An α-2,3-sialyltransferase mutant and its application

Abstract

This invention belongs to the field of microbial genetic engineering technology, specifically relating to an α-2,3-sialic acid transferase mutant and its application. This invention involves the modification of a mutant derived from Campylobacter jejuni (…). Campylobacter jejuni Six amino acids in the amino acid sequence of α-2,3-sialyltransferase encoded by the gene OH4384 were mutated at specific sites to obtain α-2,3-sialyltransferase mutants V175N, P198A, G173D, D174Q, F196K, and A315H. Among them, the single-point mutation V175N significantly increased the yield of 3'-sialyl lactose. The optimal recombinant microbial strain 3'-SL-09 (V175N / P198A / D174Q) was obtained through the combination of multiple mutation sites, with a yield of up to 5.91 g / L. This effectively solved the bottleneck problem of low catalytic efficiency of sialyltransferase and provided a high-performance enzyme element for the efficient and low-cost biomanufacturing of 3'-SL.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to an α-2,3-sialic acid transferase mutant and its application. Background Technology

[0002] Sialyllactose is an important oligosaccharide in human milk and one of the most abundant oligosaccharide components in breast milk. Chemically, it is a trisaccharide formed by sialic acid linked to the galactose group of a lactose molecule (glucose-β-1,4-galactose) via an α-2,3 or α-2,6 glycosidic bond. Depending on the position of the sialic acid linkage, it mainly exists in two isomers: 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL).

[0003] α-2,3-sialyltransferase (ST3Gal-T enzyme) is a class of glycosyltransferases that catalyze the linkage of sialic acid to the glycan terminus via α-2,3 glycosidic bonds. PmST1, in particular, is derived from Neisseria meningitidis (…). Neisseria meningitidis A typical α-2,3-sialic acid transferase identified in the study, its name "Pm" is derived from the abbreviation of the genus name (Pm). Pathogenic meningococcus "ST1" represents the member number in the sialyltransferase family.

[0004] The core function of these enzymes in organisms is to participate in the sialylation modification of glycans. Sialic acid, as an important terminal group of glycans, is widely present in cell surface glycoproteins and glycolipids, and its modified state directly affects physiological processes such as intercellular recognition, signal transduction, and immune escape. α-2,3-sialyltransferases use cytidine monophosphate sialic acid (CMP-sialicacid) as a donor to transfer sialic acid to the C3 hydroxyl position of a recipient glycan (such as a galactose residue), forming an α-2,3-linked sialic acid glycan structure.

[0005] Currently, the production of 3′-sialic acid lactose mainly relies on microbial fermentation, which involves optimizing the sialic acid synthesis pathway and transport system of host bacteria such as E. coli or yeast through genetic engineering, thereby achieving efficient, large-scale, and low-cost biomanufacturing. In addition, enzymatic catalysis (using sialic acid transferases, etc.) and chemical synthesis methods are also used, but the former is limited by enzyme costs, while the latter is difficult to industrialize due to its cumbersome steps and low yield. Summary of the Invention

[0006] To address the shortcomings of the prior art, the purpose of this invention is to provide an α-2,3-sialic acid transferase mutant and its applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an α-2,3-sialyltransferase mutant, wherein the amino acid sequence of the mutant is selected from any of the following:

[0009] (1) Based on the amino acid sequence shown in SEQ ID NO:1, there is any mutation or combination of V175N, P198A, G173D, D174Q, F196K or A315H, wherein the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid.

[0010] (2) is a protein that has more than 98% identity with the protein in (1) and has the same function, obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the mutant described in (1).

[0011] (3) A fusion protein with the same function obtained by linking a tag protein to the N-terminus and / or C-terminus of the mutant described in (1) and / or (2).

[0012] In some embodiments, the amino acid sequence of the α-2,3-sialyltransferase mutant contains both V175N and P198A mutations based on the amino acid sequence shown in SEQ ID NO: 1.

