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 that has excellent LNFPI productivity; and a method for producing LNFPI. The present invention pertains to: a protein that has an activity of transferring fucosyl groups to lacto-N-tetraose (LNT) and that has an amino acid sequence represented by SEQ ID NO. 2, 4, 6, 8, 10, 14, 18, or 26; or a mutant protein or a homologous protein thereof.

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 inhibitory effects against meningitis-causing group B streptococcus (GBS) and norovirus (Non-Patent Documents 3 and 4). Furthermore, Bifidobacterium infantis, which is highly prevalent in the intestines of newborns, has been shown to grow preferentially in LNFPI, and 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 is widely used. Patent Documents 1 and 2 and 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 (Patent Document 1, 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] International Publication No. WO 2017 / 106864 International Publication No. WO 2019 / 008133

[0010] 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

[0011] 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.

[0012] On the other hand, in order to produce LNFPI more efficiently using lactose as the initial raw 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.

[0013] 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.

[0014] 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 consisting of a specific amino acid sequence, and have completed the present invention. Furthermore, they have discovered for the first time a fucosyltransferase derived from the genus Neisseria or Francisella that is suitable for producing LNFPI or fucosylated oligosaccharides such as fucosyllactose.

[0015] That is, the present invention is as follows: 1. A protein according to any one of the following [1] to [3], which has the activity of transferring fucosyl groups to lacto-N-tetraose (LNT): [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26. [2] A mutant protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26, in which 1 to 20 amino acids have been deleted, substituted, inserted, or added, and which has α1,2-fucosyltransferase activity. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26, and which has α1,2-fucosyltransferase activity. 2. A DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17 or 25 or a homologous sequence thereof, and encoding the protein according to any one of [1] to [3] described in 1 above. 3. A recombinant DNA containing the DNA described in 2 above. 4. A transformant obtained by transforming a host cell with the recombinant DNA described in 3 above. 5. The transformant according to 4 above, which is a microorganism in which the activity of the protein of any one of [1] to [3] described in 1 above and the productivity of a fucose-containing saccharide are enhanced. 6. The transformant according to 5 above, wherein the microorganism is Escherichia coli. 7. A method for producing a fucose-containing saccharide, comprising preparing the transformant according to any one of 4 to 6 above, and producing a fucose-containing saccharide in a culture using the transformant. 8. The production method according to 7 above, wherein the fucose-containing saccharide is lacto-N-fucopentaose I (LNFPI).

[0016] 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.

[0017] Figure 1 shows the biosynthetic pathway of LNFPI in one embodiment of the present invention. Figure 2 shows the combined LNFPI production amounts in the supernatant and intracellular fraction (Example 2).

[0018] <Protein, DNA, and Transformant> The protein of the present invention is a protein described in any one of the following [1] to [3], which has the activity of transferring fucosyl groups to lacto-N-tetraose (LNT): [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26. [2] A mutant protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26, in which 1 to 20 amino acids have been deleted, substituted, inserted, or added, and which has α1,2-fucosyltransferase activity. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26, and which has α1,2-fucosyltransferase activity.

[0019] From the viewpoint of further enhancing the fucosylation activity on LNT, among the proteins described in [1] above, a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 14, 18, or 26 is preferred, and a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 18 is more preferred.

[0020] The protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is the α1,2-fucosyltransferase GsFucT derived from Gramella sp. MAR_2010_147 strain described below in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 4 is the α1,2-fucosyltransferase FsFucT derived from Francisella sp. FSC1006 strain described below in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 6 is the α1,2-fucosyltransferase NbFucT1 derived from Neisseriaceae bacterium DSM 100970 strain described below in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 8 is the α1,2-fucosyltransferase MtFucT derived from Methylobacter tundripaludum strain described below in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 10 is the α1,2-fucosyltransferase AjFucT derived from the Amphritea japonica strain described below in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 14 is the α1,2-fucosyltransferase SbFucT derived from the Sterolibacteriaceae bacterium J5B strain described below in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 18 is the α1,2-fucosyltransferase NbFucT2 derived from the Neisseriales bacterium strain described below in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 26 is the α1,2-fucosyltransferase HMFT derived from the Helicobacter mustelae ATCC43772 strain described below in the Examples.

[0021] As used herein, a mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in an original protein, or by inserting or adding amino acid residues into the protein.

[0022] In the mutant protein of [2] above, the deletion, substitution, insertion, or addition of amino acids may mean the deletion, substitution, insertion, or addition of 1 to 20 amino acids at any position within the same sequence. The number of deleted, substituted, inserted, or added amino acids is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 8, and most preferably 1 to 5.

[0023] The amino acids to be deleted, substituted, inserted, or added may be naturally occurring or non-naturally occurring. Naturally occurring amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0024] 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

[0025] In the mutant protein of the above [2], the amino acid residue to be substituted is, for example, the 17th asparagine residue.

[0026] As used herein, a homologous protein refers to a protein that is found in organisms in nature and is similar in structure and function to the original protein, such that the gene encoding the protein is considered to have the same evolutionary origin as the gene encoding the original protein.

[0027] Examples of homologous proteins include amino acid sequences that have an identity of preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, and particularly preferably 97% or more with the amino acid sequence of the target protein.

[0028] 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.

[0029] As used herein, the fucosyltransferase activity to LNT refers to the activity of transferring a fucose residue from GDP-fucose, which is a donor substrate, to the N-acetylglucosamine hydroxyl group of LNT, which is a carbohydrate, which is an acceptor substrate (hereinafter referred to as "acceptor carbohydrate").

