Glycosyltransferase and its application in preparing rhodioloside

Abstract

The application provides a glycosyltransferase and its application in preparing rhodiolin, and relates to the technical field of enzyme engineering. Fraxinus pennsylvanica The glycosyltransferase FpUGT in the application is derived from Malpighia emarginata DC. There is no report about the glycosyltransferase gene capable of directly catalyzing synthesis of rhodiolin in the plants of the genus Malpighia, the application provides a new biological route for searching for the glycosyltransferase with high efficiency in synthesizing rhodiolin, and also provides a new biological material for preparing rhodiolin.

Description

Technical Field

[0001] This application relates to the field of enzyme engineering technology, and in particular to glycosyltransferases and their application in the preparation of rhodioloside. Background Technology

[0002] Salidroside is the main active ingredient of Rhodiola rosea, a traditional and precious Chinese medicine, and possesses significant pharmacological effects such as anti-fatigue, anti-hypoxia, anti-microwave radiation, anti-toxicity, anti-tumor, and neuroprotective properties. With the surge in demand from the pharmaceutical, health product, and cosmetic industries, wild Rhodiola rosea resources are nearing depletion due to over-harvesting. While chemical synthesis is feasible, it suffers from cumbersome procedures, low yields, and environmental pollution. Therefore, utilizing synthetic biology methods to achieve green and efficient biomanufacturing of salidroside has become a research hotspot.

[0003] Currently reported uridine diphosphate glycosyltransferases (UGTs) that can catalyze the production of rhodioloside from tyrosol mainly originate from the Rhodiola genus itself (such as UGT85A1) or the Ligustrum genus (such as UGT85AF8). However, on the one hand, existing technologies have the following problems: 1. Limited enzyme sources: Existing enzyme sources are mainly concentrated in a few species, which limits the diversity of enzyme properties; 2. Poor catalytic efficiency and substrate specificity: The catalytic activity or affinity for substrates of some wild-type enzymes still has room for improvement, making it difficult to meet the needs of large-scale industrial fermentation; On the other hand, when studying glycosyltransferases in existing technologies, it was found that not all glycosyltransferases (UGTs) have the function of catalyzing the production of rhodiola glycosides. Michael P. Torrens-Spence et al. conducted functional analysis on 34 candidate UGT genes and found that only a few enzymes have the function of catalyzing the production of rhodiola glycosides. This result also proves the difficulty and importance of finding glycosyltransferases with specific functions (Reference: Torrens-Spence MP, Pluskal, Tomá, Li FS, et al. Complete Pathway Elucidation and Heterologous Reconstitution of Rhodiola Salidroside Biosynthesis[J]. Molecular Plant (English Edition)). 2017:S1674205217303775.DOI:10.1016 / j.molp.2017.12.007.).

[0004] In summary, there is an urgent need to discover new glycosyltransferases that can catalyze the production of rhodioloside from substrates. Summary of the Invention

[0005] This invention aims to address the lack of enzyme elements in the existing biosynthesis of rhodiolosides by providing a novel glycosyltransferase (named FpUGT or UGT8) derived from *Fraxinus pennsylvanica*. The inventors utilized a deep learning model combined with structural modeling and substrate (tyrosol) affinity prediction to screen potential candidate sequences from massive amounts of genomic prediction data. Through heterologous expression and in vitro enzyme activity experiments, they demonstrated for the first time that a protein from *Fraxinus pennsylvanica* possesses glycosyltransferase activity, capable of transferring glucose groups to the phenolic hydroxyl groups of tyrosol or its analogues to generate rhodiolosides. This enzyme exhibits highly efficient catalytic ability to catalyze the glycosylation modification of tyrosol or its analogues to generate rhodiolosides, and can be used for the industrial production of rhodiolosides using engineered strains.

[0006] The purpose of this invention is to provide a novel glycosyltransferase with highly efficient catalytic function in generating rhodioloside.

