WO2019154217A1 - 柳杉二醇合成酶、其编码基因及包含该编码基因的重组酵母菌 - Google Patents

柳杉二醇合成酶、其编码基因及包含该编码基因的重组酵母菌 Download PDF

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WO2019154217A1
WO2019154217A1 PCT/CN2019/073905 CN2019073905W WO2019154217A1 WO 2019154217 A1 WO2019154217 A1 WO 2019154217A1 CN 2019073905 W CN2019073905 W CN 2019073905W WO 2019154217 A1 WO2019154217 A1 WO 2019154217A1
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yeast
gene
strain
twcs
expression vector
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高伟
黄璐琦
童宇茹
胡添源
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首都医科大学
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    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention relates to a cedar diol synthase and a gene encoding the same, and a recombinant yeast containing the gene encoding the cedar diol synthase, belonging to the field of synthetic biology of medicinal components.
  • Sesquiterpenoids are a class of natural compounds with a variety of physiological and ecological functions, such as antimicrobials, defense against insects and herbivores. At present, a large number of sesquiterpenoids have been found in higher plants and lower plants, and are widely used in the fields of industry, agriculture and medicine. The biosynthesis of sesquiterpene only comes from the simple C5 isoprene structural unit, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP).
  • IPP isopentenyl pyrophosphate
  • DMAPP isomer dimethylallyl pyrophosphate
  • the three-molecule C5 unit undergoes a cascade reaction with the participation of the isopentenyl transferase, and the carbon chain is extended to form an arsylyl pyrophosphate (FPP) containing 15 carbon atoms, and the FPP can be obtained in sesquiterpene.
  • FPP arsylyl pyrophosphate
  • Cyclization catalyzes the formation of structurally sesquiterpene compounds.
  • Cedardiol was first discovered in Cryptomeria japonica plants, and then a small amount of cedardiol was detected in the volatile oils of various plants.
  • Cedardiol has a certain antispasmodic effect and is an anticonvulsant drug.
  • Activity of 08 In view of the low content and separation of cedar diol in plants, some researchers have designed their chemical synthesis and semi-synthetic routes, such as (-)-elemol through metal ion-catalyzed oxidation reaction. The multi-step reaction synthesizes cedar diol.
  • anisic acid can be used as a starting material, and a relatively simple and efficient semi-synthesis process can be used to obtain cedardiol in a three-step reaction.
  • the invention provides a cedar diol synthetase having the following amino acid sequence:
  • Another aspect of the invention provides a gene (Tripterygium wilfordii Cryptomeridiol Synthase, TwCS) encoding the cedar diol synthase of the invention: the gene, which is one of the following nucleotide sequences:
  • nucleotide sequence in which the nucleotide molecule of SEQ ID NO: 1 is substituted, deleted or added to one or more nucleotides and expresses the same functional protein
  • nucleotide sequence which hybridizes under stringent conditions to the nucleotide molecule of SEQ ID NO: 1, the stringent conditions being: 0.1 ⁇ SSPE containing 0.1% SDS or 0.1 ⁇ containing 0.1% of SDS Hybridization in SSC solution.
  • the "stringency" of the hybridization reaction can be readily determined by one of ordinary skill in the art and is typically calculated empirically based on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization typically relies on the ability of denatured DNA to reanneal when the complementary strand is present in an environment below its melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is inferred that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanation of hybridization stringency, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, 1995.
  • Stringent conditions or “high stringency conditions”, as defined herein, can be identified as follows: (1) Washing with low ionic strength and high temperature, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% ten Sodium dialkyl sulfate, 50 ° C; (2) using a denaturing agent such as formamide during the hybridization process, such as 50% (v/v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyethylene Pyrrolidone / 50 mM sodium phosphate buffer pH 6.5, containing 750 mM sodium chloride, 75 mM sodium citrate, 42 ° C; or (3) using 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM Sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS,
  • an expression vector comprising a promoter, a gene encoding the cedardiol synthase of the invention, and a transcription terminator.
  • the promoter, the cedardiol synthase encoding gene, the terminator and the episomal vector are spliced by yeast homologous recombination, such as pYX212, pYES2.0, pRS425, pRS426 or P424.
  • a recombinant expression vector pYX212-TwCS comprising a promoter TPIp, a cedar diol synthase expression gene TwCS, a terminator pYX12t;
  • Recombinant expression vector pYX212-(IDI-EGR20)+TwCS it contains promoter TPIp, IDI gene, fusion protein linker peptide, EGR20 gene, yeast terminator FBA1t, yeast promoter TEF1p, cedardiol synthase expression Gene TwCS, terminator pYX12t;
  • Recombinant expression vector pYX212-(EGR20-IDI)+TwCS it contains promoter TPIp, EGR20 gene, fusion protein linker, IDI gene, yeast terminator FBA1t, yeast promoter TEF1p, cedardiol synthase expression Gene TwCS, terminator pYX12t;
  • the fusion protein is any one of GGGS, GSG, GSGGGGS, GSGEAAAK, GSGEAAAKEAAAK or GSGMGSSSN, preferably GGGS, and the coding gene is ggtggtggttct.
  • an engineered bacterium comprising the expression vector of the present invention, which may be selected from a yeast cell or a plant cell, preferably a yeast cell, and the yeast may be a GEN.PK line.
  • the recombinant expression vector p424-tHMG1 comprises: a yeast promoter TDH3p, a truncated HMG-CoA reductase gene tHMG1, and a yeast terminator TDH3t.
  • a more efficient engineered bacteria ie, a recombinant yeast comprising the cedar of the present invention
  • the alcohol synthase encodes a gene, and the yeast knocks out at least one of the erg9 or rox1 genes.
  • a more preferred method is the recombinant yeast, which knocks out the erg9 gene, and more preferably, simultaneously knocks out the erg9 gene and the rox1 gene.
  • the more efficient recombinant yeast expressing cedardiol comprises at least the following gene fragments:
  • Recombinant expression vector pYX212-IDI+TwCS constructed into plasmid pYX212 by promoter TPIp, IDI gene, yeast terminator FBA1t, yeast promoter TEF1p, cedardiol synthase expression gene TwCS, terminator pYX12t;
  • the recombinant expression vector p424-tHMG1 was constructed from the yeast promoter TDH3p, the truncated HMG-COA reductase gene tHMG1, and the yeast terminator TDH3t, into plasmid p424.
  • the construction of the recombinant yeast of the present invention for more efficient expression of cedardiol comprises the following steps:
  • the recombinant expression vectors pYX212-IDI+TwCS and p424-tHMG1 were transformed into the mutant yeast strain BY4741erg9:: ⁇ -200-176rox1::mut to obtain recombinant yeast TE9.
  • the cedar diol synthase of the present invention or the cedar diol synthase encoding gene of the present invention, or the recombinant expression vector of the present invention, or the engineered bacteria of the present invention
  • the fermentation broth was extracted with n-hexane.
  • the product of sesquiterpene was detected by GC-MS.
  • the structure of cedar diol was determined by structure identification. The invention can be used to generate cedar diol by biosynthesis technology, and the problem of lack of drug source is alleviated, and has a good application prospect.
  • cedardiol synthase of the present invention or the cedardiol synthase gene encoding the present invention for plant breeding comprising a chemical component of cedardiol.
  • the use of the cedar diol synthase of the present invention, or a gene encoding the same, can be used to improve the content of cedar diol in a plant by applying it to plant cells.
  • Figure 1 is a schematic diagram of the plasmid type of the TE1-TE7 strain.
  • Figure 2 is a GC-MS analysis of the fermentation product, in which A is the fermentation product of strain TE1, and the product with peak position at 1, 2 is a trace of sesquiterpene component (peak position 1 is eucalyptol), peak position The product at 3, identified as cedar diol, with a retention time of 21.43 min; B is an empty vector expression product containing no TwCS-expressing gene, and found no sesquiterpenoid product; C is the standard cedar The GC-MS diagram of the diol has the same peak position as the peak 3 of Figure 1; D and E are the products of the peak 3 position of TE1 and the mass spectrum of the standard cedar diol, respectively.
