Fluorolactose whole-cell synthesis method and application thereof in fermentative preparation of GM3-F

Abstract

A whole-cell synthesis method for fluorolactose and its application in the fermentation preparation of GM3-F, relating to the field of fluorolactose preparation technology. The whole-cell synthesis method for fluorolactose of this invention utilizes overexpression of the UDP-glucose-4-epimerase gene. galE β-1,4-galactosyltransferase gene GalTpm1141 UDP-glucose pyrophosphorylase gene GalT and sucrose phosphorylase gene SPase A lactose synthesis system co-expresses engineered strains and yeast to synthesize fluorolactose through coupled fermentation using glucose and sucrose as substrates. This invention also discloses the application of the fluorolactose synthesis method in the fermentation preparation of GM3-F. This invention provides a whole-cell fluorolactose synthesis method, co-expressing engineered strains and yeast to synthesize fluorolactose through coupled fermentation. galE, GalTpm1141, GalT, SPase The lactose synthesis system co-expresses engineered strains and yeast for coupled fermentation, which is safe and efficient, and improves the efficiency and yield of whole-cell synthesis of fluorolactose.

Description

Technical Field

[0001] This invention relates to the field of fluorolactose preparation technology, and in particular to a whole-cell synthesis method for fluorolactose and its application in the fermentation preparation of GM3-F. Background Technology

[0002] Gangliosides are a type of glycosphingolipid compound characterized by the presence of sialic acid in their oligosaccharide chain portion. They are widely found in mammalian nerve cells, especially brain cells, and have neuroprotective and anti-inflammatory effects, making them a class of drugs with great market potential.

[0003] Gangliosides are complex, consisting of ceramides and oligosaccharide chains. The core oligosaccharide sequence of gangliosides is a tetrasaccharide structure: β-Gal-(1-3)-β-GalNAc-(1-4)-β-Gal-(1-4)-β-Glc-(1-1) 1-Cer. Lactose, as the core structural unit of ganglioside oligosaccharides, is one of the key starting substrates for ganglioside synthesis. It can be linked with sphingosine to form lactosylsphingosine, and with sialic acid to form ganglioside oligosaccharides. Fluorinated lactose is a glycoside compound, such as... Figure 1 As shown, the hydroxyl group at position 1 of glucose is replaced by a fluorine atom, and the chemical formula is C. 12 H 21 FO 10 With a molecular weight of 344.29 g / mol, it is an excellent glycosyl donor.

[0004] Li Yuyang. Improved Synthesis Method of Fluorogalactose and Its Application in Glycosylation Modification Catalyzed by Galactosidase [D]. Supervisor: Liu Li; Zeng Xianming. Nanjing Agricultural University, 2022. This paper discloses a method for synthesizing fluorogalactose, the glycosyl donor of galactosidase, through three steps: total acetylation, fluorine substitution, and deacetylation protection. However, it does not disclose a whole-cell synthesis method for fluorogalactose.

[0005] CN 111500660 A discloses a method for synthesizing monosialotetrahexosylganglioside, which uses a first combination enzyme to catalyze the synthesis of GM3-F from fluorolactose, sialic acid, and cytidine triphosphate. The first combination enzyme is NmCSS, PmST1, and PmPpA. The molar concentration of fluorolactose is 10-40 mM; the molar ratio of fluorolactose, sialic acid, and cytidine triphosphate is 1:(1.2-2):(1.3-2); the amount of NmCSS is 80-140 U; the amount of PmST1 is 80-220 U; the reaction temperature is 10-40℃; and the pH value is 7-9. The key to the above method is the need for efficient synthesis of fluorolactose.

[0006] Therefore, finding an efficient method for synthesizing fluorolactose is extremely important, especially the whole-cell synthesis method, which would make the synthesis of fluorolactose more efficient and safe. The expression level of key enzymes in the synthesis of fluorolactose using glucose and sucrose as substrates is an effective solution to improve the yield of fluorolactose by whole-cell synthesis and lays the foundation for the efficient synthesis of GM3-F using fluorolactose as a substrate. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a new, efficient and safe whole-cell synthesis method for fluorolactose in view of the shortcomings of the prior art. This invention also discloses the application of fluorolactose as a substrate in the fermentation preparation of GM3-F.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A method for whole-cell synthesis of fluorolactose includes the following steps: fermentation of an engineered *E. coli* strain overexpressing the β-1,4-galactosyltransferase gene GalTpm1141 for whole-cell catalytic synthesis of fluorolactose; or, co-expression of an engineered strain in a lactose synthesis system overexpressing the UDP-glucose-4-epimerase gene galE, the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose pyrophosphorylase gene GalT, and the sucrose phosphorylase gene SPase for whole-cell catalytic synthesis of fluorolactose; or, co-expression of an engineered *E. coli* strain overexpressing the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose-4-epimerase gene galE, the UDP-glucose pyrophosphorylase gene GalT, and the sucrose phosphorylase gene SPase, respectively, followed by four-strain coupled fermentation for whole-cell catalytic synthesis of fluorolactose.

[0010] In this invention, the gene GalTpm1141 is a gene from Pasteurella multocida that encodes β-1,4-galactosyltransferase. This enzyme has high specificity and can catalyze the synthesis of lactose from D-glucose and UDP-D-galactose. Using fluoroglucose and UDP-galactose as substrates, and by using fluoroglucose to replace glucose in the lactose synthesis system, the β-1,4-galactosyltransferase produced by engineered Escherichia coli overexpressing the gene GalTpm1141 successfully catalyzes the whole-cell synthesis of fluorolactose.

[0011] Using sucrose and fluorinated glucose as substrates, coupled fermentation was performed using four engineered *E. coli* strains overexpressing the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose-4-epimerase gene galE, the UDP-glucose pyrophosphorylase gene GalT, and the sucrose phosphorylase gene SPase. Among these, the strains overexpressing the UDP-glucose-4-epimerase gene galE, the UDP-glucose pyrophosphorylase gene GalT, and the sucrose phosphorylase gene SPase were used. Enzymes produced by the coupled fermentation of engineered E. coli strains containing the phosphorylase gene SPase (i.e., including UDP-glucose-4-epimerase galE, overexpressed UDP-glucose pyrophosphorylase gene GalT, and sucrose phosphorylase SPase) can promote the conversion of sucrose to UDP-galactose. Then, β-1,4-galactosyltransferase produced by engineered strains overexpressing the β-1,4-galactosyltransferase gene GalTpm1141 can synthesize fluorolactose from UDP-galactose and fluoroglucose.

[0012] The nucleotide sequences of the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose-4-epimerase gene galE, the UDP-glucose pyrophosphorylase gene GalT, and the sucrose phosphorylase gene SPase are shown in SEQ NO.1, SEQ NO.2, SEQ NO.3, and SEQ NO.4, respectively.

[0013] Preferably, an engineered strain is co-expressed in a lactose synthesis system that overexpresses the UDP-glucose-4-epimerase gene galE, the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose pyrophosphorylase gene GalT, and the sucrose phosphorylase gene SPase. This reduces the number of transmembrane crossings and improves reaction efficiency and yield. The mechanism of its synthesis of fluorolactose is the same as the mechanism of the above four engineered strains coupled with fermentation.

[0014] In one exemplary embodiment, a method for whole-cell synthesis of fluorolactose includes the following steps:

[0015] S1. Preparation of fluoroglucose: Fluoroglucose is synthesized from glucose as a starting material through acetylation protection, fluorine substitution reaction and deacetylation reaction.

[0016] S2. Constructing an engineered strain for co-expression of lactose synthesis system: using E. coli Using JM109 as the starting strain, the UDP-glucose-4-epimerase gene galE and the β-1,4-galactosyltransferase gene GalTpm1141 were overexpressed using the pETDuet1 vector, and the UDP-glucose pyrophosphorylase gene GalT and the sucrose phosphorylase gene SPase were overexpressed using the pRSFDuet1 vector. Plasmids pETDuet1-galE-GalTpm1141, pETDuet1-GalTpm1141-galE, pRSFDuet1-SPase-GalT, and pRSFDuet1-GalT-SPase were introduced to construct various lactose synthesis systems for co-expression. Specifically, GalT encodes UDP-glucose pyrophosphorylase; galE encodes UDP-glucose-4-epimerase; SPase encodes sucrose phosphorylase; and GalTpm1141 is derived from Pasteurella. The gene encoding β-1,4-galactosyltransferase of *Pasteurella multocida*.

[0017] S3. Synthesis of fluorolactose by coupled fermentation of two strains: Using glucose and sucrose as substrates, the engineered strains co-expressing the lactose synthesis system constructed in S2 are coupled with yeast for fermentation to synthesize fluorolactose.

