Method for producing a carbohydrate containing lacto-N-triose II as the core trisaccharide and method for producing crystals of said carbohydrate

Abstract

The present invention relates to microorganisms that have enhanced, reduced, or inactivated activity of any of [1] a protein composed of an amino acid sequence represented by SEQ ID NO: 2 or 4, [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 or 4 have been deleted, substituted, inserted, or added and that has oligosaccharide transport activity, and [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 or 4 and that has oligosaccharide transport activity, the microorganisms also having improved productivity of lacto-N-triose II or an oligosaccharide having a lacto-N-triose II skeleton over that of a parent strain.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a saccharide containing lacto-N-triose II as a core trisaccharide, such as lacto-N-triose II, lacto-N-tetraose, or lacto-N-neotetraose, and a method for producing crystals of the saccharide. [Background technology]

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

[0003] Lacto-N-triose II (hereinafter referred to as LNTII) is a type of HMO found in trace amounts in human colostrum, and is known as the oligosaccharide that constitutes lacto-N-tetraose (hereinafter referred to as LNT), which is found in large amounts in colostrum (Non-Patent Document 2).

[0004] LNTII has been reported to have strong antibacterial activity against pathogenic Streptococcus, immunoregulatory function via TLRs, and the ability of bifidobacteria to grow using LNTII as a carbon source, and its functionality has recently attracted attention among HMOs (Non-patent documents 3, 4, 5).

[0005] Microbial fermentation methods using N-acetylglucosaminyltransferase are widely used to produce oligosaccharides such as LNTII or LNT containing LNTII as the core trisaccharide. For example, Non-Patent Document 6 discloses a method for producing oligosaccharides such as LNTII or lacto-N-neotetraose (hereinafter referred to as LNnT) containing LNTII as the core trisaccharide by fermentation using lactose and UDP-N-acetylglucosamine as substrates and N-acetylglucosaminyltransferase derived from Neisseria meningitides or the like.

[0006] Figure 1 shows a representative biosynthetic pathway for the uptake of glucose and the synthesis of LNTII, LNT, and LNnT. Glucose is taken up into cells from the medium via a phosphoenolpyruvate-dependent phosphotransferase system (PTS). LgtA links N-acetylglucosamine to lactose to produce LNTII. As further shown in Figure 1, GalT produces LNT or LNnT from LNTII. LNTII is exported from cells via an LNTII transporter. LNT or LNnT is exported from cells into the medium via an oligosaccharide transporter.

[0007] As transporters involved in the extracellular export of LNTII, Patent Document 1 discloses the YjhB protein derived from Escherichia coli, the ProP protein derived from Mannheimia succiniciproducens, and the SetA protein derived from Sedecea neteri (Patent Document 1).

[0008] Patent Document 1 also discloses that the amount of LNTII produced can be improved by overexpressing these proteins involved in LNTII transport, and that deletion of the yjhB gene on the E. coli genome suppresses leakage of LNTII and improves the yield of LNT.

[0009] HMO transporters other than LNTII have been reported, including the Escherichia coli-derived SetA protein, MdfA protein, and Neurospora crassa-derived CDT-2 protein, which are involved in the excretion of 2-fucosyllactose and 3-fucosyllactose (Patent Documents 2 and 3, Non-Patent Document 7).

[0010] As a method for obtaining LNTII crystals, a method based on inverse crystallization has been disclosed in which an LNTII-containing aqueous solution is added to methanol or acetone to obtain LNTII crystals (Patent Document 4). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japan Special Publication No. 2018-526019 [Patent Document 2] Japanese Patent No. 5580408 [Patent Document 3] International Publication No. 2021 / 013708 [Patent Document 4] Japanese Patent No. 4347042 [Non-patent literature]

[0012] [Non-Patent Document 1] Int J Pediatrics(2019)2390240:1-8 [Non-patent document 2] Curr Dev Nutr(2020)4:nzaa113 [Non-patent document 3] Acc Chem Res(2019)52,760 [Non-patent document 4] J Functional Foods(2019)59,174 [Non-Patent Document 5] Front Microbiol(2020)11,569700 [Non-patent document 6] Syst Microbiol Biomanufact(2021)1, 291 [Non-Patent Document 7] Metabolic Engineering(2019)52:232-242 Summary of the Invention [Problem to be solved by the invention]

[0013] As mentioned above, the functionality of LNTII or oligosaccharides having an LNTII backbone containing LNTII as the core trisaccharide has been attracting attention in recent years, and an efficient production method is needed. Patent Document 1 also discloses that the amount of LNTII produced can be improved by overexpressing the above-mentioned proteins involved in LNTII transport, and that deletion of the yjhB gene in the E. coli genome suppresses LNTII leakage and improves LNT yield. However, since some E. coli strains do not have yjhB or its homologous sequence, there is a need to discover new proteins that are widely conserved among various E. coli strains and have excellent LNTII transport activity.

[0014] Patent documents 2 and 3, and non-patent document 7 report HMO transporters other than LNTII, such as the Escherichia coli-derived SetA protein, MdfA protein, and Neurospora crassa-derived CDT-2 protein, which are involved in the excretion of 2-fucosyllactose and 3-fucosyllactose, but it is not known that these proteins have the transport activity of LNTII.

[0015] Therefore, an object of the present invention is to provide a microorganism that is excellent in productivity of LNTII or oligosaccharides having an LNTII backbone containing LNTII as the core trisaccharide.

[0016] Furthermore, no method is known for efficiently producing LNTII crystals from the culture medium obtained by the above-mentioned microbial fermentation method, and there is a need for a simple method for purifying and crystallizing high-purity LNTII from a culture medium containing a mixture of various water-soluble substances and carbohydrates with structures similar to LNTII.

[0017] Therefore, an object of the present invention is to provide a method for easily producing high-purity LNTII crystals. [Means for solving the problem]

[0018] The present inventors discovered that a microorganism in which the activity of the YdeA protein or MdfA protein has been enhanced has improved LNTII productivity compared to the parent strain, thereby completing a first invention. Furthermore, the present inventors discovered that a microorganism in which the activity of the protein has been reduced or inactivated has improved productivity of oligosaccharides having an LNTII backbone compared to the parent strain, thereby completing a second invention.

[0019] Furthermore, the present inventors have perfected a method for producing LNTII crystals using a culture obtained by culturing the above microorganism.

[0020] 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 [3] below has been enhanced and in which productivity of lacto-N-triose II (LNTII) has been improved compared to that of the parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4. [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport 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 or 4 and that has oligosaccharide transport activity. 2. A microorganism in which the activity of a protein described in any one of [1] to [3] below is reduced or inactivated, and which has improved productivity of oligosaccharides having a lacto-N-triose II (LNTII) skeleton compared to the parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4. [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport 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 or 4 and that has oligosaccharide transport activity. 3. The microorganism according to 2 above, wherein the oligosaccharide having an LNTII backbone is lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT). 4. A method for producing oligosaccharides, comprising preparing the microorganism according to any one of 1 to 3 above, and producing oligosaccharides in a culture using the microorganism. 5. A method for producing crystals of LNTII from a culture obtained by culturing the microorganism described in 1 above, comprising the following steps (i) to (iv): (i) centrifuging the culture to obtain a supernatant from which the bacterial cells have been removed; (ii) subjecting the supernatant obtained in step (i) to cation exchange and anion exchange to obtain a solution from which ions have been removed from the supernatant; (iii) concentrating the solution obtained in step (ii) using an evaporator; and (iv) cooling the solution obtained in step (iii) or adding an anti-solvent dropwise to the solution obtained in step (iii) to obtain crystals of LNTII. [Effects of the Invention]

[0021] In the microorganism according to the first invention, the activity of a specific protein is enhanced, which promotes extracellular export of LNTII and can improve LNTII productivity. Furthermore, in the microorganism according to the second invention, the activity of the protein is reduced or inactivated, which inhibits extracellular export of LNTII and can improve productivity of oligosaccharides having an LNTII backbone that are produced using LNTII as a substrate. Therefore, by using the microorganisms according to the first and second inventions, LNTII or oligosaccharides having an LNTII backbone that contain LNTII as the core trisaccharide can be produced more efficiently than conventional methods.

