Protein with 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

[Technical Field]

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

[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 inhibiting 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 selectively grow preferentially on LNFPI, drawing attention to its prebiotic function (Non-Patent Document 5).

[0006] Widely used methods for producing LNFPI include microbial fermentation and enzymatic reaction methods (one-pot multienzyme (OPME) systems) using α1,2-fucosyltransferase. Patent documents 1 and 2 and non-patent documents 4, 5, and 6 disclose methods for producing oligosaccharides such as LNFPI by overexpressing α1,2-fucosyltransferase derived from microorganisms such as Thermosynechococcus elongatus, Sideroxydans lithotrophicus, or Helicobacter pylori in Escherichia coli, and using LNT and GDP-fucose as substrates through fermentation or continuous enzymatic reaction.

[0007] However, in the fermentation method or continuous enzymatic reaction method, a problem with LNFPI production is that α1,2-fucosyltransferase reacts not only with the desired substrate, LNT, but also with the coexisting lactose, resulting in the production of 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). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2017 / 106864 [Patent Document 2] International Publication No. 2019 / 008133 [Non-patent literature]

[0010] [Non-Patent Document 1] Int.J.Pediatrics(2019),Article ID 2390240 [Non-patent document 2] Nutr.Rev.(2017)75,920-933 [Non-patent document 3] J.Biol.Chem.(2017)292(27)11243-11249 [Non-patent document 4] J.Biotechnol.(2020)318,31-38 [Non-Patent Document 5] Chem.Commun.(2016)52,3899-3902 [Non-patent document 6] Bioorganic&Medicinal Chemistry 23(2015)6799-6806 Summary of the Invention [Problem to be solved by the invention]

[0011] As mentioned 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, which poses a problem in the production of LNFPI: the generation of 2'FL as a by-product.

[0012] On the other hand, to produce LNFPI more efficiently using lactose as the starting material, an α1,2-fucosyltransferase that does not transfer sugars to lactose but can selectively transfer sugars to the non-reducing terminal galactose site of LNT is required.

[0013] Therefore, an object of the present invention is to provide a protein having α1,2-fucosyltransferase activity that is highly productive for LNFPI, and a method for producing LNFPI. [Means for solving the problem]

[0014] The present inventors discovered that LNFPI can be produced more efficiently than conventional methods by using a microorganism capable of producing a protein having α1,2-fucosyltransferase activity consisting of a specific amino acid sequence, and thus completed the present invention. Furthermore, we have discovered for the first time a fucosyltransferase derived from the genus Neisseria or Francisella that is suitable for producing fucosylated oligosaccharides such as LNFPI or fucosyllactose.

[0015] That is, the present invention is as follows. 1. A protein according to any one of [1] to [3] below, which has a fucosylation transfer activity onto 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 an 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 having 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 having 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] 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 according to 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] above and the productivity of the fucose-containing carbohydrate 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 the fucose-containing saccharide in a culture using the transformant. 8. The method according to 7 above, wherein the fucose-containing carbohydrate is lacto-N-fucopentaose I (LNFPI). [Effects of the Invention]

[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, LNFPI can be produced more efficiently with less by-product formation than conventional methods. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the biosynthetic pathway of LNFPI in one embodiment of the present invention. [Figure 2] FIG. 2 shows the combined LNFPI production amounts in the supernatant and intracellular fractions (Example 2). DETAILED DESCRIPTION OF THE INVENTION

[0018] <Protein, DNA, transformants> The protein of the present invention is a protein having a fucosylation activity onto lacto-N-tetraose (LNT) and is described in any one of [1] to [3] below. [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 an 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 having 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 having α1,2-fucosyltransferase activity.

[0019] From the viewpoint of further enhancing the fucosylation activity to 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 α1,2-fucosyltransferase GsFucT derived from Gramella sp. strain MAR_2010_147, which will be described later in the Examples. The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is α1,2-fucosyltransferase FsFucT derived from Francisella sp. strain FSC1006, which will be described later in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 6 is α1,2-fucosyltransferase NbFucT1 derived from Neisseriaceae bacterium DSM 100970 strain, which will be described later in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 8 is α1,2-fucosyltransferase MtFucT derived from a Methylobacter tundripaludum strain, which will be described later 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, which will be described later in the Examples. The protein consisting of the amino acid sequence shown in SEQ ID NO: 14 is α1,2-fucosyltransferase SbFucT derived from Sterolibacteriaceae bacterium J5B strain, which will be described later in the Examples. The protein consisting of the amino acid sequence represented by SEQ ID NO: 18 is α1,2-fucosyltransferase NbFucT2 derived from a Neisseriales bacterium strain, which will be described later in the Examples. The protein consisting of the amino acid sequence shown in SEQ ID NO: 26 is α1,2-fucosyltransferase HMFT derived from Helicobacter mustelae ATCC43772 strain, which will be described later 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 amino acids deleted, substituted, inserted, or added 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 D group: 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 [2] above, an example of the amino acid residue to be substituted is the 17th asparagine residue.

