Genetically modified microorganism and method for producing sugar

Abstract

Provided is a genetically modified microorganism that has enhanced GlcNAc-β1,6 transferase activity and can be used to produce a branched HMO. The genetically modified microorganism has enhanced activity with respect to a protein represented by at least one of [1]–[3] and produces a sugar that has a GlcNAc-β1,6-Gal structure. [1] A protein that includes an amino acid sequence represented by one of SEQ ID NO:47–53 and 55–60. [2] A protein that: includes an amino acid sequence that results from the deletion, substitution, insertion, or addition of 1–40 amino acids to an amino acid sequence represented by one of SEQ ID NO:47–53 and 55–60; and has GlcNAc-β1,6 transferase activity with respect to a receptor substrate that includes galactose. [3] A protein that: includes an amino acid sequence that has at least 50% identity with an amino acid sequence represented by one of SEQ ID NO:47–53 and 55–60; and has GlcNAc-β1,6 transferase activity with respect to a receptor substrate that includes galactose.

Description

Genetically modified microorganisms and methods for producing sugars

[0001] The present disclosure relates to genetically modified microorganisms and methods for producing sugars.

[0002] Human milk oligosaccharides (HMOs) contained in human breast milk have attracted attention as prebiotic materials and have been shown to be effective in the development of cognitive function in infants, defense against infection, and improvement of the intestinal environment (Non-Patent Document 1). HMOs can have a linear or branched chain structure.

[0003] Typical HMOs that serve as the backbone of HMOs having a branched chain structure (hereinafter also referred to as branched-chain HMOs) include lacto-N-hexaose (CAS No. 64003-51-6, hereinafter also referred to as LNH) and lacto-N-neohexaose (CAS No. 64003-52-7, hereinafter also referred to as LNnH), and these can serve as the basic structure of even longer branched-chain HMOs.

[0004] β-1,6-N-Acetylglucosaminyltransferase (hereinafter also referred to as GlcNAc-β1,6 transferase or β1,6-GlcNAcT), which is necessary for the biosynthesis of LNH and LNnH, which are the basic structures of the longer branched-chain HMOs, is essential for expanding the structural diversity of branched-chain HMOs.

[0005] Known GlcNAc-β1,6-transferases reported to be involved in the biosynthesis of branched-chain HMOs include a GlcNAc-β1,6-transferase involved in the biosynthesis of human blood group I antigen (human β1,6N-acetylglucosaminyltransferase, hereinafter also referred to as hIGnT), and a GlcNAc-β1,6-transferase involved in the biosynthesis of mouse blood group I antigen (mouse β1,6N-acetylglucosaminyltransferase, hereinafter also referred to as mIGnT).

[0006] It has been reported that hIGnT and mIGnT have GlcNAc-β1,6-transfer activity toward lacto-N-triose II (hereinafter also referred to as LNTII) and lacto-N-neotetraose (hereinafter also referred to as LNnT) (Non-Patent Documents 2 and 3).

[0007] Int. J. Pediatrics (2019), 2390240PNAS 2017, 114, 6954-6959. Glycobiology, Volume 10, Issue 10, 1 October 2000, Pages 1001-1011

[0008] As mentioned above, hIGnT and mIGnT are known GlcNAc-β1,6-transferases that have been reported to be involved in the biosynthesis of branched-chain HMOs. However, efficient methods for producing oligosaccharides with specific structures have not yet been established, and there is still a need to discover enzymes with GlcNAc-β1,6-transferase activity.

[0009] The present disclosure relates to providing genetically modified microorganisms that have enhanced activity of an enzyme having GlcNAc-β1,6 transfer activity that can be used to produce oligosaccharides.

[0010] The present disclosure includes the following: 1. A genetically modified microorganism in which the activity of a protein represented by at least one selected from the following [1] to [3] is enhanced, and which produces a sugar having a structure in which N-acetylglucosamine (hereinafter also referred to as GlcNAc) is linked to the 6-position of galactose (hereinafter also referred to as Gal) (hereinafter also referred to as GlcNAc-β1,6-Gal structure). [1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 2. The genetically modified microorganism according to 1 above, which produces a sugar having a GlcNAc-β1,6-Gal structure using lactose (Lac) or lacto-N-biose II (LNBII) (CAS: 63121-25-5, structural name GlcNAcβ1→3Gal) as an acceptor substrate. 3. 2. The genetically modified microorganism according to 1 above, wherein the activity of at least one protein selected from the following [a1] to [a3] is enhanced, and the genetically modified microorganism produces a sugar having a GlcNAc-β1,6-Gal structure using Lac, LNBII, or LNTII (lacto-N-triose II) as an acceptor substrate:[a1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53; [a2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose; [a3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 4. The genetically modified microorganism according to 1 above, in which the activity of a protein represented by at least one selected from the following [b1] to [b3] has been enhanced, and which produces a sugar having a GlcNAc-β1,6-Gal structure using at least one acceptor substrate selected from LNnT, LNT, Galactose-β1,4-LNBII, and Galactose-β1,3LNBII: [b1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60; [b2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose; [b3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 5. A method for producing a sugar having a GlcNAc-β1,6-Gal structure using a protein represented by at least one selected from the following [1] to [3].[1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60; [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose. 6. A method for producing the sugar described in 5 above, comprising preparing a genetically modified microorganism in which the activity of at least one protein represented by at least one selected from [1] to [3] above has been enhanced, and using the genetically modified microorganism to produce a sugar having a GlcNAc-β1,6-Gal structure in at least one of a culture supernatant and intracellular space. A method for producing the sugar according to 5 above, comprising bringing a protein represented by at least one selected from the above [1] to [3], an acceptor substrate, and UDP-GlcNAc into the presence of an aqueous medium and producing a sugar having a GlcNAc-β1,6-Gal structure by an enzymatic reaction. 8. A method for producing GlcNAc-β1,6-Gal using a protein represented by at least one selected from the following [1] to [3], and using Lac, LNBII, or LNTII (lacto-N-triose II) as an acceptor substrate:[1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 9. A method for producing GlcNAc-β1,6-Gal using at least one protein selected from the following [1] to [3], and at least one acceptor substrate selected from LNnT (lacto-N-neotetraose), LNT (lacto-N-tetraose), Gal-β1,4-LNBII, and Gal-β1,3-LNBII: [1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 10. The genetically modified microorganism according to 3 above, in which the activity of a protein represented by at least one selected from the following [a1] to [a3] has been enhanced, and which produces GlcNAc-β1,6-LNTII using LNTII (lacto-N-triose II) as an acceptor substrate.[a1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53; [a2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose; [a3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 11. The genetically modified microorganism according to 4 above, in which the activity of a protein represented by at least one selected from the following [b1] to [b3] has been enhanced, and which produces GlcNAc-β1,6-LNnT using LNnT as an acceptor substrate. [b1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 51 and 55 to 60; [b2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 51 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose; [b3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 12. A method for producing GlcNAc-β1,6-LNTII using LNTII (lacto-N-triose II) as an acceptor substrate, using a protein represented by at least one selected from the following [1] to [3]:[1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 13. A method for producing GlcNAc-β1,6-LNnT using LNnT (lacto-N-neotetraose) as an acceptor substrate, using a protein represented by at least 1 selected from the following [1] to [3]. [1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

[0011] The present disclosure provides a novel enzyme (β1,6-GlcNAcT) having GlcNAc-β1,6 transfer activity that can be used to produce sugars having a GlcNAc-β1,6-Gal structure.

[0012] FIG. 1 shows an example of a sugar having a GlcNAc-β1,6-Gal structure. FIG. 2 is a schematic diagram illustrating the biosynthetic pathway of a sugar having a GlcNAc-β1,6-Gal structure in one aspect of this embodiment. FIG. 3 is a structural schematic diagram of an acceptor substrate having galactose. FIG. 4 shows the results of evaluating GlcNAc-β1,6 transferase activity for each acceptor substrate. FIG. 5 shows the results of evaluating GlcNAc-β1,6 transferase activity for each acceptor substrate. FIG. 6 shows the results of evaluating GlcNAc-β1,6 transferase activity for each acceptor substrate. FIG. 7 shows the results of evaluating GlcNAc-β1,6 transferase activity for each acceptor substrate. FIG. 8 shows the results of evaluating GlcNAc-β1,6 transferase activity for each acceptor substrate. FIG. 9 shows the results of evaluating the GlcNAc-β1,6 transfer activity in strains in which β1,6-GlcNAcTs derived from 10 types of bacteria were introduced into an LNnT-producing bacterium.

[0013] Hereinafter, the present disclosure will be described based on an embodiment, but the present disclosure is not limited to the embodiment.

[0014] 1. Genetically Modified Microorganism The genetically modified microorganism of this embodiment is a genetically modified microorganism in which the activity of a protein represented by at least one selected from the following [1] to [3] is enhanced and which produces a sugar having a GlcNAc-β1,6-Gal structure: [1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60; [2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; or [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose.

[0015] In this embodiment, "producing a sugar having a GlcNAc-β1,6-Gal structure" includes both producing a sugar having a GlcNAc-β1,6-Gal structure as a final product and producing a sugar having a GlcNAc-β1,6-Gal structure as an intermediate product.

[0016] When a sugar having a GlcNAc-β1,6-Gal structure is produced as an intermediate product, a sugar having an LNH structure or a sugar having an LNnH structure may be produced as a final product as a branched-chain HMO containing a sugar having a GlcNAc-β1,6-Gal structure as its backbone.

[0017] The long-chain branched-chain HMO shown in FIG. 2 refers to a saccharide having an LNH structure or an LNnH structure, and may refer to, in addition to LNH or LNnH, a saccharide further bound to LNH or LNnH, such as GlcNAc, galactose, sialic acid, or fucose.

[0018] In this embodiment, the expression "the activity of a protein represented by at least one selected from the following [1] to [3]" means that the productivity of the protein is improved compared to that of the parent strain.

[0019] As used herein, "the activity of a protein is enhanced" may mean that the activity of the protein is enhanced compared to that of a parent strain. Specifically, as used herein, "the activity of a protein is enhanced" may mean that the activity of the protein per cell is enhanced compared to that of a parent strain.

[0020] In this specification, the term "parent strain" refers to a type strain (i.e., a type strain of a species to which a microorganism belongs) that is the target of genetic modification and transformation, and a strain to which modifications other than those that enhance the activity of a target protein have already been added to the type strain. Examples of such strains include, but are not limited to, the strains exemplified in the description of "parent strain" below.

[0021] That is, in one embodiment, the activity of the protein may be enhanced compared to the parent strain.

[0022] As used herein, "enhanced protein activity" may more specifically mean that the number of molecules of the protein per cell is increased and / or the function of the protein per molecule is enhanced compared to that of the parent strain. That is, the "activity" in "enhanced protein activity" is not limited to the catalytic activity of the protein, but may also mean the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein.

[0023] Furthermore, "enhanced protein activity" encompasses not only enhancing the activity of a target protein in a strain that originally has the activity of that protein, but also imparting the activity of that protein to a strain that does not originally have the activity of that protein. Furthermore, as long as the protein activity is enhanced as a result, the target protein activity may be imparted after reducing or eliminating the activity of the target protein originally possessed by the host.

[0024] In this embodiment, the degree of enhancement of protein activity is not particularly limited as long as the protein activity is enhanced compared to that of the parent strain. For example, the protein activity may be increased by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to that of the parent strain. Furthermore, if the parent strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein, provided that the protein is produced to an extent that its activity can be measured.