  • Figure 3 is a standard curve for the quantitative determination of cedar diol standard.
  • Figure 4 is a graph showing the yield of the cedar diol production product of the TE1-TE7 strain.
  • Figure 5 is a graph showing the comparison of the expression yields of the mutant strains TE8 and TE9 cedardiol and eucalyptol obtained by editing the CRISP/Cas9 gene.
  • p426-SNR52p-gRNA eukaryotic expression vector and p414-TEF1p-Cas9-CYC1t eukaryotic expression vector Addgene purchase
  • pESC-LEU eukaryotic expression vector purchased from Agilent Technologies
  • SC-His yeast medium, SC-Trp-His yeast medium, SC-URA-Trp-His yeast medium all purchased from Beijing Fenino Technology Limited.
  • CTAB Buffer 2% CTAB (W/V); 100 mmol ⁇ L -1 Tris-HCl (pH 8.0); 25 mmol ⁇ L -1 EDTA; 2.0 mol ⁇ L -1 NaCl; 0.5 g ⁇ L - 1 spermidine) extracts total RNA from tripterygium wilfordii suspension cells.
  • the extracted RNA was purified using an RNA purification kit (Tiangen Biochemical Technology Co., Ltd.).
  • the full-length sequence fragments of the gene were screened according to the transcriptome data of Tripterygium wilfordii. 5'RACE and 3'RACE primers were designed.
  • the primer sequences are as follows:
  • RNA obtained in Example 1 was reverse transcribed into RACE Ready first strand cDNA using a PrimeScript 1 st Strand cDNA Synthesis Kit (Takara) kit. Referring SMARTer TM RACE kit instructions, rapid amplification of cDNA ends.
  • TwCS-R TTAATCTTGCATTGGTATTTGTTG (SEQ ID NO: 6)
  • PCR amplification was carried out using RACE Ready first strand cDNA as a template.
  • the PCR reaction conditions were 98 ° C for 30 s; 98 ° C for 10 s, 60 ° C for 15 s, 72 ° C for 1 min, 35 cycles; 72 ° C, 7 min.
  • the sequencing result showed that the sequence of the PCR amplification product was as shown in SEQ ID No. 1, and the gene shown in the sequence 1 was named TwCS, and the DNA sequence encoded a protein consisting of 553 amino acids, and the protein was named TwCS.
  • the amino acid sequence is SEQ ID No. 2.
  • yeast promoter and terminator used in this example are publicly available on the SGD website (https://www.yeastgenome.org).
  • the total DNA of yeast BY4741 was extracted using a yeast genome extraction kit (Tiangen Biochemical Technology Co., Ltd.). Then use this DNA as a template to design the following primers:
  • TEF1p-F ATAGCTTCAAAATGTTTCTACTC SEQ ID NO: 7
  • TEF1p-R TTTGTAATTAAAACTTAGATTAG (SEQ ID NO: 8)
  • a fragment of the promoter TEF1p (TEF1 SGD ID: S000006284) and the terminator FBA1t (FBA1 SGD ID: S000001543) were obtained by PCR amplification (specific procedure as in Example 2).
  • the promoter TPIp and the terminator pYX212t were obtained by PCR amplification (specific procedure as in Example 2).
  • the amplification primers are as follows:
  • the primers were designed according to the yeast EGR20 gene sequence (SGD ID: S000003703) as follows:
  • ERG20-F ATGGCTTCAGAAAAAGAAATTAG (SEQ ID NO: 15)
  • ERG20-R CTATTTGCTTCTCTTGTAAAC (SEQ ID NO: 16)
  • the yeast EGR20 gene sequence was obtained by PCR amplification (specific procedure as in Example 2).
  • the primers were designed according to the yeast IDI gene sequence (SGD ID: S000006038) as follows:
  • IDI-F ATGACTGCCGACAACAATAGTATGC (SEQ ID NO: 17)
  • the yeast IDI gene sequence was obtained by PCR amplification (specific procedure as in Example 2).
  • Construction method (1) mixing DNA fragments: the promoter, the gene, the terminator, the promoter... mixed in a molar ratio of 1:3:5:7:XX:7:5:3:1, the ratio is 1.
  • the amount of DNA is 50-100 ng/kb.
  • the second step of PCR Take 2 ⁇ L of the PCR product in (2), use as a template, use the forward primer of the starter promoter, the terminal terminator or the reverse primer of the promoter for PCR amplification (specific steps are the same as in Example 2). ).
  • the PCR product was purified using an EZNA Gel Extraction Kit (OMEGA), referring to the instructions.
  • EZNA Gel Extraction Kit OEGA
  • Purified product according to the instruction of pEASY-Blunt Simple Cloning Kit (Beijing Quanjin Biotechnology Co., Ltd.), ligated, transformed, and sequenced to obtain the corresponding module DNA.
  • GGGS GGT GGTGGT TCT
  • the constructed module was ligated into the expression vector pYX212 by yeast homologous recombination method, as follows:
  • the expression vector pYX212 was digested with BamH I endonuclease (NEB).
  • the TPIp-TwCS-pYX212t module was mixed with the linear expression vector pYX212 obtained in (1), wherein the module molar concentration was (100 ng/kb) and the molar concentration of the carrier was (60-80 ng/kb). Then, the yeast BY4741 competent state was electrically transferred together, and the electroporation conditions were 2.5 kV, 25 ⁇ F and 200 ⁇ (Bio-Rad Gene Pulsers).
  • yeast BY4741 competent state was prepared by the lithium acetate conversion method.
  • Yeast strains were cultured in respective screening-deficient medium for 2-3 days at 30 °C. A single colony was picked and the yeast plasmid was extracted using an E.Z.N.A. Yeast Plasmid Mini Kit (OMEGA) with reference to the instructions.
  • OEGA Yeast Plasmid Mini Kit
  • TEF1p-TwCS-pYX212t module was constructed into the vector pYX212 to obtain the following recombinant plasmid: pYX212-TPIp-ERG20-FBA1t-TEF1p-TwCS-pYX212t, abbreviated as pYX212-ERG20+TwCSpYX212-TPIp-IDI-FBA1t-TEF1p-TwCS- pYX212t, abbreviated as pYX212-IDI+TwCSpYX212-TPIp-IDI/ERG20-FBA1t-TEF1p-TwCS- pYX212t, abbreviated as pYX212-IDI+TwCSpYX212-TPIp-IDI/ERG20-FBA1t-TEF1p-T
  • Plasmid p424-tHMG1 The yeast promoter TDH3p, the gene tHMG1, and the yeast terminator TDH3t were constructed into plasmid p424 carrying the HIS3 marker.
  • the detailed construction method is in the literature "Zhou, YJ; Gao, W.; Rong, Q.; Jin, G.; Chu, H.; Liu, W.; Yang, W.; Zhu, Z.; Li, G.; Zhu, GJAm.Chem.Soc.2012, 134, 3234-3241.” has been published in the literature, and the public can also obtain it from the Laboratory of Molecular Biopharmaceuticals and Chinese Medicine Resources of Capital Medical University.
  • gRNA sequence refer to published paper " T.;Bonde, I.; M.; Harrison, SJ; Kristensen, M.; Pedersen, LE; Jensen, MK; Keasling, JDM etab. Eng. 2015, 28, 213-222.”, designing the gRNA sequences of the rox1 and erg9 promoters, as follows:
  • the p426-SNR52p-gRNA vector (Addgene) was engineered to insert two opposite restriction enzymes, AarI, at the 20 bp single RNA site.
  • AarI opposite restriction enzymes
  • the primers are as follows:
  • the p426-SNR52p-gRNA vector was constructed into the first-class universal vector pTY-U01.
  • the gRNA site was designed to be a 24 nt Oligo with a cohesive end complementary to the vector, forming a double strand under an annealing procedure.
  • the Oligo sequence is shown in the table below.
