Oxidosqualene cyclase, recombinant vector, recombinant engineering bacteria and application thereof, and parkol and preparation method thereof

Abstract

The present application relates to oxidized squalene cyclase, recombinant vector, recombinant engineering bacteria and application thereof, and preparation method of parkol. The oxidized squalene cyclase comprises: a polypeptide consisting of the amino acid sequence shown as SEQ ID No. 1; or a polypeptide consisting of the amino acid sequence shown as SEQ ID No. 1 with one or more amino acids being deleted, substituted or added; or a polypeptide having at least 80% homology with the polypeptide consisting of the amino acid sequence shown as SEQ ID No. 1. The above-mentioned oxidized squalene cyclase can catalyze 2, 3 oxidized squalene to produce parkol, and the enzyme can be expressed according to the coding sequence of the cyclase, and then the substrate is catalyzed to improve the yield of parkol.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an oxidized squalene cyclase, a recombinant vector, a recombinant engineering bacteria, and an application thereof, and a parkerol and a preparation method thereof. BACKGROUND

[0002] Research shows that after the intake of different concentrations of parkerol, the contents of triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) in cells all show a downward trend, and the effect is more significant as the concentration of parkerol increases. When the concentration of parkerol increases to 20 μmol / L, the contents of TG, TC and LDL-c in cells decrease by 59.54%, 33.69% and 40.03% respectively compared with the model group, and the improvement effect on cell lipid accumulation is very significant. When the concentration of parkerol is lower than 20 μmol / L, there is no significant cytotoxicity, and within this concentration range, parkerol has a significant improvement effect on cell lipid accumulation and oxidative stress, and the improvement effect increases significantly as the concentration of parkerol increases. The intake of parkerol significantly changes the expression of genes related to lipid synthesis and metabolism such as FASN, ACSL1, CPT1A and PIK3CB in cells, and also changes the expression of genes related to oxidative stress such as NQO-1, PRXL2A, NF-kB2 and TXNRD1. These genes with expression differences in the model group are mainly involved in metabolic pathways such as amino acid metabolism, carbohydrate metabolism, energy metabolism, glycan synthesis and metabolism, lipid metabolism, coenzyme factor and vitamin metabolism, aging and cardiovascular disease. Moreover, parkerol is relatively expensive, and currently 5 mg of parkerol costs about 3200 yuan. Therefore, the preparation of parkerol has very high practical value and economic value. At present, parkerol is mainly extracted from plants and animals, and the extraction rate is low, which is not conducive to large-scale industrial production. How to improve the yield of parkerol is a difficult problem that needs to be solved urgently. SUMMARY

[0003] Therefore, it is necessary to provide an oxidized squalene cyclase capable of catalyzing substrates to produce parkerol and an application thereof. The enzyme can be expressed by using genetic engineering means according to the coding sequence of the cyclase, and then the substrate is catalyzed to improve the yield of parkerol.

[0004] In addition, it is also necessary to provide a recombinant vector, a recombinant engineering bacteria, a preparation method and an application thereof, a parkerol and a preparation method thereof.

[0005] An oxidized squalene cyclase, the oxidized squalene cyclase comprising:

[0006] a polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 1; or

[0007] A polypeptide consisting of one or more amino acids deleted, substituted, or added as shown in SEQ ID No. 1; or,

[0008] A polypeptide that has at least 80% homology with a polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 1.

[0009] This study found that the aforementioned squalene cyclase can catalyze the production of parkol from squalene 2,3-oxide. Based on the coding sequence of this cyclase, genetic engineering techniques can be used to express the enzyme, which can then catalyze substrates to increase parkol production. Experimental verification showed that a eukaryotic expression vector was constructed based on the coding sequence of the aforementioned squalene cyclase, and this eukaryotic expression vector was transformed into GIL77 yeast to induce expression of the cyclase. This cyclase can catalyze the production of parkol from the recombinant yeast substrate squalene 2,3-oxide.