[0030] LNFPI is produced by the transfer of a fucose residue from GDP-fucose to the hydroxyl group of N-acetylglucosamine. The biosynthetic pathway of LNFPI in one embodiment of the present invention is shown in Figure 1.

[0031] As used herein, α1,2-fucosyltransferase activity refers to the activity of transferring a fucose residue from the donor substrate GDP-fucose to the hydroxyl group of N-acetylglucosamine in an acceptor saccharide via an α1,2-bond to produce a fucose-containing saccharide. The acceptor saccharide is preferably LNT. The fucose-containing saccharide is preferably LNFPI.

[0032] The α1,2-fucosyltransferase activity of the above-mentioned mutant protein or homologous protein can be confirmed, for example, by the following method. First, a recombinant DNA containing DNA encoding the mutant protein or homologous protein whose activity is to be confirmed is prepared by the method described below. Next, a parent strain is transformed with the recombinant DNA to prepare a transformant having higher activity of the protein than the parent strain, and the amount of fucose-containing carbohydrate produced and accumulated in the culture medium of the parent strain or the transformant can be compared to confirm the activity. A specific example of a fucose-containing carbohydrate is LNFPI.

[0033] As used herein, the term "parent strain" refers to the original strain that is the subject of genetic modification, transformation, and the like.

[0034] In the present specification, the parent strain is preferably a prokaryote or a yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, ...MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli MG1655, Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli MG1655 KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 codon plus (Stratagene), Escherichia coli 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.

[0035] 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.

[0036] 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; 2) a microorganism to which the ability to supply LNT, a reaction substrate for α1,2-fucosyltransferase, has been artificially imparted or enhanced. These will be explained below.

[0037] 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).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 2) A microorganism used as a parent strain that has been artificially imparted or enhanced with the ability to supply LNT, a reaction substrate for α1,2-fucosyltransferase. 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 described below.

[0053] 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).

[0054] 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.

[0055] 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).

[0056] Furthermore, as described above, it is preferable that the parent strain has reduced or no lacZ activity and / or colanic acid synthesis activity.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] Examples of a microorganism in which the activity of the protein according to any one of [1] to [3] above is enhanced compared to that of a parent strain of a microorganism include a microorganism obtained by transforming a parent strain of a microorganism with a recombinant DNA containing a DNA encoding the protein, and in which the copy number of the gene is increased compared to that of the parent strain.

[0061] Examples of microorganisms that have an increased copy number of a gene compared to a parent strain, and that can be obtained by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding a protein according to any one of [1] to [3] above, include microorganisms in which the copy number of the gene on chromosomal DNA has been increased by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding a protein according to any one of [1] to [3] above, and microorganisms in which the gene is carried outside of chromosomal DNA as plasmid DNA.

[0062] The DNA encoding the protein according to any one of [1] to [3] above may be any DNA encoding a protein having the activity of the protein according to any one of [1] to [3] above, and specifically includes one DNA selected from the group consisting of [4] to [7] below. [4] DNA encoding the protein according to any one of [1] to [3] above. [5] DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17 or 25. [6] DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17 or 25, and encodes a homologous protein having α1,2-fucosyltransferase activity. [7] DNA consisting of a nucleotide sequence that has 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17 or 25, and encodes a homologous protein having α1,2-fucosyltransferase activity.

[0063] In the above [6], "hybridize" means that DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part of the DNA can be used as a probe in Northern or Southern blot analysis, and can also be used as an oligonucleotide primer in PCR analysis.

[0064] The DNA used as a probe may be at least 100 bases long, preferably at least 200 bases long, more preferably at least 500 bases long.The DNA used as a primer may be at least 10 bases long, preferably at least 15 bases long.

[0065] Methods for DNA hybridization experiments are well known, and those skilled in the art can determine hybridization conditions according to the present specification. The hybridization conditions can be determined according to the methods described in Molecular Cloning, 4th Edition (2012), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology Methods Manual, Academic Press (1996), and many other standard textbooks.

[0066] 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.

[0067] The above-mentioned stringent conditions include incubating a DNA-immobilized filter and probe DNA overnight at 42°C in a solution containing 50% formamide, 5xSSC (750 mmol / L sodium chloride, 75 mmol / L sodium citrate), 50 mmol / L sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, followed by washing the filter in a 0.2xSSC solution at approximately 65°C.

[0068] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA that has at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17, or 25, when calculated based on the above-mentioned parameters using, for example, BLAST or FASTA.

[0069] The DNA encoding the protein of [1] above can be obtained, for example, by Southern hybridization of a chromosomal DNA library of a microorganism, preferably a microorganism, using a probe DNA that can be designed based on the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17, or 25, or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the nucleotide sequence and the chromosomal DNA of the above microorganism as a template. The origin of the chromosomal DNA of the microorganism used in the above procedure is not particularly limited, and examples include bacteria of the genus Neisseria (Neisseriaceae, Neisseriales), Francisella, Methylobacter, Amphritea, Sterolibacteriaceae, or Helicobacter. Among these, Francisella sp. FSC1006 strain, Neisseriaceae bacterium DSM 100970 strain, Methylobacter tundripaludum strain, Amphritea japonica strain, Sterolibacteriaceae bacterium J5B strain, Neisseriales bacterium strain, and Helicobacter mustelae ATCC43772 strain are preferred.

