CN111518817A - Hemsleya amabilis triterpene synthetase HcOSC6 gene, engineering bacteria thereof and application thereof in preparation of cucurbitadienol - Google Patents

Abstract

The invention discloses a hemsleya amabilis triterpene synthetase HcOSC6 gene, engineering bacteria thereof and application thereof in preparation of cucurbitadienol. The cucurbitacin triterpene synthetase HcOSC6 gene has a nucleotide sequence shown as SEQ ID No.1, and the engineering bacteria can express the cucurbitacin triterpene synthetase HcOSC 6. In a culture medium with D-glucose and D-galactose as carbon sources, the transgenic engineering bacteria can express cucurbitacin triterpene synthetase HcOSC6 in cells, under the catalysis of the cucurbitacin triterpene synthetase HcOSC6, a substrate 2, 3-oxidized squalene endogenous to yeast can carry out cyclization reaction to generate cucurbitadienol, the yield of the cucurbitadienol is about 5.5mg/L, an important way for producing the cucurbitadienol is provided, and abundant raw materials are provided for biosynthesis of cucurbitacins such as cucurbitacin and the like.

Description

Hemsleya amabilis triterpene synthetase HcOSC6 gene, engineering bacteria thereof and application thereof in preparation of cucurbitadienol

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hemsleya amabilis triterpene synthetase HcOSC6 gene, engineering bacteria thereof and application thereof in preparation of cucurbitadienol.

Background

Cucurbits contain numerous secondary metabolites that are important for human life, such as: (1) cucurbitacins (cucurbitacins) are bitter substances separated from cucurbitaceae plants at first, and subsequently discovered to exist in cruciferae plants and other plants, on one hand, cucurbitacins have remarkable anticancer activity and are potential new drug candidate compounds, wherein the most representative cucurbitacins IIa (25-acetoxy-23,24-Dihydrocucurbitacin F) and cucurbitacins IIb (23,24-Dihydrocucurbitacin F) separated and extracted from cucurbitacins are the most effective novel antibacterial drugs in cucurbitacin development and utilization, and meanwhile, cucurbitacins IIa can inhibit the proliferation of tumor cells by inhibiting Survivin (Survivin) at the downstream of JAK2/STAT3, so that the cucurbitacins become novel anticancer drugs; on the other hand, as bitter substances in cucurbitaceae, cucurbitacin with extremely low content (0.1mg/L) can cause obvious bitter taste, thereby improving the disease resistance of melon plants; (2) mogrosides, which is separated from momordica grosvenori belonging to the Cucurbitaceae family, has strong sweet taste and is a potential sugar substitute for diabetics.

Cucurbitacin and mogroside are tetracyclic triterpenoids with cucurbitadienol as a skeleton, and researches suggest that the synthesis route of the compounds starts from 2,3-oxidosqualene, firstly forms cucurbitadienol (Cuol) with the triterpene skeleton through oxidosqualene cyclase (OSCs), and then forms different cucurbitacin or mogroside under the action of CYP450 oxidoreductases, methyltransferases, acyltransferase (BAHD-AT) and glycosyltransferase. It can be seen that cucurbitadienol is an important intermediate for biosynthesis of cucurbitacin and tetracyclic triterpenoids such as mogroside, and moreover, cucurbitadienol has significant anti-inflammatory and anti-tumor effects.

Although cucurbitadienol, cucurbitacin, mogroside and other human production and life play an important role, many of the tetracyclic triterpenoids, such as cucurbitacin (cucurbitacin IIa and cucurbitacin II b), have extremely low content in cucurbitacin plants. The separation and extraction of the hemsleyadin require the collection of a large amount of plant raw materials, the extraction process is complex, the planting period of the plant raw materials is long, the requirements on the planting land and the planting technology are high, and the development and utilization of the hemsleyadin are seriously hindered.

At present, for natural products with high added values, homologous or heterologous expression systems established by adopting modern biotechnology for efficiently producing medicinal active ingredients are widely regarded as important technical means for solving the shortage of medicinal resources in the future. However, the biosynthesis pathway of cucurbitacin in cucurbitacin plants has not been reported, and particularly, the gene function of OSCs responsible for forming a cucurbitacin carbon skeleton is unclear, so that the promotion of the biosynthesis work of cucurbitacin is influenced.