[0026] As used herein, a homologous protein refers to a protein that is found in organisms that exist 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, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing nucleotide sequences using BLASTN based on BLAST, parameters are, for example, score = 100 and word length = 12. When analyzing amino acid sequences using BLASTX based on BLAST, parameters are, for example, score = 50 and word length = 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 the donor substrate GDP-fucose to the N-acetylglucosamine hydroxyl group of the acceptor substrate carbohydrate (hereinafter referred to as the "acceptor carbohydrate"), LNT.

[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 N-acetylglucosamine hydroxyl group of an acceptor saccharide via an α1,2-linkage to produce a fucose-containing saccharide. The acceptor saccharide is preferably LNT. The fucose-containing saccharide is preferably LNFPI.

[0032] Whether the above mutant protein or a homologous protein has α1,2-fucosyltransferase activity can be confirmed, for example, by the following method. First, a recombinant DNA encoding a mutant protein or a 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 produce a transformant having higher activity of the protein than the parent strain, and the activity can be confirmed by comparing the amounts of fucose-containing carbohydrates produced and accumulated in the culture medium of the parent strain or the transformant. Specific examples of fucose-containing carbohydrates include 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, Escherichia coli 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, Serratiamarcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum Examples of suitable strains include prokaryotes such as Bacillus subtilis ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, and Pseudomonas sp. D-0110, and yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and 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 in which the ability to supply GDP-fucose, the reaction substrate of α1,2-fucosyltransferase, has been artificially imparted or enhanced. 2) A microorganism that has been artificially conferred or enhanced with the ability to supply LNT, the reaction substrate of α1,2-fucosyltransferase. The following is an explanation.

[0037] 1) A microorganism used as a parent strain, which has been artificially imparted or enhanced with the ability to supply GDP-fucose, which is a reaction substrate for α1,2-fucosyltransferase. 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 for alleviating or eliminating at least one of the mechanisms controlling the biosynthetic pathway for producing GDP-fucose from sugars. (1b) A method for enhancing the expression of at least one enzyme involved in the biosynthetic pathway for producing GDP-fucose from sugars. (1c) A method for increasing the copy number of at least one gene encoding an enzyme involved in a biosynthetic pathway for producing GDP-fucose from sugars. (1d) A method for weakening or blocking at least one metabolic pathway branching off from a biosynthetic pathway that produces GDP-fucose from sugar to a metabolic product other than the target substance.

[0039] Specific examples of mechanisms for regulating the biosynthetic pathway that produces GDP-fucose from sugars include known mechanisms, such as a regulatory mechanism by a transcriptional regulatory factor (e.g., RcsA) involved in the regulation 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 that produces 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 are 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 number 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 expression of fucI and / or fucK may be impaired. Eliminating the intracellular synthesis of FucI and / or FucK eliminates fucose metabolism in the cells, thereby enabling an increased amount of fucose to be 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), which 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 sugars (2b) A method for enhancing expression of at least one enzyme involved in a biosynthetic pathway for producing LNT from sugars. (2c) A method for increasing the copy number of at least one enzyme gene involved in a biosynthetic pathway for producing LNT from sugars. (2d) A method for alleviating or eliminating at least one of the mechanisms that decompose LNT or its substrate sugar (2e) A method for enhancing the expression of at least one enzyme involved in the cellular uptake of LNT or its substrate sugar. (2f) A method for increasing the copy number of at least one gene encoding an enzyme involved in the cellular uptake of LNT or its substrate sugar (2g) A method for weakening or blocking at least one metabolic pathway branching off from a biosynthetic pathway that produces LNT from sugar to a metabolic product other than the target substance. (2h) A method for selecting cell lines that are more resistant to LNT analogs than wild-type strains

[0045] Specific examples of enzymes involved in the biosynthetic pathway that produces LNT from sugars include known enzymes, such as enzymes with β1,4-galactosyltransferase (hereinafter referred to as galT) activity and enzymes with 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 exhibit, for example, 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 / glucosamine-1-phosphate uridyltransferase (GLT) activity, in order to supply LNT. It is preferable that the enzyme has at least one activity selected from the group consisting of phosphate acetyltransferase (hereinafter referred to as glmU) activity, phosphoglucomutase (hereinafter referred to as pgm) activity, UTP glucose-1-phosphate uridylyltransferase (hereinafter referred to as galU) activity, UDP glucose-4-epimerase (hereinafter referred to as galE) activity, UTP glucose-1-phosphate uridylyltransferase (hereinafter referred to as galF) activity, and glucose-6-phosphate isomerase (hereinafter referred to as pgi) activity, and it is more preferable that the activity is enhanced.

