DNA molecule for coding 20-hydroxyecdysone biosynthetic protein, 20-hydroxyecdysone biosynthetic protein and application of 20-hydroxyecdysone biosynthetic protein

Abstract

The invention discloses a DNA (deoxyribonucleic acid) molecule for coding 20-hydroxyecdysone biosynthetic protein and the 20-hydroxyecdysone biosynthetic protein, the 20-hydroxyecdysone biosynthetic protein obtained by expression of the DNA molecule catalyzes pagodone A to generate 20-hydroxyecdysone, and the method is high in efficiency and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a DNA molecule encoding a 20-hydroxyecdysone biosynthetic protein, a 20-hydroxyecdysone biosynthetic protein, and the application of the 20-hydroxyecdysone biosynthetic protein in the preparation of 20-hydroxyecdysone. Background Technology

[0002] Ecdysones are widely found in animals, plants, and microorganisms in nature. To date, more than 520 ecdysones have been isolated from natural sources, among which 20-hydroxyecdysone (20E) is an important representative, and its research started earliest and is the most extensive and comprehensive.

[0003] 20-Hydroxyecdysone, an important hormone in all arthropods, maintains their normal growth, development, and reproduction, and is widely used in animal science and aquaculture. Furthermore, numerous in vivo and in vitro studies have shown that 20-hydroxyecdysone also possesses broad pharmacological activities in mammals, such as enhancing anabolism, lowering blood lipids, anti-inflammation, and liver protection. This has led to its increasingly widespread application in pharmaceuticals and health products. Currently, a wide variety of health products and cosmetics with 20-hydroxyecdysone as a main ingredient have been developed on the market, indicating a huge market potential. Therefore, the market demand for this molecule has been continuously increasing in recent years.

[0004] The current industrial production of 20-hydroxyecdysone is highly dependent on the extraction of rhizomes from Yunnan's specialty plant, Dewdrop. However, its production system faces three major bottlenecks: (1) Limited supply of raw materials: Dewdrop has a long growth cycle (2-3 years), a narrow geographical distribution (1600-2500 meters above sea level in Yunnan), and significant interannual fluctuations in effective components (>30%); (2) Low process efficiency: The existing extraction and purification process requires multiple stages of organic solvent extraction, column chromatography, and recrystallization, with a total yield of only 1.1-1.3%, and generates highly toxic waste liquid (methanol / ethyl acetate ratio >85%); (3) Product purity constraints: Plant-derived 20-hydroxyecdysone contains many isomer impurities and requires multiple purifications to meet the production and application standards (HPLC purity >98%).

[0005] It is evident that existing processes for preparing 20-hydroxyecdysone face challenges such as limited raw materials, inefficient extraction techniques, and insufficient purity. Developing novel, efficient, and industrially viable production methods presents new challenges for researchers. This invention proposes a technical pathway for the biosynthesis of 20-hydroxyecdysone, filling a gap in domestic and international research on the biosynthesis of ecdysone. Identifying the biosynthetic genes involved in the synthesis of 20-hydroxyecdysone in *Desmodium styracifolium* and applying them to synthetic biology-based production development is of significant importance. Summary of the Invention

[0006] The primary objective of this invention is to provide a DNA molecule encoding a 20-hydroxyecdysone biosynthetic protein, thereby addressing the shortcomings of existing technologies in the preparation of 20-hydroxyecdysone, such as limited raw materials and inefficient extraction processes.

[0007] A second objective of this invention is to provide a carrier.

[0008] A third objective of this invention is to provide a 20-hydroxyecdysone biosynthetic protein.

[0009] The fourth objective of this invention is to provide an application of a 20-hydroxyecdysone biosynthetic protein in the preparation of 20-hydroxyecdysone.

[0010] To achieve the above objectives, the following technical solution is adopted: A DNA molecule encoding a 20-hydroxyecdysone biosynthetic protein, the nucleotide sequence of which is shown in SEQ ID No: 1.

[0011] A vector comprising the aforementioned DNA molecule encoding the 20-hydroxyecdysone biosynthetic protein.

[0012] Furthermore, the vector is one of plasmid, engineered bacteria, cell line, and expression cassette.

