Method for producing oligosaccharides having Lewis X skeleton

Abstract

The present invention relates to a microorganism having an enhanced activity of any one of the following proteins :[1] a protein composed of an amino acid sequence represented by SEQ ID NO: 2; [2] a mutant protein that is composed of an amino acid sequence in which 1-20 amino acids in the amino acid sequence represented by SEQ ID NO: 2 have been deleted, substituted, inserted or added and that has an α1,3-fucosyltransferase activity; [3] a homologous protein that is composed of an amino acid sequence having 90% or greater identity with the amino acid sequence represented by SEQ ID NO: 2 and that has an α1,3-fucosyltransferase activity; and [4] a protein composed of an amino acid sequence represented by SEQ ID NO: 24, 18 or 26. This microorganism also has improved productivity of an oligosaccharide having a Lewis X skeleton compared with the parent strain.

Description

[Technical Field]

[0001] The present invention relates to a method for producing oligosaccharides having a Lewis X backbone. [Background technology]

[0002] Human oligosaccharides (HMOs) contained in human breast milk have attracted attention as prebiotic materials, and their effects on the development of cognitive function in infants, protection against infection, and improvement of the intestinal environment have been disclosed (Non-Patent Document 1).

[0003] Known oligosaccharides with a Lewis X backbone include lacto-N-fucopentaose III (LNFPIII) and lacto-N-neodifucohexaose II (LNnDFHII). LNFPIII is a pentasaccharide HMO in which fucose is α1,3-linked to the 3-position of N-acetylglucosamine in lacto-N-neotetraose (LNnT).

[0004] LNFPIII is found in high amounts in human breast milk, following lacto-N-fucopentaose I (hereinafter referred to as LNFPI) and lacto-N-fucopentaose II (hereinafter referred to as LNFPII), which are also pentasaccharides and are known as isomers of LNFPIII (Non-Patent Document 2).

[0005] LNFPIII has been suggested to have the ability to activate macrophages and NK cells, and to induce the secretion of cytokines such as IL-10 and TNFα (Non-Patent Document 3), and its functionality has attracted attention.

[0006] Known methods for producing oligosaccharides having a Lewis X backbone, including LNFPIII, include enzymatic reactions using α1,3-fucosyltransferases and microbial fermentation. Examples of α1,3-fucosyltransferases include FucTIII derived from Helicobacter pylori (Non-Patent Document 4) and Bf13FT derived from Bacteroides fragilis (Non-Patent Document 5).

[0007] Furthermore, Non-Patent Document 6 discloses a method for producing LNFPIII by fermentation using Escherichia coli in which α1,3-fucosyltransferase derived from Helicobacter pylori, FutA or FutB, is overexpressed. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Int J Pediatrics(2019)2390240:1-8 [Non-patent document 2] Nutrients(2019)11,1282 [Non-patent document 3] Journal of Functional Foods 72(2020)104074 [Non-patent document 4] ACS Catal.(2019)9,10712-10720 [Non-patent document 5] ACS Catal.(2019)9,12,11794-11800 [Non-patent document 6] Biotechnol.Prog.(2004)20,412-419 [Non-Patent Document 7] Metabolic Engineering 41(2017)23-38 Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, known methods for producing oligosaccharides having a Lewis X backbone include enzymatic reaction methods using α1,3-fucosyltransferase and microbial fermentation methods. However, the known α1,3-fucosyltransferases described in Non-Patent Documents 4, 5, and 6 have low substrate specificity, and therefore, for example, in the production of LNFPIII, there is a problem that lacto-N-fucopentaose VI (hereinafter referred to as LNFPVI) and lacto-N-neodifucohexaose II (hereinafter referred to as LNnDFHII), in which fucose is bound to the 3-position of glucose on the reducing end of LNnT, are produced as by-products.

[0010] Furthermore, the α1,3-fucose transferase derived from Helicobacter pylori described in Non-Patent Document 6 can utilize not only LNnT but also lactose as a substrate, and therefore produces 3-fucosyllactose (hereinafter referred to as 3FL) as a by-product (Non-Patent Document 7), making it unsuitable for producing LNFPIII using lactose as the initial raw material. To produce oligosaccharides with Lewis X backbones such as LNFPIII more efficiently, it is necessary to search for α1,3-fucosyltransferases that can selectively transfer fucose to the N-acetylglucosamine site of substrates such as LNnT.

[0011] Therefore, an object of the present invention is to provide a microorganism that is excellent in productivity of oligosaccharides having a Lewis X backbone, particularly a microorganism that is excellent in productivity of oligosaccharides having an LNFPIII backbone. [Means for solving the problem]

[0012] The present inventors have discovered that microorganisms in which the activity of GDP-fucosyltransferase 1 (hereinafter referred to as FucT1) from Parabacteroides goldsteinii JCM 13446, FucT-D from Gramella sp. BOM4, FucT-A from Parabacteroides sp. BX2, or FucT-E from Lachnospiraceae bacterium NLAE-zl-G231 is enhanced have improved productivity of oligosaccharides having a Lewis X skeleton compared to the parent strains, and have completed the present invention.

[0013] That is, the present invention is as follows. 1. A microorganism in which the activity of a protein according to any one of [1] to [6] below is enhanced and which has improved productivity of oligosaccharides having a Lewis X skeleton compared to the parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2. [2] A mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, and having α1,3-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 and having α1,3-fucosyltransferase activity. [4] A protein consisting of the amino acid sequence represented by SEQ ID NO: 24. [5] A protein consisting of the amino acid sequence represented by SEQ ID NO: 18. [6] A protein consisting of the amino acid sequence represented by SEQ ID NO: 26. 2. The microorganism described in 1 above, wherein the oligosaccharide having a Lewis X backbone is an oligosaccharide having a lacto-N-fucopentaose III (LNFPIII) backbone. 3. The microorganism described in 2 above, wherein the oligosaccharide having an LNFPIII backbone is at least one of LNFPIII and lacto-N-neodifucohexaose II (LNnDFHII). 4. The microorganism described in 1 above, wherein the oligosaccharide having a Lewis X skeleton is an oligosaccharide in which L-fucose is α1,3-linked to the 3-position of at least one N-acetylglucosamine contained in a paralacto-N-neohexaose (Para-LNnH) skeleton. 5. A method for producing oligosaccharides, comprising preparing a microorganism according to any one of 1 to 4 above, and producing oligosaccharides in a culture using the microorganism. [Effects of the Invention]

