Application of safflower glycosyltransferase and related biological materials in biosynthesis of hydroxysafflor yellow A

Abstract

The invention discloses glycosyltransferase in safflower and a related biological material and application thereof. The invention firstly discloses any one of the following proteins: A1) a protein consisting of an amino acid sequence as shown in a sequence 2; a2) a fusion protein obtained by connecting the N end or / and C end of the protein as shown in the sequence 2 with a protein tag. The invention further discloses the protein related biological material and application thereof. The HSYA biosynthesis key glycosyl transferase gene UGT708U5 is obtained from safflower for the first time, it is proved that the protein UGT708U5 coded by the gene can catalyze phloretin to form HSYA precursor compounds, namely phloretin-mono-C-glucoside and phloretin-di-C-glucoside, and the gene plays an important role in biosynthesis and heterologous production of HSYA; the method has important theoretical and practical significance for regulating and producing the quinonechalcone C-glycoside compounds of plants and cultivating high-quality safflower carthamus.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal plant genetic engineering, and particularly relates to the functional characterization of safflower glycosyltransferase UGT708U5 and the application of related biomaterials. Background Art

[0002] Safflower is the dried flower of the Asteraceae plant Carthamus tinctorius L., known for its effects on promoting blood circulation, relieving menstruation, and alleviating pain (Chinese Pharmacopoeia Committee. Chinese Pharmacopoeia [M]. Beijing: China Medical Science and Technology Press, 2020:157). As a traditional Chinese medicine for promoting blood circulation and removing blood stasis, safflower has been used for over 2,500 years. It also has clinical benefits such as lowering cholesterol, lowering blood pressure, dilating coronary arteries, providing antioxidant benefits, and protecting the myocardium. Currently, several injections (such as Safflower Injection and Danhong Injection) have been developed and are widely used to treat coronary heart disease, angina pectoris, myocardial infarction, ischemic encephalopathy, and cerebral thrombosis.

[0003] Hydroxysafflor yellow A (HSYA) is a specific component of safflower with a high content. It is the most effective water-soluble component in safflower's blood-activating and blood-stasis-removing effects (Bai X, et al. Front Pharmacol, 2020, 11:01265). Pharmacological studies have shown that HSYA has a series of functions that can inhibit platelet aggregation and release induced by platelet activating factor, and plays an antioxidant role in traumatic brain injury (Tian Y, et al. J Ethnopharmacol, 2010, 129(1): 1-4; Sun L, et al. Nitric Oxide, 2013, 35: 144-51; Wang X, et al. ASN Neuro, 2016, 8(2): 1759091416642345); HSYA can reduce hyperlipidemic myocardial ischemia-reperfusion injury by inhibiting the inflammatory response triggered by the TLR4 signaling pathway (Han D, et al. Sci Rep, 2016, 6: 35319); HSYA can also promote angiogenesis by stimulating the expression of the angiopoietin signaling pathway Ang1 / Tie2. On December 19, 2023, Yuekang Pharmaceutical's new drug application (NDA) for Hydroxysafflor Yellow A for Injection (API), an innovative Class 1.2 traditional Chinese medicine for the treatment of acute ischemic stroke, was accepted. The purity of HSYA is expected to be no less than 95%. Acute ischemic stroke is the most common type of stroke, with extremely high morbidity, mortality, and disability rates. The patient population is large, and the drug market is vast. Hydroxysafflor Yellow A for Injection will provide a new therapeutic strategy to address this clinically challenging situation.

