Microorganisms with improved productivity of 3'-sialyl lactose and methods for producing 3'-sialyl lactose

Abstract

The present invention relates to a microorganism which has an enhanced activity of a protein consisting of an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 by deletion of 20-37 amino acid residues on the N-terminal side, and which has an improved productivity of 3'-sialyllactose compared to the parent strain.

Description

Technical Field

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

Background Art

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

[0003] Although 3'SL is contained in human breast milk, it is contained only slightly in the milk of other mammals such as cow's milk (Non-Patent Document 5). Since infant milk is mainly produced by adding necessary nutrients to raw materials such as cow's milk, the content of human milk oligosaccharides containing 3'SL is lower than that of breast milk, and in order to give the same effect as breast milk to infant milk, the addition of 3'SL to infant milk has been desired (Non-Patent Document 6).

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

[0005] As disclosed in Non-Patent Document 7 and Patent Document 1, a method for producing 3'SL using recombinant microorganisms is generally a method in which CMP-sialic acid is synthesized from inexpensive carbon sources such as glycerol and glucose in cells, and the sialic acid residue of CMP-sialic acid is transferred to lactose added from the outside by α2,3-sialyltransferase to produce 3'SL.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-licensed literature

[0007]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0008] In this recombinant microorganism-based production method, the activity of α2,3-sialyltransferase, which transfers sialic acid residues to lactose, is considered important for 3'SL productivity. Various types of α2,3-sialyltransferase have already been cloned from microorganisms, and expression of α2,3-sialyltransferase has been successfully achieved in E. coli and other organisms (Non-patent documents 8, 9, 10, 11, 12, 13). Therefore, in order to improve 3'SL productivity compared to conventional methods, there is a need for an α2,3-sialyltransferase that is highly active and can efficiently transfer sialic acid residues to lactose.

[0009] Sialyltransferases are generally known to have a region at the N-terminus of their amino acid sequence that is not related to their activity. Non-patent documents 14 and 15 disclose a method for expressing exogenous sialyltransferase in E. coli by cleaving the N-terminus of the amino acid sequence. Although it is known that this method alters the expression and activity of sialyltransferase, it is not clear which specific region should be cleaved to obtain useful activity.

[0010] Therefore, an object of the present invention is to provide a microorganism in which the activity of a protein lacking specific amino acid residues is enhanced and the productivity of 3'SL is improved as compared with the parent strain. Another object is to provide a method for producing 3'SL using the microorganism. **Means for Solving the Problems**

[0011] As a result of intensive studies, the inventors of the present invention have found that a microorganism in which the activity of a protein consisting of an amino acid sequence lacking 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is enhanced has improved productivity of 3'SL as compared with the parent strain, and thus completed the present invention.

[0012] That is, the present invention is as follows. <1> A microorganism in which the activity of a protein consisting of an amino acid sequence lacking 20 to 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 as compared with the parent strain. <2> The microorganism according to <1> above, wherein the protein consisting of an amino acid sequence lacking 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is the protein according to any one of the following [1] to [3]. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 4, 6, 8 or 10. [2] A mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, 6, 8 or 10 and having α2,3-sialyltransferase 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: 4, 6, 8 or 10 and having α2,3-sialyltransferase activity. <3> A method for producing 3'-sialyllactose, comprising preparing the microorganism according to <1> or <2> above and producing 3'-sialyllactose in a culture using the microorganism. **Effects of the Invention**

[0013] In one aspect of the present invention, the microorganism has enhanced activity of a protein lacking specific amino acid residues, thereby improving the productivity of 3'SL. According to the production method of one aspect of the present invention, 3'SL can be produced by using the culture obtained by culturing the microorganism.

Brief Description of Drawings

[0014] [Figure 1] Figure 1 is a diagram showing an example of the 3'SL biosynthetic pathway.

Modes for Carrying Out the Invention

[0015] 1. Microorganism with Improved Productivity of 3'SL Examples of the microorganism of one aspect 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. [A] A protein consisting of an amino acid sequence lacking 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2. <Productivity> In the present specification, productivity refers to the ability of a microorganism to accumulate an oligosaccharide of the target product produced by the microorganism in the culture of the microorganism. The productivity of oligosaccharides by a microorganism can be confirmed by detecting the oligosaccharides in the culture of the microorganism using an analyzer or the like described below.

[0016] The protein consisting of an amino acid sequence lacking 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 preferably has higher α2,3-sialyltransferase activity than the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The protein consisting of an amino acid sequence lacking 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 means a protein consisting only of the amino acid sequence lacking 20 to 37 amino acid residues on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2.

[0017] α2,3-sialyltransferase activity refers to the activity of transferring a sialic acid residue to the galactose at the end of a carbohydrate molecule via an α2,3 linkage, using CMP-sialic acid and a receptor carbohydrate as substrates, thereby obtaining a sialic acid-containing carbohydrate.

[0018] Examples of CMP-sialic acids include CMP-N-acetylneuraminic acid (hereinafter also referred to as "CMP-NeuAc"). The receptor carbohydrate used as a substrate can be any carbohydrate that is a substrate for sialyl lactose, such as oligosaccharides, polysaccharides, and complex carbohydrates such as glycoproteins and glycolipids.

[0019] Examples of oligosaccharides or polysaccharides that serve as substrates for sialyl lactose include oligosaccharides or polysaccharides having galactose at the non-reducing end, or oligosaccharides or polysaccharides having N-acetylneuraminic acid (hereinafter also referred to as "NeuAc") at the non-reducing end. Preferably, the non-reducing end is a oligosaccharide having a structure selected from the group consisting of lactose, globotrioose, N-acetyllactosamine, lacto-N-tetraose, lacto-N-neotetraose, Lewis a, and Lewis X, or more preferably, lactose. Examples of complex carbohydrates include those in which proteins or lipids are bound to the oligosaccharides and polysaccharides mentioned above.

[0020] As the sialic acid-containing carbohydrate, NeuAc-containing carbohydrates are preferred. Examples of NeuAc-containing carbohydrates include carbohydrates to which NeuAc is attached to the above-mentioned receptor carbohydrate, preferably carbohydrates containing oligosaccharides having NeuAcα2-3Galβ1-4Glc at the non-reducing end, and more preferably 3'SL.

[0021] An example of the 3'SL biosynthesis pathway is shown in Figure 1. It is hypothesized that the enhanced α2,3-sialyltransferase activity of microorganisms promotes the α2,3 binding of the NeuAc residue to the galactose residue of lactose, using CMP-NeuAc and lactose as substrates, thereby improving 3'SL productivity.

