CN117866933A - Oxidation squalene cyclase NiOSC5, encoding gene and application thereof - Google Patents

Abstract

The invention discloses an oxidation squalene cyclase NiOSC5 and its coding productβ‑The application of amyrin alcohol belongs to the technical fields of synthetic biology and natural medicines. The invention is prepared from cucurbitaceae plant cucurbita pepoNeoalsomitra integrifoliola) Starting, cloning and functionally identifying a synthesisβ‑The triterpene synthase NiOSC5 coding gene of the amyrin alcohol has a nucleotide sequence shown as the sequence ID No.2, is connected with an expression vector pYES2 after gene cloning, is constructed into a recombinant plasmid capable of being expressed in escherichia coli, and is transformed into saccharomyces cerevisiae to be constructed into engineering cells, thus realizing the heterologous efficient synthesis of the compound by the saccharomyces cerevisiaeβ‑The gene engineering cell constructed by the invention is safe and stable, has short production period and shows great value in application and development. The invention providesNiOSC5Product(s)β‑The amyrin can be used for preparing medicines for protecting cardiovascular and cerebrovascular, enhancing immunity, reducing blood sugar, relieving inflammation, resisting oxidation, relieving fatigue, resisting AIDS, and resisting cancer.

Description

Oxidation squalene cyclase NiOSC5, encoding gene and application thereof

Technical Field

The invention belongs to the technical fields of synthetic biology and natural medicines. In particular to an oxidation squalene cyclase NiOSC5 and a coded product beta-amyrin thereof, and application of the product beta-amyrin and the product beta-amyrin in preparing medicaments for protecting cardiovascular and cerebrovascular diseases, improving immunity, reducing blood sugar, resisting inflammation, resisting oxidation, resisting fatigue, resisting AIDS and resisting cancer.

Background

Pentacyclic triterpene is an important plant secondary metabolite, is formed by connecting 6 isoprene units, and is classified into various types according to aglycone differences. Pentacyclic triterpene not only participates in communication, defense and sensory regulation of plants, but also has wide pharmacological actions and important biological activities. Research shows that pentacyclic triterpene plays an important role in protecting cardiovascular and cerebrovascular, improving immunity, reducing blood sugar, resisting inflammation, resisting oxidation, resisting fatigue, resisting AIDS, resisting cancer, etc. However, pentacyclic triterpene compounds have the disadvantages of lack of natural resources, low content in plant bodies, long growth cycle, complex structure, difficulty in large-scale extraction from natural plant bodies and serious limitation of development and application of pentacyclic triterpene. Therefore, a new way for large-scale production of pentacyclic triterpene needs to be developed.

Disclosure of Invention

The invention provides an oxidation squalene cyclase gene NiOSC5 and a coding product thereof, wherein the gene is a key enzyme gene participating in the synthesis of triterpenoid sapogenin of the mallow melon, can be used as a biosynthesis regulatory gene of beta-amyrin alcohol and is applied to the preparation of the beta-amyrin alcohol. Thus providing a novel method for biosynthesis of beta-amyrin.

In order to achieve the above object of the present invention, the present invention adopts the following technical scheme:

an oxidosqualene cyclase nioc 5, which is:

(1) A protein comprising the amino acid sequence shown in Seq ID No. 1;

(2) The amino acid sequence shown in the Seq ID No.1 is a derivative protein with the same function by substituting and/or deleting and/or adding one or more amino acid residues.

The coding gene of the oxidation squalene cyclase NiOSC5 is as follows:

(a) A nucleotide sequence shown as Seq ID No.2;

(b) The nucleotide sequence shown in Seq ID No.2 is a nucleotide sequence which is substituted and/or deleted and/or added with one or several nucleotides and expresses the same functional protein.

A recombinant vector containing the gene encoding the oxidation squalene cyclase NiOSC 5.

Recombinant bacteria containing the gene encoding the oxidation squalene cyclase NiOSC 5.

The oxidation squalene cyclase NiOSC5 or the coding gene thereof is applied to the preparation of recombinant vectors, expression cassettes, transgenic cell lines and recombinant bacteria containing beta-amyrin.

The application of the oxidation squalene cyclase NiOSC5 or the coding gene thereof in preparing fermentation liquor containing the compound beta-amyrin is characterized in that the application adopts: constructing an expression vector containing the coding gene, converting the recombinant vector into saccharomyces cerevisiae cells, and fermenting and culturing the obtained genetically engineered saccharomycetes to obtain fermentation liquor containing beta-amyrin alcohol.

