Blumea Balsamifera Monoterpene Synthase BBTPS3 And Related Biological Materials Thereof and Use Thereof
20220348691 · 2022-11-03
Assignee
Inventors
- Luqi Huang (Beijing, CN)
- Ping Su (Beijing, CN)
- Rui MA (Beijing, CN)
- Wei Gao (Beijing, CN)
- Guanghong Cui (Beijing, CN)
- Baolong Jin (Beijing, CN)
- Yating Hu (Beijing, CN)
- Jichen Bao (Beijing, CN)
- Juan Guo (Beijing, CN)
Cpc classification
C07K19/00
CHEMISTRY; METALLURGY
International classification
C07K19/00
CHEMISTRY; METALLURGY
C12N15/10
CHEMISTRY; METALLURGY
Abstract
Provided are a Blumea balsamifera monoterpene synthase BbTPS3 and related biological materials thereof and use thereof. BbTPS3 is: A1) a protein having the amino acid sequence shown in SEQ ID NO: 2; A2) a fusion protein obtained by linking protein-tags at the N-terminus or/and the C-terminus of the protein shown in SEQ ID NO: 2; and A3) a protein having at least 90% identity and the same function as the protein shown in A1), which is obtained by performing substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence shown in SEQ ID NO: 2. BbTPS3 can catalyze GPP to form l-borneol, and can be used to regulate and produce plant monoterpene compounds and cultivate Blumea balsamifera (L.) DC.
Claims
1. A protein, wherein the protein is represented by any one of the followings: A1) a protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; A2) a fusion protein obtained by linking protein-tags at the N-terminus or/and the C-terminus of the protein shown in SEQ ID NO: 2 in the sequence listing; A3) a protein having at least 90% identity and the same function as the protein shown in A1), which is obtained by performing substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
2. A related biological material of the protein according to claim 1, wherein the related biological material is represented by any one of the followings: A1) a nucleic acid molecule encoding the protein according to claim 1; A2) an expression cassette containing the nucleic acid molecule of A1); A3) a recombinant vector containing the nucleic acid molecule of A1); A4) a recombinant vector containing the expression cassette of A2); A5) a recombinant microorganism containing the nucleic acid molecule of A1); A6) a recombinant microorganism containing the expression cassette of A2); A7) a recombinant microorganism containing the recombinant vector of A3); A8) a recombinant microorganism containing the recombinant vector of A4); A9) a transgenic plant cell line containing the nucleic acid molecule of A1); A10) a transgenic plant cell line containing the expression cassette of A2); All) a transgenic plant cell line containing the recombinant vector of A3); A12) a transgenic plant cell line containing the recombinant vector of A4).
3. The related biological material according to claim 2, wherein the nucleic acid molecule of A1) is represented by any one of the followings: B1) a DNA molecule shown in SEQ ID NO: 1 in the sequence listing; B2) a DNA molecule encoding the sequence shown in SEQ ID NO: 1 in the sequence listing; B3) a DNA molecule which is hybridized with the DNA molecule defined in B1) or 2) under a strict condition and encodes the protein according to claim 1.
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. A method for preparing the protein according to claim 1, wherein the method comprises the steps of: introducing the encoding gene of the protein according to claim 1 into a recipient microorganism to obtain a recombinant microorganism expressing the protein according to claim 1, and culturing the recombinant microorganism to express the protein according to claim 1.
9. A method for preparing (−)-borneol, wherein the method comprises the step of catalyzing geranyl pyrophosphate with the protein according to claim 1.
10. A method for biosynthesizing (−)-borneol, wherein the method comprises the steps of: introducing the encoding gene of the protein according to claim 1 into Saccharomyces cerevisiae to obtain recombinant Saccharomyces cerevisiae, and fermenting the recombinant Saccharomyces cerevisiae to obtain (−)-borneol.
11. A monoterpene synthase, wherein the monoterpene synthase is selected from the proteins according to claim 1.
12. A method for preparing the monoterpene synthase according to claim 11, wherein the method comprises the step of using the related biological material according to claim 2 to prepare the monoterpene synthase.
13. A method for preparing or synthesizing a monoterpene compound, wherein the method comprises the step of using the protein according to claim 1 to prepare or synthesize the monoterpene compound.
