Phloridzin glycosyltransferase gene dougt885 in dendrobium officinale and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genes, in particular to a phloridzin glycosyltransferase gene in dendrobium officinale DoUGT885 and application thereof. BACKGROUND

[0002] Phloretin is a chemical name of 2', 4', 6'-Trihydroxy-3-(4-hydroxyphenyl) propiophenone. Its molecular formula is C 15 H 14 O5, white crystal powder, soluble in organic solvents such as methanol, ethanol and DMSO. Phloretin was first discovered in 1835 and widely exists in the rhizomes or root barks of dendrobium officinale, apples, pears and various vegetable plants, and has many biological activities such as bactericidal, anti-inflammatory, antioxidant, inhibition of cancer cell proliferation, hypoglycemic and tyrosinase inhibition.

[0003] Phloridzin is a 2'-O-glucoside of phloretin in structure. This unique structure endows it with special biological activity. Its hypoglycemic mechanism is mainly to inhibit the activity of sodium-glucose cotransporter (SGLT) which is responsible for glucose reabsorption in the human body. Under normal circumstances, SGLT will reabsorb glucose in the kidney back to the blood, and the intervention of phloridzin can effectively inhibit this process, so that the excess glucose is excreted out of the body with urine, thereby reducing the blood glucose level.

[0004] Due to the excellent hypoglycemic effect of phloridzin, the hypoglycemic drugs taking it as the basic skeleton have become the research focus of scientific researchers and pharmaceutical enterprises, and have been widely developed. Among them, Dapagliflozin, Empagliflozin and Canagliflozin are three drugs that have attracted much attention. They are used as drugs for treating type II diabetes and have achieved remarkable clinical effects. These drugs effectively reduce the blood glucose level of patients by simulating the mechanism of action of phloridzin, and improve the quality of life of diabetic patients, providing an important means for the treatment of type II diabetes.

[0005] The glycosylation mode of phloretin to phloridzin is O-glycosylation, that is, a glucose molecule is specifically connected to the 2'-OH of the A ring of phloretin by the action of phloretin 2'-O-glycosyltransferase, thereby directly synthesizing phloridzin. At present, five glycosyltransferase genes in apples have been identified to have the ability to catalyze phloretin to generate phloridzin, which are MdPGT1 (UGT88F1), UGT71K1, UGT71A15, UGT75L17 and MdUGT88F4. The sequence alignment result of MdPGT1 (UGT88F1) gene sequence with plant genome database shows that, in addition to one sequence in Fagaceae Quercus plants, the gene sequences with similarity higher than 60% to the MdPGT1 gene sequence are all concentrated in Rosaceae. There are also genes with similar functions in other plants, such as GhUGT88F3 recombinant protein in Gossypium hirsutum, which specifically catalyzes phloretin substrate to generate phloridzin and has no effect on other flavonoid substrates; seven glycosyltransferases that can catalyze phloretin glycosylation are found in in vitro enzymatic reaction of Lithocarpus litseifolius. There are also related enzyme reports in bacteria, and UDP-glycosyltransferase from Bacillus licheniformis is used for phloretin glycosylation. In vitro glycosylation reaction proves the production of five phloretin glucosides, including phloridzin, trilobatin and three novel glucosides.

[0006] Dendrobium candidum, as a perennial epiphyte of Orchidaceae, occupies a pivotal position in traditional Chinese medicine. As early as in ancient times, "Qianjin Baoyao" recorded that "it tastes bitter and sweet, and is cold in nature. It is used to treat chronic diarrhea, cough, five diseases and seven injuries", fully showing the wide application of Dendrobium candidum in traditional medicine. Phloretin mainly exists in the roots and stems of Dendrobium candidum. Wild Dendrobium candidum has been extremely scarce due to long-term over-harvesting and destruction of the living environment. Therefore, it is of great significance to clarify the phloridzin glycosyltransferase gene of Dendrobium candidum for artificially cultivating high medicinal value varieties. SUMMARY

[0007] The purpose of the present application is to provide a phloridzin glycosyltransferase gene in Dendrobium candidum DoUGT885 and its application, which breaks through the source limitation of existing phloridzin synthesis enzymes and provides new high-quality enzyme element selection for biosynthesis system.

