The 3apos is improved; -microorganisms and 3apos for the productivity of sialic acid lactose; method for producing-sialyllactose

Abstract

The present invention pertains to a microorganism in which the activity of a protein comprising an amino acid sequence obtained by deleting 20-37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is enhanced, and the productivity of 3 '-sialyllactose is improved compared to a parent strain.

Description

Technical Field

[0001] The present invention relates to a microorganism having improved productivity of 3'-sialyllactose and a method for producing 3'-sialyllactose. Background Art

[0002] 3'-Sialyl lactose (hereinafter referred to as 3'SL) is an acidic oligosaccharide contained in human breast milk and is the main acidic human milk oligosaccharide in breast milk together with 6'-Sialyl lactose (Non-Patent Document 1). 3'SL is known to play an important role in the language development, cognitive function and various health of infants (Non-Patent Documents 2, 3, 4).

[0003] 3'SL is contained in human breast milk, and only in trace amounts in the milk of other mammals such as cow's milk (Non-Patent Document 5). Infant formula milk powder is mainly processed and manufactured by adding necessary nutrients to raw materials such as cow's milk, so the content of human milk oligosaccharides including 3'SL is lower than that of breast milk. In order to make infant formula milk powder achieve the same effect as breast milk, it is preferred to add 3'SL to infant formula milk powder (Non-Patent Document 6).

[0004] Non-Patent Document 6 discloses various methods for obtaining human milk oligosaccharides, such as extraction, chemical synthesis, and fermentation using recombinant microorganisms. Among these, the fermentation using recombinant microorganisms is considered to be the most economical and reasonable method.

[0005] As disclosed in Non-Patent Document 7 and Patent Document 1, the method for producing 3'SL using recombinant microorganisms is generally as follows: cytidine-5'-monophosphate (hereinafter also referred to as "CMP")-sialic acid is synthesized from cheap carbon sources such as glycerol and glucose within the cell, and α2,3-sialyltransferase is used to transfer the sialic acid residue of CMP-sialic acid to lactose added from the outside to produce 3'SL.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2015 / 037698

[0009] Non-patent literature

[0010] Non-patent literature 1: Nutrition Reviews 75 (2017) 920-933

[0011] Non-patent literature 2: The American Journal of Clinical Nutrition 114 (2021) 588-597

[0012] Non-patent literature 3: Nutrients 13 (2021) 4191

[0013] Non-patent literature 4: Nature Metabolism 2 (2020) 678-687

[0014] Non-patent document 5: Journal of Dairy Science 86 (2003) 52-59

[0015] Non-patent literature 6: Nutrition Review 74 (2016) 635-644

[0016] Non-patent document 7: Journal of Biotechnology 134 (2008) 261-265

[0017] Non-patent document 8: Journal of Biological Chemistry 271 (1996) 28271-28276

[0018] Non-patent document 9: Journal of Biological Chemistry 275 (2000) 3896-3906

[0019] Non-patent document 10: Journal of Applied Glycoscience 56 (2009) 77-82

[0020] Non-patent document 11: Journal of Biological Chemistry 282 (2007) 29274-29802

[0021] Non-patent document 12: Journal of the American Chemical Society 127 (2005) 17618-17619

[0022] Non-patent document 13: Molecular Microbiology 39 (2001) 341-350

[0023] Non-patent document 14: Biotechnology Letters 30 (2008) 671-676

[0024] Non-patent document 15: Journal of Biological Chemistry 286 (2011) 37237-37248 Summary of the invention

[0025] Problems to be solved by the invention

[0026] In the production method using recombinant microorganisms, the activity of α2,3-sialyltransferase that transfers sialic acid residues to lactose is considered to be important for the productivity of 3'SL. Various α2,3-sialyltransferases have been cloned from microorganisms and successfully expressed in Escherichia coli and the like (Non-patent Documents 8, 9, 10, 11, 12, 13). In addition, in order to improve the productivity of 3'SL compared to the past, a highly active α2,3-sialyltransferase that can efficiently transfer sialic acid residues to lactose is required.

[0027] It is known that sialyltransferases usually have a region at the N-terminus of the amino acid sequence that is not related to activity. Non-patent literatures 14 and 15 disclose methods of cutting the N-terminus of the amino acid sequence and expressing it when an exogenous sialyltransferase is expressed in Escherichia coli. It is known that the expression and activity of the sialyltransferase will be changed by using this method, but it is not clear which region will be cut to obtain useful activity.

[0028] Therefore, an object of the present invention is to provide a microorganism in which the activity of a protein lacking a specific amino acid residue is enhanced and the productivity of 3'SL is improved compared with the parent strain. Another object is to provide a method for producing 3'SL using the microorganism.

[0029] Methods used to solve problems

[0030] The present inventors conducted intensive studies and found that a microorganism having enhanced activity of a protein consisting of an amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 are deleted has improved productivity of 3'SL compared with the parent strain, thereby completing the present invention.

[0031] That is, the present invention is as follows.

[0032] <1> A microorganism, wherein the activity of a protein consisting of an amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 are deleted is enhanced, and the productivity of 3'-sialyllactose is increased compared with that of a parent strain.

[0033] <2> The microorganism according to <1> above, wherein the protein consisting of the amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 are deleted is a protein described in any one of the following [1] to [3].

[0034] [1] A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10.

[0035] [2] A mutant protein having α2,3-sialyltransferase activity, which comprises an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added to the amino acid sequence represented by SEQ ID NO: 4, 6, 8 or 10.

[0036] [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10 and having α2,3-sialyltransferase activity.

[0037] <3> A method for producing 3'-sialyllactose, comprising the steps of preparing the microorganism described in <1> or <2> above and producing 3'-sialyllactose in a culture using the microorganism.

[0038] Effects of the Invention

[0039] The microorganism of one embodiment of the present invention can improve the productivity of 3'SL due to the enhanced activity of the protein lacking specific amino acid residues. According to the production method of one embodiment of the present invention, 3'SL can be produced by using a culture obtained by culturing the above microorganism. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram showing an example of the 3'SL biosynthetic pathway. DETAILED DESCRIPTION

[0041] 1. Microorganisms that improve the productivity of 3'SL

[0042] Examples of microorganisms according to one embodiment of the present invention include microorganisms in which the activity of the protein described in [A] below is enhanced and the productivity of 3'SL is improved compared to the parent strain.

[0043] [A] A protein consisting of an amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 are deleted.

[0044] <Productivity>

[0045] In this specification, productivity refers to the ability to accumulate the target oligosaccharide produced by the microorganism in the culture of the microorganism. The productivity of oligosaccharides produced by the microorganism can be confirmed by detecting the oligosaccharides in the culture of the microorganism using an analysis device described later.

[0046] Preferably, a protein consisting of an amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 are deleted has higher α2,3-sialyltransferase activity than a protein consisting of an amino acid sequence shown in SEQ ID NO: 2. A protein consisting of an amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 are deleted refers to a protein consisting of an amino acid sequence in which only 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 are deleted.

[0047] α2,3-sialyltransferase activity refers to an activity of transferring a sialic acid residue to the galactose at the terminal of the saccharide via an α2,3 bond using CMP-sialic acid and an acceptor saccharide as substrates to obtain a sialic acid-containing saccharide.

[0048] Examples of CMP-sialic acid include CMP-N-acetylneuraminic acid (hereinafter also referred to as "CMP-NeuAc").

[0049] The acceptor sugar used as a substrate may be any of oligosaccharides, polysaccharides, and complex sugars such as glycoproteins and glycolipids, as long as they can serve as a substrate for sialyllactose.