[0010] In one embodiment, the coding sequence of the oxidized squalene cyclase includes:

[0011] The nucleotide sequence shown in SEQ ID No. 2;

[0012] Alternatively, a nucleotide sequence that has 80% homology with the nucleotide sequence shown in SEQ ID No. 2;

[0013] Alternatively, a nucleotide sequence obtained by deleting, substituting, or adding one or more bases in the nucleotide sequence shown in SEQ ID No. 2.

[0014] A recombinant vector carrying the coding sequence of the above-mentioned squalene cyclase.

[0015] In one embodiment, the recombinant vector is a eukaryotic cell expression vector carrying the coding sequence of the oxidized squalene cyclase.

[0016] In one embodiment, the recombinant vector is a pYES2.0 expression vector carrying the coding sequence of the oxidized squalene cyclase.

[0017] In one embodiment, the method includes the following steps: performing PCR amplification on the coding sequence of the oxidized squalene cyclase using amplification primer pairs with nucleotide sequences as shown in SEQ ID No. 3 and SEQ ID No. 4, and ligating it into an empty vector to obtain the recombinant vector.

[0018] A recombinant engineered bacterium, wherein the recombinant engineered bacterium is transformed with the above-mentioned recombinant vector.

[0019] In one embodiment, the host bacteria of the recombinant engineered bacteria contain squalene 2,3-oxide.

[0020] In one embodiment, the host strain of the recombinant engineered bacteria is a lanosterol-deficient yeast strain.

[0021] In one embodiment, the host strain of the recombinant engineered bacteria is the lanosterol-deficient yeast strain Gil77.

[0022] The application of the above-mentioned squalene cyclase, the above-mentioned recombinant vector, or the above-mentioned recombinant engineered bacteria in the preparation of Parkol.

[0023] A method for preparing pacol includes the following steps: expanding the culture of recombinant engineered bacteria to obtain pacol, wherein the recombinant engineered bacteria are transformed with the above-mentioned recombinant vector, and the recombinant engineered bacteria contain 2,3-squalene oxide.

[0024] In one embodiment, the recombinant engineered bacteria is a lanosterol-deficient yeast strain Gil77 transformed with the recombinant vector, and the step of scaling up the recombinant engineered bacteria to obtain pakol includes:

[0025] The recombinant engineered bacteria were cultured on a large scale, and the bacterial cells from the large-scale culture were separated into solid and liquid components and collected.

[0026] The expanded bacterial culture was subjected to induced culture to induce the expression of the lanosterol synthase gene in the expanded bacterial culture.

[0027] After induction, the induced bacterial cells were separated into solid and liquid phases and collected. Then, the induced bacterial cells were lysed to obtain the paclitaxel.

[0028] A parkol, prepared by the above-described method for preparing parkol. Attached Figure Description

[0029] Figure 1 The results are from the GC-MS detection in Example 1. Detailed Implementation

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples and accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] One embodiment of this study provides an oxidized squalene cyclase, which includes:

[0032] A polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 1; or

[0033] A polypeptide consisting of one or more amino acids deleted, substituted, or added as shown in SEQ ID No. 1; or,

[0034] A polypeptide having at least 80% homology with a polypeptide comprising the amino acid sequence shown in SEQ ID No. 1. This study found that the aforementioned squalene cyclase can catalyze the production of parkol from squalene 2,3-oxide. The enzyme can be expressed using genetic engineering based on its coding sequence, and then used to catalyze substrates to increase parkol production. Experimental verification showed that a eukaryotic expression vector was constructed based on the coding sequence of the aforementioned squalene cyclase, and this eukaryotic expression vector was transformed into GIL77 yeast to induce expression of the cyclase. This cyclase can catalyze the production of parkol from the recombinant yeast substrate squalene 2,3-oxide.

[0035] The amino acid sequence shown in SEQ ID No. 1 is derived from deep-sea sea cucumber.

[0036] Specifically, the sequence shown in SEQ ID No. 1 is as follows:

[0037] *

[0038] In some embodiments, the coding sequence for oxidative squalene cyclase includes:

[0039] The nucleotide sequence shown in SEQ ID No. 2;

[0040] Alternatively, a nucleotide sequence that has 80% homology with the nucleotide sequence shown in SEQ ID No. 2;

[0041] Alternatively, a nucleotide sequence obtained by deleting, substituting, or adding one or more bases in the nucleotide sequence shown in SEQ ID No. 2.