[0025] Enhanced protein activity can also be confirmed by confirming that the expression of the gene encoding the protein is increased compared to the parent strain. Increased gene expression can be confirmed by confirming that the transcription level of the gene is increased compared to the parent strain, or by confirming that the amount of the protein expressed from the gene is increased compared to the parent strain.

[0026] Modifications that enhance protein activity can also be achieved, for example, by enhancing the specific activity of the protein. Enhancement of specific activity may also include desensitization to feedback inhibition. That is, when a protein is subject to feedback inhibition by metabolites, the activity of the protein can be enhanced by having the host harbor a gene encoding a mutant protein in which feedback inhibition is desensitized.

[0027] Unless otherwise specified, "desensitization to feedback inhibition" may include cases where feedback inhibition is completely released and cases where feedback inhibition is reduced. Furthermore, "desensitization to feedback inhibition" (i.e., feedback inhibition is reduced or released) is also referred to as "resistance to feedback inhibition."

[0028] Proteins with enhanced specific activity can be obtained, for example, by searching various organisms. Alternatively, highly active proteins can be obtained by introducing mutations into existing proteins. The mutations introduced may be, for example, substitutions, deletions, insertions, or additions of one or several amino acids at one or several positions in the protein.

[0029] Furthermore, when enhancing protein activity, efforts may be made to express the protein in a soluble fraction and ensure correct folding of the protein, etc. Specific examples include co-expression with a chaperone, investigation of gene inducers, refolding techniques, deletion of the N-terminus or C-terminus of the amino acid sequence of the protein, selection of a protein expression vector, optimization of purification conditions, and optimization of codons.

[0030] (Proteins described in [1] to [3]) In the above [1], the origins and names of the amino acid sequences represented by SEQ ID NOs: 47 to 53 and 55 to 60 are shown in Table 1.

[0031]

[0032] In the above [2], "amino acids are deleted, substituted, inserted or added" means that 1 to 40 amino acids are deleted, substituted, inserted or added at any position in the same sequence. In the above [2], the number of amino acids to be deleted, substituted, inserted or added is 1 to 40, preferably 1 to 30, 1 to 20, 1 to 10, more preferably 1 to 8, and most preferably 1 to 5, in that order.

[0033] The amino acids to be deleted, substituted, inserted, or added may be naturally occurring or non-naturally occurring. Naturally occurring 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.

[0034] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can 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

[0035] The protein of [3] above consists of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60. The identity is preferably 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 99% or more, in the following order.

[0036] 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 so that the residues contained in the two sequences are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm.

[0037] 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 similarity search method of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, and an improved version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, although mathematical algorithms are not limited to these examples.

[0038] Alignment for determining the percentage of sequence identity can be performed using programs based on these mathematical algorithms, and the programs can be executed by a computer, if appropriate. Examples of such programs include, but are not limited to, the PC / Gene program 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 / ), and MUSCLE (Edgar R. C. (2004). Nucleic acids research, 32(5), 1792-1797., http: / / www.ebi.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA.

[0039] Alignment using these programs can be performed, for example, using default 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.

[0040] BLAST is described in Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). 215(3), 403-410., Mount D. W. (2007). CSH protocols, 2007, pdb. top17., etc. Programs called BLASTP and BLASTN have been developed based on BLAST, and the percentage of sequence identity can be calculated using these programs with default settings.

[0041] The proteins described in [1] to [3] above are proteins that have the activity of transferring GlcNAc (N-acetylglucosamine) to an acceptor substrate having galactose via a β1,6 linkage (also referred to as GlcNAc-β1,6 transfer activity). The microorganism of this embodiment has enhanced activity of the proteins described in [1] to [3] above, thereby improving the productivity of HMOs, including LNH and LNnH, which are the basic structures of branched-chain HMOs, and thereby expanding the structural diversity of branched-chain HMOs.

[0042] As used herein, "GlcNAc-β1,6 transfer activity toward a galactose-containing acceptor substrate" refers to the activity of transferring GlcNAc to a galactose-containing acceptor substrate. Whether a protein has GlcNAc-β1,6 transfer activity toward a galactose-containing acceptor substrate can be assessed by preparing a recombinant DNA containing DNA encoding the protein by the method described below, transforming a microorganism in which the activity cannot be confirmed with the recombinant DNA, culturing the resulting microorganism, preparing a cell extract containing the protein from the resulting culture, diluting the culture appropriately, and centrifuging the culture. The supernatant is reacted with the galactose-containing acceptor substrate, and the reaction product is analyzed using a liquid chromatograph mass spectrometer (e.g., Shimadzu LCMS-8040) to confirm that a fragment similar to that of an authentic sample is detected. As used herein, the generation of a compound is also referred to as "producing."

[0043] Specifically, whether a microorganism is capable of producing a sugar having a GlcNAc-β1,6-Gal structure can be confirmed by, for example, transforming the microorganism with a recombinant DNA having a DNA encoding a protein consisting of any one of the amino acid sequences represented by SEQ ID NOs: 47 to 53 and 55 to 60, culturing the transformed microorganism in a medium, and detecting the sugar having a GlcNAc-β1,6-Gal structure accumulated in the culture using an analytical device described below.

[0044] (Sugars Having a GlcNAc-β1,6-Gal Structure) The genetically modified microorganism of this embodiment produces sugars having a GlcNAc-β1,6-Gal structure. In this embodiment, examples of the GlcNAc-β1,6-Gal structure include GlcNAc-β1,6-Lac, GlcNAc-β1,6-LNBII, GlcNAc-β1,6-LNTII, GlcNAc-β1,6-LNnT, Gal-β1,4-GlcNAc-β1,3-Gal-β1,6-GlcNAc (hereinafter also referred to as Gal-β1,4-LNBII-β1,6-GlcNAc), Gal-β1,3-GlcNAc-β1,3-Gal-β1,6-GlcNAc (hereinafter also referred to as Gal-β1,3-LNBII-β1,6-GlcNAc), and GlcNAc-β1,6-LNT.

[0045] Figure 1 shows an example of a GlcNAc-β1,6-Gal structure, and the structure enclosed by a dotted circle in Figure 1 is the GlcNAc-β1,6-Gal structure. Figure 2 shows a schematic diagram illustrating the biosynthetic pathway of a sugar having a GlcNAc-β1,6-Gal structure in one aspect of this embodiment.

[0046] In this embodiment, the sugar having a GlcNAc-β1,6-Gal structure is preferably at least one selected from GlcNAc-β1,6-Lac, GlcNAc-β1,6-LNBII, GlcNAc-β1,6-LNTII, GlcNAc-β1,6-LNnT, Gal-β1,4-LNBII-β1,6-GlcNAc, Gal-β1,3-LNBII-β1,6-GlcNAc, and GlcNAc-β1,6-LNT, and more preferably at least one of a sugar having an LNnH structure and a sugar having an LNH structure. Sugars having an LNnH structure and sugars having an LNH structure are also referred to as long-chain branched-chain HMOs.

[0047] As used herein, "GlcNAc-β1,6-Lac" refers to a structure in which GlcNAc is bound to the 6-position of galactose in lactose via a β1,6 bond (CAS No. 68665-69-0, structural name: GlcNAcβ1→6Galβ1→4Glc). As used herein, "GlcNAc-β1,6-LNBII" refers to a structure in which GlcNAc is bound to the 6-position of galactose in LNBII via a β1,6 bond (CAS No. 55612-66-3, structural name: GlcNAcβ1→3[GlcNAcβ1→6]Gal). As used herein, "GlcNAc-β1,6-LNTII" refers to a structure in which GlcNAc is bound to the 6-position of the galactose of LNTII via a β1,6 bond (CAS No. 88264-62-4, structural name: GlcNAcβ1→3[GlcNAcβ1→6]Galβ1→4Glc).

[0048] GlcNAc-β1,6-LNTII is produced by the action of β1,3-N-acetylglucosaminyltransferase on GlcNAc-β1,6-Lac. GlcNAc-β1,6-LNTII is produced by the action of glucosyltransferase on the GlcNAc-β1,6-LNBII structure.

[0049] As shown in FIG. 2, LNnH is produced by the action of β1,4-galactosyltransferase (hereinafter also referred to as β1,4GalT) on GlcNAc-β1,6-LNTII, and LNH is produced by the action of β1,3-galactosyltransferase (hereinafter also referred to as β1,3GalT) and β1,4GalT.

[0050] The term "GlcNAc-β1,6-LNnT structure" refers to a structure in which GlcNAc is bound via a β1,6 bond to the 6-position of the galactose of the lactose backbone containing the reducing end in LNnT (CAS No. 1949763-93-2, structural name: Galβ1→4GlcNAcβ1→3[GlcNAcβ1→6]Galβ1→4Glc). As shown in Figure 2, LNnH is produced by the action of β1,4GalT on GlcNAc-β1,6-LNnT.

[0051] The "Gal-β1,4-GlcNAc-β1,3-Gal-β1,6-GlcNAc structure" is also called a Gal-β1,4-LNBII-β1,6GlcNAc structure, and refers to a Galβ1→4GlcNAcβ1→3[GlcNAcβ1→6] Gal structure, which is produced by the action of glucosyltransferase to produce GlcNAc-β1,6-LNnT.

[0052] The "Gal-β1,3-GlcNAc-β1,3-Gal-β1,6-GlcNAc structure" is also called a Gal-β1,3-LNBII-β1,6-GlcNAc structure and refers to a Galβ1→3GlcNAcβ1→3[GlcNAcβ1→6]Gal structure, from which GlcNAc-β1,6-LNT is produced by the action of glucosyltransferase.

[0053] The GlcNAc-β1,6-LNT structure refers to a structure in which GlcNAc is bound via a β1,6 bond to the 6-position of the galactose of the lactose backbone, including the reducing end of LNT (CAS No. 1397285-79-8, structural name: Galβ1→3GlcNAcβ1→3[GlcNAcβ1→6]Galβ1→4Glc). As shown in Figure 2, LNH is produced by the action of β1,4GalT on GlcNAc-β1,6-LNT.

[0054] Examples of sugars having an LNH structure include LNH (lacto-N-hexaose), DFLNH (difucosyllacto-N-hexaose), FLNH-I (fucosyllacto-N-hexaose I), FLNH-II (fucosyllacto-N-hexaose II), DSLNH-I (disialyllacto-N-hexaose I), DSLNH-II (disialyllacto-N-hexaose II), FDSLNH-I (fucosyldisiallacto-N-hexaose I), FDSLNH-II (fucosyldisiallacto-N-hexaose II), and FDSLNH-III (fucosyldisiallacto-N-hexaose III).

[0055] Examples of sugars having an LNnH structure include LNnH (lacto-N-neohexaose), F-LNnH-II (fucosyllacto-N-neohexaose II), F-LNnH-I (fucosyllacto-N-neohexaose-I), DF-LNnH (difucosyllacto-N-neohexaose), S-LNnH-I (sialyllacto-N-neohexaose-I), S-LNnH-II (sialyllacto-N-neohexaose II), FS-LNnH-I ( Examples of such fucosyldiaryllacto-N-neohexaose include FS-LNnH-II (fucosialyllacto-N-neohexaose II), DFS-LNnH (difucosyldiaryllacto-N-neohexaose), DS-LNnH (disialyllacto-N-neohexaose), FDS-LNnH (fucosyldisialyllacto-N-neohexaose), and TF-LNnH-III (trifucosyl-lacto-N-neohexaose-III).

