Microorganisms having an altered lactose permease and method for producing lactose-containing oligosaccharides

Abstract

The purpose of the present invention is to provide a more efficient method for producing a lactose-containing oligosaccharide by fermentation production. According to the present invention, a microorganism capable of producing a protein having the activity of a lactose permease that is modified in such a manner that a specific amino acid residue is substituted by another amino acid residue is used, whereby it becomes possible to produce a lactose-containing oligosaccharide, e.g., 2'-fucosyllactose, more efficiently compared with a case where a microorganism capable of producing a protein having the activity of the lactose permease of the wild type is used.

Description

Technical Field

[0001] The present invention relates to a method for producing lactose-containing oligosaccharides, which can produce lactose-containing oligosaccharides with high efficiency.

Background Art

[0002] It has been reported that milk oligosaccharides (HMO) contained in human breast milk have functions such as infection defense against pathogenic bacteria and functions as prebiotics, and have attracted attention as additives to infant formula milk due to their physiological activities (Non-Patent Document 1).

[0003] To date, more than 130 types of HMO have been known, and most of them are lactose-containing oligosaccharides having a free lactose unit at the reducing end.

[0004] In the method for producing lactose-containing oligosaccharides by fermentation production, a method of adding relatively inexpensive lactose as a substrate is known (Non-Patent Document 2). In addition, it is known that the uptake of lactose from outside microbial cells is carried out via lactose permease (Non-Patent Document 3).

[0005] On the other hand, a method for producing lactose-containing oligosaccharides using a microorganism having a mutant lactose permease and the influence of mutations in lactose permease on the productivity of lactose-containing oligosaccharides are not known. On the other hand, due to its high attention, a more efficient production method for lactose-containing oligosaccharides is required.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] As described above, for lactose-containing oligosaccharides, a more efficient production method is required. The present invention aims to provide a method for producing lactose-containing oligosaccharides by more efficient fermentation production.

Means for Solving the Problems

[0008] The present invention relates to the following. 1. In the amino acid sequence of the protein described in any one of the following [1] to [3], a protein consisting of an amino acid sequence containing a substitution of an amino acid residue corresponding to the 319th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 with another amino acid residue, and A microorganism having a higher ability to produce lactose-containing oligosaccharides than the parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2, and which is a mutant protein having lactose permease activity. [3] A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and which is a homologous protein having lactose permease activity. 2. The microorganism according to 1 above, having a protein consisting of an amino acid sequence containing a substitution of an amino acid residue corresponding to the 319th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 with L-aspartic acid or L-glutamic acid, in the amino acid sequence of the protein described in any one of the above [1] to [3]. 3. The microorganism according to 1 or 2 above, having a protein consisting of an amino acid sequence containing a substitution of an amino acid residue corresponding to the 319th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 with L-glutamic acid, in the amino acid sequence of the protein described in any one of the above [1] to [3]. 4. A microorganism according to any one of [1] to [3], which is obtained by transforming a parent strain with a recombinant DNA containing a DNA encoding a protein consisting of an amino acid sequence containing a substitution of an amino acid residue at the 319th position of the amino acid sequence represented by SEQ ID NO: 2 with another amino acid residue in the amino acid sequence of the protein according to any one of [1] to [3]. 5. A microorganism according to claim 4, which is obtained by integrating into the chromosome a recombinant DNA containing a DNA encoding a protein consisting of an amino acid sequence containing a substitution of an amino acid residue at the 319th position of the amino acid sequence represented by SEQ ID NO: 2 with another amino acid residue in the amino acid sequence of the protein according to any one of [1] to [3]. 6. A microorganism according to any one of [1] to [5], which is obtained by transforming a parent strain with a recombinant DNA containing a DNA having the nucleotide sequence represented by SEQ ID NO: 3. 7. A microorganism according to claim 6, which is obtained by integrating into the chromosome a recombinant DNA containing a DNA having the nucleotide sequence represented by SEQ ID NO: 3. 8. A microorganism according to any one of [1] to [7], wherein the parent strain is a microorganism having the ability to produce lactose-containing oligosaccharides. 9. A method for producing a lactose-containing oligosaccharide, which comprises culturing the microorganism according to any one of [1] to [8] in a medium to produce a lactose-containing oligosaccharide in the culture. 10. The production method according to claim 9, wherein the lactose-containing oligosaccharide is 2'-fucosyllactose.

Advantages of the Invention

[0009] According to the present invention, there is provided a method for producing a lactose-containing oligosaccharide, which can produce the lactose-containing oligosaccharide with high efficiency.

Modes for Carrying Out the Invention

[0010] 1. The microorganism of the present invention The microorganism of the present invention is a microorganism having the protein described in the following (1) or (2), and is a microorganism having a higher ability to produce lactose-containing oligosaccharides than the parent strain. (1) A protein consisting of an amino acid sequence containing a substitution of an amino acid residue at the 319th position of the amino acid sequence represented by SEQ ID NO: 2 with another amino acid residue, in the amino acid sequence of the protein described in any one of the following [1] to [3]. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 [2] A mutant protein which is a protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2, and which has lactose permease activity. [3] A homologous protein which is a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and which has lactose permease activity. (2) The protein according to (1) above, consisting of an amino acid sequence containing a substitution with L-glutamic acid at the amino acid residue corresponding to the 319th position of the amino acid sequence represented by SEQ ID NO: 2, in the amino acid sequence of the protein described in any one of the above [1] to [3].

