Gastrodin and arbutin are prepared by using a bifunctional glycosyltransferase GaUGT015 gene

Abstract

The present application relates to a kind of gastrodia elata bifunctional glycosyltransferase GaUGT015 Gene and application in preparing gastrodin and arbutin belong to the field of biotechnology.The gastrodia elata bifunctional glycosyltransferase GaUGT015 Gene nucleotide sequence is as shown in SEQ ID NO.1, and sequence full length is 1407bp;The amino acid sequence of coding protein is as shown in SEQ ID NO.2, and 469 amino acid residues are encoded.The gastrodia elata bifunctional glycosyltransferase GaUGT015 Gene can be used as the biosynthesis control gene of gastrodin and arbutin, and be applied to prepare gastrodin and arbutin, and application prospect is remarkable, easy to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bifunctional glycosyltransferase from Gastrodia elata. GaUGT015 Genes and their application in the preparation of gastrodin and arbutin. Background Technology

[0002] Gastrodia elata ( Gastrodia elata Bl . Gastrodia elata, also known as Dingfengcao or Chijian, belongs to the genus Gastrodia elata in the family Orchidaceae. Gastrodia Gastrodia elata is a perennial parasitic herb. It has the effects of calming wind and stopping spasms, suppressing liver yang, dispelling wind and unblocking collaterals. It is often used to treat infantile convulsions, epilepsy, tetanus, headache and dizziness, hemiplegia, numbness of limbs, and rheumatic pain.

[0003] Gastrodin (GAS) is one of the main active ingredients of Gastrodia elata, also known as gastrodin, chemical name: 4-hydroxymethylphenyl- β -D-glucopyranoside is a phenolic glycoside with the molecular formula: C 13 H 18 O7, molecular weight: 286.28 kDa. Gastrodin has long been used clinically in my country for its analgesic and hypnotic effects, with no obvious side effects observed. Known as a "miracle drug for treating wind-related ailments," it is used in the central nervous system. Primarily used for sedation, hypnosis, and analgesia, gastrodin can inhibit excitatory amino acid-induced cell death and apoptosis, possesses free radical scavenging ability, and can counteract free radical-induced damage to PC12 cells, exhibiting neuroprotective effects. Gastrodin has significant effects in the treatment of sclerosis, presumably related to its promotion of angiogenesis. Gastrodin has a good therapeutic effect on patients with functional dyspepsia accompanied by anxiety and depression symptoms, and can dilate blood vessels, reduce cardiac hypertension, and reduce fibrosis, among other pharmacological effects on the central nervous system. Gastrodin enhances its effect through spinal cord synapses, inhibiting inflammatory pain. Furthermore, some studies have found that gastrodin has various biological effects, including anti-obesity, anti-inflammation, memory improvement, and acetylcholinesterase inhibition.

[0004] Early methods for obtaining gastrodin mainly involved direct extraction from the tubers of Gastrodia elata or chemical processing. However, chemical synthesis of gastrodin is energy-intensive and environmentally unfriendly; artificial cultivation and extraction methods suffer from problems such as inconsistent Gastrodia elata quality, long cultivation cycles, and difficulty in extracting high-purity gastrodin. Therefore, the biosynthesis of gastrodin has attracted increasing attention in recent years.

