A forsythia-derived glycosyltransferase fs20491 and its use in synthesis of forsythoside

Abstract

The application provides a forsythia suspense-derived glycosyltransferase Fs20491 and application thereof in synthesis of forsythoside, and relates to the technical field of enzyme engineering. The glycosyltransferase Fs20491 sequence is novel, can efficiently catalyze glycosylation of forsythoside to generate forsythoside, and is beneficial to subsequent separation and purification; the technical bottleneck that a forsythoside specific synthesis enzyme lacking of function verification in the prior art is broken, and a brand-new enzyme resource is provided for green biosynthesis of forsythoside.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a forsythia-derived glycosyltransferase Fs20491 and its application in the synthesis of forsythosides. Background Technology

[0002] Forsythia suspensa is a traditional and widely used Chinese medicinal herb. Forsythoside, its characteristic lignan glycoside active ingredient, possesses a variety of pharmacological activities, including anti-inflammatory, antioxidant, antibacterial, antiviral, hepatoprotective, and immunomodulatory effects. It has significant application value in new drug development, functional foods, and the modernization of traditional Chinese medicine, and market demand continues to grow. Currently, forsythoside is mainly prepared through two methods: plant extraction and chemical synthesis. Plant extraction is easily affected by the environment and growth cycle, resulting in poor raw material stability and significant resource consumption. Chemical synthesis involves cumbersome processes, stringent reaction conditions, and significant pollution, making large-scale production difficult. Glycosyltransferases can specifically catalyze glycosylation reactions under mild conditions. Exploring these functional enzymes through multi-omics and metabolomics has become the mainstream direction for the green preparation of natural products.

[0003] Existing patents and literature have reported the identification of glycosyltransferases with forsythoside synthesis activity through in vitro enzyme activity verification and in vivo functional complementation experiments. These glycosyltransferases can catalyze the conversion of forsythoside from forsytholipin, indicating that there are readily available enzyme resources for the glycosylation step in forsythoside biosynthesis. However, the number of publicly disclosed glycosyltransferases related to forsythoside synthesis remains limited. Enzyme resources from different sources, with different sequence families and tissue expression patterns still need further exploration, especially enzymes with high substrate specificity, low byproducts, and good heterologous soluble expression capabilities. Research gaps also exist regarding related functional gene resources. Conducting research on the discovery and functional analysis of this enzyme can provide novel enzyme resources and technical solutions for the in vitro enzymatic synthesis of forsythoside, which is of great significance for promoting its green industrial production. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a Forsythia-derived glycosyltransferase Fs20491 and its application in the synthesis of forsythosides. Through a combination of transcriptome sequencing and analysis of differences in forsythoside content (metabolite analysis) in different Forsythia tissues, heterologous expression, and in vitro enzyme activity verification, its biological function of specifically catalyzing the conversion of forsythosides from forsythosides was successfully identified and confirmed. Based on existing reported glycosyltransferases for forsythoside synthesis, this invention provides a novel enzyme resource with unique sequence and expression characteristics, enriching the gene element library for the green biosynthesis of forsythosides.

[0005] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a glycosyltransferase Fs20491 derived from Forsythia suspensa, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1 MEETKELVFIPGPGLGHLVSAVETGRKIISRHQHLSVTYLLMDTIPNDKHLYNYTQLLHSAATSRLRFTNLHRAQPDYSEELKSKNPAILITEFIDSHKQLVREAVLEIINFESSQVA GIIVDIFCVNMIDVADEFKIPSYVFFPSSAGFLAVMLQVQVITDEFKRDITEYKDTDKELLIPGFFNQVPAKVLPTGMLNKHGTLDLDVATARRFRESKGIIVNTFMELETNAIKSML SDGKIPPVFPVGPVINLKQKPDEDEGIRRWLDNQPTSSVIFLCFGSWGSFKKEQVKEIAVALEHSGYRFLWSLRRPPMDVDFSSDYEYREEVVPEGFLERTSSVGKVIGWAPQLAVLS HPAVGGFISHCGWNSILESLWFGVPIAAWPMYAEQHFNAFEMVVEFGMAVDINMDSKIIVTCEEIERGIRQLMNGNEIRMKVKEMKEKSHITLMEHGSSYDFLGRLIDGIMHNSISSGT The present invention also provides a polynucleotide encoding the above-mentioned glycosyltransferase Fs20491, the polynucleotide sequence of which is shown in SEQ ID NO.2.

