Whole-cell catalytic body for synthesizing epothilone B glucoside compound as well as preparation and application of whole-cell catalytic body

Abstract

The invention discloses a whole-cell catalytic body for synthesizing an epothilone B glucoside compound as well as preparation and application of the whole-cell catalytic body, and belongs to the field of microbial technology application. According to the present invention, the fusion protein of the N terminal of the ice nucleation protein and the glycosyl transferase BsGT-1 is displayed on the cell surface of Escherichia coli BL21 (DE3) so as to construct the INP-BsGT-1 whole-cell catalysis body; in a Tris-HCl buffer solution, the catalytic body is used for catalyzing epothilone B and UDP-glucose to carry out glycosylation reaction, and epothilone B 7-O-beta-D-glucoside (compound 1) is efficiently synthesized in one step. After the reaction is finished, the catalytic body is recovered and recycled for multiple times through simple centrifugation, and the activity retention rate is still higher than 80% after 8 times of circulation. The activity retention rate of the INP-BsGT-1 cell catalytic body is still higher than 97% after the INP-BsGT-1 cell catalytic body is stored at-80 DEG C for 180 days.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology application, specifically relating to a whole-cell catalyst for the synthesis of epothilone B glucosinolates and its preparation and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Fibrous cysts ( Sorangium cellulosum *Cyclocarya*, belonging to the order Cyclocaryales, family Cyclocaryaceae, and genus *Cyclocarya*, is a group of Gram-negative bacteria widely distributed in soil. It produces abundant secondary metabolites. Epothilone is a 16-membered macrocyclic lactone compound derived from *Cyclocarya fibrinosus*. In terms of mechanism of action, epothilone is similar to paclitaxel, both promoting tubulin polymerization and stabilizing microtubules, thereby inhibiting tumor cell proliferation. However, epothilone exhibits stronger inhibitory activity against multidrug-resistant tumor cells and has the potential for large-scale fermentation production, thus being considered a powerful alternative to paclitaxel. This class of compounds mainly includes two natural products: epothilone A and epothilone B, with the following structural formulas: The structural formula for epothilone is (R = H, epothilone A; R = CH3, epothilone B).

[0004] Epothilone has a simpler chemical structure than paclitaxel, with its core structure consisting of a 16-membered macrocyclic lactone, a three-membered oxygen ring, and a thiazole side chain. Analogs of this compound typically share the same basic skeleton, but epothilone B has an additional methyl group at the 12-carbon position compared to epothilone A, significantly enhancing its antitumor activity. In 2007, the epothilone B derivative, ixabepilone, was approved by the US FDA for the treatment of breast cancer. However, epothilone B and its analogs still have significant limitations, including strong neurotoxicity, hematologic toxicity, and poor water solubility, severely restricting their clinical application.

[0005] The inventors previously successfully synthesized epothilone B glucoside in vitro using the glycosyltransferase BsGT-1 via enzymatic catalysis. This method offers advantages over traditional organic synthesis, which is cumbersome, produces numerous byproducts, and is environmentally unfriendly. However, this method still relies on the use of free enzymes, presenting several significant bottlenecks: First, enzyme purification is cumbersome and costly, and the activity and stability of the purified enzyme are easily reduced. Second, free enzymes are difficult to recover and reuse, leading to low catalytic efficiency and increased production costs. Furthermore, the free enzyme BsGT-1 readily catalyzes the formation of large amounts of low-biological-activity disaccharides (compounds 2 and 3) or trisaccharides (compound 4), reducing the target yield of highly active monosaccharides and impacting production efficiency. These limitations restrict the large-scale preparation and application of epothilone B glucoside.

