WO2022007542A1 - 紫杉二烯合酶TcTS2、编码核苷酸序列及其应用 - Google Patents

紫杉二烯合酶TcTS2、编码核苷酸序列及其应用 Download PDF

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WO2022007542A1
WO2022007542A1 PCT/CN2021/097561 CN2021097561W WO2022007542A1 WO 2022007542 A1 WO2022007542 A1 WO 2022007542A1 CN 2021097561 W CN2021097561 W CN 2021097561W WO 2022007542 A1 WO2022007542 A1 WO 2022007542A1
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tcts2
nucleotide sequence
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sequence
amino acid
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闫建斌
苟君波
廖庆刚
梁海菲
李冲
杜然
熊兴耀
黄三文
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中国农业科学院深圳农业基因组研究所
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    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03017—Taxadiene synthase (4.2.3.17)

Definitions

  • the present application relates to the technical field of plant genetic engineering, in particular to a taxadiene synthase TcTS2, an encoding nucleotide sequence and its application in synthesizing baccatin III or paclitaxel.
  • Paclitaxel is a diterpenoid alkaloid, which was first isolated from the bark of Taxus brevifolia. It is a widely used anticancer drug and is widely used in the clinical treatment of various cancers. Cancer is one of the top ten causes of human death. At present, the annual incidence of cancer in the world is still more than 10 million (WHO). Paclitaxel and its preparations are important first-line anticancer drugs.
  • the main production method of paclitaxel is semi-synthetic method, that is, firstly by extracting natural precursors baccatin III (baccatin III) and 10-deacetylbaccatin III (10-deacetylbaccatin III, 10-DAB), and then chemically synthesizing Paclitaxel (Li et al., 2015; Liu et al., 2016), but the precursor substances used in this method still rely on plant extraction and are subject to plant or cell resources, which cannot completely solve the supply problem.
  • baccatin III baccatin III
  • 10-DAB 10-deacetylbaccatin III
  • Taxadiene synthase was first cloned from Taxus brevis, and taxadiene synthase (TS) catalyzes the cyclization of Geranylgeranyl Diphosphate (GGPP) to generate taxus Diene, taxadiene undergoes a series of functional group reactions to form baccatin III, TS plays an indispensable role in the synthesis of taxol (Wildung et al., 1996).
  • Taxadiene synthase is the first-step enzyme that catalyzes the formation of the parent taxane skeleton of paclitaxel, and its cyclization efficiency of GGPP is very low compared with that of other gymnosperm cyclases. This step is the limit of the paclitaxel synthesis pathway. Fast steps, TS was confirmed to be a slow enzyme. Therefore, it is of great significance to discover the paclitaxel biosynthesis genes and their encoded enzymes with higher enzymatic activity.
  • the present application is devoted to providing a taxadiene synthase TcTS2, which improves the efficiency of catalyzing the cyclization of GGPP, so that the intermediate taxadiene, baccatin III and product taxol in the process of synthesizing paclitaxel are greatly produced. It can significantly reduce the production cost of paclitaxel, and effectively solve the current situation that paclitaxel is expensive and in short supply in the market.
  • a first aspect of the present application provides a taxadiene synthase TcTS2, the amino acid sequence of the taxadiene synthase TcTS2 comprises or consists of the following sequences:
  • TcTS2 novel taxadiene synthase gene in Taxus chinensis var.maire, which was named TcTS2, and successfully obtained the encoded protein of the gene (that is, taxadiene synthase).
  • TcTS2 also named TcTS2 in this application.
  • the inventors of the present application found that the efficiency of TcTS2 catalyzing the substrate GGPP was significantly higher than that of another type of taxadiene synthase gene TcTS1, and it showed inducible expression under the stress of the plant hormone methyl jasmonate (MeJA). model.
  • the amino acid sequence of the taxadiene synthase TcTS2 is the amino acid sequence shown in SEQ ID NO. 1, with a total of 758 amino acids.
  • the amino acid sequence of the taxadiene synthase TcTS2 is a functional homologous sequence having at least 80% sequence similarity with the amino acid sequence shown in SEQ ID NO.1.
  • the functional homologous sequence of the identity includes, but is not limited to, the amino acids shown in SEQ ID NO.1 have about 80% or more, 82% or more, 84% or more, 85% or more, 88% or more. , 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more , an amino acid sequence of 99.9% or more identity.
  • the amino acid sequence of the taxadiene synthase TcTS2 is one or more additions, deletions, and substitutions in the amino acid sequence shown in SEQ ID NO. 1 (for example, it can be one , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid sequences that have TcTS2 activity.
  • the amino acid sequence of the taxadiene synthase TcTS2 is the amino acid sequence shown in SEQ ID NO.2, with a total of 815 amino acids, which is the same as the amino acid sequence shown in SEQ ID NO.1 In contrast, the extra 57 amino acids at the N-terminus are signal peptide sequences.
  • the amino acid sequence of the taxadiene synthase TcTS2 is a functional homologous sequence having at least 80% sequence similarity with the amino acid sequence shown in SEQ ID NO. 2.
  • the functional homologous sequence of the identity includes, but is not limited to, the amino acids shown in SEQ ID NO. 2 have about 80% or more, 82% or more, 84% or more , 85% or more, 88% or more, 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more , 99% or more, 99.5% or more, 99.9% or more identical amino acid sequences.
  • the amino acid sequence of the taxadiene synthase TcTS2 is one or more additions, deletions, and substitutions (for example, it can be one) in the amino acid sequence shown in SEQ ID NO.2 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid sequences that have TcTS2 activity.
  • the taxadiene synthase TcTS2 of the present application significantly improves the cyclization efficiency of GGPP, reduces the rate-limiting effect, and further increases the yields of intermediate taxadiene, baccatin III and product taxol in the process of synthesizing taxol. It can significantly reduce the production cost of paclitaxel, and can effectively solve the current situation that paclitaxel is expensive and in short supply in the market.
  • taxadiene synthase TcTS2 in plant cells is significantly regulated by methyl jasmonate (MeJA), which is beneficial to the application of synthetic biology technology.
  • MeJA methyl jasmonate
  • Other synthetic biology techniques can better regulate the expression of taxadiene synthase TcTS2, which can be used in plant genetic engineering and metabolic engineering to produce paclitaxel and its intermediates in the future.
  • the second aspect of the present application provides a nucleotide sequence encoding the taxadiene synthase TcTS2.
  • nucleotide sequence encoding taxadiene synthase TcTS2 comprises or consists of the following sequences:
  • the nucleotide sequence encoding taxadiene synthase TcTS2 is the nucleotide sequence shown in SEQ ID NO. 3, with a full length of 5138 bases.
  • the nucleotide sequence encoding taxadiene synthase TcTS2 is a complementary sequence formed by the nucleotide sequence shown in SEQ ID NO. 3 according to the principle of base complementary pairing.
  • the nucleotide sequence encoding taxadiene synthase TcTS2 is a degenerate sequence of the nucleotide sequence shown in SEQ ID NO.3.
  • Degenerate sequence means that after one or more nucleotide sequences of SEQ ID NO.3 are changed, the type of amino acid encoded by the changed nucleotide sequence position remains unchanged, and will not affect taxadiene synthase TcTS2.
  • the nucleotide sequence encoding taxadiene synthase TcTS2 is the homologous sequence of the nucleotide sequence shown in SEQ ID NO.3.
  • Said homologous nucleotide sequence includes adding and/or replacing and/or deleting one or several nucleotides in the nucleotide sequence shown in SEQ ID NO. Mutant gene, allele or derivative of enzymatic activity.
  • the homologous sequence is about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more of the nucleotide sequence shown in SEQ ID NO. 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, Polynucleotides of 99.7% or more, 99.8% or more, or 99.9% or more identity.
  • the nucleotide sequence encoding taxadiene synthase TcTS2 hybridizes with the nucleotide sequence of SEQ ID NO.3 under stringent conditions, and can encode taxadiene Nucleotide sequence of synthase TcTS2.
