Method for producing sialyllacto-N-neotetraose c

Abstract

To provide a novel method for producing sialyllacto-N-neotetraose c with minimal by-products. [Solution] A method for producing sialyl lacto-N-neotetraose c (LST-c) from lacto-N-neotetraose (LNnT) in the presence of at least one protein selected from the group consisting of [1] to [3] below; [1] A protein containing the amino acid sequence shown in one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, which includes an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity.

Description

Technical Field

[0001] The present invention relates to a method for producing sialyllacto-N-neotetraose c. Further, the present invention also relates to a method for suppressing the production of 6'-sialyllactose, sialylated lacto-N-trioose II, and sialyllacto-N-neotetraose c isomers in the production of sialyllacto-N-neotetraose c, a genetically modified cell having a protein, or a culture of the genetically modified cell.

Background Art

[0002] Human milk oligosaccharides (HMOs) are a general term for oligosaccharides contained in breast milk, and are the third most abundant component among the solid components contained in breast milk, after lactose and lipids. More than 200 types of HMOs have been discovered from breast milk so far. Among them, HMOs containing N-acetylneuraminic acid, a type of sialic acid, in their structure are called sialylated HMOs. 3'-sialyllactose (hereinafter sometimes referred to as 3SL; the same applies to other abbreviated descriptions below), 6'-sialyllactose (hereinafter referred to as 6SL), which are already commercially produced sialylated HMOs, have been reported to play various beneficial roles such as the formation of the intestinal environment of infants and the regulation of immune function (Non-Patent Documents 1 and 2). Furthermore, research and development on the uses and production methods of dialsyllacto-N-tetraose (hereinafter referred to as DSLNT), sialyllacto-N-tetraose a (hereinafter referred to as LST-a), sialyllacto-N-tetraose b (hereinafter referred to as LST-b), and sialyllacto-N-neotetraose c (hereinafter referred to as LST-c), which have a longer sugar chain length, are also underway. For example, there is a report that LST-c is the most preferentially secreted among sialylated HMOs in the quantitative analysis of human colostrum on the 1st to 3rd day after the start of secretion (Non-Patent Document 3), and it may have functions for the development and health of newborns. LST-c can be synthesized by adding N-acetylneuraminic acid (SIA) to the galactose at the terminal end of lacto-N-neotetraose (hereinafter referred to as LNnT) using α2,6-sialyltransferase. While enzymes with α2,6-sialyltransferase activity have been widely identified, ranging from those derived from microorganisms such as Helicobacter (Patent Documents 1 and 2, Non-Patent Document 4), Photobacterium (Patent Document 3, Non-Patent Document 5), and Pasteurella (Non-Patent Document 6), to those derived from eukaryotes such as humans and mice, there is still little knowledge about α2,6-sialyltransferases with activity suitable for LST-c production. Although 6SL and LNnT are commercially produced, there are no reported examples of industrial-scale fermentation production of LST-c. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent Application Publication No. 2019 / 0218582 [Patent Document 2] International Publication No. 2023 / 247537 [Patent Document 3] International Publication No. 2021 / 123113 [Non-patent literature]

[0004] [Non-Patent Document 1] Wusun Li et al.,Trends in Food Science & Technology,118:374-387,2021 [Non-Patent Document 2] Lis Kubrak, J. & Orczyk-Pawilowicz, M., Nutrients,11(2),306,2019 [Non-Patent Document 3] Tadao.S et al., The Chemical Times, 215(1), 3-8, 2010 [Non-Patent Document 4] Melissa J Schur et al., Glycobiology, 22,997-1006,2012 [Non-Patent Document 5] Nhung Huynh et al., FEBS Letters,558(24),4720-4729,2014 [Non-Patent Document 6] Gulati S. et al.,infection and immunity,73(11),7390-7397,2005 [Overview of the project] [Problems that the invention aims to solve]

[0005] In their diligent efforts to enable industrial-scale LST-c production, the inventors discovered that known α2,6 sialyltransferases have low substrate selectivity and unintentionally add N-acetylneuraminic acid to unintended substrates, resulting in the generation of multiple by-products during LST-c production.

[0006] Therefore, the present invention aims to provide a novel method for producing LST-c with minimal by-products. [Means for solving the problem]

[0007] The inventors have discovered that an α2,6 sialyltransferase having a specific amino acid sequence is effective in reducing by-products in the production of LST-c, and have completed an invention relating to a novel method for producing LST-c.

[0008] In other words, the present invention encompasses the following inventions. (1) A method for producing sialyl lacto-N-neotetraose c (LST-c) from lacto-N-neotetraose (LNnT) in the presence of at least one protein selected from the group consisting of [1] to [3] below; [1] A protein containing the amino acid sequence shown in one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, which includes an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity. (2) The method for producing a protein represented by at least one selected from the group consisting of [1] to [3] in genetically modified cells, further comprising the step of producing a protein represented by at least one selected from the group consisting of [1] to [3] above, according to (1). (3) A method for producing LST-c according to (1) or (2), comprising producing LST-c in an aqueous medium containing a protein represented by at least one selected from the group consisting of [1] to [3], cytidine 5'-phosphate-N-acetylneuraminic acid, and LNnT. (4-1) A method for producing LST-c according to (1) or (2), wherein LST-c is produced in the presence of at least one protein selected from the group consisting of β-1,3-N-acetylglucosaminyltransferase (LgtA), β-1,4-galactosyltransferase (GalT), and proteins involved in the biosynthetic pathway of cytidine 5'-phosphate-N-acetylneuraminic acid, and at least one protein represented by the group consisting of [1] to [3]. (4-2) The method for producing LST-c according to (3), wherein LST-c is produced in the presence of at least one protein selected from the group consisting of β-1,3-N-acetylglucosaminyltransferase (LgtA), β-1,4-galactosyltransferase (GalT), and proteins involved in the biosynthetic pathway of cytidine 5'-phosphate-N-acetylneuraminic acid, and at least one protein represented by the group consisting of [1] to [3]. (5-1) LgtA derived from Neisseria polysaccharea ATCC43768 (NpLgtA), GalT derived from Helicobacter pylori (HpGalT), UDP-N-acetylglucosamine 2-epimerase (FpNeuC) derived from Flavobacterium psychrophilum, N-acetylneuraminic acid synthase (CjNeuB) derived from Campylobacter jenuni, and Cytidine 5'-phosphate-N-acetylneuraminic acid synthase (PmNeuA) derived from Pasteurella multocida The production method according to (1) or (2), which produces LST-c in the presence of at least one protein selected from the group consisting of and at least one protein represented by at least one selected from the group consisting of the above [1] to [3]. (5-2) LgtA derived from Neisseria polysaccharea ATCC43768 (NpLgtA), GalT derived from Helicobacter pylori (HpGalT), UDP-N-acetylglucosamine 2-epimerase (FpNeuC) derived from Flavobacterium psychrophilum, N-acetylneuraminic acid synthase (CjNeuB) derived from Campylobacter jenuni, and Cytidine 5'-phosphate-N-acetylneuraminic acid synthase (PmNeuA) derived from Pasteurella multocida The production method according to (3), which produces LST-c in the presence of at least one protein selected from the group consisting of and at least one protein represented by at least one selected from the group consisting of the above [1] to [3]. (5-3) LgtA derived from Neisseria polysaccharea ATCC43768 (NpLgtA), GalT derived from Helicobacter pylori (HpGalT), UDP-N-acetylglucosamine 2-epimerase (FpNeuC) derived from Flavobacterium psychrophilum, N-acetylneuraminic acid synthase (CjNeuB) derived from Campylobacter jenuni, and Cytidine 5'-phosphate-N-acetylneuraminic acid synthase (PmNeuA) derived from Pasteurella multocida The production method according to (4-1), which produces LST-c in the presence of at least one protein selected from the group consisting of and at least one protein represented by at least one selected from the group consisting of [1] to [3] above. (5-4) LgtA (NpLgtA) derived from Neisseria polysaccharea ATCC43768, GalT (HpGalT) derived from Helicobacter pylori, UDP-N-acetylglucosamine 2-epimerase (FpNeuC) derived from Flavobacterium psychrophilum, N-acetylneuraminic acid synthase (CjNeuB) derived from Campylobacter jenuni, and Cytidine 5'-phosphate-N-acetylneuraminic acid synthase (PmNeuA) derived from Pasteurella multocida The production method according to (4-2), which produces LST-c in the presence of at least one protein selected from the group consisting of and at least one protein represented by at least one selected from the group consisting of [1] to [3] above. (6) In the production of sialyllacto-N-neotetraose c (LST-c) from lacto-N-neotetraose (LNnT), a method for suppressing the production of at least one selected from the group consisting of 6'-sialyllactose, sialyllacto-N-triose II, and sialyllacto-N-neotetraose c isomers, using at least one protein represented by at least one selected from the group consisting of [1] to [3] below; [1] A protein containing the amino acid sequence shown in one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, which includes an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity. (7) Genetically modified cells having a protein represented by at least one selected from the group consisting of [1] to [3] below. [1] A protein containing the amino acid sequence shown in one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, which includes an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity. (8) A genetically modified cell according to (7), having at least one protein selected from the group consisting of β-1,3-N-acetylglucosaminyltransferase (LgtA), β-1,4-galactosyltransferase (GalT), and proteins involved in the biosynthetic pathway of cytidine 5'-phosphate-N-acetylneuraminic acid, and at least one protein represented by the group consisting of [1] to [3] above. (9) LgtA (NpLgtA) from Neisseria polysaccharea ATCC43768, GalT (HpGalT) from Helicobacter pylori, UDP-N-acetylglucosamine 2-epimerase (FpNeuC) derived from Flavobacterium psychrophilum, N-acetylneuraminic acid synthase (CjNeuB) derived from Campylobacter jenuni, and Cytidine 5'-phosphate-N-acetylneuraminate synthase (PmNeuA) derived from Pasteurella multocida A genetically modified cell according to (8), having at least one protein selected from the group consisting of and at least one protein represented by the group consisting of [1] to [3]. (10) Contains sialyllacto-N-neotetraose c, The relative abundance of 6'-sialyl lactose to sialyl lactose-N-neotetraose c is 800% or less. The relative abundance of the sialyllacto-N-neotetraose c isomer to sialyllacto-N-neotetraose c is 20% or less. The ratio of sialyl lacto-N-triose II to sialyl lacto-N-neotetraose c is 6% or less. A culture of genetically modified cells. In (1) to (9) above, in [1] to [3], the protein that "contains an amino acid sequence" may also be a protein that "consists of an amino acid sequence". [Effects of the Invention]

[0009] According to the present invention, a novel method for producing sialyllacto-N-neotetraose c can be provided. [Brief explanation of the drawing]

[0010] [Figure 1] The results of LC-MS analysis of LST-c, 6SL, and LST-c structural isomers are shown. [Figure 2] This shows sequence numbers 1 through 4. [Figure 3] Sequence numbers 5-8 are shown. [Figure 4]This shows sequence numbers 9 through 12. [Figure 5] This shows sequence numbers 13-17. [Figure 6] This shows sequence numbers 18-23. [Figure 7] This shows sequence numbers 24-31. [Figure 8] This shows sequence numbers 32-38. [Figure 9] This shows sequence numbers 39-46. [Figure 10] This shows sequence numbers 47-52. [Figure 11] This shows sequence numbers 53-110. [Figure 12] This shows sequence numbers 111-135. [Modes for carrying out the invention]

[0011] The following describes embodiments of the present invention (hereinafter referred to as "these embodiments"), but the scope of the present invention is not limited to these embodiments.

