Alpha-2,6-sialyltransferase mutant and application

Abstract

This invention belongs to the field of microbial genetic engineering technology, specifically relating to an α-2,6-sialic acid transferase mutant and its application. This invention utilizes a mutant derived from *Lithocarpus lucida* (…). Photobacterium Five amino acids in the amino acid sequence of α-2,6-sialyl transferase encoded by the gene of sp. JT-ISH-224 were mutated at specific sites to obtain α-2,6-sialyl transferase mutants M133L, E140D, D228H, N289D, and G322Q. Among them, the E140D and G322Q single-site mutants of α-2,6-sialyl transferase showed increases in 6'-SL yield of 87.80% and 78.01%, respectively, compared with wild-type α-2,6-sialyl transferase. The optimal recombinant microbial strain 6'-SL-7 (E140D / G322Q) obtained by combining the mutation sites achieved a yield of 1.471 g / L, effectively solving the bottleneck problem of low catalytic efficiency of sialyl transferase and providing a high-performance enzyme element for the efficient and low-cost biomanufacturing of 6'-SL.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to an α-2,6-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] Traditional degradation methods (such as acid / alkali treatment, high temperature and high pressure) suffer from high energy consumption, significant pollution, and product inhibition in the preparation of 6′-SL. In contrast, enzymatic degradation has become a research hotspot due to its advantages such as mild conditions, high specificity, and environmental friendliness, and sialyl transferase is the core catalyst in this process.

[0004] The attachment of sialic acid residues is a crucial final step in the biosynthesis of complex oligosaccharides, primarily occurring on glycoproteins and gangliosides. Sialic acid-coated oligosaccharide structures are core elements of cell recognition. Sialyltransferases (STs) are glycosyltransferases that transfer N-acetylneuraminic acid (NeuAc) from the common donor substrate cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) to the acceptor substrate. Sialyltransferases are widely distributed in animal tissues but also exist in other organisms such as Vibrio photobacteria and bacteria.

[0005] α-2,6-Sialyltransferase is a key enzyme in the synthesis of 6'-SL, but its low catalytic efficiency limits the large-scale production of 6'-sialyl lactose. Therefore, achieving low-cost and efficient production of 6'-SL is a critical problem that urgently needs to be solved. Summary of the Invention

[0006] To address the shortcomings of the prior art, the purpose of this invention is to provide an α-2,6-sialylate 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-2,6-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 are mutations of M133L, E140D, D228H, N289D, G322Q or E140D / G322Q, where the letters before the numbers represent the original amino acids and the letters after the numbers represent the mutated amino acids.

[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] A second aspect of the invention provides a nucleic acid molecule encoding the α-2,6-sialylate mutant described in this invention.

[0013] A third aspect of the invention provides a recombinant expression vector containing the nucleic acid molecule described herein.

[0014] A fourth aspect of the invention provides recombinant cells containing the recombinant expression vector described in this invention.

[0015] A fifth aspect of the invention provides the use of the α-2,6-sialyltransferase mutant, nucleic acid molecule, recombinant expression vector, or recombinant cell described herein in any of the following:

[0016] (a1) Preparation of 6'-sialyl lactose;

[0017] (a2) Prepare a product containing 6'-sialyl lactose;

[0018] (a3) Increase the yield of α-2,6-sialyltransferase;

[0019] (a4) Construct recombinant microorganisms for the production of 6'-sialyl lactose;

[0020] (a5) Regulate the production of 6'-sialic acid lactose by utilizing microorganisms.

[0021] A sixth aspect of the present invention provides a method for producing 6'-sialyl lactose, comprising the following steps:

[0022] S1. Construct recombinant cells or recombinant microorganisms that can express the α-2,6-sialic acid transferase mutant described in this invention;

[0023] S2. Culture the recombinant cells or recombinant microorganisms to obtain 6'-sialic acid lactose.

[0024] 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.

[0025] In some embodiments of the present invention, culturing the recombinant cells or recombinant microorganisms includes inducing culture by inoculating the recombinant cells or recombinant microorganisms into a liquid culture medium and culturing for 3-5 hours.

[0026] In some embodiments of the present invention, the liquid culture medium is LB medium.

