Protein having α1,2-fucosyltransferase activity and method for producing lacto-n-fucopentaose i (LNFPI)

Abstract

The purpose of the present invention is to provide: a protein that has an α1,2-fucosyltransferase activity and has excellent LNFPI productivity; and a method for producing LNFPI. The present invention provides a protein that has an activity to transfer a fucosyl group into lacto-N-tetraose (LNT) and has a specific mutation introduced therein (excluding a protein having a property such that an amino acid sequence for the protein to which a mutation is not introduced yet and an amino acid sequence for the protein to which the mutation has been introduced are the same as each other).

Description

Protein having α1,2-fucosyltransferase activity and method for producing lacto-N-fucopentaose I (LNFPI)

[0001] The present invention relates to a protein having α1,2-fucosyltransferase activity and a method for producing lacto-N-fucopentaose I (LNFPI).

[0002] Human milk oligosaccharides (HMOs) contained in human breast milk have attracted attention as prebiotic materials and have been shown to be effective in improving cognitive function development, infection prevention, and intestinal environment in infants (Non-Patent Document 1).

[0003] Lacto-N-fucopentaose I (hereinafter referred to as LNFPI) is a type of HMO, and is a pentasaccharide HMO in which fucose is α1,2-linked to the 2-position of galactose in lacto-N-tetraose (hereinafter referred to as LNT).

[0004] LNFPI is the third most abundant sugar in breast milk after 2'-fucosyllactose (hereinafter referred to as 2'FL) and lacto-N-difucohexaose (hereinafter referred to as LNDFHI), and is known to be present in a higher amount in breast milk than lacto-N-fucopentaose II (hereinafter referred to as LNFPII) and lacto-N-fucopentaose III (hereinafter referred to as LNFPIII), which are also pentasaccharides and are known to be isomers of LNFPI (Non-Patent Document 2).

[0005] LNFPI is known to have functions such as an inhibitory effect against meningitis-causing group B Streptococcus (GBS) and norovirus (Non-Patent Documents 3 and 4). In addition, Bifidobacterium infantis, which is highly prevalent in the intestines of newborns, has been shown to grow preferentially in LNFPI, and thus its prebiotic function has also attracted attention (Non-Patent Document 5).

[0006] As a method for producing LNFPI, a microbial fermentation method or an enzymatic reaction method [one-pot multienzyme (OPME) system] using α1,2-fucosyltransferase (hereinafter also abbreviated as FucT) is widely used. Non-Patent Documents 4, 5, and 6 disclose a method for producing oligosaccharides such as LNFPI by overexpressing α1,2-fucosyltransferase derived from a microorganism such as Thermosynechococcus elongatus, Sideroxydans lithotrophicus, or Helicobacter pylori in Escherichia coli, and using LNT and GDP-fucose as substrates, by a fermentation method or a continuous enzymatic reaction method.

[0007] However, in the fermentation method or continuous enzymatic reaction method, a problem with the production of LNFPI is that α1,2-fucosyltransferase reacts not only with the desired substrate LNT but also with the coexisting lactose, producing 2'FL as a by-product.

[0008] Methods for reducing by-products include an enzymatic reaction method using purified, highly purified LNT as a substrate (Non-Patent Document 5), and a method for producing LNFPI by inducing the expression of α1,2-fucosyltransferase when the initial raw material, lactose, is depleted (Non-Patent Document 6).

[0009] Int. J. Pediatrics (2019), Article ID 2390240Nutr. Rev. (2017) 75, 920-933J. Biol. Chem. (2017) 292 (27) 11243-11249J. Biotechnol. (2020) 318, 31-38Chem. Commun. (2016) 52, 3899-3902 Bioorganic & Medicinal Chemistry 23 (2015) 6799-6806

[0010] As described above, microbial fermentation methods and enzymatic reaction methods using α1,2-fucosyltransferase are known. However, the microbial α1,2-fucosyltransferases described in Patent Documents 1 and 2 and Non-Patent Documents 4, 5, and 6 can tolerate a wide range of sugar substrates, and therefore, the production of 2'FL as a by-product in the production of LNFPI poses a problem.

[0011] On the other hand, in order to produce LNFPI more efficiently using lactose as the starting material, an α1,2-fucosyltransferase that does not transfer sugars to lactose but can selectively transfer sugars to the non-reducing terminal galactose site of LNT is required.

[0012] Therefore, an object of the present invention is to provide a protein having α1,2-fucosyltransferase activity that is excellent in LNFPI productivity, and a method for producing LNFPI.

[0013] The present inventors have discovered that LNFPI can be produced more efficiently than by conventional methods by using a microorganism capable of producing a protein having α1,2-fucosyltransferase activity and consisting of a specific amino acid sequence, and have thus completed the present invention.

[0014] That is, the present invention is as follows: 1. A protein having activity of transferring a fucosylation group to lacto-N-tetraose (LNT), into which at least one of the following mutations [1] and [2] has been introduced (excluding those in which the amino acid sequences constituting the protein before and after the mutation are identical): [1] A mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 98 is inserted [2] A mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 99 is inserted 2. The protein according to 1 above, which is a protein in which at least one of the mutations [1] and [2] has been introduced into an amino acid sequence having 50% or more full-length sequence identity to at least one of the amino acid sequences represented by SEQ ID NOs: 63, 94, and 95. 3. The protein according to 1 or 2, wherein the mutation [1] is a mutation introduced at a position corresponding to a region contributing to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 101 when aligned with the amino acid sequence represented by SEQ ID NO: 101, and the mutation [2] is a mutation introduced at a position corresponding to a region contributing to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 131 when aligned with the amino acid sequence represented by SEQ ID NO: 131. 4. The protein according to 3, wherein the region contributing to interaction with a substrate is an amino acid sequence corresponding to positions 162 to 176 of the amino acid sequence represented by SEQ ID NO: 101 when aligned with the amino acid sequence represented by SEQ ID NO: 101. 5. The protein according to any one of the above 1 to 4, wherein the mutation [1] is a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO: 101, the amino acid sequence corresponding to positions 162 to 176 is substituted with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 98, and the mutation [2] is a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO: 131, the amino acid sequence corresponding to positions 168 to 184 is substituted with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 99. 6. The mutation [1] is at least one mutation selected from the following [1a] to [1o] at positions corresponding to positions 162 to 176 when aligned with the amino acid sequence represented by SEQ ID NO: 101,The protein according to any one of 1 to 5, wherein the mutation [2] is at least one mutation selected from the following [2a] to [2q], located at positions corresponding to positions 168 to 184 when aligned with the amino acid sequence represented by SEQ ID NO: 131: [1a] At position 162, it is threonine. [1b] At position 163, it is aspartic acid. [1c] At position 164, it is leucine. [1d] At position 165, it is serine. [1e] At position 166, it is alanine. [1f] At position 167, it is asparagine. [1g] At position 168, it is asparagine. [1h] At position 169, it is isoleucine. [1i] At position 170, it is histidine. [1j] At position 171, it is glycine. [1k] At position 172, it is isoleucine. [1l] At position 173, it is cysteine. [1m] At position 174, it is serine. [1n] At position 175, it is glutamic acid. [1o] At position 176, it is glutamic acid. [2a] At position 168, it is phenylalanine. [2b] At position 169, it is valine. [2c] At position 170, it is glutamine. [2d] At position 171, it is aspartic acid. [2e] At position 172, it is proline. [2f] At position 173, it is valine. [2g] At position 174, it is valine. [2h] At position 175, it is arginine. [2i] At position 176, it is arginine. [2j] At position 177, it is valine. [2k] At position 178, it is histidine. [2l] At position 179, it is glycine. [2m] At position 180, it is valine. [2n] At position 181, it is aspartic acid. [2o] At position 182, it is leucine. [2p] At the position corresponding to position 183, it is threonine. [2q] At the position corresponding to position 184, it is alanine. 7. A protein having fucosyl group transfer activity to LNT,A protein in which, when aligned with the amino acid sequence represented by SEQ ID NO: 63, at least one mutation selected from the following (1) to (31) has been introduced at each of the positions corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270, and which has a higher fucosylation group transfer activity to LNT than the protein before the introduction of the mutation: (1) at position 7, it is alanine or serine; (2) at position 42, it is isoleucine; (3) at position 94, it is histidine; (4) at position 95, it is tyrosine; (5) at position 159, it is phenylalanine; (6) at position 160, it is valine; (7) at position 161, it is glutamine or threonine; (8) at position 162, it is aspartic acid or asparagine; (9) at position 163, it is leucine; (10) at position 164, it is isoleucine, lysine, serine, or valine; (11) at position 165, it is alanine or valine; (12) at position 166, it is arginine or asparagine; (13) at position 167, it is arginine or asparagine. (14) Valine at position 168. (15) Aspartic acid at position 167 and isoleucine at position 168, with isoleucine, histidine, and glycine inserted between positions 167 and 168. (16) Histidine at position 169. (17) Leucine, glycine, or serine at position 170. (18) Threonine, valine, or glutamic acid at position 171. (19) Glutamic acid or alanine at position 172. (20) Aspartic acid at position 172 and tyrosine at position 173, with leucine, threonine, and alanine inserted between positions 172 and 173. (21) Lysine at position 259. (22) At the position corresponding to position 260, it is glutamine. (23) At the position corresponding to position 262, it is histidine. (24) At the position corresponding to position 263, it is lysine. (25) At the position corresponding to position 264, it is valine. (26) At the position corresponding to position 265, it is arginine.(27) Aspartic acid at position 266 (28) Phenylalanine at position 267 (29) Aspartic acid at position 268 (30) Threonine at position 269 (31) Arginine at position 270 8. The protein according to 7 above, wherein the protein has at least one mutation selected from (1) to (31) introduced into an amino acid sequence having a full-length sequence identity of 50% or more with at least one of the amino acid sequences shown in SEQ ID NOs: 63, 94, and 95, at positions corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence shown in SEQ ID NO: 63. The protein according to 7 or 8, wherein the mutations introduced at positions corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence represented by SEQ ID NO: 63 are any one of the following [3] to

[12] : [3] At position 159, there is phenylalanine; at position 160, there is valine; at position 161, there is glutamine; at position 162, there is aspartic acid; at position 164, there is valine; at position 165, there is valine; at position 166, there is arginine; at position 167, there is arginine; at position 168, there is valine; at position 169, there is histidine; at position 170, there is glycine; at position 171, there is valine; at position 172, there is aspartic acid; and at position 173, there is tyrosine; and leucine, tyrosine, and alanine are inserted between positions 172 and 173. [4] At position 94, it is histidine; at position 95, it is tyrosine; at position 159, it is phenylalanine; at position 160, it is valine; at position 161, it is glutamine; at position 162, it is aspartic acid; at position 164, it is valine; and at position 165, it is valine.At the position corresponding to position 166, it is arginine; At the position corresponding to position 167, it is arginine; At the position corresponding to position 168, it is valine; At the position corresponding to position 169, it is histidine; At the position corresponding to position 170, it is glycine; At the position corresponding to position 171, it is valine; At the position corresponding to position 172, it is aspartic acid; At the position corresponding to position 173, it is tyrosine; Leucine, tyrosine, and alanine are inserted between the positions corresponding to positions 172 and 173; At the position corresponding to position 259, it is lysine; At the position corresponding to position 260, it is glutamine; At the position corresponding to position 263, it is lysine; At the position corresponding to position 264, it is valine; At the position corresponding to position 265, it is arginine; At the position corresponding to position 266, it is aspartic acid; At the position corresponding to position 267, it is phenylalanine; At position 268, it is aspartic acid, at position 269, it is threonine, and at position 270, it is arginine. [5] At position 161, it is threonine, at position 162, it is aspartic acid, at position 163, it is leucine, at position 164, it is serine, at position 165, it is alanine, at position 166, it is asparagine, at position 167, it is aspartic acid, at position 168, it is isoleucine, isoleucine, histidine, and glycine are inserted between positions 167 and 168, it is serine, at position 170, it is glutamic acid, and at position 172, it is glutamic acid. [6] At the position corresponding to position 164, it is isoleucine. [7] At the position corresponding to position 162, it is aspartic acid, and at the position corresponding to position 164, it is lysine. [8] At the position corresponding to position 262, it is histidine. [9] At the position corresponding to position 42, it is isoleucine.