[0011] In the amino acid sequence of the original protein described in any one of the above [1] to [3], the amino acid residue corresponding to the 319th position of the amino acid sequence represented by SEQ ID NO: 2 refers to the amino acid residue aligned at the same position as the 319th amino acid residue in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence of the original protein and the amino acid sequence of SEQ ID NO: 2 are aligned.

[0012] In the amino acid sequence of the protein described in (1) or (2) above, the fact that the amino acid residue corresponding to the 319th position of the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue can be confirmed, for example, by aligning the amino acid sequence of the protein described in (1) or (2) above for which the amino acid residue is to be confirmed with the amino acid sequence of the original protein described in any one of the above [1] to [3].

[0013] The alignment of amino acid sequences can be created, for example, using the known alignment program ClustalW [Nucelic Acids Research 22, 4673, (1994)]. ClustalW is available, for example, from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). As parameters for creating an alignment using ClustalW, for example, default values can be used.

[0014] The other amino acid residues described in (1) above may be either natural or non-natural. Examples of natural 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, L-cysteine, and the like.

[0015] Examples of amino acids that can be mutually substituted are shown below. Amino acids included in the same group can be 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

[0016] As the other amino acid residue described in the above (1), preferably, one selected from the amino acid residues (aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid) described in the above Group B is preferred, and it is more preferable that the amino acid residue is in the L-form. As the other amino acid residue described in the above (1), L-glutamic acid is even more preferable.

[0017] A mutant protein refers to a protein obtained by artificially deleting or substituting an amino acid residue in the original protein, or artificially inserting or adding an amino acid residue into the protein.

[0018] In a mutant protein, when an amino acid is deleted, substituted, inserted or added, 1 to 20 amino acids may be deleted, substituted, inserted or added at any position in the same sequence.

[0019] The amino acid to be deleted, substituted, inserted or added may be either a natural type or a non-natural type. Examples of natural type amino acids include the above-mentioned natural type amino acids.

[0020] Examples of mutually substitutable amino acids are as described above. Amino acids included in the same group are mutually substitutable.

[0021] The term "homologous protein" refers to a group of proteins that are present in organisms existing in nature and are derived from proteins with the same evolutionary origin. Homologous proteins are similar in structure and function to each other. The identity of the amino acid sequence or nucleotide sequence can be determined using the algorithms BLAST [Pro. Nat. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] developed 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 a nucleotide sequence using BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

[0022] That the above-mentioned mutant protein or homologous protein has lactose permease activity can be confirmed, for example, by the following method. First, a recombinant DNA having a DNA encoding the mutant protein or homologous protein whose activity is to be confirmed is prepared by the method described below. Next, a microorganism that does not have lactose permease activity, for example, Escherichia coli W3110 strain lacking lactose permease, is transformed with the recombinant DNA. Finally, by culturing the microorganism in a medium containing lactose as a glycogen and confirming that the growth is improved as compared with the parent strain, it can be confirmed that the mutant protein or homologous protein has lactose permease activity.

[0023] In the amino acid sequence of the protein described in the above (1) or (2), specific examples of the protein containing a substitution of an amino acid residue at the 319th position of the amino acid sequence represented by SEQ ID NO: 2 with another amino acid residue include the protein consisting of the amino acid sequence represented by SEQ ID NO: 32.

[0024] The parental 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 referred to as the host strain.

[0025] The parental strain in the microorganism of the present invention may be any microorganism as long as it has the ability to produce lactose-containing oligosaccharides.

[0026] Lactose-containing oligosaccharides refer to oligosaccharides having a lactose unit at the reducing end. Examples of lactose-containing oligosaccharides include 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3'-sialyl-3-fucosyllactose, and the like.

[0027] As the microorganism having the ability to produce lactose-containing oligosaccharides, preferably, a breeding strain in which the ability to produce lactose-containing oligosaccharides is artificially imparted or enhanced can be used.

[0028] Methods for artificially imparting or enhancing the ability to produce lactose-containing oligosaccharides to the microorganism used as the parental strain include: (a) a method of enhancing the expression of at least one enzyme involved in the biosynthetic pathway for generating lactose-containing oligosaccharides from sugars; (b) a method of increasing the copy number of at least one gene encoding an enzyme involved in the biosynthetic pathway for generating lactose-containing oligosaccharides from sugars; (c) a method of relaxing or releasing at least one mechanism for controlling the biosynthetic pathway for generating lactose-containing oligosaccharides from sugars; (d) a method of weakening or blocking at least one metabolic pathway that branches from the biosynthetic pathway for generating lactose-containing oligosaccharides from sugars to metabolites other than the target substance, and the like. The above-known methods can be used alone or in combination.

[0029] Specific examples of the enzyme involved in the biosynthetic pathway for producing lactose-containing oligosaccharides from the above-mentioned sugar include, for example, an enzyme having an α1,2-fucosyltransferase activity that produces 2'-fucosyllactose using GDP-fucose and lactose as substrates, an enzyme having an α1,3-fucosyltransferase activity that produces 3-fucosyllactose using GDP-fucose and lactose as substrates, an enzyme having an α2,3-sialyltransferase activity that produces 3'-sialyllactose using CMP-sialic acid and lactose as substrates, and an enzyme having an α2,6-sialyltransferase activity that produces 6'-sialyllactose using CMP-sialic acid and lactose as substrates, etc., known enzymes.

