Hawthorn carbon glycosyl transferase CGT52202 as well as coding gene and application thereof

Abstract

The invention discloses an amino acid sequence of C-glycosyltransferase. The amino acid sequence of the C-glycosyltransferase is as follows: 1) an amino acid sequence as shown in SEQ ID NO.2 in a sequence table; or 2) the amino acid sequence as shown in SEQ ID NO.2 in the sequence table is conventionally modified, or a label for detection or purification is also connected to the amino acid sequence; the invention also discloses a nucleic acid molecule for coding the C-glycosyl transferase, a vector containing the nucleic acid molecule, an expression cassette, a host cell or a transformant. The invention also discloses application of the compound in catalyzing C-glycosylation reaction of a substrate. According to the invention, a new glycosyl transferase gene (CGT52202) is successfully cloned from hawthorn, and the gene can be used for preparing nothofagin by taking phloretin as a substrate, preparing nothofagin from phloretin through an in-vitro enzymatic reaction, or synthesizing nothofagin in a plant or microorganism system as a glycosylation original, so that the application prospect is very good.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to hawthorn carbon glycosyltransferase CGT52202 and a coding gene and application thereof. Background Art

[0002] Hawthorn (Crataegus pinnatifida Bunge) is a deciduous tree plant of the genus Crataegus in the Rosaceae family. Hawthorn has always been loved by people as a high-quality tonic that is both a medicine and a food. Flavonoids are one of its main components. Studies have shown that flavonoids in hawthorn have pharmacological activities such as anti-tumor, anti-inflammatory, antioxidant, and hypotensive. At present, the flavonoid aglycones isolated from hawthorn mainly include apigenin, luteolin, kaempferol, quercetin, and bi-hydroflavonoids, most of which exist in the form of glycosides. Compared with other types of glycosides (O-, N-, or S-glycosides), C-glycosides are not hydrolyzed by acid and glycosidase, so they have stronger stability and higher bioavailability.

[0003] With the development of synthetic biology technology, mining the key enzyme genes of the biosynthetic pathway of target ingredients from plants and reconstructing their biosynthetic pathways in heterologous organisms for direct production has become a more promising approach. Carbon glycosyltransferase (CGT) is a key biosynthetic enzyme that catalyzes the formation of C-glycosidic bonds, and has the characteristics of being green, efficient, and highly selective. However, due to low gene homology and other reasons, there are relatively few reports of CGT found in plants. Phloretin, also known as 2,4,6-trihydroxy-3-(4-hydroxyphenyl) phenylpropionone, is a new type of natural skin whitening agent recently developed. It is mainly distributed in the peel and root bark of juicy fruits such as apples and pears, and can be used in beauty and skin care products. Nothofagin (Cas No: 11023-94-2) is a dihydrochalcone compound, and its Chinese name is 1-(3-BETA-D-pyranosyl-2,4,6-trihydroxyphenyl)-3-(4-hydroxyphenyl)-1-propanone. Nothofagin improves various inflammatory responses, such as sepsis reactions and vascular inflammation.

[0004] Most of the flavonoid CGTs identified from plants have high substrate heterozygosity and low catalytic efficiency, which limits their application in the synthesis of C-glycosides. Among them, only a few CGTs such as GgCGT, TcCGT1, FcCGT, CuCGT and MiCGTb can catalyze phloretin to produce the mono-carbon glycoside nothofagin, but their catalytic end products are mostly phloretin di-C glycoside, which to some extent limits the industrial production of nothofagin. Identifying new high-efficiency glycosyltransferases from plants or obtaining more active and specific glycosyltransferases through enzyme engineering based on protein structure and structure-activity relationship is a hot topic in current biosynthesis research. Summary of the invention

[0005] In order to solve the above existing technical problems, the purpose of the present invention is a new hawthorn carbon glycosyltransferase CGT52202 and its encoding gene and application.

[0006] In order to achieve the purpose, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a C-glycosyltransferase, the amino acid sequence of which is any of the following amino acid sequences:

[0008] 1) the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0009] 2) The amino acid sequence shown in SEQ ID NO. 2 in the sequence list is conventionally modified, or a tag for detection or purification is connected thereto;

[0010] Preferably, the conventional modification includes acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, or immobilization modification; the tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or Profinity eXact.

