An alpha-2,6-sialyltransferase mutant, and methods of making and using the same

Abstract

The present application relates to a kind of alpha-2,6-sialyltransferase mutant and its preparation method and application in the preparation of 6 '-sialyl lactose, and the biosynthesis of 6 '-sialyl lactose has important significance, suitable for the needs of industrial production, with wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an α-2,6-sialic acid transferase mutant, its preparation method, and its application. Background Technology

[0002] Human milk oligosaccharides (HMOs) are a group of structurally diverse non-conjugated polysaccharides, the third most abundant nutrient in breast milk after lactose and lipids. Among them, 6'-sialyl lactose (6'-SL) accounts for approximately 6% of the total HMOs, making it one of the most abundant. Studies have shown that 6'-SL possesses various functions, including anti-adhesion and antibacterial activity, antiviral activity, prevention of necrotizing enterocolitis (NEC), and immunomodulatory activity. It can protect the infant's intestinal tract from infection, promote the growth and maturation of the immune system, promote brain maturation in infants, and enhance learning abilities. The chemical synthesis of 6'-SL involves complex protection and deprotection steps, making large-scale industrial production difficult. Microbial fermentation, on the other hand, offers advantages such as low raw material costs, mild reaction conditions, and ease of large-scale production, making it the most common method for producing 6'-SL.

[0003] 6'-SL is essentially formed by the binding of n-acetylneuraminic acid (Neu5Ac) to a lactose molecule via an α-(2,6)-bond. Lactose and Neu5Ac are direct precursors in the synthesis of sialyl lactose. First, Neu5Ac is converted to cytidine-5'-monophosphate-n-acetylneuraminic acid (CMP-Neu5Ac) under the catalysis of CMP-NeuAc synthase. Subsequently, 6'-SL is produced using CMP-NeuAc as the donor and lactose as the acceptor, catalyzed by α-2,6-sialyltransferase (α-2,6-pdst).

[0004] α-2,6-Sialyltransferase is a key enzyme in the synthesis of 6'-sialyl lactose, but its low catalytic efficiency is currently a bottleneck problem in the synthesis of 6'-sialyl lactose, which limits the large-scale production of 6'-sialyl lactose. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an α-2,6-sialyltransferase mutant that can effectively improve the catalytic activity of α-2,6-sialyltransferase and can efficiently synthesize 6'-sialyl lactose.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first objective of this invention is to provide an α-2,6-sialyltransferase mutant, wherein the amino acid sequence of the mutant is any one of the following:

[0008] (1) Compared with the wild-type α-2,6-sialyltransferase from Photobacterium sp. JT–ISH–224 shown in amino acid sequence SEQ ID NO:2, the mutant includes one or more of the following mutation sites:

[0009] T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L or F499C;

[0010] (2) Compared with the amino acid sequence described in (1), an amino acid sequence having one or more amino acid residues replaced, deleted, added or any combination thereof, wherein the replacement is a conservative replacement;

[0011] (3) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the amino acid sequence described in (1).

[0012] Preferably, the mutation site is one or a combination of T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L or F499C.

[0013] More preferably, the mutation site is one or a combination of T360S, K403Q, N412D, V464I, T468S or M486L.

[0014] More preferably, the mutation site is T360S and / or M486L.

[0015] A second objective of this invention is to provide a nucleic acid encoding the mutant described above.

[0016] A third object of the present invention is to provide a vector comprising the nucleic acid encoding the α-2,6-sialic acid transferase mutant described above.

[0017] A fourth object of the present invention is to provide a recombinant engineered bacterium or recombinant engineered cell comprising a nucleic acid encoding an α-2,6-sialyltransferase mutant as described above or a vector as described above.

[0018] Preferably, the background strain of the recombinant engineered bacteria is Escherichia coli.

[0019] More preferably, the background strain of the recombinant engineered bacteria is Escherichia coli BL21(DE3).

[0020] More preferably, the recombinant engineered bacteria or recombinant engineered cells express CMP-Neu5Ac synthase neuA, encoding n-acetylneuraminic acid synthase neuB, and udp-n-acetylglucosamine 2-diaminease neuC derived from Campylobacter jejuni.