[0008] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0009] The present application provides a phloridzin glycosyltransferase gene in Dendrobium candidum DoUGT885 , and the sequence of the phloridzin glycosyltransferase gene in Dendrobium candidum is shown as SEQ ID NO. 1.

[0010] Preferably, the root bark glycosyltransferase gene in the Dendrobium officinale... DoUGT885 The sequence of the expressed protein is shown in SEQ ID NO.2.

[0011] This invention also provides a root bar glycosyltransferase gene from Dendrobium officinale. DoUGT885 Application in the production of phlorizin.

[0012] This invention also provides a root bar glycosyltransferase gene from Dendrobium officinale. DoUGT885 Application in the preparation of drugs for treating diabetes.

[0013] The beneficial effects of this invention compared to the prior art are as follows:

[0014] 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

[0015] 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.

[0016] Figure 1 As described in the embodiments of the present invention DoUGT885 Gene cloning results;

[0017] Figure 2 As described in the embodiments of the present invention DoUGT885 SDS-Page gel image of recombinant protein;

[0018] Figure 3 As described in the embodiments of the present inventionDoUGT885 UPLC chart for catalytic activity of phloretin;

[0019] Figure 4 For the embodiments of the present application DoUGT885 MS and MS / MS identification chart for catalytic product of phloretin, wherein peak I is phloridzin;

[0020] Figure 5 For the molecular structural formula of phloretin and phloridzin in the embodiments of the present application, wherein A is the molecular structural formula of phloretin; B is the molecular structural formula of phloridzin. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but rather as a description of certain aspects, features and embodiments of the present application.

[0022] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or stated range is also included within the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to A description of the state of the art.

[0024] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the present application. Other implementations of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the present application. The specification and examples are illustrative only.

[0025] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", and the like, are open-ended terms that are intended to mean including but not limited to. Example 1

[0026] The embodiment 1 of the present application provides a screening method of phloridzin glycosyltransferase gene in Dendrobium candidum, and the specific steps are as follows:

[0027] (1) Gene cloning

[0028] (1.1) Based on the genome data of D. candidum, 132 DoUGTs sequences of 305aa-529aa were screened out by combining the conserved domain PSPG box of UGTs. By comparing the phylogenetic tree of functional genes, the following genes were focused on DoUGT885 Gene.

[0029] The RNA of the roots, stems and leaves of D. candidum (AT311, Beijing Quanshijin Biotechnology Co., Ltd.) was extracted and reverse transcribed into cDNA. The primer sequences were designed based on the screened genes, as shown in Table 1. Dendrobium officinale DoUGT885

[0030] Note: Only the primer sequences of one gene successfully expressing recombinant proteins are listed.

[0031] (1.2) A gene fragment was cloned from the mixed cDNA of the roots, stems and leaves of D. candidum using KOD high-fidelity enzyme (KMM-101, Toyo Roshi (Shanghai) Biotechnology Co., Ltd.).

[0032] DoUGT The total volume of KOD high-fidelity enzyme PCR system was 50

[0033] L: 5 μ L 10x Buffer, 3 μ L MgSO4, 5 μ L dNTP (2mM), 4 μ L primer (10mM), 1 μ L template and 32 μ L water, and the program is shown in Table 2. μ

[0034]

[0035] (1.3) The DoUGT885 fragment was ligated into the vector using pEASY-Blunt (CB101, Beijing Quanshijin Biotechnology Co., Ltd.) as the carrier.

[0036] The total volume of the ligation system was 2.5 μ L: 0.5 μ L Mix Buffer with enzyme and 2 μ L template, and the reaction was carried out at 25°C for 2 h.

[0037] The ligation system was directly transformed TransT1 ​​​Competent cells (Beijing Zoman Biotech Co., Ltd.), the cells of positive clones were selected for sequencing.

[0038] The total volume of colony PCR system was 20 μ L: 10 μ L Mix Buffer, 1 μ L template, 1 μ L primer and 8 μ L water, the program was shown in Table 3.

[0039]

[0040] The nucleotide sequence of the clone was aligned with the CDS sequence, and the nucleotide sequence was 99.585% similar to the original data, and the actual sequencing result was used as the standard, DoUGT885 The gene sequence was shown as SEQ ID NO. 1, DoUGT885 The amino acid sequence was shown as SEQ ID NO. 2.