The application of the oxidation squalene cyclase NiOSC5 or the coding gene thereof in the synthesis or preparation of the compound beta-amyrin.

The application of the oxidation squalene cyclase NiOSC5 or the coding gene thereof in preparing medicaments for protecting cardiovascular and cerebrovascular diseases, improving immunity, reducing blood sugar, resisting inflammation, resisting oxidization, resisting fatigue, resisting AIDS and resisting cancer.

A method for preparing a compound beta-amyrin, comprising the steps of:

constructing an expression vector containing a coding gene for coding an oxidation squalene cyclase NiOSC5, converting the recombinant vector into saccharomyces cerevisiae, fermenting and culturing the obtained genetically engineered saccharomycetes to obtain a fermentation liquor containing beta-amyrin alcohol, extracting the fermentation liquor with petroleum ether, ethyl acetate, dichloromethane or chloroform to obtain an extract containing beta-amyrin alcohol, separating and purifying the extract by a silica gel column chromatography method to finally obtain the compound beta-amyrin alcohol shown in the following structural formula,

the application of the compound beta-amyrin in preparing medicaments for protecting cardiovascular and cerebrovascular, improving immunity, reducing blood sugar, resisting inflammation, resisting oxidation, resisting fatigue, resisting AIDS and resisting cancer.

The Open Reading Frame (ORF) of the gene encoding the oxidation squalene cyclase gene NiOSC5 provided by the invention is 2295bp (Seq ID No. 2), 764 amino acids (Seq ID No. 1) are encoded, and the gene is placed in NCBI for BLASTN analysis and comparison, so that the homology with SgBAS1 of Momordica grosvenori (Siraitia grosvenorii) of Cucurbitaceae is 76%.

The coding gene of the oxidation squalene cyclase NiOSC5 provided by the invention is a gene cloned from the cucumis metuliferus (Neoalsomitra integrifoliola), and can cyclize 2, 3-oxidation squalene to form a compound beta-amyrin. The NiOSC5 is cloned from the plant for the first time, and the discovery of the oxidation squalene cyclase NiOSC5 and the coding gene thereof enriches the diversity of triterpene synthase.

The invention clones and functionally identifies the oxidation squalene cyclase NiOSC5 from cucurbitaceae plant mallow, and uses saccharomyces cerevisiae to generate beta-amyrin.

The invention starts from cucurbitaceae plant clavicle cucurbit (Neoalsomitra integrifoliola), clones and functionally identifies a coding gene of oxidation squalene cyclase NiOSC5 for synthesizing beta-amyrin alcohol, the nucleotide sequence is shown as Seq ID No.2, the coding gene is connected with an expression vector pYES2 after gene cloning, the recombinant plasmid is constructed to be a recombinant plasmid capable of being expressed in escherichia coli, and then the recombinant plasmid is transformed into saccharomyces cerevisiae to be constructed as engineering cells, thus realizing the heterologous efficient synthesis of compound beta-amyrin alcohol by saccharomyces cerevisiae. The NiOSC5 product beta-amyrin provided by the invention is applied to the preparation of medicaments for protecting cardiovascular and cerebrovascular diseases, improving the immunity, reducing the blood sugar, resisting inflammation, resisting oxidization, resisting fatigue, resisting AIDS and resisting cancer.

Drawings

FIG. 1 is a three-dimensional structure prediction diagram of the oxidosqualene cyclase NiOSC5 in example 1.

FIG. 2 shows the analysis of the expression level of the oxidosqualene cyclase NiOSC5 in roots, stems, leaves and flowers in example 2.

FIG. 3 is a chromatogram of GC-MS analysis of an engineered yeast extract expressing the oxidosqualene cyclase NiOSC5 in example 2.

FIG. 4 is a mass spectrum of beta-amyrin, a catalytic product of the oxidation squalene cyclase NiOSC5 in example 2.

FIG. 5 is a chromatogram of the β -amyrin standard of example 2.

FIG. 6 is a standard quality spectrum of beta-amyrin in example 2.

FIG. 7 is a schematic diagram showing the construction of recombinant expression plasmid pYES2-NiOSC5 in example 2.

Detailed description of the preferred embodiments

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise indicated, the examples were conducted under conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: albometer Manual, 2001), or under conditions recommended by the manufacturer's instructions.

Example 1

Cloning of the Oxidation squalene cyclase Gene NiOSC5 in Calophyllum Instrongylus.