[0050] Examples of the oligosaccharide or polysaccharide that can be a substrate for sialyllactose include an oligosaccharide or polysaccharide having galactose at the non-reducing end, or an oligosaccharide or polysaccharide having N-acetylneuraminic acid (hereinafter also referred to as "NeuAc") at the non-reducing end. Preferably, an oligosaccharide having a structure selected from the group consisting of lactose, globotriose, N-acetyllactosamine, lacto-N-tetraose, lacto-N-neotetraose, Lewis a and Lewis X at the non-reducing end can be mentioned. More preferably, lactose can be mentioned.

[0051] Examples of complex carbohydrates include those in which proteins, lipids, and the like are bonded to the above-mentioned oligosaccharides and polysaccharides.

[0052] As the saccharide containing sialic acid, a saccharide containing NeuAc is preferred. Examples of the saccharide containing NeuAc include saccharides obtained by adding NeuAc to the above-mentioned acceptor saccharides, and preferably saccharides containing oligosaccharides having NeuAcα2-3Galβ1-4Glc at the non-reducing end, and 3'SL is more preferred.

[0053] An example of the 3'SL biosynthetic pathway is shown in Figure 1 It is speculated that due to the enhancement of the α2,3-sialyltransferase activity of the microorganism, the α2,3 bonding of the NeuAc residue to the galactose residue of lactose using CMP-NeuAc and lactose as substrates was promoted, and the productivity of 3'SL was improved.

[0054] The protein consisting of an amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 are deleted as described in [A] above may be a protein as described in any one of the following [1] to [3].

[0055] [1] A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10.

[0056] [2] A mutant protein having α2,3-sialyltransferase activity, which comprises an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added to the amino acid sequence represented by SEQ ID NO: 4, 6, 8 or 10.

[0057] [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10 and having α2,3-sialyltransferase activity.

[0058] A mutant protein refers to a protein in which amino acid residues in a base protein are artificially deleted or substituted, or in which amino acid residues are inserted or added into the protein.

[0059] In the mutant protein of [2] above, the deletion, substitution, insertion or addition of amino acids may be deletion, substitution, insertion or addition of 1 to 20 amino acids at any position in 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.

[0060] The amino acid to be deleted, substituted, inserted or added may be either natural or non-natural. As natural amino acids, 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, L-cysteine ​​etc. may be mentioned.

[0061] Examples of amino acids that can be substituted for each other are shown below. Amino acids included in the same group can be substituted for each other.

[0062] Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, o-methylserine, tert-butylglycine, tert-butylalanine, cyclohexylalanine

[0063] Group B: Aspartic acid, Glutamic acid, Isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

[0064] Group C: Asparagine, Glutamine

[0065] Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid

[0066] Group E: Proline, 3-hydroxyproline, 4-hydroxyproline

[0067] Group F: serine, threonine, homoserine

[0068] Group G: Phenylalanine, Tyrosine

[0069] In this specification, a homologous protein refers to a protein that is similar in structure and function to a base protein and the gene encoding the protein is considered to have the same evolutionary origin as the gene encoding the base protein, and is a protein possessed by organisms existing in nature.

[0070] Examples of homologous proteins include amino acid sequences having an identity of preferably 90% or more, particularly preferably 95% or more, to the amino acid sequence of the target protein.

[0071] The identity of the amino acid sequence and the base sequence can be determined using the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] of Karlin and Altschul. Based on the algorithm BLAST, programs called BLASTN and BLASTX were developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing the base sequence by BLASTN based on BLAST, the parameters are set to, for example, Score=100 and wordlength=12. In addition, when analyzing the amino acid sequence by BLASTX based on BLAST, the parameters are set to, for example, score=50 and wordlength=3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific techniques of these analysis methods are well known.

[0072] <Parent strain>

[0073] In this specification, the parent strain refers to the original strain that is the target of genetic modification, transformation, etc. The original strain that is the target of transformation by gene introduction is also called the host strain.

[0074] In the present specification, the parent strain is preferably a prokaryotic organism or a yeast strain, more preferably a prokaryotic organism belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwann yeast, Pichia or Candida, and most preferably Escherichia coli BW25113 (available from the National Institute of Genetics), 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 (manufactured by Stratagenes Co., Ltd.), Escherichia coli W3110S3GK (NBRC114657), Escherichia coli ATCC9637, Serratia aficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes), Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354 or Pseudomonas sp.D-0110 or other prokaryotes, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.

[0075] The parent strain may be a wild strain. When the wild strain does not have the ability to produce substrates for producing 3'SL, such as CMP-NeuAc and / or lactose, the parent strain may be artificially endowed with the ability to produce CMP-NeuAc and / or lactose.

[0076] The production of CMP-NeuAc and / or lactose by the microorganism can be confirmed by, for example, culturing the microorganism in a culture medium and detecting CMP-NeuAc and / or lactose accumulated in the culture using a known method such as HPLC described later.

[0077] Examples of methods for artificially imparting or enhancing the ability to produce CMP-NeuAc and / or lactose include known methods such as the following (a) to (d), and these methods can be used alone or in combination.

[0078] (a) Method for enhancing the expression of at least one enzyme involved in the biosynthetic pathway for producing CMP-NeuAc and / or lactose from sugar

[0079] (b) A method for increasing the copy number of at least one enzyme gene involved in the biosynthetic pathway for producing CMP-NeuAc and / or lactose from sugar

[0080] (c) A method for weakening or eliminating at least one mechanism controlling the biosynthetic pathway for producing CMP-NeuAc and / or lactose from sugars

[0081] (d) A method for weakening or blocking at least one metabolic pathway that branches from the biosynthetic pathway for producing CMP-NeuAc and / or lactose to a metabolite other than the target substance

[0082] <<Activity that is preferably enhanced in the parent strain>>

[0083] The parent strain preferably has at least one activity selected from the following: uridine diphosphate N-acetylglucosamine (hereinafter also referred to as "UDP-GlcNAc") epimerase activity, NeuAc synthase activity, pyruvate carboxylase activity, L-glutamine-D-fructose 6-phosphate aminotransferase activity, NanT activity, CMP-NeuAc synthase activity, lactose permease activity, glucosamine transacetylase activity, N-acetylmannosamine epimerase activity, and more preferably the activity is enhanced. Among them, it is preferred to have CMP-NeuAc synthase activity, and it is further preferred that the activity is enhanced.

[0084] UDP-GlcNAc epimerase is an enzyme responsible for the reaction of generating N-acetylmannosamine (hereinafter also referred to as "ManNAc") from UDP-GlcNAc, and is encoded by the neuC gene. The neuC gene is preferably derived from Flavobacterium psychrophilum strain NBRC100250.

[0085] NeuAc synthase is an enzyme responsible for the reaction of generating NeuAc using ManNAc and phosphoenolpyruvate (hereinafter also referred to as "PEP") as substrates. Examples of DNA encoding NeuAc synthase include DNA encoding CjneuB preferably derived from prokaryotes such as bacteria or yeast, particularly preferably from prokaryotes, particularly preferably from Campylobacter jejuni ATCC43438 strain, or DNA encoding neuB from Rhodobacter capsulatus.

[0086] Pyruvate carboxylase is responsible for the enzyme of the reaction of carboxylating pyruvate to generate oxaloacetate, and is encoded by the pyc gene. PEP is produced by decarbonating oxaloacetate, so Pyc can strengthen the supply of PEP. The pyc gene is preferably from Sinorhizobium meliloti or Corynebacterium glutamicum.

[0087] L-glutamine-D-fructose 6-phosphate aminotransferase is an enzyme responsible for the reaction of generating glucosamine 6-phosphate from fructose 6-phosphate, and is encoded by the glmS gene. The glmS gene is preferably derived from Escherichia coli. Glucosamine 6-phosphate is an intermediate in the NeuAc synthesis pathway, so Glms can enhance the supply of NeuAc.

[0088] NanT is a NeuAc transporter that can recycle NeuAc by taking up NeuAc excreted from the body into the bacteria. The nanT gene encoding the NeuAc transporter is preferably derived from Escherichia coli.