[0022] A protein consisting of an amino acid sequence in which the 20-37 amino acid residues from the N-terminus of the amino acid sequence represented by Sequence ID No. 2 described in [A] above is deleted may also be the protein described in any one of the following [1]-[3]. [1] A protein consisting of an amino acid sequence represented by sequence numbers 4, 6, 8, or 10. [2] A mutant protein having α2,3-sialyltransferase activity, consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 4, 6, 8, or 10. [3] A homologous protein having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NOs: 4, 6, 8, or 10, and possessing α2,3-sialyltransferase activity.

[0023] A mutant protein is a protein obtained by artificially deleting or substituting amino acid residues in an original protein, or by inserting or adding amino acid residues to that protein.

[0024] In the mutant protein described in [2] above, the deletion, substitution, insertion, or addition of amino acids means that 1 to 20 amino acids may be deleted, substituted, inserted, or added 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.

[0025] The amino acids that are deleted, substituted, inserted, or added may be native or unnatural forms. Examples of native 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.

[0026] Below are examples of amino acids that are mutually substituted. Amino acids belonging to the same group are mutually substituted. Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic Acid, Methionine, O-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: Proline, 3-hydroxyproline, 4-hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, tyrosine

[0027] In this specification, homologous proteins are proteins that, due to their similarity in structure and function to the original protein, are thought to have the same evolutionary origin as the gene encoding the original protein, and are found in organisms that exist in nature.

[0028] Examples of homologous proteins include amino acid sequences that have, preferably 90% or more, and particularly preferably 95% or more, identity with the amino acid sequence of the target protein.

[0029] The identity of amino acid sequences and base sequences can be determined using algorithms such as BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] by Karlin and Altschul. Based on this BLAST algorithm, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing base sequences using BLASTN based on BLAST, the parameters should be, for example, Score=100 and wordlength=12. When analyzing amino acid sequences using BLASTX based on BLAST, the parameters should be, for example, score=50 and wordlength=3. When using the BLAST and Gapped BLAST programs, the default parameters of each program should be used. The specific methods for these analysis methods are publicly known.

[0030] <Parent stock> In this specification, "parent plant" refers to the original plant that is the subject of genetic modification and transformation. The original plant that is the subject of transformation by gene introduction is also called the host plant.

[0031] In this specification, the parent strain is preferably a prokaryotic or yeast strain, more preferably a prokaryotic organism belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genera Saccharomyces, Schizosaccharomyces, Kluiveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, 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 (Stratagene), Escherichia coli W3110S3GK (NBRC114657), Escherichia coli ATCC9637, 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 include prokaryotes such as ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, or Pseudomonas sp. D-0110, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.

[0032] The parent plant may be a wild-type plant, or, if the wild-type plant does not have the ability to produce substrates for 3'SL production, such as CMP-NeuAc and / or lactose, it may be a bred plant that has been artificially given the ability to produce CMP-NeuAc and / or lactose.

[0033] The production of CMP-NeuAc and / or lactose by microorganisms can be confirmed, for example, by culturing the microorganisms in a culture medium and detecting the CMP-NeuAc and / or lactose accumulated in the culture using known methods such as HPLC, as described later.

[0034] Methods for artificially conferring or enhancing the ability to produce CMP-NeuAc and / or lactose include, for example, known methods such as (a) to (d) below, which can be used individually or in combination. (a) A method to enhance the expression of at least one enzyme involved in the biosynthetic pathway that produces CMP-NeuAc and / or lactose. (b) A method for increasing the copy number of at least one enzyme gene involved in the biosynthetic pathway that produces CMP-NeuAc and / or lactose. (c) A method of relaxing or releasing at least one mechanism that controls the biosynthetic pathway that produces CMP-NeuAc and / or lactose. (d) A method of weakening or blocking at least one metabolic pathway that branches off from the biosynthetic pathway producing CMP-NeuAc and / or lactose to metabolites other than the target substance.

[0035] <<Preferably enhanced activity in the parent plant>> The parent strain preferably possesses at least one activity selected from 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 permyase activity, glucosamine acetyltransferase activity, and N-acetylmannosamine epimerase activity, and it is more preferable that this activity is enhanced. Of these, it is preferable that it possesses CMP-NeuAc synthase activity, and it is even more preferable that this activity is enhanced.

[0036] UDP-GlcNAc epimerase is an enzyme responsible for the reaction that produces 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 the Flavobacterium psychrophilum NBRC100250 strain.

[0037] NeuAc synthase is an enzyme responsible for the reaction that produces NeuAc using ManNAc and phosphoenolpyruvate (hereinafter also referred to as "PEP") as substrates. The DNA encoding NeuAc synthase is preferably derived from prokaryotes such as bacteria or yeast, particularly preferably from prokaryotes, and especially preferably from the DNA encoding CjneuB from Campylobacter jejuni strain ATCC 43438 or from the DNA encoding neuB from Rhodobacter capsulatus.

[0038] Pyruvate carboxylase is an enzyme responsible for the carboxylation of pyruvate to produce oxaloacetate, and is encoded by the pyc gene. Since PEP is produced by the decarboxylation of oxaloacetate, Pyc enhances the supply of PEP. The pyc gene is preferably derived from Sinorhizobium meliloti or Corynebacterium glutamicum.

[0039] L-glutamine-D-fructose-6-phosphate aminotransferase is an enzyme responsible for the reaction that produces glucosamine-6-phosphate from fructose-6-phosphate, and is encoded by the glmS gene. The glmS gene is preferably derived from E. coli. Since glucosamine-6-phosphate is an intermediate in the NeuAc synthesis pathway, Glms enhances the supply of NeuAc.

[0040] NanT is a NeuAc transporter that reabsorbs NeuAc that has been excreted into the bacterial cell, thereby enabling the reuse of NeuAc. The nanT gene encoding the NeuAc transporter is preferably derived from E. coli.

[0041] CMP-NeuAc synthase is an enzyme that carries out the reaction to produce CMP-NeuAc using cytidine-5'-triphosphate (hereinafter also referred to as "CTP") and NeuAc as substrates. The DNA encoding CMP-NeuAc synthase is preferably derived from a prokaryote such as bacteria or yeast, particularly preferably from a prokaryote, and most preferably from the neuA gene of Pasteurella maltosida PM70 strain.

[0042] Lactose permylase is a membrane protein that takes lactose into cells and is encoded by the lacY gene. The lacY gene is preferably derived from E. coli. When lactose is added from an external source, lactose permylase promotes the uptake of lactose into the bacterial cell.

[0043] Glucosamine acetyltransferase is an enzyme responsible for the reaction that produces GlcNAc from glucosamine 6-phosphate. Glucosamine acetyltransferase from budding yeast is encoded by the GNA1 gene.

[0044] 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.

