Genetically engineered bacteria for biosynthesis of loganin and method

Abstract

The present invention provides a genetically engineered bacterium and method for biosynthesizing lochnericine. The method includes: using a glycosyltransferase having the amino acid sequence shown in SEQ ID NO: 1 to catalyze a reaction between the substrate lochnerinine and a glycosyl donor, thereby obtaining lochnericine; wherein, the glycosyl donor is UDP - arabinose. The technical solution of the present invention realizes high - yield biosynthesis of lochnericine, has a wide application prospect, and has high economic value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a genetically engineered bacterium and method for biosynthesizing rosavin. Background Art

[0002] Rhodiola rosea L., a perennial herb of the genus Rhodiola in the family Crassulaceae, grows in alpine and high-altitude areas. As a traditional rare Chinese medicinal material, it is recorded in the "Dictionary of Chinese Medicinal Materials". Research shows that Rhodiola rosea L. extracts have functions such as anti-fatigue, anti-hypoxia, anti-aging, anti-cancer, and protecting the cardiovascular system, and have been widely used in the pharmaceutical, food, and health product industries, and there are no reports of side effects or drug interactions.

[0003] Rhodiola rosea L. extracts are functional mixtures, and there are significant differences in the types of active ingredients among different species. The main active ingredients of Rhodiola crenulata extracts are salidroside and tyrosol, while the main active ingredients of Rhodiola rosea L. extracts include total rosavin (rosin, rosavin, roselle), salidroside, and tyrosol. As a unique ingredient of Rhodiola rosea L., the content of total rosavin determines the market price of Rhodiola rosea L. extracts, and rosavin has the highest content. In addition, rosavin has functions such as anti-fatigue, anti-hypoxia, and stress relief, and has broad application prospects and high application value in the fields of medicine, food, and cosmetics.

[0004] Currently, the production of Rhodiola rosea L. extracts mainly relies on direct extraction from the plant Rhodiola rosea L., but its yield is limited by both the low content in the plant and the growth time (slow growth cycle) and space (special high-altitude growth environment) of the plant. Although salidroside and tyrosol have been widely studied due to their clear natural synthesis pathways and relatively mature production processes, there are few reports on the synthesis of rosavin. Microbial fermentation methods have received increasing attention due to their high efficiency, sustainability, and economy, and efficient and specific enzymes are the core biological components for improving the yield of target products.

[0005] The existing methods for synthesizing rosavin include: using UDP-arabinose and rosin as substrates, and the glycosyltransferase transfers the arabinose group in the sugar donor UDP-arabinose to the 6-position glucose hydroxyl group of the sugar acceptor rosin, finally generating rosavin. Among them, the glycosyltransferase required for rosavin synthesis is arabinose glycosyltransferase and it belongs to the sugar chain elongation transferase.

[0006] Although glycosyltransferases are widely present in nature, they have high specificity for the glycosyl groups (glucose, arabinose or xylose) of glycosyl donors. In the prior art, there are about 300 glycosyltransferases with known functions, but only 6 are involved in arabinose transfer (Liu Yuqian. Construction and Application of Plant UDP-Glycosyltransferase Database [D]. South China University of Technology, 2021). For the synthesis of rosavin, only Li et al. obtained four glycosyltransferases from Solanum lycopersicum, Nicotiana tabacum, Camellia sinensis and Solanum pennellii respectively by screening glycosyltransferases of 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). Using these glycosyltransferases to synthesize rosavin has the defect of low yield. Therefore, it is of great significance to develop a method for efficiently producing rosavin. Summary of the Invention

[0007] The main object of the present invention is to provide a genetically engineered bacterium and method for biosynthesizing rosavin to solve the problem of low yield of biosynthesized rosavin in the prior art.

[0008] To achieve the above object, according to the first aspect of the present invention, a method for biosynthesizing rosavin is provided. The method includes: catalyzing a reaction of a substrate, rosavin, and a glycosyl donor with a glycosyltransferase having the amino acid sequence shown in SEQ ID NO: 1 to obtain the rosavin; wherein the glycosyl donor is UDP-arabinose.

[0009] Further, the glycosyltransferase is selected from the crude enzyme solution or the purified enzyme solution of the glycosyltransferase.