[0089] CMP-NeuAc synthase is an enzyme responsible for the reaction of generating CMP-NeuAc from cytidine-5'-triphosphate (hereinafter also referred to as "CTP") and NeuAc as substrates. DNA encoding CMP-NeuAc synthase preferably includes neuA gene derived from prokaryotes such as bacteria or yeast, particularly preferably from prokaryotes, and most preferably from Pasteurella multocida PM70 strain.

[0090] Lactose permease is a membrane protein that takes lactose into cells and is encoded by the lacY gene. The lacY gene is preferably derived from Escherichia coli. When lactose is added from the outside, lactose permease can promote the uptake of lactose into the bacteria.

[0091] Glucosamine transacetylase is an enzyme responsible for the reaction of generating GlcNAc from glucosamine 6-phosphate. Glucosamine transacetylase from budding yeast is encoded by the GNA1 gene.

[0092] N-acetylmannosamine epimerase is involved in the epimerization of GlcNAc to ManNAc. N-acetylmannosamine epimerase from the cyanobacterium Synechocystis sp. PCC6803 is encoded by the slr1975 gene.

[0093] Therefore, in one embodiment of the present invention, it is preferred that a genetically modified microorganism containing at least one base sequence selected from the group consisting of neuC gene (SEQ ID NO: 19), neuB gene (SEQ ID NO: 18 or ADE86687.1), pyc gene (WP_010970538.1 or WP_208400776.1), glmS gene (Accession No. BAE77559.1), nanT gene (SEQ ID NO: 15 or BAE77267.1), neuA gene (SEQ ID NO: 20), lacY gene (Accession No. BAE76125.1), GNA1 gene (NP_116637.1), and slr1975 gene (BAK50383.1) is used as a parent strain. In one embodiment of the present invention, the above-mentioned genetically modified microorganism preferably has an improved productivity of 3'SL compared to a parent strain that has not been genetically modified.

[0094] As a method for manufacturing an Escherichia coli having at least one activity selected from UDP-GlcNAc epimerase activity, NeuAc synthase activity, pyruvate carboxylase activity, L-glutamine-D-fructose 6-phosphate aminotransferase activity, NanT activity, CMP-NeuAc synthase activity, lactose permease activity, glucosamine transacetylase activity, and N-acetylmannosamine epimerase activity or the activity is strengthened, a known method can be used. Specifically, for example, methods based on various genetic manipulations (Syst Microbiol Biomanufact, 2021, 1, 291) etc. can be cited.

[0095] <<Activities that are preferably decreased or absent in the parent strain>>

[0096] In addition, the parent strain preferably has at least one activity selected from the group consisting of N-acetylglucosamine (hereinafter also referred to as "GlcNAc") transporter activity, N-acetylmannosamine kinase activity, N-acetylglucosamine-6-phosphate-2-isomerase activity, RNase adapter protein activity, transcriptional control factor activity that negatively regulates the transcription of a gene group involved in the assimilation of NeuAc, NeuAc lyase activity, β-galactosidase activity and galactoside transacetylase activity decreased or absent, among which the decreased or absent activities of β-galactosidase and NeuAc lyase are particularly preferred.

[0097] The GlcNAc transporter is involved in the uptake and assimilation of GlcNAc into the bacteria and is encoded by the nagE gene and the nagB gene. By reducing or deleting the activity of the GlcNAc transporter, the production of GlcNAc, which is a substrate of ManNAc, can be promoted.

[0098] N-acetylmannosamine kinase is an enzyme responsible for the reaction of generating N-acetylmannosamine 6-phosphate (hereinafter also referred to as "ManNAc-6P") from ManNAc, and is encoded by the nanK gene. By inhibiting the generation of substances other than NeuAc from ManNAc, the supply of NeuAc can be promoted.

[0099] N-acetylglucosamine-6-phosphate-2-epimerase is an enzyme responsible for the reaction of converting ManNAc-6P to N-acetyl-D-glucosamine 6-phosphate (hereinafter also referred to as "GlcNAc-6P"), and is encoded by the nanE gene.

[0100] The RNase adaptor protein is a transcription factor that negatively regulates the activity of genes encoding enzymes involved in the biosynthesis of NeuAc and is encoded by the yhbJ gene. The yhbJ gene is also called the rapZ gene. YhbJ is a transcriptional control factor that negatively regulates the activity of the glmS gene encoding L-glutamine-D-fructose 6-phosphate aminotransferase involved in the biosynthesis of NeuAc.

[0101] The transcriptional control factor that negatively regulates the transcription of the gene group involved in the assimilation of NeuAc is a transcriptional control factor involved in the synthesis and metabolism of sialic acid and is encoded by the nanR gene.

[0102] NeuAc lyase is an enzyme that decomposes NeuAc into ManNAc and pyruvate and is encoded by the nanA gene.

[0103] β-Galactosidase is an enzyme that hydrolyzes lactose and is encoded by the lacZ gene.

[0104] Galactoside transacetylase is an enzyme having the activity of acetylation of lactose and its analogs, and is encoded by the lacA gene.

[0105] Therefore, in one embodiment of the present invention, it is more preferred to use a genetically modified microorganism that does not contain at least one base sequence selected from the group consisting of nagE gene, nagB gene, nanK gene, nanE gene, yhbJ gene, nanR gene, nanA gene, lacZ gene and lacA gene as the parent strain. In one embodiment of the present invention, the above-mentioned genetically modified microorganism preferably has an improved productivity of 3'SL compared to the parent strain that has not been genetically modified.

[0106] As a method for producing Escherichia coli in which at least one activity selected from among GlcNAc transporter activity, N-acetylmannosamine kinase activity, N-acetylglucosamine-6-phosphate-2-epimerase activity, Rnase adaptor protein activity, transcriptional control factor activity that negatively regulates the transcription of a gene group involved in the assimilation of NeuAc, NeuAc lyase activity, β-galactosidase activity and galactoside transacetylase activity is reduced or deleted, a known method can be used. Specifically, for example, methods based on various gene manipulations (Metabolic Engineering, 2017, 41: 23-38) and the like can be cited.

[0107] <Obtaining microorganisms with enhanced activity of specific proteins>

[0108] As a microorganism having an enhanced activity of a protein described in any one of [A] and [1] to [3] above as compared to a parental microorganism, there can be mentioned a microorganism obtained by transforming a parental microorganism with a recombinant DNA containing a DNA encoding the protein, and having an increased number of copies of the gene as compared to the parental microorganism. In the present specification, the increased number of copies of the gene may be a new carrying of the gene in a parental strain that does not carry the gene on a chromosome or a plasmid, or a greater carrying of the gene in a strain that carries the gene on a chromosome or a plasmid.

[0109] Examples of microorganisms obtained by transforming a parent microorganism with a recombinant DNA containing a DNA encoding a protein described in any one of [A] and [1] to [3] above, in which the number of copies of the gene is increased compared to the parent microorganism, include microorganisms in which the number of copies of the gene on chromosomal DNA is increased by transforming a parent microorganism with a recombinant DNA containing a DNA encoding a protein described in any one of [A] and [1] to [3] above, and microorganisms carrying the gene outside the chromosomal DNA in the form of a plasmid DNA.

[0110] The DNA encoding the protein described in any one of [A] and [1] to [3] above may be any DNA as long as it encodes a protein having the activity of the protein described in any one of [A] and [1] to [3] above. Specifically, the DNA may be a DNA selected from the group consisting of the following [4] to [7].