[0014] The microorganism according to the present invention has enhanced activity of a specific protein, and thereby selectively transfers fucose to the N-acetylglucosamine site of a substrate, thereby reducing the production of by-products other than oligosaccharides having a Lewis X backbone and improving the productivity of oligosaccharides having a Lewis X backbone. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows schematic diagrams of the structures of Lewis X, LNFPIII, and paralacto-N-neohexaose (hereinafter referred to as Para-LNnH). [Figure 2] FIG. 2 shows a schematic diagram of the biosynthetic pathways of LNFPIII, LNnDFHII, etc. in a microorganism according to one embodiment of the present invention. [Figure 3] FIG. 3 shows examples of oligosaccharides in which L-fucose is α1,3-linked to the 3-position of at least one N-acetylglucosamine contained in the Para-LNnH backbone. [Figure 4] FIG. 4 shows an alignment of the amino acid sequences represented by SEQ ID NOs: 18, 20, 22, 24, and 26, which are encoded by the PgsFucT1 homolog genes represented by SEQ ID NOs: 17, 19, 21, 23, and 25, respectively. [Figure 5] FIG. 5 shows a schematic diagram of the biosynthetic pathways of 3FL and LewisX. [Figure 6]FIG. 6 shows an alignment of the amino acid sequences encoding PgsFucT1, BfFucT1, and HpFutA, which are represented by SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 98, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1. Microorganisms with improved productivity of oligosaccharides containing Lewis X skeletons Examples of the microorganisms of the present invention that have improved productivity of oligosaccharides having a Lewis X skeleton include the following microorganisms. A microorganism in which the activity of the protein according to any one of [1] to [6] below is enhanced and the productivity of oligosaccharides having a Lewis X skeleton is improved compared to that of a parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2. [2] A mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, and having α1,3-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 and having α1,3-fucosyltransferase activity. [4] A protein consisting of the amino acid sequence represented by SEQ ID NO: 24. [5] A protein consisting of the amino acid sequence represented by SEQ ID NO: 18. [6] A protein consisting of the amino acid sequence represented by SEQ ID NO: 26.

[0017] As used herein, the oligosaccharide having a Lewis X backbone may be any oligosaccharide as long as it has a Lewis X backbone consisting of Gal-β1,4(Fuc-α1,3)GlcNAc as shown in Figure 1. However, an oligosaccharide having an LNFPIII backbone or an oligosaccharide having L-fucose linked via an α1,3-linkage to the 3-position of at least one N-acetylglucosamine contained in a Para-LNnH backbone is preferred.

[0018] As used herein, the oligosaccharide having an LNFPIII backbone may be any oligosaccharide having an LNFPIII backbone, but is preferably LNFPIII or LNnDFHII, and more preferably LNFPIII. A schematic diagram of the structure of LNFPIII is shown in Figure 1. Figure 2 shows a schematic diagram of the biosynthetic pathway of LNFPIII, LNnDFHII, etc. in a microorganism according to one embodiment of the present invention.

[0019] As used herein, an oligosaccharide in which L-fucose is α1,3-linked to the 3-position of at least one N-acetylglucosamine in the Para-LNnH backbone includes an oligosaccharide having the structure shown in Figure 3. A schematic diagram of the structure of Para-LNnH is shown in Figure 1.

[0020] 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.

[0021] 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 1 to 20, preferably 1 to 10, more preferably 1 to 8, and most preferably 1 to 5.

[0022] 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.

[0023] 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

[0024] In the mutant protein of [2] above, an example of the amino acid residue to be substituted is the 17th asparagine residue.

[0025] 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.

[0026] Examples of homologous proteins include amino acid sequences that have an identity of 90% or more, preferably 93% or more, more preferably 95% or more, and particularly preferably 97% or more with the amino acid sequence of the target protein.

[0027] 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.

[0028] As used herein, α1,3-fucosyltransferase activity refers to the activity of transferring fucose from the donor substrate GDP-fucose to the 3-hydroxyl group of N-acetylglucosamine of the acceptor substrate carbohydrate (hereinafter referred to as "acceptor carbohydrate") via an α1,3-bond to produce a fucose-containing carbohydrate.

[0029] Acceptor carbohydrates include compounds having an N-acetyllactosamine (hereinafter referred to as LacNAc) skeleton, such as LNnT, LNFPVI, para-LNnH, or combinations thereof, and sugar chains containing these as partial structures. Among these, LNnT is preferred.

[0030] As used herein, the term "acceptor substrate" refers to a substance or combination of substances on which α1,3-fucosyltransferase can act to generate oligosaccharides having a Lewis X backbone.

[0031] As used herein, the term "parent strain" refers to the original strain that is the target of genetic modification, transformation, etc. In particular, the parent strain of the microorganism of the present invention with improved productivity of oligosaccharides having a Lewis X backbone refers to the strain prior to genetic modification, transformation, etc. that enhances the activity of the protein described in any one of [1] to [6].

[0032] 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.

[0033] The parent strain may be a wild-type strain as long as it is a microorganism that produces GDP-fucose and / or an acceptor carbohydrate. If the wild-type strain does not have the ability to produce GDP-fucose and / or an acceptor carbohydrate, the parent strain may be a bred strain that has been artificially imparted with the ability to supply GDP-fucose and / or an acceptor carbohydrate.

[0034] 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 α1,3-fucosyltransferase. The parent strain is preferably a microorganism to which the ability to supply GDP-fucose, a reaction substrate for α1,3-fucosyltransferase, has been artificially imparted or enhanced. Specific examples of methods for imparting or enhancing the ability to 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).

[0035] The ability to produce GDP-fucose includes the ability to produce GDP-fucose from sugar. Methods for artificially imparting or enhancing the ability to supply 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 2) A microorganism used as a parent strain that has been artificially conferred or enhanced with the ability to supply an acceptor carbohydrate, which is a reaction substrate for α1,3-fucosyltransferase. Methods for artificially imparting the ability to supply acceptor carbohydrates 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 that produces an acceptor carbohydrate from a sugar. (2b) A method for enhancing expression of at least one enzyme involved in a biosynthetic pathway that produces an acceptor carbohydrate from a sugar. (2c) A method for increasing the copy number of at least one enzyme gene involved in a biosynthetic pathway that produces an acceptor carbohydrate from a sugar. (2d) A method for alleviating or eliminating at least one of the mechanisms for decomposing an acceptor carbohydrate or its substrate sugar. (2e) A method for enhancing the expression of at least one enzyme involved in the cellular uptake of an acceptor carbohydrate 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 an acceptor carbohydrate or its substrate sugar. (2g) A method for weakening or blocking at least one metabolic pathway branching off from a biosynthetic pathway that produces an acceptor carbohydrate from a sugar to a metabolic product other than the target substance. (2h) A method for selecting cell lines that are more resistant to receptor carbohydrate analogs than wild-type cells

[0040] Specific examples of enzymes involved in the biosynthetic pathway that produces acceptor carbohydrates 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 LNnT from glucose and lactose.

[0041] Specific examples of mechanisms for decomposing acceptor carbohydrates or their substrate sugars include known enzymes such as β-galactosidase, which catalyzes the hydrolysis of lactose, a substrate of LNnT, to produce glucose and galactose. Specifically, β-galactosidase (hereinafter referred to as lacZ), which hydrolyzes lactose, a substrate of LNTII, can be mentioned. Loss of lacZ activity can suppress the decrease in lactose supply.

[0042] Specific examples of enzymes involved in the cellular uptake of acceptor carbohydrates or sugars that serve as their substrates include known enzymes such as lactose permease, which is involved in the cellular uptake of lactose, a substrate for LNnT.

[0043] Specifically, the microorganism to which the ability to supply an acceptor carbohydrate has been imparted or enhanced has lactose permease (hereinafter referred to as lacY) activity, β1,4-galactosyltransferase (hereinafter referred to as galT) activity, β1,3-N-acetylglucosamine transferase (hereinafter referred to as 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 transaminase (hereinafter referred to as glmS) activity, in order to supply LNnT. It is preferable that the enzyme has at least one activity selected from the group consisting of thiamin-1-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.

