Recombinant vector for expressing bisabolol synthetase, engineering bacterium and application thereof

Abstract

The invention relates to the technical field of genetic engineering, in particular to a recombinant vector for expressing bisabolol synthetase, engineering bacteria and application of the recombinant vector and the engineering bacteria. According to the invention, the ORBBS gene is mutated, connected to a pGEX-6P-1 vector and introduced into Escherichia coli to obtain an engineering bacterium, after engineering is subjected to induced expression, a strip, a protein molecular weight and a label can be clearly seen, and the total protein molecular weight is about 92kDa, so that the ORBBS gene is successfully expressed on the pGEX-6P-1 vector. The bisabolol synthase can be produced by using a microbial fermentation method, and further the farnesyl pyrophosphoric acid is catalyzed to generate the bisabolol, so that the method has the advantages that the method is environment-friendly, and the produced bisabolol is a single component.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant vector expressing bisabolol synthase, an engineering bacterium and applications thereof. Background Art

[0002] Current methods for preparing bisabolol include isolating it from natural plants, such as by distilling the leaves of Eremanthus erythropappus, and then distilling the essential oil from steam. [1] However, this method of extraction will damage the local environment and is an unsustainable means of obtaining. In addition to the direct extraction method, Han, GH et al. [2] Bisabolol was produced by fermentation of an engineered Escherichia coli containing bisabolol synthase via a biosynthetic mevalonate pathway. de Meireles et al. used a chemical approach to synthesize bisabolol by heteropolyacid-catalyzed cyclization of nerol and farnesol. [3] However, chemical synthesis methods produce two isomers of bisabolol, which means that a single target product cannot be produced.

[0003] [1] DE SOUZA AT, BENAZZI TL, GRINGS MB, et al. Supercritical extraction process and phase equilibrium of Candeia (Eremanthus erythropappus) oil using supercritical carbon dioxide [J]. Journal of Supercritical Fluids, 2008, 47(2): 182-187.

[0004] [2]HAN GH,KIM SK,YOON PKS,et al.Fermentative production and direct extraction of(-)-α-bisabolol in metabolically engineered Escherichiacoli[J].Microbial Cell Factories,2016,15:1293401605882.

[0005] [3]DE MEIRELES ALP, COSTAM D, ROCHA KA D, et al. Heteropoly acidcatalyzed cyclization ofnerolidol and farnesol:Synthesis of α-bisabolol[J].Applied Catalysis a-General,2015,502:271-275. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a recombinant vector expressing bisabolol synthase, an engineered bacterium and its application. The recombinant vector provided by the present invention can produce bisabolol synthase by microbial fermentation method, and then catalyze farnesyl pyrophosphate to produce bisabolol, which has the advantages of being environmentally friendly and the produced bisabolol is a single component.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a recombinant vector for expressing bisabolol synthase, comprising a starting vector and a coding gene inserted into the starting vector, wherein the nucleotide sequence of the primer pair for amplifying the coding gene is as shown in SEQ ID NO.4 and SEQ ID NO.5; and the template used for amplifying the coding gene comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.1.

[0009] Preferably, the starting vector is pGEX-6P-1 vector.

[0010] Preferably, the coding gene is located between the BmgBI restriction sites of the pGEX-6P-1 vector.

[0011] Preferably, the nucleotide sequence of the template used to amplify the encoding gene is shown as SEQ ID NO.30.

[0012] The present invention provides an engineered bacterium for synthesizing bisabolol synthase, wherein the engineered bacterium comprises an original strain and a recombinant vector introduced into the original strain, and the recombinant vector is the recombinant vector described in the above technical solution.

[0013] Preferably, the original strain comprises Escherichia coli.

[0014] Preferably, the Escherichia coli is a BL21 (DE3) strain.

[0015] The present invention provides the use of the recombinant vector described in the above technical solution or the engineering bacteria described in the above technical solution in synthesizing bisabolol synthase.

[0016] The present invention provides a method for synthesizing bisabolol synthase, comprising the following steps:

[0017] The engineered bacteria described in the above technical solution are cultured in a culture medium containing IPTG to obtain a culture solution containing bisabolol synthase.