[0018] The whole-cell synthesis method for fluorolactose of this invention uses two compatible plasmids, pETduet-1 and pRSFduet-1, with different copy numbers as co-expression vectors to construct a lactose synthesis system co-expression engineered strain expressing four enzymes: sucrase phosphorylase (SPase), GalT, galE, and GalTpm1141. This reduces the number of transmembrane steps and increases the reaction yield. This lactose synthesis system co-expression engineered strain can catalyze sucrose to obtain Glc-1-p via sucrase phosphorylase SPase, obtain UDP-Glc using UDP-glucose pyrophosphorylase GalT, obtain UDP-Gal using UDP-glucose-4-epimerase galE, and finally, connect the Gal unit of UDP-Gal to the 1-fluoroglucose unit via β-1,4-galactosyltransferase GalTpm1141 to form fluorolactose. Yeast provides the UTP cycle system, further improving the reaction efficiency in a dual-strain coupled fermentation system.

[0019] The steps involved in S2, constructing the lactose synthesis system co-expression engineered strain, are as follows:

[0020] S21. Acquisition and amplification of the target gene

[0021] The sequence of the gene GalTpm1141 was optimized using E. coli preferred codons and synthesized by Tianlin Biotechnology Co., Ltd., to obtain the recombinant plasmid pET28a-GalTpm1141. The target gene was then obtained from the laboratory-preserved experimental strain E4 and the recombinant plasmid pET28a-GalTpm1141 using PCR.

[0022] S22, agarose gel electrophoresis and gel recovery

[0023] S23. Construction of recombinant plasmids

[0024] Recombinant plasmids were constructed using the ClonExpress MultiS One Step Cloning Kit (a seamless cloning kit based on homologous recombination technology).

[0025] S24. Preparation and transformation of competent Escherichia coli cells

[0026] (1) Streak the preserved Escherichia coli cells onto antibiotic-free LB plates and incubate overnight at 37°C. Inoculate single colonies into 10 mL of LB liquid medium and incubate at 37°C and 200 rpm in a shaker for 10-12 h.

[0027] (2) Inoculate 2 mL of bacterial culture into 100 mL of LB medium and incubate at 37°C with shaking at 200 rpm for about 3 h;

[0028] (3) Place the cultured bacterial solution on ice to pre-cool for 5 min, centrifuge at 8000 rpm for 5 min to collect the bacterial cells, and repeat once;

[0029] (4) Add 25 mL of pre-cooled Solution A to resuspend the bacteria, centrifuge at 8000 rpm for 5 min to collect the cells, and repeat once;

[0030] (5) Add 5 mL Solution B to resuspend the bacteria, take 100 µL of the resuspension and dispense it into 1.5 mL centrifuge tubes, place on ice for later use or store at -80℃.

[0031] Chemical transformation method: Add an appropriate amount of plasmid vector to competent cells, mix well by pipetting, incubate on ice for 30 min, then heat shock at 42℃ for 90 s, incubate on ice for 2 min, and add 1 mL of antibiotic-free LB liquid medium. Incubate at 37℃ for 1 h, centrifuge the incubated bacterial solution at 12000 rpm for 2 min, retain about 100 µL of supernatant, mix well by pipetting, and spread onto LB plates containing the corresponding antibiotic.

[0032] S25, recombinant protein induced expression

[0033] After streaking the engineered bacteria onto plates, incubate overnight at 37°C. Pick a single colony and inoculate it into 10 mL of LB liquid medium, incubating overnight at 37°C with a shaker at 200 rpm. Then, inoculate the seed culture at a 2% inoculation rate into 100 mL of LB medium and incubate at 37°C with a shaker at 200 rpm for 2-3 hours. When OD... 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.3 mmol / L and incubate in a shaker at 15℃ and 200 rpm for 20 h.

[0034] S26, SDS-PAGE detection

[0035] Take a certain amount of culture medium, centrifuge, discard the supernatant, add protein loading buffer, resuspend the bacterial cells, boil in a water bath for 5 min, then remove and add 10 µL of sample to the protein loading wells. Turn on the electrophoresis apparatus at 80 V for 30 min, then switch to 150 V and continue for 1.5-2 h. When the protein marker and sample bands are close to the bottom of the protein gel, turn off the electrophoresis, remove the protein gel, and end the electrophoresis. Transfer the gel to a culture dish, add Coomassie Brilliant Blue staining solution, and shake to stain for 1 h. After staining, place the gel in a culture dish containing destaining solution and shake to destain. Replace the destaining solution after several hours until the background is clear.

[0036] S27. Construction and culture of co-expression engineered strains

[0037] Plasmids pETDuet1-galE-GalTpm1141, pETDuet1-GalTpm1141-galE, pRSFDuet1-SPase-GalT, and pRSFDuet1-GalT-SPase were constructed using a one-step cloning method. These four plasmids were then introduced into *E. coli* JM109 according to Table 3 to obtain bacterial strains. The strains were plated onto LB agar plates containing Amp and Kans and incubated overnight at 37°C. Positive strains were selected and induced. SDS-PAGE analysis of the induced culture medium was performed to screen for co-expressing engineered strains. Based on the SDS-PAGE results, suitable induced culture media were centrifuged, the supernatant was discarded, and the bacterial sludge was stored at -80°C.

[0038] The preparation of fluoroglucose, step S1, includes the following steps:

[0039] S11, Synthesis of 1,2,3,4,6-penta-O-acetyl-D-glucopyranose

[0040] Anhydrous glucose, acetic anhydride solution, and anhydrous pyridine solution were mixed in a concentration ratio of 1:32:3. 108 mg (600 μmol) of anhydrous glucose was accurately weighed into a reaction flask using an analytical balance. 1.8 mL (19.2 mmol) of acetic anhydride solution and 145 µL (1.8 mmol) of anhydrous pyridine solution were added. After adding the rotor, the mixture was stirred in an oil bath at 110°C for approximately 2 hours until completely dissolved. The mixture was then stirred at room temperature for 12 hours to obtain an α-configuration product. Finally, thin-layer chromatography (TLC) was used to determine if the reaction was complete.

[0041] After the reaction is complete, turn on the rotary evaporator, set the temperature to 80℃ and the rotation speed to 30 rpm. Place the flask containing the sample onto the interface, close the vent valve, turn on the cooling water and vacuum pump, and after the pressure stabilizes, lower the liquid level in the flask below the water level in the water bath. Turn on the rotary evaporator to start the rotary evaporation. After complete evaporation, turn off the rotary evaporator and remove the sample.

[0042] S12, Synthesis of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose

[0043] The rotary evaporated sample was dissolved in 1 mL of pre-cooled dichloromethane, transferred to a 10 mL PP centrifuge tube, and then 3 mL of 70% pyridine hydrogen fluoride solution was added. The rotor was then added, and the mixture was in an ice bath for 38 h. The reaction progress was detected by TLC.

[0044] Synthesis of S13 and fluoroglucose

[0045] A known weight of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose was dissolved in anhydrous methanol to prepare a 200 mM 2,3,4,6-tetra-O-acetyl-D-fluoroglucose methanol solution. Sodium methoxide was dissolved in anhydrous methanol to prepare a 300 mM sodium methoxide methanol solution. The 2,3,4,6-tetra-O-acetyl-D-fluoroglucose methanol solution and sodium methoxide methanol solution were mixed in a volume ratio of 1:1.35. A rotor was added to the reaction flask, and the mixture was stirred in an ice-water bath for 3 h to remove the acetyl groups. The reaction progress was monitored by TLC. The eluent was ethyl acetate:anhydrous methanol:water in a ratio of 7:2:1. Finally, the reaction solution was concentrated by rotary evaporation at 30 °C and 10 rpm.

[0046] In one exemplary embodiment, the specific operation of S3, the dual-strain coupled fermentation for the synthesis of fluorolactose, is as follows: 400 mmol / L sucrose, 50 mmol / L fluoroglucose, 20 mmol / L UMP, 550 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L ethanol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether 100 g / L, engineered bacteria (wet weight), and 100 g / L Saccharomyces cerevisiae (dry weight) are mixed in proportion and reacted at 30°C and 200 rpm for 22 h.

[0047] Another technical solution adopted by the present invention to solve its technical problem is:

[0048] An application of a fluorolactose synthesis method in the fermentation preparation of GM3-F, wherein the above-mentioned fluorolactose synthesis method is used to provide fluorolactose for the preparation of GM3-F.

[0049] An application of a fluorolactose synthesis method in the fermentation preparation of GM3-F, the process of fermentation preparation of GM3-F includes the following steps:

[0050] S1. Construct the recombinant plasmid PETDuet1 neuA-nst3 using homologous recombination, and construct an engineered E. coli strain co-expressing the N-acylneuraminic acid cytidine transferase gene neuA and the α-2,3-sialidyl transferase gene nst3.