  • the "stringent conditions” refer to conditions under which a probe will hybridize to its target sequence to a detectable degree over hybridization to other sequences (eg, at least 2-fold over background). Stringent conditions are sequence-dependent and vary with the environment. By controlling the stringency of hybridization and/or wash conditions, target sequences that are 100% complementary to the probe can be identified. Alternatively, stringency conditions can be adjusted to allow some sequence mismatches such that lower degrees of similarity are detected, while such sequence mismatches result in a nucleotide sequence that does not affect the nucleotide sequence encoding the taxadiene synthase TcTS2. its normal enzymatic activity.
  • sequence of the cDNA comprises or consists of the following sequence:
  • the homologous sequence is about 80% or more of the nucleotide sequence shown in SEQ ID NO.4 , 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more , 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more , or a polynucleotide of 99.9% or greater identity.
  • the nucleotide sequence encoding taxadiene synthase TcTS2 is the cDNA sequence of the nucleotide sequence shown in SEQ ID NO. 4, with a full length of 2448 bases.
  • the third aspect of the present application provides the use of the taxadiene synthase TcTS2, or the nucleotide sequence encoding the taxadiene synthase TcTS2, in synthesizing baccatin III and/or paclitaxel.
  • the application of the taxadiene synthase TcTS2 in synthesizing baccatin III and/or paclitaxel includes the following aspects: (1) the taxadiene synthase TcTS2 provided in this application or containing at least part of SEQ The polypeptide of the amino acid sequence shown in ID NO.1 may still have the biological activity of TcTS2 or even have new biological activities after removing or replacing some amino acids, or improve the yield or optimize the protein kinetic characteristics or other efforts to obtain properties; (2) involved in the biosynthesis of taxadiene; (3) involved in the biosynthesis of paclitaxel and its intermediates (eg baccatin III).
  • the application of the nucleotide sequence encoding taxadiene synthase TcTS2 in synthesizing baccatin III or paclitaxel includes the following aspects: (1) the nucleotide sequence or at least part of the nucleosides provided in this application
  • the acid sequence is modified or mutated by means of insertion, deletion, polymerase chain reaction (PCR), error-prone PCR, rejoining of different sequences, different parts of the sequence or targeting with homologous sequences from other sources evolution, or mutagenesis by chemical agents, etc.
  • the cloned gene of the nucleotide sequence or at least part of the nucleotide sequence provided in this application is expressed in an exogenous host through a suitable expression system to obtain the corresponding TcTS2 enzyme or a TcTS2 enzyme with higher biological activity or yield.
  • the gene or gene cluster of the nucleotide sequence or at least part of the nucleotide sequence provided in this application can be used to construct a recombinant plasmid through genetic recombination to obtain a novel biosynthetic pathway, or through insertion, replacement, deletion or inactivation. Thus, new biosynthetic pathways were obtained.
  • the fourth aspect of the present application provides primers for detecting the nucleotide sequence.
  • nucleotide sequence of the PCR product that amplifies the nucleotide sequence encoding taxadiene synthase TcTS2, which can meet the requirements of specific amplification or specific detection of the encoding taxadiene synthase TcTS2.
  • a primer for the nucleotide sequence of ene synthase TcTS2 is sufficient.
  • the primer includes an upstream primer and/or a downstream primer; wherein, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.5: 5'-CGAGGCTTGCAAGTTACACA-3 '; and/or, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6: 5'-CAGGGCATTTGAAACCTCAT-3'.
  • a fifth aspect of the present application provides a vector into which the nucleotide sequence is introduced.
  • the type of the carrier is not specifically limited, and it can be a carrier commonly used in the art.
  • vectors include, but are not limited to, pET28b, pIJ702, pUCP19, pYMB03 or pHT43, preferably pET28b.
  • a sixth aspect of the present application provides a host cell into which the nucleotide sequence or the vector is introduced.
  • the host cells include plant cells and/or microbial cells
  • the plant cells include but are not limited to yew cells, tobacco cells, white yew cells, and Artemisia annua cells;
  • the microbial cells include, but are not limited to, Streptomyces, Pseudomonas, Bacillus, yeast cells, and Escherichia coli.
  • in vivo and in vitro methods for introducing the nucleotide sequence encoding taxadiene synthase TcTS2, or the recombinant plasmid, or the expression vector into host cells include but are not limited to electroporation, polyethylene glycol (PEG) transformation, lipofection, heat shock, calcium phosphate precipitation, virus mediation and microinjection.
  • PEG polyethylene glycol
  • a seventh aspect of the present application provides a method for expressing taxadiene synthase TcTS2 in a plant, by transferring the nucleotide sequence, or the vector, or the host cell into a plant , to obtain the taxadiene synthase TcTS2.
  • the plants include but are not limited to: Taxus chinensis, White yew, tobacco, and Artemisia annua.
  • the plant body also includes plant parts such as explants, including, but not limited to, cuttings, tissue cultures, cell suspensions, and callus.
  • the plant is more preferably yew and/or tobacco.
  • the eighth aspect of the present application provides a method for producing paclitaxel and its intermediates, comprising expressing the taxadiene synthase TcTS2 in a plant.
  • the method includes: transforming the nucleotide sequence, or the vector, or the host cell into a plant to express the taxadiene synthase TcTS2 to obtain paclitaxel and its intermediates body.
  • the application provides a taxadiene synthase TcTS2, which significantly improves the efficiency of catalyzing the cyclization of GGPP, so that the intermediate taxadiene, baccatin III and the product taxol in the process of synthesizing taxol can be produced greatly. It can significantly reduce the production cost of paclitaxel, and can effectively solve the current situation that paclitaxel is expensive and in short supply in the market.
  • taxadiene synthase TcTS2 provided in this application in plant cells is significantly induced and regulated by MeJA. Combined with the induction of small molecule MeJA and other synthetic biology techniques, it can be used in plant genetic engineering and plant genetic engineering in the future. Metabolic engineering for the production of paclitaxel and its intermediates.
  • nucleotide sequence encoding taxadiene synthase TcTS2 provided in this application provides a new gene resource for improving the yield of taxol, and the nucleotide sequence and TcTS2 can be used for the transformation of plant genetic engineering and metabolic engineering strategies Chassis host, production of paclitaxel and its intermediates, etc., has significant economic and social value.
  • FIG. 1 shows the analysis of the in vitro biochemical activity of TcTSs.
  • A is the chromatogram of TcTSs enzymatic reaction product detected by GC-MS, and the characteristic ion m/z 122 of taxadiene is extracted
  • B is the schematic diagram of TcTSs-catalyzed GGPP to generate taxadiene
  • C is the mass spectrum of TcTSs enzymatic reaction product picture.
  • Figure 2 shows the changes in metabolite content and the expression pattern of TcTSs genes under MeJA stress.
  • A is the relative change of Baccatin III and paclitaxel production under MeJA stress;
  • B is the expression pattern of TcTS1 gene under MeJA stress;
  • C is the expression pattern of TcTS2 gene under MeJA stress;
  • MeJA+ represents 100 ⁇ M MeJA treatment group;
  • MeJA- Indicates the solvent control group.
  • Figure 3 shows the fermentation growth curve induced by engineering strains E.coli TbTS and E.coli TcTS2 in Example 4.
  • Fig. 4 shows in Example 4, the yield comparison of the synthetic taxadiene product of engineering strain E.coli TbTS and E.coli TcTS2.
  • TcTS2 may refer to taxadiene synthase, or taxadiene synthase gene, or a nucleotide sequence encoding taxadiene synthase, and the specific meaning can be determined according to the context.
  • gene is defined as a genetic unit (usually represented by a DNA sequence) that occupies a specific location in a chromosome and contains genetic instructions that contribute to an underlying phenotypic trait or trait in a plant.
  • nucleotide is used in its ordinary meaning as understood by those skilled in the art.
  • amino acid refers to any amino acid (both standard and non-standard amino acids), including but not limited to alpha-amino acids, beta-amino acids, gamma-amino acids, and delta-amino acids.
  • suitable amino acids include, but are not limited to, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine , histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.