[0045] Accordingly, in one embodiment of the present invention, it is preferable to use a genetically modified microorganism as the parent strain, which preferably contains at least one nucleotide sequence selected from the following: 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). In one embodiment of the present invention, it is preferable that the genetically modified microorganism has improved 3'SL productivity compared to the parent strain that is not genetically modified.

[0046] Any known method can be used to produce 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 permyase activity, glucosamine acetyltransferase activity, and N-acetylmannosamine epimerase activity, or having enhanced such activity. Specifically, for example, methods involving various genetic manipulations can be used.

[0047] <<Preferred activity levels that are reduced or absent in the parent plant>> Furthermore, it is preferable that the parent strain exhibits reduced or deleted activity of at least one selected from N-acetylglucosamine (hereinafter also referred to as "GlcNAc") transporter activity, N-acetylmannosamine kinase activity, N-acetylglucosamine-6-phosphate-2-epimerase activity, RNase adapter protein activity, transcription factor activity that negatively regulates the transcription of genes involved in NeuAc assimilation, NeuAc lyase activity, β-galactosidase activity, and galactoside acetyltransferase activity, with particular preference for reduced or deleted activity of β-galactosidase and NeuAc lyase.

[0048] The GlcNAc transporter is involved in the uptake and assimilation of GlcNAc into bacterial cells and is encoded by the nagE and nagB genes. Reduced or deleted GlcNAc transporter activity can promote the production of GlcNAc, a substrate for ManNAc.

[0049] N-acetylmannosamine kinase is an enzyme responsible for the reaction that produces N-acetylmannosamine 6-phosphate (hereinafter also referred to as "ManNAc-6P") from ManNAc, and is encoded by the nanK gene. By suppressing the production of substances other than NeuAc from ManNAc, the supply of NeuAc can be promoted.

[0050] N-acetylglucosamine-6-phosphate-2-epimerase is an enzyme responsible for the conversion of ManNAc-6P to N-acetyl-D-glucosamine-6-phosphate (hereinafter also referred to as "GlcNAc-6P"), and is encoded by the nanE gene.

[0051] The RNase adapter protein is a transcription factor that negatively regulates the activity of genes encoding enzymes involved in NeuAc biosynthesis, and is encoded by the yhbJ gene. The yhbJ gene is also called the rapZ gene. YhbJ is a transcription factor that negatively regulates the activity of the glmS gene, which encodes L-glutamine-D-fructose 6-phosphate aminotransferase, an enzyme involved in NeuAc biosynthesis.

[0052] The transcription factor that negatively regulates the transcription of the genes involved in NeuAc assimilation is a transcription factor involved in the synthesis and metabolism of sialic acid, and is encoded by the nanR gene.

[0053] NeuAc lyase is an enzyme that breaks down NeuAc into ManNAc and pyruvate, and is encoded by the nanA gene.

[0054] β-galactosidase is an enzyme that hydrolyzes lactose and is encoded by the lacZ gene.

[0055] Galactoside acetyltransferase is an enzyme that has the activity to acetylate lactose and its analogues, and is encoded by the lacA gene.

[0056] Accordingly, in one embodiment of the present invention, it is preferable to use a genetically modified microorganism as the parent strain that does not contain at least one base sequence selected from the nagE gene, nagB gene, nanK gene, nanE gene, yhbJ gene, nanR gene, nanA gene, lacZ gene, and lacA gene. In one embodiment of the present invention, it is preferable that the genetically modified microorganism has improved 3'SL productivity compared to the unmodified parent strain.

[0057] Known methods can be used to produce Escherichia coli in which at least one activity selected from GlcNAc transporter activity, N-acetylmannosamine kinase activity, N-acetylglucosamine-6-phosphate-2-epimerase activity, RNase adapter protein activity, transcription factor activity that negatively regulates the transcription of genes involved in NeuAc assimilation, NeuAc lyase activity, β-galactosidase activity, and galactoside acetyltransferase activity is reduced or deleted. Specifically, for example, methods involving various genetic manipulations can be used. (Metabolic Engineering, 2017, 41:23-38)

[0058] <Obtaining microorganisms with enhanced activity of specific proteins> Microorganisms in which the activity of any one of [A] and [1] to [3] is enhanced compared to the parent microorganism include microorganisms obtained by transforming the parent microorganism with recombinant DNA containing the DNA encoding the protein, resulting in a microorganism with an increased copy number of the gene compared to the parent. In this specification, an increase in the copy number of the gene may mean newly acquiring the gene in a parent strain that does not possess the gene on a chromosome or plasmid, or additionally acquiring the gene in a strain that already possesses the gene on a chromosome or plasmid.

[0059] Microorganisms obtained by transforming a parent microorganism with recombinant DNA containing DNA encoding the protein described in any one of [A] and [1] to [3] above, in which the copy number of the gene is increased compared to the parent microorganism, include microorganisms in which the copy number of the gene is increased on chromosomal DNA by transforming a parent microorganism with recombinant DNA containing DNA encoding the protein described in any one of [A] and [1] to [3] above, and microorganisms in which the gene is carried outside of chromosomal DNA as plasmid DNA.

[0060] The DNA encoding the protein described in any one of [A] and [1] to [3] above may be any DNA encoding a protein having the activity of the protein described in any one of [A] and [1] to [3] above, but specifically, one DNA selected from the group consisting of [4] to [7] below is an example. [4] DNA encoding the protein described in any one of [A] and [1] to [3] above [5] DNA consisting of the base sequence represented by SEQ ID NOs: 3, 5, 7, or 9 [6] DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NOs: 3, 5, 7, or 9, and encodes a homologous protein having α2,3-sialyltransferase activity. [7] DNA encoding a homologous protein having α2,3-sialyltransferase activity, 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 with the base sequence represented by SEQ ID NOs: 3, 5, 7, or 9.

[0061] In the above [6], "hybridize" means that DNA hybridizes with DNA having a specific base sequence or a portion of said DNA. Therefore, DNA having a specific base sequence or a portion of said DNA can be used as a probe for Northern or Southern blot analysis, and can also be used as oligonucleotide primers for PCR analysis.

[0062] Examples of DNA used as a probe include DNA with at least 100 bases, preferably 200 bases, and more preferably 500 bases. Examples of DNA used as a primer include DNA with at least 10 bases, preferably 15 bases.

[0063] Methods for DNA hybridization experiments are well known, and for example, those skilled in the art can determine the hybridization conditions according to this specification. These hybridization conditions can be 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.

[0064] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions included with commercially available hybridization kits. Examples of commercially available hybridization kits include the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which uses the Random Prime method to prepare probes and performs hybridization under stringent conditions.

[0065] The stringent conditions described above include incubating the DNA-immobilized filter and probe DNA 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× Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA, followed by washing the filter in a 0.2×SSC solution at approximately 65°C.