[0051] After optimizing the sequences of the neuA and nst3 genes using E. coli preferred codons, Tianlin Biotechnology Co., Ltd. synthesized the recombinant plasmid PETDuet1 neuA-nst3 and cloned it into the multiple cloning site of the corresponding vector through homologous recombination.

[0052] The successfully constructed plasmid PETDuet1 neuA-nst3 was transformed into competent E. coli JM109(DE3) cells. Amp was used as a selection marker for plate plating. After colony PCR verification, single colonies were picked and cultured in shake flasks. The plasmid was extracted from the bacterial culture for PCR verification. Expression of PETDuet1 neuA-nst3 in the engineered E. coli JM109(DE3) cells was induced and verified by SDS-PAGE analysis.

[0053] S2. GM3-F was synthesized by coupled fermentation of engineered Escherichia coli JM109(DE3) / PETDuet1 neuA-nst3 and yeast.

[0054] Using the fluorolactose and sialic acid prepared in Example 2 as substrates, the co-expressed Escherichia coli engineered bacteria constructed above were coupled with yeast for fermentation to synthesize GM3-F.

[0055] The nucleotide sequences of the N-acylneuraminic acid cytidine transferase gene neuA and the α-2,3-sialidyl transferase gene nst3 are shown in SEQ NO.5 and SEQ NO.6, respectively.

[0056] GM3-F was synthesized by co-expressing the engineered strain and yeast through coupled fermentation: 80 mmol / L fluorolactose, 70 mmol / L LMP, 60 mmol / L Neu5Ac, 300 mmol / L glucose, 250 mmol / L K2HPO4, 150 mmol / L Tris, 20 mmol / L MgCl2, 1 mmol / L DTT, 20 mL / L glycerol, 4 g / L ethanol, 4 g / L octadecylamine polyoxyethylene ether, 100 g / L co-expressed engineered strain (wet weight) and 100 g / L Saccharomyces cerevisiae (dry weight), and the reaction was carried out at 30℃ and 200 rpm for 24 h.

[0057] The engineered strain was co-expressed with yeast for fermentation. The reaction progress was detected by TLC. The reaction solution showed the Rf value of the product GM3-F, which preliminarily confirmed that GM3-F was successfully prepared.

[0058] An appropriate amount of the coupled fermentation broth was sieved and analyzed by LC-MS to determine that the fermentation product was GM3-F.

[0059] The beneficial effects of the whole-cell synthesis method for fluorolactose of the present invention are as follows:

[0060] The present invention discloses a method for whole-cell synthesis of fluorolactose, using *Escherichia coli* EcN(DE3) as the starting strain. It utilizes a vector to overexpress the UDP-glucose-4-epimerase gene galE, the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose pyrophosphorylase gene GalT, and the sucrose phosphorylase gene SPase to obtain engineered strains that overexpress a single gene galE, GalTpm1141, GalT, or SPase, thereby achieving whole-cell synthesis of lactose using fluoroglucose as a substrate. Furthermore, recombinant *E. coli* strains co-expressing galE, GalTpm1141, GalT, and SPase are used as co-expression engineered strains in the lactose synthesis system, enabling whole-cell synthesis of lactose using glucose and sucrose as substrates. Simultaneously, the yeast's own enzymes effectively catalyze the conversion of UMP to UTP, and the synthesized UTP enters the lactose synthesis system, participating in the synthesis of UDP-Gal, fully meeting the cofactor supply requirements, thereby improving the efficiency and yield of whole-cell synthesis of fluorolactose. In addition, a method for synthesizing fluoroglucose from glucose as a starting material via acetylation protection, fluorine substitution reaction, and deacetylation reaction has been proposed.

[0061] The whole-cell synthesis method of fluorolactose of the present invention provides a new method for the synthesis of fluorolactose, which is safe and efficient, and provides a new synthetic route for the preparation of fluorolactose, the raw material of GM3-F, which can further promote the whole-cell catalytic synthesis of GM3-F. Attached Figure Description

[0062] Figure 1 —The chemical structural formula of fluorolactose in this invention;

[0063] Figure 2 —A diagram illustrating the synthetic pathway of fluoroglucose in this invention;

[0064] Figure 3 —This invention relates to a schematic diagram of the synthetic pathways for lactose and fluorolactose;

[0065] Where A: lactose synthesis pathway; B: fluorolactose synthesis pathway;

[0066] Figure 4 —SDS-PAGE and TLC analysis of fluorolactose synthesized by engineered bacteria E1 in Example 1 of this invention;

[0067] Wherein, A: (a) SDS-PAGE results of the induction solution of strain E1; M: Protein Marker; 1: Pre-induction protein of strain E1; 2: Post-induction protein of strain E1; B: TLC detection and analysis diagram;

[0068] Figure 5—TLC analysis of the product of glucose acetylation protection reaction in Example 2 of this invention;

[0069] Where a: glucose standard; 1: reaction solution;

[0070] Figure 6 —Mass spectrum of 1,2,3,4,6-penta-O-acetyl-D-pyranose in Example 2 of this invention;

[0071] Figure 7 —TLC analysis chromatogram of fluorine substitution in Example 2 of this invention;

[0072] Wherein, a: pentaacetylglucose standard; b: tetraacetylglucose standard; 1, 2, 3, and 4 are reaction solutions for 5 h, 17 h, 25 h, and 41 h, respectively;

[0073] Figure 8 —Mass spectrum of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose, the product of the fluorine substitution reaction in Example 2 of this invention;

[0074] Figure 9 —TLC analysis chromatogram of fluoroglucose in Example 2 of this invention;

[0075] Wherein, a: glucose standard; b: fluoroglucose standard; 1: reaction solution;

[0076] Figure 10 —LC-MS detection and analysis diagram of fluoroglucose in Example 2 of this invention;

[0077] Figure 11 —Flowchart of the construction of co-expression plasmid in Example 2 of this invention;

[0078] Figure 12 —A schematic diagram of four plasmid combinations of co-expressed engineered strains E6, E7, E8, and E9 in Example 2 of this invention;

[0079] Figure 13 —SDS-PAGE analysis diagram of four co-expressed engineered strains after IPTG induction in Example 2 of this invention;

[0080] Where M: protein marker; 1, 3, 6, 8: bacterial proteins after induction of strains E6, E7, E8, E9; 2, 4, 5, 7: bacterial proteins before induction of strains E6, E7, E8, E9.

[0081] Figure 14 —TLC analysis of whole-cell synthesis of fluorolactose by co-expressed engineered strain and yeast in Example 2 of this invention;

[0082] Wherein, a: lactose standard; b: fluorolactose standard; c: fluoroglucose standard; 1: E6 coupled fermentation broth; 2: E7 coupled fermentation broth; 3: E8 coupled fermentation broth; 4: E9 coupled fermentation broth;

[0083] Figure 15 —LC-MS detection and analysis of whole-cell synthesis of fluorolactose by co-expressed engineered strains and yeast in Example 2 of this invention;

[0084] Figure 16 —A schematic diagram of the pathway for the synthesis of fluorolactose by co-expressed engineered strains and yeast through whole-cell fermentation in Example 2 of this invention;

[0085] Figure 17 —TLC analysis of the whole-cell synthesis of fluorolactose by four engineered strains coupled with yeast in Example 3 of this invention;

[0086] Wherein, a: UDP-gal standard; b: fluoroglucose standard; c: fluorolactose standard; d: lactose standard; 1: coupled fermentation broth.

[0087] Figure 18 —SDS-PAGE analysis of Escherichia coli induced expression in Example 5 of this invention;

[0088] Where M: protein marker; 1: bacterial culture before induction; 2: bacterial culture after induction;

[0089] Figure 19 —TLC analysis of GM3-F prepared by fermentation in Example 5 of this invention;

[0090] 1: Catalytic stock solution with sialic acid and fluorolactose as substrates; 2: 3'-sialyl lactose (3'-SL); 3: Reaction solution for coupled fermentation of GM3-F; 4: Lactose standard; 5: Fluorolactose standard. Detailed Implementation

[0091] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0092] The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were all commercially available products, and the specific operations were performed according to the kit instructions. Routine procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0093] The following is a description of the sources of the materials involved in this invention:

[0094] (1) Engineered bacteria: Escherichia coli JM109 was purchased from Tiangen Biotech (Beijing) Co., Ltd.;

[0095] (2) Reagents used in organic experiments: glucose, anhydrous pyridine, acetic anhydride, dichloromethane, n-hexane, petroleum ether, ethyl acetate, N,N-dimethylformamide (DMF), 70% pyridine hydrogen fluoride solution, anhydrous methanol, 0.3 M sodium methoxide solution, saturated sodium bicarbonate solution, and saturated copper sulfate solution.

