Glycosyltransferase protein of physalis and its encoding gene and application

Abstract

The present invention discloses a glycosyltransferase protein of stonewort and its encoding gene and application. The glycosyltransferase protein of stonewort provided by the present invention is a protein of a) or b) as follows: a) a protein composed of the amino acid sequence shown in sequence 2 in the sequence list; b) a protein derived from a) in which the amino acid sequence shown in sequence 2 in the sequence list is substituted and / or deleted and / or added with one or more amino acid residues and has stonewort glycosyltransferase activity. The present invention is based on the full-length transcriptome data of the third generation of hanging stonewort, and uses a reverse genetics method to find and identify the specific glycosylation modification enzyme LpUGT95 synthesized by stonewort-7-O-β-D-glucoside and stonewort-5-O-β-D-glucoside, and provides a glycosyltransferase protein and its encoding sequence for the biosynthesis of stonewort-7-O-β-D-glucoside and stonewort-5-O-β-D-glucoside.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chelidonium glycosyltransferase protein and a coding gene and application thereof. Background Art

[0002] Lysionotin (also known as Nevadensin) is chemically named 5,7-dihydroxy-6,8-dimethoxy-2-(4-methoxyphenyl)chromen-4-one. Its molecular formula is C18H16O7. It is soluble in DMSO, slightly soluble in methanol, and insoluble in water. Lysionotin is a relatively specific compound in Gesneriaceae plants. It has been established as a promising natural bioactive substance and may become a new "natural lead" in drug design. Its representative plant, Lysionotus pauciflorus, has abundant wild resources and a wide range of sources, and the extraction method and purification process are relatively simple. Lysionotin is a naturally occurring selective inhibitor of human carboxylesterase 1 (hCE1). Pharmacological studies have shown that chlorella has liver protection, blood pressure reduction, anti-allergy, antibacterial, antiviral, anti-tumor, and neuroprotective effects. At the same time, it is also beneficial for the treatment of lymphatic tuberculosis, pulmonary tuberculosis, bone tuberculosis, and bronchitis in clinical practice. Lena Müller et al. reported the effect of chlorella on topoisomerase (TOPO) in vitro. The results showed that chlorella induced endogenous apoptosis pathways by activating caspase-9 and caspase-3, revealing that cell cycle disruption and apoptosis events play a key role in the cell response of HT29 cells TOPO I poisoning caused by chlorella, and revealing the DNA embedding properties of chlorella. Zhang Yafen found that chlorella may reduce the expression of mast cell stem cell growth factor (c-Kit) receptor and has the effect of alleviating food allergic reactions in mice. Wang et al. found that chlorella is a highly selective inhibitor of hCE1 over hCE2 through preliminary screening of a series of natural flavonoid compounds. However, due to the poor water solubility of chlorella, the promotion and application of chlorella is greatly restricted. After glycosylation, the solubility of chelidonin can be greatly improved, and its efficacy can also be improved. Taking chelidonin as the basic structure, it is an important research work to modify the structure to enhance the activity of chelidonin and expand its application range.

[0003] The research on chlorella in China and abroad remains at the stage of chemical extraction, synthesis and pharmacological activity, and there are few reports on the structural modification of chlorella. As early as 1966, chlorella was first isolated by Farkas in the Iva species and its structure was proved using a 14-step synthesis method. Subsequently, researchers from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences simplified the design route, and the total yield increased by more than 10 times. Chlorella is insoluble in water, and its sodium salt is water-soluble, but clinical injection will cause pain to patients. Therefore, the researcher synthesized a chlorella methylamine glycoside salt for intramuscular injection, which has good antibacterial activity. In summary, there is an urgent need for a highly specific enzyme to specifically synthesize chlorella glycosides to provide a new reference for drug synthesis design. Summary of the invention

[0004] Although there are reports on the chemical extraction of chelidonin glycosides, there is no published method for the biosynthesis of chelidonin glycosides. The present invention is mainly based on the third-generation full-length transcriptome of chelidonin, obtains the chelidonin glycosyltransferase sequence, and through prokaryotic expression of recombinant proteins, confirms that LpUGT95 can catalyze the glycosylation of chelidonin to produce chelidonin-7-O-β-D-glucoside and / or chelidonin-5-O-β-D-glucoside.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention provides a protein, which is the protein of a) or b) as follows:

[0007] a) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing;

[0008] b) A protein derived from a) having the activity of chelidonium glycosyltransferase, wherein one or more amino acid residues of the amino acid sequence shown in Sequence 2 in the sequence list are substituted and / or deleted and / or added.