14. A method for preparing or synthesizing a monoterpene compound, wherein the method comprises the step of using the related biological material according to claim 2 to prepare or synthesize the monoterpene compound.
15. A method for catalyzing the formation of (−)-borneol from geranyl pyrophosphate, wherein the method comprises the step of catalyzing the formation of (−)-borneol from geranyl pyrophosphate with the protein according to claim 1.
16. A method for catalyzing the formation of (−)-borneol from geranyl pyrophosphate, wherein the method comprises the step of catalyzing the formation of (−)-borneol from geranyl pyrophosphate with the related biological material according to claim 2.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention is further described in detail hereinafter with reference to specific embodiments, and the given examples are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. All the materials and reagents used in the following examples are commercially available unless otherwise specified.
[0069] The Phusion® High-Fidelity DNA Polymerase and the restriction endonuclease BamHI in the following examples are products of New England Biolabs Company.
[0070] Quick RNA isolation kit is a product of Huayueyang Biotechnology (Beijing) Co., Ltd.
[0071] TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, Trans2K DNA Marker, pEASY-Uni Seamless Cloning and Assembly Kit and Escherichia coli competent cell Transetta (DE3) are products of Beijing TransGen Biotech Co., Ltd.
[0072] Premixed Protein Marker (Low) is a product of Takara Company.
[0073] pET32a(+) vector is a product of Novagen Company.
[0074] pESC-Leu vector is a product of Agilent Company.
[0075] SD-Ura and SD-Ura-Leu are products of Beijing FunGenome Company.
[0076] ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit is a product of Zymo Research Company.
[0077] BY4741 yeast strain (genotype: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) is purchased from Huayueyang Biotechnology (Beijing) Co., Ltd.
[0078] Geranyl pyrophosphate (GPP) is a product of Sigma Company, with a product catalog number G6772 and a CAS number 763-10-0.
[0079] l-Borneol ((−)-borneol) is a product of Sigma Company, with a product catalog number CRM40456 and a CAS number 464-45-9.
Example 1 Full-Length cDNA Sequence Clone of Bbtps3 Gene of Blumea balsamifera (L.) DC
[0080] 1. Extraction of Total RNA
[0081] According to the instructions of the Quick RNA isolation kit of Huayueyang Biotechnology (Beijing) Co., Ltd., the total RNA of Blumea balsamifera leaves was extracted.
[0082] 2. Synthesis of First-Strand cDNA
[0083] According to the instructions of the first-strand cDNA synthesis kit TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix of Beijing TransGen Biotech Co., Ltd., the cDNA was obtained by reverse transcription.
TABLE-US-00001 The reverse transcription reaction system was as follows: Total RNA 5.0 μg Anchored Oligo(dT).sub.18 Primer 1.0 μL 2 × TS Reaction Mix 10.0 μL TransScript ® RT/RI Enzyme Mix 1.0 μL gDNA Remover 1.0 μL RNase-free Water added to a final volume of 20.0 μL Total volume 20.0 μL
[0084] The steps of reverse transcription were as follows:
[0085] (1) in order to improve the synthesis efficiency, the total RNA, Anchored Oligo(dT).sub.18 Primer and RNase-free Water were evenly mixed in a PCR tube at 65° C. for 5 minutes;
[0086] (2) 10.0 μL of 2×TS Reaction Mix, 1.0 μL of TransScript RT/RI Enzyme Mix and 1.0 μL of gDNA Remover were added into the above PCR tube, and mixed evenly and gently;
[0087] (3) the reverse transcription reaction was performed at “42° C. for 30 minutes, 85° C. for 5 seconds” to obtain the first-strand cDNA;
[0088] (4) the first-strand cDNA was stored at −20° C.
[0089] 3. Design of Primers
[0090] According to the transcriptome data of Blumea balsamifera leaves, the open reading frame (ORF) sequence was obtained. Based on this, cloning primers BbTPS3-F1 and BbTPS3-R1 were designed. The sequences of the primers were as follows:
TABLE-US-00002 BbTPS3-F1: 5′-ATGGTTGGATTTCAAAAACACTCATG-3′; BbTPS3-R1: 5′-CTAGGTTTTAGGCTTCAAAAGTAATGAGT-3′
[0091] 4. PCR Amplification
[0092] The PCR amplification was performed with high-fidelity enzyme Phusion® High-Fidelity DNA Polymerase, with the first-strand cDNA obtained in step 2 as template, as well as BbTPS3-F1 and BbTPS3-R1 are primers. The results are shown in
[0093] The PCR amplification procedure was as follows:
[0094] PCR reaction procedure: pre-denaturation at 98° C. for 3 minutes; 35 cycles of (98° C. for 20 seconds, 55° C. for 20 seconds, 72° C. for 1 minute); and extension at 72° C. for 5 minutes.
