Protein having 1,3-fucosyltransferase activity and method for producing fucose-containing sugar

Abstract

The purpose of the present invention is to provide a means for producing a fucose-containing sugar more efficiently as compared with conventional techniques. The present invention relates any protein of [1]-[3] below.　[1] A protein comprises the amino acid sequence represented by SEQ ID NO: 4 with at least the 5th to 9th amino acids from the N-terminus lost, the protein further having an α 1,3-fucosyltransferase activity.　[2] A protein comprises the amino acid sequence represented by SEQ ID NO: 4 with at least the 5th to 9th amino acids from the N-terminus lost, where 1-20 amino acids are deleted, substituted, inserted or added, the protein further having an α 1,3-fucosyltransferase activity.　[3] A protein has at least 90% identity with the amino acid sequence represented by SEQ ID NO: 4 with at least the 5th to 9th amino acids from the N-terminus lost, the protein further having an α 1,3-fucosyltransferase activity.

Description

Method for producing proteins having 1,3-fucosyltransferase activity and fucose-containing carbohydrates

[0001] The present invention relates to a protein having 1,3-fucosyltransferase activity and a method for producing fucose-containing carbohydrates such as 3-fucosyllactose.

[0002] Human milk oligosaccharides (HMOs) contained in human breast milk have been reported to have health functions such as improving the intestinal environment as a prebiotic, activating the immune system, and promoting cognitive development in infants. Due to their physiological activities, they are expected to be used as additives to infant formula and as health functional materials for adults (Non-Patent Document 1).

[0003] Among the oligosaccharides known as HMOs, fucose-containing carbohydrates such as 3-fucosyllactose and lactodifucotetraose account for approximately 60% of the total HMOs contained in breast milk in terms of substance amount (Non-Patent Document 2), and their functionality has attracted particular attention among HMOs.

[0004] Methods for producing 3-fucosyllactose and lactodifucotetraose include methods using fucosyltransferase. Examples of methods for producing 3-fucosyllactose using fucosyltransferase include methods using α1,3-fucosyltransferase derived from Helicobacter pylori (Non-Patent Document 3) and α1,3-fucosyltransferase derived from Bacteroides fragilis (Non-Patent Document 4, Patent Document 1).

[0005] Furthermore, it has been reported that the productivity of 3-fucosyllactose has been improved by modifying the α1,3-fucosyltransferase derived from Helicobacter pylori, specifically by improving enzyme activity by introducing amino acid mutations (Patent Document 2, Non-Patent Document 5), and by improving protein expression by deleting or adding amino acid sequences on the C-terminal side (Non-Patent Document 6).

[0006] Japanese Patent No. 6097691 U.S. Patent No. 10336990

[0007] Int J Pediatrics, 2019, 2390240, 1-8 Curr Opin Biotechnol, 2019, 56, 130-137 Metabolic Engineering, 2017, 41, 23-38 Biotechnol Bioproc Eng, 2013, 18, 843-849Biotechnol Bioeng, 2016, 113, 1666-1675 Metabolic Engineering, 2018, 48, 269-278

[0008] However, the productivity of fucose-containing saccharides by the α1,3-fucosyltransferases described in the above prior art documents is insufficient, and there is a need for a more efficient method for producing fucose-containing saccharides.

[0009] Therefore, an object of the present invention is to provide a means for producing fucose-containing saccharides more efficiently than conventional methods.

[0010] The inventors have discovered that by deleting a portion of the N-terminal amino acid sequence of a protein having 1,3-fucosyltransferase activity containing a specific amino acid sequence, 3-fucosyllactose or lactodifucotetraose can be produced more efficiently than before the deletion, and have completed the present invention.

[0011] That is, the present invention is as follows: 1. A protein according to any one of [1] to [3] below. [1] A protein consisting of an amino acid sequence obtained by deleting at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4, and which has α1,3-fucosyltransferase activity. [2] A protein consisting of an amino acid sequence obtained by deleting at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4, and which has 1 to 20 amino acids deleted, substituted, inserted or added, and which has α1,3-fucosyltransferase activity. [3] A protein consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 4, and which has at least the fifth to ninth amino acids from the N-terminus deleted, and which has α1,3-fucosyltransferase activity. 2. The protein according to 1 above, which is any one of [1A] to [1C] below. [1A] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2. [1B] A protein consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 20 amino acids with respect to the amino acid sequence represented by SEQ ID NO: 2, and having α1,3-fucosyltransferase activity. [1C] A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 2, and having α1,3-fucosyltransferase activity. 3. DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a sequence homologous thereto, and encoding the protein described in 1 above. 4. Recombinant DNA containing the DNA described in 3 above. 5. A transformant obtained by transforming a parent strain with the recombinant DNA described in 4 above. 6. The transformant described in 5 above, which is a microorganism in which the activity of the protein described in 1 or 2 above has been enhanced. 7. The transformant described in 6 above, wherein the microorganism is Escherichia coli. 8. A method for producing a fucose-containing saccharide, comprising culturing the transformant described in 5 above in a medium to produce the fucose-containing saccharide. 9. A method for producing a fucose-containing saccharide, comprising: culturing the transformant according to 5 above in a medium to obtain a culture or a processed product of the culture; and using the culture or the processed product of the culture as an enzyme source, causing the enzyme source, GDP-fucose and an acceptor saccharide to be present in an aqueous medium, and producing a fucose-containing saccharide in the aqueous medium.10. The method according to 8 or 9 above, wherein the fucose-containing saccharide is 3-fucosyllactose or lactodifucotetraose.

[0012] By using the protein of this embodiment, fucose-containing carbohydrates can be produced more efficiently than conventional methods.

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

[0014] 1. Protein The protein of this embodiment is a protein described in any one of [1] to [3] below. [1] A protein consisting of an amino acid sequence obtained by deleting at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4, and having α1,3-fucosyltransferase activity. [2] A protein consisting of an amino acid sequence obtained by deleting at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4, and having 1 to 20 amino acids deleted, substituted, inserted, or added, and having α1,3-fucosyltransferase activity. [3] A protein consisting of an amino acid sequence that shares 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4, and having at least the fifth to ninth amino acids from the N-terminus, and having α1,3-fucosyltransferase activity.

[0015] One aspect of the protein of this embodiment is preferably any one of the proteins [1A] to [1C] below. [1A] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2. [1B] A protein consisting of an amino acid sequence represented by SEQ ID NO: 2 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and which has α1,3-fucosyltransferase activity. [1C] A protein consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 2 and which has α1,3-fucosyltransferase activity.

[0016] The amino acid sequences of [1] to [3] above will be explained below. The amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence in which the 2nd to 11th amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 (BfFucT derived from Bacteroides fragilis ATCC 25285) are deleted. One embodiment of the protein of [1] above is the protein of [1A] above.

