Rock white menu bifunctional glycosyltransferase BpUGT1 gene and application thereof in preparing arbutin and gastrodin

Abstract

The present application relates to a rock white bifunctional glycosyltransferase BpUGT1 Gene and application in preparation of arbutin and gastrodin, belong to the field of biotechnology. The rock white bifunctional glycosyltransferase BpUGT1 The gene nucleotide sequence is as shown in SEQ ID NO.1, and the sequence full length is 1431bp; the amino acid sequence of the encoded protein is as shown in SEQ ID NO.2, and 476 amino acid residues are encoded. The rock white bifunctional glycosyltransferase BpUGT1 The gene can be used as the biosynthesis regulation gene of arbutin and gastrodin, and is applied to the preparation of arbutin and gastrodin, and the application prospect is remarkable, and easy to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bifunctional glycosyltransferase from Bergenia crassifolia. BpUGT1 Genes and their application in the preparation of arbutin and gastrodin. Background Technology

[0002] Bergenia serratifolia ( Bergenia purpurascens *Gnaphalium affine* is a perennial medicinal plant belonging to the genus *Gnaphalium* of the family Saxifragaceae. It is mainly distributed in high-altitude areas (2200-4800m) in Yunnan, Guizhou, and Sichuan provinces of my country. Its dried rhizome, used in traditional Chinese medicine, possesses astringent, antidiarrheal, hemostatic, antitussive, and meridian-clearing properties, and is widely used clinically to treat respiratory, digestive, and traumatic diseases. Modern pharmacological studies reveal that its chemical composition is mainly phenolic compounds, including characteristic components such as bergenin, arbutin, gallic acid, and catechins, with arbutin being a particularly prominent key active ingredient.

[0003] Arbutin, a natural hydroquinone glycoside, works by reversibly inhibiting tyrosinase activity, competitively binding to and blocking the melanin production pathway. It also possesses multiple biological activities, including antioxidant, anti-inflammatory, and antibacterial properties, making it a preferred safe whitening agent in the global cosmetics industry. In the pharmaceutical field, this compound also demonstrates various potential applications.

[0004] Currently, the commercial production of arbutin mainly relies on plant extraction and chemical synthesis. The former is limited by the endangered wild resources of Bergenia sanguinea (long growth cycle and narrow distribution area) and low extraction efficiency; the latter relies on the petroleum-based raw material hydroquinone, which has problems with highly toxic byproducts and environmental pollution. Although synthetic biology strategies have achieved heterologous synthesis of arbutin in engineered bacteria such as Escherichia coli, the catalytic efficiency of the key rate-limiting enzyme—glycosyltransferase—is low (currently, Arbutin synthase derived from Ophiopogon japonicus is mainly used), which seriously restricts the increase in yield.

[0005] Gastrodin, the core active ingredient of the traditional and precious Chinese herb Gastrodia elata, possesses significant sedative, neuroprotective, and anticonvulsant pharmacological effects and is widely used clinically in the treatment of central nervous system diseases. The acquisition of this 4-hydroxymethylphenyl glucoside has long relied on extraction from Gastrodia elata tubers, facing industrialization bottlenecks such as long raw material cultivation cycles (3-5 years) and low effective ingredient content. While chemical synthesis can circumvent plant-source limitations, it suffers from drawbacks such as difficulty in chiral control and high energy consumption.

[0006] The key step in the biosynthesis of phenolic glycosides lies in the glycosylation reaction catalyzed by glycosyltransferases (UGTs). While the UGT family is known to possess significant substrate breadth and catalytic diversity, highly efficient UGT resources specific to hydroquinone / p-hydroxybenzyl alcohol remain extremely scarce. Although studies have confirmed the possible existence of related glycosylation enzyme systems in Bergenia schreberi, their specific genetic information, enzymatic characteristics, and bifunctional catalytic potential remain unclear, severely restricting metabolic engineering and the construction of in vitro enzyme catalytic systems. Summary of the Invention

[0007] To address the above problems, this invention provides a bifunctional glycosyltransferase from Bergenia crassifolia. BpUGT1 The gene and its application in the preparation of arbutin and gastrodin can serve as a regulatory gene for the biosynthesis of arbutin and gastrodin.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] The first aspect of this invention provides a bifunctional glycosyltransferase from Bergenia crassifolia. BpUGT1

[0010] The second aspect of this invention provides the above-mentioned Bergenia schreberi bifunctional glycosyltransferase. BpUGT1 The gene encodes a protein whose amino acid sequence is shown in SEQ ID NO.2, encoding 476 amino acid residues.