[0012] This embodiment includes a method for producing sialyl lacto-N-neotetraose c (LST-c) from lacto-N-neotetraose in the presence of a protein having α2,6-sialyltransferase activity.

[0013] <Sialyllacto-N-neotetraose c and its by-products> Siallyl lacto-N-neotetraose c (LST-c, Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, CAS No. 64003-55-0) is an HMO having a structure in which N-acetylneuraminic acid is α(2-6) linked to the terminal galactose (Gal) residue of lacto-N-neotetraose (LNnT). Sialyllacto-N-neotetraose c may form salts. The salts formed by LST-c are not particularly limited as long as they are pharmaceutically acceptable, and examples include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, organic amine salts such as ammonium salts, triethanolamine salts and triethylamine salts, and basic amino acid salts such as lysine salts and arginine salts. Lacto-N-neotetraose (LNnT, Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, CAS No. 14116-68-8) is an HMO having a structure in which Gal, N-acetylglucosamine (GlcNAc), Gal, and glucose (Glu) are linked by β(1-4) bonds, β(1-3) bonds, and β(1-4) bonds, respectively. In this specification, sialylated lacto-N-triose II (sialylated LNTII) is a tetrasaccharide represented by the following formula (1) or (2), having a structure in which N-acetylneuraminic acid is α(2-6) bonded to the hydroxyl group at position 6 of the galactose residue or N-acetylglucosamine residue of LNTII. (1)O-(N-Acetyl-α-neuraminosyl)-(2→6)-O-2-(acetylamino)-2-deoxy-β-D-glucopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→4)-D-glucose(CAS No.130457-84-0) (2)O-2-(Acetylamino)-2-deoxy-β-D-glucopyranosyl-(1→3)-O-[N-acetyl-α-neuraminosyl-(2→6)]-O-β-D-galactopyranosyl-(1→4)-D-glucose(CAS No.1799312-77-8) In this specification, the sialyllacto-N-neotetraose c isomer is a structural isomer of LST-c, having a structure in which N-acetylneuraminic acid is α(2-6) bonded to the hydroxyl group at position 6 of the non-terminal galactose residue or N-acetylglucosamine residue of LNnT, and is represented by the following formula (1) or (2). (1)O-(N-Acetyl-α-neuraminosyl)-(2→6)-O-[β-D-galactopyranosyl-(1→4)]-O-2-(acetylamino)-2-deoxy-β-D-glucopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→4)-D-glucose (2)O-β-D-galactopyranosyl-(1→4)-O-2-(acetylamino)-2-deoxy-β-D-glucopyranosyl-(1→3)-O-[N-Acetyl-α-neuraminosyl)-(2→6)]-O-β-D-galactopyranosyl-(1→4)-D-glucose 6'-Sialyl lactose (6SL, Neu5Ac(α2-6)Gal(β1-4)Glc) is an HMO having a structure in which N-acetylneuraminic acid is α(2-6) linked to galactose.

[0014] <Protein> The inventors have found that a protein represented by at least one selected from the group consisting of [1] to [3] below has α2,6-sialyltransferase activity toward lacto-N-neotetraose (LNnT).

[0015] The protein in this embodiment is a protein represented by at least one selected from the group consisting of [1] to [3] below. [1] A protein containing the amino acid sequence shown in one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having an amino acid sequence represented by any one of sequence numbers 5, 9, 20, 26, 28, or 30, wherein the amino acid sequence contains 1 to 30 amino acids that have been deleted, substituted, inserted, or added, and which has α2,6-sialyltransferase activity. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity. The protein of this embodiment further comprises a protein represented by at least one selected from the group consisting of [4] to [6] below. [4] Proteins containing the amino acid sequence shown in one of the following sequence numbers: 5, 9, 20, 26, 28, 30, 43, 45, 47, 49, or 51. [5] A protein having an amino acid sequence represented by any one of sequence numbers 5, 9, 20, 26, 28, 30, 43, 45, 47, 49, or 51, wherein the amino acid sequence contains 1 to 30 amino acids that are deleted, substituted, inserted, or added, and the protein has α2,6-sialyltransferase activity. [6] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, 30, 43, 45, 47, 49, or 51, and which has α2,6-sialyltransferase activity.

[0016] The protein in this embodiment may be a protein represented by at least one selected from the group consisting of [1] to [6] below. [1] A protein consisting of the amino acid sequence shown in one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added, and which has α2,6-sialyltransferase activity, in which the amino acid sequence is represented by any one of sequence numbers 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity. [4] A protein consisting of one of the amino acid sequences shown in sequence numbers 5, 9, 20, 26, 28, 30, 43, 45, 47, 49, or 51. [5] A protein comprising an amino acid sequence represented by any one of sequence numbers 5, 9, 20, 26, 28, 30, 43, 45, 47, 49, or 51, wherein 1 to 30 amino acids are deleted, substituted, inserted, or added, and the protein has α2,6-sialyltransferase activity. [6] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, 30, 43, 45, 47, 49, or 51, and which has α2,6-sialyltransferase activity.

[0017] In this specification, α2,6-sialyltransferase activity refers to the activity of transferring an N-acetylneuraminic acid residue from cytidine 5'-phosphate-N-acetylneuraminic acid (CMP-N-acetylneuraminic acid) to the hydroxyl group at position 6 of the terminal galactose of the receptor substrate LNnT via an α2,6 bond. Examples of proteins possessing α2,6-sialyltransferase activity include sialyltransferases. While the microorganisms from which sialyltransferases originate are not particularly limited, examples include Photobacterium damselae, Photobacterium phosphoreum, Photobacterium carnosum, Haemophilus haemolyticus, Shewanella piezotolerans, Shewanella sp. PS-2, Alphaproteobacteria bacterium, Helicobacter acinonychis str. Sheeba, Pasteurella multocida, Helicobacter sp. 10-6591, Helicobacter bizzozeronii, Campylobacter lari, Streptococcus suis TL13, Streptococcus suis, and Streptococcus suis 89 / 1591. suis, Streptococcus suis 89 / 1591 is preferred. The presence of α2,6-sialyltransferase activity in the protein of this embodiment can be confirmed, for example, by a method including the following steps 1) to 4). 1) First, recombinant DNA containing the DNA encoding the protein of this specification whose activity is to be confirmed is prepared by the method described below. 2) Microorganisms lacking α2,6-sialyltransferase activity, such as Escherichia coli strain W3110, are transformed with the recombinant DNA, and the resulting microorganisms are cultured. A protein preparation containing the protein or a processed product of the culture is then prepared from the resulting culture. 3) A protein preparation or culture containing the aforementioned protein is brought into contact with an aqueous solution containing the substrates CMP-N-acetylneuraminic acid and LNnT to obtain a reaction solution. 4) By detecting LST-c in the reaction solution using the analytical device described later, it is confirmed that the protein of this embodiment has α2,6-sialyltransferase activity.

[0018] In this specification, "80% or more identity" means that when the amino acid sequence represented by the specific sequence number is aligned with the amino acid sequence whose identity is to be confirmed, the identity value calculated based on the number of matching amino acid residues is 80% or more. For example, when the amino acid sequence represented by a particular sequence number consists of 100 amino acids, 80% or more identity means that the sequence may contain 80 amino acids where 80 of them are identical (80% identity), or it may contain 120 amino acids where 100 of them are identical (100% identity). It may also contain 100 amino acids where 80 of them are identical (80% identity). In this specification, when it is stated that "the amino acid sequence has 80% or more identity with the sequence number," the identity is preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The phrase "having 80% or more identity with the amino acid sequence represented by the sequence number" includes cases where there is 100% identity, so unless otherwise specified, it may be the amino acid sequence represented by the sequence number itself. If a sequence has "80% or more identity with the amino acid sequence represented by the sequence number," then 1 to 30 amino acids may be deleted, substituted, inserted, and / or added to the amino acid sequence represented by that particular sequence number.

[0019] In this embodiment, the percentage of sequence identity between two amino acid sequences or two nucleotide sequences is calculated as the ratio of matching residues when the two sequences are aligned to the extent that they are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm. Examples of such mathematical algorithms include the local homology algorithm of Smith et al (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and the improved algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. However, mathematical algorithms are not limited to the examples above. Programs based on these mathematical algorithms can be used to perform alignment to determine the percentage of sequence identity. This program can be executed by a computer as appropriate.Examples of such programs, though not limited to them, include the PC / Gene programs CLUSTAL (available from Intelligenetics, Mountain View, Calif.), MAFFT (Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002), 30(14), 3059-3066., http: / / mafft.cbrc.jp / alignment / server / ), MUSCLE (Edgar RC (2004). Nucleic acids research, 32(5), 1792-1797., http: / / www.ebi.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA, with BLAST being preferred. Alignment using these programs can be performed, for example, using initial parameters. The CLUSTAL program is described in Higgins et al. (1988) Gene 73:237-244, Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol.Biol. 24:307-331. ClustalW is available from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When creating alignments using ClustalW, parameters can be set to default values, for example. BLAST is described in Altschul, SF, Gish, W., Miller, W., Myers, EW, & Lipman, DJ (1990). 215(3), 403-410., Mount DW (2007). CSH protocols, 2007, pdb.top17., etc. Based on BLAST, programs called BLASTP and BLASTN have been developed, and the percentage of sequence identity can be calculated using these programs with default settings.

[0020] In this specification, "in the amino acid sequence represented by the SEQ ID NO: 1 to 30 amino acids are deleted, substituted, inserted, and / or added" means that amino acids are deleted, substituted, inserted, and / or added at the terminal or non-terminal end of the amino acid sequence represented by the SEQ ID NO: 1. The number of amino acids deleted, substituted, inserted, and / or added is not particularly limited as long as the resulting protein exhibits the effects of the present invention. It may be 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, or 3 or less amino acids, and it may be 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids. Preferably, it is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably about 1, 2, 3, 4, or 5. Furthermore, the sites that are deleted, replaced, inserted, and / or added may be one or more. "Amino acid deletion" means the absence or disappearance of an amino acid residue in a sequence; "amino acid substitution" means that an amino acid residue in a sequence is replaced by another amino acid residue; "amino acid insertion" means that a new amino acid residue is inserted into a sequence; and "amino acid addition" means that a new amino acid residue is added to a sequence in such a way that it is inserted. Specific examples of "1 to 30 amino acids being deleted, substituted, inserted, and / or added" include cases where 1 to 30 amino acids are replaced with other chemically similar amino acids. For example, this could involve substituting one hydrophobic amino acid with another hydrophobic amino acid, or substituting one polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known in the relevant art for each amino acid. Specific examples of nonpolar (hydrophobic) amino acids include alanine, valine, glycine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid. The amino acids that are substituted, inserted, or added may be native or non-native amino acids. Examples of native amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine. Examples of mutually substituted amino acids are shown below. Amino acids belonging to the same group may be substituted for each other. Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic acid, Methionine, o-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: proline, 3-hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

[0021] In this specification, that some amino acid residues of an amino acid sequence represented by a specific SEQ ID are substituted with other amino acid residues can be confirmed, for example, by aligning the amino acid sequence represented by the specific SEQ ID with the amino acid sequence to be confirmed which has been substituted with other amino acid residues.