[0017] The present inventors have found that a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 has superior productivity of 3-fucosyllactose or lactodifucotetraose compared to a protein consisting of the amino acid sequence represented by SEQ ID NO: 4. In addition to this finding, the present inventors have concluded the following (1) and (2) by analyzing the amino acid sequences represented by SEQ ID NOs: 2 and 4. Based on these findings, it is believed that deleting at least the fifth to ninth amino acids in the amino acid sequence represented by SEQ ID NO: 4 will increase 3-fucosyllactose or lactodifucotetraose productivity compared to a protein consisting of the amino acid sequence represented by SEQ ID NO: 4. (1) The amino acid sequence from the fourth cysteine ​​to the ninth leucine from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 (CLSIIL, SEQ ID NO: 36) is thought to form a helix structure, thereby affecting enzymatic activity. (2) It is thought that a rare codon in the amino acid sequence from the fifth leucine to the tenth leucine in the amino acid sequence represented by SEQ ID NO: 4 (LSIILL, SEQ ID NO: 35) inhibits the expression of a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 in Escherichia coli. Here, a rare codon refers to a codon that is used less frequently than other codons in endogenous genes of E. coli.

[0018] The amino acid sequence of the protein [1] is such that at least the 5th to 9th amino acids from the N-terminus are deleted from the amino acid sequence represented by SEQ ID NO: 4. The deletion preferably extends from at least the 5th to 9th amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4, and extends up to the 2nd to 16th amino acids from the N-terminus. For example, it is more preferable that the deletion extend from the 2nd, 3rd, or 4th amino acid from the N-terminus to the 10th, 11th, 12th, 13th, 14th, 15th, or 16th amino acid from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4.

[0019] Specific examples of the range of the deletion are as follows for (1) and (2): From the viewpoint of the helix structure described in (1), the range of the deletion is preferably the 4th to 9th amino acids, more preferably the 5th to 9th amino acids, from the N-terminus of the amino acid sequence shown in SEQ ID NO: 4. From the viewpoint of the rare codon that affects the expression of α1,3-fucosyltransferase in Escherichia coli described in (2), the range of the deletion is preferably the 5th to 10th amino acid sequences, more preferably the 5th to 9th amino acids, from the N-terminus of the amino acid sequence shown in SEQ ID NO: 4.

[0020] Furthermore, the present inventors have speculated through amino acid sequence analysis that the amino acid sequence from the 17th amino acid from the N-terminus of the amino acid sequence represented by SEQ ID NO:4 forms a secondary structure separate from the aforementioned helix structure (the helix structure formed by the 4th to 9th amino acids at the N-terminus), and that this secondary structure is necessary for maintaining α1,3-fucosyltransferase activity. Therefore, by limiting the deletion to a maximum of the 2nd to 16th amino acids from the N-terminus, a decrease in α1,3-fucosyltransferase activity can be suppressed.

[0021] The protein of [2] above is one embodiment of a mutant protein of the protein of [1]. One aspect of the protein of [2] is the protein of [1B] above. A mutant protein is a protein obtained by artificially deleting or substituting amino acid residues in a protein before mutation, or by artificially inserting or adding amino acid residues into the protein. In a mutant protein, "deleted, substituted, inserted, or added amino acids" may mean that 1 to 20 amino acids are deleted, substituted, inserted, or added at any position in the same sequence.

[0022] The amino acids to be substituted, inserted, or added may be natural or non-natural. Examples of natural amino acids include the natural amino acids described below. Examples of amino acids that can be substituted for each other are described below. Amino acids within the same group can be substituted for each other.

[0023] The fact that the amino acid sequence constituting the protein of [2] "has 1 to 20 amino acids deleted, substituted, inserted or added in an amino acid sequence in which at least the 5th to 9th amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 have been deleted" can be confirmed, for example, by aligning the amino acid sequence of the protein to be confirmed to have deletions, substitutions, insertions or additions with the amino acid sequence in which at least the 5th to 9th amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 have been deleted.

[0024] The range of "1 to 20" in "deletion, substitution, insertion or addition of 1 to 20 amino acids" is not particularly limited, but for example, if 100 amino acids in an amino acid sequence is considered to be one unit, this means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and more preferably about 1, 2, 3, 4, or 5, per unit.

[0025] "Amino acid deletion" means the absence or disappearance of an amino acid residue in a sequence; "amino acid substitution" means that an amino acid residue in a sequence is replaced with another amino acid residue; "amino acid insertion" means that a new amino acid residue is inserted into a sequence; and "amino acid addition" means that a new amino acid residue is added to a sequence in an inserting manner.

[0026] Specific embodiments of "deletion, substitution, insertion, or addition of 1 to 20 amino acids" include embodiments in which 1 to 20 amino acids are replaced with other chemically similar amino acids. Examples include substitution of a hydrophobic amino acid with another hydrophobic amino acid, or substitution of a polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known for each amino acid in the art.

[0027] Specific examples of nonpolar (hydrophobic) amino acids include alanine, valine, glycine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid.

[0028] When 1 to 20 amino acids are substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4 in which at least the 5th to 9th amino acids from the N-terminus are deleted, the amino acid residues to be substituted, inserted, or added may be either naturally occurring or non-naturally occurring.

[0029] Examples of naturally occurring amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0030] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3-hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

[0031] An amino acid sequence having a deletion, substitution, insertion, or addition of 1 to 20 amino acids in the amino acid sequence of a target protein includes an amino acid sequence that has a certain level of sequence identity with the amino acid sequence of the target protein, such as an amino acid sequence that has preferably 90% or more identity with the amino acid sequence of the target protein, more preferably 91, 92, 93, or 94% or more identity, further preferably 95% or more identity, most preferably 98% or more identity, and particularly preferably 99% or more identity.

[0032] The protein of [3] above is one embodiment of a homologous protein to the protein of [1]. One embodiment of the protein of [3] is the protein of [1C] above. Homologous proteins are proteins found in organisms in nature, and refer to a group of proteins that are derived from the same protein in evolutionary origin. Homologous proteins are similar to each other in structure and function.

[0033] The fact that the amino acid sequence constituting the protein of [3] "has 90% or more identity with the amino acid sequence of SEQ ID NO: 4 from which at least the fifth to ninth amino acids from the N-terminus have been deleted" can be confirmed by aligning the amino acid sequence of the protein whose identity is to be confirmed with the target amino acid sequence (the amino acid sequence of SEQ ID NO: 4 from which at least the fifth to ninth amino acids from the N-terminus have been deleted), and examining the identity.

[0034] The identity between the amino acid sequence constituting the protein of [3] and the amino acid sequence of SEQ ID NO: 4 in which at least the 5th to 9th amino acids from the N-terminus are deleted is 90% or more, preferably 91, 92, 93, or 94% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0035] An alignment of amino acid sequences can be created using, for example, the known alignment program ClustalW [Nucleic Acids Research 22, 4673, (1994)]. ClustalW is available, for example, at http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When creating an alignment using ClustalW, for example, default parameters can be used.

[0036] The α1,3-fucosyltransferase activity refers to the activity of transferring fucose from the donor substrate GDP-fucose to the 3-hydroxyl group of N-acetylglucosamine or the 3-hydroxyl group of glucose of the acceptor substrate carbohydrate (also referred to as "acceptor carbohydrate") via an α1,3-bond to generate a fucose-containing carbohydrate.

[0037] Examples of the acceptor carbohydrate include N-acetylglucosamine, N-acetyllactosamine, galactose, fucose, sialic acid, glucose, or lactose, or a combination thereof, and a sugar chain containing any of these as a partial structure. Among these, lactose or 2'-fucosyllactose is preferred as the acceptor carbohydrate.

[0038] The fucose-containing carbohydrates that can be obtained include, for example, 3-fucosyllactose, lactodifucotetraose, Lewis X, and lacto-N-fucopentaose III, and among these, 3-fucosyllactose or lactodifucotetraose is preferred.