[0010] Further, the method includes: inoculating the overnight cultured recombinant cells into a fermentation medium for enlarged culture, culturing until the OD600 reaches 0.6-0.8, adding an inducer for induced fermentation for 48-72 h, and collecting the supernatant product to obtain the rosavin, wherein the recombinant cells can express the glycosyltransferase and can biosynthesize the UDP-arabinose in vivo; the rosavin is added together with the inducer or the rosavin is added to the fermentation medium.

[0011] Furthermore, the above-mentioned recombinant cell contains the UDP-glucose 6-dehydrogenase encoding gene UGD derived from Escherichia coli, the UDP-glucuronic acid decarboxylase encoding gene UXS derived from Sinorhizobium meliloti and the glucose-4-epimerase encoding gene galE derived from Escherichia coli.

[0012] Furthermore, the UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34, the UDP-glucuronic acid decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35; and the glucose-4-isomerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36.

[0013] Furthermore, the conditions for the above-mentioned expanded culture are 30°C-37°C and 200rpm-220rpm.

[0014] Furthermore, the above-mentioned induction fermentation conditions are 28°C-30°C, 200rpm-220rpm.

[0015] Furthermore, the above-mentioned inducer is IPTG.

[0016] Furthermore, the final concentration of the above inducer is 0.1mM-1mM.

[0017] Furthermore, the addition amount of the above-mentioned chord is 0.5-1 g / L.

[0018] Furthermore, the formula of the above fermentation medium includes: 15-20 g / L glycerol, 10-15 g / L glucose, 5-10 g / L yeast extract, 5-8 g / L disodium hydrogen phosphate, 2-5 g / L potassium dihydrogen phosphate, 0.3-0.8 g / L sodium chloride, 0.5-1.5 g / L ammonium chloride, 0.01-0.03 g / L calcium chloride, 0.2-0.5 g / L magnesium sulfate, 40-50 g / L MOPS and 50 mg / L-100 mg / L antibiotics.

[0019] In order to achieve the above-mentioned object, according to the second aspect of the present invention, a genetically engineered bacterium is provided, which contains a glycosyltransferase encoding gene having a nucleotide sequence shown in SEQ ID NO: 2, a UDP-glucose 6-dehydrogenase encoding gene UGD derived from Escherichia coli, a UDP-glucuronic acid decarboxylase encoding gene UXS derived from Sinorhizobium alfalfa, and a glucose-4-isomerase encoding gene galE derived from Escherichia coli.

[0020] Furthermore, the above-mentioned UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34, and the above-mentioned UDP-glucuronate decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35; the above-mentioned glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36.

[0021] Applying the technical solution of the present invention, the glycosyltransferase of the present invention (having the amino acid sequence shown in SEQ ID NO: 1) catalyzes the reaction of the glycosyl donor UDP-arabinose and the substrate loquatide, and can obtain loganin, and the yield of loganin is higher than that of other glycosyltransferases in the prior art for catalyzing the synthesis of loganin from loquatide, achieving high-yield biosynthesis of loganin, with broad application prospects and high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 Shows the synthetic route diagram of the biosynthesis of loganin of the present invention.

[0024] Figure 2A Shows the protein electrophoresis patterns of 11 strains of Escherichia coli containing glycosyltransferases (UGT2, UGT6, UGT7, UGT9, UGT11, UGT13, UGT14, UGT15, UGT16, UGT31 or UGT36) from different sources according to Example 3 of the present invention after induced expression. Among them, ↑ represents the soluble protein electrophoresis pattern after induction, and ↓ represents the insoluble protein electrophoresis pattern after induction.

[0025] Figure 2B Shows the protein electrophoresis patterns of 4 strains of Escherichia coli containing glycosyltransferases (UGT44, UGT38, UGT41 or AtUGT78D3) from different sources according to Example 3 of the present invention after induced expression. Among them, ↑ represents the soluble protein electrophoresis pattern after induction, and ↓ represents the insoluble protein electrophoresis pattern after induction.

[0026] Figure 2C Shows the protein electrophoresis patterns of 9 strains of Escherichia coli containing glycosyltransferases (AcUGT, GmUGT-1, PtUGT, AtUGT-1, FaUGT, AtUGT-2, CGTb, UGT73P10 or UGT99D1) from different sources according to Example 3 of the present invention after induced expression. Among them, ↑ represents the soluble protein electrophoresis pattern after induction, and ↓ represents the insoluble protein electrophoresis pattern after induction.