[0022] According to the production method of the present invention, highly pure LNTII crystals can be easily produced by using a culture obtained by culturing the microorganism. [Brief explanation of the drawings]

[0023] [Figure 1]FIG. 1 shows a schematic diagram of the biosynthetic pathways of LNTII, LNT, LNnT, etc. in a microorganism according to one embodiment of the present invention. [Figure 2] Figure 2 shows the results of culturing the INTC / pLNTII strain and 12 LNTII-producing strains in which the expression of each transporter gene was enhanced, diluting the culture solution appropriately after cultivation, and then centrifuging it. The LNTII contained in the supernatant was analyzed using the ICS-6000 sugar analyzer. [Figure 3] 3 shows the results of sugar analysis of LNTII crystals obtained in Example 6. The vertical axis represents intensity, and the horizontal axis represents analysis time. [Figure 4] 4 shows the results of sugar analysis of LNTII crystals obtained in Example 7. The vertical axis represents intensity, and the horizontal axis represents analysis time. [Figure 5] Figure 5 shows the Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of the LNTII·6.0 hydrate crystal. [Figure 6] 6 shows the results of sugar analysis of LNTII crystals obtained in Example 8. The vertical axis represents intensity, and the horizontal axis represents analysis time. DETAILED DESCRIPTION OF THE INVENTION

[0024] 1. Microorganisms with improved LNTII productivity Examples of microorganisms with improved LNTII productivity according to the present invention include the following microorganisms: A microorganism in which the activity of the protein according to any one of [1] to [3] below is enhanced and the productivity of LNTII is improved compared to that of the parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4. [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport 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 or 4 and that has oligosaccharide transport activity.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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

[0029] As used herein, a homologous protein refers to a protein that is found in organisms that exist in nature and is similar in structure and function to the original protein, such that the gene encoding the protein is considered to have the same evolutionary origin as the gene encoding the original protein.

[0030] Examples of homologous proteins include amino acid sequences that have an identity of preferably 90% or more, and particularly preferably 95% or more, to the amino acid sequence of the target protein.

[0031] 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.

[0032] As used herein, "oligosaccharide transport activity" refers to the activity of transporting intracellular oligosaccharides to the outside of cells. Whether a protein has oligosaccharide transport activity can be confirmed by preparing a recombinant DNA containing DNA encoding the protein using the method described below, transforming a microorganism in which oligosaccharide transport activity cannot be confirmed with the recombinant DNA, culturing the resulting microorganism, preparing a cell extract containing the protein from the resulting culture, diluting the culture appropriately, centrifuging it, and analyzing the oligosaccharides contained in the supernatant using a sugar analyzer. When using Escherichia coli W3110 as a microorganism in which oligosaccharide transport activity cannot be confirmed, for example, the ability to produce LNTII can be artificially imparted during transformation using the method described below.

[0033] As used herein, "productivity" refers to the ability of a microorganism to accumulate the target oligosaccharide produced by the microorganism in a culture medium. The productivity of oligosaccharides by a microorganism can be confirmed by detecting oligosaccharides in the culture medium of the microorganism using a sugar analyzer described below. 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 LNTII productivity refers to the strain prior to genetic modification, transformation, etc. that enhances the activity of the protein described in any one of [1] to [3].

[0034] In the present specification, the parent strain is preferably a prokaryote or a yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 codon plus (Stratagene), Escherichia coli W3110S3GK (NBRC114657), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratiamarcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum Examples of suitable strains include prokaryotes such as Bacillus subtilis ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, and Pseudomonas sp. D-0110, and yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and Candida utilis.

[0035] The parent strain may be a wild-type strain as long as it is a microorganism that produces LNTII, or if the wild-type strain does not have the ability to produce LNTII, it may be a bred strain that has been artificially imparted with the ability to produce LNTII.

[0036] Methods for artificially imparting the ability to produce LNTII to a microorganism include, for example, the following methods (1a) to (1e), which can be used alone or in combination. (1a) A method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway that produces LNTII. (1b) A method for enhancing expression of at least one enzyme involved in the biosynthetic pathway that produces LNTII. (1c) A method for increasing the copy number of at least one enzyme gene involved in the biosynthetic pathway that produces LNTII. (1d) A method for weakening or blocking at least one metabolic pathway branching off from the biosynthetic pathway that produces LNTII to a metabolite other than the target substance. (1e) A method for selecting a cell line that is more resistant to an LNTII analog than a wild-type cell line

[0037] Whether a microorganism is capable of producing LNTII can be confirmed by transforming the microorganism with recombinant DNA containing DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4, culturing the transformed microorganism in a medium, and detecting LNTII accumulated in the culture using a sugar analyzer described below.

[0038] The parent strain preferably has at least one activity selected from lactose permease (hereinafter referred to as lacY), β1,3-N-acetylglucosamine transferase (hereinafter referred to as LgtA), glutamine-fructose-6-phosphate transaminase (hereinafter referred to as glmS), phosphoglucosamine mutase (hereinafter referred to as glmM), and N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase (hereinafter referred to as glmU), and more preferably has enhanced activity. Of these, the parent strain preferably has lacY and LgtA activities, and even more preferably has enhanced activity.

[0039] LacY is a membrane protein that imports lactose, the substrate for LNTII, into cells. LgtA is an enzyme involved in the production of LNTII from lactose and uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), as shown in Figure 1. GlmS, glmM, and glmU are enzymes involved in the biosynthetic pathway that produces LNTII, as shown in Figure 1.

[0040] Therefore, in one embodiment of the present invention, it is preferable to use a genetically modified microorganism as a parent strain, preferably comprising at least one nucleotide sequence selected from the group consisting of a nucleotide sequence encoding lacY (accession number BAE76125.1), a nucleotide sequence encoding LgtA (SEQ ID NO: 5), a nucleotide sequence encoding glmS (accession number BAE77559.1), a nucleotide sequence encoding glmM (accession number BAE77220.1), and a nucleotide sequence encoding glmU (accession number BAE77558.1). It is particularly preferable to use a genetically modified microorganism comprising a nucleotide sequence encoding lacY 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 LNTII compared to a non-genetically modified parent strain.

[0041] E. coli having at least one activity selected from lacY activity, LgtA activity, glmS activity, glmM activity, and glmU activity, or having enhanced activity, can be produced by known methods, such as various genetic engineering methods (Syst Microbiol Biomanufact, 2021, 1, 291).

[0042] Furthermore, it is preferable that the parent strain has reduced or absent β-galactosidase (hereinafter referred to as lacZ) activity and / or the activity of the regulatory factor YhbJ. lacZ is an enzyme that hydrolyzes lactose, the substrate of LNTII. Therefore, by eliminating lacZ activity, the decrease in lactose supply can be suppressed. As shown in Figure 1, YhbJ is a negative transcriptional regulator of the glmS gene in the LNTII biosynthetic pathway. Therefore, by eliminating YhbJ activity, LNTII biosynthesis can be promoted.

[0043] Therefore, in one embodiment of the present invention, the parent strain is preferably a genetically modified microorganism in which the activity of lacZ and / or YhbJ is reduced or deleted, and more preferably does not contain a nucleotide sequence encoding lacZ and / or a nucleotide sequence encoding YhbJ. In one embodiment of the present invention, the genetically modified microorganism preferably has an increased ability to produce LNTII compared to a non-genetically modified parent strain.

[0044] E. coli in which β-galactosidase activity and / or the activity of the regulatory factor YhbJ is reduced or eliminated can be produced by known methods, such as by various genetic engineering methods (Metabolic Engineering, 2017, 41:23-38).

[0045] Examples of microorganisms in which the activity of the protein described in any one of [1] to [3] above is enhanced compared to that of a parent strain of a microorganism include microorganisms in which the copy number of the gene is increased compared to that of the parent strain, and which are obtained by transforming a parent strain of a microorganism with a recombinant DNA containing DNA encoding the protein.

[0046] Examples of microorganisms that have an increased copy number of a gene compared to a parent strain, and that can be obtained by transforming a parent strain microorganism with recombinant DNA containing DNA encoding a protein described in any one of [1] to [3] above, include microorganisms in which the copy number of the gene on chromosomal DNA has been increased by transforming a parent strain microorganism with recombinant DNA containing DNA encoding a protein described in any one of [1] to [3] above, and microorganisms in which the gene is carried outside of chromosomal DNA as plasmid DNA.

[0047] The DNA encoding the protein described in any one of [1] to [3] above may be any DNA encoding a protein having the activity of the protein described in any one of [1] to [3] above, and specifically includes one DNA selected from the group consisting of [4] to [7] below. [4] A DNA encoding the protein according to any one of [1] to [3] above. [5] DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3 [6] A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 3 and encodes a homologous protein having oligosaccharide transport activity. [7] 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 or 3, and encoding a homologous protein having oligosaccharide transport activity.

[0048] In the above [6], "hybridize" means that DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part of the DNA can be used as a probe in Northern or Southern blot analysis, or as an oligonucleotide primer in PCR analysis.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA that has at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3, when calculated based on the above-mentioned parameters using, for example, BLAST or FASTA.