[0039] As used herein, the term "substrate" refers to a substance or combination of substances on which α1,3-fucosyltransferase can act to produce a fucose-containing carbohydrate.

[0040] The percentage of sequence identity between two amino acid sequences or two nucleotide sequences is calculated as the ratio of matching residues when the residues contained in the two sequences are aligned so that they are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm. Non-limiting examples of such mathematical algorithms include the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, and the algorithm of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and a modified version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0041] Alignment for determining the percentage of sequence identity can be performed using programs based on these mathematical algorithms. The programs can be executed by a computer as appropriate. Such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.), MAFFT (http: / / mafft.cbrc.jp / alignment / server / ), MUSCLE (http: / / www.ebi.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA. Alignment using these programs can be performed, for example, using default parameters.

[0042] The CLUSTAL program is described in Higgins et al. (1988) Gene 73:237-244, Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. Programs called BLASTP and BLASTN have been developed based on BLAST (see http: / / www.ncbi.nlm.nih.gov), and the percentage of sequence identity may be calculated using these programs with default settings. Alternatively, the percentage of sequence identity may be calculated using, for example, software GENETYX Ver. 16 from Genetics Corporation, which employs the Lipman-Pearson method. The lowest value of the percentage of sequence identity derived by these calculations may be used.

[0043] The presence of α1,3-fucosyltransferase activity in a protein of this embodiment can be confirmed, for example, by a method including the following steps 1) to 4). 1) First, a recombinant DNA containing DNA encoding the protein of this embodiment whose activity is to be confirmed is prepared by the method described below. 2) A microorganism lacking α1,3-fucosyltransferase activity, such as Escherichia coli W3110 strain, is transformed with the recombinant DNA to produce a microorganism, and a cell extract containing the protein is prepared from the resulting culture. 3) The cell extract containing the protein is contacted with an aqueous solution containing the substrate GDP-fucose and an acceptor carbohydrate to obtain a reaction solution in which fucose-containing carbohydrates are produced in the aqueous solution. 4) The fucose-containing carbohydrates in the reaction solution are detected using a sugar analyzer described below, and the presence of α1,3-fucosyltransferase activity in the protein of this embodiment is confirmed.

[0044] 2. DNA The DNA of this embodiment consists of the nucleotide sequence represented by SEQ ID NO: 1 or a homologous sequence thereof, and encodes the protein described in any one of [1] to [3] above. In this embodiment, a "homologous sequence of the nucleotide sequence represented by SEQ ID NO: 1" includes not only a sequence identical to the nucleotide sequence represented by SEQ ID NO: 1, but also a sequence that differs due to the substitution, deletion, or addition of several nucleotides. The degree of identity of a homologous sequence varies depending on the length of the polynucleotide, but can be, for example, at least 80% or more, preferably at least 85% or more, more preferably at least 90% or more, even more preferably 95% or more, and most preferably 100%.

[0045] Specific examples of the DNA of this embodiment include the following DNAs [D1] to [D3]. [D1] DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1. [D2] DNA that hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and encodes a homologous protein having α1,3-fucosyltransferase activity. [D3] DNA that consists of a nucleotide sequence that has at least 95% identity, preferably 97% identity, more preferably 98% identity, and most preferably 99% identity to the nucleotide sequence represented by SEQ ID NO: 1 and encodes a homologous protein having α1,3-fucosyltransferase activity.

[0046] In the above, hybridization refers to a process in which DNA hybridizes to a DNA having a specific base sequence or a part of the DNA. Therefore, the base sequence of the DNA having the specific base sequence or the DNA hybridizing to a part of the DNA may be a DNA of a length that is useful as a probe for Northern or Southern blot analysis or can be used as an oligonucleotide primer for PCR analysis.

[0047] Examples of DNA used as a probe include DNA having at least 100 bases, preferably at least 200 bases, and more preferably at least 500 bases. Examples of DNA used as a primer include DNA having at least 10 bases, preferably at least 15 bases.

[0048] Methods for DNA hybridization experiments are well known, and hybridization conditions can be determined and experiments can be performed according to, for example, Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Methods for General and Molecular Bacteriology (ASM Press, 1994), Immunology Methods Manual (Academic Press, 1997), and many other standard textbooks.

[0049] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with a commercially available hybridization kit, such as the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which prepares a probe by the random prime method and hybridizes under stringent conditions.

[0050] The stringent conditions include, for example, incubating a DNA-immobilized filter and probe DNA overnight at 42°C in a solution containing 50% formamide, 5xSSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20 µg / L of denatured salmon sperm DNA, followed by washing the filter in a 0.2xSSC solution at about 65°C.

[0051] The various conditions described above can also be achieved by adding or changing blocking reagents used to suppress background in hybridization experiments. The addition of blocking reagents described above may be accompanied by changes in hybridization conditions to suit the conditions.

[0052] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA consisting of a base sequence that has at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to the base sequence represented by SEQ ID NO: 1, when calculated based on the above parameters using, for example, the above-mentioned programs such as BLAST and FASTA.

[0053] The DNA of this embodiment can be obtained, for example, by using DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and introducing mutations by site-directed mutagenesis as described in, for example, Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press, 2012) and Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), thereby substituting a base sequence encoding another amino acid residue. Alternatively, the DNA of this embodiment can also be obtained using a PrimeSTAR Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) or the like.

[0054] DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 can be obtained, for example, by Southern hybridization to a chromosomal DNA library of a microorganism, preferably a bacteroides genus, more preferably Bacteroides fragilis, using a probe that can be designed based on the base sequence of DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 2; or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and the chromosomal DNA of Bacteroides fragilis as a template.

[0055] A specific example of the DNA encoding the protein consisting of the amino acid sequence shown in SEQ ID NO:2 is a DNA having the base sequence shown in SEQ ID NO:1.

[0056] A specific example of the DNA encoding the protein consisting of the amino acid sequence shown in SEQ ID NO:4 is DNA having the base sequence shown in SEQ ID NO:3.

[0057] The DNA described in [2] above, which encodes a protein having α1,3-fucosyltransferase activity, and which is composed of an amino acid sequence in which at least the 5th to 9th amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 have been deleted, substituted, inserted or added with 1 to 20 amino acids, can be obtained, for example, by subjecting DNA consisting of the base sequence represented by SEQ ID NO: 3 as a template to error-prone PCR or the like.

[0058] The DNA encoding a protein having α1,3-fucosyltransferase activity and consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added from the amino acid sequence represented by SEQ ID NO: 2 described in [1B] above can be obtained, for example, by subjecting DNA consisting of the base sequence represented by SEQ ID NO: 1 as a template to error-prone PCR or the like.

[0059] Alternatively, DNA encoding the protein of [2] above can be obtained by site-directed mutagenesis using a pair of PCR primers each having a nucleotide sequence at its 5' end designed to introduce a desired mutation (deletion, substitution, insertion, or addition) (Gene, 77, 51, 1989).

[0060] Alternatively, the DNA can be obtained by following the instructions provided with a commercially available site-directed mutagenesis kit, such as the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce a mutation (deletion, substitution, insertion, or addition) at the desired site.