[0030] Specific examples of the method for imparting or enhancing the ability to produce lactose-containing oligosaccharides include, for example, a method for enhancing the ability to produce 2'-fucosyllactose or 3-fucosyllactose by various genetic manipulations (Metabolic Engineering (2017) 41:23-38), etc., known methods.

[0031] In addition, a breeding strain obtained by artificially imparting or enhancing the ability to supply lactose, which is a precursor, to a microorganism having the ability to produce lactose-containing oligosaccharides can also be used.

[0032] As a method for artificially imparting or enhancing the ability of a microorganism used as a parent strain to supply lactose from sugar, examples include (a) a method of relaxing or releasing at least one mechanism for controlling the biosynthetic pathway for generating lactose from sugar, (b) a method of enhancing the expression of at least one enzyme involved in the biosynthetic pathway for generating lactose from sugar, (c) a method of increasing the copy number of at least one gene encoding an enzyme involved in the biosynthetic pathway for generating lactose from sugar, (d) a method of relaxing or releasing at least one mechanism for degrading lactose, (e) a method of weakening or blocking at least one metabolic pathway that branches from the biosynthetic pathway for generating lactose from sugar to a metabolite other than the target substance, and the like. The above-known methods can be used alone or in combination.

[0033] Specific examples of the enzyme involved in the biosynthetic pathway for generating lactose from sugar include known enzymes such as an enzyme having lactose synthase activity that generates lactose using glucose and UDP-galactose as substrates.

[0034] Specific examples of the method for imparting or enhancing the ability to supply lactose include known methods such as a method of reducing or inactivating the activity of β-galactosidase involved in the degradation of lactose (Metabolic Engineering (2017) 41:23-38).

[0035] Any microorganism having the ability to produce lactose-containing oligosaccharides may be used, but preferably a prokaryote or yeast strain is used, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, or Candida, most preferably Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (manufactured by Merck Millipore), 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 Inc.), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli W3110S (Kyow34, National BioResource Project), Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli ATCC9637, Escherichia coli KY3591 (Accession number: NITE BP-03062), Serratia ficaria, 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.Examples include prokaryotes such as D-0110, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.

[0036] The above-mentioned Escherichia coli KY3591 was deposited at the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 5-8 Kazusa Kamashidome 2-chome, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818). The receipt date (deposit date) was November 18, Reiwa 1 (2019 AD), and the deposit number is NITE BP-03062.

[0037] Microorganisms having the protein of (1) or (2) above prepared using the above host strain, to which the ability to produce lactose-containing oligosaccharides has been subsequently imparted or enhanced, and microorganisms to which the ability to supply lactose has been further imparted or enhanced, and which have a higher ability to produce lactose-containing oligosaccharides than the parent strain, are also the microorganisms of the present invention.

[0038] Examples of the microorganism having the protein described in (1) or (2) above include, for example, a microorganism obtained by transforming a parent strain with a recombinant DNA having the DNA described in any one of the following (3) to (6), and a microorganism obtained by integrating the recombinant DNA into a chromosome. (3) DNA encoding the protein described in (1) or (2) above (4) DNA encoding a protein consisting of an amino acid sequence containing a substitution of an amino acid residue corresponding to the 319th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 with another amino acid residue in the amino acid sequence of the protein encoded by the DNA described in any one of the following [4] to [6] [4] DNA having the base sequence represented by SEQ ID NO: 1 [5] DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a homologous protein having lactose permease activity [6] DNA consisting of a base sequence having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 1 and encodes a homologous protein having lactose permease activity (5) The DNA according to (4) above, which encodes a protein consisting of an amino acid sequence including a substitution to L-glutamic acid from the amino acid residue corresponding to the 319th position of the amino acid sequence represented by SEQ ID NO: 2 in the amino acid sequence encoded thereby (6) DNA having the base sequence represented by SEQ ID NO: 3

[0039] In the above, hybridization means a step in which DNA hybridizes with DNA having a specific base sequence or a part of the DNA. Therefore, the base sequence of the DNA that hybridizes with the DNA having the specific base sequence or a part of the DNA may be DNA having a length useful as a probe for Northern or Southern blot analysis or can be used as an oligonucleotide primer for PCR analysis.

[0040] Examples of the DNA used as a probe include DNA of at least 100 bases or more, preferably 200 bases or more, and more preferably 500 bases or more. Examples of the DNA used as a primer include DNA of at least 10 bases or more, preferably 15 bases or more.

[0041] Methods for DNA hybridization experiments are well-known. For example, in addition to Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press (2012)), Methods for General and Molecular Bacteriology (ASM Press (1994)), Immunology methods manual (Academic press (1997)), hybridization conditions can be determined and experiments can be conducted according to many other standard textbooks.

[0042] Also, DNA that hybridizes under stringent conditions can be obtained by following the instructions attached to commercially available hybridization kits. Examples of commercially available hybridization kits include the Random Prime DNA Labeling Kit (manufactured by Roche Diagnostics) that prepares probes by the random prime method and performs hybridization under stringent conditions.

[0043] The above-mentioned stringent conditions include, for example, incubating a filter immobilized with DNA and probe DNA overnight at 42°C in a solution containing 50% formamide, 5×SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg / L of denatured salmon sperm DNA, and then washing the filter in, for example, a 0.2×SSC solution at about 65°C.

