Genetically engineered bacterium and method for biosynthesizing rosavin

Abstract

Provided are a genetically engineered bacterium for biosynthesizing rosavin and a method therefor. The method comprises: using a glycosyltransferase having the amino acid sequence set forth in SEQ ID NO: 1 to catalyze a reaction between a substrate rosin and a glycosyl donor to obtain rosavin, wherein the glycosyl donor is UDP-arabinose.

Description

Genetically engineered bacteria and methods for biosynthesizing lorsevier

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510382499.5, filed on March 28, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of biotechnology, and more specifically, to a genetically engineered bacterium and method for the biosynthesis of crosavior. Background Technology

[0004] Rhodiola rosea L., a perennial herb belonging to the genus Rhodiola in the family Crassulaceae, grows in high-altitude, cold regions. As a traditional and rare Chinese medicinal herb, it is recorded in the *Dictionary of Traditional Chinese Medicine*. Studies have shown that Rhodiola rosea extract possesses anti-fatigue, anti-hypoxia, anti-aging, anti-cancer, and cardiovascular protective functions, and has been widely used in the pharmaceutical, food, and health product industries with no reported side effects or drug interactions.

[0005] Rhodiola rosea extract is a functional mixture, and the types of active ingredients vary considerably among different species. The main active ingredients of Rhodiola grandiflora extract are rhodioloside and tyrosol, while the main active ingredients of Rhodiola rosea extract include total crocetin (crocetin, crocetin, crocetin), rhodioloside, and tyrosol. As a unique component of Rhodiola rosea, the content of total crocetin determines the market price of Rhodiola rosea extract, with crocetin being the most abundant component. Furthermore, crocetin has anti-fatigue, anti-hypoxia, and stress-relieving effects, and has broad application prospects and high application value in the pharmaceutical, food, and cosmetic fields.

[0006] Currently, the production of Rhodiola rosea extract mainly relies on direct extraction from the Rhodiola rosea plant. However, its yield is limited by the low content within the plant and by the plant's growth time (slow growth cycle) and location (specific high-altitude growing environment). Although rhodioloside and tyrosol have been extensively studied and their production processes are relatively mature due to their well-established natural synthetic pathways, the synthesis of crosavior is rarely reported. Microbial fermentation is receiving increasing attention due to its high efficiency, sustainability, and economic viability, and highly efficient and specific enzymes are the core biological components for increasing the yield of target products.

[0007] Existing methods for synthesizing roxithion include: using UDP-arabinose and cyclohexane as substrates, a glycosyltransferase transfers the arabinose group from the UDP-arabinose donor to the 6-position glucose hydroxyl group of the cyclohexane acceptor, ultimately generating roxithion. The glycosyltransferase required for roxithion synthesis is an arabinose glycosyltransferase, which belongs to the glycan elongation transferase family.

[0008] Although glycosyltransferases are widely distributed in nature, they exhibit high specificity for the glycosyl groups (glucose, arabinose, or xylose) of glycosyl donors. Currently, approximately 300 glycosyltransferases are known to have specific functions, but only six are involved in arabinose transfer (Liu Yuqian. Construction and Application of Plant UDP-Glycosyltransferase Database [D]. South China University of Technology, 2021.). Regarding the synthesis of rosavin, only Li et al. have obtained four glycosyltransferases derived from *Solanum lycopersicum*, *Nicotiana tabacum*, *Camellia sinensis*, and *Solanum pennellii* by screening the UGT91 family (Li, Lijun, et al. "High-level production of Rhodiola rosea characteristic component rosavin from D-glucose and L-arabinose in engineered Escherichia coli." *Metabolic Engineering* 82(2024):274-285.). However, the synthesis of rosavin using these glycosyltransferases suffers from low yields. Therefore, developing an efficient method for producing roximate is of great significance. Summary of the Invention

[0009] The main objective of this invention is to provide a genetically engineered bacterium and method for biosynthesizing crocetin, in order to solve the problem of low yield of biosynthesized crocetin in the prior art.