[0056] (Acceptor substrate having galactose) In this embodiment, an acceptor substrate having galactose refers to a substance that can serve as a direct substrate for the reaction. For example, when Lac (lactose) is the acceptor substrate, a sugar having a GlcNAc-β1,6-Lac structure can be produced by the activity of a protein having GlcNAc-β1,6 transfer activity.

[0057] In this embodiment, examples of acceptor substrates having galactose include Lac (lactose), LNBII (lacto-N-biose II), LNTII (lacto-N-triose II), LNnT (lacto-N-neotetraose), LNT (lacto-N-tetraose), Gal-β1,4-GlcNAc-β1,3-Gal (hereinafter also referred to as Gal-β1,4-LNBII), and Gal-β1,3-GlcNAc-β1,3-Gal (hereinafter also referred to as Gal-β1,3-LNBII). A structural schematic diagram of an acceptor substrate having galactose is shown in FIG. 3.

[0058] "Gal-β1,4-GlcNAc-β1,3-Gal" is also called Gal-β1,4-LNBII, and refers to a trisaccharide structure containing the non-reducing end of LNnT, that is, a Galβ1→4 GlcNAcβ1→3 Gal structure.

[0059] "Gal-β1,3-GlcNAc-β1,3-Gal" is also called Gal-β1,3-LNBII, and refers to a trisaccharide structure containing the non-reducing end of LNT, that is, a Galβ1→3 GlcNAcβ1→3 Gal structure.

[0060] As shown in Figure 2, in one aspect, when the acceptor substrate having galactose is LNTII, the genetically modified microorganism of this embodiment can produce, as a sugar having a GlcNAc-β1,6-Gal structure, at least one selected from a sugar having an LNnH structure and a sugar having an LNH structure.

[0061] [Aspect 1] As one aspect of the genetically modified microorganism of this embodiment, a genetically modified microorganism is preferred that has enhanced activity of a protein represented by at least one selected from the following [a1] to [a3] and that produces a sugar having a GlcNAc-β1,6-Gal structure using Lac, LNBII, or LNTII (lacto-N-triose II) as an acceptor substrate (hereinafter referred to as Aspect 1). [a1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53; [a2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and that has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; or [a3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, and that has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose.

[0062] In Aspect 1, the acceptor substrate is preferably at least one selected from Lac, LNBII, and LNTII, and more preferably LNTII. In Aspect 1, the saccharide having a GlcNAc-β1,6-Gal structure is preferably at least one selected from GlcNAc-β1,6-Lac, GlcNAc-β1,6-LNBII, and GlcNAc-β1,6-LNTII, and more preferably GlcNAc-β1,6-LNTII.

[0063] [1] in Figure 2 shows a reaction in which GlcNAc-β1,6-LNTII is produced using LNTII as an acceptor substrate, as an example of Aspect 1. In Aspect 1, the protein whose activity is enhanced is more preferably [a1] above. Furthermore, in Aspect 1, mGCNT1 (SEQ ID NO: 52) and mGCNT3 (SEQ ID NO: 53) are particularly preferred proteins whose activity is enhanced.

[0064] [Aspect 2] As one aspect of the genetically modified microorganism of this embodiment, a genetically modified microorganism in which the activity of at least one protein selected from the following [b1] to [b3] is enhanced and which produces a sugar having a GlcNAc-β1,6-Gal structure using at least one acceptor substrate selected from LNnT (lacto-N-neotetraose), LNT (lacto-N-tetraose), Gal-β1,4-LNBII, and Gal-β1,3-LNBII is preferred (hereinafter, Aspect 2). [b1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60. [b2] A protein consisting of an amino acid sequence in which 1 to 40 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [b3] A protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

[0065] In embodiment 2, the acceptor substrate is preferably at least one selected from LNT, LNnT, Gal-β1,4-LNBII, and Gal-β1,3-LNBII, and among these, the acceptor substrate is more preferably LNnT.

[0066] In Aspect 2, the saccharide having a GlcNAc-β1,6-Gal structure is preferably at least one selected from GlcNAc-β1,6-LNT, GlcNAc-β1,6-LNnT, Gal-β1,4-LNBII-β1,6-GlcNAc, and Gal-β1,3-LNBII-β1,6-GlcNAc, and among these, GlcNAc-β1,6-LNnT is more preferred. [2] in Figure 2 shows a reaction for producing GlcNAc-β1,6-LNnT from LNnT as an example of Aspect 2. [3] in Figure 2 shows a reaction for producing GlcNAc-β1,6-LNT from LNT as an example of Aspect 2.

[0067] In Aspect 2, the protein whose activity is enhanced is more preferably [b1] above. Furthermore, in Aspect 2, the proteins whose activity is enhanced are particularly preferably CseGlcNAcT (SEQ ID NO: 55), CsoGlcNAcT (SEQ ID NO: 56), RsGlcNAcT (SEQ ID NO: 57), ArGlcNAcT (SEQ ID NO: 58), and AeGlcNAcT (SEQ ID NO: 60), which have particularly high activity.

[0068] Furthermore, in Aspect 2, the proteins whose activity is enhanced are particularly preferably BbGlcNAcT (SEQ ID NO: 47), AbGlcNAcT (SEQ ID NO: 48), SpGlcNAcT (SEQ ID NO: 49), SpGlcNAcT (SEQ ID NO: 50), CseGlcNAcT (SEQ ID NO: 55), CsoGlcNAcT (SEQ ID NO: 56), RsGlcNAcT (SEQ ID NO: 57), ArGlcNAcT (SEQ ID NO: 58), NrGlcNAcT (SEQ ID NO: 59), and AeGlcNAcT (SEQ ID NO: 60), because they react with LNnT but not with LNTII, they can be said to have substrate selectivity for LNnT.

[0069] (Parent Strain) The parent strain used to construct the microorganisms described herein is not particularly limited.

[0070] In this embodiment, the parent strain is preferably a prokaryote or a yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, ...MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli MG1655, Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli MG1655, KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 codon plus (Stratagene), Escherichia coli BL21 (DE3), Escherichia coli W3110S3GK (NBRC114657), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870,Examples of suitable bacteria include prokaryotes such as Microbacterium ammoniaphilum ATCC15354 or Pseudomonas sp. D-0110, and yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and Candida utilis.

[0071] In this embodiment, the parent strain used in the (in vivo method) may be a wild-type strain as long as it is a microorganism that produces an acceptor substrate having galactose, which is a reaction substrate for GlcNAc-β1,6-transferase. If the wild-type strain does not have the ability to produce an acceptor substrate having galactose, the parent strain may be a bred strain that has been artificially imparted with the ability to produce an acceptor substrate having galactose.

[0072] The parent strain is preferably a microorganism to which the ability to produce an acceptor substrate having galactose, which is a reaction substrate for GlcNAc-β1,6-transferase, has been artificially imparted or enhanced. Specific examples of methods for imparting or enhancing the ability to produce an acceptor substrate having galactose to a microorganism used as a parent strain include known methods such as various genetic engineering methods (Metabolic Engineering (2017) 41:23-38).

[0073] Examples of the ability to produce a galactose-containing acceptor substrate include the ability to generate sugars such as glucose, galactose, N-acetylglucosamine, or sugar donors (e.g., sugar nucleotides) that are constituent sugars of a galactose-containing acceptor substrate using a carbon source that the microorganism can assimilate, such as glucose, fructose, sucrose, raffinose, methanol, or molasses containing these sugars, starch, or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol, or propanol, and thereby produce a galactose-containing acceptor substrate. Methods for artificially imparting or enhancing the ability of a microorganism used as a parent strain to produce a galactose-containing acceptor substrate from sugar include, for example, methods (1a) to (1h) below. These methods may be used alone or in combination. (1a) A method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway that produces a galactose-containing acceptor substrate from a sugar. (1b) A method for enhancing the activity of at least one enzyme involved in the biosynthetic pathway that produces a galactose-containing acceptor substrate or its precursor sugar from a sugar. (1c) A method for increasing the copy number of at least one enzyme gene involved in the biosynthetic pathway that produces a galactose-containing acceptor substrate from a sugar. (1d) A method for alleviating or deactivating at least one of the mechanisms that decompose a galactose-containing acceptor substrate or its precursor sugar. (1e) A method for enhancing the activity of at least one protein involved in the cellular uptake of a galactose-containing acceptor substrate or its precursor sugar. (1f) A method for increasing the copy number of at least one gene encoding a protein involved in the cellular uptake of a galactose-containing acceptor substrate or its precursor sugar. (1g) A method for weakening or blocking at least one metabolic pathway that branches off from the biosynthetic pathway that produces a galactose-containing acceptor substrate to a metabolite other than the target substance. (1h) A method for selecting a cell line that has a higher tolerance to an analogue of a galactose-containing acceptor substrate compared to a wild-type strain. (1i) A method for enhancing hydrolytic activity of a long-chain sugar having a galactose-containing acceptor substrate structure in its backbone, compared to a wild-type strain, to produce a galactose-containing acceptor substrate structure.

[0074] As used herein, the term "hydrolytic activity" refers to hydrolase activity. Examples of hydrolases include galactosidase, glucosidase, and sialidase.

[0075] In (1b), specific examples of enzymes involved in a biosynthetic pathway that produces an acceptor substrate having galactose or a sugar that serves as a precursor thereof include known enzymes such as an enzyme having β1,4-galactosyltransferase (hereinafter referred to as β1,4-galT) activity, which is an enzyme involved in the biosynthetic pathway that produces LNnT from glucose and lactose, an enzyme having β1,3-N-acetylglucosaminetransferase (hereinafter referred to as LgtA) activity, Pgi, glmS, glmM, and glmU, which are enzymes involved in the biosynthetic pathway of LNTII, and Pgm, galU, galE, and galF, which are enzymes involved in the biosynthetic pathway of uridine diphosphate galactose (hereinafter referred to as UDP-Gal).

[0076] In (1d) above, specific examples of the mechanism for decomposing a galactose-containing acceptor substrate or its precursor sugar include known enzymes such as β-galactosidase, which catalyzes the hydrolysis of lactose, a precursor of LNnT, to produce glucose and galactose. Specifically, for example, β-galactosidase (hereinafter referred to as lacZ), which hydrolyzes lactose, a precursor of LNTII, can be mentioned; reducing or deleting the activity of lacZ can suppress a decrease in lactose supply. Another example is YhbJ, a negative transcriptional regulator of the glmS gene in the LNTII biosynthetic pathway; reducing or deleting the activity of YhbJ can promote LNTII biosynthesis.

[0077] In the above (1e) and (1f), examples of proteins involved in the intracellular uptake of galactose-containing receptor substrates or sugars that are precursors thereof include known proteins such as lactose permease, which is involved in the intracellular uptake of lactose, a precursor of LNnT, and lacY, a membrane protein that takes up lactose, a precursor of LNTII, into cells.

[0078] In one aspect of this embodiment, the parent strain preferably has reduced or deleted β-galactosidase (hereinafter referred to as lacZ) activity and / or regulatory factor YhbJ activity. lacZ is an enzyme that hydrolyzes lactose, a precursor of LNTII. Therefore, reducing or deleting lacZ activity can suppress a decrease in lactose supply. Therefore, in one aspect of this embodiment, the parent strain is preferably a genetically modified microorganism in which lacZ and / or YhbJ activity is reduced or deleted, and more preferably does not contain a nucleotide sequence encoding lacZ and / or a nucleotide sequence encoding YhbJ.

[0079] Examples of methods for artificially imparting or enhancing the ability to produce an acceptor substrate having galactose from sugar to a microorganism used as a parent strain by the methods (1a) to (1h) above include various genetic engineering methods (Metabolic Engineering, 2017, 41: 23-38, Syst Microbiol Biomanufact, 2021, 1, 291).