[0044] The various conditions described above can also be set by adding or changing the blocking reagent used to suppress the background of the hybridization experiment. The addition of the above-mentioned blocking reagent may be accompanied by a change in the hybridization conditions to adapt the conditions.

[0045] Examples of DNA that can hybridize under the stringent conditions described above include DNA consisting of a nucleotide sequence having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 when calculated based on the above parameters using programs such as BLAST and FASTA described above.

[0046] The parent strain, a microorganism obtained by transforming the recombinant DNA having the DNA described in any one of (3) to (6) above, and a microorganism obtained by integrating the recombinant DNA into the chromosome can be created by the following method.

[0047] The DNA described in any one of (3) to (6) above can be obtained, for example, by using DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and introducing a mutation into the nucleotide sequence portion encoding the amino acid residue corresponding to the 319th position of the amino acid sequence represented by SEQ ID NO: 2 located on the DNA by a site-directed mutagenesis method described in, for example, Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press (2012)) and Current Protocols in Molecular Biology (JOHN WILEY & SONS, INC.) and substituting it with a nucleotide sequence encoding an arbitrary amino acid residue. Alternatively, the DNA of the present invention can also be obtained by using PrimeSTAR Mutagenesis Basal Kit (manufactured by Takara Bio Inc.).

[0048] In the same manner, a mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having lactose permease activity, and DNA encoding the mutant protein are used. When the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence of the mutant protein are aligned by the above method, a mutation can also be introduced into the nucleotide sequence of the portion encoding the amino acid residue corresponding to the 319th amino acid of the amino acid sequence represented by SEQ ID NO: 2 in the amino acid sequence of the mutant protein.

[0049] Further, DNA encoding a homologous protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having lactose permease activity is used. When the amino acid sequence of the homologous protein and the amino acid sequence represented by SEQ ID NO: 2 are aligned by the above method, a mutation can also be introduced into the nucleotide sequence of the portion encoding the amino acid residue corresponding to the 319th amino acid of the amino acid sequence represented by SEQ ID NO: 2 in the amino acid sequence of the homologous protein.

[0050] The DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 can be obtained, for example, by Southern hybridization to a chromosomal DNA library of a microorganism, preferably a genus Escherichia, more preferably Escherichia coli W3110 strain, using a probe designed based on the nucleotide sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or by PCR [PCR Protocols, Academic Press (1990)] using chromosomal DNA of the Escherichia coli W3110 strain as a template and using primer DNA designed based on the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

[0051] The Escherichia coli W3110 (ATCC27325) strain can be obtained from the American Type Culture Collection (ATCC).

[0052] Examples of the DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO:2 include DNA consisting of the nucleotide sequence represented by SEQ ID NO:1.

[0053] The DNA encoding a mutant protein which is a mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO:2 described in the above [2] and which has lactose permease activity can be obtained, for example, by subjecting DNA consisting of the nucleotide sequence represented by SEQ ID NO:1 to error-prone PCR or the like using the DNA as a template.

[0054] Alternatively, DNA encoding a mutant protein which is 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:2 described in the above [2] and which has lactose permease activity can also be obtained by PCR [Gene, 77, 51 (1989)] using a set of PCR primers each having a nucleotide sequence designed to introduce a desired mutation (deletion, substitution, insertion or addition) at the 5' end.

[0055] In addition, the DNA can also be obtained by following the instructions attached to a commercially available site-directed mutagenesis kit. Examples of the commercially available site-directed mutagenesis kit include PrimeSTAR (registered trademark) Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) which can introduce a mutation (deletion, substitution, insertion or addition) at a position where a desired mutation is to be introduced.

[0056] That is, first, a pair of mutagenesis primers with a 15-base overlap on the 5' side is designed using a plasmid having a nucleotide sequence designed to introduce a desired mutation (deletion, substitution, insertion or addition) as a template. At this time, the desired mutation is included in the overlapping portion. Next, PCR is performed using the mutagenesis primers with a plasmid having a nucleotide sequence into which the desired mutation is to be introduced as a template. When the amplified fragment thus obtained is transformed into Escherichia coli, a plasmid having a nucleotide sequence into which the desired mutation has been introduced can be obtained.

[0057] The DNA encoding a homologous protein that consists of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and has lactose permease activity as described in [3] above can be obtained, for example, by searching various gene sequence databases for a nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and using a probe DNA or primer DNA that can be designed based on the nucleotide sequence or amino acid sequence obtained by the search, and a microorganism having the DNA, in a method similar to the method for obtaining the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 above. The identity of the nucleotide sequence and amino acid sequence can be determined by the same method as described above.

[0058] The recombinant DNA having the DNA described in any one of (3) to (6) above refers to, for example, an expression vector in which the DNA is a DNA capable of autonomous replication in the parent strain and contains a promoter at a position where the DNA described in any one of (3) to (6) above can be transcribed, and the DNA described in any one or more of (3) to (6) above is incorporated.

[0059] A DNA that can be integrated into the chromosome in the parent strain, and the recombinant DNA described in any one of (3) to (6) above is also a recombinant DNA having the DNA described in any one of (3) to (6) above.

[0060] When the recombinant DNA is a recombinant DNA capable of integration into the chromosome, it may not contain a promoter.