[0066] Examples of DNA that can hybridize under the stringent conditions described above include DNA that, when calculated using BLAST, FASTA, or the like based on the parameters described above, has at least 95% identity, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the DNA consisting of the base sequence represented by sequence numbers 3, 5, 7, or 9.

[0067] The DNA encoding the protein described in [1] above, and the DNA described in [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, more preferably Escherichia coli strain BW25113, using probe DNA that can be designed based on the base sequence represented by sequence numbers 3, 5, 7, or 9, or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the said base sequence, with the chromosomal DNA of the above microorganism as a template.

[0068] The DNA encoding the mutant protein described in [2] above can be obtained, for example, by subjecting a DNA consisting of the base sequence represented by SEQ ID NOs: 3, 5, 7, or 9 to error-prone PCR or the like as a template.

[0069] Alternatively, the DNA encoding the mutant protein described in [2] above can also be obtained by PCR [Gene, 77, 51 (1989)] using a set of PCR primers with a nucleotide sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition) at each of their 5' ends.

[0070] Alternatively, the DNA can be obtained by following the instructions provided with a commercially available partially specific mutagenesis kit. An example of a commercially available partially specific mutagenesis kit is the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce mutations (deletions, substitutions, insertions, or additions) at the desired location.

[0071] Specifically, first, a plasmid containing a nucleotide sequence designed to introduce the target mutation (deletion, substitution, insertion, or addition) is used as a template to design a pair of mutation-introducing primers with a 15-nucleotide overlap at the 5' end. The overlapping portion contains the target mutation. Next, PCR is performed using these mutation-introducing primers on a plasmid containing the nucleotide sequence into which the target mutation is to be introduced. When the resulting amplified fragment is transformed into E. coli, a plasmid containing the nucleotide sequence with the target mutation introduced is obtained.

[0072] The DNA encoding the homologous protein described in [3] above, and the DNA described in [6] and [7] above, can be obtained, for example, by searching various gene sequence databases for a base sequence that has 95% or more identity with the base sequence represented by SEQ ID NOs: 3, 5, 7, or 9, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more, or by searching various protein sequence databases for an amino acid sequence that has 90% or more identity with the amino acid sequence represented by SEQ ID NOs: 4, 6, 8, or 10, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and most preferably 99% or more, and then 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 said DNA, in a manner similar to the method for obtaining the DNA described above.

[0073] The DNA obtained from any one of the above [4] to [7] can be incorporated into a vector by conventional methods, either as is or after being cut with an appropriate restriction enzyme. The resulting recombinant DNA is then introduced into host cells, and its base sequence can be determined by analyzing it using commonly used base sequence analysis methods, such as the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analyzer such as the 3700 DNA analyzer (Applied Biosystems).

[0074] The host cells that can be used when determining the DNA base sequence can be any cells that can be introduced into the vector and grow, for example, Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam - / dcm - Examples include 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 (both manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, and Escherichia coli NM522.

[0075] Examples of the vectors mentioned above 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).

[0076] Any method for introducing recombinant DNA into host cells can be used, such as the calcium ion method [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Publication No. 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0077] If the DNA obtained as a result of sequencing is only a partial length, the full-length DNA can be obtained by using the partial-length DNA as a probe and applying methods such as Southern hybridization to a chromosomal DNA library.

[0078] Furthermore, based on the determined DNA base sequence, the target DNA can also be prepared by chemical synthesis using an NTS M-series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd.

[0079] Recombinant DNA containing DNA encoding a protein described in any one of [A] and [1] to [3] above means recombinant DNA in which the DNA is autonomously replicated or can be incorporated into a chromosome in the parent strain and is incorporated into an expression vector containing a promoter at a position where the DNA can be transcribed.

[0080] If the recombinant DNA is recombinant DNA that can be incorporated into a chromosome, it does not need to contain a promoter.

[0081] Microorganisms in which the copy number of the gene is increased compared to the parent strain, obtained by transforming a parent microorganism with recombinant DNA containing the DNA encoding the protein described in any one of [A] and [1] to [3] above, can be obtained by the following method.

[0082] Based on the DNA encoding the protein described in any one of [A] and [1] to [3] obtained by the method described above, a DNA fragment of appropriate length containing the protein-coding portion is prepared as needed. Furthermore, by substituting bases in the base sequence of the protein-coding portion to obtain codons optimized for expression in host cells, a transformant with improved productivity can be obtained.

[0083] Recombinant DNA is produced by inserting the aforementioned DNA fragment downstream of the promoter of a suitable expression vector. By transforming the parent strain with this recombinant DNA, a microorganism can be obtained in which the copy number of the gene encoding the protein is increased compared to the parent strain.

[0084] When using a prokaryote such as bacteria as the parent strain, the recombinant DNA is preferably composed 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 also include a gene that controls the promoter.

[0085] It is preferable 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, a transcription termination sequence is not necessarily required for the expression of the DNA, but it is preferable to place the transcription termination sequence directly below the structural gene.

[0086] Furthermore, the expression level of the protein having α2,3-sialyltransferase activity can be increased by substituting bases in the base sequence of the portion encoding the protein having α2,3-sialyltransferase activity so that the codon is optimal for host expression. Examples of proteins having α2,3-sialyltransferase activity include the proteins described in any one of [A] and [1] to [3] above. Information on codon usage frequency in the parent strain used in the manufacturing method of the present invention can be obtained through public databases.

[0087] 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 in a host. This includes not only plasmids, but also, for example, artificial chromosomes, vectors using transposons, and cosmids.

[0088] When using a microorganism belonging to the genus Escherichia as the parent strain, the expression vectors include, for example, pColdI, pSTV28, pSTV29, pUC118 (all from Takara Bio), pMW119 (from Nippon Gene), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (all from Merck Millipore), pMAL-c5x (from New England Biolabs), pGEX-4T-1, pTrc99A (all from GE Healthcare Biosciences), pTrcHis, pSE280 (all from Thermo Fisher Scientific), pGEMEX-1 (from Promega), pQE-30, pQE80L (all from Qiagen), pET-3, pBluescriptII SK(+), and pBluescriptII. KS(-) (both manufactured by Agilent Technologies), pKYP10 (Japanese Patent Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescriptII SK(+), pBluescript II KS(-) (manufactured by Stratagene), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [Escherichia coli JM109 / pTrS32 (FERM Examples include pTK31 (prepared from BP-5408), pTK31 (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385), pPAC31 (International Publication No. 1998 / 12343), pUC19 (Gene, 33, 103 (1985)), pPA1 (Japanese Patent Publication No. 63-233798), pKD46 (Proc. Natl. Acad. Sci., USA, 97, 6640-6645 (2000)), etc.