[0080] In this embodiment, specific examples of parent strains include LNTII-producing bacteria (WO 2023 / 120615) and LNnT-producing bacteria (WO 2023 / 153461) in the in vivo method, which will be described in detail below, and SHuffle T7 Express Competent E. coli (New England Biolabs), BL21 (DE3), and the like in the in vitro method.

[0081] (Method for Producing Genetically Modified Microorganisms) In a method for producing a genetically modified microorganism according to this embodiment, for example, a genetically modified microorganism having enhanced activity of a protein represented by at least one selected from the above [1] to [3] can be achieved by transforming a parent strain of a microorganism with recombinant DNA containing DNA encoding the protein, thereby increasing the expression of DNA encoding the protein compared to the parent strain.

[0082] As used herein, "gene expression is increased" may mean that the expression of the gene is enhanced compared to that of the parent strain. As used herein, "gene expression is increased" may specifically mean that the expression level of the gene per cell is enhanced compared to that of the parent strain. As used herein, "gene expression is increased" may more specifically mean that the transcription level (mRNA level) of the gene is enhanced and / or the translation level (protein level) of the gene is enhanced.

[0083] Note that "increased gene expression" is also referred to as "enhanced gene expression." In this embodiment, gene expression may be increased, for example, by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to the parent strain. Furthermore, "increased gene expression" encompasses not only increasing the expression level of a target gene in a strain in which the target gene is originally expressed, but also expressing the gene in a strain in which the target gene is not originally expressed. In other words, "increased gene expression" may mean, for example, introducing the target gene into a strain that does not harbor the gene and expressing the gene.

[0084] Increased gene expression can be achieved, for example, by increasing the copy number of the gene, selecting a promoter with a high frequency of transcription initiation, or disrupting or enhancing the expression of a transcriptional regulatory factor involved in controlling the expression of the target gene. That is, in the case of a transcriptional regulatory factor that contributes to suppressing the expression of the target gene, this can be achieved by disruption, and in the case of a transcriptional regulatory factor that contributes to promoting the expression of the target gene, this can be achieved by enhancement.

[0085] Examples of the microorganisms in which the copy number of the DNA encoding the protein is increased compared to the parent strain include the following (Ia) and (Ib): (Ia) a microorganism in which the copy number of the DNA encoding the protein on the chromosomal DNA is increased by transforming a parent strain microorganism with a recombinant DNA containing a gene encoding the protein according to any one of the above [1] to [3]; (Ib) a microorganism in which the DNA encoding the protein is carried outside the chromosomal DNA as a plasmid DNA.

[0086] Specific examples of DNA encoding the protein described in any one of [1] to [3] above include DNA selected from the group consisting of (D1) to (D4) below: (D1) DNA encoding the protein described in any one of [1] to [3] above; (D2) DNA consisting of at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46; (D3) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46, and encodes the homologous protein described in [3] above; and (D4) DNA that consists of a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46, and encodes the homologous protein described in [3] above.

[0087] Examples of recombinant DNAs containing DNA encoding the protein according to any one of [1] to [3] include recombinant DNAs containing DNA according to any one of (D1) to (D4) above. The recombinant DNA containing DNA according to any one of (D1) to (D4) above refers to recombinant DNA in which the DNA is incorporated into an expression vector that is capable of autonomous replication in a parent strain or integration into a chromosome and contains a promoter at a position where the DNA can be transcribed.

[0088] In the above (D3), "hybridizing" means that DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part of the DNA can be used as a probe in Northern or Southern blot analysis, and can also be used as an oligonucleotide primer in PCR analysis.

[0089] The DNA used as a probe may be at least 100 bases long, preferably at least 200 bases long, more preferably at least 500 bases long. The DNA used as a primer may be at least 10 bases long, preferably at least 15 bases long.

[0090] Methods for DNA hybridization experiments are well known, and those skilled in the art can determine hybridization conditions according to the present specification. The hybridization conditions can be determined according to the methods described in Molecular Cloning, 4th Edition (2012), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology Methods Manual, Academic Press (1996), and many other standard textbooks.

[0091] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with a commercially available hybridization kit, such as the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which prepares a probe by the random prime method and hybridizes under stringent conditions.

[0092] The above-mentioned stringent conditions include incubating the DNA-immobilized filter and the probe DNA overnight at 42°C in a solution containing 50% formamide, 5xSSC (750 mmol / L sodium chloride, 75 mmol / L sodium citrate), 50 mmol / L sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, followed by washing the filter in a 0.2xSSC solution at approximately 65°C.

[0093] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA that has at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to DNA consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46, when calculated based on the above-mentioned parameters using, for example, BLAST or FASTA.

[0094] A microorganism having an increased copy number of the gene compared to the parent strain, which is obtained by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding the protein according to any one of [1] to [3] above, can be obtained by the following method.

[0095] The DNA encoding the protein of [1] described in (D1) above and the DNA described in (D2) above can be detected by using a probe DNA that can be designed based on at least one base sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46, for example, against organisms that originally have SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46, such as Beijerinckiaceae bacterium, Alphaproteobacteria bacterium, Sphingorhabdus profundilacus, Sphingobium sp. AS12, Caulobacter segnis, Caulobacter soli, Rhizobium sp. The DNA fragment can be obtained by Southern hybridization of chromosomal DNA libraries of PP-WC-1G-195, Aurantiacibacter rhizosphaerae, Novosphingobium resinovorum, Altericroceibacterium endophyticum, Homo sapiens, or Mus musculus, or by PCR using primer DNAs that can be designed based on the base sequence and the chromosomal DNA of the above microorganisms as a template [PCR Protocols, Academic Press (1990)].

[0096] Furthermore, based on the determined DNA base sequence, the desired DNA (artificially synthesized gene) can be obtained by entrusting the task to Eurofins, or an artificially synthesized gene can be prepared by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd.

[0097] The DNA encoding the mutant protein of [2] above can be obtained, for example, by subjecting a DNA consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46 to error-prone PCR or the like using as a template.

[0098] Alternatively, DNA encoding the mutant protein of the above [2] can also be obtained by PCR using a pair of PCR primers each having a nucleotide sequence at its 5' end designed to introduce a desired mutation (deletion, substitution, insertion, or addition) [Gene, 77, 51 (1989)].

[0099] Alternatively, the DNA can be obtained by following the instructions provided with a commercially available site-directed mutagenesis kit, such as the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce a mutation (deletion, substitution, insertion, or addition) at the desired site.

[0100] That is, first, a pair of mutagenesis primers is designed with a template of a plasmid having a base sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition), with a 15-base overlap on the 5' side. The overlapping portion contains the desired mutation. Next, PCR is performed using the mutagenesis primers and a template of a plasmid having the base sequence into which the desired mutation is to be introduced. The amplified fragment obtained is transformed into Escherichia coli, yielding a plasmid having the base sequence into which the desired mutation has been introduced.

[0101] Mutations may also be introduced by, for example, mutagenesis. Examples of mutagenesis include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Random mutations may also be induced in vitro by directly treating DNA with hydroxylamine.

[0102] The DNA encoding the homologous protein of [3] above and the DNAs of (D3) and (D4) above can be obtained by, for example, searching various gene sequence databases for a base sequence having an identity of 50% or more, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more with at least one base sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46, or searching various protein sequence databases for a base sequence having an identity of 50% or more, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more with at least one base sequence selected from the group consisting of SEQ ID NOs: 11 to 14. , 23 to 25, and 41 to 46, and an amino acid sequence having an identity of 50% or more, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more, in the following order, can be searched for, and a probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search, and a microorganism having the DNA can be used to obtain the DNA by a method similar to the method for obtaining the DNA described above.

[0103] Based on the DNA encoding the protein according to any one of [1] to [3] above, preferably one DNA selected from the group consisting of (D1) to (D4) above, obtained by the above method, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as needed. Furthermore, by substituting bases in the base sequence of the portion encoding the protein so that it contains codons optimal for expression in host cells, a transformant with improved production efficiency can be obtained. Information on codon usage frequencies in parent strains used in the production method of this embodiment is available from public databases.

[0104] The DNA fragment is inserted downstream of a promoter in an appropriate expression vector to prepare a recombinant DNA, which is then transformed into a parent strain to obtain a microorganism in which the copy number of the gene encoding the protein is increased compared to the parent strain.

[0105] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA is preferably a recombinant DNA comprising a promoter, a ribosome binding sequence, the DNA described in any one of (D1) to (D4) above, and a transcription termination sequence. A gene that controls the promoter may also be included.

[0106] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (ribosome binding sequence) and the initiation codon has been adjusted to an appropriate distance (for example, 6 to 18 bases). In the recombinant DNA, a transcription termination sequence is not necessarily required for expression of the DNA, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

[0107] When a microorganism belonging to the genus Escherichia is used as the parent strain, examples of the expression vector include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio Inc.), pMW118, pMW119 (all manufactured by Nippon Gene Co., Ltd.), pET21a, pCOLADuet-1, pCDFDuet-1, pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pCDF-1b, pRSF-1b (all manufactured by Novagen), pMAL-c2x, and pMA L-c5x (both manufactured by New England Biolabs), pGEX-4T-1 (manufactured by GE Healthcare Biosciences), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (both manufactured by Qiagen), pET-3, pTrc99A (manufactured by GE Healthcare Biosciences), pET-3 (manufactured by Novagen), pKYP10 (Japanese Patent Laid-Open 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)], pBluescript II SK(+), pBluescript II KS(-) (Stratagene), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, 20, p. 6378-6385], pPAC31 (WO 1998 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Bio), pUC118 (Takara Bio), pPA1 (JP 63-233798 A), pKD46 [Datsenko, K. A., Warner, B. L., Proc. Natl. Acad. Sci., USA, Vol. 97,6640-6645 (2000)], etc.

[0108] When using the above expression vector, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia. For example, the trp promoter (P ｔｒｐ ), lac promoter (P ｌａｃ ), P Ｌ Promoter, P Ｒ Promoter, P ＳＥ Examples of promoters include promoters derived from Escherichia coli or phages, such as the promoter having two Ptrp promoters in tandem, the tac promoter, the lacT7 promoter, and the letI promoter, which are artificially designed and modified.

[0109] When a microorganism belonging to the genus Corynebacterium is used as the parent strain, examples of the expression vector include pCG1 (Japanese Patent Application Laid-Open No. 57-134500), pCG2 (Japanese Patent Application Laid-Open No. 58-35197), pCG4 (Japanese Patent Application Laid-Open No. 57-183799), pCG11 (Japanese Patent Application Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Application Laid-Open No. 58-105999), pCE51, pCE52, and pCE53 (all of which are described in Molecular and General Genetics, 196, 175 (1984)).

[0110] When the above expression vector is used, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Corynebacterium, and an example thereof is the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)].

[0111] When a yeast strain is used as the parent strain, examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0112] When using the above expression vector, any promoter may be used as long as it functions in the cells of a yeast strain, and examples include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0113] The recombinant DNA used in the production method of this embodiment can be prepared by inserting the DNA fragment described in any one of (D1) to (D4) above downstream of the promoter of an appropriate expression vector.

[0114] Furthermore, when two or more genes are introduced, it is sufficient that each gene is retained in a usable state in the host. For example, all of the genes may be retained on a single expression vector, or all may be retained on a chromosome. Alternatively, each gene may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. Furthermore, two or more genes may constitute an operon and be introduced. "Introducing two or more genes" may include, for example, introducing genes encoding two or more proteins (e.g., enzymes), introducing genes encoding two or more subunits that constitute a single protein complex (e.g., an enzyme complex), and combinations thereof.