  • the ligation product was transformed, the positive clone was screened, and the sample was sequenced and identified to obtain an erg9p-gRNA vector.
  • the gRNA sequence is as follows:
  • the erg9p-rox1-gRNA vector was obtained.
  • the dsOligo of the rox1 gene was synthesized by synthesizing a 120 nt long-chain Oligo, and then annealed to form a DNA double-stranded, and then purified by using EZNA Gel Extraction Kit (OMEGA), referring to the instructions, to obtain dxOligo of rox1.
  • OEGA EZNA Gel Extraction Kit
  • Erg9p directly adopts the method of synthesizing double-stranded DNA (Beijing Ruibo Biotechnology Co., Ltd.), and then dsOligo of erg9p can be amplified by PCR.
  • Oligo sequences and amplification primers are as follows:
  • the screening marker TRP of p414-TEF1p-Cas9-CYC1t vector is not suitable for BY4741 yeast
  • the TRP screening marker was replaced with LEU by seamless splicing in this experiment, and the LEU sequence template was eukaryotic expression vector pESC. -LEU (Agilent Technologies).
  • (i) Amplify the LEU sequence with the pESC-LEU plasmid as a template, and amplify the upstream and downstream sequences of the TRP with the p414-TEF1p-Cas9-CYC1t plasmid as a template.
  • the primers are LEU-F/R, UF/R, DF, respectively. /R.
  • a 148 bp SnaBI restriction endonuclease site upstream of the TRP1 ORF and a downstream 323 bp DraIII restriction endonuclease site were selected.
  • the transformed Cas9 plasmid was transformed into the BY4741 yeast strain, and the transformation method was carried out according to the Frozen-EZ Yeast Transformation IITM (Zymo Research) specification to obtain the strain BY4741-Cas9.
  • gRNA About 500 ng of gRNA, 2 ⁇ g of erg9p dsOligo, and 1 ⁇ g of rox1dsOligo were added to 100 ⁇ L of BY4741-Cas9 competent cells according to different knockout purposes, and electroporated.
  • the strain modified in the step (6) was tested with the unmodified strain as a control, and the primer sequence was as follows:
  • the BY4741 mutant strain was divided into YPD solid medium (Beijing Fenino Technology Co., Ltd.), and cultured at 42 ° C for 3 days to grow a single colony;
  • the plasmid pYX212-TwCS in Example 3 was transformed into the BY4741 strain, and the transformation method was carried out in accordance with the Frozen-EZ Yeast Transformation IITM (Zymo Research) specification to obtain the engineered bacteria TE1 as shown in Table 1 and FIG.
  • the plasmid pYX212-ERG20+TwCS in Example 3 was transformed into the BY4741 strain, and the transformation method was carried out in accordance with the Frozen-EZ Yeast Transformation IITM (Zymo Research) specification to obtain the engineered bacteria TE2 as shown in Table 1 and FIG.
  • the plasmid pYX212-IDI+TwCS in Example 3 was transformed into the BY4741 strain, and the transformation method was carried out in accordance with the instructions of Frozen-EZ Yeast Transformation IITM (Zymo Research) to obtain the engineered bacteria TE3 as shown in Table 1 and FIG.
  • the plasmid pYX212-(IDI-ERG20)+TwCS in Example 3 was transformed into the BY4741 strain, and the transformation method was carried out according to the instructions of Frozen-EZ Yeast Transformation IITM (Zymo Research) to obtain the engineered bacteria TE4, as shown in Table 1 and FIG. Shown.
  • the plasmid pYX212-(ERG20-IDI)+TwCS in Example 3 was transformed into the BY4741 strain, and the transformation method was carried out according to the Frozen-EZ Yeast Transformation IITM (Zymo Research) specification to obtain the engineered bacteria TE5, as shown in Table 1 and FIG. Shown.
  • the plasmid pYX212-(ERG20-IDI)+TwCS and p424-tHMG1 in Example 3 were transformed into the BY4741 strain, and the transformation method was carried out according to the instructions of Frozen-EZ Yeast Transformation IITM (Zymo Research) to obtain the engineered strain TE6, as shown in Table 1. And Figure 1 shows.
  • the plasmids pYX212-IDI+TwCS and p424-tHMG1 in Example 3 were transformed into the BY4741 strain, and the transformation method was carried out according to the instructions of Frozen-EZ Yeast Transformation IITM (Zymo Research) to obtain the engineered bacteria TE7, as shown in Table 1 and FIG. Shown.
  • the plasmids pYX212-IDI+TwCS and p424-tHMG1 in Example 3 were transformed into the BY4741erg9:: ⁇ -200-176 mutant strain, and the transformation method was carried out according to the instructions of Frozen-EZ Yeast Transformation IITM (Zymo Research) to obtain the engineered bacteria TE8. ,As shown in Table 1.
  • the plasmids pYX212-IDI+TwCS and p424-tHMG1 in Example 3 were transformed into the BY4741erg9:: ⁇ -200-176rox1::mut mutant strain, and the transformation method was carried out according to the instructions of Frozen-EZ Yeast Transformation IITM (Zymo Research). Engineering bacteria TE9, as shown in Table 1.
  • the sesquiterpene-producing strain is fermented by a bioreactor.
  • the corresponding defective medium (Beijing Fukino Co., Ltd.) was used to pre-culture the corresponding auxotrophic strain.
  • the medium used for the 3L bioreactor consisted of 8g/L synthetic denitrogenation medium without uracil and histidine, 10g/L (NH 4 ) 2 SO 4 , 10g/L KH 2 PO 4 , 1.0g /L MgSO 4 ⁇ 7H 2 O. 50% NH 3 ⁇ H 2 O was used as a pH adjuster.
  • the strain was pre-incubated in a shake flask at 30 ° C for 48 h at 230 rpm. Then, 1 L of the fermentation medium was inoculated with the precultured cells in a 3 L stirred bioreactor (Eppendorf BioFlo/CelliGen 115). The 500 g/L glucose solution was fed regularly to maintain the growth of the strain. A concentrated medium lacking uracil and histidine and (NH 4 ) 2 SO 4 containing 40 g/L of synthetic denitrogenation medium was subjected to fermentation.
  • the fermentation product is a sesquiterpenoid component and is easily soluble in n-hexane, so n-hexane is selected as an extraction reagent.
  • the fermentation broth was centrifuged into two parts: the bacterial body and the bacterial liquid, and an equal volume of n-hexane was added to the bacterial liquid for extraction three times. After the bacterial cells were broken, the cells were ultrasonically extracted three times with 3 volumes of n-hexane.
  • the organic layers were combined, and an appropriate amount of anhydrous sodium sulfate was added thereto, and the mixture was allowed to stand for a while to remove water in the extract.
  • the extract was concentrated on a rotary evaporator, taking care that the bath temperature did not exceed 35 ° C (volatile components) and was finally transferred to a glass collection bottle.
  • the structure of the compound was analyzed by NMR spectroscopy. All data was collected from a BRUKER ACANCE III 600 MHz spectrometer. The solvent was deuterated chloroform containing TMS, and the final compound was identified as cedar diol. The results are shown in Fig. 2.
  • each strain In order to determine the sesquiterpene production amount of each strain, it was inoculated at a ratio of 1:100, and 50 mL of the bacterial liquid was preliminarily cultured as a strain.
  • the strain was cultured in a defective medium containing 20 g/L of glucose at 230 rpm and 30 °C. After 72 hours of shaking culture, the OD600 of all strains was examined.
  • the culture was added to an equal volume of n-hexane and kept shaking at 200 rpm for 2 h, then ultrasonically extracted twice with an equal volume of n-hexane.
  • the organic layers were combined and concentrated by rotary evaporation.
  • the concentrated sample was finally made up to 1.0 mL, and then 100 ⁇ L of the GC-MS sample was prepared for GC-MS analysis.
