Screening and application of a gene encoding glycosyltransferase involved in biosynthesis of triterpenoid saponins

Abstract

The application provides a screening and identification of a glycosyltransferase gene involved in biosynthesis of triterpene saponins and application, and belongs to the technical field of genetic engineering. The glycosyltransferase gene is screened based on the omics research of the radix of traditional Chinese medicine Danxueliu, i.e. Saussurea gossypiphila Maxim, a prokaryotic expression vector pET28a-MBP-CcUGT-g57755 is constructed, and the glycosyltransferase is successfully obtained. It is found that the glycosyltransferase can catalyze Prosaikogenin A to generate Saikosaponin B1, Saikosaponin c1 and Saikosaponin X by using a glycosyl donor UDP-glucose, the catalytic function of the glycosyltransferase is disclosed, gene components are provided for the biological research of triterpene saponin components, and the problem of biological resources of rare saponins is solved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to the screening, identification and application of glycosyltransferase-encoding genes involved in the biosynthesis of triterpenoid saponins. Background Technology

[0002] Triterpenoid saponins are key pharmacodynamic substances in the traditional Chinese medicines Duanxueliu (mainly containing saikosaponin and Buddleja saponin) and Chaihu (with saikosaponin as the core active ingredient). The content of triterpenoid saponins in natural medicinal materials is low, and the high cost of chemical extraction and severe pollution during purification processes limit their potential for new drug development and clinical application. Therefore, the biosynthesis of triterpenoid saponins has become a research hotspot.

[0003] Triterpenoid saponins are a class of natural compounds whose basic structure includes a triterpenoid skeleton and a sugar chain. The sugar chain is linked to the triterpenoid skeleton via glycosidic bonds. The type, number, and position of the sugar chain determine the diversity and function of triterpenoid saponins. Glycosylation modification of triterpenoid saponins (especially at the C-3 position) directly determines their structural diversity and pharmacological activities, such as anti-inflammatory, immunomodulatory, and antitumor effects. Current research indicates that uridine diphosphate-dependent glycosyltransferases (UGTs) are the core enzymes catalyzing the glycosylation modification of triterpenoid saponins. They are responsible for transferring activated sugar donors, such as UDP-fucose, UDP-glucose, and UDP-rhamnose, to the triterpenoid skeleton to form glycosidic bonds. Different glycosyltransferases can recognize different sugar donors and acceptors, resulting in structurally diverse 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 hinders the elucidation of their biosynthetic pathways and makes it impossible to achieve large-scale production through synthetic biology methods. Systematic research is urgently needed.

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

[0006] In view of this, the present invention provides a screening, identification and application of glycosyltransferase encoding genes involved in the biosynthesis of triterpenoid saponins. It successfully discovered a glycosyltransferase that can catalyze C-3 glucosylation modification, providing gene components for the biosynthetic biology research of triterpenoid saponins and solving the problem of rare saponins in biological resources.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

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

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

[0010] Another object of the present invention is to provide a primer set for amplifying the above-mentioned glycosyltransferase gene, said 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, expression cassette, transgenic cell line or recombinant bacteria.

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

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

[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 C-3 glucosylation modification of triterpenoid saponins;

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

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

[0021] Preferably, in steps (4)-(6), the glycosyltransferase utilizes the glycosyl donor UDP-glucose to catalyze the generation of Saikosaponin B1, Saikosaponin c1 and Saikosaponin X from Prosaikogenin A.

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

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

[0024] Using the aforementioned gene, the prokaryotic expression vector pET28a-MBP-CcUGT-g57755 was constructed, and a glycosyltransferase was successfully obtained. Using Prosaikogenin A as a substrate, the catalytic function of the enzyme was verified in vitro. The study found that the glycosyltransferase of this invention can utilize the glycosyl donor UDP-glucose to catalyze the generation of Saikosaponin B1, Saikosaponin c1, and Saikosaponin X from Prosaikogenin A. In summary, the glycosyltransferase of this invention catalyzes the C-3 glucosylation modification of triterpenoid compounds, providing genetic components for the synthetic biology research of triterpenoid saponins and solving the problem of rare saponin biological resources. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 Metabolomics study of triterpenoid saponins in different tissues of *Gnaphalium affine*; 1A-1F represent the contents of triterpenoid saponins in different tissues of *Gnaphalium affine*.