[0054] The DNA encoding the protein of [1] above and the DNA of [5] 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 (ydeA gene) or the nucleotide sequence shown in SEQ ID NO: 3 (mdfA gene), or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the nucleotide sequence and the chromosomal DNA of the above microorganism as a template.

[0055] The Escherichia coli W3110 strain is available from the National Institute of Technology and Evaluation (NITE) Biological Resource Center.

[0056] 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 or 3 as a template to error-prone PCR or the like.

[0057] 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).

[0058] 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.

[0059] 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.

[0060] The DNA encoding the homologous protein of [3] above, and the DNAs of [6] and [7] above can be obtained, for example, by searching various gene sequence databases for base sequences that have an identity of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more to the base sequence represented by SEQ ID NO: 1 or 3, or by searching various protein sequence databases for amino acid sequences that have an identity of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more to the amino acid sequence represented by SEQ ID NO: 2 or 4, and using probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search, and a microorganism containing the DNA, in a manner similar to the method for obtaining the above DNA.

[0061] The obtained DNA described in any one of [4] to [7] above can be used as is or cleaved with an appropriate restriction enzyme or the like, and then inserted into a vector by a conventional method. The resulting recombinant DNA can then be introduced into a host cell, and the base sequence of the DNA can be determined by a commonly used 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 a 3700 DNA Analyzer (manufactured by Applied Biosystems).

[0062] The host cells that can be used for determining the DNA base sequence may be any cells that can be transformed with the vector and grow, and examples thereof include Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam, and the like. - / dcm - Examples of such a mutated Escherichia coli include Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, and Escherichia coli TH2 (all manufactured by Takara Bio Inc.), Escherichia coli XL1-Blue, and Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, and Escherichia coli NM522.

[0063] 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).

[0064] 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)].

[0065] 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.

[0066] 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.

[0067] The recombinant DNA containing the DNA encoding the protein according to any one of [1] to [3] above refers to a recombinant DNA in which the DNA is incorporated into an expression vector that is capable of autonomous replication in a parent strain or of being integrated into a chromosome and that contains a promoter at a position where the DNA can be transcribed.

[0068] When the recombinant DNA is capable of being integrated into a chromosome, it does not need to contain a promoter.

[0069] A microorganism having an increased copy number of the gene compared to the parent strain, which is obtained by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding the protein described in any one of [1] to [3] above, can be obtained by the following method.

[0070] Based on the DNA encoding the protein according to any one of [1] to [3] above obtained by the above method, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as needed. Furthermore, by substituting bases in the base sequence of the portion encoding the protein so that it contains optimal codons for expression in host cells, a transformant with improved productivity can be obtained.

[0071] 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.

[0072] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA is preferably a recombinant DNA comprising a promoter, a ribosome binding sequence, the DNA described in any one of [4] to [7] above, and a transcription termination sequence. A gene that controls the promoter may also be included.

[0073] 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.

[0074] Furthermore, by substituting bases in the base sequence of the portion encoding the protein having oligosaccharide transport activity so that it has optimal codons for expression in the host, the expression level of the protein having oligosaccharide transport activity can be improved. Examples of proteins having oligosaccharide transport activity include the proteins described in any one of [1] to [3] above. Information on codon usage frequencies in parent strains used in the production methods of the present invention is available from public databases.

[0075] 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.

[0076] 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.), pMW119 (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), pKYP10 (Japanese Patent Application Laid-Open 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 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385], 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)].

[0077] 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.

[0078] 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)).

[0079] 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)].

[0080] When a yeast strain is used as the parent strain, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0081] 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.

[0082] The recombinant DNA used in the production method of the present invention can be prepared by inserting the DNA fragment described in any one of [4] to [7] above downstream of the promoter of an appropriate expression vector.

[0083] 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)].

[0084] 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)].

[0085] 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)].

[0086] 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.

[0087] Whether the microorganism produced by the above method is a microorganism in which the activity of the protein described in any one of [1] to [3] above is enhanced compared to the parent strain can be confirmed by culturing the microorganism, diluting the culture medium appropriately, centrifuging it, and analyzing the LNTII contained in the supernatant using a sugar analyzer, and comparing it with that of the parent strain.

[0088] The above-mentioned microorganisms have enhanced activity of the protein described in any one of [1] to [3] above compared to the parent strain, which can promote extracellular export of LNTII and improve LNTII productivity. An example of such a microorganism is the INTC / pLNTII strain, which has enhanced expression of the ydeA gene or mdfA gene, as described below in the Examples.

[0089] Examples of such microorganisms include microorganisms in which the activity of the YdeA protein or MdfA protein is enhanced, and the transporter that exports LNTII is overexpressed, which increases LNTII export and allows for improved LNTII productivity.

[0090] Microorganisms with improved LNTII productivity also include microorganisms in which the activity of any one of the proteins described in [8] to

[10] below is enhanced, instead of any one of the proteins described in [1] to [3] above. [8] A protein consisting of the amino acid sequence represented by SEQ ID NO: 65 or 67. [9] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 65 or 67 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport activity.

[10] A homologous protein having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 65 or 67 and that has oligosaccharide transport activity.

[0091] 2. Microorganisms with improved productivity of oligosaccharides containing the LNTII backbone Examples of the microorganisms of the present invention that have improved productivity of oligosaccharides having an LNTII backbone include the following microorganisms. A microorganism in which the activity of a protein described in any one of [1] to [3] below is reduced or inactivated, and which has improved productivity of oligosaccharides having a lacto-N-triose II (LNTII) skeleton compared to a parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4. [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport 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 or 4 and that has oligosaccharide transport activity.

[0092] A microorganism in which the activity of a protein described in any one of [1] to [3] is reduced or inactivated compared to a parent strain can be obtained by introducing a base deletion, substitution, or addition into the base sequence of a gene encoding the protein described in any one of [1] to [3] above that does not contain a mutation present on chromosomal DNA, and examples thereof include (a) a microorganism in which the specific activity of the protein is reduced to 80% or less, preferably 50% or less, more preferably 30% or less, even more preferably 20% or less, particularly preferably 10% or less, and most preferably 0% compared to the parent strain, and (b) a microorganism in which the transcription level of the gene or the production level of the protein is reduced to 80% or less, preferably 50% or less, more preferably 30% or less, even more preferably 20% or less, particularly preferably 10% or less, and most preferably 0% compared to the parent strain. More preferred examples include a microorganism in which the gene is partially or completely deleted.

[0093] Methods for producing a microorganism in which the activity of any one of the proteins described in [1] to [3] is reduced or inactivated compared to the parent strain include known methods such as knocking out a specific gene using PCR [Baba T. et al., Mol Systems Biol (2006)], using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)], inhibiting the expression of a specific gene using RNAi, and introducing a mutation into a specific gene and its promoter region using a mutagen such as nitrosoguanidine or ultraviolet light.

[0094] In the mutant protein of [2] above, the deletion, substitution, insertion or addition of amino acids has the same meaning as the deletion, substitution, insertion or addition of amino acids described above in 1.

[0095] Whether the mutant protein or a homologous protein has oligosaccharide transport activity can be confirmed by the method described above in 1.

[0096] The oligosaccharide having an LNTII backbone is preferably a human milk oligosaccharide containing LNTII as a core trisaccharide. Note that an oligosaccharide having a "core trisaccharide" refers to a specific trisaccharide that corresponds to the reducing end of a desired oligosaccharide, and optionally includes other sugar moieties in addition to the specific trisaccharide that corresponds to the main portion.

[0097] Examples of oligosaccharides having an LNTII backbone include LNT, LNnT, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc, disialyllacto-N-tetraose, disialyllacto-N-neotetraose, etc. In this specification, oligosaccharides having an LNTII backbone do not include LNTII.

[0098] In one embodiment of the present invention, the oligosaccharide having an LNTII backbone is preferably LNT or LNnT, and its precursor is more preferably LNTII.

[0099] The parent strain of the microorganism of the present invention with improved productivity of oligosaccharides having an LNTII backbone refers to a strain prior to genetic modification, transformation, or the like that reduces or inactivates the activity of the protein described in any one of the above [1] to [3]. The parent strain may be a wild-type strain as long as it is a microorganism that produces oligosaccharides having an LNTII backbone, or if the wild-type strain does not have the ability to produce LNTII, it may be a bred strain to which the ability to produce LNTII has been artificially imparted.