[0089] When using the above expression vector, any promoter that functions in the cells of microorganisms belonging to the genus Escherichia may be used. For example, promoters of genes involved in amino acid biosynthesis, such as the trp promoter and the ilv promoter, or promoters derived from Escherichia coli or phages, such as the uspA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter, can be used. In addition, artificially designed and modified promoters such as a promoter with two trp promoters in series, the tac promoter, the trc promoter, the lacT7 promoter, and the letI promoter can also be used.

[0090] When using a microorganism belonging to the genus Corynebacterium as the parent strain, examples of expression vectors include pCG1 (Japanese Patent Publication No. 57-134500), pCG2 (Japanese Patent Publication No. 58-35197), pCG4 (Japanese Patent Publication No. 57-183799), pCG11 (Japanese Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (both Japanese Patent Publication No. 58-105999), pCE51, pCE52, pCE53 [all from Molecular and General Genetics, 196, 175 (1984)].

[0091] When using the above expression vector, any promoter that functions in the cells of microorganisms belonging to the genus Corynebacterium may be used, but for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)] can be used.

[0092] When using a yeast strain as the parent strain, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, etc.

[0093] When using the above expression vector, any promoter that functions in the cells of the yeast strain may be used, but examples of promoters include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0094] Recombinant DNA used in the manufacturing method of the present invention can be produced by inserting one of the DNA fragments described in any one of [4] to [7] above downstream of the promoter of a suitable expression vector.

[0095] Methods for introducing recombinant DNA into parental strains as self-replicating plasmids include, for example, the calcium ion method [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Publication No. 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0096] One method for incorporating recombinant DNA into the chromosomes of a host cell is homologous recombination. Homologous recombination methods include, for example, the use of homologous recombination plasmids, which can be produced by linking plasmid DNA containing a drug resistance gene that cannot autonomously replicate within the host cell to be introduced. A frequently used homologous recombination method in Escherichia coli is the introduction of recombinant DNA using a lambda phage homologous recombination system [Proc.Natl.Acad.Sci.USA,97,6640-6645(2000)].

[0097] Furthermore, by using selection methods that utilize the fact that E. coli becomes sucrose-sensitive due to Bacillus subtilis levanseucrase incorporated into the chromosome along with recombinant DNA, or by using selection methods that utilize the fact that E. coli becomes streptomycin-sensitive by incorporating the wild-type rpsL gene into E. coli having a streptomycin-resistant mutant rpsL gene [Mol.Microbiol.,55,137(2005), Biosci.Biotechnol.Biochem.,71,2905(2007)], E. coli in which the target region on the chromosomal DNA of the host cell has been replaced with recombinant DNA can be obtained.

[0098] The fact that the recombinant DNA has been introduced into the parent strain as an autonomously replicating plasmid, or incorporated into the parent strain's chromosome, can be confirmed, for example, by observing the amplification product of the gene introduced by transformation using a PCR with a primer set capable of amplification, even though the microorganism cannot amplify the gene that it originally possesses on its chromosomal DNA. Furthermore, an increase in the transcription level of the DNA or the production level of the protein encoded by the DNA can be confirmed by comparing the transcription level of the gene in the microorganism with that of the parent strain using Northern blotting, or the production level of the protein in the microorganism with that of the parent strain using Western blotting.

[0099] The fact that the microorganisms produced by the above method have enhanced protein activity compared to the parent strain can be confirmed by analyzing the 3'SL contained in the supernatant using HPLC or the like after culturing the microorganisms, diluting the culture medium appropriately, centrifuging it, and comparing it with that of the parent strain.

[0100] The microorganisms described above exhibit enhanced activity of the proteins described in [A] and any one of [1] to [3] above compared to the parent strain, thereby promoting the transfer of sialic acid residues from CMP-sialic acid to galactose at the receptor carbohydrate terminus via α2,3 bonds, and potentially improving 3'SL productivity. 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.

[0101] In microorganisms with enhanced α2,3-sialyltransferase activity, for example, the high activity of α2,3-sialyltransferase can improve 3'SL productivity.

[0102] 2.3'SL Manufacturing Method One embodiment of the present invention includes the following method for manufacturing 3'SL. A method for producing 3'SL, comprising preparing the microorganisms described in 1. above, and generating 3'SL in a culture using the said microorganisms.

[0103] The method for culturing the microorganisms described in 1. above can be carried out according to the usual methods used for culturing microorganisms.

[0104] As a culture medium for the microorganism, either a natural medium or a synthetic medium may be used, as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that the microorganism can utilize and allows for efficient cultivation of the transformant.

[0105] Any carbon source that the microorganism can utilize is acceptable, and examples include glucose, fructose, sucrose, molasses containing these, sugars such as starch or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol or propanol.

[0106] Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as 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 hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation microorganisms and their digests.

[0107] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0108] As the microorganism of the present invention used in the method for producing 3'SL, a microorganism capable of producing receptor carbohydrates such as glucose, lactose, or lactose monohydrate may be used.

[0109] In the method for producing 3'SL, glucose, liquid sugar, sucrose, lactose, NeuAc, etc., may be added to the culture medium during cultivation.

[0110] Furthermore, in the method for producing 3'SL, instead of adding lactose, NeuAc, etc. to the culture medium during cultivation, a microorganism capable of producing lactose and NeuAc may be cultivated simultaneously with the microorganism of the present invention to supply lactose, NeuAc, etc. to the microorganism of one embodiment of the present invention.

[0111] In a method for producing oligosaccharides, it is preferable that the culture medium does not contain GlcNAc transporter, N-acetylmannosamine kinase, N-acetylglucosamine-6-phosphate-2-epimerase, RNase adapter protein, transcription factors that negatively regulate the transcription of genes involved in the assimilation of NeuAc, NeuAc lyase, β-galactosidase, and galactoside acetyltransferase.

[0112] The cultivation is usually preferably carried out under aerobic conditions by aeration and agitation culture. The cultivation temperature is usually 28 to 37 °C, and the cultivation time is usually 36 hours to 4 days. The pH of the culture broth during cultivation is usually maintained at 6.4 to 7.4. The pH adjustment is carried out using an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, etc.

[0113] By the above cultivation, 3'SL is generated and accumulated in the culture, and 3'SL can be produced by collecting 3'SL from the culture.