[0010] To achieve the above objective, according to a first aspect of the present invention, a method for biosynthesizing rosivate is provided, the method comprising: catalyzing a substrate rosivate and a glycosyl donor using a glycosyltransferase having the amino acid sequence shown in SEQ ID NO: 1 to obtain the aforementioned rosivate; wherein the aforementioned glycosyl donor is UDP-arabinose.

[0011] Furthermore, the glycosyltransferase mentioned above is selected from the crude enzyme solution or purified enzyme solution of the glycosyltransferase mentioned above.

[0012] Further, the above method includes: inoculating overnight recombinant cells into a fermentation medium for expansion culture, culturing until OD600 reaches 0.6-0.8, adding an inducer for induced fermentation for 48-72 hours, and collecting the supernatant product to obtain the above-mentioned rosivate, wherein the above-mentioned recombinant cells can express the above-mentioned glycosyltransferase and can synthesize the above-mentioned UDP-arabinose in vivo; the above-mentioned rosivate is added together with the above-mentioned inducer or the above-mentioned rosivate is added to the above-mentioned fermentation medium.

[0013] Furthermore, the recombinant cells contain the UDP-glucose 6-dehydrogenase encoding gene UGD from Escherichia coli, the UDP-glucuronide decarboxylase encoding gene UXS from Rhizobium sinense from alfalfa, and the glucose-4-epimerase encoding gene galE from Escherichia coli.

[0014] Furthermore, the UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34, the UDP-glucuronide decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35, and the glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36.

[0015] Furthermore, the conditions for the above-mentioned expanded culture are 30℃-37℃ and 200rpm-220rpm.

[0016] Furthermore, the conditions for the above-mentioned induced fermentation are 28℃-30℃ and 200rpm-220rpm.

[0017] Furthermore, the aforementioned inducer is IPTG.

[0018] Furthermore, the final concentration of the aforementioned inducer is 0.1 mM-1 mM.

[0019] Furthermore, the amount of the above-mentioned cypermethrin added is 0.5-1 g / L.

[0020] Furthermore, the above-mentioned fermentation medium formulation includes: glycerol 15-20 g / L, glucose 10-15 g / L, yeast extract 5-10 g / L, disodium hydrogen phosphate 5-8 g / L, potassium dihydrogen phosphate 2-5 g / L, sodium chloride 0.3-0.8 g / L, ammonium chloride 0.5-1.5 g / L, calcium chloride 0.01-0.03 g / L, magnesium sulfate 0.2-0.5 g / L, MOPS 40-50 g / L, and antibiotics 50 mg / L-100 mg / L.

[0021] To achieve the above objectives, according to a second aspect of the present invention, a genetically engineered bacterium is provided, which contains a glycosyltransferase encoding gene having the nucleotide sequence shown in SEQ ID NO: 2, a UDP-glucose 6-dehydrogenase encoding gene UGD derived from Escherichia coli, a UDP-glucuronide decarboxylase encoding gene UXS derived from Rhizobium sinense of alfalfa, and a glucose-4-epimerase encoding gene galE derived from Escherichia coli.

[0022] Furthermore, the UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34, the UDP-glucuronide decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35, and the glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36.

[0023] By applying the technical solution of this invention, the glycosyltransferase of this invention (having the amino acid sequence shown in SEQ ID NO: 1) catalyzes the reaction of the glycosyl donor UDP-arabinose and the substrate cyclohexane to obtain cyclohexane. The yield of cyclohexane is higher than that of other glycosyltransferases in the prior art that catalyze the synthesis of cyclohexane from cyclohexane. This achieves high-yield biosynthetic cyclohexane, has broad application prospects, and has high economic value. Attached Figure Description

[0024] The accompanying drawings, which form part of this specification, 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 undue limitation of the invention. In the drawings:

[0025] Figure 1 shows the synthetic route of the biosynthetic roxavir of the present invention.

