Bifunctional gastrodia elata glycosyltransferase GaUGT015 gene and application thereof in preparation of gastrodin and arbutin

Abstract

The invention relates to a difunctional gastrodia elata glycosyl transferase GaUGT015 gene and application thereof in preparation of gastrodin and arbutin, and belongs to the technical field of biology. The nucleotide sequence of the difunctional glycosyl transferase GaUGT015 gene of the gastrodia elata is shown as SEQ ID NO.1, and the total length of the sequence is 1407 bp; the amino acid sequence of the encoded protein is as shown in SEQ ID NO.2, and 469 amino acid residues are encoded. The gastrodia elata bifunctional glycosyl transferase GaUGT015 gene disclosed by the invention can be used as a biosynthesis regulation gene of gastrodin and arbutin, is applied to preparation of gastrodin and arbutin, and is remarkable in application prospect and easy to popularize and apply.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a gastrodia bifunctional glycosyltransferase GaUGT015 gene and its application in the preparation of gastrodin and arbutin. Background Art

[0002] Gastrodia elata Blume ( Gastrodia elata Bl . ), also known as Dingfengcao and Chijian, is a perennial parasitic herb of the genus Gastrodia in the family Orchidaceae ( Gastrodia ). Gastrodia elata has the effects of calming endogenous wind and stopping convulsions, suppressing hyperactive liver-yang, expelling wind and dredging collaterals, etc., and is often used to treat infantile convulsions, epilepsy convulsions, tetanus, headache and dizziness, flaccidity of hands and feet, numbness of limbs, and rheumatic arthralgia.

[0003] Gastrodin (GAS) is one of the main active components of Gastrodia elata, also known as gastrodioside, chemical name: 4-hydroxymethylphenyl- β -D-glucopyranoside, which is a phenolic glycoside with the molecular formula: C 13 H 18 O7 and a molecular weight of 286.28 kDa. Gastrodin has long been used clinically in China for analgesic and hypnotic effects without obvious side effects. As the "divine medicine for treating wind", it is a drug for the central nervous system. It is mainly used for sedation, sleep aid, and analgesia. Gastrodin can inhibit the cell death and apoptosis induced by excitatory amino acids, has the ability to scavenge free radicals, can counteract the damage of free radicals to PC12 cells, and has a neuroprotective effect. Gastrodin has obvious effects in the treatment of sclerosis, presumably related to promoting angiogenesis. Gastrodin has a good therapeutic effect on patients with functional dyspepsia accompanied by anxiety and depression symptoms, and can dilate blood vessels and reduce the pharmacological effects of the central nervous system such as hypertensive heart disease and fibrosis. Gastrodin enhances spinal cord synapses and inhibits inflammatory pain. In addition, some studies have also found that gastrodin has various biological effects such as anti-obesity, anti-inflammatory, memory improvement, and acetylcholinesterase inhibitor.

[0004] The main methods for obtaining gastrodin in the early stage were direct extraction from Gastrodia elata tubers or chemical processing. However, the chemical synthesis of gastrodin is energy-consuming and environmentally unfriendly; the artificial cultivation extraction method has problems such as the inability to guarantee the quality of Gastrodia elata, the long cultivation period of Gastrodia elata, and the difficulty in extracting high-purity gastrodin. Therefore, the biosynthesis of gastrodin has attracted more and more attention in recent years.

[0005] Arbutin (Arb), also known as arbutin, arbutin glycoside, and arbutin phenol glycoside, its chemical name is p-hydroxyphenyl- β-D-glucopyranoside, is a glycoside substance naturally present in green plants and is a solute rich in many freeze-dried tolerant plants such as wheat, pears, and bearberries in the Ericaceae family. Arbutin has pharmacological effects such as whitening, pigment removal, antioxidant, anti-microbial, and anti-inflammatory activities. With the in-depth research and further expansion of the research scope in recent years, more functions of arbutin have emerged, such as antioxidant, anti-inflammatory, antibacterial, antitussive, expectorant, antiasthmatic, anticancer, antineoplastic, and treatment of acute lung injury. In recent years, arbutin has also been applied to some extent in the food field such as improving the aroma of wine, and there are more and more research reports on its safety, nutritional value, etc. With the increasing emphasis on diet therapy and health care by people, many drugs are also developing towards the direction of homology between medicine and food. Therefore, arbutin has broad development prospects in the fields of food and health products. The production of arbutin by plant extraction method has problems such as long plant growth cycle, complex extraction process, and low yield, while the chemical synthesis method of arbutin has disadvantages such as harsh reaction conditions, poor stereoselectivity, and low yield. In recent years, the biosynthesis method has gradually become a popular research direction for synthesizing arbutin due to its advantages such as simple and mild reaction conditions and economical and environmentally friendly production process.