[0018] Preferably, the concentration of IPTG in the culture medium is 0.1-0.5 mM.

[0019] Beneficial effects:

[0020] The present invention provides a recombinant vector for expressing bisabolol synthase, including a starting vector and a coding gene inserted into the starting vector, the nucleotide sequence of the primer pair for amplifying the coding gene is shown in SEQ ID NO.4 and SEQ ID NO.5; the template used for amplifying the coding gene includes a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO.1. The present invention mutates the ORBBS gene and connects it to the pGEX-6P-1 vector, and introduces it into Escherichia coli to obtain an engineered bacterium. After the engineered bacteria are induced to express, the bands can be clearly seen. The protein molecular weight is added with a label, and the overall protein molecular weight is about 92kDa. Therefore, the ORBBS gene is successfully expressed on the pGEX-6P-1 vector. The microbial fermentation method can be used to produce bisabolol synthase, and then catalyze farnesyl pyrophosphate to generate bisabolol, which has the advantages of being environmentally friendly and the produced bisabolol is a single component. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 is the plasmid map of pHA8688-1 vector;

[0023] Figure 2 The results of multiple comparisons of the amino acid sequence of OS-BBS with TPS of other species;

[0024] Figure 3 The results of pHA8688-1-Ecoli BL21(DE3)-ORBBS agarose gel electrophoresis; wherein, M is a 5000bp marker, 1 is a positive control of the pHA8688-1 vector containing the target gene, 2 is a negative control of Ecoli BL21(DE3) without the vector, and lanes 3 to 5 are parallel samples of pHA8688-1-Ecoli BL21(DE3)-ORBBS;

[0025] Figure 4The results of agarose gel electrophoresis of pGEX-6P-1-Ecoli BL21(DE3)-ORBBS; M is a 5000bp marker, 1 is a positive control of the pGEX-6P-1 vector containing the target gene, 2 to 4 are parallel samples of pGEX-6P-1-Ecoli BL21(DE3)-ORBBS, and 5 is a negative control of Ecoli BL21(DE3) without the vector;

[0026] Figure 5 The results of agarose gel electrophoresis verification of site-directed mutation of the bisabolol synthase gene; Note: M is a 5000bp marker, 1 is a positive control of the pGEX-6P-1 vector containing the mutant target gene, 2-6 and 8-12 are SSM-L101 mutant strains, and 7 is a negative control of Ecoli BL21 (DE3) without the vector;

[0027] Figure 6 The results of sequencing of some mutant strains;

[0028] Figure 7 The SDS-PAGE gel electrophoresis diagram of the target gene after expression in the pHA8688-1 vector; wherein, M is a 180 kDa protein pre-stained marker, and 1 to 4 are mutant strains;

[0029] Figure 8 The figure is the SDS-PAGE gel electrophoresis diagram after the target gene is expressed in the pGEX-6P-1 vector; M is the 180kDa protein pre-stained marker, the first lane is the 0mM IPTG concentration control group, the second lane is the IPTG final concentration of 0.1mM, the third lane is the IPTG final concentration of 0.2mM, and the fourth lane is the IPTG final concentration of 0.5mM;

[0030] Fig. 9 GC-MS chromatogram of the enzymatic reaction product. DETAILED DESCRIPTION

[0031] The present invention provides a recombinant vector for expressing bisabolol synthase, comprising a starting vector and a coding gene inserted into the starting vector, wherein the nucleotide sequence of the primer pair for amplifying the coding gene is as shown in SEQ ID NO.4 and SEQ ID NO.5; and the template used for amplifying the coding gene comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.1.

[0032] As an embodiment, the starting vector is a pGEX-6P-1 vector.

[0033] As an embodiment, the coding gene is located between the BmgBI restriction sites of the pGEX-6P-1 vector.

[0034] As an embodiment, the nucleotide sequence of the template used to amplify the encoding gene is shown as SEQ ID NO.30.

[0035] The present invention provides an engineered bacterium for synthesizing bisabolol synthase, wherein the engineered bacterium comprises an original strain and a recombinant vector introduced into the original strain, and the recombinant vector is the recombinant vector described in the above technical solution.