[0044] 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.

[0045] lacY is a membrane protein that imports lactose, the acceptor carbohydrate substrate, into the cell. galT is an enzyme involved in the production of LNnT from lacto-N-triose II (LNTII). LNnT is a precursor of LNFPIII. 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 a precursor of LNnT.

[0046] 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.

[0047] Whether a microorganism is capable of producing an acceptor carbohydrate and / or GDP-fucose can be confirmed by culturing the microorganism in a medium and detecting the acceptor carbohydrate and / or GDP-fucose accumulated in the culture using a general technique such as a sugar analyzer or a high-performance liquid chromatograph mass spectrometer described below.

[0048] 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 an acceptor carbohydrate, which are reaction substrates for α1,3-fucosyltransferase, has been artificially imparted or enhanced. Therefore, 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: 101), the nucleotide sequence encoding lacY (accession number BAE76125.1), the nucleotide sequence encoding galT (SEQ ID NO: 102), the nucleotide sequence encoding lacY (accession number BAE76125.1), the nucleotide sequence encoding galT (SEQ ID NO: 103), the nucleotide sequence encoding lacY (accession number BAE76125.1), the nucleotide sequence encoding galT (SEQ ID NO: 104), the nucleotide sequence encoding lacY (accession number BAE76125.1), the nucleotide sequence encoding galT (SEQ ID NO: 105), the nucleotide sequence encoding lacY (accession number BAE76125.1), the nucleotide sequence encoding galT (SEQ ID NO: 106), the nucleotide sequence encoding lacY (accession number 29), a nucleotide sequence encoding LgtA (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).

[0049] 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 an acceptor substrate compared to a non-genetically modified parent strain.

[0050] 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).

[0051] Furthermore, as described above, it is preferable that the parent strain has reduced or no lacZ activity and / or colanic acid synthesis activity.

[0052] 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 gene, which is a base sequence encoding a colanic acid production-related protein.

[0053] In one embodiment of the present invention, the genetically modified microorganism preferably has an increased ability to produce an acceptor carbohydrate and / or GDP-fucose compared to a parent strain that has not been genetically modified.

[0054] 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).

[0055] Examples of microorganisms in which the activity of the protein described in any one of [1] to [6] 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.

[0056] 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 [6] 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 [6] above, and microorganisms in which the gene is carried outside of chromosomal DNA as plasmid DNA.

[0057] The DNA encoding the protein described in any one of [1] to [6] above may be any DNA encoding a protein having the activity of the protein described in any one of [1] to [6] above, and specifically includes one DNA selected from the group consisting of [7] to

[10] below. [7] A DNA encoding the protein according to any one of [1] to [6] above. [8] DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, 23, 17, or 25 [9] 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, 23, 17, or 25, and encodes a homologous protein having α1,3-fucosyltransferase activity.

[10] 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 with the nucleotide sequence represented by SEQ ID NO: 1, 23, 17, or 25, and encoding a homologous protein having α1,3-fucosyltransferase activity.

[0058] In the above [9], "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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] DNA that can hybridize under the above-mentioned stringent conditions includes 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, 23, 17, or 25, when calculated based on the above-mentioned parameters using, for example, BLAST or FASTA.

[0064] The DNA encoding the protein of [1], [4], [5], or [6] above, and the DNA of [8] above can be obtained, for example, by Southern hybridization of a chromosomal DNA library of a microorganism, preferably a microorganism belonging to the genus Escherichia coli, more preferably the Escherichia coli W3110 strain, using probe DNA that can be designed based on the nucleotide sequence shown in SEQ ID NO: 1 (FucT1 gene), the gene shown in SEQ ID NO: 23 (FucT-D gene), the gene shown in SEQ ID NO: 17 (FucT-A gene), or the nucleotide sequence shown in SEQ ID NO: 25 (FucT-E gene); or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the nucleotide sequences and the chromosomal DNA of the above microorganism as a template.

[0065] The Escherichia coli W3110 strain is available from the National Institute of Technology and Evaluation (NITE) Biological Resource Center.

[0066] The DNA encoding the mutant protein of [2] above can be obtained, for example, by subjecting the DNA consisting of the base sequence shown in SEQ ID NO: 1, 23, 17 or 25 as a template to error-prone PCR or the like.

[0067] Alternatively, the DNA described in [2] above can be obtained by PCR [Gene, 77, 51 (1989)] 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).

[0068] 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.

[0069] 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.

[0070] The DNA encoding the homologous protein of [3] above, and the DNAs of [9] and

[10] above can be obtained, for example, by searching various gene sequence databases for a base sequence that has 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 1, 23, 17 or 25, or by searching various protein sequence databases for an amino acid sequence that has 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 2, 24, 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 containing the DNA, in a manner similar to the method for obtaining the above DNA.

[0071] The obtained DNA described in any one of [7] to

[10] 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 sequence can be determined by a conventional base sequence analysis method, such as the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)], or by analysis using 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).

[0072] The host cells that can be used for determining the DNA sequence may be any cells that can be grown by introducing the vector, and examples thereof 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, 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, Escherichia coli NM522, etc.

[0073] 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).

[0074] 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)].

[0075] 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.

[0076] 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.

[0077] The recombinant DNA containing the DNA encoding the protein according to any one of [1] to [6] 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.

[0078] When the recombinant DNA is capable of being integrated into a chromosome, it does not need to contain a promoter.

[0079] 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 [6] above, can be obtained by the following method.

[0080] Based on the DNA encoding any one of the proteins described in [1] to [6] 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.

[0081] 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.

[0082] 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 [7] to

[10] above, and a transcription termination sequence. A gene that controls the promoter may also be included.

[0083] 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.

[0084] Furthermore, by substituting bases in the nucleotide sequence encoding a protein having α1,3-fucosyltransferase activity so that the codons are optimal for expression in the host, the expression level of the protein having α1,3-fucosyltransferase activity can be improved. Examples of proteins having α1,3-fucosyltransferase activity include the proteins described in any one of [1] to [6] above. Information on codon usage in the parent strain used in the present invention is available from public databases.

[0085] 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.

[0086] 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 such 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)].

[0087] 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.

[0088] 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)).

[0089] 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)].

[0090] When a yeast strain is used as the parent strain, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0091] 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.

[0092] 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 [7] to

[10] above downstream of the promoter of an appropriate expression vector.

[0093] 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)].

[0094] 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)].

[0095] 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)].

[0096] 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.

[0097] The microorganisms constructed by the above methods have enhanced activity of the protein described in any one of [1] to [6] above and improved productivity of oligosaccharides having a Lewis X backbone compared to the parent strain. This can be confirmed by culturing the microorganism, appropriately diluting the culture medium, centrifuging it, and analyzing the oligosaccharides having a Lewis X backbone contained in the supernatant or the cells using a sugar analyzer or high-performance liquid chromatograph mass spectrometer described below, and comparing them with those of the parent strain. In this specification, the term "productivity of oligosaccharides having a Lewis X backbone" refers to the ability of the microorganism to accumulate oligosaccharides having a Lewis X backbone produced by the microorganism intracellularly and / or extracellularly.