[0061] When using a prokaryote such as bacteria as the host strain, the recombinant DNA capable of autonomous replication in the parent strain is preferably a recombinant DNA composed of a promoter, a ribosome binding sequence, the DNA described in any one or more of (3) to (6) above, and a transcription termination sequence. A gene controlling the promoter may be included.

[0062] It is preferable to adjust the distance between the Shine-Dalgarno sequence, which is a ribosome-binding sequence, and the start codon to an appropriate distance, for example, 6 to 18 bases.

[0063] In a recombinant DNA capable of autonomous replication in a parental strain, a transcription termination sequence is not necessarily required for the expression of the DNA, but it is preferable to arrange a transcription termination sequence immediately downstream of the structural gene.

[0064] When using a microorganism belonging to the genus Escherichia as a parent strain, examples of expression vectors include pColdI, pSTV28, pUC118 (all manufactured by Takara Bio Inc.), pET21a, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE-60, pQE80L (all manufactured by Qiagen), pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (all manufactured by Agilent Technologies), pKYP10 (Japanese Patent 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)], 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, p6378-6385], pPE167 (Appl. Environ. Microbiol. 2007, 73:6378-6385), pPAC31 (International Publication No. 98 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (Japanese Patent Laid-Open No. 63-233798), and the like.

[0065] When using the above expression vector, any promoter can be used as long as it functions in the cells of microorganisms belonging to the genus Escherichia. For example, promoters derived from Escherichia coli, phages, etc., such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, PSE promoter, etc. can be used. Also, artificially designed and modified promoters such as a promoter with two trp promoters in series, tac promoter, trc promoter, lacT5 promoter, lacT7 promoter, let I promoter can also be used.

[0066] When using a coryneform bacterium as the parent strain, examples of the expression vector include pCG1 (Japanese Patent Laid-Open No. 57-134500), pCG2 (Japanese Patent Laid-Open No. 58-35197), pCG4 (Japanese Patent Laid-Open No. 57-183799), pCG11 (Japanese Patent Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all of which are Japanese Patent Laid-Open No. 58-105999), pCE51, pCE52, pCE53 [all of which are Molecular and General Genetics, 196, 175 (1984)], etc.

[0067] When using the above expression vector, any promoter can be used as long as it functions in the cells of coryneform bacteria. For example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, p674-679 (2000)] can be used.

[0068] When using a yeast strain as the parent strain, examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, etc.

[0069] When using the above expression vector, any promoter can be used as long as it functions in the cells of the yeast strain. For example, promoters such as the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP1 promoter, etc. can be mentioned.

[0070] The microorganism obtained by transforming the parent strain with the recombinant DNA refers to a microorganism in which the recombinant DNA is introduced as a plasmid capable of autonomous replication in the parent strain or integrated into the chromosome of the parent strain, so that the DNA is transcribed and the protein encoded by the DNA is produced.

[0071] As a method for confirming that the DNA described in the above (3) to (6) is transcribed and that the protein encoded by the DNA is produced, for example, the transcription level of the DNA can be measured by Northern blotting, or the production level of the protein can be measured by Western blotting.

[0072] The DNA described in the above (3) to (6) obtained can be used as it is, or cut with an appropriate restriction enzyme, etc., integrated into a vector by a conventional method, and after introducing the obtained recombinant DNA into a host cell, the nucleotide sequence of the DNA can be determined by using a commonly used nucleotide sequence analysis method, such as the dideoxy method [Proc. Nat. Acad. Sci., USA, 74, 5463 (1977)], or a nucleotide sequence analyzer such as the Applied Biosystems 3500 Genetic Analyzer or the Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific).

[0073] Examples of host cells that can be used when determining the nucleotide sequence of the DNA of the present invention 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 Inc.), 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, Escherichia coli NM522, and the like.

[0074] Examples of the above vectors include pBluescriptII KS(+), pPCR-Script Amp SK(+) (both manufactured by Agilent Technologies), pT7Blue (manufactured by Merck Millipore), pCRII (manufactured by Thermo Fisher Scientific), pCR-TRAP (manufactured by GeneHunter), and pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], and the like.

[0075] As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into a host cell. Examples include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Laid-Open No. 63-248394), an electroporation method [Nucleic Acids Res., 16, 6127 (1988)], and the like.

[0076] As a result of determining the nucleotide sequence, when the obtained DNA is a partial length, the full-length DNA can be obtained by the Southern hybridization method or the like with respect to a chromosomal DNA library using the partial length DNA as a probe.

[0077] Furthermore, based on the determined nucleotide sequence of the DNA, the target DNA can also be prepared by chemical synthesis using a type 8905 DNA synthesizer manufactured by Perceptive Biosystems or the like.

[0078] Here, by substituting the bases so that the nucleotide sequence of the DNA described in the above (3) to (6) becomes a codon optimal for the expression of the parental strain, the expression level of the protein encoded by the DNA can also be improved. Information on the codon usage frequency in host cells can be obtained through public databases.

[0079] By inserting the DNA fragment prepared as described above downstream of the promoter of the appropriate expression vector, a recombinant DNA possessed by the microorganism of the present invention can be prepared.

[0080] Examples of such recombinant DNA include pYHA2 described later in the Examples.

[0081] Examples of methods for introducing recombinant DNA as a plasmid capable of autonomous replication in the parental strain include the methods using calcium ions, the protoplast method, the electroporation method, and the like described above.