[0111] [4] A DNA encoding the protein described in any one of [A] and [1] to [3] above

[0112] [5] A DNA consisting of the base sequence shown in SEQ ID NO: 3, 5, 7 or 9

[0113] [6] A DNA that hybridizes under stringent conditions with a DNA consisting of a complementary base sequence to the base sequence represented by SEQ ID NO: 3, 5, 7 or 9 and encodes a homologous protein having α2,3-sialyltransferase activity

[0114] [7] A DNA comprising a base sequence having 95% or more identity, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to the base sequence represented by SEQ ID NO: 3, 5, 7 or 9, and encoding a homologous protein having α2,3-sialyltransferase activity

[0115] In the above [6], "hybridization" means that DNA hybridizes with DNA having a specific base sequence or a portion of the DNA. Therefore, the DNA having a specific base sequence or a portion of the DNA is a DNA that can be used as a probe for Northern blot or Southern blot analysis, and can be used as an oligonucleotide primer for PCR analysis.

[0116] Examples of DNA used as a probe include DNA having at least 100 bases, preferably at least 200 bases, and more preferably at least 500 bases. Examples of DNA used as a primer include DNA having at least 10 bases, preferably at least 15 bases.

[0117] The method of DNA hybridization experiment is well known, for example, those skilled in the art can determine the conditions of hybridization according to this specification. The conditions of this hybridization can be carried out according to the records of molecular cloning 4th edition (2012), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press (1996) and various other standard textbooks.

[0118] Alternatively, DNA hybridized under stringent conditions may be obtained according to the instructions provided with a commercial hybridization kit. Commercially available hybridization kits include, for example, a random primer DNA labeling kit (manufactured by Roche Diagnostics) that prepares probes by the random primer method and hybridizes under stringent conditions.

[0119] The above-mentioned stringent conditions can be listed as follows: the filter membrane fixed with DNA and the probe DNA are incubated overnight at 42°C in a solution containing 50% formamide, 5×SSC (750 mmol / l sodium chloride, 75 mmol / l sodium citrate), 50 mmol / l sodium phosphate (pH 7.6), 5×Denhart's solution, 10% dextran sulfate and 20 μg / l denatured salmon sperm DNA, and then the filter membrane is washed in a 0.2×SSC solution at, for example, about 65°C.

[0120] Examples of DNAs capable of hybridizing under the above-mentioned stringent conditions include DNAs having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the DNA consisting of the base sequence shown in sequence number 3, 5, 7 or 9 when calculated based on the above-mentioned parameters, etc. using BLAST, FASTA, etc.

[0121] The DNA encoding the protein of the above-mentioned [1] and the DNA of the above-mentioned [5] can be obtained, for example, as follows: by Southern hybridization of a chromosomal DNA library of a microorganism, preferably a microorganism belonging to the genus Escherichia coli, more preferably Escherichia coli BW25113 strain, using a probe DNA that can be designed based on the base sequence shown in sequence number 3, 5, 7 or 9; or by PCR [PCR Protocols, Academic Press (1990)] using a primer DNA that can be designed based on the base sequence and the chromosomal DNA of the above-mentioned microorganism as a template.

[0122] The DNA encoding the mutant protein of [2] can be obtained, for example, by subjecting the DNA consisting of the base sequence shown in SEQ ID NO: 3, 5, 7 or 9 as a template to error-prone PCR or the like.

[0123] Alternatively, DNA encoding the mutant protein of [2] described above can also be obtained by PCR using a set of PCR primers having a base sequence designed to introduce the target mutation (deletion, substitution, insertion or addition) at each 5' end [Gene, 77, 51 (1989)].

[0124] In addition, the DNA can also be obtained according to the instructions attached to a commercially available site-directed mutagenesis kit. As a commercially available site-directed mutagenesis kit, for example, a PrimeSTAR (registered trademark) mutagenesis basal kit (Takara Biotechnology Co., Ltd.) can be cited that can introduce a mutation (deletion, substitution, insertion or addition) into the position where the target mutation is to be introduced.

[0125] That is, first, a plasmid having a base sequence designed in a manner that can introduce a target mutation (deletion, substitution, insertion or addition) is used as a template, and a pair of primers for mutation introduction with 15 bases overlapping on the 5' side are designed. At this time, the target mutation is contained in the overlapping portion. Then, using the primer for mutation introduction, PCR is performed using a plasmid having a base sequence into which the target mutation is to be introduced as a template. The amplified fragment thus obtained is transformed into Escherichia coli to obtain a plasmid having a base sequence into which the target mutation is introduced.

[0126] The DNA encoding the homologous protein of the above-mentioned [3] and the DNA of the above-mentioned [6] and [7] can be obtained, for example, as follows: searching various gene sequence databases for a base sequence having an identity of 95% or more, preferably 97% or more, further preferably 98% or more, and most preferably 99% or more with the base sequence shown in sequence number 3, 5, 7 or 9, or searching various protein sequence databases for an amino acid sequence having an identity of 90% or more, preferably 95% or more, more preferably 97% or more, further preferably 98% or more, and most preferably 99% or more with the amino acid sequence shown in sequence number 4, 6, 8 or 10, and obtaining them by the same method as the above-mentioned method for obtaining DNA using a probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search and a microorganism having the DNA.

[0127] The obtained DNA described in any one of [4] to [7] above can be directly or cleaved with an appropriate restriction enzyme or the like and then integrated into a vector by a conventional method, and the obtained recombinant DNA can be introduced into a host cell. The DNA can then be analyzed using a commonly used base sequence analysis method, such as the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analysis apparatus such as 3700 DNA Analyzer (manufactured by Applied Biosystems Co., Ltd.), thereby determining the base sequence of the DNA.

[0128] As host cells that can be used for determining the base sequence of the DNA, any host cell can be used as long as it can be introduced with the vector and grow, and examples thereof include Escherichia coli DH5α, Escherichia coli HST08 Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm - , Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, Escherichia coli NM522, etc.

[0129] Examples of the vector include pBluescriptII KS(+), pPCR-Script Amp SK(+) (all manufactured by Agilent Technologies Inc.), pT7Blue (manufactured by Merck Millipore Corporation), pCRII (manufactured by Thermo Fisher Scientific Inc.), pCR-TRAP (manufactured by Din Handar Co., Ltd.), and pDIRECT (Nucleic Acids Res., 18, 6069, 1990).

[0130] As a method for introducing recombinant DNA, any method can be used as long as it is a method that can introduce DNA into host cells, and examples thereof include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Application Publication No. 63-248394), and an electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0131] When the DNA obtained as a result of base sequence determination is partially long, the full-length DNA can be obtained by Southern hybridization or the like with a chromosomal DNA library using the partially long DNA as a probe.

[0132] Furthermore, the target DNA can also be prepared by chemical synthesis based on the determined DNA base sequence using an NTS M series DNA synthesizer manufactured by Nippon Technosapiens Co., Ltd. or the like.

[0133] The recombinant DNA containing a DNA encoding a protein described in any one of [A] and [1] to [3] above refers to a recombinant DNA in which the DNA is integrated into an expression vector capable of autonomously replicating in the parent strain or capable of integrating into a chromosome and containing a promoter at a position capable of transcribing the DNA.

[0134] When the recombinant DNA is a recombinant DNA that can be integrated into chromosomes, it may not contain a promoter.

[0135] A microorganism obtained by transforming a parent microorganism with a recombinant DNA containing a DNA encoding a protein described in any one of [A] and [1] to [3] above, wherein the copy number of the gene is increased compared to the parent microorganism, can be obtained by the following method.

[0136] Based on the DNA encoding the protein described in any one of [A] and [1] to [3] above obtained by the above method, a DNA fragment of appropriate length containing a portion encoding the protein is prepared as required. In addition, by base substitution of a portion of the base sequence encoding the protein in a manner that produces a codon most suitable for expression in a host cell, a transformant with improved productivity can be obtained.

[0137] By inserting the DNA fragment into the downstream of a promoter of an appropriate expression vector, a recombinant DNA is prepared, and by transforming a parent strain with the recombinant DNA, a microorganism having an increased copy number of a gene encoding the protein compared to the parent strain can be obtained.