[0011] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned C-glycosyltransferase.

[0012] The nucleic acid molecule is C-glycosyltransferase gene CGT52202, and its nucleotide sequence is shown in SEQ ID NO.1.

[0013] The third aspect of the present invention provides a vector, an expression cassette, a host cell or a transformant containing the above-mentioned nucleic acid molecule;

[0014] Preferably, the host cell is selected from bacteria, yeast, plant cells, and animal cells;

[0015] Preferably, the bacteria is selected from Escherichia coli.

[0016] The fourth aspect of the present invention provides the use of the above-mentioned C-glycosyltransferase or the above-mentioned nucleic acid molecule or the above-mentioned vector, expression cassette, host cell or transformant containing the above-mentioned nucleic acid molecule in catalyzing the C-glycosylation reaction of a substrate.

[0017] In the above-mentioned application technology scheme, the C-glycosylation reaction refers to reacting a glycosyl acceptor compound and a glycosyl donor under the action of a C-glycosyltransferase, and obtaining a product C-glycoside compound by extraction and separation means.

[0018] In the above application technology scheme, the glycosyl acceptor compound includes flavonoid compounds, and the flavonoid compounds include phloretin.

[0019] Preferably, the glycosyl donor is selected from uridine diphosphate-α-D-glucose, uridine diphosphate-α-D-xylose, and uridine diphosphate-α-D-galactose.

[0020] The fifth aspect of the present invention provides a method for synthesizing nothofagin, using the above-mentioned C-glycosyltransferase to catalyze the reaction of a glycosyl acceptor compound and a glycosyl donor to produce nothofagin;

[0021] The reaction is preferably carried out at 15 to 55°C, and more preferably at 25 to 35°C;

[0022] Preferably, the glycosyl acceptor compound comprises a flavonoid compound, and the flavonoid compound comprises phloretin; the glycosyl donor is selected from uridine diphosphate-α-D-glucose, uridine diphosphate-α-D-xylose, and uridine diphosphate-α-D-galactose.

[0023] The sixth aspect of the present invention provides a method for producing the above-mentioned C-glycosyltransferase, comprising culturing the above-mentioned host cell in a suitable culture medium, and extracting and purifying the obtained C-glycosyltransferase;

[0024] Preferably, the culture medium is LB medium, and the host cell is Escherichia coli.

[0025] In order to facilitate the purification of the glycosyltransferase CGT52202 of the present invention, a tag as shown in Table 1 can be connected to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence list:

[0026] Table 1: Sequences of tags

[0027] Label Residue sequence SEQ ID NO Poly-Arg 5-6 (usually 5) RRRRR SEQ ID NO.5 Poly-His 2-10 (usually 6) HHHHHH SEQ ID NO.6 FLAG 8 DYKDDDDK SEQ ID NO.7 Strep-tag II 8 WSHQ SEQ ID NO.8 c-myc 10 EQKLISEEDL SEQ ID NO.9

[0028] The beneficial effects of the present invention are:

[0029] The present invention successfully cloned a new glycosyltransferase gene (CGT52202) from hawthorn. The enzyme can be used to prepare nothofagin using phloretin as a substrate, can prepare nothofagin from phloretin through an in vitro enzymatic reaction, or can be used as a glycosylation element to synthesize nothofagin in a plant or microbial system, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the phylogenetic tree of CGTs (neighbor-joining method).

[0031] Figure 2 Schematic diagram of CGT52202 recombinant expression vector and expression product detection (SDS-PAGE 10%): A: prokaryotic expression vector diagram; B: pET28a-HIS-MBP-CGT52202 expression product detection, V in the figure represents an empty vector.

[0032] Figure 3 UPLC detection of CGT52202 enzymatic reaction products.

[0033] Figure 4 Q-tof detection of CGT52202 enzymatic product (ES - ,MS 2 spectra). DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were set up for three repeated experiments, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0036] The main sources of reagents in the embodiments of the present invention are as follows:

[0037] Tool enzyme expression backbone vector pET28a-HIS-MBP: commercially available.

[0038] Escherichia coli competent cells Trans1-T1 and E. coli Rossetta (DE3), pEASY-Uni Seamless Cloning and Assembly Kit, and DNA gel recovery kit: products of Beijing Quanshijin Biotechnology Co., Ltd.