[0036] As one embodiment, the original strain includes Escherichia coli.

[0037] As an embodiment, the Escherichia coli is a BL21 (DE3) strain.

[0038] The present invention provides the use of the recombinant vector described in the above technical solution or the engineering bacteria described in the above technical solution in synthesizing bisabolol synthase.

[0039] The present invention provides a method for synthesizing bisabolol synthase, comprising the following steps:

[0040] The engineered bacteria described in the above technical solution are cultured in a culture medium containing IPTG to obtain a culture solution containing bisabolol synthase.

[0041] As an embodiment, the concentration of IPTG in the culture medium is 0.1-0.5 mM.

[0042] Farnesyl pyrophosphate (FPP) generates bisabolol under the catalytic action of bisabolol synthase. The present invention uses a gene sequence of bisabolol synthase, which is obtained from rice. The gene is constructed on a gene sequence and allowed to cyclize itself to form a vector with a target gene, which is then introduced into a competent cell to induce expression. After homology analysis of the target gene, a semi-rational design method is used to achieve directed evolution of enzyme molecules. Then, the target gene is mutated and connected to the pGEX-6P-1 vector, and introduced into Escherichia coli to obtain an engineered bacterium. The engineered bacteria can clearly see the bands after induced expression. The protein molecular weight is added with a label, and the overall protein molecular weight is about 92kDa. Therefore, the ORBBS gene is successfully expressed on the pGEX-6P-1 vector. Bisabolol synthase can be produced by microbial fermentation method, and then catalyzes farnesyl pyrophosphate to generate bisabolol, which has the advantages of being environmentally friendly and producing bisabolol as a single component.

[0043] To further illustrate the present invention, a recombinant vector expressing bisabolol synthase, an engineered bacterium and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Farnesyl pyrophosphate (FPP) generates bisabolol under the catalysis of bisabolol synthase. The present invention obtains a gene sequence (SEQ ID NO.1) of bisabolol synthase from rice [ZHAN CS, LEI L, GUO H, et al. Disease resistance conferred by components of essential chrysanthemum oil and the epigenetic regulation of OsTPS1 [J]. Science China-Life Sciences, 2023, 66 (5): 1108-1118.], and entrusts Suzhou Anshengda Biotechnology Co., Ltd. to synthesize it, constructs the gene on a gene sequence and allows it to self-cyclize to form a vector with the target gene, and obtains an expression vector containing the target gene, which is recorded as pHA8688-1 vector, and the nucleotide sequence is shown in SEQ ID NO.30, as follows: Among them, the bold sequence (SEQ ID NO.1) is the gene sequence of bisabolol synthase.

[0046] 1) Introduce the pHA8688-1 vector into E. coli DH5α competent cells, and evenly spread the bacterial solution on LB resistant solid medium containing spectinomycin (100 μg / mL) (if the pGEX-6P-1 vector is introduced into competent cells, use ampicillin sodium as the antibiotic at a concentration of 50 μg / mL), let it stand for 30 minutes, and invert the plate in a 37°C constant temperature incubator for 14 to 16 hours.

[0047] 2) Use a sterile toothpick to pick up a single colony from the coated plate and place it in 20 μL of sterile deionized water, heat it in a 95°C metal bath for 5 minutes, and centrifuge it at 13,000 rpm for 2 minutes. The supernatant can be used as a template for verification PCR. Design PCR primers based on the gene sequence. The PCR verification primers are as follows: upstream primer: 5'-atgcctgctgatcatatg-3', SEQ ID NO.2; downstream primer: 5'-tcacacttgtattggaacca-3', SEQ ID NO.3.

[0048] The PCR amplification system was: Premix Taq 25 μL, upstream primer 1 μL, downstream primer 1 μL, PCR template (plasmid) 1 ng, deionized water to 50 mL. The PCR amplification program was: 94°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 15 s, 72°C extension for 30 s, 30 cycles; 72°C final extension for 2 min.

[0049] 3) After the PCR process is completed, the PCR product is subjected to agarose gel electrophoresis experiment, and the colonies with electrophoresis bands are expanded and cultured, and the plasmid is extracted using the Plasmid Mini KitI plasmid extraction kit to obtain the expanded pHA8688-1 vector.

