Beta-1, 3-acetyl glucosamine transferase mutant and application thereof

Abstract

The invention relates to a beta-1, 3-acetyl glucosamine transferase mutant and an application thereof, and the beta-1, 3-acetyl glucosamine transferase mutant is subjected to amino acid residue mutation at one or more sites in the 163rd, 233rd, 291st and 294 of a wild type beta-1, 3-acetyl glucosamine transferase as shown in SEQ ID NO.1. The invention also relates to a preparation method of the beta-1, 3-acetyl glucosamine transferase mutant. The mutant reduces the generation of long-chain derivatives such as LNnH and LNnO in the process of synthesizing lactose-N neotetrasaccharide in microbial synthesis, thereby improving the yield and purity of LNnT.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a beta-1,3-acetylglucosamine transferase mutant and application thereof. BACKGROUND

[0002] Human milk oligosaccharides (HMOs) are the third largest nutrient in breast milk, and lacto-N-neotetraose (LNnT) is a very important neutral core structure in HMOs, accounting for about 6% (w / w) of total HMOs. LNnT has multiple biological functions, such as increasing the abundance of intestinal probiotics, maintaining intestinal microecological balance, inhibiting pathogen adhesion, and regulating immune response. With the continuous confirmation of the biological value of LNnT, it is gradually added to infant formula milk powder to make up for the lack of HMOs in traditional formula milk, and also used as a prebiotic or immunomodulator for adult intestinal health or specific disease adjuvant therapy.

[0003] With the progress of synthetic biology technology, the microbial fermentation method of genetically engineered bacteria is the most promising method to realize large-scale production of LNnT at present, and its main synthesis path is as follows: beta-1,3-N-acetylglucosaminyltransferase (beta1,3-GlcNAcT, lgtA) catalyzes the connection of uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and lactose in the form of beta-1,3 bond to form lacto-N-triose (LNT II), and then LNT II is connected with uridine 5'-diphosphate galactose (UDP-gal) in the form of beta-1,4 bond to form LNnT under the action of beta-1,4-galactosyltransferase (beta1,4-GalT). This synthesis path is relatively simple, without complicated catalytic steps and complex substrate requirements, which can greatly reduce the synthesis difficulty. Especially the beta-1,3-N-acetylglucosaminyltransferase lgtA from Neisseria meningitidis is widely used for the biosynthesis of LNnT due to its high catalytic activity.

[0004] In the process of synthesizing LNnT using microbial fermentation method, the substrate of glycosyltransferase is mixed, which can cause the production of oligosaccharide derivatives with longer sugar chains, LNnT can be used as the substrate of lgtA to generate pentasaccharide, and the pentasaccharide is catalyzed by β1,4-GalT to generate lacto-N-neohexaose (LNnH), and lgtA and β1,4-GalT catalyze the generation of lacto-N-neooctaose (LNnO) and lacto-N-neodecaose (LNnD) in the same way to generate long-chain derivatives, which can cause the consumption of the target product LNnT and increase the difficulty of downstream separation and purification, and therefore a suitable method is needed to reduce the synthesis of long-chain LNnT derivatives. lgtA plays a crucial role in the synthesis of long-chain LNnT derivatives, and it initiates the first step of LNnT carbon chain extension, which causes the appearance of LNnH and LNnO and other long-chain derivatives in the process of LNnT synthesis, and therefore it needs to be designed to enhance the substrate specificity so as to reduce the synthesis of long-chain LNnT derivatives. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a β-1,3-acetylglucosamine transferase mutant and its use in the synthesis of lacto-N-neotetraose in microbial synthesis using the mutant and reducing the production of LNnH and LNnO and other long-chain derivatives, thereby improving the yield and purity of LNnT.

[0006] Solution to the problem In one aspect, the present application provides a β-1,3-acetylglucosamine transferase mutant, wherein one or more of the amino acid residues at positions 163, 233, 291 and 294 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO. 1 are mutated.