[10] At the position corresponding to position 162, it is asparagine.

[11] At the position corresponding to position 7, it is serine.

[12] At the position corresponding to position 7, it is alanine. 10. A DNA encoding the protein according to any one of 1 to 9 above.11. Recombinant DNA containing the DNA according to 10. 12. A transformant obtained by transforming a host cell with the recombinant DNA according to 11. 13. The transformant according to 12, which is a microorganism in which the activity of the protein according to any one of 1 to 9 and the productivity of a fucose-containing saccharide are enhanced. 14. The transformant according to 13, wherein the microorganism is Escherichia coli. 15. A method for producing a fucose-containing saccharide, comprising preparing the transformant according to any one of 13 to 14, and producing a fucose-containing saccharide in a culture using the transformant. 16. The production method according to 15, wherein the fucose-containing saccharide is lacto-N-fucopentaose I (LNFPI).

[0015] The protein of the present invention, which is composed of a specific amino acid sequence, has α1,2-fucosyltransferase activity capable of transferring sugars to the non-reducing terminal galactose site of LNT. By using a microorganism capable of producing the protein of the present invention, the production of by-products can be suppressed compared to conventional methods, and LNFPI can be produced efficiently.

[0016] Figure 1 shows the biosynthetic pathway of LNFPI in one embodiment of the present invention. Figure 2 shows the amino acid sequences of FucT (FsFucT) derived from Francisella sp. strain FSC1006, S3 α1,2-fucosyltransferase, and α1,2-fucosyltransferase derived from Amphritea japonica. Figure 3 is a graph showing the total amount of LNFP1 (g / L). Figure 4 is a graph showing the total amount of LNFP1 (g / L). Figure 5 is a graph showing the total amount of LNFP1 (g / L).

[0017] The present invention will be described below with reference to embodiments, including embodiment 1 and embodiment 2.

[0018] In this embodiment, the identity of amino acid sequences and nucleotide sequences can be determined using the algorithm BLAST by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing nucleotide sequences using BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0019] In this embodiment, amino acid sequence alignments can be created using known alignment programs such as ClustalW [Nucleic Acids Research 22, 4673, (1994)], MAFTT [Nucleic Acids Research 30, 3059, (2002)], and Clustal Omega. These alignment programs are available from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When creating alignments using these alignment programs, for example, default parameters are used.

[0020] In this embodiment, the fucosyltransferase activity to LNT refers to the activity of transferring a fucose residue from GDP-fucose, which is a donor substrate, to the galactose hydroxyl group of LNT, which is a carbohydrate, which is an acceptor substrate (hereinafter referred to as "acceptor carbohydrate").

[0021] In this embodiment, α1,2-fucosyltransferase activity refers to the activity of transferring a fucose residue from the donor substrate GDP-fucose to the galactose hydroxyl group of an acceptor saccharide via an α1,2-bond to produce a fucose-containing saccharide. In this embodiment, the acceptor saccharide is preferably LNT. In this embodiment, the fucose-containing saccharide is preferably LNFPI.

[0022] In this embodiment, the protein having the activity of transferring a fucosyl group to LNT is not particularly limited as long as it has the activity of transferring a fucose residue from the donor substrate GDP-fucose to the galactose hydroxyl group of LNT, and examples thereof include α1,2-fucosyltransferase. Specific examples include α1,2-fucosyltransferase derived from Francisella sp. FSC1006 strain, α1,2-fucosyltransferase derived from Sideroxydans lithotrophicus ES-11, α1,2-fucosyltransferase derived from Neisseriaceae bacterium DSM 100970 strain, and α1,2-fucosyltransferase derived from Gramella sp. α1,2-fucosyltransferase derived from MAR_2010_147 strain, α1,2-fucosyltransferase derived from Methylobacter tundripaludum strain, α1,2-fucosyltransferase derived from Amphritea japonica strain, α1,2-fucosyltransferase derived from Sterolibacteriaceae bacterium J5B strain, α1,2-fucosyltransferase derived from Neisseria bacterium strain, α1,2-fucosyltransferase derived from Helicobacter mustelae ATCC43772 strain, and Pseudohalocynthiibacter Examples of the α1,2-fucosyltransferase include α1,2-fucosyltransferase derived from Pedobacter sp. CF074 strain, α1,2-fucosyltransferase derived from Candidatus Methylobacter favarea strain, α1,2-fucosyltransferase derived from Escherichia coli O86 strain, and α1,2-fucosyltransferase derived from Escherichia coli O127 strain.

[0023] In this embodiment, each mutation may be substituted with the following mutually substitutable amino acids: 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

[0024] <Biosynthetic pathway of LNFPI> The biosynthetic pathway of LNFPI in this embodiment is shown in Figure 1. LNFPI is produced by the transfer of a fucose residue from GDP-fucose to the galactose hydroxyl group.

[0025] The present inventors have confirmed that by substituting a portion of the nucleotide sequence encoding the α1,2-fucosyltransferase derived from Francisella sp. FSC1006 with at least one of the corresponding portions of the nucleotide sequence encoding the α1,2-fucosyltransferase derived from Amphritea japonica and the α1,2-fucosyltransferase derived from Sterolibacteriaceae bacterium J5B, the by-production of 2'FL can be reduced compared to the enzyme before substitution. In other words, they have newly discovered an α1,2-fucosyltransferase protein with higher substrate selectivity for LNT.

[0026] Furthermore, the present inventors have similarly confirmed that the production of 2'FL by-production is reduced by substituting a portion of the base sequence encoding the α1,2-fucosyltransferase derived from Sideroxidans lithotrophicus ES-11 (WO 2019 / 008133) and the α1,2-fucosyltransferase derived from Neisseriaceae bacterium DSM 100970 with the base sequence of a corresponding portion of the base sequence encoding the α1,2-fucosyltransferase derived from Amphritea japonica.

[0027] From this, it is believed that the amino acid sequences encoded by the base sequences used for the substitution in the α1,2-fucosyltransferase derived from Amphritea japonica and the α1,2-fucosyltransferase derived from Sterolibacteriaceae bacterium J5B contribute to reducing 2'FL as a by-product in α1,2-fucosyltransferase homologs, i.e., to improving substrate selectivity for LNT.

[0028] <Embodiment 1: Protein Having Activity of Transferring Fucosyl Groups to LNT> The protein of embodiment 1 is a protein having activity of transferring fucosyl groups to LNT (hereinafter also simply abbreviated as "transfucosyl group activity"), into which at least one of the following mutations [1] and [2] has been introduced: [1] A mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 98 is inserted; [2] A mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 99 is inserted.

[0029] In embodiment 1, mutations do not mean that the amino acid sequences constituting the protein before and after the mutation are identical. Examples of the insertion include insertion mutations and substitution mutations. The protein of embodiment 1 is preferably a protein in which the substrate selectivity for LNT of the protein after the mutation is improved compared to the protein before the mutation. More preferably, the protein of embodiment 1 is a protein in which, in addition to improved substrate selectivity for LNT, the transfucosylation activity of the protein after the mutation is improved compared to the protein before the mutation.

[0030] The substrate selectivity of a protein for LNT can be confirmed, for example, by the following procedures (A1) to (A3). (A1) A recombinant DNA is prepared containing DNA encoding the protein whose activity is to be confirmed. (A2) A parent strain is transformed with the recombinant DNA to prepare a transformant, and the amounts of LNFPI and 2'FL produced and accumulated in the culture medium of the transformant are measured. (A3) The smaller the ratio of the amount of 2'FL to the amount of LNFPI, the higher the substrate selectivity for LNT is determined to be.

[0031] The transfucosylation activity of a protein can be confirmed, for example, by the following procedures (B1) to (B3). (B1) A recombinant DNA is prepared having a DNA encoding the protein whose activity is to be confirmed. (B2) A parent strain is transformed with the recombinant DNA to prepare a transformant, and the amount of fucose-containing saccharide produced and accumulated in the culture medium of the transformant is measured. (B3) The transfucosylation activity is evaluated based on the amount of the fucose-containing saccharide. A specific example of a fucose-containing saccharide is LNFPI.

[0032] The protein of embodiment 1 is preferably a protein in which at least one of the mutations described in [1] and [2] above has been introduced into an amino acid sequence having a full-length sequence identity of 50% or more with at least one of the amino acid sequences represented by SEQ ID NOs: 63, 94, and 95. The full-length sequence identity is preferably 50% or more, more preferably 60% or more, 70% or more, 80% or more, 90% or more, even more preferably 95% or more, particularly preferably 97% or more, and most preferably 100%.

[0033] The amino acid sequence represented by SEQ ID NO: 63 is the amino acid sequence of FucT derived from Francisella sp. FSC1006 (hereinafter also abbreviated as FsFucT). The amino acid sequence represented by SEQ ID NO: 94 is the amino acid sequence of FucT derived from Sideroxydans lithotrophicus ES-11 (hereinafter also abbreviated as FucT54). The amino acid sequence represented by SEQ ID NO: 95 is the amino acid sequence of FucT derived from Neisseriaceae bacterium DSM 100970 (hereinafter also abbreviated as NbFucT1).

[0034] [1] and [2] in the first embodiment will be explained below.

[0035] <<[1] Mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 98 is inserted>> In [1], the amino acid sequence of SEQ ID NO: 98 is a partial sequence of the amino acid sequence constituting the amino acid sequence of Amphritea japonica-derived α1,2-fucosyltransferase (SEQ ID NO: 101), and is an amino acid sequence corresponding to the amino acid sequence from positions 162 to 176 in the amino acid sequence represented by SEQ ID NO: 101.

[0036] The amino acid sequence inserted in [1] has 80% or more identity with the amino acid sequence of SEQ ID NO: 98. The identity is preferably 90% or more, more preferably 93% or more, even more preferably 95%, particularly preferably 97% or more, and most preferably 99% or more.

[0037] The mutation [1] is preferably a mutation introduced at a position corresponding to a region that contributes to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 101 when aligned with the amino acid sequence represented by SEQ ID NO: 101. The region that contributes to interaction with the substrate consists of a binding site with the substrate via hydrogen bonds or the like and a region in the vicinity thereof. Examples of the region that contributes to interaction with the substrate include a region that forms a helix structure. The region that contributes to interaction with the substrate is preferably a region corresponding to positions 140 to 280 when aligned with the amino acid sequence represented by SEQ ID NO: 101, more preferably a region corresponding to positions 150 to 200, even more preferably a region corresponding to positions 160 to 180, and particularly preferably a region corresponding to positions 162 to 176.

