Transferase and its application in preparing rhodioloside

Abstract

The present application provides transferase and its application in preparing rhodiolin, and relates to the technical field of enzyme engineering. The present application discloses UGT30, UGT50 and UGT55 proteins for the first time, and finds that the proteins have glycosyltransferase function, can be used as glycosyltransferase to efficiently catalyze substrate to synthesize rhodiolin, and provide new bioactive material for preparing rhodiolin. The present application also provides engineering bacteria expressing the glycosyltransferase, and a method for preparing rhodiolin by using the glycosyltransferase or the engineering bacteria, the method is simple and easy to operate, the yield and conversion rate of rhodiolin are high, and the method has good prospect in industrial production of rhodiolin.

Description

Technical Field

[0001] This application relates to the field of enzyme engineering technology, and in particular to transferases 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] Glycosyltransferases are key enzymes affecting the yield or conversion rate of rhodioloside. However, on the one hand, existing enzyme sources are mainly concentrated in a few species, which limits the diversity of enzyme properties and results in poor catalytic efficiency and substrate specificity. The catalytic activity or substrate affinity 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, existing research on glycosyltransferases has revealed that not all glycosyltransferases (UGTs) have the function of catalyzing the production of rhodiola glycosides from substrates. Michael P. Torrens-Spence et al. conducted functional analysis on 34 candidate glycosyltransferase genes and found that only a few enzymes have the function of catalyzing the production of rhodiola glycosides. This result also demonstrates 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. Summary of the Invention

[0005] This invention aims to address the lack of enzyme elements in the current biosynthesis of rhodioloside. Utilizing a deep learning model combined with structural modeling and substrate affinity prediction, the inventors, through heterologous expression and in vitro enzyme activity experiments, have for the first time discovered three novel protein sequences with glycosyltransferase activity capable of catalyzing the production of rhodioloside—that is, the discovery of new transferases. These three newly discovered transferases possess the ability to efficiently catalyze the glycosylation modification of substrates to generate rhodioloside, and can be used for the industrial production of rhodioloside through enzyme catalysis or engineered strains.

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

[0007] On the one hand, this application provides a transferase containing the amino acid sequence described in SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3 or an amino acid sequence that has 85% or more of the same identity as the protein and has the same function.

[0008] The transferase includes at least one of the following: A1) It contains the amino acid sequences described in SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3; A2) is a transferase with 85% or more amino acid sequence identity and the same function as the one shown in A1), specifically 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%, and 99.9%. A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0009] Furthermore, the nucleic acid molecule encoding the transferase contains the nucleic acid molecule shown in SEQ ID NO.4 and / or SEQ ID NO.5 and / or SEQ ID NO.6, or a nucleic acid molecule having 80% or more of the same identity as the nucleic acid molecule.

[0010] The nucleic acid molecule encoding the transferase contains the nucleic acid molecule shown in SEQ ID NO.4 and / or SEQ ID NO.5 and / or SEQ ID NO.6, or a nucleic acid molecule with 80% or more identity with the nucleic acid molecule shown in SEQ ID NO.6. Specifically, it can be a nucleic acid molecule with 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.

[0011] Optionally, the amino acid sequence of the transferase UGT30 is shown in SEQ ID NO.1, and the nucleotide sequence encoding the transferase UGT30 is shown in SEQ ID NO.4.

[0012] Optionally, the amino acid sequence of the transferase UGT50 is shown in SEQ ID NO.2, and the nucleotide sequence encoding the transferase UGT30 is shown in SEQ ID NO.5.

[0013] Optionally, the amino acid sequence of the transferase UGT55 is shown in SEQ ID NO.3, and the nucleotide sequence encoding the transferase UGT30 is shown in SEQ ID NO.6.

[0014] Those skilled in the art will understand that reasonable sequence alterations or modifications that do not affect the transferase activity can be made based on the sequences shown in SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3, 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 transferase function equivalent to the sequences in SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3. 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.

[0015] Those skilled in the art will understand that the label 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 peptide can be easily purified, for example, from conditioned medium by chelation chromatography or affinity chromatography.

[0016] Optionally, the transferase is a glycosyltransferase.

[0017] On the other hand, this application also provides biological materials, said biological materials comprising at least one or more of the following B1)-B6): B1) A nucleic acid molecule, said nucleic acid molecule containing a nucleic acid molecule encoding said transferase; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B5) Recombinant cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B6) A whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in B1), the expression cassette described in B2), the recombinant vector described in B3), the recombinant microorganism described in B4), and / or the recombinant cell described in B5).