[0004] HSYA is a quinone chalcone C-glycoside compound currently obtained primarily through extraction and isolation from the medicinal plant safflower. With the further development and utilization of the medicinal value of safflower HSYA and the industrialization of drugs for the treatment of cardiovascular and cerebrovascular diseases, the shortage of medicinal resources will become a primary issue. Despite the continuous expansion of safflower cultivation area, it still cannot meet market demand. In recent years, synthetic biology has attracted widespread attention in the research on the sustainable utilization of traditional Chinese medicine resources. The production of natural active products using synthetic biology technology has become a highly promising method for obtaining natural product resources. Successful cases include different types of natural active products such as artemisinic acid (Paddon CJ, et al. Nature, 2013, 496:528-532), the paclitaxel precursor taxadiene (Parayil Kumaran Ajikumar, et al. 2010, Science, 330:70-74), and vinblastine (Zhang J, et al. 2022, Nature, 609, 341-347). Therefore, by mining and characterizing the key glycosyltransferase genes in HSYA biosynthesis, we constructed and optimized the metabolic pathway in microorganisms ( Figure 1 ) to efficiently produce HSYA. At present, the HSYA biosynthesis pathway in safflower has not been fully elucidated, and the discovery and functional characterization of glycosyltransferases are the bottlenecks to break through the HSYA biosynthesis pathway. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to obtain C-glycosyltransferase involved in the biosynthesis of safflower HSYA, and to provide precursor compounds for the later synthesis or preparation of HSYA.

[0006] To solve the above problems, the present invention first provides a protein, which is UGT708U5, derived from safflower (Carthamus tinctorius L.), named safflower C-glycosyltransferase UGT708U5, and is shown in any one of the following A1)-A3):

[0007] A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

[0008] A2) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein shown in Sequence 2 in the sequence listing;

[0009] A3) A protein having more than 90% identity and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

[0010] Among them, sequence 2 consists of 471 amino acid residues.

[0011] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0012] In the above-mentioned proteins, a protein tag refers to a polypeptide or protein that is fused and expressed with a target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0013] In the above-mentioned proteins, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0014] Biological materials related to UGT708U5 are also within the scope of protection of the present invention.

[0015] The biomaterial related to UGT708U5 provided by the present invention is any one of the following A1) to A12):

[0016] A1) Nucleic acid molecule encoding UGT708U5;

[0017] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0018] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0019] A4) a recombinant vector containing the expression cassette described in A2);

[0020] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0021] A6) a recombinant microorganism containing the expression cassette described in A2);

[0022] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0023] A8) a recombinant microorganism containing the recombinant vector described in A4);

[0024] A9) a transgenic plant cell line containing the nucleic acid molecule described in A1);

[0025] A10) a transgenic plant cell line containing the expression cassette described in A2);

[0026] A11) a transgenic plant cell line containing the recombinant vector described in A3);

[0027] A12) A transgenic plant cell line containing the recombinant vector described in A4).

[0028] In the above biological material, the nucleic acid molecule described in A1) is as shown in B1) or B2) or B3) below:

[0029] B1) DNA molecule shown in Sequence 1 in the sequence listing;

[0030] B2) The coding sequence is the DNA molecule shown in Sequence 1 in the sequence listing;

[0031] B3) A DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in B1) or B2) and encodes UGT708U5.

[0032] Among them, Sequence 1 in the sequence listing consists of 1416 nucleotides and encodes the protein shown in Sequence 2.

[0033] The stringent conditions are hybridization in a 2×SSC, 0.1% SDS solution at 68°C and washing the membrane twice for 5 minutes each time, and hybridization in a 0.5×SSC, 0.1% SDS solution at 68°C and washing the membrane twice for 15 minutes each time.

[0034] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA, or may be RNA, such as mRNA or hnRNA.

[0035] In the above-mentioned biological materials, the expression cassette containing the gene encoding UGT708U5 described in A2) refers to DNA capable of expressing UGT708U5 in a host cell. This DNA may include not only a promoter for initiating transcription of UGT708U5 but also a terminator for terminating transcription of UGT708U5. Furthermore, the expression cassette may also include an enhancer sequence.

[0036] In the above biological materials, the vector may be a plasmid, cosmid, phage or viral vector.

[0037] In the above biological materials, the microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0038] Among the above-mentioned biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include reproductive materials.

[0039] The present invention further provides applications of the above protein or related biological materials.

[0040] The application is specifically as follows:

[0041] 1) Use of the above protein as a glycosyltransferase;

[0042] 2) Application of the above-mentioned related biological materials for the preparation of glycosyltransferases;

[0043] 3) Use of the above-mentioned proteins or related biomaterials in the preparation or synthesis of phloretin-mono-C-glucoside (1a);

[0044] 4) Use of the above protein or related biomaterials in catalyzing phloretin to form phloretin-di-C-glucoside (1b).