[0049] Among these, it is preferable that the lacY, galT and lgtA activities are present, and it is even more preferable that these activities are enhanced.

[0050] lacY is a membrane protein that imports lactose, the substrate of LNT, into cells. galT is an enzyme involved in the production of LNT from lacto-N-triose II (LNTII). LNT is the precursor of LNFPI. LgtA is an enzyme involved in the production of LNTII from lactose and uridine diphosphate-N-acetylglucosamine (hereinafter referred to as UDP-GlcNAc). LNTII is the 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. Accordingly, in one embodiment of the present invention, the nucleotide sequence encoding rcsA (accession number BAA15776.1), the nucleotide sequence encoding mannose-6-phosphate isomerase (accession number BAA15361.1), the nucleotide sequence encoding phosphomannomutase (accession number BAA15901.1), the nucleotide sequence encoding mannose-1-phosphate guanylyltransferase (accession number BAA15905.1), the nucleotide sequence encoding GDP mannose-4,6-dehydratase (accession number BAA15909.1), the nucleotide sequence encoding GDP-L-fucose synthase (accession number BAA15908.1), the nucleotide sequence encoding lacY (accession number BAE76125.1), the nucleotide sequence encoding galT (SEQ ID NO: 104 ... Preferably, the parent strain is a genetically modified microorganism comprising 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 the 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] Microorganisms 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 having enhanced activity, may be produced by known methods. 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 a colanic acid production-related protein, such as the wcaJ, wzxC, wcaK, wcaL, or wcaM gene.

[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] E. coli with reduced or lost β-galactosidase activity and / or colanic acid synthesis activity can be produced by known methods, such as by various genetic engineering methods (Metabolic Engineering, 2017, 41:23-38).

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

[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 recombinant DNA containing DNA encoding a protein described in 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 recombinant DNA containing DNA encoding a protein described in 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 described in any one of [1] to [3] above may be any DNA encoding a protein having the activity of the protein described in any one of [1] to [3] above, and specifically includes one DNA selected from the group consisting of [4] to [7] below. [4] A DNA encoding the protein according to any one of [1] to [3] above. [5] DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17, or 25 [6] A DNA that hybridizes under stringent conditions with a 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] A DNA consisting of a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 13, 17, or 25, and encoding 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, or as an oligonucleotide primer in PCR analysis.

[0064] 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. 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, such as those 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 (Roche Diagnostics), which uses a random primed probe to prepare a probe and hybridize it under stringent conditions.

[0067] The above-mentioned stringent conditions include incubating the DNA-immobilized filter and probe DNA overnight at 42°C in a solution containing 50% formamide, 5x SSC (750 mmol / L sodium chloride, 75 mmol / L sodium citrate), 50 mmol / L sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, followed by washing the filter in a 0.2x SSC 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% identity, preferably 97% identity, more preferably 98% identity, and most preferably 99% 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 probe DNA that can be designed based on the nucleotide sequence shown in 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 genera Neisseria (Neisseriaceae, Neisseriales), Francisella, Methylobacter, Amphritea, Sterolibacteriaceae, and 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 Defence Research Agency, Neisseriaceae bacterium DSM 100970 strain is available from the University of Malaya, and Methylobacter tundripaludum strain, Amphritea japonica strain, and Helicobacter mustelae ATCC43772 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 shown in SEQ ID NO: 1, 3, 5, 7, 9, 13, 17 or 25 as a template to error-prone PCR or the like.

[0072] Alternatively, DNA encoding the mutant protein described above in [2] can be obtained by PCR using a pair of PCR primers each having a base sequence at the 5' end designed to introduce the 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 plasmid having a base sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition), with a 15-base overlap at the 5' end. The overlapping portion contains the desired mutation. Next, PCR is performed using the mutagenesis primers and a template plasmid having the base sequence into which the desired mutation is to be introduced. The resulting amplified fragment 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 base sequences that have 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 amino acid sequences that have 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 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 containing the DNA, in a manner similar to the method for obtaining the above DNA.