Disclosure of Invention

The invention aims to provide a hemsleya amabilis triterpene synthetase HcOSC6 gene, engineering bacteria thereof and application thereof in preparation of cucurbitadienol, provides a preparation way of cucurbitadienol, and provides abundant raw materials for biosynthesis of cucurbitacin Iia.

In order to achieve the above purpose, the technical solution of the present application is as follows:

the hemsleya amabilis triterpene synthetase HcOSC6 gene has a nucleotide sequence shown as SEQ ID No. 1. The hemsleya amabilis triterpene synthetase HcOSC6 gene is obtained by screening after a large number of experiments from tubers of hemsleya amabilis through transcriptome sequencing and bioinformatics technology.

During extraction, total RNA of hemsleya amabilis tubers is extracted firstly, then reverse transcription is carried out to obtain cDNA, and the cDNA is amplified by PCR to obtain the gene of the hemsleya amabilis triterpene synthetase HcOSC 6; the specific primers used for PCR amplification were:

an upstream primer: 5'-ATGTGGAAGTTAAAGATAGGAGGAG-3' (SEQ ID No. 3);

a downstream primer: 5'-TCAGAATAAAGCGGCCGGATG-3' (SEQ ID No. 4).

The invention also provides a recombinant vector containing the hemsleya amabilis triterpene synthetase HcOSC6 gene. The recombinant vector can be a recombinant plasmid, and the original plasmid of the recombinant plasmid can be pYES 2.

The invention also provides a hemsleya amabilis triterpene synthetase HcOSC6, wherein the hemsleya amabilis triterpene synthetase HcOSC6 has an amino acid sequence shown as SEQ ID No. 2.

The invention also provides a transgenic engineering bacterium for expressing the hemsleya amabilis triterpene synthetase HcOSC 6.

In a culture medium with D-glucose and D-galactose as carbon sources, the transgenic engineering bacteria can express cucurbitacin triterpene synthase HcOSC6 in cells, and under the catalysis of the cucurbitacin triterpene synthase HcOSC6, a substrate 2,3-oxidosqualene endogenous to yeast can carry out cyclization reaction to generate cucurbitadienol; the yield of cucurbitadienol is about 5.5mg/L of yeast culture, which is far higher than that of cucurbitadienol plants, thereby not only providing an important way for producing cucurbitadienol, but also providing abundant raw materials for biosynthesis of cucurbitacin such as cucurbitacin.

The structural formula of the cucurbitadienol is as follows:

in the transgenic engineering bacterium for expressing the cucurbitacin triterpene synthase HcOSC6, the cucurbitacin triterpene synthase HcOSC6 gene can exist in a recombinant plasmid or can be directly integrated into the genome of an original bacterium.

That is, the genetically engineered bacterium may contain the recombinant vector containing the cucurbitane triterpene synthase HcOSC6 gene, or may have the cucurbitane triterpene synthase HcOSC6 gene integrated into its genome.

In the invention, the original strain of the transgenic engineering bacterium for expressing the hemsleya amabilis triterpene synthetase HcOSC6 is saccharomyces cerevisiae, and saccharomyces cerevisiae GIL77 strain can be selected.

The invention also provides application of the cucurbitadienol preparation by using the cucurbitacin triterpene synthase HcOSC6 gene and application of the transgenic engineering bacterium for expressing the cucurbitadienol preparation by using the cucurbitadienol triterpene synthase HcOSC6 gene.

Wherein, the application of the cucurbitadienol preparation by the cucurbitacin triterpene synthase HcOSC6 gene is to construct transgenic engineering bacteria, and the application of the transgenic engineering bacteria expressing the cucurbitadienol synthase HcOSC6 in the cucurbitadienol preparation comprises the following steps:

(1) inoculating the transgenic engineering bacteria for expressing the hemsleya amabilis triterpene synthetase HcOSC6 into a culture medium taking D-glucose and D-galactose as carbon sources, and performing fermentation culture;

(2) centrifuging the culture, taking engineering bacteria cells, and performing wall breaking treatment to obtain intracellular product mixed liquor;

(3) separating and purifying the mixed solution of the intracellular products to obtain the cucurbitadienol.