[0061] That is, first, a pair of mutagenesis primers is designed with a template of a plasmid having a base sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition), with a 15-base overlap on the 5' side. The overlapping portion contains the desired mutation. Next, PCR is performed using the mutagenesis primers and a template of a plasmid having the base sequence into which the desired mutation is to be introduced. The amplified fragment obtained is transformed into Escherichia coli, yielding a plasmid having the base sequence into which the desired mutation has been introduced.

[0062] The DNA encoding a protein having α1,3-fucosyltransferase activity and consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:4 in which at least the 5th to 9th amino acids from the N-terminus are deleted, as described in [3] above, can be obtained, for example, by the following method. Specifically, for example, various gene sequence databases are searched for nucleotide sequences having preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more identity with the nucleotide sequence of SEQ ID NO:3, and DNA encoding the homologous protein can be obtained by a method similar to the method for obtaining DNA encoding the protein consisting of the amino acid sequence of SEQ ID NO:4, using a probe DNA or primer DNA that can be designed based on the nucleotide sequence or amino acid sequence obtained by the search, and a microorganism containing the DNA.

[0063] The DNA encoding a protein having α1,3-fucosyltransferase activity and consisting of an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:2 as described in [1C] above can be obtained, for example, by the following method. Specifically, for example, various gene sequence databases are searched for nucleotide sequences having preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more identity to the nucleotide sequence represented by SEQ ID NO:1, and DNA encoding the homologous protein can be obtained by a method similar to that for obtaining DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO:2 using a probe DNA or primer DNA that can be designed based on the nucleotide sequence or amino acid sequence obtained by the search and a microorganism containing the DNA.

[0064] The identity of the nucleotide sequence or amino acid sequence can be determined by the same method as in 1. The DNA of this embodiment obtained by the above method can be used as is or cleaved with an appropriate restriction enzyme or the like, inserted into a vector by a standard method, and the resulting recombinant DNA can be introduced into a parent strain. The nucleotide sequence of the DNA can then be determined by a commonly used nucleotide sequence analysis method, for example, the dideoxy method (Proc. Nat. Acad. Sci., USA, 74, 5463, 1977), or by analysis using a nucleotide sequence analyzer such as an Applied Biosystems 3500 Genetic Analyzer or an Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific).

[0065] Examples of vectors that can be used to determine the base sequence of the DNA of this embodiment 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).

[0066] The parent strain may be any strain that can be propagated by introducing the vector, and examples thereof include Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, and Escherichia coli HST04 dam. － / dcm －, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, 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 Examples of the strain include Escherichia coli W3110, Escherichia coli MP347, and Escherichia coli NM522.

[0067] As a method for introducing recombinant DNA obtained by incorporating the DNA of this embodiment into a parent strain, any method for introducing DNA into a parent strain 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).

[0068] If the DNA obtained as a result of determining the base sequence is a partial length, full-length DNA can be obtained by Southern hybridization or the like against a chromosomal DNA library using the partial length DNA as a probe.

[0069] 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.

[0070] 3. Recombinant DNA The recombinant DNA of this embodiment is DNA that can autonomously replicate in a parent strain, and is DNA in which the DNA of this embodiment is incorporated into an expression vector containing a promoter at a position where the DNA of this embodiment described in 2 above can be transcribed.

[0071] DNA that can be integrated into a chromosome in a parent strain and that contains the DNA of this embodiment is also the recombinant DNA of this embodiment. When the recombinant DNA is recombinant DNA that can be integrated into a chromosome, it does not need to contain a promoter.

[0072] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA of this embodiment is preferably a recombinant DNA composed of a promoter, a ribosome binding sequence, the DNA of this embodiment described in 2 above, and a transcription termination sequence. Furthermore, the recombinant DNA of this embodiment may contain a gene that controls the promoter.

[0073] Here, it is preferable to adjust the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon to an appropriate distance, for example, 6 to 18 bases. In addition, in the recombinant DNA of this embodiment, a transcription termination sequence is not necessarily required for expression of the DNA of this embodiment, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

[0074] When a microorganism belonging to the genus Escherichia is used as a parent strain into which the recombinant DNA of this embodiment is introduced, examples of the expression vector include pColdI, pSTV28, pUC118 (all manufactured by Takara Bio Inc.), pET21a, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE-60, pQE80L (all manufactured by Qiagen), pET-3, pBluescriptII SK(+), and pBluescriptII. KS(-) (both manufactured by Agilent Technologies), pKYP10 (Japanese Patent Laid-Open Publication No. 58-110600), pKYP200 (Agric. Biol. Chem., 48, 669, 1984), pLSA1 (Agric. Biol. Chem., 53, 277, 1989), pGEL1 (Proc. Natl. Acad. Sci., USA, 82, 4306 (1985), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385], pPE167 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pPAC31 (WO 1998 / 12343), pUC19 (Gene, 33, 103, 1985), pPA1 (JP 63-233798 A), and the like.

[0075] When using the above expression vectors, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples thereof include promoters derived from Escherichia coli or phages, such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter. Further examples thereof include artificially designed and modified promoters such as a promoter in which two promoters are connected in tandem, the tac promoter, the trc promoter, the lacT5 promoter, the lacT7 promoter, and the letI promoter.

[0076] When a coryneform bacterium is used as a parent strain into which the recombinant DNA of this embodiment is introduced, 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 Molecular and General Genetics, 196, 175, 1984).

[0077] When using the above expression vector, any promoter may be used as long as it functions in cells of coryneform bacteria, and an example thereof is the P54-6 promoter (Appl. Microbiol. Biotechnol., 53, pp. 674-679, 2000).

[0078] When a yeast strain is used as a parent strain into which the recombinant DNA of this embodiment is introduced, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0079] When using the above-mentioned expression vector, any promoter may be used as long as it functions in the cells of a yeast strain, and examples thereof include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0080] The recombinant DNA of this embodiment can be prepared, for example, by treating the DNA fragment prepared by the method 2 above with a restriction enzyme, and then inserting it downstream of the promoter of the appropriate expression vector.

[0081] Here, by substituting bases in the base sequence constituting the DNA of this embodiment so that the codons are optimal for expression in the parent strain, the expression level of the protein encoded by the DNA can be improved. Information on codon usage in the parent strain is available through public databases.

[0082] 4. Transformant The transformant of this embodiment is a transformant obtained by transforming a parent strain with recombinant DNA containing the DNA of this embodiment described in 2 above. As used herein, "parent strain" refers to a strain that is the target of genetic modification and transformation, etc., before genetic modification and transformation, etc. The parent strain may be a wild-type microorganism, or may be an industrially useful improved mutant strain, a cell fusion strain, a transduced strain, or a recombinant strain constructed using genetic recombination technology.

[0083] Specific examples of the transformant of this embodiment include a microorganism in which the activity of any one of the proteins [1] to [3] above is enhanced, such as a microorganism obtained by transforming a parent strain with a recombinant DNA having any one of the DNAs [D1] to [D3] above, and which has enhanced productivity of fucose-containing saccharides compared to the parent strain.

[0084] Examples of microorganisms that are obtained by transforming a parent strain with the recombinant DNA and that have enhanced productivity of fucose-containing saccharides compared to the parent strain include the microorganisms i) to iii) below: i) a microorganism in which a recombinant DNA having any one of the DNAs [D1] to [D3] above is introduced into the parent strain as an autonomously replicable plasmid or integrated into the chromosome of the parent strain, thereby increasing the transcription amount of the DNA or the production amount of the protein encoded by the DNA; ii) a microorganism in which the productivity of fucose-containing saccharides is enhanced by producing a protein having an enhanced specific activity of a protein with α1,3-fucosyltransferase activity as the mutant protein of [2] above; and iii) a microorganism in which the productivity of fucose-containing saccharides is enhanced by producing a protein having an enhanced specific activity of a protein with α1,3-fucosyltransferase activity as the homologous protein of [3] above.