[0022] Alignment of amino acid sequences can be performed using the above-mentioned known programs. These proteins may be produced using naturally occurring microorganisms or the genetically modified cells described below, or may be produced using a cell-free protein synthesis system.

[0023] <Method for producing LST-c> The method for producing LST-c in this embodiment includes the method described below. A method for producing sialyllacto-N-neotetraose c (LST-c) including a step of producing sialyllacto-N-neotetraose c (LST-c) from lacto-N-neotetraose (LNnT) in the presence of at least one protein selected from the group consisting of the following [1] to [3]; [1] A protein containing an amino acid sequence represented by any one of SEQ ID NO: 5, 9, 20, 26, 28, 30. [2] A protein containing an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by any one of SEQ ID NO: 5, 9, 20, 26, 28, 30, and having α2,6-sialyltransferase activity. [3] A protein containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by any one of SEQ ID NO: 5, 9, 20, 26, 28, 30, and having α2,6-sialyltransferase activity. In this embodiment, the method for producing LST-c refers to a method for producing LST-c by transferring an N-acetylneuraminic acid residue from a donor substrate, CMP-N-acetylneuraminic acid, to a receptor substrate, LNnT, using, for example, a protein represented by at least one selected from the group consisting of [1] to [3] above. The protein used in this embodiment may be obtained from naturally occurring microorganisms or genetically modified cells (hereinafter collectively referred to as genetically modified cells, etc.), or it may be obtained from a cell-free protein synthesis system. Furthermore, the protein used in this embodiment may be present inside the genetically modified cells, etc., or it may be extracted from the genetically modified cells, etc., and purified as necessary. The manufacturing method of this embodiment includes the method described below. (I) further comprising the step of producing a protein represented by at least one selected from the group consisting of [1] to [3] above in genetically modified cells, and / or, (II) The above method for producing LST-c in an aqueous medium containing a protein represented by at least one selected from the group consisting of [1] to [3] above, CMP-N-acetylneuraminic acid, and LNnT. In the manufacturing method of this embodiment, both or either of the donor substrate CMP-N-acetylneuraminic acid and the receptor substrate LNnT may be added, or another cell capable of producing both or either of CMP-N-acetylneuraminic acid and the receptor substrate LNnT may be cultured together with the genetically modified cells described later. Furthermore, the manufacturing methods of this embodiment include (i) fermentation, (ii) enzymatic methods, and (iii) microbial reaction methods. (I) above includes fermentation, enzymatic methods, and microbial reaction methods, and (II) above includes enzymatic methods and microbial reaction methods.

[0024] (i) Fermentation method One fermentation method involves reacting the aforementioned proteins with CMP-N-acetylneuraminic acid and LNnT inside the genetically modified cells described later, thereby producing LST-c in the culture of the genetically modified cells.

[0025] If the genetically modified cells in this embodiment do not have the ability to produce CMP-N-acetylneuraminic acid, CMP-N-acetylneuraminic acid may be added to the culture medium, or the cells may be cultured together with other cells that have the ability to produce CMP-N-acetylneuraminic acid. Alternatively, substrates of CMP-N-acetylneuraminic acid such as SIA and cytidine 5'-triphosphate (CTP) may be added to the culture medium, or the cells may be cultured together with other cells that have the ability to produce SIA, CTP, etc. If the genetically modified cells in this embodiment do not have the ability to produce LNnT, LNnT may be added to the culture medium, or they may be cultured together with other cells that have the ability to produce LNnT.

[0026] The method for culturing genetically modified cells in this embodiment can be carried out according to the usual methods used for cell culture.

[0027] As a culture medium for genetically modified cells, either a natural medium or a synthetic medium may be used, as long as it contains a carbon source, nitrogen source, and / or inorganic salts that the genetically modified cells can utilize, and is capable of efficiently culturing the genetically modified cells.

[0028] Any carbon source that can be utilized by genetically modified cells is acceptable, and examples include glucose, fructose, sucrose, lactose, molasses containing these, sugars such as starch or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol or propanol.

[0029] Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate or ammonium phosphate, other inorganic acids, ammonium salts of organic acids, other nitrogen-containing compounds, or peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation microorganisms or their digests.

[0030] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, or calcium carbonate.

[0031] Furthermore, it is preferable to add lactose to the culture medium as a substrate for LNnT production.

[0032] Culturing is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is usually 15 to 40°C, and the culture time is usually 5 hours to 7 days. The pH of the culture medium during cultivation is usually maintained at 3.0 to 9.0. pH adjustment is performed using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0033] Furthermore, antibiotics such as ampicillin or tetracycline may be added to the culture medium as needed during cultivation. When culturing microorganisms transformed with an expression vector using an inducible promoter, an inducer may be added to the culture medium as needed. For example, when culturing cells transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) may be added to the culture medium, and when culturing cells transformed with an expression vector using a trp promoter, indole-acrylic acid may be added to the culture medium.

[0034] By culturing as described above, LST-c is generated and accumulated in the culture, and sialyllacto-N-neotetraose c can be produced by collecting LST-c from the culture.

[0035] The obtained LST-c can be analyzed by conventional analytical methods using glycans, such as ion exchange chromatography or gel filtration chromatography. Sialyllacto-N-neotetraose c can be collected or purified from the culture by conventional methods using filtration, activated carbon, and / or ion exchange resin. If sialyllacto-N-neotetraose c accumulates inside the cells, LST-c can be collected by, for example, disrupting the cells by sonication or drug treatment, removing cellular components by centrifugation, and purifying the supernatant using activated carbon or ion exchange resin.

[0036] <Genetically modified cells> The genetically modified cells in this embodiment are cells that have been genetically modified to have at least one protein selected from the group consisting of [1] to [3] below. [1] A protein containing the amino acid sequence shown in one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, which includes an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity. The genetically modified cells in this embodiment have the ability to produce the protein by containing the DNA encoding the protein of this embodiment described above. The genetically modified cells in this embodiment can be obtained, for example, by transforming a host cell with recombinant DNA containing the DNA encoding the protein of this embodiment and its expression units. The DNA encoding the protein of this embodiment can be obtained, for example, by PCR using the genomic DNA of a microorganism having the DNA encoding the protein as a template. The transformation method is not particularly limited, and conventionally known methods can be used. The DNA encoding the proteins described in [2] or [3] above can be obtained, for example, by introducing a mutation into the DNA encoding a protein containing one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30 using site-directed mutagenesis methods described in Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press (2012)) and Current Protocols in Molecular Biology (JOHN WILEY & SONS, INC.), and substituting it with a base sequence encoding another amino acid residue. The DNA encoding the protein described in [2] or [3] above can also be obtained by subjecting DNA encoding a protein containing the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30 to error-prone PCR or the like as a template. Alternatively, DNA encoding the proteins described in [2] or [3] above can also be obtained by a partially specific mutagenesis method using PCR [Gene, 77, 51 (1989)], which uses a set of PCR primers having a nucleotide sequence designed to introduce the desired mutation (deletion, substitution, insertion, and / or addition) at each 5' end. Alternatively, the DNA can be obtained by following the instructions included with commercially available partially specific mutagenesis kits. Specifically, first, a plasmid containing a nucleotide sequence designed to introduce the target mutation (deletion, substitution, insertion, or addition) is used as a template to design a pair of mutation-introducing primers with a 15-base overlap at the 5' end. The overlapping portion contains the target mutation. Next, PCR is performed using these mutation-introducing primers on a plasmid containing the nucleotide sequence into which the target mutation is to be introduced. This yields a plasmid containing the nucleotide sequence into which the target mutation has been introduced.

[0037] In this specification, “expression unit” means the smallest unit that enables the transcription of a given DNA to be expressed as a protein and, consequently, the production of the protein encoded by that DNA, including a promoter operably linked to it. An expression unit may further include elements such as a terminator, a ribosome binding site, and a drug resistance gene.

[0038] In this embodiment, the host cell may be any of the following: prokaryotes, yeast, animal cells, insect cells, plant cells, etc., but preferably a prokaryote or yeast strain, more preferably a prokaryote belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genera Saccharomyces, Schizosaccharomyces, Kluiveromyces, Trichosporon, Siwaniomyces, Pitia, or Candida, and even more preferably Escherichia coli [for example, Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (manufactured by Merck Millipore), Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm-, Escherichia coli JM109, Escherichia coliHB101, Escherichia coliCJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio Inc.), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli ATCC9637, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillussubtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum [e.g. Brevibacterium immariophilum ATCC14068], Brevibacterium saccharolyticum [e.g. Brevibacterium saccharolyticum ATCC14066], Corynebacterium ammoniagenes, Corynebacterium glutamicum [Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869], Corynebacterium acetoacidophilum [Corynebacterium acetoacidophilum ATCC13870], Microbacteriummmoniaphilum [Microbacterium ammoniaphilum Examples include prokaryotes such as ATCC15354 or Pseudomonas (e.g., Pseudomonas sp.D-0110), or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis, with Escherichia coli being the most preferred.

[0039] The promoter constituting the expression unit is not particularly limited as long as it can express the protein encoded by the polynucleotide linked downstream in the host cell. For example, the promoter may be homogeneous or heterogeneous with respect to the host cell. For example, a promoter commonly used for recombinant protein production or an inducible promoter can be used. Examples of such promoters include the PhoA promoter, PhoC promoter, T7 promoter, T5 promoter, T3 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter, PL promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, and tetracycline-inducible promoter. Preferably, a promoter that has potent transcriptional activity in the host cell can be used. Examples of promoters that have potent transcriptional activity in the host cell include promoters of genes that are highly expressed in the host cell and virus-derived promoters. When the host cell is a microorganism belonging to the genus Escherichia, promoters derived from Escherichia coli or phages, such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, PSE promoter, ilv promoter, and uspA promoter, can be used. Additionally, artificially designed and modified promoters, such as two trp promoters in series, the tac promoter, the trc promoter, the lacT5 promoter, the lacT7 promoter, and the let I promoter, can also be used.

[0040] Genetically modified cells can be obtained by transforming the host cells described above with recombinant DNA containing the DNA encoding the protein of this embodiment and its expression units. The number of copies of the DNA incorporated into the host cell by transformation may be only one copy, or it may be two copies or more.

[0041] The genetically modified cells in this embodiment can be produced by any method known in the art. For example, the expression units described above are contained in the host cell either in a form integrated into the host cell's genomic DNA or in a form not integrated into the host cell's genomic DNA (for example, in a form where the expression vector exists independently of the host cell's genomic DNA). Host cells containing the expression units can be obtained by transforming the host cell with the expression vector using any method known in the art (for example, the competent cell method or the electroporation method).

[0042] If the expression vector is an integrative vector that undergoes homologous recombination with the host cell's genomic DNA, the expression units can be incorporated into the host cell's genomic DNA through transformation. On the other hand, if the expression vector is a non-integrative vector that does not undergo homologous recombination with the host cell's genomic DNA, the expression units are not incorporated into the host cell's genomic DNA through transformation and can exist independently of the genomic DNA within the host cell in the state of the expression vector. Alternatively, the expression units can be incorporated into the host cell's genomic DNA using genome editing technology [e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)].