According to the invention, through recombinant plasmids, a target protein cucurbitacin triterpene synthetase HcOSC6 is expressed in a saccharomyces cerevisiae body, and the cucurbitacin triterpene synthetase HcOSC6 further performs cyclization reaction on a yeast endogenous high accumulation substrate 2,3-oxidosqualene to directly generate cucurbitadienol.

Compared with the prior art, the invention has the beneficial effects that:

the method identifies and obtains a cucurbitacin triterpene synthetase HcOSC6 gene, constructs and obtains a transgenic engineering bacterium for expressing the cucurbitacin triterpene synthetase HcOSC6, the transgenic engineering bacterium can express the cucurbitacin triterpene synthetase HcOSC6 in cells in a culture medium taking D-glucose and D-galactose as carbon sources, and a substrate 2, 3-oxidation squalene endogenous to yeast can generate cyclization reaction under the catalysis of the cucurbitacin triterpene synthetase HcOSC6 to generate cucurbitacin alcohol; the yield of the cucurbitadienol is about 5.5mg/L, and the yeast culture not only provides an important way for producing the cucurbitadienol, but also provides rich raw materials for the biosynthesis of cucurbitacins such as cucurbitacin.

Drawings

FIG. 1 shows the result of electrophoresis detection of recombinant plasmid containing the hemsleya amabilis triterpene synthetase HcOSC6 gene of the present invention;

wherein, Marker represents standard substance, the same is as below;

FIG. 2 is a schematic structural view of a recombinant plasmid containing the hemsleya amabilis triterpene synthase HcOSC6 gene of the present invention;

FIG. 3 is a TCL detection chart of intracellular extracts of genetically engineered bacteria of the present invention;

wherein, 1: ergosterol as Ergosterol supplement in culture medium, 2,3-Oxidosqualene as endogenous 2,3-Oxidosqualene substrate in yeast, 2 and 3 as empty plasmid controls; 4 and 5 are HcOSC6 gene extract, black arrows are the target products;

FIG. 4 is a GC-MS detection chart of intracellular extracts of genetically engineered bacteria of the present invention;

wherein, standard: the peak time of the cucurbitadienol standard product; EV: reaction results with empty plasmid of control pYES2 as positive control; HcOSC6 shows the cyclization reaction result of hemsleya amabilis triterpene synthetase HcOSC6 in yeast, and retentitime (min) shows the peak time;

FIG. 5 shows the result of NMR detection of cucurbitadienol, a target product of the genetically engineered bacterium of the present invention;

wherein HMBC represents the cucurbitadienol H-C relationship;

FIG. 6 is a schematic diagram of the synthesis of cucurbitadienol by cyclization of 2,3-oxidosqualene catalyzed by cucurbitacin triterpene synthase HcOSC 6;

wherein 2,3-oxidosqualene represents 2,3-oxidosqualene, cucurbitenol represents cucurbitadienol.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.

Example 1 Gene identification

(1) Candidate Gene screening

Based on the basic functional annotation information of cucurbit transcriptome Unigene, screening of OSCs candidate genes was performed in the sequencing annotation results, and simultaneously, with cucurbitadienol synthase (CBS) identified in cucurbitaceae plants, SgCBS identified mainly in luo han guo (Siraitiagrosvenorii), CsBi identified in cucumber (cuumis sativus), CsBi identified in melon (Cucumismelo), and cppq identified in Cucurbita pepo, etc., by sequence local BLAST analysis, the screening results were collated and finally 6 oxidized squalene cyclase (OSCs ) genes were found, which were respectively named HcOSC1, HcOSC2, HcOSC3, HcOSC4, HcOSC5, and HcOSC 6. The HcOSC6 was functionally annotated as cucurbitadienol synthase (CBS), and finally Unigene nucleotide sequences were extracted from fasta files for subsequent analysis based on the corresponding ID numbers of Unigene.

(2) RNA extraction and cDNA template preparation

A fresh sample of hemsleya tuber is taken, sliced and quickly frozen by liquid nitrogen, and RNA extraction is carried out by adopting a HiPure Plant RNA Mini Kit of magenta (Guangzhou Meiji Biotechnology Co., Ltd.). After the extracted RNA is qualified by detection, the TAKARA reverse transcription kit is used for reverse transcription of the RNA into cDNA, and the cDNA is stored at the temperature of-20 ℃ for later use.