[0026] Figure 2A shows the electrophoretic images of proteins from 11 strains of *Escherichia coli* containing glycosyltransferases (UGT2, UGT6, UGT7, UGT9, UGT11, UGT13, UGT14, UGT15, UGT16, UGT31, or UGT36) according to Example 3 of the present invention after induced expression. In the figure, ↑ indicates the electrophoretic image of soluble proteins after induction, and ↓ indicates the electrophoretic image of insoluble proteins after induction.

[0027] Figure 2B shows the electrophoretic images of the proteins from four strains of *E. coli* containing glycosyltransferases (UGT44, UGT38, UGT41, or AtUGT78D3) from different sources after induction expression according to Example 3 of the present invention. In the figure, ↑ represents the electrophoretic image of soluble proteins after induction, and ↓ represents the electrophoretic image of insoluble proteins after induction.

[0028] Figure 2C shows the electrophoretic images of proteins from nine strains of *Escherichia coli* containing glycosyltransferases (AcUGT, GmUGT-1, PtUGT, AtUGT-1, FaUGT, AtUGT-2, CGTb, UGT73P10, or UGT99D1) according to Example 3 of the present invention after induced expression. In the figure, ↑ indicates the electrophoretic image of soluble proteins after induction, and ↓ indicates the electrophoretic image of insoluble proteins after induction.

[0029] Figure 2D shows the electrophoretic images of eight strains of *Escherichia coli* containing glycosyltransferases (GmUGT-2, OsUGT, NtUGT, CsUGT, SpUGT, SIUGT, CIUGT, or ZmUGT) from different sources after induction expression according to Example 3 of the present invention. In the figure, ↑ indicates the electrophoretic image of soluble protein after induction, and ↓ indicates the electrophoretic image of insoluble protein after induction.

[0030] Figure 3 shows the liquid chromatogram of roximate synthesized using the glycosyltransferase FaUGT according to Example 3 of the present invention, wherein (1) is roximate standard, (2) is roximate standard, and (3) is roximate product synthesized using the glycosyltransferase FaUGT of the present invention.

[0031] Figure 4 shows the mass spectrum of cyclophosphamide synthesized using the glycosyltransferase FaUGT according to Example 3 of the present invention, wherein (1) is the cyclophosphamide standard and (2) is the cyclophosphamide product synthesized using the glycosyltransferase FaUGT of the present invention.

[0032] Figure 5 shows the liquid chromatogram of Ros01 synthesized according to the engineered strains Ros01 and Ros02 of Example 5 of the present invention, wherein (1) is Ros01 and (2) is Ros02.

[0033] Figure 6 shows the mass spectrum of Ros01 synthesized according to the engineered strains Ros01 and Ros02 of Example 5 of the present invention, wherein (1) is Ros01 and (2) is Ros02. Detailed Implementation

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0035] Terminology Explanation:

[0036] UGT: Abbreviation for UDP-dependent glycosyltransferase, a type of glycosyltransferase that can transfer glycosyl groups from UDP-sugars to other molecules, thus playing a role in glycosyltransfer.

[0037] As mentioned in the background section, crosene has high application value, but existing methods for synthesizing crosene suffer from low yield. In this invention, the inventors attempted to screen for a highly active glycosyltransferase with strong substrate specificity for efficient crosene production, and thus proposed a series of protective schemes for this invention.

[0038] In a first typical embodiment of the present invention, a method for biosynthesizing crosaviin is provided, the method comprising: catalyzing a reaction between a substrate crosaviin and a glycosyl donor using a glycosyltransferase having the amino acid sequence shown in SEQ ID NO: 1, to obtain the aforementioned crosaviin (see Figure 1).

