Gynostemma pentaphylla oxidized squalene cyclase GpOSC2 and application thereof

Abstract

The invention relates to a gynostemma pentaphylla oxidized squalene cyclase gene GpOSC2 and application thereof, and belongs to the technical field of genes. The amino acid sequence of the gynostemma pentaphylla oxidized squalene cyclase GpOSC2 is as shown in SEQ ID NO. 1. A coding sequence for coding the gynostemma pentaphylla oxidized squalene cyclase GpOSC2 gene is a nucleotide sequence as shown in SEQ ID NO. 2. According to the present invention, the gene is adopted to carry out fermentation so as to obtain Dammarenediol-II, lupeol, lanosterol, cycloartenol and alpha-amyrin alcohol, such that the application prospect is good, and the promotion and the application are easy.

Description

Technical Field

[0001] The invention belongs to the field of gene technology, and particularly relates to Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2 and applications thereof. Background Art

[0002] Gynostemma pentaphyllum[ Gynostemma Pentaphyllum (Thunb) Mak is a perennial herbaceous climbing plant of the genus Gynostemma in the Cucurbitaceae family. It is also known as bitter medicine, fairy grass, seven-leaf gall, and five-leaf ginseng. It is primarily found in sparse woods, grassy areas, and shrublands in Hunan, Yunnan, and Guangxi. It prefers shade and avoids direct sunlight, preferring diffused and scattered light, ideally 40% to 60% of natural sunlight. It thrives in a humid, mild climate, with an optimal growth temperature of 14-25°C. Growth slows at temperatures between 4-6°C. When temperatures exceed 35°C, in the absence of shade, and with low soil moisture, stem growth slows, and leaves can wilt due to heat burns. Growth typically begins in late March, with peak growth in June, July, and August. Gynostemma has strict water requirements, ideally requiring soils with a maximum water holding capacity of 55% to 85%. Gynostemma pentaphyllum is an important plant resource in my country with a long history of growth. It was first recorded as a wild vegetable eaten during the Spring and Autumn Period and the Warring States Period, and was gradually developed into a medicinal plant. Gynostemma pentaphyllum grows mostly in the south, and its above-ground parts are often used as medicine. It is also the only known plant outside the genus Panax in the Araliaceae family that contains ginsenosides, and is also known as "Southern Ginseng." Gynostemma pentaphyllum is cold in nature, sweet, and slightly bitter in taste, and enters the lung, spleen, and kidney meridians. It has the effects of clearing the lungs and resolving phlegm, strengthening the spleen and stomach, replenishing qi and nourishing yin, lowering blood pressure, and lowering blood lipids. It is mainly used to treat various inflammatory conditions such as physical weakness, hyperlipidemia, and chronic gastroenteritis. It is a dual-purpose medicinal and edible plant with broad development prospects. Therefore, research on the medicinal value and biological activity of Gynostemma pentaphyllum is of great significance.