[0027] Figure 2D The protein electrophoresis patterns after induced expression of 8 strains of Escherichia coli containing glycosyltransferases (GmUGT-2, OsUGT, NtUGT, CsUGT, SpUGT, SIUGT, CIUGT or ZmUGT) from different sources according to Embodiment 3 of the present invention are shown. Among them, ↑ represents the electrophoresis pattern of soluble proteins after induction, and ↓ represents the electrophoresis pattern of insoluble proteins after induction.

[0028] Figure 3 The liquid chromatography pattern of rosavin synthesized using glycosyltransferase FaUGT according to Embodiment 3 of the present invention is shown. Among them, (1) is the rosavin standard, (2) is the rosavin standard, and (3) is the rosavin product synthesized using glycosyltransferase FaUGT of the present invention.

[0029] Figure 4 The mass spectrometry pattern of rosavin synthesized using glycosyltransferase FaUGT according to Embodiment 3 of the present invention is shown. Among them, (1) is the rosavin standard, and (2) is the rosavin product synthesized using glycosyltransferase FaUGT of the present invention.

[0030] Figure 5 The liquid chromatography pattern of rosavin synthesized by engineering strains Ros01 and Ros02 according to Embodiment 5 of the present invention is shown. Among them, (1) is Ros01 and (2) is Ros02.

[0031] Figure 6 The mass spectrometry pattern of rosavin synthesized by engineering strains Ros01 and Ros02 according to Embodiment 5 of the present invention is shown. Among them, (1) is Ros01 and (2) is Ros02. Detailed implementation manners

[0032] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0033] Term explanation:

[0034] UGT: Abbreviation for UDP-dependent glycosyltransferase, a kind of glycosyltransferase that can transfer the glycosyl group in UDP-sugar to other molecules to play a role in glycosyl transfer.

[0035] As mentioned in the background art, rosavin has high application value, but the methods for synthesizing rosavin in the prior art have the problem of low yield. In the present invention, the inventors attempted to screen a glycosyltransferase with high activity and strong substrate specificity for the efficient production of rosavin, and thus proposed a series of protection schemes of the present invention.

[0036] In the first typical embodiment of the present invention, a method for biosynthesizing rosavin is provided. The above method includes: using a glycosyltransferase having the amino acid sequence shown in SEQ ID NO: 1 to catalyze a reaction of the substrate rosavin and a glycosyl donor to obtain the above rosavin (see Figure 1 ).

[0037] Among them, the amino acid sequence SEQ ID NO: 1 of the glycosyltransferase FaUGT is as follows:

[0038] MSSSSATKRKKLHIALFPWLAFGHIIPFLEVAKHIARKGHKVSFISTPRNIQRLPKIPETLTPLINLVQIPLPHVENLPENAEATMDVPHDVIPYLKIAHDGLEQGISEFLQAQSPDWIIHDFAPHWLPPIATKLGISNAHFSIFNASSMCFFGSTSPNRVSRYAPRKKLEQFTSPPEWIPFPSKIYHRPFEAKRLMDGTLTPNASGVTDRFRLESTIQGCQVYFIRSCREIEGEWLDLLEDLHEKPIVLPTGLLPPSLPRSDEDGGKDSNWSKIAVWLDKQEKGKVVYAAFGSELNLSQEVFNELALGLELSGLPFFWVLRKPSHGSGDGDSVKLPDGFEDRVKGRGLVWTTWAPQLKILSHESVGGFLTHCGWSSIIESLQYGCPLIMLPFMYDQGLIARFWDNKIGAEVPRDEETGWFTRNELANSLKLIVVDEEGKQYRDGANEYSKLFRDKELHDRYMDECVEYLETHAHHEV.

[0039] The nucleotide sequence SEQ ID NO: 2 of the gene encoding the glycosyltransferase FaUGT is as follows:

[0040]

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

[0042] The method for biosynthesizing rosavin of the present invention can utilize either the crude enzyme solution or be carried out in vivo by fermenting recombinant cells containing the coding gene of the above glycosyltransferase of the present invention (having the nucleotide sequence shown in SEQ ID NO: 2) and capable of synthesizing the above UDP-arabinose in vivo. In a preferred embodiment of the present invention, the overnight-cultured recombinant cells are inoculated into a fermentation medium for scale-up culture until the OD600 reaches 0.6 - 0.8, then an inducer is added for induced fermentation for 48 - 72 h, and the supernatant product is collected to obtain the above rosavin. Among them, the above recombinant cells can express the above glycosyltransferase and can synthesize the above UDP-arabinose in vivo; the above rosamine is added together with the above inducer or the above rosamine is added to the above fermentation medium.