[0018] 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 transferase can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.

[0019] 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, and the tag can be an MBP protein tag.

[0020] 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 actual situation; no excessive restrictions are imposed here. Optionally, the vector may include a nucleic acid molecule encoding the aforementioned transferase, a promoter, and transcription and translation termination signals. When preparing the recombinant vector, the nucleic acid molecule encoding the aforementioned transferase can be located within the vector so that it can be operatively linked to an appropriate expression regulatory sequence.

[0021] In one alternative embodiment, the recombinant vector is pET-28a(+).

[0022] 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 it enables the expression of the nucleic acid molecule or the synthesis of the protein.

[0023] Further, the nucleic acid molecule described in B1) contains the nucleic acid molecule shown in SEQ ID NO.4 and / or SEQ ID NO.5 and / or SEQ ID NO.6 or a nucleic acid molecule that has 80% or more of the same identity as the nucleic acid molecule.

[0024] Wherein, the nucleic acid molecule described in B1) contains the nucleic acid molecule shown in SEQ ID NO.4 and / or SEQ ID NO.5 and / or SEQ ID NO.6, 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.

[0025] Furthermore, the recombinant microorganism comprises any one of the following C1)-C2): C1) Escherichia coli bacteria; C2) Escherichia coli.

[0026] 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 transferase described in this application. No specific strain is limited here.

[0027] Optionally, the recombinant microorganism is Escherichia coli.

[0028] In one optional embodiment, the *E. coli* is *Escherichia coli*. E. coli BL21 (DE3).

[0029] The whole-cell catalyst may include suspensions, metabolites, extracts, etc. of recombinant microorganisms or recombinant cells.

[0030] 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.

[0031] On the other hand, this application also provides the use of the said transferase as a glycosyltransferase.

[0032] Optionally, the transferase is uridine diphosphate glycosyltransferase.

[0033] Optionally, the glycosyltransferase catalyzes the glycosylation of tyrosol or its analogues.

[0034] 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.

[0035] Alternatively, the glycosyltransferase can be prepared and / or used to generate rhodioloside by catalyzing the transfer of glucose to the phenolic hydroxyl group of tyrosol.

[0036] On the other hand, this application also provides the use of transferases in the preparation of rhodiolosides, wherein the transferases comprise at least one or more of the following: D1)-D3): D1) Transferases derived from cassava (Manihot esculenta); D2) Transferases derived from Osmanthus delavayi; D3) A transferase derived from Lathraea japonica.

[0037] Further, the transferase includes at least the amino acid sequence described in SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3, or an amino acid sequence having 85% or more identity with the transferase and having the same function, specifically 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%.

[0038] Optionally, the cassava-derived (Manihot esculenta) transferase is protein UGT30, which contains the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence encoding protein UGT30 contains the nucleotide sequence shown in SEQ ID NO.4.

[0039] Optionally, the transferase from Osmanthus delavayi is protein UGT50, which contains the amino acid sequence shown in SEQ ID NO.2, and the nucleotide sequence encoding protein UGT30 contains the nucleotide sequence shown in SEQ ID NO.5.

[0040] Optionally, the transferase from *Lathraea japonica* is protein UGT55, which contains the amino acid sequence shown in SEQ ID NO.3, and the nucleotide sequence encoding protein UGT30 contains the nucleotide sequence shown in SEQ ID NO.6.

[0041] Preferably, the transferase catalyzes the glycosylation reaction of tyrosol or its analogues.

[0042] More preferably, the transferase catalyzes the transfer of glucose groups to the phenolic hydroxyl groups of tyrosol or its analogues, thereby achieving glycosylation of tyrosol or its analogues.

[0043] More preferably, the transferase is prepared and / or generates rhodioloside by catalyzing the transfer of glucose to the phenolic hydroxyl group of tyrosol or its analogues.

[0044] Specifically, the tyrosol analogue can be at least one or more of hydroxytyrosol, tyroamine, phenylethyl alcohol, tyrosol phenolic ester, and tyrosol fatty acid ester.

