A glycosyltransferase ugt73dy2, its coding gene and application

Abstract

This invention belongs to the field of natural product biosynthesis technology, specifically relating to a glycosyltransferase UGT73DY2, its encoding gene, and its applications. The diosgenin-3-O-α-L-pyranorhamnetose-(1→4)-[α-L-pyranorhamnetose]-(1→2)-β-D-glucoside and pennosapogenin-3-O-α-L-pyranorhamnetose-(1→4)-[α-L-pyranorhamnetose]-(1→2)-β-D-glucoside synthesized by the glycosyltransferase UGT73DY2 of this invention are key intermediate compounds in the biosynthesis of Paris polyphylla saponins II or VII. These compounds are synthesized in vitro under the catalytic action of glycosyltransferase UGT73DY2, laying the foundation for the industrial production of Paris polyphylla saponins II and VII. Meanwhile, a new Ophiopogon japonicus saponin compound can also be synthesized under the action of glycosyltransferase UGT73DY2, providing a new direction for further research on the application of glycosyltransferase UGT73DY2.

Description

Technical Field

[0001] This invention belongs to the field of natural product biosynthesis technology, specifically relating to a glycosyltransferase UGT73DY2, its encoding gene, and its applications. Background Technology

[0002] Paris polyphylla, also known as seven-leafed Paris, is a plant belonging to the genus Paris in the family Melanthiaceae. my country is one of the distribution centers of this genus, with approximately 17 species and 10 varieties, distributed in most provinces. The diversity center is located in the Yunnan-Guizhou Plateau and the Qionglai Mountains of China. *Paris polyphylla* Smith var. *Yunnanensis* and *Paris polyphylla* Smith var. *Chinensis* (French.) Hara are listed in the Chinese Pharmacopoeia. Their rhizomes are mainly used medicinally and are included in the *Pharmacopoeia of the People's Republic of China*. Their main effects are clearing heat and detoxifying, reducing swelling and relieving pain, cooling the liver and calming convulsions. They are used for boils, carbuncles, snake and insect bites, traumatic injuries, and infantile convulsions.

[0003] Paris saponins are the main active components of Paris polyphylla, possessing a wide range of pharmacological activities including antitumor, anti-inflammatory and analgesic, antibacterial, hemostatic, antioxidant, and immunomodulatory effects. However, Paris saponins have complex structures, making chemical synthesis difficult, and their formation mechanism within the plant is not yet fully understood. Heterologous biosynthesis is one of the most reliable methods to provide a stable source of these active substances.

[0004] Glycosyltransferases participate in the synthesis, modification, and transport of plant secondary metabolites. Glycosylation alters the water solubility, stability, and transport characteristics of compounds within plants, both cellularly and plant-wise. Furthermore, glycosyltransferases regulate hormone homeostasis, influencing plant growth and development. Glycosylation is a crucial step in the biosynthesis of Paris polyphylla saponins, requiring glycosyltransferase catalysis. However, the biosynthetic pathways of the main active components of Paris polyphylla, Paris polyphylla saponin II and Paris polyphylla saponin VII, remain unsolved. Clarifying the biosynthetic pathways of Paris polyphylla saponin II and Paris polyphylla saponin VII holds promise for achieving heterologous biosynthesis and resolving the increasingly difficult problem of obtaining Paris polyphylla saponins. Summary of the Invention

[0005] The purpose of this invention is to provide a key glycosyltransferase in the biosynthetic pathway of Paris polyphylla saponin II and Paris polyphylla saponin VII, which is used to catalyze the synthesis of key intermediate compounds in the biosynthetic process of Paris polyphylla saponin II or VII, and can also catalyze the synthesis of other steroidal saponin compounds such as Ophiopogon japonicus saponin compounds, thereby achieving heterologous synthesis of Paris polyphylla saponin compounds and Ophiopogon japonicus saponin compounds and other steroidal saponin compounds.

[0006] This invention provides a glycosyltransferase UGT73DY2, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0007] The present invention also provides a gene encoding the aforementioned glycosyltransferase UGT73DY2, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0008] The present invention also provides recombinant vectors, expression cassettes or recombinant bacteria containing the aforementioned genes.

[0009] Furthermore, the recombinant vector is a plasmid that links a gene with a nucleotide sequence as shown in SEQ ID NO. 1; the plasmid includes pCold-TF, pESC-URA, or pEAQ-HT.

[0010] Furthermore, the recombinant bacteria include recombinant Escherichia coli, recombinant Saccharomyces cerevisiae, or recombinant Agrobacterium tumefaciens;

[0011] The recombinant Saccharomyces cerevisiae includes Saccharomyces cerevisiae that integrate the aforementioned genes, as well as the DHCR7 gene, DHCR24 gene, RHM gene, CYP90B27 gene, CYP90G4 gene, CYP94D109 gene, UGT80A41 gene, UGT73CE1 gene, and cas9 gene; the expression of the HMGR gene and ERG9 gene in the Saccharomyces cerevisiae is enhanced; the Saccharomyces cerevisiae includes Saccharomyces cerevisiae BY4742;

[0012] The recombinant Agrobacterium includes Agrobacterium that integrates the aforementioned genes, as well as the CAS gene, HMGR gene, CYP94D109 gene, C5-SD2 gene, CPI-5 gene, CYP51 gene, SMO1-3 gene, SMO2-2 gene, SSR1-3 gene, CYP90B71 gene, CYP90G6 gene, CYP94N8 gene, CYP72A616 gene, CYP90G4 gene, CYP90B27 gene, UGT80A41 gene, and UGT73CE1 gene; the Agrobacterium includes Agrobacterium LBA4404;