(3) Gene amplification and recovery

The target fragment of gene amplification was the open reading frame of the candidate gene HcOSCs, and the cDNA prepared as described above was used. Firstly, an open reading frame of candidate genes HcOSCs is found out by using NCBI online software (https:// www.ncbi.nlm.nih.gov/orffinder /), and then a primer with a homology arm (the homology arm is on a yeast expression vector pYES 2-ura) is designed by using primer Design software (CE Design) v 1.04):

an upstream primer:

5'-ttggtaccgagctcggatccATGTGGAAGTTAAAGATAGGAGGAG-3'(SEQ ID No.5)；

a downstream primer: 5'-cactggcggccgttactagtTCAGAATAAAGCGGCCGGATG-3' (SEQ ID No. 6).

Thereafter using

High-Fidelity DNA Polymerase (NEB: M0491) for gene amplification, the PCR reaction program is: 94 ℃ for 5 min; 94 deg.C, 30S, 56 deg.C, 1.5min, 72 deg.C, 1min, 35 cycles; 72 deg.C, 7 min.

After the PCR is finished, the gel is run, and the target band is recovered after the successful amplification is confirmed. Gene cutting recovery Using EasyPure Quick Gel Extraction Kit from Beijing all-terrain gold Biotechnology Ltd, recovery of the target gene was performed. After recovery, the concentration of the recovered water is measured on a NanoReady ultramicro ultraviolet-visible spectrophotometer, and finally the water is stored in a refrigerator at the temperature of-20 ℃ for later use. Through sequencing, the nucleotide sequence of the hemsleya amabilis triterpene synthetase HcOSC6 gene is shown as SEQ ID No. 1.

Example 2 recombinant plasmid construction

(1) Construction of recombinant plasmids

Firstly, the vector pYES2 was linearized, and the linearized vector was obtained by a single cleavage with BamH I enzyme. Recovering by using an easy PureQuick Gel Extraction Kit, determining the concentration of the recovered product after recovery is finished, and finally storing the product in a refrigerator at the temperature of-20 ℃ for later use; secondly, carrying out homologous recombination, assembling according to the operation instruction of a homologous recombinase kit during homologous recombination, and then calculating the use amount of each component according to the concentrations of the insert and the vector and the recombination instruction; finally, the components were added to the PCR reaction tube on ice. The recombination procedure was carried out as follows in the system of table 1:

TABLE 1 recombination reaction System

Where, X ═ (base pair 0.02 × HcOSC 6) ng/linearized pYES2 concentration ng/. mu.l.

After assembly, the result is detected and sent to a company for sequencing, and the assembled electrophoresis detection result is shown in figure 1, which indicates that the assembly is successful; the schematic structure of the successfully assembled recombinant plasmid is shown in FIG. 2.

(2) Extraction of recombinant plasmid

Sucking 10 μ L of positive monoclonal stock solution detected by sequencing, placing in 6mL LB liquid medium (100mg/mLAmp), and culturing overnight in a shaker (220r/min) at 37 ℃; extracting plasmids according to the instructions in the plasmid DNA miniprep kit centrifugation column type (GenStar, Shenzhen, China), determining the recovery concentration of the extracted plasmid DNA on a NanoReady ultramicro ultraviolet-visible spectrophotometer, and finally storing in a refrigerator at-20 ℃ for later use.

Example 3 construction of transgenic engineering bacteria

(1) Competent preparation of GIL77 Yeast Strain (lanosterol synthase deficient)

The GIL77 strain on YPD plates was picked and inoculated into 100mL YPD medium supplemented with ergosterol (20. mu.g/mL), heme (13. mu.g/mL) and Tween 80(5mg/mL), cultured at 30 ℃ and 220rpm until OD ═ 1.3-1.5, and ice-cooled for 30 min; the yeast cells were collected by centrifugation.

Yeast cells were washed repeatedly with 25mL of water, 2mL of 1M sorbitol, 2mL of 0.1M lithium acetate, and 2mL of sorbitol. Finally, the cells were dispersed in 250. mu.L of 1M sorbitol and dispensed into pre-cooled 1.5ml sterile centrifuge tubes in 100. mu.L.