[0013] In some embodiments, the amino acid sequence of the α-2,3-sialyltransferase mutant contains mutations of V175N, P198A, and D174Q simultaneously based on the amino acid sequence shown in SEQ ID NO: 1.

[0014] A second aspect of the invention provides a nucleic acid molecule encoding the α-2,3-sialic acid transferase mutant described in this invention.

[0015] A third aspect of the invention provides a recombinant expression vector containing the nucleic acid molecule described herein.

[0016] A fourth aspect of the invention provides recombinant cells containing the recombinant expression vector described in this invention.

[0017] In some embodiments, the starting strain of the recombinant cells is selected from any one of Escherichia coli, yeast, Corynebacterium glutamicum, and Serratia marcescens.

[0018] In some embodiments, the starting strain of the recombinant cells is Escherichia coli.

[0019] In some embodiments, the starting strain of the recombinant cells is Escherichia coli BL21(DE3)-LH.

[0020] A fifth aspect of the invention provides the use of the α-2,3-sialyltransferase mutant, nucleic acid molecule, recombinant expression vector, or recombinant cell described herein in any of the following:

[0021] (a1) Preparation of 3'-sialyl lactose;

[0022] (a2) Prepare a product containing 3'-sialyl lactose;

[0023] (a3) Increase α-2,3-sialyltransferase;

[0024] (a4) Construct recombinant microorganisms for the production of 3'-sialyl lactose;

[0025] (a5) Regulate the production of 3'-sialic acid lactose by utilizing microorganisms.

[0026] A sixth aspect of the present invention provides a method for producing 3'-sialic acid lactose, comprising the following steps:

[0027] S1. Construct recombinant cells or recombinant microorganisms that can express the α-2,3-sialic acid transferase mutant described in this invention;

[0028] S2. Culture the recombinant cells or recombinant microorganisms to obtain 3'-sialic acid lactose.

[0029] In some embodiments of the present invention, the recombinant cells or recombinant microorganisms are selected from at least one of Escherichia coli, yeast, Corynebacterium glutamicum, and Serratia marcescens.

[0030] In some embodiments of the present invention, culturing the recombinant cells or recombinant microorganisms includes: inoculating the recombinant cells or recombinant microorganisms into a liquid culture medium and culturing for 10-14 hours to obtain a seed culture; taking the seed culture and inoculating it into a fermentation culture medium and culturing until the OD600 is 0.6-0.8, then inducing culture for 70-74 hours.

[0031] In some embodiments of the present invention, the liquid culture medium is LB culture medium and the fermentation culture medium is DM fermentation culture medium.

[0032] In some embodiments of the present invention, the conditions for induction culture are as follows: lactose with a final concentration of 2.5~3.5 g / L and IPTG of 0.4~0.6 mM are added to the fermentation medium for induction culture.

[0033] In some embodiments of the present invention, the induction culture temperature is 25°C and the rotation speed is 200 rpm.

[0034] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0035] This invention utilizes the method derived from Campylobacter jejuni ( Campylobacter jejuni Six amino acids in the amino acid sequence of α-2,3-sialyltransferase encoded by the gene (GenBank: AAF13495.1, OH4384) were mutated at specific sites to obtain α-2,3-sialyltransferase mutants V175N, P198A, G173D, D174Q, F196K, and A315H. Among them, the single-point mutation V175N significantly increased the yield of 3-sialyl lactose. The optimal recombinant microbial strain 3'-SL-09 (V175N / P198A / D174Q) was obtained through the combination of multiple mutation sites, with a yield of up to 5.91 g / L. This effectively solved the bottleneck problem of low catalytic efficiency of sialyltransferase and provided a high-performance enzyme element for the efficient and low-cost biomanufacturing of 3'-SL. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the recombinant plasmid PETduet-Psp2,3ST in an embodiment of the present invention;

[0037] Figure 2 This is a bar chart showing the yield of the α-2,3-sialic acid transferase mutant strain 3'-SL in an embodiment of the present invention. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0040] The culture medium formulation used in this embodiment of the invention is as follows:

[0041] LB+Amp medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 75 μg / mL ampicillin, pH 7.0.