[0076] The obtained DNA described in any one of [4] to [7] above 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 can then be introduced into a host cell, and the DNA can be analyzed using a conventional base sequence analysis method, such as 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). The base sequence of the DNA can be determined.

[0077] Any host cells can be used for determining the base sequence of the DNA as long as they can be grown by introducing the vector. 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 TH2 (all manufactured by Takara Bio Inc.), Escherichia coli XL1-Blue, and Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, and Escherichia coli. W3110, Escherichia coli MP347, Escherichia coli NM522, etc.

[0078] Examples of the above vectors 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 host cells can be used, including, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Application Laid-Open No. 63-248394), and the 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, full-length DNA can be obtained by Southern hybridization or the like against a chromosomal DNA library using the partial length DNA as a probe.

[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 the 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 described in 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 base sequence of the portion encoding the protein so that it contains optimal codons for expression in host cells, a transformant with improved productivity can be obtained.

[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 (ribosome binding sequence) and the initiation codon is adjusted to an appropriate distance (e.g., 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 encoding a protein having α1,2-fucosyltransferase activity so that it contains codons optimal for expression in the host, the expression level of the protein having α1,2-fucosyltransferase activity can be improved. Examples of proteins having α1,2-fucosyltransferase activity include the proteins described in any one of [1] to [3] above. Information on codon usage in the parent strain 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 the parent strain, examples of the expression vector include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio Inc.), pMW118, pMW119 (all manufactured by Nippon Gene Co., Ltd.), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (all manufactured by Qiagen), pET-3, pBluescriptII SK(+), and pBluescriptII. 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(-) (manufactured by Stratagene), pTrS30 [Escherichia coli JM109 / pTrS30 (FERM Examples of suitable vectors include pTK31 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385], pPAC31 (WO 1998 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (JP 63-233798 A), and pKD46 [Proc. Natl. Acad. Sci., USA, 97, 6640-6645 (2000)].

[0092] When using the above-mentioned expression vectors, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples of such promoters include promoters of genes involved in amino acid biosynthesis, such as the trp promoter and the ilv promoter, and promoters derived from Escherichia coli or phages, such as the uspA promoter, the lac promoter, the PL promoter, the PR promoter, and the PSE promoter.Other examples include artificially designed and modified promoters, such as a promoter with 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 from Molecular and General Genetics, 196, 175 (1984)).

[0094] When using the above expression vector, any promoter that functions in the cells of a microorganism belonging to the genus Corynebacterium can be used, for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)].

[0095] When a yeast strain is used as the parent strain, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0096] When using the above expression vector, any promoter that functions in the cells of a yeast strain may be used, 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 Application Laid-Open 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 a plasmid DNA carrying a drug resistance gene that cannot autonomously replicate in the host cell to be introduced. A method using homologous recombination that is frequently used in Escherichia coli includes, for example, 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 sensitive to sucrose due to Bacillus subtilis levansucrase incorporated into the chromosome along with recombinant DNA, or a selection method that utilizes the fact that E. coli becomes sensitive to streptomycin when a wild-type rpsL gene is incorporated into E. coli that has a mutant rpsL gene that confers streptomycin resistance [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 integrated into the chromosome of the parent strain can be confirmed, for example, by a method in which the gene originally present on the chromosomal DNA of a microorganism cannot be amplified, but the gene introduced by transformation can be amplified, and the amplified product can be confirmed by PCR. Furthermore, an increase in the amount of transcription of the DNA or the amount of production of the protein encoded by the DNA can be confirmed by a method in which the amount of transcription of the gene in the microorganism is compared with that of the parent strain by Northern blotting, or the amount of production of the protein in the microorganism is compared with that of the parent strain by Western blotting.

[0102] Whether the microorganism constructed by the above method is a microorganism in which the activity of the protein described in any one of [1] to [3] above is enhanced 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 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 thus can selectively transfer fucose to the N-acetylglucosamine site of LNT, thereby improving LNFPI productivity. 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 are described below in the Examples.