[0013] The HMGR gene has an accession number of OP243233.1 on NCBI;

[0014] The accession number of the ERG9 gene on NCBI is WNM97123.1;

[0015] The DHCR7 gene has an accession number of NP_958487.2 on NCBI;

[0016] The DHCR24 gene has an NCBI accession number of NP_001008645.1;

[0017] The RHM gene has the NCBI accession number CAD92667.1;

[0018] The CYP90B27 gene has an accession number of KX904822.1 on NCBI;

[0019] The CYP90G4 accession number on NCBI is QDS03628.1;

[0020] The NCBI accession number for CYP94D109 is QDS03630.1;

[0021] The NCBI accession number for the UGT80A41 is WIL59759.1;

[0022] The NCBI accession number for the UGT73CE1 is WIL59760.1;

[0023] The CAS gene has an accession number of MN368727.1 on NCBI;

[0024] The accession number of the C5-SD2 gene on NCBI is OP243261.1;

[0025] The accession number of the CPI-5 gene on NCBI is OP243252.1;

[0026] The CYP51 gene has an accession number of OP243253.1 on NCBI;

[0027] The SMO1-3 gene has the NCBI accession number OP243242.1.

[0028] The SMO2-2 gene has an accession number of OP243245.1 on NCBI;

[0029] The SSR1-3 gene has an accession number of OP243236.1 on NCBI;

[0030] The CYP90B71 gene has the NCBI accession number QPZ88854.1;

[0031] The CYP90G6 gene has the NCBI accession number QPZ88855.1;

[0032] The CYP94N8 gene has the NCBI accession number QPZ88856.1;

[0033] The CYP72A616 gene has an accession number of MK636705.1 on NCBI;

[0034] The nucleotide sequence of the cas9 gene is shown in SEQ ID NO. 11.

[0035] The present invention also provides the use of the aforementioned glycosyltransferase UGT73DY2 or the aforementioned gene or the aforementioned recombinant vector, expression cassette or recombinant bacteria in the synthesis of steroidal saponin compounds, wherein the steroidal saponin compounds include Paris polyphylla saponin compounds and Ophiopogon japonicus saponin compounds.

[0036] Furthermore, the Paris saponin compounds include diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside and phenobarbitin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside;

[0037] The ophiopogonin saponins include ophiopogonin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside;

[0038] The chemical structural formula of diosgenin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside is shown in Formula I; the chemical structural formula of pimonoside-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside is shown in Formula II; and the chemical structural formula of Ophiopogon saponins, including Ophiopogon saponin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside, is shown in Formula III.

[0039] Formula I

[0040] Formula II

[0041] Formula III.

[0042] This invention also provides a method for synthesizing steroidal saponin compounds, comprising the following steps:

[0043] Using Paris polyphylla saponin or Ophiopogon japonicus saponin-3-O-α-L-pyrano-rhamnoside-(1→2)-β-D-glucoside as substrates and UDP-rhamnoside as sugar donor, the reaction can be carried out through the aforementioned glycosyltransferase UGT73DY2 catalytic reaction.

[0044] Alternatively, ferment the aforementioned recombinant Saccharomyces cerevisiae in YPDA medium.

[0045] Furthermore, when the substrate is Paris polyphylla saponin V, the catalytic reaction generates diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside; when the substrate is Paris polyphylla saponin VI, the catalytic reaction generates pimonoside-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside; when the substrate is Ophiopogon saponin-3-O-α-L-rhamnopyranose-(1→2)-β-D-glucoside, the catalytic reaction generates Ophiopogon saponin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside.

[0046] The present invention also provides the application of the aforementioned glycosyltransferase UGT73DY2 or the aforementioned gene or the aforementioned recombinant vector, expression cassette or recombinant bacteria in the preparation of transgenic organisms with the function of biosynthesizing Paris polyphylla saponins.

[0047] Furthermore, the genetically modified organism includes genetically modified tobacco;

[0048] The transgenic tobacco is *Nicotiana benthamiana* whose genome integrates the aforementioned genes, as well as the CAS gene, HMGR gene, CYP94D109 gene, C5-SD2 gene, CPI-5 gene, CYP51 gene, SMO1-3 gene, SMO2-2 gene, SSR1-3 gene, CYP90B71 gene, CYP90G6 gene, CYP94N8 gene, CYP72A616 gene, CYP90G4 gene, CYP90B27 gene, UGT80A41 gene, and UGT73CE1 gene.

[0049] The saponin compounds of Paris polyphylla include diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside.

[0050] Using Paris polyphylla as the research material, this invention discovered a glycosyltransferase gene, named UGT73DY2. It was verified that the UGT73DY2 protein expressed by this gene can catalyze the glycosylation of Paris polyphylla saponin V, Paris polyphylla saponin VI, and Ophiopogonin-3-O-α-L-rhamnopyranose-(1→2)-β-D-glucoside to generate diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside, phenobarbitin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside, and Ophiopogonin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside.

[0051] The diosgenin-3-O-α-L-pyranorhamnetose-(1→4)-[α-L-pyranorhamnetose]-(1→2)-β-D-glucoside and pennosapogenin-3-O-α-L-pyranorhamnetose-(1→4)-[α-L-pyranorhamnetose]-(1→2)-β-D-glucoside, synthesized via glycosyltransferase UGT73DY2, are key intermediates in the biosynthesis of Paris polyphylla saponins II or VII. The in vitro catalytic synthesis of these compounds under the action of glycosyltransferase UGT73DY2 lays the foundation for the industrial production of Paris polyphylla saponins II and VII. Simultaneously, a novel Ophiopogon japonicus saponin compound can also be synthesized under the action of glycosyltransferase UGT73DY2, providing direction for further research and application of glycosyltransferase UGT73DY2.

