Screening identification and application of glycosyl transferase coding gene participating in triterpenoid saponin biosynthesis

Abstract

The invention provides screening identification and application of a glycosyl transferase coding gene participating in triterpenoid saponin biosynthesis. Belongs to the technical field of gene engineering. A glycosyl transferase gene is screened based on the multi-omics research of a traditional Chinese medicine clinopodium polycephalum-based original plant-calamint, a pET28a-MBP-CcUGT-g57755 prokaryotic expression vector is constructed, the glycosyl transferase is successfully obtained, the research finds that the glycosyl transferase can utilize a glycosyl donor UDP-glucose to catalyze Prosagogenin A to generate Saikosaponin B1, Saikosaponin c1 and Saikosaponin X, the catalytic function of the glycosyl transferase is disclosed, and the Saikosaponin B1, the Saikosaponin c1 and the Saikosaponin X can be used for preparing the glycosyl transferase. A gene component is provided for the biological research on synthesis of triterpenoid saponin components, and the biological resource problem of rare saponin is solved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and more particularly to the screening, identification and application of a glycosyltransferase encoding gene involved in the biosynthesis of triterpenoid saponins. Background Art

[0002] Triterpenoid saponins are key pharmacological compounds in the traditional Chinese medicines Duanxueliu (primarily containing saikosaponins and buddlejasin) and Bupleurum (with saikosaponins as the core active ingredient). However, their low concentrations in natural medicinal materials hinder their development and clinical application due to high chemical extraction costs and significant pollution during the purification process. Consequently, their biosynthesis has become a research hotspot.

[0003] Triterpenoid saponins are a class of natural compounds whose basic structure consists of a triterpenoid backbone and a sugar chain, with the sugar chain linked to the triterpenoid backbone via glycosidic bonds. The type, number, and attachment position of the sugar chain determine the diversity and function of triterpenoid saponins. Glycosylation modifications of triterpenoid saponins, particularly at the C-3 position, directly determine their structural diversity and pharmacological activities, such as anti-inflammatory, immunomodulatory, and anti-tumor effects. Current research indicates that uridine diphosphate-dependent glycosyltransferases (UGTs) are the core enzymes catalyzing the glycosylation of triterpenoid saponins. They are responsible for transferring activated sugar donors, such as UDP-fucose, UDP-glucose, and UDP-rhamnose, to the triterpenoid backbone to form the glycosidic bond. Different glycosyltransferases can recognize different sugar donors and acceptors, resulting in a diverse range of triterpenoid saponins. Therefore, glycosyltransferases are crucial for the synthesis of triterpenoid saponins.

[0004] However, the key UGT enzymes and their encoding genes for the C-3 glycosylation modification of triterpenoid saponins have not yet been clearly identified, which has hindered the analysis of their biosynthetic pathway and made it impossible to achieve large-scale production through synthetic biology. Systematic research is urgently needed.

[0005] Therefore, how to provide a glycosyltransferase that can catalyze the glycosylation modification of the C-3 position of triterpenoid compounds is a problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for screening, identifying and applying a gene encoding a glycosyltransferase involved in the biosynthesis of triterpenoid saponins. The invention successfully discovered a glycosyltransferase that can catalyze the glucosylation modification at the C-3 position, providing genetic components for the synthetic biology research of triterpenoid saponins and solving the biological resource problem of rare saponins.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A glycosyltransferase involved in the biosynthesis of triterpenoid saponins, the glycosyltransferase consisting of the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having the same enzymatic catalytic performance after substitution, deletion and / or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO. 6.

[0009] Another object of the present invention is to provide: a gene encoding the above-mentioned glycosyltransferase, wherein the gene is composed of the nucleotide sequence shown in SEQ ID NO.3 or a nucleotide sequence shown in SEQ ID No.3 that still has the same enzymatic catalytic performance after substitution, deletion and / or addition of one or more bases.

[0010] Another object of the present invention is to provide: a primer set for amplifying the above-mentioned glycosyltransferase gene, the primer set comprising CcUGT-g57755-F and CcUGT-g57755-R, the specific sequences of which are as follows:

[0011] CcUGT-g57755-F: 5'-ATGGAGATGGAGATGGAGAATTACGAACT-3', SEQ ID No. 1;

[0012] CcUGT-g57755-R: 5'-TTAAGCATTATTAATGTTACTAATAACATCC TC-3', SEQ ID No. 2.