  • a Thermo TRACE 1310/TSQ8000 gas chromatograph (no split; syringe temperature 250 ° C), TG-5MS (30 m ⁇ 0.25 mm ⁇ 0.25 ⁇ m) capillary column; GC conditions are as follows: first keep the oven temperature at 50 ° C for 2 minutes, It was then raised to 280 ° C at a rate of 8 ° C / min and held at the final temperature for 10 minutes. The temperature of the syringe and detector was 50 °C. A standard curve was established using the cedardiol analog ⁇ -eudesmol, and the standard curve equation was obtained as: y 3E+06x-3E+07, as shown in FIG. Calculate the specific yield as:

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Abstract

提供一种柳杉二醇合成酶及其编码基因，以及柳杉二醇合成酶及编码基因、含有所述柳杉二醇编码基因的酵母工程菌，以及其在植物育种及生物合成中的运用，通过聚合酶链式反应克隆得到雷公藤中柳杉二醇合成酶基因的cDNA全长序列，然后利用合成生物学手段，构建了包含所述柳杉醇合成酶基因的酵母工程菌，实现在酵母中生产柳杉二醇。

Description

柳杉二醇合成酶、其编码基因及包含该编码基因的重组酵母菌 技术领域
本发明涉及一种柳杉二醇合成酶及其编码基因，以及含有该柳杉二醇合成酶编码基因的重组酵母菌，属于药用成分合成生物学领域。
通过聚合酶链式反应首次克隆得到了雷公藤(Tripterygium wilfordii Hook.F.)中新型柳杉二醇合成酶基因(TwCS)的cDNA全长序列，然后利用合成生物学手段，构建酵母工程菌，在酵母中生产柳杉二醇。
背景技术
倍半萜类化合物是一类天然化合物，具有多种生理和生态功能，如抗微生物，防御昆虫和草食动物等。目前，在高等植物和低等植物中都发现了大量的倍半萜类化合物，广泛用于工农业和医药领域。倍半萜的生物合成仅来自简单的C5异戊二烯结构单元，异戊烯基焦磷酸(isopentenyl pyrophosphate，IPP)及其异构体二甲基烯丙基焦磷酸(dimethylallyl pyrophosphate，DMAPP)。三分子的C5单元在异戊烯基转移酶参与下，发生级联反应，碳链延伸，生成含有15个碳原子的焦磷酸法呢酯(arnesyl pyrophosphate，FPP)，FPP既而可在倍半萜合酶催化下，环化生成结构多样的倍半萜化合物。
柳杉二醇首先在Cryptomeria japonica植物中被发现，随后在多种植物的挥发油中均有微量的柳杉二醇被检出。柳杉二醇具有一定的解痉作用，是抗痉挛药物
Figure PCTCN2019073905-appb-000001
的活性成08鉴于柳杉二醇在植物中含量较低且分离困难，一些研究者设计了其化学合成和半合成的路线，如通过金属离子催化的氧化反应，可以将(-)-elemol历经多步反应合成柳杉二醇。此外，还可以茴香酸(ilicic acid)为起始原料，采用相对简单高效的半合成工艺，经三步反应获得柳杉二醇。
然而，使用多步合成步骤相较植物体内，柳杉二醇合成途径的酶而言，是较为繁琐的。目前，通过细胞工厂的微生物发酵在单体化合物的工业生产方面具有高度竞争力。将基因元件(启动子、转录调控区域、核糖体结合位点、开放阅读框、终止子等)依据工程化目标需要，有机重构和连接起来，便形成了功能基因模块。通过对已有生物网络加以利用，同时引入新的功能基因模块，表达出天然细胞不能合成或含量极低的产物。如利用完整的青蒿酸生物合成途径在酵母工程菌中生产抗疟药青蒿素，每升发酵液可生产青蒿酸25克。
发明内容
本发明一方面提供了一种柳杉二醇合成酶，所述合成酶具有如下氨基酸序列：
(1)SEQ ID NO：2所示氨基酸序列；
(2)SEQ ID NO：2所示的氨基酸序列经取代、缺失或增加一个或多个氨基酸且功能相同的蛋白。
本发明的另一方面提供了一种编码本发明所述柳杉二醇合成酶的基因(Tripterygium wilfordii Cryptomeridiol Synthase,TwCS)：所述基因，它是下列核苷酸序列之一：
(1)SEQ ID NO：1第所示的核苷酸分子；或
(2)SEQ ID NO：1第所示的核苷酸分子经取代、缺失或增加一个或多个核苷酸且表达相同功能蛋白的核苷酸序列；或
(3)在严谨条件下与SEQ ID NO：1所示核苷酸分子杂交的核苷酸序列，所述严谨条件为：在含0.1％SDS的0.1×SSPE或含0.1％的SDS的0.1×SSC溶液中杂交。
杂交反应的“严谨度”可以容易的由本领域普通技术人员确定，而且通常根据探针长度、洗涤温度、和盐浓度凭经验计算。通常，较长的探针要求较高的温度以正确退火，而较短的探针需要较低的温度。杂交通常依赖于当互补链存在于低于其解链温度的环境中时变性DNA重新退火的能力。探针和可杂交序列之间的期望同源性程度越高，可使用的相对温度也越高。结果是，推断出较高相对温度将趋向于使反应条件更为严格，而较低温度也就较不严格。关于杂交反应严谨度的另外细节和解释，参见Ausubel等人，《Current Protocols in Molecular Biology》，Wiley Interscience Publishers，1995。
“严谨条件”或“高严谨条件”，如本文中所定义的，可如下鉴别：(1)采用低离子强度和高温进行洗涤，例如0.015M氯化钠/0.0015M柠檬酸钠/0.1％十二烷基硫酸钠，50℃；(2)在杂交过程中采用变性剂，诸如甲酰胺，例如50％(v/v)甲酰胺及0.1％牛血清清蛋白/0.1％Ficoll/0.1％聚乙烯吡咯烷酮/50mM磷酸钠缓冲液pH 6.5，含750mM氯化钠,75mM柠檬酸钠，42℃；或(3)在采用50％甲酰胺,5x SSC(0.75M NaCl,0.075M柠檬酸钠),50mM磷酸钠(pH 6.8),0.1％焦磷酸钠,5x Denhardt氏溶液,超声波处理的鲑鱼精DNA(50μg/ml),0.1％SDS,和10％硫酸右旋糖苷的溶液中于42℃杂交过夜，以及于42℃在0.2x SSC(氯化钠/柠檬酸钠)中洗涤10分钟，接着在含EDTA的0.1x SSC中于55℃进行10分钟高严格性洗涤。
本发明的再一方面，提供了一种表达载体，所述表达载体包含启动子、编码本发明所述柳杉二醇合成酶的基因、以及转录终止子。将启动子、柳杉二醇合成酶编码基因、终止子与附加型载体采用酵母同源重组的方法拼接起来，所述附加型载体为酵母表达载体，如pYX212、pYES2.0、pRS425、pRS426或p424。
在本发明的一具体实施方案中，提供了以下一些具体的表达载体：
(1)重组表达载体pYX212-TwCS：其包含启动子TPIp、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
(2)重组表达载体pYX212-ERG20+TwCS：其包含启动子TPIp、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
(3)重组表达载体pYX212-IDI+TwCS：其包含启动子TPIp、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
(4)重组表达载体pYX212-(IDI-EGR20)+TwCS：其包含启动子TPIp、IDI 基因、融合蛋白连接肽、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
(5)重组表达载体pYX212-(EGR20-IDI)+TwCS：其包含启动子TPIp、EGR20基因、融合蛋白连接肽、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
其中所述融合蛋白为GGGS、GSG、GSGGGGS、GSGEAAAK、GSGEAAAKEAAAK或GSGMGSSSN中的任意一种，优选为GGGS，编码基因为ggtggtggttct。
本发明的又一方面，提供了一种包含本发明所述表达载体的工程菌，所述工程菌可以是选自酵母细胞或植物细胞，优选为酵母细胞，所述酵母可以是GEN.PK系酿酒酵母或BY系酿酒酵母，如BY4741酿酒酵母。