[0043] The expression vectors in this disclosure may further include, in addition to the minimum units described above, elements such as terminators that function in host cells, ribosome binding sites, and drug resistance genes. Examples of drug resistance genes include resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0044] The expression vector may also further include a region that enables homologous recombination with the host cell's genome for homologous recombination with the host cell's genomic DNA. For example, the expression vector may be designed so that the expression units contained therein are located between a pair of homologous regions (e.g., homology arms, loxP, FRT homologous to a specific sequence in the host cell's genome). The genomic region of the host cell to which the expression units should be introduced (the target of the homologous region) is not particularly limited, but may be a locus of a gene that is highly expressed in the host cell. Alternatively, the target of the homologous region may be a suitable nucleotide sequence on the chromosome, such as a gene that is not necessary for the production of the target substance, LST-c. Furthermore, the gene can also be randomly introduced onto the chromosome using a transposon or Mini-Mu (Japanese Patent Publication No. 2-109985, US5,882,888, EP805867B1). It should be noted that this chromosome modification method using homologous recombination is not limited to the introduction of target genes, but can be used for any modification of the chromosome, such as modification of expression regulatory sequences.

[0045] Expression vectors may be plasmids, viral vectors, phages, or artificial chromosomes. Expression vectors may also be integrative or non-integrative vectors. Integrative vectors may be vectors in which the entire vector is integrated into the host cell's genome. Alternatively, integrative vectors may be vectors in which only a portion of the vector (e.g., expression units) is integrated into the host cell's genome.

[0046] The expression vector may also be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may also be a commonly used expression vector. Examples of such expression vectors include pMAL (e.g., pMALcx, pMALcx2), pET (e.g., pET-His6-MBP-TEV-LIC), pCDFDuet-1, pTrc99A, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), and pQE (e.g., pQE30), as well as their derivatives.

[0047] When selecting microbial host cells belonging to the genus Escherichia, the following expression vectors can be used: pColdI, pSTV28, pUC118 (all from Takara Bio), pET21a, pCDF-1b, pRSF-1b (all from Merck Millipore), pMAL-c5x (from New England Biolabs), pGEX-4T-1, pTrc99A (all from GE Healthcare Biosciences), pTrcHis, pSE280 (all from Thermo Fisher Scientific), pGEMEX-1 (from Promega), pQE-30, pQE-60, pQE80L (all from Qiagen), pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (both manufactured by Agilent Technologies), pKYP10 (Japanese Patent Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL Examples include MICROBIOLOGY, 2007, Vol.73, No.20, p6378-6385, pPE167 (Appl. Environ. Microbiol. 2007, 73:6378-6385), pPAC31 (International Publication No. 1998 / 12343), pUC19 [Gene, 33, 103 (1985)], and pPA1 (Japanese Patent Publication No. 63-233798). When selecting Corynebacterium glutamicum as the host cell, examples of expression vectors include high-copy vectors such as pPK4, pCS299P, and pRI109.

[0048] The recombinant DNA in this embodiment can be prepared, for example, by restricting the preparation of a DNA fragment and inserting it downstream of the promoter of a suitable expression vector. If the recombinant DNA is capable of being incorporated into a chromosome, it does not need to have a promoter.

[0049] The expression level of the protein encoded by the DNA of this embodiment can also be improved by substituting bases in the DNA sequence to create codons that are optimal for expression in the host cell. Information on codon usage frequency in host cells can be obtained through public databases.

[0050] In this embodiment, the genetically modified cells preferably have the ability to produce CMP-N-acetylneuraminic acid and / or LNnT in addition to the proteins of this embodiment. If the host cells do not have the ability to produce CMP-N-acetylneuraminic acid and / or LNnT, or if the ability is insufficient, the genetically modified cells can be created by artificially conferring or enhancing this ability.

[0051] Methods for artificially conferring or enhancing the ability of host cells to produce CMP-N-acetylneuraminic acid include (a) methods for relaxing or releasing at least one mechanism that controls the CMP-N-acetylneuraminic acid biosynthesis pathway, (b) methods for enhancing the expression of at least one protein involved in the CMP-N-acetylneuraminic acid biosynthesis pathway, (c) methods for increasing the copy number of at least one gene encoding a protein involved in the CMP-N-acetylneuraminic acid biosynthesis pathway, and (d) methods for weakening or blocking at least one metabolic pathway that branches off from the CMP-N-acetylneuraminic acid biosynthesis pathway to metabolites other than the target substance. The known methods can be used individually or in combination.

[0052] Specific examples of proteins involved in the biosynthesis pathway of CMP-N-acetylneuraminic acid include known enzymes such as glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase (NeuC), N-acetylneuraminic acid synthase (NeuB), and CMP-N-acetylneuraminic acid synthetase (NeuA), glucokinase (glk), glucose phosphate isomerase (pgi), glucosamine-6-phosphate synthase (glmS), phosphoglucosamine mutase (glmM), and N-acetylglucosamine-1-phosphate uridyltransferase (glmU). Proteins involved in the biosynthesis pathway of CMP-N-acetylneuraminic acid include, preferably, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine 2-epimerase, NeuB and NeuA, or NeuC, NeuB and NeuA, and more preferably NeuC, NeuB and NeuA.

[0053] One example of a metabolic pathway that branches off from the CMP-N-acetylneuraminic acid biosynthesis pathway to metabolites other than the target substance is the N-acetylneuraminic acid metabolic pathway. Proteins involved in the N-acetylneuraminic acid metabolic pathway include NanR (DNA-binding transcriptional dual regulator), NanA (DNA-binding transcriptional dual regulatorDNA), NanT (sialic acid transporter), NanE (N-acetylglucosamine 6-phosphate epilerase), and NanK (N-acetylglucosamine kinase). Glucosamine-6-phosphate deaminase (nagB) is another protein involved in the metabolic pathway that branches off from the CMP-N-acetylneuraminic acid biosynthesis pathway to metabolites other than the target substance.

[0054] Other proteins that are preferable to be weakened or deficient in host cells include PTS transporters encoded by genes such as manXYZ and nagE.

[0055] Methods for artificially conferring or enhancing the ability of host cells to produce LNnT include (a) relaxing or releasing at least one mechanism that controls the biosynthetic pathway that generates LNnT, (b) enhancing the expression of at least one protein involved in the LNnT biosynthetic pathway, (c) increasing the copy number of at least one gene encoding a protein involved in the LNnT biosynthetic pathway, and (d) weakening or blocking at least one metabolic pathway that branches off from the LNnT biosynthetic pathway to metabolites other than the target substance. The known methods can be used individually or in combination.

[0056] Specific examples of proteins involved in the LNnT biosynthesis pathway include, for example, known enzymes such as glucokinase (glk), phosphoglucocomutase (pgm), UDP-glucose pyrophosphorylase (galU), galF, UDP-galactose-4-epimerase (galE), glucose phosphate isomerase (pgi), glucosamine-6-phosphate synthase (glmS), glucosamine-6-phosphate deaminase (nagB), phosphoglucosamine mutase (glmM), and N-acetylglucosamine-1-phosphate uridyltransferase (glmU), β1,3-N-acetylglucosamine transferase (LgtA), and β1,4-galactosyltransferase (GalT). Preferably, LgtA and GalT are included as proteins involved in the LNnT biosynthesis pathway.

[0057] The genetically modified cells in this embodiment may be prepared, for example, by introducing DNA encoding at least one protein selected from the group consisting of LgtA, GalT, and proteins involved in the biosynthesis pathway of CMP-N-acetylneuraminic acid into Escherichia coli, thereby conferring the ability to produce CMP-N-acetylneuraminic acid and LNnT. Alternatively, the genetically modified cells in this embodiment may be prepared, for example, by introducing DNA encoding at least one protein selected from the group consisting of LgtA, GalT, NeuC, NeuB, and NeuA into Escherichia coli, thereby conferring the ability to produce CMP-N-acetylneuraminic acid and LNnT. More specifically, the genetically modified cells in this embodiment may be prepared by introducing DNA encoding at least one protein selected from the group consisting of LgtA (NpLgtA) from Neisseria polysaccharea ATCC43768, GalT (HpGalT) from Helicobacter pylori, NeuC (FpNeuC) from Flavobacterium psychrophilum, NeuB (CjNeuB) from Campylobacter jenuniRhizobium capsulatus, and NeuA (PmNeuA) from Pasteurella multocida into Escherichia coli as a host cell, thereby conferring the ability to produce CMP-N-acetylneuraminic acid and LNnT. In other words, the genetically modified cells of this embodiment may have at least one protein selected from the group consisting of LgtA, GalT, and proteins involved in the biosynthesis pathway of CMP-N-acetylneuraminic acid, and at least one protein represented by the group consisting of [1] to [3] above. Alternatively, the genetically modified cells of this embodiment may have at least one protein selected from the group consisting of NpLgtA, HpGalT, FpNeuC, CjNeuB, and PmNeuA, and at least one protein represented by the group consisting of [1] to [3] above.

[0058] (ii) Enzymatic method, bacterial cell reaction method Enzymatic or cellular reaction methods include a method comprising reacting CMP-N-acetylneuraminic acid and LNnT with at least one protein selected from the group consisting of [1] to [3] above in an aqueous medium to produce LST-c. The protein used in the enzymatic method may be unpurified, crudely purified, or purified. Specifically, a method for producing LST-c by an enzymatic method or a microbial reaction method includes placing a protein produced by a cell-free protein synthesis system, or a protein contained in a culture of genetically modified cells or a protein preparation which is a processed product of said culture, along with CMP-N-acetylneuraminic acid and LNnT, in an aqueous medium to generate LST-c in the aqueous medium. The production method may also include, for example, generating LST-c in an aqueous medium, accumulating it, and then collecting or purifying the LST-c from the aqueous medium.

[0059] CMP-N-acetylneuraminic acid and LNnT may be derived from any substrate that serves as a substrate for the aforementioned proteins, but a culture or processed product of a microorganism capable of producing CMP-N-acetylneuraminic acid and / or LNnT may be used as is, or CMP-N-acetylneuraminic acid and / or LNnT collected from said culture or processed product may be used.

[0060] Examples of processed products of the culture include concentrated culture, dried culture, cells obtained by centrifugation of the culture, dried cells, freeze-dried cells, cells treated with surfactants, cells treated with sonication, cells treated with mechanical grinding, cells treated with solvents, cells treated with enzymes, protein fractions of cells, or immobilized cells or isolated and purified enzymes obtained by extraction from cells. Among these, sonicated cells, solvent-treated cells, and isolated and purified enzymes obtained by extraction from cells are preferred.

[0061] Of these methods, the method of producing LST-c using a treated material containing bacteria and in which the cell plasma membrane of the bacteria has been made permeable to substances is specifically called the bacterial cell reaction method. A cell plasma membrane being permeable to substances means that various molecules, both small (ions, etc.) and large (proteins, etc.), can freely enter and exit the cell membrane by diffusion. The bacteria used in the bacterial cell reaction method are preferably dormant cells that have lost their ability to proliferate due to treatment to impart membrane permeability.

[0062] It is preferable that the treatment for imparting membrane permeability allows for the incorporation of CMP-N-acetylneuraminic acid and LNnT into the bacterial cells without completely disrupting them, while also maintaining the aforementioned proteins produced by the bacterial cells.