[0013] Another object of the present invention is to provide: a biological material containing the above-mentioned glycosyltransferase gene, wherein the biological material is a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

[0014] Another object of the present invention is to provide the application of the above-mentioned glycosyltransferase or the above-mentioned gene encoding glycosyltransferase or the above-mentioned biomaterial, wherein the application is in any of the following directions:

[0015] (1) Application in the preparation of glycosyltransferases and / or glycosyltransferase-related products;

[0016] (2) Application in the preparation of glycosyltransferase mutants and / or products containing glycosyltransferase mutants;

[0017] (3) Application in the preparation of recombinant glycosyltransferases and / or related products containing recombinant glycosyltransferases;

[0018] (4) Application in the glucosylation modification of the C-3 position of triterpenoid saponins;

[0019] (5) Application in glycosylation modification of triterpenoid saponins;

[0020] (6) Application in the biosynthesis of triterpenoid saponins.

[0021] Preferably, in the application of steps (4) to (6), the glycosyltransferase catalyzes Prosaikogenin A to produce Saikosaponin B1, Saikosaponin c1 and Saikosaponin X using the glycosyl donor UDP-glucose.

[0022] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses liquid chromatography-mass spectrometry (LC-MS) to detect differences in the content of eight target components in the roots, stems, and leaves of Brassica rapa. Then, based on the Brassica rapa genome, members of the Brassica glycosyltransferase (GT) gene family were identified. A phylogenetic tree was constructed to analyze the evolutionary relationship between Brassica rapa and GTs from other species, and key enzymes involved in the glycosylation modification of triterpenoid saponins were initially screened. Finally, differential gene expression was analyzed based on transcriptome data from different Brassica tissues to further screen for key enzyme genes involved in the glycosylation modification of triterpenoid saponins. The above method successfully screened for glycosyltransferase genes.

[0024] The pET28a-MBP-CcUGT-g57755 prokaryotic expression vector was constructed using the aforementioned gene, and the glycosyltransferase was successfully obtained. The catalytic function of the enzyme was verified in vitro using prosaikogenin A as a substrate. The study found that the glycosyltransferase of the present invention can catalyze the conversion of prosaikogenin A to Saikosaponin B1, Saikosaponin C1, and Saikosaponin X using the glycosyl donor UDP-glucose. In summary, the glycosyltransferase of the present invention catalyzes the C-3 glucosylation modification of triterpenoid compounds, providing genetic components for the synthetic biology research of triterpenoid saponins and addressing the bioresource challenge of rare saponins. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0026] Figure 1 Figure 1: Metabolomics study of triterpenoid saponins in different tissues of Herba Cyperi. 1A-1F are the contents of triterpenoid saponins in different tissues of Herba Cyperi.

[0027] Figure 2Figure 2 is: Screening and phylogenetic analysis of key glycosyltransferases in the biosynthesis of triterpenoid saponins from Cyperus rotundus; 2A is the 11 candidate genes screened out in Example 2; 2B is the ML phylogenetic tree.

[0028] Figure 3 Figure 3: In vitro functional verification of CcUGT-g57755 catalyzing the production of Saikosaponin B1, Saikosaponin C1, and Saikosaponin X from Prosaikogenin A; 3A, a schematic diagram of the production of Saikosaponin B1, Saikosaponin C1, and Saikosaponin X from Prosaikogenin A catalyzed by CcGT-g57755; 3B, liquid chromatography detection results of the production of Saikosaponin B1, Saikosaponin C1, and Saikosaponin X from Prosaikogenin A catalyzed by CcGT-g57755; 3C, mass spectrometry fragmentation pattern of the catalytic product Saikosaponin B1.

[0029] Figure 4 Saikosaponin c1 1 H NMR and 13 C NMR mass spectrometry results; 4A, Saikosaponin c1 1 H NMR mass spectrometry results (600 MHz, measured inpyridine-d5); 4B, Saikosaponin c1 13 CNMR mass spectrometry detection results (150 MHz, measured in pyridine-d5).

[0030] Figure 5 5A, HSQC mass spectrometry results of Saikosaponin c1 (measured in pyridine-d5); 5B, HMBC mass spectrometry results of Saikosaponin c1 (measured in pyridine-d5).