[0085] The increase in the transcription amount of the DNA described in any one of [D1] to [D3] above or the production amount of the protein encoded by the DNA can be confirmed, for example, by measuring the transcription amount of the DNA by Northern blotting or the production amount of the protein by Western blotting and comparing it with that of the parent strain.

[0086] A method for confirming that the specific activity of a protein having α1,3-fucosyltransferase activity has been enhanced can be, for example, by purifying the mutant protein from a transformant obtained by transforming a parent strain with DNA encoding the mutant protein, causing the protein, GDP-fucose, and an acceptor carbohydrate to be present in an aqueous medium, measuring the specific activity from the amount of fucose-containing carbohydrates produced and accumulated in the aqueous medium and the amount of the protein, and comparing this with the specific activity of a protein having fucose-containing carbohydrate-producing activity and to which no mutation has been introduced, measured in the same manner.

[0087] An example of such a transformant of this embodiment is BfFucTΔN, which will be described later in the Examples. ２－１１ Examples include stocks.

[0088] The parent strain into which the recombinant DNA of this embodiment is introduced may be any of prokaryotes, yeast, animal cells, insect cells, plant cells, etc., but is preferably a prokaryote or yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli [e.g., Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue ... XL2-Blue (both manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (manufactured by Merck Millipore), Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam － / dcm －, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio Inc.), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli ATCC9637, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum [e.g., Brevibacterium immariophilum ATCC14068], Brevibacterium saccharolyticum [e.g., Brevibacterium saccharolyticum ATCC14066], Corynebacterium ammoniagenes, Corynebacterium glutamicum [Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869], Corynebacterium acetophilum [Corynebacterium acetophilum ATCC13870], Microbacterium ammoniphilum [Microbacterium ammoniphilum ATCC15354] or Pseudomonas (e.g.,Examples of suitable yeast strains include prokaryotes such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and Candida utilis.

[0089] Examples of parent strains include the following 1) and 2): 1) A microorganism used as a parent strain in which the ability to produce GDP-fucose, which is a reaction substrate for fucosyltransferase, has been artificially imparted or enhanced. 2) A microorganism used as a parent strain in which the ability to supply an acceptor carbohydrate, which is a reaction substrate for fucosyltransferase, has been artificially imparted or enhanced. Each parent strain will be described below.

[0090] 1) A microorganism used as a parent strain in which the ability to produce GDP-fucose, a reaction substrate for fucosyltransferase, has been artificially imparted or enhanced. The parent strain is preferably a microorganism in which the ability to produce GDP-fucose, a reaction substrate for α1,3-fucosyltransferase, has been artificially imparted or enhanced. Specific examples of methods for imparting or enhancing the ability to produce GDP-fucose in a microorganism used as a parent strain include known methods such as various genetic engineering methods (Metabolic Engineering (2017) 41:23-38).

[0091] The ability to produce 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.

[0092] Specific examples of mechanisms that control 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.

[0093] 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.

[0094] 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.

[0095] 2) A microorganism used as a parent strain in which the ability to supply an acceptor carbohydrate, which is a reaction substrate for fucosyltransferase, has been artificially imparted or enhanced. Methods for artificially imparting or enhancing the ability to supply an acceptor carbohydrate to a microorganism used as a parent strain include, for example, the following methods (2a) to (2g). These methods may be used alone or in combination. (2a) A method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway that produces an acceptor carbohydrate from a sugar. (2b) A method for enhancing the expression of at least one of the enzymes involved in the biosynthetic pathway that produces an acceptor carbohydrate from a sugar. (2c) A method for increasing the copy number of at least one of the genes encoding the enzymes involved in the biosynthetic pathway that produces an acceptor carbohydrate from a sugar. (2d) A method for alleviating or deactivating at least one of the mechanisms that degrades an acceptor carbohydrate. (2e) A method for enhancing the expression of at least one of the enzymes involved in the cellular uptake of an acceptor carbohydrate. (2f) A method for increasing the copy number of at least one of the genes encoding the enzymes involved in the cellular uptake of an acceptor carbohydrate. (2g) A method for weakening or blocking at least one of the metabolic pathways that branch off from an acceptor carbohydrate to a metabolite other than the target substance.

[0096] Specific examples of enzymes involved in the biosynthetic pathway that produces an acceptor carbohydrate from a sugar include known enzymes such as an enzyme with lactose synthase activity that produces lactose using glucose and UDP-galactose as substrates. Specific examples of mechanisms that degrade acceptor carbohydrates include known enzymes such as β-galactosidase that catalyzes the hydrolysis of lactose to produce glucose and galactose.

[0097] Specific examples of enzymes involved in the cellular uptake of acceptor carbohydrates include known enzymes such as lactose permease, which is involved in the cellular uptake of lactose. Specific examples of methods for imparting or enhancing the ability to supply acceptor carbohydrates include known methods such as reducing or inactivating the activity of β-galactosidase by genetic engineering (Metabolic Engineering, 2017, 41: 23-38).

[0098] Methods for introducing the above-mentioned three recombinant DNAs into a parent strain as an autonomously replicable plasmid include, for example, the above-mentioned calcium ion method, the protoplast method, the electroporation method, the spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889, 1984), the lithium acetate method (J. Bacteriol., 153, 163, 1983), and the like.

[0099] Methods for integrating recombinant DNA into the chromosome of a parent strain 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 parent strain 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, 6641-6645, 2000).

[0100] Furthermore, E. coli in which a target region on the chromosomal DNA of a parent strain 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)].

[0101] For example, when the transformant obtained by the above method is a transformant obtained by transforming a parent strain having the ability to produce GDP-fucose and an acceptor carbohydrate from sugars, whether the transformant has the DNA of this embodiment described in 2 above can be confirmed by culturing the transformant in a medium and comparing the amount of fucose-containing carbohydrates produced and accumulated in the culture with that of the parent strain. Alternatively, it can be confirmed by preparing an extract containing the protein of this embodiment from the culture, causing the extract, GDP-fucose, and acceptor carbohydrate to exist in an aqueous medium, and comparing the amount of fucose-containing carbohydrates produced and accumulated in the aqueous medium with that of the parent strain.

[0102] 5. Method for Producing Fucose-Containing Carbohydrate The method for producing the fucose-containing saccharide of this embodiment is the method described in 5-1 and 5-2 below.

[0103] 5-1. Method for producing fucose-containing saccharides by fermentation The method for producing fucose-containing saccharides of this embodiment includes a method for producing fucose-containing saccharides by fermentation, which comprises culturing the transformant described in 4 above in a medium to produce the fucose-containing saccharides.

[0104] The transformant of this embodiment used in the method for producing fucose-containing saccharides by fermentation is preferably a transformant capable of producing GDP-fucose from sugar.

[0105] Furthermore, a transformant having the ability to produce an acceptor carbohydrate may be used as the transformant of this embodiment used in the method for producing a fucose-containing carbohydrate by fermentation.