[0013] Furthermore, the plasmid is pCAMBIA35S-4×Myc-CMV-3×FLAG plasmid.

[0014] Furthermore, the engineered bacteria is one of Escherichia coli or Agrobacterium.

[0015] A 20-hydroxyecdysone biosynthetic protein was obtained by expression of the above-mentioned DNA molecule.

[0016] Furthermore, the amino acid sequence of the 20-hydroxyecdysone biosynthetic protein is shown in SEQ ID No: 2.

[0017] Application of the above-mentioned 20-hydroxyecdysone biosynthetic protein in the preparation of 20-hydroxyecdysone.

[0018] Furthermore, the preparation process of 20-hydroxyecdysone includes the following steps: using ponasterone A as a substrate, and catalyzing with 20-hydroxyecdysone biosynthetic protein, to obtain the product 20-hydroxyecdysone.

[0019] Furthermore, it also includes the following steps: injecting ponasterone A compound and engineered bacteria containing DNA molecules encoding a 20-hydroxyecdysone biosynthetic protein into tobacco.

[0020] The present invention has the following beneficial effects: This invention proposes a 20-hydroxyecdysone biosynthesis gene and its encoded protein, which can catalyze the formation of 20-hydroxyecdysone from ponasterone A. Furthermore, the preparation of 20-hydroxyecdysone is achieved through heterologous expression in tobacco, solving the technical problem that the current production of 20-hydroxyecdysone heavily relies on the extraction of Yunnan dew grass raw materials, and improving production efficiency. Attached Figure Description

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0022] Figure 1 This is a schematic diagram of the reaction mechanism for the formation of 20-hydroxyecdysone from Ponasterone A as a substrate under the catalysis of CaCYP571 protein; Figure 2 This is an agarose gel electrophoresis image of the CaCYP571 gene clone from an embodiment of the present invention; Figure 3 This is a mass spectrometry image of Ponasterone A, the substrate of CaCYP571 protein. Figure 4 Mass spectrometry identification of the intermediate compound catalyzed by CaCYP571 protein to form 20-hydroxyecdysone. Detailed Implementation

[0023] Example 1 Obtaining the full-length cDNA sequence of the CaCYP571 protein-coding gene described in this invention 1. Extraction of RNA from the root tissue of *Dewdrop* plant. The specific method is as follows: Fresh root tissue from healthy *Dewdrop* plants was collected and total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The quality of the extracted RNA was assessed using a spectrophotometer, and RNA integrity was verified by 1% agarose gel electrophoresis. First-strand cDNA synthesis was performed using the PrimeScript™ II First-Strand cDNA Synthesis Kit (TaKaRa, Kusatsu, Japan) with random hexameric primers. The reaction was carried out at 30°C for 10 min, followed by heat inactivation at 70°C for 15 min. The RNA template was removed by treatment with RNase H at 50°C for 20 min. The resulting cDNA library was used as a template for candidate gene cloning.

[0024] 2. The CaCYP571 gene was cloned from a cDNA library of *Dewdrop* root tissue.

[0025] Gene cloning was performed using the primers listed in Table 1 and a high-fidelity enzyme in 2 × Phanta Max Master Mix (Vazyme, Nanjing, China). The 50 μL PCR reaction mixture consisted of: 2 μL cDNA template, 2 μL each of primers CaCYP571-F and CaCYP571-R, 25 μL 2 × Phanta Max Master Mix, and 19 μL ddH2O.

[0026] PCR reaction conditions: 95℃ pre-denaturation for 3 min; cycling program: 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 2 min, 34 cycles, with a final extension at 72℃ for 5 min. The reaction product was recovered using a standard agarose gel DNA recovery kit (Omega Bio-Tek) and ligated into the pCAMBIA35S-4×Myc-CMV-3×FLAG plasmid vector. The ligation product was then transformed into *E. coli* TOP10 competent cells and plated on kanamycin-containing selective plates, incubated at 37℃ for 12–16 h.

