Plorizin glycosyl transferase gene DoUGT885 in dendrobium officinale and application thereof

Abstract

The invention provides a phlorizin glycosyl transferase gene DoUGT885 in dendrobium officinale and application of the phlorizin glycosyl transferase gene DoUGT885, and belongs to the technical field of gene cloning. Based on dendrobium officinale genome data, a phlorizin synthesis key enzyme-phlorizin glycosyl transferase gene DoUGT885 is successfully explored and identified through a reverse genetics method, it is confirmed that DoUGT885 glycosyl transferase can specifically catalyze glycosylation of phloretin to generate phlorizin, and it is confirmed that DoUGT885 glycosyl transferase can specifically catalyze glycosylation of phloretin to generate phlorizin; the problem of scarcity of efficient specific glycosyl transferase elements in phlorizin biosynthesis is solved. The discovery of the enzyme breaks through the source limitation of the existing phlorizin synthetase, provides a new high-quality enzyme element selection for a biosynthesis system, not only enriches a glycosyltransferase resource library, but also reduces the dependence of phlorizin biosynthesis on the traditional enzyme element, and lays a key foundation for constructing an efficient and stable phlorizin biosynthesis pathway.

Description

Technical Field

[0001] This invention relates to the field of gene technology, and in particular to a root bark glycosyltransferase gene from Dendrobium officinale. DoUGT885 And its applications. Background Technology

[0002] Phloretin, chemically named 2',4',6'-trihydroxy-3-(4-hydroxyphenyl)propiophenone, has the molecular formula C2. 15 H 14 O5 is a white crystalline powder, soluble in organic solvents such as methanol, ethanol, and DMSO. Phloretin was first discovered in 1835 and is widely found in the rhizomes or root bark of Dendrobium officinale, apples, pears, and various vegetables. It has a variety of biological activities, including antibacterial, anti-inflammatory, antioxidant, inhibition of cancer cell proliferation, hypoglycemic, and inhibition of tyrosinase.

[0003] Phlorizin, structurally a 2'-O-glucoside of phlorizin, possesses a unique structure that endows it with special biological activities. Its hypoglycemic mechanism primarily involves inhibiting the activity of sodium-glucose cotransporters (SGLTs), which are responsible for glucose reabsorption in the body. Normally, SGLTs reabsorb glucose from the kidneys back into the bloodstream; phlorizin effectively inhibits this process, allowing excess glucose to be excreted in the urine, thereby lowering blood glucose levels.

[0004] Due to the remarkable hypoglycemic effects of phlorizin, hypoglycemic drugs based on it have become a research hotspot for scientists and pharmaceutical companies, and are currently under extensive development. Among them, dapagliflozin, empagliflozin, and canagliflozin have attracted considerable attention, demonstrating significant clinical efficacy as treatments for type 2 diabetes. These drugs, by mimicking the mechanism of action of phlorizin, effectively lower patients' blood glucose levels, improve the quality of life for diabetic patients, and provide an important means of treating type 2 diabetes.

[0005] The glycosylation pattern from phlorin to phloridine is O-glycosylation, meaning that glucose molecules are specifically linked to the 2'-OH group of the phlorin A ring via the action of phlorin 2'-O-glycosyltransferase, thereby directly synthesizing phloridine. Five glycosyltransferase genes in apple have been identified as having the ability to catalyze the conversion of phlorin to phloridine: MdPGT1 (UGT88F1), UGT71K1, UGT71A15, UGT75L17, and MdUGT88F4. Alignment of the MdPGT1 (UGT88F1) gene sequence with plant genome databases shows that, except for one sequence in the *Quercus* genus of the Fagaceae family, gene sequences with a sequence similarity higher than 60% to the MdPGT1 gene are concentrated in the Rosaceae family. Similar genes with similar functions exist in other plants. For example, the recombinant protein GhUGT88F3 in *Gossypium hirsutum* specifically catalyzes the production of phlorizin from phlorin substrates, without affecting other flavonoid substrates. In vitro enzymatic reactions in *Lithocarpus litseifolius* have identified seven glycosyltransferases that can catalyze the glycosylation of phlorin. Related enzymes have also been reported in bacteria. Using UDP-glycosyltransferase from *Bacillus licheniformis* for phlorin glycosylation, in vitro glycosylation reactions confirmed the production of five phlorin glucosides, including phlorizin, trifolin, and three novel glucosides.