[0082] Examples of methods for integrating recombinant DNA into the chromosome of a parental strain include, for example, homologous recombination methods. Examples of homologous recombination methods include, for example, a method using a plasmid for homologous recombination that can be prepared by ligating with a plasmid DNA having a drug resistance gene that cannot autonomously replicate in the host cell to be introduced. In addition, as a method using homologous recombination frequently used in Escherichia coli, for example, a method of introducing recombinant DNA using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)] can be mentioned.

[0083] Furthermore, a selection method utilizing the fact that Escherichia coli becomes sucrose-sensitive due to Bacillus subtilis levansucrase integrated onto the chromosome together with the recombinant DNA, or a selection method utilizing the fact that Escherichia coli becomes streptomycin-sensitive by integrating the wild-type rpsL gene into Escherichia coli having a mutant rpsL gene resistant to streptomycin [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)] etc. can be used to obtain Escherichia coli in which the target region on the chromosomal DNA of the host cell has been replaced with the recombinant DNA.

[0084] That the microorganism created by the above method is a microorganism with a higher ability to produce lactose-containing oligosaccharides than the parental strain can be confirmed, for example, by culturing the parental strain and the created microorganism in a medium and comparing the production amounts of lactose-containing oligosaccharides. An example of such a microorganism is the KFL / pYHA2 strain described later in the examples.

[0085] 2. Method for producing fucose-containing oligosaccharides of the present invention The method for producing fucose-containing oligosaccharides of the present invention includes a method for producing fucose-containing oligosaccharides, characterized by culturing the microorganism of 1 above in a medium and generating fucose-containing oligosaccharides in the culture.

[0086] The method for culturing the microorganism of 1 above can be carried out according to the usual methods used for culturing microorganisms. As the medium for culturing the microorganism, any of natural media and synthetic media may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that the microorganism can assimilate and can efficiently culture the microorganism.

[0087] As the carbon source, any substance that the microorganism can assimilate may be used. For example, sugars such as glucose, fructose, sucrose, molasses containing these, starch or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol or propanol can be used.

[0088] As the nitrogen source, for example, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate or ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermented microbial cells and their digests, etc. can be used.

[0089] Examples of the inorganic salts include potassium dihydrogen phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc.

[0090] The culture is usually preferably carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is usually 15 to 40 °C, and the culture time is usually 5 hours to 7 days. The pH of the culture solution during culture is usually maintained at 3.0 to 9.0. The pH adjustment is carried out using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0091] Also, if necessary during cultivation, antibiotics such as ampicillin and tetracycline may be added to the medium. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added to the medium, and when culturing a microorganism transformed with an expression vector using the trp promoter, indoleacrylic acid or the like may be added to the medium.

[0092] In the above cultivation, if necessary, a precursor necessary for the production of lactose-containing oligosaccharides such as lactose and N-acetyl lactosamine may be added to the medium.

[0093] By the above cultivation, lactose-containing oligosaccharides are produced and accumulated in the culture, and lactose-containing oligosaccharides can be produced by collecting lactose-containing oligosaccharides from the culture.

[0094] The produced lactose-containing oligosaccharides can be analyzed by ordinary methods using carbohydrate ion chromatography or the like. The collection of lactose-containing oligosaccharides from the above culture or a processed product of the culture can be carried out by ordinary methods using activated carbon, ion exchange resin or the like. When lactose-containing oligosaccharides accumulate in the cells, for example, the cells are disrupted by ultrasonic waves or the like, and the cells are removed by centrifugation, and lactose-containing oligosaccharides can be collected from the supernatant obtained by using activated carbon, ion exchange resin or the like.

[0095] [Analysis Example] In the examples, the analysis and quantification of 2'-fucosyllactose were performed according to the following procedure. The culture solution containing the microorganism after cultivation was centrifuged, and the supernatant was collected. The 2'-fucosyllactose contained in the supernatant was analyzed using a sugar analyzer ICS-5000 (manufactured by Thermo Fisher Scientific). [Analysis Conditions] Column: CarboPAC PA1 Column temperature: 25 °C Mobile phase: (Mobile phase A) water (Mobile phase B) 500 mmol / L sodium hydroxide (Mobile phase C) 300 mmol / L sodium acetate Mixing ratio of mobile phase A, mobile phase B and mobile phase C: (0 - 10 minutes) Gradient from 80:20:0 to 70:20:10 (10 - 18 minutes) 70:20:10 (18 - 25 minutes) 80:20:0 Flow rate: 0.8 mL / min Detector: Pulsed amperometry detector

Example

[0096] Details of the examples are shown below, but the present invention is not limited to these examples.

[0097] [Example 1] Construction of microorganisms used for the production of 2'-fucosyllactose (1) Acquisition of DNA fragment used as a marker during gene deletion Using the DNA consisting of the base sequence represented by the "primer set" in Table 1 as a primer set, PCR was performed using the DNA described in the "template" in Table 1 as a template to obtain each amplified DNA fragment.

[0098]

Table 1

[0099] The genomic DNA of Bacillus subtilis 168 strain was prepared by a conventional method. Cat of the amplified DNA fragment contains approximately 200 bp upstream and approximately 50 bp downstream of the cat gene on pHSG396. SacB of the amplified DNA fragment contains approximately 300 bp upstream and approximately 100 bp downstream of the sacB gene on the genomic DNA of Bacillus subtilis 168 strain.