[0007] SEQ ID NO.2 The present invention also provides a recombinant expression vector comprising the above-mentioned polynucleotide sequence.

[0008] The present invention also provides a host cell comprising the above-mentioned polynucleotide or recombinant expression vector.

[0009] Furthermore, the host cell is a prokaryotic cell or a eukaryotic cell.

[0010] The present invention also provides a recombinant engineered bacterium containing the above-mentioned polynucleotides or recombinant expression vector.

[0011] This invention also provides the application of the above-mentioned glycosyltransferase Fs20491 in the catalytic synthesis of forsythoside.

[0012] The present invention also provides a method for preparing glycosyltransferase Fs20491, wherein the above-mentioned recombinant engineered bacteria are cultured, expression is induced, the bacterial cells are collected and broken, and the glycosyltransferase is purified from the broken material.

[0013] Furthermore, the optimal induction conditions for Fs20491 glycosyltransferase expression were: culturing the recombinant strain at 37 °C until OD2000 was achieved. 600 When the concentration of the sample is 0.8, add IPTG to a final concentration of 0.4 mM and induce culture at 16°C for 20 hours.

[0014] The present invention also provides an enzymatic method for synthesizing forsythoside, using forsythoside as a substrate and the above-mentioned glycosyltransferase Fs20491 for enzymatic catalysis to generate forsythoside.

[0015] The present invention has the following technical effects: (1) A novel glycosyltransferase Fs20491 is provided. The glycosyltransferase Fs20491 has excellent substrate specificity and can efficiently catalyze the glycosylation of forsythosides to generate forsythoside.

[0016] (2) The in vitro enzyme-catalyzed synthesis system based on this enzyme can achieve efficient conversion of forsythoside under mild reaction conditions, effectively avoiding the problems of unstable raw materials, large resource loss and complicated chemical synthesis process and serious pollution in plant extraction method. (3) The recombinant engineered strain and protein purification scheme provided by this invention have enabled the soluble heterologous expression and efficient preparation of the Fs20491 enzyme, laying a solid foundation for the large-scale enzymatic production of forsythoside. This invention provides key gene elements and technical support for the green and sustainable industrial application of forsythoside.

[0017] (4) The screening strategy of combining transcriptomics and metabolomics analysis breaks through the limitations of single-mic screening and improves the efficiency and accuracy of target enzyme discovery. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 HPLC analysis of forsythoside content in different tissues of Forsythia suspensa; Figure 2 Three-dimensional principal component analysis of transcriptome samples from roots, stems, leaves, and fruits of Forsythia suspensa was performed. The three-dimensional coordinate axes PC1, PC2, and PC3 explained 40.5%, 33.3%, and 17.7% of the gene expression variations, respectively. The orange, yellow, purple, and blue scatter points correspond to the root, stem, leaf, and fruit tissue samples, respectively. Figure 3 Phylogenetic analysis results of glycosyltransferase genes in roots, stems, leaves, and fruits of Forsythia suspensa. In the analysis, the color gradient on the right represents gene expression abundance, with red indicating high expression and blue indicating low expression. The inner ring represents the gene phylogenetic tree, and the outer ring bands, from the inside out, represent roots, stems, leaves, and fruits, respectively. The target gene Fs20491 and GWHTERDT030408 ​​are marked with triangles, which are reported control genes. Figure 4 SDS-PAGE results of crude enzyme and purified enzyme of Fs20491; Figure 5 Standard curve of forsythoside; Figure 6 A is the HPLC chromatogram of the in vitro catalytic product of Fs20491; B is the enzyme catalytic activity analysis. Figure 7 Mass spectrometry analysis of the products of in vitro enzyme-catalyzed reaction of Fs20491, where the horizontal axis represents the mass-to-charge ratio (m / z) and the vertical axis represents the ion count (Count). Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0021] Example 1: Obtaining Forsythia suspensa glycosyltransferase Fs20491 Using wild Forsythia suspensa from the Taihang Mountains in Huixian County, Xinxiang City, Henan Province as material, and combining transcriptome sequencing analysis and metabolic differences in forsythoside content in different Forsythia suspensa tissues, the glycosyltransferase encoding gene Fs20491 was screened and further sequence optimized. Specific primers were designed using SnapGene software to amplify the target gene. The vector was linearized by restriction enzyme digestion with Nde I (TaKara, Code No: 1621) and Xho I (TaKara, Code No: 1635), and the gene and vector were recombined through seamless cloning, successfully constructing an engineered strain.