[0003] Gynostemma pentaphyllum saponin ( GypenosidesGynostemma pentaphyllum (GP) is one of the most important functional ingredients in Gynostemma pentaphyllum. Gynostemma pentaphyllum saponins have the same aglycone skeleton structure as ginsenosides, and some of the gynostemma pentaphyllum saponins can also be converted into rare ginsenosides. Most of the gynostemma pentaphyllum saponins are dammarane-type, and they have attracted much attention because they have a tetracyclic triterpenoid dammarane-type basic structure similar to ginsenosides. In 1976, Japanese scholars obtained ginsenodiol and 2α-hydroxy ginsenodiol from the hydrolyzate of total gynostemma pentaphyllum saponins, proving for the first time that Gynostemma pentaphyllum contains dammarane-type saponin components. Since then, scientific researchers have successively isolated more than 200 types of gynostemma pentaphyllum saponins from Gynostemma pentaphyllum, and divided them into 12 categories according to the degree of similarity of the gypenosin aglycone structure. It has now been found that the content of total gynostemma pentaphyllum saponins is about 3 times that of ginseng. The structures of gypenosides Gyp-III, Gyp-IV, Gyp-VIII, Gyp-XII, Gyp-I, and Gyp-A-AH are identical to those of ginsenosides Rb1, Rb3, Rd, F2, K, and Rg3, respectively. The common saponin components of ginseng and gypenoside are Re, Rg2, Rc, F1, malonyl-Rbl, malonyl-Rd, and Rf. Dammarane-type ginsenosides primarily include protopanaxadiol (PPD) and protopanaxatriol (PPT). Both PPT and PPD ginsenosides belong to the dammarane-type tetracyclic triterpenoid family, formed through modifications based on a dammarenediol skeleton. The most important medicinally active ingredients in Gynostemma pentaphyllum are saponins and polysaccharides. Modern pharmacological research has revealed that these saponins and polysaccharides possess antioxidant and anti-aging properties. Combined use with other medications can enhance lipid-lowering effects. Furthermore, they have blood sugar-lowering, liver-protecting, sleep-improving, anti-tumor, and neuroprotective properties. Therefore, Gynostemma pentaphyllum holds broad development prospects as a medicine and health supplement. It can also serve as an economical alternative to ginseng, holding significant research and development value.

[0004] Oxidosqualene cyclase (OSC) is the first rate-limiting enzyme in the downstream synthesis of triterpenoid saponins. It guides the protonation, cyclization, rearrangement, and deprotonation of 2,3-oxidosqualene to complete the cyclization process to form the triterpenoid backbone. OSC also serves as a branch point in the biosynthetic pathways for triterpenoid saponins and sterols. The cyclization of 2,3-oxidosqualene is the first step in the synthesis of triterpenoid saponins in plants and the first diversification step in triterpenoid saponin biosynthesis. This cyclization reaction is catalyzed by OSC and produces over 100 different triterpenoid saponin backbones. Plant OSCs can be broadly divided into two categories based on the intermediate structures formed during substrate binding and folding: the P group forms a protosteryl cation via a chair-boat-chair (CBC) conformation, ultimately producing sterols; the D group forms a dammaryl cation via a chair-chair-chair (CCC) conformation, ultimately producing triterpenes. Therefore, OSCs are key enzymes in the biosynthesis of sterols and triterpenes. Over 110 OSCs have been identified in plants, with five types found in Panax ginseng: β-Amyrin synthase (β-AS), dammaranediol synthase (DS), cycloartenol synthase (CAS), lupeol synthase (LUS), and lanosterol synthase (LS). OSCs also serve as the first key enzyme in the biosynthesis pathway of gypenosidin.

[0005] Dammarane saponins are important bioactive components found in valuable Chinese medicinal materials such as ginseng and Gynostemma pentaphyllum. They are the main active ingredients in popular Chinese medicines such as Xuesetong and Xueshuantong, and possess anti-cancer and central nervous system protective properties. Their structure is a dammarane backbone formed from epoxysqualene in a fully chair-like configuration. Gynostemma pentaphyllum saponins are a class of tetracyclic triterpenoid saponins based on the dammarane aglycone structure. Rare ginsenosides can be synthesized through xenobiotic synthesis, leveraging synthetic biology techniques. Using plant tissues and Escherichia coli expression systems as a platform, genes encoding enzymes such as oxidosqualene cyclase and glycosyltransferases are introduced. Using the methylerythritol phosphate (MEP) and mevalonate acid (MVA) pathways, diverse rare ginsenosides are synthesized from the triterpenoid precursor squalene through the action of enzymes such as oxidosqualene cyclase and glycosyltransferase. To synthesize rare ginsenosides in Gynostemma pentaphyllum, plant tissues with strong differentiation abilities can be selected, including hairy roots, adventitious roots, and other plant tissues. Plant tissue culture of Gynostemma pentaphyllum is an effective way to produce saponins, and the hairy roots and adventitious roots produced by Gynostemma pentaphyllum can be used for culture. Hairy roots can be produced directly from explants infected with Agrobacterium rhizogenes. Hairy roots produced through Agrobacterium rhizogenes-mediated genetic transformation are genetically stable, grow rapidly, and have a high capacity for synthesizing secondary metabolites. They can be used to mass-produce the secondary metabolites required for medicinal ingredients in Gynostemma pentaphyllum. Adventitious roots can also be induced from explant callus. Heterologous plants such as tobacco and Platycodon grandiflorum can also be used as expression systems. For example, Shin et al. achieved heterologous synthesis of rare ginsenosides through transgenic tobacco.