[0011] MEMENQPPPPHIVIIPSPGMGHLIPLSEFAKRLVHHHNFSITFIVPTDGPPSKAQKSVLKQLPERISHVFLPPVNFDDLPETSMIETRISLMVTRSLSSLHDAMRPLAENSNLVALVVDL FGVDAFLVAREMNISPYVFYPSTAMNLSLFLYLPTLDKAVSCEYRDLTEPVQIPGCIPIHGRDLLDPVQDRKDEAYKWVLHNANMYRSAEGIMVNSFIDLEPGAIKALQEIEPGKPPIYP IGPLINMDPSSGVDGSECLKWLDDQPHGSVLFVSFGSGGTLSSEQLNELALGLDMSEQRFLWVVRSPNDQAANATYFSAQSISDPLSFLPKGFLEKTKGKGLVVPNWAPQAQILSHGSTG GFLTHCGWNSTLESVVNGIPLITWPLYAEQKMNAVMLTQDIKVALRPKSSENGGLVEREEIARVVRGLMEGEEGKNLRFRMKELKDAAADVLSENGSSSKALAELAHKWKNHQKST; (SEQ ID NO.2)

[0012] The third aspect of the present invention provides a bifunctional glycosyltransferase containing the above-mentioned Bergenia schreberi. BpUGT1 Recombinant plasmids of genes.

[0013] Preferably, the recombinant plasmid is prepared by transfecting the above-mentioned Bergenia spp. bifunctional glycosyltransferase. BpUGT1 The gene was obtained through homologous recombination with the pET28a vector and named pET28a- BpUGT1 .

[0014] A fourth aspect of the present invention provides a transgenic engineered bacterium containing the recombinant plasmid described above, or wherein the genome of the transgenic engineered bacterium is integrated with an exogenous bifunctional glycosyltransferase of Bergenia spp. described above. BpUGT1 Gene.

[0015] Preferably, the genetically engineered bacteria is Escherichia coli. BL21 (DE3) strain.

[0016] The fifth aspect of this invention provides the above-mentioned Bergenia schreberi bifunctional glycosyltransferase. BpUGT1 Application of genes in the preparation of gastrodin.

[0017] Preferably, p-hydroxybenzyl alcohol and the glycosyl donor UDP-Glc are used as raw materials, and the above-mentioned Bergenia bifunctional glycosyltransferase is used. BpUGT1 Under the catalysis of the gene-encoded Bergenia glycosyltransferase, glycosylation occurs at the p-hydroxyl position of p-hydroxybenzyl alcohol to generate gastrodin.

[0018] The sixth aspect of this invention provides the above-mentioned Bergenia schreberi bifunctional glycosyltransferase. BpUGT1 Application of genes in the preparation of arbutin.

[0019] Preferably, hydroquinone and the glycosyl donor UDP-Glc are used as raw materials, and the above-mentioned Bergenia bifunctional glycosyltransferase is used. BpUGT1 Under the catalysis of the gene-encoded Bergenia glycosyltransferase, glycosylation of the hydroxyl group of hydroquinone is carried out to generate arbutin.

[0020] 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.