[0007] In one aspect, the present application provides a β-1,3-acetylglucosamine transferase mutant, wherein the amino acid residue at position 163 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO. 1 is mutated, and optionally one or more of the amino acid residues at positions 233, 291 and 294 are mutated.

[0008] Preferably, the β-1,3-acetylglucosamine transferase mutant has one or more of the following amino acid residue mutations in the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO. 1: F163W, H233F, Q291F, K294F.

[0009] Preferably, the β-1,3-acetylglucosamine transferase mutant has mutations in any one of the following groups of amino acid residues in the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO. 1: (1) F163W and H233F; (2) F163W, H233F and Q291F; (3) F163W, H233F, Q291F and K294F.

[0010] In one aspect, the present application provides an isolated polynucleotide encoding the β-1,3-acetylglucosamine transferase mutant of any one of the above.

[0011] In one aspect, the present application provides a vector containing the polynucleotide of the above.

[0012] In one aspect, the present application provides a genetically engineered host cell containing the vector of the above, or the polynucleotide of the above integrated in the genome.

[0013] Preferably, the host cell Escherichia coli E. coli BL21 (DE3) is the starting strain, in which the β-galactosidase gene lacZ, the uridine diphosphate-N-acetylglucosamine-2-epimerase gene wecB, and the glucosamine-6-phosphate deaminase gene nagB are knocked out from the genome; and the β-1,4-galactosyltransferase gene Ags, the uridine diphosphate glucose-4-epimerase gene galE derived from MG1655, and the β-1,3-acetylglucosamine transferase mutant of any one of the above are expressed. Aggregatibacter kilianii β-1,4-galactosyltransferase gene Ags, the uridine diphosphate glucose-4-epimerase gene galE derived from E. coli MG1655, and the β-1,3-acetylglucosamine transferase mutant of any one of the above are expressed.

[0014] In one aspect, the present application provides a method for producing lacto-N neotetraose, which uses the host cell of the above as a fermentation strain to produce lacto-N neotetraose.

[0015] In one aspect, the present application provides the use of the β-1,3-acetylglucosamine transferase mutant of any one of the above, or the vector of the above, or the host cell of the above in the preparation of lacto-N neotetraose.

[0016] Preferably, the use can improve the purity, the production efficiency and / or the yield of lacto-N neotetraose.

[0017] Effects of the application The application enhances the substrate specificity of the beta-1,3-N-acetylglucosamine transferase lgtA from Neisseria meningitidis by single-point mutation or combined mutation of the key site in the substrate binding pocket, reduces the generation of long-chain derivatives in the biosynthesis of LNnT, and realizes the synthesis of high-purity LNnT. The method can effectively improve the yield of the target product LNnT, and especially the lgtA mutant containing F163W, H233F, Q291F and K294F, the content of long-chain derivative lacto-N-neohexaose (LNnH) in the fermentation product is reduced by 72.79% compared with the wild type, and the content of LNnT is increased by 89.71% compared with the wild type, which greatly reduces the difficulty of downstream separation and purification. The beta-1,3-N-acetylglucosamine transferase lgtA mutant provided by the application has important significance for promoting the large-scale production of lacto-N-neotetraose. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is the plasmid map of the recombinant plasmid pAC-Ags-lgtA-galE; Figure 2 It is the column chart of the yield of lacto-N-neotetraose and long-chain derivative lacto-N-neohexaose of each mutant strain. DETAILED DESCRIPTION

[0019] In order to make the technical solutions and beneficial effects of the application more obvious and easy to understand, the following will be described in detail by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meaning as the technical and scientific terms in the technical field to which the application belongs.

[0020] In the present specification, the amino acids at the corresponding sites are represented by the recognized IUPAC one-letter abbreviations, wherein each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0021] In the present specification, regarding the mutation of amino acid, it is expressed as "original amino acid, site, substituted amino acid". For example, the mutation of phenylalanine at site 163 to tryptophan is expressed as F163W.

[0022] The plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercially available products, and the specific operations are carried out according to the kit instructions.