[0009] The glycosyltransferase activity of stipetoxin catalyzes the glycosylation of stipetoxin to generate stipetoxin-7-O-β-D-glucoside and / or stipetoxin-5-O-β-D-glucoside.

[0010] The coding gene of the protein also belongs to the protection scope of the present invention.

[0011] The coding gene is as shown in 1) or 2) or 3) below:

[0012] 1) Its nucleotide sequence is the DNA molecule shown in Sequence 1 in the sequence list;

[0013] 2) A DNA molecule that hybridizes with the DNA molecule defined in 1) under stringent conditions;

[0014] 3) A DNA molecule having 90% or more homology with the DNA molecule defined in 1) or 2).

[0015] The gene was named LpUGT95, and the protein encoded by it was named LpUGT95. The specific information is as follows: The sequence of the LpUGT95 gene is shown in sequence 1 in the sequence list, which contains 1416 nucleotides and encodes the protein shown in sequence 2 in the sequence list, and sequence 2 consists of 471 amino acids.

[0016] Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant microorganisms containing the encoding gene also fall within the protection scope of the present invention.

[0017] The use of the protein as a glycosyltransferase for phytosyltransferase also falls within the protection scope of the present invention.

[0018] The glycosyltransferase activity of stipetoxin catalyzes the glycosylation of stipetoxin to generate stipetoxin-7-O-β-D-glucoside and / or stipetoxin-5-O-β-D-glucoside.

[0019] The use of the protein and the encoding gene in catalyzing UDP-glucose and chelidonin to produce chelidonin-7-O-β-D-glucoside and / or chelidonin-5-O-β-D-glucoside also falls within the protection scope of the present invention.

[0020] Based on the full-length transcriptome data of the third generation of Clerodendrum sutchuenensis, the present invention uses a reverse genetics method to find and identify the specific glycosylation modification enzyme LpUGT95 for the synthesis of Clerodendrum sutchuenensis-7-O-β-D-glucoside and Clerodendrum sutchuenensis-5-O-β-D-glucose, and provides a glycosyltransferase protein and a coding gene thereof for the biosynthesis of Clerodendrum sutchuenensis-7-O-β-D-glucoside and Clerodendrum sutchuenensis-5-O-β-D-glucose. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For purposes of illustration and not limitation, the present invention will now be described in terms of preferred embodiments thereof, with particular reference to the accompanying drawings, in which:

[0022] Figure 1 Results of LpUGT95 gene cloning and pMAL-c2X ligation vector construction.

[0023] Figure 2 Results for Novablue transformation of pMAL-c2X-LpUGT95.

[0024] Figure 3 This is the SDS-Page gel image of LpUGT95 recombinant protein.

[0025] Figure 4UPLC diagram for the identification of the catalytic activity of LpUGT95 on chelidonium nitrate.

[0026] Figure 5 This is the MS and MS / MS identification diagram of the product catalyzed by LpUGT95 on chelidonium.

[0027] Figure 6 The molecular structures of chlorella solani (left), chlorella solani-5-O-β-D-glucoside (middle) and chlorella solani-7-O-β-D-glucoside (right). DETAILED DESCRIPTION

[0028] Example 1: Cloning of the gene encoding the chelidonin glycosyltransferase protein

[0029] 1. Methods and steps for obtaining genes

[0030] Lysionotus pauciflorus (Non-patent literature that records Lysionotus pauciflorus is: Huang Mei, Li Meijun, Huang Hong, et al. Diversity and geographical distribution of wild Gesneriaceae species in Guizhou Province [J]. Guangxi Plants, 2022, 42(2): 210-219. DOI: 10.11931 / guihaia.gxzw202008055.) Sample plants were collected from Xianheping Town, Anlong County, Guizhou Province, China. Four tissue samples of roots, stems, leaves, and flowers were quickly stored at -80℃ for multi-omics experiments. Based on the third-generation full-length transcriptome of Lysionotus pauciflorus and combined with the PSPG box of the conserved domain of UGTs, 187 LpUGTs sequences ranging from 302aa to 560aa were obtained. Through the joint analysis of the second-generation + third-generation transcriptome and the broad-target metabolome, LpUGT95 was focused on.