[0042] DM fermentation medium: 20 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, and 10 mL / L trace metal elements; 100× trace metal element stock solution formula: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate. To prepare 1 L of DM fermentation medium, add 10 mL of the above stock solution (i.e., dilute 100 times). Adjust the pH to 6.8 with sodium hydroxide, sterilize at 115℃ for 30 min, and add 4.5 mg / L thiamine before use.

[0043] The experimental materials and main reagents used in the following examples are from the following sources:

[0044] The plasmid pETDuet-1 was purchased from Novagen / Merck, catalog number: 71146-3; Dpn I. The enzyme was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: 1609; E.coli DH5α was purchased from Beijing Qingke Biotechnology Co., Ltd., catalog number: DLC114-100; DL-BL21(DE3) strain was purchased from Daling Biotechnology, catalog number: DLC201-100; BL21(DE3)-LH strain was obtained by modifying DL-BL21(DE3) strain, specifically by knocking out the nanA and Lacz genes of strain DL-BL21(DE3) and then heterologously expressing the neuA and neuD genes. The public can obtain BL21(DE3)-LH strain from the laboratory of the Department of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences). This biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0045] The detection methods involved in the following embodiments:

[0046] Methods for detecting 3'-sialic acid lactose:

[0047] The content of 3'-sialic acid lactose was determined by high-performance liquid chromatography (HPLC) (Shimadzu, LC-20A). Specifically, 1 mL of fermentation broth was boiled for 5 min, centrifuged at 12,000 rpm for 5 min at room temperature, the supernatant was collected, and then filtered through an aqueous membrane with a pore size of 0.22 μm. The product concentration was then detected by HPLC. The HPLC instrument used a UV detector, a Carbomix H-NP column, 2.5 mM dilute sulfuric acid as the mobile phase, a detection temperature of 55℃, a flow rate of 0.6 mL / min, and an injection volume of 10 μL.

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0049] Example 1

[0050] 1. Acquisition and optimization of wild-type genes:

[0051] First, the unmutated Psp2,3ST gene was constructed. The gene sequence was then queried and compared using the NCBI database, and Campylobacter jejuni was selected. Campylobacter jejuni α-source of OH4384 2,3 Psp2,3ST, plasmid map as follows Figure 1 As shown, through thermal shock conversion to E.coli After overnight incubation at 37°C, plasmid PETduet was extracted. Psp2,3ST.

[0052] 2. Design of mutation sites and site-directed mutagenesis:

[0053] First, we obtain Campylobacter jejuni ( Campylobacter jejuni, The gene sequence of α-2,3-sialyltransferase (OH4384) was obtained, and the enzyme was modeled using Alphafold3. Molecular docking with the substrates lactose and CMP was performed using AutoDock and AutoDock Vina software. Based on intermolecular interactions and hydrogen bonds, potential key sites were identified around the acceptor-substrate binding pocket. Based on this, site-directed mutagenesis was performed on amino acids 175, 198, 173, 174, 196, and 315. Specifically, amino acid V at position 175 was changed to N (V175N), amino acid P at position 198 was changed to A (P198A), amino acid G at position 173 was changed to D (G173D), amino acid D at position 174 was changed to Q (D174Q), amino acid F at position 196 was changed to K (F196K), and amino acid A at position 315 was changed to H (A315H). Primers were designed as shown in Table 1.