[0027] Colony PCR identification was then performed. After the reaction, 5 μL of the reaction product was taken for agarose gel electrophoresis to identify positive clones. Plasmids from the positive clones were extracted and sent to a sequencing company (Shanghai Sangon Biotech) for sequencing. The selected positive clones were then identified by PCR as follows. Figure 2 As shown, Figure 2 The middle arrow points to the band of the amplified target gene. According to the gene sequencing results, its deoxyribonucleotide sequence is confirmed as shown in SEQ ID NO: 1, and its encoded amino acid sequence is shown in SEQ ID NO: 2.

[0028] Table 1. Primers used in the examples to clone CaCYP571 from cDNA in *Dewdrop* root tissue. nucleotide sequence SEQ ID No. 1: amino acid sequence SEQ ID No. 2: MDVILSLFLLVSSLFFLLRRGRPSDKNFAPLKPYPIVGNLPHFFANSHRWQQWSSELISLSPSATTVTVAPLVFTANPDVIEHVTKTNLSNYVKITGVDHVMECFGRSAVTTNGEEWRIHRKD AVSEISNKSFRAHILSTVNNKLANGLAPILRRASQSGQILDLQETLERFAFDNMCNVVFGTLDLDSLNRGGSQGERLFRAFEEAMDLSINRSSEALAIVWKLKKLLALGSERLRLRDCVRAIDD FVQDRIIYANNSRRDGLPIGPDLVSRLVSNHDHKYIRDMAATFVLAGRDTTPSALTWFFWTLCSNPPVLKKIRQEIKQIRSEKQGEEVMFSLEDIRKMNYLHAAISESMRLHPPVTLISRDA VASDDLPDGSRVDKNWTIMISTLAMTRRQEIWGTDSNEFKPERWIEEGVYEPKSAFVNPTFLCGPRICIGKDMAYVQMKAVAASVLESFDVEVVEERGKHEYSMVTKMVGGLPVRFKAPTQNV Example 2 Agrobacterium transformation and Agrobacterium-mediated transient expression in tobacco A. Transformation of Agrobacterium tumefaciens using a tobacco heterologous expression vector TOP10 *E. coli* strains expressing the *pCAMBIA35S-4×Myc-CMV-3×FLAG* plasmid containing the *CaCYP571* gene were cultured in 10 ml of selective LB medium supplemented with kanamycin at 37°C for 12–16 h. Plasmids were extracted using the Omega Bio-Tek Plasmid Mini Kit II. 100 μL of competent cells from a -80°C freezer were placed on ice until partially thawed, then inserted into ice while in an ice-water mixture. The recombinant plasmid was added and gently mixed. The mixture was then incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min. 700 μL of fresh LB medium (without antibiotics) was added, and the cells were incubated at 28°C and 150 rpm for 2–3 h for recovery. After resuscitation, the bacterial cells were collected by centrifugation at 6000 rpm for 1 min. 50 μL of supernatant was retained and the bacterial block was gently resuspended by pipetting. The resuspended block was then spread on LB solid medium containing 50 μg / mL rifampicin, 50 μg / mL gentamicin, and 25 μg / mL kanamycin. After incubation at 28°C for 2-3 days, single Agrobacterium clones were picked and colony PCR was performed for verification. Agrobacterium with correct verification results were preserved.

[0029] B. Agrobacterium-mediated transient expression in tobacco Agrobacterium containing the recombinant expression vector was cultured overnight at 28°C until OD... 600 The bacterial cells were centrifuged at 4000 rpm for 10 min at a value of 2.5, 4 °C, and resuspended in tobacco injection buffer (50 mM MES, 22.2 mM glucose, 10 mM MgCl2, 100 μM acetylsylcholine) for washing. After centrifugation, the cells were centrifuged again at 4 °C and 4000 rpm for 10 min. After centrifugation, the bacterial cells were resuspended in tobacco injection buffer and the OD value was adjusted. 600 The value was 0.6. After incubating at room temperature in the dark for 2-3 hours, Agrobacterium was injected into tobacco leaves with good growth conditions using a needleless injector. After the injection of Agrobacterium, the tobacco was cultured for 4 days. Then, Ponasterone A was injected into the tobacco leaves injected with Agrobacterium using a needleless injector. After standing for 1 day, the tobacco leaves were collected and their metabolites were detected by high-resolution liquid chromatography-mass spectrometry.