[0052] The glycosyltransferase UGT73DY2 gene of this invention was introduced into Saccharomyces cerevisiae or Agrobacterium tumefaciens, and further introduced with corresponding genes, such as DHCR7, DHCR24, RHM, CYP90B27, CYP90G4, CYP94D109, UGT80A41, UGT73CE1, cas9, etc. The resulting recombinant Saccharomyces cerevisiae, after fermentation culture, can produce Paris polyphylla saponins. The recombinant Agrobacterium tumefaciens obtained by transfecting transgenic tobacco through Agrobacterium-mediated transformation can also synthesize Paris polyphylla saponins on its own, thus realizing the heterologous biosynthesis of Paris polyphylla saponins.

[0053] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0054] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0055] Figure 1 The image shows the PCR agarose gel electrophoresis of the amplified product of the gene encoding glycosyltransferase UGT73DY2, where lane M is the DL2000 DNA Marker and lane 1 is the target band (1485bp).

[0056] Figure 2 This is a schematic diagram of the Escherichia coli expression plasmid UGT73DY2, which expresses the glycosyltransferase UGT73DY2 that synthesizes diosgenin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside, pimonosiderin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside and ophiopogonin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside.

[0057] Figure 3 A schematic diagram of the recombinant yeast expression plasmid UGT73DY2, which is the glycosyltransferase for expressing diosgenin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside.

[0058] Figure 4 A schematic diagram of the tobacco heterologous synthesis expression plasmid UGT73DY2, which is the glycosyltransferase for expressing diosgenin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside.

[0059] Figure 5 The images show the UPLC analysis of the enzyme-catalyzed reaction products; where A represents the catalytic reaction of glycosyltransferase UGT73DY2; B represents the HPLC and mass spectrometry analysis of the enzyme activity product of UGT73DY2 with Paris polyphylla saponin V(1) as substrate and UDP-rhamnose as sugar donor; C represents the HPLC and mass spectrometry analysis of the enzyme activity product of UGT73DY2 with Paris polyphylla saponin VI(2) as substrate and UDP-rhamnose as sugar donor; and D represents the HPLC and mass spectrometry analysis of the enzyme activity product of UGT73DY2 with Ophiopogon japonicus saponin-3-O-α-L-pyranoside-(1→2)-β-D-glucoside (3) as substrate and UDP-rhamnose as sugar donor.

[0060] Figure 6 To fit the nonlinear Michaelis-Menten regression curves of initial rate and sugar receptor concentration using Origin, the Michaelis constant (Km) was calculated; A and B are the nonlinear Michaelis-Menten regression curves of UGT73DY2 with Paris polyphylla saponin V and Paris polyphylla saponin VI as substrates, respectively.

[0061] Figure 7 A schematic diagram of the production pathway of diosgenin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside in Saccharomyces cerevisiae.

[0062] Figure 8 The transient expression of different co-expressed gene combinations in Nicotiana benthamiana resulted in the yield of diosgenin-3-O-α-L-pyranorhamnosyl-(1→4)-[α-L-pyranorhamnosyl]-(1→2)-β-D-glucoside. Gray boxes indicate the absence of biosynthetic genes, while green boxes indicate the addition of biosynthetic genes in the co-expression experiment. Detailed Implementation

[0063] Unless otherwise specified, the materials and reagents used in the specific embodiments of this invention are all obtained from commercial sources or conventional formulation methods in the field. The specific structural formulas of the steroidal saponins involved are as follows:

[0064] Paris saponin V

[0065]

[0066] Paris saponin VI

[0067]

[0068] Ophiopogonin-3-O-α-L-rhamnopyranose-(1→2)-β-D-glucoside

[0069]

[0070] Diosgenin-3-O-α-L-rhamnopyranose-(1-4)-[α-L-rhamnopyranosyl]-(1-2)-β-D-glucoside

[0071]

[0072] Pyrnosapogenin-3-O-α-L-rhamnopyranose-(1→4)[α-L-rhamnopyranosyl](1→2)-β-D-glucoside

[0073]

[0074] Ophiopogonin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside

[0075]

[0076] Example 1: Discovery, cloning, and expression of the gene encoding glycosyltransferase UGT73DY2

[0077] In previous studies on Paris polyphylla, using *Paris polyphylla* yunnanensis as research material, a glycosyltransferase gene was discovered in the transcriptome of *Paris polyphylla* Smith var. *yunnanensis* (Franch.) Hand.-Mazz. through Blast homology comparison. This gene has an open reading frame containing 1485 bases and is named UGT73DY2. Its nucleotide sequence is shown in SEQ ID NO.1.