[0041] DoUGT885 Gene sequence, SEQ ID NO. 1:

[0042]

[0043] DoUGT885 Amino acid sequence, SEQ ID NO. 2:

[0044] MDNGARQPHIVFFPSAGMGHILPMAELAKLLVDRHHFTVTFITFSEHSNKTQDAFLASLPSSITSISLPPIPLSDLPENSAVETRMSIAAARSVPHLRSLLLPLLSSTRLVAFIADLFTTTGCDAAKALKIQHFIFIPTNLLFVTLMLHLPALNAELSCDFWELEQPVLLPGYPPIPGTEILHPLQDRKNECYRWMLEHAKRYREAEGILVNTFDAIEPEAANLLKKEEPGRATVYAVGPLIRAHAVSGEEGAHCLRWLDSQPTGSVLFVSFGSVGSHSTEQLGELALGLEASGQRFLWVVRTPVDLNSVGSNYIEAQSADNPLAYLPEGFLERTKGVGLVVPSWAPQVDILAHSSTGGFLSHCGWNSTLESMARGVPMIVWPLFAEQRMNAVMMVEGAKVAMRLKARKDGIFDRKKISRVVKNLMEGEEGERLRKRAKELQAEAAAAMTEGGSSSVALAAFAEKLKSFPTV.

[0045] (2) Verification of gene function

[0046] The system and procedure shown in step (1.3) were taken, and the DoUGT885 fragment was constructed into the expression vector pMAL-c2X (New England Biolabs (Beijing) LTD. NEB (Beijing) Co., Ltd.) to obtain the pMAL-c2X-DoUGT885 vector. DoUGT885 The fragment was added with an enzyme cutting site linker, and the primer information is shown in Table 4; after the DoUGT885 fragment (with an enzyme cutting site) was cut, T4-DNA ligase (East Asia Spinning (Shanghai) Biological Technology Co., Ltd.) was used to construct it into the expression vector pMAL-c2X (New England Biolabs (Beijing) LTD. NEB (Beijing) Co., Ltd.) to obtain the pMAL-c2X-DoUGT885 vector.

[0047] The total volume of the ligation system was 7 μ L: 3.5 μ L Mix Buffer, 2.8 μ L DoUGT885 fragment and 0.7 μ L pMAL-c2X; 4°C reaction overnight;

[0048] The ligation system was transformed into DH5α competent cells, and the positive clones were selected by PCR and enzyme digestion. TransT1After the competent cells, positive clone cells were selected; after extracting the plasmid, it was transformed into the expression strain Novablue (Beijing Huayueyang Biological Technology Co., Ltd.) for in vitro verification, and the results are shown in Figure 1 .

[0049]

[0050] Note: The lower case letters in the sequence are protection bases and enzyme cutting sites.

[0051] Figure 1 It is shown that DoUGT885 the gene is successfully transformed into the expression strain Novablue.

[0052] (3) Induction, purification, enzyme activity analysis and product identification of recombinant protein as follows:

[0053] (3.1) Induction of recombinant protein

[0054] Respectively pick pMAL-c2X-UGT and pMAL-c2X (as a control) monoclonal colonies in 2 mL LB (containing Amp 100 mg / L) liquid medium (peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, all reagents are purchased from Beijing Solaybao Technology Co., Ltd.) 37℃ in the shaking (200 rpm) overnight culture.

[0055] Take 1 mL of overnight culture and add 100 mL of fresh LB culture solution (containing Amp 100 mg / L, 0.2% membrane sterilized glucose) and shake culture at 37℃ when the OD 600nm value is 0.5-0.6, add 30 μ L IPTG (1 M, isopropyl- β -D-thio-galactoside) to 100 mL of bacterial solution, the final concentration is 0.3 mM, and culture at 16℃ for 24 h. 4℃, 8,000× g centrifugation for 3 min, collect the bacterial cells.

[0056] (3.2) Purification of recombinant protein

[0057] According to the pMAL fusion protein and purification system (New England BioLab Inc.) manual, the recombinant DoUGT885 protein was purified.

[0058] Column buffer (2 L system: 40 mL 1.0 M Tris-HCl (pH 7.5), 23.4 g sodium chloride, 4 mL EDTA (0.5 M), 308 mg DTT) was used to resuspend the bacterial pellet expressing pMAL-c2X-UGT and pMAL-c2X plasmids collected above and stored at -20 °C overnight. The next day, after the sample was thawed, the cells were broken by sonication and the proteins were released. The sample was centrifuged at 9,000 x g for 30 mins. g After centrifugation, the sample was loaded.