本发明的一具体实施例中，提供了构建本发明所述工程菌的方法，所述方法为：
(1)将重组表达载体pYX212-TwCS转化到酵母BY4741菌株中，得到工程菌TE1；或
(2)将重组表达载体pYX212-ERG20+TwCS转化到酵母BY4741菌株中，得到工程菌TE2；或
(3)将重组表达载体pYX212-IDI+TwCS转化到酵母BY4741菌株中，得到工程菌TE3；或
(4)将重组表达载体pYX212-(IDI-EGR20)+TwCS转化到酵母BY4741菌株中，得到工程菌TE4；或
(5)将重组表达载体pYX212-(EGR20-IDI)+TwCS转化到酵母BY4741菌株中，得到工程菌TE5；或
(6)将重组表达载体pYX212-(EGR20-IDI)+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中，得到工程菌TE6；或
(7)将重组表达载体pYX212-IDI+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中，得到工程菌TE7；
其中所述重组表达载体p424-tHMG1包括：酵母启动子TDH3p、截断的HMG-CoA还原酶基因tHMG1，酵母终止子TDH3t。
为了在酵母菌中获得更高的柳杉二醇表达水平，本发明的再一方面，提供了一种更高效工程菌，即重组酵母菌，所述重组酵母菌包含本发明所述柳杉二醇合成酶编码基因，并且所述酵母菌敲除了erg9或rox1基因中的至少一种。
一个更为优先的方式是，所述的重组酵母菌，敲除了erg9基因，更为优选的是，同时敲除了erg9基因和rox1基因。
所述更为高效的表达柳杉二醇的重组酵母菌，至少包含有如下基因片段：
重组表达载体pYX212-IDI+TwCS：由启动子TPIp、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t，构建到质粒pYX212中；
重组表达载体p424-tHMG1：由酵母启动子TDH3p，截断的HMG-COA还原酶基因tHMG1，酵母终止子TDH3t，构建到质粒p424中。
构建本发明所述的更为高效表达柳杉二醇的重组酵母菌，包括如下步骤：
(1)构建突变菌株：敲除BY4741酵母菌株中的erg9基因，得到突变菌株BY4741erg9::△-200—176；或者
敲除BY4741酵母菌株中的erg9基因和rox1基因，得到突变菌株BY4741erg9::△-200—176rox1::mut；
(2)构建重组表达载体pYX212-IDI+TwCS及p424-tHMG1；
(3)将重组表达载体pYX212-IDI+TwCS及p424-tHMG1转化到突变酵母菌株BY4741erg9::△-200—176，得到重组酵母菌TE8；或者
将重组表达载体pYX212-IDI+TwCS及p424-tHMG1转化到突变酵母菌株BY4741erg9::△-200—176rox1::mut中，得到重组酵母菌TE9。
本发明的又一方面，提供了本发明所述柳杉二醇合成酶、或本发明所述柳杉二醇合成酶编码基因、或本发明所述重组表达载体、或本发明所述工程菌，在合成柳杉二醇及桉叶醇中的运用。发酵培所得菌株2-3d后，用正己烷提取发酵液，经GC-MS检测，可检测到倍半萜产物，经结构鉴定确定柳杉二醇为主产物。利用本发明可以通过生物合成技术来生成柳杉二醇，缓解药源缺乏问题，具有很好的应用前景。
本发明的还一方面，提供了本发明所述柳杉二醇合成酶或编码本发明所述柳杉二醇合成酶基因，在含有柳杉二醇化学成分的植物育种中的运用。运用本发明所述柳杉二醇合成酶、或其编码基因，通过将其运用到植物细胞中，可改善植物体内柳杉二醇的含量。
附图说明
图1为TE1-TE7菌株质粒类型简图。
图2为GC-MS分析发酵产物图，其中A为菌株TE1的发酵产物，出峰位置在1,2的产物为微量的倍半萜成分(出峰位置1是桉叶醇)，出峰位置在3处的产物，经鉴定为柳杉二醇，其保留时间为21.43min；B为不含TwCS表达基因的空载体表达产物图，发现没有任何倍半萜类产物；C为标准品柳杉二醇的GC-MS图，出峰位置与图1的峰3相同；D和E分别为TE1的峰3位置的产物以及标准品柳杉二醇的质谱图。
图3为柳杉二醇标准品的定量用标准曲线图。
图4为TE1-TE7菌株表达产物柳杉二醇产量测定图。
图5为经CRISP/Cas9基因编辑获得的突变菌株TE8和TE9柳杉二醇及桉叶醇表达产量对比图。
具体实施方式
以下通过优选实施例并结合附图具体说明本发明的各个方面和特征，本领域的技术人员应该理解，这些实施例只是用于说明，而不是限制本发明的范围。在不背离权利要求书范围的条件下，本领域的技术人员可以对本发明的各个方面进 行各种修改和改进，这些修改和改进也属于本发明的保护范围。例如，将实施例中所实用的启动子和表达载体替换为本领域中常用的其它启动子和表达载体，是本领域的普通技术人员所能够理解并实现的。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
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，比如，p426-SNR52p-gRNA真核表达载体及p414-TEF1p-Cas9-CYC1t真核表达载体，均从Addgene购买；pESC-LEU真核表达载体，购买自安捷伦科技；SC-His酵母培养基，SC-Trp-His酵母培养基，SC-URA-Trp-His酵母培养基，均购自北京泛基诺科技有限公司。
下述实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的雷公藤(Tripterygium wilfordii Hook.f.)悬浮细胞在文献“雷公藤4-(5’-二磷酸胞苷)-2-C-甲基-D-赤藓醇激酶基因的全长克隆与表达分析.中国中药杂志,2015,40(21):4165-4170”中公开过，公众可从首都医科大学分子生药与中药资源实验室获得。
实施例1雷公藤悬浮细胞总RNA提取、纯化
采用改良CTAB法(CTAB Buffer:2％CTAB(W/V)；100mmol·L -1Tris-HCl(pH 8.0)；25mmol·L -1EDTA；2.0mol·L -1NaCl；0.5g·L -1亚精胺)提取雷公藤悬浮细胞的总RNA。利用RNA纯化试剂盒(天根生化科技有限公司)，对所提RNA进行纯化。
实施例2 TwCS基因全长cDNA克隆
1.引物设计
根据雷公藤转录组数据注释筛选得到基因全长序列片段，设计5’RACE和3’RACE引物，引物序列如下：
5’RACE GTACCGTAAGCATCGTATGTGTCG(SEQ ID NO：3)
3’RACE CTATGAAGAGGACGAGTCTCGG(SEQ ID NO：4)
2.PCR扩增
利用PrimeScript 1 st Strand cDNA Synthesis Kit(Takara公司)试剂盒将实施例1所得RNA反转录成RACE Ready第一链cDNA。参照SMARTer TM RACE试剂盒说明书，进行cDNA末端快速扩增。
通过RACE方法得到SEQ ID No.1DNA序列的3’和5’端，然后依据序列信息设计引物，引物序列如下：
TwCS-F ATGGCAGCGACCACCCAATCCAC(SEQ ID NO：5)
TwCS-R TTAATCTTGCATTGGTATTTGTTG(SEQ ID NO：6)
以RACE Ready第一链cDNA为模板，进行PCR扩增。
PCR反应条件为98℃30s；98℃ 10s,60℃ 15s,72℃ 1min,35个循环；72℃,7min。
测序结果表明，PCR扩增产物的序列如SEQ ID No.1所示，将序列1所示的基因命名为TwCS，此DNA序列编码553个氨基酸组成的蛋白质，该蛋白命名 为TwCS，该蛋白的氨基酸序列为SEQ ID No.2。
实施例3质粒构建
1、启动子和终止子的克隆
本实施例中使用的酵母启动子和终止子均可在SGD网站(https://www.yeastgenome.org)上公开获取。
采用酵母基因组提取试剂盒(天根生化科技有限公司)提取酵母BY4741的总DNA。然后以此DNA为模板，设计如下引物：
TEF1p-F ATAGCTTCAAAATGTTTCTACTC(SEQ ID NO：7)
TEF1p-R TTTGTAATTAAAACTTAGATTAG(SEQ ID NO：8)
FBA1t-F GTTAATTCAAATTAATTGATATAG(SEQ ID NO：9)
FBA1t-R AGTAAGCTACTATGAAAGACTTT(SEQ ID NO：10)