[0027] Figure 2The results are as follows: Screening and phylogenetic analysis of key glycosyltransferases for the biosynthesis of triterpenoid saponins in *Gnaphalium affine*; 2A shows 11 candidate genes screened in Example 2; 2B is the ML phylogenetic tree.

[0028] Figure 3 3A shows the in vitro functional validation of the CcGT-g57755 catalysis of Prosaikogenin A to Saikosaponin B1, Saikosaponinc1, and Saikosaponin X; 3B shows the liquid chromatography detection results of the CcGT-g57755 catalysis of Prosaikogenin A to Saikosaponin B1, Saikosaponin c1, and Saikosaponin X; 3C shows the mass spectrometry fragmentation pattern of the catalytic product Saikosaponin B1.

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

[0030] Figure 5 5A, HSQC and HMBC 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 For: NOESY of Saikosaponin c1 and 1 H- 1 6A, NOESY mass spectrometry results for Saikosaponinc1 (measured in pyridine-d5); 6B, NOESY mass spectrometry results for Saikosaponinc1.1 H- 1 H COSY mass spectrometry results (measured inpyridine-d5).

[0032] Figure 7 For: Saikosaponin X 1 H NMR and 13 10⁻⁶ NMR mass spectrometry results; 7A, Saikosaponin X 1 1H NMR mass spectrometry results (600MHz, measured inpyridine-d5); 7B, Saikosaponin X 13 150 MHz NMR mass spectrometry results (measured in pyridine-d5).

[0033] Figure 8 8A, HSQC and HMBC 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 For: NOESY of Saikosaponin X and 1 H- 1 9A, NOESY mass spectrometry results of Saikosaponin X (measured in pyridine-d5); 9B, NOESY mass spectrometry results of Saikosaponin X. 1 H- 1 H COSY mass spectrometry results (measured inpyridine-d5). Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] Metabolomics study of triterpenoid saponins from different tissues of *Gnaphalium affine*

[0038] Samples of roots, stems, and leaves of *Saikosaponin* stored at -80℃ were removed and placed in liquid nitrogen, ground into a fine powder, and 1.0 g was quickly weighed into a centrifuge tube. 10 mL of methanol was added for ultrasonic extraction. After concentration, 1.0 mL of methanol was added to redissolve the extract. The relative contents of eight compounds (Saikogenin A, Saikogenin F, Prosaikogenin A, Prosaikogenin F, Saikosaponin B1, Saikosaponin A, Buddlejasaponin IV, and Buddlejasaponin IVb) in various tissues of *Saikosaponin* were determined and analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0039] The measurement conditions were as follows: the instruments were an Agilent Technologies 1290 Infinity II and a 6545Q-TOF liquid chromatography-mass spectrometry system; the measurement was performed using Waters Acquity... BEH C18 column (1.7μm, 100×2.1mm), column temperature 40℃;

[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: dry gas temperature 350℃ (flow rate 8.0 L / min), sheath gas temperature 250℃ (flow rate 11.0 L / min), nebulizer 45 psig, VCap 4000 V;

[0042] The relative contents of eight compounds in different tissues were compared and analyzed using Agilent MassHunter Qualitative Analysis 10.0.

[0043] Results analysis: The results showed that Buddlejasaponin IVb was found in high concentrations in the leaves of *Buddlejasaponin var. fasciata*, and its biosynthetic precursors Prosaikogenin A and Saikosaponin B1 were present in the roots of *Buddlejasaponin var. fasciata*. Figure 1A-1C); Buddlejasaponin IV is found in higher concentrations in the leaves of *Sedum aizoon*, while its direct biosynthetic precursor Saikosaponin A mainly accumulates in the leaves, and its indirect biosynthetic precursor Prosaikogenin F is found in higher concentrations in the roots of *Sedum aizoon*. Figure 1 D-1F); Saikogenin A and Saikogenin F were not detected.

[0044] Example 2

[0045] Based on multi-omics research on *Gnaphalium affine*, the gene encoding the key CcUGT enzyme in the biosynthesis of triterpenoid saponins was screened.

[0046] Based on the genome data of *Arabidopsis thaliana*, UGT protein sequences from the TAIR database were extracted and BLASTP sequences were used to identify homologous genes in *Arabidopsis thaliana*. Protein sequences were aligned using Mega software to construct an ML phylogenetic tree, with 1000 bootstrap replicates. RNA-Seq transcriptome data from *Arabidopsis thaliana* roots, stems, and leaves were aligned to the *Arabidopsis thaliana* genome using HiSAT2. Gene expression FPKM values ​​were calculated using Cufflinks to further screen for UGT genes specifically highly expressed in *Arabidopsis thaliana*.