[0006] Dendrobium officinale, a perennial epiphytic herb of the Orchidaceae family, holds a pivotal position in traditional Chinese medicine. As early as ancient times, the *Qianjin Baoyao* (a classic Chinese medical text) recorded its "sweet and bitter taste, cool in nature, and used to treat chronic diarrhea, cough, and various ailments," fully demonstrating its widespread application in traditional medicine. Phloretin is mainly found in the roots and stems of Dendrobium officinale. Due to long-term over-harvesting and habitat destruction, wild Dendrobium officinale resources are extremely scarce. Therefore, identifying the phloretin glycosyltransferase gene in Dendrobium officinale is of great significance for the artificial cultivation of varieties with high medicinal value. Summary of the Invention

[0007] The purpose of this invention is to provide a root bar glycosyltransferase gene from Dendrobium officinale. DoUGT885 Its application breaks through the source limitations of existing phlorizin synthases and provides a new selection of high-quality enzyme elements for biosynthetic systems.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a root bar glycosyltransferase gene from Dendrobium officinale. DoUGT885 The sequence of the root bark glycosyltransferase gene in the Dendrobium officinale is shown in SEQ ID NO.1.

[0009] Preferably, the root bark glycosyltransferase gene in the Dendrobium officinale... DoUGT885 The sequence of the expressed protein is shown in SEQ ID NO.2.

[0010] This invention also provides a root bar glycosyltransferase gene from Dendrobium officinale. DoUGT885 Application in the production of phlorizin.

[0011] This invention also provides a root bar glycosyltransferase gene from Dendrobium officinale. DoUGT885 Application in the preparation of drugs for treating diabetes.

[0012] The beneficial effects of this invention compared to the prior art are as follows: Based on the genomic data of Dendrobium officinale, this invention successfully discovered and identified the gene of phlorizin glycosyltransferase, a key enzyme in phlorizin synthesis, using reverse genetics. DoUGT885 The protein sequence was compared with the NCBI database. Protein sequences with a similarity greater than 70% all originated from Dendrobium species, predicting it to be a glycosyltransferase catalyzing hydroquinone glycosylation. Recombinant protein was then expressed in prokaryotes, confirming its identity. DoUGT885 The discovery of glycosyltransferases that specifically catalyze the glycosylation of phlorizin to phloridine has solved the problem of the scarcity of highly efficient and specific glycosyltransferase elements in phloridine biosynthesis. This discovery overcomes the source limitations of existing phloridine synthases, providing a new and high-quality enzyme element selection for biosynthetic systems. It not only enriches the glycosyltransferase resource library but also reduces the dependence of phloridine biosynthesis on traditional enzyme elements, laying a crucial foundation for constructing an efficient and stable phloridine biosynthetic pathway. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 As described in the embodiments of the present invention DoUGT885 Gene cloning results; Figure 2 As described in the embodiments of the present invention DoUGT885 SDS-Page gel image of recombinant protein; Figure 3 As described in the embodiments of the present invention DoUGT885 UPLC diagram for identifying the catalytic activity of phloretin; Figure 4As described in the embodiments of the present invention DoUGT885 MS and MS / MS identification chromatograms of phlorizin catalytic products, where peak I is phlorizin; Figure 5 The following are the molecular structural formulas of phlorizin and phlorizin in the embodiments of the present invention, wherein A is the molecular structural formula of phlorizin and B is the molecular structural formula of phlorizin. Detailed Implementation

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0016] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0018] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to. Example 1

[0020] Example 1 of this invention provides a method for screening phlorizin glycosyltransferase genes in Dendrobium officinale, the specific steps of which are as follows: (1) Gene cloning (1.1) Based on the genome data of *Dendrobium officinale* and the conserved PSPG box of UGTs, 132 DoUGTs sequences ranging from 305aa to 529aa were screened. Through functional gene phylogenetic tree comparison, particular attention was paid to… DoUGT885 Gene.