[0063] Treatments to make the bacterial cell plasma membrane permeable to substances include surfactant treatment, solvent treatment, freeze-thaw treatment, enzyme treatment, sonication treatment, and mechanical grinding treatment. As surfactants, nonionic surfactants are preferred because they have a milder effect on proteins, etc. (compared to ionic surfactants). Examples of surfactants include digitonin, saponin, TritonX100 (hereinafter, Triton is a registered trademark), TritonX114, Tween20 (hereinafter, Tween is a registered trademark), Tween80, N,N-Bis(3-D-gluconamidopropyl)cholamide [BIGCHAP], N,N-Bis(3-D-gluconamidopropyl)deoxycholamide [Deoxy-BIGCHAP], NIKKOLBL-9EX [Polyoxyethylene(9)LaurylEther], Octanoyl-N-methylglucamide [MEGA-8], and benzalkonium chloride. Examples of organic solvents include Benzene, Toruene, Xylene, and other alcohols. Examples of enzymes include lysozyme and achromopeptidase. The concentration, temperature, time, and other conditions for treatment with the above substances vary depending on the cell type, and appropriate conditions must be set to perform the desired analysis. However, typical treatment concentrations are 10 to 1000 μg / ml, more commonly 20 to 200 μg / ml, with temperatures of 2 to 37°C and durations of 1 to 30 minutes. Examples of mechanical treatments include ultrasonic treatment and mechanical grinding.

[0064] When using purified enzymes isolated and purified as protein preparations, the purified enzymes can be obtained using the genetically modified cells described above, but the following method is more preferable, for example.

[0065] Recombinant DNA is prepared, which includes DNA encoding a protein represented by at least one selected from [1] to [3] above, and a tag sequence for protein purification. Next, genetically modified cells obtained by transforming with the recombinant DNA are cultured, and the protein is prepared from the resulting culture as a purified enzyme.

[0066] The tag sequences for protein purification described above are documented in the literature and are known to those skilled in the art. Examples include sequences that can be used to purify (affinity) polypeptide chains (e.g., see Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purification. Curr Protoc Protein Sci. 2013;73:9.9.1-9.9.23. Published 2013 Sep 24. doi:10.1002 / 0471140864.ps0909s73), calmodulin-binding peptides, His tags such as 6His tags, and / or maltose-binding protein sequences.

[0067] For example, a His tag (polyhistidine tag) is a polyhistidine amino acid motif in proteins that typically consists of at least six histidine (His) residues and is often located at the N-terminus or C-terminus of a protein. Polyhistidine tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli and other prokaryotic expression systems by incubation with affinity resins containing bound divalent nickel or cobalt ions, which are commercially available in various types.

[0068] These resins are generally Sepharose / agarose functionalized with chelating agents such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel, and carboxymethyl aspartic acid (Co-CMA) for cobalt, to which polyhistidine tags are bound with micromolar affinity. The resins are then typically washed with phosphate buffer to remove proteins that do not specifically interact with cobalt or nickel ions. In the case of Ni-based methods, washing efficiency can be improved by adding 20 mM imidazole (proteins are usually eluted with 150-300 mM imidazole).

[0069] Examples of the aqueous medium include water, buffers such as phosphates, carbonates, acetates, borates, citrates, or Tris, alcohols such as methanol or ethanol, esters such as ethyl acetate, ketones such as acetone, or amides such as acetamide. Further, for example, a medium for culturing a genetically modified microorganism used for the production of a protein preparation is mentioned as an aqueous medium.

[0070] The method for analyzing, collecting, or purifying LST-c produced in an aqueous medium is the same as the fermentation method. From the above, the production method of this embodiment includes the methods described below. The above production method for producing LST-c in the presence of at least one protein selected from the group consisting of β-1,3-N-acetylglucosaminyltransferase (LgtA), β-1,4-galactosyltransferase (GalT), and proteins involved in the biosynthetic pathway of cytidine 5'-phosphate N-acetylneuraminic acid, and at least one protein represented by at least one selected from the group consisting of the above [1] to [3]. The above production method for producing LST-c in the presence of at least one protein selected from the group consisting of LgtA derived from Neisseria polysaccharea ATCC43768 (NpLgtA, SEQ ID NO: 34), GalT derived from Helicobacter pylori (HpGalT, SEQ ID NO: 32), UDP-N-acetylglucosamine 2-epimerase (FpNeuC, SEQ ID NO: 37) derived from Flavobacterium psychrophilum, N-acetylneuraminic acid synthase (CjNeuB, SEQ ID NO: 39) derived from Campylobacter jenuni, and cytidine 5'-phosphate-N-acetylneuraminic acid synthase (PmNeuA, SEQ ID NO: 41) derived from Pasteurella multocida, and at least one protein represented by at least one selected from the group consisting of the above [1] to [3].

[0071] <Method for Suppressing By-products in the Production of LST-c> This embodiment further includes a method for suppressing the production of at least one selected from the group consisting of 6'-sialyl lactose, sialyl lacto-N-neotetraose c, and sialyl lacto-N-neotetraose c isomers in the production of sialyl lacto-N-neotetraose c using a protein represented by at least one selected from the group consisting of [1] to [3] above. In this embodiment, "suppressing the generation" of the above-mentioned sugars means suppressing the generation of at least one selected from the group consisting of 6'-sialyl lactose, sialyl lacto-N-triose II, and sialyl lacto-N-neotetraose c isomer, and relatively reducing the amount of at least one selected from the group consisting of 6'-sialyl lactose, sialyl lacto-N-triose II, and sialyl lacto-N-neotetraose c isomer in the product. Specifically, the use of at least one protein selected from the group consisting of [1] to [3] above results in a reduced production of at least one selected from the group consisting of 6'-sialyl lactose, sialyl lacto-N-triose II, and sialyl lacto-N-neotetraose c isomers compared to the use of a known α2,6 sialyltransferase (e.g., PdsiaT△N15△C176 (Crystal structures of sialyltransferase from Photobacterium damselae - Huynh - 2014 - FEBS Letters - Wiley Online Library) (SEQ ID NO: 1)). In the method of this embodiment, compared to the case in which PdsiaT△N15△C176 is used, the ratio of 6'-sialyl lactose to sialyl lactose-N-neotetraose c contained in the solution after the enzymatic reaction or bacterial reaction, or in the culture of genetically modified cells or the processed product of said culture (hereinafter referred to as "culture, etc.") is 50% or less, 40% or less, 30% or less, 20% or less, 17% or less, 12% or less, 7% or less, 5% or less, or 2% or less. Furthermore, the method of this embodiment is preferable to the case where PdsiaT△N15△C176 is used, in which the ratio of LST-c structural isomers to sialyllacto-N-neotetraose c contained in the culture is 50% or less, 40% or less, 30% or less, 20% or less, 17% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 1% or less, 0.5% or less, 0.1% or less, and 0%.Furthermore, the method of this embodiment preferably has a ratio of sialylated lacto-N-triose II to sialyl lacto-N-neotetraose c contained in the culture, etc., of 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, and 5% or less, compared to the case where PdsiaT△N15△C176 is used. Furthermore, in this embodiment, "suppressing the production" of the above-mentioned sugars may mean that, by using a protein represented by at least one selected from the group consisting of [1] to [3] above, the content of at least one selected from the group consisting of 6'-sialyl lactose, sialylated lacto-N-triose II, and sialyl lacto-N-neotetraose c isomers relative to the content of sialyl lacto-N-neotetraose c in the product is below a certain value. Preferably, in the method of this embodiment, the ratio of 6'-sialyl lactose to sialyl lacto-N-neotetraose c contained in the culture is 800% or less, 700% or less, 600% or less, 500% or less, 400% or less, 300% or less, 200% or less, 150% or less, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, and 12% or less. In the method of this embodiment, the abundance ratio of LST-c structural isomers to sialyllacto-N-neotetraose c contained in the culture is preferably 20% or less, 15% or less, 10% or less, 7% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.7% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, and 0%. In the method of this embodiment, the abundance ratio of sialyl lacto-N-triose II to sialyllacto-N-neotetraose c contained in the culture is preferably 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.7% or less, 0.5% or less, 0.4% or less, and 0.3% or less. This embodiment preferably involves a method that suppresses the formation of at least two isomers selected from the group consisting of 6'-sialyl lactose, sialyl lacto-N-triose II, and sialyl lacto-N-neotetraose c isomers, and more preferably involves a method that suppresses the formation of all three. The method of this embodiment preferably includes the step of producing a protein represented by at least one selected from the group consisting of [1] to [3] above using genetically modified cells, and is a method for suppressing the production of at least one selected from the group consisting of 6'-sialyl lactose, sialyl lacto-N-triose II, and sialyl lacto-N-neotetraose c isomer in the culture of the genetically modified cells. In this embodiment, the suppression of the above-mentioned sugar production can be confirmed, for example, by analyzing the content of sialyl lacto-N-neotetraose c and at least one selected from the group consisting of 6'-sialyl lactose, siallylated lacto-N-triose II, and sialyl lacto-N-neotetraose c isomers in the product, according to the analysis example described later.

[0072] <Culture of genetically modified cells> This embodiment includes a culture of genetically modified cells containing sialcrit-N-neotetraose c, wherein the 6'SL ratio to sialyllacto-N-neotetraose c (e.g., HPLC peak intensity ratio, hereafter the same) is 800% or less, the sialyllacto-N-neotetraose c isomer ratio to sialyllacto-N-neotetraose c is 20% or less, and the sialylated lacto-N-triose II ratio to sialyllacto-N-neotetraose c is 6% or less. In this embodiment, the culture preferably has a 6'SL abundance ratio to sialyllacto-N-neotetraose c of 800% or less, 700% or less, 600% or less, 500% or less, 400% or less, 300% or less, 200% or less, 150% or less, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, and 12% or less. In addition, in this embodiment, the culture preferably has a LST-c structural isomer abundance ratio to sialyllacto-N-neotetraose c of 20% or less, 15% or less, 10% or less, 7% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.7% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, and 0%. Furthermore, the culture of this embodiment preferably has a ratio of sialylated lacto-N-triose II to sialyl lacto-N-neotetraose c of 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.7% or less, 0.5% or less, 0.4% or less, and 0.3% or less. The fact that the content of each sugar relative to the content of sialyllacto-N-neotetraose c in the culture of this embodiment is the value described above can be confirmed, for example, by analyzing the content of sialyllacto-N-neotetraose c and at least one selected from the group consisting of 6'-sialyl lactose, sialyl lacto-N-triose II, and sialyllacto-N-neotetraose c isomers in the culture, according to the analysis example described later. [Examples]

[0073] Examples of the present invention are shown below, but the present invention is not limited to these examples.