[0106] The transformant of the above item 4 can be cultured according to a conventional method used for culturing microorganisms.

[0107] The medium for culturing the transformant may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the transformant and allows the transformant to be cultured efficiently.

[0108] The carbon source may be any that can be utilized by the transformant, 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.

[0109] 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.

[0110] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. Acceptor carbohydrates such as lactose may be added to the medium as precursors of fucose-containing carbohydrates, and GTP, mannose, etc. may be added to the medium as precursors of GDP-fucose.

[0111] In the method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce GDP-fucose, GDP-fucose is added to the medium during culture. Alternatively, in the method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce GDP-fucose, GDP-fucose may be supplied to the transformant of this embodiment by culturing a microorganism capable of producing GDP-fucose from sugar simultaneously with the transformant of this embodiment, instead of adding GDP-fucose to the medium during culture.

[0112] Furthermore, in order to supply or enhance GTP, a precursor of GDP-fucose, microorganisms capable of producing GTP may be simultaneously cultured. Examples of microorganisms capable of producing GTP include known microorganisms such as microorganisms in which the expression of enzymes involved in the GTP biosynthetic pathway has been enhanced by various genetic manipulations (Biotechnol Bioeng, Sep 3: 2019, 2412-2417).

[0113] In the method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce an acceptor carbohydrate such as lactose, an acceptor carbohydrate such as lactose is added to the medium during cultivation.

[0114] Furthermore, in the method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce an acceptor carbohydrate such as lactose, instead of adding an acceptor carbohydrate such as lactose to the medium during cultivation, an acceptor carbohydrate such as lactose may be supplied to the transformant of this embodiment by culturing a microorganism that has the ability to produce an acceptor carbohydrate such as lactose from sugar simultaneously with the transformant of this embodiment.

[0115] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. The culture temperature is usually 15 to 40°C, and the culture time is usually 5 hours to 7 days. The pH of the culture solution during cultivation is usually maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0116] Furthermore, antibiotics such as ampicillin or tetracycline may be added to the medium during culture as needed. When culturing a microorganism transformed with an expression vector using an inducible promoter as the promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added to the medium when culturing a microorganism transformed with an expression vector using the lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using the trp promoter.

[0117] By producing fucose-containing carbohydrates by producing fucose-containing carbohydrates in the culture by the above-mentioned culture, the fucose-containing carbohydrates can be obtained, for example, by collecting the fucose-containing carbohydrates accumulated in the transformant or the culture.

[0118] The obtained fucose-containing saccharides can be analyzed by a conventional method using saccharide ion chromatography, etc. Collection of fucose-containing saccharides from the above-mentioned culture or a processed product of the culture can be carried out by a conventional method using activated carbon, ion exchange resin, etc. When fucose-containing saccharides accumulate within the cells, for example, the cells can be disrupted by ultrasound, etc., and the cells can be removed by centrifugation, and the fucose-containing saccharides can be collected from the resulting supernatant using activated carbon, ion exchange resin, etc.

[0119] 5-2. Method for producing fucose-containing saccharides using GDP-fucose and an acceptor saccharide as substrates The method for producing fucose-containing saccharides of this embodiment also includes a method for producing fucose-containing saccharides using GDP-fucose and an acceptor saccharide as precursors. Specifically, a culture obtained by culturing the transformant described in 4 above or a treated product of the culture is used as an enzyme source, and the enzyme source, GDP-fucose and an acceptor saccharide are allowed to exist in an aqueous medium to produce fucose-containing saccharides in the aqueous medium, and the fucose-containing saccharides produced in the aqueous medium can be collected as needed.

[0120] The origin of GDP-fucose and the acceptor saccharide is not important as long as they serve as substrates for the protein of this embodiment possessed by the transformant of this embodiment. A culture of a microorganism capable of producing GDP-fucose or an acceptor saccharide or a processed product of the culture may be used as is, or GDP-fucose and an acceptor saccharide collected from the culture or a processed product of the culture may be used.

[0121] Examples of processed culture products include concentrates of the culture, dried cultures, bacterial cells obtained by centrifuging the cultures, dried products of the bacterial cells, freeze-dried products of the bacterial cells, surfactant-treated products of the bacterial cells, ultrasonically treated products of the bacterial cells, mechanically ground products of the bacterial cells, solvent-treated products of the bacterial cells, enzyme-treated products of the bacterial cells, protein fractions of the bacterial cells, immobilized products of the bacterial cells, and enzyme preparations obtained by extraction from the bacterial cells.

[0122] Examples of aqueous media include water, phosphates, carbonates, acetates, borates, citrates, buffer solutions such as Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, amides such as acetamide, etc. The culture medium of the microorganism used as the enzyme source can also be used as the aqueous medium.

[0123] The methods for analyzing and collecting the fucose-containing saccharides produced in the aqueous medium are the same as those in 5-1.

[0124] The above embodiment allows for efficient production of fucose-containing carbohydrates. Specifically, the efficiency can be judged by increasing the amount of 3-fucosyllactose or lactodifucotetraose produced, and shortening the time required for producing 3-fucosyllactose or lactodifucotetraose, but is not limited to these criteria.

[0125] As explained above, the present specification discloses the following configurations. 1. A protein according to any one of [1] to [3] below. [1] A protein consisting of an amino acid sequence obtained by deleting at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4, and having α1,3-fucosyltransferase activity. [2] A protein consisting of an amino acid sequence obtained by deleting at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4, and having 1 to 20 amino acids deleted, substituted, inserted or added, and having α1,3-fucosyltransferase activity. [3] A protein consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 4, and having at least the fifth to ninth amino acids from the N-terminus, and having α1,3-fucosyltransferase activity. 2. A protein according to 1 above, which is any one of [1A] to [1C] below. [1A] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2. [1B] A protein consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 20 amino acids with respect to the amino acid sequence represented by SEQ ID NO: 2, and having α1,3-fucosyltransferase activity. [1C] A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 2, and having α1,3-fucosyltransferase activity. 3. DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a sequence homologous thereto, and encoding the protein described in 1 or 2 above. 4. Recombinant DNA containing the DNA described in 3 above. 5. A transformant obtained by transforming a parent strain with the recombinant DNA described in 4 above. 6. The transformant described in 5 above, which is a microorganism in which the activity of the protein described in 1 or 2 above has been enhanced. 7. The transformant described in 6 above, wherein the microorganism is Escherichia coli. 8. A method for producing a fucose-containing saccharide, which comprises culturing the transformant described in 5 or 6 above in a medium to produce the fucose-containing saccharide.9. A method for producing a fucose-containing saccharide, comprising culturing the transformant according to 5 or 6 above in a medium to obtain a culture or a processed product of the culture, and using the culture or the processed product of the culture as an enzyme source, causing the enzyme source, GDP-fucose and an acceptor carbohydrate to be present in an aqueous medium, and producing a fucose-containing saccharide in the aqueous medium. 10. The production method according to 8 or 9 above, wherein the fucose-containing saccharide is 3-fucosyllactose or lactodifucotetraose.