[0095] Sequencing results show that: the sequence of the PCR amplification product is consistent with SEQ ID NO: 1, the gene shown in SEQ ID NO: 1 is named Bbtps3, which encodes a protein consisting of 556 amino acid residues, wherein the protein is named as BbTPS3 and the amino acid sequence of the protein is shown in SEQ ID NO: 2.
Example 2 Acquisition and Functional Analysis of BbTPS3 of Blumea balsamifera (L.) DC
[0096] I. Acquisition of BbTPS3 Protein of Blumea balsamifera (L.) DC.
[0097] 1. Construction of Recombinant Vector
[0098] The Bbtps3 gene shown in SEQ ID NO: 1 was inserted at the BamHI restriction enzyme site of the pET32a(+) vector (Novagen Company) by using the pEASY-Uni Seamless Cloning and Assembly Kit of Beijing TransGen Biotech Co., Ltd., and the rest sequence of the pET32a(+) vector remains unchanged to obtain a recombinant plasmid pET32a::BbTPS3.
[0099] Specific steps were as follows:
[0100] 1) the PCR amplification product obtained in Example 1 was used as template, PCR amplification was performed with primers BbTPS3-F2 and BbTPS3-R2, finally, the purified PCR product was obtained through recovering and purifying steps. The sequences of the primers were as follows (the underlined sequences were vector homologous regions):
TABLE-US-00003 BbTPS3-F2: 5′-CCATGGCTGATATCGGAATGGTTGGATTTCAAAAACACTCA-3′; BbTPS3-R2: 5′-ACGGAGCTCGAATTCGGCTAGGTTTTAGGCTTCAAAAGTA-3′;
[0101] 2) the pET32a(+) vector (Novagen Company) was digested with the restriction endonuclease BamHI, and then the linearized vector backbone was recovered;
[0102] 3) according to the instructions of the pEASY-Uni Seamless Cloning and Assembly Kit of Beijing TransGen Biotech Co., Ltd., the purified PCR product obtained in step 1) was combined with the linearized vector backbone in step 2) to obtain a recombinant plasmid pET32a::BbTPS3.
[0103] 2. Acquisition of Recombinant Bacteria
[0104] The recombinant plasmid pET32a::BbTPS3 was transformed into expression strain Escherichia coli Transetta (DE3) (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain pET32a::BbTPS3 recombinant bacteria. Meanwhile, E. coli Transetta (DE3) was transformed with the pET32a(+) vector without the target gene (i.e., the Bbtps3 gene) and this recombinant strain was used as control bacteria.
[0105] 3. Acquisition of Recombinant Protein BbTPS3
[0106] The pET32a::BbTPS3 recombinant bacteria and the control bacteria were respectively inoculated into 2 mL of LB liquid medium (containing 100 mg/L ampicillin), shaken and cultured overnight at 37° C. The next day, the cells were diluted in 200 mL LB liquid medium in the ratio of 1:100, shaken and cultured at 37° C. until the OD.sub.600 reached 0.6˜0.8, and then shaken at 18° C. for 1 hour. IPTG was added to a final concentration of 0.5 mM, and the mixture was continuously cultured in a shaking table at 18° C. for 24 hours to induce the expression of the target protein. The bacterial solution was centrifuged at 8000 g for 5 minutes, the supernatant was discarded, the cells of pET32a::BbTPS3 recombinant bacteria and the control bacteria were collected, and stored at −80° C. for later use.