[0096] Table 1. Main Reagents for Biological Experiments

[0097] Table 1 Experimental Reagents

[0098]

[0099] All other reagents used are commercially available.

[0100] (3) Source of the gene required for the experiment: The sequence of the gene GalTpm1141 was optimized by the E. coli preferred codons and synthesized by Tianlin Biotechnology Co., Ltd. to obtain the recombinant plasmid pET28a-GalTpm1141.

[0101] (4) Culture medium and main solutions

[0102] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl. For solid LB medium, add an additional 20 g / L agar powder.

[0103] When using the above culture media, add the corresponding antibiotics according to the following working concentrations based on the plasmids contained in the recombinant strain: ampicillin (Amp) 100 µg / mL, kanamycin (Kan) 50 µg / mL.

[0104] (5) Primer design

[0105] Table 2. Main primers

[0106]

[0107] (6) Strains and plasmids

[0108] The plasmid vectors pETDuet1 and pRSFDuet1 plasmid system were used for genome assembly and expression. E. coli JM109 was used for plasmid construction, preservation, and amplification. The strains and plasmids involved in this invention are shown in Table 3. The nucleotide sequences of the genes involved in plasmid construction are shown in Table 4.

[0109] Table 3. Strains and plasmids used in this invention

[0110] Table 4. Nucleotide sequence listing of the gene involved in this invention.

[0111] (7) The detection method used in this invention

[0112] (7-1) Validation by thin-layer chromatography (TLC)

[0113] Dilute the fermentation broth with ddH2O to a suitable concentration, cut out a suitable TLC plate, streak a line at the bottom 1.5 cm, and spot 1.5 µL of standard and test solution every 1 cm. After air drying or drying with a hot air gun, develop the plate with acetonitrile:water (volume ratio) = 4:1, stain with anisaldehyde, and develop at 165℃ for 5 min.

[0114] (7-2) LC-MS detection of fermentation broth

[0115] A certain amount of fermentation broth sample was sieved and placed in a liquid chromatography vial, then sent to the testing center for LC-MS analysis to ensure that the final product was fluorolactose.

[0116] Example 1

[0117] This embodiment of a whole-cell synthesis method for fluorolactose includes the following steps: using fluoroglucose and UDP-galactose as substrates, constructing an engineered Escherichia coli strain overexpressing the β-1,4-galactosyltransferase gene GalTpm1141, and synthesizing fluorolactose through whole-cell catalysis.

[0118] (1) The recombinant plasmid Pet28-a-GalTpm1141 was constructed using homologous recombination, and an engineered E. coli strain E1 overexpressing the β-1,4-galactosyltransferase gene GalTpm1141 was constructed.

[0119] The sequence of the gene GalTpm1141 (the nucleotide sequence of the β-1,4-galactosyltransferase gene GalTpm1141 is shown in SEQ NO.1) was optimized using E. coli preferred codons and synthesized by Tianlin Biotechnology Co., Ltd., to obtain the recombinant plasmid pET28a-GalTpm1141. The target gene was obtained by PCR from the laboratory-preserved experimental strain E4 and the recombinant plasmid pET28a-GalTpm1141. The PCR system and reaction conditions are shown in Tables 5 and 6 below.

[0120] Table 5 PCR reaction system

[0121]

[0122] Table 6 PCR Reaction System

[0123]

[0124] The corresponding fragment was obtained by PCR amplification. The gene fragment GalTpm1141 was amplified by primers pm1141-F and PM1141-R. After verification by nucleic acid electrophoresis, the fragment was excised and recovered. The fragment was then ligated by homologous recombination.

[0125] The successfully constructed recombinant plasmid Pet28-a-GalTpm1141 was transformed into competent E. coli JM109(DE3) cells. Amp was used as a selection marker for plate plating. After colony PCR verification, single colonies were picked and cultured in shake flasks. The plasmid was extracted from the bacterial culture for PCR verification. The engineered strain E. coli JM109(DE3) Pet28-a-GalTpm1141 (abbreviated: E1) was induced to express the plasmid, and SDS-PAGE analysis showed that the plasmid size was consistent with the actual molecular weight. Figure 4 As shown in -A, this indicates that the target protein was successfully expressed.

[0126] (2) Fluorolactose was synthesized in whole cells of E. coli engineered strain E1, which overexpressed the β-1,4-galactosyltransferase gene GalTpm1141.

[0127] The engineered strain E1 was used to synthesize fluorolactose in a 1 mL system using fluoroglucose and UDP-galactose as substrates. The reaction progress was detected by TLC. The stretching agent was acetonitrile:water = 4:1, and the colorimetric agent was anisaldehyde.

[0128] In this embodiment, the biosynthetic pathway of fluorolactose involves the synthesis of fluorolactose from fluoroglucose and UDP-galactose via β-1,4-galactosyltransferase produced by fermentation of engineered strain E1. Figure 4 As shown in -B, the lactose synthesis system constructed in this embodiment successfully synthesized fluorolactose by co-expressing engineered strain E1.

[0129] Example 2

[0130] This embodiment of a method for whole-cell synthesis of fluorolactose includes the following steps:

[0131] S1. Preparation of fluoroglucose

[0132] Following Yokoyama's method for synthesizing fluorinated sugars, fluorinated glucose was synthesized from glucose as a starting material via acetylation protection, fluorine substitution, and deacetylation. The specific synthetic route is as follows: Figure 2 As shown.

[0133] S11, Synthesis of 1,2,3,4,6-penta-O-acetyl-D-glucopyranose

[0134] 108 mg (600 μmol) of anhydrous glucose was accurately weighed into a reaction flask using an analytical balance. 1.8 mL (19.2 mmol) of acetic anhydride solution and 145 µL (1.8 mmol) of anhydrous pyridine solution were added. After adding the rotor, the mixture was stirred in an oil bath at 110 °C for about 2 h until the mixture was completely dissolved. Then, the mixture was stirred at room temperature for 12 h to obtain the α-configuration of the product. Finally, the reaction was detected by thin-layer chromatography (TLC). The TLC detection method is described in (7-1).

[0135] The sample was diluted 10-fold with N,N-dimethylformamide (DMF), and a suitable TLC plate was cut. A line was streaked 1 cm from the bottom. 0.5 µL of glucose standard and the sample solution were spotted every 0.7 cm. After air drying or hot air drying, the plate was placed in a chromatography tank with hexane:ethyl acetate = 1:1 as the stretching agent. The plate was stretched to 1 cm from the top, removed, dried, stained with lichenin, and heated in a 110°C oven until color development. The results are as follows: Figure 5 As shown, the glucose standard is located at the origin, the reaction sample does not show color at the origin, and the lichenol shows color at only one point, which suggests that acetylation protection is complete.

[0136] After the reaction is complete, turn on the rotary evaporator, set the temperature to 80℃ and the rotation speed to 30 rpm. Place the flask containing the sample onto the interface, close the vent valve, turn on the cooling water and vacuum pump, and after the pressure stabilizes, lower the liquid level in the flask below the water level in the water bath. Turn on the rotary evaporator to start the rotary evaporation. After complete evaporation, turn off the rotary evaporator and remove the sample.

[0137] To further clarify the molecular weight of the product, MALDI-TOF-MS, with higher measurement accuracy, was used for detection. A small amount of the concentrated sample was taken, pipetted onto it with a 1 mL pipette, diluted 20-fold with DMF, and then 1 μL was dropped onto the target plate to determine its molecular weight. After the sample crystallized, 1 μL of DHB matrix (prepared by dissolving 20 mg of 2,5-dihydroxyformic acid in a 30% acetonitrile solution containing 0.1% TFA to a concentration of 1 mL) was dropped onto the sample, recrystallized, and then used for MALDI-TOF-MS detection. Simultaneously, 1 µL of mixed peptide standards was placed on the target plate for instrument calibration. The instrument was calibrated using the above-mentioned mixed standards to achieve an accuracy of 0.1 Da before the sample was measured. The signal values ​​from at least five measurements were superimposed to obtain the final mass spectrum. The detection mode was positive ion mode, the accelerating voltage was set to 20 k, and the scan range was m / z 100–400 Da. The mass spectrum was analyzed using Bruker Flexanalysis 3.3 software.

[0138] Mass spectrometry detection results as follows Figure 6 As shown, the main peak in the mass spectrum with a mass-to-charge ratio of 412.9 [M+Na]+ corresponds to the molecular ion peak of the sodium adduct of 1,2,3,4,6-penta-O-acetyl-D-pyranose, confirming that... Figure 5 The TLC results shown are correct.

