Epimedium sagittatum flavonol glycosyltransferase EsGT_F and application thereof

Abstract

This invention discloses a flavonol glycosyltransferase EsGT_F from *Epimedium sagittatum* and its applications. The EsGT_F gene provided by this invention is 1398 bp in length, encoding 466 amino acids. After cloning the *Epimedium sagittatum* flavonol glycosyltransferase gene EsGT_F, a prokaryotic expression system was constructed. The expressed protein exhibits flavonol glycosyltransferase activity, catalyzing the conversion of cymosin I to icariin. The *Epimedium sagittatum* flavonol glycosyltransferase and its encoding gene provided by this invention offer important gene elements and corresponding technical methods for the microbial cell factory production of the active ingredients of *Epimedium*, and have significant application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant gene and metabolic engineering technology, specifically relating to a flavonol glycosyltransferase derived from Epimedium sagittatum and its application. Background Technology

[0002] Epimedium is one of the most commonly used bulk Chinese medicinal herbs. Its origin belongs to the genus Epimedium in the family Berberidaceae. Epimedium Besides the five species listed in the Chinese Pharmacopoeia, more than ten other dried leaves of Epimedium are used in local and folk medicine. Epimedium not only possesses traditional effects such as tonifying kidney yang, strengthening muscles and bones, and dispelling wind and dampness, but has also been proven to have a wide range of pharmacological activities, including anti-aging, immune regulation, cardiovascular protection, anti-inflammation, and anti-tumor effects, making it a research hotspot in modern medicine. Its core active ingredients are flavonol glycosides (such as icariin, icariin A, B, and C), which are currently mainly extracted from plants. However, production efficiency faces industrial bottlenecks due to the depletion of wild resources and high cultivation costs. Microbial cell factory technology can achieve heterologous synthesis of active ingredients, and due to its advantages of resource independence, process controllability, and environmental friendliness, it is considered a key strategy to overcome industrial bottlenecks. Against this backdrop, this study newly identified a glycosyltransferase gene derived from Epimedium sagittatum. EsGT_F After cloning, expression, and enzyme activity verification, it was confirmed that it is a flavonol 7-O-glycosyltransferase, which can catalyze the production of icariin from baicalin I using UDPG as a donor. This enzyme can serve as an efficient tool for synthesizing flavonol glycosides unique to icariin, and is of great significance for promoting the biosynthesis of such components. Summary of the Invention

[0003] The purpose of this invention is to provide an icariin glycosyltransferase EsGT_F and its application in the synthesis of icariin. The glycosyltransferase EsGT_F can catalyze the production of icariin from cytosine I using UDPG as a glycosyl donor.

[0004] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0005] The present invention clones the glycosyltransferase gene according to the following method. EsGT_F And perform in vitro protein expression: EsGT_F Primers were designed for the coding region, and the glycosyltransferase gene was obtained by PCR amplification using Epimedium sagittatum cDNA as a template. EsGT_FThe coding sequence is shown in SEQ ID No. 1. The prokaryotic expression recombinant plasmid EsGT_F-pMAL-c2x was constructed, transformed into Rosetta (DE3) competent E. coli cells, and the expression of the target protein was induced by isopropyl-β-D-thiogalactoside (IPTG). The expression of the target protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0006] Application of glycosyltransferase EsGT_F in the in vitro synthesis of icariin, a unique flavonol glycoside active ingredient of Epimedium: In a specific embodiment of the present invention, the substrate cyproteroside I and the glycosyl donor UDPG are provided to the in vitro recombinant protein of glycosyltransferase EsGT_F. Combined with HPLC results, it is determined that glycosyltransferase EsGT_F can catalyze the production of cyproteroside I into icariin. Attached Figure Description

[0007] Figure 1 SDS-PAGE electrophoresis image of EsGT_F protein. From left to right: protein marker, empty vector control, and crude EsGT_F enzyme solution.

[0008] Figure 2 HPLC results of the in vitro catalytic activity analysis of EsGT_F. The peak chromatograms are as follows: icariin standard (elevation time 37.462 min), cymosin I standard (elevation time 49.961 min), and the EsGT_F enzyme-catalyzed reaction product (elevation time 37.462 min). Detailed Implementation

[0009] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention. 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 invention pertains.

[0010] Example 1: Epimedium sagittatum flavonol glycosyltransferase gene EsGT_F Cloning

[0011] Based on the metabolomics and transcriptomics data of various tissues of Epimedium sagittatum collected by the inventors' team, one candidate gene for flavonol glycosyltransferase was identified through analysis and screening. EsGT_FPrimers for full-length cDNA amplification were designed based on the reference genome sequence: EsGT_F_MBP_F: 5'-ATGGGAGAAGTACATCAAACACCCC-3', EsGT_F_MBP_R: 5'-TTACTTTTTCCACTTAAGCGCCACCT-3'. Using Epimedium sagittatum cDNA as a template, PCR amplification was performed using the above primers. The amplified products were detected by 1.2% agarose gel electrophoresis, and the product size was consistent with the reference sequence.

