Rhamnosyl transferase ScRhaGT and application thereof

Abstract

The invention provides a rhamnosyl transferase (derived from Streptomyces cirrhosus B2), and the rhamnosyl transferase is named as the rhamnosyl transferase ScRhaGT. The invention further provides a preparation method of the rhamnosyl transferase ScRhaGT. The invention also provides an expression vector containing the rhamnosyl transferase ScRhaGT, and a cloning and expression method of the rhamnosyl transferase ScRhaGT. On the basis, the invention provides an application of the rhamnosyl transferase ScRhaGT in preparation of a glycosylation modified podophyllum compound, and a method for preparing podophyllum rhamnoside by adopting the rhamnosyl transferase ScRhaGT. The rhamnosyl transferase ScRhaGT can be used for preparing the rhamnosylated podophyllum compound through enzyme reaction by taking various podophyllum compounds as substrates, so that the technical blank of preparing the rhamnose modified podophyllum compound through enzyme catalysis in the prior art is filled; and the method is also applicable to various donors such as glucose, galactose and the like, has a certain degree of donor universality, and has an important practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and biopharmaceuticals, and relates to a glycosyltransferase, specifically to a rhamnosyltransferase and its application in the glycosylation modification of podophyllotoxin derivatives. Background Art

[0002] Glycosylation modification is an effective strategy to improve the water solubility of anticancer drugs, improve pharmacokinetic properties, and reduce toxicity. However, glycosylated compounds are easily hydrolyzed in organisms and have poor metabolic stability; but when the glycosylation modification is rhamnosyl, since there is no rhamnosidase in the human body except for intestinal microorganisms, the in vivo stability of rhamnosylated drugs is ensured. Moreover, rhamnosylation modification can endow compounds with potential targeting to tumor cells and reduce toxicity to normal cells, which is an effective strategy for the structural modification of anticancer drugs.

[0003] Rhamnosyltransferase is a class of glycosyltransferases that achieve rhamnosylation modification by catalyzing the formation of rhamnoside bonds, and is widely distributed in plants and microorganisms. Although rhamnosylation modification plays an important role in the structural diversity and biological activity enhancement of natural products, there are still the following technical bottlenecks: (1) Narrow substrate spectrum: mainly glycosylates flavonoid compounds and has low catalytic activity for other types of natural products. (2) Single source: mainly from plants, and there are difficulties in heterologous expression and large-scale preparation. (3) Single product structure: unable to meet the needs of drug research and development for rhamnoside compounds with diverse structures. (4) Insufficient catalytic efficiency: the conversion rate for specific substrates is not high, making it difficult to meet the requirements of industrial production. In response to the above problems, researchers have made some improvement attempts. Currently, there are studies on modifying glycosyltransferases by methods such as site-directed mutagenesis and domain replacement to improve their performance. For example, there is a study on site-directed mutagenesis (G421T and A1144T) of α-L-rhamnosidase, which improves the heat stability of the enzyme. Patent application 202311247504.9 discloses "Application of a rhamnosyltransferase in the rhamnosylation modification of natural products". This patent application recombinantly expresses a rhamnosyltransferase Ss-RhaT derived from Actinosynnema mirum, which can use dTDP-Rha as the rhamnose donor and various natural products as glycosyl acceptors to synthesize corresponding rhamnosylated natural products.

[0004] Podophyllotoxin glycosides are an important class of natural product derivatives formed by introducing sugar groups (such as glucose) into different positions of the podophyllotoxin parent nucleus structure. Due to the significant alteration of the pharmacological activity and mechanism of action of podophyllotoxin by glycosylation modification, this class of compounds exhibits good antitumor pharmacological activity and plays an important role in the development of antitumor drugs. Currently, the podophyllotoxin glycoside drugs that have been developed and applied clinically include: Etoposide (VP-16), Teniposide (VM-26), and Etopophos. The aforementioned podophyllotoxin glycoside drugs are all prepared by chemical glycosylation methods, involving the protection and deprotection of multiple hydroxyl groups on the sugar group, which not only makes the reaction process complex but also is environmentally unfriendly. Although enzymatic methods have advantages such as high reaction efficiency, strong regioselectivity, mild reaction conditions, and environmental friendliness, currently, only two podophyllo-glucosyltransferases have been reported: UGT78D2 and AbGT5, which catalyze the glucosylation of the 4-OH in the C-ring and the 4′-OH in the E-ring of 4'-demethylepipodophyllotoxin respectively, and no rhamnosyltransferase that can catalyze the rhamnosylation modification of podophyllum has been found.