[0126] [Analysis Examples] In the Examples, analysis and quantification of 3-fucosyllactose (3FL) and lactodifucotetraose (LDFT) were carried out according to the following procedures. After cultivation, the culture medium containing the microorganism was centrifuged, and the culture supernatant was collected. 3FL or LDFT contained in the culture supernatant was analyzed using a sugar analyzer ICS-5000 (manufactured by Thermo Fisher Scientific). 3FL and LDFT manufactured by Biosynth were used as standards. [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: (0-10 min) 80:20:0 (10-15 min) Gradient from 80:20:0 to 70:20:10 (15-17 min) Gradient from 70:20:10 to 0:20:80 (17-25 min) 0:20:80 (25-35 min) 80:20:0 Flow rate: 1.0 mL / min Detector: Pulsed amperometry detector

[0127] Example 1: Isolation of α1,3-fucosyltransferase useful for 3FL or LDFT production Using the productivity of 3FL or LDFT as an indicator, α1,3-fucosyltransferases showing high reactivity to lactose or 2'-fucosyllactose were screened.

[0128] (1) Construction of host strain for evaluation <Obtaining 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: 5 and 6 as a primer set and pCatSac (Appl Environ Microbiol (2013) 79, 3033-3039) as a template to obtain a cat-sacB fragment containing the chloramphenicol-resistant cat gene and the sucrose-sensitive sacB gene.

[0129] <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.

[0130] 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.

[0131]

[0132] 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.

[0133] 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: 8 and 10 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.

[0134] 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: 8 and 10 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.

[0135] The lacZY::cat-sacB fragment was introduced by electroporation into the W3110 strain 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, to obtain a transformant that exhibited chloramphenicol resistance and sucrose sensitivity (a transformant in which the lacZY gene had been replaced with lacZY::cat-sacB).

[0136] The ΔlacZY fragment was introduced into the transformant by electroporation to obtain transformants that were sensitive to chloramphenicol and resistant to sucrose (transformants in which lacZY::cat-sacB had been replaced with ΔlacZY). From these, a transformant that was sensitive to ampicillin (transformant from which pKD46 had been lost) was further obtained. This transformant was designated W3110ΔlacZY.

[0137] Similarly, PCR was carried out using the genomic DNA of the W3110 strain as a template and DNAs consisting of the base sequences represented by the "primer set" shown in Table 2 as primer sets to obtain amplified DNA fragments.

[0138]

[0139] 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.

[0140] 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: 14 and 16 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).

[0141] 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: 14 and 16 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.

[0142] The wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110ΔlacZY strain constructed above by electroporation to obtain a transformant that exhibited chloramphenicol resistance and sucrose sensitivity (a transformant in which wcaJ-wzxC-wcaKLM was replaced with wcaJ-wzxC-wcaKLM::cat-sacB).

[0143] 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 W3110ΔlacZYΔwcaJM strain.

[0144] <Construction of a microorganism with enhanced expression of a transporter gene> Escherichia coli carrying a plasmid for expressing a gene encoding a transporter gene belonging to the drug:H+ antiporter-1 family (hereinafter referred to as MdfA) derived from the W3110 strain and consisting of the amino acid sequence represented by SEQ ID NO:20 was constructed by the following method.

[0145] PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs:21 and 22 as a primer set and genomic DNA of the W3110 strain prepared by standard methods as a template to obtain an mdfA fragment. PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs:23 and 24 as a primer set and plasmid pMW118 (manufactured by Nippon Gene Co., Ltd.) as a template to obtain a vector fragment of approximately 4.1 kb. The nucleotide sequences represented by SEQ ID NOs:21 and 23, and SEQ ID NOs:22 and 24 each contain a complementary sequence at the 5' end.

[0146] The mdfA fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the MdfA expression plasmid pMW118_mdfA. The W3110ΔlacZYΔwcaJM strain constructed in Example 1(1) was transformed with the expression plasmid pMW118_mdfA to construct an E. coli strain harboring pMW118_mdfA, which was designated the FUC strain.

[0147] (2) Construction of Microorganisms Having α1,3-Fucosyltransferase Activity Escherichia coli carrying a plasmid for expressing various α1,3-fucosyltransferase-encoding genes placed under the uspA promoter was constructed by the following method.

[0148] <Construction of Expression Vector> Using genomic DNA of the W3110 strain prepared by a conventional method as a template, PCR was performed using DNAs consisting of the base sequences shown in "Primer Set" in Table 3 as a primer set to obtain amplified DNA fragments.

[0149]

[0150] PCR was performed using an equimolar mixture of the rcsA fragment and the lacY fragment as a template and DNAs consisting of the base sequences represented by SEQ ID NOs: 25 and 28 as a primer set to obtain a DNA fragment in which the two fragments were ligated (hereinafter referred to as rcsA-lacY).

[0151] PCR was performed using a primer set consisting of DNAs with the nucleotide sequences represented by SEQ ID NOs: 29 and 30 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: 25 and 29, and SEQ ID NOs: 28 and 30 each contain a complementary sequence at their 5' ends.

[0152] The rcsA-lacY fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain the expression vector pUAKQE-rcsA-lacY.

[0153] <Construction of Plasmid for Expression of α1,3-Fucosyltransferase> PCR was performed using DNAs consisting of the base sequences shown in "Primer Set" in Table 4 as a primer set and DNAs shown in "Template" in Table 4 as templates to obtain amplified DNA fragments. Genomic DNA of Bacteroides fragilis ATCC 25285 was prepared by standard methods.

[0154]

[0155] PCR was performed using the expression vector pUAKQE-rcsA-lacY constructed by the method described above as a template and DNAs consisting of the nucleotide sequences represented by SEQ ID NOs: 29 and 34 as a primer set to obtain a vector fragment of approximately 6.7 kb. The nucleotide sequences represented by SEQ ID NOs: 31, 33, and 29, and SEQ ID NOs: 32 and 34 each contain a complementary sequence at the 5' end.

[0156] The amplified DNA fragments and vector fragments obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to generate plasmids expressing various α1,3-fucosyltransferases, pBfFucT and pBfFucTΔN. ２－１１ was created.

[0157] <Construction of Escherichia coli carrying a plasmid for expressing α1,3-fucosyltransferase> The plasmids for expressing α1,3-fucosyltransferase obtained above, pBfFucT and pBfFucTΔN ２－１１ The FUC strain constructed in Example 1(1) was transformed with pUAKQE-rcsA-lacY as a vector control to construct E. coli strains carrying various plasmids, and these strains were designated FUC / pBfFucT and FUC / pBfFucTΔN, respectively. ２－１１ The strains were designated as FUC / Ctrl strain and FUC / Ctrl strain.

[0158] (3) Evaluation of productivity of 3FL and LDFT The FUC / pBfFucT strain and FUC / pBfFucTΔN strain obtained in (2) above were used. ２－１１ The productivity of 3FL and LDFT was evaluated for the strain and the FUC / Ctrl strain.

[0159] Each strain was inoculated into a 14 mL plastic tube containing 2 mL of LB medium containing 100 mg / L kanamycin and 100 mg / L ampicillin, and cultured overnight at 30°C with shaking.

[0160] The resulting culture solution was then transferred to a production medium containing 100 mg / L of kanamycin and 100 mg / L of ampicillin [glucose 30 g / L, lactose monohydrate or 2'-fucosyllactose 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, 2'-fucosyllactose 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, 2'-fucosyllactose and magnesium sulfate heptahydrate were prepared separately, then sterilized and mixed) 0.2 mL was inoculated into a large test tube containing 4 mL and cultured with shaking at 30 ° C. for 29 hours. 7 hours after the start of culture, IPTG was added to a final concentration of 1 mM. At this time, when evaluating the productivity of 3FL, 10 g / L of lactose monohydrate was added at the start of the culture, and when evaluating the productivity of LDFT, 10 g / L of 2'-fucosyllactose was added.