  • TbTS Taxus brevis taxadiene synthase
  • TcTS1 amino acid sequence of the reading frame of TcTS1 is 98% homologous to TbTS
  • TcTS2 is a newly discovered taxadiene synthase gene
  • genomic DNA sequence of TcTS2 is as shown in SEQ ID NO.3
  • the nucleotide similarity with TbTS is 42%; the nucleotide sequence and amino acid sequence of the TcTS2 reading frame are shown in SEQ ID NO.4 and SEQ ID NO.1, respectively, and the nucleotide similarity and amino acid sequence with TbTS are shown in The homology is both 79%.
  • the applicant designed primers P1 and P2, P3 and P4 for respectively amplifying the TcTS1 and TcTS2 genes according to the sequences after deleting the position of the signal peptide.
  • the primers contained Part of the prokaryotic expression vector pET28b sequence, construct a vector whose N-terminus is fused and expressed with 6 histidines, which is convenient for subsequent purification.
  • PCR was used to amplify the TcTS1 and TcTS2 genes. After the PCR product was recovered by gel cutting, it was recombined with the linear pET28b vector double-digested by Sal I and Not I. Hieff Clone TM one-step cloning kit (YEASEN, China) cloned and sequenced, and the positive recombinant plasmids were named pET28b-TcTS1 and pET28b-TcTS2, respectively.
  • P1 and P2 were used to amplify TcTS1
  • P3 and P4 were used to amplify TcTS2
  • the primer sequences were as follows:
  • TcTSs-His6 fusion protein The constructed fusion expression vectors pET28b-TcTS1 and pET28b-TcTS2 were respectively transferred into E. coli BL21(DE3) to obtain BL21(DE3)/pET28b-TcTS1 and BL21(DE3)/pET28b- TcTS2 transgenic strain; pick positive single clones and inoculate them in LB medium (containing 50 ⁇ g ⁇ mL -1 of Kanamycin), and cultivate overnight at 37°C; take the overnight culture and inoculate it in 300 mL of fresh LB medium at a ratio of 1:100 ( Contain 50 ⁇ g ⁇ mL -1 of Kanamycin), expand the culture at 37°C to an OD600 value of 0.4-0.6; add IPTG (isopropylthiogalactoside) to a final concentration of 1.0mmol ⁇ L -1 , 16°C, 160rpm for induction Expressed for 12h; 12,000rpm, centr
  • TcTSs-His6 fusion protein was based on the HisPur Ni NTA Resin and desalting ultrafiltration tube method of Thermo Scientific Company in the United States: the collected cells were resuspended in 20 mL of lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole) ), use ultrasonic degradation method to break the wall, centrifuge at 4,000g for 20 minutes at 4°C, load the supernatant into a 1.5mL HisPur Ni-NTA resin packing column (Thermo Scientific, USA), remove impurities and use elution buffer solution (50 mM sodium phosphate, 300 mM NaCl, 50 mM imidazole).
  • lysis buffer 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole
  • Recombinant TcTSs-His6 purified using 30kDa ultrafiltration desalted, stored in enzyme reaction buffer (25mM HEPES, pH8.5,10% glycerol, 5mM DTT, 5mM sodium ascorbate, sodium metabisulfite 5mM and 1mM MgCl 2), the recombinant
  • enzyme reaction buffer 25mM HEPES, pH8.5,10% glycerol, 5mM DTT, 5mM sodium ascorbate, sodium metabisulfite 5mM and 1mM MgCl 2
  • TcTS1-His6 fusion protein and fusion protein are TcTS2-His6 soluble expression, purified protein purity over 98% at concentrations of 3.4 ⁇ g ⁇ ⁇ L -1 and 1.8 ⁇ g ⁇ ⁇ L -1.
  • the in vitro enzymatic reaction system was 500 ⁇ L containing 100 ⁇ g purified protein, 100 ⁇ M GGPP (Sigma-Aldrich) and enzyme reaction buffer (25 mM, pH 8.5, 10% glycerol, 5 mM DTT, 5 mM ascorbate, 5 mM sodium metabisulfite and 1 mM MgCl 2 ), the reaction mixture was covered with 500 ⁇ L of pentane (Macklin, GC-MS grade) and reacted in a 32°C water bath for 2 hours, vortexed for 2 minutes, centrifuged at 5000 rpm for 10 minutes, the pentane layer was taken out and put into 2 mL of the sample, and concentrated using a nitrogen blower at low temperature , the product was analyzed by gas chromatography mass spectrometry (GC-MS) instrument.
  • GC-MS gas chromatography mass spectrometry
  • the control group was the reaction product of purified recombinant protein TcTSs-His6 after being boiled at 100 °C for 10 min.
  • the GC-MS instrument was Agilent 7890B/7000C (Agilent Technologies, Waldbronn, USA), mass detector parameters: 70eV, helium flow rate 1.2mL ⁇ min -1 , chromatographic column Agilent HP-5MS (5% phenylmethyl silica, 30m ⁇ 250 ⁇ m inner diameter, 0.25 ⁇ m film thickness), the injection volume is 1-5 ⁇ L; the temperature program of the column oven is as follows: 45°C for 1 minute, then 10°C ⁇ min -1 gradient to 250°C, and 250°C for 5 minutes. The temperature of the injection port was 250°C; the mass-to-charge ratio (m/z) of the scan was 40-350, and the qualitative analysis of the enzymatic product was carried out with the taxadiene standard as the reference.
  • FIG. 1 The results of in vitro biochemical function analysis are shown in Figure 1.
  • the results show that both TcTS1 and TcTS2 can catalyze the substrate GGPP to generate the main products taxa-4(5), 11(12)-diene (taxene 4(5), 11(12 )-diene, accounting for 94%) and the secondary product taxa-4(20),11(12)-diene (taxene 4(20),11(12)-diene, accounting for 4%), showing the same Taxadiene synthase activity.
  • Figure 1C shows the mass spectrum of the product of the enzymatic reaction of TcTSs, which is used to characterize the enzymatic product.
  • Kinetic parameter determination standard enzymatic reaction total volume 100 ⁇ L, containing enzyme reaction buffer (25 mM, pH 8.5, 10% glycerol, 5 mM DTT, 5 mM ascorbate, 5 mM sodium metabisulfite and 1 mM MgCl 2 ), 34 ⁇ g (TcTS1-His6 ) or 18 ⁇ g (TcTS2-His6) recombinant protein and 7 different concentrations of GGPP (0.2 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, 25 ⁇ M, 50 ⁇ M) mixed with [ 1-3 H]-GGPP substrate (American Radiolabeled Chemicals, Inc, 30 Ci ⁇ mM ⁇ 1 ).
  • the kinetic constant results showed that the K m of TcTS1-His6 and TcTS2-His6 were 5.5 ⁇ 1.6 and 8.6 ⁇ 1.5 (pM ⁇ g -1 ⁇ min -1 ), respectively, while the K cat values of TcTS1-His6 and TcTS2-His6 were are 0.00292(1 ⁇ s -1 ) and 0.00901(1 ⁇ s -1 ), respectively. From their K m and K cat values, despite TcTS1-His6 GGPP binding strength is 1.6 times TcTS2-His6, but the catalytic efficiency TcTS2-His6 three times (K cat value) of TcTS1-His6. The kinetic constant results show that the catalytic efficiency of TcTS2 is significantly better than that of TcTS1, and the application prospect of synthetic biology is also significantly better than that of TcTS1.
  • Taxus chinensis 211 cell lines were divided into two groups: the experimental group (MeJA+) was soaked with 100 ⁇ M methyl jasmonate; the control group (MeJA-) was soaked with 0.05% ethanol solution. The two groups were carried out at the same time, and the samples were collected after 0h, 2h, 3h and 4h, respectively, for the expression pattern of TcTSs gene and the analysis of the content of baccatin III and paclitaxel, with three biological replicates.
  • the metabolites were extracted by the modified Wolfender method.