[0005] Therefore, exploring podophyllo-rhamnosyltransferase is of great significance for the research and development of podophyllotoxin glycosides, which are lead compounds for antitumor drugs. Moreover, as high-value pharmaceutical intermediates, podophyllotoxin glycosides will surely bring considerable economic benefits and have broad prospects for industrial application. However, currently, there is no relevant report on podophyllo-rhamnosyltransferase. Summary of the Invention

[0006] To solve the technical blank of rhamnosylation modification of podophyllum compounds by enzymatic reaction in the prior art, the present invention provides a rhamnosyltransferase (derived from Streptomyces cirratus B2), named rhamnosyltransferase ScRhaGT. The rhamnosyltransferase ScRhaGT can not only use a variety of podophyllum compounds as substrates to prepare rhamnosylated podophyllum compounds through enzymatic reaction, but also be applicable to a variety of donors such as glucose and galactose, having a certain degree of donor generality and important practical application value.

[0007] The technical solution of the present invention: The amino acid sequence of rhamnosyltransferase ScRhaGT is selected from the following (1), (2), or (3):

[0008] (1) The amino acid sequence shown in SEQ ID NO:1;

[0009] (2) An amino acid sequence shown in SEQ ID NO: 1, which has been substituted, deleted, or added with one or several amino acids and has the activity of catalyzing the binding of a rhamnosyl donor to the 4'-OH of the E ring or the 4-OH of the C ring of a podophyllotoxin compound;

[0010] (3) An amino acid sequence with a homology of ≥ 90% to the amino acid sequence shown in SEQ ID NO: 1, and the expressed protein has the activity of catalyzing the binding of a rhamnosyl donor to the 4'-OH of the E ring or the 4-OH of the C ring of a podophyllotoxin compound.

[0011] The present invention first provides a rhamnosyltransferase ScRhaGT derived from Streptomyces. Using the rhamnosyltransferase ScRhaGT, rhamnosylated podophyllotoxin derivatives - podophyllotoxin rhamnoside, which take a series of podophyllotoxin derivatives as acceptors, can be prepared, filling the technical gap in the enzymatic synthesis of rhamnosylated podophyllotoxin derivatives, providing a new strategy for improving the water solubility and stability of podophyllotoxin candidate drugs, and having important application value and social significance.

[0012] The nucleotide sequence encoding the rhamnosyltransferase ScRhaGT is selected from the following (1), (2), (3), or (4):

[0013] (1) The nucleotide sequence shown in SEQ ID NO: 2;

[0014] (2) Different from the nucleotide sequence shown in SEQ ID NO: 2, but a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1;

[0015] (3) A nucleotide sequence with a homology of ≥ 90% to the nucleotide sequence shown in SEQ ID NO: 2, and the expressed protein has the activity of catalyzing the binding of a rhamnosyl donor to the 4'-OH of the E ring or the 4-OH of the C ring of a podophyllotoxin compound;

[0016] (4) A nucleotide sequence complementary to the nucleotide sequence described in any one of (1), (2), or (3).

[0017] An expression vector containing the nucleotide sequence encoding the rhamnosyltransferase ScRhaGT. The expression vector is a vector suitable for expression in Escherichia coli.

[0018] The application of the expression vector in expressing the rhamnosyltransferase ScRhaGT.