[0104] Examples of such microorganisms include those with enhanced expression of GsFucT, FsFucT, NbFucT1, MtFucT, AjFucT, SbFucT, NbFucT2, or HMFT. This enhances the activity of α1,2-fucose transferase, which selectively transfers fucose to N-acetylglucosamine sites, potentially improving LNFPI productivity. Therefore, these microorganisms can be used to efficiently produce LNFPI. 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 carbohydrates> 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 example] (1) Analysis and quantification of LNFPI, 2'FL, or lactose In the Examples, analysis and quantification of LNFPI, 2'FL or lactose were carried out according to the following procedures. After cultivation, the culture medium containing the microorganisms was centrifuged, and the supernatant was collected. The precipitated cells were suspended in an equal volume of water to the original culture medium, disrupted with an equal volume of chloroform, and then centrifuged. The resulting supernatant water 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 (Thermo Fisher Scientific). [Analysis conditions] Column: CarboPAC PA1 Column temperature: 25℃ Mobile phase: (Mobile phase A) water (Mobile phase B) 500mmol / L sodium hydroxide (Mobile phase C) 300mmol / L sodium acetate Mixing ratio of mobile phase A, mobile phase B and mobile phase C: (0~10 minutes)80:20:0 (10-18 minutes) Gradient from 80:20:0 to 70:20:10 (18-35 minutes) Gradient from 70:20:10 to 0:20:80 (35~40 minutes) 0:20:80 (40~50 minutes)80:20:0 Flow rate: 1.0mL / mIn Detector: Pulsed amperometric detector

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

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

[0122] [Example 1] Construction of microorganisms expressing various α1,2-fucosyltransferases (1) Construction of host strain <Identifying DNA fragments to be used as markers 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 the chloramphenicol-resistant cat gene and the sucrose-sensitive sacB gene.

[0123] <Creation of E. coli strains lacking β-galactosidase activity, lactose permease activity, and colanic acid synthesis activity> E. coli strains lacking the DNA encoding β-galactosidase (hereinafter referred to as the lacZ gene), the DNA encoding lactose permease (hereinafter referred to as the lacY gene), and the DNA encoding proteins involved in colanic acid production (hereinafter referred to as the wcaJ, wzxC, wcaK, wcaL, or wcaM genes) were constructed by the following method. Note that lacZ and lacY (hereinafter referred to as lacZY), as well as wcaJ, wzxC, wcaK, wcaL, and wcaM (hereinafter referred to as wcaJ-wzxC-wcaKLM), each form an operon on the E. coli genome.

[0124] PCR was performed using genomic DNA of Escherichia coli W3110 strain prepared by a conventional method as a template and DNA primer sets consisting of the base sequences shown in "Primer set" in Table 1 to amplify each DNA fragment.

[0125] [Table 1]

[0126] LacZ upstream 1 and lacZ upstream 2 comprise the region from the initiation codon of the lacZ gene to approximately 1000 bp upstream of the initiation codon. LacY downstream 1 and lacY downstream 2 comprise the region from approximately 50 bp to approximately 1000 bp downstream of the termination codon of the lacY gene.

[0127] PCR was performed using a mixture of lacZ upstream 1, lacY 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: 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 surrounding 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 and downstream regions of lacZ are directly linked.

[0129] The lacZY::cat-sacB fragment was introduced by electroporation into the W3110S3GK strain (NBRC114657) harboring the plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)], which contains the gene encoding the λ recombinase. Transformants that exhibited chloramphenicol resistance and sucrose sensitivity (transformants in which the lacZY gene had been replaced with lacZY::cat-sacB) were obtained.

[0130] The ΔlacZY fragment was introduced into the transformant by electroporation to obtain transformants that were sensitive to chloramphenicol and resistant to sucrose (transformants in which lacZY::cat-sacB had been replaced by ΔlacZY). From these, an ampicillin-sensitive transformant (transformant in which pKD46 had been lost) was further obtained and designated W3110S3GKΔlacZY.

[0131] Similarly, PCR was performed using the genomic DNA of the W3110 strain as a template and DNA consisting of the base sequences shown in "Primer set" in Table 2 as a primer set, to obtain each amplified DNA fragment.

[0132] [Table 2]

[0133] wcaJ upstream 1 and wcaJ upstream 2 comprise the region from the initiation codon of the wcaJ gene to approximately 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 approximately 1000 bp downstream of the termination codon.

[0134] PCR was performed using an equimolar mixture of wcaJ upstream 1, wcaM downstream 1, and cat-sacB fragments 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 a mixture of wcaJ upstream 2 and wcaM downstream 2 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 (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, and a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (a transformant in which wcaJ-wzxC-wcaKLM was replaced by wcaJ-wzxC-wcaKLM::cat-sacB) was obtained.