[0138] When a prokaryotic organism such as bacteria is used as the parent strain, the recombinant DNA is preferably a recombinant DNA consisting of a promoter, a ribosome binding sequence, the DNA described in any one of [4] to [7] above, and a transcription termination sequence. It may contain a gene that controls the promoter.

[0139] It is preferred to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases). In the recombinant DNA, the expression of the DNA does not necessarily require a transcription termination sequence, but it is preferred to arrange the transcription termination sequence immediately downstream of the structural gene.

[0140] In addition, by base substitution of a partial base sequence encoding a protein having α2,3-sialyltransferase activity in a manner that becomes a codon most suitable for expression in a host, the expression level of a protein having α2,3-sialyltransferase activity can be increased. Proteins having α2,3-sialyltransferase activity include proteins described in any one of [A] and [1] to [3] above. The codon usage frequency information of the parent strain used in the production method of the present invention can be obtained from a public database.

[0141] The expression vector is not particularly limited as long as it is an appropriate nucleic acid molecule for introducing the target DNA into a host, growing it, and expressing it. Not only plasmids but also artificial chromosomes, vectors using transposons, and cosmids can be used.

[0142] When a microorganism belonging to the genus Escherichia is used as the parent strain, examples of expression vectors include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio), pMW119 (manufactured by Nippondin), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5 x (manufactured by Neurograf Biolabs), pGEX-4T-1, pTrc99A (manufactured by Dihales Biosciences), pTrcHis, pSE280 (manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (manufactured by Chiagen), pET-3, pBluescriptII SK(+), 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)], pBluescriptII SK(+), pBluescript II KS(-) (manufactured by Stratagene Co., Ltd.), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p.6378-6385], pPAC31 (International Publication No. 1998 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (Japanese Patent Application Publication No. 63-233798)), pKD46 [Proc. Natl. Acad. Sci., USA, 97, 6640-6645 (2000)], etc.

[0143] As a promoter when using the above expression vector, any promoter may be used as long as it functions in the cells of microorganisms belonging to the genus Escherichia, and for example, promoters of amino acid biosynthesis-related genes such as trp promoter and ilv promoter, uspA promoter, lac promoter, PL promoter, PR promoter, PSE promoter, etc., from Escherichia coli or bacteriophage, etc., can be used. In addition, artificially designed and modified promoters such as a promoter composed of two trp promoters in tandem, a tac promoter, a trc promoter, a lacT7 promoter, and a letI promoter can be mentioned.

[0144] When a microorganism belonging to the genus Corynebacterium is used as the parent strain, examples of expression vectors 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 see Japanese Patent Application Laid-Open No. 58-105999), pCE51, pCE52, and pCE53 [all see Molecular and General Genetics, 196, 175 (1984)].

[0145] When the expression vector is used, any promoter may be used as long as it functions in cells of microorganisms belonging to the genus Corynebacterium, and for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)] can be used.

[0146] When a yeast strain is used as a parent strain, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0147] As a promoter when using the above expression vector, any promoter may be used as long as it functions in cells of a yeast strain, and examples thereof include promoters such as PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0148] By inserting the DNA fragment described in any one of [4] to [7] above into the downstream of a promoter of an appropriate expression vector, a recombinant DNA used in the production method of the present invention can be prepared.

[0149] Examples of methods for introducing recombinant DNA in the form of a plasmid that can replicate autonomously in the parent strain include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Application Laid-Open No. 63-248394), and an electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0150] As a method for integrating recombinant DNA into the chromosome of a host cell, for example, homologous recombination can be cited. As a homologous recombination method, for example, a method using a homologous recombination plasmid can be cited, and the homologous recombination plasmid can be prepared by connecting with a plasmid DNA having a drug resistance gene that cannot replicate autonomously in the host cell to be introduced. As a method for utilizing homologous recombination frequently used in Escherichia coli, for example, a method for introducing recombinant DNA using the homologous recombination system of lambda phage can be cited [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)].

[0151] Furthermore, by using a selection method utilizing the fact that Escherichia coli is made sucrose sensitive by the Bacillus subtilis levansucrase integrated into the chromosome together with the recombinant DNA, or a selection method utilizing the fact that Escherichia coli is made streptomycin sensitive by integrating the wild-type rpsL gene into Escherichia coli having a mutant rpsL gene having streptomycin resistance [Mol. Microbiol., 55, 137(2005), Biosci. Biotechnol. Biochem., 71, 2905(2007)], it is possible to obtain Escherichia coli in which the target region on the chromosomal DNA of the host cell is replaced with the recombinant DNA.

[0152] The fact that the recombinant DNA has been introduced in the form of a plasmid capable of autonomously replicating in the parent strain or that the recombinant DNA has been integrated into the chromosome of the parent strain can be confirmed, for example, by performing PCR using a primer set that cannot amplify the gene originally present on the chromosomal DNA of the microorganism but can amplify the gene introduced by transformation and confirming the amplification product, etc. In addition, the fact that the transcription amount of the DNA or the production amount of the protein encoded by the DNA has increased can be confirmed by comparing the transcription amount of the gene of the microorganism with that of the parent strain by Northern blotting or by comparing the production amount of the protein of the microorganism by Western blotting, etc.

[0153] The fact that the microorganism produced by the above method is a microorganism in which the activity of the protein described in any one of [A] and [1] to [3] above is enhanced compared to the parent strain can be confirmed as follows: after culturing the microorganism, the culture solution is appropriately diluted and then centrifuged, and the 3'SL contained in the supernatant is analyzed by HPLC or the like and compared with the parent strain.

[0154] The above-mentioned microorganism has enhanced activity of the protein described in any one of [A] and [1] to [3] above compared to the parent strain, and the transfer of sialic acid residues from CMP-sialic acid to galactose at the terminal of the acceptor sugar in the form of α2,3 bond is promoted, thereby improving the productivity of 3'SL. Examples of such microorganisms include tNlsiaTΔN20AA, tNlsiaTΔN23AA, tNlsiaTΔN27AA and tNlsiaTΔN37AA in which the expression of the α2,3-sialyltransferase gene described later in the Examples is enhanced.

[0155] As an example of such a microorganism, a microorganism having enhanced α2,3-sialyltransferase activity can improve 3'SL productivity due to high α2,3-sialyltransferase activity.

[0156] 2. Method for producing 3'SL

[0157] As a method for producing 3'SL according to one embodiment of the present invention, the following method can be mentioned.

[0158] A method for producing 3'SL, comprising the steps of: preparing the microorganism described in 1.; and producing 3'SL in culture using the microorganism.

[0159] The method for culturing the microorganism described in 1. can be carried out according to a method generally used for culturing microorganisms.

[0160] As a medium for culturing the microorganism, any natural medium or synthetic medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic salt, etc. that can be assimilated by the microorganism and can efficiently culture the transformant.

[0161] The carbon source may be any carbon source as long as it can be assimilated by the microorganism, and examples thereof include sugars such as glucose, fructose, sucrose, molasses containing these sugars, starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, and alcohols such as glycerol, ethanol or propanol.

[0162] Examples of nitrogen sources include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate or ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented bacteria and their digests.

[0163] Examples of the inorganic salt include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0164] As the microorganism of the present invention used in the method for producing 3'SL, a microorganism having the ability to produce an acceptor sugar such as glucose, lactose or lactose monohydrate can be used.

[0165] In the method for producing 3'SL, glucose, sugar solution, sucrose, lactose, NeuAc, or the like may be added to the medium during the culture.

[0166] In the method for producing 3'SL, instead of adding lactose, NeuAc, etc. to the medium during culture, lactose, NeuAc, etc. may be supplied to the microorganism of one embodiment of the present invention by culturing a microorganism capable of producing lactose and NeuAc simultaneously with the microorganism of the present invention.