[0019] The method for cloning and expressing the rhamnosyltransferase ScRhaGT includes the following steps: cloning the nucleotide sequence encoding the rhamnosyltransferase ScRhaGT into an expression vector to construct an expression vector; then transferring the expression vector into an expression system for protein expression; and finally obtaining the rhamnosyltransferase ScRhaGT through purification.

[0020] The application of the rhamnosyltransferase ScRhaGT as described above in the preparation of glycosylated podophyllotoxin compounds. Among them, the glycosylation modification is rhamnosylation modification, glucosylation modification or galactosylation modification. Compared with the rhamnosyltransferases in the prior art, the rhamnosyltransferase ScRhaGT of the present application can be applied to various donors such as rhamnosylation modification, glucose, and galactose, has a certain degree of donor generality, and has important practical application value.

[0021] Preferably, the rhamnosylation modification is to bind a rhamnose donor to the 4'-OH of ring E or 4-OH of ring C of the podophyllotoxin compound. Among them, the rhamnose donor is dTDP-L-rhamnose. The podophyllotoxin compounds are dehydrodesmethylpodophyllotoxin, dehydropodophyllotoxin, podophyllopicrin, podophyllotoxin, demethylepipodophyllotoxin, etoposide, teniposide, 4α-S-5-fluorobenzoxazole-demethylepipodophyllotoxin, 4β-S-5-fluorobenzoxazole-demethylepipodophyllotoxin, 4α-S-5-fluorobenzothiazole-demethylepipodophyllotoxin, 4β-S-5-fluorobenzothiazole-demethylepipodophyllotoxin, 4α-S-thiadiazole-demethylepipodophyllotoxin, 4β-S-thiadiazole-demethylepipodophyllotoxin, 4α-S-triazole-demethylepipodophyllotoxin, 4β-S-triazole-demethylepipodophyllotoxin. By using the rhamnosyltransferase ScRhaGT of the present application, a variety of α-substituted rhamnosylated podophyllotoxin derivatives are prepared with a variety of podophyllotoxin compounds as substrates, filling the technical gap in the enzymatic catalysis for the preparation of rhamnose-modified podophyllotoxin compounds in the prior art, and having outstanding substantive features and remarkable progress. This will provide more podophyllotoxin compound entities for the new drug research and development in China, and has important theoretical significance and practical application value for promoting the new drug research and development with independent intellectual property rights in China.

[0022] The method for preparing podophyllotoxin rhamnoside by using the rhamnosyltransferase ScRhaGT as described above. The specific operation is as follows: taking an appropriate amount of rhamnosyltransferase ScRhaGT, adding it to a reaction system containing a rhamnose donor and a receptor substrate, centrifuging after the reaction is completed, taking the supernatant and purifying it to obtain podophyllotoxin rhamnoside. The preparation method of the present invention not only avoids the cumbersome steps of hydroxyl protection and deprotection in the chemical synthesis route in the prior art, but also has mild conditions and is environmentally friendly, and has important industrial application value and good economic benefit prospects.

[0023] Advantages of the present invention:

[0024] (1) The present invention provides for the first time a rhamnosyltransferase ScRhaGT derived from Streptomyces, which can use dTDP-L-rhamnose as a donor and a series of podophyllotoxin derivatives as acceptors to synthesize rhamnosylated podophyllotoxin derivatives, filling the technical gap in the enzymatic synthesis of rhamnosylated podophyllotoxin derivatives.

[0025] (2) The rhamnosyltransferase ScRhaGT described in the present application is not only applicable to rhamnosylation modification, providing a tool enzyme for the synthesis of rhamnosylated compounds; it is also applicable to glucosylation modification and galactosylation modification, having a certain degree of donor generality and important practical application value.