[0098] The above-mentioned microorganisms have enhanced activity of the protein described in any one of [1] to [6] above compared to the parent strain, and thus can selectively transfer fucose to the N-acetylglucosamine site of a substrate, thereby improving productivity of oligosaccharides having a Lewis X backbone. Examples of such microorganisms include the TROS / pPgsFucT1 or FUC / pPgsFucT1 strains in which expression of the FucT1 gene is enhanced, the FUC / pFucT-D strain in which expression of the FucT-D gene is enhanced, the FUC / pFucT-A strain in which expression of the FucT-A gene is enhanced, and the FUC / pFucT-E strain in which expression of the FucT-E gene is enhanced, as described below in the Examples.

[0099] Examples of such microorganisms include microorganisms in which the activity of the FucT1 protein, FucT-D protein, FucT-A protein, or FucT-E protein is enhanced, and the activity of α1,3-fucose transferase, which can selectively transfer fucose to the N-acetylglucosamine site, is enhanced, thereby improving the productivity of oligosaccharides having a Lewis X backbone.

[0100] 2. Oligosaccharide manufacturing method Examples of the method for producing oligosaccharides of the present invention (hereinafter also referred to as the method of the present invention) include the following methods. A method for producing oligosaccharides, comprising preparing the microorganism described above in 1. and producing oligosaccharides in a culture using the microorganism.

[0101] In the method of the present invention, the desired oligosaccharide is preferably an oligosaccharide having a Lewis X backbone, more preferably an oligosaccharide having an LNFPIII backbone or an oligosaccharide having an L-fucose α1,3-linked to the 3-position of at least one N-acetylglucosamine contained in a Para-LNnH backbone.

[0102] In the method of the present invention, when the desired oligosaccharide is an oligosaccharide having an LNFPIII backbone, the oligosaccharide is preferably at least one of LNFPIII and LNnDFHII.

[0103] In the method of the present invention, when the desired oligosaccharide is an oligosaccharide in which L-fucose is α1,3-linked to the 3-position of at least one N-acetylglucosamine contained in the Para-LNnH backbone, examples of the oligosaccharide include those having the structure shown in Figure 3.

[0104] The method for culturing the microorganisms described above in 1. can be carried out according to a conventional method used for culturing microorganisms.

[0105] The medium for culturing the microorganism may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the microorganism and allows the transformant to be cultured efficiently.

[0106] 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.

[0107] 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.

[0108] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0109] The microorganism of the present invention used in the method for producing oligosaccharides may be a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like.

[0110] In the method for producing oligosaccharides, glucose, lactose, lactose monohydrate, or the like may be added to the medium during cultivation.

[0111] When the microorganism of the present invention used in the method for producing oligosaccharides does not have the ability to produce GDP-fucose and / or an acceptor carbohydrate, which are substrates for oligosaccharides having a Lewis X backbone, GDP-fucose and / or an acceptor carbohydrate may be added to the medium.

[0112] Furthermore, in the method for producing oligosaccharides, instead of adding glucose, lactose, lactose monohydrate, or an acceptor carbohydrate to the medium during cultivation, glucose, lactose, lactose monohydrate, or an acceptor carbohydrate may be supplied to the microorganism of the present invention by simultaneously culturing a microorganism capable of producing glucose, lactose, lactose monohydrate, or an acceptor carbohydrate from sugar with the microorganism of the present invention.

[0113] In the method for producing oligosaccharides, it is preferable that β-galactosidase and WcaJ are not present in the medium.

[0114] 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.

[0115] By the above-mentioned cultivation, oligosaccharides can be produced in the culture, thereby producing oligosaccharides.

[0116] Typically, oligosaccharides can be collected from the supernatant after centrifugation of the culture. When oligosaccharides accumulate within the cells, the cells can be disrupted by ultrasonication or the like, and then centrifuged to remove the cells. The resulting supernatant can then be used to collect the oligosaccharides by ion exchange resin methods or the like.

[0117] Furthermore, the desired oligosaccharide can be produced by adding other sugars to the oligosaccharides in the culture or the collected oligosaccharides.

[0118] [Analysis example] (1) Analysis and quantification of LNFPIII, 3FL, LNFPVI or lactose In the Examples, analysis and quantification of LNFPIII, 3FL, LNFPVI, 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 supernatant water phase was used as the intracellular fraction. The LNFPIII, 3FL, LNFPVI, or lactose contained in the supernatant and / or intracellular fraction was analyzed using a sugar analyzer, ICS-5000 (Thermo Fisher Scientific).

[0119] [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 LewisX or LNnDFHII In the Examples, analysis and quantification of LewisX or LNnDFHII were carried out according to the following procedures. As in (1) above, supernatants and / or intracellular fractions were prepared from the culture broth containing the microorganisms after cultivation. LewisX or LNnDFHII contained in the supernatants and / or intracellular fractions was analyzed using a UFLC&LCMS-8040 (Shimadzu Corporation).

[0121] [Analysis conditions] Column: Coregel 87H3 (7.8 x 300 mm) Column temperature: 40℃ Mobile phase: 0.1% formic acid in water Gradient conditions: Isocratic elution Analysis time: 25 minutes Flow rate: 0.4mL / min Injection volume: 10μL Detection: SIM mode [Example]

[0122] Examples of the invention are shown below, but the present invention is not limited to these examples. [Example 1] Isolation of α1,3-fucosyltransferase useful for LNFPIII production Using the productivity of 3FL or LewisX as an indicator, we screened for α1,3-fucosyltransferases that exhibit high substrate specificity for N-acetyllactosamine.

[0123] (1) Construction of host strains for evaluation <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: 33 and 34 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.

[0124] <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.

[0125] Using genomic DNA of Escherichia coli W3110 strain prepared by a conventional method as a template, PCR was performed using DNA primer sets consisting of the base sequences shown in "Primer Set" in Table 1 to amplify each DNA fragment.

[0126] [Table 1]

[0127] 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.

[0128] PCR was performed using a mixture of equimolar amounts of the lacZ upstream 1, lacY downstream 1, and cat-sacB fragments as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 36 and 38 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.

[0129] 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: 36 and 38 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 lacZ and downstream lacY are directly linked.

[0130] The lacZY::cat-sacB fragment was introduced by electroporation into the W3110 strain 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, to obtain transformants that were chloramphenicol resistant and sucrose sensitive (transformants in which the lacZY gene had been replaced with lacZY::cat-sacB).

[0131] 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 W3110ΔlacZY.

[0132] 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.

[0133] [Table 2]

[0134] 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.

[0135] 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: 42 and 44 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).

[0136] 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: 42 and 44 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.

[0137] The wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110ΔlacZY strain constructed above by electroporation, and a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (transformant in which wcaJ-wzxC-wcaKLM was replaced by wcaJ-wzxC-wcaKLM::cat-sacB) was obtained.

[0138] The ΔwcaJ-wzxC-wcaKLM fragment was introduced into the transformant by electroporation to obtain a transformant that exhibited chloramphenicol sensitivity and sucrose resistance (transformant in which wcaJ-wzxC-wcaKLM::cat-sacB was replaced by ΔyhbJ). Furthermore, a transformant exhibiting ampicillin sensitivity (transformant in which pKD46 had been lost) was obtained. This transformant was designated W3110ΔlacZYΔwcaJM.