[0023] The following experimental operations such as preparation of competent cells, colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, and extraction and preservation of bacterial genomes are carried out according to the conventional method. The sequencing of the following plasmids and DNA products is completed by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0024] The present inventors have conducted extensive and in-depth research and obtained a group of novel β-1,3-acetylglucosamine transferase mutants through a large number of experimental screenings, which can significantly reduce the generation of derivatives in the synthesis process of lacto-N-neotetraose, thereby improving the purity of lacto-N-neotetraose and the yield of lacto-N-neotetraose. Based on this, the present application is completed.

[0025] The mutant of the present application The present application provides a β-1,3-acetylglucosamine transferase mutant, wherein one or more of the amino acid residues at positions 163, 233, 291 and 294 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO. 1 are mutated.

[0026] SEQ ID NO. 1 (lgtA): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* The present application provides a β-1,3-acetylglucosamine transferase mutant, which has an amino acid residue mutation at position 163 and optionally one or more of the following positions: 233, 291 and 294 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO. 1.

[0027] In some embodiments, the β-1,3-acetylglucosamine transferase mutant has one or more of the following amino acid residue mutations: F163W, H233F, Q291F, K294F in the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO. 1.

[0028] In some embodiments, the β-1,3-acetylglucosamine transferase mutant has a phenylalanine at position 163 mutated to a tryptophan in the wild-type β-1,3-acetylglucosamine transferase, designated as lgtA-F163W, and the amino acid sequence is shown in SEQ ID NO. 4.

[0029] SEQ ID NO. 4 (lgtA-F163W): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPWGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* In some embodiments, the β-1,3-acetylglucosamine transferase mutant has a histidine at position 233 mutated to a phenylalanine in the wild-type β-1,3-acetylglucosamine transferase, designated as lgtA-H233F, and the amino acid sequence is shown in SEQ ID NO. 5.

[0030] SEQ ID NO. 5 (lgtA-H233F): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYFCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* In some embodiments, the β-1,3-acetylglucosaminyltransferase mutant is a mutant in which the glutamine at position 291 of wild-type β-1,3-acetylglucosaminyltransferase is mutated to phenylalanine, designated as lgtA-Q291F, and the amino acid sequence is shown in SEQ ID NO. 6.

[0031] SEQ ID NO. 6 (lgtA-Q291F): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYFCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* In some embodiments, the β-1,3-acetylglucosaminyltransferase mutant is a mutant in which the lysine at position 294 of wild-type β-1,3-acetylglucosaminyltransferase is mutated to phenylalanine, designated as lgtA-K294F, and the amino acid sequence is shown in SEQ ID NO. 7.

[0032] SEQ ID NO. 7 (lgtA-K294F): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYQCFFRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* In some embodiments, the β-1,3-acetylglucosaminyltransferase mutant is a mutant in which the phenylalanine at position 163 is mutated to tryptophan and the histidine at position 233 is mutated to phenylalanine in wild-type β-1,3-acetylglucosaminyltransferase, designated as lgtA-F163W / H233F, and the amino acid sequence is shown in SEQ ID NO. 8.

[0033] SEQ ID NO. 8 (lgtA-F163W / H233F): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPWGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQFEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* In certain embodiments, the beta-1,3-acetylglucosaminyltransferase mutant is a mutant of wild-type beta-1,3-acetylglucosaminyltransferase in which phenylalanine at position 163 is mutated to tryptophan, histidine at position 233 is mutated to phenylalanine, and glutamine at position 291 is mutated to phenylalanine, designated as lgtA-F163W / H233F / Q291F, and the amino acid sequence is shown in SEQ ID NO. 9.

[0034] SEQ ID NO. 9 (lgtA-F163W / H233F / Q291F): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPWGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQFEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYFCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* In certain embodiments, the beta-1,3-acetylglucosaminyltransferase mutant is a mutant of wild-type beta-1,3-acetylglucosaminyltransferase in which phenylalanine at position 163 is mutated to tryptophan, histidine at position 233 is mutated to phenylalanine, glutamine at position 291 is mutated to phenylalanine, and lysine at position 294 is mutated to phenylalanine, designated as lgtA-F163W / H233F / Q291F / K294F, and the amino acid sequence is shown in SEQ ID NO. 10.