[0026] (3) The present invention uses the rhamnosyltransferase ScRhaGT, which can use a variety of podophyllotoxin compounds as substrates to produce a variety of α-substituted rhamnosylated podophyllotoxin derivatives, enriching the members of the rhamnosyltransferase family and providing a new strategy for improving the water solubility and stability of podophyllotoxin candidate drugs, having important application value and social significance. Description of the drawings

[0027] Figure 1 It is the SDS-PAGE analysis electrophoresis diagram of the purified rhamnosyltransferase ScRhaGT in Example 1 of the present invention;

[0028] Figure 2 It is the HPLC chromatogram of the reaction of ScRhaGT with 15 podophyllotoxin compounds in Example 2 of the present invention;

[0029] Figure 3 It is the HR-MS spectrum of the reaction product of ScRhaGT and dehydrodesmethylpodophyllotoxin in Example 2;

[0030] Figure 4 It is the HR-MS spectrum of the reaction product of ScRhaGT and dehydro-podophyllotoxin in Example 2;

[0031] Figure 5 It is the HR-MS spectrum of the reaction product of ScRhaGT and picropodophyllin in Example 2;

[0032] Figure 6 It is the HR-MS spectrum of the reaction product of ScRhaGT and podophyllotoxin in Example 2;

[0033] Figure 7 It is the HR-MS spectrum of the reaction product of ScRhaGT and desmethyl-epipodophyllotoxin in Example 2;

[0034] Figure 8HR-MS spectrum of the reaction product of ScRhaGT and etoposide in Example 2;

[0035] Figure 9 HR-MS spectrum of the reaction product of ScRhaGT and teniposide in Example 2;

[0036] Figure 10 HR-MS spectrum of the reaction product of ScRhaGT and 4α-S-5-fluorobenzooxazole-demethyl epipodophyllotoxin in Example 2;

[0037] Figure 11 HR-MS spectrum of the reaction product of ScRhaGT and 4β-S-5-fluorobenzooxazole-demethyl epipodophyllotoxin in Example 2;

[0038] Figure 12 HR-MS spectrum of the reaction product of ScRhaGT and 4α-S-5-fluorobenzothiazole-demethyl epipodophyllotoxin in Example 2;

[0039] Figure 13 HR-MS spectrum of the reaction product of ScRhaGT and 4β-S-5-fluorobenzothiazole-demethyl epipodophyllotoxin in Example 2;

[0040] Figure 14 HR-MS spectrum of the reaction product of ScRhaGT and 4α-S-thiadiazole-demethyl epipodophyllotoxin in Example 2;

[0041] Figure 15 HR-MS spectrum of the reaction product of ScRhaGT and 4β-S-thiadiazole-demethyl epipodophyllotoxin in Example 2;

[0042] Figure 16 HR-MS spectrum of the reaction product of ScRhaGT and 4α-S-triazole-demethyl epipodophyllotoxin in Example 2;

[0043] Figure 17 HR-MS spectrum of the reaction product of ScRhaGT and 4β-S-triazole-demethyl epipodophyllotoxin in Example 2;

[0044] Figure 18 HPLC spectrum of the reaction product of ScRhaGT and dTDP-Glc (glucose), dTDP-Gal (galactose), UDP-Rha (rhamnose) and dTDP-Man (mannose) in Example 3;

[0045] Figure 19 HR-MS spectrum of the reaction product of ScRhaGT and dTDP-Glc (glucose) in Example 3;

[0046] Figure 20 HR-MS spectrum of the reaction product of ScRhaGT and dTDP-Gal (galactose) in Example 3;

[0047] Figure 21 HR-MS spectrum of the reaction product of ScRhaGT and UDP-Rha (rhamnose) in Example 3;

[0048] Figure 22 HPLC spectra of the reactions of the wild-type ScRhaGT and mutants R59A, R59F, E63A, D116A, and M223F in Example 4 of the present invention. Detailed implementation manners

[0049] The present invention will be further described below in conjunction with examples.