[0045] The present invention also provides a method for preparing UGT708U5.

[0046] The method for preparing UGT708U5 of the present invention comprises introducing a UGT708U5 encoding gene into a recipient microorganism to obtain a recombinant microorganism expressing UGT708U5, and culturing the recombinant microorganism to express UGT708U5.

[0047] In the above method, the recipient microorganism is a prokaryotic microorganism. Specifically, the prokaryotic microorganism is Escherichia coli. More specifically, the Escherichia coli expression strain Transetta (DE3).

[0048] In the above method, the UGT708U5 encoding gene can be introduced into the Escherichia coli expression strain Transetta (DE3) through the recombinant plasmid pET28-SUMO::UGT708U5; the recombinant plasmid pET28-SUMO::UGT708U5 is a recombinant expression vector constructed by using the UGT708U5 gene shown in Sequence 1 to the BamHI restriction site of the pET28a(+)-Sumo vector, while keeping other sequences of the pET28a(+)-Sumo vector unchanged.

[0049] The present invention further provides a method for preparing safflower HSYA precursor compounds phloretin-mono-C-glucoside and / or phloretin-di-C-glucoside, the method comprising the step of catalyzing phloretin to generate phloretin-mono-C-glucoside and / or phloretin-di-C-glucoside using UGT708U5.

[0050] The present invention cloned the UGT708U5 gene from safflower cDNA. This gene is the first C-glycosyltransferase isolated from safflower that participates in the biosynthesis of HSYA. Experimental studies have demonstrated that the UGT708U5 protein of the present invention can catalyze the sequential two-step C-glycosylation of phloretin to produce phloretin-mono-C-glucoside (1a) and phloretin-di-C-glucoside (1b), serving as a precursor compound in the biosynthesis pathway of safflower HSYA. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a schematic diagram of the inferred biosynthesis pathway of safflower HSYA.

[0052] Figure 2 The figure shows the agarose gel electrophoresis of the safflower UGT708U5 gene clone; M represents Trans5K DNA Marker (nucleic acid molecular weight standard, with bands of 5000, 3000, 2000, 1000, 750, 500, 250, and 100 bp from top to bottom); UGT708U5 represents the UGT708U5 gene.

[0053] Figure 3 This is polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the UGT708U5 protein expressed in E. coli. In A, M represents a protein marker (protein molecular weight standard, with bands from top to bottom representing 190, 140, 95, 70, 55, 43, 33, and 26 kDa). (B) represents the electrophoresis result of the purified protein from the recombinant plasmid pET28a-SUMO::UGT708U5. The arrow in the figure indicates the recombinant protein UGT708U5 expressed from the recombinant plasmid pET28a-SUMO::UGT708U5.

[0054] Figure 4 LC-MS analysis of the products of the UGT708U5 enzymatic reaction. A is the ion current diagram of the UGT708U5 enzymatic reaction time course, B is the primary mass spectrum of phloretin-mono-C-glucoside (1a) generated using phloretin as a substrate, C is the secondary mass spectrum of phloretin-mono-C-glucoside (1a) generated using phloretin as a substrate, D is the primary mass spectrum of phloretin-di-C-glucoside (1b) generated using phloretin as a substrate, and E is the secondary mass spectrum of phloretin-di-C-glucoside (1b) generated using phloretin as a substrate. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0056] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0057] The safflower in the following examples was collected from Henan Academy of Agricultural Sciences; High-Fidelity DNA Polymerase and BamHI restriction endonuclease are products of New England Biolabs;

[0058] The Rapid Universal Plant RNA Extraction Kit is a product of Beijing Huayueyang Biotechnology Co., Ltd.;

[0059] TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, Trans2KDNA Marker, pEASY-Uni Seamless Cloning and Assembly Kit, and Escherichia coli competent cells Transetta (DE3) are products of Beijing Quanshijin Biotechnology Co., Ltd.