[0054] Table 1 Primer Sequences

[0055]

[0056] The mutant amino acid sequence is as follows:

[0057] V175N:MTRTRMENELIVSKNMQNIIIAGNGPSLKNINYKRLPREYDVFRCNQFYFEDKYYLGKKIKAVFFNPGVFLQQYHTAKQLILKNEYEIKNIFCSTFNLPFIESNDFLHQFYNFFPDAKLGYEVIENLKEFYAYIKYNEIYFNKRITSGVYMCAIAIALGYKTIYLCGIDFYEGDNIYPFEAMSTNIKTIFPGIKDFKPSNCHSKEYDIEALKLLKSIYKVNIYALCDDSILANHFPLSININNNFTLENKHNNSINDILLTDNTPGVSFYKNQLKADNKIMLNFYNILHSKDNLIKFLNKEIAVLKKQTTQRAKARIQNHLSYKLGQALIINSKSVLGFLSLPFIILSIVISHKQEQKAYKFKVKKNPNLALPPLETYPDYNEALKEKECFTYKLGEEFIKAGKNWYGEGYIKFIFKDVPRLKREFEKGE(SEQ ID NO:15);

[0058] P198A:MTRTRMENELIVSKNMQNIIIAGNGPSLKNINYKRLPREYDVFRCNQFYFEDKYYLGKKIKAVFFNPGVFLQQYHTAKQLILKNEYEIKNIFCSTFNLPFIESNDFLHQFYNFFPDAKLGYEVIENLKEFYAYIKYNEIYFNKRITSGVYMCAIAIALGYKTIYLCGIDFYEGDVIYPFEAMSTNIKTIFPGIKDFKASNCHSKEYDIEALKLLKSIYKVNIYALCDDSILANHFPLSININNNFTLENKHNNSINDILLTDNTPGVSFYKNQLKADNKIMLNFYNILHSKDNLIKFLNKEIAVLKKQTTQRAKARIQNHLSYKLGQALIINSKSVLGFLSLPFIILSIVISHKQEQKAYKFKVKKNPNLALPPLETYPDYNEALKEKECFTYKLGEEFIKAGKNWYGEGYIKFIFKDVPRLKREFEKGE(SEQ ID NO:16);

[0059] G173D:MTRTRMENELIVSKNMQNIIIAGNGPSLKNINYKRLPREYDVFRCNQFYFEDKYYLGKKIKAVFFNPGVFLQQYHTAKQLILKNEYEIKNIFCSTFNLPFIESNDFLHQFYNFFPDAKLGYEVIENLKEFYAYIKYNEIYFNKRITSGVYMCAIAIALGYKTIYLCGIDFYEDDVIYPFEAMSTNIKTIFPGIKDFKPSNCHSKEYDIEALKLLKSIYKVNIYALCDDSILANHFPLSININNNFTLENKHNNSINDILLTDNTPGVSFYKNQLKADNKIMLNFYNILHSKDNLIKFLNKEIAVLKKQTTQRAKARIQNHLSYKLGQALIINSKSVLGFLSLPFIILSIVISHKQEQKAYKFKVKKNPNLALPPLETYPDYNEALKEKECFTYKLGEEFIKAGKNWYGEGYIKFIFKDVPRLKREFEKGE(SEQ ID NO:17);

[0060] D174Q:MTRTRMENELIVSKNMQNIIIAGNGPSLKNINYKRLPREYDVFRCNQFYFEDKYYLGKKIKAVFFNPGVFLQQYHTAKQLILKNEYEIKNIFCSTFNLPFIESNDFLHQFYNFFPDAKLGYEVIENLKEFYAYIKYNEIYFNKRITSGVYMCAIAIALGYKTIYLCGIDFYEGQVIYPFEAMSTNIKTIFPGIKDFKPSNCHSKEYDIEALKLLKSIYKVNIYALCDDSILANHFPLSININNNFTLENKHNNSINDILLTDNTPGVSFYKNQLKADNKIMLNFYNILHSKDNLIKFLNKEIAVLKKQTTQRAKARIQNHLSYKLGQALIINSKSVLGFLSLPFIILSIVISHKQEQKAYKFKVKKNPNLALPPLETYPDYNEALKEKECFTYKLGEEFIKAGKNWYGEGYIKFIFKDVPRLKREFEKGE(SEQ ID NO:18);