[0006] Therefore, developing low-cost, efficient, green, and recyclable biocatalytic strategies to achieve enzyme recycling and process economy, as well as the efficient synthesis of epothilone B glucoside, has become an urgent need in this field of research. Summary of the Invention

[0007] To address the problems of high cost, low efficiency, and difficulty in scaling up the preparation of epothilone B glucoside, this invention aims to provide a whole-cell catalyst for the synthesis of epothilone B glucoside compounds, as well as its preparation and application. This meets the urgent need for low cost, high efficiency, ease of operation, and environmental friendliness in the industrial production of epothilone B glucoside, and lays the foundation for subsequent drug development and application research. Specifically, this invention achieves the synthesis of epothilone B 7- in epothilone B glucoside compounds. O -β-D-glucosinolate (compound 1) was synthesized efficiently, reducing the yield of less bioactive disaccharides (compounds 2 and 3) or trisaccharides (compound 4).

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a whole-cell catalyst for the synthesis of epothilone B glucoside compounds, wherein the whole-cell catalyst InaB-BsGT-1 is obtained by displaying a fusion protein of the N-terminus of ice nucleation protein InaB (INP) and glycosyltransferase BsGT-1 on the surface of Escherichia coli BL21(DE3) cells.

[0009] Furthermore, the protein sequence of the ice nucleus protein (Uniprot ID: B2D1X8) has been published. Specifically, it can be obtained by extracting the genome of P. borealis DL7, designing primers (F-NdeI-InaB:CATATGAACGATGACAAAGTTTTGGT; R-BamHI-InaB:GGATCCCACCGCTGTCTCCAGCGTTT), and forming a pET28a-InaB recombinant plasmid through double enzyme digestion and ligation. The recombinant vector is then transformed into E. coli BL21(DE3) to induce expression.

[0010] Furthermore, the glycosyltransferase BsGT-1 (GenBank: CUB50191) has been disclosed in patent CN 111138444 B. The protein sequence of glycosyltransferase BsGT-1 (CUB50191) has been published. It was obtained by extracting the genome of B. subtilis JRS11, designing primers (F-BamHI: CGCGGATCCATGAAAAAGTACCATATTTCGAT; R-SalI: ACGCGTCGACTTACTGCGGGACAGCGGATTTTT), performing double enzyme digestion and ligation to form the pET28a-BsGT-1 recombinant plasmid, and transforming the recombinant vector into E. coli BL21(DE3) for induced expression.

[0011] The protein sequence of InaB-BsGT-1 is shown in SEQ ID NO:2.

[0012] Secondly, the present invention provides a method for preparing the whole-cell catalyst for the synthesis of epothilone B glucoside compounds, comprising the following steps: The N-terminus of the ice nucleoprotein InaB and the glycosyltransferase BsGT-1 were fused via gene synthesis to form the fusion protein InaB-BsGT-1, which was then linked to the multiple cloning sites NdeI and BamHI on the pET28a plasmid to form a new... pET28a- InaB-N-BsGT-1 The recombinant plasmid was obtained by transforming Escherichia coli BL21(DE3) with the recombinant vector and inducing expression.

[0013] pET28a-INP-BsGT-1 The gene sequence of the recombinant plasmid is shown in SEQ ID NO:1.

[0014] Furthermore, pET28a-INP-BsGT-1The recombinant plasmid was transformed into *E. coli* BL21(DE3), and the transformants were transferred to 1–3 mL of LB medium supplemented with kanamycin (final concentration 35–45 μg / mL) and cultured at 100–500 r / min (preferably 200 r / min) at 35–40 °C (preferably 37 °C) for 10–15 h (preferably 12 h). Then, 1–3 mL of the seed culture was transferred to LB medium (with 50–150 μL of kanamycin supplemented at a final concentration of 35–45 μg / mL) and cultured at 35–40 °C (preferably 37 °C) for expansion. When the OD value reached 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.05–0.2 mM (preferably 0.1 mM), and then transferred to a shaker at 10–20 °C (preferably 16 °C) and cultured at 100–500 r / min (preferably 200 r / min). The cells were cultured at 2000-6000 r / min for 5-15 min at 0-10℃ (preferably 4℃) for 12-30 h (preferably 24 h) and collected by centrifugation.