1. According to the sequenced data of the gloriope clavatus transcriptome, the candidate oxidation squalene cyclase gene cDNA sequence in the gloriope clavatus saponin synthesis pathway is obtained through operations such as splicing, annotation, screening and the like.

2. Designing a primer of the candidate oxidation squalene cyclase gene, wherein the primer sequence is as follows:

forward primer (NiOSC 5-F):

gggaatattaagcttggtaccATGTGGCGGCTTAAGATTGCAG，ID NO.3；

reverse primer (NiOSC 5-R):

ccctctagatgcatgctcgagTTAAAATACAGAGGAAGTAGTTGGCAATTTGAC，ID NO.4；

primers were synthesized by Kunming division, inc. of Biotech, beijing.

3. Individual tissues (root, stem, leaf and flower) of the plant of the Hamamelis mollis were taken, total RNA was extracted using TRIzol kit (Invitrogen, carlsbad, calif., USA) using PrimeScript ^TM Reverse transcription was performed using RT kit (Takara, china) to obtain cDNA, and the gene sequence of NiOSC5 was amplified using the cDNA as a template.

4. The amplified product agarose gel electrophoresis shows a specific band at about 2.3kb, the target band is subjected to gel cutting recovery, the gel recovery product is connected to a vector pYES2, and E.coli DH5 alpha is transformed, positive clones are selected for sequencing (Kunming division of Beijing qing biological science and technology Co., ltd.), and NiOSC5 gene clones with correct sequences are selected and saved for the construction of subsequent expression vectors.

Sequencing to obtain the triterpenoid saponin anabolism pathway oxidation squalene cyclase gene NiOSC5 with the length of 2295bp and nucleotide sequence as ID No.2; encodes 764 amino acids, and the amino acid sequence is ID NO.1.

By multiple sequence alignment and phylogenetic tree analysis of NiOSC5 with the identified OSCs, it was shown that NiOSC5 has the highly conserved domains QW and DCTAE of the OSCs gene family.

Example 2

Eukaryotic expression and functional analysis of NiOSC5 gene.

NiOSC5 functional preliminary analysis.

Extracting RNA of root, stem, leaf and flower of Coptis chinensis respectively, referring to PrimeScript ^TM RT kit (Takara, china) was reverse transcribed into cDNA and real-time fluorescent quantitative PCR amplification was performed on a Applied Biosystems QuantStudioTM5 platform (Life Technologies) using 2X ChamQUniversal SYBR qPCR Master Mix (Vazyme).

Forward primer (NiOSC 5-qRT-F): GCTGGTTTGCTATTGGTGGT, ID NO.5;

reverse primer (NiOSC 5-qRT-R): ACCGGTTTCCTTCAAGAGGT, ID NO.6.

From the results of the real-time fluorescent quantitative PCR analysis (FIG. 2), it was estimated that NiOSC5 was expressed in the highest amount in leaves and flowers, and that NiOSC5 was probably involved in biosynthesis of clavulanic saponin in the leaves.

2. Construction of a Yeast expression vector.

ERG 7-deficient Saccharomyces cerevisiae mutant GIL77 lacking lanosterol synthase gene can endogenously accumulate 2, 3-oxidized squalene, and 2, 3-oxidized squalene can be used as a substrate for functional verification of NiOSC 5.

By analyzing the coding sequence and cleavage site of the gene NiOSC5, primers with Kpn I and XhoI cleavage sites were designed as follows, and full-length ORF amplification of NiOSC5 was performed.

After sequencing and verifying the amplification product, connecting a target gene NiOSC5 to a yeast expression vector pYES2 by a homologous recombination method, screening a transformed colony by using an LB solid plate containing ampicillin (100 mu g/mL), selecting a monoclonal for verification, sequencing and verifying correctness to obtain a pYES2-NiOSC5 vector, inoculating the verified correct monoclonal into 5mL of LB liquid culture with the same resistance, fermenting and culturing, and extracting pYES2-NiOSC5 plasmid.

3. Yeast transformation.

The pYES2-NiOSC5 plasmid was transferred into Saccharomyces cerevisiae strain GIL77 by lithium acetate transformation, and an empty pYES2 transformation control group was set. Positive clones GIL77-pYES2-NiOSC5 and GIL77-pYES2 were selected by colony PCR.

4. Induction of expression and incubation.

Positive yeast monoclonal GIL77-pYES2-NiOSC5 and GIL77-pYES2 were picked separately and inoculated in 50ml of uracil-free synthetic complete medium [ SC-U; comprises ergosterol (20. Mu.g/ml), tween 80 (5 mg/ml) and hemin (13. Mu.g/ml), and is then cultured at 30℃for 2 days with shaking at 200 rpm.