[0042] The nucleotide sequence shown in SEQ ID No. 2 is derived from deep-sea sea cucumber.

[0043]

[0044] Since the same amino acid can be determined by several different codons, the same amino acid can correspond to different nucleotide sequences, and different amino acids can correspond to the same nucleotide sequence. Therefore, the amino acid sequence of the oxidized squalene cyclase in this application is encoded by a nucleotide sequence obtained by substituting one or more nucleotides into the nucleotide sequence shown in SEQ ID No. 2, resulting in a codon-synonymous mutation. Those skilled in the art can obtain the oxidized squalene cyclase of this application based on the amino acid sequence of the oxidized squalene cyclase disclosed in this application, using existing molecular biology techniques, cDNA cloning and site-directed mutagenesis, or other suitable methods. Therefore, the encoding of the aforementioned oxidized squalene cyclase is not limited to the nucleotide sequence shown in SEQ ID No. 2. If the encoded protein does not have a significant functional difference from the oxidized squalene cyclase, it is also included within the scope of this invention.

[0045] Furthermore, due to the polymorphism and variation of protein-coding sequences, naturally occurring proteins can undergo gene mutations, resulting in the deletion, substitution, or addition of bases in the coding sequence, or the deletion, insertion, substitution, or other variations of amino acids, leading to the deletion, substitution, or addition of one or more amino acids in the protein's amino acid sequence. Therefore, some proteins exist that are physiologically and biologically equivalent to the unmutated proteins. These polypeptides or proteins, whose structures differ from the corresponding proteins but whose functions are not significantly different, are called functionally equivalent variants.

[0046] Functionally equivalent variants also apply to polypeptides created by artificially altering one or more codons through deletion, insertion, and mutation, thereby introducing such variations into the amino acid sequence of a protein. Although this can yield more diverse variants, the prerequisite for a variant to be considered a functionally equivalent variant is that its physiological activity is essentially equivalent to that of the original, unmutated protein.

[0047] Generally, the coding sequences of functionally equivalent variants are homologous. Therefore, a polypeptide or protein obtained by at least one alteration (such as the deletion, insertion, or substitution of one or more bases in the coding sequence of a protein or the deletion, insertion, or substitution of one or more amino acids in the amino acid sequence of a protein) generally has the same activity as the protein. Therefore, a polypeptide encoded by the above nucleotide sequence or a polypeptide composed of the above amino acid sequence is also included in the scope of this application if the protein encoded by it does not have a significant functional difference from oxidized squalene cyclase.

[0048] This study discloses the coding sequence of the aforementioned squalene cyclase, which can be expressed using genetic engineering techniques based on this coding sequence, and then catalyzed to increase the yield of parkol. Experimental verification showed that a eukaryotic expression vector was constructed based on the coding sequence of the aforementioned squalene cyclase, and this eukaryotic expression vector was transformed into GIL77 yeast to induce the expression of the cyclase. This cyclase can catalyze the production of parkol from the recombinant yeast substrate 2,3-oxidized squalene.

[0049] One embodiment of this study also provides a recombinant vector carrying the coding sequence of the above-mentioned oxidized squalene cyclase.

[0050] The recombinant vector is either a recombinant cloning vector or a recombinant expression vector.

[0051] Furthermore, the recombinant vector is a eukaryotic expression vector carrying the coding sequence of the aforementioned oxidized squalene cyclase. Expressing the aforementioned oxidized squalene cyclase using a eukaryotic expression vector is beneficial for improving the enzyme's activity.

[0052] Specifically, the recombinant vector is the pYES2.0 plasmid vector carrying the coding sequence of the aforementioned oxidized squalene cyclase. Using the pYES2.0 plasmid vector facilitates the efficient expression of the aforementioned oxidized squalene cyclase gene.