[0007] On the one hand, this application provides the application of glycosyltransferases in the preparation of rhodiolosides, wherein the glycosyltransferases include those derived from American ash (Fraxinus pennsylvanica).

[0008] Optionally, the glycosyltransferase catalyzes the glycosylation of tyrosol or its analogues.

[0009] Alternatively, the glycosyltransferase catalyzes the transfer of glucose groups to the phenolic hydroxyl groups of tyrosol or its analogues, thereby achieving glycosylation of tyrosol or its analogues.

[0010] Alternatively, the glycosyltransferase is prepared and / or generates rhodioloside by catalyzing the transfer of glucose to the phenolic hydroxyl group of tyrosol or its analogues.

[0011] Specifically, the tyrosol analogue can be at least one or more of hydroxytyrosol, tyroamine, phenylethyl alcohol, tyrosol phenolic ester, and tyrosol fatty acid ester.

[0012] Optionally, the glycosyltransferase is a protein from Fraxinus pennsylvanica.

[0013] This application is the first to discover that a protein sequence derived from American ash (Fraxinus pennsylvanica) has glycosyltransferase function, which can prepare and / or generate rhodioloside by catalyzing the transfer of glucose to the phenolic hydroxyl group of tyrosol and its analogues.

[0014] Furthermore, the glycosyltransferase derived from American ash (Fraxinus pennsylvanica) contains an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having 98% or more of the same identity as SEQ ID NO.1.

[0015] The glycosyltransferase derived from *Fraxinus pennsylvanica* comprises an amino acid sequence as shown in SEQ ID NO. 1 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 1.

[0016] Those skilled in the art will understand that reasonable sequence alterations or modifications can be made to the sequence shown in SEQ ID NO.1 without affecting glycosyltransferase activity, and these altered sequences should also fall within the scope of protection of this application. Such alterations include, but are not limited to: conserved amino acid substitution, partial amino acid deletion, addition, and N-terminal or C-terminal truncation; the altered polypeptide should still retain the protein function equivalent to the sequence in SEQ ID NO.1. Modification methods include, but are not limited to: cyclization, acetylation, fatty acid modification, etc.; modifications can occur at the N-terminus, C-terminus, main chain, side chain, or specific amino acid residues of the polypeptide.

[0017] On the other hand, this application also provides biological materials, said biological materials comprising at least one or more of the following A1)-A5):

[0018] A1) A nucleic acid molecule, wherein the nucleic acid molecule contains the glycosyltransferase;

[0019] A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1);

[0020] A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2);

[0021] A4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3);

[0022] A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3).

[0023] Those skilled in the art will recognize that the expression cassette described herein may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of the nucleic acid molecule encoding the protein can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.

[0024] Those skilled in the art will recognize that other commonly used expression elements, such as tags, fluorescent protein markers, and resistance selection markers, can be adaptively added to the expression cassette described in this application. The resistance gene is used at least to screen for positive transformants; optionally, the resistance gene is a kanamycin resistance gene. Those skilled in the art will understand that the tag may include "tags" that facilitate purification, including but not limited to histidine (HIS) tags, glutathione S-transferase tags (GST), maltose-binding protein tags (MBP), calmodulin-binding peptide tags (CBP), etc. For example, the labeled polypeptide can be easily purified, for instance, from conditioned media by chelation chromatography or affinity chromatography.

[0025] The recombinant vector described herein refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, and those skilled in the art can choose according to the specific circumstances; no excessive restrictions are imposed here. Optionally, the vector may include a nucleic acid molecule encoding the aforementioned protein, a promoter, and transcription and translation termination signals. During the preparation of the recombinant vector, the nucleic acid molecule encoding the aforementioned protein can be located within the vector so that it can be operatively linked to an appropriate expression regulatory sequence.

[0026] In one alternative embodiment, the recombinant vector is pET-28a(+).