[0100] Methods for artificially imparting the ability to produce oligosaccharides having an LNTII backbone to a microorganism include the methods described below in (2a) to (2e), and these methods can be used alone or in combination. (2a) A method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway that produces oligosaccharides having an LNTII backbone. (2b) A method for enhancing expression of at least one enzyme involved in a biosynthetic pathway that produces oligosaccharides having an LNTII backbone. (2c) A method for increasing the copy number of at least one enzyme gene involved in a biosynthetic pathway that produces oligosaccharides having an LNTII backbone. (2d) A method for weakening or blocking at least one metabolic pathway branching off from a biosynthetic pathway that produces an oligosaccharide having an LNTII backbone to a metabolic product other than the target substance. (2e) A method for selecting a cell line that is more resistant to an oligosaccharide analogue having an LNTII backbone than a wild-type strain

[0101] Whether a microorganism is capable of producing oligosaccharides having an LNTII backbone can be confirmed by culturing a microorganism in a medium in which the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 has been reduced or inactivated by various genetic manipulation methods, and detecting the oligosaccharides having an LNTII backbone that have accumulated in the culture using a sugar analyzer described below.

[0102] The parent strain preferably has at least one of the following activities, and more preferably has enhanced activity: lacY activity, LgtA activity, glmS activity, glmM activity, glmU activity, β1,3-galactosyltransferase (hereinafter referred to as GalT) 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. Of these, the parent strain preferably has lacY, LgtA, and GalT activities, and even more preferably has enhanced activity.

[0103] LacY, LgtA, glmS, glmM, and glmU are the same as those described in 1. GalT is the enzyme responsible for converting LNTII to LNT or LNnT. Pgm, galU, galE, and galF are enzymes involved in the pathway that produces uridine diphosphate galactose (hereinafter referred to as UDP-Gal), as shown in Figure 1. Pgi is an enzyme involved in the pathway that produces LNTII, as shown in Figure 1.

[0104] Therefore, in one embodiment of the present invention, a genetically modified microorganism containing a nucleotide sequence encoding at least one selected from the group consisting of a nucleotide sequence encoding lacY, a nucleotide sequence encoding LgtA (SEQ ID NO: 5), a nucleotide sequence encoding glmS, a nucleotide sequence encoding glmM, a nucleotide sequence encoding glmU, a nucleotide sequence encoding GalT, a nucleotide sequence encoding Pgm (Accession No. BAA35337.1), a nucleotide sequence encoding galU (Accession No. BAA36104.1), a nucleotide sequence encoding galE (Accession No. BAA35421.1), a nucleotide sequence encoding galF (Accession No. BAA15896.1), and a nucleotide sequence encoding Pgi (Accession No. BAE78027.1) is preferably used as a parent strain. In particular, a genetically modified microorganism containing a nucleotide sequence encoding lacY, a nucleotide sequence encoding LgtA, and a nucleotide sequence encoding GalT is preferably used as a parent strain.

[0105] In one embodiment of the present invention, the genetically modified microorganism preferably has an increased ability to produce oligosaccharides having an LNTII backbone compared to a non-genetically modified parent strain.

[0106] Specific examples of methods for producing E. coli having at least one activity selected from lacY activity, LgtA activity, glmS activity, glmM activity, glmU activity, GalT activity, Pgm activity, galU activity, galE activity, galF activity, and Pgi activity, or in which such activity is enhanced, include known methods such as various gene manipulation methods (Non-Patent Document 6).

[0107] Furthermore, it is preferable that the parent strain has reduced or no β-galactosidase (hereinafter referred to as lacZ) activity and / or regulatory factor YhbJ activity.

[0108] Therefore, in one embodiment of the present invention, it is preferred to use a genetically modified microorganism as a parent strain, preferably comprising a nucleotide sequence encoding lacZ and / or a nucleotide sequence encoding YhbJ.

[0109] In one embodiment of the present invention, the genetically modified microorganism preferably has an increased ability to produce oligosaccharides having an LNTII backbone compared to a non-genetically modified parent strain.

[0110] Specific examples of methods for producing E. coli in which β-galactosidase activity and / or regulatory factor YhbJ activity has been lost include known methods such as various genetic engineering methods (Metabolic Engineering, 2017, 41:23-38).

[0111] Whether the microorganism produced by the above method is a microorganism in which the activity of any one of the proteins described in [1] to [3] above is reduced or inactivated compared to the parent strain can be confirmed by culturing the microorganism, diluting the culture medium appropriately, centrifuging it, and analyzing the LNTII contained in the supernatant using a sugar analyzer, and comparing it with that of the parent strain.

[0112] In the above-mentioned microorganisms, the activity of any one of the proteins described in [1] to [3] above is reduced or inactivated compared to the parent strain, thereby suppressing the extracellular export of LNTII and improving the productivity of oligosaccharides having an LNTII backbone that are produced using LNTII as a substrate. Examples of such microorganisms include microorganisms in which the activity of the YdeA protein or MdfA protein is reduced or inactivated, and the exporter protein that exports LNTII is deleted or inactivated, improving the productivity of oligosaccharides having an LNTII backbone, including LNT and LNnT.

[0113] Microorganisms with improved productivity of oligosaccharides having an LNTII backbone also include microorganisms in which the activity of any one of the proteins described in [8] to

[10] below has been reduced or inactivated, instead of any one of the proteins described in [1] to [3] above. [8] A protein consisting of the amino acid sequence represented by SEQ ID NO: 65 or 67. [9] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 65 or 67 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport activity.

[10] A homologous protein having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 65 or 67 and that has oligosaccharide transport activity.

[0114] 3. Oligosaccharide manufacturing method The oligosaccharides of the present invention can be produced by the following method. A method for producing oligosaccharides, comprising preparing a microorganism according to any one of 1. and 2. above, and producing oligosaccharides in a culture using the microorganism.

[0115] In the method of the present invention, the desired oligosaccharide is preferably a human milk oligosaccharide containing LNT-II as a core trisaccharide. Note that an oligosaccharide having a "core trisaccharide" refers to a specific trisaccharide that corresponds to the reducing end of the desired oligosaccharide, and optionally contains other sugar moieties in addition to the specific trisaccharide that corresponds to the main portion.

[0116] Oligosaccharide refers to a sugar polymer composed of at least three moieties, i.e., trisaccharides, tetrasaccharides, pentasaccharides, etc., and is preferably an oligosaccharide selected from at least one of LNTII, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc, disialyllacto-N-tetraose, and disialyllacto-N-neotetraose. Of these, LNTII, LNT, and LNnT are particularly preferred.

[0117] The method for culturing the microorganism described in any one of 1. and 2. above can be carried out according to a conventional method used for culturing microorganisms.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0122] The microorganism of the present invention used in the method for producing oligosaccharides may be a microorganism capable of producing an acceptor carbohydrate such as glucose, lactose, or lactose monohydrate.

[0123] In the method for producing oligosaccharides, glucose, lactose, lactose monohydrate, or the like may be added to the medium during cultivation.

[0124] Furthermore, in the method for producing oligosaccharides, instead of adding glucose, lactose, lactose monohydrate, or the like to the medium during cultivation, glucose, lactose, lactose monohydrate, or the like may be supplied to the microorganism of the present invention by simultaneously culturing a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like from sugar with the microorganism of the present invention.

[0125] In the method for producing oligosaccharides, it is preferable that β-galactosidase and YhbJ are not present in the medium.

[0126] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. 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 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0127] By the above-mentioned cultivation, oligosaccharides are produced and accumulated in the culture, and the oligosaccharides can be collected from the culture, thereby producing oligosaccharides.

[0128] 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.

[0129] 4. Method for producing LNTII crystals The following methods can be used to produce the LNTII crystals of the present invention. A method for producing crystals of LNTII from a culture obtained by culturing the microorganism described in 1 above, comprising the following steps (i) to (iv): (i) centrifuging the culture to obtain a supernatant from which the bacterial cells have been removed; (ii) subjecting the supernatant obtained in step (i) to cation exchange and anion exchange to obtain a solution from which ions have been removed from the supernatant; (iii) concentrating the solution obtained in step (ii) using an evaporator; and (iv) cooling the solution obtained in step (iii) or adding an anti-solvent dropwise to the solution obtained in step (iii) to obtain crystals of LNTII. Each step will be described below.

[0130] (i) centrifuging the culture to obtain a supernatant from which the bacterial cells have been removed; Step (i) is a step of centrifuging the culture of the microorganism described in 1 above to obtain a supernatant from which the bacterial cells have been removed. Prior to centrifugation, the culture is preferably adjusted to a pH of 3.0 to 4.0, and then heated at 60 to 80°C for 30 to 120 minutes.