[0107] 4. Purification of Recombinant Protein BbTPS3
[0108] The proteins in the pET32a::BbTPS3 recombinant bacteria and the control bacteria were extracted. Specific steps were as follows:
[0109] the pET32a::BbTPS3 recombinant bacteria and the control bacteria were resuspended with 5 mL of pre-cooled HEPES buffer (25 mM HEPES, 5 M MgCl.sub.2, 5 M DTT, pH 7.0), sonication (at 30% power for 5 seconds by an interval of 5 seconds, which lasted for 5 minutes and was repeated once) in ice bath, and centrifuged at 12,000 g and 4° C. for 30 minutes to obtain the supernatant protein solutions of the pET32a::BbTPS3 recombinant bacteria and the control bacteria respectively.
[0110] SDS-PAGE was performed on the supernatant of the pET32a::BbTPS3 recombinant bacteria and the supernatant of the control bacteria. The results are shown in
[0111] II. Enzymatic Activity Analysis of Recombinant Protein BbTPS3
[0112] 1. Enzymatic Reaction
[0113] An enzymatic reaction was performed with the supernatant of the pET32a::BbTPS3 recombinant bacteria, and to obtain an enzymatic reaction product. The specific steps of the enzymatic reaction were as follows:
[0114] the total enzymatic reaction system was 0.2 mL; including 190 μL of the supernatant of the pET32a::BbTPS3 recombinant bacteria (the supernatant of the pET32a::BbTPS3 recombinant bacteria contained an enzymatic buffer, which was namely the HEPES buffer (25 mM HEPES, 5 M MgCl.sub.2, 5 M DTT, pH 7.0)) and 10 μL of geranyl pyrophosphate (GPP) as a substrate. After evenly mixing, the overall enzymatic reaction system was sealed with 200 μL of n-hexane covering solution and placed at 30° C. for 2 hours; the n-hexane in the water phase was thoroughly removed under a stream of nitrogen (to avoid affecting the dephosphorylation reaction of the next step) to obtain an enzymatic reaction product of the supernatant of the pET32a::BbTPS3 recombinant bacteria.
[0115] 2. Dephosphorylation Reaction
[0116] A dephosphorylation reaction system was prepared, fully mixed (blown with a pipette), and dephosphorylated at 37° C. for 4 hours to obtain a dephosphorylated product.
TABLE-US-00004 The dephosphorylation reaction system was as follows: Water phase (enzymatic reaction product of the supernatant of 200 μL pET32a::BbTPS3 recombinant bacteria) 10 × CutSmart Buffer 22 μL CIP 2 μL
[0117] The dephosphorylated product was extracted with n-hexane for three times, 0.2 mL each time, and the extracted organic phases were pooled together. The extracting solution was blow-dried with nitrogen, and added with 100 μL of n-hexane for dissolution to obtain the target compound (which was namely the target compound of the supernatant of the pET32a::BbTPS3 recombinant bacteria) for GC-MS analysis.
[0118] 3. GC-MS Analysis
[0119] Gas chromatography-mass spectrometry GC-MS was used to detect the target compound of the supernatant of the pET32a::BbTPS3 recombinant bacteria: the GC-MS analysis system was Thermo TRACE 1310/TSQ 8000 gas chromatograph, with an injection volume of 1 μL, a mode of splitless, a gas chromatographic column of Agilent J&W Cyclodex-B chiral column (30 m×0.25 mm×0.25 μm) was used. And helium was used as carrier gas with flow rate of 1.0 mL/min. The injection port temperature was 220° C. and ion source temperature of 200° C., a heating program was following: hold at 50° C. for 2 minutes, increased from 50° C. to 150° C. by 3° C. min′ and hold 150° C. for 5 minutes, then increased to 220° C. by 10° C. min′. Ionization energy was set at 70 eV, and the sample was scanned in a range of 50 m/z to 500 m/z.
[0120] 190 μL of the supernatant of the pET32a::BbTPS3 recombinant bacteria in the above reaction was replaced by 190 μL of the supernatant of the control bacteria, and the above experiment was repeated to obtain the target compound of the supernatant of the control bacteria.
[0121] The results of the GC-MS analysis are shown in
Example 3 Introduction of Blumea balsamifera BbTPS3 into Yeast Strain for Fermenting and Producing (−)-Borneol
[0122] 1. Construction of Eukaryotic Expression Vector
[0123] The Bbtps3 gene shown in SEQ ID NO: 1 was insert at the BamHI restriction enzyme site of the pESC-Leu vector (Agilent Company) by using the pEASY-Uni Seamless Cloning and Assembly Kit of Beijing TransGen Biotech Co., Ltd., and the rest sequence of the pESC-Leu vector remains unchanged to obtain a recombinant plasmid pESC-Leu::BbTPS3.