  • the separation column was Waters (2.1 ⁇ 50 mm; ACQUITY UPLC TM Waters XselectHSS T3); the injection volume was 10 ⁇ L, and the flow rate was 0.2 mL ⁇ min ⁇ 1 .
  • the mobile phase composition is: A is 0.1% formic acid methanol; B is 0.1% formic acid water.
  • the LC separation procedure was: 40% A equilibration for 2 min, followed by gradient elution for 10 min to 100% B, maintained for 2 min, and then returned to 40% A equilibration for 1 min.
  • Data acquisition and processing were performed on AB SCIEXAnalyst 1.6.3 software (Applied Biosystems).
  • RNA Extraction Kit (Aidlab, Beijing) to extract and collect the total RNA of the samples with different treatment times. Take 1 ⁇ g of total RNA for the synthesis of the first-strand cDNA, and use it as the template for real-time fluorescence quantitative PCR.
  • the first-strand cDNA Synthetic use kit III 1st Strand cDNA Synthesis (YEASEN, China) and qPCR Fluorescent Dye Kit qPCR Green Master Mix (Low Rox Plus) (commercialized reagents, purchased from YEASEN, China). qPCR experiments were performed on a QuantStudio TM 3 system with 2 technical replicates and 3 biological replicates. Taxus chinensis actin1 ( TcACTIN1) gene as an internal control gene, using the calculated relative expression of genes TcTSs 2 - ⁇ Ct method.
  • TcACTIN1 Taxus chinensis actin1
  • qPCR gene-specific primers are as follows:
  • TcTS1 5'-AGCACTGGCACTAGCAAGGT-3' (TcTS1) (SEQ ID NO. 13)
  • TcTS2 5'-CGAGGCTTGCAAGTTACACA-3' (TcTS2) (SEQ ID No. 5)
  • TcTS2 5'-CAGGGCATTTGAAACCTCAT-3' (TcTS2) (SEQ ID No. 6).
  • TcTS2 was significantly up-regulated, and the production of paclitaxel and baccatin III was significantly increased.
  • the inducible expression of TcTS2 gene is more conducive to the application of synthetic biology technology, and the synthetic application prospect is significantly better than that of TcTS1.
  • novel taxadiene synthase gene TcTS2 provided in this application is significantly different from another type of taxadiene synthase gene TcTS1, and the expressed proteins are also significantly different. Inducible expression was presented in the lower part, which significantly promoted the synthesis of baccatin III and paclitaxel.
  • TcTS2 and TbTS were used to construct taxadiene-producing engineered strains E.coli TcTS2 and E.coli TbTS, respectively.
  • the growth curve was drawn according to the arithmetic mean, and the results are shown in Figure 3.
  • the detection of the synthetic taxadiene product of two kinds of engineering bacteria is detected by GC-MS, and the GC-MS detection method is the same as that in Example 2, and the target peak of its chromatogram is integrated, and converted by area integration concentration, the results are shown in Figure 4, it can be seen that the yield of the target product expressed by E.coli TcTS2 is 12 times that of E.coli TbTS.

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Abstract

提供了一种紫杉二烯合酶TcTS2，编码核苷酸序列及其应用。TcTS2的氨基酸序列包含或由如下序列组成：a)SEQ ID NO.1所示的氨基酸序列；或，b)与SEQ ID NO.1所示的氨基酸序列具有至少80％序列相似性的功能性同源序列；或c)在SEQ ID NO.1所示的氨基酸序列中添加、缺失、替换一个或多个氨基酸且具有TcTS2活性的氨基酸序列。TcTS2和编码TcTS2的核苷酸序列，为提高紫杉醇的产量提供了新的基因资源，可用于植物基因工程和代谢工程策略改造底盘宿主，生产紫杉醇及其中间体等，具有显著经济和社会价值。

Description

紫杉二烯合酶TcTS2、编码核苷酸序列及其应用 技术领域
本申请涉及植物基因工程技术领域，具体涉及一种紫杉二烯合酶TcTS2，编码核苷酸序列及在合成巴卡亭III或紫杉醇中的应用。
背景技术
紫杉醇是一种二萜类生物碱，最早分离于短叶红豆杉Taxus brevifolia树皮中，是广泛使用的抗癌药物，广泛用于多种癌症的临床治疗。癌症是人类十大死亡原因之一，目前，世界上癌症年发病人数仍在1000万以上(WHO)，紫杉醇及其制剂是重要的一线抗癌药物。当前，紫杉醇的主要生产方式为半合成法，即首先通过提取天然前体巴卡亭Ⅲ(baccatin Ⅲ)和10-去乙酰巴卡亭Ⅲ(10-deacetylbaccatin Ⅲ,10-DAB)，然后化学合成紫杉醇(Li et al.,2015；Liu et al.,2016)，但该方法所用的前体物质仍依赖于植物提取，受制于植物或细胞资源，无法完全解决供应问题。