[0021] A fifth objective of this invention is to provide a method for preparing the mutant described above, comprising: culturing the recombinant engineered bacteria or recombinant engineered cells described above, and inducing the expression of the mutant.

[0022] A sixth objective of this invention is to provide a biological agent comprising: the mutant described above, the nucleic acid described above, the vector described above, the recombinant engineered bacteria or recombinant engineered cells described above, or the product obtained by the method described above.

[0023] A seventh object of the present invention is to provide the application of the mutants, nucleic acids, vectors, recombinant engineered bacteria or recombinant engineered cells, products obtained by the methods described above, or biological agents described above in the preparation of 6'-sialyl lactose.

[0024] The eighth object of the present invention is the use of the mutants, nucleic acids, vectors, recombinant engineered bacteria or recombinant engineered cells, products obtained by the methods described above, or biological agents described above in the preparation of food or health products.

[0025] Compared with existing technologies, it has the following beneficial effects:

[0026] Through extensive practical research, the inventors of this invention have used gene-directed mutagenesis technology to modify α-2,6-sialyl transferase from Photobacterium sp. JT–ISH–224, thereby significantly improving the enzyme's activity. The resulting mutant exhibits enhanced catalytic activity for the natural substrate lactose, which is of great significance for the biosynthesis of 6'-sialyl lactose, suitable for industrial production, and has broad application prospects. Attached Figure Description

[0027] Figure 1 This is the plasmid map of the recombinant plasmid pRD-Psp2,6ST of the present invention;

[0028] Figure 2 A bar chart showing the 6'-sialyl lactose production of the mutant strain provided by this invention. Detailed Implementation

[0029] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0030] α-2,6-sialyltransferase is a key enzyme in the synthesis of 6'-SL. Numerous studies have been reported on α-2,6-sialyltransferase, particularly the resolution of its crystal structure, laying the foundation for elucidating its catalytic mechanism. Rational enzyme design utilizes various biochemical and structural biology methods to obtain information about the structure, properties, and function of enzyme molecules. Based on the known relationship between structure and function, individual amino acid residues and structural domains are modified to alter the enzyme molecule, aiming to obtain mutant enzymes with novel properties or better catalytic performance. Based on this, the inventors of this invention, through extensive and in-depth research, have proposed the technical solution of this invention.

[0031] The first objective of this invention is to provide an α-2,6-sialyltransferase mutant, wherein the amino acid sequence of the mutant is any one of the following:

[0032] (1) Compared with the wild-type α-2,6-sialyltransferase from Photobacterium sp. JT–ISH–224 shown in amino acid sequence SEQ ID NO:2, the mutant includes one or more of the following mutation sites:

[0033] T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L or F499C;

[0034] (2) Compared with the amino acid sequence described in (1), an amino acid sequence having one or more amino acid residues replaced, deleted, added or any combination thereof, wherein the replacement is a conservative replacement;

[0035] (3) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the amino acid sequence described in (1).

[0036] The amino acid sequence of the wild-type α-2,6-sialyltransferase from Photobacterium sp. JT–ISH–224 shown in SEQ ID NO:2 is as follows:

[0037] MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHASLPTLQQLMDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIV KQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNLYNWRDTLNKTDNLKIGKDYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTLEPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFLSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPGGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV*

[0038] The nucleic acid sequence encoding the wild-type α-2,6-sialyltransferase from said Photobacterium sp. JT–ISH–224 is shown as SEQ ID NO: 1:

[0039]

[0040] In some embodiments, the mutation site is one or more of the following combinations: T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0041] In some embodiments, the mutation site is any one of T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0042] In some embodiments, the mutation sites are any two of T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0043] In some embodiments, the mutation sites are any three of T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0044] In some embodiments, the mutation sites are any four of T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0045] In some embodiments, the mutation sites are any five of the following: T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0046] In some embodiments, the mutation sites are any six of the following: T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0047] In some embodiments, the mutation sites are any seven of the following: T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0048] In some embodiments, the mutation sites are any eight of the following: T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0049] In some embodiments, the mutation sites are any nine of the following: T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, or F499C.

[0050] In some embodiments, the mutation sites are T360S, K403Q, N412D, A445G, T454S, I463Y, V464I, T468S, M486L, and F499C.