[0006] In recent years, with the rapid development of the field of synthetic biology, using synthetic biology technology to produce natural medicine monomers can effectively solve the above problems. However, to clarify the biosynthetic pathways of these active ingredients, it is necessary to identify the key genes related to these pathways, and exploring these catalytic enzyme genes has become a key link in studying the biosynthetic pathways of plant metabolites. Currently, the synthetic pathways for catalyzing the glycosylation reaction of the hydroxyl group at the C -4 position of p-hydroxybenzyl alcohol to form gastrodin and the formation of arbutin by the connection of the glucose group and the aldehyde group between glucose and benzaldehyde are clear, but the function of the glycosyltransferase responsible for glycosylation excavated in Gastrodia elata has not been verified, which affects the progress of the biosynthesis of gastrodin and arbutin. Summary of the Invention

[0007] To solve the above problems, the present invention provides a Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene, which can be used as a biosynthetic regulatory gene for gastrodin.

[0008] To achieve the above object, the technical solution of the present invention is as follows: In the second aspect of the present invention, a protein encoded by the above Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene is provided. The amino acid sequence of the protein is as follows SEQ ID NO.2, encoding 469 amino acid residues.

[0009] MEKGRSGSNRPHVAMLPTPGTGHLNPIAELARLLVDRHGFTVTIVLFSESWNPVQDALLSSLPSSITSLTLPPIPLSDLPSDSRIETRISEAASRCVPSLRSLLLSLLSSTALVAFITDLFGPPACDAAEQLSIPHYILMPSNLQLLTLILHLPDIDADLTCDFHDLDRPVHLPGFPPIPGPDILHPLQDRANPCYAWILEHARRYRRSHGILVNSFNAIEPHAASLLKAGHSPVVYTVGPLIRSAAENGDRSHPLFRWLDLQPEGSVIFVSFGSGGTLSADQLAELAMGLEDSRQRFLWVVRSPMGGSASGTYFTAGSEDDALAYLPQGFLERTKGVGLVVPSWAPQVEILAHAATGGFLSHCGWNSTLESVTHGVPMIGWPLFAEQRMNAVMLAEGVKVLLRMKAREDGLFDRTEIARAVRELMEGEEGKQARKKARELQQEAAAALGEGGTSTAALAAVADRWKRV; (SEQ ID NO.2) In the third aspect of the present invention, a recombinant plasmid containing the above Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene is provided.

[0010] Preferably, the recombinant plasmid is obtained by homologous recombination of the above Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene with the pET28a vector, named pET28a- GaUGT015 .

[0011] In the fourth aspect of the present invention, a transgenic engineering bacterium is provided, which contains the above recombinant plasmid, or, the exogenous Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene is integrated into the genome of the genetic engineering bacterium.

[0012] Preferably, the transgenic engineering bacterium is Escherichia coli BL21 (DE3) strain.

[0013] In the fifth aspect of the present invention, an application of the above Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene in the preparation of gastrodin is provided.

[0014] Preferably, using 4-hydroxybenzyl alcohol and the glycosyl donor UDP-glucose as raw materials, under the catalysis of the gastrodia glycosyltransferase encoded by the above gastrodia bifunctional glycosyltransferase GaUGT015 gene, glycosylation occurs at the hydroxyl group at the -4 position of 4-hydroxybenzyl alcohol to produce gastrodin; C -4 position hydroxyl group to generate gastrodin; The sixth aspect of the present invention provides an application of the above gastrodia bifunctional glycosyltransferase GaUGT015 gene in the preparation of arbutin.

[0015] Preferably, using hydroquinone and the glycosyl donor UDP-glucose as raw materials, under the catalysis of the gastrodia glycosyltransferase encoded by the above gastrodia bifunctional glycosyltransferase GaUGT015 gene, glycosylation occurs at the hydroxyl group of hydroquinone to produce arbutin.

[0016] In the present invention, through a recombinant plasmid, the target protein is obtained after in vitro expression, and after further catalyzing the substrates 4-hydroxybenzyl alcohol and hydroquinone respectively, gastrodin and arbutin are directly generated.