[0131] The conditions for centrifugation are not particularly limited, but are generally preferably 0 to 30°C, more preferably 0 to 10°C, preferably 4000 to 12000 G or 6000 to 10000 rpm, and preferably 5 to 30 minutes.

[0132] (ii) subjecting the supernatant obtained in step (i) to cation exchange and anion exchange to obtain a solution from which ions have been removed from the supernatant; In step (ii), the supernatant obtained in step (i), from which the cells have been removed, is subjected to cation exchange and anion exchange to obtain a solution from which ions contained in the supernatant have been removed. Step (ii) may be performed at room temperature or a lower temperature, preferably at a lower temperature.

[0133] The cation exchange resin is not particularly limited, and various ion exchange resins can be used as appropriate, but a strongly acidic cation exchange resin is preferred. For example, a strongly acidic cation exchange resin has a sulfonic acid group in a cross-linked styrene skeleton. More specifically, Diaion UBK550(H + type) (Mitsubishi Chemical Corporation), Marathon C (H + Examples of resins include PEG-100 (manufactured by Dow Chemical Co.), and others.

[0134] The anion exchange resin is not particularly limited, and various ion exchange resins can be used as appropriate, but weakly basic anion exchange resins are preferred. Weakly basic anion exchange resins have, for example, primary to tertiary amine groups in a cross-linked styrene skeleton, or carboxylic acid in a cross-linked acrylic skeleton. More specifically, A111S(OH - Type) (Prolite), A845S (OH - Type) (Prolite), WA30(OH - Examples of resins include acrylic resins such as acrylic resin (manufactured by Mitsubishi Chemical Corporation).

[0135] In addition, the cation exchange and anion exchange may be performed using one type of cation exchange resin and one type of anion exchange resin. If necessary, multiple cation exchange resins and multiple anion exchange resins may be used in appropriate combination. Cation exchange may be performed first, followed by anion exchange. Alternatively, anion exchange may be performed first, followed by cation exchange.

[0136] The pH for cation exchange is not particularly limited, but is usually preferably pH 0 to 10, more preferably pH 0 to 5. The pH for anion exchange is not particularly limited, but is usually preferably pH 4 to 14, more preferably pH 9 to 14.

[0137] The column to be packed with the cation exchange resin may be any column used for purifying chemical substances, but it is preferable to select a column so that when the ion exchange resin is packed into the column, the resin layer height / column inner diameter is large, and it is more preferable to select a column so that the resin layer height / column inner diameter is 3 or more.

[0138] The column to be packed with the anion exchange resin may be any column used for purifying chemical substances, but it is preferable to select a column so that when the ion exchange resin is packed into the column, the resin layer height / column inner diameter is large, and it is more preferable to select a column so that the resin layer height / column inner diameter is 3 or more.

[0139] To pass the supernatant from which the bacterial cells have been removed in step (i) through a column packed with a cation exchange resin, the solution may be passed through the top of the column packed with the ion exchange resin, i.e., the upper layer of the column bed, or the bottom of the column, i.e., the lower layer of the column bed, preferably from the top. The speed of passage through the column is preferably a space velocity of 5 1 / hr or less, more preferably 3 1 / hr or less.

[0140] To pass the supernatant from which the bacterial cells have been removed in step (i) through a column packed with an anion exchange resin, the solution may be passed through the top of the column packed with the ion exchange resin, i.e., the upper layer of the column bed, or through the bottom of the column, i.e., the lower layer of the column bed, preferably from the top. The passing speed is preferably a space velocity of 5 1 / hr or less, more preferably 3 1 / hr or less.

[0141] The fractions obtained after cation exchange and anion exchange may be filtered using an MF membrane (HVLP06225, manufactured by Merck), an MF membrane module (PSP-003, manufactured by Asahi Kasei), a UF membrane (VVLP06225, manufactured by Merck), a UF membrane module (SIP-0013, manufactured by Asahi Kasei), or similar filtration membranes.

[0142] (iii) A step of concentrating the solution obtained in step (ii) using an evaporator. Step (iii) is a step of concentrating the solution obtained in step (ii) using an evaporator to obtain a solution containing LNTII at a high purity.

[0143] To obtain a solution containing LNTII in high purity, a concentration step is carried out. The concentration step is generally carried out under vacuum, for example, at a pressure in the range of 0 to 2 mbar, preferably 0 to 1.2 mbar. This vacuum makes it possible to reduce the temperature required for evaporation and the duration of this concentration step. It can be carried out at a temperature in the range of 5 to 60°C, preferably 5 to 50°C, and more preferably 5 to 40°C.

[0144] This concentration step can be carried out in a single stage evaporator, a multi-stage evaporator, for example a two-stage evaporator. This concentration step can be carried out continuously.

[0145] In a solution containing highly purified LNTII, the purity of LNTII is preferably 50% or more, and more preferably 80% or more.

[0146] (iv) cooling the solution obtained in step (iii) or adding an anti-solvent dropwise to the solution obtained in step (iii) to obtain crystals of LNTII. Step (iv) is a step of obtaining LNTII crystals by cooling the solution containing LNTII obtained in step (iii) or by adding a poor solvent dropwise to the solution.

[0147] LNTII crystals are added as seeds to a solution containing LNTII. The LNTII crystals added as seeds can be crystals obtained by the method of the present invention. The seed crystals may be added before the step of precipitating the crystals or during the step of precipitating the crystals, as long as they are added before the LNTII crystals are precipitated in the solution. The seed crystals are added so that the concentration in the solution is usually 0.1 to 10% by mass, preferably 0.1 to 2.0% by mass.

[0148] Examples of methods for precipitating LNTII crystals in the solution include cooling the solution, adding a poor solvent dropwise, etc. These methods can also be used in combination of one or more.

[0149] In the method for cooling the solution, water can be used as the solvent for the solution.

[0150] In the method for cooling the solution, the temperature of the solution is usually 0 to 20°C, preferably 5 to 20°C, and most preferably 5 to 10°C, and the cooling time is usually 2 to 72 hours, preferably 12 to 72 hours, and most preferably 24 to 72 hours.

[0151] A poor solvent is a solvent in which a chemical substance has low solubility. It may also be a mixture of a poor solvent and a solvent. The poor solvent may preferably be ethanol, methanol, n-propanol, isopropyl alcohol, acetone, etc. Mixing the solutions reduces the solubility of the substance in the solvent mixture, causing it to precipitate.

[0152] After precipitating LNTII crystals as described above, the precipitated crystals may be further aged for usually 0.5 to 12 hours, preferably 2 to 12 hours, and most preferably 2 to 6 hours before the step of collecting the precipitated crystals.

[0153] "Aging" refers to temporarily stopping the process of precipitating LNTII crystals and allowing the crystals to grow. After the crystals have been aged, the process of precipitating LNTII crystals may be resumed.

[0154] The method for collecting LNTII crystals is not particularly limited, but examples include filtration, pressure filtration, suction filtration, and centrifugation. Furthermore, to reduce adhesion of the mother liquor to the crystals and improve their quality, the crystals can be washed appropriately after collection. The solution used for washing the crystals is not particularly limited, but water, methanol, ethanol, acetone, n-propanol, isopropyl alcohol, and solutions in which one or more selected from these are mixed in any proportion can be used.

[0155] The thus obtained wet crystals can be dried to obtain the crystals of the present invention. That is, the method for producing the crystals of the present invention may further include a step of drying the LNTII crystals.

[0156] The drying conditions may be any method that can maintain the morphology of the LNTII crystals, and examples thereof include reduced pressure drying, vacuum drying, fluidized bed drying, and forced air drying.

[0157] The drying temperature may be any temperature within the range in which the adhering water or solution can be removed, but is preferably 80°C or lower, more preferably 60°C or lower, and even more preferably 10°C or lower.

[0158] High-purity LNTII crystals can be obtained by the above method. The preferred crystals of the present invention are LNTII·6.0 hydrate crystals.

[0159] It can be confirmed by single crystal X-ray analysis described below that the crystal of the present invention is a hexahydrate crystal. The purity of the obtained LNTII crystals is usually 80% or more, preferably 90% or more, more preferably 97% or more, 98% or more, even more preferably 99% or more, and most preferably 99.5% or more.

[0160] Methods for analyzing the structure of the obtained LNTII crystals include, for example, powder X-ray diffraction using CuKα as an X-ray source and single crystal X-ray diffraction.