[0059] The affinity column was activated with column buffer (8 column volumes) at a flow rate of 1 mL / min. The sample was diluted 5-fold and loaded. After the sample passed through the column, the column was washed with 12 column volumes of column buffer to remove the impurities. Finally, the target protein was eluted with 5 column volumes of column buffer (10 mM maltose). The eluted protein was concentrated using a Millipore (30 KDa) and exchanged into the enzyme activity reaction buffer. After SDS-PAGE, the recombinant protein was confirmed by Coomassie blue staining. The results are shown in Figure 2 .

[0060] Figure 2 As shown, the recombinant protein of the present application was successfully expressed using a prokaryotic expression system. DoUGT885 .

[0061] (3.3) Enzyme activity determination

[0062] The general enzyme activity reaction system was 50 μL, as shown in Table 5. After the reaction at 37 °C for 2 h, the reaction was terminated with three volumes of methanol, centrifuged at 13,000 rpm for 10 min, and 2 μL was loaded.

[0063]

[0064] 4) Enzyme activity product analysis and identification

[0065] The donor of the enzyme activity reaction was UDP-glucose, and the acceptor was phloretin. The UPLC spectrum of the enzyme activity product was analyzed, as shown in Figure 3 .

[0066] UPLC conditions:

[0067] UPLC model: Nexera UHPLC LC-20A system (SHIMADZU, Japan).

[0068] Chromatographic column: Agilent Poroshell 120 SB-C18 (1.9 μm, 2.1 mm x 50 mm)

[0069] Mobile phase: A phase: 0.1% formic acid in water; B phase: acetonitrile.

[0070] Flow rate: 0.3 mL / min

[0071] 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.

[0072] DAD detection wavelength: 286 nm.

[0073] Figure 3 showed, DoUGT885 can specifically catalyze the generation of phloretin substrate to generate phlorizin.

[0074] The enzyme activity product was identified by mass spectrometry, and the results are shown in Table 1, and the mass spectrometry conditions are as follows: Figure 4

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

[0076] The sample was separated by UPLC-MS / MS, the column was Agilent Poroshell 120 SB-C18 (1.9 μm, 2.1 mm × 50 mm), the mobile phase was the same as UPLC, the elution gradient was 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.

[0077] The UPLC-MS / MS mass spectrometry conditions were as follows: electrospray ionization, full ion scanning, negative ion mode (EI) mass spectrometry analysis. Atomization gas temperature: 300°C; atomization gas flow, 5.0 L / min, capillary voltage, 3500 V; nozzle voltage, 500 V; electron multiplier voltage value, 200 V. MS TOF (Expt): fragmentation voltage, 120 V; intercept voltage, 65 V; mass spectrometry range m / z: 100-1000. The characteristic ions of phloretin in negative ion mode were 273.077 and 167.035.

[0078] Figure 4 showed, DoUGT885 ​For the enzyme activity reaction product peak of the acceptor being phloretin and the donor being glucose, there is only 1, and the mass-to-charge ratio of peak I is about 162.053 more than that of the substrate (one glucose and one molecule of water removed from the hydroxyl of the substrate, the product increases in molecular weight), indicating that the product is a monoglucoside of phloretin. According to the compound structural formula (Formula I) Figure 5 ), phloretin can only be O-glycosylated at the 2' or 4' position of the A ring and the 4 position of the B ring with a hydroxyl, and the product of peak I is determined to be phloridzin according to the retention time of the standard. In summary, the in vitro enzyme activity evidence shows DoUGT885 that the enzyme encoded by the gene specifically catalyzes the glycosylation of phloretin to generate phloridzin.

[0079] The above only describes preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. The application of the phlorizin glycosyltransferase gene DoUGT885 from Dendrobium officinale in the production of phlorizin, characterized in that, The sequence of the phlorizin glycosyltransferase gene DoUGT885 in the Dendrobium officinale is shown in SEQ ID NO.1; The sequence of the protein expressed by the phlorizin glycosyltransferase gene DoUGT885 in Dendrobium officinale is shown in SEQ ID NO.

2.

2. The use of the phlorizin glycosyltransferase gene DoUGT885 from Dendrobium officinale as described in claim 1 in the preparation of a drug for treating diabetes.

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