[0038] The mutation [1] is preferably a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO: 101, the amino acid sequence corresponding to positions 140 to 280, more preferably positions 150 to 200, even more preferably positions 160 to 180, and particularly preferably positions 162 to 176 is replaced with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 98.

[0039] Specifically, the mutation [1] is preferably at least one selected from the following [1a] to [1o] at positions corresponding to positions 162 to 176 when aligned with the amino acid sequence represented by SEQ ID NO: 101: [1a] At position 162, it is threonine. [1b] At position 163, it is aspartic acid. [1c] At position 164, it is leucine. [1d] At position 165, it is serine. [1e] At position 166, it is alanine. [1f] At position 167, it is asparagine. [1g] At position 168, it is asparagine. [1h] At position 169, it is isoleucine. [1i] At position 170, it is histidine. [1j] At position 171, it is glycine. [1k] At position 172, it is isoleucine. [1l] At position 173, it is cysteine. [1m] At position 174, it is serine. [1n] At position 175, it is glutamic acid. [1o] At position 176, it is glutamic acid.

[0040] <<[2] Mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 99 is inserted>> In [2], the amino acid sequence of SEQ ID NO: 99 is a partial sequence of the amino acid sequence constituting the amino acid sequence of SbFucT derived from Sterolibacteriaceae bacterium J5B (SEQ ID NO: 131), and is an amino acid sequence corresponding to the amino acid sequence of positions 168 to 184 of the amino acid sequence represented by SEQ ID NO: 131.

[0041] The amino acid sequence inserted in [2] has 80% or more identity with the amino acid sequence of SEQ ID NO: 99. The identity is preferably 90% or more, more preferably 93% or more, even more preferably 95%, particularly preferably 97% or more, and most preferably 99% or more.

[0042] The mutation [2] is preferably a mutation introduced at a position corresponding to a region that contributes to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 131 when aligned with the amino acid sequence represented by SEQ ID NO: 131. Examples of the region that contributes to interaction with a substrate include a region that forms a helix structure. When aligned with the amino acid sequence represented by SEQ ID NO: 131, the region that contributes to interaction with a substrate is preferably a region corresponding to positions 168 to 184, more preferably positions 150 to 200, even more preferably positions 160 to 190, and particularly preferably positions 168 to 184.

[0043] The mutation [2] is preferably a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO: 131, the amino acid sequence corresponding to positions 140 to 280, more preferably positions 150 to 200, even more preferably positions 160 to 190, and particularly preferably positions 168 to 184 is replaced with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 99.

[0044] Specifically, the mutation [2] is preferably at least one selected from the following mutations [2a] to [2q] at positions corresponding to positions 168 to 184 when aligned with the amino acid sequence represented by SEQ ID NO: 131. [2a] At position 168, it is phenylalanine. [2b] At position 169, it is valine. [2c] At position 170, it is glutamine. [2d] At position 171, it is aspartic acid. [2e] At position 172, it is proline. [2f] At position 173, it is valine. [2g] At position 174, it is valine. [2h] At position 175, it is arginine. [2i] At position 176, it is arginine. [2j] At position 177, it is valine. [2k] At position 178, it is histidine. [2l] At position 179, it is glycine. [2m] At position 180, it is valine. [2n] At position 181, it is aspartic acid. [2o] At position 182, it is leucine. [2p] Threonine at position 183 [2q] Alanine at position 184

[0045] Specific examples of the amino acid sequences constituting the protein of embodiment 1 include the amino acid sequences represented by SEQ ID NOs: 66, 96, and 97.

[0046] <Embodiment 2: Protein having fucosyltransferase activity to LNT> Embodiment 2 is a protein having fucosyltransferase activity to LNT, which, when aligned with the amino acid sequence represented by SEQ ID NO: 63, has at least one mutation selected from the following (1) to (31) introduced at positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270, and which has a higher fucosyltransferase activity to LNT than the protein before the introduction of the mutation:(1) at position 7, it is alanine or serine; (2) at position 42, it is isoleucine; (3) at position 94, it is histidine; (4) at position 95, it is tyrosine; (5) at position 159, it is phenylalanine; (6) at position 160, it is valine; (7) at position 161, it is glutamine or threonine; (8) at position 162, it is aspartic acid or asparagine; (9) at position 163, it is leucine; (10) at position 164, it is isoleucine, lysine, serine, or valine; (11) at position 165, it is alanine or valine; (12) at position 166, it is arginine or asparagine; (13) at position 167, it is arginine or asparagine. (14) Valine at position 168. (15) Aspartic acid at position 167 and isoleucine at position 168, with isoleucine, histidine, and glycine inserted between positions 167 and 168. (16) Histidine at position 169. (17) Leucine, glycine, or serine at position 170. (18) Threonine, valine, or glutamic acid at position 171. (19) Glutamic acid or alanine at position 172. (20) Aspartic acid at position 172 and tyrosine at position 173, with leucine, threonine, and alanine inserted between positions 172 and 173. (21) Lysine at position 259. (22) At the position corresponding to position 260, it is glutamine. (23) At the position corresponding to position 262, it is histidine. (24) At the position corresponding to position 263, it is lysine. (25) At the position corresponding to position 264, it is valine. (26) At the position corresponding to position 265, it is arginine. (27) At the position corresponding to position 266, it is aspartic acid. (28) At the position corresponding to position 267, it is phenylalanine. (29) At the position corresponding to position 268, it is aspartic acid. (30) At the position corresponding to position 269, it is threonine. (31) At the position corresponding to position 270, it is arginine.

[0047] In an amino acid sequence having a full-length sequence identity of 50% or more with at least one of the amino acid sequences represented by SEQ ID NOs: 63, 94, and 95, the protein is preferably one in which the mutation has been introduced at a position corresponding to at least one position selected from the group consisting of positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence represented by SEQ ID NO: 63. The full-length sequence identity is preferably 50% or more, more preferably 60% or more, 70% or more, 80% or more, 90% or more, even more preferably 95% or more, particularly preferably 97% or more, and most preferably 100%.

[0048] The amino acid sequences represented by SEQ ID NOs: 63, 94, and 95 are the same as those described above in the section <Embodiment 1: Proteins having fucosylation transfer activity to LNT>.

[0049] Each mutation in embodiment 2 is preferably any one of the following [3] to

[12] . (A) At least one mutation selected from the group consisting of [3] to [6]. [3] At position 159, it is phenylalanine; At position 160, it is valine; At position 161, it is glutamine; At position 162, it is aspartic acid; At position 164, it is valine; At position 165, it is valine; At position 166, it is arginine; At position 167, it is arginine; At position 168, it is valine; At position 169, it is histidine; At position 170, it is glycine; At position 171, it is valine; At position 172, it is aspartic acid; At position 173, it is tyrosine; and between positions 172 and 173, leucine, tyrosine, and alanine are inserted.[4] At position 94, it is histidine; at position 95, it is tyrosine; at position 159, it is phenylalanine; at position 160, it is valine; at position 161, it is glutamine; at position 162, it is aspartic acid; at position 164, it is valine; at position 165, it is valine; at position 166, it is arginine; at position 167, it is arginine; at position 168, it is valine; at position 169, it is histidine; at position 170, it is glycine; at position 171, it is valine; at position 172, it is aspartic acid; and at position 173, it is tyrosine. Between positions corresponding to 172 and 173, leucine, tyrosine, and alanine are inserted; at position corresponding to 259, it is lysine; at position corresponding to 260, it is glutamine; at position corresponding to 263, it is lysine; at position corresponding to 264, it is valine; at position corresponding to 265, it is arginine; at position corresponding to 266, it is aspartic acid; at position corresponding to 267, it is phenylalanine; at position corresponding to 268, it is aspartic acid; at position corresponding to 269, it is threonine; and at position corresponding to 270, it is arginine. [5] At position 161, it is threonine, At position 162, it is aspartic acid, At position 163, it is leucine, At position 164, it is serine, At position 165, it is alanine, At position 166, it is asparagine, At position 167, it is asparagine, At position 167, it is aspartic acid, At position 168, it is isoleucine, Isoleucine, histidine, and glycine are inserted between positions 167 and 168, It is serine, At position 170, it is glutamic acid, At position 171, it is glutamic acid, At position 172, it is glutamic acid. [6] At position 164, it is isoleucine.[7] At the position corresponding to position 162, it is aspartic acid; and at the position corresponding to position 164, it is lysine. [8] At the position corresponding to position 262, it is histidine. [9] At the position corresponding to position 42, it is isoleucine.

[10] At the position corresponding to position 162, it is asparagine.

[11] At the position corresponding to position 7, it is serine.

[12] At the position corresponding to position 7, it is alanine.

[0050] Specific examples of the amino acid sequence constituting the protein of embodiment 2 include the amino acid sequences represented by any one of SEQ ID NOs: 64 to 78. Among these, the amino acid sequences represented by any one of SEQ ID NOs: 64 to 66, 68, and 72 to 77 are particularly preferred.

[0051] <DNA> The DNA of this embodiment is DNA that encodes the protein of this embodiment described above. Examples of DNA that encodes the protein of Embodiment 1 include DNA having the nucleotide sequence shown in SEQ ID NO: 49, 92, or 93. Examples of DNA that encodes the protein of Embodiment 2 include DNA having the nucleotide sequence shown in any one of SEQ ID NOs: 47 to 61.

[0052] The DNA of this embodiment can be used as is or cleaved with an appropriate restriction enzyme or the like, and then inserted into a vector by a standard method. The resulting recombinant DNA is then introduced into a host cell, and the DNA can be analyzed using a standard base sequence analysis method, for example, the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analyzer such as an Applied Biosystems 3500 Genetic Analyzer or an Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific), to determine the base sequence of the DNA.

[0053] Any host cell can be used when determining the base sequence of the DNA, as long as it can grow after the vector is introduced into it. Examples of such host cells include Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, and Escherichia coli MV1184. Examples of suitable Escherichia coli include Escherichia coli TH2 (all manufactured by Takara Bio Inc.), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, and Escherichia coli NM522.

[0054] Examples of the vector include pBluescriptII KS(+), pPCR-Script Amp SK(+) (both manufactured by Agilent Technologies), pT7Blue (manufactured by Merck Millipore), pCRII (manufactured by Thermo Fisher Scientific), pCR-TRAP (manufactured by Gene Hunter), and pDIRECT (Nucleic Acids Res., 18, 6069, 1990).

[0055] Any method for introducing recombinant DNA into a host cell can be used, and examples thereof include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Laid-Open Publication No. 63-248394), and an electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0056] If the DNA obtained as a result of determining the base sequence is a partial length, the full-length DNA can be obtained by Southern hybridization or the like to a chromosomal DNA library using the partial length DNA as a probe.

[0057] Furthermore, the desired DNA can be prepared by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd., based on the determined DNA base sequence.

[0058] The recombinant DNA containing the DNA encoding the protein of this embodiment refers to a recombinant DNA in which the DNA is incorporated into an expression vector that is capable of autonomous replication in a parent strain or can be integrated into a chromosome and contains a promoter at a position where the DNA can be transcribed.

[0059] When the recombinant DNA is capable of being integrated into a chromosome, it does not need to contain a promoter.

[0060] 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 recombinant DNA containing DNA encoding the protein of this embodiment, can be obtained by the following method.