[0043] Using this method to produce rosavin has the beneficial effects of being simple and fast. The addition time of the substrate rosamine is not specifically limited, and it can be added to the fermentation medium or added together with the inducer. In order to maximize the yield of rosavin, the addition amount of the substrate should be as sufficient as possible. In a preferred embodiment of the present invention, the addition amount of the above rosamine is 0.5 - 1 g / L. Using the substrate with this addition amount helps the efficient production of rosavin.

[0044] In order to enable the above recombinant cells to synthesize the above UDP-arabinose in vivo, in a preferred embodiment of the present invention, the above recombinant cells further contain the UDP-glucose 6-dehydrogenase coding gene UGD derived from Escherichia coli, the UDP-glucuronate decarboxylase coding gene UXS derived from Sinorhizobium meliloti, and the glucose-4-epimerase coding gene galE endogenous to Escherichia coli. Using the above recombinant cells with glucose as the substrate to generate UDP-arabinose, and then using UDP-arabinose as the glycosyl donor and the exogenous substrate rosamine to generate rosavin under the action of the glycosyltransferase.

[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, and the UDP-glucuronate decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35; 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, and the UDP-glucuronate decarboxylase UXS has the amino acid sequence shown in SEQ ID NO: 32; the glucose-4-epimerase GalE has the amino acid sequence shown in SEQ ID NO: 33.

[0046] The formula of the above fermentation medium 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. Fermenting the above recombinant cells with this fermentation medium helps the growth and reproduction of the recombinant cells, and is beneficial to the expression of glycosyltransferase and the supply of UDP-arabinose in vivo, thereby further increasing the production of rosavin.

[0047] It should be noted that the type of antibiotic is determined by the resistance gene on the plasmid introduced into the above genetically engineered bacterium. For example, when the above genetically engineered bacterium is transformed with two plasmids containing different resistance genes (for example, chloramphenicol resistance gene and kanamycin resistance gene), the fermentation medium contains 50 mg / L-100 mg / L chloramphenicol and 50 mg / L-100 mg / L kanamycin. When only one plasmid (for example, chloramphenicol resistance plasmid or kanamycin resistance plasmid) is introduced into the above genetically engineered bacterium, the fermentation medium only contains 50 mg / L-100 mg / L chloramphenicol or 50 mg / L-100 mg / L kanamycin.

[0048] After inoculating the recombinant cells into the fermentation medium, it is necessary to carry out scale-up culture to increase the number of recombinant cells. The conditions of scale-up culture are closely related to the number and growth status of recombinant cells. When the number of recombinant cells reaches a certain concentration, it is necessary to carry out induced fermentation culture on the recombinant cells. In order to make the recombinant cells produce as much lochneri as possible, it is best to culture them under the optimal induced fermentation culture conditions for the production of lochneri by recombinant cells. In another preferred embodiment of the present invention, the conditions of the above-mentioned scale-up culture are 30°C - 37°C, 200 rpm - 220 rpm. In another preferred embodiment of the present invention, the conditions of the above-mentioned induced fermentation are 28°C - 30°C, 200 rpm - 220 rpm.

[0049] According to the different expression vectors and the expression of the target protein in recombinant cells, a suitable inducer with a suitable concentration is selected. In a preferred embodiment of the present invention, the above-mentioned inducer is IPTG. In a more preferred embodiment of the present invention, the final concentration of the above-mentioned inducer is 0.1 mM - 1 mM.

[0050] In the second typical embodiment of the present invention, a genetically engineered bacterium is provided. The above-mentioned genetically engineered bacterium 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-glucuronic acid decarboxylase encoding gene UXS derived from Sinorhizobium meliloti, and a glucose-4-epimerase encoding gene galE derived from Escherichia coli.