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

[0048] Example 3

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

[0050] (1) Cloning of the target gene

[0051] RNA was extracted from the leaves and roots of *Gnaphalium affine* using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. cDNA templates were synthesized using Takara's PrimeScript™ II 1st Strand cDNA Synthesis Kit for cloning the CcUGT target gene.

[0052] Based on the bioinformatics analysis results and the sequence characteristics of CcUGT-g57755, cloning primers for CcUGT-g57755 were designed, and the target gene was cloned using Novizan 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: 25μL; CcUGT-g57755-F (10μM): 2μL; Primers CcUGT-g57755-R (10μM): 2μL; cDNA: 2μL; ddH2O: 19μL.

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

[0058] DNA electrophoresis was used to obtain the target gene band. The target gene band was then recovered using the Tiangen agarose gel DNA recovery kit. The target gene fragment was then A-tailed using the Takara DNA A-Tailing Kit. Takara's pMD... TM The target gene was ligated into a T-vector using the 18-T Vector Cloning Kit; the sequence accuracy of the amplified gene was verified by first-generation sequencing; the sequenced pMD18-T-CcUGT-g57755 was used to extract plasmids using the Tiangen Plasmid Mini-Prep Kit.

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

[0060]

[0061] (2) Construction of expression carrier

[0062] The pET28a (Cat. No. 69864-3) expression vector was purchased from Novagen. MBP·Tag was synthesized using gene chemical synthesis and inserted between the NcoI and NheI restriction enzyme sites of the pET28a vector to form the pET28a-MBP vector. The pET28a-MBP vector was then cleaved using Takara's EcoRI and XhoI restriction enzymes to obtain a linearized vector fragment. The pET28a-MBP-CcUGT-g57755 prokaryotic expression vector was constructed using the Novizan ClonExpress II One Step Cloning Kit based on a homologous recombination strategy.

[0063] The accuracy of the expression vector construction was verified by first-generation sequencing technology. The pET28a-MBP-CcUGT-g57755, which was sequenced without error, was used to extract plasmids using the Tiangen plasmid mini-prep kit.

[0064] The amplification primer sequences for 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 recombinant expression vector pET28a-MBP-CcUGT-g57755 was transformed into the BL21(DE3) *Escherichia coli* expression strain. 2 mL of the overnight culture was added to 40 mL of LB broth (containing 50 μg / mL kanamycin resistance), and activated at 37°C and 200 rpm for 3 h. IPTG was then added to a final concentration of 0.3 mM, and the culture was induced at 16°C and 160 rpm for 24 h. The cells were collected by centrifugation and sonicated to obtain the crude protein enzyme. The amino acid sequence of the obtained glycosyltransferase CcUGT-g57755 is as follows:

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

[0070] Example 4

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

[0072] The crude protein enzyme obtained in Example 3 was used to perform an in vitro 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 available standards, qualitative analysis was performed using liquid chromatography-mass spectrometry (LC-MS / MS) (as described in the metabolomics detection methods above). For unknown products, product preparation was performed, and structural characterization was conducted using nuclear magnetic resonance spectroscopy.

[0073] Results analysis: CcUGT-g57755 has the following functions: It utilizes the glycosyl donor UDP-glucose to catalyze the generation of Saikosaponin B1, Saikosaponin C1, and Saikosaponin X from Prosaikogenin A. Figures 3-9 ).

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

[0075] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A glycosyltransferase involved in the biosynthesis of triterpenoid saponins, characterized in that, The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO.

6.

2. A gene encoding the glycosyltransferase of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. A primer set for amplifying the glycosyltransferase gene of claim 2, characterized in that, The primer set is CcUGT-g57755-F and CcUGT-g57755-R, with the following specific sequences: CcUGT-g57755-F: 5'-ATGGAGATGGAGATGGAGAATTACGAACT-3', SEQ ID No. 1; CcUGT-g57755-R: 5'-TTAAGCATTATTAATGTTACTAATAACATCC TC-3', SEQ ID No.

2.

4. A biomaterial containing the glycosyltransferase gene of claim 2, characterized in that, The biological material is a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

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

6. The application according to claim 5, characterized in that, In the application of steps (4)-(6), the glycosyltransferase uses the glycosyl donor UDP-glucose to catalyze the generation of Saikosaponin B1, Saikosaponin c1 and Saikosaponin X from Prosaikogenin A.

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