通过PCR扩增(具体步骤同实施例2)得到启动子TEF1p(TEF1 SGD ID:S000006284)和终止子FBA1t(FBA1 SGD ID:S000001543)片段。
以pYX212质粒为模板，通过PCR扩增(具体步骤同实施例2)得到启动子TPIp和终止子pYX212t。扩增引物如下：
TPIp-F GAATTGGGGATCTACGTATGGTC(SEQ ID NO：11)
TPIp-R AGTTTATGTATGTGTTTTTTG(SEQ ID NO：12)
pYX212t-F GAATTGGGGATCTACGTATGGTC(SEQ ID NO：13)
pYX212t-R TGCCGTAAACCACTAAATCGGAACC(SEQ ID NO：14)
2、EGR20基因(酵母FPP和GPP合酶基因)的获得
根据酵母EGR20基因序列(SGD ID：S000003703)设计引物如下：
ERG20-F：ATGGCTTCAGAAAAAGAAATTAG(SEQ ID NO：15)
ERG20-R：CTATTTGCTTCTCTTGTAAAC(SEQ ID NO：16)
通过PCR扩增(具体步骤同实施例2)，获得酵母EGR20基因序列。
3、IDI基因的获得
根据酵母IDI基因序列(SGD ID：S000006038)设计引物如下：
IDI-F：ATGACTGCCGACAACAATAGTATGC(SEQ ID NO：17)
IDI-R：TTATAGCATTCTATGAATTTGCCTG(SEQ ID NO：18)
通过PCR扩增(具体步骤同实施例2)，获得酵母IDI基因序列。
4、表达模块构建
利用PCR方法构建以下模块：
TPIp-ERG20-FBA1t-TEF1p
TPIp-IDI-FBA1t-TEF1p
TPIp-IDI/ERG20-FBA1t-TEF1p
TPIp-ERG20/IDI-FBA1t-TEF1p
TEF1p-TwCS-pYX212t
TPIp-TwCS-pYX212t
构建方法：(1)混合DNA片段：将启动子，基因，终止子，启动子……按 照1:3:5:7:XX:7:5:3:1的摩尔比例混合，比例为1的DNA的量为50-100ng/kb。(2)第一步PCR：将(1)的混合DNA作为模板，不加入引物，通过PCR扩增，PCR反应条件为98℃ 30s；98℃ 10s,60℃ 15s,72℃ 1min,15个循环；72℃,7min。(3)第二步PCR：取2μL(2)中PCR产物，作为模板，利用始端启动子的正向引物，终端终止子或启动子的反向引物进行PCR扩增(具体步骤同实施例2)。(4)利用EZNA Gel Extraction Kit(OMEGA公司)，参照说明书，纯化PCR产物。(5)纯化产物，参照pEASY-Blunt Simple Cloning Kit(北京全式金生物技术有限公司)说明书，连接，转化，经过测序鉴定，得到相应模块DNA。
其中IDI和ERG20之间有GGGS(GGT GGTGGT TCT)linker连接。
5、同源重组法构建质粒
利用酵母同源重组方法，将构建的模块连接到表达载体pYX212中，具体操作如下：
(1)利用BamH I内切酶(NEB公司)对表达载体pYX212进行酶切。
酶切反应体系(50μL体系)
Figure PCTCN2019073905-appb-000002
37℃反应2h后琼脂糖凝胶电泳，利用EZNA Gel Extraction Kit(OMEGA公司)，参照说明书，纯化酶切产物。
(2)将TPIp-TwCS-pYX212t模块与(1)中所得线性表达载体pYX212混合，其中模块摩尔浓度为(100ng/kb)，载体的摩尔浓度为(60-80ng/kb)。然后共同电转入酵母BY4741感受态，电转条件为2.5kV,25μF和200Ω(Bio-Rad Gene Pulsers)。
其中，酵母BY4741感受态是利用醋酸锂转化法制备。
(3)酵母菌株在各自的筛选缺陷培养基中培养2-3d，30℃。挑取单菌落，用E.Z.N.A.Yeast Plasmid Mini Kit(OMEGA公司)，参照说明书，提取酵母质粒。
(4)以(3)质粒为模板，利用PCR方法筛选，筛选引物为TPIp-F和pYX212t-R(见1启动子和终止子的克隆)，经过测序鉴定，得到重组质粒pYX212-TPIp-TwCS-pYX212t，简写为pYX212-TwCS。
(5)重复(1)-(4)步骤，依次将TPIp-ERG20-FBA1t-TEF1p，TPIp-IDI-FBA1t-TEF1p，TPIp-IDI/ERG20-FBA1t-TEF1p，TPIp-ERG20/IDI-FBA1t-TEF1p，TEF1p-TwCS-pYX212t模块构建到载体pYX212中，得到以下重组质粒：pYX212-TPIp-ERG20-FBA1t-TEF1p-TwCS-pYX212t，简写为pYX212-ERG20+TwCSpYX212-TPIp-IDI-FBA1t-TEF1p-TwCS-pYX212t，简写为pYX212-IDI+TwCSpYX212-TPIp-IDI/ERG20-FBA1t-TEF1p-TwCS-pYX212t，简写为pYX212-(IDI-ERG20)+TwCSpYX212-TPIp-ERG20/IDI-FBA1t-TEF1p-TwCS-pYX212t，简写为 pYX212-(ERG20-IDI)+TwCS
(6)质粒p424-tHMG1是将酵母启动子TDH3p，基因tHMG1，酵母终止子TDH3t构建到质粒p424中，质粒携带HIS3marker。详细构建方法在文献“Zhou,Y.J.；Gao,W.；Rong,Q.；Jin,G.；Chu,H.；Liu,W.；Yang,W.；Zhu,Z.；Li,G.；Zhu,G.J.Am.Chem.Soc.2012,134,3234-3241.”中公开过，可依据文献记载方式获得，公众也可从首都医科大学分子生药与中药资源实验室获得。
实施例4酵母菌株改造
(1)gRNA序列：参考公开发表论文“
Figure PCTCN2019073905-appb-000003
T.；Bonde,I.；
Figure PCTCN2019073905-appb-000004
M.；Harrison,S.J.；Kristensen,M.；Pedersen,L.E.；Jensen,M.K.；Keasling,J.D.Metab.Eng.2015,28,213-222.”，设计rox1和erg9启动子的gRNA序列，如下：
rox1 ACAGGATCTTAATAGACGAAGTTTTAGAGCTAGAA(SEQ ID NO：19)
erg9p TTTTCCACTGCACTTTGCATGTTTTAGAGCTAGAA(SEQ ID NO：20)
(2)gRNA载体改造
p426-SNR52p-gRNA载体(Addgene公司)，对此载体进行了改造，在20bp single RNA位点处插入两个方向相反的限制性酶切位点AarI，酶切位点序列如下：
AarI:5'...CACCTGC(N)4↑...3'
3'...GTGGACG(N)8↑...5'
利用PCR扩增方法(具体步骤同实施例2)，引物如下：
pU01-F GTCA CACCTGCATCGGATCATTTATCTTTCACTGCG(SEQ ID NO：21)
pU01-R CTTG CACCTGCATCGGTTTTAGAGCTAGAAATAGCA(SEQ ID NO：22)
横线处为AarI酶切位点序列
将p426-SNR52p-gRNA载体构建成为一级通用载体pTY-U01。
(3)Single gRNA(sgRNA)载体构建
将gRNA位点设计成为带有与载体互补粘性末端的24 nt Oligo，在退火程序下形成双链。Oligo序列见下表。
erg9p-F GATCTTTTCCACTGCACTTTGCAT(SEQ ID NO：23)
erg9p-R AAACATGCAAAGTGCAGTGGAAAA(SEQ ID NO：24)
Figure PCTCN2019073905-appb-000005
条件：95℃,5min；95～25℃,-1℃/min,71cycles,10℃ hold利用Golden Gate反应，进行连接。
体系：AarI2μL
10×Buffer AarI2 μL
50×oligonucleotide(0.025mM)0.4μL
T4 Ligase(HC)1μL
T4 Ligase Buffer 2μL
pTY-U0130 fmol
Annealing oligo 2μL
ddH 2Oup to 20μL
条件：37℃,4h；50℃,5min；80℃,5min；4℃ hold
连接产物经转化，阳性克隆的筛选，送样测序鉴定，得到erg9p-gRNA载体。