[0039] Plant RNA Purification Reagent: product of Invitrogen.

[0040] PrimeScript 1st Strand cDNA Synthesis Kit: a product of TaKaRa Biotechnology (Dalian) Engineering Co., Ltd.

[0041] BamH I and other restriction endonucleases, Prestained Protein Ladder (protein gradient): products of New England Biotechnology (NEB) Beijing Co., Ltd.

[0042] KOD-Plus-Neo high-fidelity PCR enzyme: product of Toyobo (Shanghai) Biotechnology Co., Ltd.

[0043] Plasmid Mini Kit (plasmid mini-extraction kit): product of OMEGA.

[0044] The inducer isopropyl-β-D-thiogalactoside IPTG and the protease inhibitor phenylmethylsulfonyl fluoride PMSF were both products of Sigma Company, USA.

[0045] Phloretin: CAS number is 60-82-2: Beijing BetterKang Biopharmaceutical Technology Co., Ltd.

[0046] Uridine diphosphate-α-D-glucose: CAS number is 28053-08-9: Shanghai Yuanye Biotechnology Co., Ltd.

[0047] Nothofagin standard: CasNo: 11023-94-2, the manufacturer is Beijing Betterkang Biotechnology Co., Ltd.

[0048] Example 1. Cloning of glycosyltransferase gene CGT52202

[0049] Total RNA from hawthorn leaves was extracted using Trizol RNA extraction kit from Takara (Japan), and total RNA was used as a template to synthesize total cDNA from hawthorn leaves using PrimeScript 1st Strand cDNA Synthesis Kit from Takara. Glycosyltransferase gene CGT52202 was amplified using total cDNA from hawthorn leaves as a template. The nucleotide sequence of glycosyltransferase gene CGT52202 is shown in SEQ ID NO.1. Glycosyltransferase gene CGT52202 encodes protein CGT52202, and the amino acid sequence of protein CGT52202 is shown in SEQ ID NO.2. Phylogenetic tree analysis was performed on the cloned amino acid sequence of glycosyltransferase CGT52202 and the publicly published glycosyltransferases. The results showed that glycosyltransferase CGT52202 and CGTs from other species were clustered into one category, indicating that glycosyltransferase CGT52202 may be a C-glycosyltransferase ( Figure 1 ).

[0050] Example 2: Hawthorn glycosyltransferase gene CGT52202 has the activity of specifically catalyzing phloretin to produce nothofagin

[0051] 1. Construction of prokaryotic expression vector

[0052] PCR amplification was performed using cDNA from hawthorn leaves as a template. The PCR reaction system was: 25 μL 2× FastPfu PCR SuperMix (Beijing Quanshijin Biological Company), 5μL forward primer aqueous solution (concentration of 10μmol / L), 5μL reverse aqueous solution (concentration of 10μmol / L), 1μL hawthorn leaf cDNA aqueous solution (containing 20ng hawthorn leaf cDNA) and 14μL sterile double distilled water. PCR reaction conditions are: 94℃ pre-denaturation for 2min; 98℃ denaturation for 10s, 55℃ annealing for 30s, 72℃ extension for 1.5min, 34 cycles; 72℃ extension for 7min. The primers used for PCR (restriction sites BamHI / SalI) are as follows:

[0053] Forward primer (SEQ ID NO.3):

[0054] 5'-AGGGGCCC GAATTC GGATCCATGTCAAACACCGCAGCTCCT-3',

[0055] Reverse primer (SEQ ID NO.4):

[0056] 5'-GCGGCCGC AAGCTTGTCGACTTAGTTCTTCTGCGTCAAATATTCCA-3'.

[0057] The obtained PCR product was connected to the pET28a-HIS-MBP vector backbone obtained by double digestion with BamHI and SalI by seamless splicing, and the obtained recombinant vector with correct sequence was recorded as pET28a-HIS-MBP-CGT52202.

[0058] pET28a-HIS-MBP-CGT52202 is a recombinant vector obtained by replacing the DNA fragment between the BamHI and SalI recognition sequences of the pET28a-HIS-MBP vector with the hawthorn glycosyltransferase CGT52202 gene shown in SEQ ID NO.1 in the sequence list. The recombinant vector can express the protein shown in SEQ ID NO.2 in the sequence list (i.e., hawthorn glycosyltransferase CGT52202).