[0012] Example 2: Epimedium sagittatum flavonol glycosyltransferase gene EsGT_F Construction of recombinant vectors and prokaryotic expression

[0013] The prokaryotic expression vector pMAL-c2x was double-digested with restriction endonucleases BamHI and SalI, and the digestion products were recovered. The PCR amplification product from Example 1 was cloned into the prokaryotic expression vector pMAL-c2x using homologous recombination to obtain the recombinant plasmid EsGT_F-pMAL-c2x. The recombinant plasmid was transformed into *E. coli* DH5α competent cells, and single clones that tested positive by colony PCR were sent to a sequencing company for sequencing. The results showed that... EsGT_F The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2.

[0014] The EsGT_F-pMAL-c2x plasmid was extracted and transformed into the prokaryotic expression strain Rosetta (DE3). 2 mL of LB broth containing Amp resistance was added to a 5 mL centrifuge tube, and the mixture was incubated overnight at 37°C and 200 rpm with shaking. The plasmid was then transferred to an Erlenmeyer flask at a 1:100 volume ratio and incubated at 37°C and 200 rpm with shaking until OD (October Expiratory Time). 600= 0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM for protein induction, and induce at 18℃ and 150 rpm for 18-24 h. After induction, centrifuge the bacterial culture at 4℃ and 5000 rpm for 10 min, discard the supernatant, and collect the precipitate. Resuspend the bacterial cells in pre-cooled lysis buffer (100 mM K2HPO4, 100 mM KH2PO4, 1 mM DTT, 5% PMSF, pH 8.0), and sonicate (sonication power 15%, 1 s sonication, 1 s pause, total sonication time 20 min). Centrifuge at 4℃ and 12000 rpm for 15 min, and collect the supernatant in a new centrifuge tube, which is the crude enzyme solution. Take 100 μL of the supernatant and add protein loading buffer. After boiling in water for 5 minutes, centrifuge at 12000 rpm for 2 minutes, and then detect the expression of the target protein in the supernatant by SDS-PAGE. Figure 1 ).

[0015] Example 3: In vitro catalytic reaction of recombinant flavonol glycosyltransferase

[0016] The catalytic activity of the EsGT_F protein from Example 2 was analyzed using tylosin I as the substrate. The reaction system consisted of a total volume of 500 μL, containing 0.1 mM tylosin I, 0.1 mM UDPG, 0.2 mM S-adenosylmethionine (SAM), and 470 μL of crude EsGT_F enzyme solution. The reaction was carried out at 30°C for 6 h, and the reaction was terminated by adding an equal volume of methanol. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected in a new centrifuge tube and dried. The solution was reconstituted with 100 μL of methanol, filtered through a 0.22 μm organic filter into a liquid chromatography vial, and analyzed by LC-MS. The results showed that the peak time of the cymopogonin I standard was 49.961 min; the peak time of the icariin standard was 37.462 min; and the peak time of the EsGT_F protein-catalyzed reaction product was also 37.462 min. The peak time of the product was consistent with that of the icariin standard, indicating that the 7-O glycosyltransferase EsGT_F of *Epimedium sagittatum* has the activity to catalyze the conversion of cymopogonin I to icariin. Figure 2 ).

[0017] The LC-MS analysis conditions are as follows:

[0018] The reaction products were analyzed by UPLC-QTOF-MS using a triple quadrupole mass spectrometer. An Agilent ZORBAX EP-C18 column (1.8 μm, 2.1 × 50 mm) was used, with an injection volume of 1 μL, column temperature of 30℃, and flow rate of 0.4 mL / min. The aqueous phase was 0.1% formic acid, and the organic phase was 100% acetonitrile. The isogradient elution program was as follows: 0–5 min, 90% acetonitrile. The mass spectrometer detector parameters were set as follows: electrospray ionization (ESI) source, positive ion mode; nebulizer temperature, 350℃; fragmentation voltage, 175 V; gas flow rate, 8 L / min; electron spray voltage, 3.5 kV; nebulizer pressure, 35 psi; scan range, 300–800 m / z.

Claims

1. The gene encoding the glycosyltransferase protein EsGT_F, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

2. A glycosyltransferase protein EsGT_F, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An expression vector or recombinant bacteria containing the encoding gene of claim 1.

4. The use of the gene of claim 1 or the glycosyltransferase protein EsGT_F of claim 2 in the preparation of flavonol glycosides.

5. The application according to claim 4, characterized in that, The glycosyltransferase protein EsGT_F catalyzes the 7-O glycosylation reaction of cymosin I and UDPG to generate icariin.

6. A method for synthesizing icariin, the main flavonol glycoside active ingredient of Epimedium, in vitro using 7-O-glycosyltransferase EsGT_F, characterized in that: The 7-O glycosylation reaction of cytosine I was catalyzed by the glycosyltransferase protein EsGT_F as described in claim 1.

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