[0051] In some embodiments, the mutation site is one or more of T360S, K403Q, N412D, V464I, T468S, or M486L.

[0052] In some preferred embodiments, the mutation site is any one of T360S, K403Q, N412D, V464I, T468S, or M486L.

[0053] In some preferred embodiments, the mutation site is any two of T360S, K403Q, N412D, V464I, T468S, or M486L.

[0054] In some preferred embodiments, the mutation sites are any three of T360S, K403Q, N412D, V464I, T468S, or M486L.

[0055] In some preferred embodiments, the mutation sites are any four of T360S, K403Q, N412D, V464I, T468S, or M486L.

[0056] In some preferred embodiments, the mutation sites are any five of T360S, K403Q, N412D, V464I, T468S, or M486L.

[0057] In some preferred embodiments, the mutation sites are T360S, K403Q, N412D, V464I, T468S, and M486L.

[0058] In some preferred embodiments, the mutation sites are T360S and / or M486L.

[0059] A second objective of this invention is to provide a nucleic acid encoding the mutant described above.

[0060] A third object of the present invention is to provide a vector comprising the nucleic acid encoding the α-2,6-sialic acid transferase mutant described above.

[0061] A fourth object of the present invention is to provide a recombinant engineered bacterium or recombinant engineered cell comprising a nucleic acid encoding an α-2,6-sialyltransferase mutant as described above or a vector as described above.

[0062] In some embodiments, the background strain of the recombinant engineered bacteria is Escherichia coli.

[0063] In some embodiments, the background strain of the recombinant engineered bacteria is Escherichia coli BL21(DE3).

[0064] In some embodiments, the recombinant engineered bacteria or recombinant engineered cells express CMP-Neu5Ac synthase neuA, encoding n-acetylneuraminic acid synthase neuB, and udp-n-acetylglucosamine 2-diaminease neuC derived from Campylobacter jejuni.

[0065] In some embodiments, the nucleic acid sequence encoding the CMP-Neu5Ac synthase neuA is shown in SEQ ID NO:3.

[0066] SEQ ID NO:3:

[0067] ATGTCTCTGGCTATCATCCCGGCTCGTGGTGGTTCTAAAGGTATCAAAAACAAAAACCTGGTTCTGCTGAACAACAAACCGCTGATCTACTACACCATCAAAGCTGCTCTGAACGCTAAATCTATCTCTAAAGTTGTTGTTTCTTCTGACTCTGACGAAATCCTGAACTACGCTAAATCTCAGAACGTTGACATCCTGAAACGTCCGATCTCTCTGGCTCAGGACGACACCACCTCTGACAAAGTTCTGCTGCACGCTCTGAAATTCTACAAAGACTACGAAGACGTTGTTTTCCTGCAGCCGACCTCTCCGCTGCGTACCAACATCCACATCAACGAAGCTTTCAACCTGTACAAAAACTCTAACGCTAACGCTCTGATCTCTGTTTCTGAATGCGACAACAAAATCCTGAAAGCTTTCGTTTGCAACGACTGCGGTGACCTGGCTGGTATCTGCAACGACGAATAC CCGTTCATGCCGCGTCAGAAACTGCCGAAAACCTACATGTCTAACGGTGCTATCTACATCCTGAAAATCAAAGAATTCCTGAACAACCCGTCTTTCCTGCAGTCTAAAACCAAACACTTCCTGATGGACGAATCTTCTTCTCTGGACATCGACTGCCTGGAAGACCTGAAAAAAGTTGAACAGATCTGGAAAAAATAA

[0068] In some embodiments, the nucleic acid sequence encoding said n-acetylneuraminate synthase neuB is set forth in SEQ ID NO: 4.

[0069] SEQ ID NO: 4:

[0070]

[0071] In some embodiments, the nucleic acid sequence encoding the udp-n-acetylglucosamine 2-diiminease neuC is shown in SEQ ID NO:5.