[0039] The amino acid sequence of the glycosyltransferase FaUGT, SEQ ID NO: 1, is shown below:

[0040] The nucleotide sequence of the gene encoding the glycosyltransferase FaUGT, SEQ ID NO: 2, is shown below:

[0041] In a preferred embodiment of the present invention, the glycosyltransferase is selected from the crude enzyme solution or purified enzyme solution of the glycosyltransferase. Using the crude enzyme solution to synthesize roximate eliminates the protein purification step, reduces the production cost of roximate, and is suitable for industrial production.

[0042] The method for biosynthesizing rosivate of the present invention can utilize crude enzyme solution or be synthesized in vivo by fermentation of recombinant cells containing the encoding gene of the glycosyltransferase described above (having the nucleotide sequence shown in SEQ ID NO: 2) and capable of synthesizing the UDP-arabinose in vivo. In a preferred embodiment of the present invention, recombinant cells cultured overnight are inoculated into a fermentation medium for expansion culture until the OD600 reaches 0.6-0.8. An inducer is added for induced fermentation for 48-72 hours, and the supernatant product is collected to obtain the rosivate described above. The recombinant cells are capable of expressing the glycosyltransferase described above and synthesizing the UDP-arabinose in vivo; the rosivate is added together with the inducer or added directly to the fermentation medium.

[0043] This method for producing crosavior is simple, rapid, and beneficial. The timing of the addition of the substrate chromium is not specifically limited; it can be added directly to the fermentation medium or together with the inducer. To maximize the yield of crosavior, the amount of substrate added should be as sufficient as possible. In a preferred embodiment of this invention, the amount of chromium added is 0.5-1 g / L. Using this amount of substrate contributes to the efficient production of crosavior.

[0044] To enable the recombinant cells to synthesize UDP-arabinose in vivo, in a preferred embodiment of the present invention, the recombinant cells further contain the UDP-glucose 6-dehydrogenase encoding gene UGD from *E. coli*, the UDP-glucuronide decarboxylase encoding gene UXS from *Rhizobium sinense* from alfalfa, and the endogenous glucose-4-epimerase encoding gene galE from *E. coli*. Using the recombinant cells as a substrate, UDP-arabinose is generated from glucose, and then, using UDP-arabinose as a glycosyl donor and exogenous substrate cyclohexane, cyclohexane is used to generate cyclohexane under the action of glycosyltransferases.

[0045] In a preferred embodiment of the present invention, the UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34; the UDP-glucuronide decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35; and the glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36. The UDP-glucose 6-dehydrogenase UGD has the amino acid sequence shown in SEQ ID NO: 31; the UDP-glucuronide decarboxylase UXS has the amino acid sequence shown in SEQ ID NO: 32; and the glucose-4-epimerase GalE has the amino acid sequence shown in SEQ ID NO: 33.

[0046] The above-mentioned fermentation medium formulation includes: glycerol 15-20 g / L, glucose 10-15 g / L, yeast extract 5-10 g / L, disodium hydrogen phosphate 5-8 g / L, potassium dihydrogen phosphate 2-5 g / L, sodium chloride 0.3-0.8 g / L, ammonium chloride 0.5-1.5 g / L, calcium chloride 0.01-0.03 g / L, magnesium sulfate 0.2-0.5 g / L, MOPS 40-50 g / L, and antibiotics 50 mg / L-100 mg / L. Fermentation of the above-mentioned recombinant cells using this medium promotes cell growth and proliferation, and is beneficial for the expression of glycosyltransferases and the supply of UDP-arabinose in vivo, thereby further increasing the yield of croseviper.

[0047] It is important to note that the type of antibiotic used depends on the resistance gene on the plasmid transferred into the genetically engineered bacteria. For example, when the genetically engineered bacteria are transferred with two plasmids containing different resistance genes (e.g., chloramphenicol resistance gene and kanamycin resistance gene), the fermentation medium will contain 50 mg / L-100 mg / L of chloramphenicol and 50 mg / L-100 mg / L of kanamycin. When only one plasmid (e.g., chloramphenicol resistance plasmid or kanamycin resistance plasmid) is transferred into the genetically engineered bacteria, the fermentation medium will contain only 50 mg / L-100 mg / L of chloramphenicol or 50 mg / L-100 mg / L of kanamycin.