[0074] [Example of analysis] In the examples, the analysis of 6SL, sialylated LNTII, LST-c, and LST-c structural isomers was performed according to the following procedure. The supernatant obtained by centrifugation of the enzyme reaction solution, or the culture medium obtained by heat treatment at 70°C for 40 minutes after stopping the culture and extracting the intracellular fraction, was analyzed by LC-MS, a type of high-performance liquid chromatography (HPLC). [Analysis conditions 1] LC-MS SIM analysis Analyzer: LC-MS 8050 (SIMADZU) Column: HILICpakVN-50 4D (SHODEX) Column temperature: 60℃ Analysis time: 37 minutes Mobile phase: (Mobile phase A) Acetonitrile (Mobile phase B) 25 mM ammonium formate Gradient method: The mixing ratio of mobile phase A and mobile phase B is as follows: a gradient of 90:10 to 72:28 from 0 to 24 minutes, a gradient of 72:28 to 62:38 from 24 to 30 minutes, a gradient of 62:38 to 90:10 from 30 to 32 minutes, and a constant 62:38 from 32 to 37 minutes. Flow rate: 1.0mL / min Detector: MS SIM analysis conditions in 6SL analysis: m / z 632.2038 [MH] SIM analysis conditions in sialylated LNTII analysis: m / z 835.2837 [MH] SIM analysis conditions for LST-c / LST-c structural isomer analysis: m / z 997.33655 [MH] [Analysis conditions 2] LC-MSMS Analyzer: LC-MS 8050 (SIMADZU) Column: HILICpakVN-50 4D (SHODEX) Column temperature: 60℃ Analysis time: 37 minutes Mobile phase: (Mobile phase A) Acetonitrile (Mobile phase B) 25 mM ammonium formate Gradient method: The mixing ratio of mobile phase A and mobile phase B is as follows: a gradient of 90:10 to 72:28 from 0 to 24 minutes, a gradient of 72:28 to 62:38 from 24 to 30 minutes, a gradient of 62:38 to 90:10 from 30 to 32 minutes, and a constant 62:38 from 32 to 37 minutes. Flow rate: 1.0mL / min Detector: MS MSMS analysis conditions in LST-c analysis: Product ion scan Precursor ion m / z 999.3514 [M+H] CE -30.0, dwell time 100 msec MSMS analysis conditions for sialylated LNTII analysis: Product ion scan Precursor ion m / z 837.298 [M+H] CE -35.0, dwell time 100 msec

[0075] [Example 1] Selection of enzymes to be evaluated (1) From the amino acid sequence of PdsiaT (Patent Document 3) (Accession No. BAA25316.1, SEQ ID NO. 127), a known α2,6 sialyltransferase from Photobacterium damselae with 6SL production activity, the amino acid sequence of PdsiaT△N15△C176 (Reference: Crystal structures of sialyltransferase from Photobacterium damselae - Huynh - 2014 - FEBS Letters - Wiley Online Library) (SEQ ID NO. 1), obtained by deleting the N-terminal 15 bases presumed to be membrane anchors and 176 residues constituting the phoU domain, was used as a query for homolog search using Protein BLAST. From the results obtained, only sequences presumed to have α2,6 activity were selected, and seven sequences, SEQ ID NOs. 3, 5, 7, 9, 11, 13, and 15 shown in Table 1, were selected as enzymes to be evaluated. [Table 1]

[0076] (2) The amino acid sequence of HAC1268 (SEQ ID NO: 17), a known α2,6 sialyltransferase derived from Helicobacter acinonychis str. Sheeba, was used as a query for homolog search using protein BLAST. From the results obtained, four sequences, SEQ ID NOs: 18, 20, 22, and 24, shown in Table 2, were selected as enzymes to be evaluated. [Table 2]

[0077] (3) Three sequences, SEQ ID NOs. 26, 28, and 30, shown in Table 3, were selected as enzymes to be evaluated from known α2,6 sialyltransferases belonging to the GT52 family, for which LST-c production activity has not been confirmed (Japanese Patent Publication No. 2023-554526). [Table 3]

[0078] [Example 2] Creation of base microorganisms for evaluation of α2,6-sialyltransferase activity (1) Obtaining DNA fragments to be used as markers in the case of gene defects PCR was performed using DNA consisting of the base sequences shown in "Primer Set" in Table 4 as the primer set, and the DNA described in "Template" in Table 4 as the template, to obtain amplified DNA fragments. [Table 4] The amplified DNA fragment cat-scaB contains the chloramphenicol-resistant cat gene on pCatSac and the sucrose-sensitive sacB gene derived from Bacillus sacillus.

[0079] (2) Creation of Escherichia coli that has lost β-galactosidase activity and lactose permyase activity Escherichia coli lacking the DNA encoding β-galactosidase (hereinafter referred to as the lacZ gene) and the DNA encoding lactose permyase (hereinafter referred to as the lacY gene) was created using the following method. Note that lacZ and lacY form an operon on the Escherichia coli genome. Using the genomic DNA of Escherichia coli strain W3110, prepared by conventional methods, PCR was performed using primer sets consisting of DNA sequences represented in the "primer sets" in Table 5, and each DNA fragment was amplified. [Table 5] lacZ upstream 1 and lacZ upstream 2 are the base sequences of approximately 1000 bp upstream of the start codon of the lacZ gene. lacY downstream 1 and lacY downstream 2 include the portion of the lacY gene from approximately 40 bp downstream of the stop codon to approximately 1000 bp further downstream. Using an equimolar mixture of lacZ upstream 1, lacY downstream 1, and the cat-sacB fragment as a template, PCR was performed using a primer set consisting of DNA with the base sequences represented by SEQ ID NOs. 55 and 58 to obtain a DNA fragment (hereinafter referred to as lacZY::cat-sacB) in which the cat-sacB fragment was inserted into the sequences surrounding the lacZ and lacY genes. Using an equimolar mixture of lacZ upstream 2 and lacY downstream 2 as a template, PCR was performed using a primer set consisting of DNA with the base sequences represented by SEQ ID NOs. 59 and 62 to obtain a DNA fragment (hereinafter referred to as ΔlacZY) that does not contain lacZ or lacY, but consists of a sequence in which the lacZ upstream and lacY downstream are directly linked. The lacZY::cat-sacB fragment was introduced by electroporation into the W3110S strain, which carries plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol.97, 6640-6645(2000)] containing the gene encoding λ recombinase, to obtain a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (a transformant in which the lacZ and lacY genes were replaced with lacZY::cat-sacB). The ΔlacZY fragment was introduced into the same transformant by electroporation to obtain a transformant exhibiting chloramphenicol sensitivity and sucrose resistance (a transformant in which lacZY::cat-sacB was replaced with ΔlacZY). This transformant was named W3110SΔlacZY.

[0080] (3) Creation of gene knockout strains related to the metabolic pathway of N-acetylneuraminic acid Escherichia coli lacking the DNA encoding the N-acetylneuraminic acid metabolic pathway-related genes nanR (DNA-binding transcriptional dual regulator), nanA (DNA-binding transcriptional dual regulatorDNA), nanT (sialic acid transporter), nanE (N-acetylglucosamine 6-phosphate epilerase), and nanK (N-acetylglucosamine kinase) was created using the following method. Note that nanR, nanA, nanT, nanE, and nanK form an operon on the Escherichia coli genome. Using the genomic DNA of Escherichia coli strain W3110, prepared by conventional methods, as a template, PCR was performed using a primer set consisting of DNA with the base sequences shown in Table 6, and each DNA fragment was amplified. [Table 6] nanR upstream 1 and nanR upstream 2 are the base sequences of approximately 800 bp upstream of the start codon of the nanR gene. nanK downstream 1 and nanK downstream 2 include the nanK gene from approximately 40 bp downstream of the stop codon to approximately 1000 bp further downstream. Using a template made by mixing nanR upstream 1, nanK downstream 1, and cat-sacB fragments in equimolar proportions, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 63 and 66 to obtain a DNA fragment (hereinafter referred to as nanRATEK::cat-sacB) in which the cat-sacB fragment was inserted into the sequences surrounding the nanR and nanK genes. Using a template made by mixing nanR upstream 2 and nanK downstream 2 in equimolar proportions, PCR was performed using a primer set consisting of DNA with the base sequences represented by SEQ ID NOs. 70, and a DNA fragment (hereinafter referred to as ΔnanRATEK) was obtained that did not contain nanR, nanA, nanT, nanE, or nanK, and consisted of a sequence in which nanR upstream and nanK downstream were directly linked. The nanRATEK::cat-sacB fragment was introduced by electroporation into the W3110S strain, which carries plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol.97, 6640-6645(2000)] containing the gene encoding λ recombinase, to obtain transformants that were chloramphenicol resistant and sucrose sensitive (transformers in which the nanR~nanK genes were replaced with nanRATEK::cat-sacB). The ΔnanRATEK fragment was introduced into the transformant by electroporation to obtain a transformant that is sensitive to chloramphenicol and resistant to sucrose (a transformant in which nanRATEK::cat-sacB is substituted with ΔnanRATEK). This transformant was named W3110SΔlacZYΔnanRATEK.

[0081] [Example 3] Creation of recombinant E. coli expressing various sialyltransferases Escherichia coli strains containing a plasmid in which the gene encoding the sialyltransferase shown in Table 1, selected in Example 1, was placed under the Lac promoter were prepared by the following method. PCR was performed using the DNA represented as the "template" in Table 7 as the template and the DNA consisting of the base sequence represented as the "primer set" as the primer set to obtain each amplified DNA fragment. At this time, the base sequences represented by SEQ ID NOs. 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99, along with the base sequence represented by SEQ ID NOs. 101, and the base sequences represented by SEQ ID NOs. 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, and 100, along with the base sequence represented by SEQ ID NOs. 102, each contain a complementary sequence at their 5' end. [Table 7] Furthermore, PCR was performed using the expression vector pSTV29 (Takara Bio, Code No. 3332) as a template, with oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 101 and 102 as primers, to obtain a linearized pSTV29 vector fragment. The fragments of each sialyltransferase homolog obtained up to the above stage (PdsiaTΔN15ΔC176, 26STa, 26STb, 26STc, 26STd, 26STe, 26STf, 26STg, GT42-1, GT42-4, GT42-10, GT42-11, GT52-1, GT52-2, GT52-3) and the pSTV29 linearization vector fragment were processed using In-Fusion HD Cloning. By linking using the kit (manufactured by Takara Bio Inc.), we obtained the expression vectors pSTV29-PdsiaT, pSTV29-26STa, pSTV29-26STb, pSTV29-26STc, pSTV29-26STd, pSTV29-26STe, pSTV29-26STf, pSTV29-26STg, pSTV29-GT42-1, pSTV29-GT42-4, pSTV29-GT42-10, pSTV29-GT42-11, pSTV29-GT52-1, pSTV29-GT52-2, and pSTV29-GT52-3.By transforming the W3110SΔlacZYΔnanRATEK strain prepared in Example 2(3) using this expression vector, strains (W3110SΔlacZYΔnanRATEK / pSTV29-PdsiaT strain, W3110SΔlacZYΔnanRATEK / pSTV29-26STa strain, W3110SΔlacZYΔnanRATEK / pSTV29-26STb strain, W3110SΔlacZYΔnanRATEK / pSTV29-26STc strain, W3110SΔlacZYΔnanRATEK / pSTV29-26STd strain, W3110SΔlacZYΔnanRATEK / pSTV29-26STe strain, W3110SΔlacZY ΔnanRATEK / pSTV29-26STf strain, W3110SΔlacZYΔnanRATEK / pSTV29-26STg strain, W3110SΔlacZYΔnanRATEK / pSTV29-GT42-1 strain, W3110SΔlacZYΔnanRATEK / pSTV29-GT42-4 strain, W3110SΔlacZYΔnanRATEK / pSTV29 We obtained strains -GT42-10, W3110SΔlacZYΔnanRATEK / pSTV29-GT42-11, W3110SΔlacZYΔnanRATEK / pSTV29-GT52-1, W3110SΔlacZYΔnanRATEK / pSTV29-GT52-2, and W3110SΔlacZYΔnanRATEK / pSTV29-GT52-3.

[0082] [Example 4] Evaluation of α2,6 sialyltransferase activity by crude enzyme reaction Based on the sialyltransferase assay described in [Glycobiology vol. 24 no. 2 pp. 159-169, 2014], the following method was used to obtain a cell suspension from sonicated cells expressing α2,6 sialyltransferase, and the activity of each α2,6 sialyltransferase was evaluated.