(2) GIL77 Yeast transformation

Adding 2 mu g of recombinant yeast plasmid DNA into 100 mu L of yeast competent cells, and gently mixing uniformly; and transferred to a pre-cooled 0.2cm electroporation cuvette, ice-cooled for 10min, and electroporated using a GenePulser electroporation system (BioRad) at the following settings: 1.5kV, 600 omega, 25 muF.

The yeast cells after electroporation were immediately added with 1M sorbitol and allowed to stand at 30 ℃ for 1 hour. Then sucking the transformant on a solid synthetic complete culture medium (SC-Ura) without uracil, carrying out inverted culture at 30 ℃ for 48-72h, selecting a single clone after the transformant grows out, and extracting a genome for verification.

(3) Cloning and screening of positive strains

Add 20. mu.L of ddH to each of the 0.2ml PCR tubes₂O, picking 8 single colonies each cultured in '2.2.7.3.3'; placing on a PCR instrument, reacting at 95 deg.C for 10min, and breaking cell wall.

The following reaction systems were added to the PCR tubes: 12.5. mu.L Super 2x Mix, 10.5. mu.L sterile water, 0.5. mu.L universal forward primer (10mM), 0.5. mu.L universal reverse primer (10mM), 1. mu.L monoclonal template in water. Wherein the universal primers for detection are designed by software (SnapGene 3.2.1), and the nucleotide sequences are respectively as follows:

an upstream primer: 5'-CGGTTTGTATTACTTCTTATTC-3', respectively;

a downstream primer: 5'-GCGTGAATGTAAGCGTGAC-3', respectively;

the PCR amplification cycle parameters were: 3min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃ and 2min at 72 ℃ for 35 cycles; 8min at 72 ℃ and 5min at 16 ℃.

And adding 5 mu L of the PCR product into 1.0 mu L of Loading buffer, uniformly mixing, and detecting an amplification result through 1% agarose gel electrophoresis, wherein if the amplification result is similar to the size of a target fragment, the yeast strain is a true positive yeast strain and is marked as successful transformation.

(4) Detection and sequencing

Transferring the bacterial liquid identified as positive clone into LB culture medium containing 1mL of liquid (with Amp as resistance) in an ultra-clean workbench ⁺100 mu g/mL), culturing for about 5 hours at 37 ℃ and 220r/min, taking about 50 mu L of bacterial liquid after the bacterial liquid is sufficiently turbid, and sending the bacterial liquid to a sequencing company for sequencing. And (5) preserving the seeds after the sequencing confirms that no errors exist. The method comprises the steps of adding 50% of glycerol and bacterial liquid into a seed preservation pipe according to the ratio of 1:1, fully and uniformly mixing, and then placing into an ultra-low temperature refrigerator at minus 80 ℃ for preservation for later use.

EXAMPLE 4 preparation of cucurbitadienol

(1) Inducible expression

Selecting a normally growing positive GIL77 yeast strain to be cultured in a medium (50mL) without uracil (SC-U), supplemented with ergosterol (20. mu.g/mL), heme (13. mu.g/mL) and Tween 80(5mg/mL) at 30 ℃, and shake-culturing at 220 rpm; culturing for 2 days, changing glucose into galactose, and inducing at 30 deg.C for 48 hr; centrifuging at 8000rpm for 5min to collect cells, resuspending with 0.5mL of 1M potassium phosphate buffer solution of pH 7.0, supplementing 50 μ L/mL glucose and 1 μ L/mL heme, and culturing at 30 deg.C in a constant temperature incubator for 24 h; centrifuging at 8000rpm for 5min, collecting cells, incubating with 2mL of lysis buffer (20% KOH, 50% EtOH) at 55 deg.C for 1h, and shaking every 20 min; extracting with n-hexane of the same volume for three times; the concentrated extract was spotted onto TCL plates, developed with the solvent cyclohexane/ethyl acetate (12:1 by volume) and stained for spotting with 5% ethanolic sulfate developer (see FIG. 3 for results of thin layer chromatography).

Then 200. mu. L N-methyl-N- (trimethylsilyl) trifloacetamide was added to the concentrated extract and derivatized at 70 ℃ for 1 h. The metabolites were analyzed by GC-MS.