[0072] SEQ ID NO:5:

[0073] ATGAAAAAAATCCTGTTCATCACCGGTTCTCGTGCTGACTACTCTAAAATCAAATCTCTGATGTACCGTGTTCAGAACTCTTCTGAATTCGAACTGTACATCTTCGCTACCGGTATGCACCTGTCTAAAAACTTCGGTTACACCGTTAAAGAACTGTACAAAAACGGTTTCAAAAACATCTACGAATTCATCAACTACGACAAATACTACCAGACCGACAAAGCTCTGGCTACCACCATCGACGGTTTCTCTCGTTACGCTAACGAACTGAAACCGGACCTGATCGTTGTTCACGGTGACCGTATCGAACCGCTGGCTGCTGCTATCGTTGGTGCTCTGAACAACATCCTGGTTGCTCACATCGAAGGTGGTGAAATCTCTGGTACCATCGACGACTCTCTGCGTCACGCTATCTCTAAACTGGCTCACATCCACCTGGTTAACGACGAATTCGCTAAACGTCGTCTGATGCAGCTGGGTGAAGACGAAAAATCTATCTTCATCATCGGTTCTCCGGACCTGGAACTGCTGAACGACAACAAAATCTCTCTGTCTGAAGCTAAAAAATACTACGACATCAACTACGAAAACTACGCTCTGCTGATGTTCCACCCGGTTACCACCGAAATCACCTCTATCAAAAACCAGGCTGACAACCTGGTTAAAGCTCTGATCCAGTCTAACAAAAACTACATCGTTATCTACCCGAACAACGACCTGGGTTTCGAACTGATCCTGCAGTCTTACGAAGAATTCAAAAACAACCCGCGTTTCAAACTGTTCCCGTCTCTGCGTTTCGAATACTTCATCACCCTGCTGAAAAACGCTGACTTCATCATCGGTAACTCTTCTTGCATCCTGAAAGAAGCTCTGTACCTGAAAACCGCTGGTATCCTGGTTGGTTCTCGTCAGAACGGTCGTCTGGGTAACGAAAACACCCTGAAAGTTAACGCTAACTCTGACGAAATCCTGAAAGCTATCAACACCATCCACAAAAAACAGGACCTGTTCTCTGCTAAACTGGAAATCCTGGACTCTTCTAAACTGTTCTTCGAATACCTGCAGTCTGGTGACTTCTTCAAACTGTCTACCCAGAAAGTTTTCAAAGACATCAAATAA

[0074] A fifth objective of this invention is to provide a method for preparing the mutant described above, comprising: culturing the recombinant engineered bacteria or recombinant engineered cells described above, and inducing the expression of the mutant.

[0075] A sixth objective of this invention is to provide a biological agent comprising: the mutant described above, the nucleic acid described above, the vector described above, the recombinant engineered bacteria or recombinant engineered cells described above, or the product obtained by the method described above.

[0076] A seventh object of the present invention is to provide the application of the mutants, nucleic acids, vectors, recombinant engineered bacteria or recombinant engineered cells, products obtained by the methods described above, or biological agents described above in the preparation of 6'-sialyl lactose.

[0077] The eighth object of the present invention is the use of the mutants, nucleic acids, vectors, recombinant engineered bacteria or recombinant engineered cells, products obtained by the methods described above, or biological agents described above in the preparation of food or health products.

[0078] Furthermore, this invention provides a method for preparing 6'-sialyl lactose via an in vitro enzymatic process, the method comprising:

[0079] (1) Provide the above-mentioned α-2,6-sialic acid transferase mutant;

[0080] (2) Provide donor and acceptor substrates, and culture host cells under suitable nutritional conditions that allow the production of the 6'-sialyl lactose and allow the expression of α-2,6-sialyl transferase, so that the α-2,6-sialyl transferase mutant in step (1) is contacted with the donor and acceptor substrates to prepare 6'-sialyl lactose.

[0081] Terminology Explanation:

[0082] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0083] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. Chem., 1968, 243, 3558.

[0084] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the biological activity of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0085] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0086] The terms involved in this invention have been defined above. Those skilled in the art can also understand the above terms in conjunction with the prior art. The following is a further description based on the content of this invention and the definition of the terms.

[0087] The culture media involved in the embodiments of the present invention are as follows:

[0088] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0089] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L.