[0006] The dammarenediol synthase gene (DDS) is a key enzyme that promotes the formation of triterpenoid saponins and is a key enzyme in the synthesis of dammarane-type triterpenoid saponins. The biosynthetic pathway for ginsenosides primarily involves PgDS and PNY (β-AS) catalyzing the conversion of 2,3-oxidosqualene to dammarenediol and β-amyrin, respectively. Dammarenediol is further catalyzed by CYP450 and UGT enzymes to dammarane-type ginsenosides, while β-amyrin generates oleanane-type ginsenosides. Since most triterpenoid saponins are derived from oleanane and dammarane, the enzymes β-AS and DS play a crucial role in the synthesis of triterpenoid saponins.

[0007] The protein encoded by GpOSC1 reported previously can only catalyze the synthesis of Dammarenediol-II and cannot produce a wider range of products. Therefore, how to overcome the shortcomings of existing technologies is an urgent problem to be solved in the field of genetic technology. Summary of the Invention

[0008] The purpose of the present invention is to solve the deficiencies of the prior art and provide Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2 and its application.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] The second aspect of the present invention provides a gene encoding the above-mentioned Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2.

[0011] Furthermore, the coding sequence of the gene is the nucleotide sequence shown in SEQ ID NO.2.

[0012] The third aspect of the present invention provides a recombinant vector containing the above gene.

[0013] A fourth aspect of the present invention provides a recombinant genetically engineered bacterium transformed with the above-mentioned recombinant vector.

[0014] In a fifth aspect, the present invention provides a use of the Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2 in the preparation of Dammarenediol-II, lupeol, lanosterol, cycloartenol and α-amyrinol.

[0015] The protein encoded by GpOSC1 reported previously can only catalyze the synthesis of Dammarenediol-II. However, the protein encoded by GpOSC2 in this study can catalyze the formation of five products: Dammarenediol-II, lupeol, lanosterol, cycloartenol, and α-amyrinol.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a protein encoded by the GpOSC2 gene of Gynostemma pentaphyllum, which can catalyze the formation of five products: Dammarenediol-II, lupeol, lanosterol, cycloartenol and α-amyrinol. The protein has good application prospects and is easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison chart of HPLC detection of an application example of the present invention. DETAILED DESCRIPTION

[0018] The present invention is described in further detail below with reference to the embodiments.

[0019] Those skilled in the art will understand that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications were used. Materials or equipment used without manufacturer identification are commercially available conventional products.

[0020] 1. Source of GpOSC gene DNA sequence and construction of target gene expression vector RNA was extracted from tender leaves and stems of Gynostemma pentaphyllum plants using the TIANGEN polysaccharide and polyphenol plant total extraction kit, and reverse transcribed into cDNA using the TIANGEN reverse transcription kit. (1) Perform PCR using specific primers (Table 1) to amplify the full-length GpOSC2 gene. The reaction system was prepared according to the instructions for Phanta-Max Super-Fidelity DNA Polymerase.

[0021] The primers used in the PCR reaction are shown in Table 1, and the PCR reaction system is shown in Table 2. The PCR reaction program was as follows: pre-denaturation at 95°C for 180 s; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 100 s, for a total of 35 cycles; extension at 72°C for 300 s, and 4°C +∞.