[0045] The inventors of this application have, for the first time, obtained three novel transferases through screening, derived from cassava (Manihot esculenta), sweet osmanthus (Osmanthus delavayi), and scabra (Lathraea japonica), respectively. Furthermore, they have demonstrated for the first time that these three novel transferases possess glycosyltransferase function. This provides a new biological information repository for glycosyltransferase screening and lays the foundation for subsequent glycosyltransferase research.

[0046] On the other hand, this application also provides the application of the biomaterial in the preparation of rhodioloside.

[0047] On the other hand, this application also provides a method for preparing the transferase or glycosyltransferase, the method comprising: culturing the recombinant microorganism to obtain the protein or glycosyltransferase.

[0048] Furthermore, the recombinant microorganism comprises any one of the following C1)-C2): C1) Escherichia coli bacteria; C2) Escherichia coli.

[0049] Optionally, the recombinant microorganism is Escherichia coli.

[0050] In one optional embodiment, the *E. coli* is *Escherichia coli*. E. coli BL21 (DE3).

[0051] In one optional implementation, the method includes the following steps: Step 1: Construct a recombinant microorganism expressing the transferase or glycosyltransferase; the transferase or glycosyltransferase contains the amino acid sequence of SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3 or an amino acid sequence that has 85% or more of the same identity as the transferase or glycosyltransferase and has the same function; Step 2: Fermentation culture of the recombinant microorganisms.

[0052] Optionally, the method includes the following steps: Step 1: Construct a recombinant microorganism expressing the glycosyltransferase; the transferase or glycosyltransferase contains the amino acid sequence of SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3 or an amino acid sequence that has 85% or more of the same identity as the transferase or glycosyltransferase and has the same function; 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.

[0053] Alternatively, the culture medium is 2YT liquid culture medium.

[0054] Alternatively, the method may include the following steps: Step 1: Construct a recombinant microorganism expressing the glycosyltransferase; the transferase or glycosyltransferase contains the amino acid sequence of SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3 or an amino acid sequence that has 85% or more of the same identity as the transferase or glycosyltransferase and has the same function; 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; 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.

[0055] Alternatively, the method may further include a purification step.

[0056] Those skilled in the art can choose conventional methods for purification.

[0057] In one alternative implementation, the purification includes cell disruption, centrifugation, and affinity chromatography purification.

[0058] Optionally, the conditions for cell disruption include 1000-1500 bar, 0℃-5℃, and cell disruption 4-5 times.

[0059] Optionally, the centrifugation includes centrifugation at 0℃-5℃ and 10000-15000 rpm for 0.5-2 h.

[0060] Optionally, the affinity chromatography purification is performed using Ni2+ Purification was performed using an agarose affinity chromatography column.

[0061] 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.

[0062] 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.

[0063] In one alternative implementation, the concentration includes concentration using a 30 kDa ultrafiltration tube.

[0064] On the other hand, this application also provides a method for preparing rhodioloside, the method comprising preparing rhodioloside using the transferase or the biological material.

[0065] Optionally, the method includes preparing rhodioloside by catalyzing the glycosylation reaction of tyrosol or its analogues using the aforementioned transferase or the aforementioned biomaterial.

[0066] 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 protein or the biomaterial.

[0067] Optionally, the glucose group may be present on UDP-glucose or other substances containing glucose groups.

[0068] Optionally, the reaction concentration of the transferase (glycosyltransferase) is 50-200 μg / mL; optionally, 100 μg / mL.

[0069] The upper or lower limit of the reaction concentration or concentration range of the protein (glycosyltransferase) may 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.

[0070] Optionally, the molar ratio of the reaction concentration of the tyrosol or its analogue with the glucose-containing substance is 1:(1-3); 1:1.

[0071] Optionally, the reaction concentration of the tyrosol or its analogue is 1-5 mM; optionally, 2 mM.

[0072] Optionally, the reaction concentration of the glucose-containing substance is 1-5 mM; optionally, 2 mM.

[0073] Optionally, the pH of the reaction is 7.0-8.0; optionally, 8.0.

[0074] 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.

[0075] Optionally, the reaction temperature is 30℃-40℃; alternatively, it is 35℃.

[0076] Optionally, the reaction time is 0.1-5 h; optionally, 1 h.

[0077] In one optional embodiment, the method includes: mixing tyrosol or its analogue with a substance containing glucose at a reaction concentration molar ratio of 1:(1-3), adding the transferase (glycosyltransferase) as a catalyst to catalyze the glycosylation reaction of tyrosol or its analogue, wherein the reaction pH is 7.0-8.0, the reaction temperature is 30℃-40℃, and the reaction time is 0.1-5 h.