[0161] When water is used as a solvent when cooling the solution obtained in step (iii) is selected in step (iv), the LNTII crystals obtained by the above method are preferably LNTII crystals having a peak at the diffraction angle (2θ) described in (i) below in powder X-ray diffraction using CuKα as an X-ray source, and more preferably LNTII crystals having a peak at the diffraction angle (2θ) described in (ii) below in addition to the diffraction angle (2θ) described in (i) below. (i)19.3, 19.6, 8.2, 19.9, 23.1 (ii)23.6, 21.4, 9.9, 26.1, 25.0

[0162] When selecting a step in step (iv) in which a poor solvent is added dropwise to the solution obtained in step (iii), if a poor solvent other than water, such as ethanol, methanol, n-propanol, isopropyl alcohol, or acetone, is used, the LNTII crystals obtained by the above method are preferably LNTII crystals having a peak at the diffraction angle (2θ) described in (iii) below in powder X-ray diffraction using CuKα as an X-ray source, and more preferably LNTII crystals having a peak at the diffraction angle (2θ) described in (iv) below in addition to the diffraction angle (2θ) described in (iii) below. (iii) 18.6, 19.8, 9.5, 20.9, 21.5 (iv)26.9, 22.0, 12.7, 8.3, 29.4

[0163] Of the above LNTII crystals, LNTII crystals having a peak at the diffraction angle (2θ) described in (i) below are preferred, and LNTII crystals having a peak at the diffraction angle (2θ) described in (ii) below in addition to the diffraction angle (2θ) described in (i) below are more preferred.

[0164] [Analysis example] (1) Analysis and quantification of LNTII or LNT In the Examples, analysis and quantification of LNTII or LNT were carried out according to the following procedures. After the cultivation, the culture medium containing the microorganisms was centrifuged, and the supernatant was collected. The LNTII or LNT contained in the supernatant was analyzed using a sugar analyzer ICS-6000 (manufactured by Thermo Fisher Scientific).

[0165] [Analysis conditions] Column: CarboPAC PA10 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: (0-18 min) Gradient from 80:20:0 to 70:20:10 (18~20 minutes)70:20:10 (20~25 minutes)80:20:0 Flow rate: 0.8mL / min Detector: Pulsed amperometric detector

[0166] (2) Single crystal X-ray structure analysis Single crystal X-ray structural analysis was carried out using a single crystal X-ray diffractometer Synergy-R (manufactured by Rigaku Corporation) according to the instruction manual.

[0167] (3) Powder X-ray Diffraction Powder X-ray diffraction was performed using a powder X-ray diffractometer (XRD) Ultima IV (manufactured by Rigaku Corporation) according to the manufacturer's instructions. [Example]

[0168] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0169] [Example 1] Construction of microorganisms used to produce lacto-N-triose II (LNTII) (1) Obtaining 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: 9 and 10 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.

[0170] (2) Construction of E. coli lacking β-galactosidase (lacZ), lactose permease (lacY), and regulatory factor YhbJ activities E. coli 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 the regulatory factor YhbJ protein (hereinafter referred to as the yhbJ gene) was constructed using the following method. Note that lacZ and lacY form an operon on the E. coli genome.

[0171] 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.

[0172] [Table 1]

[0173] 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 40 bp to approximately 1000 bp downstream of the termination codon of the lacY gene.

[0174] PCR was performed using a mixture of lacZ upstream 1, lacY downstream 1, and cat-sacB fragments in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 12 and 14 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.

[0175] PCR was performed using a mixture of lacZ upstream 2 and lacY downstream 2 in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 12 and 14 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.

[0176] The lacZY::cat-sacB fragment was introduced by electroporation into the W3110S3GK strain (NBRC114657) harboring the plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)], which contains the gene encoding the λ recombinase. Transformants that exhibited chloramphenicol resistance and sucrose sensitivity (transformants in which the lacZY gene had been replaced with lacZY::cat-sacB) were obtained.

[0177] The ΔlacZY fragment was introduced into the transformant by electroporation to obtain transformants that were sensitive to chloramphenicol and resistant to sucrose (transformants in which lacZY::cat-sacB had been replaced by ΔlacZY). From these, an ampicillin-sensitive transformant (transformant in which pKD46 had been lost) was further obtained and designated W3110S3GKΔlacZY.

[0178] 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.

[0179] [Table 2]

[0180] yhbJ upstream 1 and yhbJ upstream 2 comprise the region from the initiation codon of the yhbJ gene to about 1000 bp upstream of the initiation codon. yhbJ downstream 1 and yhbJ downstream 2 comprise the region from the termination codon of the yhbJ gene to about 1000 bp downstream of the termination codon.

[0181] PCR was performed using a mixture of yhbJ upstream 1, yhbJ downstream 1, and cat-sacB fragments in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 18 and 20 as a primer set to obtain a DNA fragment (hereinafter referred to as yhbJ::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the yhbJ gene.

[0182] PCR was performed using a mixture of yhbJ upstream 2 and yhbJ downstream 2 in an equimolar ratio as a template and DNA consisting of the base sequences represented by sequence numbers 18 and 20 as a primer set, to obtain a DNA fragment (hereinafter referred to as ΔyhbJ) that does not contain yhbJ and consists of a sequence in which the upstream and downstream parts of yhbJ are directly linked.

[0183] The yhbJ::cat-sacB fragment was introduced into the W3110S3GKΔlacZY strain constructed above by electroporation, and a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (a transformant in which yhbJ had been replaced by yhbJ::cat-sacB) was obtained.

[0184] The ΔyhbJ fragment was introduced into the transformant by electroporation to obtain a transformant that exhibited chloramphenicol sensitivity and sucrose resistance (a transformant in which yhbJ::cat-sacB had been replaced with ΔyhbJ). Furthermore, a transformant that exhibited ampicillin sensitivity (a transformant in which pKD46 had been lost) was obtained. This transformant was designated the INTC strain.

[0185] (3) Creation of a microorganism with β1,3-N-acetylglucosamine transferase activity (hereinafter referred to as LgtA activity). Escherichia coli carrying a plasmid for expressing a gene encoding β1,3-N-acetylglucosamine transferase (hereinafter referred to as NpLgtA) derived from Neisseria polysaccharea ATCC43768 strain, which consists of the amino acid sequence shown in SEQ ID NO:6, was constructed by the following method. PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Table 3 as a primer set and DNAs shown in "Template" in Table 3 as templates to obtain each amplified DNA fragment.

[0186] [Table 3]

[0187] The DNA represented by SEQ ID NO:5 is a codon-optimized DNA for expression in Escherichia coli of the nucleotide sequence of the gene encoding β1,3-N-acetylglucosamine transferase from Neisseria polysaccharina ATCC43768 strain represented by SEQ ID NO:6. The genomic DNA of Escherichia coli W3110 strain was prepared by standard methods. The nucleotide sequences represented by SEQ ID NOs:24 and 25 each contain complementary sequences at their 5' ends.

[0188] PCR was performed using a mixture of the NplgtA fragment and the lacY fragment in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 23 and 26 as a primer set to obtain a DNA fragment (hereinafter referred to as NplgtA-lacY) in which the two fragments were linked together.

[0189] PCR was performed using a primer set consisting of DNAs with the nucleotide sequences represented by SEQ ID NOs: 27 and 28 and the plasmid pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385) as a template to obtain a vector fragment of approximately 4.7 kb. In this case, the base sequences represented by SEQ ID NOs: 23 and 27, and SEQ ID NOs: 26 and 28 each contain a complementary sequence at the 5' end.

[0190] The NplgtA-lacY fragment and the vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the NplgtA expression plasmid pLNTII.

[0191] The INTC strain constructed in Example 1(2) was transformed with the expression plasmid pLNTII to construct an E. coli strain carrying pLNTII, which was designated INTC / pLNTII strain.

[0192] (4) Creation of microorganisms with enhanced expression of transporter genes E. coli carrying a plasmid for expressing the transporter gene derived from the W3110 strain was constructed as follows. PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Table 4 as a primer set and the chromosomal DNA of the W3110 strain as a template to obtain each amplified DNA fragment.

[0193] [Table 4]

[0194] Chromosomal DNA of Escherichia coli W3110 strain was prepared by standard methods. Using DNA consisting of the base sequences represented by SEQ ID NOs: 29 and 30 as a primer set, PCR was carried out using the plasmid pSTV29 (manufactured by Takara Bio Inc.) as a template to obtain a vector fragment of approximately 3 kb.

[0195] In this case, the base sequences represented by SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 29, and SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 30 each contain a complementary sequence at the 5' end. The amplified DNA fragments and vector fragments obtained above were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain 12 types of plasmids for expressing the transporter genes derived from the W3110 strain.

[0196] The INTC / pLNTII strain constructed in Example 1(3) was transformed with the above 12 transporter gene expression plasmids to construct 12 LNTII-producing strains in which expression of each transporter gene was enhanced.