[0021] Extraction of Dendrobium officinale ( Dendrobium officinale RNA from the roots, stems, and leaves of [a specific plant] was reverse transcribed into cDNA (AT311, Beijing TransGen Biotech Co., Ltd.). Based on the selected [a specific type of cDNA]... DoUGT885 The primer sequences for gene design are shown in Table 1.

[0022]

[0023] Note: Only the primer sequence of one gene that successfully expressed the recombinant protein is listed.

[0024] (1.2) Using a mixture of cDNA from the roots, stems, and leaves of Dendrobium officinale as a template, one molecule was cloned using KOD high-fidelity enzyme (KMM-101, Toyobo (Shanghai) Biotechnology Co., Ltd.). DoUGT Gene fragments.

[0025] The total volume of the KOD high-fidelity enzyme PCR system is 50. m L:5 m L 10×Buffer, 3 m L MgSO4,5 m L dNTP (2mM), 4 m L primer (10mM), 1 m L template and 32 m L water, the procedure is shown in Table 2.

[0026]

[0027] (1.3) Using pEASY-Blunt (CB101, Beijing TransGen Biotech Co., Ltd.) as a carrier, the following were introduced: DoUGT885 The fragment is attached to the carrier.

[0028] The total volume of the connection system is 2.5. m L: 0.5 m L Mix Buffer with enzyme and 2 m L template, react at 25℃ for 2 h.

[0029] Direct conversion of connection system TransT1 Competent cells (Beijing TransGen Biotech Co., Ltd.), and positive clones were selected for sequencing.

[0030] The total volume of the colony PCR system was 20. m L: 10 m L Mix Buffer, 1 m L template, 1 m L primer and 8 m L water, the procedure is shown in Table 3.

[0031]

[0032] The cloned nucleotide sequence was compared with the CDS sequence. The nucleotide sequence showed 99.585% similarity to the original data. The actual sequencing results shall prevail. DoUGT885 The gene sequence is shown in SEQ ID NO.1. DoUGT885 The amino acid sequence is shown in SEQ ID NO.2.

[0033] DoUGT885 Gene sequence, SEQ ID NO.1:

[0034] DoUGT885 Amino acid sequence, SEQ ID NO.2: MDNGARQPHIVFFPSAGMGHILPMAELAKLLVDRHHFTVTFITFSEHSNKTQDAFLASLPSSITSISLPPIPLSDLPENSAVETRMSIAAARSVPPHLRSLLLPLLSSTRLVAFIADLF TTTGCDAAKALKIQHFIFIPPTNLLFVTLMLHLPALNAELSCDFWELEQPVLLPGYPPIPGTEILHPLQDRKNECYRWMLEHAKRYREAEGILVNTFDAIEPEAANLLKKEEPGRATVY AVGPLIRAHAVSGEEGAHCLRWLDSQPTGSVLFVSFGSVGSHSTEQLGELALGLEASGQRFLWVVRTPVDLNSVGSNYIEAQSADNPLAYLPEGFLERTKGVGLVVPSWAPQVDILAH SSTGGFLSHCGWNSTLESMARGVPMIVWPLFAEQRMNAVMMVEGAKVAMRLKARKDGIFDRKKISRVVKNLMEGEEGERLRKRAKELQAEAAAAMTEGGSSSVALAAFAEKLKSFPTV.