[0015] Thirdly, the present invention provides the application of the above-mentioned whole-cell catalyst for synthesizing epothilone B glucosides in the whole-cell conversion to prepare epothilone B glucosides or products containing epothilone B glucosides.

[0016] Epothilone B glucosides include compound 1, compound 2, compound 3, and compound 4.

[0017] Epothilone B glucosides are monosaccharides and disaccharides of epothilone B glucose. The trisaccharide compound, wherein the epothilone B glucoside compound is compound 1: epothilone B 7-O-β-D glucoside; or compound 2: epothilone B 7-O-β-D-glucosyl-(1→3)-β-D glucoside; or compound 3: epothilone B 7-O-β-D-glucosyl-(1→2)-β-D glucoside; or compound 4: epothilone B 7-O-β-D-glucosyl-(1→2)-β-D-glucosyl-(1→4)-β-D glucoside; the structural formulas, linkages, and corresponding names of the compounds are as follows: .

[0018] Table 1

[0019] Fourthly, the present invention provides a method for preparing epothilone B glucoside compounds by whole-cell transformation, comprising the following steps: The whole-cell catalyst used to synthesize epothilone B glucosides was added to a system containing the substrates epothilone B and UDP-glucose to prepare epothilone B glucosides by in vitro glycosylation reaction.

[0020] In one or more embodiments, an in vitro glycosylation reaction of epothilone B with UDP-glucose was catalyzed using a cell catalyst in a Tris-HCl buffer solution environment.

[0021] Furthermore, the buffer system contains Mg 2+ The Tris-HCl buffer solution has a concentration of 40–60 mM, a pH of 7–7.5, and contains 5–15 mM MgCl2.

[0022] Furthermore, the conditions for the glycosylation reaction are as follows: the reaction temperature is 30~45℃, preferably 35~40℃, more preferably 37±1℃, and the reaction time is 0.5~5 h, preferably 1~3 h, more preferably 2±0.5 h.

[0023] Furthermore, the molar ratio of epothilone B to UDP-glucose is (0.5~1.5):(1.5~3), preferably (0.8~1.2):(1.8~2.2), and most preferably 1:2.

[0024] Furthermore, the ratio of Tris-HCl buffer to epothilone B is (1~3 ml):(5~15 mM).

[0025] Furthermore, the amount of the whole-cell catalyst used is 5-15 OD. 600 Preferably, 8~12 OD 600 The optimal value is 10 OD. 600 .

[0026] In one or more embodiments, after the in vitro glycosylation reaction is completed, the whole-cell catalyst and the product are rapidly separated by centrifugation, and the glycoside product is obtained by simple and rapid purification from the reaction solution.

[0027] Furthermore, this invention does not specifically limit the centrifugation conditions, with the aim of achieving centrifugal recovery of cell catalysts. Further, the centrifugation conditions can be: a rotation speed of 2000~6000 rpm / min and a time of 5~20 min.

[0028] Further, after centrifugation, the supernatant sample was evaporated to dryness by rotary evaporation, and the product was resuspended in acetonitrile. After centrifugation again, the product was separated and purified by semi-preparative liquid chromatography. The chromatographic column used was a YMC-Pack Pro C18, 250 mm × 10.0 mm, 5 μm; the mobile phase consisted of a gradient elution system of 10%–90% acetonitrile-water. This invention does not specify the conditions for the second centrifugation; the purpose is to achieve centrifugal recovery of cellular catalysts. The conditions for the second centrifugation were: rotation speed of 2000–6000 rpm / min, and time of 5–20 min.

[0029] Furthermore, the elution times of epothilone B glucoside were as follows: compound 1: 15–15.5 min, compound 2: 13.6–14.2 min, compound 3: 12.5–12.8 min, and compound 4: 12.2–12.45 min. The separated compounds were evaporated to dryness, dissolved in CD3OD, and then identified using UHPLC-ESI-Q-TOF high-resolution mass spectrometry and nuclear magnetic resonance, respectively.

[0030] Fifthly, the present invention provides the use of the epothilone B glucoside compounds prepared by the above-described whole-cell transformation method in the preparation of medicaments for the prevention and / or treatment of cancer.