[0053] This study provides a recombinant vector carrying the coding sequence of the aforementioned oxidized squalene cyclase, which facilitates the cloning or expression of this enzyme through genetic engineering, and then catalyzes substrates to increase the yield of parkol. The recombinant vector presented in this study can be used for the preparation of parkol.

[0054] One embodiment of this study also provides a method for preparing the above-mentioned recombinant vector, comprising the following steps: performing a PCR reaction on the coding sequence of the oxidized squalene cyclase and the empty vector using amplification primers with nucleotide sequences as shown in SEQ ID No. 3 and SEQ ID No. 4, so as to ligate the coding sequence of the oxidized squalene cyclase into the empty vector to obtain the recombinant vector.

[0055] The empty vector can be a cloning vector or an expression vector. More specifically, the empty vector can be a eukaryotic expression vector. In a specific example, the empty vector is the pYES2.0 plasmid vector.

[0056] Specifically, the sequence of SEQ ID No. 3 is GG GGTACC TACACAATGTCCTCCATTA; the sequence GC is as shown in SEQ ID No. 4. TCTAGATTAGGCTTGGGAAGTCAAA. Here, the underlined portion GGTACC represents the KpnI restriction site, and TCTAGA represents the XbaI restriction site. The oxidized squalene cyclase gene and the PYES2.0 expression vector were digested with KpnI and XbaI. The oxidized squalene cyclase gene was ligated into the pYES2.0 vector using T4 ligase to obtain the recombinant plasmid.

[0057] One embodiment of this study provides a recombinant engineered bacterium, which is transformed with the above-mentioned recombinant vector.

[0058] In some embodiments, the host bacteria of the recombinant engineered bacteria contain 2,3-squalene oxide. Expression of squalene cyclase in the recombinant engineered bacteria can directly catalyze the expression of 2,3-squalene oxide within the strain, thereby directly expressing parkol.

[0059] In some embodiments, the host strain of the recombinant engineered bacteria is a lanosterol-deficient yeast strain. The lanosterol-deficient yeast strain, used as the host organism, cannot synthesize ergosterol, an important component of the yeast cell membrane, thus accumulating the substrate 2,3-oxidized squalene within the cell. Therefore, the transformation and expression of the lanosterol-deficient yeast strain with the OSC gene leads to the conversion of the substrate 2,3-oxidized squalene into the corresponding reaction product.

[0060] Furthermore, the host strain of the recombinant engineered bacteria is the lanosterol-deficient yeast strain Gil77. The lanosterol-deficient yeast strain Gil77 contains the substrate 2,3-squalene oxide, and by expressing squalene cyclase, it can directly catalyze the expression of 2,3-squalene oxide within the strain to directly express parkol.

[0061] This study provides a recombinant engineered bacterium capable of cloning or expressing the squalene cyclase described in this study, which can then catalyze substrates to increase the yield of pacolol, and can be used for the preparation of pacolol.

[0062] This study provides a method for preparing parkol according to one embodiment, comprising the following steps: expanding the culture of recombinant engineered bacteria to obtain parkol, wherein the recombinant engineered bacteria are transformed with the above-mentioned recombinant vector and contain 2,3-squalene oxide.

[0063] This study creatively discovered that the oxidative squalene cyclase catalyzed by this study can be used to produce parkol from 2,3-oxidized squalene.

[0064] In some embodiments, the recombinant engineered bacteria is a lanosterol-deficient yeast strain Gil77 transformed with a recombinant vector. The steps for scaling up the recombinant engineered bacteria to obtain pakol include S110-S130:

[0065] S110. The recombinant engineered bacteria are cultured on a large scale, and the solid-liquid separation and collection of the cultured bacterial cells are performed.

[0066] The culture medium used for scaling up the recombinant engineered bacteria was SD-URA SC liquid medium containing 2% (w / v) glucose, 20 μg / mL supplement, 13 μg / mL heme, and 5 mg / mL Tween80. The supplement was ergosterol, for example, ergosterol from Fluka. The heme was, for example, heme from Sigma-Aldrich. Tween80 was, for example, Tween80 from Sigma-Aldrich.

