Gypenoside oxidized squalene cyclase gposc2 and application thereof

Abstract

The present application relates to Gypenoside oxidized squalene cyclase gene GpOSC2 and its application, and belongs to the technical field of gene. The amino acid sequence of the Gypenoside oxidized squalene cyclase GpOSC2 is shown as SEQ ID NO. 1. The coding sequence of the Gypenoside oxidized squalene cyclase GpOSC2 gene is the nucleotide sequence shown as SEQ ID NO. 2. The present application can obtain Dammarenediol-II, lupine alcohol, lanosterol, cyclotene and alpha-amyrin alcohol by fermentation using the gene, has a good application prospect, and is easy to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, specifically relating to Gynostemma pentaphyllum oxidase GpOSC2 and its applications. Background Technology

[0002] Gynostemma pentaphyllum Gynostemma pentaphyllum [Thunb. Mak] is a perennial herbaceous climbing plant belonging to the genus Gynostemma of the Cucurbitaceae family. It is also known as bitter herb, fairy herb, seven-leaf ginseng, and five-leaf ginseng. It is mainly distributed in sparse forests, grasslands, and shrublands in Hunan, Yunnan, and Guangxi provinces. Gynostemma is a shade-loving plant, incompatible with strong direct sunlight, preferring diffused light, ideally 40%-60% of natural light. It thrives in shady, humid, and mild climates, with the optimal growth temperature being 14-25℃. Growth slows at 4-6℃, and above 35℃, without shade and with low soil moisture, stem growth is slow, and leaves may scorch and wilt due to the high temperature. Gynostemma generally begins to grow in late March, with the fastest growth occurring in June, July, and August. It has relatively strict water requirements, with soil moisture content ideally between 55% and 85%. Gynostemma pentaphyllum is an important plant resource in my country with a long history of cultivation. It was first recorded as a wild vegetable during the Spring and Autumn and Warring States periods, and its medicinal use gradually developed. Gynostemma pentaphyllum mainly grows in the south, and its above-ground parts are commonly used medicinally. It is also the only known non-Araliaceae plant in the Panax genus that contains ginsenosides, hence its nickname "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 weakness, hyperlipidemia, chronic gastroenteritis, and other inflammations. It is a medicinal and edible plant with broad development prospects, therefore, research on the medicinal value and bioactivity of Gynostemma pentaphyllum is of great significance.

[0003] Gynostemma pentaphyllum saponins ( GypenosidesGynostemma pentaphyllum (Gynostemma pentaphyllum saponins) is one of the most important functional components of Gynostemma pentaphyllum. Gynostemma pentaphyllum saponins share the same aglycone skeleton structure as ginsenosides, and some Gynostemma pentaphyllum saponins can be converted into rare ginsenosides. Most Gynostemma pentaphyllum saponins are dammarane-type, and it has attracted much attention due to its similar tetracyclic triterpenoid dammarane-type basic structure to ginsenosides. In 1976, Japanese scholars obtained ginsenoside diol and 2α-hydroxyginsenoside diol from the hydrolysis products of total Gynostemma pentaphyllum saponins, proving for the first time that Gynostemma pentaphyllum contains dammarane-type saponin components. Since then, researchers have successively isolated more than 200 kinds of Gynostemma pentaphyllum saponins, classifying them into 12 categories according to the similarity of their aglycone structures. It has now been found that the total saponin content of Gynostemma pentaphyllum is about three times that of ginseng. The structures of Gynostemma pentaphyllum saponins 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. Common saponin components of ginseng and Gynostemma pentaphyllum include Re, Rg2, Rc, F1, malonyl-Rbl, malonyl-Rd, and Rf. Dammarane-type ginsenosides mainly include protopanaxadiol type (PPD) and protopanaxtriol type (PPT). Both PPT and PPD type ginsenosides belong to the dammarane-type tetracyclic triterpenoid compounds, which are formed by modification of dammarene diol as the basic skeleton. The most important active medicinal components of Gynostemma pentaphyllum are saponins and polysaccharides. Modern pharmacological studies have found that Gynostemma pentaphyllum saponins and polysaccharides have certain antioxidant activity, playing a role in anti-oxidation and anti-aging. When used in combination with other drugs, they can enhance the lipid-lowering effect. It also has hypoglycemic, liver-protective, sleep-improving, anti-tumor, and neuroprotective effects. Therefore, Gynostemma pentaphyllum has broad development prospects as both a medicine and a health product. At the same time, Gynostemma pentaphyllum can also serve as an economical substitute for ginseng, possessing significant research and utilization value.