[0167] In the method for producing oligosaccharides, it is preferred that the culture medium does not contain GlcNAc transporter, N-acetylmannosamine kinase, N-acetylglucosamine-6-phosphate-2-isomerase, RNase adaptor protein, transcription control factor that negatively regulates the transcription of a gene group involved in the assimilation of NeuAc, NeuAc lyase, β-galactosidase and galactoside transacetylase.

[0168] Cultivation is usually preferably carried out under aerobic conditions using ventilation and stirring. The culture temperature is usually 28 to 37°C, and the culture time is usually 36 hours to 4 days. The pH of the culture solution during cultivation is usually maintained at 6.4 to 7.4. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0169] By the above-mentioned cultivation, 3'SL is produced and accumulated in the culture, and 3'SL can be produced by collecting 3'SL from the culture.

[0170] Usually, the culture is centrifuged and 3'SL is collected from the supernatant. When 3'SL accumulates in the cells, the cells are disrupted by ultrasound or the like, the cells are removed by centrifugation, and 3'SL is collected from the obtained supernatant by ion exchange resin or the like.

[0171] [Analysis example]

[0172] In the Examples, the analysis and quantification of 3'SL were performed by the following procedure: The culture solution containing the cultured microorganisms was appropriately diluted, sterilized by a filter, and the supernatant was collected. The 3'SL contained in the supernatant was analyzed using an analysis device SPD-20A (manufactured by Shimadzu Corporation).

[0173] [Analysis conditions]

[0174] Column: Shodex SUGAR SH1011

[0175] Column temperature: 50°C

[0176] Eluent composition: 5 mM sulfuric acid aqueous solution

[0177] Flow rate: 0.6ml / min

[0178] Detector: SPD-20A

[0179] Example

[0180] [Example 1] Preparation of microorganisms used in the production of 3'SL

[0181] (1) Preparation of a host cell for 3'SL production

[0182] <Escherichia coli with yhbJ gene defect>

[0183] Escherichia coli in which the transcriptional control factor yhbJ gene (SEQ ID NO: 11) that negatively regulates the activity of the glmS gene encoding L-glutamine-D-fructose 6-phosphate aminotransferase involved in the biosynthesis of N-acetylneuraminic acid (hereinafter also referred to as NeuAc) was disrupted was prepared according to the following steps.

[0184] Using BW25113 strain (Keio collection (Systematic single-gene knock-out mutants of E. coli K-12) ) as a host, the strain BW25113ΔyhbJ with complete deletion of the yhbJ gene was obtained by the same method as Baba et al. (Baba T. et al. (2006) Molsystems Biol).

[0185] <Obtaining DNA fragments used as markers for gene defects>

[0186] PCR was performed using a DNA consisting of the base sequence shown in "Primer Set" in Table 1 as a primer set and a DNA described in "Template" in Table 1 as a template to obtain each amplified DNA fragment.

[0187] [Table 1]

[0188]

[0189] The genomic DNA of Bacillus subtilis 168 was prepared by conventional methods. The cat of the amplified DNA fragment contained about 200 bp upstream to about 50 bp downstream of the cat gene (encoding chloramphenicol acetyltransferase) on pHSG396. The sacB of the amplified DNA fragment contained about 300 bp upstream to about 100 bp downstream of the sacB gene (encoding levansucrase) on the genomic DNA of Bacillus subtilis 168.

[0190] Then, a substance obtained by mixing cat and sacB of the amplified DNA fragment in an equimolar ratio is used as a template, and PCR is performed using a DNA composed of the base sequence shown in sequence numbers 21 and 24 as a primer set to obtain a DNA fragment containing the cat gene and the sacB gene (hereinafter also referred to as cat-sacB).

[0191] <Escherichia coli with lacZ gene deficiency>

[0192] Using the BW25113ΔyhbJ strain prepared above as a parent strain, Escherichia coli in which the lacZ gene (SEQ ID NO: 12) encoding β-galactosidase involved in the decomposition of lactose was disrupted was prepared according to the following procedure.

[0193] PCR was performed using chromosomal DNA of the BW25113 strain prepared by a conventional method as a template and DNA consisting of the base sequence shown in "Primer Set" in Table 2 as a primer set to obtain each amplified DNA fragment.

[0194] [Table 2]

[0195]

[0196] lacZupstream1 and lacZupstream2 contain the start codon of the lacZ gene to about 700 bp upstream thereof, and lacZdownstream1 and lacZdownstream2 contain the stop codon of the lacZ gene to about 800 bp downstream thereof.

[0197] A substance obtained by mixing lacZ upstream 1, lacZ downstream 1 and cat-sacB fragments in an equimolar ratio was used as a template, and PCR was performed using a DNA consisting of the base sequence shown in sequence numbers 33 and 34 as a primer set to obtain a DNA fragment consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the lacZ gene peripheral region (hereinafter also referred to as lacZ::cat-sacB).

[0198] A substance obtained by mixing lacZ upstream 2 and lacZ downstream 2 in an equimolar ratio was used as a template, and PCR was performed using a DNA composed of the base sequence shown in sequence numbers 33 and 34 as a primer set to obtain a DNA fragment that does not contain lacZ and is composed of a sequence directly connected between lacZ upstream and lacZ downstream (hereinafter also referred to as ΔlacZ).

[0199] The lacZ::cat-sacB fragment was introduced into the BW25113ΔyhbJ strain carrying the plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol.97, 6640-6645 (2000)] containing the gene encoding λ recombinase by electroporation, and transformants showing chloramphenicol resistance and sucrose sensitivity (transformants in which the lacZ gene was replaced by lacZ::cat-sacB) were obtained.

[0200] The ΔlacZ fragment was introduced into the transformant by electroporation to obtain a transformant showing chloramphenicol sensitivity and sucrose resistance (a transformant in which lacZ::cat-sacB was replaced by ΔlacZ). The transformant was named BW25113ΔyhbJΔlacZ strain.

[0201] <Escherichia coli with defects in nanR, nanA, nanT, nanE and nanK genes>

[0202] Using the BW25113ΔyhbJΔlacZ strain as a host, the following steps were followed to prepare Escherichia coli in which the nanR gene (sequence number 13) encoding a transcriptional control factor that negatively regulates the transcription of a gene group involved in the assimilation of NeuAc, the nanA gene (sequence number 14) encoding a NeuAc lyase, the nanT gene (sequence number 15) encoding a NeuAc transporter, the nanE gene (sequence number 16) encoding N-acetylglucosamine-6-phosphate-2-epimerase, and the nanK gene (sequence number 17) encoding N-acetylmannosamine kinase were destroyed. It should be noted that nanR, nanA, nanT, nanE, and nanK (hereinafter also referred to as nanRATEK) formed an operon on the Escherichia coli genome.

[0203] PCR was performed using chromosomal DNA of the BW25113 strain prepared by a conventional method as a template and a DNA consisting of the base sequence shown in "Primer Set" in Table 3 as a primer set to obtain each amplified DNA fragment.

[0204] [Table 3]

[0205]

[0206] nanR upstream 1 and nanR upstream 2 contain the start codon of the nanR gene to about 800 bp upstream thereof, and nanK downstream 1 and nanK downstream 2 contain the stop codon of the nanK gene to about 850 bp downstream thereof.

[0207] A substance obtained by mixing nanR upstream 1, nanK downstream 1 and cat-sacB fragment in an equimolar ratio was used as a template, and PCR was performed using a DNA composed of the base sequence shown in sequence numbers 43 and 44 as a primer set to obtain a DNA fragment composed of a sequence in the region surrounding the nanRATEK gene with the cat-sacB fragment inserted therein (hereinafter also referred to as nanRATEK::cat-sacB).

[0208] A substance obtained by mixing nanR upstream 2 and nanK downstream 2 in an equimolar ratio is used as a template, and PCR is performed using a DNA composed of the base sequence shown in sequence numbers 43 and 44 as a primer set to obtain a DNA fragment that does not contain nanRATEK and is composed of a sequence directly connected between nanR upstream and nanK downstream (hereinafter also referred to as ΔnanRATEK).