[0124] Specific steps were as follows:
[0125] 1) the PCR amplification product obtained in Example 1 was used as a template, PCR amplification was performed with primers BbTPS3-F3 and BbTPS3-R3, and the purified PCR product was obtained through recovering and purifying steps; the sequences of the primers were as follows (the underlined sequences were vector homologous regions):
TABLE-US-00005 BbTPS3-F3: 5′-AAGGAGAAAAAACCCCGATGGTTGGATTTCAAAAACACTC-3′; BbTPS3-R3: 5′-AGTGAGTCGTATTACGGCTAGGTTTTAGGCTTCAAAAGTA-3′;
[0126] 2) the pESC-Leu vector was digested with the restriction endonuclease BamHI, and then the linearized vector backbone was recovered;
[0127] 3) according to the instructions of the pEASY-Uni Seamless Cloning and Assembly Kit of Beijing TransGen Biotech Co., Ltd., the purified PCR product obtained in step 1) was combined with the linearized vector backbone in step 2) to obtain a recombinant plasmid pESC-Leu::BbTPS3.
[0128] 2. Construction of BY-Mono Yeast Strain
[0129] YPD solid medium: 1% of yeast extract+2% of peptone+2% of glucose+1.5% of agar; the corresponding liquid medium (YPD liquid medium) was prepared without adding the agar.
[0130] YPL induction medium: 1% of yeast extract+2% of peptone+2% of galactose.
[0131] SD-Ura solid plate: SD-Ura+2% of glucose+2% of agar; the corresponding liquid medium (SD-Ura liquid medium) was prepared without adding the agar.
[0132] SD-Ura-Leu solid plate: SD-Ura-Leu+2% of glucose+2% of agar; the corresponding liquid medium (SD-Ura-Leu liquid medium) was prepared without adding the agar.
[0133] BY4741 yeast strain (genotype: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was coated on the YPD solid plate, and cultured upside down at 30° C. for 48 hours to 72 hours to obtain a newly activated BY4741 yeast colony. Ura3 marker, yeast-derived tHMGR1 (containing promoter sequence P.sub.TDH3 and terminator sequence T.sub.TPI1, which was namely P.sub.TDH3-tHMGR1-T.sub.TPI1), yeast-derived IDI1 (containing promoter sequence P.sub.ADH1 and terminator sequence T.sub.PGI, which was namely P.sub.ADH1-IDI1-T.sub.PGI), yeast-derived tHMGR1 (containing promoter sequence P.sub.PGK1 and terminator sequence T.sub.ADH1, which was namely P.sub.PGK1-tHMGR1-T.sub.ADH1), and yeast-derived ERG20.sup.F96W-N127W (containing promoter sequence P.sub.TEF2 and terminator sequence T.sub.CYC1, which was namely P.sub.TEF2-ERG20.sup.F96W-N127W-T.sub.CYC1) were integrated at the YPRCΔ15 site (chromosome XVI long_terminal_repeat and Autonomously Replicating Sequence, YPRCΔ15) of the BY4741 yeast strain. Specific steps were as follows:
[0134] 1) inoculated 5 ml of YPD with an aliquot of an overnight culture or a colony from a BY4741 fresh plate, grew at 30° C. and 200 rpm until OD.sub.600 of 0.6 to 1.0;
[0135] 2) taken a cuvette (0.2 cm) soaked in ethanol and then be cleaned with ultra-pure water and air-dried, put upside down on filter paper, and finally placed in an ultra-clean table for sterilization;
[0136] 3) harvested 1 mL to 2 mL solution at 10,000 g for 1 minute at room temperature;
[0137] 4) washed by resuspending the pellet in 1 ml of ice-cold sterile water, and centrifuged as above;
[0138] 5) repeated step 4) and discarded the supernatant, resuspended in 1 ml of ice-cold buffer (10 mM LiAc, 10 mM DTT, 0.6 M sorbitol, and 10 mM Tris-HCl (pH 7.5)), and cultured at 25° C. for 20 minutes;
[0139] 6) centrifuged as above and discarded the supernatant;