[0139] S12, Synthesis of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose

[0140] 70% pyridine hydrogen fluoride solution is a good fluorinating agent, capable of directly substituting the hydroxyl group on monosaccharides with fluorine. However, for acetyl-protected monosaccharides, only the acetyl group at the first position can be fluorinated. Therefore, using 1,2,3,4,6-penta-O-acetyl-D-pyranose glucose obtained in the previous step as a substrate, adding 70% pyridine hydrogen fluoride solution allows for the fluorination of the acetyl group at the first position, thus achieving the synthesis of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose.

[0141] Synthesis of S121, 2,3,4,6-tetra-O-acetyl-D-fluoroglucose

[0142] The rotary evaporated sample was dissolved in 1 mL of pre-cooled dichloromethane, transferred to a 10 mL PP centrifuge tube, and then 3 mL of 70% pyridine hydrogen fluoride solution was added. The rotor was then added, and the mixture was in an ice bath for 38 h. The reaction progress was detected by TLC.

[0143] The sample to be tested was diluted 10 times with N,N-dimethylformamide (DMF), and a suitable TLC plate was cut out. A line was streaked 1 cm from the bottom, and 0.5 µL of standard and test solution were spotted every 0.7 cm. After air drying or drying with a hot air gun, the plate was placed in a chromatography tank with petroleum ether:ethyl acetate = 2:1 as the stretching agent. The plate was stretched to 1 cm from the top, removed, dried, stained with lichenol, and heated to 110 °C until the color developed.

[0144] Because a fluorine substitution reaction occurs, the polarity of the product decreases, suggesting that the product should be positioned above the substrate. The result is as follows... Figure 7 As shown in the figure, a product with the same Rf value as the 2,3,4,6-tetra-O-acetyl-D-fluoroglucose standard was present, and the reaction reached near equilibrium after 25 h, at which point the byproducts began to increase. Simultaneously, TLC detected multiple components, requiring silica gel column purification to separate the different components and obtain relatively pure 2,3,4,6-tetra-O-acetyl-D-fluoroglucose.

[0145] To further determine the molecular weight of different components, MALDI-TOF-MS, which offers higher measurement accuracy, was used. Small amounts of samples with different Rf values ​​were diluted 20-fold with DMF and then analyzed by MALDI-TOF-MS. The detection method was the same as in S11, and the results are as follows. Figure 8 As shown, the mass-to-charge ratio is 373 [M+Na] + and 413 [M+Na] + The two peaks are the ion peaks of the sodium adduct molecules of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose and 1,2,3,4,6-penta-O-acetyl-D-pyranose, respectively, which confirms the TLC results.

[0146] After TLC verification, the reaction solution was transferred to a PP separatory funnel. The centrifuge tube was rinsed several times with dichloromethane and transferred to the separatory funnel. 100 mL of ultrapure water was added, the cap was tightened, and the mixture was shaken vigorously. The vent valve was slowly opened to release gas. This process was repeated 3-4 times. The mixture was then placed on a triple iron ring, the cap was opened, and the mixture was allowed to stand for 1-2 minutes. After separation, the lower organic phase was collected in a beaker, and the upper aqueous phase was discarded. The organic phase was poured back into the separatory funnel, a certain amount of saturated copper sulfate solution was added, the cap was tightened, and the mixture was shaken to release gas 3-4 times. The lower organic phase was collected. The organic phase was poured back into the separatory funnel again, a certain amount of saturated sodium bicarbonate solution was added, and the mixture was shaken and released gas 3-4 times. The lower organic phase was collected. Finally, the organic phase was poured back into the separatory funnel, a certain amount of saturated sodium chloride solution was added, and the mixture was shaken and released gas 3-4 times. After separation, the lower organic phase was collected, an appropriate amount of anhydrous magnesium sulfate was added, the bottle was sealed, and the mixture was allowed to stand for 15-20 minutes to remove moisture. If the sample volume is too large, rotary evaporation can be performed at 35℃ and 10 rpm to achieve an appropriate volume.

[0147] Silica gel column purification of S122, 2,3,4,6-tetra-O-acetyl-D-fluoroglucose

[0148] The residual pyridine hydrogen fluoride in the reaction solution was removed by extraction, and the solution was purified in a silica gel column. The purified tetraacetyl fluoroglucose was collected and concentrated by rotary evaporation.

[0149] Mix petroleum ether and ethyl acetate in a 2:1 ratio to prepare a certain amount of stretching solution. Select a SepaFlash column of appropriate size and slowly pass 100 mL of methanol and dichloromethane in batches to activate the adsorption column. After activation, pass the extracted sample into the column to complete the sample loading. Then, pass the stretching solution in for elution. Collect the sample in clean 10 mL centrifuge tubes, 5 mL per tube, for a total of 60 tubes.

[0150] Spot the sample onto a TLC plate every 2-3 tubes for initial observation of product elution, followed by detailed TLC analysis. The TLC detection method is the same as in S11.

[0151] The eluent with the same Rf value and containing only the product spot was placed in a conical-bottom flask and concentrated on a rotary evaporator at 30°C and 10 rpm. The rotary evaporation method was the same as in S11.

[0152] Transfer the concentrated sample to a 10 mL centrifuge tube of known mass, place the centrifuge tube on the positioning rack and support it with a tray, adjust the needle valve to blow nitrogen gas onto the liquid sample, and continuously adjust the height of the tray and positioning rack and the gas velocity of nitrogen gas according to the liquid level of the experimental sample to make ripples on the sample surface without splashing, until the solvent is basically evaporated.

[0153] After nitrogen blowing, the tube opening was sealed with sealing film and placed in a vacuum drying oven at 25°C for 12 hours, and then weighed.

[0154] Synthesis of S13 and fluoroglucose

[0155] After fluorine substitution, purification via silica gel column chromatography yields pure 2,3,4,6-tetra-O-acetyl-D-fluoroglucose. To obtain fluoroglucose, the acetyl groups at other positions in 2,3,4,6-tetra-O-acetyl-D-fluoroglucose need to be removed. Anhydrous solvents should be used in the deacetylation reaction to avoid the removal of the fluorine atom.

[0156] A known weight of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose was dissolved in anhydrous methanol to prepare a 200 mM 2,3,4,6-tetra-O-acetyl-D-fluoroglucose methanol solution. Sodium methoxide was dissolved in anhydrous methanol to prepare a 300 mM sodium methoxide methanol solution. The 2,3,4,6-tetra-O-acetyl-D-fluoroglucose methanol solution and sodium methoxide methanol solution were mixed in a volume ratio of 1:1.35. A rotor was added to the reaction flask, and the mixture was stirred in an ice-water bath for 3 h to remove the acetyl groups. The reaction progress was monitored by TLC. The eluent was ethyl acetate:anhydrous methanol:water in a ratio of 7:2:1, and the detection method was the same as in S11.

[0157] TLC test results are as follows Figure 9 As shown, glucose is more polar than fluoroglucose and is located at a lower position. The original point of the reaction sample did not show color, and the only point after the lichenol showed color was the same as the Rf value of the fluoroglucose standard. Therefore, it can be inferred that the deacetylation reaction was completed and fluoroglucose was synthesized.

[0158] Because fluoroglucose has a small molecular weight, it is difficult to detect using MALDI-TOF-MS, so LC-MS was used instead. The fluoroglucose solution obtained in the previous step was diluted 50 times with Wahaha purified water, then centrifuged at 12,000 rpm for 10 minutes. 40 μL of the supernatant was placed in a sample vial and sent to the testing center for LC-MS detection.

[0159] LC-MS detection and analysis results are as follows Figure 10 As shown, the main peak is [M+HCOO]⁻ with a mass-to-charge ratio of 227, which corresponds to the formate adduct of fluoroglucose, confirming the TLC results, i.e., the fluoroglucose was successfully synthesized.

[0160] Subsequently, the reaction solution was concentrated by rotary evaporation at 30°C and 10 rpm, using the same method as S11.

[0161] The concentrated sample was transferred to a 10 mL centrifuge tube of known mass. The centrifuge tube was placed on a positioning rack and supported by a tray. The needle valve was adjusted to blow nitrogen gas onto the liquid sample. The height of the tray and positioning rack and the gas velocity of the nitrogen gas were continuously adjusted according to the liquid level of the experimental sample to make ripples on the sample surface without splashing until the solvent was basically evaporated.

[0162] After nitrogen blowing, the tube opening was sealed with sealing film and placed in a vacuum drying oven at 25°C for 12 hours, and then weighed.

[0163] Finally, the reaction solution was concentrated by rotary evaporation at 30°C and 10 rpm.