[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 was replaced with ΔwcaJ-wzxC-wcaKLM). Furthermore, a transformant exhibiting ampicillin sensitivity (a transformant in which pKD46 had been lost) was obtained. This transformant was designated W3110S3GKΔlacZYΔwcaJM.

[0138] <Creation of microorganisms with enhanced expression of β1,3-galactosyltransferase and β1,3-N-acetylglucosaminetransferase> Escherichia coli carrying a plasmid for expressing the genes was constructed using the following method: a gene encoding β1,3-galactosyltransferase (hereinafter referred to as Cvβ3GalT) derived from Chromobacterium violaceum ATCC553 strain, consisting of the amino acid sequence shown in SEQ ID NO: 34; a gene encoding β1,3-N-acetylglucosamine transferase (hereinafter referred to as NpLgtA) derived from Neisseria polysaccharea ATCC43768, shown in SEQ ID NO: 36; and the lacY gene derived from the W3110 strain, all located under the uspA promoter.

[0139] PCR was performed 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] [Table 3]

[0141] The DNA represented by SEQ ID NO: 33 is a codon-optimized DNA for expression in Escherichia coli of the nucleotide sequence of the gene encoding β1,3-galactosyltransferase Cvβ3GalT derived from the Chromobacterium violaceum ATCC553 strain, which is 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 codon-optimized DNA for expression in Escherichia coli of 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, 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 complementary sequences at their 5' ends.

[0143] PCR was performed using a mixture of the Cvβ3galT fragment, NplgtA fragment, and lacY fragment in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 51 and 56 as a primer set to obtain a DNA fragment in which the three fragments were linked (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 the 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 complementary sequences at their 5' ends.

[0145] The Cvβ3galT-NplgtA-lacY fragment and vector fragment obtained above were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pUAKQE-Cvβ3galT-NplgtA-lacY. The W3110S3GKΔlacZYΔwcaJM strain constructed above was transformed with the expression plasmid pUAKQE-Cvβ3galT-NplgtA-lacY to construct an E. coli strain carrying pUAKQE-Cvβ3galT-NplgtA-lacY, which was designated the NNN strain.

[0146] (2) Creation of microorganisms with 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, including rcsA derived from the W3110 strain, placed under the lac promoter, was constructed in the following manner.

[0147] <Construction of expression vector> PCR was performed using a W3110 strain prepared by standard methods as a template and DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 59 and 60 as a primer set to obtain an rcsA fragment. PCR was performed using plasmid pSTV29 (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. 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 a plasmid for expressing 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 each amplified DNA fragment.

[0150] [Table 4]

[0151] The DNA represented by sequence number 1 is a DNA in which the base sequence of the gene encoding α1,2-fucosyltransferase GsFucT derived from Gramella sp. MAR_2010_147 strain represented by sequence number 2 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

[0152] The DNA represented by sequence number 3 is a DNA in which the base sequence of the gene encoding α1,2-fucosyltransferase FsFucT derived from Francisella sp. strain FSC1006 represented by sequence number 4 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

[0153] The DNA represented by sequence number 5 is a DNA in which the base sequence of the gene encoding α1,2-fucosyltransferase NbFucT1 derived from Neisseriaceae bacterium DSM 100970 strain represented by sequence number 6 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

[0154] The DNA represented by sequence number 7 is a DNA in which the base sequence of the gene encoding the α1,2-fucosyltransferase MtFucT derived from the Methylobacter tundripaludum strain represented by sequence number 8 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

[0155] The DNA represented by sequence number 9 is a DNA in which the base sequence of the gene encoding the α1,2-fucosyltransferase AjFucT derived from the Amphritea japonica strain represented by sequence number 10 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

[0156] The DNA represented by sequence number 11 is a DNA in which the base sequence of the gene encoding the α1,2-fucosyltransferase PaFucT derived from the Pseudohalocynthiibacter aestuariivivens strain represented by sequence number 12 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

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

[0158] The DNA represented by sequence number 15 is a DNA in which the base sequence of the gene encoding the α1,2-fucosyltransferase PsFucT derived from Pedobacter sp. CF074 strain represented by sequence number 16 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

[0159] The DNA represented by sequence number 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 sequence number 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 in which the base sequence of the gene encoding the α1,2-fucosyltransferase CMfFucT derived from the Candidatus Methylobacter favarea strain represented by SEQ ID NO: 20 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.