[0005] Arbutin (Arb), also known as bearberry glycoside, is chemically named p-hydroxyphenyl- β-D-glucopyranoside is a naturally occurring glycoside derived from green plants and is a abundant solute in many freeze-drying resistant plants, such as wheat, pear, and bearberry (Arbutin from the Ericaceae family). Due to its whitening and pigment-removing properties, as well as its antioxidant, antimicrobial, and anti-inflammatory activities, arbutin has seen its effects become increasingly apparent with the deepening and expansion of research in recent years. These effects include antioxidant, anti-inflammatory, antibacterial, antitussive, expectorant, antiasthmatic, anticancer, antitumor, and treatment of acute lung injury. In recent years, arbutin has also seen some applications in the food industry, such as improving the aroma of wine, and there are increasing reports on its safety and nutritional value. With increasing emphasis on dietary therapy and health maintenance, many pharmaceuticals are developing towards the direction of food and medicine homology, thus arbutin has broad development prospects in the food and health product sectors. Plant extraction methods for producing arbutin suffer from problems such as long plant growth cycles, complex extraction processes, and low yields, while chemical synthesis methods have disadvantages such as stringent reaction conditions, poor stereoselectivity, and low yields. In recent years, biosynthesis has gradually become a popular research direction for the synthesis of 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 synthetic biology, the production of natural drug monomers using synthetic biology techniques can effectively solve the aforementioned problems. However, to elucidate the biosynthetic pathways of these active ingredients, it is essential to identify the key genes involved in these pathways, and discovering these catalytic enzyme genes has become a crucial step in studying the biosynthetic pathways of plant metabolites. Currently, p-hydroxybenzyl alcohol... C The synthetic pathways for the formation of gastrodin and arbutin by glycosylation at the -4 hydroxyl group and the formation of glucose and benzaldehyde molecules through the linkage of glucose and aldehyde groups have been clarified. However, the function of the glycosyltransferase responsible for glycosylation discovered in Gastrodia elata has not yet been verified, which has affected the progress of the biosynthesis of gastrodin and arbutin. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a bifunctional glycosyltransferase gene from Gastrodia elata, GaUGT015, which can serve as a regulatory gene for the biosynthesis of gastrodin.

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

[0009] MEKGRRSGSNRPHVAMLPTPGTGHLNPIAELARLLVDRHGFTVTIVLFSESWNPVQDALLSSLPSSITSLTLPPIPLSDLPSDSRIETRISEAASRCVPSLRSLLLSLLSSTALVAFIT DLFGPPACDAAEQLSIPHYILMPSNLQLLTLILHLPDIDADLTCDFHDLDRPVHLPGFPPIPGPDILHPLQDRANPCYAWILEHARRYRRSHGILVNSFNAIEPHAASLLKAGHSPVVY TVGPLIRSAAENGDRSHPLFRWLDLQPEGSVIFVSFGSGGTLSADQLAELAMGLEDSRQRFLWVVRSPMGGSASGTYFTAGSEDDALAYLPQGFLERTKGVGLVVPSWAPQVEILAHA ATGGFLSHCGWNSTLESVTHGVPMIGWPLFAEQRMNAVMLAEGVKVLLRMKAREDGLFDRTEIARAVRELMEGEEGKQARKKARELQQEAAAALGEGGTSTAALAAVADRWKRV; (SEQ ID NO.2) A third aspect of the present invention provides a recombinant plasmid containing the above-mentioned Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene.

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

[0011] The fourth aspect of the present invention provides a transgenic engineered bacterium containing the recombinant plasmid described above, or wherein the genome of the genetically engineered bacterium is integrated with an exogenous Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene described above.

[0012] Preferably, the genetically engineered bacteria is Escherichia coli. BL21 (DE3) strain.

[0013] The fifth aspect of this invention provides an application of the above-mentioned Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene in the preparation of gastrodin.

[0014] Preferably, p-hydroxybenzyl alcohol and the glycosyl donor UDP-glucose are used as raw materials, and under the catalysis of the gastrodin glycosyltransferase encoded by the aforementioned gastrodin bifunctional glycosyltransferase GaUGT015 gene, p-hydroxybenzyl alcohol is produced. C Glycosylation of the hydroxyl group at the -4 position produces gastrodin. The sixth aspect of this invention provides an application of the above-mentioned Gastrodia elata bifunctional glycosyltransferase GaUGT015 gene in the preparation of arbutin.

[0015] Preferably, hydroquinone and UDP-glucose, a glycosyl donor, are used as raw materials. Under the catalysis of the gastrodin glycosyltransferase encoded by the gastrodin bifunctional glycosyltransferase GaUGT015 gene, hydroquinone is glycosylated at the hydroxyl group to generate arbutin.