[0050] Example 1: Cloning and in vitro expression of the rhamnosyltransferase ScRhaGT gene

[0051] 1. Extraction of genomic DNA

[0052] Streptomyces cirratus B2 was inoculated into TSBY liquid medium and cultured at 30 °C. The cells were collected by centrifugation and washed with 1 mL of STE buffer; 500 μL of lysozyme solution at 3 - 5 mg / ml prepared with STE buffer was added to fully suspend the cells, and the mixture was incubated in a water bath at 37 °C for 30 min until the cells became translucent; 250 μL of 6% SDS was added, and the mixture was gently mixed up and down and continued to be incubated in a water bath at 37 °C until it became clear; after adding 1 / 10 volume of 3M NaAc (pH = 4.8), 200 μL of phenol:chloroform:isoamyl alcohol (25:24:1; v / v / v) was added, mixed, and centrifuged at 12000 rpm; the upper layer supernatant was taken and extracted repeatedly with phenol:chloroform:isoamyl alcohol until there were no protein impurities in the middle layer. The supernatant was transferred, and an equal volume of isopropanol was added and mixed until white flocculent DNA precipitates were formed; the flocculent precipitates were picked out and washed once with 70% ethanol; after air drying at room temperature, the genomic DNA was dissolved in an appropriate amount of TE for standby.

[0053] 2. Construction of the recombinant vector

[0054] The T-DNA of Streptomyces cirratus B2 prepared above was diluted 10 times as a template for PCR, and primer pair ScRhaGT-FP / RP was designed to amplify the functional gene of rhamnosyltransferase ScRhaGT:

[0055] ScRhaGT-FP: 5’-GGAATTCCATATGCGCATACTCTTCACCGGACCG-3’ /

[0056] ScRhaGT-RP: 5’-CCGCTCGAGCTACGAGGCGGCCAGGGCC-3’.

[0057] PCR reaction system: 5 μl (50 pmol) each of primer pair ScRhaGT-FP / RP, 5 μl of template, 10 μl of 10× Reaction Buffer, 10 μl of 2.5 mM dNTP, 6 μl of 25 mM MgCl2, 1 μl of pfu DNA Polymerase (5 U / μl), add ddH2O to 100 μl.

[0058] PCR conditions: Functional gene amplification conditions, denaturation at 98 °C for 2 min; 95 °C for 10 s, 66.3 °C for 15 s, 72 °C for 10 s, 25 cycles; 72 °C for 5 min. Clone it into the expression vector pET28a to construct the recombinant plasmid pET28a-ScRhaGT.

[0059] 3. Expression and purification of ScRhaGT in Escherichia coli

[0060] The constructed recombinant vectors were respectively introduced into Escherichia coli BL21(DE3). The overnight bacterial solution was inoculated into 1 L of LB liquid medium containing 50 μg / mL -1 kanamycin, cultured at 37 °C until the OD600 was about 0.8, added 0.05 mM isopropyl β-D-thiogalactoside (IPTG), and continued to culture at 16 °C for 16 h. Centrifuge to collect the bacterial cells, resuspend the bacterial cells with buffer (0.05 M Tris-HCl, 0.5 M NaCl, pH 7.5, containing cOmpleteTM protease inhibitor cocktail), ultrasonically disrupt the bacterial cells, purify through a nickel column, and detect by SDS-PAGE (as Figure 1 shown), to obtain ScRhaGT with a size of 38.8 kDa. Concentrate and replace the enzyme with higher purity into buffer (0.025 M Tris-HCl, 0.02 M NaCl, and 10% glycerol, pH 7.5), and store at -80 °C for later use.

[0061] Example 2: Detection of the acceptor promiscuity of rhamnosyltransferase ScRhaGT

[0062] An in vitro enzyme activity reaction system was used to verify the acceptor promiscuity of ScRhaGT. The specific operation was as follows:

[0063] Prepare the ScRhaGT reaction system: 50 mM Tris-HCl (pH 8.0) buffer, 0.1 mM dTDP-Rha, 15 μM ScRhaGT, 0.1 mM receptor substrate. The receptor substrate is 15 podophyllotoxin compounds, and the specific information is as follows. At the same time, use the boiled ScRhaGT catalytic group as the control group.