[0031] The roots, stems, leaves and flowers of the genus Eupatorium were collected in Xianheping, Anlong County, Guizhou Province, and RNA was extracted and reverse transcribed into cDNA. The primer sequences were designed (as shown in Table 1). The mixed cDNA of roots, stems, leaves and flowers was used as a template and cloned into a LpUGT gene fragment using KOD high-fidelity enzyme (the total volume of the KOD high-fidelity enzyme PCR system was 50 μL: 5 μL 10X Buffer, 3 μL MgSO4, 5 μL dNTP (2mM), 3 μL primer (10mM), 1 μL template and 32 μL water, and the procedure was shown in Table 2). With the help of pEASY-Blunt vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product catalog number CB101-01), the LpUGT95 fragment was successfully connected to the vector (the total volume of the connection system was 2.5 μL: 0.5 μL Mix Buffer with enzyme and 2 μL template, and the reaction was carried out at 25°C for 2h). The ligation system was directly transformed into TransT1 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product catalog number CB101-01), and positive clones were selected for sequencing (the total volume of the colony PCR system was 20 μL: 13 μL Mix Buffer, 1 μL template, 1 μL primer and 5 μL water, the procedure is shown in Table 3), and compared with the transcript sequence. The nucleotide sequence had a 98% similarity with the original data, and the actual sequencing result was the standard.

[0032] Table 1 Primer sequences used for cloning genes

[0033]

[0034] Note: Only the primer sequence of one gene that successfully expressed the recombinant protein is listed.

[0035] Table 2 KOD high-fidelity enzyme PCR reaction program

[0036]

[0037] Table 3 Colony PCR reaction procedure

[0038]

[0039]

[0040] 2. Obtaining gene sequences and their encoded protein sequences

[0041] The obtained LpUGT95 gene contains 1416 nucleotides, as shown in sequence 1 in the sequence list; it encodes a protein of 471 amino acids, as shown in sequence 2 in the sequence list, and the encoded protein is named LpUGT95.

[0042] 3. Gene Function Verification

[0043] We used the prokaryotic system to verify the gene function, constructed the pMAL-c2X-LpUGT vector, and after sequencing confirmed the sequence was correct, successfully transferred the vector into the prokaryotic expression strain Novablue (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) for in vitro verification ( Figure 1 ).

[0044] First, a restriction site linker was added to the LpUGT fragment (the system and procedure were consistent with the above-mentioned gene cloning method, and the primer information was shown in Table 4); after the LpUGT fragment (with the restriction site) was digested, it was constructed into the expression vector pMAL-c2X using T4-DNA ligase (provided by Yin, and the non-patent literature describing the expression vector pMAL-c2X is: Yin Q, Han X, Han Z, Chen Q, Shi Y, Gao H, Zhang T, Dong G, Xiong C, Song C, Sun W, Chen S. Genome-wide analyses reveals aglucosyltransferase involved in rutin and emodin glucoside biosynthesis in tartary buckwheat. Food Chem. 2020 Jul 15; 318:126478. doi:10.1016 / j.foodchem.2020.126478. Epub 2020 Feb 25.PMID:32126466.) (the total ligation system is 7 μL: 3.5 μL Mix Buffer, 2.8 μL LpUGT fragment and 0.7 μL pMAL-c2X; react overnight at 4°C); secondly, after the ligation system is transformed into TransT1, positive clones are selected ( Figure 1 ); after extracting the plasmid, it was transformed into the expression strain Novablue.