[0035] SEQ ID NO. 10 (lgtA-F163W / H233F / Q291F / K294F): MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPWGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQFEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFELARRFLYFCFFRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR* In some embodiments, the β-1,3-acetylglucosaminyltransferase mutant has any one of the following mutations in the amino acid residues of the wild-type β-1,3-acetylglucosaminyltransferase shown in SEQ ID NO. 1: (1) F163W and H233F; or (2) F163W, H233F and Q291F; or (3) F163W, H233F, Q291F and K294F.

[0036] In some embodiments, the β-1,3-acetylglucosaminyltransferase mutant has an amino acid sequence as shown in any one of SEQ ID NO. 4~10.

[0037] The amino terminal or carboxyl terminal of the protein of the present application can also contain one or more polypeptide fragments as a protein tag. Any suitable tag can be used in the present application. For example, the tag can be FLAG, HA, c-Myc, Poly-His, Poly-Arg, StrepII, etc. These tags can be used for purifying the protein.

[0038] Polynucleotides, vectors and host cells of the present application The present application provides an isolated polynucleotide encoding the β-1,3-acetylglucosaminyltransferase mutant of any one of the above.

[0039] The polynucleotide of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0040] The present application also relates to variants of the above polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present application. The variants of the polynucleotides can be isovariants or non-isovariants, but do not substantially change the function of the encoded polypeptides.

[0041] The polypeptides and polynucleotides of the present application are preferably provided in isolated form, and more preferably purified to homogeneity. The full-length sequence of the β-1, 3-acetylglucosamine transferase mutant nucleotide of the present application or fragments thereof can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present application, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence.

[0042] Once the relevant sequence is obtained, a recombinant protein can be prepared using a biological engineering method. This generally involves cloning the polynucleotide sequence encoding the recombinant protein into an expression vector, transforming the expression vector into cells for expression, and harvesting and purifying the recombinant protein.

[0043] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing plasmids (or vectors) known in the art, and transformed into cells for expression and purification to obtain the protein.

[0044] The present application provides a vector containing the above-mentioned polynucleotide.

[0045] The present application provides a genetically engineered host cell containing the above-mentioned vector, or the above-mentioned polynucleotide integrated into the genome.

[0046] In some embodiments, the host cell Escherichia coli E. coli BL21 (DE3) is the starting strain, in which the β-galactosidase gene lacZ, the uridine diphosphate-N-acetylglucosamine-2-epimerase gene wecB, and the glucosamine-6-phosphate deaminase gene nagB are knocked out from the genome of the starting strain; and the β-1, 4-galactosyltransferase gene Ags, the uridine diphosphate glucose-4-epimerase gene galE derived from MG1655, and the β-1, 3-acetylglucosamine transferase mutant of any one of the above are expressed. Aggregatibacter kilianii The β-1, 4-galactosyltransferase gene Ags, the uridine diphosphate glucose-4-epimerase gene galE derived from MG1655, and the β-1, 3-acetylglucosamine transferase mutant of any one of the above are expressed. E. coli

[0047] In some embodiments, the amino acid sequence of Ags is shown in SEQ ID NO. 2.​

[0048] SEQ ID NO. 2 (Ags): MHFIENKNFVISIPTADKRRNHIIQQFGQKKIPFEFFDAFTPSERLNDHLQRYLPNVATIPKLTMGEKGCLMSHFMLWKKCVDDDLDFITLFEDDILLGENAEQFLAEDEWLKVRFNFQEIFVLRLETFLMPVKVEKQQQILPFQQREIDILRSKHFGTAGYVISHGAAKYLMEVFEKFSSEEIKPIDEIIFNQLIDVPRYRVYQLNPAICVQELQLNQENSVLTSGLQQERKKNTASHTKK In some embodiments, the amino acid sequence of the galE is as set forth in SEQ ID NO. 3.