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

[0162] The DNA represented by SEQ ID NO: 23 is a DNA in which the base sequence of the gene encoding α1,2-fucosyltransferase WbiQ derived from Escherichia coli O127 strain represented by SEQ ID NO: 24 has been codon-optimized 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] PCR was carried out using the expression vector pSTV29-rcsA constructed above as a template and 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 the 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 E. 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 Escherichia coli strains containing various plasmids. These strains 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 α1,2-fucosyltransferase, which is 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] [Table 5]

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

[0172] The DNA represented by sequence number 29 is a codon-optimized DNA for expression in Escherichia coli of the base sequence of the gene encoding α1,2-fucosyltransferase Te2FT derived from Thermosynechococcus elongatus BP-1 strain represented by sequence number 30, and was prepared by artificial synthesis.

[0173] The DNA shown in SEQ ID NO: 31 is the nucleotide 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 a primer set consisting of DNAs with the nucleotide sequences shown in SEQ ID NOs: 61 and 95 to obtain a vector fragment of approximately 3.5 kb. The nucleotide sequences shown in 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 the In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various known α1,2-fucosyltransferases, 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 named NNN / pFucT54 strain, NNN / pTe2FT strain, and NNN / pFutC strain, respectively.

[0177] [Example 2] Productivity evaluation of LNFPI The LNFPI productivity 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 strains obtained in Example 1(2) above. The NNN / pTe2FT strain and NNN / pFutC strain constructed in the comparative example were used as positive controls. The NNN / pCtrl strain constructed in Example 1(2) was used as a negative control.

[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 and cultured with shaking at 30°C for 15 hours. Subsequently, 0.2 mL of the resulting culture broth was inoculated into a large test tube containing 4 mL of production medium containing 100 mg / L kanamycin and 25 mg / L 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, and manganese sulfate pentahydrate 10 mg / L (all components except glucose, lactose monohydrate, and magnesium sulfate heptahydrate were adjusted to pH 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 mixed)] and cultured with shaking at 30°C for 29 hours.

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

[0180] [Table 6]

[0181] As a result, compared with strains expressing FutC or Te2FT, which are known to be capable of producing LNFPI through α1,2-fucosyltransferases, it was found that 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 Neisseriales bacterium, which showed significantly high LNFPI productivity, were selected as candidates for α1,2-fucosyltransferases useful for LNFPI production.

[0183] [Example 3] Preparation of LNFPI The NNN / pFsFucT and NNN / pNbFucT2 strains 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 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 (Mitsuwa Frontech Co., Ltd.) containing 760 mL of 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 cultured with shaking at 30°C and 800 rpm for 72 hours. The pH during culture was adjusted to 6.9 by adding 14% aqueous ammonia.

[0185] When all the initial glucose was consumed, IPTG was added to a final concentration of 0.5 mM, and in the subsequent culture, 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 culture 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] [Table 7]

[0188] As shown in Table 7, the NNN / pFsFucT and NNN / pNbFucT2 strains produced the fucosylated oligosaccharides 2'FL and LNFPI, suggesting that FsFucT and NbFucT2 may be used to produce these oligosaccharides. Furthermore, the NNN / pFsFucT and NNN / pNbFucT2 strains accumulated significantly more LNFPI in both the supernatant and intracellular cultures than the NNN / pFucT54 strain, a known α1,2-fucosyltransferase-expressing strain. In particular, the NNN / pNbFucT2 strain produced approximately twice as much LNFPI as the NNN / pFucT54 strain. Furthermore, the NNN / pNbFucT2 strain produced significantly less 2'FL, a by-product, than 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 entire contents of which are incorporated by reference. All references cited herein are incorporated in their entirety. [Sequence List Free Text]