The GC-MS detection conditions were as follows:

the dry sample after derivatization was resuspended in 200. mu.L of extraction solvent, i.e.redissolved with 1mL of n-hexane, and then transferred to a glass insert in a glass autosampler vial. 1 μ L of each sample was taken and directly injected into a quantitative GC ultra gas chromatograph (THERMO Science) used in conjunction with an ISQ type mass spectrometer for detection. GC-MS analysis was performed using 7890B GC (Agilent) and electron bombardment (EI)5977AMSD (Agilent) equipped with ZebronZB5-HT chromatography column (Phenomenex). Briefly, 1 μ Ι _ of sample (inlet 250 ℃) was injected in non-split mode (pulse pressure 30psi), a procedure involving a 2 minute column box temperature of 170 ℃, ramping up to 300 ℃ at a rate of 20 ℃/min, 11.5 minutes at 300 ℃; after a solvent delay of 8 minutes, the detection was carried out in scanning mode (60-800 mass units) and set at 7.2. Data analysis was performed using MassHunter workstation (Agilent) software and the results are shown in fig. 4.

(2) Inducible expression

10L of GIL77 yeast cells (pYES2) containing the HcOSC1, HcOSC5 and HcOSC6 genes, respectively, were cultured and each centrifuged to give 48.25g of cell biomass.

Yeast cells were mixed with 0.5L of saponification reagent (50% v/v) ethanol, 20% KOHw/v) and incubated at 70 ℃ for 2 hours, followed by extraction with an equal volume of hexane three times; the hexane extract was applied to a silica gel column (zcx II, particle size 200-; Qingdao, Haiyang, China) having a length of 30cm and a diameter of 2.4cm, and purified by using hexane: ethyl acetate (15: 1, v/v); fractions were collected in 10ml tubes and analyzed by Thin Layer Chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS).

The fraction containing the triterpene material was dried on a rotary evaporator and the detectable triterpene compound was further purified by reverse phase High Performance Liquid Chromatography (HPLC) on an Agilent 1260 series liquid chromatograph equipped with a semi-preparative column C18(eclipse XDB-C18, 5lm, 9.4mm 9250 mm, Santa Clara, Calif., USA). Flowing at a flow rate of 2.5ml/min for 35 minutes at 40 ℃ using a gradient of 95% to 100% methanol; the semi-preparative fractions were collected manually based on UV absorbance at 210nm and monitored by GC-MS using the method described above; concentrating the target compound to dryness at low temperature; cucurbitadienol (55mg) was purified only from 10L of GIL77 yeast cultures expressing HcOSC 6.

The purified compounds were identified by Nuclear Magnetic Resonance (NMR) analysis (including 1H, 13C, HMBC and HSQC) under the following conditions:

NMR spectra were recorded in fourier transform mode at nominal frequencies of 800MHz for 1H and 800C NMR or 600MHz for 1HNMR and 600MHz for 13C NMR using the indicated deuterated solvents, with Nuclear Magnetic Resonance (NMR) including 1H, 13C, HMBC and HSQC. Chemical shifts are reported in parts per million (ppm) and are referenced to residual solvent peaks. Multiplicity is described as: s, singlet; d, double peak; dd, a doublt of a doublt; dt, doublet of triplet; t, triplet; q, quartet; m, multiple; br, broad; appt, appent; the coupling constants are associated with transported in hertz.

The results of the nmr spectroscopy are shown in table 2 and fig. 5.

TABLE 2¹³C&¹Hassignments for cucurbitadienol (HcOSC6 Product)

A schematic diagram of the synthesis of cucurbitadienol by catalyzing cyclization of 2,3-oxidosqualene with cucurbitacin triterpene synthase HcOSC6 is shown in FIG. 6.