[0059] 2. Induced expression of engineered bacteria

[0060] Take 1 μL of the target plasmid pET28a-HIS-MBP-CGT52202 to transform E. coli Rossetta (DE3) competent cells, select transformants on LB / Kan plates, and culture at 37°C overnight. Pick a single clone colony (i.e., the recombinant bacteria containing the target plasmid, recorded as E-pET28a-HIS-MBP-CGT52202) and then transfer it to a 50 μg mL -1 Kan was cultured in LB liquid medium at 37°C overnight with shaking; the culture medium was inoculated at a ratio of 1:100 in a 50 μg mL -1 Kan's LB liquid medium was cultured at 37°C and 250 rpm until OD 600 ≈ 0.6 (logarithmic growth phase); 10 μL of isopropylthio-β-D-galactoside (IPTG, 1 M·L -1 ) and cultured at 16°C with shaking for 18 h to induce the expression of the target protein.

[0061] According to the above steps, pET28a-HIS-MBP-CGT52202 was replaced with the pET28a-HIS-MBP empty vector as a negative control, and the resulting recombinant bacteria was recorded as E-pET28a-HIS-MBP.

[0062] The results of SDS-polyacrylamide gel electrophoresis showed that an obvious specific protein expression band appeared at a molecular weight of about 100 kD, which was consistent with the theoretical prediction (e.g. Figure 2 ).

[0063] 3. In vitro enzyme function verification

[0064] 1. Preparation of crude enzyme extract:

[0065] The E-pET28a-HIS-MBP-CGT52202 LB culture obtained in step 2 was centrifuged at 4°C, 5000g for 5 min, the bacterial precipitate was collected, the bacterial cell was resuspended in pre-cooled pure water, washed once, and 3mL20mM PB Buffer (pH7.4) buffer containing 0.5mM EDTA, 0.5mM PMSF, and 10% glycerol (pH 7.4) by volume was added to each 50mL bacterial cell obtained by centrifugation. The bacterial cell was disrupted using a cell disruptor, and the cell was disrupted at 65% power for 2s and 2s, and the cell was disrupted for 20min; after the disruption, the resulting liquid was centrifuged at 4°C, 13000rpm for 15min, the supernatant was taken, 30μL 4×Loadingbuffer was added to 90μL supernatant, and the supernatant was bathed in a 100°C water bath for 5min as an SDS-PAGE analysis sample, and the remaining supernatant was used as a CGT52202 crude enzyme extract and placed at -80°C for use.

[0066] E-pET28a-HIS-MBP-CGT52202 was replaced with E-pET28a-HIS-MBP as a negative control, and the resulting crude enzyme extract was recorded as the control crude enzyme extract.

[0067] 2. Enzyme function verification:

[0068] The crude enzyme extract obtained from E-pET28a-HIS-MBP-CGT52202 (the crude enzyme extract obtained from E-pET28a-HIS-MBP was used as a negative control) was used to perform in vitro enzyme function detection using phloretin as a reaction substrate.

[0069] The enzymatic reaction system was as follows: 100 μL crude enzyme extract, 0.04 μM phloretin, 0.08 μM uridine diphosphate-α-D-glucose, and after reacting at 30°C overnight, 200 μL pre-cooled pure methanol was directly added to the system to terminate the reaction, shaken, centrifuged at 13,000 rpm for 15 min, filtered through a 0.22 μm PTFE filter membrane, and the filtrate was collected for UPLC / Q-TOF-MS analysis. UPLC-MS analysis conditions were as follows: chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 mm×50 mm, 1.7 μm); mobile phase: 0.1% formic acid-water (A), 0.1% formic acid-acetonitrile (B), 0-0.5 min, 90%-80% A; 0.5-5 min, 80%-75% A; 5-7 min, 75%-30% A; 7-7.2 min, 30%-5% A; 7.2-9.2 min, 5% A; 9.2-10 min, 5%-90% A; 10-13 min, 90% A; flow rate: 0.4 mL min -1; Full scan 190nm-500nm, column temperature 40℃, injection volume 2μL.

[0070] The results showed that the peak time of the crude enzyme reaction product of CGT52202 was consistent with that of the reference substance nothofagin, while no corresponding characteristic peak was detected in the negative control sample ( Figure 3 ).