[0049] SEQ ID NO. 3 (galE): MRVLVTGGSGYIGSHTCVQLLQNGHDVIILDNLCNSKRSVLPVIERLGGKHPTFVEGDIRNEALMTEILHDHAIDTVIHFAGLKAVGESVQKPLEYYDNNVNGTLRLISAMRAANVKNFIFSSSATVYGDQPKIPYVESFPTGTPQSPYGKSKLMVEQILTDLQKAQPDWSIALLRYFNPVGAHPSGDMGEDPQGIPNNLMPYIAQVAVGRRDSLAIFGNDYPTEDGTGVRDYIHVMDLADGHVVAMEKLANKPGVHIYNLGAGVGNSVLDVVNAFSKACGKPVNYHFAPRREGDLPAYWADASKADRELNWRVTRTLDEMAQDTWHWQSRHPQGYPD Applications The present application provides a method for producing lacto-N neotetraose, which uses the above-mentioned host cell as a fermentation strain to ferment and produce lacto-N neotetraose.

[0050] The present application provides the use of the above-mentioned β-1, 3-acetylglucosamine transferase mutant, or the above-mentioned vector, or the above-mentioned host cell in the preparation of lacto-N neotetraose. In some embodiments, the use can improve the purity of lacto-N-neotetraose, improve the production efficiency of lacto-N-neotetraose, and / or improve the yield of lacto-N-neotetraose.

[0051] In some embodiments, the fermentation medium used comprises the following components and in the following amounts: 10-20 g / L glucose, 1-2 g / L citric acid, 10-20 g / L potassium dihydrogen phosphate, 1-10 g / L ammonium phosphate, 1-2 g / L magnesium sulfate heptahydrate, 5-15 mL / L trace metal solution; The trace metal solution comprises: 5-15 g / L ferric citrate, 1-5 g / L zinc sulfate heptahydrate, 0.5-2 g / L copper sulfate pentahydrate, 0.1-1 g / L manganese sulfate monohydrate, 0.1-1 g / L sodium borate decahydrate, 0.05-0.5 g / L ammonium molybdate heptahydrate, 1-5 g / L calcium chloride dihydrate 2.0 g / L.

[0052] In some embodiments, the fermentation medium used comprises the following components and in the following amounts: 12-16 g / L glucose, 1.5-2 g / L citric acid, 10-15 g / L potassium dihydrogen phosphate, 2-6 g / L ammonium phosphate, 1.2-1.6 g / L magnesium sulfate heptahydrate, 8-12 mL / L trace metal solution; The trace metal solution comprises: 8-12 g / L ferric citrate, 2-3 g / L zinc sulfate heptahydrate, 0.5-1.5 g / L copper sulfate pentahydrate, 0.1-0.5 g / L manganese sulfate monohydrate, 0.1-0.5 g / L sodium borate decahydrate, 0.05-0.2 g / L ammonium molybdate heptahydrate, 1-3 g / L calcium chloride dihydrate.

[0053] In some embodiments, the fermentation medium used comprises the following components and in the following amounts: 15.07 g / L glucose, 1.7 g / L citric acid, 13.5 g / L potassium dihydrogen phosphate, 4 g / L ammonium phosphate, 1.4 g / L magnesium sulfate heptahydrate, 10 mL / L trace metal solution; The trace metal solution comprises: 10 g / L ferric citrate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate heptahydrate, 2 g / L calcium chloride dihydrate.

[0054] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate but not limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sam brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0055] General Materials and Methods The culture media involved in the following examples are as follows: LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0056] LB solid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 20 g / L.

[0057] Glucose fermentation medium: Glucose 15.07 g / L, citric acid 1.7 g / L, potassium dihydrogen phosphate 13.5 g / L, ammonium phosphate 4 g / L, magnesium sulfate heptahydrate 1.4 g / L, trace metal solution 10 mL / L (ferric citrate 10 g / L, zinc sulfate heptahydrate 2.25 g / L, copper sulfate pentahydrate 1.0 g / L, manganese sulfate monohydrate 0.35 g / L, sodium borate decahydrate 0.23 g / L, ammonium molybdate heptahydrate 0.11 g / L, calcium chloride dihydrate 2.0 g / L).