[0061] Based on the DNA encoding the protein of this embodiment 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 optimal codons for expression in host cells, a transformant with improved productivity can be obtained.

[0062] 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.

[0063] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA is preferably composed of a promoter, a ribosome binding sequence, a DNA encoding the protein of this embodiment, and a transcription termination sequence. A gene that controls the promoter may also be included.

[0064] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is 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.

[0065] Furthermore, by substituting bases in the base sequence of the portion encoding the protein of this embodiment so that it contains codons optimal for expression in the host, the expression level of the protein having α1,2-fucosyltransferase activity can be improved. Information on codon usage in the parent strain used in this embodiment is available through public databases.

[0066] The expression vector is not particularly limited as long as it is a suitable nucleic acid molecule for introducing, amplifying, and expressing the target DNA into a host, and not only plasmids but also, for example, artificial chromosomes, vectors using transposons, and cosmids may be used.

[0067] When a microorganism belonging to the genus Escherichia is used as a 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, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), and pM AL-c5x (New England Biolabs), pGEX-4T-1, pTrc99A (both manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (both manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (both manufactured by Qiagen), pET-3, pBluescript II SK(+), pBluescript II KS(-) (all manufactured by Agilent Technologies), pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pKYP10 (JP Patent Publication No. 58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. , USA, 82, 4306 (1985)], 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, No. 20, p. 6378-6385], pPAC31 (WO 1998 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (JP 63-233798 A), pKD46 [Proc. Natl. Acad. Sci., USA, 97, 6640-6645 (2000)], and the like.

[0068] When using the above-mentioned expression vector, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples of such promoters include promoters of genes involved in amino acid biosynthesis, such as the trp promoter and ilv promoter, and promoters derived from Escherichia coli or phages, such as the uspA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter. Other examples include artificially designed and modified promoters, such as a promoter consisting of two trp promoters in tandem, the tac promoter, the trc promoter, the lacT7 promoter, and the letI promoter.

[0069] 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)).

[0070] 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)].

[0071] 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.

[0072] 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.

[0073] <Transformant> The transformant of this embodiment can be obtained by transforming a host cell with the recombinant DNA of this embodiment. In this embodiment, the term "parent strain" refers to the original strain to be transformed.

[0074] 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, or 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 DH1, Escherichia coli MC1000, Escherichia coli MG1655, Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli MG1655, Escherichia coli XL1-Blue, Es 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 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, Microbacterium ammoniaphilum ATCC15354,or Pseudomonas sp. D-0110, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.

[0075] The parent strain may be a wild-type strain as long as it is a microorganism that produces GDP-fucose and / or LNT. If the wild-type strain does not have the ability to produce GDP-fucose and / or LNT, the parent strain may be a bred strain that has been artificially imparted with the ability to supply GDP-fucose and / or LNT.

[0076] Examples of microorganisms that can be used as parent strains include the following 1) and 2): 1) a microorganism to which the ability to supply GDP-fucose, a reaction substrate for α1,2-fucosyltransferase, has been artificially imparted or enhanced; and 2) a microorganism to which the ability to supply LNT, a reaction substrate for α1,2-fucosyltransferase, has been artificially imparted or enhanced. 1) and 2) are described below.

[0077] 1) Microorganisms used as parent strains to which the ability to supply GDP-fucose, a reaction substrate for α1,2-fucosyltransferase, has been artificially imparted or enhanced. The parent strain is preferably a microorganism to which the ability to supply GDP-fucose, a reaction substrate for α1,2-fucosyltransferase, has been artificially imparted or enhanced. Specific examples of methods for imparting or enhancing the ability to supply GDP-fucose to a microorganism used as a parent strain include known methods such as various genetic engineering methods (Metabolic Engineering (2017) 41:23-38).

[0078] The ability to supply GDP-fucose includes the ability to produce GDP-fucose from sugar. Methods for artificially imparting or enhancing the ability to produce GDP-fucose from sugar to a microorganism used as a parent strain include, for example, the following methods (1a) to (1d). These methods may be used alone or in combination. (1a) A method of alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway that produces GDP-fucose from sugar. (1b) A method of enhancing the expression of at least one enzyme involved in the biosynthetic pathway that produces GDP-fucose from sugar. (1c) A method of increasing the copy number of at least one gene encoding an enzyme involved in the biosynthetic pathway that produces GDP-fucose from sugar. (1d) A method of weakening or blocking at least one metabolic pathway that branches off from the biosynthetic pathway that produces GDP-fucose from sugar to a metabolic product other than the target substance.

[0079] Specific examples of mechanisms for controlling the biosynthetic pathway that produces GDP-fucose from sugars include known mechanisms, such as a control mechanism by a transcriptional regulatory factor (e.g., RcsA) involved in the control of the biosynthetic pathway. RcsA is a regulatory factor that upregulates the entire colanic acid biosynthetic pathway, which uses GDP-fucose as an intermediate. As described below, by strengthening rcsA while blocking the pathway downstream of GDP-fucose in the colanic acid biosynthetic pathway, it is possible to accumulate a large amount of GDP-fucose.

[0080] Specific examples of enzymes involved in the biosynthetic pathway for producing GDP-fucose from sugars include known enzymes such as mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase.

[0081] Specific examples of metabolic pathways that branch off from the biosynthetic pathway that produces GDP-fucose from sugars to metabolites other than the target substance include known metabolic pathways, such as the metabolic pathway from GDP-fucose to colanic acid. In particular, blocking WcaJ, WzxC, WcaK, WcaL, or WcaM, which is a pathway downstream of GDP-fucose in the colanic acid biosynthetic pathway, can increase the supply of GDP-fucose.

[0082] The microorganism used as the parent strain may be modified to promote the import of exogenous L-fucose across its cell membrane. For example, by expressing or overexpressing a nucleotide sequence encoding FucP (Accession No. AIZ90162), the uptake of exogenous L-fucose across the cell membrane into the cell can be improved, thereby increasing the amount of fucose for producing GDP-fucose.

[0083] The microorganism used as the parent strain may be deleted in the fucI and / or fucK genes encoding L-fucose isomerase and L-fuculose kinase, respectively, or the nucleotide sequences of fucI and / or fucK may be altered so as to irreversibly inactivate the enzymatic activity of the corresponding polypeptides, or the microorganism may be modified so as to impair the expression of fucI and / or fucK. Eliminating the intracellular synthesis of FucI and / or FucK eliminates fucose metabolism in the cells, thereby enabling an increase in the amount of fucose used to produce GDP-fucose.

[0084] 2) A microorganism used as a parent strain to which the ability to supply LNT, a reaction substrate for α1,2-fucosyltransferase, has been artificially imparted or enhanced. Methods for artificially imparting the ability to supply LNT to a microorganism used as a parent strain include, for example, the following methods (2a) to (2h), and these methods can be used alone or in combination. (2a) A method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway for producing LNT from sugar. (2b) A method for enhancing the expression of at least one of the enzymes involved in the biosynthetic pathway for producing LNT from sugar. (2c) A method for increasing the copy number of at least one of the enzyme genes involved in the biosynthetic pathway for producing LNT from sugar. (2d) A method for alleviating or deactivating at least one of the mechanisms for decomposing LNT or its substrate sugar. (2e) A method for enhancing the expression of at least one of the enzymes involved in the cellular uptake of LNT or its substrate sugar. (2f) A method for increasing the copy number of at least one of the genes encoding the enzymes involved in the cellular uptake of LNT or its substrate sugar. (2g) A method for weakening or blocking at least one of the metabolic pathways branching off from the biosynthetic pathway for producing LNT from sugar to a metabolite other than the target substance. (2h) A method for selecting a cell line having a higher resistance to an LNT analogue compared to a wild-type strain.

[0085] Specific examples of enzymes involved in the biosynthetic pathway that produces LNT from sugars include known enzymes, such as enzymes having β1,4-galactosyltransferase (hereinafter referred to as galT) activity and enzymes having β1,3-N-acetylglucosamine transferase (hereinafter referred to as lgtA) activity, which are involved in the biosynthetic pathway that produces LNT from glucose and lactose.

[0086] Specific examples of mechanisms for decomposing LNT or its substrate sugars include known enzymes such as β-galactosidase, which catalyzes the hydrolysis of lactose, a substrate of LNT, to produce glucose and galactose. Specific examples include β-galactosidase (hereinafter referred to as lacZ), which hydrolyzes lactose, a substrate of LNT. Loss of lacZ activity can suppress a decrease in lactose supply.

[0087] Specific examples of enzymes involved in the cellular uptake of LNT or its substrate sugar include known enzymes such as lactose permease, which is involved in the cellular uptake of lactose, a substrate of LNT.

[0088] Specifically, the microorganism to which the ability to supply LNT has been imparted or enhanced may, for example, exhibit lactose permease (hereinafter referred to as lacY) activity, β1,4-galactosyltransferase (galT) activity, β1,3-N-acetylglucosamine transferase (lgtA) activity, glutamine-fructose-6-phosphate transaminase (hereinafter referred to as glmS) activity, phosphoglucosamine mutase (hereinafter referred to as glmM) activity, and N-acetylglucosamine-1-phosphate uridyltransferase / ... It is preferable that the enzyme has at least one activity selected from the group consisting of phosphate acetyltransferase (hereinafter referred to as glmU), phosphoglucomutase (hereinafter referred to as pgm), UTP glucose-1-phosphate uridylyltransferase (hereinafter referred to as galU), UDP glucose-4-epimerase (hereinafter referred to as galE), UTP glucose-1-phosphate uridylyltransferase (hereinafter referred to as galF), and glucose-6-phosphate isomerase (hereinafter referred to as pgi), and it is more preferable that the activity is enhanced.

[0089] Among these, it is preferable that the activity of lacY, galT and lgtA is present, and it is more preferable that the activity is enhanced.

[0090] LacY is a membrane protein that imports lactose, the substrate for LNT, into the cell. GalT is an enzyme involved in the production of LNT from lacto-N-triose II (LNT II). LNT is a precursor of LNFPI. LgtA is an enzyme involved in the production of LNT II from lactose and uridine diphosphate-N-acetylglucosamine (hereinafter referred to as UDP-GlcNAc). LNT II is a precursor of LNT.

[0091] glmS, glmM, and glmU are enzymes involved in the biosynthetic pathway that produces LNTII. Pgm, galU, galE, and galF are enzymes involved in the pathway that produces uridine diphosphate galactose (hereinafter referred to as UDP-Gal). Pgi is an enzyme involved in the pathway that produces LNTII.

[0092] Whether a microorganism is capable of producing GDP-fucose and / or LNT can be confirmed by culturing the microorganism in a medium and detecting the GDP-fucose and / or LNT accumulated in the culture using a general technique such as a sugar analyzer or a high-performance liquid chromatograph mass spectrometer, which will be described later. Commercially available samples can also be used as standard samples.