[0017] The glycosyltransferase described in the present invention GaUGT015 gene was identified through transcriptome sequencing and bioinformatics techniques from the tubers of Gastrodia elata, and was obtained by PCR amplification after reversing the RNA of Gastrodia elata tubers with RNA reagent into cDNA. The amplification primers of the described glycosyltransferase GaUGT015 gene are as follows: F: ATGGAGAAGGGCAGGAGC; (SEQ ID NO.3) R: CTACACACGCTTCCACCG; (SEQ ID NO.4) In addition, when performing homologous recombination with the vector pET28a, GaUGT015 the gene needs to be amplified and recovered using primers with homologous arms. The primers with homologous arms are as follows: F: gtggacagcaaatgggtcgcggatccATGGAGAAGGGCAGGAGC; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACACACGCTTCCACCG. (SEQ ID NO.6) The glycosyltransferase isolated and identified from Gastrodia elata GaUGT015The gene can be used as an important marker gene for molecular assisted breeding of Gastrodia elata, and can also be used as an important candidate gene for producing gastrodin in the construction of yeast chassis cells.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention provides the Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene, which can be used as a biosynthetic regulatory gene for gastrodin and arbutin, and is applied to the preparation of gastrodin and arbutin.

[0019] (2) With the rapid development of bioinformatics technology, the excavation of key enzyme genes in the biosynthetic pathways of gastrodin and arbutin has been greatly promoted. The biosynthetic regulatory gene for gastrodin and arbutin in the present invention, namely the Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene, is identified and successfully verified for the first time, opening up a new biosynthetic method for producing gastrodin. The present invention obtains the target product by means of in vitro enzymatic catalysis of heterologously expressed proteins in Escherichia coli, adopts in vitro biosynthesis for directional production, and has the advantages of few by-products.

[0020] (3) The present invention also provides a recombinant plasmid, a genetically engineered bacterium and a recombinant protein containing the glycosyltransferase GaUGT015 gene, laying a foundation for the large-scale synthesis of gastrodin and arbutin by bioengineering methods and further for the research on constructing cell factories producing gastrodin and arbutin.

[0021] (4) By in vitro biosynthesis of gastrodin and arbutin, the controllability is strong, the demand for raw material planting can be reduced, the production product is single, which is convenient for the separation and purification of gastrodin and arbutin in the later stage, and can also reduce problems such as difficulties in chemical synthesis and complex synthesis pathways. The Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene, as a key gene for the biosynthesis of gastrodin and arbutin, can also be used for the breeding research of plants rich in gastrodin and arbutin such as Mirabilis jalapa. Description of the Drawings

[0022] Figure 1 Schematic diagram of the deduced synthesis pathway for gastrodin; Figure 2 Schematic diagram of the deduced synthesis pathway for arbutin; Figure 3 Schematic diagram of the construction of the recombinant expression plasmid pET28a-GaUGT015; Figure 4 Electrophoresis detection result after recombination of GaUGT015; Figure 5 SDS-PAGE protein electrophoresis map of GaUGT015; where M is the protein molecular weight standard; Figure 6To detect the glycosylation of p - hydroxybenzyl alcohol by glycosyltransferase GaUGT015 using HPLC. The abscissa is time, with the unit of min, and the ordinate is the response value, with the unit of mAU. Among them, CK: the enzymatic inactivation reaction result of the control group (p - hydroxybenzyl alcohol + uridine diphosphate glucose + inactivated gastrodia bifunctional glycosyltransferase GaUGT015); Standard: p - hydroxybenzyl alcohol standard + gastrodin standard; GaUGT015: the enzymatic activity reaction result of the experimental group (p - hydroxybenzyl alcohol + uridine diphosphate glucose + gastrodia bifunctional glycosyltransferase GaUGT015). Figure 7 To detect the glycosylation of hydroquinone by glycosyltransferase GaUGT015 using HPLC. The abscissa is time, with the unit of min; the ordinate is the response value, with the unit of mAU. Among them, CK: the enzymatic inactivation reaction result of the control group (hydroquinone + uridine diphosphate glucose + inactivated gastrodia bifunctional glycosyltransferase GaUGT015); Standard: hydroquinone + arbutin standard; GaUGT015: the enzymatic activity reaction result of the experimental group (hydroquinone + uridine diphosphate glucose + gastrodia bifunctional glycosyltransferase GaUGT015). Figure 8 It is the fragment ion diagram (theoretical molecular weight 331) of the reaction product gastrodin (LC / MS / MS); Figure 9 It is the fragment ion diagram (theoretical molecular weight 331) of the standard gastrodin (LC / MS / MS); Figure 10 It is the fragment ion diagram (theoretical molecular weight 271) of the reaction product arbutin (LC / MS / MS); Figure 11 It is the fragment ion diagram (theoretical molecular weight 271) of the standard arbutin (LC / MS / MS). Detailed implementation manners

[0023] The present invention will be further described in detail below in conjunction with the embodiments.