[0030] Example 3 Functional identification of the CaCYP571 gene Agrobacterium containing the CaCYP571 gene expression vector from Example 2 was injected into tobacco. Four days later, Ponasterone A was injected, and the mixture was allowed to stand for 24 hours. Leaf discs (6 leaf discs with a diameter of 1.0 cm were collected from each experimental group) were then collected using a 1.0 cm diameter cork drill. The leaf discs were transferred to 2 mL centrifuge tubes and lyophilized overnight. The dried tissue was extracted with 600 μL of methanol and homogenized. The homogenate was centrifuged at 15,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm PES filter into a liquid chromatography vial for subsequent high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) analysis. Parallel control experiments were also conducted: blank control group 1, blank control group 2, and blank control group 3.

[0031] Blank control group 1: Tobacco Benedict leaves; Blank control group 2: Tobacco Benzovia leaves were injected with Agrobacterium expressing the CaCYP571 gene only; Blank control group 3: Ponasterone A was injected only into tobacco leaves; The blank control group and the experimental group were subjected to experiments and tests according to the same treatment steps and methods.

[0032] Results: The expression of the 20-hydroxyecdysone biosynthesis-related protein CaCYP571 from *Nicotiana benthamiana* was detected in *Nicotiana benthamiana*, enabling the conversion of Ponasterone A into 20-hydroxyecdysone. Specifically, no 20-hydroxyecdysone was detected in the three control groups (blank *Nicotiana benthamiana* leaves, tobacco leaves injected with *Agrobacterium* expressing the CaCYP571 gene, and tobacco leaves injected with Ponasterone A). 20-hydroxyecdysone was only produced in the experimental group expressing CaCYP571 protein and simultaneously injected with Ponasterone A. Standards for both Ponasterone A and 20-hydroxyecdysone were available. The retention times and mass spectrometry identification of the experimental and control groups on high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) are shown below. Figure 3 , Figure 4 As shown.

[0033] Therefore, this invention successfully identified a functional gene derived from *Ponasterone A* that can catalyze the conversion of Ponasterone A intermediate into 20-hydroxyecdysone.

[0034] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A DNA molecule encoding a 20-hydroxyecdysone biosynthetic protein, characterized in that, The nucleotide sequence of the DNA molecule is shown in SEQ ID No:

1.

2. A carrier, characterized in that, Includes the DNA molecule encoding the 20-hydroxyecdysone biosynthetic protein as described in claim 1.

3. The carrier according to claim 2, characterized in that, The vector is one of plasmid, engineered bacteria, cell line, and expression cassette.

4. The carrier according to claim 3, characterized in that, The plasmid is pCAMBIA35S-4×Myc-CMV-3×FLAG plasmid.

5. The carrier according to claim 3, characterized in that, The engineered bacteria is one of Escherichia coli or Agrobacterium.

6. A 20-hydroxyecdysone biosynthetic protein, characterized in that, It is obtained by expressing the DNA molecule as described in claim 1.

7. The 20-hydroxyecdysone biosynthetic protein according to claim 6, characterized in that, The amino acid sequence of the 20-hydroxyecdysone biosynthetic protein is shown in SEQ ID No:

2.

8. The use of the 20-hydroxyecdysone biosynthetic protein of claim 6 or 7 in the preparation of 20-hydroxyecdysone.

9. The application of the 20-hydroxyecdysone biosynthetic protein according to claim 8 in the preparation of 20-hydroxyecdysone, characterized in that, The preparation of 20-hydroxyecdysone includes the following steps: using ponasterone A as a substrate, and catalyzing with 20-hydroxyecdysone biosynthetic protein, the product 20-hydroxyecdysone is obtained.

10. The application of the 20-hydroxyecdysone biosynthetic protein according to claim 9 in the preparation of 20-hydroxyecdysone, characterized in that, It also includes the following steps: Engineered bacteria containing DNA molecules encoding the biosynthetic protein of 20-hydroxyecdysone and ponasterone A compound were injected into tobacco.

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