[0078] atggagtccc aaccagagct cctccaccta gttttcttcc ccttcctcgc tcgcagccacatgatcccca tgctggagac cgcccgcctc gccgtcgagc gcggtgtcaa aaccaccctc gtcaccacccctgccaacgc ccacctcatc cgccccgccc tccaccgcac caactcctct ctcctcccct cccatcccccgatgcagctc caactcattc ccttcccctc ggccgagttc ggcatccccg aggggtgcga gaacctcacctctctccccc tccccctcgc cgccgccttc ttcaacgcca tcttcgccct gcgtgccccg ctcggcgcgctgttgcggga gctcagcgct cacgccctgg tcgccgacgc gctgttcccg tgggccacgg ggctggcggccgagatgggg

[0079] atcccgaggc ttatcttcca ggtcacgggt ctgttcccgc tctgcgctgc ccacgatctcgatgcacatc ggccgcacga ggccgttggt ggggatgacg aggagtttac catcccgggg tttccggacccggtgacgct taccaggggg cagatccccg aggtcttcag gcacaatttc atgctggccc tcctccgcgacgcagagctc accagctacg gcgtgatagt gaacagcttc tacgccctcg agcccagcta cgcggagcactactacaagg tggccccccg taaggtcttc ctcctcggcc cggttgccct cgccggctcc aatccctcgccgccgccatt ggagagtggc gacccctgca tcacctggct cgactccaag cccgacgact cggtcctttacctgagcttc

[0080] ggcaccctct gtcgattcag caacgagcag ctcgtcgagc tcgctgaggg cctcgcgtcctccggccaca actttgtgtg ggtcgtggcc cgccccgaga gcagcggtgg gcccggcgag gagtggcttccggaaggcta cgagcgtaat gtggcaggcc gggggcttct cgtaagtggt tgggctccgc agaccgcgatcctgaaccac cgggcggtgg gtgggttcgt gatccactgc gggtggaact ccgtcatgga ggcggtggcagcggaggtgc ccatggccac gtggccgctt cactcagagc aattcgtcat tgagaagctg ctctgcgatgtgctgcacgt ggcggtgcca atgtgggagg ggtggaaaag catttgggac gatcagaagg aggtggtgcgggcgggaaca

[0081] gtggcagcgt cggtgaagcg gcttatgggg ggcggggacg aggtggaggc gatgcgaaggagagtgaggg agctcgggga attaggacgg gccgcggtgg cagaaagcgg atcatcccac tccgacatgggccgtcttat cgacgtgttg acggaggagc gaagcaaggc taacgaaatg atagacaaga agggtgttagttatgattgt tctggttgca attga

[0082] RNA was extracted from different tissues (including rhizomes, fibrous roots, leaves, and stems) of *Paris polyphylla* using the TRIzol Plus RNA Purification Kit. Total RNA was then reverse transcribed into cDNA according to the HiScript® III 1st Strand cDNA Synthesis Kit instructions. Using the synthesized cDNA as a template, PCR amplification was performed using the designed specific primers for the glycosyltransferase UGT73DY2: UGT73DY2-F:5'-ATGGAGTCCCAACCAGAG-3' (SEQ ID NO. 3) and UGT73DY2-R:5'-TCAATTGCAACCAGAACAATCATAA3' (SEQ ID NO. 4), and 2×Phanta MaxMaster Mix high-fidelity enzyme (vazyme, catalog number: 515-02). The amplified UGT73DY2 gene fragment (…) was… Figure 1 The ligation product was ligated into pClone007 Blunt Simple Vector (Beijing Qingke Biotechnology, catalog number: TSV-007BS), and the ligation product was transformed into E. coli DH5α. The pClone007-UGT73DY2 plasmid was extracted using the Oriscience Plasmid Mini Extraction Kit (Oriscience, catalog number: NB101-200T).

[0083] Using pClone007-UGT73DY2 plasmid as a template, the designed primer pair was: pCold-UGT73DY2-F (SEQ ID NO. 5): 5'-ATGGAGCTCGGTACCCTCGAGA

[0084] TGGAGTCCCAACCAGAG-3'; pCold-UGT73DY2-R (SEQ ID NO.6): 5'-CTA

[0085] The UGT73DY2 gene fragment was amplified by PCR using TCTAGACTGCAGGTCGACTCAATTGCAACCAGAACAATCATAA-3' and 2×Phanta MaxMaster Mix high-fidelity enzyme (vazyme, catalog number: 515-02). The amplified UGT73DY2 gene fragment was ligated into a pCold-TF expression vector containing a His tag at the N-terminus using 2×Clonexpress Mix (vazyme, catalog number: C115-02) (this vector was double-digested with restriction endonucleases XhoI (TaKaRa, catalog number: 1094A) and SalI (TaKaRa, catalog number: 1080A)). The vector was then transformed into E. coli DH5α, and single colonies were picked. After colony PCR identification, positive single colonies were sequenced, and the plasmid of correctly sequenced pCold-UGT73DY2 was extracted.

[0086] pCold-UGT73DY2 plasmid ( Figure 2The culture was transformed into E. coli expression strain BL21(DE3). Single colonies were selected for PCR verification. Correct single colonies were inoculated into 6 mL of LB liquid medium containing Amp resistance and incubated overnight at 37°C and 200 rpm with a shaker. The activated bacteria were then inoculated into 100 mL of LB liquid medium containing Amp resistance and incubated at 37°C and 200 rpm with a shaker until OD600 = 0.6. IPTG was added to a final concentration of 0.3 mM, and protein expression was induced at 16°C and 200 rpm for 12-16 h. Collect bacterial cells and reselect them using 8 mL of Buffer 1 (0.5 M NaCl, 10 mM imidazole, 5 mM DTT, 20 mM Tris-HCl, pH 8.0). Then, sonicate the cells and take 100 μL as a control. Centrifuge the remaining samples at 4°C and 12000 rpm for 10 min. Collect the supernatant and purify the protein. Adsorb the crude enzyme with Ni-NTA agarose (Qiagen). Elute the protein sequentially with Buffer 2 (0.5 M NaCl, 10 mM imidazole, 5 mM DTT, 20 mM Tris-HCl, 20 mM imidazole, pH 8.0) and Buffer 3 (0.5 M NaCl, 10 mM imidazole, 5 mM DTT, 20 mM Tris-HCl, 250 mM imidazole, pH 8.0) while collecting the buffers. Buffer 3 is the purified protein. The protein eluted with buffer 3 was concentrated using a 30 kDa ultrafiltration tube, and its concentration was determined using a BCA protein assay kit. An equal volume of glycerol was added to the eluted protein, and it was stored at -80°C. Protein solubility and size were analyzed by 10% SDS-PAGE. The results showed that UGT73DY2 protein was well soluble, with a protein size of approximately 101 kDa. The amino acid sequence of UGT73DY2 protein is shown in SEQ ID NO. 2.