[0045] Table 4 Primer sequences used to construct prokaryotic expression

[0046]

[0047] Notes: Lowercase letters in the sequence represent protected bases and restriction sites.

[0048] The Novablue conversion steps are as follows:

[0049] 1. Take 5 μL of plasmid (about 200 μg / μL) into 50 μL Novablue competent medium (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) and mix well;

[0050] 2. After 20-30 minutes of ice bath, heat shock in 42°C water bath for 1 minute; ice bath for 2 minutes;

[0051] 3. Add 500 μL of liquid LB medium (without antibiotics) and culture at 37°C with shaking for 1 hour (200 rpm);

[0052] 4. Take 1 / 2 volume of bacterial solution, mix well by pipetting, and then spread on LB solid medium (containing ampicillin);

[0053] 5. Incubate at 37℃ overnight and select single clones for PCR verification (e.g. Figure 2 ). Three pMAL-c2X-LpUGT95 monoclones were selected, and all of them had positive bands at 1500 bp.

[0054] The induction, purification, enzyme activity analysis and product identification of the recombinant protein are as follows:

[0055] 1) Induction of recombinant protein

[0056] The pMAL-c2X-UGT and pMAL-c2X monoclonal colonies were picked up respectively and cultured in 2 mL LB (containing Carb 100 mg / L) liquid culture medium at 37°C with shaking (200 rpm) overnight.

[0057] Take 1 mL of overnight cultured bacterial solution and add it to 100 mL of fresh LB culture medium (containing 100 mg / L Carb and 0.2% sterilized glucose). Cultivate on a shaking platform at 37°C until the OD value at 600 nm is 0.5-0.6. Take 1 mL of bacterial solution and collect the cells as a control.

[0058] Add 30 μL IPTG (1 M, isopropyl-β-D-thiogalactoside) to 100 mL of bacterial solution to a final concentration of 0.3 mM and culture at 16°C for 24 h.

[0059] The cells were collected by centrifugation at 8,000 × g for 3 min at 4°C.

[0060] 2) Purification of recombinant protein

[0061] The recombinant UFGT protein was purified according to the manual of pMAL fusion protein and purification system (New England BioLab Inc.). In short, the collected bacterial precipitate was resuspended in column buffer and stored at -20°C overnight. The next day, after the sample was thawed, the cells were disrupted with an ultrasonic disruptor to release the protein. After centrifugation at 9,000×g for 30 minutes, the sample was loaded; the affinity column filler was activated with column buffer (8 times column volume) (flow rate of 1mL / min), and the sample was diluted 5 times before loading. After the sample flowed through the affinity column filler, the impurities were washed with 12 times column volume of column buffer, and finally the target protein was eluted with 5 times column volume of column buffer (10mM maltose). It was concentrated using Millpore (30KDa) and replaced with enzyme activity reaction buffer. After SDS-PAGE electrophoresis, Coomassie Brilliant Blue staining was used to confirm the recombinant protein ( Figure 3 ).

[0062] 3) Determination of enzyme activity

[0063] The general enzyme activity reaction system is 50 μL, as shown in Table 5. After reacting for 2 hours at 37°C, the reaction is terminated with three volumes of methanol and centrifuged at 13,000 rpm for 10 minutes. Take 2 μL for sample loading.

[0064] Table 5 Recombinant protease activity reaction system

[0065]

[0066] 4) Analysis and identification of enzyme activity products

[0067] The recombinant protein of LpUGT95 was successfully expressed using a prokaryotic expression system, and further enzyme activity analysis was performed to identify their functions. The donor of the enzyme activity reaction was UDP-glucose (purchased from Sigma), and the acceptor was phytosporin (purchased from Chengdu Pusi Biotechnology Co., Ltd., product batch number PS010177).

[0068] UPLC conditions:

[0069] UPLC model: Nexera UHPLC LC-20A system (SHIMADZU, Japan).

[0070] Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.

[0071] Elution gradient: 0-7 min, 5%-100% B; 7-9 min, 100% B; 9-10.5 min, 100%-5% B; 10.5-11.5 min, 5% B.

[0072] DAD detection wavelength: 335nm.