[0114] Usually, after centrifugation of the culture, 3'SL can be collected from the supernatant. When 3'SL accumulates in the cells, for example, the cells are disrupted by ultrasonic waves or the like, and 3'SL can be collected from the supernatant obtained by removing the cells by centrifugation by an ion exchange resin method or the like. [Analysis Example] In the examples, the analysis and quantification of 3'SL were carried out according to the following procedure. The culture broth containing microorganisms after cultivation was appropriately diluted, filter sterilized, and the supernatant was recovered. 3'SL contained in the supernatant was analyzed using an analyzer SPD-20A (manufactured by Shimadzu Corporation). [Analysis Conditions] Column: Shodex SUGAR SH1011 Column temperature: 50 °C Eluent composition: 5 mM sulfuric acid aqueous solution Flow rate: 0.6 ml / min Detector: SPD-20A

Examples

[0115] [Example 1] Construction of microorganisms used for the production of 3'SL (1) Construction of a host for 3'SL production <Escherichia coli lacking the yhbJ gene> Escherichia coli in which the transcriptional regulator 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 N-acetylneuraminic acid (hereinafter referred to as NeuAc) biosynthesis was disrupted was prepared by the following procedure.

[0116] Using the BW25113 strain (Keio collection (Systematic single-gene knock-out mutants of E. coli K-12)) as the host, a strain BW25113ΔyhbJ in which the yhbJ gene was completely deleted was obtained by the same method as Baba et al. (Baba T. et al. (2006) Mol systems Biol).

[0117] <Obtaining DNA fragments used as markers during gene deletion> Using the DNA consisting of the base sequences represented by the "primer set" in Table 1 as a primer set and the DNA described in the "template" in Table 1 as a template, PCR was performed to obtain each amplified DNA fragment.

[0118]

Table 1

[0119] The genomic DNA of Bacillus subtilis 168 strain was prepared by a conventional method. The cat of the amplified DNA fragment contains approximately 200 bp upstream and approximately 50 bp downstream of the cat gene (encoding chloramphenicol acetyltransferase) on pHSG396. The sacB of the amplified DNA fragment contains approximately 300 bp upstream and approximately 100 bp downstream of the sacB gene (encoding levansucrase) on the genomic DNA of Bacillus subtilis 168 strain.

[0120] Next, using the mixture of cat and sacB of the amplified DNA fragments at an equimolar ratio as a template and the DNA consisting of the base sequences represented by SEQ ID NOs: 21 and 24 as a primer set, PCR was performed to obtain a DNA fragment containing the cat gene and the sacB gene (hereinafter referred to as cat-sacB).

[0121] <Escherichia coli lacking the lacZ gene> Using the BW25113ΔyhbJ strain created above as the parent strain, E. coli strains in which the lacZ gene (SEQ ID NO: 12), which encodes β-galactosidase involved in lactose degradation, was disrupted were prepared using the following procedure.

[0122] Using chromosomal DNA from strain BW25113, prepared by conventional methods, as a template, PCR was performed using DNA consisting of the base sequences shown in Table 2 as primer sets to obtain each amplified DNA fragment.

[0123] [Table 2]

[0124] lacZ upstream 1 and lacZ upstream 2 include the start codon of the lacZ gene and approximately 700 bp upstream from it. lacZ downstream 1 and lacZ downstream 2 include the stop codon of the lacZ gene and approximately 800 bp downstream from it.

[0125] Using a template made by mixing lacZ upstream 1, lacZ downstream 1, and the cat-sacB fragment in equimolar proportions, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 33 and 34 to obtain a DNA fragment (hereinafter referred to as lacZ::cat-sacB) in which the cat-sacB fragment was inserted into the region surrounding the lacZ gene.

[0126] Using a template made by mixing lacZ upstream 2 and lacZ downstream 2 in equimolar ratios, PCR was performed using a primer set consisting of DNA with the base sequences represented by SEQ ID NOs. 33 and 34 to obtain a DNA fragment (hereinafter referred to as ΔlacZ) that does not contain lacZ and consists of a sequence in which lacZ upstream and lacZ downstream are directly linked.

[0127] The lacZ::cat-sacB fragment was introduced into the BW25113ΔyhbJ strain harboring the plasmid pKD46 [Datsenko, K. A., Warner, B. L., Proc. Natl. Acad. Sci., USA, Vol. 97, 6640 - 6645 (2000)] containing the gene encoding λ recombinase by electroporation, and a transformant showing chloramphenicol resistance and sucrose sensitivity (a transformant in which the lacZ gene was replaced with lacZ::cat-sacB) was obtained.

[0128] The ΔlacZ fragment was introduced into the transformant by electroporation, and a transformant showing chloramphenicol sensitivity and sucrose resistance (a transformant in which lacZ::cat-sacB was replaced with ΔlacZ) was obtained. This transformant was named the BW25113ΔyhbJΔlacZ strain.

[0129] <Escherichia coli lacking the nanR, nanA, nanT, nanE, and nanK genes> Using the BW25113ΔyhbJΔlacZ strain as a host, the nanR gene (SEQ ID NO: 13) encoding a transcriptional regulator that negatively controls the transcription of the gene group involved in the assimilation of NeuAc, the nanA gene (SEQ ID NO: 14) encoding NeuAc lyase, the nanT gene (SEQ ID NO: 15) encoding a NeuAc transporter, the nanE gene (SEQ ID NO: 16) encoding N-acetylglucosamine-6-phosphate-2-epimerase, and the nanK gene (SEQ ID NO: 17) encoding N-acetylmannosamine kinase were disrupted in Escherichia coli by the following procedure. Note that nanR, nanA, nanT, nanE, and nanK (hereinafter referred to as nanRATEK) form an operon on the Escherichia coli genome.

[0130] Using the chromosomal DNA of the BW25113 strain prepared by a conventional method as a template, PCR was performed using a DNA consisting of the base sequences represented by the "primer set" in Table 3 as a primer set, and each amplified DNA fragment was obtained.

[0131] [Table 3]

[0132] nanR upstream 1 and nanR upstream 2 include the start codon of the nanR gene and approximately 800 bp upstream from it. nanK downstream 1 and nanK downstream 2 include the stop codon of the nanK gene and approximately 850 bp downstream from it.

[0133] Using a template made by mixing nanR upstream 1, nanK downstream 1, and cat-sacB fragments in equimolar proportions, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 43 and 44 to obtain a DNA fragment (hereinafter referred to as nanRATEK::cat-sacB) in which the cat-sacB fragment was inserted into the sequence surrounding the nanRATEK gene.

[0134] Using a template made by mixing nanR upstream 2 and nanK downstream 2 in equimolar proportions, PCR was performed using a primer set consisting of DNA with the base sequences represented by SEQ ID NOs. 43 and 44 to obtain a DNA fragment (hereinafter referred to as ΔnanRATEK) that does not contain nanRATEK and consists of a sequence in which nanR upstream and nanK downstream are directly linked.

[0135] The nanRATEK::cat-sacB fragment was introduced by electroporation into the BW25113ΔyhbJΔlacZ strain, which carries plasmid pKD46 containing the gene encoding λ recombinase, to obtain a transformant that was chloramphenicol resistant and sucrose sensitive (a transformant in which the nanRATEK gene was replaced with nanRATEK::cat-sacB).