[0061] F196K: MTRTRMENELIVSKNMQNIIIAGNGPSLKNINYKRLPREYDVFRCNQFYFEDKYYLGKKIKAVFFNPGVFLQQYHTAKQLILKNEYEIKNIFCSTFNLPFIESNDFLHQFYNFFPDAKLGYEVIENLKEFYAYIKYNEIYFNKRITSGVYMCAIAIALGYKTIYLCGIDFYEGDVIYPFEAMSTNIKTIFPGIKDKKPSNCHSKEYDIEALKLLKSIYKVNIYALCDDSILANHFPLSININNNFTLENKHNNSINDILLTDNTPGVSFYKNQLKADNKIMLNFYNILHSKDNLIKFLNKEIAVLKKQTTQRAKARIQNHLSYKLGQALIINSKSVLGFLSLPFIILSIVISHKQEQKAYKFKVKKNPNLALPPLETYPDYNEALKEKECFTYKLGEEFIKAGKNWYGEGYIKFIFKDVPRLKREFEKGE (SEQ ID NO: 19);

[0062] A315H: MTRTRMENELIVSKNMQNIIIAGNGPSLKNINYKRLPREYDVFRCNQFYFEDKYYLGKKIKAVFFNPGVFLQQYHTAKQLILKNEYEIKNIFCSTFNLPFIESNDFLHQFYNFFPDAKLGYEVIENLKEFYAYIKYNEIYFNKRITSGVYMCAIAIALGYKTIYLCGIDFYEGDVIYPFEAMSTNIKTIFPGIKDFKPSNCHSKEYDIEALKLLKSIYKVNIYALCDDSILANHFPLSININNNFTLENKHNNSINDILLTDNTPGVSFYKNQLKADNKIMLNFYNILHSKDNLIKFLNKEIAVLKKQTTQRAKHRIQNHLSYKLGQALIINSKSVLGFLSLPFIILSIVISHKQEQKAYKFKVKKNPNLALPPLETYPDYNEALKEKECFTYKLGEEFIKAGKNWYGEGYIKFIFKDVPRLKREFEKGE (SEQ ID NO: 20).

[0063] 3. α 2,3 Construction of sialyltransferase mutants:

[0064] Taking the mutant V175N as an example, the plasmid PETduet was used. Using Psp2 and 3ST as templates, PCR amplification was performed using forward primer V175N-F and reverse primer V175N-R. High-fidelity polymerase 2× Phanta Max Master Mix was used. The PCR reaction system consisted of 25 μL of 2× Phanta Max Master Mix, 19 μL of ddH2O, 2 μL of forward primer, 2 μL of reverse primer, and 2 μL of template, for a total volume of 50 μL. The PCR reaction conditions were: 95℃ for 10 min, one cycle; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 30 s, 35 cycles; and 72℃ for 5 min, one cycle. After the reaction, the PCR product was analyzed by gel electrophoresis and recovered. 25 μL of the gel was mixed with 2 μL of DpnI enzyme and 5 μL of Cutone Buffer, and 18 μL of ddH2O, and incubated at 37℃ for 30 min. Dpn I. The digested product was transformed into DH5α and plated on LB+CL plates. The plates were incubated upside down at 37°C overnight. After colonies appeared, single colonies were picked using a 50 μL pipette tip and transferred to 50 mL of LB+Amp medium. Amp was added to each medium to a final concentration of 75 μg / mL. The plates were incubated at 37°C and 220 rpm for approximately 12 h. 1 mL of bacterial culture and 1 mL of 30% glycerol were added to sterile glycerol tubes for preservation. Plasmids were extracted using a plasmid kit and sequenced. The correctly sequenced positive transformant was identified as Psp2,3ST-V175N. Using the same method, plasmids Psp2,3ST-V175N, Psp2,3ST-P198A, Psp2,3ST-G173D, Psp2,3ST-D174Q, Psp2,3ST-F196K, and Psp2,3ST-A315H were finally constructed. DNA sequencing revealed that the site-directed mutations V175N, P198A, G173D, D174Q, F196K, and A315H were completely consistent with the intended mutations.