[0022] Table 1

[0023] Table 2

[0024] Note: The reaction system was prepared according to the instructions of Phanta-Max Super-Fidelity DNA Polymerase. Phanta is the reagent in Novozymes high-fidelity PCR Phanta-Max Super-Fidelity DNA Polymerase.

[0025] (2) SnapGene software was then used to screen the full-length sequence of the gene coding region through data analysis, and primers with homology arms were designed for the gene (see Table 3) to facilitate homologous recombination between the target gene and the vector.

[0026] (3) The amplified product obtained in step (1) was subjected to PCR reaction using primers with homology arms to obtain the target gene fragment GpOSC2 with homology arms; the target gene fragment GpOSC2 with homology arms was then recovered by gel recovery using a kit from GenStar, the bright band was cut out, and the recovery was carried out in sequence according to the kit steps. Finally, the recovered target gene was measured for concentration and stored in a -20°C refrigerator. The PCR reaction system was the same as that in Table 2, except that the template was replaced by the amplified product obtained in step (1) instead of Gynostemma pentaphyllum cDNA; the PCR reaction procedure was the same as that in step (1).

[0027] The pYES2 plasmid was digested with the restriction endonuclease BamHI to generate the linearized vector pYES2. Successful digestion was confirmed by agarose gel electrophoresis. A single band indicated successful digestion. The digestion system is shown in Table 4. The digestion reaction conditions were: 37°C for 1 hour, 65°C for 15 minutes, and 10°C +∞. The linearized vector was recovered using the DNA gel recovery kit from Jerui Biotech Co., Ltd. Follow the kit's instructions, determine the concentration, and store at -20°C until needed.

[0028] Table 3

[0029] Table 4

[0030] (5) The linearized vector pYES2 and the target gene fragment GpOSC2 with homology arms were recombined using the pEASY®-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the recombinant plasmid pYES2-OSC2. The recombination system is: 0.7 μL linearized vector pYES2, 2.3 μL target gene fragment GpOSC2 with homology arms, 3 μL 2×SDMM (homologous recombination enzyme).

[0031] The recombination reaction procedure was 50°C for 20 min, 10°C +∞.

[0032] (6) Transform the recombinant plasmid pYES2-OSC2 into competent DH5a E. coli and culture on LB (Table 5) plates containing 100 mg / L Amp at 37°C overnight. The specific method is as follows: (6.1) Add 10 μL of recombinant plasmid pYES2-OSC2 to 50 μL of competent E. coli cells and mix thoroughly (carefully pipette). Place the resulting mixture on ice for 30 minutes (to maintain the competent state of the E. coli and facilitate the entry of the plasmid into the E. coli).

[0033] (6.2) After heat shock at 42°C for 90 seconds, remove the tube and place it in an ice bath for 2–5 minutes.

[0034] (6.3) Add 450 μL of LB liquid medium (see Table 5 for the recipe) and incubate at 37°C, 220 rpm, and shake for 50 min.

[0035] (6.4) After 50 minutes, take 100 μL of the bacterial culture obtained in step (6.3) and spread it on LB solid medium containing 100 mg / L Amp. Incubate in a constant temperature incubator at 37°C for 8-12 hours. The formula of LB solid medium is shown in Table 5. (6.5) When a single colony is observed, perform a bacterial picking and shaking operation (add 500 μL of LB liquid medium containing 100 mg / L Amp to a 2 mL centrifuge tube) and shake at 220 rpm at 37°C to obtain a bacterial suspension. This involves taking a plate containing a single colony (i.e., a monoclonal colony) to a sterile operating table and using a dedicated bacterial picking device to select a single colony. The tube is then sealed and placed on a shaker to shake the suspension until the bacterial suspension becomes turbid. This will yield a monoclonal bacterial suspension.

[0036] (7) Monoclonal colony suspension was selected for PCR detection. The primers used in PCR were shown in Table 6, the reaction system was shown in Table 7, and the reaction procedure was as follows: pre-denaturation at 95°C for 300s; denaturation at 95°C for 15s, annealing at 60°C for 15s, and extension at 72°C for 100s, for a total of 35 cycles; extension at 72°C for another 300s, and 4°C +∞.