[0136] The ΔnanRATEK fragment was introduced into the transformant by electroporation to obtain a transformant that was sensitive to chloramphenicol and resistant to sucrose (a transformant in which nanRATEK::cat-sacB was replaced with ΔnanRATEK). This transformant was named strain BW25113ΔyhbJΔlacZΔnanRATEK.

[0137] (2) Creation of plasmids for NeuAc production Plasmids expressing the NeuAc synthase gene CjneuB (SEQ ID NO: 18) derived from Campylobacter jejuni ATCC43438 strain and the UDP-GlcNAc epimerase gene FpneuC (SEQ ID NO: 19) derived from Flavobacterium psychrophilum NBRC100250 strain were constructed using the following procedure.

[0138] PCR was performed using DNA consisting of the base sequences shown in "Primer Set" in Table 4 as the primer set, and the DNA described in "Template" in Table 4 as the template, to obtain amplified DNA fragments.

[0139] Since the start codon of the wild-type sequence of FpneuC (SEQ ID NO: 19) is GTG, in order to express it in E. coli, ATG was added to the primer sequence so that the start codon of the amplified DNA fragment FpneuC obtained by PCR becomes ATG.

[0140] [Table 4]

[0141] Genomic DNA from Campylobacter jejuni strain ATCC43438 and Flavobacterium psychrophilum strain NBRC100250 was prepared by conventional methods.

[0142] The nucleotide sequences represented by sequence numbers 46 and 47 each contain a complementary sequence at their respective 5' ends. Using an equimolar mixture of the CjneuB and FpneuC fragments obtained above as a template, PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs. 45 and 48 as primers to obtain a DNA fragment of approximately 2.2 kb (hereinafter referred to as the CjneuB-FpneuC fragment) by ligating the CjneuB and FpneuC fragments.

[0143] The expression plasmid pTrc99a-CjneuB-FpneuC (hereinafter referred to as pBC) was obtained by ligating the CjneuB-FpneuC fragment with the expression vector pTrc99a (manufactured by GE Healthcare Biosciences) using the In-Fusion HD Cloning Kit (manufactured by Takara Bio).

[0144] (3) Creation of plasmids for 3'SL production Plasmids expressing the sialylatesase gene NlsiaT (SEQ ID NOs. 1, 3, 5, 7, or 9) from the full-length or truncated Neisseria lactamica ATCC23970 strain and the CMP-NeuAc synthase gene PmneuA (SEQ ID NOs. 20) from Pasteurella multocida subsp. multocida str. Pm70 ATCC BAA-1909 strain were prepared using the following procedure.

[0145] Using chromosomal DNA from Neisseria lactamica, prepared by conventional methods, as a template, PCR was performed using DNA consisting of the base sequences shown in Table 5 as primer sets to obtain each amplified DNA fragment.

[0146] [Table 5]

[0147] Using chromosomal DNA from Pasteurella multocida subsp. multocida str. Pm70 ATCC BAA-11909, prepared by conventional methods, PCR was performed using a primer set consisting of DNA sequences represented by sequence numbers 55 and 56 to obtain the PmneuA fragment.

[0148] The nucleotide sequences represented by sequence numbers 50 and 55 each contain a complementary sequence at their respective 5' ends. Using an equimolar mixture of the NlsiaT fragment, tNlsiaTΔN20AA fragment, tNlsiaTΔN23AA fragment, tNlsiaTΔN27AA fragment, or tNlsiaTΔN37AA fragment, and PmneuA fragment obtained above as a template, PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs. 49, 51, 52, 53 or 54, and 56 as a primer set to obtain DNA fragments obtained by ligating each NlsiaT fragment and PmneuA fragment.

[0149] The DNA fragments obtained by ligating each of the NlsiaT and PmneuA fragments obtained above, along with the expression vector pSTV29 (Takara Bio Inc.), were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain expression plasmids pSTV-NlsiaT, pSTV-tNlsiaTΔN20AA, pSTV-tNlsiaTΔN23AA, pSTV-tNlsiaTΔN27AA, and pSTV-tNlsiaTΔN37AA.

[0150] (4) Creation of microorganisms possessing plasmids for NeuAc production Using the NeuAc production plasmid pBC obtained in (2) above, the BW25113ΔyhbJΔlacZΔnanRATEK strain created in (1) above was transformed to create an Escherichia coli strain containing pBC, which was named the BW25113ΔyhbJΔlacZΔnanRATEK / pBC strain.

[0151] (5) Creation of microorganisms possessing plasmids for 3'SL production Using the 3'SL production plasmids obtained in (2) above, pSTV-NlsiaT, pSTV-tNlsiaTΔN20AA, pSTV-tNlsiaTΔN23AA, pSTV-tNlsiaTΔN27AA, and pSTV-tNlsiaTΔN37AA, the BW25113ΔyhbJΔlacZΔnanRATEK / pBC strain created in (4) above was transformed to create Escherichia coli strains possessing various plasmids, which were named NlsiaT strain, tNlsiaTΔN20AA strain, tNlsiaTΔN23AA strain, tNlsiaTΔN27AA strain, and tNlsiaTΔN37AA strain, respectively.

[0152] [Example 2] Production of 3'SL by fermentation method The productivity of 3'SL was evaluated for the NlsiaT strain, tNlsiaTΔN20AA strain, tNlsiaTΔN23AA strain, tNlsiaTΔN27AA strain, and tNlsiaTΔN37AA strain obtained in Example 1.

[0153] Each bacterial strain was incubated overnight at 30°C on LB plates containing 100 mg / L ampicillin and 25 mg / L chloramphenicol. The cells were then inoculated into large test tubes containing 5 ml of LB medium with 100 mg / L ampicillin and 25 mg / L chloramphenicol, and incubated with shaking at 30°C for 16 hours.

[0154] Subsequently, 0.3 mL of the culture medium containing 3 ml of a production medium containing 100 mg / L ampicillin and 25 mg / L chloramphenicol [30 g / L glycerol, 10 g / L lactose, 2.0 g / L magnesium sulfate heptahydrate, 16 g / L dipotassium hydrogen phosphate, 14 g / L potassium dihydrogen phosphate, 2.0 g / L ammonium sulfate, 1.0 g / L citric acid monohydrate, 5.0 g / L casamino acid (Difco), 10 mg / L vitamin B1, 50 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate heptahydrate, and sodium hydroxide aqueous solution was adjusted to pH 7.2 and then autoclaved] [glycerol, lactose, and magnesium sulfate heptahydrate aqueous solutions were prepared separately, autoclaved, and mixed after cooling] was inoculated into a large test tube and cultured with shaking at 30°C for 24 hours. Five hours after the start of culture, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM.