[0035] (2) Gene function verification Adopt the system and procedure shown in step (1.3), in DoUGT885 The fragment was fitted with an enzyme restriction site adapter, and the primer information is shown in Table 4. After the DoUGT885 fragment (with restriction sites) was digested, it was constructed into the expression vector pMAL-c2X (New England Biolabs (Beijing) LTD.NEB) using T4-DNA ligase (Toyobo (Shanghai) Biotechnology Co., Ltd.) to obtain the pMAL-c2X-DoUGT885 vector.

[0036] The overall connection system consists of 7 m L: 3.5 m L Mix Buffer, 2.8 m L DoUGT885 fragment and 0.7 m LpMAL-c2X; react overnight at 4°C; Transform the connection system TransT1After obtaining competent cells, positive clones were selected; plasmids were extracted and transformed into the expression strain Novablue (Beijing Huayueyang Biotechnology Co., Ltd.) for in vitro validation. The results are as follows: Figure 1 As shown.

[0037]

[0038] Note: Lowercase letters in the sequence are protective bases and restriction enzyme sites.

[0039] Figure 1 show, DoUGT885 The gene was successfully transformed into the expression strain Novablue.

[0040] (3) The induction, purification, enzyme activity analysis, and product identification of the recombinant protein are as follows: (3.1) Induction of recombinant proteins Single colonies of pMAL-c2X-UGT and pMAL-c2X (as a control) were picked and cultured overnight at 37°C with shaking (200 rpm) in 2 mL LB broth (containing 100 mg / L Amp) (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, all reagents purchased from Beijing Solarbio Science & Technology Co., Ltd.).

[0041] Add 1 mL of overnight culture to 100 mL of fresh LB broth (containing 100 mg / L Amp and 0.2% membrane-sterilized glucose), and incubate at 37°C on a shaker until OD reaches 100 mg / L. 600nm When the value is 0.5-0.6, add 30 to 100 mL of bacterial culture. m L IPTG (1 M, isopropyl-) β -D-thiogalactoside), with a final concentration of 0.3 mM, was incubated at 16℃ for 24 h. 4℃, 8,000× g Centrifuge for 3 minutes and collect the bacterial cells.

[0042] (3.2) Purification of recombinant protein According to the pMAL fusion protein and purification system (New England BioLab Inc.) manual, recombinant DoUGT885 The protein was purified.

[0043] The bacterial pellet expressing pMAL-c2X-UGT and pMAL-c2X plasmids collected above was resuspended in column buffer (2 L system: 40 mL 1.0 M Tris-HCl (pH 7.5), 23.4 g sodium chloride, 4 mM LEDTA (0.5 M), 308 mg DTT) and incubated overnight at -20°C. The next day, after thawing the samples, the cells were disrupted using an ultrasonic disruptor to release the proteins at a high speed of 9,000×. g Centrifuge for 30 minutes, then load the sample.

[0044] The affinity column packing was activated using column buffer (8 column volumes) at a flow rate of 1 mL / min. The sample was diluted 5-fold and loaded onto the column. After the sample had completely passed through the affinity column packing, contaminating proteins were washed with 12 column volumes of column buffer. Finally, the target protein was eluted with 5 column volumes of column buffer (10 mM maltose). The solution was concentrated using a Millipore (30 kDa) electrophoresis apparatus and replaced with enzyme activation buffer. After SDS-PAGE electrophoresis, Coomassie Brilliant Blue staining was performed to confirm the recombinant protein. The results are as follows: Figure 2 As shown.

[0045] Figure 2 The results show that this invention successfully expressed [the substance] using a prokaryotic expression system. DoUGT885 Recombinant proteins.

[0046] (3.3) Determination of enzyme activity The typical enzyme activity reaction system is 50 μL, as shown in Table 5. After reacting at 37℃ for 2 h, the reaction is terminated with three times the volume of methanol, centrifuged at 13,000 rpm for 10 min, and 2 μL of the sample is loaded.