重复(1)-(3)步骤，将rox1的gRNA插入到载体上，gRNA序列如下：
rox1-F GATCACAGGATCTTAATAGACGAA(SEQ ID NO：25)
rox1-R AAACTTCGTCTATTAAGATCCTGT(SEQ ID NO：26)
得到erg9p-rox1-gRNA载体。
(4)dsOligo获得
rox1基因的dsOligo采用合成120nt长链Oligo，再退火形成DNA双链，然后利用EZNA Gel Extraction Kit(OMEGA公司)，参照说明书，纯化得到rox1的dsOligo。合成序列如下：
rox1-Oligo-F
Figure PCTCN2019073905-appb-000006
rox1-Oligo-R
Figure PCTCN2019073905-appb-000007
erg9p则直接采取合成双链DNA的方法(北京睿博生物科技有限公司)，然后可用PCR方法扩增得到erg9p的dsOligo。Oligo序列和扩增引物如下：
erg9p-OLIGO：
Figure PCTCN2019073905-appb-000008
ERG9p-OLIGO-F CTAGAGACCCTGCGAGCGTGTC(SEQ ID NO：30)
ERG9p-OLIGO-R TATACGTTTTCCGCTTCTGCTCTTC(SEQ ID NO：31)
(5)Cas 9载体改造和转化
由于p414-TEF1p-Cas9-CYC1t载体(Addgene公司)的筛选标记TRP不适用于BY4741酵母，因此本实验中采用无缝拼接的方式将TRP筛选标记替换为LEU，LEU序列模板为真核表达载体pESC-LEU(安捷伦科技)。
Cas9载体改造引物
Figure PCTCN2019073905-appb-000009
(i)分以pESC-LEU质粒为模板扩增LEU序列、以p414-TEF1p-Cas9-CYC1t质粒为模板扩增TRP上游序列、下游序列，引物分别是LEU-F/R、U-F/R、D-F/R。
Figure PCTCN2019073905-appb-000010
Figure PCTCN2019073905-appb-000011
(ii)p414-TEF1p-Cas9-CYC1t质粒双酶切
选取TRP1ORF上游148bp的SnaBI限制性内切酶位点和下游323bp的DraIII限制性内切酶位点。
Figure PCTCN2019073905-appb-000012
条件：37℃ 2h
(iii)切胶回收各片段。
(iv)In-Fusion反应
Figure PCTCN2019073905-appb-000013
Figure PCTCN2019073905-appb-000014
注：n(载体)：n(片段)＝1:2
条件：50℃孵育15min；冰上放置。
(v)将10μL拼接产物转化到50μLTrans1-T1感受态细胞中，30℃复苏，涂LB+Amp固体培养基(北京泛基诺科技有限公司)，30℃培养过夜。
(vi)挑取单菌落，LB+Amp液体培养基30℃，250rpm摇培4～6h，菌液PCR验证，引物如下，若PCR产物经琼脂糖凝胶电泳检测出现1740bp附近的条带，则将对应菌液送公司测序。
Cas9载体改造菌液PCR引物
Figure PCTCN2019073905-appb-000015
(vii)将测序正确的菌液摇培，提取得改造好的cas9质粒(Leu2-TEF1p-Cas9-CYC1t)。
(viii)将此改造好的Cas9质粒转化到BY4741酵母菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到菌株BY4741-Cas9。
(6)gRNA和dsOligo转化BY4741-Cas9
取gRNA约500ng，erg9p dsOligo 2μg，rox1dsOligo 1μg，根据不同敲除目的配成混合体系加入到100μL BY4741-Cas9感受态细胞中，电转。
(7)突变菌株的获得
将步骤(6)中改造的菌株，以未改造的菌株作为对照，用筛选引物对进行检测，引物序列如下：
rox1-D-F TCCTCGTATTGTCTTGCCGG(SEQ ID NO：40)
rox1-D-R CTAGACCACCTGCGCCTAAC(SEQ ID NO：41)
erg9p-D-F CTAGAGACCCTGCGAGCGTG(SEQ ID NO：42)
erg9p-D-R CAGCTACGTAGTGACAGTAC(SEQ ID NO：43)
PCR筛选后，将阳性克隆的送测序鉴定，得到突变成功的菌株。
(8)Cas9和gRNA质粒的去除
(i)将BY4741突变菌株划YPD固体培养基(北京泛基诺科技有限公司)，42℃培养3天，至长出单菌落；
(ii)挑取单菌落，置于相同成分的液体培养基中42℃摇培，传代两次；
(iii)将(ii)中摇培的BY4741突变菌株分别划YPD、SC-LEU、SC-URA固体培养基(北京泛基诺科技有限公司)，30℃培养3天,若BY4741改造菌株在YPD固体培养基上能正常生长，在SC-LEU和SC-URA固体培养基上均无法生长，则证明Cas9和gRNA质粒均已除去。
(iv)将不含质粒的突变菌株液体培养基摇培，然后再次按照(7)中方法进行 测序检测，确保突变正确，得到BY4741erg9::△-200—176和BY4741erg9::△-200—176rox1::mut两种突变的菌株，。
实施例4生产柳杉二醇工程菌构建
将实施例3中的质粒pYX212-TwCS转化到BY4741菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE1，如表1和附图1所示。
将实施例3中的质粒pYX212-ERG20+TwCS转化到BY4741菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE2，如表1和附图1所示。
将实施例3中的质粒pYX212-IDI+TwCS转化到BY4741菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE3，如表1和附图1所示。
将实施例3中的质粒pYX212-(IDI-ERG20)+TwCS转化到BY4741菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE4，如表1和附图1所示。
将实施例3中的质粒pYX212-(ERG20-IDI)+TwCS转化到BY4741菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE5，如表1和附图1所示。
将实施例3中的质粒pYX212-(ERG20-IDI)+TwCS和p424-tHMG1转化到BY4741菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE6，如表1和附图1所示。
将实施例3中的质粒pYX212-IDI+TwCS和p424-tHMG1转化到BY4741菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE7，如表1和附图1所示。
将实施例3中的质粒pYX212-IDI+TwCS和p424-tHMG1转化到BY4741erg9::△-200—176突变菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE8，如表1所示。
将实施例3中的质粒pYX212-IDI+TwCS和p424-tHMG1转化到BY4741erg9::△-200—176rox1::mut突变菌株中，转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书，得到工程菌TE9，如表1所示。
表1本发明所涉及到的菌株的基因型和重组质粒
Figure PCTCN2019073905-appb-000016
Figure PCTCN2019073905-appb-000017
实施例5工程菌培养和产物鉴定
(1)工程菌培养
通过生物反应器来发酵生产倍半萜的菌株。用20g/L浓度的葡萄糖作为碳源，相应的缺陷培养基(北京泛基诺有限公司)用于预培养对应的营养缺陷型菌株。用于3L生物反应器的培养基由不含尿嘧啶和组氨酸的8g/L合成脱氮培养基组成，10g/L(NH 4) 2SO 4，10g/L KH 2PO 4，1.0g/L MgSO 4·7H 2O。50％NH 3·H 2O用作pH调节剂。将菌株在摇瓶中在30℃条件下以230rpm转速预培养48h。然后在3L搅拌式生物反应器(Eppendorf BioFlo/CelliGen 115)中，用预培养细胞接种1L发酵培养基。定期喂养500g/L葡萄糖溶液以保持菌株的生长。将含有40g/L合成脱氮培养基的缺乏尿嘧啶和组氨酸和(NH 4) 2SO 4的浓缩培养基进行发酵。