[0027] In one optional embodiment, the expression cassette of the nucleic acid molecule is located on a recombinant vector or introduced into a recombinant microorganism using a recombinant vector. It should be noted that, as will be known to those skilled in the art, the nucleic acid molecule can be selectively inserted into the genome of the starting strain or can exist on a free plasmid, as long as the expression of the nucleic acid molecule or the synthesis of the glycosyltransferase protein can be achieved.

[0028] Furthermore, the recombinant microorganism comprises any one of the following B1)-B2):

[0029] B1) Escherichia coli bacteria;

[0030] B2) Escherichia coli.

[0031] The recombinant microorganisms include Escherichia coli or Escherichia coli.

[0032] Optionally, the recombinant microorganism is Escherichia coli.

[0033] In one optional embodiment, the *E. coli* is *Escherichia coli*. E. coli BL21 (DE3).

[0034] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain to construct recombinant microorganisms, as long as they can complete the expression of the mutants described in this application. No specific strains are limited here.

[0035] Furthermore, the nucleic acid molecule comprises the nucleic acid molecule shown in SEQ ID NO.2 or a nucleic acid molecule having 80% or more of the same identity as the nucleic acid molecule.

[0036] The nucleic acid molecule includes the nucleic acid molecule shown in SEQ ID NO.2 or a nucleic acid molecule having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with the nucleic acid molecule.

[0037] It is understood that those skilled in the art can select appropriate gene editing systems and gene editing methods to obtain the above-mentioned biological materials based on the actual situation.

[0038] On the other hand, this application also provides a whole-cell catalyst for preparing rhodioloside, the whole-cell catalyst comprising the recombinant microorganisms and / or recombinant cells described above.

[0039] The whole-cell catalyst may include suspensions, metabolites, extracts, etc. of recombinant microorganisms or recombinant cells.

[0040] Furthermore, the recombinant microorganism comprises any one of the following B1)-B2):

[0041] B1) Escherichia coli bacteria;

[0042] B2) Escherichia coli.

[0043] The recombinant microorganisms include Escherichia coli or Escherichia coli.

[0044] Optionally, the recombinant microorganism is Escherichia coli.

[0045] In one optional embodiment, the *E. coli* is *Escherichia coli*. E. coli BL21 (DE3).

[0046] On the other hand, this application also provides a method for preparing the glycosyltransferase, the method comprising: culturing the recombinant microorganisms and / or recombinant cells to obtain the glycosyltransferase.

[0047] Optional, recombinant microorganisms.

[0048] Alternatively, the recombinant microorganism is Escherichia coli.

[0049] In one optional embodiment, the *E. coli* is *Escherichia coli*. E. coli BL21 (DE3).

[0050] In one optional implementation, the method includes the following steps:

[0051] Step 1: Construct a recombinant microorganism expressing the glycosyltransferase; the glycosyltransferase contains the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having 98% or more identity with SEQ ID NO. 1;

[0052] Step 2: Fermentation culture of the recombinant microorganisms.

[0053] Optionally, the method includes the following steps:

[0054] Step 1: Construct a recombinant microorganism expressing the glycosyltransferase; the glycosyltransferase contains the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having 98% or more identity with SEQ ID NO. 1;

[0055] Step 2: Inoculate the recombinant microorganisms into the culture medium and culture at 25℃-37℃ and 100-300 rpm until OD. 600 The concentration is approximately 0.6-0.8. Cool the temperature to 15℃-20℃, add IPTG to a final concentration of 0.1-0.5 mM, and induce expression for 14-16 h.

[0056] Alternatively, the culture medium is 2YT medium.