[0060] PageRuler TM Prestained Protein Ladder is a product of ThermoFisher Scientific;

[0061] The pET28a(+)-Sumo vector is a product of Novagen;

[0062] Phloretin is a product of Shanghai Yuanye Biotechnology Co., Ltd., catalog number S31393, CAS number 60-82-2;

[0063] UDP-Glc is a product of Shanghai Yuanye Biotechnology Co., Ltd. with catalog number S18074 and CAS number 28053-08-09.

[0064] (I) Cloning of the full-length cDNA of the safflower UGT708U5 gene

[0065] 1. Extraction of Total RNA

[0066] Total RNA was extracted from fresh safflower petals using the TransZol method and analyzed by 1.0% agarose gel electrophoresis. 260 / 280 ≧1.8, indicating that the total RNA extracted by the above method has high quality and purity and can be used for subsequent reverse transcription experiments to synthesize cDNA.

[0067] Total RNA was extracted according to the instructions of the Rapid Universal Plant RNA Extraction Kit of Beijing Huayueyang Biotechnology Co., Ltd.

[0068] 2. Synthesis of first-strand cDNA

[0069] The operation was performed according to the instructions of the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix first-strand cDNA synthesis kit from Beijing Quanshijin Biotechnology Co., Ltd., and cDNA was finally obtained by reverse transcription.

[0070] The reverse transcription reaction system is as follows:

[0071]

[0072]

[0073] The steps of reverse transcription are as follows:

[0074] (1) To obtain higher synthesis efficiency, Total RNA, Anchored Oligo (dT) 18 Primerh and RNase-free water were placed in a PCR tube, mixed, and incubated at 65°C for 5 min.

[0075] (2) Add 10.0 μL 2×TS Reaction Mix, 1.0 μL RT / RI Enzyme Mix, 1.0 μL gDNA Remover, and gently mix;

[0076] (3) Reverse transcription was performed at 42°C for 30 min, 85°C for 5 s to obtain the first-strand cDNA.

[0077] (4) The first-strand cDNA was stored at -20°C.

[0078] 3. Primer design

[0079] Based on the safflower transcriptome data, the open reading frame (ORF) sequence was obtained, and based on this, the cloning primers UGT708U5-F1 and UGT708U5-R1 were designed. The primer sequences are as follows:

[0080] UGT708U5-F1:5'-ATGCCGACCGCCACCGTC-3'; (SEQ ID NO: 3)

[0081] UGT708U5-R1:5'-TTATGGTTTTACATTGTTGTCCTTGCT-3'. (SEQ ID NO: 4)

[0082] 4. PCR amplification

[0083] Using the first-strand cDNA obtained in step 2 as a template, PCR amplification was performed using the high-fidelity enzyme Prime Start Max, UGT708U5-F1 and UGT708U5-R1 primers to obtain the PCR amplification product. Figure 2 The PCR amplification products were sequenced.

[0084] The PCR amplification procedure is as follows:

[0085] Pre-denaturation at 98°C for 5 min; 40 cycles of 98°C for 15 s, 55°C for 15 s, and 72°C for 50 s; extension at 72°C for 16 min.

[0086] The sequencing results showed that the sequence of the PCR amplification product was shown in Sequence 1. The gene shown in Sequence 1 was named UGT708U5, encoding a protein composed of 471 amino acid residues. The protein was named UGT708U5, and the amino acid sequence of the protein was shown in Sequence 2.

[0087] (II) Expression and functional characterization of safflower UGT708U5 protein

[0088] 1. Construction of recombinant vector

[0089] The UGT708U5 gene shown in sequence 1 was constructed into the BamHI restriction site of the pET28a(+)-Sumo vector using the pEASY-Uni Seamless Cloning and Assembly Kit from Beijing Quanshijin Biotechnology Co., Ltd., while keeping other sequences of the pET28a(+)-Sumo vector unchanged to obtain the recombinant plasmid pET28-SUMO::UGT708U5.