[0070] These strains are available from public institutions, etc. For example, Francisella sp. FSC1006 strain is available from the Swedish Defense Research Agency, and Neisseriaceae bacterium DSM 100970 strain is available from the University of Malaya. Additionally, Methylobacter tundripaludum strain, Amphritea japonica strain, and Helicobacter mustelae ATCC 43772 strain are available from the American Type Culture Collection (ATCC).

[0071] The DNA encoding the mutant protein of [2] above can be obtained, for example, by subjecting DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17 or 25 to error-prone PCR or the like using the DNA as a template.

[0072] Alternatively, DNA encoding the mutant protein of the above item [2] can be obtained by PCR using a pair of PCR primers each having a nucleotide sequence at its 5' end designed to introduce a desired mutation (deletion, substitution, insertion, or addition) [Gene, 77, 51 (1989)].

[0073] 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.

[0074] 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.

[0075] The DNA encoding the homologous protein of [3] above, and the DNAs of [6] and [7] above can be obtained, for example, by searching various gene sequence databases for a base sequence having 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, 3, 5, 7, 9, 13, 17 or 25, or by searching various protein sequence databases for an amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18 or 26, and using a probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search, and a microorganism having the DNA, in a manner similar to the method for obtaining the above-mentioned DNA.

[0076] The obtained DNA according to any one of the above [4] to [7] can be used as is or cleaved with an appropriate restriction enzyme or the like, and then inserted into a vector by a conventional method. The resulting recombinant DNA is then introduced into a host cell, and the DNA can be analyzed using a commonly used 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.

[0077] 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.

[0078] 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).

[0079] 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)].

[0080] 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.

[0081] 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.

[0082] The recombinant DNA containing a DNA encoding the protein according to any one of [1] to [3] above 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 of being integrated into a chromosome and that contains a promoter at a position where the DNA can be transcribed.

[0083] When the recombinant DNA is capable of being integrated into a chromosome, it does not need to contain a promoter.

[0084] A microorganism having an increased copy number of the gene compared to the parent strain, which is obtained by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding the protein according to any one of [1] to [3] above, can be obtained by the following method.

[0085] Based on the DNA encoding the protein according to any one of [1] to [3] above obtained by the above method, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as needed. Furthermore, by substituting bases in the nucleotide 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.

[0086] 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.

[0087] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA is preferably a recombinant DNA comprising a promoter, a ribosome binding sequence, the DNA described in any one of [4] to [7] above, and a transcription termination sequence. A gene that controls the promoter may also be included.

[0088] 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.

[0089] Furthermore, by substituting bases in the nucleotide sequence of the portion encoding a protein having α1,2-fucosyltransferase activity so that the codons are optimal for expression in the host, the expression level of the protein having α1,2-fucosyltransferase activity can be improved. Examples of proteins having α1,2-fucosyltransferase activity include the proteins described in any one of [1] to [3] above. Information on codon usage frequencies in parent strains used in the present invention is available from public databases.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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)).

[0094] 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. For example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)] can be used.

[0095] 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.

[0096] 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.

[0097] The recombinant DNA used in the production method of the present invention can be prepared by inserting the DNA fragment described in any one of [4] to [7] above downstream of the promoter of an appropriate expression vector.

[0098] 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)].

[0099] 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)].

[0100] 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)].

[0101] 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.

[0102] The fact that the microorganism constructed by the above method is a microorganism in which the activity of the protein described in any one of [1] to [3] above is enhanced and the productivity of LNFPI is improved compared to the parent strain can be confirmed by culturing the microorganism, appropriately diluting the culture solution, centrifuging it, and analyzing the LNFPI contained in the supernatant or the bacterial cells using a sugar analyzer or high-performance liquid chromatograph mass spectrometer described below, and comparing it with that of the parent strain.

[0103] The above-mentioned microorganisms have enhanced activity of the protein described in any one of [1] to [3] above compared to the parent strain, and therefore can selectively transfer fucose to the N-acetylglucosamine site of LNT, thereby improving the productivity of LNFPI. Examples of such microorganisms include the NNN / pGsFucT strain with enhanced expression of the GsFucT gene, the NNN / pFsFucT strain with enhanced expression of the FsFucT gene, the NNN / pNbFucT1 strain with enhanced expression of the NbFucT1 gene, the NNN / pMtFucT strain with enhanced expression of the MtFucT gene, the NNN / pAjFucT strain with enhanced expression of the AjFucT gene, the NNN / pSbFucT strain with enhanced expression of the SbFucT gene, the NNN / pPsFucT strain with enhanced expression of the PsFucT gene, the NNN / pNbFucT2 strain with enhanced expression of the NbFucT2 gene, and the NNN / pHMFT strain with enhanced expression of the HMFT gene, which will be described later in the Examples.

[0104] Examples of such microorganisms include microorganisms in which expression of GsFucT, FsFucT, NbFucT1, MtFucT, AjFucT, SbFucT, NbFucT2, or HMFT is enhanced, resulting in enhanced α1,2-fucose transferase activity, which can selectively transfer fucose to the N-acetylglucosamine site, thereby improving LNFPI productivity. Therefore, LNFPI can be efficiently produced using these microorganisms. Furthermore, these microorganisms can also be used to produce fucosylated oligosaccharides other than LNFPI, such as fucosyllactoses such as 2'FL and 3'FL.

[0105] <Method for producing fucose-containing saccharide> The method for producing a fucose-containing saccharide of the present invention (hereinafter also referred to as the method of the present invention) includes a method for producing a fucose-containing saccharide, which comprises preparing the above-mentioned transformant and producing oligosaccharides in a culture using the transformant. In the method of the present invention, the fucose-containing saccharide is preferably LNFPI.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] As the transformant used in the method for producing fucose-containing saccharides, a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like may be used.