[0022] Main reagents: Primes STAR HS high-fidelity enzyme, Nde I, Xho I restriction endonuclease, 2×Seamless Cloning Mix, Plant Genome Extraction Kit (Cat#B518231), Plasmid Mini-Prep Kit (Lot: 1N702249002), DNA Gel Recovery Kit (B610353-0200), IPTG, Kanamycin, Coomassie Brilliant Blue G-250, Methanol, Glacial Acetic Acid, etc.

[0023] Primer sequences (10 pmol / µL): Primer 1: 5'-GTGCCGCGCGGCAGCCATATGGAAGAAACCAAAGAGCTG-3'; Primer 2: 5'-CAGTGGGTGGTGGTGGTGGTGCTCGAGTCATGTACCGGAGGAGATCGAG-3'.

[0024] The Fs20491 gene sequence is shown below: The amino acid sequence is shown below: MEETKELVFIPGPGLGHLVSAVETGRKIISRHQHLSVTYLLMDTIPNDKHLYNYTQLLHSAATSRLRFTNLHRAQPDYSEELKSKNPAILITEFIDSHKQLVREAVLEIINFESSQVA GIIVDIFCVNMIDVADEFKIPSYVFFPSSAGFLAVMLQVQVITDEFKRDITEYKDTDKELLIPGFFNQVPAKVLPTGMLNKHGTLDLDVATARRFRESKGIIVNTFMELETNAIKSML SDGKIPPVFPVGPVINLKQKPDEDEGIRRWLDNQPTSSVIFLCFGSWGSFKKEQVKEIAVALEHSGYRFLWSLRRPPMDVDFSSDYEYREEVVPEGFLERTSSVGKVIGWAPQLAVLS HPAVGGFISHCGWNSILESLWFGVPIAAWPMYAEQHFNAFEMVVEFGMAVDINMDSKIIVTCEEIERGIRQLMNGNEIRMKVKEMKEKSHITLMEHGSSYDFLGRLIDGIMHNSISSGT Prepare a 0.2 M, pH 7.0 phosphate buffer solution: Weigh 4.35 g of disodium hydrogen phosphate dodecahydrate and 1.22 g of sodium dihydrogen phosphate dihydrate, dissolve them in ultrapure water, adjust the pH to 7.0, and bring the volume to 100 mL.

[0025] The specific experimental steps include: 1.1 Forsythia sample collection 1) Fresh wild Forsythia samples were collected in 2025 from the Taihang Mountains (407.8 m above sea level, 113°75′ E, 35°631′ N) in Huixian County, Xinxiang City, Henan Province; 2) Use sterile tools for sampling, seal in bags, transport with dry ice, and freeze in the laboratory at -80°C.

[0026] 1.2 Detection of forsythoside content in different tissues of Forsythia suspensa 1) Take the roots, stems, leaves and fruits of Forsythia, wash them with sterile water, dry them at 50 ℃ to constant weight, pulverize them and pass them through a 60 mesh sieve; 2) Accurately weigh 1.5 g of powder into a 100 mL Erlenmeyer flask, add 50 mL of methanol and weigh; 3) Extract by ultrasonication at 300 W and 40 kHz for 30 min, then add methanol to the original weight after cooling; 4) Centrifuge the extract at 4000 rpm for 10 min, and filter the supernatant through a 0.45 μm filter membrane to obtain the test solution; 5) HPLC detection: A Thermo column was used, with acetonitrile-0.4% glacial acetic acid aqueous solution as the mobile phase for gradient elution. The elution program was 16:84 (20 min), 35:65 (10 min), 90:10 (10 min), and 16:84 (10 min). Methanol was used as the diluent. The sample concentration was 0.1 mg / mL, the injection volume was 10 μL, and the column temperature was 35 ℃.

