Dendrobium huoshanense glycosyl transferase DhUGT44 and application thereof

Abstract

The invention discloses application of glycosyl transferase coded by a dendrobium huoshanense DhUGT44 gene in biosynthesis of cosmosiin, and belongs to the technical field of biology. The nucleotide sequence of the DhUGT44 gene encodes a nucleotide sequence of an amino acid sequence as shown in SEQ ID NO. 2. The cosmosiin is obtained by a biosynthesis method for carrying out enzyme catalysis by utilizing the DhUGT44 protein in vitro, and a new method is provided for synthesizing the cosmosiin.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to Dendrobium huoshanense glycosyltransferase and its applications. Background Art

[0002] Cosmosiin, also known as apigenin-7-glucoside, is a glycoside form of a flavonoid compound. Its molecular formula is C21H20O10, with a molecular weight of approximately 432.38 and a CAS number of 578-74-5. This compound is commonly present in various plants such as artichoke, chamomile, olive, Pogostemon cablin, Agrimonia pilosa var. japonica, etc. As a natural product, cosmosiin plays a protective role in plants and also shows potential application value in human health. Research has shown that it has various biological activities such as antioxidant, anti-inflammatory, and anti-cancer effects. For example, cosmosiin may help inhibit inflammatory responses, counteract the growth of certain types of cancer cells, and provide cardiovascular protection. In addition, due to its high purity and stability, it is also used as an analytical standard and reference substance in scientific research.

[0003] The preparation methods of cosmosiin usually include the following: 1. Plant extraction: This is the most direct and common method. It involves extracting and purifying cosmosiin from plants containing it, such as chamomile, Agrimonia pilosa, etc. This method relies on natural resources, so the yield may be affected by seasonal changes, geographical distribution, and plant growth conditions. 2. Semi-synthesis method: In some cases, a semi-synthesis method can be adopted, that is, using a structurally similar compound as the starting material and obtaining the target product through chemical modification. For example, cosmosiin can be synthesized by introducing a glucose group through glycosylation of apigenin. This requires specific enzymes or chemical catalysts to achieve glycosyl transfer or addition. 3. Biosynthesis / fermentation technology: With the development of biotechnology, using microbial fermentation or transgenic plants to produce specific natural products has become a trend. Scientists have explored using engineered strains or other biological systems to express the relevant genes responsible for synthesizing cosmosiin to achieve industrial production. This method can not only reduce the dependence on natural resources but also improve the stability and consistency of the product. 4. Enzyme-catalyzed synthesis: Enzymatic synthesis is a green chemistry method that uses specific enzymes as catalysts to carry out selective chemical reactions. For cosmosiin, glycosyltransferase can be used to transfer a glucose group to apigenin to form cosmosiin. This synthesis method has high regioselectivity and stereoselectivity, can effectively reduce the generation of by-products, and is environmentally friendly. So far, the enzymes available for the enzymatic synthesis of cosmosiin are very limited. Therefore, it is of great significance to explore new enzymes and genes for the enzymatic synthesis of cosmosiin. Summary of the Invention

[0004] The present invention mainly aims at the above technical problems and provides a glycosyltransferase gene for catalytically generating cosmosiin compounds and a method for biosynthesizing cosmosiin compounds to solve the deficiencies of the existing chemical synthesis of cosmosiin.

[0005] Specifically, the present invention provides the following technical solutions: On the one hand, the present invention provides a glycosyltransferase, the amino acid sequence of which contains the sequence shown in SEQ ID NO.2.

[0006] On the other hand, the present invention provides a glycosyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence containing the sequence shown in SEQ ID NO.2, or based on the principle of complementary pairing, the glycosyltransferase gene provided by the present invention can be a sequence that is fully complementary paired with the nucleotide sequence encoding an amino acid sequence containing the sequence shown in SEQ ID NO.2.

[0007] In a preferred embodiment, the nucleotide sequence of the above-mentioned glycosyltransferase gene comprises the nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1.

[0008] As is well known to those skilled in the art, a gene sequence can also contain introns, promoters, and various regulatory elements. Therefore, the nucleotide sequence of the above-mentioned glycosyltransferase gene can also contain introns, promoters, and various regulatory elements.