[0048] After inoculating recombinant cells into the fermentation medium, scale-up culture is required to increase the number of recombinant cells. The scale-up culture conditions are closely related to the number and growth status of the recombinant cells. Once the number of recombinant cells reaches a certain concentration, induced fermentation culture is necessary. To maximize the production of crosene by the recombinant cells, it is best to culture them under the optimal induced fermentation culture conditions for crosene production. In another preferred embodiment of the present invention, the scale-up culture conditions are 30℃-37℃ and 200rpm-220rpm. In another preferred embodiment of the present invention, the induced fermentation conditions are 28℃-30℃ and 200rpm-220rpm.

[0049] Depending on the expression vector and the expression status of the target protein in recombinant cells, an appropriate concentration of the inducer is selected. In a preferred embodiment of the present invention, the inducer is IPTG. In a more preferred embodiment of the present invention, the final concentration of the inducer is 0.1 mM-1 mM.

[0050] In a second typical embodiment of the present invention, a genetically engineered bacterium is provided, which contains a glycosyltransferase encoding gene having the nucleotide sequence shown in SEQ ID NO: 2, a UDP-glucose 6-dehydrogenase encoding gene UGD derived from Escherichia coli, a UDP-glucuronide decarboxylase encoding gene UXS derived from Rhizobium sinense of alfalfa, and a glucose-4-epimerase encoding gene galE derived from Escherichia coli.

[0051] In a preferred embodiment of the present invention, the UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34, the UDP-glucuronide decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35, and the glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36. The yield of Loosevera produced using the genetically engineered bacteria of the present invention is higher, which is conducive to the industrial production of Loosevera.

[0052] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0053] Example 1: Construction of recombinant Escherichia coli BL21(DE3) / pET28a-UGT

[0054] Looseveratrol is mainly derived from Rhodiola rosea. The inventors considered that Rhodiola rosea likely contains a unique glycosyltransferase that synthesizes looseveratrol. Therefore, the inventors downloaded Rhodiola rosea transcriptome data from NCBI for CDS prediction and performed docking analysis on the predicted glycosyltransferases (a total of 13,000) with lobesin and UDP-arabinose, screening out 14 glycosyltransferases that may synthesize looseveratrol. Based on the codon usage preferences of E. coli, the genes of these 14 glycosyltransferases were optimized and synthesized in the pET28a vector, resulting in 14 pET28a-UGT plasmids (Table 1). All nucleic acids used in this invention were synthesized by Genewiz Biotechnology Co., Ltd.

[0055] Table 1. Glycosyltransferase expression vectors derived from Rhodiola rosea

[0056] Furthermore, through preliminary research and experiments, the inventors discovered that although glycosyltransferases have a certain specificity for glycosyl donors and aglycone acceptors, this specificity is not absolute. Therefore, a search was conducted on glycosyltransferases in Uniprot (a total of 2720), and docking analysis was performed with cyclohexane and UDP-arabinose to screen out 10 glycosyltransferases that may synthesize cyclohexane. Based on the codon usage preferences of *E. coli*, the genes of the above 10 glycosyltransferases were optimized and synthesized in the pET28a vector, resulting in 10 pET28a-UGT plasmids (Table 2).

[0057] Table 2 Uniprot-derived glycosyltransferase expression vectors

[0058] To further expand the possibility of screening for glycosyltransferases that catalyze the synthesis of rosivate, four glycosyltransferases that can catalyze the transfer of arabinose reported in relevant literature and four glycosyltransferases that can catalyze the synthesis of rosivate disclosed in existing technologies were also screened. Based on the codon usage preference of *E. coli*, the above eight glycosyltransferases were optimized and synthesized in the pET28a vector, resulting in eight pET28a-UGT plasmids (Table 3).