紫杉二烯合酶(Taxadiene synthase,TS)最早在短叶红豆杉中被克隆出来，紫杉二烯合酶(TS)催化牛儿基二磷酸(Geranylgeranyl Diphosphate,GGPP)环化反应生成紫杉二烯，紫杉二烯经过一系列的官能团反应生成巴卡亭Ⅲ，TS对紫杉醇的合成具有不可或缺的作用(Wildung et al.,1996)。
紫杉二烯合酶是催化紫杉醇母体紫杉烷骨架形成的第一步酶，其催化GGPP环化的效率和其他裸子植物的环化酶效率相比很低，此步骤是紫杉醇合成途径的限速步骤，TS被证实为慢速酶。因此，发掘具有更高酶活性的紫杉醇生物合成基因及其编码的酶具有十分重要的意义。
发明内容
有鉴于此，本申请致力于提供一种紫杉二烯合酶TcTS2，提高催化GGPP环化的效率，使合成紫杉醇过程的中间体紫杉二烯、巴卡亭Ⅲ以及产物紫杉醇产量得到大幅度提高，显著降低紫杉醇药物的生产成本，有效解决市场上紫杉醇价格昂贵、供不应求的现状。
本申请第一方面提供了一种紫杉二烯合酶TcTS2，所述紫杉二烯合酶TcTS2的氨基酸序列包含或由如下序列组成：
a)SEQ ID NO.1所示的氨基酸序列；或，
b)与SEQ ID NO.1所示的氨基酸序列具有至少80％序列相似性的功能性同源序列；或
c)在SEQ ID NO.1所示的氨基酸序列中添加、缺失、替换一个或多个氨基酸且具有TcTS2活性的氨基酸序列。
本申请的发明人在南方红豆杉Taxus chinensis var.maire中发现了一种新型紫杉二烯合酶基因，自定义命名为TcTS2，并成功获得该基因的编码蛋白(即紫杉二烯合酶TcTS2，本申请中也命名为TcTS2)。本申请的发明人经过研究分析发现TcTS2催化底物GGPP的效率显著高于另一类型的紫杉二烯合酶基因TcTS1，且在植物激素茉莉酸甲酯(MeJA)胁迫下表现为诱导型表达模式。
在本申请的一种实施方式中，所述紫杉二烯合酶TcTS2的氨基酸序列为SEQ ID NO.1所示的氨基酸序列，共有758个氨基酸。
在本申请的一种实施方式中，所述紫杉二烯合酶TcTS2的氨基酸序列为与SEQ ID NO.1所示的氨基酸序列具有至少80％序列相似性的功能性同源序列。所述的同一性的功能性同源序列包括但不限于SEQ ID NO.1所示的氨基酸具有约80％或以上、82％或以上、84％或以上、85％或以上、88％或以上、90％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.5％或以上、99.9％或以上同一性的氨基酸序列。
在本申请的一种实施方式中，所述紫杉二烯合酶TcTS2的氨基酸序列为在SEQ ID NO.1所示的氨基酸序列中添加、缺失、替换一个或多个(例如可以为1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个)氨基酸且具有TcTS2活性的氨基酸序列。
在本申请的一种实施方式中，所述紫杉二烯合酶TcTS2的氨基酸序列为SEQ ID NO.2所示的氨 基酸序列，共有815个氨基酸，与SEQ ID NO.1所示的氨基酸序列相比，其N端多出的57个氨基酸为信号肽序列。
在本申请的一种实施方式中，所述紫杉二烯合酶TcTS2的氨基酸序列为与SEQ ID NO.2所示的氨基酸序列具有至少80％序列相似性的功能性同源序列。
在本申请的一种实施方式中，所述的同一性的功能性同源序列包括但不限于SEQ ID NO.2所示的氨基酸具有约80％或以上、82％或以上、84％或以上、85％或以上、88％或以上、90％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.5％或以上、99.9％或以上同一性的氨基酸序列。
在本申请的一种实施方式中，所述紫杉二烯合酶TcTS2的氨基酸序列为在SEQ ID NO.2所示的氨基酸序列中添加、缺失、替换一个或多个(例如可以为1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个)氨基酸且具有TcTS2活性的氨基酸序列。
本申请的紫杉二烯合酶TcTS2催化GGPP环化的效率明显提高，减轻了限速的影响，进而使合成紫杉醇过程的中间体紫杉二烯、巴卡亭Ⅲ以及产物紫杉醇产量得到大幅度提高，显著降低了紫杉醇药物的生产成本，能够有效解决市场上紫杉醇价格昂贵、供不应求的现状。
此外，紫杉二烯合酶TcTS2在植物细胞内的表达受茉莉酸甲酯(Methyl Jasmonate，简称MeJA)的显著诱导调控，这种调控方式有利于合成生物学技术应用，结合小分子MeJA诱导及其他合成生物学技术手段能够更好地调控紫杉二烯合酶TcTS2的表达情况，可以实现在未来用于植物基因工程和代谢工程改造生产紫杉醇及其中间体等多个方面。
本申请第二方面提供了编码所述的紫杉二烯合酶TcTS2的核苷酸序列。
进一步，在本申请提供的技术方案的基础上，编码紫杉二烯合酶TcTS2的核苷酸序列包含或由如下序列组成：
a)SEQ ID NO.3所示的核苷酸序列；或，
b)SEQ ID NO.3所示的核苷酸序列的互补序列、简并序列或同源序列(优选同源性70％以上)；或，
c)在严格条件下与SEQ ID NO.3所示的核苷酸序列杂交，且能够编码紫杉二烯合酶TcTS2的核苷酸序列；或，
d)所述序列a)-c)中任一核苷酸序列的cDNA序列。
在本申请的一种实施方式中，所述编码紫杉二烯合酶TcTS2的核苷酸序列为SEQ ID NO.3所示的核苷酸序列，全长5138个碱基。
在本申请的一种实施方式中，所述编码紫杉二烯合酶TcTS2的核苷酸序列为SEQ ID NO.3所示的核苷酸序列按照碱基互补配对原则形成的互补序列。
在本申请的一种实施方式中，所述编码紫杉二烯合酶TcTS2的核苷酸序列为SEQ ID NO.3所示的核苷酸序列的简并序列。简并序列是指改变SEQ ID NO.3核苷酸某个或多个核苷酸序列后，改变的核苷酸序列位置对应编码的氨基酸种类不变，不会影响紫杉二烯合酶TcTS2基因的功能和紫杉二烯合酶TcTS2的表达水平。
在本申请的一种实施方式中，所述编码紫杉二烯合酶TcTS2的核苷酸序列为SEQ ID NO.3所示的核苷酸序列的同源序列。
所述同源核苷酸序列包括在SEQ ID NO.3所示的核苷酸序列中添加和/或取代和/或缺失一个或几个核苷酸而生成的可编码具有紫杉二烯合酶活性的突变基因、等位基因或衍生物。
进一步优选同源序列为与SEQ ID NO.3所示的核苷酸序列约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上同一性的多核苷酸。
在本申请的一种实施方式中，所述编码紫杉二烯合酶TcTS2的核苷酸序列为在严格条件下与SEQ ID NO.3的核苷酸序列杂交，且能够编码紫杉二烯合酶TcTS2的核苷酸序列。
示例性地，所述“严格条件”是指探针将与其靶序列杂交至可探测程度超过与其它序列杂交(如至少2倍于背景)的条件。严格条件具有序列依赖性，且因环境的不同而不同。通过控制杂交和/或洗涤条件的严格性，可以鉴定与探针100％互补的靶序列。可选择地，可以调节严格条件以允许一些序列错配，使得探测到较低程度的相似性，同时这种序列错配获得的核苷酸序列，编码紫杉二 烯合酶TcTS2时不会影响其正常酶活。
进一步，所述cDNA的序列包含或由如下序列组成：
a)SEQ ID NO.4所示的核苷酸序列；或，
b)SEQ ID NO.4所示的核苷酸序列的简并序列或同源序列；优选地，所述同源序列为与SEQ ID NO.4所示的核苷酸序列约80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.5％或以上、或99.9％或以上同一性的多核苷酸。
在本申请的一种实施方式中，所述编码紫杉二烯合酶TcTS2的核苷酸序列为SEQ ID NO.4所示的核苷酸序列的cDNA序列，全长2448个碱基。
本申请第三方面提供了所述紫杉二烯合酶TcTS2，或，所述的编码紫杉二烯合酶TcTS2的核苷酸序列在合成巴卡亭III和/或紫杉醇中的应用。
其中，所述紫杉二烯合酶TcTS2在合成巴卡亭III和/或紫杉醇中的应用包括以下几个方面：(1)本申请所提供的紫杉二烯合酶TcTS2或含有至少部分SEQ ID NO.1所示氨基酸序列的多肽可能在去除或替代某些氨基酸之后仍具有TcTS2的生物活性甚至有新的生物学活性，或者提高了产量或优化了蛋白动力学特征或其他致力于得到的性质；(2)涉及紫杉二烯的生物合成；(3)涉及紫杉醇及其中间体(如巴卡亭III)的生物合成。