[0065] 4. Construction of α-2,3-sialyltransferase mutant strains:

[0066] First, a recombinant strain capable of producing 3′-SL was constructed. The selected strains were BL21(DE3)-LH, derived from K87 and containing neuA (encoding CMP-sialic acid synthase), neuD (encoding sialic acid acetylesterase with gene knockout), NanA (degrading sialic acid), and LacZ (degrading lactose). The plasmid PETduet-Psp2,3ST from Example 1 was transformed into the BL21(DE3)-LH strain. The transformed strain was plated on chloramphenicol and ampicillin-resistant plates, and positive clones were screened. The correctly identified positive transformants were 3′-SL-1 (…). E.coli BL21(DE3)-LH-PETduet-Psp2,3ST). Using the same method, 3′-SL-2 is finally constructed ( E.coli BL21(DE3)-LH-Psp2,3ST-V175N), 3′-SL-3( E.coli BL21(DE3)-LH-Psp2,3ST-P198A), 3′-SL-4( E.coli BL21(DE3)-LH-Psp2,3ST-G173D), 3′-SL-5( E.coli BL21(DE3)-LH-Psp2,3ST-D174Q), 3′-SL-6( E.coli BL21(DE3)-LH-Psp2,3ST-F196K), 3′-SL-7( E.coli BL21(DE3)-LH-Psp2,3ST-A315H).

[0067] Example 2

[0068] 1. Construction of α-2,3-sialyltransferase combinatorial mutants:

[0069] Taking the combined mutant V175N / P198A as an example, PCR amplification was performed using plasmid Psp2,3ST-P198A as a template and V175N-F and V175N-R as forward and reverse primers, respectively. PCR amplification was completed using the high-fidelity polymerase 2×PhantaMax MasterMix reaction system described in Example 1. After the reaction, the PCR product was analyzed by gel electrophoresis and the gel was recovered. 25 μL of the product was then added to the gel. Dpn I. The enzyme, 5 μL of Cutone Buffer, and 18 μL of ddH2O were mixed and incubated at 37°C for 30 min. The reaction proceeded... DpnI. The digested product was transformed into DH5α and plated on LB+Amp plates. It was incubated upside down at 37°C overnight. After colonies appeared, a single colony was picked using a 50 μL pipette tip and transferred to 50 mL of LB+Amp medium. Amp was added to each medium to a final concentration of 75 μg / mL. The medium was incubated at 37°C and 220 rpm for approximately 12 h. 1 mL of bacterial culture and 1 mL of 30% glycerol were added to sterile glycerol tubes for preservation. The plasmid (Psp2,3ST-V175N / P198A) was extracted using a plasmid kit and sequenced. The correctly sequenced positive transformant was Psp2,3ST-V175N / P198A. Using the same method, the plasmid Psp2,3ST-V175N / P198A / D174Q was finally constructed. DNA sequencing revealed that the combined mutations V175N / P198A and V175N / P198A / D174Q were completely consistent with the intended design mutations.

[0070] 2. Construction of α-2,3-sialyltransferase combinatorial mutant strains

[0071] Plasmid Psp2,3STV-175N / P198A was transformed into BL21(DE3)-LH, plated on chloramphenicol plus ampicillin plates, and positive clones were screened. The correct positive transformant was identified as 3'-SL-8 ( E.coli BL21(DE3)-LH-PETduet-Psp2,3ST-V175N / P198A). The same method was used to construct 3'-SL-9 ( E.coli BL21(DE3)-LH-PETduet-Psp2,3ST-V175N / P198A / D174Q).