[0059] Table 3. Glycosyltransferase expression vectors from literature sources

[0060] The 32 recombinant plasmids shown in Tables 1-3 were transformed into Escherichia coli BL21(DE3) to obtain 32 recombinant Escherichia coli BL21(DE3) / pET28a-UGT strains. The specific transformation method was as follows: BL21(DE3) competent cells (50 μL of competent cells in 1.5 ml EP tubes) were thawed on ice, 50 ng of plasmid was added to the competent cells, mixed well, incubated on ice for 30 min, heat-shocked in a 42℃ water bath for 90 s, 600 μL of recovery medium (LB medium) was added, and incubated at 37℃ for 50 min. After incubation, all bacterial cultures were spread on plates (LB medium) containing 50 mg / L kanamycin and incubated overnight at 37℃.

[0061] Example 2: Verification of Glycosyltransferase Protein Expression

[0062] To determine whether the synthesized glycosyltransferase was expressed normally, protein expression verification was performed. The specific method was as follows: Glycerol strains of 32 engineered strains expressing glycosyltransferase in the BL21(DE3) chassis strain were inoculated at 0.2% volume into test tubes containing 5 mL of LB liquid medium (with 50 mg / L kanamycin). After overnight incubation at 37°C, 1% volume was inoculated into 250 mL shake flasks containing 50 mL of LB liquid medium (with 50 mg / L kanamycin). The flasks were shaken at 37°C until the OD600 reached 0.8. After adding IPTG to a final concentration of 0.1 mM, the flasks were induced and cultured in a shaker at 30°C for 20 hours. 3 mL of bacterial culture was then collected by centrifugation at 12,000 rpm at 4°C. The bacterial cells were resuspended in 1 mL of PBS buffer and sonicated for 5 min. After centrifugation at 12,000 rpm for 10 min, the supernatant protein and precipitated protein were separated and analyzed by SDS-PAGE. The SDS-PAGE results of the 32 engineered strains are shown in Figures 2A, 2B, 2C, and 2D.

[0063] Example 3: Synthesis of Looseveratrol by Liquid Ex vivo Catalysis of Crude Glycosyltransferase

[0064] A crude enzyme solution containing UGT protein was obtained using the 32 recombinant Escherichia coli BL21(DE3) / pET28a-UGT strains constructed in Example 1. The specific method is as follows: Glycerol-containing strains of 32 engineered strains expressing glycosyltransferase in the BL21(DE3) chassis strain were inoculated at a volume of 0.2% into test tubes containing 5 mL of LB liquid medium (with 50 mg / L kanamycin added). After overnight incubation at 37°C, a volume of 1% was inoculated into test tubes containing 50 mL of LB liquid medium. In a 250 mL shake flask containing LB liquid medium (with 50 mg / L kanamycin), the bacteria were shaken at 37 °C until the OD600 reached 0.8. After adding IPTG to a final concentration of 0.1 mM, the culture was induced and cultured in a shaker at 30 °C for 20 hours. 3 mL of the bacterial culture was then collected by centrifugation at 12,000 rpm at 4 °C. The bacterial cells were resuspended in 1 mL of PBS buffer and sonicated for 5 min. After centrifugation at 12,000 rpm for 10 min, the supernatant protein and precipitated protein were separated. The supernatant was the crude enzyme solution.

[0065] The crude enzyme solution was used to prepare the following reaction system: 50 mg / L crosene, 100 mg / L UDP-arabinose, 2 mM MgCl2, 100 μL crude enzyme solution, and 50 mM Tris-HCl (pH 7.5) to a final volume of 500 μL. The system was placed in a 30°C water bath and reacted for 2 hours. Then, 100 μL of ice-cold methanol was added to terminate the reaction. The mixture was then cooled on ice for 2 minutes, centrifuged at 12000 rpm for 5 minutes, and the reaction solution was filtered through a 0.22 μm organic phase filter membrane to prepare a sample. The amount of crosene synthesized in the reaction system was determined by HPLC.