[0071] The characteristic peaks in the crude enzyme reaction product of CGT52202 were qualitatively analyzed by Q-TOF-MS. The mass spectrometry conditions were as follows: Waters Xevo G2-S QTOF-MS mass spectrometer with electrospray ionization source (ESI) and negative ion detection mode; scanning range m / z 50-1500, scanning time 0.2 s, capillary voltage 2000 V, cone voltage 40 V, and solvent removal gas nitrogen 900 L·h -1 , solvent removal temperature 450°C, ion source temperature 100°C.

[0072] The results are as follows Figure 4 As shown, the product molecular ion peak is m / z 435.1294[MH] - With substrate phloretin m / z273.2700[MH] - The molecular weight difference is 162, indicating that the product is a glucose molecule added to phloretin, and the product molecule contains 315.0870 [M-120-H] - The ion fragment peak indicates that the product is phloretin-C-β-D-glucoside. In addition, the product molecule and the reference substance nothofagin have the same peak time, molecular weight and fragment ion, indicating that the product m / z 435.1294 [MH] - is nothofagin (see Figure 4 ).

[0073] The above experiment shows that CGT52202 has the activity of specifically catalyzing phloretin to produce nothofagin without the generation of byproducts. The product of CGT catalyzing phloretin reported so far is mainly phloretin di-carbon glycoside, and nothofagin is a byproduct. However, CGT52202 of the present invention can catalyze phloretin to produce only nothofagin.

[0074] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

[0075] The nucleotide sequence of the C-glycosyltransferase gene CGT52202 of the present invention (SEQ ID NO.1) is as follows :

[0076]

[0077] The amino acid sequence of the C-glycosyltransferase of the present invention (SEQ ID NO.2) is as follows:

[0078] *。

Claims

1. A C-glycosyltransferase, characterized in that: Its amino acid sequence is any of the following: 1) the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; 2) The amino acid sequence shown in SEQ ID NO. 2 in the sequence list is conventionally modified, or a tag for detection or purification is connected thereto; Preferably, the conventional modification includes acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, or immobilization modification; the tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or Profinity eXact.

2. A nucleic acid molecule encoding the C-glycosyltransferase according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is C-glycosyltransferase gene CGT52202, and its nucleotide sequence is shown in SEQ ID NO.

1.

4. A vector, expression cassette, host cell or transformant containing the nucleic acid molecule according to claim 2 or 3; Preferably, the host cell is selected from bacteria, yeast, plant cells, and animal cells; Preferably, the bacteria is selected from Escherichia coli.

5. Use of the C-glycosyltransferase according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the vector, expression cassette, host cell or transformant containing the nucleic acid molecule according to claim 2 or 3 according to claim 4 in catalyzing a C-glycosylation reaction of a substrate.

6. The use according to claim 5, characterized in that: The C-glycosylation reaction refers to the reaction of a glycosyl acceptor compound and a glycosyl donor under the action of a C-glycosyltransferase, and obtaining a product C-glycoside compound by extraction and separation.

7. The use according to claim 6, characterized in that: The glycosyl acceptor compound comprises flavonoid compounds, and the flavonoid compounds comprise phloretin.

8. The use according to claim 6, characterized in that: The glycosyl donor is selected from uridine diphosphate-α-D-glucose, uridine diphosphate-α-D-xylose, and uridine diphosphate-α-D-galactose.

9. A method for synthesizing nothofagin, characterized in that: Using the C-glycosyltransferase of claim 1 to catalyze the reaction between a glycosyl acceptor compound and a glycosyl donor to produce nothofagin; The reaction is preferably carried out at 15 to 55°C, and more preferably at 25 to 35°C; Preferably, the glycosyl acceptor compound comprises a flavonoid compound, and the flavonoid compound comprises phloretin; the glycosyl donor is selected from uridine diphosphate-α-D-glucose, uridine diphosphate-α-D-xylose, and uridine diphosphate-α-D-galactose.

10. A method for producing the C-glycosyltransferase according to claim 1, characterized in that: Culturing the host cell according to claim 4 in a suitable culture medium, and extracting and purifying the obtained C-glycosyltransferase; Preferably, the culture medium is LB medium, and the host cell is Escherichia coli.

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