[0090] The detection methods involved in the following embodiments are as follows:

[0091] High performance liquid chromatography method for the determination of 6'-sialyl lactose:

[0092] The content of 6'-sialyl lactose was determined by high-performance liquid chromatography (HPLC) (Agilent 1260 series, USA). 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 determined by HPLC. The HPLC instrument was equipped with a UV detector (195 nm), a differential refractive index detector (RID-10A), an Aminex HPX-87H column (300 mm × 7.8 mm), a mobile phase of 25 mM dilute sulfuric acid, a detection temperature of 65 °C, and a flow rate of 0.6 mL / min.

[0093] The present invention will be further described below through specific embodiments. Unless otherwise specified, "%" represents a mass percentage. The materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments without specified conditions are generally performed according to conventional experimental conditions or the conditions recommended by the manufacturer of the relevant reagent (kit).

[0094] Example 1

[0095] Construction of α-2,6-sialyltransferase Psp2,6ST single-point mutant plasmid

[0096] First, the unmutated Psp2,6ST gene was constructed. The gene sequence was searched and aligned using the NCBI database. Based on literature reports, α-2,6-sialyltransferase from *Photobacterium sp. JT–ISH–224* (GenBank: BAF92026.1) was selected and its full sequence was synthesized at Genewiz (gene sequence is SEQ ID No. 1, amino acid sequence is SEQ ID No. 2). Primers were designed and PCR amplification was performed. The recombinant plasmid pRD was constructed using a one-step cloning method, resulting in the plasmid pRD-Psp2,6ST. The plasmid map is shown below. Figure 1 As shown, the plasmid was extracted after heat shock transformation into E. coli DH5α and overnight culture at 37°C.

[0097] To screen for mutation sites, the HotSpot Wizard algorithm was used on two substrates. Hotspot residue analysis was performed on amino acids within the range, and the 10 mutation sites with the highest scores and the best predicted mutation results were selected and primers were designed as shown in Table 1.

[0098] Table 1

[0099]

[0100]

[0101] Taking the mutant T360S as an example, plasmid pRD-Psp2,6ST was used as a template. PCR amplification was performed using forward primer T360S-F and reverse primer T360S-R. High-fidelity polymerase PrimeSTAR Max DNA Polymerase was used. The PCR reaction system consisted of 25 μL PrimestarMax Mix, 23 μL ddH2O, 1 μL forward primer (5.0 μM), 1 μL reverse primer (5.0 μM), template <200 ng, and a total volume of 50 μL. The PCR reaction conditions were: 98℃ for 1 min, one cycle; 98℃ for 10 s, 55℃ for 10 s, 72℃ for 30 s, 35 cycles; and 72℃ for 5 min, one cycle. After the reaction, 5 μL of the PCR product was subjected to gel electrophoresis, and 10 μL was digested (since E. coli DH5α is a dam+ E. coli, the enzyme DpnI, which targets specific methylation sites, can digest the plasmid template used in PCR). 10 μL of the PCR product was then added to 1 μL of DpnI enzyme and 2 μL of 10×SpeedyOne Buffer, and the mixture was incubated at 37°C for 30 min. The DpnI-digested product was then transferred into DH5α competent cells, plated on ampicillin-resistant plates, and positive clones were screened and sequenced. The correctly sequenced positive transformants were identified as E. coli DH5αpRD-Psp2,6ST. T360S Using the same method described above, ten expression plasmids pRD-Psp2,6ST were finally constructed. T360S pRD-Psp2,6ST K403Q pRD-Psp2,6ST N412D pRD-Psp2,6ST A445G pRD-Psp2,6ST T454S pRD-Psp2,6ST I463Y pRD-Psp2,6ST V464I pRD-Psp2,6ST T468S pRD-Psp2,6ST M486L pRD-Psp2,6ST F499C .