[0031] In a sixth aspect, the present invention provides a method for preventing and / or treating cancer, wherein the epothilone B glucoside compound prepared by the above-described whole-cell transformation method is applied to the recipient.

[0032] One or more of the above technical solutions have the following advantages or beneficial effects: This invention utilizes ice nucleation protein to display the glycosyltransferase BsGT-1 on the cell surface of *E. coli* BL21(DE3), constructing a whole-cell catalyst of INP-BsGT-1. In Tris-HCl buffer (pH 7.5, 10 mM MgCl2), at 37°C, this catalyst catalyzes the glycosylation reaction of epothilone B with UDP-glucose, achieving a one-step, highly efficient synthesis of epothilone B 7- O-β-D-glucoside (compound 1). After the reaction, the catalyst can be recovered and recycled multiple times by simple centrifugation. After 8 cycles, the activity retention rate is still higher than 80%. The INP-BsGT-1 cell catalyst retains an activity retention rate of more than 97% after being stored at -80℃ for 180 days. This invention overcomes the bottlenecks of poor stability and difficulty in recovery of free enzymes, avoids the cumbersome steps of chemical synthesis, and has outstanding advantages such as simple operation, mild conditions, reusability, easy separation and purification of products, and environmental friendliness. It provides a reliable solution for the large-scale, low-cost, and green manufacturing of epothilone B glucoside and has the potential for industrial application.

[0033] Compared to the inventor's previous patent CN 111138444 B, this invention discloses a whole-cell preparation method for epothilone B glucoside based on recyclability. This method utilizes the cell surface display of the glycosyltransferase BsGT-1 to form a whole-cell catalyst, achieving efficient synthesis of epothilone B glucoside in a single step under mild conditions. It eliminates the need for cumbersome chemical reactions in organic synthesis and avoids the bottlenecks of poor stability and difficulty in recycling of free enzymes, achieving simple and efficient whole-cell biotransformation. The reaction system is carried out under controlled in vitro conditions, effectively avoiding the complex regulatory interference of in vivo synthesis. Furthermore, the small number of components involved in the reaction greatly promotes raw material recovery and product separation and purification. The overall process is green and economical, suitable for future large-scale industrial applications. The cell catalyst constructed in this invention can achieve the simple, rapid, and large-scale synthesis of epothilone B glucoside. Faced with the current severe situation of cancer and drug-resistant bacterial infections, the large-scale production of highly active anticancer drugs with novel mechanisms of action has become an urgent clinical need. The efficient whole-cell preparation method of epothilone B glucoside provided by this invention is expected to further promote the industrialization of novel epothilone glycoside drugs, and has significant social and economic benefits. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 The HPLC chromatogram shows the reaction solution of the cellular catalyst INP-BsGT-1 of this invention glycosylation modification with epothilone B; wherein, the peak time of compound 1 is 15.2 min; and the peak time of epothilone B standard is 19.4 min. Figure 2 This is a curve showing the change in the activity retention rate of the INP-BsGT-1 catalytic cell body over time. Figure 3This is a bar chart evaluating the recyclability of the INP-BsGT-1 catalytic cell body of the present invention. Figure 4 Compound 1 (epotassium B 7-) prepared in this invention O High-resolution mass spectrum of β-D-glucosinolate, with quasi-molecular ion peak [M + H]. + for m / z 670.3131. Detailed Implementation