[0083] (1) Culturing of α2,6 sialyltransferase-expressing strains and acquisition of bacterial cells The strains obtained in Example 3 were W3110SΔlacZYΔnanRATEK / pSTV29-PdsiaT, W3110SΔlacZYΔnanRATEK / pSTV29-26STa, W3110SΔlacZYΔnanRATEK / pSTV29-26STb, W3110SΔlacZYΔnanRATEK / pSTV29-26STc, W3110SΔlacZYΔnanRATEK / pSTV29-26STd, W3110SΔlacZYΔnanRATEK / pSTV29-26STe, W3110SΔlacZYΔnanRATEK / pSTV29-26STf, and W3110SΔlacZYΔnanRATEK / pSTV29-26STg. For strains W3110SΔlacZYΔnanRATEK / pSTV29-GT42-1, W3110SΔlacZYΔnanRATEK / pSTV29-GT42-4, W3110SΔlacZYΔnanRATEK / pSTV29-GT42-10, W3110SΔlacZYΔnanRATEK / pSTV29-GT42-11, W3110SΔlacZYΔnanRATEK / pSTV29-GT52-1, W3110SΔlacZYΔnanRATEK / pSTV29-GT52-2, and W3110SΔlacZYΔnanRATEK / pSTV29-GT52-3, LB medium containing 25 mg / L chloramphenicol [yeast The culture solution was inoculated into a plastic test tube containing 2 mL of extract [0.5%, plypeptone 1%, sodium chloride 1%] and cultured with shaking at 30°C for 19 hours. Subsequently, 0.5 mL of the resulting culture solution was inoculated into a large test tube containing 5 mL of production medium [glucose 30 g / L, magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, casamino acid 5 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L, pH 7.2] containing 25 mg / L of chloramphenicol, and cultured at 30°C. 1 mM IPTG was added at 6 hours of culture and cultured until 24 hours.After the culture was complete, the test tubes were collected on ice and the culture solution was measured into a 1.5 mL plastic tube so that the OD (660 nm) was 50 when suspended in 0.2 mL of buffer. After centrifugation at 13,000 rpm at 4°C for 3 minutes, the supernatant was removed to obtain individual bacterial cells for crude enzyme extraction.

[0084] (2) Obtaining crude enzyme extract Each bacterial cell collected in (1) was suspended in 0.2 mL of 50 mM Tris-HCl buffer (pH 8.0) with 10 mM magnesium chloride added. Three sets of 10 ultrasonic cell disruption were performed using an ultrasonic disruptor (Bioraptor II BR2012A TYPE12) with output: High, and ultrasonic irradiation time and stop time each amounting to one cycle, to obtain each cell disruption solution. The supernatant obtained by centrifugation at 13,000 rpm at 4°C for 3 minutes was used as the crude enzyme extract.

[0085] (3) Crude enzyme reaction using LNnT as a substrate (2) 20 μL of each crude enzyme extract obtained in (2), 5 μL of 100 mM cytidine 5'-monophosphate-N-acetylneuraminate sodium salt (Sigma-Aldrich Japan, no. C8271), 5 μL of 100 mM LNnT, and 20 μL of 50 mM Tris-HCl buffer (pH 8.0) were mixed to prepare crude enzyme reaction solutions. Each crude enzyme reaction solution was incubated at 30°C for 16 hours. After the reaction was complete, a mixture of acetonitrile:25 mM ammonium formate = 3:2 was added to stop the reaction, and the supernatant after centrifugation was used as a sample for analysis. First, using the method of analysis condition 1, LC-MS analysis was performed in SIM mode, specifying m / z 997.3366 [MH], which corresponds to the proton-desorbed product of LST-c. A new peak was detected at the same retention time (hereinafter, RT) as the LST-c standard (Funakoshi Co., Ltd. no. 55 / 52) in samples obtained from the crude enzyme reaction solutions of PdsiaT (sequence number 1), 26STa (sequence number 3), 26STb (sequence number 5), 26STc (sequence number 7), 26STd (sequence number 9), 26STe (sequence number 11), 26STf (sequence number 13), GT42-4 (sequence number 20), GT52-1 (sequence number 26), GT52-2 (sequence number 28), and GT52-3 (sequence number 30). To identify the newly detected peaks, these samples were subjected to LC-MSMS analysis using the method of analytical condition 2, and fragment ions were analyzed by product ion scanning of the precursor ion m / z 999.3514 [M+H] corresponding to the proton adduct of LST-c. Fragment ions m / z 657,528,292 were detected at the peak at RT25.86, and since they were detected at the same RT as the LST-c standard and their fragment patterns matched, this peak was identified as LST-c. Furthermore, a peak with the same molecular weight as LST-c detected at RT24.9 in LC-MS SIM mode analysis at m / z 997.3366 [MH] was identified as a structural isomer of LST-c because its fragment pattern differed from that of the LST-c standard.Table 8 shows the relative production (%) of LST-c and LST-c structural isomers produced by each crude enzyme reaction solution, based on the peak intensity detected by analyzing each sample under analysis condition 1, with the LST-c production amount by the PdsiaT crude enzyme reaction solution set to 100. Furthermore, the production ratio (%) of LST-c structural isomers relative to LST-c production, calculated based on the peak intensity ratio of LST-c and LST-c structural isomers for the same enzyme under evaluation, is shown. [Table 8-1] [Table 8-2]

[0086] As shown in Table 8, LST-c production activity was confirmed for PdsiaT, 26STa, 26STb, 26STc, 26STd, 26STe, 26STf, GT42-4, GT52-1, GT52-2, and GT52-3. Furthermore, PdsiaT, 26STb, 26STd, 26STf, GT42-4, GT52-1, GT52-2, and GT52-3 showed higher LST-c production activity compared to the other enzymes evaluated. In particular, compared to PdsiaT, a known α2,6 sialyltransferase, 26STb, 26STd, GT42-4, GT52-1, GT52-2, and GT52-3 were shown to be sialyltransferases with useful activity in LST-c production, meaning they are less likely to produce LST-c structural isomers that are difficult to remove in the purification process, i.e., they have high specificity for galactose at the LNnT terminus.

[0087] (4) Crude enzymatic reaction using LNTII as a substrate (2) 20 μL of crude enzyme extract obtained in (2), 5 μL of 100 mM cytidine 5'-monophosphate-N-acetylneuraminate sodium salt (Sigma-Aldrich Japan, no. C8271), 5 μL of 100 mM LNTII, and 20 μL of 50 mM Tris-HCl buffer (pH 8.0) were mixed and incubated at 30°C for 16 hours. After the reaction was complete, a mixture of acetonitrile:25 mM ammonium formate = 3:2 was added to stop the reaction, and the supernatant after centrifugation was subjected to analysis. Analysis was performed by LC-MS using the method of analytical condition 1, specifying m / z 835.2837 [MH] corresponding to the protonated product of sialylated LNTII in SIM mode, and novel peaks were detected in multiple samples. To identify the newly detected peaks, these samples were subjected to LC-MSMS analysis using the method of analytical condition 2, and fragment ions were analyzed by product ion scanning of precursor ions corresponding to the proton adduct of sialylated LNTII with m / z 837.2983 [M+H]. Since fragment ions 204, 292, 366, and 546 were detected from the new peaks with the same molecular weight as the sialylated LNTII detected in RT, it was determined that sialylated LNTII was being produced. The relative production amounts of sialylated LNTII for each sample quantified using the method of analytical condition 1 are shown in Table 9, with the production amount of sialylated LNTII from the PdsiaT crude enzyme reaction solution set to 100. Among the enzymes that showed LST-c production activity in Example 4-(3), 26STb (SEQ ID NO: 5), 26STc (SEQ ID NO: 7), 26STd (SEQ ID NO: 9), 26STf (SEQ ID NO: 13), GT42-4 (SEQ ID NO: 20), GT52-1 (SEQ ID NO: 26), GT52-2 (SEQ ID NO: 28), and GT52-3 (SEQ ID NO: 30) were shown to be sialyltransferases that produce less sialylated LNTII, which is considered difficult to remove in the purification process during LST-c production, compared to the known α2,6 sialyltransferase PdsiaT (SEQ ID NO: 1). [Table 9] The results of the crude enzymatic reactions using LNnT and LNTII as substrates in (3) and (4) showed that 26STb, 26STd, GT42-4, GT52-1, GT52-2, and GT52-3 are the most useful sialyltransferases, as they do not readily produce both LST-c structural isomers and sially hydrated LNTII, which are by-products in LST-c production, and yet possess LST-c production activity.

[0088] [Example 5] Creation of Escherichia coli possessing β1,4-galactosyltransferase, β1,3-N-acetylglucosaminetransferase, and lactose permyase activity Escherichia coli W3110 lactose permease (lacY) plasmid containing a gene encoding Helicobacter pylori-derived β1,4-galactosyltransferase (hereinafter referred to as Hpβ4GalT) consisting of the amino acid sequence represented by SEQ ID NO: 32, a gene encoding Neisseria polysaccharea-derived β1,3-N-acetylglucosaminetransferase (hereinafter referred to as NplgtA) represented by SEQ ID NO: 34, and a gene encoding Escherichia coli W3110-derived lactose permease (hereinafter referred to as lacY) was created using the following method. PCR was performed using the DNA represented as the "template" in Table 10 as the template and the DNA consisting of the base sequence represented as the "primer set" as the primer set to obtain each amplified DNA fragment. At this time, the base sequences represented by SEQ ID NOs. 128 and 103, SEQ ID NOs. 104 and 105, SEQ ID NOs. 106 and 107, and SEQ ID NOs. 108 and 129 each contain a complementary sequence at their 5' end. [Table 10] Using a template made by mixing Hpβ4galT, NplgtA, and lacY fragments in equimolar proportions, PCR was performed using a primer set consisting of DNA with the base sequences represented by SEQ ID NOs: 103 and 108 to obtain a DNA fragment consisting of a sequence in which Hpβ4galT, NplgtA, and lacY are directly linked (hereinafter referred to as Hpβ4galT-NplgtA-lacY). Furthermore, PCR was performed using the expression vector pTK31 (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol.73, No.20, pp.6378-6385) as a template, with oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 128 and 129 as primers, to obtain a linearized pTK31 vector fragment. The Hpβ4galT-NplgtA-lacY fragment obtained above and the pTK31 linearization vector fragment were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pTK31-Hpβ4galT-NplgtA-lacY. Using this expression vector pTK31-Hpβ4galT-NplgtA-lacY, the W3110SΔlacZYΔnanRATEK strain prepared in Example 2 (3) was transformed to obtain a transformant with LNnT production activity. This transformant was named W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY.