[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 FsFucT from Francisella sp. FSC1006 SEQ ID NO: 5: Nucleotide sequence of NbFucT1 derived from Neisseriaceae bacterium DSM 100970 SEQ ID NO: 6: Amino acid sequence of NbFucT1 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 from Methylobacter tundripaludum SEQ ID NO: 9: Nucleotide sequence of AjFucT derived from Amphritea japonica SEQ ID NO: 10: Amino acid sequence of AjFucT from Amphritea japonica SEQ ID NO: 11: Nucleotide sequence of PaFucT derived from Pseudohalocynthiibacter aestuariivivens SEQ ID NO: 12: Amino acid sequence of PaFucT from Pseudohalocynthiibacter aestuariivivens SEQ ID NO: 13: Nucleotide sequence of SbFucT derived from Sterolibacteriaceae bacterium J5B SEQ ID NO: 14: Amino acid sequence of SbFucT 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 PsFucT from Pedobacter sp. CF074 SEQ ID NO: 17: Nucleotide sequence of NbFucT2 derived from Neisseriales bacterium SEQ ID NO: 18: Amino acid sequence of NbFucT2 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 from Candidatus Methylobacter favarea SEQ ID NO: 21: Nucleotide sequence of WbwK derived from Escherichia coli O86 SEQ ID NO: 22: Amino acid sequence of WbwK 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 from Escherichia coli O127 SEQ ID NO: 25: Base 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 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 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 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 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 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 2 SEQ 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 wcaM downstream 1 amplification primer SEQ ID NO: 48: Nucleotide sequence of wcaM downstream 1 amplification primer SEQ ID NO: 49: Nucleotide sequence of primer for amplifying wcaJ upstream 2 SEQ ID NO: 50: Nucleotide sequence of wcaM downstream 2 amplification primer 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 primer for amplifying pUAKQE fragment SEQ ID NO: 59: Nucleotide sequence of primer for amplifying rcsA fragment SEQ ID NO: 60: Nucleotide sequence of primer for amplifying rcsA fragment SEQ ID NO: 61: Nucleotide sequence of primer for amplifying pSTV29 fragment SEQ ID NO: 62: Nucleotide sequence of primer for amplifying pSTV29 fragment SEQ ID NO: 63: Nucleotide sequence of primer for amplifying GsFucT fragment SEQ ID NO: 64: Nucleotide sequence of primer for amplifying GsFucT fragment SEQ ID NO: 65: Nucleotide sequence of primer for amplifying FsFucT fragment SEQ ID NO: 66: Nucleotide sequence of primer for amplifying FsFucT fragment SEQ ID NO: 67: Nucleotide sequence of primer for amplifying NbFucT1 fragment SEQ ID NO: 68: Nucleotide sequence of primer for amplifying NbFucT1 fragment SEQ ID NO: 69: Nucleotide sequence of primer for amplifying MtFucT fragment SEQ ID NO: 70: Nucleotide sequence of primer for amplifying MtFucT fragment SEQ ID NO: 71: Nucleotide sequence of primer for amplifying AjFucT fragment SEQ ID NO: 72: Nucleotide sequence of primer for amplifying AjFucT fragment SEQ ID NO: 73: Nucleotide sequence of primer for amplifying PaFucT fragment SEQ ID NO: 74: Nucleotide sequence of primer for amplifying PaFucT fragment SEQ ID NO: 75: Nucleotide sequence of primer for amplifying SbFucT fragment SEQ ID NO: 76: Nucleotide sequence of primer for amplifying SbFucT fragment SEQ ID NO: 77: Nucleotide sequence of primer for amplifying PsFucT fragment SEQ ID NO: 78: Nucleotide sequence of primer for amplifying PsFucT fragment SEQ ID NO: 79: Nucleotide sequence of primer for amplifying NbFucT2 fragment SEQ ID NO: 80: Nucleotide sequence of primer for amplifying NbFucT2 fragment SEQ ID NO: 81: Nucleotide sequence of primer for amplifying CMfFucT fragment SEQ ID NO: 82: Nucleotide sequence of primer for amplifying CMfFucT fragment SEQ ID NO: 83: Nucleotide sequence of primer for amplifying WbwK fragment SEQ ID NO: 84: Nucleotide sequence of primer for amplifying WbwK fragment SEQ ID NO: 85: Nucleotide sequence of primer for amplifying WbiQ fragment SEQ ID NO: 86: Nucleotide sequence of primer for amplifying WbiQ fragment SEQ ID NO: 87: Base sequence of primer for amplifying HMFT fragment SEQ ID NO: 88: Base sequence of primer for amplifying HMFT fragment SEQ ID NO: 89: Nucleotide sequence of primer for amplifying FucT54 fragment SEQ ID NO: 90: Nucleotide sequence of primer for amplifying FucT54 fragment SEQ ID NO: 91: Base sequence of primer for amplifying Te2FT fragment SEQ ID NO: 92: Base 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 method for producing lacto-N-fucopentaose I (LNFPI), comprising: preparing a transformant by transforming a host cell with recombinant DNA containing DNA encoding a protein described in any one of [1] to [3] below, which has fucosyl group transfer activity to lacto-N-tetraose (LNFPI); and producing lacto-N-fucopentaose I (LNFPI) in a culture using the transformant. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO:

18. [2] A mutant protein having the above amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added, and having α1,2-fucosyltransferase activity. [3] A homologous protein having 90% or more identity with the above amino acid sequence and having α1,2-fucosyltransferase activity.

Images