[0115] Examples of methods for integrating recombinant DNA into the chromosome of a host cell include homologous recombination. Examples of homologous recombination include a method using a homologous recombination plasmid, which can be prepared by ligating a plasmid DNA carrying a drug resistance gene that cannot autonomously replicate in the host cell to be introduced. Examples of methods using homologous recombination that are frequently used in Escherichia coli include a method using the lambda phage homologous recombination system to introduce recombinant DNA [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)]. Other selection methods include a method that utilizes the sucrose sensitivity of Escherichia coli mediated by Bacillus subtilis levansucrase integrated into the chromosome together with the recombinant DNA, and a selection method that utilizes the streptomycin sensitivity of Escherichia coli mediated by integration of a wild-type rpsL gene into Escherichia coli carrying a streptomycin-resistant mutant rpsL gene [Mol. Microbiol. , 55, 137 (2005), Biosci. Biotechnol. Biochem. , 71, 2905 (2007)], or the like, it is possible to obtain E. coli in which a target region on the chromosomal DNA of the host cell has been replaced with recombinant DNA.

[0116] Whether the recombinant DNA has been introduced into the parent strain as an autonomously replicable plasmid or incorporated into the chromosome of the parent strain can be confirmed, for example, by a method in which the gene originally present on the chromosomal DNA of a microorganism cannot be amplified, but the gene introduced by transformation can be amplified, and the amplified product can be confirmed by PCR.

[0117] Furthermore, whether the transcription amount of the DNA or the production amount of the protein encoded by the DNA has been increased can be confirmed by comparing the transcription amount of the gene in the microorganism with that of the parent strain by Northern blotting, or the production amount of the protein in the microorganism by Western blotting or polyacrylamide electrophoresis.

[0118] Whether the microorganism constructed by the above method is a microorganism in which the activity of the protein described in any one of [1] to [3] above is enhanced compared to the parent strain can be confirmed by culturing the microorganism, diluting the culture solution appropriately, centrifuging it, and analyzing the sugars having a GlcNAc-β1,6-Gal structure contained in at least one of the supernatant and the bacterial cells using an analyzer or the like, and comparing them with those of the parent strain.

[0119] Furthermore, whether the microorganism constructed by the above method is a microorganism in which the activity of the protein described in any one of [1] to [3] above is enhanced compared to the parent strain can also be confirmed by measuring the amount of the protein produced by the microorganism and the amount of the protein produced by the parent strain of the microorganism by Western blotting and comparing the results.

[0120] The above-mentioned microorganisms have enhanced activity of the protein described in any one of [1] to [3] above compared to the parent strain, and thus can improve productivity of sugars having a GlcNAc-β1,6-Gal structure. An example of such a microorganism is the strain obtained by introducing 10 types of bacterial β1,6GlcNAcT into an LNnT-producing bacterium, as described below in Example 3.

[0121] 2. Method for Producing a Saccharide Having a GlcNAc-β1,6-Gal Structure The method for producing a saccharide having a GlcNAc-β1,6-Gal structure of this embodiment (hereinafter also referred to as the method of this embodiment) is a method for producing a saccharide having a GlcNAc-β1,6-Gal structure using a protein represented by at least one selected from the following [1] to [3]: [1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids are deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

[0122] One aspect of the method of this embodiment is a method in which the sugar having a GlcNAc-β1,6-Gal structure is at least one selected from GlcNAc-β1,6-Lac, GlcNAc-β1,6-LNBII, and GlcNAc-β1,6-LNTII, and a protein represented by at least one selected from the following [a1] to [a3] is used: [a1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53; [a2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; [a3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose.

[0123] Another aspect of the method of the present embodiment includes a method using a protein represented by at least one protein selected from the group consisting of GlcNAc-β1,6-LNT, GlcNAc-β1,6-LNnT, Gal-β1,4-LNBII-β1,6GlcNAc, and Gal-β1,3-LNBII-β1,6-GlcNAc, and at least one protein selected from the following [b1] to [b3]: [b1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60. [b2] A protein consisting of an amino acid sequence in which 1 to 40 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [b3] A protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

[0124] Specific aspects of the method of this embodiment include an in vivo method and an in vitro method, which will be described below.

[0125] (In vivo method) In the method of this embodiment, the in vivo method is a method in which a target product is produced within a microorganism using a microorganism in a growing or growing state. The microorganism used in the in vivo method is preferably the genetically modified microorganism of this embodiment described above. An example of an in vivo method is a fermentation method. The fermentation method produces a target substance by utilizing the functions of the growth process of a microorganism, and involves bacterial cell growth.

[0126] In this embodiment, the in vivo method is preferably a method comprising the following steps (x1) and (x2): (x1) preparing a genetically modified microorganism in which the activity of at least one protein selected from the above [1] to [3] is enhanced; and (x2) producing a sugar having a GlcNAc-β1,6-Gal structure in at least one of the culture supernatant and the intracellular space using the genetically modified microorganism prepared in (x1). In the method of this embodiment, the galactose-containing acceptor substrate used may be the galactose-containing acceptor substrate itself added to the medium during culture, or, when a microorganism capable of producing a galactose-containing acceptor substrate from its precursor sugar is used, the galactose-containing acceptor substrate may be supplied by adding the precursor sugar.

[0127] Sugars that can be precursors to galactose-containing acceptor substrates include, but are not limited to, sugars such as glucose, fructose, lactose, sucrose, molasses containing these, starch, or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol, or propanol.

[0128] In this embodiment, the microorganism can be cultured according to a conventional method for culturing a microorganism. The medium for culturing the microorganism may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism and allows the transformant to be cultured efficiently.

[0129] The carbon source may be any that can be assimilated by the microorganism, and examples thereof include sugars such as glucose, fructose, sucrose, molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as glycerol, ethanol, and propanol.

[0130] Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria and digested products thereof, and the like.

[0131] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like.

[0132] The microorganism of this embodiment used in the method of this embodiment may be a microorganism capable of producing an acceptor carbohydrate such as glucose, lactose, lactose monohydrate, etc. In this production method, glucose, lactose, lactose monohydrate, etc. may be added to the medium during cultivation.

[0133] In the method of this embodiment, instead of adding glucose, lactose, lactose monohydrate, or the like to the medium during cultivation, glucose, lactose, lactose monohydrate, or the like may be supplied to the microorganism of this embodiment by simultaneously culturing a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like from sugar with the microorganism of this embodiment.

[0134] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. The culture temperature is usually 30 to 37°C, and the culture time is usually 24 hours to 3 days. The pH of the culture solution during cultivation is usually maintained at 6.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0135] By culturing as described above, sugars having a GlcNAc-β1,6-Gal structure can be produced in the culture, thereby producing sugars having a GlcNAc-β1,6-Gal structure. Furthermore, by analyzing the culture with a liquid chromatograph mass spectrometer (e.g., LCMS-8040 manufactured by Shimadzu Corporation), it is possible to confirm and quantify the production of sugars having a GlcNAc-β1,6-Gal structure.

[0136] By the above-mentioned cultivation, sugars having a GlcNAc-β1,6-Gal structure are produced and accumulated in the culture, and by collecting the sugars having a GlcNAc-β1,6-Gal structure from the culture, sugars having a GlcNAc-β1,6-Gal structure can be produced.

[0137] Usually, after centrifugation of the culture, sugars having a GlcNAc-β1,6-Gal structure can be collected from the supernatant. When sugars having a GlcNAc-β1,6-Gal structure accumulate in the cells, the cells can be disrupted by, for example, ultrasonication, and centrifuged to remove the cells. From the resulting supernatant, sugars having a GlcNAc-β1,6-Gal structure can be collected by an ion exchange resin method or the like.

[0138] (In vitro method) In this embodiment, the in vitro method refers to a method in which a culture obtained by culturing a microorganism or a processed product of the culture is used as an enzyme source, the enzyme source, an acceptor substrate, and UDP-GlcNAc are present in an aqueous medium, and a sugar having a GlcNAc-β1,6-Gal structure is produced by an enzymatic reaction. Among these methods, the in vitro method is called a microbial cell reaction method, which uses a reaction system in which microbial cells in a dormant or stationary state without growth are used as a catalyst (also called an enzyme source). However, the form of the reaction between the microorganism and the acceptor substrate is not limited to the specific method described below.

[0139] The in vitro method preferably comprises the following steps (y1) and (y2): (y1) preparing an enzyme source containing at least one protein selected from the group consisting of [1] to [3], an acceptor substrate, and UDP-GlcNAc; and (y2) allowing the enzyme source containing at least one protein selected from the group consisting of [1] to [3], the acceptor substrate, and UDP-GlcNAc prepared in (y1) to exist in an aqueous medium, and producing a sugar having a GlcNAc-β1,6-Gal structure by an enzymatic reaction.

[0140] The enzyme source containing at least one protein selected from [1] to [3] in step (y1) is a culture obtained by culturing the genetically modified microorganism of the present embodiment described above, or a processed product of the culture. Examples of the culture or processed product of the culture include a concentrate of the culture, a dried product of the culture, bacterial cells obtained by centrifuging the culture, a dried product of the bacterial cells, a lyophilized product of the bacterial cells, a surfactant-treated product of the bacterial cells, an ultrasonically-treated product of the bacterial cells, a mechanically ground product of the bacterial cells, a solvent-treated product of the bacterial cells, an enzyme-treated product of the bacterial cells, a protein fraction of the bacterial cells, an immobilized product of the bacterial cells, and an isolated and purified purified enzyme obtained by extraction from the bacterial cells. Among these, a solvent-treated product of the bacterial cells and an isolated and purified purified enzyme obtained by extraction from the bacterial cells are preferred.

[0141] Examples of aqueous media include water, buffer solutions such as phosphates, carbonates, acetates, borates, citrates, Tris, etc., alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, amides such as acetamide, etc. Another example of an aqueous medium is the culture medium of the microorganism used as the enzyme source.

[0142] In this embodiment, the microorganisms can be cultured according to a conventional method used for culturing microorganisms, similar to the method described above in the (in vivo method).

[0143] When extracting an enzyme source containing a protein represented by at least one selected from the above [1] to [3] from a culture of a microorganism, for example, the microorganism can be separated from the medium by centrifugation, and the microorganism can be disrupted to extract the protein represented by at least one selected from the above [1] to [3].

[0144] The isolated and purified enzyme used in the "in vitro method" can be obtained using the genetically modified microorganism of this embodiment described above, but the following method is more preferred. Recombinant DNA is prepared, which contains DNA encoding a protein represented by at least one of [1] to [3] above and a tag sequence for enzyme purification. Next, the microorganism obtained by transforming the recombinant DNA is cultured, and the protein is prepared from the resulting culture as a purified enzyme.

[0145] Tag sequences for the purification of such enzymes are described in the literature and known to those skilled in the art. Examples are sequences that can be used to (affinity) purify the polypeptide chain (see, e.g., 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.

[0146] For example, a His tag (polyhistidine tag) is a polyhistidine amino acid motif in proteins, typically consisting of at least six histidine (His) residues, often located at the N- or C-terminus of the 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, of which various types are commercially available.