[0139] <Creation of microorganisms with enhanced expression of transporter genes> Drug H derived from the W3110 strain consisting of the amino acid sequence represented by SEQ ID NO: 28 + Escherichia coli carrying a plasmid for expressing a gene encoding a transporter gene belonging to the antiporter-1 family (hereinafter referred to as MdfA) was constructed by the following method.

[0140] PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 47 and 48 as a primer set and genomic DNA of the W3110 strain prepared by standard methods as a template to obtain an mdfA fragment. PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 49 and 50 as a primer set and plasmid pMW118 (Nippon Gene Co., Ltd.) as a template to obtain a vector fragment of approximately 4.1 kb. The nucleotide sequences represented by SEQ ID NOs: 47 and 49 and SEQ ID NOs: 48 and 50 each contain complementary sequences at their 5' ends.

[0141] The mdfA fragment and vector fragment obtained above were ligated using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the MdfA expression plasmid pMW118_mdfA. The W3110ΔlacZYΔwcaJM strain constructed in Example 1(1) was transformed with the expression plasmid pMW118_mdfA to construct an E. coli strain harboring pMW118_mdfA, which was designated the FUC strain.

[0142] (2) Creation of microorganisms with α1,3-fucosyltransferase activity Escherichia coli carrying a plasmid for expressing a gene encoding various α1,3-fucosyltransferases placed under the uspA promoter was constructed by the following method.

[0143] <Construction of expression vector> PCR was performed using genomic DNA of the 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 3, to obtain amplified DNA fragments.

[0144] [Table 3]

[0145] PCR was performed using a mixture of the rcsA fragment and the lacY fragment in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 53 and 56 as a primer set to obtain a DNA fragment (hereinafter referred to as rcsA-lacY) in which the two fragments were linked.

[0146] PCR was performed using a primer set consisting of DNAs with the nucleotide sequences represented by SEQ ID NOs: 51 and 52 and the plasmid pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385) as a template to obtain a vector fragment of approximately 4.7 kb.

[0147] The base sequences represented by SEQ ID NOs: 51 and 53, and SEQ ID NOs: 52 and 56 each contain a complementary sequence at the 5' end.

[0148] The rcsA-lacY fragment and the vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pUAKQE-rcsA-lacY.

[0149] <Construction of a plasmid for expressing α1,3-fucosyltransferase> PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Table 4 as a primer set and DNAs shown in "Template" in Table 4 as templates to obtain each amplified DNA fragment.

[0150] [Table 4]

[0151] Various genomic DNAs were prepared by standard methods. The DNA represented by SEQ ID NO: 3 is the nucleotide sequence of the gene encoding the α1,3-fucosyltransferase derived from Helicobacter pylori strain 26695 represented by SEQ ID NO: 4, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 15 is the DNA obtained by optimizing the nucleotide sequence of the gene encoding the α1,3-fucosyltransferase derived from Mediterranea sp. strain An20 represented by SEQ ID NO: 16 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0152] PCR was performed using the expression vector pUAKQE-rcsA-lacY constructed by the above-mentioned method as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 51 and 57 as a primer set to obtain a vector fragment of approximately 6.7 kb.

[0153] The base sequences represented by SEQ ID NOs: 51, 58, 60, 62, 64, 66, 68, 70 and 72, and SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, 71 and 73 each contain a complementary sequence at the 5' end.

[0154] The various 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 α1,3-fucosyltransferases: pHpFutA, pBnFucT, pBsFucT, pPgsFucT1, pPgsFucT2, pBfFucT1, pBfFucT2, and pMFucT.

[0155] <Construction of E. coli carrying a plasmid for expressing α1,3-fucosyltransferase> The FUC strain constructed in Example 1(1) was transformed with the α1,3-fucosyltransferase expression plasmids pHpFutA, pBnFucT, pBsFucT, pPgsFucT1, pPgsFucT2, pBfFucT1, pBfFucT2, and pMFucT obtained above, as well as pUAKQE-rcsA-lacY as a vector control, to construct E. coli strains carrying various plasmids. These strains were designated FUC / pHpFutA strain, FUC / pBnFucT strain, FUC / pBsFucT strain, FUC / pPgsFucT1 strain, FUC / pPgsFucT2 strain, FUC / pBfFucT1 strain, FUC / pBfFucT2 strain, FUC / pMFucT strain, and FUC / Ctrl strain, respectively.

[0156] (3) Productivity evaluation of 3FL or LewisX The FUC / pHpFutA, FUC / pBnFucT, FUC / pBsFucT, FUC / pPgsFucT1, FUC / pPgsFucT2, FUC / pBfFucT1, FUC / pBfFucT2, FUC / pMFucT, and FUC / Ctrl strains obtained in (2) above were evaluated for productivity of 3FL and LewisX. The biosynthetic pathways of 3FL and LewisX are shown in Figure 5.

[0157] Each strain was cultured on an LB plate containing 100 mg / L kanamycin and 100 mg / L ampicillin at 37°C for 17 hours, then inoculated into a 14 mL plastic tube containing 2 mL of LB medium containing 100 mg / L kanamycin and 100 mg / L ampicillin, and cultured with shaking at 30°C for 15 hours. The resulting culture solution was then diluted to a production medium containing 100 mg / L of kanamycin and 100 mg / L of ampicillin [glucose 30 g / L, lactose monohydrate or N-acetyllactosamine 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 (pH adjusted with sodium hydroxide solution for ingredients other than glucose, lactose monohydrate, N-acetyllactosamine, and magnesium sulfate heptahydrate). 0.2 mL of the bacteria was inoculated into a large test tube containing 4 mL of the above solution (adjusted to a temperature of 7.2 and then autoclaved) (aqueous solutions containing glucose, lactose monohydrate, N-acetyllactosamine, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and mixed), and cultured with shaking at 30°C for 29 hours. 6.5 hours after the start of culture, IPTG was added to a final concentration of 1 mM.

[0158] When evaluating the productivity of 3FL, a production medium containing lactose monohydrate was used, and when evaluating the productivity of LewisX, a production medium containing 10 g / L of N-acetyllactosamine was used.

[0159] After the culture was completed, the culture solution was centrifuged and diluted appropriately, and the 3FL or LewisX contained in the supernatant was analyzed using a sugar analyzer ICS-5000 or UFLC&LCMS-8040. The results are shown in Table 5.

[0160] [Table 5]

[0161] Most strains were able to produce either 3FL or LewisX or neither, but the FUC / pPgsFucT1 strain was the only one that produced significant amounts of LewisX but no 3FL at all, suggesting that the α1,3-fucosyltransferase PgsFucT1 from Parabacteroides goldsteinii exhibits high substrate specificity for N-acetyllactosamine and cannot utilize lactose as a substrate.

[0162] Based on the above results, PgsFucT1 was selected as the α1,3-fucosyltransferase for producing LNFPIII and subjected to the following tests.

[0163] [Example 2] Evaluation of PgsFucT1 homologue genes The PgsFucT1 homologous genes selected in Example 1 were evaluated for their usefulness in producing LNFPIII.