[0093] The microorganism used as the parent strain of the present invention is preferably a microorganism to which the ability to supply GDP-fucose and / or LNT, which are reaction substrates for α1,2-fucosyltransferase, has been artificially imparted or enhanced. Therefore, in one embodiment of the present invention, a nucleotide sequence encoding rcsA (accession number BAA15776.1), a nucleotide sequence encoding mannose-6-phosphate isomerase (accession number BAA15361.1), a nucleotide sequence encoding phosphomannomutase (accession number BAA15901.1), a nucleotide sequence encoding mannose-1-phosphate guanylyltransferase (accession number BAA15905.1), a nucleotide sequence encoding GDP mannose-4,6-dehydratase (accession number BAA15909.1), a nucleotide sequence encoding GDP-L-fucose synthase (accession number BAA15908.1), a nucleotide sequence encoding lacY (accession number BAE76125.1), a nucleotide sequence encoding galT (SEQ ID NO: 104), a nucleotide sequence encoding galT (accession number BAA15908.1), a nucleotide sequence encoding lacY (accession number BAE76125.1), a nucleotide sequence encoding galT (accession number BAA15908 ... Preferably, the parent strain is a genetically modified microorganism containing at least one nucleotide sequence selected from the group consisting of a nucleotide sequence encoding IgtA (SEQ ID NO: 31), a nucleotide sequence encoding glmS (accession number BAE77559.1), a nucleotide sequence encoding glmM (accession number BAE77220.1), a nucleotide sequence encoding glmU (accession number BAE77558.1), a nucleotide sequence encoding Pgm (accession number BAA35337.1), a nucleotide sequence encoding galU (accession number BAA36104.1), a nucleotide sequence encoding galE (accession number BAA35421.1), a nucleotide sequence encoding galF (accession number BAA15896.1), and a nucleotide sequence encoding pgi (accession number BAE78027.1).

[0094] In particular, it is more preferable to use a genetically modified microorganism as a parent strain, preferably comprising a nucleotide sequence encoding lacY, a nucleotide sequence encoding rcsA, a nucleotide sequence encoding galT, and a nucleotide sequence encoding lgtA. In one embodiment of the present invention, it is preferable that the genetically modified microorganism has an increased ability to produce GDP-fucose and / or LNT compared to a non-genetically modified parent strain.

[0095] A known method can be used to produce a microorganism having at least one activity selected from lacY activity, rcsA activity, galT activity, lgtA activity, glmS activity, glmM activity, glmU activity, pgm activity, galU activity, galE activity, galF activity, and pgi activity, or an activity in which the activity is enhanced. Specific examples include methods using various genetic manipulations (Syst Microbiol Biomanufact, 2021, 1, 291).

[0096] Furthermore, as described above, it is preferable that the parent strain has reduced or no lacZ activity and / or colanic acid synthesis activity.

[0097] Therefore, in one embodiment of the present invention, it is preferable to use a genetically modified microorganism as a parent strain, which preferably has reduced or absent lacZ activity and / or colanic acid synthesis activity, and more preferably does not contain a base sequence encoding lacZ and / or a base sequence encoding the wcaJ, wzxC, wcaK, wcaL or wcaM genes, which are base sequences encoding colanic acid production-related proteins.

[0098] In one embodiment of the present invention, the genetically modified microorganism preferably has an increased ability to produce GDP-fucose and / or LNT compared to a non-genetically modified parent strain.

[0099] A method for producing Escherichia coli having reduced or lost β-galactosidase activity and / or colanic acid synthesis activity may be a known method, specifically, for example, a method using various genetic manipulations (Metabolic Engineering, 2017, 41: 23-38).

[0100] Examples of microorganisms in which the activity of the protein of this embodiment is enhanced compared to that of a parent strain of a microorganism include microorganisms obtained by transforming a parent strain of a microorganism with recombinant DNA containing DNA encoding the protein, and in which the copy number of the recombinant DNA is increased compared to that of the parent strain.

[0101] Microorganisms in which the copy number of the recombinant DNA is increased compared to the parent strain, obtained by transforming a parent strain microorganism with recombinant DNA containing DNA encoding the protein of this embodiment, include microorganisms in which the copy number of the recombinant DNA on the chromosomal DNA is increased by transforming a parent strain microorganism with recombinant DNA containing DNA encoding the protein of this embodiment, and microorganisms in which the gene is carried outside the chromosomal DNA as plasmid DNA.

[0102] Methods for introducing recombinant DNA into a parent strain as an autonomously replicable plasmid include, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open Publication No. 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0103] Methods for integrating recombinant DNA into the chromosome of a host cell include, for example, homologous recombination. Examples of homologous recombination include a method using a plasmid for homologous recombination, which can be prepared by ligating with 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 in which recombinant DNA is introduced using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)].

[0104] Furthermore, E. coli in which a target region on the chromosomal DNA of a host cell has been replaced with recombinant DNA can be obtained using a selection method that utilizes the fact that E. coli becomes sucrose-sensitive due to Bacillus subtilis levansucrase that has been incorporated onto the chromosome together with recombinant DNA, or a selection method that utilizes the fact that E. coli becomes streptomycin-sensitive by incorporating a wild-type rpsL gene into E. coli that has a mutant rpsL gene that is resistant to streptomycin [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)].

[0105] 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 an amplified gene introduced by transformation is amplified by PCR to confirm the amplified product. Furthermore, an increase in the transcription level of the DNA or the production level of the protein encoded by the DNA can be confirmed by a method in which the transcription level of the gene in the microorganism is compared with that of the parent strain by Northern blotting, or the production level of the protein in the microorganism is compared with that of the parent strain by Western blotting.

[0106] The microorganisms constructed by the above method can be confirmed to have enhanced activity of the protein of this embodiment and improved LNFPI productivity compared to the parent strain by culturing the microorganism, appropriately diluting the culture solution, centrifuging it, and analyzing the LNFPI contained in the supernatant or the cells using a sugar analyzer or high-performance liquid chromatograph mass spectrometer described below, and comparing it with that of the parent strain. Furthermore, commercially available samples can be used as appropriate as standard samples.

[0107] <Method for producing fucose-containing saccharide> The method for producing a fucose-containing saccharide of this embodiment (hereinafter also referred to as the present production method) includes a method for producing a fucose-containing saccharide, which comprises preparing the transformant of this embodiment described above and producing oligosaccharides in a culture using the transformant. In this production method, the fucose-containing saccharide is preferably LNFPI.

[0108] The above-mentioned transformant can be cultured according to a method commonly used for culturing microorganisms. As a medium for culturing the transformant, either a natural medium or a synthetic medium may be used 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] The transformant used in this production method may be a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like.

[0113] In this production method, glucose, lactose, lactose monohydrate, or the like may be added to the medium during cultivation.

[0114] When the transformant used in this production method does not have the ability to produce GDP-fucose and / or LNT, GDP-fucose and / or LNT may be added to the medium.

[0115] Furthermore, in the present production method, instead of adding glucose, lactose, lactose monohydrate, LNT, or the like to the medium during culture, glucose, lactose, lactose monohydrate, LNT, or the like may be supplied to the transformant of the present invention by simultaneously culturing a microorganism capable of producing glucose, lactose, lactose monohydrate, LNT, or the like from sugar with the transformant of the present invention.

[0116] In this production method, it is preferable that β-galactosidase and WcaJ are not present in the medium.

[0117] Cultivation is preferably carried out under aerobic conditions, such as by shaking culture, submerged aeration agitation culture, or jar fermenter. 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 8.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0118] By the above-mentioned culture, fucose-containing saccharides are produced in the culture, and thus fucose-containing saccharides can be produced.

[0119] Usually, the culture is centrifuged, and the fucose-containing saccharide can be collected from the supernatant. When the fucose-containing saccharide accumulates in the cells, the cells can be disrupted by ultrasonication or the like, and the cells can be removed by centrifugation. The fucose-containing saccharide can be collected from the supernatant by an ion exchange resin method or the like.

[0120] Furthermore, the desired fucose-containing saccharide can also be produced by adding other sugars to the fucose-containing saccharide in the culture or the collected fucose-containing saccharide.

[0121] [Analysis Examples] In the Examples, analysis and quantification of LNFPI and 2'FL were performed according to the following procedures. After cultivation, the culture solution containing the microorganisms was centrifuged, and the culture supernatant was recovered. In addition, the precipitated bacterial cells were suspended in the same volume of water as the original culture solution, and then an equal volume of chloroform was added to disrupt the bacterial cells, followed by centrifugation, and the supernatant aqueous phase was used as the intracellular fraction. LNFPI and 2'FL contained in the culture supernatant and the intracellular fraction were analyzed using a sugar analyzer ICS-5000 (manufactured by Thermo Fisher Scientific).

[0122] [Analysis conditions] Column: CarboPAC PA1 Column temperature: 25°C Mobile phase: (Mobile phase A) Water (Mobile phase B) 500mmol / L Sodium hydroxide (Mobile phase C) 300mmol / L Sodium acetate Mixing ratio of mobile phase A, mobile phase B and mobile phase C: (0 to 10 minutes) 80:20:0 (10 to 15 minutes) Gradient from 80:20:0 to 70:20:10 (15 to 17 minutes) Gradient from 70:20:10 to 0:20:80 (17 to 25 minutes) 0:20:80 (25 to 35 minutes) 80:20:0 Flow rate: 1.0 mL / min Detector: Pulsed amperometry detector

[0123] Examples of the invention are shown below, but the present invention is not limited to these examples.

[0124] Example 1: Creation of microorganisms expressing various α1,2-fucosyltransferases (1) Creation of host strains <Obtaining a DNA fragment to be used as a marker for gene deletion> PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 102 and 103 as a primer set and pCatSac (Appl Environ Microbiol (2013) 79, 3033-3039) as a template to obtain a cat-sacB fragment containing a chloramphenicol-resistant cat gene and a sucrose-sensitive sacB gene.

[0125] <Construction of Escherichia coli lacking β-galactosidase activity, lactose permease activity, and colanic acid synthesis activity> Escherichia coli lacking DNA encoding β-galactosidase (hereinafter referred to as the lacZ gene), DNA encoding lactose permease (hereinafter referred to as the lacY gene), and DNA encoding proteins related to colanic acid production (hereinafter referred to as the wcaJ, wzxC, wcaK, wcaL, or wcaM genes) was constructed by the following method. Note that lacZ and lacY (hereinafter referred to as lacZY), and wcaJ, wzxC, wcaK, wcaL, and wcaM (hereinafter referred to as wcaJ-wzxC-wcaKLM) each form an operon on the E. coli genome.

[0126] Using genomic DNA of Escherichia coli W3110 strain prepared by a conventional method as a template, PCR was carried out using DNA primer sets consisting of the base sequences shown in "Primer Set" in Table 1 to amplify each DNA fragment.

[0127]

[0128] LacZ upstream 1 and lacZ upstream 2 comprise the region from the initiation codon of the lacZ gene to about 1000 bp upstream of the initiation codon. LacY downstream 1 and lacY downstream 2 comprise the region from about 50 bp to about 1000 bp downstream of the termination codon of the lacY gene.

[0129] PCR was performed using an equimolar mixture of lacZ upstream 1, lacY downstream 1, and cat-sacB fragments as a template and DNAs consisting of the base sequences shown in SEQ ID NOs: 105 and 107 as a primer set to obtain a DNA fragment (hereinafter referred to as lacZY::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the peripheral regions of the lacZ and lacY genes.

[0130] PCR was performed using an equimolar mixture of lacZ upstream 2 and lacY downstream 2 as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 105 and 107 as a primer set to obtain a DNA fragment (hereinafter referred to as ΔlacZY) that does not contain lacZY and consists of a sequence in which the upstream of lacZ and the downstream of lacY are directly linked.

[0131] The lacZY::cat-sacB fragment was introduced by electroporation into the W3110S3GK strain (NBRC114657) harboring the plasmid pKD46 [Datsenko, K. A., Warner, B. L., Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)] containing a gene encoding λ recombinase, and a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (a transformant in which the lacZY gene had been replaced with lacZY::cat-sacB) was obtained.