[0016] This invention obtains the target protein by expressing it in vitro using a recombinant plasmid, and then directly generates gastrodin and arbutin by catalyzing the substrates p-hydroxybenzyl alcohol and hydroquinone, respectively.

[0017] The glycosyltransferase described in this invention GaUGT015 The gene was identified from the tubers of Gastrodia elata through transcriptome sequencing and bioinformatics techniques, after extensive experimental screening. RNA from the tubers was extracted using RNA reagents, reversed to cDNA, and then amplified by PCR. The glycosyltransferase mentioned... GaUGT015 The primers for gene amplification are shown below: F: ATGGAGAAGGGCAGGAGC; (SEQ ID NO.3) R: CTACACACGCTTCCACCG; (SEQ ID NO.4) Furthermore, when homologous recombination occurs with the vector pET28a, GaUGT015 Genes need to be amplified and recovered using primers with homology walls. The primers with homology walls are as follows: F: gtggacagcaaatgggtcgcggatccATGGAGAAGGGCAGGAGC; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACACACGCTTCCACCG. (SEQ ID NO.6) Glycosyltransferase isolated and identified from Gastrodia elata GaUGT015The gene can serve as an important marker gene for molecular-assisted breeding of Gastrodia elata, and can also serve as an important candidate gene for the production of gastrodin in yeast chassis cell construction.

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

[0019] (2) With the rapid development of bioinformatics technology, the discovery of key enzyme genes in the biosynthetic pathways of gastrodin and arbutin has been greatly advanced. In this invention, the regulatory gene for the biosynthesis of gastrodin and arbutin, namely the gastrodin bifunctional glycosyltransferase GaUGT015 gene, is identified and successfully verified for the first time, opening up a new method for the biosynthesis of gastrodin. This invention obtains the target product by in vitro enzymatic catalysis through heterologous expression of the protein in Escherichia coli. It adopts in vitro biosynthesis for targeted production, which has the advantages of fewer by-products.

[0020] (3) The present invention also provides a glycosyltransferase containing this glycosyltransferase. GaUGT015 Recombinant plasmids, genetically engineered bacteria, and recombinant proteins lay the foundation for the large-scale synthesis of gastrodin and arbutin through bioengineering methods, and further for the research on constructing cell factories that produce gastrodin and arbutin.

[0021] (4) In vitro biosynthesis of gastrodin and arbutin offers strong controllability, reduces the need for raw material cultivation, produces a single product, facilitates the subsequent separation and purification of gastrodin and arbutin, and reduces difficulties in chemical synthesis and complex synthetic pathways. The aforementioned gastrodin bifunctional glycosyltransferase GaUGT015 gene, as a key gene for the biosynthesis of gastrodin and arbutin, can also be used for breeding research on plants rich in gastrodin and arbutin, such as Mirabilis jalapa. Attached Figure Description