[0004] Oxido squalene cyclase (OSC) is the first rate-limiting enzyme in the downstream synthesis of triterpenoid saponins. It guides the cyclization of 2,3-oxidized squalene through protonation, cyclization, rearrangement, and deprotonation to form the triterpenoid skeleton. OSC also serves as a branch point in the biosynthetic pathways of triterpenoid saponins and sterols. The cyclization of 2,3-oxidized squalene is the first step in the synthesis of triterpenoid saponins in plants and the first step in diversifying triterpenoid saponin biosynthesis. This cyclization reaction is catalyzed by OSC and produces more than 100 different triterpenoid saponin skeletons. Based on the intermediate structures formed during substrate binding and folding, plant OSCs can be broadly classified into two groups: Group P forms a protosteryl cation via a chair-boat-chair (CBC) conformation, ultimately producing sterols; Group D forms a dammarenyl cation via a chair-chair-chair (CCC) conformation, ultimately producing triterpenoids. Therefore, OSCs are key enzymes determining the biosynthesis of sterols and triterpenoids. Currently, more than 110 OSCs have been identified in plants. Five types of OSCs have been found in ginseng: β-Amyrin synthase (β-AS), dammarenediol synthase (DS), cycloartenol synthase (CAS), lupeol synthase (LUS), and lanosterol synthase (LS). OSCs have also become the first key enzyme in the biosynthetic pathway of Gynostemma pentaphyllum saponins.

[0005] Dammarane-type saponins are important characteristic bioactive components in precious Chinese medicinal herbs such as ginseng and gynostemma pentaphyllum. They are the main active ingredients in major Chinese medicine products such as Xuesaitong and XueShuantong, and have functions such as anti-cancer and protection of the central nervous system. Their structure is formed by the formation of a dammarane skeleton from squalene through a full-chair conformation. Gynostemma pentaphyllum saponins are a class of tetracyclic triterpenoid saponins based on the dammarane aglycone structure. Rare ginsenosides can be synthesized through heterologous biosynthesis using synthetic biology techniques. Plant tissue and Escherichia coli expression systems are used as chassis, and enzyme genes such as squalene cyclase and glycosyltransferase are introduced. Relying on the methylerythritol phosphate pathway (MEP) and mevalonate acid pathway (MVA), different kinds of rare ginsenosides are synthesized from squalene, the precursor of triterpenoid compounds, under the action of enzymes such as squalene cyclase and glycosyltransferase. The synthesis of rare ginsenosides from Gynostemma pentaphyllum can be achieved by selecting plant tissues with strong differentiation capabilities, including hairy roots, adventitious roots, and other plant tissues. Plant tissue culture is an effective method for saponin production, utilizing hairy roots and adventitious roots generated from Gynostemma pentaphyllum. Hairy roots can be directly produced 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 secondary metabolite synthesis, making them suitable for the large-scale production of secondary metabolites required for medicinal use in Gynostemma pentaphyllum. Adventitious roots can also be induced from explant callus tissue. Heterologous plants such as tobacco and Platycodon grandiflorus can also be used as expression systems. For example, Shin et al. achieved heterologous synthesis of rare ginsenosides through transgenic tobacco.

[0006] The dammarene-11 synthase gene (DDS) is a crucial synthase gene promoting the formation of triterpenoid saponins and a key enzyme in the synthesis of dammarane-type triterpenoid saponins. The biosynthetic pathway of ginsenosides mainly involves PgDS and PNY (β-AS) catalysis of 2,3-oxidized squalene to generate dammarene-11 and β-amyrin, respectively. Dammarene-11 is further catalyzed by CYP450 and UGT enzymes to generate dammarane-type ginsenosides, and β-amyrin to generate oleanane-type ginsenosides. Since most triterpenoid saponins are derived from oleanane and dammarene, β-AS and DS enzymes play a vital role in triterpenoid saponin synthesis.