[0050] 4) After the expanded pHA8688-1 vector was introduced into E. coli.BL21 (DE3) competent cells, single clones were picked for PCR verification (see step 2 for the method), and the colonies with electrophoresis bands were cultured in LB medium (containing 100 μg / mL spectinomycin), and cultured at 37°C, 180 rpm for 12 h, and then 500 μL was mixed with 500 μL competent cell freezing solution, stored in a 1.5 mL EP tube, and stored at -80°C; the preparation method of the competent cell freezing solution is as follows: take 17 mL of 0.1 M CaCl2 in a 20 mL volumetric flask, and add 3 mL of 100% glycerol.

[0051] 5) According to the inoculation volume of 2% (v / v), the bacteria containing the pHA8688-1 vector were taken out from the EP tube preserved in glycerol and added to the LB medium containing spectinomycin (50 μg / mL); the constant temperature of the shaking incubator was set to 37°C and the shaking culture was carried out at 1800 rpm for 12 hours to obtain the seed solution;

[0052] 6) Inoculate the cultured seed solution into a conical flask containing spectinomycin resistance at a 2% (v / v) inoculation volume to expand the culture; culture in a constant temperature shaker at 37°C, 180 rpm for 2-3 hours, and continuously draw the bacterial solution to measure OD during the process. 600 , until the absorbance value OD 600 When the pH value was 0.5-0.6, IPTG was added and the final concentration was adjusted to 0.25 mM, and the expression was induced overnight (14 h-16 h) at 18°C ​​and 180 rpm.

[0053] 7) Pour the bacterial solution after IPTG induction into 50 mL centrifuge tubes in batches, place in a high-speed centrifuge, and centrifuge at 4000 r / min for 20 min at 4°C. Pour out the upper culture medium, add PBS buffer solution with a pH of 7.4 in batches to adjust the OD 600=1.0, resuspend the bacteria (recorded as pHA8688-1-Ecoli BL21(DE3)-ORBBS) (if protein concentration is required, record the volume of PBS buffer added, centrifuge again, and add the previous PBS buffer volume divided by the concentration multiple. For example, OD 600 =0.1, 50 mL PBS buffer was added, 25 mL buffer was added when the concentration was 2 times, 10 mL buffer was added when the concentration was 5 times, 5 mL buffer was added when the concentration was 10 times, and so on).

[0054] 8) Place the resuspended bacterial solution in an ice water bath and put it into an ultrasonic cell disruptor. At 30% power (total power of 750W), work for 3s, pause for 5s, and perform cell ultrasonic disruption for about 30min until the bacterial solution is clear and transparent. After the disruption is completed, centrifuge it in a high-speed refrigerated centrifuge at 4°C and 4000rpm for 20min. The supernatant obtained is the crude enzyme solution. The crude enzyme solution is stored at -20°C to maintain the activity of the enzyme.

[0055] 9) SDS-PAGE electrophoresis: Take 5μL 5×Protein Loading Buffer and mix with 20μL crude enzyme solution, blow and mix in a PCR tube, boil at 100℃ for 10min, centrifuge quickly for 30s, and aspirate 10μL of supernatant for sample loading. Add 5mL of separation gel to the fixed clean electrophoresis gel glass plate, and immediately add 1mL of distilled water to flatten the separation gel. Pour out the distilled water after a clear dividing line between the separation gel and the distilled water. Prepare 2mL of concentrated gel and quickly add the concentrated gel solution to the gel glass plate, immediately insert the gel comb of the electrophoresis tank, let it stand for 5min, and carefully pull out the gel comb. Put the prepared gel into the protein electrophoresis buffer, aspirate 10μL of the prepared sample containing protein loading buffer and add it to the gel well. Turn on the gel electrophoresis instrument, run at 90V for 30min, then switch the voltage to 120V and continue running for 90min.