[0100] Next, using cat and sacB of the amplified DNA fragment as templates, SEQ ID NO 4 and 7PCR was performed using DNA consisting of the nucleotide sequence represented by as a primer set to obtain a DNA fragment (hereinafter referred to as cat-sacB) containing the cat gene and the sacB gene.

[0101] (2) Construction of Escherichia coli lacking β-galactosidase activity, lactose permease activity, and cholanic acid production activity Escherichia coli lacking DNA encoding β-galactosidase (hereinafter referred to as the lacZ gene), DNA encoding lactose permease (hereinafter referred to as the lacY gene), and DNA encoding a cholanic acid production-related protein (hereinafter referred to as the wcaJ, wzxC, wcaK, wcaL, or wcaM gene) was constructed by the following method. Note that lacZ and lacY (hereinafter referred to as lacZY), and wcaJ, wzxC, wcaK, wcaL, and wcaM (hereinafter referred to as wcaJ-wzxC-wcaKLM) form operons on the Escherichia coli genome, respectively.

[0102] Using the genomic DNA of Escherichia coli KY3591 strain prepared by a conventional method as a template, PCR was performed using DNA consisting of the nucleotide sequence represented by "Primer Set" in Table 2 as a primer set to obtain each amplified DNA fragment.

[0103]

Table 2

[0104] lacZ upstream 1 and lacZ upstream 2 contain approximately 900 bp upstream from the start codon of the lacZ gene. lacY downstream 1 and lacY downstream 2 contain approximately 800 bp downstream from the stop codon of the lacY gene.

[0105] Using a mixture of lacZ upstream 1, lacY downstream 1, and the cat-sacB fragment in an equimolar ratio as a template, SEQ ID NO 9 and 11PCR was performed using DNA consisting of the nucleotide sequence represented by as a primer set, and a DNA fragment consisting of a sequence in which the cat-sacB fragment was inserted into the sequences around the lacZ and lacY genes (hereinafter referred to as lacZY::cat-sacB) was obtained.

[0106] A mixture of equimolar ratios of 2 upstream of lacZ and 2 downstream of lacY was used as a template, and PCR was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NO. 9 and 11 as a primer set to obtain a DNA fragment consisting of a sequence in which the upstream of lacZ and the downstream of lacY are directly linked without including lacZ and lacY (hereinafter referred to as ΔlacZY).

[0107] The lacZY::cat-sacB fragment was introduced into Escherichia coli W3110 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 and lacY genes were replaced with lacZY::cat-sacB) was obtained.

[0108] The ΔlacZY fragment was introduced into the transformant by electroporation to obtain a transformant showing chloramphenicol sensitivity and sucrose resistance (a transformant in which lacZY::cat-sacB was replaced with ΔlacZY). Among them, a transformant showing ampicillin sensitivity (a transformant in which pKD46 was lost) was further obtained. The transformant was named W3110ΔlacZY.

[0109] Similarly, using the genomic DNA of Escherichia coli KY3591 strain (accession number: NITE BP-03062) as a template, PCR was performed using DNA consisting of the nucleotide sequences represented by "primer set" in Table 3 as a primer set to obtain each amplified DNA fragment.

[0110]

Table 3

[0111] wcaJ upstream 1 and wcaJ upstream 2 contain approximately 900 bp upstream from the start codon of the wcaJ gene. wcaM downstream 1 and wcaM downstream 2 contain approximately 800 bp downstream from the stop codon of the wcaM gene.

[0112] Using as a template a mixture of wcaJ upstream 1, wcaM downstream 1, and the cat-sacB fragment in an equimolar ratio, PCR was performed using a DNA consisting of the nucleotide sequences represented by SEQ ID NO. 15 and 17 as a primer set, and a DNA fragment (hereinafter referred to as wcaJ-wzxC-wcaKLM::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence around the wcaJ-wzxC-wcaKLM operon was obtained.

[0113] Using as a template a mixture of wcaJ upstream 2 and wcaM downstream 2 in an equimolar ratio, PCR was performed using a DNA consisting of the nucleotide sequences represented by SEQ ID NO. 15 and 17 as a primer set, and a DNA fragment (hereinafter referred to as ΔwcaJ-wzxC-wcaKLM) consisting of a sequence in which the upstream of wcaJ and the downstream of wcaM are directly linked without including wcaJ-wzxC-wcaKLM was obtained.

[0114] The wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110ΔlacZY strain constructed above by the electroporation method, and a transformant (a transformant in which wcaJ-wzxC-wcaKLM was replaced with wcaJ-wzxC-wcaKLM::cat-sacB) showing chloramphenicol resistance and sucrose sensitivity was obtained.

[0115] The ΔwcaJM fragment was introduced into the transformant by electroporation, and a transformant showing chloramphenicol sensitivity and sucrose resistance (a transformant in which wcaJ-wzxC-wcaKLM::cat-sacB was replaced with ΔwcaJ-wzxC-wcaKLM) was obtained. Furthermore, a transformant showing ampicillin sensitivity (a transformant in which pKD46 was lost) was obtained. The transformant was named the KFL strain.

[0116] (3) Preparation of a lactose permease (LacY) expression vector derived from Escherichia coli Using the DNA consisting of the base sequences represented by the "primer set" in Table 4 as a primer set and the DNA described in the "template" in Table 4 as a template, PCR was performed to obtain each amplified DNA fragment.