[0072] Example 3

[0073] 1. Production of 3'-sialic acid lactose by shake-flask fermentation

[0074] The strains constructed in Examples 1 and 2 were inoculated into LB liquid medium containing the corresponding resistance and cultured overnight for 12 h to obtain seed culture. The seed culture was then inoculated into DM fermentation medium and cultured at 37°C and 200 rpm until the OD600 reached 0.6–0.8. Lactose and 0.5 mM IPTG were added to a final concentration of 3 g / L, and the culture was further induced at 25°C and 200 rpm for 72 h. 1 mL of fermentation broth was collected, and the supernatant was used for HPLC analysis. 1 mL of fermentation broth was centrifuged at 12,000 rpm for 5 min, and the supernatant was used for HPLC analysis. The results are shown in Table 2 and... Figure 2As shown, the control strain (3'-SL-1) produced 2.10 g / L of 3'-sialic acid lactose. The 3'-SL-02 strain had the highest 3'-SL yield, reaching 3.504 g / L, which was 66.86% higher than the control strain (3'-SL-1). The 3'-SL-3 strain produced 3.11 g / L of 3'-SL, which was 48.10% higher than the control strain (3'-SL-1). The 3'-SL-5 strain produced 2.91 g / L of 3'-SL, which was 38.58% higher than the control strain (3'-SL-1). The 3'-SL-06 strain produced 1.88 g / L of 3'-SL, which was 10% lower than the control strain (3'-SL-1). Among the single mutations, V175N, P198A, and D174Q were the optimal mutation points. The yield of 3'-SL in the double mutant strain 3'-SL-8 reached 4.10 g / L, which was 95.24% higher than that of the control strain (3'-SL-1). The yield of 3'-SL in the triple mutant strain 3'-SL-9 reached 5.91 g / L, which was 181.42% higher than that of the control strain (3'-SL-1).

[0075] Table 2. Yield of α-2,3-sialyltransferase mutant strain 3'-SL

[0076]

[0077] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An α-2,3-sialyltransferase mutant, characterized in that, The amino acid sequence of the α-2,3-sialyltransferase mutant contains mutations of V175N, P198A, and D174Q simultaneously, based on the amino acid sequence shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, It encodes the α-2,3-sialic acid transferase mutant of claim 1.

3. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 2.

4. Recombinant cells, characterized in that, It contains the recombinant expression vector as described in claim 3.

5. The use of the α-2,3-sialyltransferase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the recombinant cell of claim 4 in any of the following: (a1) Preparation of 3'-sialyl lactose; (a2) Prepare a product containing 3'-sialyl lactose; (a3) Improve the catalytic efficiency of α-2,3-sialyltransferase; (a4) Constructing recombinant microorganisms for the production of 3'-sialyl lactose; (a5) Regulate the production of 3'-sialic acid lactose by utilizing microorganisms.

6. A method for producing 3'-sialic acid lactose, characterized in that, Includes the following steps: S1. Construct recombinant cells or recombinant microorganisms capable of expressing the α-2,3-sialic acid transferase mutant of claim 1; S2. Culture the recombinant cells or recombinant microorganisms to obtain 3'-sialic acid lactose.

7. The method as described in claim 6, characterized in that, The recombinant cells or recombinant microorganisms are selected from at least one of Escherichia coli, yeast, Corynebacterium glutamicum, and Serratia marcescens.

8. The method as described in claim 6, characterized in that, The process of culturing the recombinant cells or recombinant microorganisms includes: inoculating the recombinant cells or recombinant microorganisms into a liquid culture medium and culturing them for 10-14 h to obtain a seed culture; then inoculating the seed culture into a fermentation culture medium and culturing it until the OD600 is 0.6-0.8, and then inducing culture for 70-74 h.

9. The method as described in claim 8, characterized in that, The liquid culture medium is LB medium, and the fermentation medium is DM fermentation medium.

10. The method as described in claim 8, characterized in that, The induction culture conditions are as follows: lactose with a final concentration of 2.5~3.5 g / L and IPTG with a final concentration of 0.4~0.6 mM are added to the fermentation medium for induction culture.

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