[0009] On the other hand, the present invention provides the use of a glycosyltransferase gene in the synthesis of cosmosinoid compounds. The nucleic acid sequence of the glycosyltransferase gene is a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO.2, or a sequence that is fully complementary to the nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO.2.

[0010] In a preferred embodiment, the above-mentioned glycosyltransferase gene sequence is a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO.2, or a nucleotide sequence that is completely complementary to the nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO.2.

[0011] In a preferred embodiment, the nucleotide sequence of the above-mentioned glycosyltransferase gene is as shown in SEQ ID NO.1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1.

[0012] On the other hand, the present invention provides the use of the aforementioned glycosyltransferase in the synthesis of cosmosinoid compounds.

[0013] On the other hand, the present invention provides a method for synthesizing cosmosinoid compounds, the method comprising the following steps: Obtain the aforementioned glycosyltransferase; Use the aforementioned glycosyltransferase to catalytically synthesize cosmosin in an enzymatic reaction system.

[0014] In a preferred embodiment, the above-mentioned glycosyltransferase is obtained by prokaryotic expression.

[0015] In a preferred embodiment, the above-mentioned glycosyltransferase is obtained by chemical synthesis.

[0016] In a preferred embodiment, the enzymatic reaction system contains the above-mentioned glycosyltransferase, UDP-Glc, a substrate for synthesizing cosmosinoid compounds, and a buffer.

[0017] On the other hand, the present invention provides a method for synthesizing cosmosinoid compounds, characterized in that the method comprises the following steps: 1) Obtain a glycosyltransferase comprising the amino acid sequence shown in SEQ ID NO.2; 2) Use the above glycosyltransferase to catalytically synthesize cosmosin compounds in an enzymatic reaction system.

[0018] In a preferred embodiment, the above glycosyltransferase is obtained by prokaryotic expression.

[0019] In a preferred embodiment, the above glycosyltransferase is obtained by chemical synthesis.

[0020] In a preferred embodiment, the enzymatic reaction system contains the above glycosyltransferase, UDP-Glc, a substrate for synthesizing cosmosin compounds, and a buffer.

[0021] In a preferred embodiment, the enzymatic reaction system further contains divalent cations.

[0022] In a preferred embodiment, the reaction temperature of the enzymatic reaction system is 4 - 70 °C, more preferably 4 - 70 °C.

[0023] In a preferred embodiment, the pH of the enzymatic reaction system is 6.0 - 9.0, more preferably 6.0 - 8.0.

[0024] Compared with the prior art, the present invention has the following advantages: Compared with the prior art, the present invention uses an enzyme-catalyzed biosynthesis method to obtain cosmosin, which has the advantages of simpler steps, less pollution, and more single products compared with the chemical synthesis method.

[0025] The glycosyltransferase in the present invention has a relatively wide temperature range suitable for the reaction.

[0026] The glycosyltransferase in the present invention does not depend on metal ions.. Description of the Drawings

[0027] The following combines the drawings and specific embodiments to detail the method of the present invention and its beneficial effects.

[0028] Figure 1 Results of colony PCR, where M in the figure represents DNA Marker, and lane 1 is DhUGT44.

[0029] Figure 2 It is an SDS-PAGE gel electrophoresis diagram of the purified pET-28a(+)-DhUGT44 fusion protein.

[0030] Figure 3 It is the result of the reaction of DhUGT44 with the substrate and a schematic diagram of catalytic activity, Figure 3A: Liquid phase diagram of the reaction of DhUGT44 with apigenin; Figure 3 B: MS diagram of the product.

[0031] Figure 4 The following are the test results of the enzymatic reaction conditions, where Figure 4 A is the temperature condition, Figure 4 B is the reaction time, Figure 4 C is the metal ion, Figure 4 D is the pH condition. Detailed implementation mode

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0034] Example 1 Cloning of the target glycosyltransferase gene 1. Total RNA was extracted from Dendrobium huoshanense using a total RNA extraction kit, and single-stranded cDNA was generated using a reverse transcription kit with gDNA wiper. Using this as a template, polymerase chain reaction (PCR) was performed to amplify the full-length fragment of the DhUGT44 gene. The specific primers are as follows: DhUGT44-F1: ATGGCTTCGCCTCTCAGCA; DhUGT44-R1: CTATTTGTGGAATTCCATGACCC.