[0037] Table 5

[0038] Table 6

[0039] Table 7

[0040] After the PCR reaction, sample 5 μL of the PCR product for agarose gel electrophoresis at 185 V, 180 mA, and 20 min. Remove the run and visualize the gel using a gel imager. If a distinct band is observed within the 2000-3000 bp range, send the positive isolates for sequencing at a sequencing company. Confirm that the sequence is consistent with GpOSC2, confirming the successful construction of the pYES2-OSC2 expression plasmid.

[0041] 2. Products extracted from BY4742 strain The pYES2-OSC2 expression plasmid was transformed into the BY4742 yeast expression strain by lithium acetate transformation.

[0042] Table 8

[0043] Note: Add 400ng pYES2-OSC2 expression plasmid and mix well To screen BY4742 transformants expressing the pYES2-OSC2 plasmid, solid synthetic complete medium without uracil (SC-Ura medium, the formula of this medium is shown in Table 9 ) was used; The BY4742 transformants expressing the pYES2-OSC2 expression plasmid were evenly spread onto SC-Ura solid medium supplemented with 2% glucose by mass and cultured. After incubation at 30°C for 4-5 days, single colonies were selected. The positive single colonies were then placed in SC-Ura liquid medium supplemented with 2% glucose by mass and cultured at 30°C with shaking at 220 rpm for 2 days.

[0044] Table 9

[0045] Yeast cells were collected and induced with 100 mL SC-Ura liquid medium containing 2% galactose, and cultured at 30°C with shaking at 220 rpm for 5 days.

[0046] The cultured cells were resuspended in 0.1 M potassium dihydrogen phosphate buffer (pH 7.0) containing 2% glucose and stirred at 220 rpm at 30°C for one day. Yeast cells were harvested and lysed using ultrasonic extraction with methanol for one hour to obtain a lysed yeast solution. Ultrasonic extraction was performed using an ultrasonic disruptor with an output power of 500 W and an operating frequency of 20 kHz.

[0047] The lysed yeast solution was centrifuged at 3900 rpm for 20 min, and the supernatant was extracted three times with n-hexane. The three n-hexane extraction solutions were combined and evaporated by rotary evaporation at 50°C.

[0048] The evaporated solid extract was dissolved in 200 mL of analytical methanol. The resulting sample was filtered through a 0.22 mm organic phase filter and analyzed using an Agilent 1260 high-performance liquid chromatograph. The HPLC elution program is shown in Table 10. HPLC analysis was performed using an Agilent phenomenex-00D-4462-EC-C18 (100 × 4.6 mm, 2.6 μm, Agilent Technologies, Santa Clara, CA, USA).

[0049] Table 10

[0050] Note: During elution, the eluent gradient changes directly after the time is up.

[0051] 2 Results 2.1 Characterization of gene function The recombinant plasmid pYES2-OSC2 was transformed into the BY4742 strain by the lithium acetate method. The fermentation products were extracted after fermentation and then analyzed by HPLC. The HPLC results showed that the expression product of GpOSC1 yeast was Dammarenediol-II; the expression products of GPOSC2 yeast were Dammarenediol-II, lupeol, lanosterol, cycloartenol and α-amyrinol ( Figure 1 ).

[0052] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2, characterized in that The amino acid sequence of the Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2 is shown in SEQ ID NO.

1.

2. A gene encoding the Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2 according to claim 1.

3. The gene according to claim 2, characterized in that The coding sequence of the gene is the nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant vector containing the gene according to claim 2 or 3.

5. A recombinant genetically engineered bacterium transformed with the recombinant vector according to claim 4.

6. Use of the Gynostemma pentaphyllum oxidosqualene cyclase GpOSC2 according to claim 1 in the preparation of Dammarenediol-II, lupeol, lanosterol, cycloartenol and α-amyrinol.

Images