[0140] 7) resuspended in 1 mL of ice-cold sorbitol (1 M), and centrifuged as above;
[0141] 8) repeated step 7) and discarded the supernatant. Resuspended cells in 100 μL of ice-cold sorbitol (1 M) solution and then BY4741 yeast competent cells were prepared;
[0142] 9) five DNA fragments of Ura3 marker, P.sub.TDH3-tHMGR1-T.sub.TPI1, P.sub.ADH1-IDI1-T.sub.PGI, P.sub.PGK1-tHMGR1-T.sub.ADH1 and P.sub.TEF2-ERG20.sup.F96W-N127W-T.sub.CYC1 were mixed in equal molar ratio, with a total mass of 500 ng (the total volume was no more than 1/10 of the volume of the competent cells), added into the BY4741 yeast competent cells, mixed and transferred to an cuvette (0.2 cm), and incubated on ice for 2 minutes to 5 minutes; electrotransformation was performed under 2.7 kV, 25 μF and 200Ω (Bio-Rad, Hercules, Calif.), and after electric shock, added 1 mL of sorbitol (1 M) solution in an ultra-clean working table, then transferred into a sterile 1.5 mL EP tube, cultured at 30° C. for 1 to 2 hours, and mixed up and down for 2 to 3 times;
[0143] 10) the mixture was centrifuged at 10,000 g for 1 minute at room temperature, discard the supernatant, and the cells were resuspended with the remaining 100 μL of solution; the solution was dropwise added in the center of the synthetic dropout medium SD-Ura solid plate, evenly coated by using a coater until all the coated solution was completely absorbed, and placed in an incubator at 30° C. for inverted culture for 2 to 3 days; the obtained strain was named as BY-Mono yeast strain, and the genotype of BY-Mono yeast was MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0, YPRCΔ15 Ura3-P.sub.TDH3-tHMGR1-T.sub.TPI1-P.sub.ADH1-IDI1-T.sub.PGI-P.sub.PGK1-tHMGR1-T.sub.ADH1-P.sub.TEF2-ERG20.sup.F96W-N127W-T.sub.CYC1.
[0144] 3. Preparation of BY-Mono Yeast Competent Cells
[0145] Yeast competent cells were prepared by using the ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit:
[0146] (1) picked the refresh BY-Mono single colony from the SD-Ura plate, inoculated into 10 mL of SD-Ura liquid medium, and shaken and cultured at 30° C. until OD.sub.600 of 0.8 to 1.0;
[0147] (2) pelleted the cells at 500 g for 4 minutes and discarded the supernatant.
[0148] (3) added 10 mL of Frozen-EZ Solution 1 to wash pellet, and centrifuged as above, and discarded the supernatant;
[0149] (4) added 1 mL of Frozen-EZ Solution 2 to resuspend the pellet to obtain the BY-Mono yeast competent cells, and the BY-Mono yeast competent cells were sub-packaged into sterile 1.5 mL EP tubes, with 50 μL in each tube;
[0150] (5) BY-Mono competent cells were slowly cooled to −70° C. (4° C. for 1 hour; −20° C. for 1 hour; −40° C. for 1 hour; stored at −70° C.), and it was forbidden to quick freeze the competent cells with liquid nitrogen.
[0151] 4. Transformation of Plasmid pESC-Leu::BbTPS3 into BY-Mono Competent Cells
[0152] (1) Mixed 50 μL of BY-Mono yeast competent cells with 0.2-1 μg plasmid pESC-Leu::BbTPS3 (in less than 5 μL).
[0153] (2) Added 500 μL of Frozen-EZ Solution 3, and mixed thoroughly.
[0154] (3) Incubated at 30° C. for 1 to 2 hours, and mix for 2 to 3 times.
[0155] (4) Spreaded 50-150 μL of the above transformation mixture on the SD-Ura-Leu solid plate, air-dried, and then invertedly incubated at 30° C. for 48 to 96 hours to obtain a recombinant yeast transformed with the recombinant plasmid pESC-Leu::BbTPS3, which was named as BY-Mono/pESC-Leu::BbTPS3.