所述的编码紫杉二烯合酶TcTS2的核苷酸序列在合成巴卡亭III或紫杉醇中的应用包括以下几个方面：(1)本申请所提供的核苷酸序列或至少部分核苷酸序列被修饰或突变，修饰或突变的途径包括插入、缺失，聚合酶链式反应(PCR)，易错PCR，不同序列的重新连接，序列的不同部分或与其他来源的同源序列进行定向进化，或通过化学试剂诱变等。(2)本申请所提供的核苷酸序列或至少部分核苷酸序列的克隆基因通过合适的表达体系在外源宿主中表达以得到相应的TcTS2酶或更高的生物活性或产量的TcTS2酶。(3)本申请所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以通过遗传重组来构建重组质粒以获得新型生物合成途径，也可以通过插入、置换、缺失或失活进而获得新型生物合成途径。
本申请第四方面提供了用于检测所述的核苷酸序列的引物。
此处对扩增编码紫杉二烯合酶TcTS2核苷酸序列的PCR产物的核苷酸序列不做具体的限定，能够满足特异性扩增出或特异性检测出所述的编码紫杉二烯合酶TcTS2核苷酸序列的引物即可。
在本申请的一种优选地实施方式中，所述引物包括上游引物和/或下游引物；其中，所述上游引物的核苷酸序列如SEQ ID NO.5所示：5’-CGAGGCTTGCAAGTTACACA-3’；和/或，所述下游引物的核苷酸序列如SEQ ID NO.6所示：5’-CAGGGCATTTGAAACCTCAT-3’。
本申请第五方面提供了一载体，其导入了所述的核苷酸序列。
对于载体的种类不作具体限定，可以为本领域常规使用的载体。例如载体包括但不限于pET28b、pIJ702、pUCP19、pYMB03或pHT43，优选pET28b。
本申请第六方面提供了一种宿主细胞，其导入了所述的核苷酸序列，或导入了所述的载体。
进一步，所述的宿主细胞包括植物细胞和/或微生物细胞；
进一步，所述植物细胞包括但不限于红豆杉细胞、烟草细胞、白豆杉细胞、青蒿细胞；
进一步，所述微生物细胞包括但不限于链霉菌、假单孢菌、芽孢杆菌、酵母细胞、大肠杆菌。
进一步，在体内和体外将编码紫杉二烯合酶TcTS2的核苷酸序列，或所述的重组质粒，或所述的表达载体引入宿主细胞的方法包括但不限于电穿孔、聚乙二醇(PEG)转化、脂质转染、热休克、磷酸钙沉淀、病毒介导和显微注射。
本申请第七方面提供了一种在植物中表达紫杉二烯合酶TcTS2的方法，将所述核苷酸序列，或将所述的载体，或将所述的宿主细胞转入植物体中，获得紫杉二烯合酶TcTS2。
进一步，所述植物包括但不限于：红豆杉、白豆杉、烟草、青蒿。
进一步，所述植物体也包括如外植体的植物部分，包括但不限于：插条、组织培养物、细胞悬浮液和愈伤组织。
进一步，所述植物更优选为红豆杉和/或烟草。
本申请第八方面提供了一种生产紫杉醇及其中间体的方法，包括使植物体表达所述的紫杉二烯合酶TcTS2。
进一步，所述方法包括：将所述核苷酸序列，或将所述的载体，或将所述的宿主细胞转入植物体中，使紫杉二烯合酶TcTS2表达，获得紫杉醇及其中间体。
本申请采用上述技术方案具有以下有益效果：
(1)本申请提供了一种紫杉二烯合酶TcTS2，显著提高了催化GGPP环化的效率，使合成紫杉醇过程的中间体紫杉二烯、巴卡亭III以及产物紫杉醇产量得到大幅度提高，显著降低了紫杉醇药物的生产成本，能够有效解决市场上紫杉醇价格昂贵、供不应求的现状。
(2)本申请提供的紫杉二烯合酶TcTS2在植物细胞内的表达受MeJA的显著诱导调控，结合小分子MeJA诱导及其他合成生物学技术手段，可以实现在未来用于植物基因工程和代谢工程改造生产紫杉醇及其中间体等多个方面。
(3)本申请提供的编码紫杉二烯合酶TcTS2的核苷酸序列，为提高紫杉醇的产量提供了新的基因资源，该核苷酸序列和TcTS2可用于植物基因工程和代谢工程策略改造底盘宿主，生产紫杉醇及其中间体等，具有显著经济和社会价值。
附图说明
图1所示为TcTSs体外生化活性分析图。其中，A为GC-MS检测TcTSs酶促反应产物，提取紫杉二烯特征离子m/z 122色谱图；B为TcTSs催化GGPP生成紫杉二烯反应示意图；C为TcTSs酶促反应产物的质谱图。
图2所示为MeJA胁迫下代谢产物含量变化及TcTSs基因的表达模式图。其中，A为MeJA胁迫下巴卡亭III和紫杉醇产量相对变化图；B为MeJA胁迫下TcTS1基因的表达模式图；C为MeJA胁迫下TcTS2基因的表达模式图；MeJA+表示100μM MeJA处理组；MeJA-表示溶剂对照组。
图3所示为实施例4中，工程菌株E.coli TbTS和E.coli TcTS2诱导发酵生长曲线。
图4所示为实施例4中，工程菌株E.coli TbTS和E.coli TcTS2合成紫衫二烯产物的产量比较。
具体实施方式
本申请中，术语“TcTS2”可以表示紫杉二烯合酶，或紫杉二烯合酶基因，或编码紫杉二烯合酶的核苷酸序列，具体所指的含义可以结合上下文判断。
本申请中，术语“基因”被定义为在染色体中占据特定位置并包含对植株潜在的表型特征或性状作出贡献的遗传指令的遗传单位(通常用DNA序列表示)。”
本申请中，术语“核苷酸”以本领域技术人员理解的一般含义。
本申请中，术语“氨基酸”是指任何氨基酸(标准和非标准氨基酸二者)，包括但不限于α-氨基酸、β-氨基酸、γ-氨基酸和δ-氨基酸。适合的氨基酸的实例包括但不限于丙氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、丝氨酸、酪氨酸、精氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、蛋氨酸、苯基丙氨酸、苏氨酸、色氨酸和缬氨酸。
除非另有定义，本申请中所使用的所有科学和技术术语具有与本申请涉及技术领域的技术人员通常理解的相同的含义。
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下面结合具体实施例详细描述本申请，这些实施例用于理解而不是限制本申请。
实施例1 紫杉二烯合酶TcTSs基因的克隆及表达纯化
1.紫杉二烯合酶TcTSs基因的克隆和序列分析
以功能已知的短叶红豆杉紫杉二烯合酶TbTS(GeneBank NO.U48796)为靶序列，在南方红豆杉T.chinensis var.mairei基因组中搜索，找到两条紫杉二烯合酶基因，分别命名为TcTS1和TcTS2。其中，经过序列比对分析发现：TcTS1读码框氨基酸序列与TbTS同源性为98％，而TcTS2为新发现的紫杉二烯合酶基因，TcTS2的基因组DNA序列为SEQ ID NO.3所示，与TbTS核苷酸相似性为42％；TcTS2读码框核苷酸序列和氨基酸序列分别为SEQ ID NO.4和SEQ ID NO.1所示，与TbTS的核苷酸相似性和氨基酸同源性均为79％。
以前研究证实在大肠杆菌系统中异源表达紫杉二烯合酶时，其5’末端信号肽序列会影响它的催化活性(Wildung et al.,1996)，因此，申请人选用三个在线软件Plant-Ploc,ChloroP和TargetP，预 测TcTS1和TcTS2叶绿体转运肽位置，结果表明TcTS1的5’端58个氨基酸为信号肽序列，TcTS2的5’端57个氨基酸为信号肽序列。
为了后续生化功能鉴定，以TcTS1和TcTS2基因的核苷酸序列为依据，申请人根据删除信号肽位置后的序列，设计分别扩增TcTS1和TcTS2基因的引物P1和P2，P3和P4，引物包含部分原核表达载体pET28b序列，构建N端与6个组氨酸融合表达的载体，便于后续纯化。
以南方红豆杉细胞系104的cDNA为模板，采用PCR方法扩增TcTS1和TcTS2基因，PCR目的产物经切胶回收后，与经Sal I和Not I双酶切的线性pET28b载体重组连接，采用Hieff Clone TM一步克隆试剂盒(YEASEN,China)克隆并测序，阳性重组质粒分别命名为pET28b-TcTS1和pET28b-TcTS2。
所用引物中，P1和P2用于扩增TcTS1，P3和P4用于扩增TcTS2，其中引物序列如下：
P1:5’-CCGAATTCGAGCTCCG TCGACATGAGCAGCAGCACTGGCACTAGC-3’(下划线为Sal I酶切位点)(SEQ ID NO.7)
P2:5’-GTGGTGCTCGAGT GCGGCCGCTCATACTTGAATTGGATCAATATA-3’(下划线为Not I酶切位点)(SEQ ID NO.8)