[0164] S2. Constructing an engineered strain for co-expression of a lactose synthesis system.

[0165] UDP-Gal and fluoroglucose are key precursors in lactose synthesis. They can be synthesized from sucrose under the catalysis of sucrose phosphorylase (SPase), GalT, and galE, while fluoroglucose and UDP-Gal can be synthesized under the catalysis of β-1,4-galactosyltransferase (GalTpm1141). Four genes—UDP-glucose pyrophosphorylase (GalT), sucrose phosphorylase (SPase), UDP-glucose-4-epimerase (galE), and β-1,4-galactosyltransferase (GalTpm1141)—were combined. The nucleotide sequences of these four genes are shown in SEQ NO.1, SEQ NO.2, SEQ NO.3, and SEQ NO.4, respectively. Using two compatible plasmids with different copy numbers, pETduet-1 and pRSFduet-1, as co-expression vectors, these genes were introduced into a single engineered bacterium to construct an engineered bacterium expressing sucrose phosphorylase (SPase), GalT, galE, and GalTpm1141. This reduces the number of transmembrane steps and increases the reaction yield.

[0166] Using *E. coli* JM109 as the starting strain, the UDP-glucose-4-epimerase gene *galE* and the β-1,4-galactosyltransferase gene *GalTpm1141* were overexpressed using the pETDuet1 vector, and the UDP-glucose pyrophosphorylase gene *GalT* and the sucrose phosphorylase gene *SPase* were overexpressed using the pRSFDuet1 vector. Plasmids *pETDuet1-galE-GalTpm1141*, *pETDuet1-GalTpm1141-galE*, *pRSFDuet1-SPase-GalT*, and *pRSFDuet1-GalT-SPase* were introduced to construct various lactose synthesis systems co-expressing engineered strains through different plasmid combinations. Specifically, *GalT* encodes UDP-glucose pyrophosphorylase; *galE* encodes UDP-glucose-4-epimerase; *SPase* encodes sucrose phosphorylase; and *GalTpm1141* is derived from *Pasteurella*. The gene encoding β-1,4-galactosyltransferase of *Pasteurella multocida*.

[0167] S21. Acquisition and amplification of the target gene

[0168] The sequence of the gene GalTpm1141 was optimized using E. coli preferred codons and synthesized by Tianlin Biotechnology Co., Ltd., to obtain the recombinant plasmid pET28a-GalTpm1141. The target gene was obtained from the laboratory-preserved experimental strain E4 and the recombinant plasmid pET28a-GalTpm1141 using PCR. The PCR system and reaction conditions were the same as in Example 1.

[0169] S22, agarose gel electrophoresis and gel recovery

[0170] Take an appropriate amount of 1×TAE buffer and add agarose at a ratio of 1% (w / v). Heat in a microwave oven until completely dissolved. Add 1 µL of Gel Red staining agent per 10 mL of buffer, mix well, and pour into a gel casting apparatus. Insert a comb of appropriate size into the apparatus. After cooling and solidification, remove the comb to create sample wells. Place the gel in an electrophoresis tank, add electrophoresis buffer, and inject 20 µL of PCR product into the sample wells. Then connect the electrodes, set the voltage to 4-10 V / cm, and begin electrophoresis.

[0171] After electrophoresis, the gel containing the target DNA fragment was quickly cut off under UV light and placed in an empty centrifuge tube. An appropriate amount of Buffer GDP was added. The gel was incubated at 55°C for approximately 8 minutes to completely dissolve the gel before being added to the adsorption column. The column was centrifuged at 12000 rpm for 1 minute, and the filtrate was discarded. Then, 300 µL of Buffer GDP was added to the adsorption column, and after standing for 1 minute, it was centrifuged at 12000 rpm for 1 minute, and the filtrate was discarded. Next, 700 µL of Buffer GW was added to the adsorption column, and after centrifugation at 12000 rpm for 1 minute, the filtrate was discarded. This step was repeated. The adsorption column was then placed in a collection tube and centrifuged at 12000 rpm for 2 minutes. Finally, the adsorption column was placed in a 1.5 mL centrifuge tube, and approximately 30 µL of Elution Buffer was added to the center of the column. After standing for approximately 2 minutes, the column was centrifuged at 12000 rpm for 1 minute to extract the desired DNA.

[0172] S23. Construction of recombinant plasmids

[0173] Recombinant plasmids were constructed using the ClonExpress MultiS One Step Cloning Kit (a seamless cloning kit based on homologous recombination technology).

[0174] The reaction conditions are shown in Table 7.

[0175] Table 7 One-step cloning system

[0176]

[0177] S24. Preparation and transformation of competent Escherichia coli cells

[0178] (1) Streak the preserved Escherichia coli cells onto antibiotic-free LB plates and incubate overnight at 37°C. Inoculate single colonies into 10 mL of LB liquid medium and incubate at 37°C and 200 rpm in a shaker for 10-12 h.

[0179] (2) Inoculate 2 mL of bacterial culture into 100 mL of LB medium and incubate at 37°C with shaking at 200 rpm for about 3 h;

[0180] (3) Place the cultured bacterial solution on ice to pre-cool for 5 min, centrifuge at 8000 rpm for 5 min to collect the bacterial cells, and repeat once;

[0181] (4) Add 25 mL of pre-cooled Solution A to resuspend the bacteria, centrifuge at 8000 rpm for 5 min to collect the cells, and repeat once;

[0182] (5) Add 5 mL Solution B to resuspend the bacteria, take 100 µL of the resuspension and dispense it into 1.5 mL centrifuge tubes, place on ice for later use or store at -80℃.

[0183] Chemical transformation method: Add an appropriate amount of plasmid vector to competent cells, mix well by pipetting, incubate on ice for 30 min, then heat shock at 42℃ for 90 s, incubate on ice for 2 min, and add 1 mL of antibiotic-free LB liquid medium. Incubate at 37℃ for 1 h, centrifuge the incubated bacterial solution at 12000 rpm for 2 min, retain about 100 µL of supernatant, mix well by pipetting, and spread onto LB plates containing the corresponding antibiotic.

[0184] S25, recombinant protein induced expression

[0185] After streaking the engineered bacteria onto plates, incubate overnight at 37°C. Pick a single colony and inoculate it into 10 mL of LB liquid medium, incubating overnight at 37°C with a shaker at 200 rpm. Then, inoculate the seed culture at a 2% inoculation rate into 100 mL of LB medium and incubate at 37°C with a shaker at 200 rpm for 2-3 hours. When OD... 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.3 mmol / L and incubate in a shaker at 15℃ and 200 rpm for 20 h.

[0186] S26, SDS-PAGE detection

[0187] Take a certain amount of culture medium, centrifuge, discard the supernatant, add protein loading buffer, resuspend the bacterial cells, boil in a water bath for 5 min, then remove and add 10 µL of sample to the protein loading wells. Turn on the electrophoresis apparatus at 80 V for 30 min, then switch to 150 V and continue for 1.5-2 h. When the protein marker and sample bands are close to the bottom of the protein gel, turn off the electrophoresis, remove the protein gel, and end the electrophoresis. Transfer the gel to a culture dish, add Coomassie Brilliant Blue staining solution, and shake to stain for 1 h. After staining, place the gel in a culture dish containing destaining solution and shake to destain. Replace the destaining solution after several hours until the background is clear.

[0188] S27. Construction and culture of co-expression engineered strains

[0189] Using two compatible plasmids with different copy numbers, pETduet-1 and pRSFduet-1, as co-expression vectors, four plasmids were constructed via a one-step cloning method: pETDuet1-galE-GalTpm1141, pETDuet1-GalTpm1141-galE, pRSFDuet1-SPase-GalT, and pRSFDuet1-GalT-SPase. The construction process of the co-expression plasmids is as follows: Figure 11 As shown in Table 3, the recombinant plasmids pETduet1 and pRSFduet1 were introduced into E. coli JM109(DE3) in pairs to obtain co-expression engineered strains (hereinafter referred to as co-expression engineered strains) E6, E7, E8, and E9 of the lactose synthesis system. The four plasmid combinations of co-expression engineered strains E6, E7, E8, and E9 are shown in Table 3. Figure 12 As shown; the culture was spread onto LB agar plates containing Amp and Kan, and incubated overnight at 37°C. Positive strains were picked and induced, and the induced culture medium was analyzed by SDS-PAGE to screen for co-expressing engineered strains. Based on the SDS-PAGE results, suitable induced culture medium was centrifuged, the supernatant was discarded, and the bacterial sludge was stored at -80°C.

[0190] SDS-PAGE analysis was performed on four engineered strains after induction, and the results are as follows: Figure 13 As shown: In the four co-expression engineered strains, all four introduced genes were expressed normally.