[0035] The PCR reaction system was: 2 × Phanta Max Master Mix (Dye Plus) (P515, Nanjing Novoprotein Scientific Co., Ltd.) 25 μL, 1 μL each of the upstream and downstream primers, 50 - 100 ng of the template, and supplemented with dd H2O to 50 μL. The PCR amplification program was: ① React at 95 °C for 3 min; ② React at 95 °C for 15 s, 60 °C for 45 s, and 72 °C for 60 s, for a total of 35 cycles; ③ Extend at 72 °C for 5 min. After the reaction, the size of the product was detected by 1% agarose gel electrophoresis.

[0036] 2. Ligate the amplified product to the T-vector (C601, Nanjing Novoprotein Scientific Inc.), and transform it into Escherichia coli DH5α. Pick single colonies and culture them in LB medium, and perform colony PCR verification ( Figure 1 : M: DL 2000 marker; Lane1: DhUGT44 gene). The reaction system for colony PCR is as follows: 12.5 μL Green Taq Mix (P131, Nanjing Novoprotein Scientific Inc.), 1 μL each of the upstream and downstream primers, 2 μL of the template (bacterial solution), and make up to 25 μL with dd H2O. Send the samples with correct colony PCR results for sequencing.

[0037] The nucleotide sequence obtained by sequencing is as shown in SEQ ID NO.1. This gene sequence contains 1368 nucleotides and encodes 452 amino acids (shown in SEQ ID NO.2).

[0038] >SEQ ID NO.1 >SEQ ID NO.2 MEPSEKKAIGHALLLPYPSQGHINPMLQFGKRIAAHGRLATAAITRFIASTTAHNADQVAIETISDGFDEAGFPSAASISAYLTSLETIGSQTLDDLLSSLADRGRPVSLLIYDSFFPWAVDVAKRHGAAAASFFTQSVAVDVIYCHVWEGRLKFPASAAVKLPGLPEMEVEDLPTFLAAPEVVYVAYLEMVLNQYKNLEKADIMVINSFYELESEEMNWLTSVRRAITIGPTVPSTYLDNRIPDDHKYAIDLYPPETSACKSWISSLPLSSAIFVSVGSMAVLSASQMVELAVGLAATDRPFLWVVRASEADKLPAGFAETTKKNGSLVVSWAPQLEILATGKFGCFVTHCGWNSTMEALALGVPMVALPQWTDQTTDAKYIEEVWGMGIRARKGEEGIVRREEVERCVREVMEGERSEGIRRRAREWKEASRRAVDEGGSSDKNIADLLDKFC Example 2 Induced Expression and Purification of DhUGT44 Protein Enlarge the culture of the target bacterial solution with correct sequencing and extract the recombinant plasmid pET-28a-DhUGT44 from it. Transform the recombinant plasmid into Escherichia coli BL21(DE3) competent cells, spread the cells on a plate, and pick out the clones into LB medium. Incubate at 37 °C and 200 rpm for 16 h to obtain the mother solution. Inoculate the mother solution into LB medium containing 50 μg / mL KaN at a ratio of 1:100, and culture at 37 °C until the OD 600 = 0.6 - 0.8 (using sterile LB medium as a control), add IPTG with a final concentration of 0.5 mM, and finally incubate at 16 °C and 200 rpm for 18 - 20 h.

[0039] Centrifuge to collect the induced bacterial liquid, resuspend the cells with pre-cooled Tris-HCl (50 mM) buffer at pH = 7.5, centrifuge again to collect the cells, and repeat once to remove the residual medium. Finally, add 15 mL of the above Tris-HCl buffer to each gram of wet bacteria. The obtained cell suspension can be frozen overnight in a -20°C refrigerator and then broken at low temperature on an ultrasonic cell disruptor with a power of 300 W, working for 3 s and intermittent for 3 s. Stop the disruption when the cell suspension becomes clear. Finally, centrifuge the disrupted solution at 4°C and 14,000 rpm for 4 min, and the supernatant is the crude enzyme solution, which is stored in a -20°C refrigerator.