[0066] As shown in Table 4, after the in vitro catalytic reaction, only AcUGT, FaUGT, and ZmUGT showed significant locivime synthesis among the 32 glycosyltransferases. Among them, the glycosyltransferase with the highest activity reported in the prior art is SlUGT from *Solanum lycopersicum*, which can synthesize 15.74 mg / L locivime after 2 hours of reaction. In contrast, FaUGT screened in this invention can synthesize 34.31 mg / L locivime, representing a 2.2-fold increase.

[0067] The liquid chromatography-mass spectra of crosavirin synthesized using FaUGT of this invention are shown in Figure 3, where the retention time of crosavirin is 13.5 min, consistent with the retention time of the crosavirin standard. To further confirm the synthesis of crosavirin, the liquid chromatography sample was concentrated and then subjected to mass spectrometry identification. The LC-MS spectrum is shown in Figure 4, where the molecular weight of crosavirin is 473, consistent with the crosavirin standard. Therefore, it can be determined that the glycosyltransferase FaUGT screened in this invention can catalyze the synthesis of crosavirin from UDP-arabinose and crosavirin, and compared with previously reported glycosyltransferases, the FaUGT screened in this invention has higher catalytic efficiency. Additionally, it should be noted that the relative molecular weight of crosavirin is 428, but due to the presence of formic acid in the mass spectrometry conditions, the crosavirin actually detected is crosavirin with added formate, hence the [M+HCOOH] molecule. - ] - 473.

[0068] Table 4. Synthesis of roxavir from different sources of UGT

[0069] Example 4: Construction of recombinant Escherichia coli producing clopidogrel

[0070] To achieve in vivo UDP-arabinose supply in *E. coli*, the UDP-glucose 6-dehydrogenase encoding gene UGD (from *E. coli*), the UDP-glucuronide decarboxylase encoding gene UXS (from *Rhizobium sinense*), and the glucose-4-epimerase gene galE (from *E. coli*) were placed under the T7 promoter and constructed in the pACYC-Duet-1 vector to obtain the UDP-arabinose-producing plasmid pACYC-UGD-UXS-galE. This plasmid was then introduced into engineered *E. coli* BL21(DE3) / pET28a-FaUGT and BL21(DE3) / pET28a-SlUGT to obtain rosevilate-producing engineered *E. coli* strains Ros01 and Ros02.

[0071] Example 5: Synthesis of Loosevermectin from Recombinant Escherichia coli

[0072] Engineered strains Ros01 and Ros02 were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 1% and cultured at 37°C and 220 rpm for 2 hours. Induction was then initiated with 0.5 mM IPTG and 0.5 g / L cypermethrin as a precursor. Fermentation was induced at 30°C and 220 rpm for 70 hours. The yield of cypermethrin was detected using high-performance liquid chromatography (HPLC). The fermentation medium formulation included: 20 g / L glycerol, 10 g / L glucose, 5 g / L yeast extract, 7 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 0.01 g / L calcium chloride, 0.3 g / L magnesium sulfate, 50 g / L MOPS, 50 mg / L kanamycin, and 50 mg / L chloramphenicol.

[0073] The experimental results are shown in Table 5. The results indicate that after 72 hours of fermentation, the engineered strain Ros01 containing SlUGT could synthesize 162.31 mg / L of locivitafen, while the engineered strain Ros02 containing FaUGT obtained through screening in this patent could synthesize 503.64 mg / L of locivitafen. This demonstrates that FaUGT screened in this invention can catalyze the synthesis of locivitafen from succinate more efficiently. The liquid chromatography results are shown in Figure 5, and further mass spectrometry identification results are shown in Figure 6.