[0047]

[0048] 4) Analysis and identification of enzyme active products The donor for the enzyme activity reaction is UDP-glucose, and the acceptor is phlorizin. Analysis of the UPLC spectrum of the enzyme activity product is shown below. Figure 3 As shown.

[0049] UPLC conditions: UPLC model: Nexera UHPLC LC-20A system (SHIMADZU, Japan).

[0050] Column: Agilent Poroshell 120 SB-C18 (1.9 μm, 2.1 mm × 50 mm) Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.

[0051] Flow rate: 0.3 mL / min Elution gradient: 0-1 min, 5%-15% B; 1-7 min, 15-25% B; 7-9 min, 25%-100% B; 9-10 min, 100% B; 10-10.5 min, 100%-5% B; 10.5-13.5 min, 5% B.

[0052] DAD detection wavelength: 286 nm.

[0053] Figure 3 show, DoUGT885 It can specifically catalyze the formation of phlorizin from phlorizin substrate.

[0054] Enzyme activity products were identified using mass spectrometry, and the results are as follows: Figure 4 As shown, the mass spectrometry conditions are: Sample preparation before mass spectrometry is the same as sample preparation before UPLC.

[0055] Samples were separated using UPLC-MS / MS with an Agilent Poroshell 120 SB-C18 column (1.9 μm, 2.1 mm × 50 mm). The mobile phase was the same as that used in UPLC, and the elution gradient was as follows: 0–1 min, 5%–15% B; 1–7 min, 15–25% B; 7–9 min, 25%–100% B; 9–10 min, 100% B; 10–10.5 min, 100%–5% B; 10.5–13.5 min, 5% B. The flow rate was 0.30 mL / min, and the detection wavelength was the same as above.

[0056] UPLC-MS / MS conditions: electrospray ionization, full ion scan, negative ion (EI) mass spectrometry. Nebulizer gas temperature: 300℃; nebulizer gas flow rate: 5.0 L / min; capillary voltage: 3500 V; nozzle voltage: 500 V; electron multiplier voltage: 200 V. MS TOF (Expt): fragmentation voltage: 120 V; cutoff voltage: 65 V; acquisition mass spectrum range: m / z: 100-1000. The characteristic ions of phlorizin in negative ion mode were 273.077 and 167.035.

[0057] Figure 4 show, DoUGT885 For the enzyme activity reaction with phlorizin as the acceptor and glucose as the donor, the product peak is only 1. The mass-to-charge ratio of peak I is approximately 162.053 higher than that of the substrate (the increase in molecular weight after the removal of one molecule of water from a glucose molecule and a substrate hydroxyl group), indicating that the product is a monoglucoside of phlorizin. Based on the compound structural formula ( Figure 5Phlorizin can undergo O-glycosylation only when there are hydroxyl groups at the 2' or 4' position of ring A and the 4' position of ring B. Based on the retention time of the standard, peak I was identified as phlorizin. In summary, in vitro enzyme activity evidence shows... DoUGT885 It encodes a glycosyltransferase that specifically catalyzes the glycosylation of phlorizin to phlorizin.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A phloridzin glycosyltransferase gene in Dendrobium candidum DoUGT885 characterized in that, The sequence of the phloridzin glycosyltransferase gene in the Dendrobium officinale is shown as SEQ ID NO.

1.

2. The gene of phloridzin glycosyltransferase in Dendrobium candidum according to claim 1. DoUGT885 characterized in that, Phlorizin glycosyltransferase gene in the dendrobium officinale DoUGT885 The sequence of the expressed protein is shown as SEQ ID NO.

2.

3. A phloridzin glycosyltransferase gene from Dendrobium candidum according to any one of claims 1-2 DoUGT885 for use in the production of phloridzin.

4. A gene of phloridzin glycosyltransferase in Dendrobium candidum according to any one of claims 1-2 DoUGT885 use in the preparation of a medicament for the treatment of diabetes.

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