[0027] In some embodiments of the present invention, the induction culture conditions are as follows: induction culture is induced by adding lactose to a final concentration of 5 g / L and 0.2 mM IPTG.

[0028] In some embodiments of the present invention, the induction culture temperature is 25°C and the rotation speed is 200 rpm.

[0029] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0030] This invention utilizes the method derived from photobacteria ( Photobacterium Five amino acids in the amino acid sequence of α-2,6-sialyltransferase encoded by the gene (GenBank: BAF92026.1) of sp. JT-ISH-224 were mutated at a specific point to obtain α-2,6-sialyltransferase mutants M133L, E140D, D228H, N289D, and G322Q. Among them, the single-point mutations E140D and G322Q significantly increased the yield of 6'-sialyl lactose. Compared with wild-type α-2,6-sialyltransferase, the yield of 6'-SL in the E140D and G322Q single-point mutants was increased by 87.80% and 78.01%, respectively. The optimal recombinant microbial strain 6'-SL-7 (E140D / G322Q) was obtained by combining mutation sites, with a yield of up to 1.471 g / L. This effectively solved the bottleneck problem of low catalytic efficiency of sialyl transferase and provided a high-performance enzyme element for the efficient and low-cost biomanufacturing of 6'-SL. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the recombinant plasmid PETduet-Psp2,6ST in an embodiment of the present invention;

[0032] Figure 2 The bar chart shows the yield of the α-2,6-sialic acid transferase mutant strain 6'-SL in the embodiments of the present invention. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] As mentioned earlier, α-2,6-sialyltransferase is a key enzyme in the synthesis of 6'-SL, but its low catalytic efficiency is currently a bottleneck problem in the synthesis of 6'-sialyl lactose, limiting its large-scale production. This invention, based on the crystal structure of α-2,6-sialyltransferase (PDB: 2Z4T), uses Autodock Vina software for molecular docking analysis to identify potential key sites. Simultaneously, it combines the HotSpot Wizard algorithm and FireProt for hotspot residue analysis of two substrates, aiming to obtain mutant enzymes with novel properties or better catalytic performance. Based on this, the technical solution of this invention is proposed.

[0036] In one specific embodiment of the present invention, an α-2,6-sialyltransferase mutant is provided, wherein the amino acid sequence of the mutant is selected from any of the following:

[0037] (1) Based on the amino acid sequence shown in SEQ ID NO:1, there are mutations of M133L, E140D, D228H, N289D or G322Q, where the letters before the numbers represent the original amino acids and the letters after the numbers represent the mutant amino acids.

[0038] (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).

[0039] (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).

[0040] The amino acid sequence shown in SEQ ID NO: 1 is derived from *Lithocarpus lucida* (…). Photobacterium wild-type α-2,6-sialyl transferase (sp. JT-ISH-224) has the following amino acid sequence:

[0041] MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHASLPTLQQLMDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNLYNWRDTLNKTDNLKIGKDYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTLEPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFLSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPGGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV (SEQ ID NO: 1).

[0042] The coding sequence of the wild-type α-2,6-sialyltransferase of Photobacterium Photobacterium sp. JT-ISH-224) is shown in SEQ ID NO: 2.

[0043]

[0044] In some embodiments, the mutation site is a combination of M133L, E140D, D228H, N289D, or G322Q.

[0045] In some embodiments, the mutation site is any one of M133L, E140D, D228H, N289D, or G322Q.

[0046] In some preferred embodiments, the mutation sites are E140D and / or G322Q.

[0047] In another specific embodiment of the present invention, a nucleic acid molecule encoding the above-mentioned mutant is provided.

[0048] In another specific embodiment of the present invention, a recombinant expression vector is provided, which contains the above-mentioned nucleic acid molecule encoding α-2,6-sialyltransferase.

[0049] In another specific embodiment of the present invention, a recombinant cell is provided, which comprises the above-described recombinant expression vector.

[0050] In some embodiments, the starting strain of the recombinant cells is selected from any one of Escherichia coli, yeast, Corynebacterium glutamicum, and Serratia marcescens.

[0051] In some embodiments, the starting strain of the recombinant cells is Escherichia coli.