[0040] According to the His-Tag on the fusion protein, nickel affinity chromatography column is used to purify the target protein, and the target protein is eluted with imidazole solutions of different concentrations. Finally, 10% SDS-PAGE electrophoresis is used to detect the molecular weight and purity of the target protein. As Figure 2 shown (M: protein Marker; Lane 1: purified protein of DhUGT44), it indicates that the protein is successfully expressed in the supernatant, and the purified protein can be used for subsequent experiments.

[0041] Example 3 In vitro enzyme activity detection 1. Enzyme activity reaction system: 10 μg of pure enzyme, 0.5 mM substrate apigenin, 5 mM sugar donor UDP-Glc, 14 mM β-mercaptoethanol, and made up to 200 μL with 50 mM Tris-HCl buffer at pH = 7.5. After the reaction solution is incubated in a water bath at 37°C for 12 h, an equal volume of pre-cooled methanol is added, vortexed and mixed evenly, and then centrifuged at 4°C and 14,000 rpm for 4 min. Take the supernatant for detection by high performance liquid chromatography.

[0042] 2. HPLC conditions Phase A is 0.1% formic acid in water, phase B is acetonitrile, the flow rate of the mobile phase is 0.8 mL / min, the injection volume is 20 μL, the column temperature is 30°C, and the gradient elution system is 5% - 20% B (0 - 5 min), 20% - 22% B (5 - 8 min), 22% - 25% B (8 - 17 min), 25% - 35% B (17 - 23 min), 35% - 50% B (23 - 25 min), 50% - 95% B (25 - 32 min), 95% B (32 - 37 min), 95% - 5% B (37 - 38 min), 5% B (38 - 42 min).

[0043] 3. LC-MS conditions: The detection mode is negative ion mode; the composition of the mobile phase is the same as that of HPLC, the detection source is an electrospray ionization source, and the acquisition mode is AutoMS2 ; capillary voltage 3500 V; sheath gas temperature 350 °C, flow rate 11 L / min; drying gas temperature 325 °C, flow rate 8 L / min; mass-to-charge ratio scanning range 100 - 1700 m / z; collision voltage 175 V; collision energy 15 - 50 eV.

[0044] 4. Detection results: The results are shown in Figure 3 , when using apigenin as the substrate, 1 product peak can be observed in the reaction of DhUGT44. By comparing the retention time and mass-to-charge ratio m / z of the standard product, it is found that the product is cosmosin.

[0045] Example 4: Detection of enzymatic reaction condition parameters Using apigenin as the glycosyl acceptor and UDP-Glc as the glycosyl donor, the effects of pH, temperature, metal ions, and reaction time on catalytic activity were investigated.

[0046] 1. Temperature: Using apigenin as the glycosyl acceptor and UDP-Glc as the glycosyl donor, the variation law of enzyme activity at different reaction temperatures (4 °C, 30 °C, 37 °C, 40 °C, 42 °C, 50 °C, 60 °C, and 70 °C) was investigated. The reaction system contained 10 μg of pure protein, 0.5 mM of the substrate, 5 mM of the glycosyl donor, 14 mM of β-mercaptoethanol, and was made up to 200 μL with 50 mM, pH = 7.0 Tris-HCl buffer. The reaction solution was reacted at each temperature for 6 h. After the reaction, an equal volume of pre-cooled methanol was immediately added to the reaction system to terminate the reaction. After centrifugation, the supernatant was taken for HPLC detection, and three parallel tests were set for each group of reactions. The conversion rate of the substrate was calculated based on the peak area ratio in the chromatogram.