[0027] Experimental results are as follows Figure 1 As shown in the figure, combined with the quantitative analysis of the corresponding column data, the forsythoside content in the roots and leaves of Forsythia suspensa was close to 900 μg / mL, with no significant difference between the two, and was significantly higher than that in the stems and fruits. The forsythoside content in the stems was approximately 230-250 μg / mL, while that in the fruits was only about 200 μg / mL, the lowest among the four tissue types. This result confirms that forsythoside has significant tissue-specific accumulation characteristics, with roots and leaves being the main organs for forsythoside accumulation, and the accumulation levels in the stems and fruits being significantly lower. This result clarifies the spatial distribution pattern of forsythoside in wild Forsythia suspensa plants, providing reliable histological experimental evidence for screening genes encoding highly active glycosyltransferases and elucidating the regulatory mechanism of forsythoside biosynthesis.

[0028] 1.3 Transcriptome Sequencing Analysis of Forsythia Tissue 1) Set up 3 groups of biological samples and commissioned Paisenno Biotechnology to complete the transcriptome sequencing; 2) The sample was ground in liquid nitrogen to extract total RNA, and its purity, concentration, and integrity were tested. 3) Qualified RNA is enriched with mRNA, fragmented, cDNA synthesized, adapter ligated, and PCR amplified to construct a library for high-throughput paired-end sequencing; 4) Fastp completes data quality control filtering, and HISAT2 aligns the valid reads to the Forsythia reference genome; 5) Quantitative gene expression was performed using HTSeq combined with FPKM method to analyze sample expression characteristics; 6) Deseq2 was used to screen differentially expressed genes based on the criteria of |log2FoldChange|>1 and (P-value<0.05), and clustering, trend, protein-protein interaction, and alternative splicing analyses were performed. 7) Use clusterProfiler to perform enrichment analysis on GO, KEGG, and GSEA; 8) Conduct transcription factor prediction, transcript splicing, SNP and InDel detection and annotation, and analyze gene expression patterns in conjunction with mapping.

[0029] Experimental results are as follows Figures 2-3 As shown, from Figure 2 The results show that the spatial distribution of biological replicates from the same tissue is highly clustered, while samples from different tissues are separated from each other, indicating that the samples in each group have good repeatability. Furthermore, there are significant differences in gene expression patterns among the roots, stems, leaves, and fruits of Forsythia suspensa, which can distinguish the transcriptional expression characteristics of different tissues. This verifies the rationality of the sample grouping setting and provides a reliable basis for subsequent differential gene screening and functional analysis.

[0030] Figure 3 Phylogenetic analysis revealed that all glycosyltransferase genes exhibited differentiated expression patterns in different tissues of Forsythia suspensa, demonstrating significant tissue expression bias. The target gene Fs20491 showed higher expression abundance in root and leaf tissues, and lower expression levels in stem and fruit tissues. This expression pattern closely matched the tissue distribution characteristics of forsythosides determined by HPLC previously. Furthermore, Fs20491 was significantly distant from the previously reported gene GWHTERDT030408 ​​on the phylogenetic clustering tree, indicating weak correlation in their expression patterns and significant differences in their tissue expression profiles. This confirms that Fs20491 is a novel glycosyltransferase encoding gene with unique sequence and expression characteristics, providing important transcriptomics evidence for subsequent in vitro functional identification and research on its catalytic synthesis of forsythosides.

[0031] 1.4 PCR amplification of the Fs20491 gene 1) Forsythia suspensa genomic DNA was extracted using the Sangon Biotech Plant Genomics Kit as a template; 2) 25 μL PCR system: 1 μL genomic DNA, 1 μL each of primer 1 and primer 2 (10 pmol / µL), 12.5 μL Primers STAR HS enzyme, 9.5 μL ddH2O; Mg2+ in the system. 2+ Concentration 2 mM, dNTP concentration 0.4 mM; 3) PCR program: 94 ℃ pre-denaturation for 5 min; 30 cycles (94 ℃ for 30 s, 52 ℃ for 5 s, 72 ℃ for 1 min and 40 s); 72 ℃ final extension for 10 min; store at 16 ℃. 4) After electrophoresis, the product was purified using a DNA gel extraction kit and stored at -20 ℃.

[0032] 1.5 pET28a (+) vector linearization 1) DH5α strain containing pET28a (+) was cultured in resistant LB liquid medium at 37 ℃ for 12-16 h, and plasmids were extracted. 2) 50 μL enzyme digestion system: ≤1 μg plasmid, 1 μL each of Nde I and Xho I, 5 μL of 10× buffer, and ddH2O to make up the difference; 3) Digest the enzyme at 37 ℃ for 30 min, recover the linear vector by gel electrophoresis, and store at -20 ℃.