[0102] SEQ ID No. 1:

[0103]

[0104] SEQ ID No.2:

[0105] MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQTCGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVYVDHASLPTLQQLMDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADLVQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNLYNWRDTLNKTDNLKIGKDYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTLEPSLHEL RDYIGDSLKQMQWDGFKKFNSKQQELFLSIVNFDKQKLQNEYNSSNLPNFVFTGTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPGGGIINTLIMQNYPSMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRSPLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV*

[0106] Example 2

[0107] Construction of single-point mutant strain of α-2,6-sialyltransferase Psp2,6ST

[0108] First, a recombinant strain capable of producing 6'-SL was constructed. NeuA (CMP-Neu5Ac synthase), NeuB (encoding n-acetylneuraminic acid synthase), and NeuC (udp-n-acetylglucosamine 2-diiminoase) from *Campylobacter jejuni* were selected and their full sequences were synthesized at Genewiz (neuA gene sequence: SEQ ID No. 3, neuB gene sequence: SEQ ID No. 4, neuC gene sequence: SEQ ID No. 5). Using the T7 promoter and terminator, the full sequences were synthesized at Genewiz, and the DNA sequences are SEQ ID No. 6 and SEQ ID No. 7. Primers were designed and PCR amplification was performed. The PCR reaction system consisted of 25 μL PrimerMax Mix, 23 μL ddH2O, 1 μL upstream primer (5.0 μM), 1 μL downstream primer (5.0 μM), template <200 ng, and a total volume of 50 μL. After purification using a gel extraction kit, the plasmid pET28a was ligated into E. coli DH5α using the ClonExpress II OneStep Cloning Kit. The ligation solution was then transformed into E. coli DH5α. The plasmid was verified and sequenced correctly as pET28a-neuABC. This plasmid was then transformed into E. coli BL21(DE3) competent cells, plated on kanamycin-resistant plates, and positive clones were screened. The finally verified positive transformant was E. coli BL21(DE3)-pET28a-neuABC.

[0109] The plasmid pRD-Psp2,6ST from Example 1 was transformed into E. coli BL21(DE3)-pET28a-neuABC, plated on canagaphedrine-resistant plates, and positive clones were screened. The correct positive transformant was identified as 6'-SL-01(E. coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST). Using the same method, strain 6'-SL-02(E. coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST) was finally constructed. T360S ), 6'-SL-03(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST K403Q ), 6'-SL-04(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST N412D ), 6'-SL-05(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST A445G), 6'-SL-06(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST T454S ), 6'-SL-07(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST I463Y ), 6'-SL-08(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST V464I ), 6'-SL-09(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST T468S ), 6'-SL-10(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST M486L ), 6'-SL-11(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST F499C ).

[0110] 6'-SL-01 to 6'-SL-11 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. After culture, the 6'-sialic acid lactose content was determined using high-performance liquid chromatography-differential refractive index detection to calculate the yield. Results are as follows... Figure 2 As shown, the yield of 6'-SL in the control strain (6'-SL-01) was 0.546 g / L, while the highest yield was achieved by strain 6'-SL-02 at 0.984 g / L, which was 80.08% higher than the control strain. The yield of 6'-SL in strain 6'-SL-04 was 0.759 g / L, which was 38.86% higher than the control strain. The yield of 6'-SL in strain 6'-SL-10 was 0.847 g / L, which was 54.96% higher than the control strain. The optimal site-directed mutation sites for single-point mutations were T360S and M486L, which increased the catalytic efficiency of the mutants by 80.08% and 54.96% respectively compared to the wild type.

[0111] SEQ ID No. 3:

[0112] ATGTCTCTGGCTATCATCCCGGCTCGTGGTGGTTCTAAAGGTATCAAAAACAAAAACCTGGTTCTGCTGAACAACAAACCGCTGATCTACTACACCATCAAAGCTGCTCTGAACGCTAAATCTATCTCTAAAGTTGTTGTTTCTTCTGACTCTGACGAAATCCTGAACTACGCTAAATCTCAGAACGTTGACATCCTGAAACGTCCGATCTCTCTGGCTCAGGACGACACCACCTCTGACAAAGTTCTGCTGCACGCTCTGAAATTCTACAAAGACTACGAAGACGTTGTTTTCCTGCAGCCGACCTCTCCGCTGCGTACCAACATCCACATCAACGAAGCTTTCAACCTGTACAAAAACTCTAACGCTAACGCTCTGATCTCTGTTTCTGAATGCGACAACAAAATCCTGAAAGCTTTCGTTTGCAACGACTGCGGTGACCTGGCTGGTATCTGCAACGACGAATACCCGTTCATGCCGCGTCAGAAACTGCCGAAAACCTACATGTCTAACGGTGCTATCTACATCCTGAAAATCAAAGAATTCCTGAACAACCCGTCTTTCCTGCAGTCTAAAACCAAACACTTCCTGATGGACGAATCTTCTTCTCTGGACATCGACTGCCTGGAAGACCTGAAAAAAGTTGAACAGATCTGGAAAAAATAA