[0058] The antibiotic concentration involved in the following examples: chloramphenicol 25 mg / L.

[0059] The substrate and inducer addition concentration involved in the following examples: lactose 5 g / L, IPTG 0.2 mM.

[0060] The detection method involved in the following examples is as follows: Method for detecting lacto-N-neotetraose and its long-chain derivatives by high-performance liquid chromatography: The determination of lacto-N-neotetraose content uses liquid chromatography HPLC (Agilent 1260 series, USA), the detector is a differential refractometer RID, and the chromatographic column is Rezex™ ROA-Organic Acid H +(8%, 300 mm x 7.8 mm) with 10 mM diluted sulfuric acid as mobile phase, detection temperature 82 °C, and flow rate 0.6 mL / min. Commercially available standards (CAS: 13007-32-4, 64003-52-7) were used to establish the quantitative method. Sample preparation: 1 mL of fermentation broth was heated at 100 °C for 20 min, centrifuged at 12000 rpm for 5 min at room temperature, and the supernatant was then filtered with a water phase membrane with a pore size of 0.22 μm. The total concentration of the product was detected by HPLC.

[0061] Example 1 Construction of recombinant plasmid pAC-Ags-galE-lgtA Based on the literature reports and the NCBI database query, the β-1, 3-N-acetylglucosamine transferase gene (Genbank ID: AAC44084.1) from Neisseria meningitidis and the β-1, 4-galactosyltransferase gene (Genbank ID: WP_111278564.1) from Aggregatibacter kilianii were obtained, which were named as genes lgtA and Ags, respectively. The lgtA and Ags genes were codon-optimized and designed according to the codon bias of E. coli BL21 (DE3) and then synthesized by Suzhou Jinweizhi Company. The amino acid sequence of lgtA was SEQ ID NO. 1, and the amino acid sequence of Ags was SEQ ID NO. 2. The E. coli MG1655 genome was extracted to obtain the uridine diphosphate glucose-4-epimerase gene galE, and the amino acid sequence was SEQ ID NO. 3.

[0062] Primers pAC-CP-F / R, lgtA-F / R, Ags-F / R and galE-F / R were designed, and the primer sequences are shown in Table 1. PCR reactions were performed with the expression vector pACYCDuet-1, the synthetic gene lgtA, the synthetic gene Ags and the E. coli MG1655 genome as templates, respectively. The pACYCDuet-1 linear vector, the lgtA gene fragment, the Ags gene fragment and the galE gene fragment were obtained, and the sizes of the amplified fragments were determined by gel electrophoresis. Then, the linear fragments were recovered for seamless cloning. The seamless cloning reaction solution was transformed into E. coli DH5α competent cells by heat shock, and then coated on a chloramphenicol-resistant LB solid plate. After overnight culture at 37 °C, positive clones were selected for sequencing. After correct sequencing, the plasmid was expanded and extracted. The plasmid was the recombinant plasmid pAC-Ags-galE-lgtA (the plasmid map is shown in Figure 1

[0063] Table 1 Primer sequences used for plasmid construction ​

[0064] Construction of recombinant strain LNnT-01 of Example 2 The β-galactosidase gene lacZ, the uridine diphosphate-N-acetylglucosamine 2 epimerase gene wecB, and the glucosamine 6 phosphate deaminase gene nagB on the genome of E. coli BL21 (DE3) were knocked out to obtain an engineering strain L5, and the construction method of the strain is disclosed in the Chinese patent application document with the publication number CN116640715A. The engineering strain L5 was used as a starting strain to construct a recombinant strain for producing lacto-N-neotetraose.