[0021] Bergenia saccharidase described in this invention BpUGT1 The gene was identified from Bergenia schreberi plants through transcriptome sequencing and bioinformatics techniques, after extensive experimental screening. RNA was extracted from young parts of Bergenia schreberi, reversed into cDNA, and then amplified by PCR. The Bergenia schreberi glycosyltransferase mentioned above... BpUGT1 The primers for gene amplification are shown below:

[0022] F: ATGGAAATGGAAAACCAACCACC; (SEQ ID NO.3)

[0023] R: TTAAGTGCTCTTTTGATG; (SEQ ID NO.4)

[0024] Furthermore, when homologous recombination occurs with the vector pET28a, BpUGT1 Genes need to be amplified and recovered using primers with homology walls. The primers with homology walls are as follows:

[0025] F: gtggacagcaaatgggtcgcggatccATGGAAATGGAAAACCAACCACC; (SEQ ID NO.5)

[0026] R: tgtcgacggagctcgaattcggatccTTAAGTGCTCTTTTGATG. (SEQ ID NO.6)

[0027] Glycosyltransferases isolated and identified from Bergenia serratifolia BpUGT1 The gene can serve as an important marker gene for molecular-assisted breeding of Bergenia schreberi, and also as an important candidate gene for the production of arbutin and gastrodin in yeast chassis cell construction.

[0028] Compared with the prior art, the beneficial effects of this invention are as follows:

[0029] (1) This invention provides a bifunctional glycosyltransferase from Bergenia schreberi. BpUGT1 The gene can serve as a regulator of the biosynthesis of gastrodin and arbutin, and can be applied to the preparation of gastrodin and arbutin.

[0030] (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 promoted. In this invention, the biosynthetic regulatory gene of gastrodin and arbutin is the bifunctional glycosyltransferase of Bergenia schreberi. BpUGT1 This invention, the first to identify and successfully verify a gene, opens up a new biosynthetic method for producing gastrodin. The invention obtains the target product through in vitro enzymatic catalysis of heterologous protein expression in *E. coli*, employing in vitro biosynthesis for targeted production, which has advantages such as fewer byproducts.

[0031] (3) The present invention also provides a glycosyltransferase containing this glycosyltransferase. BpUGT1 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.

[0032] (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 Bergenia bifunctional glycosyltransferase... BpUGT1 As a key gene in the biosynthesis of gastrodin and arbutin, it can also be used in breeding research of plants rich in gastrodin and arbutin, such as Mirabilis jalapa. Attached Figure Description

[0033] Figure 1 A schematic diagram illustrating the synthetic pathway of gastrodin;

[0034] Figure 2 A schematic diagram of the synthetic pathway of arbutin;

[0035] Figure 3 A schematic diagram illustrating the construction of the recombinant expression plasmid pET28a-BpUGT1;

[0036] Figure 4 for BpUGT1 Electrophoretic detection results after recombination (M: DNA Marker);

[0037] Figure 5 for BpUGT1 SDS-PAGE protein electrophoresis image; where M is the protein molecular weight standard;

[0038] Figure 6 For HPLC detection of glycosyltransferases BpUGT1 Glycosylation of hydroquinone. The x-axis represents time (min); the y-axis represents the response value (mAU); where CK: control group (hydroquinone + UDP-Glc + inactivated Bergenia spp. glycosyltransferase). BpUGT1 Enzyme inactivation reaction results; Standard: hydroquinone standard + arbutin standard; BpUGT1: experimental group (hydroquinone + UDP-Glc + Bergenia saccharidase) BpUGT1 The results of the enzyme activity reaction;

[0039] Figure 7 For HPLC detection of glycosyltransferases BpUGT1 Glycosylation of p-hydroxybenzyl alcohol. The x-axis represents time (min), and the y-axis represents the response value (mAU). Wherein, CK: control group (p-hydroxybenzyl alcohol + UDP-Glc + inactivated Bergenia spp. glycosyltransferase). BpUGT1 Enzyme inactivation reaction results; Standards: p-hydroxybenzyl alcohol standard + gastrodin standard; BpUGT1 Experimental group (p-hydroxybenzyl alcohol + UDP-Glc + Bergenia glycosyltransferase) BpUGT1 The results of the enzyme activity reaction;

[0040] Figure 8 Fragment ion diagrams of the standard arbutin (theoretical molecular weight 271) (LC / MS / MS) (Figure A) and the reaction product arbutin (theoretical molecular weight 271) (LC / MS / MS) (Figure B) are shown.