[0197] [Example 2] Production of lacto-N-triose II (LNTII) The INTC / pLNTII strain obtained in Example 1 and 12 LNTII-producing strains in which expression of each transporter gene was enhanced were cultured on an LB plate containing 100 mg / L kanamycin at 30° C. for 24 hours, and then inoculated into a large test tube containing 2 mL of LB medium containing 100 mg / L kanamycin and cultured with shaking for 15 hours at 30° C. When culturing LNTII strains in which expression of each transporter gene was enhanced, 25 mg / L chloramphenicol was added to the medium.

[0198] Then, 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 [glucose 30 g / L, lactose monohydrate 10 g / L, magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, casamino acids 5 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L (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 the medium was cultured with shaking at 30°C for 29 hours. When culturing an LNTII-producing strain in which expression of each transporter gene was enhanced, 25 mg / L of chloramphenicol was added to the medium, and 6 hours after the start of culturing, IPTG was added to a concentration of 1 mM.

[0199] After the culture was completed, the culture medium was diluted appropriately and centrifuged, and the LNTII contained in the supernatant was analyzed using the ICS-6000 sugar analyzer. The results are shown in Figure 2.

[0200] As shown in Figure 2, enhanced expression of the ydeA gene or mdfA gene resulted in significantly higher LNTII productivity than the INTC / pLNTII strain, suggesting that the proteins encoded by the ydeA gene and mdfA gene both possess LNTII transport activity.

[0201] Furthermore, although not shown in the data, enhanced expression of the setA and setB genes of the W3110 strain also improved LNTII productivity, suggesting that the proteins encoded by these genes also have LNTII transport activity.

[0202] [Example 3] Construction of microorganisms used for producing lacto-N-tetraose (LNT) (1) Construction of E. coli lacking the ydeA gene Using genomic DNA of the 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 5 to amplify each DNA fragment.

[0203] [Table 5]

[0204] ydeA upstream 1 and ydeA upstream 2 comprise the region from the initiation codon of the ydeA gene to about 1000 bp upstream of the initiation codon. ydeA downstream 1 and ydeA downstream 2 comprise the region from the termination codon of the ydeA gene to about 1000 bp downstream of the termination codon.

[0205] A mixture of ydeA upstream 1, ydeA downstream 1, and the cat-sacB fragment obtained in Example 1(1) in an equimolar ratio was used as a template, and PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 56 and 58 as a primer set to obtain a DNA fragment (hereinafter referred to as ydeA::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the ydeA gene.

[0206] PCR was performed using a mixture of ydeA upstream 2 and ydeA downstream 2 in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 56 and 58 as a primer set, to obtain a DNA fragment (hereinafter referred to as ΔydeA) that does not contain ydeA and consists of a sequence in which the upstream and downstream of ydeA are directly linked.

[0207] The INTC strain constructed in Example 1(2) was reintroduced with the plasmid pKD46, and the ydeA::cat-sacB fragment was then introduced by electroporation to obtain a transformant that exhibited chloramphenicol resistance and sucrose sensitivity (a transformant in which the ydeA gene had been replaced with ydeA::cat-sacB).

[0208] The ΔydeA fragment was introduced into the transformant by electroporation to obtain transformants that were sensitive to chloramphenicol and resistant to sucrose (transformants in which ydeA::cat-sacB had been replaced with ΔydeA). From these, a transformant that was sensitive to ampicillin (transformants in which pKD46 had been lost) was further obtained and designated INTCΔydeA.

[0209] (2) Construction of microorganisms with β1,3-galactosyltransferase (GalT) and LgtA activities Escherichia coli carrying a plasmid co-expressing a gene encoding β1,3-galactosyltransferase (hereinafter referred to as Cvβ3GalT) derived from Chromobacterium violaceum ATCC553 strain, which has the amino acid sequence shown in SEQ ID NO: 8, and a gene encoding NpLgtA, was constructed by the following method.

[0210] PCR was carried out using DNA consisting of the nucleotide sequences shown in SEQ ID NO: 61 and 62 as a primer set and DNA shown in SEQ ID NO: 7 as a template to obtain a Cvβ3galT fragment.

[0211] The DNA represented by SEQ ID NO: 7 was prepared by artificial synthesis with reference to ACS Catal. 2019, 9(12), 10721-10726. The DNA represented by SEQ ID NO: 8 is a codon-optimized DNA for expression in Escherichia coli of the base sequence of the gene encoding β1,3-galactosyltransferase derived from Chromobacterium violaceum ATCC553 strain.

[0212] Using the plasmid pLNTII obtained in Example 1(3) as a template, PCR was performed using a primer set consisting of DNAs with the nucleotide sequences shown in SEQ ID NOs: 27 and 63 to obtain a vector fragment of approximately 7.0 kb. The nucleotide sequences shown in SEQ ID NOs: 27 and 61, and SEQ ID NOs: 62 and 63 each contain a complementary sequence at their 5' ends.

[0213] The Cvβ3galT fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the Cvβ3galT and NplgtA expression plasmid pLNT.

[0214] The above expression plasmid pLNT was used to transform the INTC strain constructed in Example 1(2) and the INTCΔydeA strain constructed in Example 3(1) to construct E. coli carrying pLNT, which were named the INTC / pLNT strain and the INTCΔydeA / pLNT strain, respectively.

[0215] [Example 4] Production of lacto-N-tetraose (LNT) The INTC / pLNT strain and INTCΔydeA / pLNT strain obtained in Example 3 were cultured on an LB plate containing 100 mg / L kanamycin at 30°C for 17 hours, and then inoculated into a large test tube containing 2 mL of LB medium containing 100 mg / L kanamycin and cultured with shaking at 30°C for 15 hours.

[0216] Then, 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 [glucose 30 g / L, lactose monohydrate 10 g / L, magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, casamino acids 5 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L (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 the medium was cultured with shaking at 30°C for 29 hours.

[0217] After the culture was completed, the culture medium was diluted appropriately and centrifuged, and the LNTII and LNT contained in the supernatant were analyzed using a sugar analyzer ICS-6000. The results are shown in Table 6.

[0218] [Table 6]

[0219] As a result, it was found that the INTCΔydeA / pLNT strain exhibited higher LNT productivity than the INTC / pLNT strain, and that LNT productivity was improved by deleting the ydeA gene.

[0220] [Example 5] Preparation of an aqueous solution containing high-purity lacto-N-triose II (LNTII) The LNTII-producing Escherichia coli strain constructed in Example 1 was cultured in a medium, and the resulting culture solution was adjusted to pH 3.0 with sulfuric acid and heated at 70°C for 40 minutes. Subsequently, the cells were removed by centrifugation (4°C, 6000 G, 7000 rpm, 10 minutes), and the supernatant was recovered.

[0221] The supernatant was passed through a column packed with a cation exchange resin and then a column packed with an anion exchange resin, and a fraction containing LNTII was collected. + (Mitsubishi Chemical Corporation), and A111S (OH) as an anion exchange resin. - A 1000-kJ / kg (manufactured by Prolite) was used.

[0222] The obtained fraction was filtered using a UF membrane module (SIP-0013, manufactured by Asahi Kasei Corporation), and the obtained filtrate was concentrated using an evaporator to obtain an LNTII-containing aqueous solution with an LNTII concentration of 800 g / L and an HPLC purity of 81% (area %).

[0223] [Example 6] Obtaining crystals of lacto-N-triose II (LNTII) 25 mL of the LNTII-containing aqueous solution obtained in Example 5 was allowed to stand at 4°C for 120 hours to precipitate a crystalline solid. The solid was collected by filtration and then dried under air at room temperature for 12 hours to yield 11.7 g of crystals. Content analysis by HPLC revealed that the LNTII content of the obtained crystals was 80.8% (HPLC purity: 84.3%). The chromatogram obtained when the crystals were analyzed using an ICS-6000 sugar analyzer is shown in Figure 3.

[0224] [Example 7] Preparation of lacto-N-triose II (LNTII) crystals by cooling crystallization The LNTII-containing aqueous solution obtained in Example 5 was diluted with water to a concentration of 500 g / L. 36 mL of this solution was stirred while maintaining the temperature at 40°C, and the crystals obtained in Example 6 were added as seed crystals to precipitate crystals. The crystals were stirred and aged at room temperature for 24 hours, and then stirred at 4°C for an additional 24 hours. The crystalline slurry was then centrifuged using a basket centrifuge (manufactured by Kokusan Co., Ltd.), and the resulting wet crystals were dried under reduced pressure at 30°C to yield 2.8 g of crystals. The LNTII content in the resulting crystals was 64.4% (HPLC purity: 84.6%). The chromatogram obtained when the crystals were analyzed using an ICS-6000 sugar analyzer is shown in Figure 4.