[0209] The nanRATEK::cat-sacB fragment was introduced into the BW25113ΔyhbJΔlacZ strain carrying the plasmid pKD46 containing a gene encoding λ recombinase by electroporation, and transformants showing chloramphenicol resistance and sucrose sensitivity were obtained (transformants in which the nanRATEK gene was replaced with nanRATEK::cat-sacB).

[0210] The ΔnanRATEK fragment was introduced into the transformant by electroporation to obtain a transformant showing chloramphenicol sensitivity and sucrose resistance (a transformant in which nanRATEK::cat-sacB was replaced by ΔnanRATEK). The transformant was named BW25113ΔyhbJΔlacZΔnanRATEK strain.

[0211] (2) Preparation of plasmid for NeuAc production

[0212] Plasmids expressing the NeuAc synthetase gene CjneuB (SEQ ID NO: 18) from Campylobacter jejuni ATCC43438 and the UDP-GlcNAc epimerase FpneuC (SEQ ID NO: 19) from Flavobacterium psychrophilum NBRC100250 were prepared according to the following procedure.

[0213] PCR was performed using a DNA consisting of the base sequence shown in "Primer Set" in Table 4 as a primer set and a DNA described in "Template" in Table 4 as a template to obtain an amplified DNA fragment.

[0214] It should be noted that the start codon in the wild-type sequence of FpneuC (SEQ ID NO: 19) is GTG, and therefore ATG was added to the primer sequence for expression in E. coli so that the start codon of the amplified DNA fragment FpneuC obtained by PCR would be ATG.

[0215] [Table 4]

[0216]

[0217] The genomic DNA of Campylobacter jejuni ATCC43438 and Flavobacterium psychrophilum NBRC100250 were prepared by conventional methods.

[0218] The base sequences shown in SEQ ID NOs: 46 and 47 contain complementary sequences at their respective 5' ends.

[0219] A substance obtained by mixing the CjneuB fragment and the FpneuC fragment obtained above in an equimolar ratio is used as a template, and a DNA composed of the base sequence shown in sequence numbers 45 and 48 is used as a primer set to perform PCR, thereby obtaining a DNA fragment of approximately 2.2 kb (hereinafter also referred to as the CjneuB-FpneuC fragment) formed by connecting the CjneuB fragment and the FpneuC fragment.

[0220] The CjneuB-FpneuC fragment and expression vector pTrc99a (manufactured by GE Healthcare Life Sciences) were ligated using the In-Fusion HD Cloning Kit (manufactured by Takara Bio) to obtain the expression plasmid pTrc99a-CjneuB-FpneuC (hereinafter also referred to as pBC).

[0221] (3) Preparation of 3'SL production plasmid

[0222] Plasmids expressing the full-length or shortened sialyltransferase gene NlsiaT (SEQ ID NO: 1, 3, 5, 7 or 9) from lactose-fermenting Neisseria lactamica ATCC 23970 and the CMP-NeuAc synthetase gene PmneuA (SEQ ID NO: 20) from Pasteurella multocida subsp. multocida str. Pm70 ATCC BAA-1909 were prepared by the following procedure.

[0223] PCR was performed using chromosomal DNA of lactose-fermenting Neisseria prepared by a conventional method as a template and a DNA consisting of the base sequence shown in "Primer Set" in Table 5 as a primer set to obtain each amplified DNA fragment.

[0224] [Table 5]

[0225]

[0226] PCR was performed using chromosomal DNA of Pasteurella multocida subsp. multocida str. Pm70 ATCC BAA-11909 prepared by a conventional method as a template and DNA consisting of the base sequence shown in SEQ ID NOs: 55 and 56 as a primer set to obtain a PmneuA fragment.

[0227] The base sequences shown in SEQ ID NOs: 50 and 55 contain complementary sequences at their respective 5' ends.

[0228] A substance obtained by mixing the NlsiaT fragment, tNlsiaTΔN20AA fragment, tNlsiaTΔN23AA fragment, tNlsiaTΔN27AA fragment or tNlsiaTΔN37AA fragment, and PmneuA fragment obtained above in an equimolar ratio is used as a template, and a DNA composed of the base sequence shown in sequence numbers 49, 51, 52, 53 or 54, and 56 is used as a primer set to perform PCR to obtain a DNA fragment formed by connecting the NlsiaT fragments and the PmneuA fragment.

[0229] The DNA fragments formed by connecting the NlsiaT fragments and PmneuA fragments obtained above and the expression vector pSTV29 (manufactured by Takara Bio) were connected using the In-Fusion HD Cloning Kit (manufactured by Takara Bio) to obtain expression plasmids pSTV-NlsiaT, pSTV-tNlsiaTΔN20AA, pSTV-tNlsiaTΔN23AA, pSTV-tNlsiaTΔN27AA and pSTV-tNlsiaTΔN37AA.

[0230] (4) Preparation of microorganisms having a NeuAc-producing plasmid

[0231] The NeuAc-producing plasmid pBC obtained in (2) was used to transform the BW25113ΔyhbJΔlacZΔnanRATEK strain prepared in (1) above, thereby preparing Escherichia coli harboring pBC, which was named BW25113ΔyhbJΔlacZΔnanRATEK / pBC strain.

[0232] (5) Preparation of microorganisms having a 3'SL-producing plasmid

[0233] The 3'SL production plasmid obtained in (2) above, pSTV-NlsiaT, pSTV-tNlsiaTΔN20AA, pSTV-tNlsiaTΔN23AA, pSTV-tNlsiaTΔN27AA and pSTV-tNlsiaTΔN37AA were used to transform the BW25113ΔyhbJΔlacZΔnanRATEK / pBC strain produced in (4) above, thereby obtaining Escherichia coli containing various plasmids, which were named NlsiaT strain, tNlsiaTΔN20AA strain, tNlsiaTΔN23AA strain, tNlsiaTΔN27AA strain and tNlsiaTΔN37AA strain respectively.

[0234] [Example 2] Production of 3'SL by fermentation

[0235] The productivity of 3'SL was evaluated for the NlsiaT strain, tNlsiaTΔN20AA strain, tNlsiaTΔN23AA strain, tNlsiaTΔN27AA strain, and tNlsiaTΔN37AA strain obtained in Example 1.

[0236] Each strain was cultured on an LB plate containing 100 mg / L ampicillin and 25 mg / L chloramphenicol at 30°C overnight, inoculated into a crude test tube containing 5 ml of LB medium containing 100 mg / L ampicillin and 25 mg / L chloramphenicol, and cultured with shaking at 30°C for 16 hours.

[0237] Then, 0.3 mL of the culture solution was inoculated into a crude test tube containing 3 ml of a production medium [30 g / L of glycerol, 10 g / L of lactose, 2.0 g / L of magnesium sulfate heptahydrate, 16 g / L of dipotassium hydrogen phosphate, 14 g / L of potassium dihydrogen phosphate, 2.0 g / L of ammonium sulfate, 1.0 g / L of citric acid monohydrate, 5.0 g / L of casamino acids (manufactured by Difco Co., Ltd.), 10 mg / L of vitamin B1, 50 mg / L of iron sulfate heptahydrate, 10 mg / L of manganese sulfate heptahydrate, adjusted to pH 7.2 with an aqueous sodium hydroxide solution and then autoclaved] [glycerol, lactose and an aqueous solution of magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled and mixed], and cultured with shaking at 30°C for 24 hours. Five hours after the start of the culture, isopropyl-β-thiogalactoside (IPTG) was added to give a final concentration of 1 mM.

[0238] After the culture was completed, the culture solution was appropriately diluted, and the filter-sterilized sample was subjected to HPLC analysis to quantify the concentration of 3'SL contained in the supernatant. The results are shown in Table 6.