[0147] These resins are generally Sepharose / agarose functionalized with chelators such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxymethylaspartic acid (Co-CMA) for cobalt, to which polyhistidine tags bind with micromolar affinity. The resin is then typically washed with phosphate buffer to remove proteins that do not specifically interact with cobalt or nickel ions. For Ni-based methods, the addition of 20 mM imidazole can improve wash efficiency (proteins are typically eluted at 150-300 mM imidazole).

[0148] The solvent-treated bacterial cells used in the "in vitro method" can be obtained using the genetically modified microorganism of this embodiment described above, and it is more preferable to use, for example, xylene as the solvent.

[0149] Using the enzyme source, an acceptor substrate and UDP-GlcNAc are reacted in an aqueous medium, and the reaction product is then analyzed using a liquid chromatograph mass spectrometer (e.g., LCMS-8040 manufactured by Shimadzu Corporation), whereby it is possible to confirm and quantify the production of a sugar having a GlcNAc-β1,6-Gal structure.

[0150] The sugar having the GlcNAc-β1,6-Gal structure can be isolated and purified by an ion exchange resin method, HPLC, or the like to obtain the sugar having the GlcNAc-β1,6-Gal structure.

[0151] As explained above, the present specification discloses the following configurations: 1. A genetically modified microorganism in which the activity of a protein represented by at least one selected from the following [1] to [3] is enhanced and which produces a sugar having a GlcNAc-β1,6-Gal structure: [1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60; [2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; or [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose. 2. The genetically modified microorganism according to 1 above, which produces a sugar having a GlcNAc-β1,6-Gal structure using lactose (Lac) or lacto-N-biose II (LNBII) as an acceptor substrate. 3. The genetically modified microorganism according to 1 or 2 above, which has enhanced activity of a protein represented by at least one of the following [a1] to [a3] and produces a sugar having a GlcNAc-β1,6-Gal structure using Lac, LNBII, or LNTII (lacto-N-triose II) as an acceptor substrate.[a1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53. [a2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [a3] A protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 4. 2. The genetically modified microorganism according to 1, wherein the activity of a protein represented by at least one selected from the following [b1] to [b3] is enhanced, and the genetically modified microorganism produces a sugar having a GlcNAc-β1,6-Gal structure using at least one acceptor substrate selected from LNnT (lacto-N-neotetraose), LNT (lacto-N-tetraose), Gal-β1,4-LNBII, and Gal-β1,3-LNBII: [b1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60; [b2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose; [b3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 5. A method for producing a sugar having a GlcNAc-β1,6-Gal structure using a protein represented by at least one selected from the following [1] to [3].[1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60; [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose. 6. A method for producing the sugar described in 5 above, comprising preparing a genetically modified microorganism in which the activity of at least one protein represented by at least one selected from [1] to [3] above has been enhanced, and using the genetically modified microorganism to produce a sugar having a GlcNAc-β1,6-Gal structure in at least one of a culture supernatant and intracellular space. A method for producing the sugar according to 5 above, comprising bringing an enzyme source containing a protein represented by at least one selected from [1] to [3] above, an acceptor substrate, and UDP-GlcNAc into the presence of an aqueous medium, and producing a sugar having a GlcNAc-β1,6-Gal structure by an enzymatic reaction. 8. A method for producing a sugar having a GlcNAc-β1,6-Gal structure using at least one protein represented by at least one selected from [1] to [3] below, and using Lac, LNBII, or LNTII (lacto-N-triose II) as an acceptor substrate:[1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. 9. A method for producing a sugar having a GlcNAc-β1,6-Gal structure using at least one protein selected from the following [1] to [3], and at least one acceptor substrate selected from LNnT (lacto-N-neotetraose), LNT (lacto-N-tetraose), Gal-β1,4-LNBII, and Gal-β1,3-LNBII: [1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

[0152] [Analysis Examples] In the Examples, β1,6-GlcNAcT activity was evaluated according to the following procedure. The analytical fractions after the reaction were analyzed using a liquid chromatograph mass spectrometer LCMS-8040 (Shimadzu Corporation). In Examples 1 and 2, the reaction solution after the enzyme reaction was centrifuged, and the supernatant was recovered and diluted 50-fold, followed by filtration to obtain the analytical fraction. In Example 3, the reaction solution after cultivation was collected, treated with an ultrasonic homogenizer (Biorputor II [(BM Kiki Co., Ltd.) (hotting conditions: Power High, ON / OFF 10 sec, total 30 min)], and then centrifuged. The reaction supernatant was collected, diluted 50-fold, and filtered to obtain an analytical fraction. The standard used for the qualitative analysis of GlcNAc-β1,6-LNTII was prepared by treating GlcNAc-β1,6-LNnT (synthesis contracted to Medical Science Pharmaceutical Co., Ltd.) with β1-4 Galactosidase.

[0153] [Analysis conditions] Column: ICSep Coregel-87H3 Column 7.8 x 300 mm Column temperature: 40°C Mobile phase: 0.1% formic acid water, isocratic elution Measurement time: 25 min Flow rate: 0.4 mL / min Injection volume: 10 μL Detection: SIM mode [M+H]+ positive, [M-H]- negative (peaks used in the analysis results are detected values ​​only in negative mode)

[0154] Examples of the invention are shown below, but the present invention is not limited to these examples.

[0155] Example 1: Construction of evaluation strains for four types of bacterial β1,6-GlcNAcT and activity evaluation (1) Construction of a strain expressing bacterial β1,6-GlcNAcT Using the expression vector pColdI (Takara) containing an N-terminal His tag sequence controlled by the cspA promoter and lac operator as a template, and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 1 and 2 as a primer set, a PCR reaction was performed to obtain fragment 1. Genes encoding the four types of bacterial β1,6-GlcNAcT (BbGlcNAcT, AbGlcNAcT, SpGlcNAcT, SaGlcNAcT) were purchased from Eurofins as artificially synthesized genes optimized for E. coli codons.

[0156] To obtain each gene fragment, PCR reactions were performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 3 to 10 as primer sets. Primers 3, 5, 7, and 9 have the nucleotide sequence of 15 residues from the 3' end of pColdI fragment 1, including the N-terminal His tag, added to the 5' end of the gene region of SEQ ID NOs: 11, 12, 13, and 14. Primers 4, 6, 8, and 10 have the nucleotide sequence complementary to the 15 residues from the 5' end of pColdI fragment 1 added to the 5' end of the nucleotide sequence complementary to the sequence containing the stop codon of the gene region of SEQ ID NOs: 11, 12, 13, and 14. A description of the primer sets is shown in Table 2.

[0157]

[0158] The amplification product obtained by PCR and pColdI fragment 1 were each reacted at 50°C for 15 minutes using an In-fusion Kit (Takara Bio Inc.), and ligated to the expression vector pColdI to obtain expression plasmids pColdI-BbGlcNAcT, pColdI-AbGlcNAcT, pColdI-SpGlcNAcT, and pColdI-SaGlcNAcT, which have the nucleotide sequences shown in SEQ ID NOs: 11 to 14 (amino acid sequences 47 to 50).

[0159] The expression plasmids obtained above were used to transform Escherichia coli BL21(DE3) as a host, to obtain BL21(DE3) / pColdI-BbGlcNAcT, BL21(DE3) / pColdI-AbGlcNAcT, BL21(DE3)pColdI-SpGlcNAcT, and BL21(DE3) / pColdI-SaGlcNAcT.

[0160] (2) Obtaining Bacterial β1,6-GlcNAcT Enzyme: BL21(DE3) / pColdI-BbGlcNAcT, BL21(DE3) / pColdI-AbGlcNAcT, BL21(DE3) / pColdI-SpGlcNAcT, and BL21(DE3) / pColdI-SaGlcNAcT obtained by the above procedure were inoculated into test tubes containing 2 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C for 16 hours. The culture broth was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of LB medium containing 100 mg / L ampicillin and cultured at 37°C for 5 hours, after which IPTG was added to a final concentration of 0.5 mM and the mixture was further cultured at 15°C for 19 hours. The culture medium was centrifuged to obtain wet cells, and each His-tagged recombinant enzyme was purified using TALON (registered trademark) Metal Affinity Resin (Clontech).

[0161] (3) Evaluation of β1,6-GlcNAc transfer activity toward each acceptor substrate (in vitro) A reaction mixture was prepared containing 0.1 g / L of each His-tagged recombinant enzyme obtained by the procedure in (2) above, 50 mM MES (pH 6.5), 10 mM KCl, 1 mM MgCl, 5 g / L of donor substrate UDP-GlcNAc, and 10 g / L of LNnT or LNTII as an acceptor substrate, and the reaction was carried out at 37°C, 650 rpm, and for 16 hours. As a control, a reaction mixture in which the enzyme was replaced with MiliQ was prepared.

[0162] After the reaction was completed, analytical fractions were collected and the reaction products were analyzed using the same method as in the analytical example. Branched-chain HMOs (GlcNAc-β1,6-LNTII and GlcNAc-β1,6-LNnT) were detected by MS at m / z 747.30(-) and m / z 909.30(-), respectively. The analytical results are shown in Tables 3 and 4 and Figures 4 and 5.

[0163]

[0164]

[0165] As shown in Table 3 and Figure 4, four bacterial β1,6-GlcNAcTs, BbGlcNAcT, AbGlcNAcT, SpGlcNAcT, and SaGlcNAcT, exhibited GlcNAc-β1,6 transfer activity toward LNnT, producing GlcNAc-β1,6-LNnT.

[0166] On the other hand, as shown in Table 4 and Figure 5, four bacterial β1,6-GlcNAcTs, BbGlcNAcT, AbGlcNAcT, SpGlcNAcT, and SaGlcNAcT, did not exhibit GlcNAc-β1,6-transferase activity toward LNTII. Therefore, it was revealed that the four bacterial β1,6-GlcNAcTs (SEQ ID NOS: 47, 48, 49, and 50) exhibit GlcNAc-β1,6-transferase activity with substrate selectivity toward LNnT.

[0167] Example 2: Construction of hIGnT, mGCNT1, mGCNT3, and mIGnT evaluation strains and activity evaluation (1) Construction of hIGnT, mGCNT1, mGCNT3, and mIGnT expression strains. Using the expression vector pColdI (Takara) containing an N-terminal His tag sequence controlled by the cspA promoter and lac operator as a template, and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 1 and 2 as a primer set, PCR was performed to obtain fragment 1. The hIGnT gene was an artificially synthesized gene purchased from Eurofins. SEQ ID NO: 15 represents the addition of a 15-residue base sequence from the 3' end of pColdI fragment 1, including the N-terminal His tag, to the 5' end of the region containing the start codon of the hIGnT gene.

[0168] SEQ ID NO: 16 is obtained by adding, to the 5' end of a nucleotide sequence complementary to a sequence including the stop codon of the hIGnT gene, a nucleotide sequence complementary to 15 residues from the 5' end of pColdI fragment 1. To obtain the hIGnT gene fragment, a PCR reaction was carried out using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 15 and 16 as a primer set.

[0169] mGCNT1 was purchased from Eurofins after E. coli codon optimization of an artificially synthesized gene. The mIGnT and mGCNT3 genes were amplified using brain-derived mouse cDNA (Cosmo Bio) and duodenum-derived mouse cDNA (Cosmo Bio), respectively. SEQ ID NOs: 17, 21, and 19 represent sequences in which a 15-residue base sequence from the 3' end of pColdI fragment 1, including an N-terminal His tag, is added to the 5' end of the mGCNT1, mGCNT3, and mIGnT gene regions, respectively. SEQ ID NOs: 18, 22, and 20 represent sequences in which a base sequence complementary to the 15-residue base sequence from the 5' end of pColdI fragment 1 is added to the 5' end of a base sequence complementary to a sequence containing the stop codon of the mGCNT1, mGCNT3, and mIGnT genes, respectively.