[0132] The ΔlacZY fragment was introduced into the transformant by electroporation to obtain a transformant that exhibited chloramphenicol sensitivity and sucrose resistance (a transformant in which lacZY::cat-sacB was replaced with ΔlacZY). From these, a transformant exhibiting ampicillin sensitivity (a transformant from which pKD46 had been lost) was further obtained. This transformant was designated W3110S3GKΔlacZY.

[0133] Similarly, PCR was carried out using the genomic DNA of the W3110 strain as a template and DNAs consisting of the base sequences shown in "Primer set" in Table 2 as primer sets to obtain amplified DNA fragments.

[0134]

[0135] wcaJ upstream 1 and wcaJ upstream 2 comprise the region from the initiation codon of the wcaJ gene to about 1000 bp upstream of the initiation codon. wcaM downstream 1 and wcaM downstream 2 comprise the region from the termination codon of the wcaM gene to about 1000 bp downstream of the termination codon.

[0136] PCR was performed using a mixture of wcaJ upstream 1, wcaM downstream 1, and cat-sacB fragments in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 111 and 113 as a primer set to obtain a DNA fragment consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the wcaJ-wzxC-wcaKLM operon (hereinafter referred to as wcaJ-wzxC-wcaKLM::cat-sacB).

[0137] PCR was performed using an equimolar mixture of wcaJ upstream 2 and wcaM downstream 2 as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 111 and 113 as a primer set to obtain a DNA fragment (hereinafter referred to as ΔwcaJ-wzxC-wcaKLM) consisting of a sequence in which the upstream of wcaJ and the downstream of wcaM are directly linked, without containing wcaJ-wzxC-wcaKLM.

[0138] The wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110S3GKΔlacZY constructed above by electroporation, and a transformant that exhibited chloramphenicol resistance and sucrose sensitivity (a transformant in which wcaJ-wzxC-wcaKLM was replaced with wcaJ-wzxC-wcaKLM::cat-sacB) was obtained.

[0139] The ΔwcaJ-wzxC-wcaKLM fragment was introduced into the transformant by electroporation to obtain a transformant that exhibited chloramphenicol sensitivity and sucrose resistance (a transformant in which wcaJ-wzxC-wcaKLM::cat-sacB had been replaced with ΔwcaJ-wzxC-wcaKLM). Furthermore, a transformant that exhibited ampicillin sensitivity (a transformant from which pKD46 had been lost) was obtained. This transformant was designated W3110S3GKΔlacZYΔwcaJM.

[0140] <Creation of a microorganism with enhanced expression of β1,3-galactosyltransferase and β1,3-N-acetylglucosamine transferase> Escherichia coli carrying a plasmid for expressing the genes, in which a gene encoding β1,3-galactosyltransferase derived from Chromobacterium violaceum ATCC553 strain and consisting of the amino acid sequence represented by SEQ ID NO: 124 (hereinafter referred to as Cvβ3GalT), a gene encoding β1,3-N-acetylglucosamine transferase derived from Neisseria polysaccharea ATCC43768 represented by SEQ ID NO: 125 (hereinafter referred to as NpLgtA), and the lacY gene derived from the W3110 strain were placed under the control of the uspA promoter, was constructed by the following method.

[0141] PCR was carried out using DNAs consisting of the base sequences shown in "Primer set" in Table 3 as a primer set and DNAs shown in "Template" in Table 3 as templates to obtain each amplified DNA fragment.

[0142]

[0143] The DNA represented by SEQ ID NO: 122 is a codon-optimized DNA for expressing in Escherichia coli the nucleotide sequence of the gene encoding β1,3-galactosyltransferase Cvβ3GalT derived from the Chromobacterium violaceum ATCC553 strain, as described in ACS Catal. 2019, 9(12), 10721-10726, and was prepared by artificial synthesis.

[0144] The DNA represented by SEQ ID NO:123 is a DNA prepared by artificial synthesis in which the nucleotide sequence of the gene encoding β1,3-N-acetylglucosamine transferase NpLgtA derived from Neisseria polysaccharea ATCC43768 strain represented by SEQ ID NO:125 has been codon-optimized for expression in Escherichia coli. The nucleotide sequences represented by SEQ ID NOs:117 and 118, and SEQ ID NOs:119 and 120 each contain a complementary sequence at the 5' end.

[0145] PCR was performed using an equimolar mixture of the Cvβ3galT fragment, the NplgtA fragment, and the lacY fragment as a template and DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 116 and 121 as a primer set to obtain a DNA fragment in which the three fragments were ligated (hereinafter referred to as Cvβ3galT-NplgtA-lacY).

[0146] PCR was performed using a primer set of oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 126 and 127 and plasmid pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385) as a template to obtain a vector fragment of approximately 4.7 kb. The nucleotide sequences represented by SEQ ID NOs: 116 and 126, and SEQ ID NOs: 121 and 127 each contain a complementary sequence at the 5' end.

[0147] The Cvβ3galT-NplgtA-lacY fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pUAKQE-Cvβ3galT-NplgtA-lacY. The expression plasmid pUAKQE-Cvβ3galT-NplgtA-lacY was used to transform the W3110S3GKΔlacZYΔwcaJM strain constructed above, thereby constructing an E. coli strain carrying pUAKQE-Cvβ3galT-NplgtA-lacY, which was designated the NNN strain.

[0148] (2) Construction of Microorganisms Expressing α1,2-Fucosyltransferases <Construction of Expression Vectors> Using the NNN strain constructed in (1) above, Escherichia coli carrying a gene expression plasmid containing genes encoding various α1,2-fucosyltransferases and rcsA derived from the W3110 strain, placed under the lac promoter, was constructed by the following method. PCR was performed using the W3110 strain, which had been prepared by conventional methods and a primer set consisting of DNAs represented by the nucleotide sequences of SEQ ID NOs: 132 and 133, as a template, to obtain an rcsA fragment. PCR was performed using the plasmid pSTV29 (Takara Bio Inc.) as a template and a primer set consisting of DNAs represented by the nucleotide sequences of SEQ ID NOs: 134 and 135, to obtain a vector fragment of approximately 2.9 kb. The nucleotide sequences represented by SEQ ID NOs: 132 and 134 and SEQ ID NOs: 133 and 135 each contain complementary sequences at their 5' ends. The rcsA fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pSTV-rcsA.

[0149] <Construction of a Plasmid for Expressing α1,2-Fucosyltransferase by Introducing a Point Mutation> A point mutation was introduced into FsFucT (α1,2-fucosyltransferase derived from Francisella sp. FSC1006 strain). The expression vector pFsFucT, which served as a template, was constructed as follows. PCR was performed using DNAs consisting of the base sequences shown in "Primer Set" in Table 4 as a primer set and DNAs listed in "Template" in Table 4 as a template, to obtain an amplified DNA fragment.

[0150]

[0151] The DNA represented by SEQ ID NO:46 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase FsFucT derived from Francisella sp. strain FSC1006 represented by SEQ ID NO:63 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0152] Using the expression vector pSTV29-rcsA constructed above as a template, PCR was carried out using DNA consisting of the base sequences represented by SEQ ID NOs: 136 and 137 as a primer set to obtain a vector fragment of approximately 3.5 kb.

[0153] The base sequences represented by SEQ ID NOs: 128 and 129 each contain a complementary sequence at the 5' end.

[0154] The resulting amplified DNA fragment and vector fragment were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct a plasmid pFsFucT that expresses α1,2-fucosyltransferase.

[0155] PCR was performed using pFsFucT as a template and DNA primer sets consisting of the base sequences shown in various primer sets in Table 5. The resulting vector fragments were ligated using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain various constructed plasmids shown in Table 5 in which point mutations had been introduced into the FsFucT gene sequence, i.e., various plasmids expressing α1,2-fucosyltransferase.

[0156]

[0157] <<Construction of Plasmids for Expressing α1,2-Fucosyltransferase Using Synthetic DNA>> PCR was performed using the DNA represented by "Synthetic DNA" in Table 6 as a template and the DNA represented by the base sequence of "Primer Set 1" as a primer set to obtain an amplified DNA fragment. PCR was performed using the plasmid represented by "Template Plasmid" in Table 6 as a template and the DNA represented by the base sequence of "Primer Set 2" as a primer set to obtain a linearized vector fragment. The resulting amplified DNA fragment and linearized vector fragment were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the various constructed plasmids listed in Table 6, i.e., various plasmids that express α1,2-fucosyltransferase.

[0158] <<Construction of E. coli strains expressing α1,2-fucosyltransferase>> The NNN strain constructed in (1) above was transformed with various "constructed plasmids" and pFsFucT shown in Tables 5 and 6 to obtain recombinant E. coli strains expressing various α1,2-fucosyltransferases and the control NNN / pFsFucT strain. Table 7 shows the mutation sites from the amino acid sequence represented by SEQ ID NO:63 for the α1,2-fucosyltransferases expressed by each recombinant E. coli strain.

[0159] The notations in Table 7 are explained below. Y159F: Indicates that Y at the position corresponding to position 159 has been replaced with F. Mutations at other positions are expressed in the same way. D172_Y173insLTA: Indicates that D is at the position corresponding to position 172 and Y is at the position corresponding to position 173, and that L, T, and A have been inserted between the positions corresponding to positions 172 and 173. Mutations at other positions are expressed in the same way. D167_I168insIHG: Indicates that D is at the position corresponding to position 167 and I is at the position corresponding to position 168, and that I, H, and G have been inserted between the positions corresponding to positions 167 and 168. I267del: Indicates that I at the position corresponding to position 267 has been deleted.

[0160]

[0161] <Evaluation of Productivity of LNFPI and 2'FL (Test Tube)> The recombinant E. coli expressing α1,2-fucosyltransferase obtained in Example 1 and the control strain NNN / pFsFucT strain were inoculated into a plastic test tube containing 2 mL of LB medium containing 100 mg / L kanamycin and 25 mg / L chloramphenicol, and cultured with shaking at 30°C for 14 to 17 hours.

[0162] 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 (except for 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 and then autoclaved, cooled, and then mixed)] and cultured with shaking at 30 ° C. Five hours after inoculation, IPTG (final concentration 1 mM) was added, and the mixture was further cultured with shaking for 24 hours (total 29 hours).

[0163] After the cultivation was completed, the culture medium was centrifuged and diluted appropriately, and the supernatant and intracellular contents of 2'FL and LNFP1 were analyzed. The results are shown in Table 8. In Table 8, the NNN / pFsFucT strain is simply referred to as FsFucT. A graph showing the total amount of LNFP1 (g / L) is shown in Figure 3.

[0164]

[0165] As shown in Table 8 and Figure 3, all strains in which LNFP production was observed showed reduced 2'FL production compared to the NNN / pFsFucT strain. Furthermore, for strains S1 to S3, S5, and S9 to S14, LNFP production was 0.4 g / L or more, and the ratio of 2'FL production to LNFPI production was reduced. In particular, the ratio of 2'FL production to LNFPI production was significantly reduced in strains S1, S2, S3, S5, S9, S10, and S14, indicating that the substrate selectivity of the α1,2-fucosyltransferase expressed in these strains for LNT was particularly improved.