[0036] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Recombinant plasmid of ice nucleoprotein N-terminal glycosyltransferase BsGT-1 pET28a-INP-BsGT-1 Acquisition and preparation of INP-BsGT-1 catalyzed cell bodies The inventors have experimentally screened and identified the glycosyltransferase BsGT-1 (GenBank: CUB50191) and its ice nucleus protein sequence (Uniprot ID: B2D1X8) capable of efficiently glycosylating epothilone B, which has been published. Specifically, the inventors have screened and identified the glycosyltransferase BsGT-1 (GenBank: CUB50191) capable of efficiently glycosylating epothilone B, and its gene and protein sequences have been published (see SEQ ID NO:1 and SEQ ID NO:2 at the end of Example 1 for details). The inventors performed a phylogenetic analysis of 8261 glycosyltransferases involved in polyketide catalysis in the CAZy database and discovered a subbranch under the macrolide I branch, containing 160 glycosyltransferase sequences. Further studies have demonstrated that the glycosyltransferases in this subbranch possess significant glycosylation activity against epothilone. In particular, the glycosyltransferase BsGT-1 exhibited optimal enzymatic properties, capable of completely converting epothilone to epothilone glycosides, becoming the first glycosyltransferase capable of achieving complete conversion of epothilone glycosides. The inventors identified an ice nucleus protein sequence capable of efficiently guiding the target protein to the outer cell membrane, namely the N-terminal domain of the ice nucleus protein InaB from Pseudomonas borealis (its UniProt ID: B2D1X8 and protein sequence have been published, see the end of Example 1 for details). This N-terminal sequence was proven to serve as a highly efficient protein transport signal for displaying recombinant proteins on the surface of E. coli cells.

[0039] The inventor obtained and linked the gene through gene synthesis. pET28a-BsGT-1 New plasmids are formed on the recombinant plasmid. pET28a-INP-BsGT-1 The recombinant plasmid was obtained by transforming Escherichia coli BL21(DE3) with the recombinant vector and inducing expression. pET28a-INP-BsGT-1 The recombinant plasmid was transformed into *E. coli* BL21(DE3), and the transformants were transferred to 2 mL of LB medium supplemented with kanamycin (final concentration 40 μg / mL). The culture was incubated at 37°C for 12 h at 200 rpm. Then, 2 mL of the seed culture was transferred to 100 mL of LB medium (final concentration of kanamycin 40 μg / mL) for scale-up culture at 37°C. When the OD value reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM. The culture was then transferred to a shaker at 16°C and incubated at 200 rpm for 24 h. The cells were then collected by centrifugation at 4000 rpm for 10 min at 4°C. The collected cells were resuspended in Tris-HCl buffer (50 mM Tris–HCl, pH 7.5), and the *E. coli* BL21(DE3) assay was performed. pET28a-INP- BsGT-1 OD of catalyst 600 Prepared to 100 OD 600 The catalyst was prepared under the following conditions: In a solution of 2 mL Tris-HCl buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2), the cellular catalyst of glycosyltransferase INP-BsGT-1 (dosage: 10 OD) was used. 600 An in vitro glycosylation reaction catalyzed by epothilone B (10 mM) and UDP-glucose (20 mM) was conducted. The reaction mixture was incubated at 37 ± 1 °C for 2 ± 0.5 h, centrifuged at 4000 rpm for 10 min to recover the cell catalyst (the cell catalyst was washed twice with Tris-HCl buffer before use for the next round of in vitro glycosylation reaction). The supernatant sample was evaporated to dryness by rotary evaporation, the product was resuspended in acetonitrile, centrifuged again at 14000 rpm for 30 min, and the product was purified by semi-preparative liquid chromatography. Figure 1The chromatographic column size was YMC-Pack Pro C18, 250 mm × 10.0 mm, 5 μm; the mobile phase system was a gradient elution of 10%-90% acetonitrile-water system. The peak times of epothilone B glucoside (compound 1) were: compound 1: 15.2 min, compound 2: 13.9 min, compound 3: 12.6 min, and compound 4: 12.4 min. Compound 1 was evaporated to dryness, dissolved in CD3OD, and then identified using UHPLC-ESI-Q-TOF high-resolution mass spectrometry and nuclear magnetic resonance, respectively.

[0040] pET28a-InaB-N-BsGT-1 The gene sequence is shown in SEQ ID NO:1 below. The bold black text represents the InaB gene sequence, the normal black text represents the BsGT-1 gene sequence, the underlined portion represents the restriction enzyme sites, and the italicized portion represents the linker gene sequence. The assembly order is: "restriction site + InaB gene sequence + linker gene sequence + BsGT-1 gene sequence + restriction enzyme site".