[0117]

Table 4

[0118] The genomic DNA of Escherichia coli W3110 strain was prepared by a conventional method. Also, Array numbers 21 and 22, 23 and 24 the base sequences represented by contain sequences complementary to their respective 5'-ends.

[0119] Using a mixture of lacY, HMFT, and rcsA fragments in an equimolar ratio as a template and the DNA consisting of the base sequences represented by 26 and 27 as a primer set, PCR was performed to obtain a DNA fragment in which three fragments were ligated (hereinafter referred to as lacY-HMFT-rcsA).

[0120] Using the DNA consisting of the base sequences represented by 28 and 29 as a primer set, PCR was performed using plasmid pPE167 (Appl. Environ. Microbiol. 2007, 73: 6378-6385) as a template to obtain a vector fragment of about 4.4 kb. At this time, 26 and 29 , 27and 28 The nucleotide sequences represented by each contain a sequence complementary to the 5'-end thereof.

[0121] The lacY-HMFT-rcsA fragment and the vector fragment obtained above were ligated using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain the expression plasmid pYHA1.

[0122] (4) Construction of a microorganism expressing mutant LacY Using the plasmid pYHA1 obtained in (2) above as a template and the Prime STAR Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), plasmid pYHA2 was prepared in which the 319th L-lysine residue in the amino acid sequence of LacY was substituted with an L-glutamic acid residue. The DNA consisting of the nucleotide sequences represented by SEQ ID NO. 30 and 31 was used for the primer set.

[0123] Using the plasmid pYHA1 obtained in (3) above and the above pYHA2, the KFL strain constructed in (2) above was transformed to obtain the KFL / pYHA1 strain and the KFL / pYHA2 strain.

[0124] [Example 2] Production of 2'-fucosyllactose by a fermentation method using a microorganism expressing mutant LacY The KFL / pYHA1 strain and KFL / pYHA2 strain obtained in Example 1 were cultured on an LB plate at 30°C for 24 hours, inoculated into a large test tube containing 5 mL of LB medium containing 100 mg / L of kanamycin, and cultured with shaking at 30°C for 16 hours. Then, the obtained culture solution was inoculated with 0.1 mL into a large test tube containing 5 mL of a production medium [30 g / L of glucose, 5 g / L of lactose monohydrate, 2 g / L of magnesium sulfate heptahydrate, 16 g / L of dipotassium hydrogen phosphate, 14 g / L of potassium dihydrogen phosphate, 2 g / L of ammonium sulfate, 1 g / L of citric acid monohydrate, 5 g / L of casamino acids, 10 mg / L of thiamine hydrochloride, 50 mg / L of ferrous sulfate heptahydrate, 10 mg / L of manganese sulfate pentahydrate (except for glucose, lactose monohydrate, and magnesium sulfate heptahydrate, adjusted to pH 7.2 with an aqueous sodium hydroxide solution and then autoclaved) (aqueous solutions containing glucose, lactose monohydrate, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and then mixed)], and cultured with shaking at 30°C for 30 hours.

[0125] After the culture was completed, the culture solution was appropriately diluted and then centrifuged, and the 2'-fucosyllactose contained in the supernatant was analyzed by HPLC. The results are shown in Table 5.

[0126]

Table 5

[0127] As a result, the KFL / pYHA2 strain showed higher 2'-fucosyllactose productivity than the KFL / pYHA1 strain.

[0128] From the above, it was found that by using a microorganism having a mutant LacY, the productivity of 2'-fucosyllactose is improved compared to a microorganism having a wild-type LacY.

[0129] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on Japanese Patent Application No. 2019-226278 filed on Dec. 16, 2019, the entire disclosure of which is incorporated herein by reference. Also, all references cited herein are incorporated by reference in their entirety.

Industrial Applicability

[0130] The present invention provides a method for producing lactose-containing oligosaccharides using a microorganism having the ability to produce a protein having modified lactase permease activity.

Claims

1. A microorganism having a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 319th position of the amino acid sequence of the protein described in any one of the following [1] to [3] is substituted with L-glutamic acid, and A microorganism having a higher ability to produce lactose-containing oligosaccharides than the parent strain. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2, and having lactose permease activity. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having lactose permease activity.

2. The microorganism according to claim 1, obtained by transforming a parent strain with a recombinant DNA containing a DNA encoding a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 319th position of the amino acid sequence of the protein described in any one of the above [1] to [3] is substituted with L-glutamic acid.

3. The microorganism according to claim 1 or 2, obtained by integrating a recombinant DNA containing a DNA encoding a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 319th position of the amino acid sequence of the protein described in any one of the above [1] to [3] is substituted with L-glutamic acid into the chromosome.

4. The microorganism according to any one of claims 1 to 3, obtained by transforming a parent strain with a recombinant DNA containing a DNA having the base sequence represented by SEQ ID NO:

3.

5. The microorganism according to claim 4, obtained by integrating a recombinant DNA containing a DNA having the base sequence represented by SEQ ID NO: 3 into the chromosome.

6. The microorganism according to any one of claims 1 to 5, wherein the parent strain is a microorganism having the ability to produce lactose-containing oligosaccharides.

7. A method for producing lactose-containing oligosaccharides, characterized by culturing the microorganism according to any one of claims 1 to 6 in a medium to produce lactose-containing oligosaccharides in the culture.

8. The production method according to claim 7, wherein the lactose-containing oligosaccharide is 2'-fucosyllactose.

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