[0170] To obtain fragments of the three genes mGCNT1, mGCNT3 and mIGnT, PCR was carried out using oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 17, 18, 19, 20, 21 and 22 as a primer set and the artificially synthesized genes and each cDNA as templates.

[0171] The amplification product obtained by PCR and pColdI fragment 1 were each reacted at 50°C for 15 minutes using an In-fusion Kit (Takara Bio Inc.) and ligated to the expression vector pColdI to obtain expression plasmids pColdI-hIGnT, pColdI-mGCNT1, pColdI-mGCNT3, and pColdI-mIGnT having the base sequences shown in SEQ ID NOs:23 to 26 (amino acid sequences 51 to 54).

[0172] The expression plasmids obtained above were transformed into SHuffle T7 Express Competent E. coli (New England Biolabs) as a host to obtain SHuffle T7 / pColdI-hIGnT, SHuffle T7 / pColdI-mGCNT1, SHuffle T7 / pColdI-mGCNT3, and SHuffle T7 / pColdI-mIGnT.

[0173] (2) Enzyme acquisition of hIGnT, mGCNT1, mGCNT3, mIGnT The SHuffle T7 / pColdI-hIGnT, SHuffle T7 / pColdI-mGCNT1, SHuffle T7 / pColdI-mGCNT3, and SHuffle T7 / pColdI-mIGnT obtained by the above procedure were inoculated into a test tube containing 2 mL of LB medium containing 100 mg / L of ampicillin and cultured for 16 hours at 30 ° C. with shaking. The culture was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of LB medium containing 100 mg / L of ampicillin and cultured at 37 ° C. for 5 hours, after which IPTG was added to a final concentration of 0.5 mM, and the mixture was further cultured at 15 ° C. for 19 hours. The culture medium was centrifuged to obtain wet cells, and His-tagged recombinant enzymes (hIGnT, mGCNT1, mGCNT3, mIGnT) were purified using TALON (registered trademark) Metal Affinity Resin (Clontech) according to the manufacturer's instructions.

[0174] (3) Evaluation of GlcNAc-β1,6 transfer activity for each acceptor substrate (in vitro) The His-tagged recombinant enzyme obtained by the above procedure was used in a 0.2-0.5 g / L solution containing 50 mM MES (pH 6.5), 10 mM KCl, and MgCl ２ A reaction solution was prepared containing 1 mM donor substrate UDP-GlcNAc at 5 g / L and acceptor substrate LNTII or LNnT at 10 g / L, and the reaction was carried out at 37°C, 850 rpm, for 16 hours. The enzyme amounts used were 0.5 g / L for hIGnT, mGCNT1, and mIGnT, and 0.2 g / L for mGCNT3. As a control, a reaction solution in which the enzyme was replaced with MilliQ (MilliQ+ reaction solution) was prepared.

[0175] After the reaction was completed, analytical fractions were collected and the reaction products were analyzed using the same method as in the analytical example. Branched-chain HMOs (GlcNAc-β1,6-LNTII and GlcNAc-β1,6-LNnT) were detected by MS at m / z 747.30(-) and m / z 909.30(-), respectively. The analytical results are shown in Tables 5 to 7 and Figures 6 to 8.

[0176]

[0177]

[0178]

[0179] As shown in Tables 5 to 7 and Figures 6 to 8, mIGnT exhibited GlcNAc-β1,6-transferase activity toward LNnT and LNTII, producing GlcNAc-β1,6-LNnT and GlcNAc-β1,6-LNTII. Furthermore, mGCNT1 and mGCNT3 exhibited GlcNAc-β1,6-transferase activity toward LNTII, producing GlcNAc-β1,6-LNTII, but did not exhibit GlcNAc-β1,6-transferase activity toward LNnT. Furthermore, hIGnT, which has previously been reported to exhibit GlcNAc-β1,6-transferase activity toward LNnT, was shown to also exhibit GlcNAc-β1,6-transferase activity toward LNTII.

[0180] Therefore, it was revealed that mGCNT1 (SEQ ID NO: 52) and mGCNT3 (SEQ ID NO: 53) have substrate selectivity, do not exhibit GlcNAc-β1,6-transferase activity toward LNnT, but exhibit GlcNAc-β1,6-transferase activity toward LNTII. The sequence identity between mGCNT1 (SEQ ID NO: 52) and mGCNT3 (SEQ ID NO: 53), which exhibit GlcNAc-β1,6-transferase activity toward LNTII, is 53%. Furthermore, it was revealed that hIGnT (SEQ ID NO: 51) exhibits GlcNAc-β1,6-transferase activity not only toward LNnT but also toward LNTII. The full-length sequence of SEQ ID NO: 51 is SEQ ID NO: 76, the full-length sequence of SEQ ID NO: 52 is SEQ ID NO: 70, and the full-length sequence of SEQ ID NO: 53 is SEQ ID NO: 71. When enhancing proteins consisting of amino acid sequences represented by these sequences, the full-length sequence may be used, or sequences with the N-terminus or C-terminus appropriately deleted to increase expression may be used.

[0181] Proteins consisting of the amino acid sequences represented by SEQ ID NOs: 52 and 53 are particularly highly active and are particularly preferred. Proteins consisting of the amino acid sequences represented by SEQ ID NOs: 52 and 53 are particularly preferred because they react with LNTII and can be used to produce both saccharides having an LNH structure and saccharides having an LNnH structure.

[0182] Example 3 Construction of strains for evaluating the GlcNAc-β1,6-transferase activity of 10 types of bacterial-derived β1,6-GlcNAcT and evaluation of activity (1) Construction of strains for evaluating the GlcNAc-β1,6-transferase activity of bacterial-derived β1,6-GlcNAcT in LNTII and LNnT-producing bacteria Using pSTV29-rcsA of SEQ ID NO: 61 as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 27 and 28 as a primer set, a PCR reaction was performed to obtain a pSTV29 fragment. Genes encoding 10 bacterial β1,6-GlcNAcTs (BbGlcNAcT, AbGlcNAcT, SpGlcNAcT, SaGlcNAcT, CseGlcNAcT, CsoGlcNAcT, RsGlcNAcT, ArGlcNAcT, NrGlcNAcT, and AeGlcNAcT) were artificially synthesized and purchased from Eurofins.

[0183] SEQ ID NOs: 29, 31, 33, 35, 37, 39, 62, 64, 66, and 68 represent sequences in which the nucleotide sequence of the N-terminal 15 residues of the pSTV29 fragment has been added to the 5' end of the region containing the start codon of the β1,6-GlcNAcT genes derived from ten species of bacteria. SEQ ID NOs: 30, 32, 34, 36, 38, 40, 63, 65, 67, and 69 represent sequences in which the nucleotide sequence complementary to the 15 residues from the 5' end of the pSTV29 fragment has been added to the 5' end of the nucleotide sequence complementary to the sequence containing the stop codon of the β1,6-GlcNAcT genes derived from ten species of bacteria (SEQ ID NOs: 41, 42, 43, 44, 45, 46, 11, 12, 13, and 14).

[0184] To obtain β1,6-GlcNAcT gene fragments derived from 10 types of bacteria, PCR reactions were performed using oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 29, 31, 33, 35, 37, 39, 62, 64, 66, 68, 30, 32, 34, 36, 38, 40, 63, 65, 67, and 69 as a primer set and an artificially synthesized gene as a template.

[0185] The PCR amplification product and the pSTV29 fragment were each subjected to infusion. The resulting products were reacted at 50°C for 15 minutes using a PCR Kit (Takara Bio Inc.) and ligated to the expression vector pSTV29 to obtain expression plasmids pSTV29-CseGlcNAcT, pSTV29-CsoGlcNAcT, pSTV29-RsGlcNAcT, pSTV29-ArGlcNAcT, pSTV29-NrGlcNAcT, pSTV29-AeGlcNAcT, pSTV29-BbGlcNAcT, pSTV29-AbGlcNAcT, pSTV29-SpGlcNAcT, and pSTV29-SaGlcNAcT, each having the nucleotide sequences represented by SEQ ID NOs: 41 to 46 (amino acid sequences: SEQ ID NOs: 55 to 60) and the nucleotide sequences represented by SEQ ID NOs: 11 to 14 (amino acid sequences: SEQ ID NOs: 47 to 50).

[0186] The expression plasmid obtained above was used to transform an LNTII-producing bacterium (construction method according to WO2023120615) as a host, to obtain strains in which β1,6-GlcNAcT derived from 10 types of bacteria was introduced into the LNTII-producing bacterium.

[0187] Similarly, the expression plasmid obtained above was used to transform an LNnT-producing bacterium (construction method according to WO2023153461) as a host, to obtain strains in which β1,6-GlcNAcT derived from 10 types of bacteria was introduced into the LNnT-producing bacterium.

[0188] (2) Evaluation of GlcNAc-β1,6-transferase activity of β1,6-GlcNAcT derived from 10 types of bacteria in LNTII and LNnT-producing bacteria (in vivo) Each strain constructed in Example 3 (1) above was cultured on an LB plate containing 100 mg / L kanamycin and 25 mg / L chloramphenicol at 37°C for 16 hours, and then inoculated into a 14 mL plastic tube containing 2 mL of LB medium containing 100 mg / L kanamycin and 25 mg / L chloramphenicol, followed by shaking culture at 30°C for 16 hours.

[0189] Thereafter, 0.2 mL of the resulting culture broth was inoculated into a large test tube containing 4 mL of a production medium containing 100 mg / L of kanamycin and 25 mg / L of chloramphenicol [glucose 30 g / L, lactose monohydrate 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 acids 5 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L (for components other than glucose, lactose monohydrate, and magnesium sulfate heptahydrate, the pH was adjusted to 7.2 with aqueous sodium hydroxide solution and then autoclaved) (aqueous solutions containing glucose, lactose monohydrate, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and then mixed)], and the mixture was cultured with shaking at 30°C for 27 hours.

[0190] Five hours after the start of culture, IPTG was added to a final concentration of 1 mM. After completion of the reaction, analytical fractions were obtained and the reaction products were analyzed using the same method as in the analytical example. As a control for evaluation, a strain (ctrl) in which a plasmid without β1,6-GlcNAcT represented by SEQ ID NO: 61 was introduced into the LNnT-producing bacterium was used.

[0191] Branched-chain HMOs (GlcNAc-β1,6-LNTII and GlcNAc-β1,6-LNnT) were detected by MS at m / z 747.30(-) and m / z 909.30(-), respectively. No production of GlcNAc-β1,6-LNTII was confirmed in strains in which β1,6-GlcNAcTs derived from ten bacteria were introduced into LNTII-producing bacteria. The results of evaluating GlcNAc-β1,6-transferase activity in strains in which β1,6-GlcNAcTs derived from ten bacteria were introduced into LNnT-producing bacteria are shown in Table 8 and FIG. 9 .

[0192]

[0193] As shown in Table 8 and Figure 9, the production of GlcNAc-β1,6-LNnT was observed, and it was revealed that even under in vivo conditions, β1,6-GlcNAcT derived from 10 species of bacteria did not exhibit GlcNAc-β1,6-transferase activity toward LNTII, but did exhibit GlcNAc-β1,6-transferase activity toward LNnT.