[0155] After the culture was complete, the culture medium was diluted as needed, and the sample, after filter sterilization, was analyzed by HPLC to quantify the 3'SL concentration in the supernatant. The results are shown in Table 6.

[0156] [Table 6]

[0157] The results above demonstrate that when using a shortened form of sialic acid transferase NlsiaT derived from Neisseria lactamica strain ATCC23970, with the N-terminal amino acids removed, it exhibits higher 3'SL productivity than the full-length form of NlsiaT. Furthermore, the effective N-terminal deletion lengths were approximately equivalent at 20, 23, 27, and 37 amino acid residues.

[0158] These shortened glycosyltransferases were shown to be useful for the efficient production of 3'SL compared to full-length glycosyltransferases.

[0159] 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 are possible without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2022-091801, filed on June 6, 2022, which is incorporated herein by reference in its entirety. All references herein are incorporated as a whole. [Industrial applicability]

[0160] This invention makes it possible to efficiently produce 3'SL. [Sequence Listing Free Text]

[0161] Sequence ID 1: Nucleotide sequence of the sialic acid transferase gene NlsiaT from Neisseria lactamica strain ATCC23970 Sequence ID 2: Amino acid sequence of the sialic acid transferase gene NlsiaT from Neisseria lactamica strain ATCC23970 Sequence ID 3: tNlsiaTΔN20AA Amino acid sequence of Sequence ID No. 4: tNlsiaTΔN20AA Sequence ID 5: tNlsiaTΔN23AA SEQ ID NO: 6: Amino acid sequence of tNlsiaTΔN23AA Sequence ID 7: tNlsiaTΔN27AA Amino acid sequence of Sequence ID No. 8: tNlsiaTΔN27AA Sequence ID 9: tNlsiaTΔN37AA Amino acid sequence of SEQ ID NO: 10: tNlsiaTΔN37AA Sequence ID 11: yhbJ base sequence from Escherichia coli BW25113 Sequence ID 12: lacZ base sequence from Escherichia coli BW25113 Sequence ID 13: nanR base sequence from Escherichia coli BW25113 Sequence ID 14: nanA base sequence from Escherichia coli BW25113 Sequence ID 15: nanT base sequence from Escherichia coli BW25113 Sequence ID 16: nanE base sequence from Escherichia coli BW25113 Sequence ID 17: nanK base sequence from Escherichia coli BW25113 Sequence ID 18: Nucleotide sequence of the NeuAc synthase gene CjneuB from Campylobacter jejuni strain ATCC43438. Sequence ID 19: Base sequence of FpneuC, which encodes UDP-GlcNAc epimerase from Flavobacterium psychrophilum NBRC100250 strain. Sequence ID 20: Nucleotide sequence of the CMP-NeuAc synthase gene PmneuA from Pasteurella multocida subsp. multocida str. Pm70 ATCC BAA-1909 strain. Sequence ID 21: Base sequence of primer Fw for cat fragment amplification Sequence ID 22: Base sequence of primer Rv for CAT fragment amplification Sequence ID 23: Base sequence of primer Fw for sacB fragment amplification Sequence ID No. 24: Base sequence of primer Rv for sacB fragment amplification Sequence ID 25: Base sequence of primer Fw for amplification of lacZ upstream 1 fragment Sequence ID 26: Base sequence of primer Rv for amplification of lacZ upstream 1 fragment Sequence ID 27: Base sequence of primer Fw for amplification of lacZ downstream 1 fragment Sequence ID 28: Base sequence of primer Rv for amplification of lacZ downstream 1 fragment Sequence ID 29: Base sequence of primer Fw for amplification of two upstream fragments of lacZ Sequence ID 30: Base sequence of primer Rv for amplification of lacZ upstream 2 fragments Sequence ID 31: Base sequence of primer Fw for amplification of lacZ downstream 2 fragments Sequence ID 32: Base sequence of primer Rv for amplification of lacZ downstream 2 fragments Sequence ID 33: Base sequence of primer Fw for lacZ disruption fragment preparation Sequence ID 34: Base sequence of primer Rv for lacZ disruption fragment preparation Sequence ID 35: Base sequence of primer Fw for amplification of nanR upstream 1 fragment Sequence ID 36: Base sequence of primer Rv for amplification of nanR upstream 1 fragment Sequence ID 37: Base sequence of primer Fw for nanK downstream 1-fragment amplification Sequence ID 38: Base sequence of primer Rv for nanK downstream 1-fragment amplification Sequence ID 39: Base sequence of primer Fw for amplification of nanR upstream 2 fragments Sequence ID 40: Base sequence of primer Rv for amplification of nanR upstream 2 fragments Sequence ID 41: Base sequence of primer Fw for nanK downstream 2-fragment amplification Sequence ID 42: Base sequence of primer Rv for nanK downstream 2-fragment amplification Sequence ID 43: Base sequence of primer Fw for generating nanRATEK disruption fragments Sequence ID 44: Base sequence of primer Rv for nanRATEK disruption fragment preparation. Sequence ID 45: Base sequence of primer Fw for CjneuB fragment amplification Sequence ID No. 46: Base sequence of primer Rv for CjneuB fragment amplification Sequence ID 47: Base sequence of primer Fw for FpneuC fragment amplification Sequence ID 48: Base sequence of primer Rv for FpneuC fragment amplification Sequence ID 49: Base sequence of primer Fw for NlsiaT amplification Sequence ID 50: Base sequence of primer Rv for NlsiaT amplification Sequence ID 51: Base sequence of primer Fw for tNlsiaTΔN20AA amplification Sequence ID 52: Base sequence of primer Fw for amplification of tNlsiaTΔN23AA Sequence ID 53: Base sequence of primer Fw for tNlsiaTΔN27AA amplification Sequence ID 54: Base sequence of primer Fw for amplification of tNlsiaTΔN37AA Sequence ID 55: Base sequence of primer Fw for PmneuA amplification Sequence ID 56: Base sequence of primer Rv for PmneuA amplification

Claims

1. A microorganism in which the activity of a protein consisting of an amino acid sequence in which the 20-37 amino acid residues at the N-terminus of the amino acid sequence represented by Sequence ID No. 2 are deleted is enhanced, and the productivity of 3'-sialyl lactose is improved compared to the parent strain.

2. The microorganism according to claim 1, wherein the protein comprising an amino acid sequence in which the N-terminal 20 to 37 amino acid residues of the amino acid sequence represented by Sequence ID No. 2 are deleted is a protein comprising the amino acid sequence represented by Sequence ID No. 4, 6, 8, or 10.

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

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