[0067] The solid-liquid separation method is centrifugation. It should be noted that the solid-liquid separation method is not limited to centrifugation; other methods, such as filtration, can also be used.

[0068] In some embodiments, prior to the step of scaling up the recombinant engineered bacteria, a step of converting the recombinant vector into a host bacterium to obtain the recombinant engineered bacteria is included.

[0069] Further, the steps for transforming the recombinant vector into host bacteria to obtain recombinant engineered bacteria include: expanding the host bacteria culture, separating the solid and liquid phases and collecting the host bacteria; preparing competent cells from the host bacteria, mixing and incubating the competent cells, recombinant vector, and denatured salmon sperm DNA, then separating the solid and liquid phases and collecting the cells; and culturing the collected cells on a selective medium to obtain recombinant engineered bacteria. The selective medium is SC liquid medium SD-URA containing 2% (w / v) glucose, 20 μg / mL supplement, 13 μg / mL heme, 5 mg / mL Tween80, and 5 mg / mL agar. The supplement is ergosterol, for example, ergosterol from Fluka. Heme is, for example, heme from Sigma-Aldrich. Tween80 is, for example, Tween80 from Sigma-Aldrich.

[0070] S120. The expanded bacterial cells were induced to express the lanosterol synthase gene in the expanded bacterial cells.

[0071] The expanded bacterial cells were induced using SC-U selection medium containing raffinose and galactose. The induction temperature was 30℃, and the induction time was 2 days.

[0072] S130. After induction, solid-liquid separation is performed and the induced bacterial cells are collected. Then, the induced bacterial cells are lysed to obtain Parkol.

[0073] The solid-liquid separation method is centrifugation. It should be noted that the solid-liquid separation method is not limited to centrifugation; other methods, such as filtration, can also be used.

[0074] The bacterial cells induced by lysis were lysed using a lysis buffer. The lysis buffer consisted of 20% alkali and 50% anhydrous ethanol. The alkali could be, for example, KOH or NaOH. The lysis temperature was 65°C, and the lysis time was 2 hours.

[0075] In some embodiments, after the step of lysing and inducing the culture of bacterial cells, the following step is further included: collecting the supernatant from the lysed mixture obtained after solid-liquid separation lysing to obtain Parkol. Further, after the step of collecting the supernatant, a step of verifying whether the supernatant contains Parkol is included: detecting the supernatant using gas chromatography-mass spectrometry (GC-MS).

[0076] Using the above-described method for preparing parkol, parkol can be directly produced by expressing squalene cyclase to catalyze the production of 2,3-oxidized squalene in the strain, thus achieving one-step synthesis of parkol.

[0077] Saponins are secondary metabolites produced in sea cucumbers. They possess significant antibacterial, antitumor, anti-inflammatory, antiviral, hepatoprotective, and lipid-lowering biological activities. The biosynthesis process of saponins is not fully understood. This study elucidates a key step in saponin synthesis: the oxidative squalene cyclase catalyzes the oxidation of the substrate 2,3-squalene to form parkol. Increasing the amount of the intermediate parkol can benefit the increase of saponin production.

[0078] The following is a specific embodiment.

[0079] Unless otherwise specified, the reagents and instruments used in the examples are conventionally selected in the art. Experimental methods not specifying particular conditions in the examples are typically performed under standard conditions, such as those described in literature, books, or recommended by the reagent kit manufacturer. All reagents used in the examples are commercially available.

[0080] Unless otherwise specified, the selective plate is SD-URA SC liquid medium containing 2% (w / v) glucose, 20 μg / mL supplement, 13 μg / mL heme, 5 mg / mL Tween 80, and 5 mg / mL agar. The liquid selection medium is SD-URA SC liquid medium containing 2% (w / v) glucose, 20 μg / mL supplement, 13 μg / mL heme, and 5 mg / mL Tween 80. The supplement is ergosterol from Fluka. The heme is heme from Sigma-Aldrich. Tween 80 is Tween 80 from Sigma-Aldrich.