[0239] [Table 6]

[0240]

[0241] From the above results, it can be seen that when using the shortened NlsiaT obtained by deleting the amino acids at the N-terminus of the sialyltransferase NlsiaT from the lactose-fermenting Neisseria ATCC23970 strain, a higher 3'SL productivity was shown compared to the full-length NlsiaT. In addition, the effective N-terminal deletion length was almost the same when it was 20 amino acid residues, 23 amino acid residues, 27 amino acid residues, and 37 amino acid residues.

[0242] These shortened glycosyltransferases were shown to be useful for efficient production of 3'SL compared to full-length glycosyltransferases.

[0243] The present invention is described in detail with reference to specific modes, but various changes and modifications can be made without departing from the spirit and scope of the present invention, which is obvious to those skilled in the art. It should be noted that this application is based on the Japanese patent application (Japanese Patent Application No. 2022-091801) filed on June 6, 2022, and is cited in its entirety by reference. In addition, all references cited herein are introduced as a whole.

[0244] Industrial Applicability

[0245] According to the present invention, 3'SL can be produced efficiently.

[0246] Sequence Listing Free Text

[0247] SEQ ID NO: 1: Base sequence of sialyltransferase gene NlsiaT from lactose-fermenting Neisseria ATCC23970 strain

[0248] SEQ ID NO: 2: Amino acid sequence of sialyltransferase gene NlsiaT from lactose fermenting Neisseria ATCC23970 strain

[0249] SEQ ID NO: 3: base sequence of tNlsiaTΔN20AA

[0250] SEQ ID NO: 4: Amino acid sequence of tNlsiaTΔN20AA

[0251] SEQ ID NO: 5: base sequence of tNlsiaTΔN23AA

[0252] SEQ ID NO: 6: Amino acid sequence of tNlsiaTΔN23AA

[0253] SEQ ID NO: 7: base sequence of tNlsiaTΔN27AA

[0254] SEQ ID NO: 8: Amino acid sequence of tNlsiaTΔN27AA

[0255] SEQ ID NO: 9: base sequence of tNlsiaTΔN37AA

[0256] SEQ ID NO: 10: Amino acid sequence of tNlsiaTΔN37AA

[0257] SEQ ID NO: 11: yhbJ base sequence from Escherichia coli BW25113

[0258] SEQ ID NO: 12: lacZ base sequence from Escherichia coli BW25113

[0259] SEQ ID NO: 13: nanR base sequence from Escherichia coli BW25113

[0260] SEQ ID NO: 14: nanA base sequence from Escherichia coli BW25113

[0261] SEQ ID NO: 15: nanT base sequence from Escherichia coli BW25113

[0262] SEQ ID NO: 16: nanE base sequence from Escherichia coli BW25113

[0263] SEQ ID NO: 17: nanK base sequence from Escherichia coli BW25113

[0264] SEQ ID NO: 18: Base sequence of NeuAc synthetase gene CjneuB from Campylobacter jejuni strain ATCC43438

[0265] SEQ ID NO: 19: Base sequence of FpneuC encoding UDP-GlcNAc epimerase from Flavobacterium psychrophilum strain NBRC100250

[0266] SEQ ID NO: 20: Base sequence of CMP-NeuAc synthase gene PmneuA from Pasteurella multocida subsp.multocida str. Pm70 ATCCBAA-1909 strain

[0267] SEQ ID NO: 21: Base sequence of primer Fw for cat fragment amplification

[0268] SEQ ID NO: 22: Base sequence of primer Rv for cat fragment amplification

[0269] SEQ ID NO: 23: Base sequence of primer Fw for sacB fragment amplification

[0270] SEQ ID NO: 24: Base sequence of primer Rv for sacB fragment amplification

[0271] SEQ ID NO: 25: Base sequence of primer Fw for amplifying the 1st upstream fragment of lacZ

[0272] SEQ ID NO: 26: Base sequence of primer Rv for amplifying the 1st upstream fragment of lacZ

[0273] SEQ ID NO: 27: Base sequence of primer Fw for amplifying the 1st fragment downstream of lacZ

[0274] SEQ ID NO: 28: Base sequence of primer Rv for amplifying the 1st fragment downstream of lacZ

[0275] SEQ ID NO: 29: Base sequence of primer Fw for amplifying 2 fragments upstream of lacZ

[0276] SEQ ID NO: 30: Base sequence of primer Rv for amplifying 2 fragments upstream of lacZ

[0277] SEQ ID NO: 31: Base sequence of primer Fw for amplifying the 2 downstream fragments of lacZ

[0278] SEQ ID NO: 32: Base sequence of primer Rv for amplifying 2 fragments downstream of lacZ

[0279] SEQ ID NO: 33: Base sequence of primer Fw for producing a fragment for lacZ disruption

[0280] SEQ ID NO: 34: Base sequence of primer Rv for producing a fragment for lacZ disruption

[0281] SEQ ID NO: 35: Base sequence of primer Fw for amplification of nanR upstream 1 fragment

[0282] SEQ ID NO: 36: Base sequence of primer Rv for amplifying the 1st fragment upstream of nanR

[0283] SEQ ID NO: 37: Base sequence of primer Fw for amplifying the fragment 1 downstream of nanK

[0284] SEQ ID NO: 38: Base sequence of primer Rv for amplifying the fragment 1 downstream of nanK

[0285] SEQ ID NO: 39: Base sequence of primer Fw for amplifying 2 fragments upstream of nanR

[0286] SEQ ID NO: 40: Base sequence of primer Rv for amplifying 2 fragments upstream of nanR

[0287] SEQ ID NO: 41: Base sequence of primer Fw for amplifying 2 fragments downstream of nanK

[0288] SEQ ID NO: 42: Base sequence of primer Rv for amplifying 2 fragments downstream of nanK

[0289] SEQ ID NO: 43: Base sequence of primer Fw for producing nanRATEK disruption fragment

[0290] SEQ ID NO: 44: Base sequence of primer Rv for producing nanRATEK disruption fragment

[0291] SEQ ID NO: 45: Base sequence of primer Fw for amplification of CjneuB fragment

[0292] SEQ ID NO: 46: Base sequence of primer Rv for amplification of CjneuB fragment

[0293] SEQ ID NO: 47: Base sequence of primer Fw for amplifying FpneuC fragment

[0294] SEQ ID NO: 48: Base sequence of primer Rv for amplification of FpneuC fragment

[0295] SEQ ID NO: 49: Base sequence of primer Fw for NlsiaT amplification

[0296] SEQ ID NO: 50: Base sequence of primer Rv for NlsiaT amplification

[0297] SEQ ID NO: 51: Base sequence of primer Fw for tNlsiaTΔN20AA amplification

[0298] SEQ ID NO: 52: Base sequence of primer Fw for tNlsiaTΔN23AA amplification

[0299] SEQ ID NO: 53: Base sequence of primer Fw for tNlsiaTΔN27AA amplification

[0300] SEQ ID NO: 54: Base sequence of primer Fw for tNlsiaTΔN37AA amplification

[0301] SEQ ID NO: 55: Base sequence of primer Fw for PmneuA amplification

[0302] SEQ ID NO: 56: Base sequence of primer Rv for PmneuA amplification.

Claims

1. A microorganism, wherein The activity of the protein consisting of the amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 are deleted is enhanced, and the productivity of 3'-sialyllactose is improved compared with the parent strain.

2. The microorganism according to claim 1, wherein The protein consisting of an amino acid sequence in which 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 are deleted is a protein described in any one of the following [1] to [3]: [1] A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10; [2] A mutant protein having α2,3-sialyltransferase activity, which comprises an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added to the amino acid sequence of SEQ ID NO: 4, 6, 8 or 10; [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10 and having α2,3-sialyltransferase activity.

3. A method for producing 3'-sialyllactose, comprising the steps of preparing the microorganism according to claim 1 or 2 and producing 3'-sialyllactose in a culture using the microorganism.

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