[0051] In a preferred embodiment of the present invention, the above-mentioned UDP-glucose 6-dehydrogenase encoding gene UGD has the nucleotide sequence shown in SEQ ID NO: 34, the above-mentioned UDP-glucuronic acid decarboxylase encoding gene UXS has the nucleotide sequence shown in SEQ ID NO: 35; the above-mentioned glucose-4-epimerase encoding gene galE has the nucleotide sequence shown in SEQ ID NO: 36. The yield of lochneri produced by the genetically engineered bacterium of the present invention is higher, which helps to realize the industrial production of lochneri.

[0052] The following further describes the present invention in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed by the present invention.

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

[0054] Rosavin is mainly derived from Rhodiola rosea. The inventors considered that Rhodiola rosea was very likely to contain unique glycosyltransferases for synthesizing rosavin. Therefore, the inventors downloaded the transcriptome data of Rhodiola rosea from NCBI for CDS prediction, and docked the predicted glycosyltransferases (a total of 13,000) with rosavin and UDP-arabinose, and screened out 14 glycosyltransferases that might synthesize rosavin. According to the codon usage preference of Escherichia coli, the genes of the above 14 glycosyltransferases were optimized and synthesized in the pET28a vector, and a total of 14 pET28a-UGT plasmids were obtained (Table 1). All the nucleic acids of the present invention were synthesized by Genewiz Biotechnology Co., Ltd.

[0055] Table 1 Expression vectors of glycosyltransferases from Rhodiola rosea

[0056]

[0057] In addition, the inventors found through preliminary research and experiments that although glycosyltransferases have certain specificities for sugar donors and aglycone receptors, it is not absolute. Therefore, the glycosyltransferases in Uniprot were retrieved (a total of 2,720), and docked with rosavin and UDP-arabinose, and 10 glycosyltransferases that might synthesize rosavin were screened out. According to the codon usage preference of Escherichia coli, the genes of the above 10 glycosyltransferases were optimized and synthesized in the pET28a vector, and a total of 10 pET28a-UGT plasmids were obtained (Table 2).

[0058] Table 2 Expression vectors of glycosyltransferases from Uniprot

[0059]

[0060] To further expand the possibility of screening glycosyltransferases that catalyze the synthesis of rosavin, 4 glycosyltransferases that can catalyze arabinose transfer reported in relevant literature and 4 glycosyltransferases that can catalyze the synthesis of rosavin disclosed in the prior art were also screened. According to the codon usage preference of Escherichia coli, the genes of the above 8 glycosyltransferases were optimized and synthesized in the pET28a vector, and a total of 8 pET28a-UGT plasmids were obtained (Table 3).

[0061] Table 3 Expression vectors of glycosyltransferases from literature

[0062]

[0063] Thirty-two recombinant plasmids shown in the above Table 1 - Table 3 were transformed into Escherichia coli BL21(DE3) to obtain 32 strains of recombinant Escherichia coli BL21(DE3) / pET28a-UGT. The specific transformation method is as follows: Take BL21(DE3) competent cells (50 μL of competent cells in a 1.5 ml EP tube) and thaw them on ice. Add 50 ng of plasmid into the competent cells, mix well, and incubate on ice for 30 min. Then heat shock in a 42°C water bath for 90 s. Add 600 μL of recovery solution (LB medium), incubate at 37°C for 50 min. After incubation, spread all the bacterial solution on a plate (LB medium) containing 50 mg / L kanamycin, and place it in a 37°C incubator for overnight culture.

[0064] Example 2 Protein Expression Verification of Glycosyltransferase

[0065] To determine whether the synthesized glycosyltransferase is normally expressed, protein expression verification needs to be carried out. The specific method is as follows: Inoculate the glycerol bacteria of 32 engineering strains expressing glycosyltransferase in the BL21(DE3) chassis strain into a test tube containing 5 mL of LB liquid medium (added with 50 mg / L kanamycin) at an inoculation volume of 0.2% by volume. After overnight culture at 37°C, inoculate it into a 250 mL flask containing 50 mL of LB liquid medium (added with 50 mg / L kanamycin) at an inoculation volume of 1% by volume. Shake the bacteria at 37°C until the OD600 reaches 0.8. After adding IPTG with a final concentration of 0.1 mM, induce and culture in a 30°C shaker for 20 hours. Then take out 3 mL of the bacterial solution, centrifuge at 4°C and 12,000 rpm to collect the bacteria. Resuspend the bacteria in 1 mL of PBS buffer, use an ultrasonic crusher to perform ultrasonic crushing for 5 min, and then centrifuge at 12,000 rpm for 10 minutes to separate the supernatant protein and the precipitate protein, and perform SDS-PAGE analysis and detection respectively. The SDS-PAGE results of 32 engineering strains are as Figure 2A 、 Figure 2B 、 Figure 2C and Figure 2D shown.