[0041] CATATG AACGATGACAAAGTTTTGGTCTTGCGCACCTGTGCCAATAACATGGCCGATCACTGCGGCCAGATATGGCCTGTTTCCGGTGTTGTCGAATGTAAATATTGGGAACCCACCCGAAAGCTCGAGAATGGGCTGGCCGGGCTGCTATGGGGCAAAGGGGCGAGCACGCATTTGAATATGCAGGCTGACGCCCGGTGGGTTATTTGTGAAGTTGCGGTGAGCGATATCATCTTTCTGGATGCCGCAGGGC GGGGTCAAGTTTCCGCGTGCTGAAGTTGTTCACGTCGGCACAAGAAACAGCGCGGCGGGCTATATTTCGGCGAATATTGCCAGTTATGCGTCTTCCACAGTTGCGTTGAATGAAACATTTGTTTTTCCTGAAGTTCGCACAGAAACGAAGGTGGATTTCCCCGCTTCGCCCGCGACCGCTGATAGCACTTTTGATTTGATCGACACGCAACTATTCAAGGCCCACAAACGCTGGAGACAGCGGTG GGGAGC GGATCC (SEQ ID NO:1).

[0042] The InaB-BsGT-1 protein sequence is shown in SEQ ID NO:2 below. The bold black text represents the InaB protein sequence, the normal black text represents the BsGT-1 protein sequence, and the underlined portion represents the linker protein sequence. The assembly order is "InaB protein sequence + linker protein sequence + BsGT-1 protein sequence".

[0043] MNDDKVLVLRTCANNMADHCGQIWPVSGVVECKYWEPTRKLENGLAGLLWGKGASTHLNMQADARWVICEVAVSDIIFLDAQGGVKFPRAEVVHVGTRNSAAGYISANIASYASSTVALNETFVFPEVRTETKVDFPASPATADSTFDIDRHATIQGPQTLETAV GS MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDDQ PDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRFC FVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ (SEQ ID NO:2).

[0044] Example 2: Determination of the retention rate of INP-BsGT-1 catalytic cell body activity over time This embodiment aims to evaluate the stability of the INP-BsGT-1 catalytic cell bodies constructed in Example 1 during long-term storage, providing key data support for its large-scale preparation and application. The INP-BsGT-1 catalytic cell bodies prepared in Example 1 were collected, resuspended in Tris-HCl buffer (50 mM Tris-HCl, pH 7.5, 20% glycerol), and stored at -80°C. The catalytic cell bodies were retrieved at 1, 7, 30, 60, 90, 120, 150, and 180 days after storage, and glycosylated using epothilone B as a substrate, according to the catalytic reaction conditions described in Example 1. The conversion rate of the product epothilone B glucoside was determined by high-performance liquid chromatography (HPLC) as the activity retention rate at that time point. The INP-BsGT-1 catalytic cell bodies exhibited excellent storage stability at different storage times. During a six-month storage period (180 days), the activity retention rate remained above 97%, maintaining excellent catalytic activity throughout the entire testing period. Figure 2 This result demonstrates that the whole-cell catalyst constructed using cell surface display technology effectively overcomes the bottlenecks of poor stability and easy inactivation of free enzymes. Its high stability ensures that the catalyst can be readily used for efficient biotransformation over a long storage period, which is of great significance for production scheduling and catalyst storage in actual production.

[0045] This embodiment successfully verified that INP-BsGT-1 catalyzes excellent storage stability in cell bodies. This characteristic is highly consistent with the "high stability" goal pursued by this invention. Combined with its efficient and green reaction characteristics, it further highlights the great industrial application prospects of this whole-cell preparation method in the future large-scale production of epothilone B glucoside.