[0057] Alternatively, the method may include the following steps:

[0058] Step 1: Construct a recombinant microorganism expressing the glycosyltransferase; the glycosyltransferase contains the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having 98% or more identity with SEQ ID NO. 1;

[0059] Step 2: Inoculate the recombinant microbial monoclonal strain into a culture medium and incubate at 25℃-37℃ and 100-300 rpm for 8-24 h to obtain seed culture;

[0060] Step 3: Inoculate the seed culture solution at an inoculum rate of 1%-10% into the culture medium and incubate at 25℃-37℃ and 100-300 rpm until OD reaches 100%. 600 The concentration is approximately 0.6-0.8. Cool the temperature to 15℃-20℃, add IPTG to a final concentration of 0.1-0.5 mM, and induce expression for 14-16 h.

[0061] Alternatively, the method may further include a purification step.

[0062] Those skilled in the art can choose conventional methods for purification.

[0063] In one alternative implementation, the purification includes cell disruption, centrifugation, and affinity chromatography purification.

[0064] Optionally, the conditions for cell disruption include 1000-1500 bar, 0℃-5℃, and cell disruption 4-5 times.

[0065] Optionally, the centrifugation includes centrifugation at 0℃-5℃ and 10000-15000 rpm for 0.5-2 h.

[0066] Optionally, the affinity chromatography purification is performed using Ni 2+ Purification was performed using an agarose affinity chromatography column.

[0067] In one optional embodiment, the purification step includes: disrupting the cells of the fermentation broth after the induction of expression has ended, wherein the cell disruption conditions include 1000-1500 bar, 0℃-5℃ for 4-5 cycles, centrifugation at 0℃-5℃, 10000-15000 rpm for 0.5-2 h, and taking the supernatant for Ni 2+ Purification was performed using an agarose affinity chromatography column.

[0068] Alternatively, the method may further include a concentration or freeze-drying step, and those skilled in the art can choose conventional methods to process the product according to the required dosage form.

[0069] In one alternative implementation, the concentration includes concentration using a 30 kDa ultrafiltration tube.

[0070] On the other hand, this application also provides the application of the biomaterial or the whole-cell catalyst in the preparation of rhodioloside.

[0071] On the other hand, this application also provides a method for preparing rhodioloside, the method comprising preparing rhodioloside using the glycosyltransferase or the biomaterial or the whole-cell catalyst described above.

[0072] Optionally, the method includes preparing rhodioloside by catalyzing the glycosyltransferase, the biomaterial, or the whole-cell catalyst to glycosylate tyrosol or its analogues.

[0073] Alternatively, the tyrosol analogue may be at least one or more of hydroxytyrosol, tyroamine, phenylethyl alcohol, tyrosol phenolic ester, and tyrosol fatty acid ester.

[0074] Optionally, the method includes the preparation of rhodioloside by catalyzing the transfer of glucose to the phenolic hydroxyl group of tyrosol or its analogue using the glycosyltransferase, the biomaterial, or the whole-cell catalyst described above.

[0075] Optionally, the glucose group may be present on UDP-glucose or other substances containing glucose groups.

[0076] Optionally, the reaction concentration of the glycosyltransferase is 50-200 μg / mL; optionally, 100 μg / mL.

[0077] The upper or lower limit of the reaction concentration or concentration range of the glycosyltransferase can be any value among 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, and 200 μg / mL.

[0078] Optionally, the molar ratio of the reaction concentration of the tyrosol or its analogue with the substance containing glucose is 1:(1-3), or alternatively, 1:1;

[0079] Optionally, the reaction concentration of the tyrosol or its analogue is 1-5 mM; optionally, 2 mM.

[0080] Optionally, the reaction concentration of the glucose-containing substance is 1-5 mM; optionally, 2 mM.

[0081] Optionally, the pH of the reaction is 7.0-8.0; optionally, 8.0.

[0082] In one optional embodiment, the pH of the reaction is controlled by a buffer solution, which may be Tris-HCl. Those skilled in the art can adjust the amount of buffer solution according to the pH requirements, and no specific limitations are imposed here.

[0083] Optionally, the reaction temperature is 30℃-40℃; alternatively, it is 35℃.