[0111] In the method for producing fucose-containing saccharides, glucose, lactose, lactose monohydrate, or the like may be added to the medium during cultivation.

[0112] When the transformant used in the method for producing a fucose-containing saccharide does not have the ability to produce GDP-fucose and / or LNT, GDP-fucose and / or LNT may be added to the medium.

[0113] Furthermore, in the method for producing fucose-containing carbohydrates, instead of adding glucose, lactose, lactose monohydrate, LNT, or the like to the medium during cultivation, a microorganism capable of producing glucose, lactose, lactose monohydrate, LNT, or the like from sugar may be cultured simultaneously with the transformant of the present invention, thereby supplying glucose, lactose, lactose monohydrate, LNT, or the like to the transformant of the present invention.

[0114] In the method for producing fucose-containing carbohydrates, it is preferable that β-galactosidase and WcaJ are not present in the medium.

[0115] 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.

[0116] By the above-mentioned culture, fucose-containing saccharides are produced in the culture, and thus fucose-containing saccharides can be produced.

[0117] 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.

[0118] Furthermore, a desired fucose-containing saccharide can be produced by further adding other sugars to the fucose-containing saccharide in the culture or the collected fucose-containing saccharide.

[0119] [Analysis Examples] (1) Analysis and Quantification of LNFPI, 2'FL, or Lactose In the Examples, analysis and quantification of LNFPI, 2'FL, or lactose were performed according to the following procedures. After cultivation, the culture solution containing the microorganism was centrifuged, and the supernatant was collected. 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. The resulting supernatant aqueous phase was used as the intracellular fraction. LNFPI, 2'FL, and lactose contained in the supernatant or intracellular fraction were analyzed using a sugar analyzer ICS-5000 (manufactured by Thermo Fisher Scientific). [Analysis conditions] Column: CarboPAC PA1 Column temperature: 25°C Mobile phase: (Mobile phase A) Water (Mobile phase B) 500 mmol / L Sodium hydroxide (Mobile phase C) 300 mmol / L Sodium acetate Mixing ratio of mobile phase A, mobile phase B and mobile phase C: (0 to 10 min) 80:20:0 (10 to 18 min) Gradient from 80:20:0 to 70:20:10 (18 to 35 min) Gradient from 70:20:10 to 0:20:80 (35 to 40 min) 0:20:80 (40 to 50 min) 80:20:0 Flow rate: 1.0 mL / min Detector: Pulsed amperometry detector

[0120] (2) Analysis and quantification of LNFPI, LNTII, or LNT In the examples, analysis and quantification of LNFPI, LNTII, or LNT were carried out according to the following procedure. As in (1) above, a supernatant and an intracellular fraction were prepared from the culture broth containing the microorganism after cultivation. LNFPI, LNTII, and LNT contained in the supernatant or intracellular fraction were analyzed using UFLC&LCMS-8040 (manufactured by Shimadzu Corporation). [Analysis conditions] Column: Coregel 87H3 (7.8 x 300 mm) Column temperature: 40°C Mobile phase: 0.1% formic acid water, isocratic elution Measurement time: 25 minutes Flow rate: 0.4 mL / ml Injection volume: 10 μL Detection: SIM mode

[0121] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0122] 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: 37 and 38 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.

[0123] <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.

[0124] 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.

[0125]

[0126] 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.

[0127] 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: 40 and 42 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.

[0128] 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: 40 and 42 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.

[0129] 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, 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).

[0130] 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 that exhibited ampicillin sensitivity (a transformant from which pKD46 had been lost) was further obtained. This transformant was designated W3110S3GKΔlacZY.

[0131] 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.

[0132]

[0133] 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.

[0134] 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: 46 and 48 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).

[0135] 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: 46 and 48 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.

[0136] The wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110S3GKΔlacZY 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).

[0137] 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.

[0138] <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:34 (hereinafter referred to as Cvβ3GalT), a gene encoding β1,3-N-acetylglucosamine transferase derived from Neisseria polysaccharea ATCC43768 represented by SEQ ID NO:36 (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.

[0139] 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.

[0140]

[0141] The DNA represented by SEQ ID NO: 33 is a codon-optimized DNA for expressing in Escherichia coli the nucleotide sequence of a 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.

[0142] The DNA represented by SEQ ID NO:35 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding β1,3-N-acetylglucosamine transferase NpLgtA derived from Neisseria polysaccharea ATCC43768 strain represented by SEQ ID NO:36 for expression in Escherichia coli, and was prepared by artificial synthesis. The nucleotide sequences represented by SEQ ID NOs:52 and 53, and SEQ ID NOs:54 and 55 each contain a complementary sequence at the 5' end.

[0143] 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: 51 and 56 as a primer set to obtain a DNA fragment in which the three fragments were ligated (hereinafter referred to as Cvβ3galT-NplgtA-lacY).

[0144] PCR was performed using a primer set of oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 57 and 58 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: 51 and 57, and SEQ ID NOs: 56 and 58 each contain a complementary sequence at their 5' ends.

[0145] 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.

[0146] (2) Construction of microorganisms having α1,2-fucosyltransferase activity Using the NNN strain constructed in (1) above, Escherichia coli carrying a plasmid for expressing genes encoding various α1,2-fucosyltransferases and rcsA derived from the W3110 strain, placed under the lac promoter, was constructed by the following method.