[0052] In some embodiments, the starting strain of the recombinant cells is Escherichia coli BL21(DE3)-LH.

[0053] In another specific embodiment of the present invention, the application of the α-2,6-sialyltransferase mutant, nucleic acid molecule, recombinant expression vector, or recombinant cell described herein is provided in any of the following:

[0054] (a1) Preparation of 6'-sialyl lactose;

[0055] (a2) Prepare a product containing 6'-sialyl lactose;

[0056] (a3) Increase α-2,6-sialyltransferase;

[0057] (a4) Construct recombinant microorganisms for the production of 6'-sialyl lactose;

[0058] (a5) Regulate the production of 6'-sialic acid lactose by utilizing microorganisms.

[0059] In another specific embodiment of the present invention, a method for producing 6'-sialic acid lactose is provided, comprising the following steps:

[0060] S1. Construct recombinant cells or recombinant microorganisms that can express the α-2,6-sialic acid transferase mutant described in this invention;

[0061] S2. Culture the recombinant cells or recombinant microorganisms to obtain 6'-sialic acid lactose.

[0062] 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.

[0063] In some embodiments of the present invention, culturing the recombinant cells or recombinant microorganisms includes inducing culture by inoculating the recombinant cells or recombinant microorganisms into a liquid culture medium and culturing for 3-5 hours.

[0064] In some embodiments of the present invention, the liquid culture medium is LB medium.

[0065] In some embodiments of the present invention, the conditions for induction culture are: addition of lactose to a final concentration of 5 g / L and 0.2 mM IPTG for induction culture.

[0066] In some embodiments of the present invention, the induction culture temperature is 25°C and the rotation speed is 200 rpm.

[0067] The culture medium formulation used in this embodiment of the invention is as follows:

[0068] LB+Amp medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 75 μg / mL ampicillin, pH 7.0.

[0069] The experimental materials and main reagents used in the following examples are from the following sources:

[0070] 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., catalog 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 Biotechnology, 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.

[0071] The detection methods involved in the following embodiments:

[0072] Methods for detecting 6'-sialic acid lactose:

[0073] The content of 6'-sialic acid lactose was determined by high-performance liquid chromatography (HPLC) (Shimadzu, LC-20A). Specifically, 1 mL of fermentation broth was 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.

[0074] 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.

[0075] Example 1: Preparation of α-2,6-sialyltransferase mutant

[0076] 1. Acquisition and optimization of wild-type genes:

[0077] First, the unmutated Psp2,6ST gene was constructed. The gene sequence was then queried and compared using the NCBI database, and *Lithocarpus lucida* was selected. Photobacterium α-2,6-sialidyltransferase (SLT) derived from *sp. JT-ISH-224* (GenBank: BAF92026.1) was synthesized in its entirety at Beijing Qingke Biotechnology Co., Ltd. (SEQ ID NO: 2). Primers 2,6-F (gggaattcCATATGaagaattttctgctgct, SEQ ID NO: 18) and 2,6-R (ggGGTACCtcaaactgcaatacaaacacc, SEQ ID NO: 19) were designed and PCR amplification was performed. The recombinant plasmid pETDuet-1 was constructed using a one-step cloning method to obtain the plasmid PETduet-Psp2,6ST. The plasmid map is shown below. Figure 1 As shown, through thermal shock conversion to E. coli Plasmid (PETduet-Psp2,6ST) was extracted after overnight incubation at 37℃ with DH5α.

[0078] 2. Design of mutation sites and site-directed mutagenesis

[0079] Based on the crystal structure of α-2,6-sialyltransferase (PDB:2Z4T), molecular docking analysis was performed using Autodock Vina software to identify potential key sites. Simultaneously, combined HotSpot Wizard algorithm and FireProt were used for joint hotspot residue analysis of the two substrates (analysis range 2.8 Å) to perform site-directed mutagenesis on amino acids 322, 140, 289, 133, and 228, which showed good predicted mutation results. Specifically, amino acid G at position 322 was changed to Q (G322Q), amino acid E at position 140 was changed to D (E140D), amino acid N at position 289 was changed to D (N289D), amino acid M at position 133 was changed to L (M133L), and amino acid D at position 228 was changed to H (D228H). Primers were designed as shown in Table 1.