[0066] Example 3 In Vitro Catalysis of Rosavin Synthesis by Crude Enzyme Solution of Glycosyltransferase

[0067] The crude enzyme solution containing UGT protein was obtained using the 32 recombinant Escherichia coli BL21(DE3) / pET28a-UGT constructed in Example 1. The specific method is as follows: Glycerol bacteria of 32 engineering strains expressing glycosyltransferase in the BL21(DE3) chassis strain were inoculated into a test tube containing 5 mL of LB liquid medium (supplemented with 50 mg / L kanamycin) at an inoculation volume of 0.2%. After overnight culture at 37°C, they were inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium (supplemented with 50 mg / L kanamycin) at an inoculation volume of 1%. After shaking at 37°C until the OD600 reached 0.8, IPTG with a final concentration of 0.1 mM was added, and then cultured with shaking at 30°C for 20 hours. Then, 3 mL of the bacterial solution was taken out, and the cells were collected by centrifugation at 4°C and 12,000 rpm. The cells were resuspended in 1 mL of PBS buffer, sonicated for 5 min using a sonicator, and then centrifuged at 12,000 rpm for 10 minutes to separate the supernatant protein and the precipitate protein. The supernatant is the crude enzyme solution.

[0068] The following reaction system was prepared with the above crude enzyme solution: 50 mg / L rosavin, 100 mg / L UDP-arabinose, 2 mM MgCl2, 100 μL of the crude enzyme solution, and made up to 500 μL with 50 mM Tris-HCl (pH 7.5). After reacting the above system in a 30°C water bath for 2 hours, 100 μL of ice-cold methanol was added to terminate the reaction, and it was cooled on ice for 2 min and then centrifuged at 12,000 rpm for 5 min. The reaction solution was filtered through a 0.22 μm organic phase filter membrane to prepare a sample, and HPLC was used to detect the synthesis amount of rosavin in the reaction system.

[0069] As can be seen from the data in Table 4 below, after the in vitro catalytic reaction, among the 32 glycosyltransferases, only AcUGT, FaUGT, and ZmUGT could detect obvious rosavin synthesis. 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 of rosavin after 2 hours of reaction. However, FaUGT screened in the present invention can synthesize 34.31 mg / L of rosavin, which is 2.2 times higher.

[0070] The liquid phase chromatogram of using FaUGT of the present invention to synthesize rosavin is as Figure 3 shown, in which the retention time of rosavin is 13.5 min, which is consistent with the retention time of the rosavin standard product. To further confirm the synthesis of rosavin, the liquid phase detection sample was concentrated and then subjected to mass spectrometry identification. The LC-MS chromatogram is as Figure 4As shown, the molecular weight of loganin is 473, which is consistent with the loganin standard. Therefore, it can be judged that the glycosyltransferase FaUGT screened in the present invention can catalyze the synthesis of loganin from UDP-arabinose and loganic acid, and compared with the reported glycosyltransferases, the FaUGT screened in the present invention has a higher catalytic efficiency. In addition, it should be noted that the relative molecular weight of loganin is 428, but due to the presence of formic acid in the mass spectrometry conditions, the actually detected loganin is the one with formate added, so it is [M+HCOOH - - 473。

[0071] Table 4 Ability of UGTs from different sources to synthesize loganin

[0072]

[0073] Example 4 Construction of recombinant Escherichia coli producing loganin

[0074] To supply UDP-arabinose in vivo in Escherichia coli, the UDP-glucose 6-dehydrogenase encoding gene UGD from Escherichia coli endogenous, the UDP-glucuronate decarboxylase encoding gene UXS from Sinorhizobium meliloti, and the glucose-4-epimerase gene galE from Escherichia coli endogenous were placed under the T7 promoter and constructed in the pACYC-Duet-1 vector to obtain the plasmid pACYC-UGD-UXS-galE producing UDP-arabinose, which was introduced into the engineered Escherichia coli BL21(DE3) / pET28a-FaUGT and BL21(DE3) / pET28a-SlUGT to obtain the engineered Escherichia coli Ros01 and Ros02 producing loganin.