[0147] <Construction of expression vector> PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 59 and 60 as a primer set and W3110 strain prepared by a standard method as a template to obtain an rcsA fragment. PCR was performed using plasmid pSTV29 (manufactured by Takara Bio Inc.) as a template and DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 61 and 62 as a primer set to obtain a vector fragment of approximately 2.9 kb. In this case, the nucleotide sequences represented by SEQ ID NOs: 59 and 61 and SEQ ID NOs: 60 and 62 each contain complementary sequences at their 5' ends.

[0148] 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 Plasmid for Expression of α1,2-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.

[0150]

[0151] The DNA represented by SEQ ID NO: 1 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase GsFucT derived from Gramella sp. strain MAR_2010_147 represented by SEQ ID NO: 2 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0152] The DNA represented by SEQ ID NO:3 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase FsFucT derived from Francisella sp. FSC1006 strain represented by SEQ ID NO:4 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0153] The DNA represented by SEQ ID NO:5 is a DNA obtained by codon-optimizing the base sequence of the gene encoding α1,2-fucosyltransferase NbFucT1 derived from Neisseriaceae bacterium DSM 100970 strain represented by SEQ ID NO:6 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0154] The DNA represented by SEQ ID NO:7 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase MtFucT derived from the Methylobacter tundripaludum strain represented by SEQ ID NO:8 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0155] The DNA represented by SEQ ID NO:9 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase AjFucT derived from Amphritea japonica strain represented by SEQ ID NO:10 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0156] The DNA represented by SEQ ID NO:11 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase PaFucT derived from Pseudohalocynthiibacter aestuariivvens strain represented by SEQ ID NO:12 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0157] The DNA represented by SEQ ID NO: 13 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase SbFucT derived from Sterolibacteriaceae bacterium J5B strain represented by SEQ ID NO: 14 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0158] The DNA represented by SEQ ID NO:15 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase PsFucT derived from Pedobacter sp. CF074 strain represented by SEQ ID NO:16 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0159] The DNA represented by SEQ ID NO: 17 is a DNA in which the base sequence of the gene encoding the α1,2-fucosyltransferase NbFucT2 derived from the Neisseriales bacterium strain represented by SEQ ID NO: 18 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

[0160] The DNA represented by SEQ ID NO:19 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase CMfFucT derived from the Candidatus Methylobacter favarea strain represented by SEQ ID NO:20 so that it can be expressed in Escherichia coli, and was prepared by artificial synthesis.

[0161] The DNA represented by SEQ ID NO:21 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding the α1,2-fucosyltransferase WbwK derived from Escherichia coli O86 strain represented by SEQ ID NO:22 for expression in E. coli, and was prepared by artificial synthesis.

[0162] The DNA represented by SEQ ID NO:23 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase WbiQ derived from Escherichia coli O127 strain represented by SEQ ID NO:24 for expression in E. coli, and was prepared by artificial synthesis.

[0163] The DNA shown in SEQ ID NO:25 is the base sequence of the gene encoding α1,2-fucosyltransferase HMFT derived from Helicobacter mustelae ATCC43772 strain shown in SEQ ID NO:26, and was prepared by artificial synthesis.

[0164] Using the expression vector pSTV29-rcsA constructed above as a template, PCR was carried out using DNA consisting of the base sequences shown in SEQ ID NOs: 61 and 95 as a primer set to obtain a vector fragment of approximately 3.5 kb.

[0165] The base sequences represented by SEQ ID NOs: 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 and 61, and SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 95 each contain a complementary sequence at the 5' end.

[0166] The resulting amplified DNA fragments and vector fragments were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various α1,2-fucosyltransferases: pGsFucT, pFsFucT, pNbFucT1, pMtFucT, pAjFucT, pPaFucT, pSbFucT, pPsFucT, pNbFucT2, pCMfFucT, pWbwK, pWbiQ, and pHMFT.

[0167] <Construction of Escherichia coli carrying a plasmid for expressing α1,2-fucosyltransferase> The NNN strain constructed in (1) above was transformed with the α1,2-fucosyltransferase expression plasmid obtained above and pSTV29-rcsA as a vector control to construct E. coli strains carrying various plasmids, which were named NNN / pGsFucT strain, NNN / pFsFucT strain, NNN / pNbFucT1 strain, NNN / pMtFucT strain, NNN / pAjFucT strain, NNN / pPaFucT strain, NNN / pSbFucT strain, NNN / pPsFucT strain, NNN / pNbFucT2 strain, NNN / pCMfFucT strain, NNN / pWbwK strain, NNN / pWbiQ strain, NNN / pHMFT strain, and NNN / pCtrl strain, respectively.

[0168] Comparative Example: Construction of a Microorganism Expressing a Known α1,2-Fucosyltransferase A microorganism expressing an α1,2-fucosyltransferase known to be capable of producing LNFPI was constructed by the following method.

[0169] PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Table 5 as a primer set and DNAs shown in "Template" in Table 5 as templates to obtain each amplified DNA fragment.

[0170]

[0171] The DNA represented by SEQ ID NO:27 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase FucT54 derived from Sideroxydans lithotrophicus ES-11 strain represented by SEQ ID NO:28 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0172] The DNA represented by SEQ ID NO:29 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding α1,2-fucosyltransferase Te2FT derived from Thermosynechococcus elongatus BP-1 strain represented by SEQ ID NO:30 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0173] The DNA shown in SEQ ID NO: 31 is the base sequence of the gene encoding α1,2-fucosyltransferase FutC derived from Helicobacter pylori strain 26695 shown in SEQ ID NO: 32, and was prepared by artificial synthesis.