[0164] (1) Construction of a microorganism carrying a PgsFucT1 homologue gene Escherichia coli carrying PgsFucT1 homolog genes was constructed by the following method. The five genes shown in SEQ ID NOs: 17 to 26 were used as PgsFucT1 homolog genes. An alignment of the amino acid sequences encoded by each homolog gene is shown in FIG. 4. PCR was performed using the DNA listed in "Template" in Table 6 as a template and DNA consisting of the base sequences listed in "Primer set" in Table 6 as a primer set, and each DNA fragment was amplified.

[0165] [Table 6]

[0166] The DNA represented by SEQ ID NO: 17 is the nucleotide sequence of the gene encoding the α1,3-fucosyltransferase derived from Parabacteroides sp. BX2 strain represented by SEQ ID NO: 18 and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 19 is the nucleotide sequence of the gene encoding the α1,3-fucosyltransferase derived from Parabacteroides sp. HGS0025 strain represented by SEQ ID NO: 20 and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 21 is the nucleotide sequence of the gene encoding the α1,3-fucosyltransferase derived from Parabacteroides bouchesdurhonensis strain Marseille-P3763 represented by SEQ ID NO: 22 and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 23 is the nucleotide sequence of the gene encoding the α1,3-fucosyltransferase derived from Gramella sp. BOM4 strain represented by SEQ ID NO: 24 and was prepared by artificial synthesis. The DNA shown in SEQ ID NO: 25 is the base sequence of the gene encoding the α1,3-fucosyltransferase derived from Lachnospiraceae bacterium strain NLAE-zl-G231 shown in SEQ ID NO: 26, and was prepared by artificial synthesis.

[0167] In this case, the base sequences represented by SEQ ID NOs: 51, 74, 76, 78, 80 and 82, and SEQ ID NOs: 57, 75, 77, 79, 81 and 83 each contain a complementary sequence at the 5' end.

[0168] The various amplified DNA fragments obtained above were ligated to the pUAKQE-rcsA-lacY vector fragment prepared in Example 1 using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various homologous genes, pFucT-A, pFucT-B, pFucT-C, pFucT-D, and pFucT-E.

[0169] The FUC strain constructed in Example 1(1) was transformed with the five plasmids obtained above to construct Escherichia coli strains carrying various plasmids, which were named FUC / pFucT-A strain, FUC / pFucT-B strain, FUC / pFucT-C strain, FUC / pFucT-D strain, and FUC / pFucT-E strain, respectively.

[0170] (2) Productivity evaluation of 3FL or LewisX The FUC / pFucT-A, FUC / pFucT-B, FUC / pFucT-C, FUC / pFucT-D, and FUC / pFucT-E strains obtained in (1) above were evaluated for productivity of 3FL or LewisX. The FUC / pPgsFucT1 and FUC / Ctrl strains constructed in Example 1 (2) were used as controls.

[0171] The culture method and conditions were as described in Example 1 (3). After the culture was completed, the culture solution was centrifuged and diluted appropriately, and the 3FL or LewisX contained in the supernatant was analyzed using a sugar analyzer ICS-5000 or UFLC&LCMS-8040. The results are shown in Table 7.

[0172] [Table 7]

[0173] As a result, although the strains containing each homolog gene had lower LewisX productivity than PgsFucT1, none of the strains showed 3FL productivity, suggesting that the proteins encoded by each homolog gene cannot utilize lactose as a substrate, just like PgsFucT1.

[0174] [Example 3] Identification of effective mutation points The crystal structure of a known Helicobacter pylori-derived α1,3-fucosyltransferase has been disclosed, and the amino acid residues involved in interaction with N-acetyllactosamine have been predicted (J. Biol. Chem. 2007, 282, 9973-9982). Based on this, the amino acid residues interacting with N-acetyllactosamine were identified by alignment of PgsFucT1 and BfFucT1, which showed high LewisX productivity in Example 1. The identified amino acid residues were mutually substituted with the corresponding amino acid residues in PgsFucT1 and BfFucT1, and the effectiveness of these sequences in LNFPIII production was evaluated.

[0175] (1) Construction of microorganisms containing mutant PgsFucT1 and mutant BfFucT1 E. coli carrying mutant PgsFucT1 and mutant BfFucT1 were constructed by the following method. An alignment of the amino acid sequences encoding PgsFucT1, BfFucT, and Helicobacter pylori-derived α1,3-fucosyltransferase is shown in FIG.

[0176] PCR was performed using the DNA listed in the "Template" column of Table 8 as a template and DNA consisting of the base sequences listed in the "Primer Set" column of Table 8 as a primer set, and each DNA fragment was amplified.

[0177] [Table 8]

[0178] PCR was performed using an equimolar mixture of the PgsFucT1 N17D upstream and PgsFucT1 N17D downstream fragments as a template and DNA consisting of the base sequences shown in SEQ ID NOs: 64 and 65 as a primer set to obtain the PgsFucT1 N17D fragment in which the 17th asparagine in the amino acid sequence of PgsFucT1 shown in SEQ ID NO: 2 had been replaced with aspartic acid.

[0179] Similarly, PCR was performed using an equimolar mixture of the PgsFucT1 S93L upstream and PgsFucT1 S93L downstream fragments as a template and DNA consisting of the base sequences shown in SEQ ID NOs: 64 and 65 as a primer set to obtain the PgsFucT1 S93L fragment, in which serine at position 93 in the amino acid sequence of PgsFucT1 shown in SEQ ID NO: 2 had been replaced with leucine.

[0180] Similarly, PCR was performed using an equimolar mixture of the BfFucT1 D28N upstream and BfFucT1 D28N downstream fragments as a template and DNA consisting of the base sequences shown in SEQ ID NOs: 68 and 69 as a primer set to obtain the BfFucT1 D28N fragment in which the aspartic acid at position 28 in the amino acid sequence of BfFucT1 shown in SEQ ID NO: 12 had been replaced with asparagine.

[0181] Similarly, PCR was performed using an equimolar mixture of the BfFucT1 L104S upstream and BfFucT1 L104S downstream fragments as a template and DNA consisting of the base sequences shown in SEQ ID NOs: 68 and 69 as a primer set to obtain the BfFucT1 L104S fragment in which the 104th leucine in the amino acid sequence of BfFucT1 shown in SEQ ID NO: 12 had been replaced with serine.

[0182] Each of the fragments obtained above was ligated to the pUAKQE-rcsA-lacY vector fragment prepared in Example 1 using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing each mutant fragment, namely, pPgsFucT1_N17D, pPgsFucT1_S93L, pBfFucT1_D28N, and pBfFucT1_L104S.

[0183] The FUC strain constructed in Example 1(3) was transformed with the four plasmids obtained above to construct Escherichia coli strains carrying various plasmids, which were named FUC / pPgsFucT1_N17D strain, FUC / pPgsFucT1_S93L strain, FUC / pBfFucT1_D28N strain, and FUC / pBfFucT1_L104S strain, respectively.

[0184] (2) Productivity evaluation of 3FL or LewisX The FUC / pPgsFucT1_N17D strain, FUC / pPgsFucT1_S93L strain, FUC / pBfFucT1_D28N strain, and FUC / pBfFucT1_L104S strain obtained in (1) above were evaluated for productivity of 3FL or LewisX. The FUC / pPgsFucT1 strain, FUC / pBfFucT1 strain, and FUC / Ctrl strain constructed in Example 1(2) were used as controls.