[0056] 10) Determination of protein concentration: The Bradford protein quantification method is used to quantitatively analyze the protein in the crude enzyme solution, and the Bradford protein concentration determination kit is used. The principle is that after Coomassie Brilliant Blue binds to the protein, the solution will change from brown to blue, and the higher the protein concentration, the darker the color. The mixed solution has a maximum absorption peak at 595nm. The protein concentration that can be detected by this kit can be 10 to 1000μL / mL.

[0057] 11) Determination of ORBBS enzyme activity: According to the results of the measured protein concentration, the enzyme solution containing about 80μg crude protein and 50μL 1M Tris-HCl, 50μL 50mM MgCl2, and 5μL FPP solution were added to the glass sample bottle in order, carefully added to one side, and then covered with 200μL n-hexane on the top of the page. Incubate at 37℃ overnight. When adding n-hexane, slowly add the liquid along the glass bottle, and do not shake the bottle during the whole process to prevent extraction into the substrate. After the reaction is completed, transfer the liquid in the glass sample bottle to a 1.5mL EP tube, mix it with a vortex oscillator for 1min, and then centrifuge it at 4℃ for 10min, transfer the supernatant, and pass the supernatant through a 0.22μm filter membrane with a disposable syringe, and detect it by GC-MS.

[0058] Example 2

[0059] The present invention constructs a gene sequence of bisabolol synthase (OS-BBS) from rice on a gene expression vector to express it in a host cell. In order to obtain a more active bisabolol synthase, a semi-rational design is performed on the sequence, including:

[0060] 1) Homology analysis of the target gene: The gene sequence was subjected to BLAST analysis on NCBI, and homology analysis was performed with the amino acid sequences containing terpene synthases in other plants. The results are shown in Figure 1 The results showed that the bisabolol synthase gene contained highly conserved sequence elements of terpene synthases, which were highly overlapped with several other similar terpene synthases. The similarities with japonica rice (XP_025878344.1), short-antherb wild rice (XP_040375922.1), lepidium rice (QGN18494.1), sorghum (XP_002443927.2), and short-tongued wild rice (QGN18496.1) were 99.46%, 80.98%, 62.52%, 60.11%, and 62.52%, respectively.

[0061] 2) Analyze and determine key sites for site-directed mutagenesis: A method based on structural analysis and Gibbs free energy stability was used in combination, and analysis software was used to predict high-quality mutation sites. The following ten mutation sites were comprehensively selected: L101, Y158, N162, E202, A203, P204, R207, F446, S450, I491, E492, and W495.

[0062] 3) PCR amplification primer design: Based on the amino acid sequence of bisabolol synthase, 12 pairs of mutation PCR amplification primers affecting the key sites of the protein were designed and synthesized by Aiki Biotechnology (Guangzhou) Co., Ltd. The primers are shown in Table 1.

[0063] Table 1 Site-directed mutagenesis primers

[0064]

[0065]

[0066] 4) 200 μL of the resuspended bacterial suspension in step 7) of Example 1 was inoculated into 20 mL of LB medium (containing 100 μg / mL of spectinomycin), and after overnight culture at 37°C and 200 rpm in a constant temperature shaker, the plasmid was extracted using a Plasmid Mini Kit I plasmid extraction kit to obtain the pHA8688-1 plasmid.

[0067] 5) Site-directed mutagenesis PCR amplification

[0068] Site-directed mutagenesis PCR amplification system: upstream site-directed mutagenesis primer (10 μM) 1.5 μL, downstream site-directed mutagenesis primer (10 μM) 1.5 μL, 10×PCRBuffer for KOD-Plus-Neo 5 μL, 2 mM dNTPs 5 μL, 25 mM MgSO4 3 μL, pHA8688-1 original plasmid template 1 μL, KOD-Plus-Neo (1 U / μL) 1 μL, and ultrapure water to 50 μL.

[0069] The site-directed mutagenesis PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 96°C for 10 s, annealing at TM±5°C for 30 s, extension at 68°C for 4 min, 30 cycles; final extension at 68°C for 4 min. The TM is the melting temperature or the depolymerization temperature.

[0070] After the PCR reaction is completed, take 5 μL of the above PCR reaction product, add 1 μL of 6× Loading Buffer, transfer it into the wells of the prepared agarose gel with a pipette for electrophoresis, adjust the DNA gel electrophoresis instrument to make the operating voltage 140V, and run it at room temperature for 40 minutes.