[0031] Figure 6 Saikosaponin c1 NOESY and 1 H- 1 H COSY mass spectrometry results; 6A, NOESY mass spectrometry results of Saikosaponin c1 (measured in pyridine-d5); 6B, Saikosaponin c11 H- 1 H COSY mass spectrometry results (measured inpyridine-d5).

[0032] Figure 7 Saikosaponin X 1 H NMR and 13 C NMR mass spectrometry results; 7A, Saikosaponin X 1 H NMR mass spectrometry results (600 MHz, measured inpyridine-d5); 7B, Saikosaponin X 13 C NMR mass spectrometry results (150 MHz, measured in pyridine-d5).

[0033] Figure 8 8A, HSQC mass spectrometry results of Saikosaponin X (measured in pyridine-d5); 8B, HMBC mass spectrometry results of Saikosaponin X (measured in pyridine-d5).

[0034] Figure 9 Saikosaponin X NOESY and 1 H- 1 H COSY mass spectrometry results; 9A, NOESY mass spectrometry results of Saikosaponin X (measured in pyridine-d5); 9B, Saikosaponin X 1 H- 1 H COSY mass spectrometry results (measured inpyridine-d5). DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1

[0037] Metabolomics study on triterpenoid saponins in different tissues of Pinellia ternata

[0038] Root, stem, and leaf samples of Herba Cynanchifolia stored at -80°C were removed and placed in liquid nitrogen. The samples were ground into fine powder, and 1.0 g was quickly weighed into a centrifuge tube. 10 mL of methanol was added for ultrasonic extraction. The extract was concentrated and reconstituted with 1.0 mL of methanol. The relative contents of eight compounds (Saikogenin A, Saikogenin F, ProsaikogeninA, ProsaikogeninF, Saikosaponin B1, Saikosaponin A, Buddlejasaponin IV, and Buddlejasaponin IVb) in various tissues of Herba Cynanchifolia were determined and analyzed by liquid chromatography-mass spectrometry.

[0039] The measurement conditions are as follows: the instrument models are Agilent Technologies 1290InfinityⅡ and 6545Q-TOF liquid chromatography-mass spectrometer; the BEH C18 column (1.7 μm, 100 × 2.1 mm), column temperature 40 °C;

[0040] Chromatographic conditions: mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile (containing 0.1% formic acid), flow rate: 0.2 mL / min, elution program: 0-5 min (25% B to 40% B), 5-13 min (40% B to 42.5% B), 13-14 min (42.5% B to 100% B), 14-20 min (100% B), 20-21 min (100% B to 25% B), 21-26 min (25% B);

[0041] Mass spectrometry conditions: drying gas temperature of 350°C (flow rate of 8.0 L / min), sheath gas of 250°C (flow rate of 11.0 L / min), nebulizer of 45 psig, and VCap of 4000 V;

[0042] Agilent MassHunter Qualitative Analysis 10.0 was used to compare the relative contents of eight compounds in different tissue sites.

[0043] Result analysis: The results showed that Buddlejasaponin IVb was present at a high level in the leaves of Pinus oleraceus, and its biosynthetic precursor compounds Prosaikogenin A and Saikosaponin B1 were present in the roots of Pinus oleraceus. Figure 1A-1C); Buddlejasaponin IV is present at higher levels in the leaves of Pinus oleraceus, its direct biosynthetic precursor Saikosaponin A accumulates mainly in the leaves, and its indirect biosynthetic precursor Prosaikogenin F is present at higher levels in the roots of Pinus oleraceus ( Figure 1 D-1F); Saikogenin A and Saikogenin F were not detected.

[0044] Example 2

[0045] Screening of key CcUGT enzyme encoding genes for triterpenoid saponin biosynthesis based on multi-omics studies of Pinus koraiensis

[0046] Based on the genome data of the genus Eupatoria oleracea, Arabidopsis thaliana UGT protein sequences were extracted from the TAIR database and BLASTP was performed to identify homologous genes in the genus. Protein sequences were aligned using Mega software, and a ML phylogenetic tree was constructed with 1000 bootstrap replicates. RNA-Seq transcriptome data from roots, stems, and leaves of the genus Eupatoria oleracea were aligned to the genus Eupatoria oleracea genome using HiSAT2. Gene expression FPKM values ​​were calculated using Cufflinks to further identify UGT genes that are specifically and highly expressed in the genus Eupatoria oleracea.