[0089] [Example 6] Creation of recombinant E. coli producing LST-c E. coli strains were prepared by the following method, each containing a plasmid under the Lac promoter, comprising: a gene encoding UDP-N-acetylglucosamine 2-epimerase from Flavobacterium psychrophilum with the amino acid sequence represented by SEQ ID NO: 37; a gene encoding N-acetylneuraminic acid synthase from Campylobacter jejuni with the amino acid sequence represented by SEQ ID NO: 39; a gene encoding N-acetylneuraminic acid cytidylyltransferase from Pasteurella multocida with the amino acid sequence represented by SEQ ID NO: 41; and a gene encoding a sialyltransferase from which LST-c production activity was confirmed in the crude enzymatic reaction of Example 4, selected from the sialyltransferases shown in Table 1 in Example 1. [Table 11] PCR was performed using the DNA represented as the "template" in Table 12 as the template and the DNA consisting of the base sequence represented as the "primer set" as the primer set to obtain each amplified DNA fragment. At this time, the base sequences represented by SEQ ID NOs. 109, 111, 113, 115, 117, 119, 136, 121, 123, 125, 130, 132, and 134, along with the base sequence represented by SEQ ID NOs. 101, and the base sequences represented by SEQ ID NOs. 116, 118, 120, 137, 122, 124, 126, 131, 133, and 135, along with the base sequence represented by SEQ ID NOs. 109, and the base sequences represented by SEQ ID NOs. 110 and 111, 112 and 113, 114 and 115, and 116 and 102 each contain a complementary sequence at their 5' end. [Table 12] Using the fragments of each sialyltransferase obtained above (PdsiaTΔN15ΔC176, 26STa, 26STb, 26STc, 26STd, 26STf, GT42-4, GT52-1, GT52-2, GT52-3) mixed with FpneuC, CjneuB, and PmneuA in equimolar ratios as a template, PCR was performed using the primer set shown in Table 12. The resulting DNA consists of a sequence in which each sialyltransferase homolog fragment and FpneuC, CjneuB, and PmneuA are directly linked (hereinafter referred to as PdsiaT-FpneuC-CjneuB-PmneuA). The fragments obtained are named uA, 26STa-FpneuC-CjneuB-PmneuA, 26STb-FpneuC-CjneuB-PmneuA, 26STc-FpneuC-CjneuB-PmneuA, 26STd-FpneuC-CjneuB-PmneuA, 26STf-FpneuC-CjneuB-PmneuA, GT42-4-FpneuC-CjneuB-PmneuA, GT52-1-FpneuC-CjneuB-PmneuA, GT52-2-FpneuC-CjneuB-PmneuA, and GT52-3-FpneuC-CjneuB-PmneuA. Furthermore, PCR was performed using the expression vector pSTV29 (Takara Bio, Code No. 3332) as a template, with oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 101 and 102 as primers, to obtain a linearized pSTV29 vector fragment. The fragments obtained up to the above point, PdsiaT-FpneuC-CjneuB-PmneuA, 26STa-FpneuC-CjneuB-PmneuA, 26STb-FpneuC-CjneuB-PmneuA, 26STc-FpneuC-CjneuB-PmneuA, 26STd-FpneuC-CjneuB-PmneuA, 26STf-FpneuC-CjneuB-PmneuA, GT42-4-FpneuC-CjneuB-PmneuA, GT52-1-FpneuC-CjneuB-PmneuA, GT52-2-FpneuC-CjneuB-PmneuA, and GT52-3-FpneuC-CjneuB-PmneuA, along with the pSTV29 linearization vector fragment, were then processed using In-Fusion HD Cloning. By linking using the kit (manufactured by Takara Bio), each expression vector can be expressed as follows: pSTV29-Plac-PdsiaT-FpneuC-CjneuB-PmneuA, pSTV29-Plac-26STa-FpneuC-CjneuB-PmneuA, pSTV29-Plac-26STb-FpneuC-CjneuB-PmneuA, pSTV29-Plac-26STc-FpneuC-CjneuB-PmneuA, pSTV29-Plac-26STd-FpneuC- CjneuB-PmneuA, pSTV29-Plac-26STf-FpneuC-CjneuB-PmneuA, pSTV29-Plac-GT42-4-FpneuC-CjneuB-PmneuA, pSTV29-Plac-GT5 2-1-FpneuC-CjneuB-PmneuA, pSTV29-Plac-GT52-2-FpneuC-CjneuB-PmneuA, and pSTV29-Plac-GT52-3-FpneuC-CjneuB-PmneuA were obtained.By transforming the W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY strain prepared in Example 5 using these expression vectors, in vivo LST-c production activity evaluation strains (W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-PdsiaT strain, W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-26STa strain, W3110SΔlacZYΔnanRA TEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-26STb strain, W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT- NplgtA-lacY / pSTV29-26STc strain, W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-2 6STd strain, W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-26STf strain, W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-GT42-4 strain, W3110SΔlacZYΔnanRATEK / pTK31-Hpβ We obtained strains 4galT-NplgtA-lacY / pSTV29-GT52-1, W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-GT52-2, and W3110SΔlacZYΔnanRATEK / pTK31-Hpβ4galT-NplgtA-lacY / pSTV29-GT52-3.

[0090] [Example 7] In vivo activity evaluation of α2,6 sialyltransferase with LST-c production activity Each LST-c production activity evaluation strain was cultured using the following method, and the LST-c production activity by each α2,6 sialyltransferase was evaluated.

[0091] (1) Culture of LST-c production activity evaluation strains Each LST-c production activity evaluation strain obtained in Example 6 was inoculated into a plastic test tube containing 2 mL of LB medium containing 25 mg / L chloramphenicol and 100 mg / L kanamycin, and cultured with shaking at 30°C for 19 hours. Subsequently, 0.4 mL of each resulting culture was inoculated into a large test tube containing 4 mL of production medium [glucose 30 g / L, lactose 10 g / L, magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, casamino acid 5 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L, pH 7.2] containing 25 mg / L chloramphenicol and 100 mg / L kanamycin, and cultured at 30°C. At 6 hours of incubation, 1 mM IPTG was added to each large test tube, and the cells were incubated until 28 hours. After incubation, the cells were heat-treated at 70°C for 40 minutes to extract the intracellular fraction, and the resulting samples were subjected to analysis. The production amount of LST-c (mg / L) in each sample, as well as the peak intensities of LST-c, LST-c structural isomers, sially modified LNTII, and 6SL, were quantified by LC-MS SIM mode analysis using the method of analysis condition 1. Figure 1 and Table 13 show the results. For LST-c, the peak intensity is shown as a percentage (%) relative to the peak intensity of PdsiaT, a known α2,6 sialyltransferase, with the peak intensity set to 100. For LST-c structural isomers, sially modified LNTII, and 6SL, the peak intensity is shown as a percentage (%) relative to the peak intensity of LST-c in the same enzyme being evaluated, with the peak intensity set to 100. [Table 13] As shown in Table 13, LST-c production was confirmed in all LST-c production activity evaluation strains. Furthermore, strains expressing PdsiaT, 26STd, GT42-4, GT52-1, GT52-2, and GT52-3 showed higher LST-c production activity compared to other enzymes under evaluation. In particular, strains expressing 26STd, GT42-4, GT52-1, GT52-2, and GT52-3 showed reduced production of LST-c structural isomers and sialylated LNTII compared to strains expressing PdsiaT, a known α2,6 sialyltransferase. Moreover, strains expressing GT42-4, GT52-2, and GT52-3 also showed reduced production of 6SL compared to strains expressing PdsiaT, indicating that these enzymes can more efficiently convert lactose added as a substrate to LST-c. From the above, this example also demonstrated that 26STd and GT42-4, GT52-1, GT52-2, and GT52-3 are useful sialyltransferases that do not readily produce both LST-c structural isomers and sially mediated LNTII, which are by-products in LST-c production, and that possess LST-c production activity. Furthermore, GT42-4, GT52-2, and GT52-3 also do not readily produce 6SL, another by-product in LST-c production, and were shown to be the most useful sialyltransferases from the viewpoint of reducing by-products. From the viewpoint of LST-c productivity, GT52-1 was shown to be the most useful sialyltransferase. Furthermore, 26STd was shown to have higher 6SL production activity compared to PdsiaT, a known α2,6 sialyltransferase. Therefore, 26STd was shown to be a sialyltransferase useful for 6SL production.

[0092] Although the present invention has been described in detail above with reference to specific embodiments, the present invention is not limited thereto. Additions, omissions, substitutions, and other modifications to the configuration are possible without departing from the spirit of the present invention.

Claims

1. A method for producing sialyl lacto-N-neotetraose c (LST-c) from lacto-N-neotetraose (LNnT) in the presence of at least one protein selected from the group consisting of [1] to [3] below; [1] A protein containing the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, comprising an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity.

2. The method for producing a protein represented by at least one selected from the group consisting of [1] to [3] using genetically modified cells, further comprising the step of producing a protein represented by at least one selected from the group consisting of [1] to [3] above, according to claim 1.

3. The method for producing LST-c according to claim 1 or 2, comprising producing LST-c in an aqueous medium containing a protein represented by at least one selected from the group consisting of [1] to [3], cytidine 5'-phosphate-N-acetylneuraminic acid, and LNnT.

4. A method for producing LST-c according to any one of claims 1 to 3, wherein LST-c is produced in the presence of at least one protein selected from the group consisting of β-1,3-N-acetylglucosaminyltransferase (LgtA), β-1,4-galactosyltransferase (GalT), and proteins involved in the biosynthesis pathway of cytidine 5'-phosphate-N-acetylneuraminic acid, and at least one protein represented by the group consisting of [1] to [3].

5. LgtA (NpLgtA) derived from Neisseria polysaccharea ATCC43768, GalT from Helicobacter pylori (HpGalT), UDP-N-acetylglucosamine 2-epimerase (FpNeuC) derived from Flavobacterium psychrhilum, Campylobacter jenuni-derived N-acetylneuraminate synthase (CjNeuB), and Cytidine 5'-phosphate-N-acetylneuraminate synthase (PmNeuA) derived from Pasteurella multicida A method for producing LST-c according to any one of claims 1 to 4, in the presence of at least one protein selected from the group consisting of and at least one protein represented by the group consisting of [1] to [3].

6. A method for suppressing the production of at least one protein selected from the group consisting of 6'-sialyl lactose, sialyl lactose-N-neotetraose c (LST-c) from lactose-N-neotetraose (LNnT), using a protein represented by at least one selected from the group consisting of [1] to [3] below; [1] A protein containing the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, comprising an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity.

7. Genetically modified cells having a protein represented by at least one selected from the group consisting of [1] to [3] below. [1] A protein containing the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30. [2] A protein having α2,6-sialyltransferase activity, comprising an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in any one of the amino acid sequences shown in SEQ ID NOs. 5, 9, 20, 26, 28, or 30. [3] A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in any one of sequence numbers 5, 9, 20, 26, 28, or 30, and which has α2,6-sialyltransferase activity.

8. The genetically modified cell according to claim 7, having at least one protein selected from the group consisting of β-1,3-N-acetylglucosaminyltransferase (LgtA), β-1,4-galactosyltransferase (GalT), and proteins involved in the biosynthesis pathway of cytidine 5'-phosphate-N-acetylneuraminic acid, and at least one protein represented by the group consisting of [1] to [3].

9. LgtA (NpLgtA) derived from Neisseria polysaccharea ATCC43768, GalT from Helicobacter pylori (HpGalT), UDP-N-acetylglucosamine 2-epimerase (FpNeuC) derived from Flavobacterium psychrhilum, Campylobacter jenuni-derived N-acetylneuraminate synthase (CjNeuB), and Cytidine 5'-phosphate-N-acetylneuraminate synthase (PmNeuA) derived from Pasteurella multicida A genetically modified cell according to claim 7 or 8, having at least one protein selected from the group consisting of and at least one protein represented by the group consisting of [1] to [3].

10. Contains sialyllacto-N-neotetraose c, The ratio of 6'-sialyl lactose to sialyl lactose-N-neotetraose c is 800% or less. The relative abundance of the sialyllacto-N-neotetraose c isomer to sialyllacto-N-neotetraose c is 20% or less. The ratio of sialyl lacto-N-triose II to sialyl lacto-N-neotetraose c is 6% or less. A culture of genetically modified cells.

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