[0113] SEQ ID No.4:

[0114] ATGAAAGAAATCAAAATCCAGAACATCATCATCTCTGAAGAAAAAGCTCCGCTGGTTGTTCCGGAAATCGGTATCAACCACAACGGTTCTCTGGAACTGGCTAAAATCATGGTTGACGCTGCTTTCTCTGCTGGTGCTAAAATCATCAAACACCAGACCCACATCGTTGAAGACGAAATGTCTAAAGCTGCTAAAAAAGTTATCCCGGGTAACGCTAAAATCTCTATCTACGAAATCATGCAGAAATGCGCTCTGGACTACAAAGACGAACTGGCTCTGAAAGAATACACCGAAAAACTGGGTCTGGTTTACCTGTCTACCCCGTTCTCTCGTGCTGGTGCTAACCGTCTGGAAGACATGGGTGTTTCTGCTTTCAAAATCGGTTCTGGTGAATGCAACAACTACCCGCTGATCAAACACATCGCTGCTTTCAAAAAACCGATGATCGTTTCTACCGGTATGAACTCTATCGAATCTATCAAACCGACCGTTAAAATCCTGCTGGACAACGAAATCCCGTTCGTTCTGATGCACACCACCAACCTGTACCCGACCCCGCACAACCTGGTTCGTCTGAACGCTATGCTGGAACTGAAAAAAGAATTCTCTTGCATGGTTGGTCTGTCTGACCACACCACCGACAACCTGGCTTGCCTGGGTGCTGTTGTTCTGGGTGCTTGCGTTCTGGAACGTCACTTCACCGACTCTATGCACCGTTCTGGTCCGGACATCGTTTGCTCTATGGACACCAAAGCTCTGAAAGAACTGATCATCCAGTCTGAACAGATGGCTATCATCCGTGGTAACAACGAATCTAAAAAAGCTGCTAAACAGGAACAGGTTACCATCGACTTCGCTTTCGCTTCTGTTGTTTCTATCAAAGACATCAAAAAAGGTGAAGTTCTGTCTATGGACAACATCTGGGTTAAACGTCCGGGTCTGGGTGGTATCTCTGCTGCTGAATTCGAAAACATCCTGGGTAAAAAAGCTCTGCGTGACATCGAAAACGACGCTCAGCTGTCTTACGAAGACTTCGCTTAA

[0115] SEQ ID No.5:

[0116]

[0117] SEQ ID No. 6:

[0118] TAATACGACTCACTATAGG

[0119] SEQ ID No. 7:

[0120] CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG

[0121] Example 3

[0122] Construction of α-2,6-sialyltransferase Psp2,6ST combined mutant strain

[0123] Taking the combined mutant T360S / N412D as an example, the plasmid pRD-Psp2,6ST was used. T360S As a template, PCR amplification was performed using forward primer N412D-F and reverse primer N412D-R, with the high-fidelity polymerase PrimeSTAR Max DNA Polymerase. The amplification system and reaction were the same as in Example 1. After the reaction, 5 μL was used for gel electrophoresis, and 10 μL was used for digestion (since E. coli DH5α is a dam+ E. coli, the enzyme DpnⅠ, which has specific methylation sites, can digest the plasmid template used in PCR). 10 μL of the PCR product was then added to 1 μL of DpnⅠ enzyme and 2 μL of 10×SpeedyOne Buffer, and the mixture was incubated at 37°C for 30 min. The product digested with DpnⅠ was then transferred into DH5α competent cells, plated on ampicillin-resistant plates, and positive clones were screened and sequenced. The correctly sequenced positive transformant was identified as E. coli DH5αpRD-Psp2,6ST. T360S / N412D Plasmids were extracted after overnight incubation at 37°C.