[0191] S3, dual-strain coupled fermentation to synthesize fluorolactose

[0192] Using glucose and sucrose as substrates, the lactose synthesis system constructed by S2 was co-expressed with engineered strains and yeast for coupled fermentation to synthesize fluorolactose.

[0193] Fluorinated lactose was synthesized by coupled fermentation of co-expressed engineered strain and yeast: 400 mmol / L sucrose, 50 mmol / L fluoroglucose, 20 mmol / L UMP, 550 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L ethanol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L co-expressed engineered strain (wet weight) and 100 g / L Saccharomyces cerevisiae (dry weight), and the reaction was carried out at 30℃ and 200 rpm for 22 h.

[0194] Among them, the co-expression engineered strains were E6, E7, E8, and E9, obtained through S2 screening. These co-expression engineered strains were coupled with yeast for fermentation, and the reaction progress was detected by TLC. The results are as follows: Figure 14 As shown, the reaction solution only has the Rf value of the product fluorolactose, indicating that the coupled fermentation is relatively thorough and there is little substrate remaining.

[0195] After sieving an appropriate amount of the coupled fermentation broth, it was analyzed by LC-MS. The results are as follows: Figure 15 As shown in the figure. The molecular weight of fluorolactose is known to be 344.28. From the mass spectrum, it can be seen that the observed quasi-molecular ion peak m / z 343.1051 corresponds to [MH]⁻ (the deprotonated ion of the fluorolactose molecule), indicating that fluorolactose has been successfully synthesized.

[0196] Compared to the single-strain fermentation using UDP-Gal and fluoroglucose as substrates in Example 1, the co-expression engineered strain obtained by co-expressing four genes—sucrose phosphorylase (SPase), GalT, galE, and GalTpm1141—was coupled with yeast for fermentation. The pathway for synthesizing fluorolactose through two-strain coupled fermentation is as follows: Figure 16 As shown: the co-expressed engineered strain can obtain Glc-1-p from sucrose through sucrose phosphorylation enzyme (SPase), and obtain UDP-Glc using UDP-glucose pyrophosphorylase (GalT); then obtain UDP-Gal using UDP-glucose-4-epimerase (galE); finally, the Gal unit of UDP-Gal is linked to the 1-fluoroglucose unit through β-1,4-galactosyltransferase (GalTpm1141) to form fluorolactose; at the same time, yeasts such as Saccharomyces cerevisiae used in this embodiment can convert UMP to UTP using their own enzyme system. The synthesized UTP enters the engineered strain E. coli JM109(DE3), providing sufficient UTP for the synthesis of UDP-Glc, thereby promoting the synthesis of UDP-Gal, making the reaction more complete and showing obvious advantages.

[0197] Example 3

[0198] This embodiment of a method for whole-cell synthesis of fluorolactose includes the following steps:

[0199] Using sucrose and fluoroglucose as substrates, engineered E. coli strains E1, E11, E12, and E13 were constructed, overexpressing the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose-4-epimerase gene galE, the sucrose phosphorylase gene SPase, and the UDP-glucose pyrophosphorylase gene GalT, respectively. The specific construction method was the same as in Example 2. Engineered strains E1, E11, E12, and E13 and yeast were coupled and fermented in the above substrates. The reaction progress was detected by TLC. Fluorinated lactose standard was used as a control. The spreading agent was acetonitrile:water = 4:1, and anisaldehyde was used as the colorimetric reagent.

[0200] TLC test results are as follows Figure 17 As shown, four engineered strains, which overexpress the β-1,4-galactosyltransferase gene GalTpm1141, the UDP-glucose-4-epimerase gene galE, the sucrose phosphorylase gene SPase, and the UDP-glucose pyrophosphorylase gene GalT respectively, can also be coupled with yeast to ferment and synthesize fluorolactose.

[0201] Example 4

[0202] This embodiment of a method for whole-cell synthesis of fluorolactose includes the following steps:

[0203] S1. Construct a lactose synthesis system co-expressing engineered strain that overexpresses UDP-glucose-4-epimerase gene galE, β-1,4-galactosyltransferase gene GalTpm1141, UDP-glucose pyrophosphorylase gene GalT, and sucrose phosphorylase gene SPase. The construction method is the same as in Example 2.

[0204] S2, the lactose synthesis system co-expresses engineered strains that use sucrose and fluoroglucose as substrates and simultaneously supplement with UTP to ferment whole cells to synthesize fluorolactose.

[0205] Example 5

[0206] An application of a fluorolactose synthesis method in the fermentation preparation of GM3-F, wherein the above-mentioned fluorolactose synthesis method is used to provide fluorolactose for the preparation of GM3-F.

[0207] This embodiment describes the preparation of GM3-F by obtaining fluorolactose through whole-cell synthesis of fluorolactose and then using fluorolactose and sialic acid as substrates via fermentation. The process includes the following steps:

[0208] S1. Construct the recombinant plasmid PETDuet1 neuA-nst3 using homologous recombination, and construct an engineered E. coli strain co-expressing the N-acylneuraminic acid cytidine transferase gene neuA and the α-2,3-sialidyl transferase gene nst3.

[0209] After optimizing the sequences of genes neuA and nst3 using E. coli preferred codons, Tianlin Biotechnology Co., Ltd. synthesized and cloned them into the multiple cloning sites of the corresponding vectors via homologous recombination to construct the recombinant plasmid PETDuet1 neuA-nst3. The nucleotide sequences of the N-acylneuraminic acid cytidine transferase gene neuA and the α-2,3-sialidyl transferase gene nst3 are shown in SEQ NO.5 and SEQ NO.6, respectively.

[0210] The successfully constructed plasmid PETDuet1 neuA-nst3 was transformed into competent E. coli JM109(DE3) cells. Amp was used as a selection marker for plate plating. After colony PCR verification, single colonies were picked and cultured in shake flasks. The plasmid was extracted from the bacterial culture for PCR verification. The expression of PETDuet1 neuA-nst3 in the engineered E. coli JM109(DE3) cells was induced, and SDS-PAGE analysis showed that the expression was consistent with the actual molecular weight. Figure 18 As shown, this indicates that the target protein was successfully expressed.

[0211] S2. GM3-F was synthesized by coupled fermentation of engineered Escherichia coli JM109(DE3) / PETDuet1 neuA-nst3 and yeast.

[0212] Using the fluorolactose and sialic acid prepared in Example 2 as substrates, the co-expressed Escherichia coli engineered bacteria constructed above were coupled with yeast for fermentation to synthesize GM3-F.

[0213] Fermentation substrates and reaction conditions for the co-expression of engineered strains and yeast to synthesize GM3-F: 80 mmol / L fluorolactose, 70 mmol / L CMP, 60 mmol / L Neu5Ac, 300 mmol / L glucose, 250 mmol / L K2HPO4, 150 mmol / L Tris, 20 mmol / L MgCl2, 1 mmol / L DTT, 20 mL / L glycerol, 4 g / L ethanol, 4 g / L octadecylamine polyoxyethylene ether, 100 g / L S1-constructed co-expressed Escherichia coli engineered strain (wet weight) and 100 g / L Saccharomyces cerevisiae (dry weight), reacted at 30℃ and 200 rpm for 24 h.

[0214] The engineered strain was co-expressed with yeast for fermentation, and the reaction progress was detected by TLC. The results are as follows: Figure 19 As shown, the reaction solution contains the Rf value of product GM3-F, indicating that GM3-F has been successfully prepared. Further verification can be achieved by LC-MS determination after sieving a suitable amount of the coupled fermentation broth.

[0215] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for whole-cell synthesis of fluorolactose, characterized in that, The method includes the following steps: constructing a gene overexpressing β-1,4-galactosyltransferase. GalTpm1141 The engineered strain of *E. coli* was used to ferment and synthesize fluorolactose through whole-cell catalysis; or, an overexpression of the UDP-glucose-4-epimerase gene was constructed. galE β-1,4-galactosyltransferase gene GalTpm1141 UDP-glucose pyrophosphorylase gene GalT and sucrose phosphorylase gene SPase The lactose synthesis system was co-expressed with engineered strains for whole-cell fermentation to synthesize fluorolactose; or, by constructing separate structures that overexpress β-1,4-galactosyltransferase genes. GalTpm1141、 Overexpression of UDP-glucose-4-epimerase gene galE、 Overexpression of UDP-glucose pyrophosphorylase gene GalT, overexpression Sucrose phosphorylase gene SPase The engineered Escherichia coli was then used for four-strain coupled fermentation, and whole-cell catalytic synthesis of fluorolactose was achieved.