[0174] PCR was performed using the expression vector pSTV-rcsA constructed in Example 1(2) as a template and DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 61 and 95 as a primer set to obtain a vector fragment of approximately 3.5 kb. The nucleotide sequences represented by SEQ ID NOs: 89, 91, 93, and 61, and SEQ ID NOs: 90, 92, 94, and 95 each contain a complementary sequence at the 5' end.

[0175] The amplified DNA fragments and vector fragments obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various known α1,2-fucosyltransferases, namely pFucT54, pTe2FT, and pFutC.

[0176] The NNN strain constructed in Example 1(1) was transformed with the α1,2-fucosyltransferase expression plasmid obtained above to construct Escherichia coli strains carrying various plasmids, which were designated as NNN / pFucT54 strain, NNN / pTe2FT strain, and NNN / pFutC strain, respectively.

[0177] [Example 2] Evaluation of productivity of LNFPI The productivity of LNFPI was evaluated for the NNN / pGsFucT strain, NNN / pFsFucT strain, NNN / pNbFucT1 strain, NNN / pMtFucT strain, NNN / pAjFucT strain, NNN / pPaFucT strain, NNN / pSbFucT strain, NNN / pPsFucT strain, NNN / pNbFucT2 strain, NNN / pCMfFucT strain, NNN / pWbwK strain, NNN / pWbiQ strain and NNN / pHMFT strain obtained in Example 1 (2). As a positive control, the NNN / pTe2FT strain and NNN / pFutC strain constructed in the comparative example were used. As a negative control, the NNN / pCtrl strain constructed in Example 1(2) was used.

[0178] Each strain was cultured on an LB plate containing 100 mg / L kanamycin and 25 mg / L chloramphenicol at 37°C for 18 hours, and then inoculated into a plastic test tube containing 2 mL of LB medium containing 100 mg / L kanamycin and 25 mg / L chloramphenicol, followed by shaking culture at 30°C for 15 hours. Thereafter, 0.2 mL of the resulting culture broth was inoculated into a large test tube containing 4 mL of a production medium containing 100 mg / L of kanamycin and 25 mg / L of chloramphenicol [glucose 30 g / L, lactose monohydrate 10 g / L, magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, casamino acids 5 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L (for components other than glucose, lactose monohydrate, and magnesium sulfate heptahydrate, the pH was adjusted to 7.2 with aqueous sodium hydroxide solution and then autoclaved) (aqueous solutions containing glucose, lactose monohydrate, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and then mixed)], and the mixture was cultured with shaking at 30°C for 29 hours.

[0179] After the cultivation was completed, the culture medium was centrifuged and diluted appropriately, and the LNFPI, LNTII, or LNT contained in the supernatant and the intracellular fraction was analyzed by UFLC&LCMS-8040. The results are shown in Table 6. The combined amounts of LNFPI produced in the supernatant and the intracellular fraction are shown in Figure 2.

[0180]

[0181] As a result, it was found that, compared with strains expressing FutC or Te2FT, which are known to be capable of producing LNFPI by α1,2-fucosyltransferase, the NNN / pGsFucT strain, NNN / pFsFucT strain, NNN / pNbFucT1 strain, NNN / pMtFucT strain, NNN / pAjFucT strain, NNN / pSbFucT strain, NNN / pNbFucT2 strain, and NNN / pHMFT strain were able to accumulate large amounts of LNFPI both in the supernatant and within the bacterial cells.

[0182] Among these, FsFucT derived from Francisella sp. FSC1006 and NbFucT2 derived from Neisseria bacterium, which showed significantly high LNFPI productivity, were selected as candidates for α1,2-fucosyltransferase useful for LNFPI production.

[0183] Example 3 Production of LNFPI The NNN / pFsFucT strain and NNN / pNbFucT2 strain selected in Example 2, and the NNN / pFucT54 strain constructed in the Comparative Example as a positive control, 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 2-L baffled Erlenmeyer flask containing 250 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 17 hours.

[0184] Subsequently, 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 20 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 72 hours with shaking. 14% aqueous ammonia was added to adjust the pH to 6.9 during the culture.

[0185] When all the initial glucose was consumed, IPTG was added to a final concentration of 0.5 mM, and in the subsequent cultivation, 480 g / L glucose solution and 4 g / L lactose monohydrate were added at a rate of 1 to 6 mL / h.

[0186] After the cultivation was completed, the culture medium was centrifuged and diluted appropriately, and the LNFPI, 2'FL, LNTII, or LNT contained in the supernatant was analyzed using a sugar analyzer ICS-5000. The results are shown in Table 7.

[0187]

[0188] As shown in Table 7, the NNN / pFsFucT and NNN / pNbFucT2 strains produced the fucosylated oligosaccharides 2'FL and LNFPI, suggesting the possibility that FsFucT and NbFucT2 could be used to produce these oligosaccharides. Furthermore, the NNN / pFsFucT and NNN / pNbFucT2 strains were found to accumulate significantly more LNFPI in both the supernatant and intracellular space than the NNN / pFucT54 strain, a known α1,2-fucosyltransferase-expressing strain. In particular, the NNN / pNbFucT2 strain produced approximately twice the amount of LNFPI as the NNN / pFucT54 strain. In addition, the NNN / pNbFucT2 strain showed significantly reduced production of the by-product 2′FL compared to the other strains, suggesting that NbFucT2 may preferentially use LNT as a substrate.