[0024] Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For those materials or equipment without indicating the manufacturer, they are all conventional products that can be obtained by purchase. Example 1

[0025] Based on the basic functional annotation information of Gastrodia elata transcriptome Unigene, UGT candidate genes were screened from the sequencing and annotation results. At the same time, glycosyltransferases (UGTs) identified in plants were used for local BLAST analysis of sequences. Then, the screening results were sorted out and analyzed. Finally, one glycosyltransferase (UGT) gene was found. After a series of operations such as cDNA preparation, amplification and recovery of candidate genes, homologous recombination, protein expression, in vitro enzyme activity reaction, and HPLC and LC / MS detection, the target candidate gene that can catalyze the glycosylation of the hydroxyl group at the C-4 position of 4-Hydroxybenzyl alcohol was finally identified. GaUGT015 ( Figure 1 、 Figure 2 ). The operating steps at each stage of gastrodin and arbutin synthesis are as follows: (1)Preparation of cDNA template Take fresh samples of Gastrodia elata tubers, quickly freeze them in liquid nitrogen after slicing, and perform RNA extraction. The RNA extraction uses the HiPure Plant RNA Mini Kit of Magen (Guangzhou Meiji Biotechnology Co., Ltd.). Extract RNA according to the operating steps of the kit. After passing the detection, use the TAKARA reverse transcription kit to reverse transcribe RNA into cDNA and store it at -20 °C for later use.

[0026] (2)Gene amplification and recovery Use SnapGene software to design specific primers for candidate genes and entrust the Kunming branch of Beijing Tsingke Biotechnology Co., Ltd. to synthesize them. Use 2×Phanta Max Master Mix polymerase to amplify the target gene. The reaction system and reaction program are as follows. The reaction system is 25 μl of 2×Phanta Max Master Mix, 1 μl each of the forward and reverse primers of the candidate gene, 1 μl of cDNA from various tissues of Bletilla striata, 22 μl of ddH2O. The PCR reaction program is: Pre-denaturation at 95 °C for 3 min; Denaturation at 95 °C for 15 s, Annealing at 58 °C for 15 s, Extension at 72 °C for 1 min, 35 cycles; Final extension at 72 °C for 5 min; Keep warm at 10 °C.

[0027] The target fragment was recovered using 1% agarose gel and Tiangen agarose gel DNA recovery kit. After recovery, its concentration was measured on a NanoReady ultra-micro ultraviolet-visible spectrophotometer, and finally it was stored at -20 °C in the refrigerator for later use. The Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene fragment was obtained. After sequencing, its nucleic acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0028] Glycosyltransferase GaUGT015 The amplification primers for the gene are as follows: F: ATGGAGAAGGGCAGGAGC; (SEQ ID NO.3) R: CTACACACGCTTCCACCG; (SEQ ID NO.4) In addition, when performing homologous recombination with the vector pET28a, GaUGT015 the gene needs to be amplified and recovered using primers with homologous arms. The primers with homologous arms are as follows: F: gtggacagcaaatgggtcgcggatccATGGAGAAGGGCAGGAGC; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACACACGCTTCCACCG. (SEQ ID NO.6) (3) Construction and identification of gene recombinant vector The schematic diagram of homologous recombination is shown in Figure 3 . First, linearize the vector pET28a. During homologous recombination, assemble according to the operation instructions of the homologous recombination enzyme, then calculate the dosage of each component according to the concentration of the inserted fragment and the vector, and finally add each component to the PCR reaction tube on ice. After assembly, detect the result and send it to the company for sequencing. The electrophoresis detection result after assembly is shown in Figure 4 , indicating successful assembly.

[0029] (4) SDS-PAGE protein electrophoresis After small-scale protein expression experiments, it was determined that GaUGT813 the protein induction conditions are: 16 °C, 0.2 mM IPTG, 180 r / min, induce for 12 h; then perform large-scale shaking, collect bacteria, break the cell wall, obtain the protein supernatant after high-speed centrifugation, and then detect it by SDS-PAGE protein electrophoresis. The detection result is shown in Figure 5 , indicating that the supernatant protein of the target gene was obtained.

[0030] (5) Enzyme activity reaction The enzymatic activity reaction was carried out in a 1.5 mL centrifuge tube and prepared according to the components in Table 1. Then, the components were added in the order shown in the table. After mixing, the reaction solution was briefly centrifuged to collect it at the bottom of the centrifuge tube. The centrifuge tube was placed in a metal bath and reacted at 32 °C for 12 h. After completion, the reaction was terminated with 100 μL of methanol, and finally the product was detected.