[0047] Results: A total of 214 GT-encoding genes were annotated in the genome using bioinformatics techniques, and 11 candidate genes were screened out by combining gene differential expression analysis ( Figure 2 A), among which g57755 was significantly highly expressed in plant leaves; phylogenetic analysis showed that g57755 belonged to the UGT71 subfamily ( Figure 2 B).

[0048] Example 3

[0049] Synthesis of glycosyltransferase CcUGT-g57755 involved in the glycosylation of triterpenoid saponins

[0050] (1) Cloning of target genes

[0051] RNA was extracted from the leaves and roots of Pinus koraiensis using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, and cDNA templates were synthesized using Takara's PrimeScriptTMII 1st Strand cDNA Synthesis Kit for cloning the CcUGT target gene.

[0052] Based on the results of bioinformatics analysis and the sequence characteristics of CcUGT-g57755, CcUGT-g57755 cloning primers were designed, and the target gene was cloned using Novozymes 2×Phanta Flash MasterMix.

[0053] The amplification primer sequences are as follows:

[0054] CcUGT-g57755-F: 5'-ATGGAGATGGAGATGGAGAATTACGAACT-3', SEQ ID No. 1;

[0055] CcUGT-g57755-R: 5'-TTAAGCATTATTAATGTTACTAATAACATCC TC-3', SEQ ID No. 2.

[0056] The cloning system is as follows: primers: 2×Phanta Flash Master Mix: 25 μL; CcUGT-g57755-F (10 μM): 2 μL; primer CcUGT-g57755-R (10 μM): 2 μL; cDNA: 2 μL; ddH2O: 19 μL.

[0057] PCR reaction parameters: denaturation at 98°C for 30 s; 98°C for 10 s; annealing at 54°C for 10 s; extension at 72°C for 20 s; 40 cycles; final extension at 72°C for 1 min.

[0058] DNA electrophoresis was used to detect the target gene bands, and the target gene bands were recovered using the Tiangen ordinary agarose gel DNA recovery kit. The target gene fragments were A-tailed using Takara's DNA A-Tailing Kit. TM The target gene was ligated to the T vector using the 18-T Vector Cloning Kit; the sequence accuracy of the amplified gene was verified by first-generation sequencing; and the plasmid pMD18-T-CcUGT-g57755, which had been sequenced correctly, was extracted using the Tiangen Plasmid Mini Kit.

[0059] The nucleotide sequence of the target gene CcUGT-g57755 is as follows:

[0060]

[0061] (2) Construction of expression vector

[0062] The pET28a (Cat. No. 69864-3) expression vector was purchased from Novagen. The MBP tag was chemically synthesized between the NcoI and NheI restriction enzyme sites of the pET28a vector to create the pET28a-MBP vector. The pET28a-MBP vector was cut with Takara's EcoR I and Xho I restriction enzymes to obtain a linearized vector fragment. The pET28a-MBP-CcUGT-g57755 prokaryotic expression vector was constructed using the Novagen ClonExpress II One-Step Cloning Kit based on homologous recombination.

[0063] The accuracy of the expression vector construction was verified by first-generation sequencing technology, and the pET28a-MBP-CcUGT-g57755 with correct sequencing was extracted using the Tiangen Plasmid Mini Extraction Kit.

[0064] The amplification primer sequences of the expression vector are as follows:

[0065] pET28a-MBP-CcUGT-g57755-F5'-atgggtcgcggatccgaattcATGGAGATGGAGATGGAGAATTACG-3', SEQ ID No. 4;

[0066] pET28a-MBP-CcUGT-g57755-R5'-gtggtggtggtggtgctcgagTTAAGCATTATTAATGTTACTAATAACATCCTC-3', SEQ ID No. 5.

[0067] (3) Synthesis of glycosyltransferase CcUGT-g57755

[0068] The pET28a-MBP-CcUGT-g57755 recombinant expression vector was transformed into the BL21(DE3) Escherichia coli expression strain. 2 mL of the overnight culture was added to 40 mL of LB liquid culture (containing 50 μg / mL kanamycin for resistance) and incubated at 37°C, 200 rpm, for activation for 3 hours. IPTG was added to a final concentration of 0.3 mM and induced at 16°C, 160 rpm, for 24 hours. The cells were harvested by centrifugation and ultrasonic cell disruption was used to obtain a crude protein. The amino acid sequence of the resulting glycosyltransferase, CcUGT-g57755, was determined as follows:

[0069] MEMEMENYELVFIPSPGLSHLVSTVEAAKLLLLRDSRLSITVLTMQFPNDTTVEDYTTKICAASTASSRLTLTALPNLPDWTPQSKNFLFDYIDKQITSVREIISDLSKRRRGGKLAGIVL DMFCLSFIDVAREFSLPSYVFFTSGACGLGLFQYLISLKFGQNKDLSEYKNSDEHLPVPCFSLPFPAKLLPAVFLDGGDMAEIFFNYFKRIPETQGVVVNTFYELEPYAIDSMSSKTPKVY PVGPILDLSSQSNDDDVAKWLDDQPEKSVIFLCFGTMGTFVEAQVREIALALENSGCRFLWSLRKPGVKGGKKLVDEYVDFGDVLPEGFLERTRGVGKVIGWAAQKAVLGHAAVGGFVSHC GWNSTLESVWFGVPIATFPMYAEQQLNAFLLVEELGMAEKIRLDYSVDFKREREPEIVGAEDIEAAIRRVMAGESGGVREKVEEMQRKGRAALEEGGSSYKAQALFIEDVISNINNA, SEQ ID NO.6.

[0070] Example 4

[0071] Functional verification of the glycosyltransferase CcUGT-g57755 involved in the glycosylation of triterpenoid saponins

[0072] The crude protein enzyme obtained in Example 3 was used to perform an in vitro test tube reaction (200 μL reaction system: 100 mM Tris-HCl, pH 8.5, 100 μM substrate (prosaikogenin A), 5 mM glycosyl donor (UDP-glucose), 50 μL crude protein; reaction at 30°C for 3 h) to verify its catalytic activity. For substrates and products with standard samples, qualitative analysis was performed using liquid chromatography-mass spectrometry (such as the metabolomics detection method described above). For unknown products, product preparation was performed and structural characterization was performed in combination with nuclear magnetic resonance spectroscopy.

[0073] Results: CcUGT-g57755 has the following functions: using the sugar donor UDP-glucose to catalyze Prosaikogenin A to produce Saikosaponin B1, Saikosaponin c1 and Saikosaponin X ( Figure 3-Figure 9 ).

[0074] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to in detail.

[0075] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A glycosyltransferase involved in the biosynthesis of triterpenoid saponins, It is characterized in that The glycosyltransferase is composed of the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having the same enzyme catalytic performance after replacing, deleting and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.

6.

2. A gene encoding the glycosyltransferase according to claim 1, It is characterized in that The gene is composed of the nucleotide sequence shown in SEQ ID NO.3 or a nucleotide sequence having the same enzyme catalytic performance after the nucleotide sequence shown in SEQ ID No.3 is replaced, deleted and / or added with one or more bases.

3. A primer set for amplifying the glycosyltransferase gene according to claim 2, It is characterized in that The primer set includes CcUGT-g57755-F and CcUGT-g57755-R, and the specific sequences are as follows: CcUGT-g57755-F: 5'-ATGGAGATGGAGATGGAGAATTACGAACT-3', SEQ ID No. 1; CcUGT-g57755-R: 5'-TTAAGCATTATTAATGTTACTAATAACATCC TC-3', SEQ ID No.

2.

4. A biological material containing the glycosyltransferase gene according to claim 2, It is characterized in that The biological material is a recombinant vector, an expression box, a transgenic cell line or a recombinant bacterium.

5. Use of the glycosyltransferase according to claim 1 or the gene encoding the glycosyltransferase according to claim 2 or the biomaterial according to claim 4, It is characterized in that The application is any of the following: (1) Application in the preparation of glycosyltransferases and / or glycosyltransferase-related products; (2) Application in the preparation of glycosyltransferase mutants and / or products containing glycosyltransferase mutants; (3) Application in the preparation of recombinant glycosyltransferases and / or products containing recombinant glycosyltransferases; (4) Application in the glucosylation modification of the C-3 position of triterpenoid saponins; (5) Application in glycosylation modification of triterpenoid saponins; (6) Application in the biosynthesis of triterpenoid saponins.

6. The use according to claim 5, It is characterized in that In the application of steps (4)-(6), glycosyltransferase catalyzes Prosaikogenin A to produce Saikosaponin B1, Saikosaponin c1 and Saikosaponin X using the glycosyl donor UDP-glucose.

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