Yeast cells were harvested, resuspended in 50mL of SC-U medium containing 2% galactose and induced by shaking at 200rpm for 2 days at 30 ℃.

After induction for 2 days, the cells were harvested and resuspended in the same volume of 0.1M potassium phosphate buffer (pH 7.0; 2% glucose and hemin (13. Mu.g/ml) were added and incubated at 30℃for 12 hours with shaking at 200 rpm.

5. And (5) extracting and identifying a catalytic product.

After 12 hours of incubation, the cells were refluxed with the same volume of saponification reagent (20% KOH/50% EtOH) for 10 minutes at 92℃and then extracted twice with the same volume of petroleum ether, the extracts were combined, concentrated to dryness under reduced pressure and the residue was derivatised with 200. Mu.l of cyano trimethylsilane at 65℃for 30 minutes.

And (3) carrying out GC-MS analysis on the derived product, wherein a catalytic group containing the NiOSC5 gene recombinant expression vector pYES2-NiOSC5 shows a specific peak compared with a control group containing no-load pYES2, namely new substances are generated, and the specific product is identified to be beta-amyrin.

The experiment proves that the NiOSC5 gene participates in the biosynthesis of triterpenoid saponins of the mallow melon, and the NiOSC5 gene can be used for regulating the biosynthesis of beta-amyrin in the mallow melon, so that the heterologous synthesis of beta-amyrin is realized.

Example 3

And (3) preparing the compound beta-amyrin.

RNA of Hakka Swinhonis was extracted, and reference was made to PrimeScript ^TM RT kit (Takara, china) is reverse transcribed into cDNA, the cDNA is used as template to amplify the gene sequence of NiOSC5, the amplified product agarose gel electrophoresis shows specific band at about 2.3kb, the target band is cut and recovered, the recovered product is connected to vector pYES2, and E.coli DH5 alpha is transformed, positive clone is selected for sequencing (Kunming division Co., ltd. Of Beijing family biotechnology), the NiOSC5 gene with correct sequencing is selected, the target gene NiOSC5 is connected to yeast expression vector pYES2 by homologous recombination method, pYES2-NiOSC5 vector is obtained, and sequencing verifies correctness. Extracting plasmid pYES2-NiOSC5, introducing plasmid pYES2-NiOSC5 into Saccharomyces cerevisiae GIL77 strain by lithium acetate method, selecting positive clones GIL77-pYES2-NiOSC5 and GIL77-pYES2 by colony PCR method, inoculating positive yeast monoclonal GIL77-pYES2-NiOSC5Uracil-free synthetic complete medium [ SC-U ] in 50 ml; comprises ergosterol (20 μg/ml), tween 80 (5 mg/ml) and hemin (13 μg/ml)]Then incubated at 30℃for 2 days. 50ml of the bacterial liquid is inoculated to 10L of synthetic complete medium [ SC-U ] without uracil; comprises ergosterol (20 μg/ml), tween 80 (5 mg/ml) and hemin (13 μg/ml)]In the following, yeast cells were harvested by shaking culture at 200rpm for 2 days at 30℃and resuspended in 10L of SC-U medium containing 2% galactose and protein expression was induced by shaking culture at 200rpm for 2 days at 30 ℃. After induction for 2 days, the yeast cells were harvested and resuspended in the same volume of 0.1M potassium phosphate buffer (pH 7.0; 2% glucose and hemin (13. Mu.g/ml) were added and incubated at 30℃for 12 hours with shaking at 200rpm, after 12 hours of incubation the cells were refluxed with the same volume of saponification reagent (20% KOH/50% EtOH) at 92℃for 10 minutes, then extracted 3 times with the same volume of petroleum ether, the extracts were combined, the extracts were concentrated to dryness under reduced pressure to give an extract containing beta-amyrin alcohol, the weighed amount was measured, the column chromatography was performed with 1.5 times the amount of silica gel, 15 times the amount of silica gel, 3 column volumes were eluted with petroleum ether, and then with a gradient of petroleum ether: ethyl acetate (12:1, v/v-8:1, v/v), each column volume was eluted with 6-7 times, fractions were collected, fractions containing beta-amyrin alcohol were collected, concentrated to dryness under reduced pressure, and their structure was examined as beta-amyrin alcohol by nuclear magnetic resonance.