[0080] Table 1 Primer Sequences

[0081]

[0082] The mutant amino acid sequence is as follows:

[0083] G322Q:MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHAS LPTLQQLMDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNLYNWRDTLNKTDNLKIGK DYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTLEPSLHELRDYIGDSLKQMQWDQFKKFNSKQQELFLSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINE SSPHYLGNSYDLFFKGHPGGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV (SEQID NO: 13);

[0084] E140D:MKNFLLLLITTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHASLPTLQQLMDIIKSEDENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNLYNWRDTLNKTDNLKIGKDYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTLEPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPPGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV(SEQID NO: 14);

[0085] N289D:MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHASLPTLQQLMDIIKSEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNLYNWRDTLNKTDNLKIGK DYLEDVINGINEDTSNGTSSVYNWQKLYPADYHFLRKDYLTLEPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPPGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV (SEQ ID NO: 15);

[0086] M133L:MKNFLLLTLILTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHAS LPTLQQLLDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIVKQAQTKHRNLVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNLYNWRDTNLKTDNLKIGK DYLEDVINGINEDTSNGTSSVYNWQKLYPANYHFLRKDYLTLEPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPPGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV (SEQ ID NO: 16);

[0087] D228H:MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHAS LPTLQQLMDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIHLYDDGSAEYVNLYNWRDTLNKTDNLKIGK DYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTLEPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFLSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINE SSPHYLGNSYDLFFKGHPGGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV (SEQID NO: 17).

[0088] 3. Construction of α-2,6-sialyltransferase mutant

[0089] Taking the mutant E140D as an example, plasmid PETduet-Psp2,6ST was used as a template. PCR amplification was performed using forward primer E140D-F and reverse primer E140D-R. High-fidelity polymerase 2×PhantaMax Master Mix was used. The PCR reaction system consisted of 25 μL of 2×PhantaMax 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 the gel was recovered. 2 μL of the product was added to 25 μL of 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. The plates were incubated upside down at 37°C overnight. Once 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 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. Positive transformants with correct sequencing results were identified as... E. coli DH5α-Psp2,6STE140D. Using the same method as above, plasmids Psp2,6STG322Q, Psp2,6STN289D, Psp2,6STM133L, and Psp2,6STD228H were finally constructed.

[0090] 4. Construction of α-2,6-sialyltransferase mutant strains

[0091] A recombinant strain capable of producing 6'-SL was constructed. The BL21(DE3)-LH strain, derived from K87, was selected, consisting of 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-Psp2,6ST prepared in Example 1 was transformed into BL21(DE3)-LH, plated on chloramphenicol and ampicillin-resistant plates, and positive clones were screened. The correctly identified positive transformants were 6'-SL-1 (…). E. coli BL21(DE3)-LH-PETduet-Psp2,6ST). The final constructed strain was 6'-SL-2 ( E. coli BL21(DE3)-LH-PETduet-Psp2,6STE140D), 6'-SL-3( E. coli BL21(DE3)-LH-PETduet-Psp2,6STG322Q), 6'-SL-4( E. coli BL21(DE3)-LH-PETduet-Psp2,6STN289D), 6'-SL-5( E. coli BL21(DE3)-LH-PETduet-Psp2,6STM133L), 6'-SL-6( E. coli BL21(DE3)-LH-PETduet-Psp2,6STD228H).

[0092] Positive transformants 6'-SL-1, 6'-SL-2, 6'-SL-3, 6'-SL-4, 6'-SL-5, 6'-SL-6, and 6'-SL-7 were inoculated into LB medium and cultured at 37°C and 220 rpm for 4 hours. Induction was then performed with 0.2 mM IPTG, followed by the addition of 5 g / L lactose as a substrate. The culture temperature was lowered to 25°C and incubated overnight (16-20 h). After incubation, the 6'-sialic acid lactose content was determined using high-performance liquid chromatography-ultraviolet detection (HPLC-UV). Triple replicates were performed for each group, and the yield was calculated. The results are shown in Table 2 and... Figure 2 As shown, the yield of 6'-SL in the control strain (6'-SL-1) was 0.541 g / L, while the yield of 6'-SL in strain 6'-SL-2 was the highest, reaching 1.016 g / L, which was 87.80% higher than that of the control strain (6'-SL-1). The yield of 6'-SL in strain 6'-SL-3 was also improved, reaching 0.936 g / L, which was 78.01% higher than that of the control strain (6'-SL-1). The yield of 6'-SL in strain 6'-SL-5 was 0.887 g / L, which was 63.96% higher than that of the control strain (6'-SL-1). The yield of 6'-SL in strain 6'-SL-06 was 0.438 g / L, which was 19.04% lower than that of the control strain (6'-SL-1). Among the single mutations, E140D and G322Q were the optimal mutation points.