[0064]

[0065] Reaction conditions: 30 °C, 30 min. After the reaction, add twice the volume of methanol to terminate the reaction, and centrifuge at 17,000 x g for 30 min to remove the protein in the reaction system. The obtained supernatant is subjected to HPLC detection and analysis.

[0066] HPLC detection conditions: Use a reverse-phase YMC-Pack ODS-AQ C18 column (specification: 50 mm × 4.6 mm, 5 μm, ); column temperature 30 °C; mobile phases A (acetonitrile + 0.1% formic acid) and B (ddH2O + 0.1% formic acid); ScRhaGT reaction elution conditions: 0 - 5 min, 5% A phase and 95% B phase; 5 - 8 min gradient elution, 5% - 20% A phase and 95% - 80% B phase; 8.1 - 20 min gradient elution, 35% - 100% A phase and 65% - 0% B phase; detection wavelength is 254 nm, flow rate is 1 mL min -1 . The results are shown in Figure 2 . From Figure 2 it can be seen that compared with the control group, new absorption peaks appeared in the reaction group; this fully shows that ScRhaGT can accurately recognize podophyllotoxin compounds and undergo enzymatic reactions.

[0067] To further confirm the above results, the inventors characterized and analyzed the obtained reaction products by high-resolution mass spectrometry (HR-MS), as shown in Figure 3-17 . Figure 3-17 They are the mass spectrometry diagrams of the products obtained after rhamnosylation modification of dehydrodesmethylpodophyllotoxin, dehydropodophyllotoxin, podophyllopicrin, podophyllotoxin, demethylepipodophyllotoxin, etoposide, teniposide, 4α-S-5-fluorobenzoxazole-demethylepipodophyllotoxin, 4β-S-5-fluorobenzoxazole-demethylepipodophyllotoxin, 4α-S-5-fluorobenzothiazole-demethylepipodophyllotoxin, 4β-S-5-fluorobenzothiazole-demethylepipodophyllotoxin, 4α-S-thiadiazole-demethylepipodophyllotoxin, 4β-S-thiadiazole-demethylepipodophyllotoxin, 4α-S-triazole-demethylepipodophyllotoxin, 4β-S-triazole-demethylepipodophyllotoxin. From Figure 3 - Figure 17 it can be seen that the [M + H] of the differential peak +They are all consistent with the theoretical molecular weights of the corresponding receptor rhamnosides. This indicates that the above substrates have all been successfully modified with rhamnose groups.

[0068] Example 3: Detection of the acceptor promiscuity of ScRhaGT

[0069] In addition to selecting dTDP-Rha (rhamnose) as the glycosyl donor, in this example, dTDP-Glc (glucose), dTDP-Gal (galactose), UDP-Rha (rhamnose), and dTDP-Man (mannose) were used to explore the acceptor promiscuity of the glycosyl donors of ScRhaGT. The enzymatic reaction conditions and HPLC detection conditions were the same as those in Example 2, and the acceptor substrate was dehydro-podophyllotoxin. See Figure 18 . From Figure 18 It can be seen that when using dTDP-Gal, dTDP-Glc, and UDP-Rha as glycosyl donors for the reaction, new chromatographic peaks appeared in the reaction groups. This indicates that ScRhaGT can catalyze the enzymatic reaction of the aforementioned donors with podophyllotoxin compounds.