Pharmaceutical formulation examples 1-8:

in the following preparation examples, conventional reagents are selected and preparation is carried out according to the conventional method, and the application examples only show that the compound beta-amyrin can be prepared into different preparations, and specific reagents and operations are not particularly limited:

1. dissolving compound beta-amyrin with absolute ethanol, adding water for injection according to a conventional method, finely filtering, packaging and sterilizing to prepare injection, wherein the concentration of the injection is 0.5-5mg/mL.

2. Dissolving beta-amyrin with dimethyl sulfoxide, dissolving in sterile water for injection, stirring to dissolve, filtering with sterile suction filter funnel, sterile fine filtering, packaging in ampoule, freeze drying at low temperature, and sealing under sterile condition to obtain powder for injection.

3. The compound beta-amyrin alcohol is added with excipient according to the mass ratio of the compound beta-amyrin alcohol to the excipient of 9:1 to prepare powder.

4. Adding excipient into beta-amyrin compound according to the mass ratio of the beta-amyrin to the excipient of 5:1, granulating and tabletting.

5. The compound beta-amyrin is prepared into oral liquid according to the conventional oral liquid preparation method.

6. Adding excipient into beta-amyrin according to the mass ratio of the beta-amyrin to the excipient of 5:1, and making into capsules.

7. Adding excipient into beta-amyrin according to the mass ratio of the beta-amyrin to the excipient of 5:1, and preparing into granules.

8. The capsule comprises the following components: 20mg of compound beta-amyrin, 180mg of lactose and 5mg of magnesium stearate.

The preparation method comprises the following steps: the compound was mixed with a cosolvent uniformly, sieved, and the resulting mixture was filled into gelatin capsules each weighing 205mg and having an active ingredient content of 20mg.

The generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. An oxidation squalene cyclase nioc 5, characterized in that it is:

(1) A protein comprising the amino acid sequence shown in Seq ID No. 1;

(2) The amino acid sequence shown in the Seq ID No.1 is a derivative protein with the same function by substituting and/or deleting and/or adding one or more amino acid residues.

2. The gene encoding the oxidosqualene cyclase nioc 5 according to claim 1, characterized in that it is:

(a) A nucleotide sequence shown as Seq ID No.2;

(b) The nucleotide sequence shown in Seq ID No.2 is a nucleotide sequence which is substituted and/or deleted and/or added with one or several nucleotides and expresses the same functional protein.

3. A recombinant vector comprising a gene encoding the oxidosqualene cyclase nioc 5 according to claim 2.

4. A recombinant bacterium comprising a gene encoding the oxidosqualene cyclase nioc 5 of claim 2.

5. Use of an oxidosqualene cyclase nioc 5 or a gene encoding it according to claim 1 or 2 for the preparation of recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria containing beta-amyrin.

6. Use of the oxidation squalene cyclase nioc 5 or a gene encoding it according to claim 1 or 2 for the preparation of a fermentation broth comprising the compound β -amyrin, characterized in that the use is made of: constructing an expression vector containing the coding gene, converting the recombinant vector into saccharomyces cerevisiae cells, and fermenting and culturing the obtained genetically engineered saccharomycetes to obtain fermentation liquor containing beta-amyrin alcohol.

7. Use of an oxidosqualene cyclase nioc 5 according to claim 1 or 2 or a gene encoding it for the synthesis or preparation of the compound β -amyrin.

8. Use of an oxidative squalene cyclase nioc 5 or a gene encoding it according to claim 1 or 2 for the preparation of a medicament for protecting cardiovascular and cerebrovascular vessels, improving immunocompetence, lowering blood sugar, anti-inflammatory, antioxidant, anti-fatigue, anti-aids, anti-cancer.

9. The preparation method of the compound beta-amyrin is characterized by comprising the following steps:

constructing an expression vector containing a coding gene for coding an oxidation squalene cyclase NiOSC5, converting the recombinant vector into saccharomyces cerevisiae, fermenting and culturing the obtained genetically engineered saccharomycetes to obtain a fermentation liquor containing beta-amyrin alcohol, extracting the fermentation liquor with petroleum ether, ethyl acetate, dichloromethane or chloroform to obtain an extract containing beta-amyrin alcohol, separating and purifying the extract by a silica gel column chromatography method to finally obtain the compound beta-amyrin alcohol shown in the following structural formula,

10. the use of beta-amyrin as claimed in claim 9 for preparing medicines for protecting cardiovascular and cerebrovascular, improving immunity, reducing blood sugar, anti-inflammatory, antioxidant, antifatigue, anti-AIDS and anticancer.