[0189] Although the present invention has been described in detail and 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. 2022-050798) filed on March 25, 2022, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0190] SEQ ID NO: 1: Nucleotide sequence of GsFucT derived from Gramella sp. MAR_2010_147 SEQ ID NO: 2: Amino acid sequence of GsFucT derived from Gramella sp. MAR_2010_147 SEQ ID NO: 3: Nucleotide sequence of FsFucT derived from Francisella sp. FSC1006 SEQ ID NO: 4: Amino acid sequence of GsFucT derived from Francisella sp. SEQ ID NO: 5: Nucleotide sequence of NbFucT1 derived from Neisseriaceae bacterium DSM 100970 SEQ ID NO: 6: Amino acid sequence of NbFucT1 derived from Neisseriaceae bacterium DSM 100970 SEQ ID NO: 7: Nucleotide sequence of MtFucT derived from Methylobacter tundripaludum SEQ ID NO: 8: Amino acid sequence of MtFucT derived from Methylobacter tundripaludum SEQ ID NO: 9: Nucleotide sequence of AjFucT derived from Amphritea japonica SEQ ID NO: 10: Amino acid sequence of AjFucT derived from Amphritea japonica SEQ ID NO: 11: Nucleotide sequence of PaFucT derived from Pseudohalocynthiibacter aestuariivvens SEQ ID NO: 12: Amino acid sequence of PaFucT derived from Pseudohalocynthiibacter aestuariivvens SEQ ID NO: 13: Nucleotide sequence of SbFucT derived from Sterolibacteriaceae bacterium J5B SEQ ID NO: 14: Amino acid sequence of SbFucT derived from Sterolibacteriaceae bacterium J5B SEQ ID NO: 15: Nucleotide sequence of PsFucT derived from Pedobacter sp. CF074 SEQ ID NO: 16: Amino acid sequence of SbFucT derived from Pedobacter sp. SEQ ID NO: 17: Amino acid sequence of PsFucT derived from Neisseriales bacterium SEQ ID NO: 18: Amino acid sequence of NbFucT2 derived from Neisseriales bacterium SEQ ID NO: 19: Nucleotide sequence of CMfFucT derived from Candidatus Methylobacter favarea SEQ ID NO: 20: Amino acid sequence of CMfFucT derived from Candidatus Methylobacter favareaSEQ ID NO: 21: Nucleotide sequence of WbwK derived from Escherichia coli O86 SEQ ID NO: 22: Amino acid sequence of WbwK derived from Escherichia coli O86 SEQ ID NO: 23: Nucleotide sequence of wbiQ derived from Escherichia coli O127 SEQ ID NO: 24: Amino acid sequence of wbiQ derived from Escherichia coli O127 SEQ ID NO: 25: Nucleotide sequence of HMFT derived from Helicobacter mustelae ATCC43772 SEQ ID NO: 26: Amino acid sequence of HMFT derived from Helicobacter mustelae ATCC43772 SEQ ID NO: 27: Nucleotide sequence of FucT54 derived from Sideroxydans lithotrophicus ES-11 SEQ ID NO: 28: Amino acid sequence of FucT54 derived from Sideroxydans lithotrophicus ES-11 SEQ ID NO: 29: Nucleotide sequence of Te2FT derived from Thermosynechococcus elongatus BP-1 SEQ ID NO: 30: Amino acid sequence of Te2FT derived from Thermosynechococcus elongatus BP-1 SEQ ID NO: 31: Nucleotide sequence of FutC derived from Helicobacter pylori 26695 SEQ ID NO: 32: Amino acid sequence of FutC derived from Helicobacter pylori 26695 SEQ ID NO: 33: Nucleotide sequence of Cvβ3galT derived from Chromobacterium violaceum ATCC553 SEQ ID NO: 34: Amino acid sequence of Cvβ3galT derived from Chromobacterium violaceum ATCC553 SEQ ID NO: 35: Nucleotide sequence of NplgtA derived from Neisseria polysaccharea ATCC43768 SEQ ID NO: 36: Amino acid sequence of NplgtA derived from Neisseria polysaccharea ATCC43768 SEQ ID NO: 37: Nucleotide sequence of primer for amplifying catsacB fragment SEQ ID NO: 38: Nucleotide sequence of primer for amplifying catsacB fragment SEQ ID NO: 39: Nucleotide sequence of primer for amplifying lacZ upstream 1 SEQ ID NO: 40: Nucleotide sequence of primer for amplifying lacZ upstream 1 SEQ ID NO: 41: Nucleotide sequence of primer for amplifying lacY downstream 1 SEQ ID NO: 42: Nucleotide sequence of primer for amplifying lacY downstream 1 SEQ ID NO: 43: Nucleotide sequence of primer for amplifying lacZ upstream 2SEQ ID NO: 44: Nucleotide sequence of primer for amplifying lacY downstream 2 SEQ ID NO: 45: Nucleotide sequence of primer for amplifying wcaJ upstream 1 SEQ ID NO: 46: Nucleotide sequence of primer for amplifying wcaJ upstream 1 SEQ ID NO: 47: Nucleotide sequence of primer for amplifying wcaM downstream 1 SEQ ID NO: 48: Nucleotide sequence of primer for amplifying wcaM downstream 1 SEQ ID NO: 49: Nucleotide sequence of primer for amplifying wcaJ upstream 2 SEQ ID NO: 50: Nucleotide sequence of primer for amplifying wcaM downstream 2 SEQ ID NO: 51: Nucleotide sequence of primer for amplifying Cvβ3galT fragment SEQ ID NO: 52: Nucleotide sequence of primer for amplifying Cvβ3galT fragment SEQ ID NO: 53: Nucleotide sequence of primer for amplifying NplgtA fragment SEQ ID NO: 54: Nucleotide sequence of primer for amplifying NplgtA fragment SEQ ID NO: 55: Nucleotide sequence of primer for