[0007] Previous reports indicated that the protein encoded by GpOSC1 could only catalyze the synthesis of Dammarenediol-II and could not yield a wider variety of products. Therefore, overcoming the shortcomings of existing technologies is a pressing issue that needs to be addressed in the field of gene technology. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide Gynostemma pentaphyllum oxidase GpOSC2 and its applications.

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

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

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

[0012] A third aspect of the present invention provides a recombinant vector containing the above-mentioned genes.

[0013] A fourth aspect of the present invention provides a recombinant genetically engineered bacterium obtained by transformation using the above-described recombinant vector.

[0014] The fifth aspect of this invention provides the application of the Gynostemma pentaphyllum oxidase GpOSC2 in the preparation of Dammarenediol-II, lupeol, lanosterol, cycloartenol and α-amyrinol.

[0015] Previous reports indicated that the protein encoded by GpOSC1 could 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 beneficial effects of this invention are as follows: This invention provides a protein encoded by the GpOSC2 gene of Gynostemma pentaphyllum that can catalyze the formation of five products: Dammarenediol-II, lupeol, lanosterol, cycloartenol, and α-amyrinol. It has good application prospects and is easy to promote and apply. Attached Figure Description

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

[0018] The present invention will now be described in further detail with reference to the embodiments.

[0019] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[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. The TIANGEN polysaccharide and polyphenol plant total extraction kit was used for RNA extraction, and the TIANGEN reverse transcription kit was used to reverse transcribe the Gynostemma pentaphyllum RNA into cDNA. (1) The full-length GpOSC2 gene was amplified by PCR using specific primers (Table 1). The reaction system was prepared according to the instructions of 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: 95℃ pre-denaturation for 180s; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 100s, for a total of 35 cycles; 72℃ further extension for 300s, 4℃ +∞.

[0022] Table 1

[0023] Table 2

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

[0025] (2) Then, the specific primers for the full-length sequence of the gene coding region obtained by data analysis were obtained using SnapGene software. The primers for this gene were designed with homologous arms (see Table 3) so that the target gene and the vector could recombine homologously.

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

[0027] The pYES2 plasmid was digested with the restriction endonuclease BamHI to obtain the linearized vector pYES2. Agarose gel electrophoresis was then used to detect successful digestion; a single band indicated successful digestion. The digestion system is shown in Table 4. The digestion conditions were: 37℃ for 1 h, 65℃ for 15 min, and 10℃ ± ∞. The linearized vector was recovered using the DNA gel recovery kit from Jereh Biotechnology Co., Ltd. The kit steps were followed sequentially, and the concentration was determined after recovery. Finally, the vector was stored at -20℃ for later use.

[0028] Table 3

[0029] Table 4

[0030] (5) The linearized vector pYES2 and the target gene fragment GpOSC2 with homologous arms were recombined using the pEASY®-Basic Seamless Cloning and Assembly Kit (Beijing TransGen Biotechnology) to obtain the recombinant plasmid pYES2-OSC2. The recombinant system was as follows: 0.7 μL linearized vector pYES2, 2.3 μL target gene fragment GpOSC2 with homologous arms, and 3 μL 2×SDMM (homogeneous recombinase).

[0031] The recombination reaction program was 50℃ for 20 min, then 10℃ +∞.

[0032] (6) The recombinant plasmid pYES2-OSC2 was transformed into competent DH5α Escherichia coli cells and cultured overnight at 37°C on LB agar plates containing 100 mg / L Amp (Table 5). The specific method is as follows: (6.1) Take 10 μL of recombinant plasmid pYES2-OSC2 and add it to 50 μL of competent E. coli cells and mix thoroughly (carefully pipette). Place the resulting mixture on an ice box (to keep the E. coli competent cells so that the plasmid can easily enter the E. coli) for 30 minutes.