[0161] After the cultivation was completed, the culture solution was centrifuged and diluted appropriately, and the 3FL or LDFT contained in the culture supernatant was analyzed using a sugar analyzer ICS-5000. The results are shown in Table 5.

[0162]

[0163] As shown in Table 5, compared with the FUC / pBfFucT strain expressing the full-length α1,3-fucosyltransferase derived from Bacteroides fragilis, the FUC / pBfFucTΔN strain expressing a deletion form in which the 2nd to 11th amino acids from the N-terminus of the α1,3-fucosyltransferase derived from Bacteroides fragilis were deleted. ２－１１ The strain showed a significant increase in productivity of 3FL and LDFT.

[0164] Next, the deletion enzymes BfFucTΔN, in which the 2nd to 25th amino acids from the N-terminus and the 2nd to 48th amino acids from the N-terminus of the amino acid sequence of BfFucT were deleted, respectively, were prepared. ２－２５and BfFucTΔN ２－４８ The expression strain of pBfFucTΔN ２－１１ The strains were constructed in the same manner as described above, and the productivity of these 3FL strains was also evaluated. The strains were cultured in a production medium containing lactose monohydrate in the same manner as described above. After the culture was completed, the culture solution was centrifuged and diluted appropriately, and the 3FL contained in the supernatant was analyzed using a sugar analyzer ICS-5000. The results are shown in Table 6.

[0165]

[0166] As shown in Table 6, BfFucTΔN ２－２５ and BfFucTΔN ２－４８ In this case, no production of 3FL was observed, suggesting that the enzyme was inactivated. ２－１１ was found to be a useful α1,3-fucosyltransferase for producing fucosylated oligosaccharides such as 3FL and LDFT.

[0167] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2024-031588) filed on March 1, 2024, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0168] SEQ ID NO: 1: BfFucTΔN derived from Bacteroides fragilis ATCC 25285 ２－１１ SEQ ID NO: 2: Bacteroides fragilis ATCC 25285-derived BfFucTΔN ２－１１ ​SEQ ID NO: 3: Nucleotide sequence of BfFucT derived from Bacteroides fragilis ATCC 25285 SEQ ID NO: 4: Amino acid sequence of BfFucT derived from Bacteroides fragilis ATCC 25285 SEQ ID NO: 5: Nucleotide sequence of catsacB_F SEQ ID NO: 6: Nucleotide sequence of catsacB_R SEQ ID NO: 7: Nucleotide sequence of lacZY_del_N2 SEQ ID NO: 8: Nucleotide sequence of lacZY_del_N1 SEQ ID NO: 9: Nucleotide sequence of lacZY_del_C1 SEQ ID NO: 10: Nucleotide sequence of lacZY_del_C2 SEQ ID NO: 11: Nucleotide sequence of lacZY_PO_N2 SEQ ID NO: 12: Nucleotide sequence of lacZY_PO_C1 SEQ ID NO: 13: Nucleotide sequence of wcaJM_del_N2 SEQ ID NO: 14: Nucleotide sequence of wcaJM_del_N1 SEQ ID NO: 15: Nucleotide sequence of wcaJM_del_C1 SEQ ID NO: 16: Nucleotide sequence of wcaJM_del_C2 SEQ ID NO: 17: Nucleotide sequence of wcaJM_PO_N2 SEQ ID NO: 18: Nucleotide sequence of wcaJM_PO_C1 SEQ ID NO: 19: Nucleotide sequence of mdfA derived from E. coli W3110 SEQ ID NO: 20: Nucleotide sequence of E. Amino acid sequence of MdfA derived from E. coli W3110 SEQ ID NO: 21: Nucleotide sequence of mdfA_pMW118_fus_1 SEQ ID NO: 22: Nucleotide sequence of mdfA_pMW118_fus_2 SEQ ID NO: 23: Nucleotide sequence of pMW118_fus_2 SEQ ID NO: 24: Nucleotide sequence of pMW118_fus_1 SEQ ID NO: 25: Nucleotide sequence of KQE-SDrcsA_fus_1 SEQ ID NO: 26: Nucleotide sequence of rcsA-SDlacY_fus_2 SEQ ID NO: 27: Nucleotide sequence of SDlacY_1 SEQ ID NO: 28: Nucleotide sequence of lacY-KQE_fus_2 SEQ ID NO: 29: Nucleotide sequence of pUAKQE_fus_2 SEQ ID NO: 30: Nucleotide sequence of pUAKQE_fus_1 SEQ ID NO: 31: Nucleotide sequence of BffucT_KQE_fus_1 SEQ ID NO: 32: Nucleotide sequence of BffucT_TAY_fus_2 SEQ ID NO: 33: Nucleotide sequence of BffucTΔN10_KQE_fus_1 SEQ ID NO: 34: Nucleotide sequence of TAY_fus_1 SEQ ID NO: 35: 5th to 10th amino acid sequence from the N-terminus of the amino acid sequence of BfFucT derived from Bacteroides fragilis ATCC 25285SEQ ID NO: 36: 4th to 9th amino acid sequence from the N-terminus of the amino acid sequence of BfFucT derived from Bacteroides fragilis ATCC 25285

Claims

1. A protein according to any one of [1] to [3] below. [1] A protein consisting of an amino acid sequence in which at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 are deleted, and which has α1,3-fucosyltransferase activity. [2] A protein consisting of an amino acid sequence in which at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 are deleted, and in which 1 to 20 amino acids are deleted, substituted, inserted, or added, and which has α1,3-fucosyltransferase activity. [3] A protein consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence in which at least the fifth to ninth amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 are deleted, and which has α1,3-fucosyltransferase activity.

2. The protein according to claim 1, which is any one of the following [1A] to [1C]: [1A] A protein consisting of the amino acid sequence represented by SEQ ID NO:

2. [1B] A protein consisting of an amino acid sequence represented by SEQ ID NO: 2 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and which has α1,3-fucosyltransferase activity. [1C] A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 2 and which has α1,3-fucosyltransferase activity.

3. DNA consisting of the base sequence shown in SEQ ID NO: 1 or a sequence homologous thereto, and encoding the protein of claim 1.

4. A recombinant DNA containing the DNA according to claim 3.

5. A transformant obtained by transforming a parent strain with the recombinant DNA of claim 4.

6. The transformant according to claim 5, which is a microorganism in which the activity of the protein according to claim 1 or 2 is enhanced.

7. The transformant according to claim 6, wherein the microorganism is Escherichia coli.

8. A method for producing fucose-containing carbohydrates, which comprises culturing the transformant according to claim 5 in a medium to produce the fucose-containing carbohydrates.

9. A method for producing a fucose-containing saccharide, comprising: culturing the transformant according to claim 5 in a medium to obtain a culture or a processed product of the culture; using the culture or the processed product of the culture as an enzyme source, causing the enzyme source, GDP-fucose and an acceptor saccharide to exist in an aqueous medium, and producing a fucose-containing saccharide in the aqueous medium.

10. The method according to claim 8 or 9, wherein the fucose-containing carbohydrate is 3-fucosyllactose or lactodifucotetraose.

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