[0156] Meanwhile, the pESC-Leu vector without the target gene (which was namely the Bbtps3 gene) was transformed into the BY-Mono yeast competent cells by the same method as above and used as a control, to obtain a recombinant yeast transformed with the pESC-Leu vector, which was named as BY-Mono/pESC-Leu.
[0157] 5. Fermentation
[0158] (1) The BY-Mono/pESC-Leu::BbTPS3 single colony grown on the SD-Ura-Leu solid plate in step 4 was picked, and inoculated in 10 mL of SD-Ura-Leu liquid medium at 200 rpm and 30° C. for 48 hours.
[0159] (2) Harvested the cells at 5,000×g and room temperature for 5 minutes, and resuspended the cells by 20 mL of YPL liquid medium, and cultured at 200 rpm and 30° C. for 72 hours to obtain a fermentation product.
[0160] 6. Extraction of Fermentation Product
[0161] The target compound was terpenoid, which was fat-soluble and easily soluble in ethyl acetate. Therefore, ethyl acetate was selected as a solvent to extract the fermentation product, to obtain the target compound. The steps of the extraction were as follows:
[0162] (1) collected the fermented solution, which was the fermentation product, and added with an equal volume of ethyl acetate;
[0163] (2) sonicated the above mixture for 1 hour, and shaken and mixed for many times during this period;
[0164] (3) the upper organic phase was taken at 5,000×g at room temperature for 5 minutes, added in an appropriate amount of anhydrous sodium sulfate (dried at 120° C. for 30 minute), and shaken during addition to remove the water in the extract;
[0165] (4) the solution was concentrated on rotary evaporator to be nearly dry;
[0166] (5) the concentrated solution was pipetted, and filtered through a 0.22 μm PTFE needle filter, and the filtrate was stored in vial, sealed with a sealing film, and stored in a refrigerator at 4° C.
[0167] 7. GC-MS Detection of Fermentation Product
[0168] Gas chromatography-mass spectrometry GC-MS was used to detect the target compound: the GC-MS analysis system was Thermo TRACE 1310/TSQ 8000 gas chromatograph, with an injection volume of 1 μL, a mode of splitless. A gas chromatographic column of Thermo Scientific TG-5MS (30 m×0.25 mm×0.25 μm) was used. And helium was used as carrier gas with flow rate of 1.0 mL/min. The injection port temperature was 220° C. and ion source temperature of 200° C., a heating program was following: hold at 50° C. for 2 minutes, increased from 50° C. to 150° C. by 5° C..Math.min.sup.−1 and hold 150° C. for 2 minutes, then increased to 300° C. by 30° C..Math.min.sup.−1. Ionization energy was set at 70 eV, and the sample was scanned in a range of 50 m/z to 500 m/z.
[0169] In the fermentation of the above step 5, “the BY-Mono/pESC-Leu::BbTPS3 single colony grown on the SD-Ura-Leu solid plate in step 4 was picked” was replaced by “the BY-Mono/pESC-Leu single colony grown on the SD-Ura-Leu solid plate in step 4 was picked”, and the above experiment steps 5, 6 and 7 were repeated.
[0170] The results of GC-MS analysis are shown in
[0171] The present invention is described in detail above. For those technicians in the field, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present invention and unnecessary experiments. Although the present invention gives specific examples, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the present application is intended to comprise any changes, uses or improvements of the present invention, comprising changes that deviate from the scope disclosed in the present application but are made by conventional techniques known in the art. According to the scope of the following appended claims, some basic features can be applied.
INDUSTRIAL APPLICATION
[0172] The Bbtps3 gene is cloned from the cDNA of Blumea balsamifera (L.) DC. in the present invention, and the gene is a key enzyme gene for the synthesis of a monoterpene ingredient obtained from Blumea balsamifera (L.) DC. for the first time. It has been proved by experiments that: the BbTPS3 protein mentioned in the present invention can catalyze the formation of (−)-borneol (l-borneol) from GPP, and has an important role in the biosynthesis of (−)-borneol and other monoterpene compounds in Blumea balsamifera (L.) DC., and provides an important basis for increasing the content of the active ingredient (−)-borneol in Blumea balsamifera (L.) DC. by using a genetic engineering technology or directly producing (−)-borneol, thus further having important theoretical and practical significances for regulating and producing plant monoterpene compounds and culturing high-quality Blumea balsamifera (L.) DC.