[0084] Optionally, the reaction time is 0.1-5 h; optionally, 1 h.

[0085] In one optional embodiment, the method includes: mixing tyrosol with a substance containing glucose at a reaction concentration molar ratio of 1:(1-2), adding the glucose as a catalyst to catalyze the tyrosol glycosylation reaction, wherein the reaction pH is 7.0-8.0, the reaction temperature is 30℃-40℃, and the reaction time is 0.1-5 h.

[0086] The conversion rate of the glycosyltransferase-catalyzed tyrosol glycosylation reaction is greater than or equal to 15%.

[0087] The yield of rhodioloside is greater than or equal to 0.3 mM.

[0088] On the other hand, this application also provides a catalyst for the glycosylation reaction of tyrosol or its analogues, the catalyst comprising the glycosyltransferase.

[0089] On the other hand, this application also provides the use of the catalyst for the glycosylation reaction of tyrosol or its analogues in catalyzing the glycosylation reaction of tyrosol or its analogues.

[0090] Optionally, the tyrosol or analogue glycosylation reaction includes a process of catalyzing the transfer of glucose groups to the phenolic hydroxyl groups of tyrosol or analogue.

[0091] The present invention has the following beneficial effects:

[0092] The glycosyltransferase FpUGT in this patent is derived from American red ash (Fraxinus pennsylvanica). Although this plant is rich in phenylethanoid glycosides, no literature has reported the existence of a glycosyltransferase gene in this genus that can directly catalyze the specific synthesis of rhodioloside from tyrosol. This application provides a new biological pathway for finding glycosyltransferases that catalyze the specific synthesis of rhodioloside from tyrosol, and also provides a new biomaterial for the preparation of rhodioloside. Attached Figure Description

[0093] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0094] Figure 1 This is a schematic diagram illustrating the construction process of the glycosyltransferase UGT8 gene expression plasmid.

[0095] Figure 2 The image shows the electrophoretic gel images of the purified glycosyltransferases UGT8 and UGT1, where M is the marker lane, U8 is the lane for glycosyltransferase UGT8, and U1 is the lane for glycosyltransferase UGT1.

[0096] Figure 3 The HPLC standard curve of rhodioloside is shown.

[0097] Figure 4 HPLC peak diagram of rhodioloside in the catalytic system of glycosyltransferase UGT8 (FpUGT);

[0098] Figure 5 The HPLC peak diagram shows the rhodioloside catalytic system of glycosyltransferase UGT1. Detailed Implementation

[0099] Technical terms:

[0100] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0101] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0102] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0103] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0104] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.

[0105] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0106] Whole-cell catalysts: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The complete biological organism that participates in this catalytic process is called a whole-cell catalyst.

[0107] Free expression: Free expression is the expression of target genes using free plasmids. Free plasmids are independent DNA molecules that exist in cells and have the ability to replicate and be transmitted independently. They are widely used in genetic engineering and molecular biology research.

[0108] Integrated expression: Expression that occurs when a gene is integrated into the genome.

[0109] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0110] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0111] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0112] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0113] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0114] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0115] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0116] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0117] The culture media involved in the following examples are as follows:

[0118] 2YT liquid culture medium: tryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, with the remainder being water.

[0119] Example 1: Screening and Mining of Candidate Genes

[0120] In this embodiment, a deep learning-based enzyme function prediction model was used to screen plant glycosyltransferases from a public database. The focus was on examining the binding affinity of candidate protein pockets to the substrate tyrosol and the spatial conformation of the catalytic center. The resulting sequence was UGT8 (also known as FpUGT, amino acid sequence as shown in SEQ ID NO.1).

[0121] Example 2: Construction of glycosyltransferase gene expression plasmid and strain

[0122] Based on the amino acid sequence of glycosyltransferase UGT8 obtained in Example 1, using... E. coli The target gene sequence was optimized using codon bias, and the nucleotide sequence is shown in SEQ ID NO.2. The DNA was synthesized using commercial services (commissioned to Tianjin Zhonghe Gene Technology Co., Ltd.).