[0081] Example 1

[0082] 1. Obtain deep-sea sea cucumbers from the deep sea (please provide publicly available sources), take a small amount of tissue, extract RNA using TRIZOL reagent, perform transcriptome analysis, and perform quality control, assembly, integrity testing, redundancy removal, prediction, library construction, alignment, and selection of the OSC gene (i.e., oxidized squalene cyclase) on the transcriptome data. The deep-sea sea cucumber *Chiridota* sp. was collected from the South China Sea at a depth of 1900m (17°9000'N, 111°29167'E). The method for selecting OSC genes from the transcriptome is as follows: Li, Y., Wang, R., Xun, X., Wang, J., Bao, L., Thimmappa, R., … Wang, S. (2018). Seacucumber genome provides insights into saponin biosynthesis and aestivation regulation. Cell Discovery, 4(1). doi:10.1038 / s41421-018-0030-5.

[0083] 2. The PCR reaction system used to obtain the OSC gene was 50 μL, including 10 μL PrimeSTAR GXL Buffer, 4 μL dNTPs, 1 μL each of primers F and R, 1 μL template cDNA, and 2 μL PrimeSTAR GXL DNA Polymerase (TaKARa). The total volume was brought to 50 μL with H2O. The primer pair in the PCR reaction system was primer F and primer R, where primer F: GXL... GGTACC TACACAATGTCCTCCATTA; Primer R: GC TCTAGA TTAGGCTTGGGAAGTCAAA; the underlined part GGTACC represents the KpnI restriction site, and TCTAGA represents the XbaI restriction site. Restriction sites were designed, and the OSC gene and pYES2.0 expression vector were digested with KpnI and XbaI. The PCR reaction conditions used to obtain the target gene were: 98℃ for 10s, 55℃ for 15s, 68℃ for 10s, 30 cycles, and storage at 4℃. The OSC gene was ligated into the pYES2.0 vector using T4 ligase to construct the recombinant plasmid.

[0084] 3. Transform the recombinant plasmid into the lanosterol-deficient yeast strain Gil77, as follows:

[0085] Inoculate 5 ml of YPD medium with Gil77 colonies and incubate overnight in a shaker at 30°C. Collect the bacteria by centrifugation at 2500 rpm and resuspend the bacterial suspension in 1 ml of 1X TE. Centrifuge again at 2500 rpm and resuspend the bacterial cells in 1 ml of 1×LiAC / 0.5×TE. Incubate the cells at room temperature for 10 min. Centrifuge, discard the supernatant, and add 100 μL of 1×LiAC / 0.5×TE to prepare competent cells. Add 1 μg of recombinant plasmid and 100 μg of denatured salmon sperm DNA to 100 μL of yeast competent cell suspension, add 700 μL of 1X LiAc / 40% PEG-3350 / 1X TE, mix well, and incubate at 30°C for 30 min. Add 88 μL of LDMSO, stir well, and heat shock at 42°C for 7 min. Centrifuge for 10 s using a microcentrifuge and discard the supernatant. Resuspend in 1 ml of 1×TE, centrifuge, discard the supernatant, add 100 μL of 1×TE, and incubate on a selective plate at 30°C for 3 days.

[0086] 4. Pick colonies from the plate and culture them overnight in liquid selection medium to expand their size. Centrifuge again to collect the cells, and induce lanosterol synthase gene expression in SC-U selection medium containing raffinose and galactose at 30°C for 2 days. After induction, centrifuge to collect the cells, and lyse them with lysis buffer (20% NaOH and 50% anhydrous ethanol) at 65°C for 2 hours. Extract the product with n-hexane, collect the supernatant, dry it under nitrogen, dissolve the product in 1 mL of n-hexane, and analyze the product using gas chromatography-mass spectrometry (GC-MS). Gas chromatography-mass spectrometry (GC-MS) conditions: HP-5MS column (30m × 0.25mm × 0.25um, Agilent Technologies); carrier gas: helium, flow rate: 0.8ml / min; injection port temperature: 250℃; splitless mode: initial temperature 80℃, hold for 2 min, then increase to 310℃, hold for 15 min; EI ionization source: EI, electron energy 70eV; full scan mode, scan range 50-600m / z; injection volume: 1μL. Detailed results can be found [link to results]. Figure 1 . Figure 1 The results are from the GC-MS detection in Example 1. Figure 1 In the diagram, the entire curve indicated by arrow (1) represents the elution curve of the empty vector, the entire curve indicated by arrow (2) represents the oxidized squalene cyclase expression vector, and the peak indicated by arrow (3) is paparol. Figure 1 The vertical axis represents abundance, and the horizontal axis represents peak time (in seconds).