[0225] To determine the crystal structure, single-crystal X-ray analysis was performed. The diffraction data were analyzed using software (CrysAlisPro, Rigaku) ​​for integration and absorption correction. The initial structure was obtained using SHELX T (Acta Crystallogr. Sect. A 2015, 71, 3-8), and refined using SHELX L (Acta Crystallogr. Sect. C 2015, 71, 3-8). The results are shown in Table 7, and the ORTEP diagram is shown in Figure 5.

[0226] [Table 7]

[0227] As a result of the above analysis, it was confirmed that the crystal has the crystal structure of LNTII and is LNTII·6.0 hydrate, which has six water molecules in the unit cell.

[0228] Next, powder X-ray diffraction of the crystals was performed. From the diffraction results, the diffraction angles of peaks with a relative intensity ratio (I / I0) of 10 or more are shown in Table 8. In the table, "2θ" indicates the diffraction angle (2θ°), "d value" indicates the lattice spacing (dÅ), and "relative intensity" indicates the relative intensity ratio (I / I0).

[0229] [Table 8]

[0230] [Example 8] Preparation of lacto-N-triose II (LNTII) crystals by antisolvent addition method The LNTII-containing aqueous solution obtained in Example 5 was used to prepare an LNTII solution with a concentration of 400 g / L. While stirring 40 mL of this solution at 40°C, 60 mL of ethanol was added dropwise as a poor solvent. The crystals obtained in Example 6 were added as seed crystals to precipitate crystals. The crystals were stirred and aged at room temperature for 72 hours, and then stirred at 4°C for an additional 24 hours. The crystalline slurry was then centrifuged in a basket centrifuge to obtain 16.1 g of wet crystals. The obtained wet crystals were dried under reduced pressure at 30°C under vacuum conditions to obtain 7.2 g of crystals. The LNTII content in the crystals was 72.9% (HPLC purity: 89.7%). The chromatogram obtained when the crystals were analyzed using an ICS-6000 sugar analyzer is shown in Figure 6.

[0231] From the results of powder X-ray diffraction of the obtained crystals, the diffraction angles of peaks with a relative intensity ratio (I / I0) of 10 or more are shown in Table 9. In the table, "2θ" indicates the diffraction angle (2θ°), "d value" indicates the lattice spacing (dÅ), and "relative intensity" indicates the relative intensity ratio (I / I0).

[0232] [Table 9]

[0233] From the above results, it was confirmed that the LNTII crystals obtained by the antisolvent addition method described in Example 8 are identical to the crystals described in Patent Document 4.

[0234] 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. 2021-207590) filed on December 21, 2021, the contents of which are incorporated herein by reference. [Sequence List Free Text]

[0235] SEQ ID NO: 1: Nucleotide sequence of ydeA derived from E. coli W3110 SEQ ID NO: 2: Amino acid sequence of ydeA from E. coli W3110 SEQ ID NO: 3: Nucleotide sequence of mdfA derived from E. coli W3110 SEQ ID NO: 4: Amino acid sequence of mdfA from E. coli W3110 SEQ ID NO: 5: Nucleotide sequence of codon-optimized NplgtA SEQ ID NO: 6: Amino acid sequence of NpLgtA SEQ ID NO: 7: Nucleotide sequence of codon-optimized Cvβ3galT SEQ ID NO: 8: Amino acid sequence of Cvβ3GalT SEQ ID NOs: 9 and 10: Nucleotide sequences of primers for amplifying catsacB fragment SEQ ID NOs: 11 and 12: Nucleotide sequences of primers for amplifying lacZ upstream 1 SEQ ID NOs: 13 and 14: Nucleotide sequences of primers for amplifying lacY downstream 1 SEQ ID NO: 15: Nucleotide sequence of primer for amplifying lacZ upstream 2 SEQ ID NO: 16: Nucleotide sequence of primer for amplifying lacY downstream 2 SEQ ID NOs: 17 and 18: Nucleotide sequences of primers for amplifying yhbJ upstream 1 SEQ ID NOs: 19 and 20: Nucleotide sequences of primers for amplifying yhbJ downstream 1 SEQ ID NO: 21: Nucleotide sequence of primer for amplifying yhbJ upstream 2 SEQ ID NO: 22: Nucleotide sequence of primer for amplifying yhbJ downstream 2 SEQ ID NOs: 23 and 24: Nucleotide sequences of primers for amplifying the NplgtA fragment SEQ ID NOs: 25 and 26: Nucleotide sequences of primers for amplifying the lacY fragment SEQ ID NOs: 27 and 28: Nucleotide sequences of primers for amplifying pUAKQE31 SEQ ID NOs: 29 and 30: Nucleotide sequences of primers for amplifying pSTV29 SEQ ID NOs: 31 and 32: Nucleotide sequences of primers for amplifying the ydeA fragment SEQ ID NOs: 33 and 34: Nucleotide sequences of primers for amplifying the mdfA fragment SEQ ID NOs: 35 and 36: Nucleotide sequences of primers for amplifying setC fragment SEQ ID NOs: 37 and 38: Nucleotide sequences of primers for amplifying the ynfM fragment SEQ ID NOs: 39 and 40: Nucleotide sequences of primers for amplifying the mdtD fragment SEQ ID NOs: 41 and 42: Nucleotide sequences of primers for amplifying the yfcJ fragment SEQ ID NOs: 43 and 44: Nucleotide sequences of primers for amplifying the emrD fragment SEQ ID NOs: 45 and 46: Nucleotide sequences of primers for amplifying the ydhC fragment SEQ ID NOs: 47 and 48: Nucleotide sequences of primers for amplifying the ybdA fragment SEQ ID NOs: 49 and 50: Nucleotide sequences of primers for amplifying the ydeE fragment SEQ ID NOs: 51 and 52: Nucleotide sequences of primers for amplifying mhpT fragment SEQ ID NOs: 53 and 54: Nucleotide sequences of primers for amplifying kgtP fragment SEQ ID NOs: 55 and 56: Nucleotide sequences of primers for amplifying ydeA upstream 1 SEQ ID NOs: 57 and 58: Nucleotide sequences of primers for amplifying ydeA downstream 1 SEQ ID NO: 59: Nucleotide sequence of primer for amplifying ydeA upstream 2 SEQ ID NO: 60: Nucleotide sequence of primer for amplifying ydeA downstream 2 SEQ ID NOs: 61 and 62: Nucleotide sequences of primers for amplifying the Cvβ3galT fragment SEQ ID NO: 63: Primer for amplifying pLNTII fragment SEQ ID NO: 64: Nucleotide sequence of setA derived from E. coli W3110 SEQ ID NO: 65: Amino acid sequence of setA from E. coli W3110 SEQ ID NO: 66: Nucleotide sequence of setB derived from E. coli W3110 SEQ ID NO: 67: Amino acid sequence of setB from E. coli W3110

Claims

1. A microorganism in which the activity of a protein according to any one of [1] to [3] below is enhanced and the productivity of lacto-N-triose II (LNT II) is improved compared to that of a parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4. [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport activity. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having oligosaccharide transport activity.

2. A microorganism in which the activity of a protein according to any one of [1] to [3] below is reduced or inactivated, and which has improved productivity of oligosaccharides having a lacto-N-triose II (LNTII) backbone compared to a parent strain, wherein the oligosaccharides having an LNTII backbone are lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT). [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4. [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 in which 1 to 20 amino acids have been deleted, substituted, inserted or added, and having oligosaccharide transport activity. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having oligosaccharide transport activity.

3. A method for producing oligosaccharides, comprising preparing the microorganism according to claim 1 or 2 and producing oligosaccharides in a culture using the microorganism.

4. A method for producing crystals of LNTII from a culture obtained by culturing the microorganism according to claim 1, the method comprising the following steps (i) to (iv): wherein the LNTII crystals are LNTII-6.0 hydrate crystals and have peaks at diffraction angles (2θ) of 19.3, 19.6, 8.2, 19.9, and 23.1 in powder X-ray diffraction using CuKα as an X-ray source: (i) centrifuging the culture to obtain a supernatant from which the bacterial cells have been removed; (ii) subjecting the supernatant obtained in step (i) to cation exchange and anion exchange to obtain a solution from which ions have been removed from the supernatant; (iii) concentrating the solution obtained in step (ii) using an evaporator; and (iv) cooling the solution obtained in step (iii) or adding an anti-solvent dropwise to the solution obtained in step (iii) to obtain crystals of LNTII.

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