2. The whole-cell synthesis method of fluorolactose as described in claim 1, characterized in that, The method includes the following steps: S1. Constructing an engineered strain for co-expression of lactose synthesis system: Constructing a strain overexpressing the UDP-glucose-4-epimerase gene. galE β-1,4-galactosyltransferase gene GalTpm1141 UDP-glucose pyrophosphorylase gene GalT and sucrose phosphorylase gene SPase The lactose synthesis system co-expresses engineered strains; S2. Synthesis of fluorolactose by coupled fermentation of two strains: Using glucose and sucrose as substrates, the engineered strains co-expressing the lactose synthesis system constructed in S1 are coupled with yeast for fermentation to synthesize fluorolactose.

3. The whole-cell synthesis method for fluorolactose as described in claim 2, characterized in that, S1, constructing a lactose synthesis system co-expressing engineered strain: with E. coli JM109 was used as the starting strain, and the UDP-glucose-4-epimerase gene was overexpressed using the pETDuet1 vector. galE and β-1,4-galactosyltransferase gene GalTpm1141 The UDP-glucose pyrophosphorylase gene was overexpressed using the pRSFDuet1 vector. GalT and sucrose phosphorylase gene SPase; Import plasmid pETDuet1- galE - GalTpm1141 pETDuet1- GalTpm1141 - galE and pRSFDuet1- SPase - GalT pRSFDuet1- GalT - SPase Various lactose synthesis systems were co-expressed by engineered strains using different plasmid combinations.

4. The whole-cell synthesis method of fluorolactose as described in claim 3, characterized in that, S1. Constructing engineered strains for co-expression of lactose synthesis system, including the following steps: S11. Obtaining and amplifying the target gene, agarose gel electrophoresis, and gel recovery to extract the required DNA; Genes GalTpm1141 The sequence was optimized using E. coli preferred codons to obtain the recombinant plasmid pET28a- GalTpm1141 ; Using PCR, the experimental strain E4 and recombinant plasmid pET28a- were extracted from the laboratory-preserved experimental strain. GalTpm1141 To obtain the target gene; S12, Construction of recombinant plasmids; S13. Preparation and transformation of competent Escherichia coli cells, induction of recombinant protein expression and SDS-PAGE detection; S14. Construction and culture of co-expression engineered strains The plasmid pETDuet1- was constructed using a one-step cloning method. galE - GalTpm1141 pETDuet1- GalTpm1141 - galE pRSFDuet1- SPase - GalT pRSFDuet1- GalT - SPase Four plasmids were introduced into E. coli, respectively. E. coli JM109 was used to obtain an engineered strain co-expressing the lactose synthesis system. The strain was plated onto LB agar plates containing Amp and Kan and incubated overnight at 37°C. Positive strains were selected and induced. The inducing culture was then analyzed by SDS-PAGE to screen for co-expressing engineered strains. Based on the SDS-PAGE results, the appropriate inducing culture was centrifuged, the supernatant was discarded, and the bacterial sludge was stored at -80°C.

5. The whole-cell synthesis method of fluorolactose as described in claim 4, characterized in that, The recombinant plasmid was constructed using the ClonExpress MultiS One Step Cloning Kit. The specific procedures for preparing and transforming the competent E. coli cells are as follows: (1) Streak the preserved Escherichia coli cells onto antibiotic-free LB plates and incubate overnight at 37°C. Inoculate single colonies into 10 mL of LB liquid medium and incubate at 37°C and 200 rpm in a shaker for 10-12 h. (2) Inoculate 2 mL of bacterial culture into 100 mL of LB medium and incubate at 37°C with shaking at 200 rpm for about 3 h; (3) Place the cultured bacterial solution on ice to pre-cool for 5 min, centrifuge at 8000 rpm for 5 min to collect the bacterial cells, and repeat once; (4) Add 25 mL of pre-cooled Solution A to resuspend the bacteria, centrifuge at 8000 rpm for 5 min to collect the cells, and repeat once; (5) Add 5 mL Solution B to resuspend the bacteria, take 100 µL of the resuspension and dispense it into 1.5 mL centrifuge tubes, place on ice for later use or store at -80℃; Chemical transformation method: Add an appropriate amount of plasmid vector to competent cells, mix by blowing and aspiration, stand on ice for 30 min, heat shock at 42℃ for 90 s, place on ice for 2 min, and add 1 mL of antibiotic-free LB liquid medium. Incubate at 37℃ for 1 h, centrifuge the incubated bacterial solution at 12000 rpm for 2 min, retain about 100 µL of supernatant, mix by pipetting and aspiration, and then spread onto LB plates with the corresponding resistance.

6. The whole-cell synthesis method of fluorolactose as described in claim 4, characterized in that, The specific steps for inducing the expression of the recombinant protein are as follows: After streaking the engineered bacteria onto plates, incubate overnight at 37°C. Pick a single colony and inoculate it into 10 mL of LB liquid medium, then incubate overnight at 37°C and 200 rpm on a shaker. Inoculate the seed culture at a rate of 2% into 100 mL of LB medium and incubate at 37°C and 200 rpm on a shaker for 2-3 h. When the OD600 reaches 0.6-0.8, add IPTG to a final concentration of 0.3 mmol / L and incubate at 15°C and 200 rpm on a shaker for 20 h.

7. The whole-cell synthesis method of fluorolactose as described in claim 4, characterized in that, The specific operation of the SDS-PAGE detection is as follows: Take a certain amount of the culture medium obtained by recombinant protein induction expression, centrifuge and discard the supernatant, add protein loading buffer, resuspend the bacterial cells, boil in water for 5 min and then take it out, take 10 µL of sample and add it to the protein loading well, turn on the electrophoresis apparatus, voltage 80 V, maintain for 30 min and then switch to 150 V, continue for 1.5-2 h, when the protein marker and sample band are close to the bottom of the protein gel, turn off the electrophoresis, take out the protein gel and end the electrophoresis; transfer the gel to a culture dish, add Coomassie brilliant blue staining solution, shake and stain for 1 h, after staining, put the gel into a culture dish containing destaining solution, shake and destain, change the destaining solution after several hours until the background is clear.

8. The whole-cell synthesis method of fluorolactose as described in claim 2, characterized in that, S3. The specific operation for the synthesis of fluorolactose by coupled fermentation of two bacteria is as follows: 400 mmol / L sucrose, 50 mmol / L fluoroglucose, 20 mmol / L UMP, 550 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L ethanol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L co-expressed engineered strain (wet weight) and 100 g / L Saccharomyces cerevisiae (dry weight) are mixed in proportion and reacted at 30℃ and 200 rpm for more than 22 h.

9. The whole-cell synthesis method of fluorolactose according to any one of claims 2 to 8, characterized in that, The method further includes: using glucose as a starting material, synthesizing fluoroglucose through acetylation protection, fluorine substitution reaction and deacetylation reaction; Specifically, the following steps are included: S11, Synthesis of 1,2,3,4,6-penta-O-acetyl-D-glucopyranose: Anhydrous glucose, acetic anhydride solution, and anhydrous pyridine solution were mixed in a certain proportion and subjected to an acetylation protection reaction to obtain α-configured 1,2,3,4,6-penta-O-acetyl-D-pyranose. S12, Synthesis of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose: The fluorine substitution reaction of 1,2,3,4,6-penta-O-acetyl-D-pyranose prepared by S11 with pyridine hydrogen fluoride yielded 2,3,4,6-tetra-O-acetyl-D-fluoroglucose. Synthesis of S13, fluoroglucose: 2,3,4,6-tetra-O-acetyl-D-fluoroglucose prepared by S12 was dissolved in anhydrous methanol to obtain a methanol solution of 2,3,4,6-tetra-O-acetyl-D-fluoroglucose. The solution was then mixed with sodium methoxide methanol solution in a volume ratio of 1:1.35 to carry out a deacetylation reaction to obtain fluoroglucose.

10. The application of a fluorolactose synthesis method in the fermentation preparation of GM3-F, wherein the fluorolactose synthesis method as described in any one of claims 1 to 9 is used to provide fluorolactose for the preparation of GM3-F, characterized in that, The process for preparing GM3-F by fermentation includes the following steps: S1. Recombinant plasmid PETDuet1 neuA-nst3 was constructed using homologous recombination, and Escherichia coli co-expressing N-acylneuraminic acid cytidine transferase gene neuA and α-2,3-sialidyl transferase gene nst3 was constructed. S2. Using fluorolactose and sialic acid prepared by the method of fluorolactose synthesis according to any one of claims 1 to 9 as substrates, GM3-F is synthesized by coupled fermentation of Escherichia coli engineered strain JM109(DE3) / PETDuet1 neuA-nst3 constructed in step S1 and yeast.

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