[0071] 6) The site-directed mutagenesis PCR amplification product was connected to the BmgBI restriction site of the pGEX-6P-1 vector to obtain the recombinant vector pGEX-6P-1-ORBBS; the wild-type ORBBS gene was artificially synthesized and connected to the BmgBI restriction site of the pGEX-6P-1 vector.

[0072] 7) Introducing the recombinant vector pGEX-6P-1-ORBBS into E. coli BL21 (DE3) competent cells to obtain the pGEX-6P-1-Ecoli.BL21 (DE3)-ORBBS strain.

[0073] 8) Verification of mutant strain clones

[0074] SSM-L101 is preferred for mutation. Pick competent cells with a sterile toothpick in a sterile operating table, add 20 μL of sterile deionized water to a sterile PCR tube, disperse the colonies on the toothpick in deionized water, heat in a 95°C metal bath for 5 minutes, and quickly centrifuge in an instant centrifuge for 2 minutes. The supernatant in the PCR tube after centrifugation is used as a mutation template. The strain verification PCR system and procedure are shown in the following table:

[0075] Clone verification PCR system: Taq enzyme 25 μL, SSM-L101-F 1 μL, SSM-L101-R 1 μL, template 1 ng, ultrapure water to 50 μL.

[0076] PCR program for clone verification: 94°C pre-denaturation for 2 min; 96°C denaturation for 10 s, 55°C annealing for 15 s, 72°C extension for 30 s, 30 cycles; 72°C final extension for 2 min

[0077] 9) Sequencing verification

[0078] The bacteria with verified bands were picked and inoculated into LB medium containing ampicillin sodium (50 μg / mL), and cultured in a constant temperature shaker at 37°C and 180 rpm for 12 hours. 1 mL of bacteria was taken from each bottle, packaged in 1.5 mL EP tubes, and then sent to Aiki Biotechnology (Guangzhou) Co., Ltd. for sequencing. The original bacterial sequence was compared with the mutant strain sequence, and 500 μL of the successfully mutated bacterial solution in the sequencing results was mixed with 500 μL of 50% glycerol (glycerol and water were mixed in equal volumes) and stored in an ultra-low temperature refrigerator. The sequencing primers are as follows: pHA8688-1-SSM-L101-F: 5'-caacggttgggactagac-3', SEQID NO.28;

[0079] pHA8688-1-SSM-L101-R: 5'-gccattctttcgcagaag-3', SEQ ID NO. 29.

[0080] Test Example 1

[0081] The verification results of pHA8688-1-Ecoli BL21(DE3)-ORBBS and pGEX-6P-1-Ecoli.BL21(DE3)-ORBBS were as follows: Figure 3 and Figure 4 shown.

[0082] From the gel electrophoresis, it is obvious that there is a bright band between 1500 bp and 2000 bp, and the length of the target gene band is about 1665 bp. Therefore, it is proved that in the strains corresponding to lanes 4, 5, and 6, the pHA8688-1-Ecoli.BL21(DE3)-ORBBS vector and pGE X-6P-1-Ecoli.BL21(DE3)-ORBBS vector plasmids were successfully introduced into competent cells.

[0083] The results of agarose gel electrophoresis of mutant DNA are shown in Figure 5 .Depend on Figure 5 It can be seen that the strain mutation was successful.

[0084] The sequencing results were opened using SnapGene software and compared with the original sequences. The comparison results of some successfully sequenced mutant strain sequences and the original genes are shown in Figure 6 From the sequencing results, it can be seen that the original gene corresponding to the "NNK" site on the mutant primer has changed. From the 5' to 3' end of the sequence, the site corresponding to mutant 1

[0085] Amino acid "TTG" → "TCG", the corresponding amino acid at the mutant 2 site is "TTG" → "ATT", the corresponding amino acid at the mutant 3 site is "TTG" → "TGG", and the corresponding amino acid at the mutant 4 site is "TTG" → "GGG".