[0185] The culture method and conditions were as described in Example 1 (3). After the culture was completed, the culture medium was centrifuged and diluted appropriately, and the 3FL or LewisX contained in the supernatant was analyzed using a sugar analyzer ICS-5000 or UFLC&LCMS-8040. The results are shown in Table 9.

[0186] [Table 9]

[0187] Although the FUC / pPgsFucT1_N17D strain produced approximately twofold more Lewis X than the FUC / pPgsFucT1 strain containing wild-type PgsFucT1, only trace amounts of 3FL were detected, whereas the FUC / pPgsFucT1_S93L strain did not produce any Lewis X or 3FL.

[0188] The FUC / pBfFucT1_D28N and FUC / pBfFucT1_L104S strains showed a significantly reduced amount of LewisX produced compared to the FUC / pBfFucT1 strain containing wild-type BfFucT1, and a decrease in the amount of 3FL produced relative to LewisX was observed.

[0189] These results suggest that the substrate specificity for lactose or N-acetyllactosamine can be altered by substituting the asparagine residue at position 17 in the amino acid sequence of PgsFucT1, or the aspartic acid residue at position 28 or the leucine residue at position 104 in the amino acid sequence of BfFucT1 with other amino acids.

[0190] [Example 4] Construction of microorganisms used for producing LNFPIII Escherichia coli carrying a plasmid for expressing the genes, in which a gene encoding β1,4-galactosyltransferase derived from Helicobacter pylori (hereinafter referred to as HpgalT) and a gene encoding β1,3-N-acetylglucosaminetransferase derived from Neisseria polysaccharea (hereinafter referred to as NplgtA) were placed under the control of the lac promoter, was constructed using the following method.

[0191] PCR was performed using the DNA listed in the "Template" column of Table 10 as a template and DNA consisting of the base sequences shown in the "Primer set" column of Table 10 as a primer set, and each DNA fragment was amplified.

[0192] [Table 10]

[0193] Genomic DNA of Helicobacter pylori strain NCTC11637 was prepared by standard methods. The DNA represented by SEQ ID NO: 31 is a codon-optimized DNA for expression in Escherichia coli of the nucleotide sequence of the gene encoding β1,3-N-acetylglucosamine transferase derived from Neisseria polysaccharea strain ATCC43768 represented by SEQ ID NO: 32, and was prepared by artificial synthesis. Furthermore, the nucleotide sequences represented by SEQ ID NOs: 95 and 96 each contain complementary sequences at their 5' ends.

[0194] Using an equimolar mixture of the HpgalT fragment and the NplgtA fragment as a template, PCR was performed using DNA consisting of the base sequences shown in SEQ ID NOs: 94 and 97 as a primer set to obtain a DNA fragment in which the HpgalT fragment and the NplgtA fragment were linked (hereinafter referred to as HpgalT-NplgtA).

[0195] PCR was performed using a primer set consisting of DNAs with the nucleotide sequences represented by SEQ ID NOs: 92 and 93 and the plasmid pSTV29 (Takara Bio Inc.) as a template to obtain a vector fragment of approximately 2.9 kb. The nucleotide sequences represented by SEQ ID NOs: 92 and 94, and SEQ ID NOs: 93 and 97 each contain a complementary sequence at their 5' ends.

[0196] The HpgalT-NplgtA fragment and the vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pSTV_HpgalT-NplgtA.

[0197] The W3110ΔlacZYΔwcaJM strain constructed in Example 1(2) was transformed with the expression plasmid pSTV_HpgalT-NplgtA to construct an E. coli strain harboring pSTV_HpgalT-NplgtA, which was designated TROS strain.

[0198] The above-mentioned TROS strain was transformed with the pHpFutA, pPgsFucT1, pBfFucT1, and pUAKQE-rcsA-lacY plasmids constructed in Example 1(2) to obtain TROS / pHpFutA, TROS / pPgsFucT1, TROS / pBfFucT1, and TROS / Ctrl strains, respectively.

[0199] [Example 5] Production of LNFPIII The TROS / pHpFutA, TROS / pPgsFucT1, TROS / pBfFucT1, and TROS / Ctrl strains obtained in Example 4 were evaluated for productivity of LNFPIII and by-product sugars.

[0200] 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 14 mL plastic tube containing 2 mL of LB medium containing 100 mg / L kanamycin and 25 mg / L chloramphenicol, followed by shaking culture at 30°C for 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. Five hours after the start of culture, IPTG was added to a final concentration of 1 mM.

[0201] After the cultivation was completed, the culture medium was centrifuged and diluted appropriately, and the LNFPIII, 3FL, LNnDFHII, or LNFPVI contained in the supernatant or the cells was analyzed using a sugar analyzer ICS-5000 or UFLC&LCMS-8040. The results are shown in Table 11. Note that the values ​​for LNnDFHII and LNFPVI are both expressed as peak relative values ​​(%).

[0202] [Table 11]

[0203] The results showed that the PgsFucT1-expressing strain accumulated more LNFPIII in both the culture medium and cells than strains expressing the known α1,3-fucosyltransferases HpFutA or BfFucT1. Furthermore, the use of PgsFucT1 reduced the production of by-products such as 3FL, LNnDFHII, and LNFPVI.

[0204] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2022-019024) filed on February 9, 2022, the entire contents of which are incorporated by reference. All references cited herein are incorporated in their entirety. [Sequence List Free Text]