[0046] Example 3 Evaluation of the recyclability of INP-BsGT-1 catalytic cell bodies This embodiment aims to evaluate the stability of the INP-BsGT-1 catalytic cell body constructed in Example 1 during repeated use, providing crucial evidence for its industrial cyclic application. The INP-BsGT-1 catalytic cell body prepared in Example 1 was subjected to the glycosylation reaction of epothilone B under the conditions described in Example 1. After the reaction, the catalytic cell body was recovered by centrifugation at 4°C and 4000 rpm / min for 10 minutes. After washing twice with Tris-HCl buffer (50 mM Tris-HCl, pH 7.5), it was reintroduced into the next round of reaction system for cyclic catalytic experiments. After each reaction, the conversion rate of epothilone B glucoside was determined by high-performance liquid chromatography (HPLC) to assess changes in catalytic activity.

[0047] The results showed that ( Figure 3The INP-BsGT-1 catalytic cell body exhibited good reusability. In the first four cycles, its catalytic activity remained above 95% without significant decline. With increasing cycle number, the catalytic activity showed a slow decreasing trend, but the conversion rate remained above 80% even in the eighth cycle, indicating that the catalytic cell body possesses strong structural stability and tolerance, enabling it to withstand multiple catalytic reactions.

[0048] The results show that the whole-cell catalyst of the present invention not only has high catalytic efficiency, but also excellent stability. It can be efficiently recovered and reused through simple centrifugation, which significantly reduces the cost of catalyst use. This further highlights the great advantages of the whole-cell catalytic system in terms of economy and sustainability, and lays a solid foundation for its application in industrial production.

[0049] Example 4: Structural Identification of Compound 1 in accordance with Figure 4 UHPLC-ESI-Q-TOF high-resolution mass spectrometry yielded a quasi-molecular ion peak [M + H] for compound 1. + for m / z 670.3131, thus confirming that compound 1 is a monoglucoside of epothilone B. Simultaneously, the monosaccharide can also be detected from the 1H NMR spectrum of compound 1 (…). 1 H NMR) and carbon spectroscopy ( 13 This was confirmed by C1NMR. In the HMBC spectrum, H-7 correlated with C-1' and H-1' correlated with C-7, thus confirming the glucose group's attachment to the 7-hydroxyl group of the epothilone B macrolide skeleton. The large coupling constant between the protons H-1' and H-2' at the sugar end (…) J 1',2' = 7.8 Hz) and a chemical shift of 4.46 ppm at the high field of the terminal protons revealed that the glycosyl donor and epothilone B receptor are linked by a β-D glucosinolate bond. Therefore, compound 1 is Epothilone B 7- O -β-D glucoside, the specific NMR data are shown in Table 2.

[0050]

[0051] Table 2. NMR data attribution for compound 1

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A whole-cell catalyst for the synthesis of epothilone B glucosides, characterized in that, The whole-cell catalyst InaB-BsGT-1 was obtained by displaying the fusion protein of the N-terminus of the ice nucleoprotein InaB and the glycosyltransferase BsGT-1 on the surface of E. coli cells.

2. The whole-cell catalyst according to claim 1, characterized in that, The protein sequence of the whole-cell catalyst InaB-BsGT-1 is shown in SEQ ID NO:2; Preferably, Escherichia coli BL21 is used.

3. A method for preparing a whole-cell catalyst for synthesizing epothilone B glucoside compounds as described in claim 1 or 2, characterized in that, Includes the following steps: The N-terminus of the ice nucleoprotein InaB and the glycosyltransferase BsGT-1 were fused via gene synthesis to form the fusion protein InaB-BsGT-1, which was then linked to the multiple cloning sites NdeI and BamHI on the pET28a plasmid to form a new... pET28a-InaB-N- BsGT-1 The recombinant plasmid was obtained by transforming Escherichia coli with the recombinant vector and inducing expression.