[0070] Similarly, in order to further confirm the above results, the inventors characterized and analyzed the obtained reaction products by high-resolution mass spectrometry (HR-MS). See Figure 19 - Figure 21 . From Figure 19 It was observed that for the reaction group with dTDP-Gal as the substrate, the [M+H] + of the differential peak was 559.5321, which was consistent with the theoretical molecular weight of dehydro-podophyllotoxin galactoside. From Figure 20 It was observed that when using dTDP-Glc as the glycosyl donor, the [M+H] + of the new peak in the reaction was 559.5325, which was consistent with the theoretical molecular weight of dehydro-podophyllotoxin glucoside. From Figure 21 It was observed that when using UDP-Rha as the glycosyl donor, the [M+H] + of the new peak in the reaction was 557.1675, which was consistent with the theoretical molecular weight of dehydro-podophyllotoxin rhamnoside. The above results indicate that ScRhaGT not only exhibits broad acceptor promiscuity for glycosyl acceptors, but also can utilize other glycosyl donors in addition to dTDP-Rha, showing a certain degree of donor promiscuity, which has important practical application value.

[0071] Example 4: Detection of the activity of mutants of ScRhaGT

[0072] To verify whether the mutants of ScRhaGT have enzymatic activity, in this example, a site-directed mutagenesis strategy was used to mutate some amino acids of ScRhaGT. It is well known to those skilled in the art that: (1) the sites around the enzyme-substrate binding pocket are crucial for the catalytic activity of the enzyme; (2) introducing large hydrophobic side chains into the substrate binding pocket may hinder the entry of the substrate or change the binding mode. Therefore, the larger the side chain volume, the greater the negative impact on its catalytic activity. Based on this, the inventors selected R59, E63, D116, and M223, which are crucial for catalytic activity around the substrate binding pocket of ScRhaGT, for mutation, and replaced alanine with the smallest side chain in the amino acid with phenylalanine with a larger steric hindrance, a hydrophobic amino acid, to obtain mutants D116A, E63A, M223F, R59A, and R59F. Among them, the amino acid sequence of mutant D116A is shown in SEQ ID NO:3; the nucleotide sequence is shown in SEQ ID NO:4. The amino acid sequence of mutant E63A is shown in SEQ ID NO:5; the nucleotide sequence is shown in SEQ ID NO:6. The amino acid sequence of mutant M223F is shown in SEQ ID NO:7; the nucleotide sequence is shown in SEQ ID NO:8. The amino acid sequence of mutant R59A is shown in SEQ ID NO:9; the nucleotide sequence is shown in SEQ ID NO:10. The amino acid sequence of mutant R59F is shown in SEQ ID NO:11; the nucleotide sequence is shown in SEQ ID NO:12.

[0073] The activity of the ScRhaGT mutants was verified using an in vitro enzymatic reaction system. The conditions for the enzymatic reaction and HPLC detection were the same as those in Example 2, and the acceptor substrate was dehydrodesmethylpodophyllotoxin. For details, see Figure 22 . From Figure 22 it can be seen that compared with the wild type of ScRhaGT, all the aforementioned mutants retained the rhamnosylation activity towards the substrate dehydrodesmethylpodophyllotoxin. Except that mutant D116A tended to produce the C-ring rhamnoside P2, the product ratios of mutants R59A, R59F, E63A, and M223F did not change significantly compared with the wild type. Lifting the lighter to illustrate the heavier, in this example, the most important sites for catalytic activity have been selected and mutated in the most unfavorable way for catalytic activity, and the mutants still have the corresponding enzymatic activity. Therefore, those skilled in the art can draw the conclusion that substitution, deletion, or addition of one or several amino acids has little effect on the catalytic activity of the rhamnosyltransferase ScRhaGT.

[0074] In summary, the rhamnosyltransferase ScRhaGT provided by the present application realizes the transfer of rhamnose from dTDP-L-rhamnose to podophyllotoxin compounds. Moreover, the rhamnosyltransferase ScRhaGT has a broad substrate spectrum and can obtain various rhamnosylated products using a variety of podophyllotoxin compounds as substrates. Therefore, the rhamnosyltransferase ScRhaGT described in the present application fills the technical gap in the enzymatic rhamnosylation modification of the C-ring 4-OH or E-ring 4'-OH of podophyllotoxin compounds, overcomes the technical problems of hydroxyl protection and deprotection required by chemical methods, and has a milestone significance for the industrial application of the enzymatic synthesis of rhamnosylated podophyllotoxin compounds. In addition, the rhamnosyltransferase can not only be applied to rhamnosylation modification, providing a tool enzyme for the synthesis of rhamnosylated compounds, but also be applied to glucosylation modification and galactosylation modification, having a certain degree of donor generality and important practical application value.