[0205] SEQ ID NO: 1: Nucleotide sequence of FucT1 derived from Parabacteroides goldsteinii JCM 13446 SEQ ID NO: 2: Amino acid sequence of FucT1 from Parabacteroides goldsteinii JCM 13446 SEQ ID NO: 3: Nucleotide sequence of FutA derived from Helicobacter pylori 26695 SEQ ID NO: 4: Amino acid sequence of FutA from Helicobacter pylori 26695 SEQ ID NO: 5: Nucleotide sequence of FucT derived from Bacteroides nordii JCM 12987 SEQ ID NO: 6: Amino acid sequence of FucT from Bacteroides nordii JCM 12987 SEQ ID NO: 7: Nucleotide sequence of FucT derived from Bacteroides salyersiae JCM 12988 SEQ ID NO: 8: Amino acid sequence of FucT from Bacteroides salyersiae JCM 12988 SEQ ID NO: 9: Nucleotide sequence of FucT2 derived from Parabacteroides goldsteinii JCM 13446 SEQ ID NO: 10: Amino acid sequence of FucT2 from Parabacteroides goldsteinii JCM 13446 SEQ ID NO: 11: Nucleotide sequence of FucT1 derived from Bacteroides fragilis ATCC 25285 SEQ ID NO: 12: Amino acid sequence of FucT1 from Bacteroides fragilis ATCC 25285 SEQ ID NO: 13: Nucleotide sequence of FucT2 derived from Bacteroides fragilis ATCC 25285 SEQ ID NO: 14: Amino acid sequence of FucT2 from Bacteroides fragilis ATCC 25285 SEQ ID NO: 15: Nucleotide sequence of FucT derived from Mediterranea sp. An20 SEQ ID NO: 16: Amino acid sequence of FucT from Mediterranea sp. An20 SEQ ID NO: 17: Nucleotide sequence of FucT-A derived from Parabacteroides sp. BX2 SEQ ID NO: 18: Amino acid sequence of FucT-A from Parabacteroides sp. BX2 SEQ ID NO: 19: Nucleotide sequence of FucT-B derived from Parabacteroides sp. HGS0025 SEQ ID NO: 20: Amino acid sequence of FucT-B from Parabacteroides sp. HGS0025 SEQ ID NO: 21: Nucleotide sequence of FucT-C derived from Parabacteroides bouchesdurhonensis strain Marseille-P3763 SEQ ID NO: 22: Amino acid sequence of FucT-C from Parabacteroides bouchesdurhonensis strain Marseille-P3763 SEQ ID NO: 23: Nucleotide sequence of FucT-D derived from Gramella sp. BOM4 SEQ ID NO: 24: Amino acid sequence of FucT-D from Gramella sp. BOM4 SEQ ID NO: 25: Nucleotide sequence of FucT-E derived from Lachnospiraceae bacterium NLAE-zl-G231 SEQ ID NO: 26: Amino acid sequence of FucT-E from Lachnospiraceae bacterium NLAE-zl-G231 SEQ ID NO: 27: Nucleotide sequence of MdfA derived from E. coli W3110 SEQ ID NO: 28: Amino acid sequence of MdfA from E. coli W3110 SEQ ID NO: 29: Base sequence of GalT derived from Helicobacter pylori NCTC 11637 SEQ ID NO: 30: Amino acid sequence of GalT from Helicobacter pylori NCTC 11637 SEQ ID NO: 31: Nucleotide sequence of LgtA derived from Neisseria polysaccharea ATCC 43768 SEQ ID NO: 32: Amino acid sequence of LgtA from Neisseria polysaccharea ATCC 43768 SEQ ID NOs: 33 and 34: Nucleotide sequences of primers for amplifying catsacB fragment SEQ ID NOs: 35 and 36: Nucleotide sequences of primers for amplifying lacZ upstream 1 SEQ ID NOs: 37 and 38: Nucleotide sequences of primers for amplifying lacY downstream 1 SEQ ID NO: 39: Nucleotide sequence of primer for amplifying lacZ upstream 2 SEQ ID NO: 40: Nucleotide sequence of primer for amplifying lacY downstream 2 SEQ ID NOs: 41 and 42: Nucleotide sequences of primers for amplifying wcaJ upstream 1 SEQ ID NOs: 43 and 44: Nucleotide sequences of primers for amplifying wcaM downstream 1 SEQ ID NO: 45: Nucleotide sequence of primer for amplifying wcaJ upstream 2 SEQ ID NO: 46: Nucleotide sequence of wcaM downstream 2 amplification primer SEQ ID NOs: 47 and 48: Nucleotide sequences of primers for amplifying the mdfA fragment SEQ ID NOs: 49 and 50: Nucleotide sequences of primers for amplifying pMW118 fragment SEQ ID NOs: 51 and 52: Nucleotide sequences of primers for amplifying pUAKQE fragment SEQ ID NOs: 53 and 54: Nucleotide sequences of primers for amplifying the rcsA fragment SEQ ID NOs: 55 and 56: Nucleotide sequences of primers for amplifying the lacY fragment SEQ ID NO: 57: Nucleotide sequence of primer for amplifying pUAKQE-rcsA-lacY fragment SEQ ID NOs: 58 and 59: Nucleotide sequences of primers for amplifying HpFutA fragment SEQ ID NOs: 60 and 61: Nucleotide sequences of primers for amplifying the BnFucT fragment SEQ ID NOs: 62 and 63: Nucleotide sequences of primers for amplifying the BsFucT fragment SEQ ID NOs: 64 and 65: Nucleotide sequences of primers for amplifying the PgsFucT1 fragment SEQ ID NOs: 66 and 67: Nucleotide sequences of primers for amplifying the PgsFucT2 fragment SEQ ID NOs: 68 and 69: Nucleotide sequences of primers for amplifying the BfFucT1 fragment SEQ ID NOs: 70 and 71: Nucleotide sequences of primers for amplifying the BfFucT2 fragment SEQ ID NOs: 72 and 73: Nucleotide sequences of primers for amplifying MFucT fragment SEQ ID NOs: 74 and 75: Nucleotide sequences of primers for amplifying FucT-A fragment SEQ ID NOs: 76 and 77: Nucleotide sequences of primers for amplifying FucT-B fragment SEQ ID NOs: 78 and 79: Nucleotide sequences of primers for amplifying the FucT-C fragment SEQ ID NOs: 80 and 81: Nucleotide sequences of primers for amplifying FucT-D fragment SEQ ID NOs: 82 and 83: Nucleotide sequences of primers for amplifying the FucT-E fragment SEQ ID NO: 84: Nucleotide sequence of PgsFucT1 N17D upstream amplification primer SEQ ID NO: 85: Nucleotide sequence of PgsFucT1 N17D downstream amplification primer SEQ ID NO: 86: Nucleotide sequence of PgsFucT1 S93L upstream amplification primer SEQ ID NO: 87: Nucleotide sequence of PgsFucT1 S93L downstream amplification primer SEQ ID NO: 88: Nucleotide sequence of BfFucT D28N upstream amplification primer SEQ ID NO: 89: Nucleotide sequence of BfFucT D28N downstream amplification primer SEQ ID NO: 90: Nucleotide sequence of BfFucT L104S upstream amplification primer SEQ ID NO: 91: Nucleotide sequence of BfFucT L104S downstream amplification primer SEQ ID NOs: 92 and 93: Nucleotide sequences of primers for amplifying pSTV29 fragment SEQ ID NOs: 94 and 95: Nucleotide sequences of primers for amplifying HpgalT fragment SEQ ID NOs: 96 and 97: Nucleotide sequences of primers for amplifying the NplgtA fragment

Claims

1. A microorganism of the genus Escherichia, in which the activity of a protein according to any one of [1] to [6] below is enhanced by transforming a parent strain of the microorganism with a recombinant DNA containing a DNA encoding the protein, and in which productivity of oligosaccharides having a Lewis X backbone is improved compared to that of the parent strain, The oligosaccharide having a Lewis X backbone is at least one of an oligosaccharide having a lacto-N-fucopentaose III (LNFPIII) backbone and an oligosaccharide having a paralacto-N-neohexaose (Para-LNnH) backbone in which L-fucose is α1,3-linked to the 3-position of at least one N-acetylglucosamine. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO:

2. [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and which has α1,3-fucosyltransferase activity. [3] A homologous protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having α1,3-fucosyltransferase activity. [4] A protein consisting of the amino acid sequence represented by SEQ ID NO:

24. [5] A protein consisting of the amino acid sequence represented by SEQ ID NO:

18. [6] A protein consisting of the amino acid sequence represented by SEQ ID NO:

26.

2. The microorganism according to claim 1, wherein the oligosaccharide having an LNFPIII backbone is at least one of LNFPIII and lacto-N-neodifucohexaose II (LNnDFHII).

3. A microorganism described in claim 1 or 2, wherein the parent strain has reduced or lost at least one of β-galactosidase activity and colanic acid synthesis activity.

4. A method for producing oligosaccharides, comprising preparing the microorganism according to any one of claims 1 to 3, and producing oligosaccharides in a culture using the microorganism.

Images