P3:5’-CCGAATTCGAGCTCCG TCGACATGAGCGGTAGCCCGACCAAGTTGGC-3’(下划线为Sal I酶切位点)(SEQ ID NO.9)
P4:5’-GTGGTGCTCGAGT GCGGCCGCTCATACTTGAATCGGTTCAATGTAAACT-3’(下划线为Not I酶切位点)(SEQ ID NO.10)
2.紫杉二烯合酶TcTSs的异源表达及纯化
TcTSs-His6融合蛋白诱导表达：将构建好的融合表达载体pET28b-TcTS1和pET28b-TcTS2分别转入大肠杆菌BL21(DE3)中，获得BL21(DE3)/pET28b-TcTS1和BL21(DE3)/pET28b-TcTS2转基因菌株；挑取阳性单克隆分别接种于LB培养基中(含50μg·mL -1的Kanamycin)，37℃过夜培养；取过夜培养物按1:100的比例于300mL新鲜LB培养基中(含50μg·mL -1的Kanamycin)，37℃扩大培养至OD600值为0.4-0.6；加入IPTG(异丙基硫代半乳糖苷)至终浓度为1.0mmol·L -1，16℃，160rpm诱导表达12h；12,000rpm，离心收集菌体，用于纯化。
TcTSs-His6融合蛋白纯化参照美国Thermo Scientific公司HisPur Ni NTA Resin及脱盐超滤管法：将收集的菌体分别重悬于20mL裂解缓冲液中(50mM磷酸钠，pH8.0，300mM NaCl，10mM咪唑)，使用超声波降解法破壁，4,000g，4℃条件下离心20分钟，将上清加载到1.5mL HisPur Ni-NTA树脂填料柱中(Thermo Scientific,USA)，经除杂后使用洗脱缓冲液(50mM磷酸钠，300mM NaCl，50mM咪唑)洗脱。
纯化后的重组TcTSs-His6使用30kDa超滤管脱盐，保存于酶反应缓冲液(25mM HEPES，pH8.5，10％甘油，5mM DTT，5mM抗坏血酸钠，5mM偏亚硫酸钠和1mM MgCl 2)中，重组蛋白TcTSs-His6的含量和纯度分别用BCA蛋白检测试剂盒(Beyotime,China)、牛血清白蛋白(BSA)标准品和SDS-PAGE凝胶电泳检查。
结果表明TcTS1-His6融合蛋白和TcTS2-His6融合蛋白均为可溶性表达，纯化蛋白纯度高达98％以上，浓度分别为3.4μg·μL -1和1.8μg·μL -1。
实施例2 紫杉二烯合酶TcTSs生化功能分析
1.TcTSs-His6体外活性
体外酶促反应体系为500μL，包含100μg纯化蛋白，100μM GGPP(Sigma-Aldrich)和酶反应缓冲液(25mM，pH8.5，10％甘油，5mM DTT，5mM抗坏血酸盐，5mM焦亚硫酸钠和1mM MgCl 2)，反应混合物覆盖500μL戊烷(Macklin，GC-MS级)32℃水浴锅中反应2小时，漩涡2分钟，5000rpm离心10分钟，戊烷层取出放入2mL进样品，使用氮吹仪低温浓缩，产物用气相色谱质谱(GC-MS)仪器进行分析。
对照组为纯化后的重组蛋白TcTSs-His6经100℃煮沸10min后反应产物。GC-MS仪器为安捷伦7890B/7000C(美国Waldbronn安捷伦科技)，质谱检测器参数：70eV，氦气流速1.2mL·min -1，色谱柱安捷伦HP-5MS(5％苯基甲基氧化硅，30m×250μm内径，0.25μm膜厚度)，进样体积1-5μL；柱温箱温度程序如下：45℃持续1分钟，然后10℃·min -1梯度上升到250℃，250℃保持5分钟。进样口温度为250℃；扫描质荷比(m/z)为40-350，以紫杉二烯标准品为参照，进行酶促产物定性分析。
体外生化功能分析结果如图1所示，结果表明TcTS1和TcTS2均能催化底物GGPP生成主产物taxa-4(5)，11(12)-diene(紫杉烯4(5),11(12)-二烯，占94％)和次产物taxa-4(20),11(12)-diene(紫杉烯4(20),11(12)-二烯，占4％)，展示相同的紫杉二烯合成酶活性。其中图1C展示了TcTSs 酶促反应产物的质谱图，对酶促产物定性。
2.TcTSs-His6动力学常数比较
动力学参数测定：标准酶促反应总体积100μL，包含酶反应缓冲液(25mM，pH8.5，10％甘油，5mM DTT，5mM抗坏血酸盐，5mM焦亚硫酸钠和1mM MgCl 2)，34μg(TcTS1-His6)或18μg(TcTS2-His6)重组蛋白和7个不同浓度GGPP(0.2μM，0.5μM，1μM，2.5μM，5μM，10μM，25μM，50μM)混有[ 1-3H]-GGPP底物(American Radiolabeled Chemicals,Inc,30Ci·mM -1)。
标记的[ 1-3H]-GGPP用未标记的GGPP(Sigma,1mg·mL-1)稀释400倍。反应混合物在32℃反应30分钟，然后使用100μL终止缓冲液(包含1M EDTA和4M NaOH)淬火10分钟；反应混合物用800μL正己烷提取(涡旋2分钟，12,000rpm离心10分钟)，400μL正己烷提取液加入2mL液体闪烁液中混匀。采用液体闪烁计数器(Tri-Carb 2910TR,Perkin Elmer)测定反应产物的总放射性。利用Origin 8.6软件对Michaelis-Menten方程进行非线性回归拟合计算动力学常数。
动力学常数结果显示：TcTS1-His6和TcTS2-His6的K m分别为5.5±1.6和8.6±1.5(pM·μg -1·min -1)，而TcTS1-His6和TcTS2-His6的K cat值则分别为0.00292(1·s -1)和0.00901(1·s -1)。从它们的K m和K cat值来看，尽管TcTS1-His6结合GGPP的强度是TcTS2-His6的1.6倍，但TcTS2-His6的催化效率是TcTS1-His6的3倍(K cat值)。动力学常数结果表明TcTS2酶催化效率显著优于TcTS1，合成生物学应用前景也显著优于TcTS1。
实施例3 茉莉酸甲酯胁迫下紫杉二烯合酶TcTSs基因表达与紫杉醇相关性分析
1.茉莉酸甲酯处理南方红豆杉细胞系211试验
将南方红豆杉211细胞系分为两组：实验组(MeJA+)用100μM茉莉酸甲酯浸泡处理；对照组(MeJA-)用0.05％乙醇溶液浸泡处理。两组同时进行，分别在0h、2h、3h和4h后收集样品，用于TcTSs基因的表达模式和巴卡亭Ⅲ以及紫杉醇含量变化分析，三次生物重复。
2.MeJA不同处理时间巴卡亭III和紫杉醇含量变化分析
代谢物提取采用改良的沃尔夫恩德Wolfender法，具体步骤包括：称取100mg冻干细胞粉放置于2ml离心管中，加入1.5mL提取缓冲液(甲醇:水＝80:20,v/v)涡旋悬浮，包含500ng/mL地塞米松为内标，超声提取30min后，14,000g离心15min，上清液于真空浓缩器(LABCONCO)中离心干燥，最后样品重悬于200μL甲醇溶剂(80:20,v/v)中用于LCMS分析。样品使用ACQUITY UPLC I-Class/AB SCIEX(Waters)仪器进行LC-MS分析。
分离柱为Waters(2.1×50mm；ACQUITY UPLC TM Waters XselectHSS T3)；进样量10μL，流速为0.2mL·min -1。
流动相组成为：A是0.1％甲酸甲醇；B为0.1％甲酸水。
LC分离程序为：40％A平衡2min，随后梯度洗脱10min至100％B，维持2min，再回到40％A平衡1min。ESI离子源，正离子模式。数据采集和处理在AB SCIEXAnalyst 1.6.3软件(AppliedBiosystems)上进行。
图2A的代谢物定量分析结果显示，100μM MeJA处理明显促进了紫杉醇和巴卡亭III的积累，当MeJA处理2h时，相对于对照组紫杉醇和巴卡亭III的增加倍数分别为9.53和1.48倍；当MeJA处理4h时，紫杉醇和巴卡亭III的增加倍数分别为12.91和7.01倍。
3.MeJA不同处理时间TcTS2基因表达模式分析
使用EASYspin植物总RNA提取试剂盒(Aidlab，北京)提取收集上述不同处理时间的样品总RNA，分别取1μg总RNA用于第一链cDNA的合成，作为实时荧光定量PCR的模板，第一链cDNA合成使用试剂盒
Figure PCTCN2021097561-appb-000001