[0090] The specific steps are as follows:

[0091] 1) Using the PCR amplification product obtained in Example 1 as a template, PCR amplification was performed using primers UGT708U5-F2 and UGT708U5-R2, and the purified PCR product was recovered and purified. The primer sequences are as follows (the underlined sequences are vector homology regions):

[0092] UGT708U5-F2:5'- CACAGAGAACAGATTGGTGGATC AATGCCGACCGCCACCGTC-3'; (SEQ ID NO: 5)

[0093] UGT708U5-R2:5'- TCGACGGAGCTCGAATTCGGATCC TTATGGTTTTACATTGTTGTCCTTGCT-3'. (Sequence 6)

[0094] 2) Take the pET28a(+)-Sumo vector, digest it with the restriction endonuclease BamHI, and recover the linearized vector backbone.

[0095] 3) The purified PCR product obtained in step 1) was cloned into the linearized vector backbone in step 2) according to the instructions of the pEASY-UniSeamless Cloning and Assembly Kit from Beijing Quanshijin Biotechnology Co., Ltd. to obtain the recombinant plasmid pET28-SUMO::UGT708U5.

[0096] 2. Obtaining recombinant bacteria

[0097] The recombinant plasmid pET28-SUMO::UGT708U5 was introduced into the Escherichia coli expression strain Transetta (DE3) to obtain the pET28-SUMO::UGT708U5 recombinant bacteria; at the same time, the pET28a(+)-Sumo vector without the target gene was used to transform the Escherichia coli expression strain Transetta (DE3) as a control bacteria.

[0098] 3. Induce expression of recombinant protein UGT708U5

[0099] Pick the pET28-SUMO::UGT708U5 recombinant bacteria and the control bacteria and inoculate them into 2 mL of LB liquid medium (containing 50 mg / L of kanamycin) and shake culture at 37 ° C overnight. The next day, dilute them 1:100 and add them to 100 mL of LB liquid medium and shake culture at 37 ° C until OD 600 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and the culture was continued in a shaking incubator at 16°C for 18 hours to induce target protein expression. The bacterial solution was centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, and the pET28-SUMO::UGT708U5 recombinant bacteria and control bacteria were collected and stored in a -80°C refrigerator until use.

[0100] 4. Extraction and purification of recombinant protein UGT708U5

[0101] The supernatant of the pET28-SUMO::UGT708U5 recombinant bacteria was purified and subjected to SDS-PAGE. Figure 3 As shown in the figure, the recombinant protein UGT708U5 expressed by the recombinant plasmid pET28-SUMO::UGT708U5 is present in the purified supernatant of the recombinant bacteria. The size of the recombinant protein UGT708U5 is approximately 65 kDa, which is consistent with the expected size.

[0102] 5. Enzymatic Function Analysis of Recombinant Protein UGT708U5

[0103] (1) Enzymatic reaction of UGT708U5

[0104] The purified pET28-SUMO::UGT708U5 protein was subjected to an enzymatic reaction to obtain the product. The enzymatic reaction system consisted of 50 mM Na2HPO4-NaH2PO4 buffer (pH 8.0), 40 mM phloretin as the substrate, 40 mM UDP-Glc as the sugar donor, and 20 μg of the purified recombinant UGT708U5 protein. The reaction was incubated at 37°C for 4 hours, then terminated with 2 volumes of cold methanol. The enzymatic reaction product was detected using UPLC / Q-TOF.

[0105] (2) UPLC / Q-TOF detection of enzymatic reaction products

[0106] Using Waters Acquity UPLC TM Separation was performed using an I-Class system and a Waters ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 μm). Mobile phase: water (0.1% formic acid): acetonitrile (0.1% formic acid); flow rate: 0.4 mL / min; elution program: 0 min, 10% acetonitrile, 3 min, 20% acetonitrile, 4 min, 25% acetonitrile, 8 min, 70% acetonitrile, 12 min, 95% acetonitrile, 15.2 min, 10% acetonitrile. TOF MS experiments were performed using a Xevo G2-S QTOF MS system in negative ion mode, and data were acquired using MS. Econtinuum. The ion source and desolvation temperatures were set at 100°C and 450°C, respectively. The desolvation gas flow rate was set at 900 L / h, the capillary voltage was set at 0.5 kV, and the injection cone voltage was set at 40 V. The collision energy was set at 20–40 eV for high-energy scans, and the data acquisition interval was set at 50–1500 Da. Data processing and analysis were performed using UNIFI software.