[0078] The glycosyltransferase (GLT) catalyzes a conversion of tyrosol or its analogues of 4% or greater, optionally 5% or greater, and more preferably 11% or greater.

[0079] The yield of rhodioloside is greater than or equal to 0.09 mM, optionally greater than or equal to 0.11 mM, and more preferably greater than or equal to 0.23 mM.

[0080] On the other hand, this application also provides a catalyst for the glycosylation reaction of tyrosol or its analogues, the catalyst comprising the transferase or the glycosyltransferase or the biomaterial.

[0081] 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.

[0082] 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.

[0083] The present invention has the following beneficial effects: This invention discloses for the first time the UGT30, UGT50 and UGT55 proteins, and for the first time discovers that they have glycosyltransferase function, which can be used as glycosyltransferases to efficiently catalyze the specific synthesis of rhodioloside from tyrosol, providing a new bioactive material for the preparation of rhodioloside.

[0084] The present invention also provides an engineered bacterium expressing the glycosyltransferase, and a method for preparing rhodioloside using the glycosyltransferase or its engineered bacterium. The method is simple and easy to operate, and the yield and conversion rate of rhodioloside are high, showing good prospects in the industrial production of rhodioloside. Attached Figure Description

[0085] 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: Figure 1 This is a schematic diagram of the construction process of the glycosyltransferase UGT gene expression plasmid, where the UGT gene can refer to UGT30, UGT50 or UGT55; Figure 2 The image shows the electrophoretic gel image of the purified glycosyltransferase UGT, where M is the marker lane, U30 is the UGT30 lane, U50 is the UGT50 lane, and U55 is the UGT55 lane. Figure 3 The HPLC standard curve of rhodioloside is shown. Figure 4 HPLC peak diagram of rhodioloside in the catalytic system of glycosyltransferase UGT30; Figure 5 HPLC peak chromatogram of rhodioloside in the catalytic system of glycosyltransferase UGT50; Figure 6 The HPLC peak diagram shows the rhodioloside catalytic system of glycosyltransferase UGT55. Detailed Implementation

[0086] Technical terms: 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.

[0087] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0088] 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.

[0089] 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.

[0090] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.

[0091] 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.

[0092] 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.

[0093] 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. Integrated expression: Expression that occurs when a gene is integrated into the genome.

[0094] 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.

[0095] 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. 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.

[0096] 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.

[0097] 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. 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).

[0098] 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.

[0099] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0100] The culture media involved in the following examples are as follows: 2YT liquid culture medium: tryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, with the remainder being water.

[0101] Example 1: Screening and Mining of Candidate Genes 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. After screening, new sequences UGT30 (amino acid sequence as shown in SEQ ID NO.1), UGT50 (amino acid sequence as shown in SEQ ID NO.2), and UGT55 (amino acid sequence as shown in SEQ ID NO.3) were obtained.

[0102] Example 2 Construction of Glycosyltransferase Gene Expression Plasmid Based on the amino acid sequences of glycosyltransferases UGT30, UGT50, and UGT55 obtained in Example 1, using... E. coli The target gene sequence was optimized using codon bias to obtain the nucleotide sequence SEQ ID NO.4 encoding glycosyltransferase UGT30, the nucleotide sequence SEQ ID NO.5 encoding glycosyltransferase UGT50, and the nucleotide sequence SEQ ID NO.6 encoding glycosyltransferase UGT55. The DNA was then synthesized using commercial services (commissioned to Tianjin Zhonghe Gene Technology Co., Ltd.).

[0103] The process of constructing glycosyltransferase gene expression plasmids is as follows: Figure 1As shown. The three synthesized UGT (UGT30, UGT50, UGT55) encoding genes were respectively processed through... NdeI and XhoI The restriction sites were cloned into the expression vector pET-28a(+), yielding pUGT plasmids pUGT30, pUGT50, and pUGT55, respectively. The MBP protein tag sequence was then ligated using Gibson (NEB, NEBuilder). ® The plasmids pMBP-UGT30, pMBP-UGT50, and pMBP-UGT55, which are glycosyltransferase gene expression plasmids, were cloned into the N-terminus of the UGT 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. The primers used for amplification using the above method are shown in Table 1.