[0194] Furthermore, BbGlcNAcT (SEQ ID NO: 47), AbGlcNAcT (SEQ ID NO: 48), SpGlcNAcT (SEQ ID NO: 49), SaGlcNAcT (SEQ ID NO: 50), CseGlcNAcT (SEQ ID NO: 55), CsoGlcNAcT (SEQ ID NO: 56), RsGlcNAcT (SEQ ID NO: 57), ArGlcNAcT (SEQ ID NO: 58), NrGlcNAcT (SEQ ID NO: 59), and AeGlcNAcT (SEQ ID NO: 60) are particularly preferred because they have substrate selectivity for LNnT. Furthermore, the amino acid sequences of these 10 bacterial β1,6-GlcNAcTs that have substrate selectivity for LNnT share 52.2% to 78.4% identity.

[0195] Furthermore, it was shown that CseGlcNAcT (SEQ ID NO: 55), CsoGlcNAcT (SEQ ID NO: 56), RsGlcNAcT (SEQ ID NO: 57), ArGlcNAcT (SEQ ID NO: 58), and AeGlcNAcT (SEQ ID NO: 60) produced 3.8 times or more the amount of GlcNAc-β1,6-LNnT compared to AbGlcNAcT (SEQ ID NO: 48), the strain with the lowest activity, and proteins consisting of the amino acid sequences represented by SEQ ID NOs: 55, 56, 57, 58, and 60 are particularly highly active and are particularly preferred. Furthermore, the amino acid sequences of these five particularly highly active bacterial β1,6-GlcNAcTs share 52.2% to 78.4% identity.

[0196] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2024-056438) filed on March 29, 2024, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0197] SEQ ID NO: 1: Nucleotide sequence of pColdI(fw) SEQ ID NO: 2: Nucleotide sequence of pColdI(rv) SEQ ID NO: 3: Nucleotide sequence of BbClcNAcT(fw) SEQ ID NO: 4: Nucleotide sequence of BbClcNAcT(rv) SEQ ID NO: 5: Nucleotide sequence of AbClcNAcT(fw) SEQ ID NO: 6: Nucleotide sequence of AbClcNAcT(rv) SEQ ID NO: 7: Nucleotide sequence of SpGlcNAcT(fw) SEQ ID NO: 8: Nucleotide sequence of SpGlcNAcT(rv) SEQ ID NO: 9: Nucleotide sequence of SaGlcNAcT(fw) SEQ ID NO: 10: Nucleotide sequence of SaGlcNAcT(rv) SEQ ID NO: 11: Nucleotide sequence of BbGlcNAcT SEQ ID NO: 12: Nucleotide sequence of AbGlcNAcT SEQ ID NO: 13: Nucleotide sequence of SpGlcNAcT SEQ ID NO: 14: Nucleotide sequence of SaGlcNAcT SEQ ID NO: 15: Nucleotide sequence of pColdI-IGnTB(fw) SEQ ID NO: 16: Nucleotide sequence of pColdI-IGnTB(Rv) SEQ ID NO: 17: Nucleotide sequence of pcoldI_MmGCNT1_Fw SEQ ID NO: 18: Nucleotide sequence of pcoldI_MmGCNT1_Rv SEQ ID NO: 19: Nucleotide sequence of pcoldI_mIGnT_Fw SEQ ID NO: 20: Nucleotide sequence of pcoldI_mIGnT_Rv SEQ ID NO: 21: Nucleotide sequence of pcoldI_MmGCNT3_Fw SEQ ID NO: 22: Nucleotide sequence of pcoldI_MmGCNT3_Rv SEQ ID NO: 23: Nucleotide sequence of hIGnT (ΔN25) SEQ ID NO: 24: Nucleotide sequence of GCNT1 (ΔN46) SEQ ID NO: 25: Nucleotide sequence of GCNT3 (ΔN57) SEQ ID NO: 26: Nucleotide sequence of mIGnT (ΔN25) SEQ ID NO: 27: Nucleotide sequence of pSTV29_Fw SEQ ID NO: 28: Nucleotide sequence of pSTV29_Rv SEQ ID NO: 29: Nucleotide sequence of pSTV29_CseGlcNAcT_Fw SEQ ID NO: 30: Nucleotide sequence of pSTV29_CseGlcNAcT_Rv SEQ ID NO: 31: Nucleotide sequence of pSTV29_CsoGlcNAcT_Fw SEQ ID NO: 32: Nucleotide sequence of pSTV29_CsoGlcNAcT_Rv SEQ ID NO: 33: Nucleotide sequence of pSTV29_RsGlcNAcT_Fw SEQ ID NO: 34: Nucleotide sequence of pSTV29_RsGlcNAcT_Rv SEQ ID NO: 35: Nucleotide sequence of pSTV29_ArGlcNAcT_Fw SEQ ID NO: 36: Nucleotide sequence of pSTV29_ArGlcNAcT_Rv SEQ ID NO: 37: Nucleotide sequence of pSTV29_NrGlcNAcT_FwSEQ ID NO: 38: Nucleotide sequence of pSTV29_NrGlcNAcT_Rv SEQ ID NO: 39: Nucleotide sequence of pSTV29_AeGlcNAcT_Fw SEQ ID NO: 40: Nucleotide sequence of pSTV29_AeGlcNAcT_Rv SEQ ID NO: 41: Nucleotide sequence of CseGlcNAcT SEQ ID NO: 42: Nucleotide sequence of CsoGlcNAcT SEQ ID NO: 43: Nucleotide sequence of RsGlcNAcT SEQ ID NO: 44: Nucleotide sequence of ArGlcNAcT SEQ ID NO: 45: Nucleotide sequence of NrGlcNAcT SEQ ID NO: 46: Nucleotide sequence of AeGlcNAcT SEQ ID NO: 47: Amino acid sequence of BbGlcNAcT SEQ ID NO: 48: Amino acid sequence of AbGlcNAcT SEQ ID NO: 49: Amino acid sequence of SpGlcNAcT SEQ ID NO: 50: Amino acid sequence of SaGlcNAcT SEQ ID NO: 51: Amino acid sequence of hIGnT (ΔN25) SEQ ID NO: 52: Amino acid sequence of GCNT1 (ΔN46) SEQ ID NO: 53: Amino acid sequence of GCNT3 (ΔN57) SEQ ID NO: 54: Amino acid sequence of mIGnT (ΔN25) SEQ ID NO: 55: Amino acid sequence of CseGlcNAcT SEQ ID NO: 56: Amino acid sequence of CsoGlcNAcT SEQ ID NO: 57: Amino acid sequence of RsGlcNAcT SEQ ID NO: 58: Amino acid sequence of ArGlcNAcT SEQ ID NO: 59: Amino acid sequence of NrGlcNAcT SEQ ID NO: 60: Amino acid sequence of AeGlcNAcT SEQ ID NO: 61: Nucleotide sequence of pSTV29-rcsA SEQ ID NO: 62: Nucleotide sequence of pSTV29_BbClcNacT(fw) SEQ ID NO: 63: Nucleotide sequence of pSTV29_BbClcNacT(rv) SEQ ID NO: 64: Nucleotide sequence of pSTV29_AbClcNAcT(fw) SEQ ID NO: 65: Nucleotide sequence of pSTV29_AbClcNAcT(rv) SEQ ID NO: 66: Nucleotide sequence of pSTV29_SpGlcNAcT(fw) SEQ ID NO: 67: Nucleotide sequence of pSTV29_SpGlcNAcT(rv) SEQ ID NO: 68: Nucleotide sequence of pSTV29_SaGlcNAcT(fw) SEQ ID NO: 69: Nucleotide sequence of pSTV29_SaGlcNAcT(rv) SEQ ID NO: 70: Amino acid sequence of GCNT1 SEQ ID NO: 71: Amino acid sequence of GCNT3 SEQ ID NO: 72: Amino acid sequence of mIGnT SEQ ID NO: 73: Nucleotide sequence of GCNT1 SEQ ID NO: 74: Nucleotide sequence of GCNT3 SEQ ID NO: 75: Nucleotide sequence of mIGnT SEQ ID NO: 76: Amino acid sequence of hIGnTSEQ ID NO: 77: Base sequence of hIGnT

Claims

1. A genetically modified microorganism that has enhanced activity of a protein represented by at least one selected from the following [1] to [3] and produces a sugar having a GlcNAc-β1,6-Gal structure: [1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60; [2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and that has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; or [3] A protein consisting of an amino acid sequence that has 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, and that has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose.

2. The genetically modified microorganism according to claim 1, which produces a sugar having a GlcNAc-β1,6-Gal structure using lactose (Lac) or lacto-N-biose II (LNBII) as an acceptor substrate.

3. The genetically modified microorganism according to claim 1, in which the activity of at least one protein selected from the following [a1] to [a3] is enhanced and which produces a sugar having a GlcNAc-β1,6-Gal structure using Lac, LNBII, or LNTII (lacto-N-triose II) as an acceptor substrate: [a1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53. [a2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, in which 1 to 40 amino acids are deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [a3] A protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence represented by one selected from SEQ ID NOs: 51 to 53, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

4. The genetically modified microorganism according to claim 1, wherein the activity of a protein represented by at least one selected from the following [b1] to [b3] is enhanced, and the genetically modified microorganism produces a sugar having a GlcNAc-β1,6-Gal structure using at least one acceptor substrate selected from LNnT (lacto-N-neotetraose), LNT (lacto-N-tetraose), Gal-β1,4-LNBII, and Gal-β1,3-LNBII: [b1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60. [b2] A protein consisting of an amino acid sequence in which 1 to 40 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [b3] A protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence represented by one selected from SEQ ID NOs: 47 to 51 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

5. A method for producing a sugar having a GlcNAc-β1,6-Gal structure using a protein represented by at least one of the following [1] to [3]: [1] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60; [2] A protein consisting of an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; or [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence represented by one selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose.

6. A method for producing the sugar according to claim 5, comprising preparing a genetically modified microorganism in which the activity of at least one protein selected from [1] to [3] above is enhanced, and producing a sugar having a GlcNAc-β1,6-Gal structure in at least one of a culture supernatant and intracellular space using the genetically modified microorganism.

7. A method for producing sugar according to claim 5, comprising bringing an enzyme source containing at least one protein selected from [1] to [3] above, an acceptor substrate, and UDP-GlcNAc into the presence of an aqueous medium, and producing sugar having a GlcNAc-β1,6-Gal structure by an enzymatic reaction.

8. A method for producing a sugar having a GlcNAc-β1,6-Gal structure using Lac, LNBII, or LNTII (lacto-N-triose II) as an acceptor substrate, by using at least one protein selected from the following [1] to [3]: [1] A protein consisting of an amino acid sequence selected from SEQ ID NOs: 47 to 53 and 55 to 60; [2] A protein consisting of an amino acid sequence selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids have been deleted, substituted, inserted, or added, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose; or [3] A protein consisting of an amino acid sequence having 50% or more identity to an amino acid sequence selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6-transferase activity toward an acceptor substrate having galactose.

9. A method for producing a sugar having a GlcNAc-β1,6-Gal structure using at least one protein selected from the following [1] to [3], and at least one acceptor substrate selected from LNnT (lacto-N-neotetraose), LNT (lacto-N-tetraose), Gal-β1,4-LNBII, and Gal-β1,3-LNBII: [1] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60. [2] A protein consisting of an amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, in which 1 to 40 amino acids are deleted, substituted, inserted or added, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose. [3] A protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence represented by 1 selected from SEQ ID NOs: 47 to 53 and 55 to 60, and which has GlcNAc-β1,6 transfer activity toward an acceptor substrate having galactose.

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