III 1st Strand cDNA Synthesis(YEASEN,China)和qPCR荧光染料试剂盒
Figure PCTCN2021097561-appb-000002
qPCR
Figure PCTCN2021097561-appb-000003
Green Master Mix(Low Rox Plus)(商业化试剂，购于翊圣生物(YEASEN，中国)。qPCR试验在QuantStudio TM 3系统上进行，2次技术重复和3次生物学重复。南方红豆杉actin1(TcACTIN1)基因作为内参基因，使用2 -ΔΔCt法计算TcTSs基因的相对表达量。
qPCR基因特异引物如下：
P5:5’-GCACGGAATTGTTTCCAACT-3’(TcACTIN1)(SEQ ID NO.11)
P6:5’-GGCAACATACATTGCAGGTG-3’(TcACTIN1)(SEQ ID NO.12)
P7:5’-AGCACTGGCACTAGCAAGGT-3’(TcTS1)(SEQ ID NO.13)
P8:5’-TTCACAACCAGCTCATCTGC-3’(TcTS1)(SEQ ID NO.14)
P9:5’-CGAGGCTTGCAAGTTACACA-3’(TcTS2)(SEQ ID No.5)
P10:5’-CAGGGCATTTGAAACCTCAT-3’(TcTS2)(SEQ ID No.6)。
图2B，C的qPCR结果显示，MeJA胁迫下，TcTS1和TcTS2的表达均有所上调，但表现出不同表达模式。不同MeJA处理时间，TcTS1的上调趋于平稳，表现为组成型表达模式，相反，TcTS2的上调显著，最显著诱导时间节点为4小时，相对于0小时诱导倍数为7，展现为诱导型表达模式。
结合代谢物定量分析结果和基因表达模式分析，MeJA胁迫下，TcTS2表达显著上调，紫杉醇和巴卡亭III产量显著增加。上述结果表明，TcTS2在植物细胞内的表达受植物激素茉莉酸甲酯(MeJA)的显著诱导调控，促进了巴卡亭III和紫杉醇的合成。诱导型表达的TcTS2基因更有利于合成生物学技术应用，合成应用前景显著优于TcTS1。
综上所述，本申请提供的新型紫杉二烯合酶基因TcTS2与另一类型紫杉二烯合成酶基因TcTS1显著不同，表达的蛋白也有显著差异，TcTS2酶催化效率显著高，在MeJA胁迫下呈现诱导型表达，显著促进了巴卡亭III和紫杉醇的合成。
实施例4 工程菌株中TcTS2与TbTS合成紫杉二烯的比较
1.大肠工程菌株的构建
基于已报道的技术(Bian et al.,2017)，分别使用TcTS2和TbTS构建产紫杉二烯的工程菌株E.coli TcTS2和E.coli TbTS。
2.两种工程菌合成紫杉二烯的比较
(1)两种工程菌生长势的比较：工程菌株E.coli TbTS和E.coli TcTS2接种于30mL含有氨苄青霉素(100毫克/升)，卡那霉素(50毫克/升)和氯霉素(34毫克/升)的LB-液体培养基中，于37℃摇动过夜生长，取菌液低速离心去上清，使用诱导培养基重悬使OD 600＝0.3，加入IPTG进行诱导(终浓度为0.1mM)，取200μL诱导培养菌液加入96孔板(3个平行样)，使用能够连续培养的多功能酶标仪28℃摇动培养，每15分钟自动检测一次OD 600，共培养40小时，根据算数平均值绘制生长曲线，结果见图3。
图3的结果表明，E.coli TbTS和E.coli TcTS2菌株的生长趋势基本一致。
两种工程菌合成紫杉二烯产物的检测：对上述工程菌的发酵产物进行GC-MS的检测，GC-MS检测方法同实施例2，对其色谱图目标峰进行积分，通过面积积分换算浓度，结果如图4，可以看出E.coli TcTS2表达目标产物的产量为E.coli TbTS的12倍。
综合结果表明，TcTS2合成紫杉二烯的能力显著优于TbTS，利用TcTS2合成紫杉二烯可大幅提高产量。
参考文献：Bian,G.et al.Production of taxadiene by engineering of mevalonate pathway in Escherichia coli and endophytic fungus Alternaria alternata TPF6.Biotechnol.J.12(2017).
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内，所作的任何修改、等同替换等，均应包含在本申请的保护范围之内。
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Claims (16)

  1. 一种紫杉二烯合酶TcTS2，其特征在于，所述紫杉二烯合酶TcTS2的氨基酸序列包含或由如下序列组成：
    a)SEQ ID NO.1所示的氨基酸序列；或，
    b)与SEQ ID NO.1所示的氨基酸序列具有至少80％序列相似性的功能性同源序列；或，
    c)在SEQ ID NO.1所示的氨基酸序列中添加、缺失、替换一个或多个氨基酸且具有TcTS2活性的氨基酸序列。
  2. 根据权利要求1所述的紫杉二烯合酶TcTS2，其特征在于，所述紫杉二烯合酶TcTS2的氨基酸序列包含或由如下序列组成：
    a)SEQ ID NO.2所示的氨基酸序列；或，
    b)与SEQ ID NO.2所示的氨基酸序列具有至少80％序列相似性的功能性同源序列；或，
    c)在SEQ ID NO.2所示的氨基酸序列中添加、缺失、替换一个或多个氨基酸且具有TcTS2活性的氨基酸序列。
  3. 编码权利要求1或2所述的紫杉二烯合酶TcTS2的核苷酸序列。
  4. 如权利要求3所述的编码紫杉二烯合酶TcTS2的核苷酸序列，其特征在于，所述核苷酸序列包含或由如下序列组成：
    a)SEQ ID NO.3所示的核苷酸序列；或，
    b)SEQ ID NO.3所示的核苷酸序列的互补序列、简并序列或同源序列(优选同源性70％以上)；或，
    c)在严格条件下与SEQ ID NO.3所示的核苷酸序列杂交，且能够编码紫杉二烯合酶TcTS2的核苷酸序列；或，
    d)所述序列a)-c)中任一核苷酸序列的cDNA序列。
  5. 根据权利要求4所述的核苷酸序列，其特征在于，所述cDNA序列包含或由如下序列组成：
    a)SEQ ID NO.4所示的核苷酸序列；或，
    b)SEQ ID NO.4所示的核苷酸序列的简并序列或同源序列(优选同源性80％以上)。
  6. 权利要求1或2所述紫杉二烯合酶TcTS2，或，权利要求3-5任一项所述的编码紫杉二烯合酶TcTS2的核苷酸序列在合成巴卡亭III和/或紫杉醇中的应用。
  7. 用于检测权利要求3-5任一项所述的核苷酸序列的引物；
    优选地，所述引物包括上游引物和/或下游引物。
  8. 根据权利要求7所述的引物，其特征在于，所述上游引物的核苷酸序列如SEQ ID NO.5所示；和/或，所述下游引物的核苷酸序列如SEQ ID NO.6所示。
  9. 一种载体，其特征在于，包含权利要求3-5任一项所述的核苷酸序列。
  10. 一种宿主细胞，其特征在于，导入了权利要求3-5任一项所述的核苷酸序列，或导入了权利要求9所述的载体。
  11. 根据权利要求10所述的宿主细胞，其特征在于，所述宿主细胞包括植物细胞和/或微生物 细胞。
  12. 根据权利要求11所述的宿主细胞，其特征在于，所述植物细胞包括红豆杉细胞和/或烟草细胞；
    优选地，所述微生物细胞包括链霉菌、假单孢菌、芽孢杆菌、酵母细胞、大肠杆菌中的至少一种。
  13. 一种在植物中表达紫杉二烯合酶TcTS2的方法，其特征在于，将权利要求3-5任一项所述核苷酸序列，或将权利要求9所述的载体，或将权利要求10-12任一项所述的宿主细胞转入植物体中，使植物中表达紫杉二烯合酶TcTS2。
  14. 根据权利要求13所述的方法，其特征在于，所述植物体为红豆杉和/或烟草。
  15. 一种生产紫杉醇及其中间体的方法，其特征在于，使植物体高表达权利要求1或2所述的紫杉二烯合酶TcTS2。
  16. 根据权利要求15所述的方法，其特征在于，所述方法包括：将权利要求3-5任一项所述核苷酸序列，或将权利要求9所述的载体，或将权利要求10-12任一项所述的宿主细胞转入植物体中，使紫杉二烯合酶TcTS2表达，获得紫杉醇及其中间体。
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