[0022] Figure 1 A schematic diagram illustrating the synthetic pathway of gastrodin; Figure 2 A schematic diagram of the synthetic pathway of arbutin; Figure 3 A schematic diagram illustrating the construction of the recombinant expression plasmid pET28a-GaUGT015; Figure 4 The electrophoretic detection results are for GaUGT015 after recombination; Figure 5 This is an SDS-PAGE protein electrophoresis image of GaUGT015; where M is the protein molecular weight standard. Figure 6This study aimed to detect the glycosylation of p-hydroxybenzyl alcohol by the glycosyltransferase GaUGT015 using HPLC. The x-axis represents time (min), and the y-axis represents the response value (mAU). Specifically, CK: control group (p-hydroxybenzyl alcohol + uridine diphosphate glucose + inactivated gastrodin bifunctional glycosyltransferase GaUGT015); standards: p-hydroxybenzyl alcohol standard + gastrodin standard; GaUGT015: experimental group (p-hydroxybenzyl alcohol + uridine diphosphate glucose + gastrodin bifunctional glycosyltransferase GaUGT015). Figure 7 This study used HPLC to detect the glycosylation of hydroquinone by the glycosyltransferase GaUGT015. The x-axis represents time (min), and the y-axis represents the response value (mAU). CK: Control group (hydroquinone + uridine diphosphate glucose + inactivated Gastrodia elata bifunctional glycosyltransferase GaUGT015) – enzyme activity after inactivation; Standard: Hydroquinone + arbutin standard; GaUGT015: Experimental group (hydroquinone + uridine diphosphate glucose + Gastrodia elata bifunctional glycosyltransferase GaUGT015) – enzyme activity. Figure 8 Fragment ion diagram of the reaction product gastrodin (theoretical molecular weight 331) (LC / MS / MS); Figure 9 Fragment ion diagram of standard gastrodin (theoretical molecular weight 331) (LC / MS / MS); Figure 10 Fragment ion diagram of the reaction product arbutin (theoretical molecular weight 271) (LC / MS / MS); Figure 11 Fragment ion diagram of standard arbutin (theoretical molecular weight 271) (LC / MS / MS). Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the embodiments.

[0024] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase. Example 1

[0025] Based on the basic functional annotation information of the Unigene transcriptome of Gastrodia elata, candidate UGT genes were screened from the sequencing annotation results. Simultaneously, glycosyltransferases (UGTs) identified in plants were analyzed using local BLAST sequence analysis. The screening results were then analyzed and compiled, ultimately identifying one glycosyltransferase (UGT) gene. Following a series of procedures including cDNA preparation, candidate gene amplification and recovery, homologous recombination, protein expression, in vitro enzyme activity reaction, and HPLC and LC / MS detection, the target candidate gene capable of catalyzing glycosylation at the C-4 position of 4-hydroxybenzyl alcohol was finally identified. GaUGT015 ( Figure 1 , Figure 2 The steps involved in the synthesis of gastrodin and arbutin are as follows: (1) Preparation of cDNA template Fresh samples of Gastrodia elata tubers were collected, sliced, and flash-frozen in liquid nitrogen for RNA extraction. RNA extraction was performed using the Magen (Guangzhou Meiji Biotechnology Co., Ltd.) HiPure Plant RNA Mini Kit. RNA was extracted according to the kit's instructions, and after passing quality checks, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -20 ℃ for later use.

[0026] (2) Gene amplification and recovery Specific primers for candidate genes were designed using SnapGene software, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to do so. The Kunming branch synthesized the gene and used 2×PhantaMax Master Mix polymerase to amplify the target gene. The reaction system and procedure are as follows: 25 μL of 2×PhantaMax Master Mix, 1 μL each of the candidate gene pre- and post-selection, 1 μL of cDNA from each Bletilla striata tissue, and 22 μL of ddH2O. The PCR reaction procedure is as follows: Pre-denaturation at 95℃ for 3 minutes; Denaturation at 95℃ for 15 seconds Annealing at 58℃ for 15 seconds. 72℃, 1 min extension, 35 cycles; Final extension at 72℃ for 5 minutes; Keep warm at 10℃.

[0027] The target fragment was recovered using 1% agarose gel and the Tiangen agarose gel DNA recovery kit. The recovered concentration was determined using a NanoReady ultra-micro UV-Vis spectrophotometer, and the fragment was stored at -20 °C for later use. The GaUGT015 gene fragment of the bifunctional glycosyltransferase from *Gastrodia elata* was obtained. Its nucleic acid sequence is shown in SEQ ID NO. 1, and its protein amino acid sequence is shown in SEQ ID NO. 2.