[0087] MESQPELLHLVFFPFLARSHMIPMLETARLAVERGVKTTLVTTPANAHLIRPALHRTNSSLLPSHPPMQLQLIPFPSAEFGIPEGCENLTSLPLPLAAAFFNAIFALRAPLGALLRELSAHAL VADALFPWATGLAAEMGIPRLIFQVTGLFPLCAAHDLDAHRPHEAVGGDDEEFTIPGFPDPVTLTRGQIPEVFRHNFMLALLRDAELTSYGVIVNSFYALEPSYAEHYYKVAPRKVFLLGPVAL AGSNPSPPPLESGDPCITWLDSKPDDSVLYLSFGTLCRFSNEQLVELAEGLASSGHNFVWVVVARPESSGGPGEEWLPEGYERNVAGRGLLVSGWAPQTAILNHRAVGGFVIHCGWNSVMEAVA AEVPMATWPLHSEQFVIEKLLCDVLHVAVPMWEGWKSIWDDQKEVVRAGTVAASVKRLMGGGDEVEAMRRRVRELGELGRAAVAEGSSHSDMGRLIDVLTEERSKANEMIDKKGVSYDCSGCN

[0088] In the experiment, the pClone007-UGT73DY2 plasmid was used as a template, and the designed primer pair was: pESC-URA-UGT73DY2-F'(SEQ ID NO. 7):5'-GGAGAAAAAACCCCGGAT

[0089] CCAATGGAGTCCCAACCAGAG-3, pESC-URA-UGT73DY2-R (SEQ ID NO. 8):5'-CAACTTCTGTTCCATGTCGACTCAATTGCAACCAGAACAATCATAA-3', and 2×Phanta Max Master Mix high-fidelity enzyme (vazyme, catalog number: 515-02) were used for PCR amplification. The amplified UGT73DY2 gene fragment was ligated with the Saccharomyces cerevisiae expression vector pESC-URA, which was double-digested with restriction endonucleases BamHI (New England Biolabs, catalog number: R3136V) and SalI (TaKaRa, catalog number: 1080A), to obtain the recombinant vector ( Figure 3 When transformed into Saccharomyces cerevisiae, it can also express the synthetic amino acid sequence UGT73DY2 protein as shown in SEQ ID NO. 2.

[0090] Using pClone007-UGT73DY2 plasmid as a template, the designed primer pair was: pEAQ-HT-UGT73DY2-F (SEQ ID NO. 9): 5'-CTGCCCAAATTCGCGACCGGT

[0091] ATGGAGGTCCCAACCAGAG-3' and pEAQ-HT-UGT73DY2-R (SEQ ID NO.10)

[0092] The gene fragment 5'-ACCAGAGTTAAAGGCCTCGAGTCAATTGCAACCAGAACAATCATAA-3' and 2×PhantaMax Master Mix high-fidelity enzyme (vazyme, catalog number: 515-02) were used for PCR amplification. The amplified UGT73DY2 gene fragment was ligated with the tobacco expression vector pEAQ-HT, which had been double-digested with restriction endonucleases XhoI (TaKaRa, catalog number: 1094A) and AgeI (New England Biolabs, catalog number: R3136V), to obtain the recombinant vector ( Figure 4 When converted into tobacco, it can also express the synthetic amino acid sequence UGT73DY2 protein as shown in SEQ ID NO. 2.

[0093] Example 2: In vitro enzyme activity assay and product analysis of glycosyltransferase UGT73DY2

[0094] The UGT73DY2 protein obtained in Example 1 and the lysis products of the Pcold-TF empty vector were used for in vitro enzyme activity testing. Specifically, Paris polyphylla saponin V, Paris polyphylla saponin VI, and Ophiopogonin-3-O-α-L-rhamnopyranose-(1→2)-β-D-glucoside were used as substrates, and UDP-rhamnoose was used as a sugar donor. The enzyme products after the reaction were analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0095] The reaction was carried out at a final volume of 200 μL, with a formulation of 50 mM Tris-HCl, 50 μM substrate, 100 μM UDP-rhamnose, and 185 μL of UGT73DY2 protein or empty vector. The reaction mixture was incubated at 37°C for 6 hours. After concentration using a vacuum freeze dryer, the sample was dissolved in 100 μL of methanol. The sample was centrifuged at 12,000 rpm for 25 min, and 25 μL was injected into an UPLC (ultra-high performance liquid chromatography) system using an Agilent 1260 system with a ZORBAX SB-C18 analytical column at a flow rate of 1 mL / min and a column temperature of 35°C. The product of Paris polyphylla saponin V was separated from the enzyme using water / acetonitrile (0-22 min linear gradient from 10% to 100% acetonitrile), washed with 100% acetonitrile for 5 min, and detected at 195 nm. The product of Paris polyphylla saponin VI was separated from the enzyme using water / acetonitrile (0-22 min linear gradient from 30% to 70% acetonitrile, 22-23 min from 70% to 100% acetonitrile), washed with 100% acetonitrile for 5 min, and detected at 195 nm. The product of Ophiopogon japonicus saponin-3-O-α-L-rhamnopyranose-(1→2)-β-D-glucoside was separated from the enzyme using water / acetonitrile (0-22 min linear gradient from 30% to 70% acetonitrile, 22-23 min from 70% to 100% acetonitrile), washed with 100% acetonitrile for 5 min, and detected at 195 nm.