[0033] 1.6 Preparation of competent cells 1) Activate single colonies of E. coli DH5α and E. coli BL21(DE3), inoculate into 50 mL LB liquid medium, and incubate until OD500. 600 ≈0.4; 2) Take 40 mL of bacterial culture, incubate on ice for 30 min, centrifuge at 4 ℃ and 8000 rpm for 5 min, and collect the bacterial cells; 3) Resuspend the bacterial cells in 10 mL of pre-cooled 0.1 M CaCl2, and wash once by centrifugation under the same conditions; 4) Resuspend the bacterial cells in a mixture of 2 mL 0.1 M CaCl2 and 180 μL DMSO, dispense 100 μL / vial, and store at -80℃.

[0034] 1.7 Seamless Cloning, Linking, and Conversion 1) 10 μL reaction system: 50-100 ng / μL target gene, linear vector of the same concentration, 5 μL of 2×SeamlessCloning Mix, and ddH2O to make up the difference; 2) Mix well and centrifuge. React at 50 °C for 15 min. Immediately after the reaction, place in an ice bath.

[0035] 3) Add the ligation product to DH5α competent cells, incubate in ice water for 20 min, heat shock at 42 ℃ for 90 s, and then incubate in ice water again for 2 min; 4) Add 500 μL of LB liquid medium and incubate at 37 ℃ and 220 rpm for 1 h; 5) Centrifuge at 4000 rpm for 1 min, resuspend 200 μL of bacterial culture, spread on kanamycin resistance plates, incubate at 37 ℃ upright for 30 min, then invert overnight.

[0036] 1.8 Identification of positive clones 1) Pick a single colony, place it in 60 μL of sterile water, boil in a water bath for 10 min, cool and centrifuge, and use the supernatant as a template for colony PCR; 2) Positive strains were screened by electrophoresis of PCR products, and plasmids were extracted by shaking and then verified by double enzyme digestion.

[0037] 1.9 Recombinant Protein Expression and Purification 1) After verification, the plasmid was transformed into BL21(DE3) competent cells, and then a single colony was picked and inoculated into 100 mL of resistant LB medium and cultured at 37 ℃ and 220 rpm until OD. 600 ≈0.8, prepare seed solution; 2) Transfer 1% inoculum to 200 mL of fermentation medium and culture under the same conditions until OD. 600 ≈0.8, add 0.4mM IPTG, induce at 16 ℃ and 220 rpm for 20 h; 3) Centrifuge at 8000 rpm for 25 min to collect the bacterial cells, and resuspend in 200 mM, pH 7.0 Tris-HCl solution; 4) Ultrasonic disruption: 3 seconds of operation followed by 3 seconds of pause, for a total of 90 cycles; centrifuge at 4℃ and 8000 rpm for 40 minutes, and collect the crude enzyme solution; 5) The crude enzyme solution was purified by Ni column chromatography, elution, dialysis, and ultrafiltration; protein concentration was determined by Coomassie Brilliant Blue G-250 method, and purity was detected by SDS-PAGE. Experimental results are as follows Figure 4 As shown, Figure 4 The image shows the SDS-PAGE results. The left lane displays the bands of the crude enzyme extract, indicating the presence of numerous and widely distributed bands, suggesting the presence of various proteins of different molecular weights. The right lane shows the purified enzyme, revealing a single, clear, and distinct blue target protein band. All other protein bands present in the crude enzyme lane have disappeared. After purification, the crude enzyme solution, which initially contained a large number of contaminating proteins, was successfully purified, ultimately yielding a single target protein of electrophoretic purity.

[0038] 1.10 In vitro enzyme catalysis experiment 1) Set up a control group and an experimental group, with two replicates in each group, for a total volume of 200 μL; 2) Control group: 1 mM UDPG, 0.5 mM forsythoside A, 0.2 M pH 7.0 phosphate buffer, and 40 μg of boil-inactivated Fs20491 were added; 3) Experimental group: The substrate was the same as the control group, but 40 μg of Fs20491 purified enzyme was added, and the volume was adjusted with buffer. 4) Incubate at 40 ℃ and 600 rpm, and take samples at 0 h, 24 h, and 48 h respectively; 5) Boil the sample for 10 min to terminate the reaction, centrifuge at 12000 rpm for 10 min, and take the supernatant for HPLC detection. The chromatographic conditions are the same as in step 2.