amplifying lacY fragment SEQ ID NO: 56: Nucleotide sequence of primer for amplifying lacY fragment SEQ ID NO: 57: Nucleotide sequence of primer for amplifying pUAKQE fragment SEQ ID NO: 58: Nucleotide sequence of a primer for amplifying a pUAKQE fragment SEQ ID NO: 59: Nucleotide sequence of a primer for amplifying a rcsA fragment SEQ ID NO: 60: Nucleotide sequence of a primer for amplifying a rcsA fragment SEQ ID NO: 61: Nucleotide sequence of a primer for amplifying a pSTV29 fragment SEQ ID NO: 62: Nucleotide sequence of a primer for amplifying a pSTV29 fragment SEQ ID NO: 63: Nucleotide sequence of a primer for amplifying a GsFucT fragment SEQ ID NO: 64: Nucleotide sequence of a primer for amplifying a GsFucT fragment SEQ ID NO: 65: Nucleotide sequence of a primer for amplifying an FsFucT fragment SEQ ID NO: 66: Nucleotide sequence of a primer for amplifying an FsFucT fragment SEQ ID NO: 67: Nucleotide sequence of a primer for amplifying an NbFucT1 fragment SEQ ID NO: 68: Nucleotide sequence of a primer for amplifying an NbFucT1 fragment SEQ ID NO: 69: Nucleotide sequence of a primer for amplifying an MtFucT fragment SEQ ID NO: 70: Nucleotide sequence of a primer for amplifying an MtFucT fragment SEQ ID NO: 71: Nucleotide sequence of a primer for amplifying an AjFucT fragment SEQ ID NO: 72: Nucleotide sequence of a primer for amplifying an AjFucT fragment SEQ ID NO: 73: Nucleotide sequence of a primer for amplifying a PaFucT fragment SEQ ID NO: 74: Nucleotide sequence of a primer for amplifying a PaFucT fragment SEQ ID NO: 75: Nucleotide sequence of a primer for amplifying an SbFucT fragment SEQ ID NO: 76: Nucleotide sequence of a primer for amplifying an SbFucT fragment SEQ ID NO: 77: Nucleotide sequence of a primer for amplifying a PsFucT fragmentSEQ ID NO: 78: Nucleotide sequence of a primer for amplifying a PsFucT fragment SEQ ID NO: 79: Nucleotide sequence of a primer for amplifying a NbFucT2 fragment SEQ ID NO: 80: Nucleotide sequence of a primer for amplifying a NbFucT2 fragment SEQ ID NO: 81: Nucleotide sequence of a primer for amplifying a CMfFucT fragment SEQ ID NO: 82: Nucleotide sequence of a primer for amplifying a CMfFucT fragment SEQ ID NO: 83: Nucleotide sequence of a primer for amplifying a WbwK fragment SEQ ID NO: 84: Nucleotide sequence of a primer for amplifying a WbwK fragment SEQ ID NO: 85: Nucleotide sequence of a primer for amplifying a WbiQ fragment SEQ ID NO: 86: Nucleotide sequence of a primer for amplifying a WbiQ fragment SEQ ID NO: 87: Nucleotide sequence of a primer for amplifying a HMFT fragment SEQ ID NO: 88: Nucleotide sequence of a primer for amplifying a HMFT fragment SEQ ID NO: 89: Nucleotide sequence of a primer for amplifying a FucT54 fragment SEQ ID NO: 90: Nucleotide sequence of a primer for amplifying a FucT54 fragment SEQ ID NO: 91: Nucleotide sequence of a primer for amplifying a Te2FT fragment SEQ ID NO: 92: Nucleotide sequence of primer for amplifying Te2FT fragment SEQ ID NO: 93: Nucleotide sequence of primer for amplifying FutC fragment SEQ ID NO: 94: Nucleotide sequence of primer for amplifying FutC fragment SEQ ID NO: 95: Nucleotide sequence of primer for amplifying pSTV-rcsA fragment

Claims

1. A protein described in any one of [1] to [3] below, which has the activity of transferring fucosyl groups to lacto-N-tetraose (LNT). [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26. [2] A mutant protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26, in which 1 to 20 amino acids have been deleted, substituted, inserted, or added, and which has α1,2-fucosyltransferase activity. [3] A homologous protein consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26, and which has α1,2-fucosyltransferase activity.

2. A DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17 or 25 or a homologous sequence thereof, and encoding the protein according to any one of [1] to [3] of claim 1.

3. A recombinant DNA containing the DNA of claim 2.

4. A transformant obtained by transforming a host cell with the recombinant DNA according to claim 3.

5. The transformant according to claim 4, which is a microorganism in which the activity of the protein of any one of [1] to [3] according to claim 1 and the productivity of fucose-containing carbohydrates are enhanced.

6. The transformant according to claim 5, wherein the microorganism is Escherichia coli.

7. A method for producing a fucose-containing saccharide, comprising preparing the transformant according to any one of claims 4 to 6, and producing the fucose-containing saccharide in a culture using the transformant.

8. The method of claim 7, wherein the fucose-containing carbohydrate is lacto-N-fucopentaose I (LNFPI).

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