[0033] (6.2) After heat shock at 42℃ for 90s, remove and place in an ice bath for 2~5min.

[0034] (6.3) Add 450 μL of LB liquid culture medium (formulation shown in Table 5) and place it in a shaker at 37℃ and 220 r / min for 50 min.

[0035] (6.4) After 50 min, take 100 μL of the bacterial culture obtained from the shaker culture in step (6.3) and plate it in LB solid medium containing 100 mg / L Amp. Incubate at 37°C for 8-12 h. The formulation of LB solid medium is shown in Table 5. (6.5) When a single colony is observed to grow, perform pick-and-shake incubation (add 500 μL of LB liquid medium containing 100 mg / L Amp to a 2 mL centrifuge tube), and shake at 220 r / min on a shaker at 37°C to obtain the bacterial suspension. Pick-and-shake refers to taking the plate with a single colony (i.e., a monoclonal colony) to a sterile operating table, using a special picker to select a single colony and transfer it to a centrifuge tube containing LB liquid medium containing 100 mg / L Amp, sealing the tube, and shaking it on a shaker until the bacterial suspension becomes turbid, thus obtaining a monoclonal colony suspension.

[0036] (7) Select single-clone colony suspensions for PCR detection. The primers used for PCR are shown in Table 6, the reaction system is shown in Table 7, and the reaction program is as follows: 95℃ pre-denaturation for 300s; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 100s, for a total of 35 cycles; 72℃ extension for 300s, 4℃ +∞.

[0037] Table 5

[0038] Table 6

[0039] Table 7

[0040] After the PCR reaction was completed, 5 μL of the PCR product was subjected to agarose gel electrophoresis at 185 V, 180 mA for 20 min. The gel was then removed and placed in a gel imaging system for observation. If a distinct band was observed in the 2000–3000 bp range, the positive bacteria with the corresponding band were sent to a sequencing company for sequencing. If the sequencing sequence was confirmed to be consistent with GpOSC2, the construction of the pYES2-OSC2 expression plasmid was confirmed to be successful.

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

[0042] Table 8

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

[0044] Table 9

[0045] Yeast cells were collected and induced to grow in 100 mL SC-Ura liquid medium with 2% galactose. The culture was carried out at 30 °C with shaking at 220 rpm for 5 days.

[0046] The cultured cells were resuspended in 0.1M potassium dihydrogen phosphate buffer (pH 7.0) containing 2% glucose and stirred at 220 rpm for 1 day at 30°C. After collecting the yeast cells, they were lysed by ultrasonic extraction with analytical methanol for 1 hour to obtain the lysed yeast culture. The ultrasonic disruptor used for ultrasonic extraction had an output power of 500W and a working frequency of 20kHz.

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

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

[0049] Table 10

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

[0051] 2 Results 2.1 Gene function characterization The successfully recombinant plasmid pYES2-OSC2 was transformed into strain BY4742 via lithium acetate conversion. After fermentation, the fermentation products were extracted and analyzed by HPLC. 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 foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

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

2.

2. The gene encoding the GpOSC2 oxidase of Gynostemma pentaphyllum as described in 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.

1.

4. A recombinant vector containing the gene shown in claim 2 or 3.

5. A recombinant genetically engineered bacterium obtained by transforming Saccharomyces cerevisiae BY4742 strain with the recombinant vector of claim 4.

6. The use of the recombinant genetically engineered bacteria according to claim 5 in the preparation of Dammarenediol-II, lupeol, lanosterol, cycloartenol, and α-amyrinol, characterized in that, GPOSC2 was ligated into the yeast expression vector pYES2 to obtain the pYES2-GpOSC2 vector, which was then transformed into the Saccharomyces cerevisiae BY4742 strain, which can endogenously accumulate 2,3-oxidized squalene, for fermentation. Using the 2,3-oxidized squalene produced by Saccharomyces cerevisiae BY4742 as a substrate, the products Dammarenediol-II, lupeol, lanosterol, cycloartenol, and α-amyrinol were synthesized by Gynostemma pentaphyllum oxidase GPOSC2.

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