[0096] The reaction solution was detected by HPLC. Figure 5 Mass spectrometry analysis was performed using a Waters Synapt™ XS HighDefinition Mass Spectrometry system equipped with an ESI source. The mass spectrometry conditions were as follows: data acquisition in positive ion mode, capillary voltage 2.8 kV, sampling cone voltage 35.0 V, extraction cone voltage 3.0 V, mass source temperature 110 °C, desolvation temperature 350 °C, desolvation gas flow rate 650 L / h, collision energy set to 10-30 eV for low-energy scan and 20-45 eV for high-energy scan, and scan mass range set to m / z 50 to 1200.

[0097] from Figure 5As shown in B~D, compared with the control, when the glycosyltransferase UGT73DY2 used Paris polyphylla saponin V, Paris polyphylla saponin VI, and Ophiopogonin-3-O-α-L-pyranoside-(1→2)-β-D-glucoside as substrates, specific product peaks were generated. Comparison with the liquid chromatography retention time and mass spectrometry molecular weight of the standard confirmed that Paris polyphylla saponin V was synthesized by the UGT73DY2 protein into diosgenin-3-O-α-L-pyranoside-(1-4)-[α-L-pyranoside]-(1-2)-β-D-glucoside. - Glucoside; Paris saponin VI is synthesized into pennosapogenin-3-O-α-L-pyranorhamnosyl-(1-4)-[α-L-pyranorhamnosyl]-(1-2)-β-D-glucoside under the catalysis of UGT73DY2 protein, and Ophiopogon saponin-3-O-α-L-pyranorhamnosyl-(1→2)-β-D-glucoside is synthesized into Ophiopogon saponin-3-O-α-L-pyranorhamnosyl-(1→4)-[α-L-pyranorhamnosyl]-(1→2)-β-D-glucoside under the catalysis of UGT73DY2 protein.

[0098] Example 3: Analysis of enzyme kinetic parameters of glycosyltransferase UGT73DY2

[0099] Enzyme kinetic parameters are important tools for studying the rate of enzyme-catalyzed reactions. Among them, the Michaelis constant (Km) is a measure of the affinity between an enzyme and its substrate, which can be used to assess enzyme activity and determine enzyme specificity.

[0100] To determine the enzyme kinetic parameters of UGT73DY2 protein, a final volume of 100 μL was prepared containing 50 mM Tris-HCl (pH 8.0), 100 μg of the UGT73DY2 protein obtained in Example 1, 2 mM UDP-rhamnose, and different concentrations of substrate. The substrate concentration gradients were 2.5, 5, 10, 20, 40, 60, 80, 100, 200, and 400 μM for Paris polyphylla V and 2.5, 5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, and 1200 μM for Paris polyphylla VI. The mixture was incubated at 37°C for 30 minutes. The reaction was stopped with 100 μL of methanol and then concentrated. The product was dissolved in 100 μL of methanol, centrifuged at 12,000 rpm for 25 minutes, and 25 μL of the supernatant was taken for UPLC analysis.

[0101] UPLC (ultra-high performance liquid chromatography) was performed using an Agilent 1260 system with a ZORBAX SB-C18 analytical column at a flow rate of 1 mL / min and a column temperature of 35°C. The mobile phase was water (A) and acetonitrile (B). For the product of UGT73DY2, a linear gradient of B was used from 10% to 100% (0 to 22 min), followed by 100% isocratic B (22.01 to 30 min). All experiments were performed in triplicate. Calibration curves for Paris polyphylla saponin V and Paris polyphylla saponin VI were generated by UPLC at seven gradients (2.5, 5, 10, 20, 40, 80, 100 μM) using the same method. The equations and correlation coefficients obtained from the linear study are as follows: Paris polyphylla saponin V is y = 8.4808x + 6.1172 (R² = 0.99), and Paris polyphylla saponin VI is y = 8.3667x + 14.28401 (R² = 0.99). The content of the reaction products was calculated based on the equations, and a nonlinear Michaelis-Menten regression curve of the initial rate and sugar receptor concentration was fitted using Origin. Figure 6 ), calculate the Michaelis constant (Km).

[0102] The Km values ​​of glycosyltransferase UGT73DY2 with Paris polyphylla saponin V as substrate were calculated using the above linear equations to be 45.27791±5.4; and the Km value of glycosyltransferase UGT73DY2 with Paris polyphylla saponin VI as substrate was 80.04102±11.8.

[0103] The smaller the Km value, the greater the affinity between the enzyme and the substrate, and the higher the enzyme activity. The results above show that glycosyltransferase UGT73DY2 has a strong affinity for both Paris polyphylla saponin V and Paris polyphylla saponin VI, with the strongest affinity for Paris polyphylla saponin V. It exhibits high enzyme activity in the catalytic synthesis of diosgenin-3-O-α-L-pyranorhamnetose-(1-4)-[α-L-pyranorhamnetose]-(1-2)-β-D-glucoside from Paris polyphylla saponin V.

[0104] Example 4: Synthesis of recombinant Saccharomyces cerevisiae of diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside

[0105] In the early stage, the UGT73DY2 gene was integrated into Saccharomyces cerevisiae, and the resulting recombinant Saccharomyces cerevisiae could express the glycosyltransferase UGT73DY2. Further genetic modification of Saccharomyces cerevisiae was carried out on this basis in order to obtain a grain-based wine yeast that can synthesize Paris polyphylla saponins.