[0166] Of these, strains S3 and S9, which showed significant suppression of 2'FL production and LNFPI production equivalent to that of the NNN / pFsFucT strain, were selected as candidates for α1,2-fucosyltransferases effective in LNFPI production.

[0167] [Example 2] Jar evaluation The S3 and S9 strains selected in Example 1 <Productivity evaluation of LNFPI and 2'FL (test tube)> and the control strain NNN / pFsFucT were each cultured on an LB plate containing 100 mg / L kanamycin and 25 mg / L chloramphenicol at 30°C for 24 hours, and then inoculated into a 300 mL baffled Erlenmeyer flask containing 65 mL of a medium [yeast extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L] containing 100 mg / L kanamycin and 25 mg / L chloramphenicol, and cultured with shaking at 30°C for 16 hours.

[0168] Then, 40 mL of the resulting culture broth was inoculated into a 3 L jar fermenter (manufactured by Mitsuwa Frontech Co., Ltd.) containing 760 mL of a production medium containing 100 mg / L kanamycin and 25 mg / L chloramphenicol [glucose monohydrate 22 g / L, ferrous sulfate heptahydrate 0.2 g / L, magnesium sulfate heptahydrate 2 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 5 g / L, ammonium chloride 1 g / L, yeast extract 5 g / L, manganese sulfate pentahydrate 10 mg / L, and thiamine hydrochloride 10 mg / L (aqueous solutions containing glucose, ferrous sulfate heptahydrate, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and mixed)], and the resulting mixture was cultured at 30 ° C. and 800 rpm for a total of 72 hours with stirring. 20% aqueous ammonia was added to adjust the pH to 6.9 during the culture.

[0169] IPTG (final concentration 1 mM) was added 24 hours after inoculation, and in the subsequent cultivation, 480 g / L glucose monohydrate and 40 g / L lactose monohydrate solutions were added at a rate of 5 to 7 mL / h.

[0170] After the cultivation was completed, the culture medium was centrifuged and diluted appropriately, and the supernatant and intracellular contents of 2'FL and LNFP1 were analyzed. The results are shown in Table 9. In Table 9, the NNN / pFsFucT strain is simply referred to as FsFucT. A graph showing the total amount of LNFP1 (g / L) is shown in Figure 4.

[0171]

[0172] As shown in Table 9 and Figure 4, 2'FL production was suppressed and LNFPI production increased in both the S3 and S9 strains compared to the NNN / pFsFucT strain. In particular, the S3 strain showed a significant reduction in the by-production of 2'FL, with the 2'FL production rate being 0.2% of the LNFPI production rate, suggesting that LNT can be preferentially used as a substrate.

[0173] Figure 2 shows the amino acid sequences of Francisella sp. FSC1006-derived FucT (FsFucT), S3 α1,2-fucosyltransferase, and Amphritea japonica-derived α1,2-fucosyltransferase. The amino acid sequence of S3 α1,2-fucosyltransferase is one in which the amino acid residues 161 to 172 of FsFucT have been replaced with the sequence motif of Amphritea japonica-derived α1,2-fucosyltransferase (shown shaded in Figure 2) (Figure 2). From this, it was found that the sequence motif of Amphritea japonica-derived α1,2-fucosyltransferase contributes to improved LNT selectivity.

[0174] Example 3 Evaluation of Amphritea japonica-Derived Sequence Motif in FucT Homologs (1) Construction of FucT Homolog Expression Vectors PCR was performed using DNAs consisting of the base sequences shown in "Primer Set" in Table 10 as a primer set and DNAs shown in "Template" in Table 10 as templates to obtain amplified DNA fragments.

[0175]

[0176] The DNA represented by SEQ ID NO: 90 is a DNA in which the base sequence of the gene encoding α1,2-fucosyltransferase (FucT54) derived from Sideroxydans lithotrophicus ES-11 (Sideroxidans lithotrophicus ES-11) represented by SEQ ID NO: 94 (WO 2019 / 008133) has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 91 is a DNA in which the base sequence of the gene encoding α1,2-fucosyltransferase (NbFucT1) derived from Neisseriaceae bacterium DSM 100970 represented by SEQ ID NO: 95 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. As in Example 1, each of the obtained amplified DNA fragments was ligated to a vector fragment amplified using the expression vector pSTV29-rcsA as a template using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids pNbFucT1 and pFucT54 that express various α1,2-fucosyltransferases.

[0177] (2) Construction of FucT Homolog Expression Vectors Having Sequence Motifs Vectors expressing FucT in which a sequence motif was introduced into FucT54 and NbFucT1 were constructed. Using the DNA represented by "Synthetic DNA" in Table 11 as a template and the DNA represented by the base sequence of "Primer Set 1" as a primer set, PCR was performed to obtain an amplified DNA fragment. Using the plasmid represented by "Template Plasmid" in Table 11 as a template and the DNA represented by the base sequence of "Primer Set 2" as a primer set, PCR was performed to obtain a linearized vector fragment. The resulting amplified DNA fragment and linearized vector fragment were ligated using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain plasmids expressing each α1,2-fucosyltransferase shown in "Constructed Plasmid" in Table 11.

[0178]

[0179] (2) Construction of Escherichia coli strains with α1,2-fucosyltransferase activity The NNN strain constructed in Example 1(1) was transformed with each of the α1,2-fucosyltransferase expression plasmids obtained above to construct recombinant Escherichia coli strains carrying each plasmid, which were designated the A_H helix strain, the F_H helix strain, the NNN / pNbFucT1 strain, and the NNN / pFucT54 strain. The mutation sites from the amino acid sequence represented by SEQ ID NO:94 or SEQ ID NO:95 for the α1,2-fucosyltransferase expressed by each recombinant Escherichia coli strain are shown in Table 12.

[0180] The notations in Table 12 are explained below. In clone name A_H helix, N170T indicates that N at position 170 in the amino acid sequence represented by SEQ ID NO: 94 has been substituted with T. Mutations at other positions are expressed in the same manner. In clone name F_H helix, N160T indicates that N at position 160 in the amino acid sequence represented by SEQ ID NO: 95 has been substituted with T. Mutations at other positions are expressed in the same manner. In clone name F_H helix, N160_H161insDLSA indicates that DLSA has been inserted between N160 and H161. In clone name F_H helix, G165del indicates that G165 has been deleted.

[0181]

[0182] (3) Productivity Evaluation The A_H helix strain, the F_H helix strain, the S3 strain constructed in Example 1, and the control strains NNN / pFsFucT strain, NNN / pNbFucT1 strain, and NNN / pFucT54 strain were each cultured on an LB plate containing 100 mg / L kanamycin and 25 mg / L chloramphenicol at 30°C for 24 hours, and then inoculated into a 300 mL baffled Erlenmeyer flask containing 65 mL of a medium [yeast extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L] containing 100 mg / L kanamycin and 25 mg / L chloramphenicol, and cultured with shaking at 30°C for 16.5 hours.

[0183] Then, 40 mL of the resulting culture broth was inoculated into a 3 L jar fermenter (manufactured by Mitsuwa Frontech Co., Ltd.) containing 760 mL of a production medium containing 100 mg / L kanamycin and 25 mg / L chloramphenicol [glucose monohydrate 22 g / L, ferrous sulfate heptahydrate 0.2 g / L, magnesium sulfate heptahydrate 2 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 5 g / L, ammonium chloride 1 g / L, yeast extract 5 g / L, manganese sulfate pentahydrate 10 mg / L, and thiamine hydrochloride 10 mg / L (aqueous solutions containing glucose, ferrous sulfate heptahydrate, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and mixed)], and the resulting mixture was cultured at 30 ° C. and 800 rpm for a total of 72 hours with stirring. 20% aqueous ammonia was added to adjust the pH to 6.9 during the culture.

[0184] 24 hours after inoculation, IPTG (final concentration: 1 mM) was added, and in the subsequent cultivation, 480 g / L glucose solution and 40 g / L lactose monohydrate were added at a rate of 5 to 7 mL / h.

[0185] After the culture was completed, the culture medium was centrifuged and appropriately diluted, and the 2'FL and LNFP1 contained in the supernatant and intracellular cells were analyzed. The results are shown in Table 13. The total amount of LNFP1 (g / L) is shown in Figure 5.

[0186]

[0187] As shown in Table 13 and FIG. 5, 2′FL production was significantly suppressed and LNFPI production was increased in the S3, A_H helix, and F_H helix strains compared to the NNN / pFsFucT, NNN / pNbFucT1, and NNN / pFucT54 strains.

[0188] From this, it was found that the sequence motif of α1,2-fucosyltransferase derived from Amphritea japonica is widely effective in improving substrate selectivity for LNT, regardless of the origin of the α1,2-fucosyltransferase.

[0189] 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. 2023-092642) filed on June 5, 2023, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0190] SEQ ID NOs: 1 to 37: Nucleotide sequences of primers SEQ ID NO: 38: Nucleotide sequence of No. 1 SEQ ID NO: 39: Nucleotide sequence of No. 2 SEQ ID NO: 40: Nucleotide sequence of No. 3 SEQ ID NO: 41: Nucleotide sequence of No. 4 SEQ ID NO: 42: Nucleotide sequence of No. 5 SEQ ID NO: 43: Nucleotide sequence of No. 6 SEQ ID NO: 44: Nucleotide sequence of No. 7 SEQ ID NO: 45: No. 27 SEQ ID NO:46: Nucleotide sequence of FsFucT SEQ ID NO:47: Nucleotide sequence of S1 SEQ ID NO:48: Nucleotide sequence of S2 SEQ ID NO:49: Nucleotide sequence of S3 SEQ ID NO:50: Nucleotide sequence of S4 SEQ ID NO:51: Nucleotide sequence of S5 SEQ ID NO:52: Nucleotide sequence of S6 SEQ ID NO:53: Nucleotide sequence of S7 SEQ ID NO:54: Nucleotide sequence of S8 SEQ ID NO:55: Nucleotide sequence of S9 SEQ ID NO:56: Nucleotide sequence of S10 SEQ ID NO:57: Nucleotide sequence of S11 SEQ ID NO:58: Nucleotide sequence of S12 SEQ ID NO:59: Nucleotide sequence of S13 SEQ ID NO:60: Nucleotide sequence of S14 SEQ ID NO:61: Nucleotide sequence of S15 SEQ ID NO:62: Nucleotide sequence of rcsA SEQ ID NO:63: Amino acid sequence of FsFucT SEQ ID NO:64: Amino acid sequence of S1 SEQ ID NO:65: Amino acid sequence of S2 SEQ ID NO:66: Amino acid sequence of S3 SEQ ID NO:67: Amino acid sequence of S4 SEQ ID NO: 68: Amino acid sequence of S5 SEQ ID NO: 69: Amino acid sequence of S6 SEQ ID NO: 70: Amino acid sequence of S7 SEQ ID NO: 71: Amino acid sequence of S8 SEQ ID NO: 72: Amino acid sequence of S9 SEQ ID NO: 73: Amino acid sequence of S10 SEQ ID NO: 74: Amino acid sequence of S11 SEQ ID NO: 75: Amino acid sequence of S12 SEQ ID NO: 76: Amino acid sequence of S13 SEQ ID NO: 77: Amino acid sequence of S14 SEQ ID NO: 78: Amino acid sequence of S15 SEQ ID NO: 79: Amino acid sequence of rcsA SEQ ID NOs: 80 to 87: Nucleotide sequences of primers SEQ ID NO: 88: Nucleotide sequence of SA_H helix SEQ ID NO: 89: Nucleotide sequence of SF_H helix SEQ ID NO: 90: Nucleotide sequence of FucT54 SEQ ID NO: 91: Nucleotide sequence of NbFucT1 SEQ ID NO: 92: Nucleotide sequence of A_H helix SEQ ID NO: 93: Nucleotide sequence of F_H helix SEQ ID NO: 94: Nucleotide sequence of FucT54 SEQ ID NO: 95: Nucleotide sequence of NbFucT1 SEQ ID NO: 96: Amino acid sequence of A_H helix SEQ ID NO: 97: Amino acid sequence of F_H helixSEQ ID NO: 98: Amino acid sequence which is a partial sequence of AjFucT derived from Amphritea japonica SEQ ID NO: 99: Amino acid sequence which is a partial sequence of SbFucT derived from Sterolibacteriaceae bacterium J5B SEQ ID NO: 100: Nucleotide sequence of AjFucT SEQ ID NO: 101: Amino acid sequence of AjFucT SEQ ID NOs: 102 to 121: Nucleotide sequences of primers SEQ ID NO: 122: Nucleotide sequence of Cvβ3galT derived from Chromobacterium violaceum ATCC553 SEQ ID NO: 123: Nucleotide sequence of NplgtA derived from Neisseria polysaccharea ATCC43768 SEQ ID NO: 124: Chromobacterium violaceum SEQ ID NO: 125: Amino acid sequence of Cvβ3galT derived from ATCC553 SEQ ID NO: 126: Nucleotide sequence of primer for amplifying rcsA fragment SEQ ID NO: 127: Nucleotide sequence of primer for amplifying rcsA fragment SEQ ID NOs: 128, 129: Nucleotide sequences of primers SEQ ID NO: 130: Nucleotide sequence of S5' SEQ ID NO: 131: Amino acid sequence of SbFucT derived from Sterolibacteriaceae bacterium J5B SEQ ID NOs: 132 to 141: Nucleotide sequences of primers