[0087] from Figure 1As can be seen, this application constructs a eukaryotic expression vector for an oxidized squalene cyclase comprising a polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 1, and then transforms the eukaryotic expression vector into GIL77 yeast to induce the expression of the cyclase. The cyclase is able to catalyze the production of parkol from the substrate 2,3-oxidized squalene in recombinant yeast.

[0088] Saponins are secondary metabolites produced in sea cucumbers. They possess significant antibacterial, antitumor, anti-inflammatory, antiviral, hepatoprotective, and lipid-lowering biological activities. The biosynthesis process of saponins is not fully understood. This study elucidates a key step in saponin synthesis: the oxidative squalene cyclase catalyzes the oxidation of the substrate 2,3-squalene to form parkol. Increasing the amount of the intermediate parkol can benefit the increase of saponin production.

[0089] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An oxidative squalene cyclase, characterized in that, The amino acid sequence of the oxidized squalene cyclase is shown in SEQ ID No. 1; The coding sequence of the oxidized squalene cyclase is the nucleotide sequence shown in SEQ ID No.

2.

2. A recombinant vector, characterized in that, The recombinant vector carries the coding sequence of the oxidized squalene cyclase as described in claim 1.

3. The recombinant vector according to claim 2, characterized in that, The recombinant vector is a eukaryotic cell expression vector carrying the coding sequence of the oxidized squalene cyclase.

4. The recombinant vector according to claim 2, characterized in that, The recombinant vector is a pYES2.0 expression vector carrying the coding sequence of the oxidized squalene cyclase.

5. The method for preparing the recombinant vector according to any one of claims 2-4, characterized in that, The procedure includes the following steps: PCR amplification of the coding sequence of the oxidized squalene cyclase using amplification primer pairs with nucleotide sequences as shown in SEQ ID No. 3 and SEQ ID No. 4, and ligation into an empty vector to obtain the recombinant vector.

6. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria are transformed with the recombinant vector as described in any one of claims 2-4.

7. The recombinant engineered bacteria according to claim 6, characterized in that, The host bacteria of the recombinant engineered bacteria contain squalene 2,3-oxide.

8. The recombinant engineered bacteria according to claim 6, characterized in that, The host strain of the recombinant engineered bacteria is a lanosterol-deficient yeast strain.

9. The recombinant engineered bacteria according to claim 8, characterized in that, The host strain of the recombinant engineered bacteria is the lanosterol-deficient yeast strain Gil77.

10. The use of the squalene cyclase of claim 1, the recombinant vector of any one of claims 2-4, or the recombinant engineered bacteria of any one of claims 6-9 in the preparation of Parkol.

11. A method for preparing pacol, characterized in that, The method includes the following steps: expanding the culture of recombinant engineered bacteria to obtain paparol, wherein the recombinant engineered bacteria are transformed with the recombinant vector according to any one of claims 2-4, and the recombinant engineered bacteria contain 2,3-squalene oxide.

12. The method for preparing paparol according to claim 11, characterized in that, The recombinant engineered bacteria is Gil77, a lanosterol-deficient yeast strain transformed with the recombinant vector. The step of scaling up the recombinant engineered bacteria to obtain pakol includes: The recombinant engineered bacteria were cultured on a large scale, and the bacterial cells from the large-scale culture were separated into solid and liquid components and collected. The expanded bacterial culture was subjected to induced culture to induce the expression of the lanosterol synthase gene in the expanded bacterial culture. After induction, the induced bacterial cells were separated into solid and liquid phases and collected. Then, the induced bacterial cells were lysed to obtain the paclitaxel.

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