[0107] UPLC / Q-TOF analysis results are as follows Figure 4 As shown: After comparison with the control, the results showed that pET28-SUMO::UGT708U5 exhibited continuous two-step C-glycosylation catalytic activity for phloretin, and the products were phloretin-mono-C-glucoside (1a) and phloretin-di-C-glucoside (1b), indicating that the recombinant protein UGT708U5 is the key C-glycosyltransferase catalyzing the production of HSYA precursor compound phloretin-di-C-glucoside (1b).

[0108] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0109] Sequence 1-UGT708U5 (1416 bp)

[0110]

[0111] serial2-UGT708U5(471aa)

[0112] MPTATVRHHQPPHIALFSSAGMGHLTPLLRVASMLASRSCHVTLVTAEPAVSAAETAHITAFLAAYPAVNRLPFRTLPFTPPADPFFVQFEAINRSVHLLAPTLSSASPPVSAVFSDMASAAGVRRVADELRVPIYIVSTTSARFTALVASIPAALIGAGSSITATAEGASSAVFGIPGLDPFEISALPPALFVPDNLFTKTLAANALAMRKAKGILTNTFTTFEPETIAAVNG GKSLPDFPPFLPIGPLQPHKLELGDQQPLPWLDQQPPHSVAYVNFGSRTALSQPQIVELRNGLEESGRSFLWVFKSTVVDRDDTGSLGELLGGDPTKPSNGMVVKGWVNQEAILSHPAIGCFVSHCGWNSAVEAAAAAGPVVAWPLAGDQKVNAEVVAGAGLARWEKGWGWMGERLVKSGEIAEKVRMVMDDEKLREKARIGEEAIEEGGSSHKVLMEIEGLSKDNNVKP 。

Claims

1. Use of protein UGT708U5 in any of the following: P1) Application as glycosyltransferase; P2) Application of preparing or synthesizing phloretin-mono-C-glucoside; P3) Application of preparing or synthesizing phloretin-di-C-glucoside; P4) Application of preparing or synthesizing hydroxysafflor yellow A; in, The protein UGT708U5 is shown as any of the following: A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; A2) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein shown in Sequence 2 in the sequence listing; A3) A protein having more than 90% identity and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

2. The use according to claim 1, characterized in that The protein UGT708U5 is derived from safflower.

3. Use of biomaterials related to protein UGT708U5 in any of the following applications: P1) Application of preparing glycosyltransferase; P2) Application of preparing or synthesizing phloretin-mono-C-glucoside; P3) Application of preparing or synthesizing phloretin-di-C-glucoside; P4) Application of preparing or synthesizing hydroxysafflor yellow A; in, The biological material related to protein UGT708U5 is any one of the following: A1) a nucleic acid molecule encoding the protein according to claim 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule described in A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) A recombinant microorganism containing the recombinant vector described in A4).

4. The use according to claim 3, characterized in that A1) The nucleic acid molecule is as shown in B1) or B2) or B3) below: B1) the DNA molecule shown in Sequence 1 in the sequence listing; B2) The coding sequence is the DNA molecule shown in Sequence 1 in the sequence listing; B3) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in B1) or B2) and encodes the protein described in claim 1.

5. The protein UGT708U5 according to any one of claims 1 or 2.

6. The biological material related to protein UGT708U5 according to any one of claims 3 or 4.

7. A method for preparing phloretin-mono-C-glucoside and / or phloretin-di-C-glucoside, characterized in that: The method comprises the steps of using the protein according to claim 1, phloretin as a substrate, and UDP-Glc as a sugar donor to cause C-glycosylation.

8. The method for preparing the protein UGT708U5 according to any one of claims 1 or 2, characterized in that: The method comprises introducing a nucleic acid molecule encoding protein UGT708U5 into a recipient microorganism to obtain a recombinant microorganism expressing the protein of claim 1, and culturing the recombinant microorganism to express the protein of claim 1; The nucleic acid molecule is as shown in B1) or B2) or B3) below: B1) the DNA molecule shown in Sequence 1 in the sequence listing; B2) The coding sequence is the DNA molecule shown in Sequence 1 in the sequence listing; B3) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in B1) or B2) and encodes the protein described in claim 1.

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