Claims

1. A protein having fucosyl transfer activity to lacto-N-tetraose (LNT), into which at least one of the following mutations [1] and [2] has been introduced (excluding those in which the amino acid sequence constituting the protein before and after the introduction of the mutation is identical). [1] A mutation in which an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:98 is inserted. [2] A mutation in which an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:99 is inserted.

2. The protein according to claim 1, which is a protein having at least one of the mutations described in [1] and [2] in an amino acid sequence having a full-length sequence identity of 50% or more with at least one of the amino acid sequences represented by SEQ ID NOs: 63, 94 and 95.

3. The protein according to claim 1 or 2, wherein the mutation [1] is a mutation introduced at a position corresponding to a region contributing to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 101 when aligned with the amino acid sequence represented by SEQ ID NO: 101, and the mutation [2] is a mutation introduced at a position corresponding to a region contributing to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 131 when aligned with the amino acid sequence represented by SEQ ID NO:

131.

4. The protein according to claim 3, wherein the region contributing to the interaction with the substrate is an amino acid sequence corresponding to positions 162 to 176 when aligned with the amino acid sequence represented by SEQ ID NO:

101.

5. The protein according to any one of claims 1 to 4, wherein the mutation [1] is a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO:101, the amino acid sequence corresponding to positions 162 to 176 is replaced with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:98, and the mutation [2] is a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO:131, the amino acid sequence corresponding to positions 168 to 184 is replaced with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:

99.

6. The protein according to any one of claims 1 to 5, wherein the mutation [1] is at least one mutation selected from the following [1a] to [1o] at a position corresponding to positions 162 to 176 when aligned with the amino acid sequence represented by SEQ ID NO: 101, and the mutation [2] is at least one mutation selected from the following [2a] to [2q] at a position corresponding to positions 168 to 184 when aligned with the amino acid sequence represented by SEQ ID NO:

131. [1a] At the position corresponding to position 162, it is threonine. [1b] At the position corresponding to position 163, it is aspartic acid. [1c] At the position corresponding to position 164, it is leucine. [1d] At the position corresponding to position 165, it is serine. [1e] At the position corresponding to position 166, it is alanine. [1f] At the position corresponding to position 167, it is asparagine. [1g] At the position corresponding to position 168, it is asparagine. [1h] At the position corresponding to position 169, it is isoleucine. [1i] At the position corresponding to position 170, it is histidine. [1j] At the position corresponding to position 171, it is glycine. [1k] At the position corresponding to position 172, it is isoleucine. [1l] At the position corresponding to position 173, it is cysteine. [1m] At the position corresponding to position 174, it is serine. [1n] At the position corresponding to position 175, it is glutamic acid. [1o] At the position corresponding to position 176, it is glutamic acid. [2a] At the position corresponding to position 168, it is phenylalanine. [2b] At the position corresponding to position 169, it is valine. [2c] At the position corresponding to position 170, it is glutamine. [2d] At the position corresponding to position 171, it is aspartic acid. [2e] At the position corresponding to position 172, it is proline. [2f] At the position corresponding to position 173, it is valine. [2g] At the position corresponding to position 174, it is valine. [2h] At the position corresponding to position 175, it is arginine. [2i] At the position corresponding to position 176, it is arginine. [2j] At the position corresponding to position 177, it is valine. [2k] At the position corresponding to position 178, it is histidine. [2l] At the position corresponding to position 179, it is glycine. [2m] At the position corresponding to position 180, it is valine. [2n] At the position corresponding to position 181, it is aspartic acid. [2o] At the position corresponding to position 182, it is leucine. [2p] At the position corresponding to position 183, it is threonine. [2q] At the position corresponding to position 184, it is alanine.

7. A protein having fucosyl transfer activity to LNT, which, when aligned with the amino acid sequence represented by SEQ ID NO:63, has at least one mutation selected from the following (1) to (31) introduced at each position corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270, and which has a higher fucosyl transfer activity to LNT than the protein before the introduction of the mutation.(1) at position 7, it is an alanine or serine; (2) at position 42, it is an isoleucine; (3) at position 94, it is a histidine; (4) at position 95, it is a tyrosine; (5) at position 159, it is a phenylalanine; (6) at position 160, it is a valine; (7) at position 161, it is a glutamine or threonine; (8) at position 162, it is an aspartic acid or asparagine; (9) at position 163, it is a leucine; (10) at position 164, it is an isoleucine, lysine, serine or valine; (11) at position 165, it is an alanine or valine; (12) at position 166, it is an arginine or asparagine; (13) at position 167, it is an arginine or asparagine. (14) At the position corresponding to position 168, it is valine. (15) At the position corresponding to position 167, it is aspartic acid, at the position corresponding to position 168, it is isoleucine, and isoleucine, histidine and glycine are inserted between the positions corresponding to positions 167 and 168. (16) At the position corresponding to position 169, it is histidine. (17) At the position corresponding to position 170, it is leucine, glycine or serine. (18) At the position corresponding to position 171, it is threonine, valine or glutamic acid. (19) At the position corresponding to position 172, it is glutamic acid or alanine. (20) At the position corresponding to position 172, it is aspartic acid, at the position corresponding to position 173, it is tyrosine, and leucine, threonine and alanine are inserted between the positions corresponding to positions 172 and 173. (21) At the position corresponding to position 259, it is lysine. (22) At the position corresponding to position 260, it is glutamine; (23) At the position corresponding to position 262, it is histidine; (24) At the position corresponding to position 263, it is lysine; (25) At the position corresponding to position 264, it is valine; (26) At the position corresponding to position 265, it is arginine; (27) At the position corresponding to position 266, it is aspartic acid; (28) At the position corresponding to position 267, it is phenylalanine; (29) At the position corresponding to position 268, it is aspartic acid; (30) At the position corresponding to position 269, it is threonine; and (31) At the position corresponding to position 270, it is arginine.

8. The protein according to claim 7, which is an amino acid sequence having a full-length sequence identity of 50% or more with at least any one of the amino acid sequences represented by SEQ ID NO:63, 94, and 95, in which at least one mutation selected from (1) to (31) has been introduced at positions corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence represented by SEQ ID NO:

63.

9. The protein according to claim 7 or 8, wherein the mutations introduced at positions corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence represented by SEQ ID NO:63 are any one of the following [3] to [12]: [3] At position 159, it is phenylalanine; at position 160, it is valine; at position 161, it is glutamine; at position 162, it is aspartic acid; at position 164, it is valine; at position 165, it is valine; at position 166, it is arginine; at position 167, it is arginine; at position 168, it is valine; at position 169, it is histidine; at position 170, it is glycine; at position 171, it is valine; at position 172, it is aspartic acid; at position 173, it is tyrosine; and between positions 172 and 173, leucine, tyrosine, and alanine are inserted.[4] At position 94, it is histidine; at position 95, it is tyrosine; at position 159, it is phenylalanine; at position 160, it is valine; at position 161, it is glutamine; at position 162, it is aspartic acid; at position 164, it is valine; at position 165, it is valine; at position 166, it is arginine; at position 167, it is arginine; at position 168, it is valine; at position 169, it is histidine; at position 170, it is glycine; at position 171, it is valine; at position 172, it is aspartic acid; and at position 173, it is tyrosine. Between positions corresponding to positions 172 and 173, leucine, tyrosine and alanine are inserted; at position corresponding to position 259, lysine; at position corresponding to position 260, glutamine; at position corresponding to position 263, lysine; at position corresponding to position 264, valine; at position corresponding to position 265, arginine; at position corresponding to position 266, aspartic acid; at position corresponding to position 267, phenylalanine; at position corresponding to position 268, aspartic acid; at position corresponding to position 269, threonine; and at position corresponding to position 270, arginine. [5] At the position corresponding to position 161, it is threonine, at the position corresponding to position 162, it is aspartic acid, at the position corresponding to position 163, it is leucine, at the position corresponding to position 164, it is serine, at the position corresponding to position 165, it is alanine, at the position corresponding to position 166, it is asparagine, at the position corresponding to position 167, it is aspartic acid, at the position corresponding to position 168, it isoleucine, isoleucine, histidine and glycine are inserted between the positions corresponding to positions 167 and 168, it is serine, at the position corresponding to position 170, it is glutamic acid, and at the position corresponding to position 172, it is glutamic acid. [6] At the position corresponding to position 164, it is isoleucine.[7] At the position corresponding to position 162, it is aspartic acid, and at the position corresponding to position 164, it is lysine. [8] At the position corresponding to position 262, it is histidine. [9] At the position corresponding to position 42, it is isoleucine. [10] At the position corresponding to position 162, it is asparagine. [11] At the position corresponding to position 7, it is serine. [12] At the position corresponding to position 7, it is alanine.

10. A DNA encoding the protein according to any one of claims 1 to 9.

11. A recombinant DNA comprising the DNA according to claim 10.

12. A transformant obtained by transforming a host cell with the recombinant DNA according to claim 11.

13. The transformant according to claim 12, which is a microorganism in which the activity of the protein according to any one of claims 1 to 9 and the productivity of the fucose-containing carbohydrate are enhanced.

14. The transformant according to claim 13, wherein the microorganism is Escherichia coli.

15. A method for producing a fucose-containing saccharide, comprising preparing the transformant according to any one of claims 13 to 14, and producing the fucose-containing saccharide in a culture using the transformant.

16. The method according to claim 15, wherein the fucose-containing carbohydrate is lacto-N-fucopentaose I (LNFPI).

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