of dithiothreitol, MgCl2, and glycerol. Preferably, the initial catalytic reaction system contains 2.0 μg of bergamotene synthase SaBERS, a substrate concentration of 5 μM, concentrations of dithiothreitol and MgCl2 of 5 mM and 10 mM, respectively, and a glycerol volume concentration of 10%.

[0010] This invention verifies the function of the sandalwood-derived bergamotene synthase SaBERS. It was found that this sandalwood-derived enzyme can be successfully expressed in a prokaryotic expression system, generating a recombinant protein. In an in vitro enzymatic reaction system, this enzyme catalyzes FPP to generate α-bergamotene and β-bergamotene (…). Figure 2 Based on peak area calculations, the contents of the two compounds reached 62.3 ng / ul and 13.4 ng / ul, respectively. This invention enables the large-scale preparation of the enzyme using *E. coli* and its application in the in vitro synthesis of bergamotene. Furthermore, it can be applied to the synthesis of α-bergamotene or β-bergamotene, demonstrating broad industrial applicability and potential for large-scale development. Attached Figure Description

[0011] Figure 1 shows a comparison of the coding sequences of the Sabers and SaSSY genes; Figure 2 shows the in vitro enzyme activity assay of bergimide synthase SaBERS. A, the total ion peaks of the product detected by GC-MS. The SaBERS enzyme catalytic product serves as a positive control, and the empty vector serves as a negative control. Peak 1, α-santalene; 2, α-bergimene; 3, ... epi 1, β-Santalene; 4, β-Santalene; 5, β-Berberine. B, The concentrations of α-berberine (peak 2) and β-berberine (peak 5) produced from the substrate FPP by the bergimene synthase SaBERS. Detailed Implementation

[0012] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0013] Example 1: 1. Full-length clone of the Sabers gene Total RNA was extracted from sandalwood stems using a plant RNA extraction kit (Huayue Yang Technology Co., Ltd.). cDNA was obtained by reverse transcription using Promega M-MLV reverse transcriptase. Primers (SaBERS ORF primers in Table 1) were designed based on the candidate SaBERS gene sequence, and high-fidelity PCR was performed using cDNA as a template. The PCR reaction system consisted of: 5 μL 10 × PCR buffer, 1 μL each of forward and reverse primers (10 μM), 1 μL cDNA, 0.5 μL high-fidelity KOD enzyme, and water to a final volume of 50 μL. The PCR parameters were: 95℃ denaturation for 5 min, 95℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 2 min, 30 cycles; and 70℃ extension for 10 min. After the PCR reaction was completed, the product was subjected to agarose gel electrophoresis, the target band was excised and recovered, 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 SaBERS with the nucleotide sequence shown in SEQ ID NO.1 was successfully obtained. The amino acid sequence of the enzyme encoded by this gene is shown in SEQ ID NO.2.

[0014] Table 1 Primer sequences used in this invention .

[0015] 2. Construction of Sabers gene recombinant vector and engineered bacteria Primers were designed to amplify the complete coding frame based on the SaBERS gene coding sequence. Restriction endonuclease sites (BamHI upstream and NotI downstream) were introduced into the upstream and downstream primers, respectively. Specific primers (pET28a:: in Table 1) were designed. SaBERSThe full-length gene with restriction enzyme sites was amplified using cDNA from sandalwood stems. The PCR reaction system consisted of 5 μL of 10 × PCR buffer, 1 μL each of forward and reverse primers, 1 μL of cDNA, 0.5 μL of high-fidelity KOD enzyme, and water to a final volume of 50 μL. The PCR parameters were: first, denaturation at 95℃ for 5 min; second, denaturation at 95℃ for 30 sec, annealing at 60℃ for 30 sec, extension at 72℃ for 2 min, for 30 cycles; and finally, extension at 70℃ for 10 min. After PCR amplification, the product was subjected to agarose gel electrophoresis, and the target band was recovered by gel excision. The product was ligated into the pET-28a vector using in-fusion, transformed into DH5α, and single clones were selected using LB + 50 μg / mL kanamycin. Sequencing was performed to verify the correct vector construction, yielding the pET28a-SSY2 plasmid. The bacterial culture was then amplified, and the pET28a-SSY2 plasmid was extracted. Two μl of pET28a-SSY2 plasmid was transferred into 100 μl of Rosetta 2(DE3) competent cells using a heat shock method. Positive clones were selected on 50 μg / mL Kana and 100 μg / mL Chl (chloramphenicol) LB agar plates to obtain the engineered strain Rosetta 2(DE3) / pET28a-SaBERS. The SaSSY gene (whose nucleotide sequence is shown in SEQ ID NO.3, and its comparison with the SaBERS gene coding sequence is as follows) was performed. Figure 1 As shown in the figure (the encoded amino acid sequence is shown in SEQ ID NO.4) as a control, the engineered bacteria Rosetta 2 (DE3) / pET28a-SaSSY were constructed by treating them in the same way.