[0074] Table 5. Production of cyclosporine synthesized in vivo by recombinant engineered strains

[0075] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The present invention is the first to discover that a glycosyltransferase derived from *Fragaria ananassa* can efficiently catalyze the reaction of the glycosyl donor UDP-arabinose with the substrate complexin to generate a single glycosylated product, crosaviin. Simultaneously, an in vivo UDP-arabinose synthesis pathway is constructed, and through fermentation culture, the concentration of crosaviin in the supernatant can reach 503.64 mg / L. Compared with other glycosyltransferases in the prior art, this glycosyltransferase exhibits high activity and high yield of crosaviin, possessing unique advantages and promising potential for application in the microbial industrial production of crosaviin.

[0076] 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 method for biosynthesizing roximate, characterized in that, The method includes: catalyzing a reaction between a substrate cyclophosphamide and a glycosyl donor using a glycosyltransferase having the amino acid sequence shown in SEQ ID NO: 1 to obtain the cyclophosphamide; The glycosyl donor is UDP-arabinose.

2. The method according to claim 1, characterized in that, The glycosyltransferase is selected from the crude enzyme solution or purified enzyme solution of the glycosyltransferase.

3. The method according to claim 1, characterized in that, The method includes: The recombinant cells cultured overnight were inoculated into fermentation medium for expansion culture. After culturing until the OD600 reached 0.6-0.8, an inducer was added for induced fermentation for 48-72 hours. The supernatant was then collected to obtain the clostridium difficile. The recombinant cells are capable of expressing the glycosyltransferase and synthesizing the UDP-arabinose in vivo. The chromium is added together with the inducer or added to the fermentation medium.

4. The method according to claim 3, characterized in that, The recombinant cells contain the UDP-glucose 6-dehydrogenase encoding gene UGD from Escherichia coli, the UDP-glucuronide decarboxylase encoding gene UXS from Rhizobium sinense from alfalfa, and the glucose-4-epimerase encoding gene galE from Escherichia coli.

5. The method according to claim 4, characterized in that, The UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34; the UDP-glucuronide decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35; and the glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO:

36.

6. The method according to any one of claims 3-5, characterized in that, The conditions for the expanded culture were 30℃-37℃ and 200rpm-220rpm.

7. The method according to any one of claims 3-5, characterized in that, The conditions for induced fermentation are 28℃-30℃ and 200rpm-220rpm.

8. The method according to any one of claims 3-5, characterized in that, The inducer is IPTG.

9. The method according to any one of claims 3-5, characterized in that, The final concentration of the inducer is 0.1 mM-1 mM.

10. The method according to any one of claims 1-5, characterized in that, The amount of cytosine added is 0.5-1 g / L.

11. The method according to claim 5, characterized in that, The fermentation medium is formulated with the following components: glycerol 15-20 g / L, glucose 10-15 g / L, yeast extract 5-10 g / L, disodium hydrogen phosphate 5-8 g / L, potassium dihydrogen phosphate 2-5 g / L, sodium chloride 0.3-0.8 g / L, ammonium chloride 0.5-1.5 g / L, calcium chloride 0.01-0.03 g / L, magnesium sulfate 0.2-0.5 g / L, MOPS 40-50 g / L, and antibiotics 50 mg / L-100 mg / L.

12. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain a glycosyltransferase encoding gene with the nucleotide sequence shown in SEQ ID NO: 2, a UDP-glucose 6-dehydrogenase encoding gene UGD derived from Escherichia coli, a UDP-glucuronide decarboxylase encoding gene UXS derived from Rhizobium sinense of alfalfa, and a glucose-4-epimerase encoding gene galE derived from Escherichia coli.

13. The genetically engineered bacterium according to claim 12, characterized in that, The UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34; the UDP-glucuronide decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35; and the glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36.

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