[0104] Table 1 Primers for constructing glycosyltransferase expression plasmids using the Giboson method

[0105] Example 3 Expression and purification of glycosyltransferase protein The three glycosyltransferase gene expression plasmids (pMBP-UGT30, pMBP-UGT50, and pMBP-UGT55) constructed in Example 2 were transformed into... E. coli Using BL21(DE3) as the host, genetically engineered bacteria U30, U50, and U55 were obtained, and their information is shown in Table 2. Single clones of these three engineered strains 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 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.

[0106] 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 Electrophoretic gel images of the three purified glycosyltransferase proteins (UGT30, UGT50, UGT55).

[0107] Table 2 Glycosyltransferase expression plasmids

[0108] Example 4 In vitro enzyme activity experiment The glycosyltransferases (UGT30, UGT50, UGT55) purified in Example 3 were used as samples to test their in vitro enzyme activity.

[0109] Glycosyltransferase activity assay 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 was calculated.

[0110] 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 ).

[0111] The enzyme activities of UGT30, UGT50, and UGT55 were detected using the above method, and the results are as follows: A chromatographic peak with the same retention time as the standard was detected at 10.705 min in the enzymatic reaction solution of glycosyltransferase UGT30. 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 0.2347 mM, and the conversion rate of rhodioloside was 11.74% (Table 3).

[0112] A chromatographic peak with the retention time of the standard was detected at 10.705 min in the enzymatic reaction solution of glycosyltransferase UGT50. Figure 5 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 0.1174 mM, and the conversion rate of rhodioloside was 5.87% (Table 3).

[0113] A chromatographic peak with the retention time of the standard was detected at 10.705 min in the enzymatic reaction solution of glycosyltransferase UGT55. Figure 6 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 0.0920 mM, and the conversion rate of rhodioloside was 4.60% (Table 3).

[0114] Table 3. Conversion rate of rhodioloside catalyzed by glycosyltransferase

[0115] In summary, given that not all transferases have the ability to catalyze the formation of rhodioloside, the transferases UGT30, UGT50, and UGT55 screened in this invention all have the ability to catalyze the formation of rhodioloside and exhibit high catalytic activity, providing three new glycosyltransferase protein materials for rhodioloside.

[0116] The above description is merely an embodiment of this application and is not intended to limit the scope of 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 principles of this application should be included within the scope of the claims of this application.

Claims

1. A transferase, characterized in that, The transferase includes at least one of the following: A1) It contains the amino acid sequences described in SEQ ID NO.1 and / or SEQ ID NO.2 and / or SEQ ID NO.3; A2) and A1) have an amino acid sequence that is more than 98.5% identical and have the same function. A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. A biomaterial, characterized in that, The biomaterial comprises at least one or more of the following B1)-B6): B1) A nucleic acid molecule, said nucleic acid molecule containing a nucleic acid molecule encoding the transferase of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B5) Recombinant cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B6) A whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in B1), the expression cassette described in B2), the recombinant vector described in B3), the recombinant microorganism described in B4), and / or the recombinant cell described in B5).

3. The biomaterial according to claim 2, characterized in that, B1) The nucleic acid molecule described herein contains the nucleic acid molecule shown in SEQ ID NO. 4 and / or SEQ ID NO. 5 and / or SEQ ID NO. 6 or a nucleic acid molecule that has 80% or more of the same identity as the nucleic acid molecule described herein.

4. The biomaterial according to claim 2, characterized in that, The recombinant microorganism in A4) includes any one of the following C1)-C2): C1) Escherichia coli bacteria; C2) Escherichia coli.

5. Use of the transferase according to claim 1 as a glycosyltransferase.

6. The use of transferase in the preparation of rhodioloside, characterized in that, The transferase includes at least the transferase described in claim 1.

7. The use of the biomaterial according to any one of claims 2-4 in the preparation of rhodioloside.

8. A method for preparing the transferase as described in claim 1, characterized in that, The method includes: culturing the recombinant microorganism of claim 2 to obtain the protein; Preferably, the recombinant microorganism comprises any one of the following C1)-C2): C1) Escherichia coli bacteria; C2) Escherichia coli.

9. A method for preparing rhodioloside, characterized in that, The method includes preparing rhodioloside using the transferase as described in claim 1 or the biomaterial as described in any one of claims 2-4.

10. A catalyst for the glycosylation reaction of tyrosol or its analogues, characterized in that, The reaction catalyst includes the transferase described in claim 1.

Images