[0075] Example 5 Synthesis of loganin by recombinant Escherichia coli

[0076] The engineering strains Ros01 and Ros02 were inoculated into LB liquid medium and cultured overnight at 37 °C and 220 rpm to obtain seed solutions. The seed solutions were inoculated into fresh fermentation medium at an inoculation volume of 1% by volume and cultured at 37 °C and 220 rpm for 2 h, then IPTG with a final concentration of 0.5 mM was added for induction and loganic acid with a final concentration of 0.5 g / L was used as a precursor, and induced fermentation culture was carried out at 30 °C and 220 rpm for 70 h. The yield of loganin was detected using a high performance liquid chromatograph. The fermentation medium formula includes: glycerol 20 g / L, glucose 10 g / L, yeast extract 5 g / L, disodium hydrogen phosphate 7 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L, calcium chloride 0.01 g / L, magnesium sulfate 0.3 g / L, MOPS 50 g / L, kanamycin 50 mg / L and chloramphenicol 50 mg / L.​

[0077] The experimental results are shown in Table 5. The results indicate that after 72 h of fermentation, the engineered strain Ros01 containing SlUGT can synthesize 162.31 mg / L of rosavin, while the engineered strain Ros02 containing FaUGT screened in this patent can synthesize 503.64 mg / L of rosavin. This shows that the FaUGT screened in the present invention can more efficiently catalyze the synthesis of rosavin from rosin. The liquid phase results are as Figure 5 shown, and the further mass spectrometry identification results are as Figure 6 shown.

[0078] Table 5 Yield of rosavin synthesized in vivo by recombinant engineered strains

[0079]

[0080] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The present invention discovers for the first time that the glycosyltransferase derived from Fragaria ananassa can efficiently catalyze the reaction of the glycosyl donor UDP-arabinose with the substrate rosin to generate a single glycosylated product, rosavin. At the same time, an in vivo UDP-arabinose synthesis pathway is constructed. Through fermentation culture, the concentration of rosavin in the supernatant can reach 503.64 mg / L. Compared with other glycosyltransferases in the prior art, this glycosyltransferase has high activity and a high yield of rosavin produced, with unique advantages, and is expected to be applied to the industrial microbial production of rosavin.

[0081] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for biosynthesizing rosavin, characterized in that, The method includes: using a glycosyltransferase with an amino acid sequence of SEQ ID NO: 1 to catalyze a reaction on the substrate loganin and a glycosyl donor to obtain the loganinsev; Wherein, 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 the purified enzyme solution of the glycosyltransferase.

3. The method according to claim 1, wherein The method includes: Inoculating the overnight - cultured recombinant cells into a fermentation medium for scale - up culture, culturing until the OD600 reaches 0.6 - 0.8, adding an inducer for induced fermentation for 48 - 72 h, and collecting the supernatant product to obtain the loganinsev, Wherein, the recombinant cells can express the glycosyltransferase and can synthesize the UDP - arabinose in vivo; The loganin is added together with the inducer or the loganin is added to the fermentation medium.

4. The method according to claim 3, characterized in that The recombinant cell contains a UDP-glucose 6-dehydrogenase encoding gene derived from Escherichia coli UGD , a UDP-glucuronate decarboxylase encoding gene derived from Sinorhizobium meliloti UXS and a glucose-4-epimerase encoding gene derived from Escherichia coli galE .

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

36.

6. The method according to any one of claims 3 to 5, characterized in that, The conditions for the scale - up culture are 30°C - 37°C, 200 rpm - 220 rpm.

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

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

9. The method according to any one of claims 3 to 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 addition amount of the loganin is 0.5 - 1 g / L.

11. The method according to claim 5, wherein The formula of the fermentation medium 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.

12. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium contains a glycosyltransferase-encoding gene with a nucleotide sequence of SEQ ID NO: 2, a UDP-glucose 6-dehydrogenase-encoding gene derived from Escherichia coli UGD , a UDP-glucuronic acid decarboxylase-encoding gene derived from Sinorhizobium meliloti UXS and a glucose-4-epimerase-encoding gene g derived from Escherichia coli alE .

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

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