[0093] Table 2. Yield of α-2,6-sialyltransferase mutant strain 6'-SL

[0094]

[0095] Example 2: Combined mutation of α-2,6-sialyltransferase Psp2,6ST

[0096] 1. Construction of α-2,6-sialyltransferase combinatorial mutants:

[0097] Using plasmid Psp2,6STE140D as a template, PCR amplification was performed using G322Q-F and G322Q-R as forward and reverse primers, respectively. The high-fidelity polymerase 2×PhantaMax Master Mix reaction system described in Example 1 was used for PCR amplification. After the reaction, the PCR product was analyzed by gel electrophoresis and the gel was recovered. 2 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 hours. 1 mL of bacterial culture and 1 mL of 30% glycerol were added to sterile glycerol tubes for preservation. Plasmids (Psp2,6STE140D / G322Q) were extracted using a plasmid kit and sequenced. Positive transformants with correct sequencing results are... E. coli DH5 α-Psp2,6STE140D / G322Q.

[0098] 2. Construction of α-2,6-sialyltransferase combinatorial mutant strains

[0099] Plasmid Psp2,6STE140D / G322Q was transformed into strain BL21(DE3)-LH, plated on LB agar plates resistant to chloramphenicol and ampicillin, and positive clones were screened. The correct positive transformant was identified as 6'-SL-7. E. coli BL21(DE3)-LH-PETduet-Psp2,6STE140D / G322Q).

[0100] Positive transformant 6'-SL-7 was inoculated into LB medium and cultured at 37°C and 220 rpm for 4 hours. Induction was then performed with 0.2 mM IPTG, followed by the addition of 5 g / L lactose as a substrate. The culture temperature was lowered to 25°C and incubated overnight (16-20 h). After incubation, the 6'-sialic acid lactose content was determined using high-performance liquid chromatography-ultraviolet detection to calculate the yield. The results are shown in Table 2 and... Figure 2 As shown, the yield of 6'-SL in the combined mutant strain 6'-SL-7 was 1.471 g / L, which was 172.1% higher than that of the control strain (6'-SL-1).

[0101] The results show that this mutant enzyme significantly improves the catalytic efficiency of α-2,6-sialyltransferase, thereby greatly increasing the yield of 6'-SL and demonstrating broad application prospects.

[0102] 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-2,6-sialyltransferase mutant, characterized in that, The amino acid sequence of the mutant was obtained by simultaneously mutating E140D and G322Q based on the amino acid sequence shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, It encodes the α-2,6-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-2,6-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 6'-sialyl lactose; (a2) Prepare a product containing 6'-sialyl lactose; (a3) Increase the yield of α-2,6-sialyltransferase; (a4) Construct recombinant microorganisms for the production of 6'-sialyl lactose; (a5) Regulate the production of 6'-sialic acid lactose by utilizing microorganisms.

6. A method for producing 6'-sialic acid lactose, characterized in that, Includes the following steps: S1. Constructing recombinant cells or recombinant microorganisms capable of expressing the α-2,6-sialic acid transferase mutant of claim 1; S2. Culture the recombinant cells or recombinant microorganisms to obtain 6'-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 inducing culture by inoculating the recombinant cells or recombinant microorganisms into a liquid culture medium and culturing for 3-5 hours.

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

10. The method as described in claim 8, characterized in that, The induction culture conditions were as follows: lactose with a final concentration of 5 g / L and IPTG with 0.2 mM were added for induction culture.

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