4. The preparation method according to claim 3, characterized in that, pET28a-INP-BsGT-1 The recombinant plasmid was transformed into *E. coli*, and the transformants were transferred to 1–3 mL of LB medium supplemented with kanamycin at a final concentration of 35–45 μg / mL. The medium was then incubated at 35–40 °C for 10–15 h at 100–500 r / min. Subsequently, 1–3 mL of the seed culture was transferred to LB medium at a final kanamycin concentration of 35–45 μg / mL, and the medium was further cultured at 35–40 °C. When the OD value reached 0.6–0.8, isopropyl-β-D-thiogalactoside was added at a final concentration of 0.05–0.2 mM. The medium was then transferred to a shaker at 10–20 °C and incubated at 100–500 r / min for 12–30 h. The bacterial cells were collected by centrifugation at 0–10 °C. pET28a-INP-BsGT-1 The gene sequence of the recombinant plasmid is shown in SEQ ID NO:

1.

5. The use of the whole-cell catalyst for synthesizing epothilone B glucoside compounds as described in claim 1 or 2, or the whole-cell catalyst for synthesizing epothilone B glucoside compounds obtained by the preparation method described in claim 3 or 4, in the whole-cell conversion to prepare epothilone B glucoside compounds or products containing epothilone B glucoside compounds.

6. The application according to claim 6, characterized in that, Epothilone B glucosides include compound 1, compound 2, compound 3, and compound 4; Compound 1 is epothilone B 7-O-β-D glucoside; Compound 2 is epothilone B 7-O-β-D-glucosyl-(1→3)-β-D glucoside; Compound 3 is epothilone B 7-O-β-D-glucosyl-(1→2)-β-D glucoside; Compound 4 is epothilone B 7-O-β-D-glucosyl-(1→2)-β-D-glucosyl-(1→4)-β-D glucoside.

7. A method for preparing epothilone B glucoside compounds by whole-cell transformation, characterized in that, The whole-cell catalyst for synthesizing epothilone B glucoside compounds, as described in claim 1 or 2, or the whole-cell catalyst for synthesizing epothilone B glucoside compounds prepared by the method described in claim 3 or 4, comprises the following steps: The whole-cell catalyst used to synthesize epothilone B glucosides was added to a system containing the substrates epothilone B and UDP-glucose, and an in vitro glycosylation reaction was carried out to prepare epothilone B glucosides.

8. The method according to claim 7, characterized in that, An in vitro glycosylation reaction of epothilone B with UDP-glucose was catalyzed using a cell catalyst in a Tris-HCl buffer solution environment. Preferably, the buffer system contains Mg 2+ Tris-HCl buffer solution; wherein the concentration of Tris-HCl buffer solution is 40~60 mM, pH 7~7.5, and 5~15 mM MgCl2; Preferably, the conditions for the glycosylation reaction are: a reaction temperature of 30~45℃, preferably 35~40℃, and a reaction time of 0.5~5 h, preferably 1~3 h; Preferably, the molar ratio of epothilone B to UDP-glucose is (0.5~1.5):(1.5~3), more preferably (0.8~1.2):(1.8~2.2), and most preferably 1:2; Preferably, the ratio of Tris-HCl buffer to epothilone B is (1~3 ml):(5~15 mM); Preferably, the amount of the whole-cell catalyst is 5-15 OD. 600 Preferably, 8~12 OD 600 The optimal value is 10 OD. 600 .

9. The method according to claim 7, characterized in that, After the in vitro glycosylation reaction was completed, the whole-cell catalyst and the product were separated by centrifugation, and the reaction solution was purified. Preferably, after centrifugation, the supernatant solution sample is evaporated to dryness by rotary evaporation, then acetonitrile is added to resuspend the product, and after centrifugation again, the product is separated and purified by semi-preparative liquid chromatography. Preferably, the peak elution times of epothilone B glucoside are as follows: the peak elution time of compound 1 is 15~15.5 min, the peak elution time of compound 2 is 13.6~14.2 min, the peak elution time of compound 3 is 12.5~12.8 min, and the peak elution time of compound 4 is 12.2~12.45 min.

10. The use of an epothilone B glucoside compound prepared by the whole-cell transformation method according to any one of claims 7 to 9 in the preparation of a medicament for the prevention and / or treatment of cancer.

Images