[0124] plasmid pRD-Psp2,6ST T360S / N412D Transformed into E. coli BL21(DE3)-pET28a-neuABC, plated on canagaphedrine-resistant plates, and positive clones were screened. The correct positive transformant was identified as 6'-SL-12(E. coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST). T360S / N412D Using the same method described above, strain 6'-SL-13(E.coli BL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST) was finally constructed. T360S / M486L), 6'-SL-14(E.coliBL21(DE3)-pET28a-neuABC\pRD-Psp2,6ST N412D / M486L ).

[0125] 6'-SL-01 and 6'-SL-12 to 6'-SL-14 were inoculated into LB medium and cultured at 37°C and 220 rpm for 4 hours. 0.2 mM IPTG was added for induction, and lactose at a concentration of 5 g / L was added as a substrate. The culture temperature was lowered to 25°C. After the culture was completed, the content of 6'-sialic acid lactose was determined by high performance liquid chromatography-differential refractive index detector to calculate the yield. The results are shown in the figure. The yield of 6'-SL in the control strain (6'-SL-01) was 0.546 g / L, the yield of 6'-SL in the 6'-SL-12 strain was 0.965 g / L, the yield of 6'-SL in the 6'-SL-13 strain was 1.1698 g / L, and the yield of 6'-SL in the 6'-SL-14 strain was 0.46 g / L. The 6'-SL-13 strain had the highest yield, which was 114.03% higher than that of the control strain, indicating that the mutant T360S / M486L has a good synergistic effect. However, the yield of the mutant N412D / M486L was lower than that of the corresponding single-site mutant, indicating that the simultaneous mutation of mutants N412D and M486L has an antagonistic effect, affecting the expression of Psp2 and 6ST. Finally, the optimal site-directed mutation site was determined to be the combined mutant T360S / M486L, and the catalytic efficiency of the mutant was increased by 114.03% compared with the wild type.

[0126] The results showed that the mutant enzyme significantly improved the catalytic efficiency of α-2,6-sialyltransferase Psp2,6ST, thereby significantly increasing the yield of 6'-SL, and has broad application prospects.

[0127] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. An α-2,6-sialyltransferase mutant, characterized in that, The amino acid sequence of the mutant is the same as that shown in SEQ ID NO:2, derived from *Lithocarpus*. Photobacterium sp. Compared to the wild-type α-2,6-sialyltransferase JT–ISH–224, the mutant has a mutation point of T360S; or T360S and N412D; or T360S and M486L.

2. A nucleic acid encoding the mutant of claim 1.

3. A carrier, characterized in that, The vector contains the nucleic acid encoding the α-2,6-sialic acid transferase mutant as described in claim 2.

4. A recombinant engineered bacterium comprising the nucleic acid encoding the α-2,6-sialyltransferase mutant as described in claim 2 or the vector as described in claim 3.

5. The recombinant engineered bacteria according to claim 4, characterized in that, The background strain of the recombinant engineered bacteria is Escherichia coli.

6. The recombinant engineered bacteria according to claim 5, characterized in that, The Escherichia coli mentioned is Escherichia coli BL21(DE3).

7. The recombinant engineered bacteria according to any one of claims 4 to 6, characterized in that, The recombinant engineered bacteria were expressed from Campylobacter jejuni (… Campylobacter jejuni The enzymes involved include CMP-Neu5Ac synthase neuA, which encodes n-acetylneuraminic acid synthase neuB, and udp-n-acetylglucosamine 2-diimine enzyme neuC.

8. A method for preparing the mutant of claim 1, characterized in that, include: Cultivate the recombinant engineered bacteria according to any one of claims 4 to 7, and induce the expression of the mutant.

9. A biological agent, characterized in that, The biological agent comprises: the mutant of claim 1, the nucleic acid of claim 2, the vector of claim 3, the recombinant engineered bacteria of claims 4-7, or the product obtained by the method of claim 8.

10. The application of the mutant of claim 1, the nucleic acid of claim 2, the vector of claim 3, the recombinant engineered bacteria of claims 4-7, the product obtained by the method of claim 8, or the biological agent of claim 9 in the preparation of 6'-sialyl lactose.

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