Claims

1. Rhamnosyltransferase ScRhaGT, characterized in that: The amino acid sequence of the rhamnosyltransferase ScRhaGT is selected from the following (1), (2), or (3): (1) The amino acid sequence shown in SEQ ID NO:1; (2) The amino acid sequence shown in SEQ ID NO:1, which has been substituted, deleted, or added with one or several amino acids and has the activity of catalyzing the binding of a rhamnose donor to the C-ring -OH or E-ring -OH of podophyllotoxin compounds; (3) The amino acid sequence having a homology of ≥90% with the amino acid sequence shown in SEQ ID NO:1, and the expressed protein has the activity of catalyzing the binding of a rhamnose donor to the C-ring -OH or E-ring -OH of podophyllotoxin compounds.

2. The rhamnosyltransferase ScRhaGT according to claim 1, wherein: The nucleotide sequence encoding the rhamnosyltransferase ScRhaGT is selected from the following (1), (2), (3), or (4): (1) The nucleotide sequence shown in SEQ ID NO:2; (2) The nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO:2, but encoding the amino acid sequence shown in SEQ ID NO:1; (3) The nucleotide sequence having a homology of ≥90% with the nucleotide sequence shown in SEQ ID NO:2, and the expressed protein has the activity of catalyzing the binding of a rhamnose donor to the C-ring -OH or E-ring -OH of podophyllotoxin compounds; (4) The nucleotide sequence complementary to the nucleotide sequence described in any one of (1), (2), or (3).

3. An expression vector, characterized in that: An expression vector containing the nucleotide sequence encoding the rhamnosyltransferase ScRhaGT as claimed in claim 2.

4. Use of the expression vector as claimed in claim 3 in expressing the rhamnosyltransferase ScRhaGT.

5. The cloning and expression method of rhamnosyltransferase ScRhaGT according to claim 1 or 2, characterized in that: Comprising the following steps: cloning the nucleotide sequence encoding the rhamnosyltransferase ScRhaGT into an expression vector to construct an expression vector; then transferring the expression vector into an expression system for protein expression; and finally obtaining the rhamnosyltransferase ScRhaGT through purification.

6. Use of the rhamnosyltransferase ScRhaGT as claimed in claim 1 or 2 in the preparation of glycosylated podophyllotoxin compounds.

7. The application according to claim 6, wherein: The glycosylation modification is rhamnosylation modification, glucosylation modification, or galactosylation modification.

8. The application according to claim 7, wherein: The rhamnosylation modification is to bind a rhamnose donor to the C-ring -OH or E-ring -OH of a podophyllotoxin compound; the rhamnose donor is dTDP-L-rhamnose or UDP-L-rhamnose.

9. The application according to any one of claims 6-8, characterized in that: The podophyllotoxin compounds are dehydrodesmethylpodophyllotoxin, dehydropodophyllotoxin, podophyllin, podophyllotoxin, demethylepipodophyllotoxin, etoposide, teniposide, 4α-S-5-fluorobenzoxazole-demethylepipodophyllotoxin, 4β-S-5-fluorobenzoxazole-demethylepipodophyllotoxin, 4α-S-5-fluorobenzothiazole-demethylepipodophyllotoxin, 4β-S-5-fluorobenzothiazole-demethylepipodophyllotoxin, 4α-S-thiadiazole-demethylepipodophyllotoxin, 4β-S-thiadiazole-demethylepipodophyllotoxin, 4α-S-triazole-demethylepipodophyllotoxin, or 4β-S-triazole-demethylepipodophyllotoxin.

10. A method for preparing podophyllum rhamnoside by using the rhamnosyltransferase ScRhaGT as described in claim 1 or 2.

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