[0123] The process of constructing glycosyltransferase gene expression plasmids is as follows: Figure 1 As shown. The synthesized UGT8 encoding gene was processed through... NdeI and XhoI The restriction enzyme sites were cloned into the expression vector pET-28a(+) to obtain plasmid pUGT8. The MBP protein tag sequence was then ligated using Gibson (NEB, NEBuilder). ® The expression plasmid pMBP-UGT8, an expression plasmid for the glycosyltransferase gene, was cloned into the N-terminus of the UGT8 coding gene using a HiFi DNA Assembly Master Mix. The expression plasmid vector backbone fragment and the MBP protein tag sequence fragment used for Gibson ligation were obtained by PCR amplification. Using the known glycosyltransferase UGT1, which catalyzes the synthesis of rhodioloside, as a control group (its amino acid sequence is shown in SEQ ID NO. 7), the expression plasmid pMBP-UGT1 could be constructed using the same method described above. The primers used for amplification using the above method are shown in Table 1.

[0124] Table 1 Primers for constructing glycosyltransferase expression plasmids using the Giboson method

[0125]

[0126] Example 3 Expression and purification of glycosyltransferase protein

[0127] The glycosyltransferase gene expression plasmids pMBP-UGT8 and pMBP-UGT1 constructed in Example 2 were transformed into the gene using a chemical transformation method. E. coli BL21(DE3) host was used to obtain genetically engineered bacteria U8 and U1. Information on the genetically engineered bacteria is shown in Table 2. Single clones of the engineered bacteria were inoculated into 5 mL of 2YT liquid medium (with 50 mg / L kanamycin added) and cultured overnight at 37°C and 220 rpm to obtain activated seed culture. All of the activated seed culture was transferred to 0.8 L of 2YT liquid medium (with 50 mg / L kanamycin added) and cultured at 37°C and 220 rpm until OD (dose elapsed). 600 The concentration was approximately 0.6-0.8. The temperature was lowered to 18°C, and IPTG was added to a final concentration of 0.5 mM. Expression was induced for 14-16 h. Cells were collected by centrifugation for subsequent purification of glycosyltransferases.

[0128] Purification of glycosyltransferases: Cells were resuspended in 100 mL of 50 mM Tris-HCl (pH 8.0) buffer and cell lysis was performed 4-5 times using an ultra-high pressure cell disruptor at 1000 bar and 4°C. The completely disrupted cell lysates were centrifuged at 10,000 rpm for 1 h at 4°C, and the supernatant was collected for subsequent affinity chromatography purification. The supernatant was then added to Ni... 2+ An agarose affinity chromatography column was used. Impurities were washed with 50 mM imidazole Tris-HCl buffer, followed by elution of the target protein with 300 mM imidazole Tris-HCl buffer to obtain the target protein solution. The eluted target protein solution was concentrated using a 30 kDa ultrafiltration tube, and the concentrated target protein was analyzed for protein expression by SDS-PAGE. Figure 2 Electrophoresis gel images of purified glycosyltransferases UGT8 and UGT1.

[0129] Table 2 Glycosyltransferase expression plasmids

[0130]

[0131] Example 4 In vitro enzyme activity experiment

[0132] The glycosyltransferases UGT8 and UGT1 purified in Example 3 were used as samples to test their in vitro enzyme activity.

[0133] Glycosyltransferase activity detection system: Tyrosol glycosylation reaction was carried out in a reaction mixture (pH 8.0) containing 100 μg / mL purified glycosyltransferase, 2 mM tyrosol, 2 mM UDP-glucose, and 50 mM Tris-HCl. The reaction mixture was incubated at 35 °C for 1 h and quenched with methanol. After centrifugation at 12000 rpm for 3 min at 12 °C, the supernatant was obtained. The reaction supernatant was diluted and the rhodioloside content was determined by high performance liquid chromatography (HPLC), and its conversion rate (actual rhodioloside yield / theoretical yield 2 Mm) was calculated.