[0086] Test Example 2

[0087] The results of SDS-PAGE gel electrophoresis after the target gene was expressed in pHA8688-1 vector are shown in Figure 7 The SDS-PAGE gel electrophoresis results of the target gene after expression in the pGEX-6P-1 vector are shown in Figure 8 .

[0088] The results showed that the target gene was not successfully expressed on the pHA8688-1 vector, but a clear band appeared on the pGEX-6P-1 vector, indicating successful expression.

[0089] Test Example 3

[0090] pGEX-ORBBS GC-MS enzyme activity test results are shown in Fig. 9 .

[0091] The sequence of the target gene was checked on the SnapGene software. It contained about 566 amino acids in total, and the protein molecular weight was about 66 kDa. Although the pHA8688-1 vector was successfully introduced into the competent cells, and DNA gel electrophoresis confirmed the appearance of bands, after trying to induce expression at different IPTG concentrations, different temperatures, different induction expression times, and different protein concentration multiples, no bands of the target protein appeared in the end ( Figure 7 ). After the target gene was introduced into the pGEX-6P-1 vector, the bands could be clearly seen after induction expression. The molecular weight of the protein plus the label was about 92kDa. Therefore, the ORBBS gene was successfully expressed on the pGEX-6P-vector. Although the target gene was successfully introduced into the Ecoli.BL21 (DE3) host cell on the pHA8688-1 vector, the target gene was not expressed after induction expression. It is speculated that the following reasons may exist:

[0092] 1) Transcription problem: The lack of suitable promoter or enhancer sequences in the constructed vector will lead to poor transcription of exogenous genes.

[0093] 2) Lack of appropriate translation signals on the vector: If the vector does not contain appropriate translation initiation signals (such as the Kozak sequence in eukaryotes), the exogenous gene may not be translated efficiently.

[0094] 3) The inserted His-tag affects protein expression.

[0095] In addition, the production of inclusion bodies will also affect the results. Inclusion bodies are protein aggregates formed in cells, which are usually rich in unfolded or misfolded proteins. They can recognize and degrade abnormally folded exogenous proteins, which may cause the proteins produced by exogenous gene expression to be rapidly degraded, thereby reducing their expression levels. In host cells, the activity of inclusion bodies consumes intracellular resources and energy. If a large amount of translation and folding resources are required during the expression of exogenous genes, the activity of inclusion bodies may compete for these resources and affect the correct folding and stability of exogenous proteins. Other conditions in the process of inclusion body experiments in cells may lead to the rapid synthesis and folding of exogenous proteins, increasing the risk of protein aggregation to form inclusion bodies.

[0096] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant vector expressing bisabolol synthase, characterized in that: It includes a starting vector and a coding gene inserted into the starting vector, and the nucleotide sequence of the primer pair for amplifying the coding gene is shown as SEQ ID NO.4 and SEQ ID NO.5; the template used for amplifying the coding gene includes a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.

1.

2. The recombinant vector according to claim 1, characterized in that The starting vector is pGEX-6P-1 vector.

3. The recombinant vector according to claim 2, characterized in that The coding gene is located between the BmgBI restriction sites of the pGEX-6P-1 vector.

4. The recombinant vector according to claim 1, characterized in that The nucleotide sequence of the template used to amplify the coding gene is shown in SEQ ID NO.

30.

5. An engineered bacterium for synthesizing bisabolol synthase, characterized in that: The engineered bacteria include an original strain and a recombinant vector introduced into the original strain, and the recombinant vector is the recombinant vector according to any one of claims 1 to 4.

6. The engineered bacteria according to claim 5, characterized in that The original strain includes Escherichia coli.

7. The engineered bacteria according to claim 6, characterized in that The Escherichia coli is a BL21 (DE3) strain.

8. Use of the recombinant vector according to any one of claims 1 to 4 or the engineered bacteria according to any one of claims 5 to 7 in synthesizing bisabolol synthase.

9. A method for synthesizing bisabolol synthase, characterized in that: The following steps are involved: The engineered bacteria according to any one of claims 5 to 7 are cultured in a culture medium containing IPTG to obtain a culture solution containing bisabolol synthase.

10. The method according to claim 9, characterized in that The concentration of IPTG in the culture medium is 0.1-0.5 mM.

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