[0106] The experiment used CRISPR gene editing technology to recombine the genes of Saccharomyces cerevisiae BY4742, enhancing the expression of the HMGR and ERG9 genes and integrating them into the cas9 gene. Using this recombinant Saccharomyces cerevisiae as the initial strain, further CRISPR gene editing was used to integrate DHCR7, DHCR24, RHM, CYP90B27, CYP90G4, CYP94D109, UGT80A41, UGT73CE1, and UGT73DY2 into its genome, resulting in recombinant Saccharomyces cerevisiae.

[0107] The specific steps for obtaining recombinant Saccharomyces cerevisiae are as follows: Take 25 mL of initial Saccharomyces cerevisiae culture (OD25) 600 =0.8-1.0) Centrifuge (2500-3000 rpm, 4℃, 3 minutes) and resuspend the Saccharomyces cerevisiae in the same volume of sterile water. Wash the cells and centrifuge. Resuspend the cells in an appropriate volume of 1.1 × LiAc to prepare competent yeast cells. Add 500 ng sgRNA, 1 µg donor DNA, and 500 µL PEG / LiAc premix (8 mL 50% PEG3350, 1 mL 10 × TE buffer, 1 mL 10 × LiAc). Incubate the above system in a 30℃ water bath for 30 minutes and heat shock at 42℃ for 15 minutes. After the water bath, centrifuge at 5000 rpm for 2 minutes, discard the supernatant, and resuspend in 100 µL sterile ddH2O. Spread the resuspended yeast on SD-URA plates and culture for 3 days. Pick single clones for P verification. Then, expand the yeast culture after successful sequencing in YPDA medium and extract the fermentation broth by sonication. The extract was analyzed by UPLC-MS / MS under the same chromatographic conditions as in Example 3. The mass spectrometry conditions were as follows: positive ion mode, capillary voltage 2.8 kV, sampling cone voltage 35.0 V, and extraction cone voltage 3.0 V. The UPLC-MS / MS source temperature was set to 110°C, the desolvent gas temperature to 350°C, and the desolvent gas flow rate to 650 L / h. The scan mass range was set to m / z 50 to 1200. The collision energy was adjusted to 10–30 eV for low-energy scans and 20–45 eV for high-energy scans. Leucine-enkephalin was used to calibrate the data quality during analysis. All data were collected in centroid mode and controlled using Masslynx™ (V4.1) software.

[0108] CRISPR gene editing technology involves genes

[0109]

[0110] Note: Nucleotide sequence of the cas9 gene (SEQ ID NO.11):

[0111]

[0112] Analysis of the fermentation broth revealed that the recombinant Saccharomyces cerevisiae could synthesize a Paris polyphylla saponin compound during fermentation—diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside. The specific synthetic pathway is detailed in [link to relevant documentation]. Figure 7 .

[0113] Example 7: Synthesis of diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside in tobacco

[0114] Previous experiments integrated the UGT73DY2 gene into tobacco, obtaining tobacco that could express the glycosyltransferase UGT73DY2. Based on this, further genetic modifications were performed on tobacco using Agrobacterium-mediated transformation, with the aim of obtaining tobacco that could synthesize Paris polyphylla saponins on its own.

[0115] The experiment involved loading genes CAS (MN368727.1), HMGR (OP243233.1), CYP94D109 (QDS03630.1), C5-SD2 (OP243261.1), CPI-5 (OP243252.1), CYP51 (OP243253.1), SMO1-3 (OP243242.1), SMO2-2 (OP243245.1), SSR1-3 (OP243236.1), and CYP90B71 (QPZ88854) into the target cells. 1) Plasmids containing CYP90G6 (QPZ88855.1), CYP94N8 (QPZ88856.1), CYP72A616 (MK636705.1), CYP90G4 (QDS03628.1), CYP90B27 (KX904822.1), UGT80A41 (WIL59759.1), UGT73CE1 (WIL59760.1), and UGT73DY2 were transformed into Agrobacterium LBA4404 competent cells using a liquid nitrogen rapid freezing method. Transformants were screened on LB agar plates containing 50 μg / mL kanamycin, 50 μg / mL gentamicin, and 50 μg / mL rifampin at 28°C. The inoculated Agrobacterium strain was cultured in liquid LB medium containing 50 μg / mL kanamycin, 50 μg / mL gentamicin, and 50 μg / mL rifampin at 28°C and 200 rpm for 24–36 hours with shaking. The culture was then diluted 1:10 and cultured for another 24 hours. Cells were collected by centrifugation at 5000 rpm for 5 minutes and washed once with 20 mL MMA buffer (10 mM MMES (pH 5.6, adjusted with KOH), 10 mM MgCl2, 100 μM acetylsylphenone). The cells were then resuspended in MMA buffer and OD was adjusted. 600 To 0.2. When performing Agrobacterium tumefaciens infiltration, equal volumes of suspensions of each strain were mixed. Before infiltration, 6-week-old Nicotiana benthamiana plants were watered and placed in a cool, shaded area for approximately 2 hours. After infiltration, the plants were kept in a cool, shaded area and transferred to normal growth conditions the following day for continued growth in a greenhouse. Approximately 6 days later, the infiltrated leaves were collected and freeze-dried using a vacuum freeze dryer. The freeze-dried samples were analyzed by UPLC-MS / MS, with the chromatographic conditions the same as in Example 3 and the mass spectrometry conditions the same as in Example 4.