[0134] The HPLC analysis of rhodioloside was performed using a Symmetry C18 column (4.6 × 250 mm, 5 μm), at a column temperature of 30℃, a flow rate of 0.8 mL / min, a mobile phase of water / methanol (80:20, v / v), and a detection wavelength of 275 nm. Rhodioloside standards showed a characteristic peak at 10.705 min. A calibration curve was generated based on the peak areas and concentrations of the characteristic peaks at 10.705 min for six standards. Figure 3 ).

[0135] The enzyme activities of UGT8 and UGT1 were detected using the above method, and the results are as follows:

[0136] A chromatographic peak with the retention time of the standard was detected at 10.705 min in the enzymatic reaction solution of glycosyltransferase UGT8 (FpUGT). Figure 4 The results indicate that the target product, rhodioloside, was generated in the reaction system. Based on peak area normalization, the rhodioloside content in the reaction system was calculated to be 0.3127 mM, and the conversion rate of rhodioloside was 15.63% (Table 3).

[0137] A chromatographic peak with the same retention time as the standard was detected at 10.705 min in the enzymatic reaction solution of glycosyltransferase UGT1. Figure 5 The presence of rhodioloside indicates that the target product was generated in the reaction system. Based on peak area normalization, the rhodioloside content in the reaction system was calculated to be 0.1892 mM, thus the conversion rate of rhodioloside was 9.46% (Table 3).

[0138] Table 3. Conversion rate of rhodioloside catalyzed by glycosyltransferase

[0139]

[0140] In summary, given that not all glycosyltransferases (UGTs) have the function of catalyzing the production of rhodioloside, the glycosyltransferase UGT8 (FpUGT) screened in this invention not only has the ability to catalyze the production of rhodioloside, but also has higher catalytic activity compared to known glycosyltransferases.

[0141] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. The application of glycosyltransferase in the catalytic preparation of rhodioloside from tyrosol, characterized in that, The glycosyltransferase is a glycosyltransferase derived from American red ash (Fraxinus pennsylvanica), and the amino acid sequence of the glycosyltransferase derived from American red ash (Fraxinus pennsylvanica) is shown in SEQ ID NO.

1.

2. The application of biomaterials in the catalytic preparation of rhodioloside from tyrosol, characterized in that, The biomaterial comprises at least one or more of the following A1)-A5): A1) A nucleic acid molecule, said nucleic acid molecule encoding the glycosyltransferase of claim 1; A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3).

3. The application according to claim 2, characterized in that, The recombinant microorganisms contain Escherichia coli bacteria.

4. The application according to claim 3, characterized in that, The recombinant microorganisms include Escherichia coli.

5. The application according to claim 2, characterized in that, The nucleic acid molecule described is shown in SEQ ID NO.

2.

6. The application of whole-cell catalysts in the catalytic preparation of rhodioloside from tyrosol, characterized in that, The whole-cell catalyst comprises any of the recombinant microorganisms and / or recombinant cells as described in claims 2-4.

7. The application according to claim 6, characterized in that, The recombinant microorganisms contain Escherichia coli bacteria.

8. The application according to claim 7, characterized in that, The recombinant microorganisms include Escherichia coli.

9. A method for preparing rhodioloside, characterized in that, The method includes preparing rhodioloside from tyrosol by catalyzing glycosyltransferase as described in claim 1, or biomaterial as described in any of claims 2-5, or whole-cell catalyst as described in any of claims 6-8.

10. The application of a catalyst for tyrosol glycosylation in the catalytic tyrosol glycosylation reaction, characterized in that, The reaction catalyst includes the glycosyltransferase described in claim 1.

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