[0028] Glycosyltransferase GaUGT015 The primers for gene amplification are shown below: F: ATGGAGAAGGGCAGGAGC; (SEQ ID NO.3) R: CTACACACGCTTCCACCG; (SEQ ID NO.4) Furthermore, when homologous recombination occurs with the vector pET28a, GaUGT015 Genes require amplification and recovery using primers with homology walls. The primers with homology walls are as follows: F: gtggacagcaaatgggtcgcggatccATGGAGAAGGGCAGGAGC; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACACACGCTTCCACCG. (SEQ ID NO.6) (3) Construction and identification of gene recombination vectors For a detailed diagram of homologous recombination, please refer to [link / reference]. Figure 3 First, the pET28a vector was linearized. For homologous recombination, assembly was performed according to the instructions for the homologous recombinase. Then, based on the concentrations of the insert fragment and vector, and following the recombination instructions, the amounts of each component were calculated. Finally, each component was added to the PCR reaction tube on ice. After assembly, the results were detected and sent to the company for sequencing. The electrophoresis results after assembly are shown below. Figure 4 This indicates that the assembly was successful.

[0029] (4) SDS-PAGE protein electrophoresis Determined after small-scale protein expression trials GaUGT813 The protein induction conditions were: 16 ℃, 0.2 mM IPTG, 180 r / min, for 12 h; then, the cells were shaken vigorously, harvested, and the cell walls were broken. The protein supernatant was obtained after high-speed centrifugation, and then detected by SDS-PAGE protein electrophoresis. The results are shown below. Figure 5 This indicates that the supernatant of the target gene was obtained.

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

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

[0032] (6) Product testing The HPLC detection conditions are as follows: The instrument used for HPLC analysis was an Agilent 1290 ultra-high performance liquid chromatograph. The HPLC 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 ℃. The injection volume was 10 μL. The absorption wavelength was 220 nm. The detection results are shown below. Figure 6 , Figure 7 This indicates the production of gastrodin and arbutin, respectively.

[0033] The LC-MS detection conditions are as follows: Detection was performed using an Agilent 1290 UPLC / 6540 Q-Tof liquid chromatography-mass spectrometry (LC / MS) system. Mass spectrometry conditions: negative ion source, voltage 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; radio frequency voltage: 750 V; scan range: 100-1000 m / z. Chromatographic conditions: an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5.0 μm) was used, with a flow rate of 0.6 mL / min. The mobile phase was 0.01% formic acid (A) and acetonitrile (B). 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, From the results, we can see the fragment ion diagram of the reaction product gastrodin ( Figure 8 Fragment ion diagrams of gastrodin and standard ( Figure 9 The results were consistent, further confirming that the product was gastrodin; the fragment ion diagram of the reaction product arbutin ( Figure 10 Fragment ion diagram of arbutin and standard ( Figure 11 The results were consistent with those of the previous study, further confirming the product arbutin.

[0034] The foregoing has shown and described 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 to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. The application of a bifunctional glycosyltransferase gene from Gastrodia elata, GaUGT015, characterized in that... The nucleic acid sequence of the *Gastrodia elata* bifunctional glycosyltransferase GaUGT015 gene is shown in SEQ ID NO.1, and it has the function of preparing both gastrodin and arbutin. Using p-hydroxybenzyl alcohol as a substrate and UDP-glucose as a glycosyl donor, under the catalysis of the *Gastrodia elata* glycosyltransferase encoded by the aforementioned *Gastrodia elata* bifunctional glycosyltransferase GaUGT015 gene, p-hydroxybenzyl alcohol is used to prepare gastrodin. C Glycosylation of the hydroxyl group at the -4 position generates gastrodin; using hydroquinone as a substrate and UDP-glucose as a glycosyl donor, under the catalysis of gastrodin glycosyltransferase encoded by the above-mentioned gastrodin bifunctional glycosyltransferase GaUGT015 gene, glycosylation of the hydroxyl group of hydroquinone generates arbutin.

2. The application of the *Gastrodia elata* bifunctional glycosyltransferase GaUGT015 gene as described in claim 1 in the preparation of gastrodin, characterized in that... The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

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