[0039] Example 2: In vitro enzyme catalysis experiment of glycosyltransferase Fs20491 This experiment included a control group and an experimental group, with two replicates in each group, for a total reaction volume of 200 μL. The control group consisted of 1 mM UDPG and 0.5 mM forsythoside A, diluted to volume with 0.2 M phosphate buffer (pH = 7.0), without any enzyme solution. The experimental group contained the same substrate, plus 40 μg of purified Fs20491 enzyme, and was diluted to 200 μL with buffer. Both groups were incubated at 40℃ and 600 rpm, and samples were taken at 0 h, 24 h, and 48 h. Samples were boiled for 10 min, centrifuged at 12000 rpm for 10 min, and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Chromatographic conditions: A C18 column was used with acetonitrile-0.4% glacial acetic acid aqueous solution as the mobile phase for gradient elution. The elution programs were: 16:84 (v / v) for 20 min, 35:65 for 10 min, 90:10 for 10 min, and 16:84 for 10 min. Methanol was used as the sample diluent. The sample concentration was 0.1 mg / mL, the injection volume was 10 μL, and the column temperature was 35 ℃. After detection, the experimental data were collected and analyzed. The forsythoside standard curve was used as a reference. Figure 5 The content of forsythoside produced in the reaction solution was quantitatively calculated.

[0040] A standard curve was established using forsythoside standards. Figure 5 The amount of forsythoside produced in the reaction solution was absolutely quantified, and the concentration and molar conversion rate of forsythoside in the reaction solution at different time points were calculated. The molar conversion rate was calculated based on the initial molar amount of the substrate forsythoside, using the following formula: Molar conversion rate (%) = (1) In vitro enzymatic reaction analysis showed that no forsythoside was detected in the control group of Fs20491, while the conversion rate in the experimental group reached 22.49% after 12 h and increased to 30.30% after 24 h. This indicates that the forsythoside conversion rate gradually increased with increasing reaction time (Figure 6). These results confirm, from an enzymatic reaction perspective, that the glycosyltransferase encoded by Fs20491 possesses excellent catalytic activity and can effectively catalyze the conversion of substrates into forsythoside.

[0041] like Figure 7 As shown, the spectrum exhibits a highly intense characteristic main peak at m / z = 557.17, which corresponds to the sodium ion adduct [M+Na] of forsythoside according to theory. + The theoretical value (m / z approximately 557.54) is in high agreement, indicating that this main peak corresponds to the [M+Na] of forsythoside. +The adduct ion, combined with its molecular weight (M = 534.55) and chromatographic retention behavior, further confirmed that the target product was forsythoside. This result directly verifies that the Fs20491 glycosyltransferase screened in this invention possesses specific catalytic activity, capable of mediating glycosylation modification of the substrate and efficiently catalyzing the synthesis of the target product forsythoside. This directly confirms from an in vitro biochemical perspective that Fs20491 is a key functional gene regulating the biosynthesis of forsythoside.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A glycosyltransferase Fs20491 derived from Forsythia suspensa, characterized in that, The amino acid sequence of the glycosyltransferase Fs20491 is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that, The glycosyltransferase Fs20491 as described in claim 1 is encoded.

3. A recombinant expression vector, characterized in that, The recombinant vector comprises the polynucleotide sequence as described in claim 2.

4. A host cell, characterized in that, It contains the polynucleotide as described in claim 2 or the recombinant expression vector as described in claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

6. A recombinant engineered bacterium, characterized in that, It contains the polynucleotide as described in claim 2 or the recombinant expression vector as described in claim 3.

7. The application of the glycosyltransferase Fs20491 as described in claim 1 in the catalytic synthesis of forsythoside.

8. A method for preparing a glycosyltransferase Fs20491, characterized in that, The recombinant engineered bacteria as described in claim 6 are cultured, induced to express, the bacterial cells are collected and broken up, and glycosyltransferase is purified from the broken up material.

9. A method for the enzymatic synthesis of forsythoside, characterized in that, Using forsythoside as a substrate, forsythoside was generated by enzymatic catalysis with the glycosyltransferase Fs20491 as described in claim 1.

Images