Sequence listing

<110> Yunnan university of agriculture

<120> Hemsleya amabilis triterpene synthetase HcOSC6 gene, engineering bacterium thereof and application thereof in preparation of cucurbitadienol

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Arg Leu Leu Lys Thr Val Asn Asn His Leu Gly Arg Gln Val Trp Glu

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Phe Ser Asn Glu Ser Asp Ser Asp Ser Asp Ser Asp Ser His His Phe

35 40 45

Gln Ile Asp Glu Ala Arg Asn Thr Phe Tyr His Asn Arg Phe His Gln

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Lys Gln Ser Ser Asp Leu Phe Ile Arg Ile Gln Tyr Gly Lys Glu Ile

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Tyr Ser Ala Ile Gln Thr Ser Asp Gly Asn Trp Ala Ser Asp Leu Gly

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Gly Pro Met Phe Leu Leu Pro Gly Leu Ile Ile Ser Leu Tyr Val Thr

130 135 140

Gly Val Leu Asn Ser Val Leu Ser Lys Gln His Arg Gln Glu Ile Cys

145 150 155 160

Arg Tyr Ile Tyr Asn His Gln Asn Glu Asp Gly Gly Trp Gly Leu His

165 170 175

Ile Glu Gly Pro Ser Thr Met Phe Cys Ser Val Leu Asn Tyr Val Ala

180 185 190

Leu Arg Leu Leu Arg Lys Asp Pro Tyr Glu Asp Ser Met Pro Lys Ala

195 200 205

Arg Leu Trp Ile Leu Asp His Gly Gly Ala Thr Ala Ile Thr Ser Trp

210 215 220

Gly Lys Leu Trp Leu Ser Val Leu Gly Val Tyr Glu Trp Ser Gly Asn

225 230 235 240

Asn Pro Leu Pro Pro Glu Phe Trp Ile Leu Pro Tyr Phe Leu Pro Phe

245 250 255

His Pro Gly Arg Met Trp Cys His Cys Arg Met Val Tyr Leu Pro Met

260 265 270

Ser Tyr Leu Tyr Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Thr Val

275 280 285

Leu Ser Leu Arg Lys Glu Leu Tyr Thr Val Pro Tyr His Glu Ile Asp

290 295 300

Trp Asn Lys Ser Arg Asn Thr Cys Ala Lys Glu Asp Leu Tyr Tyr Pro

305 310 315 320

His Pro Lys Met Gln Asp Ile Leu Trp Gly Ser Ile His His Val Tyr

325 330 335

Glu Pro Phe Phe Thr Arg Trp Pro Gly Lys Arg Leu Arg Glu Lys Ala

340 345 350

Leu Asp Ala Ala Met Gln His Ile His Tyr Glu Asp Glu Asn Thr Arg

355 360 365

Tyr Ile Cys Leu Gly Pro Val Asn Lys Val Leu Asn Met Leu Cys Cys

370 375 380

Trp Val Glu Asp Pro His Ser Glu Ala Phe Lys Leu His Leu Glu Arg

385 390 395 400

Val His Asp Tyr Leu Trp Val Ala Glu Asp Gly Met Lys Met Gln Gly

405 410 415

Tyr Asn Gly Ser Gln Leu Trp Asp Thr Ala Phe Ser Val Gln Ala Ile

420 425 430

Ile Ser Thr Lys Leu Thr Asp Asn Phe Gly Pro Thr Leu Arg Lys Ala

435 440 445

His Asp Phe Ile Lys Asn Ser Gln Ile Arg Gln Asp Cys Pro Gly Asp

450 455 460

Pro Asn Ile Trp Tyr Arg His Ile His Lys Gly Ala Trp Pro Phe Ser

465 470 475 480

Thr Ala Asp His Gly Trp Leu Ile Ser Asp Cys Thr Ala Glu Gly Leu

485 490 495

Lys Ala Ala Leu Leu Leu Ser Lys Leu Ser Ser Glu Thr Val Gly Glu

500 505 510

Pro Leu Glu Arg Asn Arg Leu Tyr Asp Ala Val Asn Val Leu Leu Ser

515 520 525

Leu Gln Asn Glu Asn Gly Gly Ile Ala Ser Tyr Glu Leu Thr Arg Ser

530 535 540

Tyr Pro Trp Leu Glu Leu Ile Asn Pro Ala Glu Thr Phe Gly Asp Ile

545 550 555 560

Val Ile Asp Tyr Pro Tyr Val Glu Cys Thr Ser Ala Ser Ile Glu Ala

565 570 575

Leu Ala Leu Phe Lys Lys Leu His Pro Gly His Arg Thr Lys Glu Ile

580 585 590

Glu Asn Ala Val Ala Lys Ala Ala Lys Phe Leu Glu Asp Met Gln Arg

595 600 605

Glu Asp Gly Ser Trp Tyr Gly Cys Trp Gly Val Cys Phe Thr Tyr Ala

610 615 620