[0065] The engineering strain L5 was used as a starting strain, chemical competence was prepared, the plasmid pAC-Ags-galE-lgtA was transformed into the chemical competence, and the positive clones were screened after coating on a chloramphenicol-resistant LB solid plate and overnight culture at 37°C. The correct positive transformant was verified as the recombinant strain LNnT-01 (L5, pAC-Ags-galE-lgtA) capable of synthesizing lacto-N-neotetraose.

[0066] Example 3

[0067] The mutant primers for amplifying F163W, H233F, Q291F, and K294F were designed as shown in Table 2, and the mutant recombinant plasmid was constructed using pAC-Ags-galE-lgtA as a template. Taking the mutant F163W as an example, the forward primer F163W-F and the reverse primer F163W-R were used to perform PCR amplification on the template pAC-Ags-galE-lgtA, the size of the amplified fragment was determined by gel electrophoresis, and then the linear fragment was recovered for seamless cloning. The seamless cloning reaction solution was transformed into E. coli DH5α competence by heat shock, coated on a chloramphenicol-resistant LB solid plate, and screened for positive clones after overnight culture at 37°C. The positive clones were sequenced, and after correct sequencing, the plasmid was extracted by expanding culture. The plasmid was the mutant recombinant plasmid pAC-Ags-galE-lgtA / F163W.

[0068] The mutant recombinant plasmids shown in Table 3 were constructed by the same method as described above.

[0069] Table 2 Primer sequences used for plasmid construction

[0070] Table 3 Recombinant plasmids constructed by the application

[0071] Example 4

[0072] The recombinant plasmids constructed according to Example 3 were used to construct recombinant strains LNnT-02 to LNnT-08 according to the method of Example 2. The information of the recombinant strains constructed according to the application is shown in Table 4.

[0073] Table 4 Mutant recombinant strains used in the present patent

[0074] Test Example The recombinant strains LNnT-01 to LNnT-08 were inoculated into glucose fermentation medium containing chloramphenicol for small-scale shake flask fermentation tests. The culture temperature was 37°C, the rotation speed was 220 rpm, and the culture was carried out until the OD 600 was about 0.6, the culture temperature was reduced to 30°C, 0.2 mM IPTG was added to induce the expression of the target gene, and the substrate lactose 5 g / L was added. After 48 h of induction culture, samples were taken, and the contents of LNnT and its main long-chain derivative LNnH were determined by high performance liquid chromatography-differential refractive index detector.

[0075] The results are shown in Table 5. Figure 2 As shown in Table 5, the wild-type recombinant strain LNnT-01 produced 1.36 g / L of the target product LNnT after 48 h of fermentation, and the mutant strains LNnT-02 to LNnT-07 produced 1.78 g / L, 1.80 g / L, 1.71 g / L, 1.83 g / L, 2.02 g / L and 2.32 g / L of the target product LNnT, respectively. Compared with LNnT-01, LNnT-02 to LNnT-07 increased the production of LNnT by 30.88%, 32.35%, 25.74%, 34.56%, 48.53% and 70.59%, respectively. The combination mutant LNnT-08 of the four sites F163W, H233F, Q291F and K294F produced the maximum amount of LNnT, reaching 2.58 g / L, which was increased by 89.71% compared with the wild type. The results of the production of the long-chain derivative LNnH showed that the wild-type recombinant strain LNnT-01 produced 2.83 g / L of LNnH after 48 h of fermentation, and the mutant strains LNnT-02 to LNnT-07 produced 2.32 g / L, 2.26 g / L, 2.35 g / L, 2.09 g / L, 1.78 g / L and 1.24 g / L of LNnH, respectively. Compared with LNnT-01, LNnT-02 to LNnT-07 reduced the production of LNnH by 18.02%, 20.14%, 16.96%, 26.15%, 37.10% and 56.18%, respectively. The LNnH production of the combination mutant LNnT-08 of the four sites was only 0.77 g / L, which was reduced by 72.79% compared with the wild type. This mutant is of great significance for the industrial synthesis of LNnT.