[0031] Table 1 Component ratio of the UGT enzymatic activity reaction system

[0032] (6)Product detection The HPLC detection conditions are as follows: The instrument used for HPLC detection was an Agilent 1290 ultra-high performance liquid chromatograph. The liquid chromatography column was an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5.0 μm). The mobile phase was: 0.01% formic acid solution (A) and acetonitrile (B). The gradient elution program was as follows: 0 - 8 min, 99% A - 95% A; 8 - 13 min, 95% A - 90% A; 13 - 20 min, 90% A - 80% A; 20 - 25 min, 80% A - 55% A; 25 - 35 min, 55% A - 10% A; 35 - 40 min, 10% A - 10% A; 40 - 40.01 min, 10% A - 99% A; 45 min Stop. The flow rate was 0.6 mL / min. The column temperature was 30 °C. The injection volume was 10 μL. The absorption wavelength was 220 nm. The detection results are shown in Figure 6 、 Figure 7 , indicating the production of gastrodin and arbutin respectively.

[0033] The LC-MS detection conditions are as follows: Detection was carried out using an Agilent 1290 UPLC / 6540 Q-Tof liquid chromatography-mass spectrometry (LC / MS) instrument. Mass spectrometry conditions: The ion source was operated in negative ion mode, with a voltage of 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; RF voltage: 750 V, and the scanning range was 100 - 1000 m / z. Chromatography conditions: The column used was an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5.0 μm), with a flow rate of 0.6 mL / min. The mobile phase was 0.01% formic acid (A) and acetonitrile (B), and the gradient was as follows: 0 min, A:B = 99:1; 8 min, A:B = 95:5; 13 min, A:B = 90:10; 20 min, A:B = 80:20; 25 min, A:B = 55:45; 35 min, A:B = 10:90; 40 min, A:B = 10:90; 40.01 min, A:B = 99:1; 45 min, Stop. The detection results are shown in Figure 8 —11. As can be seen from the results, the fragment ion spectrum of the reaction product gastrodin ( Figure 8 ), is consistent with the fragment ion spectrum of the standard gastrodin ( Figure 9 ), further confirming that the product formed is gastrodin; the fragment ion spectrum of the reaction product arbutin ( Figure 10 ), is consistent with the fragment ion spectrum of the standard arbutin ( Figure 11 ), further confirming that the product formed is arbutin.

[0034] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene, characterized in that, The nucleic acid sequence of the gastrodia bifunctional glycosyltransferase GaUGT015 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene according to claim 1, characterized in that, The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. A recombinant plasmid containing the gastrodia bifunctional glycosyltransferase GaUGT015 gene described in claim 1.

4. The recombinant plasmid containing the gastrodia bifunctional glycosyltransferase GaUGT015 gene according to claim 3, characterized in that, It was obtained by homologous recombination of the gastrodia bifunctional glycosyltransferase GaUGT015 gene with the pET28a vector to obtain pET28a- GaUGT015 recombinant plasmid.

5. A genetically engineered bacterium, characterized in that, Containing the recombinant plasmid described in claim 3 or 4, or, the exogenous gastrodia bifunctional glycosyltransferase GaUGT015 gene described in claim 1 is integrated into the genome of the genetically engineered bacterium.

6. The genetically engineered bacterium according to claim 5, characterized in that, The genetically engineered bacterium is Escherichia coli BL21(DE3) strain.

7. Application of the gastrodia bifunctional glycosyltransferase GaUGT015 gene described in claim 1 in the preparation of gastrodin.

8. Use of the gastrodia elata bifunctional glycosyltransferase GaUGT015 gene according to claim 7 in the preparation of gastrodin, characterized in that, Using p-hydroxybenzyl alcohol as a substrate and UDP-glucose as a glycosyl donor, under the catalysis of gastrodia glycosyltransferase encoded by the above gastrodia bifunctional glycosyltransferase GaUGT015 gene, glycosylation occurs on the C hydroxyl group at the -4 position of p-hydroxybenzyl alcohol to produce gastrodin.

9. Application of the gastrodia bifunctional glycosyltransferase GaUGT015 gene described in claim 1 in the preparation of arbutin.

10. Use of the gastrodia elata bifunctional glycosyltransferase GaUGT015 gene according to claim 9 in the preparation of gastrodin, characterized in that, Using hydroquinone as a substrate and UDP-glucose as a glycosyl donor, under the catalysis of the gastrodia glycosyltransferase encoded by the above-mentioned gastrodia bifunctional glycosyltransferase GaUGT015 gene, glycosylation occurs on the hydroxyl group of hydroquinone to produce arbutin.

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