[0041] Figure 9 Figure A shows the fragment ion diagram of the standard gastrodin (theoretical molecular weight 331) (LC / MS / MS) and Figure B shows the fragment ion diagram of the reaction product gastrodin (theoretical molecular weight 331) (LC / MS / MS). Detailed Implementation

[0042] The present invention will now be described in further detail with reference to the embodiments.

[0043] 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

[0044] Based on the basic functional annotation information of the Bergenia schreberi Unigene, candidate UGT genes were screened from the sequencing annotation results. Simultaneously, glycosyltransferases (UGTs) identified in plants were analyzed using local BLAST sequences. 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 reactions, and HPLC and LC / MS detection, the target candidate gene capable of catalyzing the conversion of hydroquinone to arbutin and the conversion of p-hydroxybenzyl alcohol to gastrodin was finally identified. BpUGT1 ( Figure 1 , Figure 2 The steps involved in the synthesis of gastrodin and arbutin are as follows:

[0045] (1) Preparation of cDNA template

[0046] Fresh rhizome samples of Bergenia sanguinalis were collected, sliced, and flash-frozen in liquid nitrogen for RNA extraction. RNA extraction was performed using the Magen HiPure Plant RNA Mini Kit (Guangzhou Meiji Biotechnology Co., Ltd.). RNA was extracted according to the kit's instructions, and after passing quality tests, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -20 °C for later use.

[0047] (2) Gene amplification and recovery

[0048] Specific primers for candidate genes were designed using SnapGene software, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to do so.

[0049] 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:

[0050] Pre-denaturation at 95℃ for 3 minutes;

[0051] Denaturation at 95℃ for 15 seconds

[0052] Annealing at 58℃ for 15 seconds.

[0053] 72℃, 1 min extension, 35 cycles;

[0054] Final extension at 72℃ for 5 minutes;

[0055] Keep warm at 10℃.

[0056] The target fragment was recovered using a 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. A bifunctional glycosyltransferase from Bergenia grosvenorii was obtained. BpUGT1 The gene fragment, after sequencing, has a nucleic acid sequence as shown in SEQ ID NO.1, and a protein amino acid sequence as shown in SEQ ID NO.2.

[0057] Glycosyltransferase BpUGT1 The primers for gene amplification are shown below:

[0058] F: ATGGAAATGGAAAACCAACCACC; (SEQ ID NO.3)

[0059] R: TTAAGTGCTCTTTTGATG; (SEQ ID NO.4)

[0060] Furthermore, when homologous recombination occurs with the vector pET28a, BpUGT1 Genes require amplification and recovery using primers with homology walls. The primers with homology walls are as follows:

[0061] F: gtggacagcaaatgggtcgcggatccATGGAAATGGAAAACCAACCACC; (SEQ ID NO.5)

[0062] R: tgtcgacggagctcgaattcggatccTTAAGTGCTCTTTTGATG. (SEQ ID NO.6)

[0063] (3) Construction and identification of gene recombination vectors

[0064] For a detailed diagram of homologous recombination, please see [link / reference]. Figure 3First, 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.

[0065] (4) SDS-PAGE protein electrophoresis

[0066] Determined after small-scale protein expression trials BpUGT1 The protein induction conditions were: 16 ℃, 0.5 mM IPTG, 160 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, passed through a Ni-NTA column at 4 ℃, and the filtrate was collected under 250 mM imidazole. SDS-PAGE protein electrophoresis was then used for detection. The results are shown below. Figure 5 This indicates that the supernatant of the target gene was obtained.

[0067] (5) Enzyme activity reaction

[0068] The enzyme activity reaction was carried out in 2.0 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 2 h. The reaction was terminated with 100 μL of methanol, and the product was finally detected.