[0076] It should be understood that the above examples are exemplary and are not intended to limit the scope of the claims encompassing all possible embodiments. Various modifications and changes can also be made on the basis of the above examples without departing from the scope of the present disclosure. Similarly, various technical features of the above examples can be combined arbitrarily to form additional embodiments of the present application that can not be explicitly described. Therefore, the above examples merely express several embodiments of the present application and do not limit the scope of the patent protection of the present application.

Claims

1. A mutant of a β-1,3-acetylglucosaminyltransferase, characterized in that, The β-1,3-acetylglucosamine transferase mutant has one or more amino acid residue mutations at positions 163, 233, 291 and 294 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO.

1.

2. A mutant of a β-1,3-acetylglucosaminyltransferase, characterized in that, The β-1,3-acetylglucosamine transferase mutant has one or more amino acid residue mutations at positions 163, 233, 291 and 294 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO.

1.

3. The beta-1,3-acetylglucosaminyltransferase mutant of any one of claims 1 or 2, characterized in that, The β-1,3-acetylglucosamine transferase mutant has one or more amino acid residue mutations at positions 163, 233, 291 and 294 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO.

1.

4. The beta-1,3-acetylglucosaminyltransferase mutant of any of claim 3, characterized in that, The β-1,3-acetylglucosamine transferase mutant has one or more amino acid residue mutations at positions 163, 233, 291 and 294 of the wild-type β-1,3-acetylglucosamine transferase shown in SEQ ID NO.

1. (1) F163W and H233F; (2) F163W, H233F and Q291F; (3) F163W, H233F, Q291F and K294F.

5. An isolated polynucleotide, comprising, The polynucleotide encodes the β-1,3-acetylglucosamine transferase mutant of any one of claims 1-4.

6. A vector, characterized in that, The vector contains the polynucleotide of claim 5.

7. A genetically engineered host cell, characterized in that, The host cell contains the vector of claim 6, or the polynucleotide of claim 5 is integrated into the genome.

8. The host cell of claim 7, wherein, The host cell Escherichia coli E.coli BL21(DE3) is a starting strain, in which the β-galactosidase gene lacZ, the uridine diphosphate-N-acetylglucosamine-2-epimerase gene wecB, and the glucosamine-6-phosphate deaminase gene nagB are knocked out on the genome of the starting strain; and the β-1, 4-galactosyltransferase gene Ags, the uridine diphosphate glucose-4-epimerase gene galE derived from MG1655, and the β-1, 3-acetylglucosamine transferase mutant of any one of claims 1-4 are expressed. Aggregatibacter kilianii BL21(DE3) is a starting strain, in which the β-galactosidase gene lacZ, the uridine diphosphate-N-acetylglucosamine-2-epimerase gene wecB, and the glucosamine-6-phosphate deaminase gene nagB are knocked out on the genome of the starting strain; and the β-1, 4-galactosyltransferase gene Ags, the uridine diphosphate glucose-4-epimerase gene galE derived from MG1655, and the β-1, 3-acetylglucosamine transferase mutant of any one of claims 1-4 are expressed. E.coli BL21(DE3) is a starting strain, in which the β-galactosidase gene lacZ, the uridine diphosphate-N-acetylglucosamine-2-epimerase gene wecB, and the glucosamine-6-phosphate deaminase gene nagB are knocked out on the genome of the starting strain; and the β-1, 4-galactosyltransferase gene Ags, the uridine diphosphate glucose-4-epimerase gene galE derived from MG1655, and the β 9. A method for producing lacto- / \ / -neotetraose, characterized by, The method uses the host cell of claim 7 or 8 as a fermentation strain to produce lacto-N neotetraose by fermentation.

10. Use of the β-1,3-acetylglucosamine transferase mutant of any one of claims 1-4 or the vector of claim 6, or the host cell of claim 7 or 8 in the preparation of lacto-N neotetraose. Preferably, the use can improve the purity of lacto-N-neotetraose, improve the production efficiency of lacto-N-neotetraose and / or improve the yield of lacto-N-neotetraose.

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