[0016] 3. Protein induction, expression, and purification The engineered bacterial colonies of Rosetta 2 (DE3) / pET28a-SaBERS or Rosetta 2 (DE3) / pET28a-SaSSY obtained in step 2 were picked and placed into 3 ml of LB broth containing 50 μg / mL Kana and 100 μg / mL Chl, and cultured overnight at 37°C with shaking. The culture was then added to LB broth containing the corresponding antibiotic (50 μg / mL Kana, 100 μg / mL Chl) at a volume ratio of 1:100, and cultured at 37°C. The OD of the culture was then expanded. 600= 0.6-0.8; Add 0.5 mM IPTG, induce at 16℃ for 24 h, collect bacterial cells by centrifugation, and wash the precipitate with sterile ddH2O. Resuspend the bacterial cells in 45 ml of lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl), add 100 μl PMSF, mix well, and place on ice for 30 min; sonicate for 0.5 h, add imidazole to a final concentration of 10 mM and 1 ml of protein purification packing material, shake slowly on a small shaker for 0.5-1 h, and load the protein onto the packing material; desalt and elute with a PD-10 column to obtain Sabers or SaSSY protein.

[0017] 4. Analysis of the in vitro catalytic properties of recombinant enzyme SaBERS The prepared SaBERS or SaSSY recombinant enzyme solution (concentration 50 mg / ml, volume 10 μL) was added to Tris-HCl (pH 7.0) buffer, 5 μM substrate FPP, 10 mM MgCl2, 5 mM DTT, and 10% glycerol to form a 1 ml reaction system. The mixture was reacted at 30℃ for 2 h, and the reaction product was extracted with n-hexane. GC-MS was used for qualitative analysis of the product. Chromatographic conditions: GC-2010 Shimadzu gas chromatograph; HP-5 column; carrier gas: N2, purge flow rate 3 mL / min, splitless; column oven initial temperature 40℃, hold for 2 min, then ramp to 220℃ at 7℃ / min, hold for 5 min; injection port temperature 250℃; detector temperature 250℃. Mass spectrometry data were collected in 45-220 full scan mode. The product was analyzed by comparison with the NIST database.

[0018] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that the bergimene synthase SaBERS of the present invention catalyzes FPP to generate α-bergimene and β-bergimene ( Figure 2 Based on peak area calculations, the contents of the two compounds reached 62.3 ng / ul and 13.4 ng / ul, respectively. In contrast, the SaSSY enzyme, which differs from Sabers by only one amino acid, catalyzes the synthesis of five products from FPP, including α-santalene and α-bergamotene. epi -β-Santalene, β-Santalene and β-Berberine; among them, the retention times of compounds at peaks 2 and 5 are completely consistent with the retention times of the two products synthesized by SaBERS enzyme, and the NIST database analysis shows that the products are α-berberine and β-berberine.

Claims

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

2.

2. A limonene synthase gene encoding the limonene synthase SaBERS of claim 1. SaBERS .

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

1.

4. The limonene synthase SaBERS of claim 1, the limonene synthase gene of claim 2 or 3 SaBERS Use in the preparation of α-limonene and / or β-limonene.

5. A recombinant expression vector comprising the limonene synthase gene of claim 2 or 3. SaBERS 5. A recombinant expression vector comprising the limonene synthase gene of claim 2 or 3.

6. The recombinant expression vector of claim 5, wherein, The recombinant expression vector uses pET28a vector as a skeleton.

7. A genetically engineered bacterium comprising the recombinant expression vector of claim 5 or 6. 8.The genetically engineered bacterium of claim 7, characterized in that, The host cell used is Escherichia coli.

9. A process for the preparation of α-bergamotene and / or β-bergamotene, characterized in that, The limonene synthase SaBERS of claim 1 is used to catalyze the synthesis of α-limonene and β-limonene from farnesyl pyrophosphate in the presence of dithiothreitol, MgCl2 and glycerol.

10. The method of claim 9, wherein, In the initial catalytic reaction system for synthesizing α-limonene and β-limonene, the amount of limonene synthase SaBERS used is 2.0 μg, the concentration of substrate is 5 μM, the concentrations of dithiothreitol and MgCl2 are 5 mM and 10 mM respectively, and the volume concentration of glycerol is 10%.

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