[0069] Table 1. Component ratio of the UGT enzyme activity reaction system

[0070]

[0071] (6) Product testing

[0072] The HPLC detection conditions are as follows:

[0073] 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 for product determination was 0.1% v / v formic acid aqueous solution (A) - acetonitrile (B). The gradient elution program was as follows: 0–8 min, 1%–5% B; 8–13 min, 5%–10% B; 13–20 min, 10%–20% B; 20–25 min, 20%–45% B; 25–35 min, 45%–90% B; 35–40 min, 90%–90% B. Elution time: 40 min; injection volume: 10 μL; flow rate: 0.6 ml / min; detection wavelength for gastrodin: 220 nm, for arbutin: 270 nm; column temperature: 30 ℃; injection volume: 10 μL. Detection results are shown below. Figure 6 , Figure 7 This indicates the production of gastrodin and arbutin, respectively.

[0074] The LC-MS detection conditions are as follows:

[0075] 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), with gradient elution: 0–8 min, 1%–5% B; 8–13 min, 5%–10% B; 13–20 min, 10%–20% B; 20–25 min, 20%–45% B; 25–35 min, 45%–90% B; 35–38 min, 90%–90% B; 38–45 min, 90%–100% B. Elution time: 45 min; injection volume: 10 μL; flow rate: 0.6 mL / min. Detection results are shown below. Figure 8 —9. From the results, we can see the fragment ion diagram of the reaction product arbutin ( Figure 8 B) Fragment ion diagram of arbutin standard ( Figure 8 The results are consistent with those in A), further confirming the product arbutin; the fragment ion diagram of the reaction product gastrodin ( Figure 9 B) Fragment ion diagram of gastrodin as a standard ( Figure 9 The results in A) are consistent, further confirming that the product generated is gastrodin.

[0076] 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. A bifunctional glycosyltransferase from Bergenia schreberi BpUGT1 Genes, characterized by, The bifunctional glycosyltransferase from Bergenia spp. BpUGT1 The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

2. The Bergenia bifunctional glycosyltransferase according to claim 1 BpUGT1 Gene-encoded protein, characterized by, The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. Containing the bifunctional glycosyltransferase of Bergenia spp. as described in claim 1 BpUGT1 Recombinant plasmids of genes.

4. The Bergenia bifunctional glycosyltransferase according to claim 3 BpUGT1 Recombinant plasmids of genes are characterized by, bifunctional glycosyltransferase of Bergenia spp. BpUGT1 The gene was obtained through homologous recombination with the pET28a vector. pET28a - BpUGT1 Recombinant plasmid.

5. A genetically engineered bacterium, characterized in that, The bacteria contain the recombinant plasmid as described in claim 3 or 4, or the genome of the genetically engineered bacteria contains an exogenous Bergenia bifunctional glycosyltransferase as described in claim 1. BpUGT1 Gene.

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

7. The Bergenia bifunctional glycosyltransferase according to claim 1 BpUGT1 Application of genes in the preparation of gastrodin.

8. The Bergenia bifunctional glycosyltransferase according to claim 7 BpUGT1 The application of genes in the preparation of gastrodin is characterized by, Using p-hydroxybenzyl alcohol as a substrate and UDP-Glc as a glycosyl donor, the above-mentioned Bergenia bifunctional glycosyltransferase was used. BpUGT1 Under the catalysis of the gene-encoded Bergenia glycosyltransferase, glycosylation occurs at the p-hydroxyl position of p-hydroxybenzyl alcohol to generate gastrodin.

9. The Bergenia bifunctional glycosyltransferase according to claim 1 BpUGT1 Due to its application in the preparation of arbutin.

10. The Bergenia bifunctional glycosyltransferase according to claim 9 BpUGT1 The application of genes in the preparation of gastrodin is characterized by, Using hydroquinone as a substrate and UDP-Glc as a glycosyl donor, the above-mentioned Bergenia bifunctional glycosyltransferase... BpUGT1 Under the catalysis of the gene-encoded Bergenia glycosyltransferase, glycosylation of the hydroxyl group of hydroquinone is carried out to generate arbutin.

Images