[0116] See results Figure 8 ,from Figure 8 It is evident that, under positive ion mode, the main ionic fragment produced by the lysis of the standard has a m / z of 869.48, which can be found in transgenic tobacco samples similar to diosgenin-3-. OThe fragment ion peaks corresponding to the α-L-pyranorhamnetose-(1→4)-[α-L-pyranorhamnetose]-(1→2)-β-D-glucoside standard indicate the presence of diosgenin-3- in the sample. O -α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside was present, among which the combination of CAS, HMGR, C5-SD2, CPI-5, CYP51, SMO1-3, SMO2-2, SSR1-3, CYP94N8, CYP90G6, CYP90B27, UGT80A41, UGT73CE1 and UGT73DY2 showed the best transient expression effect, based on diosgenin-3- O The peak area of ​​the α-L-pyranorhamnetose-(1→4)-[α-L-pyranorhamnetose]-(1→2)-β-D-glucoside standard was calculated, and the diosgenin-3- in the infected tobacco leaves was finally determined. O The content of -α-L-pyranorhamnetose-(1→4)-[α-L-pyranorhamnetose]-(1→2)-β-D-glucoside is 2.63 μg / g (dry weight).

[0117] In summary, the UGT73DY2 protein expressed by the glycosyltransferase gene UGT73DY2 of this invention can catalyze the glycosylation of Paris polyphylla saponin V, Paris polyphylla saponin VI, and Ophiopogon saponin-3-O-α-L-rhamnopyranose-(1→2)-β-D-glucoside to generate diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside, phenobarbitin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside, and Ophiopogon saponin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside.

[0118] The glycosyltransferase UGT73DY2 gene of this invention was introduced into Saccharomyces cerevisiae or Agrobacterium tumefaciens, and further introduced with corresponding genes to construct recombinant Saccharomyces cerevisiae. After fermentation, the resulting recombinant Saccharomyces cerevisiae can produce Paris polyphylla saponins. The recombinant Agrobacterium tumefaciens obtained by transfecting transgenic tobacco through Agrobacterium-mediated transformation can also synthesize Paris polyphylla saponins on its own, thus realizing the heterologous biosynthesis of Paris polyphylla saponins.

Claims

1. A glycosyltransferase UGT73DY2, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the glycosyltransferase UGT73DY2 of claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

3. A recombinant vector, expression cassette, or recombinant bacteria comprising the gene of claim 2.

4. The recombinant bacteria according to claim 3, characterized in that: It includes recombinant Escherichia coli or recombinant Saccharomyces cerevisiae; The recombinant Saccharomyces cerevisiae includes a strain of Saccharomyces cerevisiae that integrates the genes described in claim 2, as well as the DHCR7 gene, DHCR24 gene, RHM gene, CYP90B27 gene, CYP90G4 gene, CYP94D109 gene, UGT80A41 gene, UGT73CE1 gene, and cas9 gene; the expression of the HMGR gene and ERG9 gene in the Saccharomyces cerevisiae is enhanced; the Saccharomyces cerevisiae includes Saccharomyces cerevisiae BY4742; The HMGR gene has an accession number of OP243233.1 on NCBI; The accession number of the ERG9 gene on NCBI is WNM97123.1; The DHCR7 gene has an accession number of NP_958487.2 on NCBI; The DHCR24 gene has an NCBI accession number of NP_001008645.1; The RHM gene has the NCBI accession number CAD92667.1; The CYP90B27 gene has an accession number of KX904822.1 on NCBI; The CYP90G4 gene has the NCBI accession number QDS03628.1; The CYP94D109 gene has the NCBI accession number QDS03630.1; The UGT80A41 gene has an accession number of WIL59759.1 on NCBI; The UGT73CE1 gene has an accession number of WIL59760.1 on NCBI; The nucleotide sequence of the cas9 gene is shown in SEQ ID NO.

11.

5. The use of the glycosyltransferase UGT73DY2 of claim 1, the gene of claim 2, or the recombinant vector, expression cassette, or recombinant bacteria of claim 3 in the synthesis of steroidal saponin compounds, characterized in that: The steroidal saponins are Paris polyphylla saponins and Ophiopogon japonicus saponins; The saponin compounds of Paris polyphylla are diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside and phenospinin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside; The ophiopogonin saponin compound is ophiopogonin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside; The chemical structural formula of diosgenin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside is shown in Formula I; the chemical structural formula of pimonoside-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside is shown in Formula II; and the chemical structural formula of Ophiopogon saponins, including Ophiopogon saponin-3-O-α-L-pyranouranose-(1→4)-[α-L-pyranouranose]-(1→2)-β-D-glucoside, is shown in Formula III. Equation I Formula II Formula III.

6. A method for synthesizing steroidal saponin compounds, characterized in that: Includes the following steps: Using Paris polyphylla saponin or Ophiopogon japonicus saponin-3-O-α-L-pyrano-rhamnoside-(1→2)-β-D-glucoside as substrates and UDP-rhamnoside as sugar donor, the reaction is catalyzed by the glycosyltransferase UGT73DY2 described in claim 1. Alternatively: ferment the recombinant Saccharomyces cerevisiae as described in claim 4 in YPDA medium; When the substrate is Paris polyphylla saponin V, the catalytic reaction generates diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside; when the substrate is Paris polyphylla saponin VI, the catalytic reaction generates pimonoside-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside; when the substrate is Ophiopogon saponin-3-O-α-L-rhamnopyranose-(1→2)-β-D-glucoside, the catalytic reaction generates Ophiopogon saponin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside.

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