(2)产物提取和分离
发酵产物为倍半萜类成分，易溶于正己烷，因此选取正己烷为提取试剂。将发酵液离心分为菌体和菌液两部分，向菌液中加入等体积正己烷，萃取3次；菌体破碎后，用3倍体积的正己烷超声提取3次。合并有机层，加入适量的无水硫酸钠，静置片刻，除去萃取液中的水分。将萃取液用旋转蒸发仪浓缩，注意水浴温度不超过35℃(挥发性成分)，最终转移到玻璃收集瓶中。
取硅胶薄层板，将浓缩产物用正己烷和乙酸乙酯以不同配比展开，显色剂为香草醛硫酸。初步分离。然后用XSelect CSH Prep C18OBD(19×150mm，5um)色谱柱分离，流动相A为0.1％(v/v)甲酸水，流动相B为乙腈.流速为20mL/min.分离浓缩富集单体化合物。
(3)结构鉴定
化合物结构通过NMR光谱解析，所有数据从BRUKER ACANCE III 600MHz spectrometer中收集，溶剂为含有TMS的氘代氯仿，最终化合物鉴定为柳杉二醇，结果如附图2所示。
实施例6工程菌生产柳杉二醇的产量比较
为了测定每种菌株的倍半萜生产量，按1：100比例接种，预培养50mL菌液作为菌种。菌株在含有20g/L葡萄糖的缺陷培养基中以230rpm转速，30℃ 条件下培养。振荡培养72h后，检测所有菌株的OD600。向培养液加入等体积的正己烷中并在200rpm下保持振荡2h小时，然后加入等体积正己烷超声波提取两次。合并有机层并旋转蒸发浓缩。浓缩样最终定容到1.0mL，然后取100uL制备GC-MS样品，进行GC-MS分析。使用Thermo TRACE 1310/TSQ8000气相色谱仪(不分流；注射器温度250℃)，TG-5MS(30m×0.25mm×0.25μm)毛细管柱；GC条件如下：首先将烘箱温度保持在50℃恒定2分钟，然后以8℃/分钟的速度升至280℃，并在最终温度下保持10分钟。注射器和检测器的温度为50℃。使用柳杉二醇类似物β-eudesmol建立标准曲线，得到标准曲线方程为：y＝3E+06x-3E+07，如附图3所示。计算得到具体的产量为：
Figure PCTCN2019073905-appb-000018
上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内，作出的变化、改型、添加或替换，也应属于本发明的保护范围，本发明的保护范围以权利要求书为准。

Claims (15)

  1. 一种柳杉二醇合成酶，所述合成酶具有如下氨基酸序列：
    (1)SEQ ID NO：2所示氨基酸序列；
    (2)SEQ ID NO：2所示的氨基酸序列经取代、缺失或增加一个或多个氨基酸且功能相同的蛋白。
  2. 编码权利要求1所述合成酶的基因，所述基因为如下中的至少一种：
    (1)SEQ ID NO：1第所示的核苷酸分子；或
    (2)SEQ ID NO：1第所示的核苷酸分子经取代、缺失或增加一个或多个核苷酸且表达相同功能蛋白的核苷酸序列；或
    (3)在严谨条件下与SEQ ID NO：1所示核苷酸分子杂交的核苷酸序列，所述严谨条件为：在含0.1％SDS的0.1×SSPE或含0.1％的SDS的0.1×SSC溶液中杂交。
  3. 重组表达载体，其包含启动子、权利要求2所示基因和转录终止子。
  4. 权利要求3所述的表达载体，其为将启动子、柳杉二醇合成酶编码基因、终止子与附加型载体采用酵母同源重组的方法拼接起来，所述附加型载体为酵母表达载体，如pYX212、pYES2.0、pRS425、pRS426或p424。
  5. 根据权利要求3所述的表达载体，其中所述载体为以下中的任意一种：
    (1)重组表达载体pYX212-TwCS：其包含启动子TPIp、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
    (2)重组表达载体pYX212-ERG20+TwCS：其包含启动子TPIp、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
    (3)重组表达载体pYX212-IDI+TwCS：其包含启动子TPIp、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
    (4)重组表达载体pYX212-(IDI-EGR20)+TwCS：其包含启动子TPIp、IDI基因、融合蛋白连接肽、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
    (5)重组表达载体pYX212-(EGR20-IDI)+TwCS：其包含启动子TPIp、EGR20基因、融合蛋白连接肽、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t；
    其中所述融合蛋白为GGGS、GSG、GSGGGGS、GSGEAAAK、GSGEAAAKEAAAK或GSGMGSSSN中的任意一种，优选为GGGS，编码基因为ggtggtggttct。
  6. 一种生产柳杉二醇工程菌，其包含权利要求2所述基因或权利要求3-5中任意一权利要求所述重组表达载体。
  7. 根据权利要求6所述的工程菌，其中所述工程菌选自酵母细胞或植物细胞。
  8. 根据权利要求7所述的工程菌，其中所述工程菌选自酵母细胞。
  9. 根据权利要求8所述的工程菌，其中所述工程菌敲除了erg9或rox1基因中的至少一种。
  10. 根据权利要求6-8中任意一权利要求所述的工程菌，其构建方法为：
    (1)将重组表达载体pYX212-TwCS转化到酵母BY4741菌株中，得到工程菌TE1； 或
    (2)将重组表达载体pYX212-ERG20+TwCS转化到酵母BY4741菌株中，得到工程菌TE2；或
    (3)将重组表达载体pYX212-IDI+TwCS转化到酵母BY4741菌株中，得到工程菌TE3；或
    (4)将重组表达载体pYX212-(IDI-EGR20)+TwCS转化到酵母BY4741菌株中，得到工程菌TE4；或
    (5)将重组表达载体pYX212-(EGR20-IDI)+TwCS转化到酵母BY4741菌株中，得到工程菌TE5；或
    (6)将重组表达载体pYX212-(EGR20-IDI)+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中，得到工程菌TE6；或
    (7)将重组表达载体pYX212-IDI+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中，得到工程菌TE7；
    其中所述重组表达载体p424-tHMG1包括：酵母启动子TDH3p、截断的HMG-CoA还原酶基因tHMG1，酵母终止子TDH3t。
  11. 根据权利要求9所述的工程菌，所述工程菌的构建方法包括如下步骤：
    (1)构建突变菌株：
    敲除BY4741酵母菌株中的erg9基因，得到突变菌株BY4741erg9::△-200-176；或者
    敲除BY4741酵母菌株中的erg9基因和rox1基因，得到突变菌株BY4741erg9::△-200-176rox1::mut；
    (2)将重组表达载体pYX212-IDI+TwCS及p424-tHMG1转化到突变酵母菌株BY4741erg9::△-200-176，得到酵母工程菌TE8；或者
    将重组表达载体pYX212-IDI+TwCS及p424-tHMG1转化到突变酵母菌株BY4741erg9::△-200-176rox1::mut中，得到酵母工程菌TE9。
  12. 根据权利要求11所述的方法，其中所述突变菌株的构建方法是采用CRISP/Cas9基因编辑技术进行改造的。
  13. 根据权利要求12所述方法，其中所述CRISP/Cas9基因编辑技术，所用的含Cas9的表达载体为p414-TEF1p-Cas9-CYC1t载体，并且p414-TEF1p-Cas9-CYC1t载体中的TRP筛选标记替换为LEU筛选标记。
  14. 权利要求1所述合成酶、或权利要求2所述基因、或权利要求3-5中任意一权利要求所述表达载体、或权利要求6-9中任意一权利要求所述工程菌，在合成柳杉二醇或桉叶醇中的运用。
  15. 权利要求1所述合成酶或权利要求2所述基因在含有柳杉二醇或桉叶醇化学成分的植物育种中的运用。
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