[0047] 2. pH: The effects of different pH buffers of 50 mM: 6.0 - 8.0, Na2HPO4-NaH2PO4 Buffer; 7.0 - 9.0, Tris-HCl Buffer on enzyme activity were investigated. The reaction system was: in a 200 μL reaction system containing 14 mM β-mercaptoethanol, 5 mM UDP-Glc, 10 μg of protein, 0.5 mM of the substrate apigenin, and supplemented to the specified volume with buffers of different pH values. The reaction solution was reacted at 37 °C for 6 h. After completion, an equal volume of pre-cooled methanol was immediately added, and after centrifugation, the supernatant was taken for HPLC detection. To ensure the reliability of the data, three replicate samples were set for each group of reactions, and finally the conversion rate was calculated through the peak area of the chromatogram.

[0048] 3. Metal ions: The receptor molecule of the reaction solution was selected as the compound apigenin, and the donor was selected as UDP-Glc. Different divalent metal ions: Mg 2+ , Ca 2+, Zn 2+ , Co 2+ , Ba 2+ , Mn 2+ The effects of , , , and EDTA on enzyme activity. In a 200 μL reaction system containing 14 mM β-mercaptoethanol, 5 mM UDP-Glc, 10 μg protein, 0.5 mM substrate apigenin, 5 mM divalent metal ions, and supplemented to the specified volume with 50 mM, pH = 7.0 Tris-HCl buffer. The reaction solution was reacted at 40 °C for 6 h. After the reaction, an equal volume of pre-cooled methanol was immediately added, and centrifuged at 14000 rpm and 4 °C for 4 min. Each reaction was set up with three replicate samples, and the supernatant was taken for HPLC detection. The catalytic efficiency of the enzyme was calculated at the maximum absorption wavelength of the substrate.

[0049] 4. Reaction time: A 200 μL Tris-HCl (50 mM, pH = 7.0) reaction system contained 14 mM β-mercaptoethanol, 5 mM UDP-Glc, 10 μg purified protein, 0.5 mM substrate apigenin, and was reacted at 40 °C in a water bath for 30 min, 60 min, 120 min, 360 min, 720 min, 960 min, and 1440 min respectively. After the reaction, an equal volume of pre-cooled methanol was quickly added to each reaction solution to terminate the reaction, and centrifuged at 14000 rpm and 4 °C for 4 min. The supernatant was taken for HPLC detection, and the conversion rate of the substrate was calculated. To ensure the accuracy of the data, three parallel experiments were set up for each group, and the substrate conversion rate was calculated according to the ratio of the peak areas in the HPLC chromatogram.

[0050] 6. Detection results: The results showed that ( Figure 4 ), DhUGT44 had the highest enzyme activity at 30 - 40 °C. As the temperature increased, its catalytic activity gradually decreased. When the temperature increased to 70 °C, the enzyme activity was still 30%. The optimum pH of the reaction solution was Tris-HCl at 7.0. This enzyme was independent of metal ions. Among the divalent metal ions, Ca 2+ , Mg 2+ could both improve the enzyme activity, and among them, Ca 2+ had the best promoting effect.

[0051] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

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

2.

2. A glycosyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.

2. A vector expressing the enzyme according to claim 1 or the gene according to claim 2.

3. Use of the glycosyltransferase according to claim 1, the gene according to claim 2 or the vector according to claim 3 in the synthesis of cosmosin compounds.

4. A method for synthesizing a cosmosin compound, the method comprising the following steps: 1) Obtaining the glycosyltransferase according to claim 1; 2) Using the glycosyltransferase in step 1) to catalytically synthesize a cosmosin compound in an enzymatic reaction system.

5. The method for synthesizing cosmosin compound according to claim 5, wherein In step 1), the glycosyltransferase is obtained by prokaryotic expression or by chemical synthesis.

6. The method for synthesizing cosmosin compound according to claim 5, wherein, In step 2), the enzymatic reaction system contains the glycosyltransferase described in step 1), UDP-Glc and flavonoid compounds.

7. A method for synthesizing cosmosin compound as claimed in claim 5, characterized in that, In step 2), the enzymatic reaction system further contains divalent cations.

8. A method for synthesizing cosmosin compound as claimed in claim 5, characterized in that, In step 2), the reaction temperature of the enzymatic reaction system is 4-70 °C.

9. The method for synthesizing cosmosinoid compounds according to claim 5, wherein, In step 2), the pH of the enzymatic reaction system is 6.0-8.0.

Images