Gly Trp Phe Gly Ile Lys Gly Leu Val Ala Ala Gly Arg Lys Tyr Asn

625 630 635 640

Asn Cys Pro Thr Ile Arg Lys Ala Cys Asn Phe Leu Leu Ser Lys Glu

645 650 655

Leu Pro Gly Gly Gly Trp Gly Glu Ser Tyr Leu Ser Cys Gln Asn Lys

660 665 670

Val Tyr Thr Asn Leu Glu Gly Asn Arg Pro His Leu Val Asn Thr Ala

675 680 685

Trp Ala Leu Met Ala Leu Ile Glu Ala Gly Gln Tyr Glu Lys Asp Pro

690 695 700

Thr Pro Leu His Arg Ala Ala Arg Leu Leu Ile Asn Ser Gln Leu Glu

705 710 715 720

Asn Gly Asp Phe Pro Gln Glu Glu Ile Met Gly Val Phe Asn Lys Asn

725 730 735

Cys Met Ile Thr Tyr Ala Ala Tyr Arg Asn Ile Phe Pro Ile Trp Ala

740 745 750

Leu Gly Glu Tyr Cys His His Val Leu Asn Glu Gln His Pro Ala Ala

755 760 765

Leu Phe

770

<210>3

<211>25

<212>DNA

<213> artificially synthesized sequence (Unknown)

<400>3

atgtggaagt taaagatagg aggag 25

<210>4

<211>21

<212>DNA

<213> artificially synthesized sequence (Unknown)

<400>4

tcagaataaa gcggccggat g 21

<210>5

<211>45

<212>DNA

<213> artificially synthesized sequence (Unknown)

<400>5

ttggtaccga gctcggatcc atgtggaagt taaagatagg aggag 45

<210>6

<211>41

<212>DNA

<213> artificially synthesized sequence (Unknown)

<400>6

cactggcggc cgttactagt tcagaataaa gcggccggat g 41

Claims (10)

1. The hemsleya amabilis triterpene synthase HcOSC6 gene is characterized by having a nucleotide sequence shown as SEQ ID No. 1.

2. A recombinant vector containing the cucurbit triterpene synthase HcOSC6 gene according to claim 1.

3. Hemsleya amabilis triterpene synthase HcOSC6 is characterized by having an amino acid sequence shown as SEQ ID No. 2.

4. A genetically engineered bacterium expressing the cucurbitacin triterpene synthase HcOSC6 according to claim 3.

5. The genetically engineered bacterium expressing cucurbitacin triterpene synthase HcOSC6 according to claim 4, comprising the recombinant vector containing the cucurbitacin triterpene synthase HcOSC6 gene according to claim 2.

6. The genetically engineered bacterium expressing cucurbitane triterpene synthase HcOSC6 according to claim 4, wherein the gene of cucurbitane triterpene synthase HcOSC6 according to claim 1 is integrated into the genome.

7. The genetically engineered bacterium of any one of claims 4 to 6 expressing cucurbitacin triterpene synthase HcOSC6, wherein the original bacterium is Saccharomyces cerevisiae.

8. Use of the cucurbitacin triterpene synthase HcOSC6 gene according to claim 1 for preparing cucurbitadienol.

9. Use of the genetically engineered bacterium expressing cucurbitacin triterpene synthase HcOSC6 according to any one of claims 4 to 6 for the preparation of cucurbitadienol.

10. The use of the genetically engineered bacterium expressing cucurbitadienol according to claim 9, which comprises:

(1) inoculating the transgenic engineering bacteria for expressing the hemsleya amabilis triterpene synthetase HcOSC6 into a culture medium taking D-glucose and D-galactose as carbon sources, and performing fermentation culture;

(2) centrifuging the culture, taking engineering bacteria cells, and performing wall breaking treatment to obtain intracellular product mixed liquor;

(3) separating and purifying the mixed solution of the intracellular products to obtain the cucurbitadienol.

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