[0073] The enzyme activity products were identified by mass spectrometry and it was found that the enzyme activity product peaks of LpUGT95 for the acceptor of phytosporin and the donor of glucose were two ( Figure 4 , Figure 5 , peak I, peak II), where the mass-to-charge ratio of peak I is 162 more than the mass-to-charge ratio of the substrate (the molecular weight of the product increases after one glucose and one substrate hydroxyl group remove one molecule of water), indicating that the product is the monoglucoside of phytochrome iodide; the primary mass spectrum of peak II detected the [MH]-ion fragment with a mass-to-charge ratio of 505.2, which is consistent with peak I, but no substrate ion fragment was detected. The secondary mass spectrum corresponding to the 505.2 parent ion on the left is consistent with peak I, so it is speculated that the peak II product is also the monoglucoside of phytochrome iodide. According to the structural formula of the compound ( Figure 6 , the left picture is the substrate chlorella, the middle picture is the product chlorella-5-O-β-D-glucoside, and the right picture is the product chlorella-7-O-β-D-glucoside). Chlorella can only be O-glycosylated if there is a hydroxyl group at the 5th or 7th position of the A ring, so the products are chlorella-7-O-β-D-glucoside and chlorella-5-O-β-D-glucoside. In summary, the in vitro enzyme activity evidence shows that LpUGT95 encodes a glycosyltransferase protein that catalyzes the glycosylation of chlorella to chlorella-7-O-β-D-glucoside and chlorella-5-O-β-D-glucoside.

[0074] Mass spectrometry conditions:

[0075] Sample preparation before mass spectrometry is the same as sample preparation before UPLC.

[0076] The samples were separated by UPLC MS / MS using an InfinityLab Poroshell 120EC-C18 column (2.1*50mm 1.9-micron; Agilent). The mobile phase was the same as that of UPLC. The elution gradient was 0 min, 5% B; 7 min, 95% B, and finally 95% B equilibrium (7-8 min). The flow rate was 0.30 mL / min, and the detection wavelength was the same as above.

[0077] UPLC MS / MS mass spectrometry conditions, electrospray ionization, full ion scan, negative-ion (EI) mass spectrometry analysis. The negative ion mode conditions are as follows: gas temperature: 300℃, gas flow: 6L / min, nebulizer: 30psi, sheath gas temperature: 300℃, sheath gas flow: 11L / min, capillary voltage: 2500V, nozzle voltage: 2000V, collision energy: 30V. The characteristic ions of scutellaria baicalensis in negative ion mode are 343.1 and 241.1.

[0078] 5) Catalytic activity of LpUGT95

[0079] To further understand the catalytic properties of LpUGT95 recombinant protein, we tested its catalytic activity for phytochrome P. truncatula. The total volume of the enzyme activity reaction system was 50 μL, which included: 10 μg LpUGT95 recombinant protein, 2 mM UDP-glucose and 10-400 μM phytochrome P. truncatula (10, 20, 40, 100, 200, 400 μM, respectively). After reacting at 30°C for 30 min, the reaction was terminated with three volumes of methanol. After high-speed centrifugation at 14,000 rpm, the reaction was passed through a 0.22 μm membrane and 2 μL was taken for UPLC detection. The experiment found that the LpUGT95 recombinant protein had a high K-value for the glucosylation of phytochrome P. truncatula. m 596.5 μM, k cat / K m 0.28s -1 μM -1 (As shown in Table 6).

[0080] Table 6 Catalytic activity of LpUGT95 recombinant protease

[0081]

[0082] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A protein, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. The gene encoding the protein according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

3. An expression cassette, recombinant expression vector, transgenic cell line or recombinant microorganism containing the encoding gene according to claim 2.

4. The protein according to claim 1 is used in catalyzing UDP-glucose and chelidonin to produce chelidonin-7-O-β-D-glucoside and / or chelidonin-5-O-β-D-glucoside.

5. The coding gene according to claim 2 is used for catalyzing UDP-glucose and chelidonin to produce chelidonin-7-O-β-D-glucoside and / or chelidonin-5-O-β-D-glucoside.

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